[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Oncol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Oncol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Oncol.</journal-id><journal-title-group><journal-title>Frontiers in Oncology</journal-title></journal-title-group><issn pub-type=\"epub\">2234-943X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850445</article-id><article-id pub-id-type=\"pmc\">PMC7431518</article-id><article-id pub-id-type=\"doi\">10.3389/fonc.2020.01383</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Oncology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Prognostic Molecular Signatures for Metastatic Potential in Clinically Low-Risk Stage I and II Clear Cell Renal Cell Carcinomas</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Shih</surname><given-names>Andrew J.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/538851/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Murphy</surname><given-names>Neal</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"author-notes\" rid=\"fn003\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Kozel</surname><given-names>Zachary</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/984403/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Shah</surname><given-names>Paras</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Yaskiv</surname><given-names>Oksana</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/954327/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Khalili</surname><given-names>Houman</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Liew</surname><given-names>Anthony</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Kavoussi</surname><given-names>Louis</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Hall</surname><given-names>Simon</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Vira</surname><given-names>Manish</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/954305/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zhu</surname><given-names>Xin-Hua</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff7\"><sup>7</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><xref ref-type=\"author-notes\" rid=\"fn003\"><sup>‡</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1019214/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Lee</surname><given-names>Annette T.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn003\"><sup>‡</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/982435/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Feinstein Institutes for Medical Research</institution>, <addr-line>Manhasset, NY</addr-line>, <country>United States</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Donald and Barbara Zucker School of Medicine at Hofstra/Northwell</institution>, <addr-line>Hempstead, NY</addr-line>, <country>United States</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Division of Hospital Medicine, LIJ Medical Center</institution>, <addr-line>New Hyde Park, NY</addr-line>, <country>United States</country></aff><aff id=\"aff4\"><sup>4</sup><institution>The Smith Institute for Urology</institution>, <addr-line>New Hyde Park, NY</addr-line>, <country>United States</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Department of Urology, Mayo Clinic</institution>, <addr-line>Rochester, MN</addr-line>, <country>United States</country></aff><aff id=\"aff6\"><sup>6</sup><institution>Northwell Health Department of Pathology</institution>, <addr-line>New Hyde Park, NY</addr-line>, <country>United States</country></aff><aff id=\"aff7\"><sup>7</sup><institution>Northwell Health Cancer Institute</institution>, <addr-line>Lake Success, NY</addr-line>, <country>United States</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Ronald M. Bukowski, Cleveland Clinic, United States</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Dharmesh Gopalakrishnan, University at Buffalo, United States; Moshe C. Ornstein, Cleveland Clinic, United States</p></fn><corresp id=\"c001\">*Correspondence: Andrew J. Shih <email>ashih@northwell.edu</email></corresp><corresp id=\"c002\">Xin-Hua Zhu <email>xzhu1@northwell.edu</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Genitourinary Oncology, a section of the journal Frontiers in Oncology</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†These authors have contributed equally to this work and share first authorship</p></fn><fn fn-type=\"other\" id=\"fn003\"><p>‡These authors have contributed equally to this work and share last authorship</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>1383</elocation-id><history><date date-type=\"received\"><day>15</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>30</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Shih, Murphy, Kozel, Shah, Yaskiv, Khalili, Liew, Kavoussi, Hall, Vira, Zhu and Lee.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Shih, Murphy, Kozel, Shah, Yaskiv, Khalili, Liew, Kavoussi, Hall, Vira, Zhu and Lee</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p><bold>Introduction:</bold> For patients with localized node-negative (Stage I and II) clear cell renal cell carcinomas (ccRCC), current clinicopathological staging has limited predictive capability because of their low risk. Analyzing molecular signatures at the time of nephrectomy can aid in understanding future metastatic potential.</p><p><bold>Objective:</bold> Develop a molecular signature that can stratify patients who have clinically low risk ccRCC, but have high risk genetic changes driving an aggressive metastatic phenotype.</p><p><bold>Patients, Materials, and Methods:</bold> Presented is the differential expression of mRNA and miRNA in 44 Stage I and Stage II patients, 21 who developed metastasis within 5 years of nephrectomy, compared to 23 patients who remained disease free for more than 5 years. Extracted RNA from nephrectomy specimens preserved in FFPE blocks was sequenced using RNAseq. MiRNA expression was performed using the TaqMan OpenArray qPCR protocol.</p><p><bold>Results:</bold> One hundred thirty one genes and 2 miRNA were differentially expressed between the two groups. Canonical correlation (CC) analysis was applied and four CCs (CC32, CC20, CC9, and CC7) have an AUC > 0.65 in our dataset with similar predictive power in the TCGA-KIRC dataset. Gene set enrichment showed CC9 as kidney development/adhesion, CC20 as oxidative phosphorylation pathway, CC32 as RNA binding/spindle and CC7 as immune response. In a multivariate Cox model, the four CCs were able to identify high/low risk groups for metastases in the TCGA-KIRC (<italic>p</italic> < 0.05) with odds ratios of CC32 = 5.7, CC20 = 4.4, CC9 = 3.6, and CC7 = 2.7.</p><p><bold>Conclusion:</bold> These results identify molecular signatures for more aggressive tumors in clinically low risk ccRCC patients who have a higher potential of metastasis than would be expected.</p></abstract><kwd-group><kwd>clear cell</kwd><kwd>molecular biomarker</kwd><kwd>renal cell carcinoma</kwd><kwd>gene expression</kwd><kwd>miRNA</kwd><kwd>canonical correlation analysis</kwd></kwd-group><counts><fig-count count=\"3\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"51\"/><page-count count=\"9\"/><word-count count=\"6456\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>In 2020, the American Cancer Society estimates 74,000 new cases of renal cell carcinoma will be diagnosed and account for ~15,000 deaths, putting renal cell carcinoma in the top ten leading cause of cancer deaths in the United States (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Of the three major subtypes (clear cell, papillary, and chromophobe) clear cell renal cell carcinoma (ccRCC) is the most prevalent comprising 75% of diagnosed cases and resulting in more deaths per year than other subtypes (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>).</p><p>For most ccRCC patients with early stage localized tumor, stage I or II, surgical resection offers achievable survival rates over 95% (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). However, for a small percentage of clinically low risk patients disease recurrence ensues in 5–10% stage I and 15–20% stage II patients. Several models exist to assess risk for disease recurrence after nephrectomy including: MSKCC postoperative prognostic nomogram (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>), UCLA UISS score (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>) and Mayo clinic SSIGN score (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). These scoring systems rely on clinical parameters such as tumor grade, size, presence of necrosis, and patient functional status that often do not accurately differentiate early stage clinically low risk disease. Only tumor size has the highest prognostic accuracy for future development of metastatic disease (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>).</p><p>While new treatments have increased survival, <20% of patients diagnosed with metastatic disease have a median survival of >27 months (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). Kuijpers et al. estimated that 71% of stage I and 52% of stage II RCC patients who developed metastatic disease following surgery were potentially curable, reasoning that early detection of metastatic disease would lead to more successful treatment (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). However, Dabestani et al. reported that intensive follow-up after nephrectomy does not improve overall survival after recurrence (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>).</p><p>Therefore, patients with clinicopathologically defined low risk ccRCC (stage I and II) who develop a distant recurrence within 5 years represent a unique cohort of patients, where understanding the biology of the key molecular factors driving progression to metastatic disease can help supplement clinical parameters. Given that there are a relatively small number of people who develop metastasis in early stage ccRCC, we chose to match a similar number to have a balanced dataset that can capture as many molecular signals as possible. Here we present an analysis of a similar number of tumor samples from stage I and II ccRCC patients who developed metastatic disease within 5 years of surgery and from patients who did not develop metastatic disease in more than 5 years to capture the molecular signatures that associate with metastatic potential at an early time point as possible. Furthermore, we validated these signatures in The Cancer Genome Atlas ccRCC (TCGA-KIRC) cohort.</p></sec><sec id=\"s2\"><title>Patients, Materials, and Methods</title><sec><title>Patient Selection</title><p>Patients from 2008-2012 who had metachronous metastases within 5 years with a primary diagnosis of stage I or II ccRCC in the Northwell Health tumor bank were selected. A similar number of patients were selected who did not have recurrence as a comparison group to maximize the molecular signatures captured. The ccRCC tissue samples in the discovery set were obtained from the Northwell Health pathology department as formalin-fixed, paraffin-embedded (FFPE) tissue blocks. Samples were initially matched on size, gender and age with two samples removed for QC issues following RNA extraction.</p><p>TNM classification and the Fuhrman grading system were used to stage and grade tumor samples (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>, <xref rid=\"B10\" ref-type=\"bibr\">10</xref>). The Northwell Health System Regional Ethics Committee granted research approval for the study with waivers of HIPAA Authorization and informed consent. The validation set consisted of 218 Stage I and 45 Stage II patients from the TCGA-KIRC Data Resource (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>).</p></sec><sec><title>RNA Extraction</title><p>A genitourinary pathologist (O. Yaskiv) reviewed FFPE tissue blocks and their corresponding H&E slides. Slides were matched up with tissue blocks and ~35 mg of the pre-identified tumor were excised. RNA was extracted using the RecoverAll<sup>TM</sup> Total Nucleic Acid Isolation Kit according to manufacturer's instructions. RNA was evaluated using an AB Bioanalyzer.</p></sec><sec><title>mRNA Sequencing and Expression Analysis</title><p>Libraries were prepared using the Illumina TruSeq RNA Access Library kit and sequenced on a NextSeq 500 following the manufacturer's instructions. Sequenced segments were aligned with STAR2 (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>) to the Human GENCODE reference (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Gene counts were assessed using ht-seq counts (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>). Differential expression was calculated using DESeq2 which is designed to analyze digital count data generated by sequencing (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). Log fold changes were shrunk using apeglm (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Pathway enrichment analysis was done using GAGE (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>).</p></sec><sec><title>Cell Subset Deconvolution</title><p>Composition of cell subsets of our bulk RNA-seq ccRCC was determined with CIBERSORT (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>) using a previously published ccRCC single cell RNA-seq dataset (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>) as a reference. Only cell populations present in >1% of ccRCC samples were considered. Correlations of cell populations to metastatic phenotype was done using MASC (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>).</p></sec><sec><title>miRNA RT-qPCR and Expression Analysis</title><p>Quantification of miRNA was done using the TaqMan OpenArray, which profiles 754 known miRNAs using qPCR. A Ct threshold of 30 was used for detectable level of expression. MiRNAs were further filtered by removing those with no expression in any samples and samples were filtered that had <10% of miRNAs detected. Housekeeping miRNAs were the top 10 most stable miRNAs from the NormqPCR package (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>), which were used to normalize raw Ct values using ΔCt normalization (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). Differential expression was calculated using limma which is designed to analyze log2 based expression assays like qPCR (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>).</p></sec><sec><title>Canonical Correlation Analysis and Validation</title><p><italic>De novo</italic> mRNA and miRNA modules or Canonical Components (CCs) were identified in the discovery set using sparse canonical correlation analysis (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>) with AUC to metastatic status calculated using pROC (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Data analysis was done using R (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>) and tidyverse (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). RNA-seq and microRNA-seq count data for validation were done in the TCGA-KIRC dataset which was downloaded, then normalized using DESeq2. Phenotypes were defined using the TCGA Pan-Cancer Clinical Data Resource (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). The specific gene and microRNA weights calculated for CCs in the discovery set were applied to the validation set. Thresholds for risk stratification in the discovery set on the discovered CCs were done using rpart (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>) and then applied to the validation set to create high and low risk groups. Cox regression of the high and low risk groups in the validation set to their disease free survival time was done using survminer (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>).</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>Demographics</title><p>The discovery set consisted of 24 patients with Stage I and 20 patients with Stage II ccRCC who underwent either partial or radical nephrectomy. In Stage I, 13 patients had more than 5.5 years of follow-up with no evidence of metastasis while 11 developed metastatic relapse within 5 years; for Stage II, 10 patients did not develop metastases, while 10 patients developed metastatic disease (see <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Two samples did not meet QC metrics for microRNA. The population was 16% female (16.7% in stage I and 15% in stage II). The average age at surgery for the entire group was 61.3 ± 10.3 years with no significant differences between stage I (62.2 ± 8.4 years) and stage II (60.3 ± 12.5 years).</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Demographics of patients in this study.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" colspan=\"2\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Stage I</bold></th><th valign=\"top\" align=\"center\" colspan=\"2\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Stage II</bold></th></tr><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Metastatic (11)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Non-metastatic (13)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Metastatic (10)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Non-metastatic (10)</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Women</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (9.1%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3 (23.1%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (20%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (20%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Size (cm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.7 ± 1.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.9 ± 1.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.7 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11 ± 3.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low grade (I–II)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9 (81.8%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11 (84.6%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (40%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5 (50%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Age at surgery</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">63.9 ± 6.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">61.8 ± 9.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">58.8 ± 11.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">64 ± 13.7</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MSKCC score</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91.5 ± 1.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91.3 ± 1.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">69.7 ± 10.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">68.5 ± 11.9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">UISS score</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">89.2 ± 4.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">89.5 ± 4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80.4 ± 0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80.4 ± 0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recurrence time</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.7 ± 20.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17.1 ± 19.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Radical Nephrectomy<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><break/>2/9 (22.2%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><break/>3/13 (23.1%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><break/>6/6 (100%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><break/>4/4 (100%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Margins<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Neg (9/9)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Neg (13/13)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Neg (6/6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Neg (4/4)</td></tr></tbody></table><table-wrap-foot><fn id=\"TN1\"><label>*</label><p><italic>Nephrectomy and margin status were not available for all patients</italic>.</p></fn></table-wrap-foot></table-wrap><p>For stage I patients, metastases were evident at 29.7 ± 20.3 months, and the average tumor size of Stage I was 4.78 ± 1.14 cm with 83.3% low grade (I–II) and 16.7% high grade (III–IV). While patients with stage II disease recurred in 17.1 ± 19.8 months, with an average tumor size of 10.82 ± 2.98 cm with 45% low grade and 55% high grade.</p><p>There was no significant difference in age, tumor size, grade, radical vs. partial nephrectomy, margin status, UISS and MSKCC recurrence score in patients who developed metastases compared to those who did not: age at surgery (<italic>p</italic>-value stage I=0.54 and stage II=0.36), tumor size (<italic>p</italic>-value stage I=0.71 and stage II=0.82), tumor grade (<italic>p</italic>-value stage I=0.57 and stage II=0.75), radical vs. partial nephrectomy (<italic>p</italic>-value stage I=1 and stage II=1), margin status (<italic>p</italic>-value stage I=1 and stage II=1), UISS recurrence score (<italic>p</italic>-value stage I=0.86 and stage II=1), or MSKCC recurrence score (<italic>p</italic>-value stage I=0.81 and stage II=0.82).</p></sec><sec><title>RNAseq Gene Expression Analysis of Stage I and II ccRCC Tumors</title><p>Group comparison of patients who developed metastatic disease vs. those who did not showed 131 genes that were significant after multiple testing correction (see <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Table 1</xref>). Of those genes, the majority of them were seen in patients that had developed metastases (125 genes) as opposed to patients cured by surgery (6 genes). Seventy two of the 125 genes upregulated in tumors from patients who developed metastatic disease were immunoglobulin genes (IGH, IGK, and IGL).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Volcano plot of patients who developed metastases within 5 years vs. those that did not. The x-axis is log 2 fold change between groups while the y-axis is -log 10 raw p-value. The top 20 genes by p-value are labeled.</p></caption><graphic xlink:href=\"fonc-10-01383-g0001\"/></fig><p>The model used to develop the molecular signature of metastasis in ccRCC used variables that took into account the contribution of stage and gender to remove those covariates from contributing to the signal for metastases. There were 267 stage specific genes with adjusted <italic>p</italic> < 0.05 with absolute value log 2 fold change > 1; 253 of these were seen up-regulated in stage II while 14 genes were seen to be up-regulated in stage I, see <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Table 2</xref>. In gender, 166 genes were found to pass similar thresholds, with 150 genes higher in males with 16 genes higher in females (see <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Table 3</xref>). Notably, many of the genes found are gender specific like Y chromosome specific genes (i.e., UTY, DDX3Y, USP9Y, and RPS4Y1) being higher in males or XIST, the X-inactive specific transcript, being higher in females.</p><p>Sub-group analysis focused on male-specific or female-specific signals found a high correlation between test statistics of the male subgroup vs. the full data (y ~ 0.94x, R<sup>2</sup>= 0.86) compared to those of the female sub-group (y~0.18x, R<sup>2</sup>= 0.039), see <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>. In the male-specific subgroup analysis 8 genes had an adjusted p-value threshold of 0.05 that was higher than 0.1 in the full group analysis: CNDB2, COL8A2, EPHB2, KRT16, PRSS21, SOGA3, SPINK5, and TMEM63C. In female-specific subgroup analysis, only three genes matched the same criteria, CTHRC1, BCL2L14, and AGBL4.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Comparison and correlation of the metastatic test statistic in the full dataset in a sub-group analysis for gender: (left) males and (right) females.</p></caption><graphic xlink:href=\"fonc-10-01383-g0002\"/></fig><p>Deconvolution of the RNA-seq data into cell subsets resulted in 28 distinct cell types (see <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Table 4</xref>. None of the cell subpopulations were associated with patients who developed metastases with <italic>p</italic> < 0.05.</p></sec><sec><title>miRNA Expression Analysis of Stage I and II ccRCC Tumors</title><p>Since miRNA is much sparser (only 214 miRNA passed QC thresholds, see <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Table 5</xref>) the adjusted p-value threshold for miRNA significance was lowered to 0.25. Only two miRNAs were found to be significant at this threshold. MiR-18a and miR-301 were found to be upregulated in patients who developed metastatic disease. Gender and stage were also taken into account in differential expression analysis of miRNA, see <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Tables 6, 7</xref>. For gender, there were no miRNA that passed adjusted <italic>p</italic> < 0.25. For stage, 157 total miRNA had adjusted <italic>p</italic> < 0.25 with 121 associated with stage II and 29 associated with stage I.</p></sec><sec><title>Canonical Correlation Analysis With mRNA and miRNA Expression Datasets</title><p>Sparse canonical correlation analysis (CCA) is able to identify similar correlation structures between two orthogonal datasets and redefine them as canonical components (CCs). It is analogous to Principal Component Analysis (PCA) with two orthogonal multi-dimensional datasets; making linear combinations of variables that have the highest correlation across datasets. CCs can be thought of as <italic>de novo</italic> modules of genes and miRNAs that have correlated expression patterns with 42 total CCs identified where expression of both can be combined into a single score (see <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Table 8</xref>). Univariate logistic regression was performed on canonical components to classify patients who developed metastatic disease. The top performing canonical component (CC32) had an AUC of 0.77 (see <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). To assign potential function to CCs, a gene enrichment analysis, GAGE, was used on Gene Ontology (GO) terms with an adjusted p-value threshold of 0.2. CC32 was enriched in GO terms related to RNA binding and spindle function, CC20 was enriched in oxidative-phosphorylation genes, CC9 was enriched in kidney development while CC7 was enriched in immune terms.</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Canonical components (CCs) with AUC > 0.65 in both the discovery and validation datasets.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Canonical Component</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Discovery AUC</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>TCGA AUC</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>GO terms</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Adjusted P-Value</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CC32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.74</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RNA BINDING</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.36E-01</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CC32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.74</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SPINDLE</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.36E-01</td></tr><tr><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">CC20</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.69</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.76</td><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">MITOCHONDRION</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">3.64E-19</td></tr><tr><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">CC20</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.69</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.76</td><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">MITOCHONDRIAL PART</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">1.29E-16</td></tr><tr><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">CC20</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.69</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.76</td><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">CELLULAR RESPIRATION</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">8.69E-15</td></tr><tr><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">CC20</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.69</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.76</td><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">SMALL MOLECULE METABOLIC PROCESS</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">1.44E-14</td></tr><tr><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">CC20</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.69</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.76</td><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">OXIDATION REDUCTION PROCESS</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">1.61E-13</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CC9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.68</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.74</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MESONEPHROS DEVELOPMENT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.68E-01</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CC9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.68</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.74</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BIOLOGICAL ADHESION</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.68E-01</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CC9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.68</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.74</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HOMOPHILIC CELL ADHESION VIA PLASMA MEMBRANE ADHESION MOLECULES</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.68E-01</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CC9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.68</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.74</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">KIDNEY EPITHELIUM DEVELOPMENT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.68E-01</td></tr><tr><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">CC7</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.65</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.68</td><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">REGULATION OF IMMUNE RESPONSE</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">8.15E-05</td></tr><tr><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">CC7</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.65</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.68</td><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">EXTERNAL SIDE OF PLASMA MEMBRANE</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">2.42E-04</td></tr><tr><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">CC7</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.65</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.68</td><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">REGULATION OF IMMUNE SYSTEM PROCESS</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">2.42E-04</td></tr><tr><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">CC7</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.65</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.68</td><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">ADAPTIVE IMMUNE RESPONSE</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">3.02E-04</td></tr><tr><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">CC7</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.65</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">0.68</td><td valign=\"top\" align=\"left\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">IMMUNOGLOBULIN COMPLEX</td><td valign=\"top\" align=\"center\" style=\"background-color:#f8f8f6\" rowspan=\"1\" colspan=\"1\">7.04E-04</td></tr></tbody></table></table-wrap></sec><sec><title>Validation in TCGA-KIRC</title><p>A subset of the publically available RNA-seq and microRNA-seq TCGA-KIRC datasets (stage I and II only) was used as validation to evaluate the accuracy of the canonical components identified. After matching RNA-seq and microRNA-seq samples and removing multiple samples from the same patient, the validation set had 263 patients total. Of those, 218 were stage I with 16 of those having recurrences, while of the 45 stage II samples 7 recurred. Gene and microRNA weights for the CC score calculated in the discovery set were applied to the validation set. Both datasets were mean normalized to 0 and standard deviation normalized to 1. The four identified CCs with AUC≥0.65 in the discovery set also had an AUC≥0.65 in TCGA-KIRC.</p><p>To determine accuracy of the CCs, the discovery dataset was used to set thresholds in order to stratify TCGA-KIRC into high risk and low risk groups. Thresholds were set using decision trees in the discovery dataset to maximize separation between groups. These thresholds were applied to TCGA-KIRC to separate them; thresholds for high risk were CC9 <0.02, CC20>0.52, CC32>0.23 and CC7 < -0.04. A cox regression was performed with these high and low risk groups in TCGA-KIRC based on their progression free survival for each CC. The recurrence rate in the validation set was ~9%; assuming an equal split between high and low risk groups (13% recurrence and 5%, respectively), an odds ratio of 2.5 has a power of 0.81. All cox regression analyses were found to be significant (p-values CC9 = 6.8E-3, CC20 = 9.4E-4, CC32 = 1.5E-4, and CC7 = 0.021, see <xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>) with an odds ratio >2.5 (OR CC32 = 5.7, CC20 = 4.4, CC9 = 3.6, and CC7 = 2.7).</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Kaplan-Meier Curves of the high and low risk groups determined by top four CCs. The 95% confidence interval plotted with each group as the lightly shaded area.</p></caption><graphic xlink:href=\"fonc-10-01383-g0003\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Here we examined tumors from clinically low risk ccRCC for molecular signatures to understand metastatic potential. Risk stratification using clinicopathological parameters in these patients has limited predictive power. In our dataset, we see no statistical significance between MSKCC and UISS scores in patients who do or do not develop metastatic disease in our cohort. Our low risk ccRCC patient cohort who develop metastasis offers an early time point where molecular signatures can supplement clinical information for better risk stratification. To increase molecular signatures found we chose to use similar numbers of patients who did and did not develop future metastatic disease to have a balanced dataset. We report several multigene and miRNA signatures generated by CCA that show clinical utility in stratifying clinically low risk ccRCC tumors.</p><p>To the best of our knowledge, this is the first report analyzing the combined differential mRNA and miRNA expression between clinically low risk stage I and II ccRCC with vastly different outcomes. Several recent studies have used gene expression analysis to create stratification scoring systems in RCC: Rini et al. found a 16 gene assay to predict recurrence in stage I-III ccRCC using gene expression across almost 1000 samples (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>), Brooks et al. developed ClearCode34 from 72 ccRCC samples (stage I-III) to identify low risk (ccA) and high risk (ccB) groups (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>), Morgan et al. examined 31 cell cycle related genes across 565 patients of clear cell, papillary, or chromophobe RCC to create the R-CCP panel (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>). However, they included stage III ccRCC patients and did not use miRNA in conjunction with gene expression. In stage III ccRCC, the tumor is no longer completely isolated to the kidney and has already spread either to the most proximal lymph node, to a major vein or to the tissue surrounding the kidney, which indicates a more aggressive phenotype and increased risk. Therefore, the results found in our discovery population and validation in TCGA-KIRC are a novel and a promising step toward identifying molecular signatures of clinicopathologically defined low risk ccRCC that develop future metastasis.</p><p>In our analysis, several immunoglobulin genes were upregulated in tumors that became metastatic. IgG is overexpressed in ccRCCs in comparison to adjacent normal tissue affecting cell proliferation, migration and invasion (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). Immunoglobulin genes have been shown to be active and expressed in many non-B epithelial cancer cells (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). MZB1, which is overexpressed in our metastatic group, has been shown to be necessary for immunoglobulin synthesis (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). It also has an immune regulatory effect that has a survival benefit in other cancers (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>), though in RCC high expression of MZB1 is unfavorable (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B39\" ref-type=\"bibr\">39</xref>). Similarly IL1R2, which has higher expression in our metastatic samples, is a mock receptor of IL1R that regulates immune response through competitive inhibition, and has an important role in cancer progression (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>). Furthermore, our CC7 module is enriched in immune response GO terms. While expression of immune genes is normally associated with immune infiltrating cells, we did not see the future development of metastasis correlate with any specific immune cell type or any cell at all when our bulk RNA-seq data was deconvoluted into cell types via CIBERSORT. These data show that the immune system may play a role in promoting a pro-tumor environment early in patients who develop metastatic disease.</p><p>In addition to the immune module (CC7), other <italic>de novo</italic> mRNA-miRNA modules have functions related to cancer growth. CC9 was enriched in GO terms for kidney development and cell adhesion. Enrichment of kidney epithelium terms directly links this module to ccRCC cells, which predominantly are kidney epithelial cells. One of the two miRNAs passed our adjusted p-value threshold, miR-18a, promotes proliferation and inhibits apoptosis in kidney cancer cell lines and is associated with worse overall survival in RCC (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>). Furthermore, this particular GO term is related to development from embryos, which implies a more stem-like state compared to a mature kidney, similar to an epithelial-mesenchymal transition. The other microRNA that passed our adjusted p-value threshold, miR-301 which had higher expression in those that developed metastasis, has been shown to be increased in microvesicles released by human renal cancer stem cells to stimulate angiogenesis to prepare the metastatic niche (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>). TGFBI is also overexpressed in our metastatic discovery set and has been shown to induce epithelial to mesenchymal transition (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>) as well as being associated with ccRCC tumor progression and poor prognosis (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>).</p><p>One of the hallmarks of ccRCC is the metabolic reprogramming of oxidative-phosphorylation pathways leading to accumulation of lipids and glycogen in the cytoplasm supporting a shift in metabolism known as the Warburg effect (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>). Analysis of TCGA-KIRC showed worse survival is associated with upregulation of fatty acid synthesis genes and pentose phosphate pathway genes while better survival was associated with Krebs cycle genes (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). CC20 in our dataset is enriched in cellular respiration and oxidative-phosphorylation terms, and we have shown has predictive value for metastatic potential. One gene related to solute transport that we also see upregulated in the metastatic group is SLC38A5, which alkalinizes tumor cells and promotes growth; it is also a transcriptional target for the oncogene c-Myc (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>).</p><p>Interestingly, there is a gender disparity in the prevalence of RCC, about 2:1 male to female, that is consistent across age, year, and region (<xref rid=\"B47\" ref-type=\"bibr\">47</xref>). Men are more likely to have a higher grade tumor and are more likely to develop metastases, while women showed a benefit in overall survival (<xref rid=\"B48\" ref-type=\"bibr\">48</xref>). Some studies have examined mutational differences between men and women in ccRCC, showing that stratification by gender showed BAP1 mutations have a female-specific poorer outcome (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>). Similarly, Tan and colleagues also showed expression levels of FABP7 and BRN2 had prognostic value in women (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>). Many of the treatment and stratification options bias toward male because of the increased prevalence, which is also present in our dataset (~84% male). We did have three genes that were differentially expressed in women that were not seen in our overall dataset: CTHRC1, BCL2L14 (overexpressed in metastatic patients) and AGBL4 (underexpressed in metastatic patients). CTHRC1 knockdown has been shown to reduce proliferation and epithelial-to-mesenchymal transition (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>) and increased expression is associated with a poorer prognosis (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B39\" ref-type=\"bibr\">39</xref>). BCL2L14 is a member of the BCL2 family and AGBL4 is an ATP/GTP binding protein, both without relevance to RCC to the best of our knowledge, but given our results further study may be warranted, particularly in women.</p><p>While our study focuses on early stage ccRCC, we have applied previous molecular panels to our dataset with mixed results, likely because of the equal number of patients who would develop metastases vs. those that did not in a low risk cohort (data not shown). We also applied our CCs to stage III patients in TCGA with two of the CCs (CC32: RNA Binding/Spindle and CC20: Oxphos) having the lowest p-value of ~0.08, implying these are worth further study for metastatic progression across all risk groups. One common limitation regarding genomic-based predictive markers is intratumor heterogeneity. However, CCA takes into account similarly correlated genes and miRNA across samples to make <italic>de novo</italic> modules or pathways that are less susceptible to cellular heterogeneity. Furthermore, bulk deconvolution using cell subset techniques did not show any cell type associated with metastases. Using TCGA-KIRC for validation may have been limited due to the fact that the TCGA-KIRC has local and distant recurrence of disease in their dataset when our cohort consisted only of distant metastases. However, 7% of stage I and 15% of stage II in TCGA-KIRC recurred which is similar to the reported distant metastatic rate, making local recurrences less likely.</p></sec><sec sec-type=\"conclusions\" id=\"s5\"><title>Conclusions</title><p>Our results highlight molecular signatures that can risk stratify patients for metastatic potential in clinically low risk ccRCC patients. Our modules provide a potential mechanistic pathway for development of metastases, of which the immune module and immunoglobulin genes are of particular interest. With further validation, the combined mRNA and miRNA modules could be used to improve treatment and survival outcomes for this group of patients.</p></sec><sec sec-type=\"data-availability\" id=\"s6\"><title>Data Availability Statement</title><p>The datasets generated and analyzed for this study can be found in the Gene Expression Omnibus at identifier GEO: <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"GSE155210\">GSE155210</ext-link>.</p></sec><sec id=\"s7\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by The Northwell Health System Regional Ethics Committee. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.</p></sec><sec id=\"s8\"><title>Author Contributions</title><p>ZK, PS, X-HZ, and ATL conceived and designed the study. OY, HK, and AL acquired the data. AS analyzed and interpreted the data. AS, NM, and ATL drafted the manuscript. AS, NM, ZK, LK, SH, MV, X-HZ, and ATL critically revised the manuscript for important intellectual content. AS did the statistical analysis. OY, HK, AL, X-HZ, and ATL provided administrative, technical, and material support. ATL obtained funding and supervised the study. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s9\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>We are exceptionally grateful to the patients and their families. We also gratefully acknowledge Peter K. Gregersen for his critical reading of the manuscript.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> Funding for this project was done with institutional funds.</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s10\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fonc.2020.01383/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fonc.2020.01383/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Data_Sheet_1.xls\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Siegel</surname><given-names>RL</given-names></name><name><surname>Miller</surname><given-names>KD</given-names></name><name><surname>Jemal</surname><given-names>A</given-names></name></person-group>\n<article-title>Cancer statistics, 2020</article-title>. <source>CA Cancer J Clin.</source> (<year>2020</year>) <volume>70</volume>:<fpage>7</fpage>–<lpage>30</lpage>. <pub-id pub-id-type=\"doi\">10.3322/caac.21590</pub-id><pub-id pub-id-type=\"pmid\">31912902</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kattan</surname><given-names>MW</given-names></name><name><surname>Reuter</surname><given-names>V</given-names></name><name><surname>Motzer</surname><given-names>RJ</given-names></name><name><surname>Katz</surname><given-names>J</given-names></name><name><surname>Russo</surname><given-names>P</given-names></name></person-group>. <article-title>A postoperative prognostic nomogram for renal cell carcinoma</article-title>. <source>J Urol.</source> (<year>2001</year>) <volume>166</volume>:<fpage>63</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0022-5347(05)66077-6</pub-id><pub-id pub-id-type=\"pmid\">11435824</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zisman</surname><given-names>A</given-names></name><name><surname>Pantuck</surname><given-names>AJ</given-names></name><name><surname>Dorey</surname><given-names>F</given-names></name><name><surname>Said</surname><given-names>JW</given-names></name><name><surname>Shvarts</surname><given-names>O</given-names></name><name><surname>Quintana</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Improved prognostication of renal cell carcinoma using an integrated staging system</article-title>. <source>J Clin Oncol.</source> (<year>2001</year>) <volume>19</volume>:<fpage>1649</fpage>–<lpage>57</lpage>. <pub-id pub-id-type=\"doi\">10.1200/JCO.2001.19.6.1649</pub-id><pub-id pub-id-type=\"pmid\">11250993</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Frank</surname><given-names>I</given-names></name><name><surname>Blute</surname><given-names>ML</given-names></name><name><surname>Cheville</surname><given-names>JC</given-names></name><name><surname>Lohse</surname><given-names>CM</given-names></name><name><surname>Weaver</surname><given-names>AL</given-names></name><name><surname>Zincke</surname><given-names>H</given-names></name></person-group>. <article-title>An outcome prediction model for patients with clear cell renal cell carcinoma treated with radical nephrectomy based on tumor stage, size, grade and necrosis: the SSIGN score</article-title>. <source>J Urol.</source> (<year>2002</year>) <volume>168</volume>:<fpage>2395</fpage>–<lpage>400</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0022-5347(05)64153-5</pub-id><pub-id pub-id-type=\"pmid\">12441925</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ku</surname><given-names>JH</given-names></name><name><surname>Moon</surname><given-names>KC</given-names></name><name><surname>Kwak</surname><given-names>C</given-names></name><name><surname>Kim</surname><given-names>HH</given-names></name></person-group>. <article-title>Metachronous metastatic potential of small renal cell carcinoma: dependence on tumor size</article-title>. <source>Urology.</source> (<year>2009</year>) <volume>74</volume>:<fpage>1271</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.urology.2009.04.072</pub-id><pub-id pub-id-type=\"pmid\">19616288</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wiechno</surname><given-names>P</given-names></name><name><surname>Kucharz</surname><given-names>J</given-names></name><name><surname>Sadowska</surname><given-names>M</given-names></name><name><surname>Michalski</surname><given-names>W</given-names></name><name><surname>Sikora-Kupis</surname><given-names>B</given-names></name><name><surname>Jonska-Gmyrek</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Contemporary treatment of metastatic renal cell carcinoma</article-title>. <source>Med Oncol.</source> (<year>2018</year>) <volume>35</volume>:<fpage>156</fpage>. <pub-id pub-id-type=\"doi\">10.1007/s12032-018-1217-1</pub-id><pub-id pub-id-type=\"pmid\">30368624</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuijpers</surname><given-names>YAM</given-names></name><name><surname>Meijer</surname><given-names>RP</given-names></name><name><surname>Jonges</surname><given-names>GN</given-names></name><name><surname>de Jong</surname><given-names>J</given-names></name><name><surname>Bosch</surname><given-names>JLHR</given-names></name><name><surname>Horenblas</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Potentially curable recurrent disease after surgically managed non-metastatic renal cell carcinoma in low-, intermediate- and high-risk patients</article-title>. <source>World J Urol.</source> (<year>2016</year>) <volume>34</volume>:<fpage>1073</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00345-016-1822-6</pub-id><pub-id pub-id-type=\"pmid\">27055532</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dabestani</surname><given-names>S</given-names></name><name><surname>Beisland</surname><given-names>C</given-names></name><name><surname>Stewart</surname><given-names>GD</given-names></name><name><surname>Bensalah</surname><given-names>K</given-names></name><name><surname>Gudmundsson</surname><given-names>E</given-names></name><name><surname>Lam</surname><given-names>TB</given-names></name><etal/></person-group>\n<article-title>Intensive imaging-based follow-up of surgically treated localised renal cell carcinoma does not improve post-recurrence survival: Results from a European Multicentre Database (RECUR)</article-title>. <source>Eur Urol.</source> (<year>2019</year>) <volume>75</volume>:<fpage>261</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.eururo.2018.10.007</pub-id><pub-id pub-id-type=\"pmid\">30318330</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><name><surname>Amin</surname><given-names>MB</given-names></name><name><surname>Edge</surname><given-names>SB</given-names></name><name><surname>Greene</surname><given-names>FL</given-names></name><name><surname>Byrd</surname><given-names>DR</given-names></name><name><surname>Brookland</surname><given-names>RK</given-names></name><name><surname>Washington</surname><given-names>MK</given-names></name><etal/></person-group>\n<source>AJCC Cancer Staging Manual</source>. Springer (<year>2018</year>). Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://books.google.com/books/about/AJCC_Cancer_Staging_Manual.html?hl=&id=O2PyjwEACAAJ\">https://books.google.com/books/about/AJCC_Cancer_Staging_Manual.html?hl=&id=O2PyjwEACAAJ</ext-link> (accessed February, 2020).</mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fuhrman</surname><given-names>SA</given-names></name><name><surname>Lasky</surname><given-names>LC</given-names></name><name><surname>Limas</surname><given-names>C</given-names></name></person-group>. <article-title>Prognostic significance of morphologic parameters in renal cell carcinoma</article-title>. <source>Am J Surg Pathol.</source> (<year>1982</year>) <volume>6</volume>:<fpage>655</fpage>–<lpage>63</lpage>. <pub-id pub-id-type=\"doi\">10.1097/00000478-198210000-00007</pub-id><pub-id pub-id-type=\"pmid\">7180965</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Network</surname><given-names>TCGAR</given-names></name></person-group>. <article-title>The cancer genome atlas research network. comprehensive molecular characterization of clear cell renal cell carcinoma</article-title>. <source>Nature.</source> (<year>2013</year>) <volume>499</volume>:<fpage>43</fpage>–<lpage>49</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature12222</pub-id><pub-id pub-id-type=\"pmid\">23792563</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dobin</surname><given-names>A</given-names></name><name><surname>Gingeras</surname><given-names>TR</given-names></name></person-group>. <article-title>Mapping RNA-seq reads with STAR</article-title>. <source>Curr Protoc Bioinformatics.</source> (<year>2015</year>) <volume>51</volume>:<fpage>11.14.1</fpage>–<lpage>19</lpage>. <pub-id pub-id-type=\"doi\">10.1002/0471250953.bi1114s51</pub-id><pub-id pub-id-type=\"pmid\">26334920</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Frankish</surname><given-names>A</given-names></name><name><surname>Diekhans</surname><given-names>M</given-names></name><name><surname>Ferreira</surname><given-names>A-M</given-names></name><name><surname>Johnson</surname><given-names>R</given-names></name><name><surname>Jungreis</surname><given-names>I</given-names></name><name><surname>Loveland</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>GENCODE reference annotation for the human and mouse genomes</article-title>. <source>Nucleic Acids Res.</source> (<year>2019</year>) <volume>47</volume>:<fpage>D766</fpage>–<lpage>73</lpage>. <pub-id pub-id-type=\"doi\">10.1093/nar/gky955</pub-id><pub-id pub-id-type=\"pmid\">30357393</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Anders</surname><given-names>S</given-names></name><name><surname>Pyl</surname><given-names>PT</given-names></name><name><surname>Huber</surname><given-names>W</given-names></name></person-group>. <article-title>HTSeq–a python framework to work with high-throughput sequencing data</article-title>. <source>Bioinformatics.</source> (<year>2015</year>) <volume>31</volume>:<fpage>166</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bioinformatics/btu638</pub-id><pub-id pub-id-type=\"pmid\">25260700</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Love</surname><given-names>MI</given-names></name><name><surname>Huber</surname><given-names>W</given-names></name><name><surname>Anders</surname><given-names>S</given-names></name></person-group>. <article-title>Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2</article-title>. <source>Genome Biol.</source> (<year>2014</year>) <volume>15</volume>:<fpage>550</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s13059-014-0550-8</pub-id><pub-id pub-id-type=\"pmid\">25516281</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhu</surname><given-names>A</given-names></name><name><surname>Ibrahim</surname><given-names>JG</given-names></name><name><surname>Love</surname><given-names>MI</given-names></name></person-group>. <article-title>Heavy-tailed prior distributions for sequence count data: removing the noise and preserving large differences</article-title>. <source>Bioinformatics.</source> (<year>2019</year>) <volume>35</volume>:<fpage>2084</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bioinformatics/bty895</pub-id><pub-id pub-id-type=\"pmid\">30395178</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luo</surname><given-names>W</given-names></name><name><surname>Friedman</surname><given-names>MS</given-names></name><name><surname>Shedden</surname><given-names>K</given-names></name><name><surname>Hankenson</surname><given-names>KD</given-names></name><name><surname>Woolf</surname><given-names>PJ</given-names></name></person-group>. <article-title>GAGE: generally applicable gene set enrichment for pathway analysis</article-title>. <source>BMC Bioinformatics.</source> (<year>2009</year>) <volume>10</volume>:<fpage>161</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1471-2105-10-161</pub-id><pub-id pub-id-type=\"pmid\">19473525</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Newman</surname><given-names>AM</given-names></name><name><surname>Liu</surname><given-names>CL</given-names></name><name><surname>Green</surname><given-names>MR</given-names></name><name><surname>Gentles</surname><given-names>AJ</given-names></name><name><surname>Feng</surname><given-names>W</given-names></name><name><surname>Xu</surname><given-names>Y</given-names></name><etal/></person-group>. <article-title>Robust enumeration of cell subsets from tissue expression profiles</article-title>. <source>Nat Methods.</source> (<year>2015</year>) <volume>12</volume>:<fpage>453</fpage>–<lpage>57</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nmeth.3337</pub-id><pub-id pub-id-type=\"pmid\">25822800</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Young</surname><given-names>MD</given-names></name><name><surname>Mitchell</surname><given-names>TJ</given-names></name><name><surname>Vieira Braga</surname><given-names>FA</given-names></name><name><surname>Tran</surname><given-names>MGB</given-names></name><name><surname>Stewart</surname><given-names>BJ</given-names></name><name><surname>Ferdinand</surname><given-names>JR</given-names></name><etal/></person-group>. <article-title>Single-cell transcriptomes from human kidneys reveal the cellular identity of renal tumors</article-title>. <source>Science.</source> (<year>2018</year>) <volume>361</volume>:<fpage>594</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1126/science.aat1699</pub-id><pub-id pub-id-type=\"pmid\">30093597</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fonseka</surname><given-names>CY</given-names></name><name><surname>Rao</surname><given-names>DA</given-names></name><name><surname>Teslovich</surname><given-names>NC</given-names></name><name><surname>Korsunsky</surname><given-names>I</given-names></name><name><surname>Hannes</surname><given-names>SK</given-names></name><name><surname>Slowikowski</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Mixed-effects association of single cells identifies an expanded effector CD4 T cell subset in rheumatoid arthritis</article-title>. <source>Sci Transl Med.</source> (<year>2018</year>) <volume>10</volume>:<fpage>eaaq0305</fpage>. <pub-id pub-id-type=\"doi\">10.1126/scitranslmed.aaq0305</pub-id><pub-id pub-id-type=\"pmid\">30333237</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Perkins</surname><given-names>JR</given-names></name><name><surname>Dawes</surname><given-names>JM</given-names></name><name><surname>McMahon</surname><given-names>SB</given-names></name><name><surname>Bennett</surname><given-names>DLH</given-names></name><name><surname>Orengo</surname><given-names>C</given-names></name><name><surname>Kohl</surname><given-names>M</given-names></name></person-group>. <article-title>ReadqPCR and NormqPCR: R packages for the reading, quality checking and normalisation of RT-qPCR quantification cycle (Cq) data</article-title>. <source>BMC Genomics.</source> (<year>2012</year>) <volume>13</volume>:<fpage>296</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1471-2164-13-296</pub-id><pub-id pub-id-type=\"pmid\">22748112</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Livak</surname><given-names>KJ</given-names></name><name><surname>Schmittgen</surname><given-names>TD</given-names></name></person-group>. <article-title>Analysis of relative gene expression data using real-time quantitative PCR and the 2<sup>−ΔΔC(T)</sup> method</article-title>. <source>Methods.</source> (<year>2001</year>) <volume>25</volume>:<fpage>402</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1006/meth.2001.1262</pub-id><pub-id pub-id-type=\"pmid\">11846609</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ritchie</surname><given-names>ME</given-names></name><name><surname>Phipson</surname><given-names>B</given-names></name><name><surname>Wu</surname><given-names>D</given-names></name><name><surname>Hu</surname><given-names>Y</given-names></name><name><surname>Law</surname><given-names>CW</given-names></name><name><surname>Shi</surname><given-names>W</given-names></name><etal/></person-group>. <article-title>limma powers differential expression analyses for RNA-sequencing and microarray studies</article-title>. <source>Nucleic Acids Res.</source> (<year>2015</year>) <volume>43</volume>:<fpage>e47</fpage>. <pub-id pub-id-type=\"doi\">10.1093/nar/gkv007</pub-id><pub-id pub-id-type=\"pmid\">25605792</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Witten</surname><given-names>DM</given-names></name><name><surname>Tibshirani</surname><given-names>RJ</given-names></name></person-group>. <article-title>Extensions of sparse canonical correlation analysis with applications to genomic data</article-title>. <source>Stat Appl Genet Mol Biol.</source> (<year>2009</year>) <volume>8</volume>:<fpage>28</fpage>. <pub-id pub-id-type=\"doi\">10.2202/1544-6115.1470</pub-id><pub-id pub-id-type=\"pmid\">19572827</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Robin</surname><given-names>X</given-names></name><name><surname>Turck</surname><given-names>N</given-names></name><name><surname>Hainard</surname><given-names>A</given-names></name><name><surname>Tiberti</surname><given-names>N</given-names></name><name><surname>Lisacek</surname><given-names>F</given-names></name><name><surname>Sanchez</surname><given-names>J-C</given-names></name><etal/></person-group>. <article-title>pROC: an open-source package for R and S to analyze and compare ROC curves</article-title>. <source>BMC Bioinformatics.</source> (<year>2011</year>) <volume>12</volume>:<fpage>77</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1471-2105-12-77</pub-id><pub-id pub-id-type=\"pmid\">21414208</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><collab>R Core Team</collab></person-group>\n<source>R: A Language and Environment for Statistical Computing</source>. Vienna, Austria: R Foundation for Statistical Computing (<year>2019</year>). Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.R-project.org/\">https://www.R-project.org/</ext-link> (accessed January, 2020).</mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wickham</surname><given-names>H</given-names></name><name><surname>Averick</surname><given-names>M</given-names></name><name><surname>Bryan</surname><given-names>J</given-names></name><name><surname>Chang</surname><given-names>W</given-names></name><name><surname>McGowan</surname><given-names>L</given-names></name><name><surname>François</surname><given-names>R</given-names></name><etal/></person-group>\n<article-title>Welcome to the Tidyverse</article-title>. <source>J Open Source Softw.</source> (<year>2019</year>) <volume>4</volume>:<fpage>1686</fpage>\n<pub-id pub-id-type=\"doi\">10.21105/joss.01686</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>J</given-names></name><name><surname>Lichtenberg</surname><given-names>T</given-names></name><name><surname>Hoadley</surname><given-names>KA</given-names></name><name><surname>Poisson</surname><given-names>LM</given-names></name><name><surname>Lazar</surname><given-names>AJ</given-names></name><name><surname>Cherniack</surname><given-names>AD</given-names></name><etal/></person-group>. <article-title>An integrated TCGA pan-cancer clinical data resource to drive high-quality survival outcome analytics</article-title>. <source>Cell.</source> (<year>2018</year>) <volume>173</volume>:<fpage>400</fpage>–<lpage>16</lpage>.e11. <pub-id pub-id-type=\"doi\">10.1158/1538-7445.AM2018-3287</pub-id><pub-id pub-id-type=\"pmid\">29625055</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><name><surname>Therneau</surname><given-names>T</given-names></name><name><surname>Atkinson</surname><given-names>B</given-names></name></person-group>\n<article-title>Rpart: Recursive Partitioning and Regression Trees</article-title>. (<year>2019</year>). Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://CRAN.R-project.org/package=rpart\">https://CRAN.R-project.org/package=rpart</ext-link> (accessed November, 2019).</mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><name><surname>Kassambara</surname><given-names>A</given-names></name><name><surname>Kosinski</surname><given-names>M</given-names></name><name><surname>Biecek</surname><given-names>P</given-names></name></person-group>\n<article-title>Survminer: drawing survival curves using ggplot2</article-title>. (<year>2019</year>). Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://CRAN.R-project.org/package=survminer\">https://CRAN.R-project.org/package=survminer</ext-link> (accessed November, 2019).</mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rini</surname><given-names>B</given-names></name><name><surname>Goddard</surname><given-names>A</given-names></name><name><surname>Knezevic</surname><given-names>D</given-names></name><name><surname>Maddala</surname><given-names>T</given-names></name><name><surname>Zhou</surname><given-names>M</given-names></name><name><surname>Aydin</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>A 16-gene assay to predict recurrence after surgery in localised renal cell carcinoma: development and validation studies</article-title>. <source>Lancet Oncol.</source> (<year>2015</year>) <volume>16</volume>:<fpage>676</fpage>–<lpage>85</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S1470-2045(15)70167-1</pub-id><pub-id pub-id-type=\"pmid\">25979595</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brooks</surname><given-names>SA</given-names></name><name><surname>Rose Brannon</surname><given-names>A</given-names></name><name><surname>Parker</surname><given-names>JS</given-names></name><name><surname>Fisher</surname><given-names>JC</given-names></name><name><surname>Sen</surname><given-names>O</given-names></name><name><surname>Kattan</surname><given-names>MW</given-names></name><etal/></person-group>\n<article-title>ClearCode34: a prognostic risk predictor for localized clear cell renal cell carcinoma</article-title>. <source>Eur Urol.</source> (<year>2014</year>) <volume>66</volume>:<fpage>77</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.eururo.2014.02.035</pub-id><pub-id pub-id-type=\"pmid\">24613583</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morgan</surname><given-names>TM</given-names></name><name><surname>Mehra</surname><given-names>R</given-names></name><name><surname>Tiemeny</surname><given-names>P</given-names></name><name><surname>Wolf</surname><given-names>JS</given-names></name><name><surname>Wu</surname><given-names>S</given-names></name><name><surname>Sangale</surname><given-names>Z</given-names></name><etal/></person-group>. <article-title>A multigene signature based on cell cycle proliferation improves prediction of mortality within 5 yr of radical nephrectomy for renal cell carcinoma</article-title>. <source>Eur Urol.</source> (<year>2018</year>) <volume>73</volume>:<fpage>763</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.eururo.2017.12.002</pub-id><pub-id pub-id-type=\"pmid\">29249291</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sheng</surname><given-names>Z</given-names></name><name><surname>Liu</surname><given-names>Y</given-names></name><name><surname>Qin</surname><given-names>C</given-names></name><name><surname>Liu</surname><given-names>Z</given-names></name><name><surname>Yuan</surname><given-names>Y</given-names></name><name><surname>Hu</surname><given-names>F</given-names></name><etal/></person-group>. <article-title>IgG is involved in the migration and invasion of clear cell renal cell carcinoma</article-title>. <source>J Clin Pathol.</source> (<year>2016</year>) <volume>69</volume>:<fpage>497</fpage>–<lpage>504</lpage>. <pub-id pub-id-type=\"doi\">10.1136/jclinpath-2015-202881</pub-id><pub-id pub-id-type=\"pmid\">26519488</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhu</surname><given-names>X</given-names></name><name><surname>Wu</surname><given-names>L</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Hao</surname><given-names>P</given-names></name><name><surname>Zhang</surname><given-names>S</given-names></name><name><surname>Huang</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Distinct regulatory mechanism of immunoglobulin gene transcription in epithelial cancer cells</article-title>. <source>Cell Mol Immunol.</source> (<year>2010</year>) <volume>7</volume>:<fpage>279</fpage>–<lpage>86</lpage>. <pub-id pub-id-type=\"doi\">10.1038/cmi.2010.13</pub-id><pub-id pub-id-type=\"pmid\">20473321</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rosenbaum</surname><given-names>M</given-names></name><name><surname>Andreani</surname><given-names>V</given-names></name><name><surname>Kapoor</surname><given-names>T</given-names></name><name><surname>Herp</surname><given-names>S</given-names></name><name><surname>Flach</surname><given-names>H</given-names></name><name><surname>Duchniewicz</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>MZB1 is a GRP94 cochaperone that enables proper immunoglobulin heavy chain biosynthesis upon ER stress</article-title>. <source>Genes Dev.</source> (<year>2014</year>) <volume>28</volume>:<fpage>1165</fpage>–<lpage>78</lpage>. <pub-id pub-id-type=\"doi\">10.1101/gad.240762.114</pub-id><pub-id pub-id-type=\"pmid\">24888588</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kanda</surname><given-names>M</given-names></name><name><surname>Tanaka</surname><given-names>C</given-names></name><name><surname>Kobayashi</surname><given-names>D</given-names></name><name><surname>Tanaka</surname><given-names>H</given-names></name><name><surname>Shimizu</surname><given-names>D</given-names></name><name><surname>Shibata</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Epigenetic suppression of the immunoregulator MZB1 is associated with the malignant phenotype of gastric cancer</article-title>. <source>Int J Cancer.</source> (<year>2016</year>) <volume>139</volume>:<fpage>2290</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1002/ijc.30286</pub-id><pub-id pub-id-type=\"pmid\">27459504</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Miyake</surname><given-names>K</given-names></name><name><surname>Mori</surname><given-names>R</given-names></name><name><surname>Homma</surname><given-names>Y</given-names></name><name><surname>Matsuyama</surname><given-names>R</given-names></name><name><surname>Okayama</surname><given-names>A</given-names></name><name><surname>Murakami</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>MZB1 in borderline resectable pancreatic cancer resected after neoadjuvant chemoradiotherapy</article-title>. <source>J Surg Res.</source> (<year>2017</year>) <volume>220</volume>:<fpage>391</fpage>–<lpage>401</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jss.2017.07.003</pub-id><pub-id pub-id-type=\"pmid\">29180208</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><collab>Human Protein Atlas</collab></person-group> Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.proteinatlas.org\">http://www.proteinatlas.org</ext-link> (accessed June, 2019).</mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mantovani</surname><given-names>A</given-names></name><name><surname>Barajon</surname><given-names>I</given-names></name><name><surname>Garlanda</surname><given-names>C</given-names></name></person-group>. <article-title>IL-1 and IL-1 regulatory pathways in cancer progression and therapy</article-title>. <source>Immunol Rev.</source> (<year>2018</year>) <volume>281</volume>:<fpage>57</fpage>–<lpage>61</lpage>. <pub-id pub-id-type=\"doi\">10.1111/imr.12614</pub-id><pub-id pub-id-type=\"pmid\">29247996</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhou</surname><given-names>L</given-names></name><name><surname>Li</surname><given-names>Z</given-names></name><name><surname>Pan</surname><given-names>X</given-names></name><name><surname>Lai</surname><given-names>Y</given-names></name><name><surname>Quan</surname><given-names>J</given-names></name><name><surname>Zhao</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Identification of miR-18a-5p as an oncogene and prognostic biomarker in RCC</article-title>. <source>Am J Transl Res.</source> (<year>2018</year>) <volume>10</volume>:<fpage>1874</fpage>–<lpage>86</lpage>.<pub-id pub-id-type=\"pmid\">30018727</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grange</surname><given-names>C</given-names></name><name><surname>Tapparo</surname><given-names>M</given-names></name><name><surname>Collino</surname><given-names>F</given-names></name><name><surname>Vitillo</surname><given-names>L</given-names></name><name><surname>Damasco</surname><given-names>C</given-names></name><name><surname>Deregibus</surname><given-names>MC</given-names></name><etal/></person-group>. <article-title>Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche</article-title>. <source>Cancer Res.</source> (<year>2011</year>) <volume>71</volume>:<fpage>5346</fpage>–<lpage>56</lpage>. <pub-id pub-id-type=\"doi\">10.1158/0008-5472.CAN-11-0241</pub-id><pub-id pub-id-type=\"pmid\">21670082</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Heldin</surname><given-names>C-H</given-names></name><name><surname>Landström</surname><given-names>M</given-names></name><name><surname>Moustakas</surname><given-names>A</given-names></name></person-group>. <article-title>Mechanism of TGF-beta signaling to growth arrest, apoptosis, and epithelial-mesenchymal transition</article-title>. <source>Curr Opin Cell Biol.</source> (<year>2009</year>) <volume>21</volume>:<fpage>166</fpage>–<lpage>76</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ceb.2009.01.021</pub-id><pub-id pub-id-type=\"pmid\">19237272</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shang</surname><given-names>D</given-names></name><name><surname>Liu</surname><given-names>Y</given-names></name><name><surname>Yang</surname><given-names>P</given-names></name><name><surname>Chen</surname><given-names>Y</given-names></name><name><surname>Tian</surname><given-names>Y</given-names></name></person-group>. <article-title>TGFBI-promoted adhesion, migration and invasion of human renal cell carcinoma depends on inactivation of von Hippel-Lindau tumor suppressor</article-title>. <source>Urology.</source> (<year>2012</year>) <volume>79</volume>:<fpage>966.e1</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.urology.2011.12.011</pub-id><pub-id pub-id-type=\"pmid\">22341602</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wettersten</surname><given-names>HI</given-names></name><name><surname>Aboud</surname><given-names>OA</given-names></name><name><surname>Lara</surname><given-names>PN</given-names></name><name><surname>Weiss</surname><given-names>RH</given-names></name></person-group>. <article-title>Metabolic reprogramming in clear cell renal cell carcinoma</article-title>. <source>Nat Rev Nephrol.</source> (<year>2017</year>) <volume>13</volume>:<fpage>410</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrneph.2017.59</pub-id><pub-id pub-id-type=\"pmid\">28480903</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bhutia</surname><given-names>YD</given-names></name><name><surname>Ganapathy</surname><given-names>V</given-names></name></person-group>. <article-title>Glutamine transporters in mammalian cells and their functions in physiology and cancer</article-title>. <source>Biochim Biophys Acta.</source> (<year>2016</year>) <volume>1863</volume>:<fpage>2531</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbamcr.2015.12.017</pub-id><pub-id pub-id-type=\"pmid\">26724577</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scelo</surname><given-names>G</given-names></name><name><surname>Li</surname><given-names>P</given-names></name><name><surname>Chanudet</surname><given-names>E</given-names></name><name><surname>Muller</surname><given-names>DC</given-names></name></person-group>. <article-title>Variability of sex disparities in cancer incidence over 30 years: the striking case of kidney cancer</article-title>. <source>Eur Urol Focus.</source> (<year>2018</year>) <volume>4</volume>:<fpage>586</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.euf.2017.01.006</pub-id><pub-id pub-id-type=\"pmid\">28753845</pub-id></mixed-citation></ref><ref id=\"B48\"><label>48.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>May</surname><given-names>M</given-names></name><name><surname>Aziz</surname><given-names>A</given-names></name><name><surname>Zigeuner</surname><given-names>R</given-names></name><name><surname>Chromecki</surname><given-names>T</given-names></name><name><surname>Cindolo</surname><given-names>L</given-names></name><name><surname>Schips</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Gender differences in clinicopathological features and survival in surgically treated patients with renal cell carcinoma: an analysis of the multicenter CORONA database</article-title>. <source>World J Urol.</source> (<year>2013</year>) <volume>31</volume>:<fpage>1073</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00345-013-1071-x</pub-id><pub-id pub-id-type=\"pmid\">23568445</pub-id></mixed-citation></ref><ref id=\"B49\"><label>49.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ricketts</surname><given-names>CJ</given-names></name><name><surname>Linehan</surname><given-names>WM</given-names></name></person-group>. <article-title>Gender specific mutation incidence and survival associations in Clear Cell Renal Cell Carcinoma (CCRCC)</article-title>. <source>PLoS ONE.</source> (<year>2015</year>) <volume>10</volume>:<fpage>e0140257</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0140257</pub-id><pub-id pub-id-type=\"pmid\">26484545</pub-id></mixed-citation></ref><ref id=\"B50\"><label>50.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tan</surname><given-names>C</given-names></name><name><surname>Takayama</surname><given-names>T</given-names></name><name><surname>Takaoka</surname><given-names>N</given-names></name><name><surname>Fujita</surname><given-names>H</given-names></name><name><surname>Miyazaki</surname><given-names>M</given-names></name><name><surname>Sugiyama</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>Impact of gender in renal cell carcinoma: the relationship of FABP7 and BRN2 expression with overall survival</article-title>. <source>Clin Med Insights Oncol.</source> (<year>2014</year>) <volume>8</volume>:<fpage>21</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.4137/CMO.S13684</pub-id><pub-id pub-id-type=\"pmid\">24653654</pub-id></mixed-citation></ref><ref id=\"B51\"><label>51.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jin</surname><given-names>XF</given-names></name><name><surname>Li</surname><given-names>H</given-names></name><name><surname>Zong</surname><given-names>S</given-names></name><name><surname>Li</surname><given-names>HY</given-names></name></person-group>. <article-title>Knockdown of collagen triple helix repeat containing-1 inhibits the proliferation and epithelial-to-mesenchymal transition in renal cell carcinoma cells</article-title>. <source>Oncol Res.</source> (<year>2016</year>) <volume>24</volume>:<fpage>477</fpage>–<lpage>85</lpage>. <pub-id pub-id-type=\"doi\">10.3727/096504016X14685034103716</pub-id><pub-id pub-id-type=\"pmid\">28281968</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Oncol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Oncol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Oncol.</journal-id><journal-title-group><journal-title>Frontiers in Oncology</journal-title></journal-title-group><issn pub-type=\"epub\">2234-943X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850464</article-id><article-id pub-id-type=\"pmc\">PMC7431519</article-id><article-id pub-id-type=\"doi\">10.3389/fonc.2020.01599</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Oncology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Quantification of Myocardial Dosimetry and Glucose Metabolism Using a 17-Segment Model of the Left Ventricle in Esophageal Cancer Patients Receiving Radiotherapy</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Sha</surname><given-names>Xue</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/949057/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Gong</surname><given-names>Guanzhong</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Han</surname><given-names>Chunlei</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Qiu</surname><given-names>Qingtao</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/667044/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Yin</surname><given-names>Yong</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/730363/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences</institution>, <addr-line>Jinan</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Turku PET Centre, Turku University Hospital</institution>, <addr-line>Turku</addr-line>, <country>Finland</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Youyong Kong, Southeast University, China</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Xu Zhiyong, Shanghai Jiao Tong University, China; Jiandong Yin, ShengJing Hospital of China Medical University, China</p></fn><corresp id=\"c001\">*Correspondence: Yong Yin, <email>yinyongsd@126.com</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Cancer Imaging and Image-directed Interventions, a section of the journal Frontiers in Oncology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>1599</elocation-id><history><date date-type=\"received\"><day>09</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>23</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Sha, Gong, Han, Qiu and Yin.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Sha, Gong, Han, Qiu and Yin</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><sec><title>Objective</title><p>Previous studies have shown that increased cardiac uptake of <sup>18</sup>F-fluorodeoxyglucose (FDG) on positron emission tomography (PET) may be an indicator of myocardial injury after radiotherapy (RT). The primary objective of this study was to quantify cardiac subvolume dosimetry and <sup>18</sup>F-FDG uptake on oncologic PET using a 17-segment model of the left ventricle (LV) and to identify dose limits related to changes in cardiac <sup>18</sup>F-FDG uptake after RT.</p></sec><sec><title>Methods</title><p>Twenty-four esophageal cancer (EC) patients who underwent consecutive oncologic <sup>18</sup>F-FDG PET/CT scans at baseline and post-RT were enrolled in this study. The radiation dose and the <sup>18</sup>F-FDG uptake were quantitatively analyzed based on a 17-segment model. The <sup>18</sup>F-FDG uptake and doses to the basal, middle and apical regions, and the changes in the <sup>18</sup>F-FDG uptake for different dose ranges were analyzed.</p></sec><sec><title>Results</title><p>A heterogeneous dose distribution was observed, and the basal region received a higher median mean dose (18.36 Gy) than the middle and apical regions (5.30 and 2.21 Gy, respectively). Segments 1, 2, 3, and 4 received the highest doses, all of which were greater than 10 Gy. Three patterns were observed for the myocardial <sup>18</sup>F-FDG uptake in relation to the radiation dose before and after RT: an increase (5 patients), a decrease (13 patients), and no change (6 patients). In a pairing analysis, the <sup>18</sup>F-FDG uptake after RT decreased by 28.93 and 12.12% in the low-dose segments (0–10 Gy and 10–20 Gy, respectively) and increased by 7.24% in the high-dose segments (20–30 Gy).</p></sec><sec><title>Conclusion</title><p>The RT dose varies substantially within LV segments in patients receiving thoracic EC RT. Increased <sup>18</sup>F-FDG uptake in the myocardium after RT was observed for doses above 20 Gy.</p></sec></abstract><kwd-group><kwd>myocardium</kwd><kwd>radiotherapy</kwd><kwd><sup>18</sup>F-FDG PET</kwd><kwd>17-segment model</kwd><kwd>esophageal cancer</kwd></kwd-group><counts><fig-count count=\"4\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"30\"/><page-count count=\"9\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Esophageal cancer (EC) is the 8th most common cancer and has the 6th highest cancer mortality rate worldwide (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). During the past decade, radiotherapy (RT) has become a primary treatment modality for patients with EC because of its effectiveness and relative safety. However, the heart lies near the middle esophagus and is inevitably irradiated during RT for middle-stage EC patients. Although advances in RT equipment and techniques have prolonged patient survival, delayed latent effects of radiation are currently being encountered in clinical practice (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B3\" ref-type=\"bibr\">3</xref>). Previous studies have reported that cardiac toxicity may even diminish the survival gains obtained from anticancer therapy (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Therefore, early observation of changes in cardiac function is extremely important for monitoring and evaluating the occurrence and development of radiation-induced heart disease (RIHD).</p><p>The heart is divided into chambers, arteries and valves, which consist of myocardial, connective, pericardial and vascular tissues. These various cardiac tissues have different radiosensitivities (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). Studies have confirmed injuries to cardiac substructures (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B8\" ref-type=\"bibr\">8</xref>), indicating that various dose constraints might be required (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). The myocardium is a vulnerable heart tissue, but organic injury from RT usually does not appear for several years when the symptoms of the injury have become irreversible, with no effective treatment (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). However, the interval between RT treatment and the detection of RIHD has been reported to range from months to years (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Functional imaging can monitor metabolic changes in myocardial activity before the occurrence of organic injury, and effective intervention measures can be taken at the initial stage of pathological changes (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>).</p><p>Currently, positron emission computed tomography (PET) imaging is considered the “gold standard” for detecting viable myocardium. Oncologic PET is usually performed in a fasting state because postprandial high blood glucose induces insulin secretion, which results in increased <sup>18</sup>F-FDG uptake by muscle and fat and decreased <sup>18</sup>F-FDG uptake by the tumor. In the fasting state, the ischemic myocardium can take up <sup>18</sup>F-FDG, whereas normal myocardium and necrotic myocardium do not take up glucose. Under a glucose load, <sup>18</sup>F-FDG is ingested by both normal and ischemic myocardium and can be used to evaluate the survival state of the myocardium (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). Although previous studies have shown that increased cardiac uptake of <sup>18</sup>F-FDG on PET may be an indicator of myocardial injury after RT, only the global left ventricle (LV) was considered, and the radiation dose and <sup>18</sup>F-FDG uptake in specific myocardial segments were not evaluated. Cardiac imaging studies have also increasingly found discrete focal changes in the heart. A better understanding of the effects of the radiation dose requires a more detailed assessment of the radiation dose for cardiac subvolumes.</p><p>This study has two objectives. First, the dosimetry and <sup>18</sup>F-FDG uptake in oncologic PET are quantified using a 17-segment model of the LV proposed by the American Heart Association (AHA). Second, the relationship between the changes in the myocardial <sup>18</sup>F-FDG uptake and the irradiated dose in EC patients who underwent radiotherapy RT is investigated (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Our hypothesis is that the myocardial segment that receives higher doses will show increased <sup>18</sup>F-FDG uptake. The confirmation of this hypothesis can be used as a preliminary basis to accurately evaluate the cardiac dose-response relationship and implement timely treatment measures in the initial stages of pathological changes.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Patient Selection</title><p>An institutional review board approved a retrospective review of the medical records for this analysis. Twenty-four EC patients who underwent consecutive oncologic <sup>18</sup>F-FDG PET/CT scans at baseline (1–2 weeks before radiotherapy) and post-RT (2–3 months after radiotherapy) were enrolled in the study. The main inclusion criteria were as follows: (1) the heart was covered by the radiation field during the scan and (2) a fasting time >12 h prior to PET was observed. The exclusion criteria were as follows: (1) prior treatment with chemotherapy, (2) history of cardiac disease, congestive heart failure or coronary artery disease, and (3) a fasting blood glucose level higher than 150 mg/dl before the <sup>18</sup>F-FDG injection.</p></sec><sec id=\"S2.SS2\"><title>Radiotherapy Design</title><p>The prescribed dose of the planning target volume (PTV) was 60 Gy, and 95% of the PTV was required to receive the prescribed dose. The gross tumor volume (GTV) consisted of the primary tumor and metastatic regional lymph nodes observed on CT and a whole body PET/CT scan. The clinical target volume (CTV) was formed by the GTV with a 1.0-cm margin in all directions. A 0.5-cm margin for CTV was expanded to delineate the PTV. Treatment was delivered as three-dimensional conformal radiation therapy (3D-CRT) or intensity-modulated radiation therapy (IMRT) at 2.0 Gy per fraction with 6-MV photon beams. The constraints of the organ at risk (OAR) were V<sub>20</sub><sub><italic>Gy</italic></sub> < 30% and V<sub>30</sub><sub><italic>Gy</italic></sub> < 20% for the total lung; a maximum dose <45 Gy for the spinal cord; and V<sub>30</sub><sub><italic>Gy</italic></sub> < 40% and V<sub>40</sub><sub><italic>Gy</italic></sub> < 30% for the heart. <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref> demonstrates the PTV and radiation fields in the IMRT plan.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Schematic diagram of the relationship between the heart and PTV. The PTV and heart are depicted in green and color, respectively.</p></caption><graphic xlink:href=\"fonc-10-01599-g001\"/></fig></sec><sec id=\"S2.SS3\"><title><sup>18</sup>F-FDG PET Image Acquisition</title><p>The <sup>18</sup>F-FDG PET/CT images were acquired from a combined PET/CT scanner (Philips Healthcare, Cleveland, OH) (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). The median fasting blood glucose level was 5.8 mmol/l (interquartile range: 5.0–6.8). Approximately 1 h after the <sup>18</sup>F-FDG injection, a spiral CT was obtained, followed by a PET emission scan from the distal femur to the top of the skull. The PET emission images were corrected by the measured attenuation and reconstructed using a conventional iterative ordered-subsets expectation maximization algorithm.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Planes of myocardial PET imaging.</p></caption><graphic xlink:href=\"fonc-10-01599-g002\"/></fig></sec><sec id=\"S2.SS4\"><title>Myocardium Delineation</title><p>The LV contouring was performed by a radiologist with 20 years of experience and reviewed by two senior cardiologists. Differences in the results were resolved by consensus. Myocardium delineation was performed by Carimas software (version 2.9)<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup> based on the AHA 17-segment model, which rearranges the myocardium circumferential profile image from the apex to the base into concentric circles from the interior to the exterior, thereby projecting the entire LV myocardial onto a bull’s eye diagram. As shown in <xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>, when contouring the LV, the myocardium was automatically divided into basal (segments 1–6), middle (segments 7–12), and apical (segments 13–16) regions.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Left myocardium segmentation <bold>(A)</bold> and polar map <bold>(B)</bold>.</p></caption><graphic xlink:href=\"fonc-10-01599-g003\"/></fig></sec><sec id=\"S2.SS5\"><title>Dosimetry Data Acquisition</title><p>The above mentioned delineation results were used to extract the radiation dose to individual segments from the planning CT images. The individual segments were combined to determine the dose to the basal, middle and apical regions. We classified the myocardium into three groups depending on the radiation dose received: lower than 10, 10–20, and 20–30 Gy. No segment received a dose greater than 20 Gy.</p></sec><sec id=\"S2.SS6\"><title>Myocardial Metabolism Measurement</title><p>The myocardial metabolism level was evaluated from the <sup>18</sup>F-FDG uptake value. The 17-segment model was used to measure the myocardial metabolic parameters, which were then used in turn to carry out a quantitative analysis. For the quantitative analysis, the mean standardized uptake values (SUVs) were obtained from the baseline and the post-RT PET images. Changes in the SUV (ΔSUV) were calculated using the following formula: ΔSUV = (SUV after RT) – (SUV before RT). The SUV ratio (SUVR) was defined as the ΔSUV divided by the baseline SUV. Finally, the change in the <sup>18</sup>F-FDG PET uptake was correlated with the radiation dose for the LV myocardium.</p></sec><sec id=\"S2.SS7\"><title>Statistical Analysis</title><p>The SPSS 22.0 software program (SPSS, Chicago, IL, United States) was used for all the statistical tests, and the quantitative parameters were presented in terms of the mean ± standard deviation (SD). The Wilcoxon signed-rank test was used to compare the differences in different patterns of patient characteristics, and differences in <sup>18</sup>F-FDG uptake values between baseline and post-RT. Only <italic>P</italic>-values < 0.05 were considered statistically significant.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Patients</title><p>Between January 2016 and December 2018, 24 patients (ranging from 51 to 73 years of age, with a mean age of 61 years) with middle thoracic EC who underwent PET scans at baseline and post-RT were retrospectively enrolled in this study. Pathology reports confirmed that all the enrolled patients had esophageal squamous cell carcinoma. The heart volumes ranged from 377.1 to 805.7 with a median of 593.7 cm<sup>3</sup>. None of the patients showed symptomatic cardiac events during the entire planned RT process or for 3 months following RT. No significant differences in the baseline patient characteristics were observed in 3 pattern cohorts, with <italic>P</italic> values ranging from 0.018 to 1.000 (see <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Patient characteristics.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristic</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">All patients</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Increased</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">No changes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Decreased</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>P</italic>-value</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Age, years, median (range)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">61 (51–73)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">63 (54–67)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60 (58–63)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">62 (51–73)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.886</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sex</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.233</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tumor location</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Middle</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tumor length, cm, median(range)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.8 (4.0–8.4)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.9 (4.3–6.9)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.7 (5.0–8.4)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.8 (4.0–7.2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.583</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Histology</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Squamous cell carcinoma</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tumor staging</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.301</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I B</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.223</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">II</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.135</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">III A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.082</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IIIB</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.018</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IIIC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.135</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IV</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.223</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IV A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.223</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mean heart volume, cm<sup>3</sup>(range)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.067</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">593.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">495.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">628.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">608.2</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(377.1–805.7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(470.1–495.7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(574.5–682.3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(377.1–805.7)</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Concurrent chemotherapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Radiotherapy technique</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.170</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IMRT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3D-CRT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Prescribed dose</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60 Gy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60 Gy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60 Gy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60 Gy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.000</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">No. of fractions</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 Gy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 Gy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 Gy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 Gy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.000</td></tr></tbody></table><table-wrap-foot><attrib><italic>IMRT, intensity-modulated radiation therapy; 3D-CRT, 3-dimensional conformal radiation therapy.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS2\"><title>Doses to the Left Myocardial Segments</title><p>The median maximum, mean, and minimum LV irradiation doses were 18.14 ± 9.08, 6.54 ± 4.07, and 2.59 ± 2.23 Gy, respectively. Valuable data were recorded for discrete areas of the LV. Significant differences were observed in the dose distribution for the LV: the median mean and median maximum doses were both higher in the basal region (9.59 and 16.12 Gy, respectively) than in the middle region (6.54 and 11.53 Gy, respectively) and the apical region (5.09 and 13.17 Gy, respectively). These results were mirrored for the segments of the left myocardium, with the basal segments receiving the highest doses (segments 1, 2, 3, and 4). <xref rid=\"T2\" ref-type=\"table\">Table 2</xref> shows the doses to the individual myocardium segments in the polar map.</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Irradiation dose and FDG uptake of individual segment in the myocardial 17-segment model.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Segment</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mean dose, Gy (range)</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">SUV<hr/></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">SUVR (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>P</italic>-value</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Baseline</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Post-RT</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Basal</bold></td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.26 (1.89–24.63)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.78 (2.13–6.81)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.82 (1.24–4.59)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–19.14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.217</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.25 (1.46–28.27)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.29 (2.65–7.57)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.33 (1.41–6.12)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–18.24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.378</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11.58 (0.62–24.54)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.28 (2.38–7.70)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.22 (1.12–7.38)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–21.91</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.304</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.07 (0.45–27.72)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.68 (2.03–6.42)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.85 (0.97–6.86)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–20.84</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.398</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.69 (0.53–23.65)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.73 (2.10–5.91)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.11 (1.48–8.14)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–17.29</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.292</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.84 (1.01–15.35)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.38 (2.07–5.80)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.65 (1.26–5.83)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–17.14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.156</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Middle</bold></td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.64 (1.14–20.79)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.40 (1.84–6.52)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.53 (1.19–4.12)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–20.12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.084</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.13 (0.96–25.88)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.67 (1.87–7.05)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.74 (1.26–5.01)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–19.67</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.090</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.48 (0.45–19.53)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.09 (2.16–7.44)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.97 (1.19–6.86)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–24.74</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.040</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.44 (0.32–19.68)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.87 (2.16–7.39)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.86 (1.04–7.16)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–24.76</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.045</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.92 (0.36–20.86)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.73 (2.11–6.59)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.94 (1.59–7.32)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–21.70</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.020</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.85 (0.64–10.89)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.68 (2.18–6.62)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.80 (1.22–5.31)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–22.79</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.033</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Apical</bold></td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.38 (0.61–8.33)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.20 (1.91–5.76)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.35 (1.11–4.53)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–24.88</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.019</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.16 (0.42–13.51)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.25 (1.85–6.19)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.39 (0.89–5.15)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–23.45</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.047</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.59 (0.24–20.64)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.20 (2.00–5.80)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.41 (1.02–5.26)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–22.59</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.024</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.78 (0.36–9.49)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.11 (1.95–5.73)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.30 (1.11–4.99)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–24.93</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.005</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.54 (0.49–6.57)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.68 (1.66–5.02)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.96 (0.86–4.13)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–24.09</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.007</td></tr></tbody></table></table-wrap></sec><sec id=\"S3.SS3\"><title><sup>18</sup>F-FDG Uptake of Left Myocardial Segments</title><p>An analysis of the changes in the <sup>18</sup>F-FDG uptake at two time points showed three patterns for the overall myocardial accumulation of <sup>18</sup>F-FDG uptake related to the radiation dose (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). A subsequent quantitative analysis demonstrated that <sup>18</sup>F-FDG uptake increased in 5 patients (average SUVR: 16.68%), decreased in 13 patients (average SUVR: −41.38%) and did not change significantly in 6 patients (average SUVR: −5.53%). However, directly focusing on specific LV segments showed that the post-RT uptake of the 17 segments tended to decrease relative to the baseline uptake: the post-RT uptake decreased in the basal, middle and apical regions by 19.09 ± 1.77, 22.30 ± 2.01, and 23.99 ± 0.89%, respectively. In agreement with these results, the <sup>18</sup>F-FDG uptake of the segmental myocardium decreased significantly in segments 9–17 in the apical region (<italic>P</italic> < 0.05). <xref rid=\"T2\" ref-type=\"table\">Table 2</xref> provides a detailed summary of baseline and post-RT <sup>18</sup>F-FDG uptake for the baseline and post-RT in the left myocardium segments.</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Baseline and post-RT PET imaging in patients with three myocardial accumulation patterns.</p></caption><graphic xlink:href=\"fonc-10-01599-g004\"/></fig></sec><sec id=\"S3.SS4\"><title>Changes of <sup>18</sup>F-FDG Uptake With Different Dose Ranges</title><p>We directly paired the radiation dose and the <sup>18</sup>F-FDG uptake of the LV segments using the AHA 17-segment model. In our study, the numbers of segments that received doses in the ranges of 0–10, 10–20, and 20–30 Gy were 296, 74, and 38, respectively. <sup>18</sup>F-FDG uptake in the segments receiving 0–10 and 10–20 Gy decreased by 28.93 and 12.12% after RT, respectively. The <sup>18</sup>F-FDG uptake in the segments receiving 20–30 Gy increased by 7.24% after RT.</p></sec></sec><sec id=\"S4\"><title>Discussion</title><p>Subacute changes following thoracic RT have been demonstrated in discrete areas of the LV, and guidelines have been recently developed to help determine the dose to subvolumes of the LV (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>, <xref rid=\"B17\" ref-type=\"bibr\">17</xref>). Therefore, the radiation dose to cardiac subvolumes needs to be more accurately quantified to better understand the effect of the radiation dose. Previous studies have calculated cardiac doses by using modeled patients with recreated 2D treatment fields and by employing set geometric rules to define cardiac subvolumes or by using anatomical landmarks to divide the heart into standard axial imaging planes (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>–<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). The primary strength of the present study is that the dosimetric values and <sup>18</sup>F-FDG uptake were evaluated by performing a segmental analysis and directly pairing the radiation dose and <sup>18</sup>F-FDG uptake in the myocardial segments using the 17-segment AHA model.</p><p>The 17-segment model method proposed by the AHA aims to accurately divide the LV according to the anatomical structure of the heart and is the closest scheme available to that visualized by clinical ultrasound and radionuclide myocardial imaging (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Erven et al. (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>) discussed the feasibility of dividing the LV into 17 segments but did not report the doses for each segment. Tang et al. (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>) used a 17-segment model to show that the dose distribution varied across LV subregions in breast cancer patients. The middle and anterior apical segments (segments 7 and 13) and the LV apical regions (segments 13, 14, 15, 16, and 17) received higher radiation doses than the other segments. The results of the present study also show a heterogeneous dose to the LV for EC patients. The basal region (segments 1, 2, 3, and 4) received a higher radiation dose than the apical and middle regions. Such reporting of specific regional dose delivery provides the most accurate spatial description of delivered radiation doses to the heart, particularly during the middle stages of EC when the LV is very frequently exposed. The use of automatic software sketching has reduced the sketching error between observers. In addition, analyzing the dose value and the <sup>18</sup>F-FDG uptake of the myocardium under the same LV-VOI conditions produced a highly accurate dose-response relationship. This analytical method accurately describes the radiation doses to the myocardium and its subspaces. Further understanding of cardiotoxicity requires the precise matching of the radiation dose to regional imaging defects, which underlines the need for determining the dose distribution in detail (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>).</p><p>Changes in the <sup>18</sup>F-FDG uptake of the segments of the myocardium were also analyzed in the current study. To suppress the physiological myocardial accumulation of <sup>18</sup>F-FDG, we selected patients who fasted for ≥18 h prior to the <sup>18</sup>F-FDG PET scan, both pre- and post-RT. Ishida et al. (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>) reported that the physiological accumulation of <sup>18</sup>F-FDG in the irradiated myocardium was suppressed in <italic>a</italic> ≥ 18-h fasting group compared with <italic>a</italic> < 18-h fasting group. Suppression of physiological myocardial <sup>18</sup>F-FDG accumulation appeared to facilitate the detection of abnormal myocardial <sup>18</sup>F-FDG accumulation. Cardiotoxicity related to external RT is a recognized phenomenon in clinical practice and has traditionally been investigated by radionuclide ventriculography or gated blood pool imaging (popularly known as the multigated acquisition [MUGA] scan) and 2D echocardiography. By comparison, the observation of <sup>18</sup>F-FDG PET/CT in the present study is novel and may have considerable significance if translated into clinical practice and utilized to monitor the effect of RT in a patient-specific manner (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). In this study, three radiation dose patterns were observed for the myocardial accumulation of <sup>18</sup>F-FDG on PET: an increase, a decrease or no change. Although the exact molecular pathway remains to be determined, in-depth research in this field can be applied to the specific diagnosis and management of different patients such that corresponding preventive or therapeutic measures can be provided.</p><p>Last, the changes in <sup>18</sup>F-FDG uptake by myocardial segments were analyzed for different dose ranges. Very few studies have explored this topic to date, and the two existing publications on RT in EC present conflicting results. Jingu et al. (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>) considered focally increased <sup>18</sup>F-FDG uptake in the basal myocardium after RT to be an indicator of radiation-induced cardiac damage, whereas Konski et al. (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>) found no correlation between the percent change in the myocardial SUV and cardiac toxicity. A recent study by Evans et al. (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>) found that in lung cancer patients treated with stereotactic body RT (SBRT), <sup>18</sup>F-FDG uptake increased when the 20 Gy isodose line exceeded 5 cm<sup>3</sup> of the heart. By comparison, the results of the present study show that <sup>18</sup>F-FDG uptake in myocardial segments receiving a low dose (0–20 Gy) decreased after RT, whereas <sup>18</sup>F-FDG uptake in the myocardial segments receiving 20–30 Gy increased after RT. Therefore, minimizing the volumes of myocardium being irradiated by more than 20 Gy can be expected to reduce the incidence of myocardial injury. However, this hypothesis needs to be evaluated in larger scale prospective studies with longer follow-up times.</p><p>The current study has several limitations. First, the 5-year incidence rate of heart disease or pericardial effusion following RT ranges from 11.1 to 13.8% (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>–<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Further follow-up is required to reveal the clinical significance of abnormal myocardial accumulation of <sup>18</sup>F-FDG following RT. Second, the heart is a mobile structure, and its location on 2 PET scans may vary both interfractionally and intrafractionally relative to the planned CT scan, rendering the regional dose distribution corresponding to <sup>18</sup>F-FDG PET a good approximation, at best. Third, strictly enrolling patients who fasted for >18 h before undergoing PET scans resulted in a limited number of patients for this retrospective study. More patients need to be evaluated in future prospective studies.</p><p>Despite these drawbacks, we consider our data on cardiac toxicity during RT to be robust. Currently, we are not suggesting that the study results should be used to modify current treatment modalities. However, we are recommending that efforts should be made to reduce the cardiac dose and irradiated volumes during thoracic RT, which may benefit patients, especially those with favorable prognoses. Very few publications on the subject of this study are currently available. Thus, this investigation is the first of its kind: specific cardiac changes on PET/CT are related to dose information to detect myocardial activity in early stage post-RT. Although none of the investigated patients experienced symptomatic cardiac events between receiving RT and 3 months following RT, we consider a longer follow-up with higher numbers of patients to be essential for assessing the clinical significance of the considered abnormalities. We recommend that considerable effort should be expended to identify means of improving RT techniques to further minimize incidental irradiation of the heart.</p></sec><sec id=\"S5\"><title>Conclusion</title><p>Radiotherapy doses vary substantially within specific LV segments in the setting of thoracic EC RT. Increased <sup>18</sup>F-FDG uptake in the myocardium after RT was observed when receiving a dose higher than 20 Gy. Determining the <sup>18</sup>F-FDG uptake and corresponding RT dose in the LV segments can help to guide focus in the diagnosis of radiation-induced cardiac toxicity.</p></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p></sec><sec id=\"S7\"><title>Ethics Statement</title><p>The retrospective study was reviewed and approved by The Ethics Committee (IRB) at Shandong Cancer Hospital and Institute. After a vote (Total 11, Agree 11, Disagree 0), the IRB agreed that the study followed the guidelines of Good Clinical Practice (GCP) and that the research could be conducted at Shandong Cancer Hospital and Institute (No. 201807013).</p></sec><sec id=\"S8\"><title>Author Contributions</title><p>XS designed the study and wrote the initial draft of the manuscript. GG and CH contributed to the design, analysis and interpretation of the data, and assisted in the preparation of the manuscript. QQ and YY contributed to the data collection and interpretation, and critically reviewed the manuscript. All authors approved the final version of the manuscript and have agreed to be accountable for all aspects of the work and for ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by the Key Technology Research and Development Program of Shandong (2018GSF118048 and 2018GSF118006).</p></fn></fn-group><fn-group><fn id=\"footnote1\"><label>1</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.turkupetcentre.fi/carimas\">www.turkupetcentre.fi/carimas</ext-link></p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ferlay</surname><given-names>J</given-names></name><name><surname>Shin</surname><given-names>HR</given-names></name><name><surname>Bray</surname><given-names>F</given-names></name><name><surname>Forman</surname><given-names>D</given-names></name><name><surname>Mathers</surname><given-names>C</given-names></name><name><surname>Parkin</surname><given-names>DM</given-names></name><etal/></person-group>\n<article-title>Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008.</article-title>\n<source><italic>Int J Cancer.</italic></source> (<year>2010</year>) <volume>127</volume>:<fpage>2893</fpage>–<lpage>917</lpage>. <pub-id pub-id-type=\"doi\">10.1002/ijc.25516</pub-id>\n<pub-id pub-id-type=\"pmid\">21351269</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hendry</surname><given-names>JH</given-names></name><name><surname>Akahoshi</surname><given-names>M</given-names></name><name><surname>Wang</surname><given-names>LS</given-names></name><name><surname>Lipshultz</surname><given-names>SE</given-names></name><name><surname>Stewart</surname><given-names>FA</given-names></name><name><surname>Trott</surname><given-names>KR.</given-names></name></person-group>\n<article-title>Radiation-induced cardiovascular injury.</article-title>\n<source><italic>Radiat Environ Biophys.</italic></source> (<year>2008</year>) <volume>47</volume>:<fpage>189</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00411-007-0155-7</pub-id>\n<pub-id pub-id-type=\"pmid\">18193445</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Darby</surname><given-names>SC</given-names></name><name><surname>Ewertz</surname><given-names>M</given-names></name><name><surname>McGale</surname><given-names>P</given-names></name><name><surname>Bennet</surname><given-names>AM</given-names></name><name><surname>Blom-Goldman</surname><given-names>U</given-names></name><name><surname>Brønnum</surname><given-names>D</given-names></name><etal/></person-group>\n<article-title>Risk of ischemic heart disease in women after radiotherapy for breast cancer.</article-title>\n<source><italic>N Engl J Med.</italic></source> (<year>2013</year>) <volume>368</volume>:<fpage>987</fpage>–<lpage>98</lpage>. <pub-id pub-id-type=\"doi\">10.1056/NEJMoa1209825</pub-id>\n<pub-id pub-id-type=\"pmid\">23484825</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Curigliano</surname><given-names>G</given-names></name><name><surname>Cardinale</surname><given-names>D</given-names></name><name><surname>Dent</surname><given-names>S</given-names></name><name><surname>Criscitiello</surname><given-names>C</given-names></name><name><surname>Aseyev</surname><given-names>O</given-names></name><name><surname>Lenihan</surname><given-names>D</given-names></name><etal/></person-group>\n<article-title>Cardiotoxicity of anticancer treatments: Epidemiology, detection, and management.</article-title>\n<source><italic>CA Cancer J Clin.</italic></source> (<year>2016</year>) <volume>66</volume>:<fpage>309</fpage>–<lpage>25</lpage>. <pub-id pub-id-type=\"doi\">10.3322/caac.21341</pub-id>\n<pub-id pub-id-type=\"pmid\">26919165</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hooning</surname><given-names>MJ</given-names></name><name><surname>Aleman</surname><given-names>BM</given-names></name><name><surname>van Rosmalen</surname><given-names>AJ</given-names></name><name><surname>Kuenen</surname><given-names>MA</given-names></name><name><surname>Klijn</surname><given-names>JG</given-names></name><name><surname>van Leeuwen</surname><given-names>FE.</given-names></name></person-group>\n<article-title>Cause-specific mortality in long-term survivors of breast cancer: A 25-year follow-up study.</article-title>\n<source><italic>Int J Radiat Oncol Biol Phys.</italic></source> (<year>2006</year>) <volume>64</volume>:<fpage>1081</fpage>–<lpage>91</lpage>.<pub-id pub-id-type=\"pmid\">16446057</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gillette</surname><given-names>EL</given-names></name><name><surname>McChesney</surname><given-names>SL</given-names></name><name><surname>Hoopes</surname><given-names>PJ.</given-names></name></person-group>\n<article-title>Isoeffect curves for radiation-induced cardiomyopathy in the dog.</article-title>\n<source><italic>Int J Radiat Oncol Biol Phys.</italic></source> (<year>1985</year>) <volume>11</volume>:<fpage>2091</fpage>–<lpage>7</lpage>.<pub-id pub-id-type=\"pmid\">4066441</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Taylor</surname><given-names>C</given-names></name><name><surname>McGale</surname><given-names>P</given-names></name><name><surname>Brønnum</surname><given-names>D</given-names></name><name><surname>Correa</surname><given-names>C</given-names></name><name><surname>Cutter</surname><given-names>D</given-names></name><name><surname>Duane</surname><given-names>FK</given-names></name><etal/></person-group>\n<article-title>Cardiac structure injury after radiotherapy for breast cancer: cross-sectional study with individual patient data.</article-title>\n<source><italic>J Clin Oncol.</italic></source> (<year>2018</year>) <volume>36</volume>:<fpage>2288</fpage>–<lpage>96</lpage>. <pub-id pub-id-type=\"doi\">10.1200/JCO.2017.77.6351</pub-id>\n<pub-id pub-id-type=\"pmid\">29791285</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Darby</surname><given-names>SC</given-names></name><name><surname>Cutter</surname><given-names>DJ</given-names></name><name><surname>Boerma</surname><given-names>M</given-names></name><name><surname>Constine</surname><given-names>LS</given-names></name><name><surname>Fajardo</surname><given-names>LF</given-names></name><name><surname>Kodama</surname><given-names>K</given-names></name><etal/></person-group>\n<article-title>Radiation-related heart disease: current knowledge and future prospects.</article-title>\n<source><italic>Int J of Radiat Oncol Biol Phys.</italic></source> (<year>2010</year>) <volume>76</volume>:<fpage>656</fpage>–<lpage>65</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijrobp.2009.09.064</pub-id>\n<pub-id pub-id-type=\"pmid\">20159360</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gagliardi</surname><given-names>G</given-names></name><name><surname>Constine</surname><given-names>LS</given-names></name><name><surname>Moiseenko</surname><given-names>V</given-names></name><name><surname>Correa</surname><given-names>C</given-names></name><name><surname>Pierce</surname><given-names>LJ</given-names></name><name><surname>Allen</surname><given-names>AM</given-names></name><etal/></person-group>\n<article-title>Radiation dose–volume effects in the heart.</article-title>\n<source><italic>Int J Radiat Oncol Biol Phys.</italic></source> (<year>2010</year>) <volume>76</volume>:<fpage>S77</fpage>–<lpage>85</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijrobp.2009.04.093</pub-id>\n<pub-id pub-id-type=\"pmid\">20171522</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lally</surname><given-names>BE</given-names></name><name><surname>Detterbeck</surname><given-names>FC</given-names></name><name><surname>Geiger</surname><given-names>AM</given-names></name><name><surname>Thomas</surname><given-names>CR</given-names></name><name><surname>Machtay</surname><given-names>M</given-names></name><name><surname>Miller</surname><given-names>AA</given-names></name><etal/></person-group>\n<article-title>The risk of death from heart disease in patients with nonsmall cell lung cancer who receive postoperative radiotherapy: analysis of the surveillance, epidemiology, and end results database.</article-title>\n<source><italic>Cancer.</italic></source> (<year>2010</year>) <volume>110</volume>:<fpage>911</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1002/cncr.22845</pub-id>\n<pub-id pub-id-type=\"pmid\">17620279</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Marks</surname><given-names>LB</given-names></name><name><surname>Yu</surname><given-names>X</given-names></name><name><surname>Prosnitz</surname><given-names>RG</given-names></name><name><surname>Zhou</surname><given-names>SM</given-names></name><name><surname>Hardenbergh</surname><given-names>PH</given-names></name><name><surname>Blazing</surname><given-names>M</given-names></name><etal/></person-group>\n<article-title>The incidence and functional consequences of RT-associated cardiac perfusion defects.</article-title>\n<source><italic>Int J Radiat Oncol Biol Phys.</italic></source> (<year>2005</year>) <volume>63</volume>:<fpage>214</fpage>–<lpage>23</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijrobp.2005.01.029</pub-id>\n<pub-id pub-id-type=\"pmid\">16111592</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sioka</surname><given-names>C</given-names></name><name><surname>Exarchopoulos</surname><given-names>T</given-names></name><name><surname>Tasiou</surname><given-names>I</given-names></name><name><surname>Tzima</surname><given-names>E</given-names></name><name><surname>Fotou</surname><given-names>N</given-names></name><name><surname>Capizzello</surname><given-names>A</given-names></name><etal/></person-group>\n<article-title>Myocardial perfusion imaging with (99 m) Tc-tetrofosmin SPECT in breast cancer patients that received postoperative radiotherapy: a case-control study.</article-title>\n<source><italic>Radiat Oncol.</italic></source> (<year>2011</year>) <volume>6</volume>:<issue>151</issue>. <pub-id pub-id-type=\"doi\">10.1186/1748-717X-6-151</pub-id>\n<pub-id pub-id-type=\"pmid\">22067743</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Karam</surname><given-names>HM</given-names></name><name><surname>Radwan</surname><given-names>RR.</given-names></name></person-group>\n<article-title>Metformin modulates cardiac endothelial dysfunction, oxidative stress and inflammation in irradiated rats: A new perspective of an antidiabetic drug.</article-title>\n<source><italic>Clin Exp Pharmacol Physiol.</italic></source> (<year>2019</year>) <volume>46</volume>:<fpage>1</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1111/1440-1681.13148</pub-id>\n<pub-id pub-id-type=\"pmid\">31357226</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schultz-Hector</surname><given-names>S</given-names></name><name><surname>Trott</surname><given-names>KR.</given-names></name></person-group>\n<article-title>Radiation-induced cardiovascular diseases: is the epidemiologic evidence compatible with the radiobiologic data?</article-title>\n<source><italic>Int J Radiat Oncol Biol Phys.</italic></source> (<year>2007</year>) <volume>67</volume>:<fpage>10</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijrobp.2006.08.071</pub-id>\n<pub-id pub-id-type=\"pmid\">17189062</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hardenbergh</surname><given-names>PH</given-names></name><name><surname>Munley</surname><given-names>MT</given-names></name><name><surname>Bentel</surname><given-names>GC</given-names></name><name><surname>Kedem</surname><given-names>R</given-names></name><name><surname>Borges-Neto</surname><given-names>S</given-names></name><name><surname>Hollis</surname><given-names>D.</given-names></name></person-group>\n<article-title>Cardiac perfusion changes in patients treated for breast cancer with radiation therapy and doxorubicin: preliminary results.</article-title>\n<source><italic>Int J Radiat Oncol Biol Phys.</italic></source> (<year>2001</year>) <volume>49</volume>:<fpage>1023</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/s0360-3016(00)01531-5</pub-id><pub-id pub-id-type=\"pmid\">11240243</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cerqueira</surname><given-names>MD</given-names></name><name><surname>Weissman</surname><given-names>NJ</given-names></name><name><surname>Dilsizian</surname><given-names>V</given-names></name><name><surname>Jacobs</surname><given-names>AK</given-names></name><name><surname>Kaul</surname><given-names>S</given-names></name><name><surname>Laskey</surname><given-names>WK</given-names></name><etal/></person-group>\n<article-title>Standardized myocardial segmentation and nomenclature for tomographic imaging of the heart. A statement for healthcare professionals from the cardiac imaging committee of the council on clinical cardiology of the american heart association.</article-title>\n<source><italic>J Nucl Cardiol.</italic></source> (<year>2002</year>) <volume>105</volume>:<fpage>539</fpage>–<lpage>42</lpage>. <pub-id pub-id-type=\"doi\">10.1067/mnc.2002.123122</pub-id>\n<pub-id pub-id-type=\"pmid\">11986572</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luijk</surname><given-names>P</given-names></name><name><surname>Faber</surname><given-names>H</given-names></name><name><surname>Meertens</surname><given-names>H</given-names></name><name><surname>Schippers</surname><given-names>JM</given-names></name><name><surname>Langendijk</surname><given-names>JA</given-names></name><name><surname>Brandenburg</surname><given-names>S</given-names></name><etal/></person-group>\n<article-title>The impact of heart irradiation on dose–volume effects in the rat lung.</article-title>\n<source><italic>Int J Radiat Oncol Biol Phys.</italic></source> (<year>2007</year>) <volume>69</volume>:<fpage>552</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijrobp.2007.05.065</pub-id>\n<pub-id pub-id-type=\"pmid\">17869668</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Taylor</surname><given-names>CW</given-names></name><name><surname>Nisbet</surname><given-names>A</given-names></name><name><surname>McGale</surname><given-names>P</given-names></name><name><surname>Darby</surname><given-names>SC.</given-names></name></person-group>\n<article-title>Cardiac exposures in breast cancer radiotherapy: 1950s–1990s.</article-title>\n<source><italic>Int J Radiat Oncol Biol Phys.</italic></source> (2007) <volume>69</volume>:<fpage>1484</fpage>–<lpage>95</lpage>.</mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wollschläger</surname><given-names>D</given-names></name><name><surname>Karle</surname><given-names>H</given-names></name><name><surname>Stockinger</surname><given-names>M</given-names></name><name><surname>Bartkowiak</surname><given-names>D</given-names></name><name><surname>Bührdel</surname><given-names>S</given-names></name><name><surname>Merzenich</surname><given-names>H</given-names></name><etal/></person-group>\n<article-title>Radiation dose distribution in functional heart regions from tangential breast cancer radiotherapy.</article-title>\n<source><italic>J Eur Soc Therap Radiol Oncol.</italic></source> (<year>2016</year>) <volume>119</volume>:<fpage>65</fpage>–<lpage>70</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.radonc.2016.01.020</pub-id>\n<pub-id pub-id-type=\"pmid\">26874543</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Johansen</surname><given-names>S</given-names></name><name><surname>Tjessem</surname><given-names>KH</given-names></name><name><surname>Fossa</surname><given-names>K</given-names></name><name><surname>Bosse</surname><given-names>G</given-names></name><name><surname>Danielsen</surname><given-names>T</given-names></name><name><surname>Malinen</surname><given-names>E</given-names></name><etal/></person-group>\n<article-title>Dose distribution in the heart and cardiac chambers following 4-field radiation therapy of breast cancer: a retrospective study.</article-title>\n<source><italic>Breast Cancer..</italic></source> (<year>2013</year>) <volume>7</volume>:<fpage>41</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.4137/BCBCR.S11118</pub-id>\n<pub-id pub-id-type=\"pmid\">23589693</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Erven</surname><given-names>K</given-names></name><name><surname>Florian</surname><given-names>A</given-names></name><name><surname>Slagmolen</surname><given-names>P</given-names></name><name><surname>Sweldens</surname><given-names>C</given-names></name><name><surname>Jurcut</surname><given-names>R</given-names></name><name><surname>Wildiers</surname><given-names>H</given-names></name><etal/></person-group>\n<article-title>Subclinical cardiotoxicity detected by strain rate imaging up to 14 months after breast radiation therapy.</article-title>\n<source><italic>Int J Radiat Oncol Biol Phys.</italic></source> (<year>2013</year>) <volume>85</volume>:<fpage>1172</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijrobp.2012.09.022</pub-id>\n<pub-id pub-id-type=\"pmid\">23149005</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tang</surname><given-names>S</given-names></name><name><surname>Otton</surname><given-names>J</given-names></name><name><surname>Holloway</surname><given-names>L</given-names></name><name><surname>Delaney</surname><given-names>PG</given-names></name><name><surname>Liney</surname><given-names>G</given-names></name><name><surname>George</surname><given-names>A</given-names></name><etal/></person-group>\n<article-title>Quantification of cardiac subvolume dosimetry using a 17 segment model of the left ventricle in breast cancer patients receiving tangential beam radiotherapy.</article-title>\n<source><italic>Radiother Oncol.</italic></source> (<year>2019</year>) <volume>132</volume>:<fpage>257</fpage>–<lpage>65</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.radonc.2018.09.021</pub-id>\n<pub-id pub-id-type=\"pmid\">30446318</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Duane</surname><given-names>F</given-names></name><name><surname>Aznar</surname><given-names>MC</given-names></name><name><surname>Bartlett</surname><given-names>F</given-names></name><name><surname>Cutter</surname><given-names>DJ</given-names></name><name><surname>Darby</surname><given-names>SC</given-names></name><name><surname>Jagsi</surname><given-names>R</given-names></name><etal/></person-group>\n<article-title>A cardiac contouring atlas for radiotherapy.</article-title>\n<source><italic>J Eur Soc Therap Radiol Oncol.</italic></source> (<year>2017</year>) <volume>122</volume>:<fpage>416</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.radonc.2017.01.008</pub-id>\n<pub-id pub-id-type=\"pmid\">28233564</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ishida</surname><given-names>Y</given-names></name><name><surname>Sakanaka</surname><given-names>K</given-names></name><name><surname>Itasaka</surname><given-names>S</given-names></name><name><surname>Nakamoto</surname><given-names>Y</given-names></name><name><surname>Togashi</surname><given-names>K</given-names></name><name><surname>Mizowaki</surname><given-names>T</given-names></name><etal/></person-group>\n<article-title>Effect of long fasting on myocardial accumulation in 18F-fluorodeoxyglucose positron emission tomography after chemoradiotherapy for esophageal carcinoma.</article-title>\n<source><italic>J Radiat Res.</italic></source> (<year>2017</year>) <volume>59</volume>:<fpage>182</fpage>–<lpage>9</lpage>.</mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Evans</surname><given-names>JD</given-names></name><name><surname>Gomez</surname><given-names>DR</given-names></name><name><surname>Chang</surname><given-names>JY</given-names></name><name><surname>Gladish</surname><given-names>GW</given-names></name><name><surname>Erasmus</surname><given-names>JJ</given-names></name><name><surname>Rebueno</surname><given-names>N</given-names></name><etal/></person-group>\n<article-title>Cardiac 18F-fluorodeoxyglucose uptake on positron emission tomography after thoracic stereotactic body radiation therapy.</article-title>\n<source><italic>Radiother Oncol.</italic></source> (<year>2013</year>) <volume>109</volume>:<fpage>82</fpage>–<lpage>8</lpage>.<pub-id pub-id-type=\"pmid\">24016676</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jingu</surname><given-names>K</given-names></name><name><surname>Kaneta</surname><given-names>T</given-names></name><name><surname>Nemoto</surname><given-names>K</given-names></name><name><surname>Ichinose</surname><given-names>A</given-names></name><name><surname>Oikawa</surname><given-names>M</given-names></name><name><surname>Takai</surname><given-names>Y</given-names></name><etal/></person-group>\n<article-title>The utility of 18F-fluorodeoxyglucose positron emission tomography for early diagnosis of radiation-induced myocardial damage.</article-title>\n<source><italic>Int J Radiat Oncol Biol Phys.</italic></source> (<year>2006</year>) <volume>66</volume>:<fpage>845</fpage>–<lpage>51</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijrobp.2006.06.007</pub-id>\n<pub-id pub-id-type=\"pmid\">17011456</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Konski</surname><given-names>A</given-names></name><name><surname>Li</surname><given-names>T</given-names></name><name><surname>Christensen</surname><given-names>M</given-names></name><name><surname>Cheng</surname><given-names>JD</given-names></name><name><surname>Yu</surname><given-names>JQ</given-names></name><name><surname>Crawford</surname><given-names>K</given-names></name><etal/></person-group>\n<article-title>Symptomatic cardiac toxicity is predicted by dosimetric and patient factors rather than changes in 18F-FDG PET determination of myocardial activity after chemoradiotherapy for esophageal cancer.</article-title>\n<source><italic>Radiother Oncol.</italic></source> (<year>2012</year>) <volume>104</volume>:<fpage>72</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.radonc.2012.04.016</pub-id>\n<pub-id pub-id-type=\"pmid\">22682539</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ishikura</surname><given-names>S</given-names></name><name><surname>Nihei</surname><given-names>K</given-names></name><name><surname>Ohtsu</surname><given-names>A</given-names></name><name><surname>Boku</surname><given-names>N</given-names></name><name><surname>Hironaka</surname><given-names>S</given-names></name><name><surname>Mera</surname><given-names>K</given-names></name><etal/></person-group>\n<article-title>Long-term toxicity after definitive chemoradiotherapy for squamous cell carcinoma of the thoracic esophagus.</article-title>\n<source><italic>J Clin Oncol.</italic></source> (<year>2003</year>) <volume>21</volume>:<fpage>2697</fpage>–<lpage>702</lpage>. <pub-id pub-id-type=\"doi\">10.1200/JCO.2003.03.055</pub-id>\n<pub-id pub-id-type=\"pmid\">12860946</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Beukema</surname><given-names>JC</given-names></name><name><surname>van Luijk</surname><given-names>P</given-names></name><name><surname>Widder</surname><given-names>J</given-names></name><name><surname>Langendijk</surname><given-names>JA</given-names></name><name><surname>Muijs</surname><given-names>CT.</given-names></name></person-group>\n<article-title>Is cardiac toxicity a relevant issue in the radiation treatment of esophageal cancer?</article-title>\n<source><italic>Radiother Oncol.</italic></source> (<year>2015</year>) <volume>114</volume>:<fpage>85</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.radonc.2014.11.037</pub-id>\n<pub-id pub-id-type=\"pmid\">25554226</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ito</surname><given-names>H</given-names></name><name><surname>Itasaka</surname><given-names>S</given-names></name><name><surname>Sakanaka</surname><given-names>K</given-names></name><name><surname>Araki</surname><given-names>N</given-names></name><name><surname>Mizowaki</surname><given-names>T</given-names></name><name><surname>Hiraoka</surname><given-names>M.</given-names></name></person-group>\n<article-title>Long-term complications of definitive chemoradiotherapy for esophageal cancer using the classical method.</article-title>\n<source><italic>J Radiat Res.</italic></source> (<year>2017</year>) <volume>58</volume>:<fpage>106</fpage>–<lpage>13</lpage>. <pub-id pub-id-type=\"doi\">10.1093/jrr/rrw078</pub-id>\n<pub-id pub-id-type=\"pmid\">27475126</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Immunol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Immunol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Immunol.</journal-id><journal-title-group><journal-title>Frontiers in Immunology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-3224</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849624</article-id><article-id pub-id-type=\"pmc\">PMC7431520</article-id><article-id pub-id-type=\"doi\">10.3389/fimmu.2020.01751</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Immunology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Molecular Evolution of Apolipoprotein Multigene Family and the Original Functional Properties of Serum Apolipoprotein (LAL2) in <italic>Lampetra japonica</italic></article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Han</surname><given-names>Qing</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1044250/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Han</surname><given-names>Yinglun</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wen</surname><given-names>Hongyan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1044208/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Pang</surname><given-names>Yue</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/676579/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Qingwei</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/751861/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>College of Life Sciences, Liaoning Normal University</institution>, <addr-line>Dalian</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Lamprey Research Center, Liaoning Normal University</institution>, <addr-line>Dalian</addr-line>, <country>China</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Collaborative Innovation Center of Seafood Deep Processing, Dalian Polytechnic University</institution>, <addr-line>Dalian</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Gyri T. Haugland, University of Bergen, Norway</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Zhihao Jia, Purdue University, United States; Jing Xing, Ocean University of China, China</p></fn><corresp id=\"c001\">*Correspondence: Yue Pang <email>pangyue01@163.com</email></corresp><corresp id=\"c002\">Qingwei Li <email>liqw@263.net</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†These authors have contributed equally to this work</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1751</elocation-id><history><date date-type=\"received\"><day>25</day><month>2</month><year>2020</year></date><date date-type=\"accepted\"><day>30</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Han, Han, Wen, Pang and Li.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Han, Han, Wen, Pang and Li</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Apolipoprotein (APO) genes represent a large family of genes encoding various binding proteins associated with plasma lipid transport. Due to the long divergence history, it remains to be confirmed whether these genes evolved from a common ancestor through gene duplication and original function, and how this evolution occurred. In this study, based on the phylogenetic tree, sequence alignment, motifs, and evolutionary analysis of gene synteny and collinearity, APOA, APOC, and APOE in higher vertebrates may have a common ancestor, lamprey serum apolipoprotein LAL1 or LAL2, which traces back to 360 million years ago. Moreover, the results of immunofluorescence, immunohistochemistry, and flow cytometry show that LAL2 is primarily distributed in the liver, kidney, and blood leukocytes of lampreys, and specifically localized in the cytoplasm of liver cells and leukocytes, as well as secreted into sera. Surface plasmon resonance technology demonstrates that LAL2 colocalizes to breast adenocarcinoma cells (MCF-7) or chronic myeloid leukemia cells (K562) associated with lamprey immune protein (LIP) and further enhances the killing effect of LIP on tumor cells. In addition, using quantitative real-time PCR (Q-PCR) and western blot methods, we found that the relative mRNA and protein expression of <italic>lal2</italic> in lamprey leukocytes and sera increased significantly at different times after stimulating with <italic>Staphylococcus aureus, Vibrio anguillarum</italic>, and Polyinosinic-polycytidylic acid (Poly I:C). Moreover, LAL2 was found to recognize and bind to gram-positive bacteria (<italic>Staphylococcus aureus</italic> and <italic>Bacillus cereus</italic>) and gram-negative bacteria (<italic>Escherichia coli</italic>) and play an important role in the antibacterial process. All in all, our data reveals a long, complex evolutionary history for apolipoprotein genes under different selection pressures, confirms the immune effect of LAL2 in lamprey sera against pathogens, and lays the foundation for further research regarding biological functions of lamprey immune systems.</p></abstract><kwd-group><kwd>apolipoprotein</kwd><kwd>LAL2</kwd><kwd>lamprey</kwd><kwd>antibacterial</kwd><kwd>immune system</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Natural Science Foundation of China<named-content content-type=\"fundref-id\">10.13039/501100001809</named-content></funding-source><award-id rid=\"cn001\">No.31772884</award-id></award-group></funding-group><counts><fig-count count=\"5\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"45\"/><page-count count=\"14\"/><word-count count=\"9431\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>As early as 1987, Pontes et al. (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>) founded two abundant apolipoproteins in the “high-density lipoprotein fraction” of ultracentrifuged plasma in <italic>Petromyzon marinus</italic>, designated lamprey apolipoproteins LAL1 and LAL2. Their amino acid compositions were similar to portions of apolipoprotein A-IV sequence in mammalian blood. However, the existing database indicates that LAL2 has no apolipoproteinA/E or C domain. Their functions are not fully elucidated, and it remains elusive whether they are the ancestors of vertebrate apolipoproteins. However, it is well-known that apolipoprotein is a protein component of plasma lipoproteins (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B3\" ref-type=\"bibr\">3</xref>). Apolipoproteins of vertebrates are primarily synthesized in the liver and small intestine, which are involved in the transport and redistribution of lipids between different tissues and cells through blood (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Research shows that several apolipoproteins also play important roles in antibacterial and antiviral processes (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>–<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). For example, APOA1-containing high-density lipoprotein particles (HDL) exert antibacterial activity by directly affecting the growth of bacteria and promoting the self-defense mechanism of normal and immunocompromised individuals (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). This function is attributed primarily to the ability of APOA1 to bind and neutralize both bacterial endotoxin lipopolysachharide (LPS) and lipoteichoic acid (LTA) (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>–<xref rid=\"B14\" ref-type=\"bibr\">14</xref>). Moreover, whether it is expressed <italic>in vivo</italic> or injected, APOA1 elictis an antiviral effect on enveloped and non-enveloped DNA and RNA viruses by directly causing viral inactivation. Specifically, APOA1 has been shown to arrest virus-induced cell fusion in the blood during human immunodeficiency virus (HIV) and Herpes virus infection, thereby preventing the virus from penetrating into the cell (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). Additionally, highly conserved alternating cationic/hydrophobic motifs have been identified in the APOC1 sequence that participate in binding to LPS and enhanced biological response to LPS via a mechanism similar to lipopolysaccharide-binding protein (LBP) (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Meanwhile, APOL-1 has pore-forming microbicidal activity that can cause lysis and death of trypanosome (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>).</p><p>The lamprey is a member of an ancient lineage of jawless fish that stem ~550 million years ago and has served as a crucial model for understanding conserved features that are relevant to biomedicine. Lampreys have adaptive immune systems with variable lymphocyte receptors (VLRs) and innate immune systems with complement related molecules to prevent the invasion of various foreign pathogens, such as mannose binding lectin (MBL), complement C1q, C3, etc. (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>–<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). Lamprey immune protein (LIP), a cytotoxic protein, has a jacalin-like domain and an aerolysin pore-forming domain previously identified in granulocytes of <italic>Lampetra japonica</italic> (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). We demonstrate the crystal structure of LIP and the mode of action involving dual selective recognition and efficient binding dependent on both N-linked glycans on GPI-anchored proteins (GPI-APs) and sphingomyelin (SM) in lipid rafts (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). LIP can kill a panel of human cancer cells yet has minimal effects on normal cells. MCF-7 and K562 cells stimulated with LIP exhibited the generation of chemokines and proinflammatory molecules, and increased the expression of genes in the calcium signaling pathway, ROS signaling pathway, and natural killer cell-mediated cytotoxicity pathways (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>, <xref rid=\"B24\" ref-type=\"bibr\">24</xref>). However, it remains unclear whether large amounts of LAL2 in serum interacts with LIP molecule and participates in the immune response.</p><p>In the present work, we elucidated the molecular evolution process of LAL2 and LAL1 and determined their relationship with vertebrate orthologs and paralogs. We further investigated LAL2 expression patterns in gill, supraneural body, heart, liver, intestine, and kidney, and also intracellular localization in liver cells and leukocytes. Simultaneously, the potential interaction between LAL2 and LIP was verified, and the addition of LAL2 was found to enhance the killing activity of LIP in lamprey. Moreover, the antibacterial and antiviral activities of LAL2 were examined to shed light on its role in immunity. Exploring the biological function of LAL2 lays the foundation for clarifying antibacterial function in lamprey and provides a reference for the research of innate immune mechanisms of lamprey.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Animals and Cell Culture</title><p>Adult <italic>L. japonica</italic> (length: 36–42 cm, weight: 75–112 g) and <italic>Lampetra morii</italic> (length: 20–25 cm, weight: 18–23 g) were obtained from the Songhua River from Heilongjiang Province, China. The lampreys were housed in fully automatic water purification tanks at 4–6°C. All animals were in good condition before the experiments.</p><p>MCF-7 cells and K562 cells, purchased from the American Type Culture Collection (Manassas, VA) were maintained in RPMI 1640 medium (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, USA), 100 U/mL penicillin (Sigma-Aldrich, USA), and 100 mg/mL streptomycin (Sigma-Aldrich, USA). Cells were cultured in an incubator humidified with 5% CO<sub>2</sub> and 95% air at 37°C.</p><p><italic>S. aureus, B. cereus, Vibrio anguillarum</italic>, and <italic>E. coli</italic> strains were isolated from the intestine of the lamprey. <italic>S. aureus</italic> (28°C), <italic>B. cereus</italic> (28°C), and <italic>E. coli</italic> (37°C) strains were cultured in Luria broth liquid medium with 1% peptone, 1% NaCl, and 0.5% yeast extract (Sangon Biotech, Shanghai, China). The <italic>V. anguillarum</italic> (28°C) strain was cultured in 2216E liquid medium with 0.5% peptone, 0.1% yeast extract, and seawater (pH = 8.0). All the strains were supplied by College of Life Science, Liaoning Normal University (Dalian, China).</p></sec><sec><title>Sequence Analysis, Sequence Alignments, and Phylogenetic Analysis</title><p>The amino acid sequences of lamprey apolipoprotein LAL1 and LAL2 were obtained from the <italic>L. japonica</italic> three-generation sequencing library and <italic>Lethenteron reissneri</italic> database from our laboratory. The amino acid sequences of the corresponding apolipoprotein family genes in other species are from NCBI (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/\">https://www.ncbi.nlm.nih.gov/</ext-link>) and Ensembl (<ext-link ext-link-type=\"uri\" xlink:href=\"http://asia.ensembl.org/index.html\">http://asia.ensembl.org/index.html</ext-link>) database for sequence alignment by Bioedit 7.0. Two comparisons of syntenic genomic regions, respectively containing <italic>lal1</italic> and <italic>lal2</italic> genes, were completed using <italic>L. reissneri</italic> databases and the Genomicus website (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.genomicus.biologie.ens.fr/genomicus-92.01/cgi-bin/search.pl\">http://www.genomicus.biologie.ens.fr/genomicus-92.01/cgi-bin/search.pl</ext-link>). Thereafter, a phylogenetic tree was constructed using the neighbor-joining (NJ) method using MEGA 7.0 software and the bootstrap test (1,000 replicates). The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Functional domains of the apolipoprotein family genes were analyzed using the NCBI website (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\">https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi</ext-link>). Motif analysis was performed using the MEME website (<ext-link ext-link-type=\"uri\" xlink:href=\"http://meme-suite.org/\">http://meme-suite.org/</ext-link>) and TB (Toolbox for Biologists) tools software.</p></sec><sec><title>Expression of Recombinant LAL2 (rLAL2) Protein and Preparation of Antibodies</title><p>The prokaryotic expression vector pET32a-<italic>lal2</italic> was constructed to obtain the recombinant LAL2 (rLAL2) protein as described previously (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Rabbit anti-LAL2 polyclonal antibody was generated through subcutaneous injection of New Zealand white rabbits with purified rLAL2 protein over 8 weeks, as described previously (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). The antibody titer in the anti-rLAL2 serum was determined via enzyme-linked immunosorbent assay (ELISA) at different dilutions, and the titer increased 640,000-fold over pre-immunization levels (pre-immunized rabbit IgG was used as a negative control). The antibody specificity was confirmed using western blot assays; rLAL2 and lamprey serum were subjected to 12% SDS-PAGE and transferred onto nitrocellulose membranes (Invitrogen, USA). The membranes were blocked with 5% non-fat powdered milk (Sangon Biotech, Shanghai, China) and incubated with rabbit anti-LAL2 (1 μg/mL) antibody overnight at 4°C, followed by incubation with 1.2 μg/mL HRP-conjugated goat anti-rabbit IgG (Sangon Biotech, Shanghai, China). Next, membranes were washed four times with tris-buffered saline Tween-20 (Sangon Biotech, Shanghai, China) and developed with enhanced chemiluminescence (ECL) substrate (Tanon, China) using Alpha FluorChem®Q (Cell Biosciences, USA).</p></sec><sec><title>Purification of Natural LAL2 Protein With Anion Exchange Chromatography</title><p>Serum from <italic>L. japonica</italic> was dialyzed in buffer A consisting of 20 mM KPB, 0.1 M KCl and 5% glycerol, at pH 7.0 at 4°C. The dialyzed fraction was filtered through a 0.22 μM membrane and was applied to a 10 mL × 2 MacroPrep Ceramic Hydroxyapatite column equilibrated with buffer A. The column was then washed with the same buffer and eluted with a linear gradient from 0 to 250 mM KPB in buffer A. The pooled fractions containing protein activity from the above column were dialyzed in buffer B consisting of 20 mM Tris-HCl and 5% glycerol, at pH 8.0 at 4°C. The dialyzed fraction was applied to a 20 mL Q Sepharose Fast Flow column equilibrated with buffer B. After washing, the sample was eluted with a linear gradient from 0 to 300 mM of KCl in buffer B. During the separation and purification of the active components of lamprey serum (TaKaRa, Dalian, China), we observed an abundance of natural LAL2 protein in the eluted sample No. 46. It was diluted with low-salt buffer (20 mM Tris-HCl, pH = 9.0), passed through the anion exchange column (HiTrap Q HP_1 mL, General Electric Company, USA) at a rate of 0.8 mL/min using ÄKTA pure (General Electric Company, USA), and impure proteins were removed with low-salt buffer at a rate of 1 mL/min. High-salt buffer (20 mM Tris-HCl, 1 M NaCl, pH = 9.0) was gradually added to the low-salt buffer, finally the target protein was eluted at a rate of 1 mL/min using mixed buffer in a time-dose dependent manner. After detection using 15% SDS-PAGE, it was dialyzed using PBS (145.3 mM NaCl, 8.4 mM Na<sub>2</sub>HPO<sub>4</sub>, and 2 mM NaH<sub>2</sub>PO<sub>4</sub>). All protein concentrations were measured using the Bicinchoninic acid (BCA) protein assay kit (Sangon biotech, Shanghai, China).</p></sec><sec><title>Immunofluorescence and Flow Cytometry Were Used to Detect the Localization of LAL2 and LIP</title><p>Lamprey liver cells and blood leukocytes were isolated, fixed, permeabilized, and blocked with fetal bovine serum as previously described (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). Briefly, cells were incubated with rabbit anti-rLAL2 antibody (0.8 μg/mL) at 4°C overnight. The next day, cells were washed twice with PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na<sub>2</sub>HPO<sub>4</sub>, and 1.8 mM KH<sub>2</sub>PO<sub>4</sub>) and incubated with Alexa Fluor 488-labeled donkey anti-rabbit IgG (Thermo Fisher, USA). Following washing with PBS, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI). After washing twice with PBS, the cover slips were mounted on glass slides with one drop of antifade solution. The immunofluorescence was visualized and captured using LSM 710 Laser Scanning Confocal Microscopy (Carl Zeiss Inc, Germany) and analyzed using Zeiss ZENLE software. The lamprey liver cells and leukocytes were fixed for 15 min in 4% paraformaldehyde with PBS at room temperature. Thereafter, cells were treated according to the method described above to detect the expression of LAL2 in liver cells and leukocytes using flow cytometry (BD Biosciences, USA). The flow cytometer was set at 488 nm (excitation wavelengths) to detect green fluorescence. Cells incubated with normal rabbit IgG were used as isotype controls.</p><p>In the same way, MCF-7/K562 cells were observed for 2 h with rLAL2 protein and rLAL2 + LIP protein incubation. When the rLAL2 + LIP incubated group exhibited bubbling, all cells were fixed, blocked using fetal bovine serum, incubated with rabbit anti-rLAL2 antibody (primary antibody), followed by Alexa Fluor 555-labeled donkey anti-rabbit IgG antibody (second antibody) to detect the localization of LAL2 protein on MCF-7/K562 cells. After labeling LIP protein with Alexa Fluor 488 (Microscale Protein Labeling Kit, Invitrogen, USA), four experimental groups: Alexa 488-LIP, rLAL2-1 μg + Alexa 488-LIP, rLAL2-2 μg + Alexa 488-LIP, and rLAL2-3 μg + Alexa 488-LIP, were established to detect the location of LIP protein on the MCF-7/K562 cells. The cells were analyzed on a FACSAria flow cytometer (BD Biosciences, USA), which was set at 488 and 555 nm (excitation wavelengths) to detect green and red fluorescence, respectively.</p></sec><sec><title>The Synergistic Effect of rLAL2 on LIP Killing</title><p>MCF-7 cells (cultured in 96 well plate) and LIP protein were incubated with rLAL2 for 3 h. According to the experimental sequence in <xref ref-type=\"fig\" rid=\"F4\">Figure 4D</xref>, MCF-7 cells were incubated with PBS, 2 μg LIP, 2.5 μg rLAL2, or 5 μg rLAL2, LIP was added to the MCF-7 cells that pre-incubated with 2.5 μg or 5 μg rLAL2, 2.5 μg/5 μg rLAL2, and LIP were pre-incubated and then the mixture was added to MCF-7 cells. Thereafter, cells were stained for 5 min using Hoechst (blue, Beyotime, Shanghai, China) and propidium iodide (PI, red, Thermo fisher, USA), finally analyzed on the Operetta™ High-content machine (PerkinElmer, USA).</p><p>MCF-7 and K562 cells were cultured and collected, the cells were divided into five groups with the addition of PBS, 5 μg rLAL2, 2 μg LIP, 2.5 μg rLAL2 + 2 μg LIP, and 5 μg rLAL2 + 2 μg LIP as shown in <xref ref-type=\"fig\" rid=\"F4\">Figure 4F</xref>, respectively. Followed by staining with Hoechst and PI for 5 min. Cells were then filtered and analyzed using a flow cytometer set at 560 nm(excitation wavelength), and analyses were performed by using Cell Quest Pro software. The appropriate FSC voltage and threshold were adjusted, inspector-gate, G1 = R1, to regulate the fluorescence voltage to set the negative control and the compensation between the fluorescence. The samples were then loaded in order, and the data files were obtained.</p></sec><sec><title>Surface Plasmon Resonance Analysis</title><p>LIP or rLAL2 protein were coupled on the second channel of a CM5 chip using buffer (pH = 4.0), while the first channel was used as a reference channel, both chips were activated with NHS/EDC and blocked with ethanolamine. LIP or rLAL2 in HBS-EP solution was flowed through the rLAL2 or LIP chip. The analyte, rLAL2 or LIP protein, was diluted in the same buffer. The Biacore T200 (General Electric Company, USA) was used for the experiment and subsequent analysis. TRX was used as a negative control, using the same experimental method.</p></sec><sec><title>Quantitative Real-Time PCR (Q-PCR)</title><p>Adult <italic>L. japonica</italic> were divided into three groups (three animals per group), each immunized with 100 μL <italic>S. aureus, V. anguillarum</italic> (suspended to 1 × 10<sup>8</sup> cells/mL in normal saline), or Poly I:C (0.1 mg/mL) for 0 h (immediately following addition of stimulus), 2, 8, 24, 48, and 72 h via intraperitoneal injections, respectively. Adult lamprey blood was collected by cutting the tail, and leukocytes were isolated from blood by Ficoll-Paque gradient centrifugation with lymphocyte separation solution (160 × <italic>g</italic>, 20 min) (TBD, China). <italic>L. morii</italic> were immunized using the same method to collect sera. The gill, supraneural body, heart, liver, intestine, kidney, and leukocytes were obtained from normal adult <italic>L. japonica</italic>. Total RNA was extracted from the tissues and cells using Trizol (Invitrogen, USA), and the RNA was treated with DNase I (TaKaRa, Dalian, China). Reverse transcription that each group of RNA is quantified to 2 μg was performed using gDNA Eraser (PrimeScript™ RT reagent Kit) as described by the manufacturer (TaKaRa, Dalian, China). Real-time quantitative PCR was conducted using a SYBR® PrimeScript™ RT-PCR Kit (TaKaRa, Dalian, China) according to the manufacturer's protocol. The PCR was performed in a 25 μL volume, consisting of 2 μL cDNA (diluting to 50 ng/μL), 12.5 μL SYBR Premix Ex Taq, 1 μL of each primer (10 μM), and ddH<sub>2</sub>O. The gene expression in each sample was normalized relative to the <italic>gapdh</italic> gene (GenBank accession no. <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"KU041137.1\">KU041137.1</ext-link>). The reaction efficiency was tested by gradual dilution of the cDNA template (1, 5, 10, 20, and 40×). The amplification efficiency of all primers was confirmed to be between 0.9 and 1.1, and the specificity of the amplification reaction was analyzed by dissociation curve analyses. The primer sequences used are as follows: <italic>lal2</italic>-F: 5′-ACGGTCCACCTGCACGAAT-3′; <italic>lal2</italic>-R: 5′-TTCACCTCCTTCATCAGTCCAA-3′. L-<italic>gapdh</italic>-F: 5′-AACCAACTGCCTGGCTCCT-3′; L-<italic>gapdh</italic>-R: 5′-GTCTTCTGCGTTGCCGTGT-3′.</p></sec><sec><title>Scanning Electron Microscopy (SEM) for Bacterial Morphology</title><p>The bacteria were incubated with PBS and 5 μM LAL2 at 4°C for 12 h, fixed with 2.5% glutaraldehyde (Kemiou, China) at 4°C overnight, and dehydrated at various ethanol gradients: 30, 50, 80, and 100%. In 100% ethanol, point samples on tables were sprayed with gold and photographed using scanning electron microscopy.</p></sec><sec><title>Enzyme Linked Immunosorbent Assay (ELISA) Analysis of the Interaction Between LAL2 Protein and Microbial Components</title><p>Plates were coated with various microbial components (0.2 μg/well) at 4°C overnight, then were washed and incubated with different concentrations of LAL2 (0, 10, 20, 50, 100, 200 nM) at 37°C for 3 h, 1% BSA was added, followed by detection with 100 μL/well rabbit anti-LAL2 antibody (4 μg/mL) and goat anti-rabbit antibody (1.5 μg/mL). ELISA substrate (100 μL/well, Solarbio, USA) was added and incubated for 15 min at 37°C, color development was halted through the addition of 2 M H<sub>2</sub>SO<sub>4</sub> (50 μL/well). The plates were washed thrice with PBST (PBS with 0.05% Tween-20) between steps. One representative experiment of three is shown. Background absorbance without LAL2 protein and with anti-LAL2 antibody was subtracted as a negative control (NC).</p></sec><sec><title>Statistical Analysis</title><p>All calculations were performed using GraphPad Prism 7 (GraphPad Software Inc, USA). The data are presented as the mean ± S.E. The significance of the differences between the mean values was determined using Microsoft Excel 2007. In all cases, <sup>*</sup><italic>P</italic> < 0.05 was considered a statistically significant difference, <sup>**</sup><italic>P</italic> < 0.01 was considered a very significant difference, and <sup>***</sup><italic>P</italic> < 0.001 was considered an extremely significant difference.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>Identification and Purification of Lamprey LAL2</title><p>In our previous study, lamprey sera exerted important cytotoxic effects on tumor cells (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). This is evidenced by the morphological changes and cell organelle damage observed in cervical cancer cells (HeLa) and acute promyelocytic leukemia cells (NB4) treated with lamprey serum during a 15 min incubation (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). To identify this cytotoxic protein in the sera (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>), lamprey sera was purified using a hydroxyapatite column and a Q Sepharose Fast Flow column. The fraction of protein activity was determined by the degree of cell membrane disruption. When these fractions with active protein were collected and analyzed via 12% SDS-PAGE, a protein band was observed at ~34 kDa molecular-weight (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). According to the liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis of tryptic-digested peptides, the purified protein was identified as LIP. In addition, we observed two protein bands positioned at ~15–20 kDa. The two proteins were identified as LAL2 by mass spectrometry (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Tables S1</xref>, <xref ref-type=\"supplementary-material\" rid=\"SM1\">S2</xref>). To detect the difference between the two LAL2 proteins (in <xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref>), N-terminal sequencing was performed by Edman degradation and online analysis of High-Performance Liquid Chromatography at the Shanghai Life Science Research Institute. As shown in <xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM2\">Figure S1</xref>, the first ten amino acids of the two bands were identical (NH2-Asp-Glu-Thr-Gln-Leu-Val-Pro-Ala-Ser-Gly), which may be the result of glycosylation. Of course, it may also be possible that the C-terminal peptide of LAL2 was degraded. The open reading frame (ORF) of LAL2 has 576 bp and encodes a total of 191 amino acid residues. The first 23 amino acids coded after the initiation ATG are characteristic of a signal peptide.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Isolation and identification of LAL2 from lamprey serum. <bold>(A)</bold> Electrophoretic patterns of lamprey serum proteins via 15% SDS-PAGE. M, low molecular-weight protein marker. Red arrow points to apolipoprotein LAL2. <bold>(B)</bold> Detection of killer components in lamprey sera via 12% SDS-PAGE. <bold>(C)</bold> Electrotransfer atlas of LAL2 from lamprey serum that dyed with Ponceau S. M, Dual marker (Bio-Rad), bands for number 1 and 2 both represent LAL2. <bold>(D)</bold> Ten amino acid test maps of LAL2 N-terminal sequence (number 1).</p></caption><graphic xlink:href=\"fimmu-11-01751-g0001\"/></fig></sec><sec><title>Evolutionary Analyses of LAL1 and LAL2</title><p>The LAL2 proteins were purified and identified in the “high-density lipoprotein fraction” of plasma from <italic>Petromyzon marinus</italic> (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). However, high homology in amino acid sequences was not observed between LAL2 and the apolipoproteins of other species, and the domain in LAL2 were not found to be similar to mammalian blood apolipoproteins. In the current study, the amino acid sequence alignment results of LAL2 revealed that LAL2 (<italic>Lampetra japonica</italic>) display more than 90% sequence similarity to blood plasma LAL2 from <italic>Petromyzon marinus, Lampetra fluviatilis</italic>, and <italic>Lethenteron reissneri</italic> (<xref ref-type=\"supplementary-material\" rid=\"SM3\">Figure S2</xref>). To better understand the evolution of the <italic>lal2</italic> gene family during the vertebrate evolutionary process, the neighboring gene environment of lamprey <italic>lal2</italic> was compared among fish, amphibian, bird, and mammals. Using the draft genome assemblies of <italic>Lethenteron reissneri</italic>, we were able to assign orthology of the lamprey genes based on conserved synteny for genes directly surrounding the <italic>lal2</italic> gene (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>). A comparison of genomic regions containing <italic>lal2</italic> genes shows that there are eight <italic>lal2</italic> loci on the <italic>L. reissneri</italic> scaffold_686, no introns, and similar gene groups as sea lamprey in its surrounding. Strong syntenic relationships among LAL2 gene orthologs were easily detected in three jawless vertebrates (<italic>L. reissneri, P. marinus</italic>, and <italic>L. japonica</italic>) genome sequences that we examined. Three genes (<italic>adar, kcnn</italic>, and <italic>rab)</italic> surrounding <italic>lal2</italic> are also found in the neighborhood of zebrafish <italic>apoa1, apoa2, apoa4, apoc1, apoc2, apoc4</italic>, and <italic>apoe</italic>. In addition, this analysis confirmed that orthologs and paralogs of mammalian <italic>apo</italic> are present in birds, amphibians, and bony fishes. Near to the <italic>lal2</italic> gene in the lamprey, the <italic>fxyd</italic> and <italic>cadm</italic> genes, although undetectable, could be examined for syntenic relationship with <italic>apo</italic> neighborhood in other vertebrates. Unfortunately, the tandem LAL2 sequences were unidentified based on the current sea lamprey genome data, thus, it is not possible to define the quantity of LAL2 on the chromosome of sea lamprey. And the adjacent genes of LAL2 are unable to determine perfectly because of poor sequencing and splicing results in <italic>Lampetra japonica</italic> genome. Furthermore, we also found <italic>lal1</italic> and <italic>tom40</italic> to be evolutionarily linked, and located on the <italic>L. reissneri</italic> scaffold_555 (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). And <italic>lal1</italic> is always accompanied by <italic>bcl</italic> in lamprey, zebrafish, frog, mouse, and human, the chromosome on which it is located is relatively stable. However, we only find <italic>lal1</italic> in scaffold_05301 with no neighboring gene environment from <italic>Petromyzon marinus</italic> Germline Genome. And there is no <italic>apoe</italic> genomic information for reptiles and birds in genomics database. In a word, it is possible that close genomic proximity of <italic>lal2</italic> and <italic>apoa</italic> evolved, while LAL1 is located in close proximity to <italic>apoe</italic> in fish and mammals (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>), <italic>apoc1, 2, 4</italic> were formed by the replication and gene deletion of <italic>apoa</italic> and translocated to the periphery of <italic>apoe</italic> (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>).</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Evolutionary analyses of LAL2. <bold>(A,B)</bold> The conservation of genes neighboring apolipoprotein family genes, the arrowheads pointing in opposite directions indicate genes located on opposite strands. Chr denotes the chromosome. <bold>(C)</bold> Model diagram of vertebrate apolipoprotein evolution. <bold>(D)</bold> The phylogenetic tree was constructed based on the 39 full-length protein sequences of APOA, APOC, APOE, LAL1, and LAL2. The optimal tree with the sum of branch length = 12.09372528 is shown and the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. The section on the right indicates the corresponding domain composition of the sequence in left. <bold>(E)</bold> A motif composition of the apolipoprotein family. The motifs, which are numbered 1–9, are shown in different colored blocks. The regions fenced by blue lines represent “apolipoprotein” domain, similarly the yellow represent “APO-CIII” domain, the green represents “SMC-pork-B” domain.</p></caption><graphic xlink:href=\"fimmu-11-01751-g0002\"/></fig><p>The phylogenetic tree based on the alignment of <italic>Branchiostoma belcheri</italic> APOA1, and other apolipoprotein amino acid sequences, involved in co-linearity was drawn using the NJ methods. <italic>Lepisosteus oculatus</italic> APOA1, APOA4, and APOEb were added to ensure the stability of the phylogenetic tree. As shown in <xref ref-type=\"fig\" rid=\"F2\">Figure 2D</xref>, the BbAPOA1 was placed apart. The overall topology supported two main clusters, which corresponded to APOA/E and APOC vertebrate families, while lamprey LAL2 sequence was considered closer to APOA/E vertebrate family, similar to DrAPOA2 and equivalent to the outgroups of distinct family 1 (DF1). Based on the results of collinear analysis, this suggests that LAL2 is likely a common ancestor of vertebrate APOA1, A4, A5, and E. Lamprey LAL1 sequence was similarly considered closer to the APOC vertebrate family and formed distinct family 2 (DF2) with an improved bootstrap value and compact structure. Notably, the branch of LAL1 is also an outgroup equivalent to DF1. The results of phylogenetic analysis demonstrated that lamprey LAL1 and LAL2 are likely to have common ancestors, and APOA/E and APOC in the vertebrate lineage arose by duplication and reorganization of LAL2 and LAL1, respectively.</p><p>Our predicted results using the PSIPRED website show that the α-helix of the LAL2 and LAL1 secondary structures account for 78.01 and 79.05% of the secondary structure, respectively (<xref ref-type=\"supplementary-material\" rid=\"SM4\">Figures S3A,B</xref>). Furthermore, circular dichroism shows the secondary structure of LAL2, similar to APOA1, to be comprised primarily of α-helices (<xref ref-type=\"supplementary-material\" rid=\"SM4\">Figures S3C,D</xref>). To analyze conservation of the amino acid sequence, after searching select representative sequences to predict motif composition (<xref ref-type=\"fig\" rid=\"F2\">Figure 2E</xref>, <xref ref-type=\"supplementary-material\" rid=\"SM1\">Table S3</xref>), we observed that when MEME selects nine motifs to analyze the apolipoproteins of each species, amphioxus and lamprey display 2–3 motifs, APOC3 possesses only two motifs, while other apolipoproteins of zebrafish, spotted gar, mouse, and human possess 5–10 motifs. From the types of motifs is was found that LrLAL1 has the same motif 1, 2, and 4 as zebrafish, spotted gar, mouse, and human, while motifs 2, 4, and 5 exist in LrLAL2 and apolipoprotein A and E family sequences of most jaw vertebrates. Furthermore, motifs 3, 6, and 7 evolved from fish apolipoproteins, and motifs 8 and 9 are unique to mammalian APOA5 and APOE, respectively. The apolipoprotein domain of APOA1, APOA4, and APOE from jawed vertebrates, which is associated with lipid particles and may function in lipoprotein-mediated lipid transport, is primarily composed of motifs 4, 5, 6, and 7. These proteins contain several 22 residue repeats which form a pair of α-helices. Meanwhile the APOC3 “APO-CIII apolipoprotein” domain is primarily composed of motifs 2 and 3, which inhibit lipoprotein lipase (LPL) activity and play roles in triglyceride metabolism. It is obvious that LrLAL1 and LrLAL2 retain parts of the same motif as the above-mentioned domain, however, they do not possess the integral apolipoprotein domain. Therefore, it is suggested that the apolipoprotein superfamily from lamprey LAL1 and LAL2 to mammal APOA, APOC, and APOE has acquired most of its structural and functional innovations throughout vertebrate evolution.</p></sec><sec><title>The Expression Pattern of LAL2 in Lamprey Tissues and Cells</title><p>The relative expression levels of <italic>lal2</italic> gene in the lamprey gill, supraneural body, heart, liver, intestine, kidney, and leukocytes were detected using Q-PCR (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>). LAL2 is expressed in these tissues or cells, and the expression levels in liver, leukocytes, and kidney were relatively high. To detect the distribution of natural LAL2 protein in different tissues of the lamprey, we purified rLAL2 migrated as a single band using a 12% SDS-PAGE gel with a molecular mass of ~38 kDa (<xref ref-type=\"supplementary-material\" rid=\"SM5\">Figure S4A</xref>), and prepared LAL2 rabbit polyclonal antibody, which specifically recognized rLAL2 and native LAL2 (<xref ref-type=\"supplementary-material\" rid=\"SM5\">Figures S4B,C</xref>). The band around 40 kDa in the serum sample was identified as LAL2-dimer using LC-MS/MS analysis (<xref ref-type=\"supplementary-material\" rid=\"SM5\">Figure S4D</xref>). The localization analysis was performed using immunohistochemistry as described previously (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>), and LAL2 was primarily expressed in the epithelial cells and blood cells of the gill, blood cells of the supraneural body, endothelial cell area of the heart, venous areas of the liver, epithelial cells of the intestine, and venous and epithelial cells of the kidney (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>). The expression was relatively high in several tissues, such as liver, kidney, and supraneural body. The lamprey liver cells and leukocytes were fixed separately to further detect LAL2 expression at the protein level. Furthermore, intracellular localization of LAL2 was revealed using flow cytometry. Results showed that LAL2 was expressed in both liver cells and leukocytes (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>, left), which was observed in the cytoplasm using confocal microscopy (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>, right).</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>The tissue distribution and cellular localization of LAL2. <bold>(A)</bold> The expression level of <italic>lal2</italic>, which was compared with <italic>gapdh</italic> in various lamprey tissues using Q-PCR. <bold>(B)</bold> Distribution and localization of LAL2 as observed by immunohistochemical staining, in which rabbit IgG is an isotype control, LAL2 with antibody incubating group (preabsorption) was used as a negative control, the upper parts of group anti-LAL2 are the corresponding enlarged view below. <bold>(C)</bold> Flow cytometry and immunofluorescence was used to analyze the localization of LAL2 in lamprey liver cells and leukocytes, as compared to IgG. Images were taken by laser confocal microscopy with the fluorescent cell-labeling dye DAPI (blue) and Alexa Fluor 488 goat anti-rabbit IgG (green). Scale bar: 5 μm.</p></caption><graphic xlink:href=\"fimmu-11-01751-g0003\"/></fig></sec><sec><title>LAL2 Can Promote the Localization and Killing Effect of LIP on MCF-7 and K562 Cells</title><p>LIP is a cytotoxic lamprey protein, which plays an important role in tumor cell survival and growth (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>–<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). Previously, during the process of purifying the cytotoxic protein in lamprey serum, it was found that LIP and LAL2 were always present in an eluted sample (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). To determine whether there is a certain interaction between LIP and LAL2, rLAL2 or LIP protein were anchored to the surface of the chip using surface plasmon resonance technology, different concentrations of LIP or rLAL2 were used as analytes, and affinity kinetics fitting analysis was performed: rLAL2 protein flowed through the anchored LIP chip, the affinity KD was 5.582E-8M, LIP flowed through the anchored rLAL2 chip, and the affinity KD was 2.11E-8M. Both KD values reflect the same magnitude. This fully demonstrates the strong interaction between rLAL2 and LIP (<xref ref-type=\"fig\" rid=\"F4\">Figures 4A,B</xref>), and suggests that LIP has no interaction with TRX (<xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>).</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>LAL2 plays a synergistic role in LIP killing the tumor cells. <bold>(A)</bold> Biacore analysis of the interactions between different concentrations of rLAL2 and immobilized LIP [in resonance units (RU)]. <bold>(B)</bold> Biacore analysis of different concentrations of LIP and immobilized rLAL2 (in RU). <bold>(C)</bold> Biacore analysis of different TRX concentrations and immobilized LIP. <bold>(D,E)</bold> High-content analyzed the killing effect of LIP on MCF-7 cells after incubation with rLAL2 (<italic>n</italic> = 3), scale bar: 100 μm. The statistical map is calculated from six fields randomly selected for each well. <bold>(F)</bold> Flow cytometry was used to analyze the killing effect of LIP on MCF-7/K562 cells after incubation with rLAL2. <bold>(G,H)</bold> Effect of rLAL2 on the localization of LIP molecule on MCF-7/K562 cells with the Alexa 488-LIP (green) and Alexa 555-goat anti-rabbit IgG (red).</p></caption><graphic xlink:href=\"fimmu-11-01751-g0004\"/></fig><p>LIP exerts a specific killing effect on certain tumor cells (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>, <xref rid=\"B24\" ref-type=\"bibr\">24</xref>). The selective killing mechanism proposes that LIP could bind to biantennary bi-sialylated non-fucosylated N-glycan of cancer cells, such as MCF-7 and K562 cells, and not affect normal cells (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). It was speculated that the interaction between rLAL2 and LIP may also influence LIP killing activity, the MCF-7 cells sensitive to LIP are plated in 96-well plates for overnight culture, according to the experimental design. Results of PI/Hoechst staining and high-content analysis indicated that rLAL2 treatment alone had no impact on MCF-7 cells. However, rLAL2 significantly promoted the killing effect of LIP on MCF-7 cells in a dose-dependent manner (<sup>*</sup><italic>P</italic> < 0.05, <xref ref-type=\"fig\" rid=\"F4\">Figures 4D,E</xref>), regardless of whether rLAL2 protein was incubated with MCF-7 cells alone before LIP protein addition or a mixture of rLAL2 and LIP was added to the cells. In order to further verify the synergy of rLAL2 on the killing activity of LIP, K562 cells were analyzed, showing results identical to those of MCF-7 cells. Furthermore, the effect of rLAL2 on the killing activity of LIP was analyzed using a combination of PI staining and flow cytometry, indicating that the results are consistent with the above results (<xref ref-type=\"fig\" rid=\"F4\">Figure 4F</xref>). In summary, our findings show that LAL2 play a major role in assisting LIP to kill tumor cells.</p><p>To investigate the localization of LIP on MCF-7 or K562 cells affected by the interaction of LAL2, immunofluorescence assays were performed with LAL2 (labeled with Alexa 555) and LIP (labeled with Alexa 488). Thereafter, MCF-7 or K562 cells were incubated with LAL2 alone, LIP alone, or the combination of LAL2 and LIP. The results revealed that rLAL2 could bind to MCF-7 or K562 cells and was not affected by LIP. When Alexa 555-LAL2 and Alexa 488-LIP were added together to MCF-7/K562 cells, compared with Alexa 488-LIP treated alone, the number of cells located by Alexa 488-LIP increased. As the concentration of LAL2 increased, the number of cells located by Alexa 488-LIP gradually increased (<xref ref-type=\"fig\" rid=\"F4\">Figures 4G,H</xref>).</p></sec><sec><title>LAL2 Involved in the Immune Response of Bacteria and Poly I:C</title><p>Based on the above experimental results, it is speculated that LAL2 plays a role in the lamprey immune response. To verify this hypothesis, by means of Q-PCR, the temporal expression of <italic>lal2</italic> genes was detected after stimulating lamprey with gram-positive bacterium, gram-negative bacterium, or Poly I:C virus mimic for 0, 2, 8, 24, 48, or 72 h (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>). The results showed that the expression of <italic>lal2</italic> mRNA was up-regulated significantly (<italic>P</italic> = 0.0005) in the <italic>S. aureus</italic> stimulation group, and reached the maximal level at 2 h post-stimulation, which was 4.0-fold compared with the 0 h group. In the <italic>V. anguillarum</italic> stimulation group, the expression of <italic>lal2</italic> was strongly up-regulated (<italic>P</italic> = 0.0214), and reached the maximal level at 24 h, which was 76.8-fold compared with the blank group. The expression of <italic>lal2</italic> was also significantly increased (<italic>P</italic> = 0.0134) with Poly I:C stimulation and reached the maximal level at 8 h, which was 12.2-fold compared with the control group. In the case of the same amount of total serum protein, the protein levels of LAL2 were significantly increased by stimulation of <italic>S. aureus, V. anguillarum</italic>, and Poly I:C over 48 and 72 h (<xref ref-type=\"fig\" rid=\"F5\">Figures 5B,C</xref>).</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>LAL2 plays an important role in the antibacterial and antiviral process. <bold>(A)</bold> Q-PCR was used to determine the relative expression of <italic>lal2</italic> genes after stimulating (<italic>n</italic> = 3). <bold>(B)</bold> After 48 and 72 h stimulation with different immunogens, western blot detected LAL2 protein expression level in lamprey serum (<italic>n</italic> = 3). <bold>(C)</bold> The summary graph of LAL2 relative expression by calculating the ratio of gray values. <bold>(D)</bold> Scanning electron microscope image of bacteria after LAL2 incubation. In the LAL2 treatment group, the right picture is an enlarged view of the left picture. Scale bar: 1 μm. <bold>(E)</bold> The binding of microorganisms by LAL2 proteins. Every living microbial strain was incubated with 10 μg natural LAL2 proteins in PBS buffer for 12 h, and the washed pellets were subjected to 15% SDS-PAGE and detected using western blot with an anti-LAL2 antibody. LAL2 protein 0.5 μg is used as positive control. <bold>(F)</bold> ELISA analysis of the interaction between LAL2 and microbial components (<italic>n</italic> = 3) *<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01, ***<italic>P</italic> < 0.001.</p></caption><graphic xlink:href=\"fimmu-11-01751-g0005\"/></fig><p>Subsequently, we used <italic>S. aureus</italic> and <italic>E. coli</italic> to detect the antibacterial activity of LAL2 (<xref ref-type=\"fig\" rid=\"F5\">Figure 5D</xref>). Different bacterial strains were incubated with the purified natural LAL2 proteins in PBS as the experimental group (<xref ref-type=\"supplementary-material\" rid=\"SM6\">Figure S5</xref>), the control group was incubated with PBS. The morphological changes of the bacteria were observed using scanning electron microscopy. After LAL2 incubation, <italic>S. aureus</italic> and <italic>E. coli</italic> had changed in morphology, the cell surface appeared wrinkly and sunken, cell contents were released into the culture media, compared with the control group (areas indicated by red arrow). It was suspected that LAL2 could play a role in antibacterial activity by combining with bacteria to perform bacterial killing and clearance. Thereafter, four different bacterial strains were incubated with natural LAL2 proteins, including <italic>S. aureus, B. cereus, V. anguillarum</italic>, and <italic>E. coli</italic>, and the bacterial pellets were washed and analyzed using western blot with anti-LAL2 polyclonal antibody, indicating that LAL2 proteins could bind to gram-positive bacteria (<italic>S. aureus</italic> and <italic>B. cereus</italic>) and gram-negative bacteria (<italic>E. coli</italic>) in the form of a trimer. However, the combination with <italic>V. anguillarum</italic> was weak (<xref ref-type=\"fig\" rid=\"F5\">Figure 5E</xref>). To explore the mechanism of combination, we used ELISA to evaluate the interaction between the natural LAL2 protein and different bacterial cell wall components (<xref ref-type=\"fig\" rid=\"F5\">Figure 5F</xref>). The results showed that LAL2 could interact with soluble peptidoglycan (PGN), LTA, and LPS in a dose-dependent manner, and LAL2 could also bind specifically to minimal PGN motif muramyl dipeptide (MDP).</p></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>In 1986, M. Pontes et al. suggested that a similar amino acid composition exists for LrLAL1, LrLAL2, and MmAPOA4 (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Later in 1996, Le Wang et al. performed a practical analysis of the systematic evolution of the apolipoprotein multigene family and found that the common ancestors of APOA1, APOA2, APOA4, and APOE may have appeared 460 million years ago in an ordovician vertebrate, which may be related to the major apolipoprotein LAL1 and LAL2 in Lamprey (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Recently, Liu et al. postulated that the ancestral members of apolipoprotein are likely APOA1 and/or APOA4, and that other apolipoproteins emerged subsequently by gene duplication (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). However, our study can trace back to their common vertebrate ancestor, lamprey, indicating that LAL1 and LAL2 are indeed apolipoproteins. Furthermore, LAL2 is located in an evolutionary original position relative to LAL1, with motifs 4 and 5 in LAL2 obtained from amphioxus APOA1, and LAL1 formed by loss of motif 5, inversion, and insertion of the transposon into another scaffold (<xref ref-type=\"fig\" rid=\"F2\">Figure 2E</xref>). We, therefore, postulate the following scenario: a series of duplication events beginning from <italic>lal2</italic> and <italic>lal1</italic> (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>), produced on zebrafish chromosome 16, resulting in the following apolipoprotein genes: <italic>apoa1, apoa2</italic>, and <italic>apoe</italic>. Subsequently, <italic>apoa1</italic> underwent tandem duplications and produced <italic>apoa4</italic>, while <italic>apoc</italic> is generated by <italic>apoa1</italic>/<italic>apoa4</italic> fragments. A DNA transposition then resulted in the insertion of the <italic>apoc3</italic> gene in between the <italic>apoa1</italic> and <italic>apoa4</italic> genes (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). The <italic>apoa2</italic> gene moved to human chromosome 1 and the genes for <italic>apoa1, a4</italic>, and <italic>c3</italic> moved to human chromosome 11. During species evolution, <italic>apoc1, apoc2</italic>, and <italic>apoe</italic> were lost in Aves. Eventually the other apolipoprotein genes, <italic>apoe, c1, c2</italic>, and <italic>c4</italic>, remained as a cluster on human chromosome 19. In the current study, it is speculated that the ancestral apolipoprotein gene may be subjected to different selection pressures at the same time during early differentiation. The order of evolution may be LAL2/LAL1—APOA1/APOA4/APOE—APOA2/APOC1/APOC2—APOC3/APOA5—APOC4 (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>, <xref rid=\"B30\" ref-type=\"bibr\">30</xref>–<xref rid=\"B34\" ref-type=\"bibr\">34</xref>).</p><p>Fitch et al. observed that human APOA1 contains multiple repeats of 22 amino acids (22-mer), each repeat is a tandem array of two 11-mers (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>, <xref rid=\"B33\" ref-type=\"bibr\">33</xref>). It is suggested that the repeat unit of 22-mer has been a structural element that builds an amphipathic α-helix. In addition, the existence of a 22-mer periodicity has also been found in other apolipoproteins, including APOA2, A4, C2, C3, and E, and an 11-mer has been found in APOC1. Lamprey LALl has a repeat pattern similar to those in human APOA1 and APOC3, while there is no clear indication for the presence of internal duplication in LAL2 sequence (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). Moreover, since there are a large number of α-helices in LAL1 and LAL2, the segments 79–99 and 100–120 of LAL2 have the potential to form an amphipathic helical structure. The hydrophilic residues on one side of the amphipathic helix keep the apolipoproteins at the surface of the lipoprotein particle to facilitate transfer between lipoprotein particles and interaction with other molecules, such as enzymes and specific cell surface receptors. New motifs are gradually evolved in LAL1 and LAL2 to form the apolipoprotein functional domain and conservative α-helix to ensure better survival, thus producing several types of apolipoproteins with significantly different structures.</p><p>Both the mRNA and protein levels of LAL2 were primarily expressed in liver, leukocytes, and kidney of the lamprey, which differed from the tissue distribution observed for apolipoprotein expression in teleost fish. For example, most apolipoprotein genes exhibited tissue-specific expression patterns in intestine, liver, and skin of channel catfish (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). Moreover, human <italic>apoa1, apoa4</italic>, and <italic>apoc3</italic> have been cloned in fetal intestine and adult liver but not in fetal liver, kidney, heart, brain, or muscle (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). Hence, with evolution, the distribution of apolipoproteins has gradually become regionalized to further perform unique functions. However, liver, leukocytes, and kidney are important immune tissues, indicating that LAL2 is likely to play a critical role in immune defense. In fact, our previous study demonstrated that LIP is primarily distributed in the supraneural body and leukocytes of the lamprey (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>), while LAL2 is abundant in the sera (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>, indicated by the red arrow), suggesting that LAL2 protein acts as a secreted protein and participates extensively in blood circulation to accomplish immune responses. Additionally, the unique recognition mechanism of LIP is dependent on binding with both N-linked glycans on GPI-Aps, and SM in lipid rafts (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). We, therefore, expect that LAL2 can assist LIP in the diagnosis and control of tumor cells via targeted human cancer therapies.</p><p>Comprehensive functional analyses revealed the role of lamprey LAL2 and immune responses (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>). Insect apolipoproteins were shown to cooperate against pathogens, such as silkworm apolipophorins (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B37\" ref-type=\"bibr\">37</xref>–<xref rid=\"B39\" ref-type=\"bibr\">39</xref>). In respect with the immune defense, LAL2 exerts unique biological functions in synergy with LIP. Moreover, <italic>lal2</italic> was up-regulated after stimulating lamprey with gram-positive bacteria, gram-negative bacteria, and Poly I:C virus mimic, respectively. Scanning electron microscope (SEM) images show that LAL2 can destroy the structure of <italic>S. aureus</italic> and <italic>E. coli</italic> and influence bactericidal activity. This is similar to the results highlighting that high-density lipoprotein (HDL) in the carp epidermis is secreted into mucus and performs antibacterial activity (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>). This lipoprotein is mainly composed of two major apolipoproteins (APOA1 and APOA2), which correspond to the most abundant plasma proteins in several bony fish and have antibacterial activity. Orange-spotted grouper <italic>E. coioides</italic> APOA1 can inhibit the replication of Singapore grouper iridovirus (SGIV), and up-regulate the expression of its immune-related genes, ISG15 and Mx-I (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>).</p><p>Microbes express signature molecules known as pathogen-associated molecular patterns (PAMPs), such as LTA, PGN, and MDP in gram-positive bacteria, and LPS in gram-negative bacteria (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>). Wang et al. found that <italic>Branchiostoma belcheri</italic> rAPOA1 can bind LPS and LTA of various gram-positive and gram-negative bacteria and exhibits antibacterial activity against gram-negative bacteria <italic>in vitro</italic> (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>). Silkworm apolipophorin protein inhibits hemolysin gene expression of <italic>S. aureus</italic> via binding to cell surface LTA (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Conserved high amphipathic α-helical content between fish and mammal apolipoproteins can neutralize LPS via the CD14/TLR4 (Toll Like Receptor 4) pathway and intercalate into lipid bilayers to resist bacterial invasion (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B43\" ref-type=\"bibr\">43</xref>, <xref rid=\"B44\" ref-type=\"bibr\">44</xref>). To investigate the antibacterial mechanism of lamprey LAL2, ELISA results show LAL2 can bind to LPS, LTA, PGN, and MDP due to highly homologous α-helical content with human APOA1 (<xref ref-type=\"supplementary-material\" rid=\"SM5\">Figure S4</xref>). In fact, APO-II/I proteins may either shuttle APO-III and other immune proteins to microbial surfaces, contribute to microbial clearance, or detoxify immune-stimulatory cell wall components (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>). The lipid particles nucleated by lipid carrier proteins in the hemolymph may serve as platforms for recruiting immunity proteins (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>). Future studies are required to elucidate the interaction between molecules in sera with LAL2 and the signaling pathway involving LAL2 in order to further unravel immune defense in lampreys.</p><p>In conclusion, this study identified the molecular evolution and tissue distribution of lamprey LAL2. Furthermore, we demonstrate that lamprey LAL2 can serve as an effector molecule in sera for immune responses, pattern recognition, and bactericidal activity. Our studies not only help to expand on the evolutionary history of the vertebrate apolipoprotein multigene family, but also provide new insight into the important and diversified functional properties of apolipoprotein.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>Data can be found on Genbank—<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"MN893307\">MN893307</ext-link> and <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"MN893306\">MN893306</ext-link>. Other raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The animal experiments were performed in accordance with the regulations of the Animal Welfare and Research Ethics Committee of the Institute of Dalian Medical University's Animal Care protocol (Permit Number: SCXK2008-0002).</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>QL, YP, and QH designed the experiments. Flow cytometry and immunohistochemistry were finished by QH. HW, QH, and YH analyzed the experimental results. QH and YP wrote the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was funded by Chinese National Natural Science Foundation Grant (No. 31772884), The Project of Department of Ocean and Fisheries of Liaoning Province (No. 201805), Program of Science and Technology of Liaoning Province (No. 2019-MS-218), and Science and Technology Innovation Fund Research Project (No. 2018J12SN079).</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s9\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fimmu.2020.01751/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fimmu.2020.01751/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Table_1.DOC\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM2\"><label>Figure S1</label><caption><p>Ten amino acid test maps of LAL2 N-terminal sequence (number 2).</p></caption><media xlink:href=\"Image_1.JPEG\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM3\"><label>Figure S2</label><caption><p>Sequence alignment results of LAL2 in <italic>Lampetra japonica, Petromyzon marinus</italic>, and <italic>Lampetra fluviatilis</italic>.</p></caption><media xlink:href=\"Image_2.JPEG\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM4\"><label>Figure S3</label><caption><p>The secondary structure of LAL2 and LAL1. <bold>(A)</bold> The predicted results on PSIPRED website of LAL2 secondary structures. Alpha helix, extended strand, beta turn, and random coil account for 78.01, 5.76, 3.14, and 13.09%, respectively. <bold>(B)</bold> The predicted results on PSIPRED website of LAL2 secondary structures. Alpha helix, beta turn, and random coil account for 79.05, 5.71, and 15.24%, respectively. <bold>(C)</bold> Circular dichroism shows two negative peaks and one positive peak in LAL2, which conform to the CD spectrum of the α-helix in the secondary structure of the protein (negative peaks at 208 and 222 nm, and positive peaks near 190 nm). <bold>(D)</bold> Sequence alignment analysis of sequence similarity between LrLAL2 and HsAPOA1. LAL2 is highly similar to the APOA1 sequence Helix3 (88–98), and it is also similar to the APOA1 sequence Helix4, 5, and 6 (99–120, 121–142, 143–164).</p></caption><media xlink:href=\"Image_3.jpg\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM5\"><label>Figure S4</label><caption><p>Expression and purification of rLAL2 protein and preparation of antibodies. <bold>(A)</bold> Prokaryotic expression and purification of rLAL2. M, low molecular-weight protein marker; <italic>lane 1</italic>, non-induced Rosetta/pET32a-<italic>lal2</italic> cells; <italic>lane 2</italic>, induced Rosetta/pET32a-<italic>lal2</italic> cells using IPTG; <italic>lane 3</italic>, purified LAL2 recombinant protein, black line points at the target protein. <bold>(B)</bold> ELISA assay to assess the serum anti-LAL2 polyclonal antibody titer from two rabbits. <bold>(C)</bold> Analysis of the specificity of the rabbit anti-LAL2 polyclonal antibody using western blot. <italic>lane 1</italic>, LAL2 recombinant protein; <italic>lane 2</italic>, lamprey serum. <bold>(D)</bold> LC-MS/MS analysis trypic-digested peptides of LAL2-Dimer.</p></caption><media xlink:href=\"Image_4.tif\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM6\"><label>Figure S5</label><caption><p>Purification of natural LAL2 protein with anion exchange chromatography. (Left) The map of natural LAL2 protein purification process with anion exchange chromatography from UNICORN 7.0; (Right) Purification of natural LAL2 protein. M, low molecular-weight protein marker; lane 1, No. 46 serum sample; lane 2, Flow-through fluid; lane 3, Equilibrium fluid; lane 4, Eluent 20; lane 5, Eluent 25; lane 6, Eluent 30.</p></caption><media xlink:href=\"Image_5.tif\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pontes</surname><given-names>M</given-names></name><name><surname>Xu</surname><given-names>X</given-names></name><name><surname>Graham</surname><given-names>D</given-names></name><name><surname>Riley</surname><given-names>M</given-names></name><name><surname>Doolittle</surname><given-names>RF</given-names></name></person-group>. <article-title>cDNA sequences of two apolipoproteins from lamprey</article-title>. <source>Biochemistry.</source> (<year>1987</year>) <volume>26</volume>:<fpage>1611</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1021/bi00380a019</pub-id><pub-id pub-id-type=\"pmid\">3593681</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morita</surname><given-names>SY</given-names></name></person-group>. <article-title>Metabolism and modification of apolipoprotein B-containing lipoproteins involved in dyslipidemia and atherosclerosis</article-title>. <source>Biol Pharm Bull.</source> (<year>2016</year>) <volume>39</volume>:<fpage>1</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1248/bpb.b15-00716</pub-id><pub-id pub-id-type=\"pmid\">26725424</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Phillips</surname><given-names>MC</given-names></name></person-group>. <article-title>New insights into the determination of HDL structure by apolipoproteins: thematic review series: high density lipoprotein structure, function, and metabolism</article-title>. <source>J Lipid Res.</source> (<year>2013</year>) <volume>54</volume>:<fpage>2034</fpage>–<lpage>48</lpage>. <pub-id pub-id-type=\"doi\">10.1194/jlr.R034025</pub-id><pub-id pub-id-type=\"pmid\">23230082</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mahley</surname><given-names>RW</given-names></name><name><surname>Innerarity</surname><given-names>TL</given-names></name><name><surname>Rall</surname><given-names>SC</given-names><suffix>Jr</suffix></name><name><surname>Weisgraber</surname><given-names>KH</given-names></name></person-group>. <article-title>Plasma lipoproteins: apolipoprotein structure and function</article-title>. <source>J Lipid Res.</source> (<year>1984</year>) <volume>25</volume>:<fpage>1277</fpage>–<lpage>94</lpage>.<pub-id pub-id-type=\"pmid\">6099394</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ramasamy</surname><given-names>I</given-names></name></person-group>. <article-title>Recent advances in physiological lipoprotein metabolism</article-title>. <source>Clin Chem Lab Med.</source> (<year>2014</year>) <volume>52</volume>:<fpage>1695</fpage>–<lpage>727</lpage>. <pub-id pub-id-type=\"doi\">10.1515/cclm-2013-0358</pub-id><pub-id pub-id-type=\"pmid\">23940067</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hanada</surname><given-names>Y</given-names></name><name><surname>Sekimizu</surname><given-names>K</given-names></name><name><surname>Kaito</surname><given-names>C</given-names></name></person-group>. <article-title>Silkworm apolipophorin protein inhibits <italic>Staphylococcus aureus</italic> virulence</article-title>. <source>J Biol Chem.</source> (<year>2011</year>) <volume>286</volume>:<fpage>39360</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M111.278416</pub-id><pub-id pub-id-type=\"pmid\">21937431</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Villarroel</surname><given-names>F</given-names></name><name><surname>Bastías</surname><given-names>A</given-names></name><name><surname>Casado</surname><given-names>A</given-names></name><name><surname>Amthauer</surname><given-names>R</given-names></name><name><surname>Concha</surname><given-names>MI</given-names></name></person-group>. <article-title>Apolipoprotein A-I, an antimicrobial protein in <italic>Oncorhynchus mykiss</italic>: evaluation of its expression in primary defence barriers and plasma levels in sick and healthy fish</article-title>. <source>Fish Shellfish Immunol.</source> (<year>2007</year>) <volume>23</volume>:<fpage>197</fpage>–<lpage>209</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fsi.2006.10.008</pub-id><pub-id pub-id-type=\"pmid\">17391986</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mohapatra</surname><given-names>A</given-names></name><name><surname>Karan</surname><given-names>S</given-names></name><name><surname>Kar</surname><given-names>B</given-names></name><name><surname>Garg</surname><given-names>LC</given-names></name><name><surname>Dixit</surname><given-names>A</given-names></name><name><surname>Sahoo</surname><given-names>PK</given-names></name></person-group>. <article-title>Apolipoprotein A-I in <italic>Labeo rohita</italic>: cloning and functional characterisation reveal its broad spectrum antimicrobial property, and indicate significant role during ectoparasitic infection</article-title>. <source>Fish Shellfish Immunol.</source> (<year>2016</year>) <volume>55</volume>:<fpage>717</fpage>–<lpage>28</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fsi.2016.06.045</pub-id><pub-id pub-id-type=\"pmid\">27368542</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wei</surname><given-names>J</given-names></name><name><surname>Gao</surname><given-names>P</given-names></name><name><surname>Zhang</surname><given-names>P</given-names></name><name><surname>Guo</surname><given-names>M</given-names></name><name><surname>Xu</surname><given-names>M</given-names></name><name><surname>Wei</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Isolation and function analysis of apolipoprotein A-I gene response to virus infection in grouper</article-title>. <source>Fish Shellfish Immunol.</source> (<year>2015</year>) <volume>43</volume>:<fpage>396</fpage>–<lpage>404</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fsi.2015.01.006</pub-id><pub-id pub-id-type=\"pmid\">25613342</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tada</surname><given-names>N</given-names></name><name><surname>Sakamoto</surname><given-names>T</given-names></name><name><surname>Kagami</surname><given-names>A</given-names></name><name><surname>Mochizuki</surname><given-names>K</given-names></name><name><surname>Kurosaka</surname><given-names>K</given-names></name></person-group>. <article-title>Antimicrobial activity of lipoprotein particles containing apolipoprotein Al</article-title>. <source>Mol Cell Biochem.</source> (<year>1993</year>) <volume>119</volume>:<fpage>171</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1007/978-1-4615-3078-7_23</pub-id><pub-id pub-id-type=\"pmid\">8455579</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Levine</surname><given-names>DM</given-names></name><name><surname>Parker</surname><given-names>TS</given-names></name><name><surname>Donnelly</surname><given-names>TM</given-names></name><name><surname>Walsh</surname><given-names>A</given-names></name><name><surname>Rubin</surname><given-names>AL</given-names></name></person-group>. <article-title><italic>In vivo</italic> protection against endotoxin by plasma high density lipoprotein</article-title>. <source>Proc Natl Acad Sci USA</source>. (<year>1993</year>) <volume>90</volume>:<fpage>12040</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.90.24.12040</pub-id><pub-id pub-id-type=\"pmid\">8265667</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grunfeld</surname><given-names>C</given-names></name><name><surname>Marshall</surname><given-names>M</given-names></name><name><surname>Shigenaga</surname><given-names>JK</given-names></name><name><surname>Moser</surname><given-names>AH</given-names></name><name><surname>Tobias</surname><given-names>P</given-names></name><name><surname>Feingold</surname><given-names>KR</given-names></name></person-group>. <article-title>Lipoproteins inhibit macrophage activation by lipoteichoic acid</article-title>. <source>J Lipid Res.</source> (<year>1999</year>) <volume>40</volume>:<fpage>245</fpage>–<lpage>52</lpage>.<pub-id pub-id-type=\"pmid\">9925653</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Omae</surname><given-names>Y</given-names></name><name><surname>Hanada</surname><given-names>Y</given-names></name><name><surname>Sekimizu</surname><given-names>K</given-names></name><name><surname>Kaito</surname><given-names>C</given-names></name></person-group>. <article-title>Silkworm apolipophorin protein inhibits hemolysin gene expression of <italic>Staphylococcus aureus</italic> via binding to cell surface lipoteichoic acids</article-title>. <source>J Biol Chem.</source> (<year>2013</year>) <volume>288</volume>:<fpage>25542</fpage>–<lpage>50</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M113.495051</pub-id><pub-id pub-id-type=\"pmid\">23873929</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parker</surname><given-names>TS</given-names></name><name><surname>Levine</surname><given-names>DM</given-names></name><name><surname>Chang</surname><given-names>JC</given-names></name><name><surname>Laxer</surname><given-names>J</given-names></name><name><surname>Coffin</surname><given-names>CC</given-names></name><name><surname>Rubin</surname><given-names>AL</given-names></name></person-group>. <article-title>Reconstituted high-density lipoprotein neutralizes gram-negative bacterial lipopolysaccharides in human whole blood</article-title>. <source>Infect Immun.</source> (<year>1995</year>) <volume>63</volume>:<fpage>253</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1128/IAI.63.1.253-258.1995</pub-id><pub-id pub-id-type=\"pmid\">7528733</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mangaraj</surname><given-names>M</given-names></name><name><surname>Nanda</surname><given-names>R</given-names></name><name><surname>Panda</surname><given-names>S</given-names></name></person-group>. <article-title>Apolipoprotein A-I: a molecule of diverse function</article-title>. <source>Indian J Clin Biochem.</source> (<year>2016</year>) <volume>31</volume>:<fpage>253</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s12291-015-0513-1</pub-id><pub-id pub-id-type=\"pmid\">27382195</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Berbée</surname><given-names>JF</given-names></name><name><surname>Coomans</surname><given-names>CP</given-names></name><name><surname>Westerterp</surname><given-names>M</given-names></name><name><surname>Romijn</surname><given-names>JA</given-names></name><name><surname>Havekes</surname><given-names>LM</given-names></name><name><surname>Rensen</surname><given-names>PC</given-names></name></person-group>. <article-title>Apolipoprotein CI enhances the biological response to LPS via the CD14/TLR4 pathway by LPS-binding elements in both its N- and C-terminal helix</article-title>. <source>J Lipid Res.</source> (<year>2010</year>) <volume>51</volume>:<fpage>1943</fpage>–<lpage>52</lpage>. <pub-id pub-id-type=\"doi\">10.1194/jlr.M006809</pub-id><pub-id pub-id-type=\"pmid\">20335569</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Catapano</surname><given-names>AL</given-names></name><name><surname>Pirillo</surname><given-names>A</given-names></name><name><surname>Bonacina</surname><given-names>F</given-names></name><name><surname>Norata</surname><given-names>GD</given-names></name></person-group>. <article-title>HDL in innate and adaptive immunity</article-title>. <source>Cardiovasc Res.</source> (<year>2014</year>) <volume>103</volume>:<fpage>372</fpage>–<lpage>83</lpage>. <pub-id pub-id-type=\"doi\">10.1093/cvr/cvu150</pub-id><pub-id pub-id-type=\"pmid\">24935428</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>F</given-names></name><name><surname>Chen</surname><given-names>L</given-names></name><name><surname>Liu</surname><given-names>X</given-names></name><name><surname>Wang</surname><given-names>H</given-names></name><name><surname>Su</surname><given-names>P</given-names></name><name><surname>Han</surname><given-names>Y</given-names></name><etal/></person-group>. <article-title>Lamprey variable lymphocyte receptors mediate complement-dependent cytotoxicity</article-title>. <source>J Immunol.</source> (<year>2013</year>) <volume>190</volume>:<fpage>922</fpage>–<lpage>30</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.1200876</pub-id><pub-id pub-id-type=\"pmid\">23293356</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nonaka</surname><given-names>M</given-names></name><name><surname>Fujii</surname><given-names>T</given-names></name><name><surname>Kaidoh</surname><given-names>T</given-names></name><name><surname>Natsuume-Sakai</surname><given-names>S</given-names></name><name><surname>Nonaka</surname><given-names>M</given-names></name><name><surname>Yamaguchi</surname><given-names>N</given-names></name><etal/></person-group>. <article-title>Purification of a lamprey complement protein homologous to the third component of the mammalian complement system</article-title>. <source>J Immunol.</source> (<year>1984</year>) <volume>133</volume>:<fpage>3242</fpage>–<lpage>9</lpage>.<pub-id pub-id-type=\"pmid\">6491286</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pei</surname><given-names>G</given-names></name><name><surname>Liu</surname><given-names>G</given-names></name><name><surname>Pan</surname><given-names>X</given-names></name><name><surname>Pang</surname><given-names>Y</given-names></name><name><surname>Li</surname><given-names>Q</given-names></name></person-group>. <article-title>L-C1qDC-1, a novel C1q domain-containing protein from <italic>Lethenteron camtschaticum</italic> that is involved in the immune response</article-title>. <source>Dev Comp Immunol.</source> (<year>2016</year>) <volume>54</volume>:<fpage>66</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.dci.2015.08.011</pub-id><pub-id pub-id-type=\"pmid\">26342581</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xue</surname><given-names>Z</given-names></name><name><surname>Liu</surname><given-names>X</given-names></name><name><surname>Pang</surname><given-names>Y</given-names></name><name><surname>Yu</surname><given-names>T</given-names></name><name><surname>Xiao</surname><given-names>R</given-names></name><name><surname>Jin</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Characterization, phylogenetic analysis and cDNA cloning of natterin-like gene from the blood of lamprey, <italic>Lampetra</italic> japonica</article-title>. <source>Immunol Lett.</source> (<year>2012</year>) <volume>148</volume>:<fpage>1</fpage>–<lpage>10</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.imlet.2012.08.005</pub-id><pub-id pub-id-type=\"pmid\">22914553</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pang</surname><given-names>Y</given-names></name><name><surname>Gou</surname><given-names>M</given-names></name><name><surname>Yang</surname><given-names>K</given-names></name><name><surname>Lu</surname><given-names>J</given-names></name><name><surname>Han</surname><given-names>Y</given-names></name><name><surname>Teng</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Crystal structure of a cytocidal protein from lamprey and its mechanism of action in the selective killing of cancer cells</article-title>. <source>Cell Commun Signal.</source> (<year>2019</year>) <volume>17</volume>:<fpage>54</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s12964-019-0358-y</pub-id><pub-id pub-id-type=\"pmid\">31133022</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pang</surname><given-names>Y</given-names></name><name><surname>Li</surname><given-names>C</given-names></name><name><surname>Wang</surname><given-names>S</given-names></name><name><surname>Ba</surname><given-names>W</given-names></name><name><surname>Yu</surname><given-names>T</given-names></name><name><surname>Pei</surname><given-names>G</given-names></name><etal/></person-group>\n<article-title>A novel protein derived from lamprey supraneural body tissue with efficient cytocidal actions against tumor cells</article-title>. <source>Cell Commun Signal.</source> (<year>2017</year>) <volume>15</volume>:<fpage>42</fpage>\n<pub-id pub-id-type=\"doi\">10.1186/s12964-017-0202-1</pub-id><pub-id pub-id-type=\"pmid\">29037260</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pang</surname><given-names>Y</given-names></name><name><surname>Wang</surname><given-names>S</given-names></name><name><surname>Ba</surname><given-names>W</given-names></name><name><surname>Li</surname><given-names>Q</given-names></name></person-group>. <article-title>Cell secretion from the adult lamprey supraneural body tissues possesses cytocidal activity against tumor cells</article-title>. <source>Springerplus.</source> (<year>2015</year>) <volume>4</volume>:<fpage>569</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s40064-015-1270-6</pub-id><pub-id pub-id-type=\"pmid\">26543704</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xu</surname><given-names>R</given-names></name><name><surname>Song</surname><given-names>X</given-names></name><name><surname>Su</surname><given-names>P</given-names></name><name><surname>Pang</surname><given-names>Y</given-names></name><name><surname>Li</surname><given-names>Q</given-names></name></person-group>. <article-title>Identification and characterization of the lamprey Flotillin-1 gene with a role in cell adhesion</article-title>. <source>Fish Shellfish Immunol.</source> (<year>2017</year>) <volume>71</volume>:<fpage>286</fpage>–<lpage>94</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fsi.2017.06.061</pub-id><pub-id pub-id-type=\"pmid\">28687359</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>D</given-names></name><name><surname>Gou</surname><given-names>M</given-names></name><name><surname>Hou</surname><given-names>J</given-names></name><name><surname>Pang</surname><given-names>Y</given-names></name><name><surname>Li</surname><given-names>Q</given-names></name></person-group>. <article-title>The role of serpin protein on the natural immune defense against pathogen infection in <italic>Lampetra japonica</italic></article-title>. <source>Fish Shellfish Immunol.</source> (<year>2019</year>) <volume>92</volume>:<fpage>196</fpage>–<lpage>208</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fsi.2019.05.062</pub-id><pub-id pub-id-type=\"pmid\">31176010</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>Z</given-names></name><name><surname>Lu</surname><given-names>J</given-names></name><name><surname>Li</surname><given-names>C</given-names></name><name><surname>Li</surname><given-names>Q</given-names></name><name><surname>Pang</surname><given-names>Y</given-names></name></person-group>. <article-title>Characterization of the LECT2 gene and its protective effects against microbial infection via large lymphocytes in <italic>Lampetra japonica</italic></article-title>. <source>Dev Comp Immunol.</source> (<year>2018</year>) <volume>79</volume>:<fpage>75</fpage>–<lpage>85</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.dci.2017.09.018</pub-id><pub-id pub-id-type=\"pmid\">29056545</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Han</surname><given-names>Y</given-names></name><name><surname>Pang</surname><given-names>Y</given-names></name><name><surname>Yu</surname><given-names>T</given-names></name><name><surname>Xiao</surname><given-names>R</given-names></name><name><surname>Shi</surname><given-names>B</given-names></name><name><surname>Su</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Lamprey serum can kill HeLa and NB4 tumor cells</article-title>. <source>Acta Biochim Biophys Sin (Shanghai).</source> (<year>2014</year>) <volume>46</volume>:<fpage>623</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1093/abbs/gmu039</pub-id><pub-id pub-id-type=\"pmid\">24850304</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>WH</given-names></name><name><surname>Tanimura</surname><given-names>M</given-names></name><name><surname>Luo</surname><given-names>CC</given-names></name><name><surname>Datta</surname><given-names>S</given-names></name><name><surname>Chan</surname><given-names>L</given-names></name></person-group>. <article-title>The apolipoprotein multigene family: biosynthesis, structure, structure-function relationships, and evolution</article-title>. <source>J Lipid Res.</source> (<year>1988</year>) <volume>29</volume>:<fpage>245</fpage>–<lpage>71</lpage>.<pub-id pub-id-type=\"pmid\">3288703</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>L</given-names></name><name><surname>Chai</surname><given-names>J</given-names></name><name><surname>Lu</surname><given-names>Y</given-names></name><name><surname>Tan</surname><given-names>CC</given-names></name></person-group>. <article-title>Studies on the molecular evolution of apolipoprotein multigene family</article-title>. <source>Yi Chuan Xue Bao.</source> (<year>1994</year>) <volume>21</volume>:<fpage>81</fpage>–<lpage>95</lpage>.<pub-id pub-id-type=\"pmid\">8080663</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>JQ</given-names></name><name><surname>Li</surname><given-names>WX</given-names></name><name><surname>Zheng</surname><given-names>JJ</given-names></name><name><surname>Tian</surname><given-names>QN</given-names></name><name><surname>Huang</surname><given-names>JF</given-names></name><name><surname>Dai</surname><given-names>SX</given-names></name></person-group>. <article-title>Gain and loss events in the evolution of the apolipoprotein family in vertebrata</article-title>. <source>BMC Evol Biol.</source> (<year>2019</year>) <volume>19</volume>:<fpage>209</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s12862-019-1519-8</pub-id><pub-id pub-id-type=\"pmid\">31722659</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huebbe</surname><given-names>P</given-names></name><name><surname>Rimbach</surname><given-names>G</given-names></name></person-group>. <article-title>Evolution of human apolipoprotein E (APOE) isoforms: gene structure, protein function and interaction with dietary factors</article-title>. <source>Ageing Res Rev.</source> (<year>2017</year>) <volume>37</volume>:<fpage>146</fpage>–<lpage>61</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.arr.2017.06.002</pub-id><pub-id pub-id-type=\"pmid\">28647612</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luo</surname><given-names>CC</given-names></name><name><surname>Li</surname><given-names>WH</given-names></name><name><surname>Moore</surname><given-names>MN</given-names></name><name><surname>Chan</surname><given-names>L</given-names></name></person-group>. <article-title>Structure and evolution of the apolipoprotein multigene family</article-title>. <source>J Mol Biol.</source> (<year>1986</year>) <volume>187</volume>:<fpage>325</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1016/0022-2836(86)90436-5</pub-id><pub-id pub-id-type=\"pmid\">3084795</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boguski</surname><given-names>MS</given-names></name><name><surname>Birkenmeier</surname><given-names>EH</given-names></name><name><surname>Elshourbagy</surname><given-names>NA</given-names></name><name><surname>Taylor</surname><given-names>JM</given-names></name><name><surname>Gordon</surname><given-names>JI</given-names></name></person-group>. <article-title>Evolution of the apolipoproteins. Structure of the rat apo-A-IV gene its relationship to the human genes for apo-A-I, C-III, E</article-title>. <source>J Biol Chem</source>. (<year>1986</year>) <volume>261</volume>:<fpage>6398</fpage>–<lpage>407</lpage>.<pub-id pub-id-type=\"pmid\">3009456</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>Y</given-names></name><name><surname>Fu</surname><given-names>Q</given-names></name><name><surname>Zhou</surname><given-names>T</given-names></name><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Liu</surname><given-names>S</given-names></name><name><surname>Zeng</surname><given-names>Q</given-names></name><etal/></person-group>. <article-title>Analysis of apolipoprotein genes and their involvement in disease response of channel catfish after bacterial infection</article-title>. <source>Dev Comp Immunol.</source> (<year>2017</year>) <volume>67</volume>:<fpage>464</fpage>–<lpage>70</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.dci.2016.09.007</pub-id><pub-id pub-id-type=\"pmid\">27640368</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Karathanasis</surname><given-names>SK</given-names></name></person-group>. <article-title>Apolipoprotein multigene family: tandem organization of human apolipoprotein AI, CIII, and AIV genes</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>1985</year>) <volume>82</volume>:<fpage>6374</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.82.19.6374</pub-id><pub-id pub-id-type=\"pmid\">3931073</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Staczek</surname><given-names>S</given-names></name><name><surname>Zdybicka-Barabas</surname><given-names>A</given-names></name><name><surname>Mak</surname><given-names>P</given-names></name><name><surname>Jasiłek</surname><given-names>AS</given-names></name><name><surname>Kedracka-Krok</surname><given-names>S</given-names></name><name><surname>Suder</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Studies on localization and protein ligands of <italic>Galleria mellonella</italic> apolipophorin III during immune response against different pathogens</article-title>. <source>J Insect Physiol.</source> (<year>2018</year>) <volume>105</volume>:<fpage>18</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jinsphys.2017.12.009</pub-id><pub-id pub-id-type=\"pmid\">29289504</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zdybicka-Barabas</surname><given-names>A</given-names></name><name><surname>Cytrynska</surname><given-names>M</given-names></name></person-group>. <article-title>Involvement of apolipophorin III in antibacterial defense of <italic>Galleria mellonella</italic> larvae</article-title>. <source>Comp Biochem Physiol B Biochem Mol Biol.</source> (<year>2011</year>) <volume>158</volume>:<fpage>90</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cbpb.2010.10.001</pub-id><pub-id pub-id-type=\"pmid\">20959145</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kamareddine</surname><given-names>L</given-names></name><name><surname>Nakhleh</surname><given-names>J</given-names></name><name><surname>Osta</surname><given-names>MA</given-names></name></person-group>. <article-title>Functional interaction between apolipophorins and complement regulate the mosquito immune response to systemic infections</article-title>. <source>J Innate Immun.</source> (<year>2016</year>) <volume>8</volume>:<fpage>314</fpage>–<lpage>26</lpage>. <pub-id pub-id-type=\"doi\">10.1159/000443883</pub-id><pub-id pub-id-type=\"pmid\">26950600</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Concha</surname><given-names>MI</given-names></name><name><surname>Smith</surname><given-names>VJ</given-names></name><name><surname>Castro</surname><given-names>K</given-names></name><name><surname>Bastías</surname><given-names>A</given-names></name><name><surname>Romero</surname><given-names>A</given-names></name><name><surname>Amthauer</surname><given-names>RJ</given-names></name></person-group>. <article-title>Apolipoproteins A-I and A-II are potentially important effectors of innate immunity in the teleost fish <italic>Cyprinus carpio</italic></article-title>. <source>Eur J Biochem.</source> (<year>2004</year>) <volume>271</volume>:<fpage>2984</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1432-1033.2004.04228.x</pub-id><pub-id pub-id-type=\"pmid\">15233794</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>G</given-names></name><name><surname>Huang</surname><given-names>S</given-names></name><name><surname>Yan</surname><given-names>X</given-names></name><name><surname>Li</surname><given-names>J</given-names></name><name><surname>Xu</surname><given-names>W</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Two apextrin-like proteins mediate extracellular and intracellular bacterial recognition in amphioxus</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2014</year>) <volume>111</volume>:<fpage>13469</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1405414111</pub-id><pub-id pub-id-type=\"pmid\">25187559</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>W</given-names></name><name><surname>Qu</surname><given-names>Q</given-names></name><name><surname>Chen</surname><given-names>J</given-names></name></person-group>. <article-title>Identification, expression analysis, and antibacterial activity of apolipoprotein A-I from amphioxus (<italic>Branchiostoma belcheri</italic>)</article-title>. <source>Comp Biochem Physiol B Biochem Mol Biol.</source> (<year>2019</year>) <volume>238</volume>:<fpage>110329</fpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cbpb.2019.110329</pub-id><pub-id pub-id-type=\"pmid\">31461686</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brouillette</surname><given-names>CG</given-names></name><name><surname>Anantharamaiah</surname><given-names>GM</given-names></name><name><surname>Engler</surname><given-names>JA</given-names></name><name><surname>Borhani</surname><given-names>DW</given-names></name></person-group>. <article-title>Structural models of human apolipoprotein A-I: a critical analysis and review</article-title>. <source>Biochim Biophys Acta.</source> (<year>2001</year>) <volume>1531</volume>:<fpage>4</fpage>–<lpage>46</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S1388-1981(01)00081-6</pub-id><pub-id pub-id-type=\"pmid\">11278170</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ma</surname><given-names>J</given-names></name><name><surname>Liao</surname><given-names>XL</given-names></name><name><surname>Lou</surname><given-names>B</given-names></name><name><surname>Wu</surname><given-names>MP</given-names></name></person-group>. <article-title>Role of apolipoprotein A-I in protecting against endotoxin toxicity</article-title>. <source>Acta Biochim Biophys Sin (Shanghai).</source> (<year>2004</year>) <volume>36</volume>:<fpage>419</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1093/abbs/36.6.419</pub-id><pub-id pub-id-type=\"pmid\">15188057</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rahman</surname><given-names>MM</given-names></name><name><surname>Ma</surname><given-names>G</given-names></name><name><surname>Roberts</surname><given-names>HL</given-names></name><name><surname>Schmidt</surname><given-names>O</given-names></name></person-group>. <article-title>Cell-free immune reactions in insects</article-title>. <source>J Insect Physiol.</source> (<year>2006</year>) <volume>52</volume>:<fpage>754</fpage>–<lpage>62</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jinsphys.2006.04.003</pub-id>\n<pub-id pub-id-type=\"pmid\">16753175</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Synaptic Neurosci</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Synaptic Neurosci</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Synaptic Neurosci.</journal-id><journal-title-group><journal-title>Frontiers in Synaptic Neuroscience</journal-title></journal-title-group><issn pub-type=\"epub\">1663-3563</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32848696</article-id><article-id pub-id-type=\"pmc\">PMC7431521</article-id><article-id pub-id-type=\"doi\">10.3389/fnsyn.2020.00033</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Neuroscience</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Neuroligins and Neurodevelopmental Disorders: X-Linked Genetics</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Nguyen</surname><given-names>Thien A.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Lehr</surname><given-names>Alexander W.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Roche</surname><given-names>Katherine W.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/3731/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Receptor Biology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health</institution>, <addr-line>Bethesda, MD</addr-line>, <country>United States</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Pharmacology and Physiology, Georgetown University</institution>, <addr-line>Washington, DC</addr-line>, <country>United States</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Kimberly M. Huber, University of Texas Southwestern Medical Center, United States</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Dilja Krueger-Burg, University Medical Center Göttingen, Germany; Michele H. Jacob, Tufts University School of Medicine, United States</p></fn><corresp id=\"c001\">*Correspondence: Thien A. Nguyen, <email>tan30@georgetown.edu</email></corresp><corresp id=\"c002\">Katherine W. Roche, <email>rochek@ninds.nih.gov</email></corresp></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>12</volume><elocation-id>33</elocation-id><history><date date-type=\"received\"><day>20</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>22</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Nguyen, Lehr and Roche.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Nguyen, Lehr and Roche</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Autism spectrum disorder (ASD) is a neurodevelopmental disorder that results in social-communication impairments, as well as restricted and repetitive behaviors. Moreover, ASD is more prevalent in males, with a male to female ratio of 4 to 1. Although the underlying etiology of ASD is generally unknown, recent advances in genome sequencing have facilitated the identification of a host of associated genes. Among these, synaptic proteins such as cell adhesion molecules have been strongly linked with ASD. Interestingly, many large genome sequencing studies exclude sex chromosomes, which leads to a shift in focus toward autosomal genes as targets for ASD research. However, there are many genes on the X chromosome that encode synaptic proteins, including strong candidate genes. Here, we review findings regarding two members of the neuroligin (NLGN) family of postsynaptic adhesion molecules, <italic>NLGN3</italic> and <italic>NLGN4</italic>. Neuroligins have multiple isoforms (NLGN1-4), which are both autosomal and sex-linked. The sex-linked genes, <italic>NLGN3</italic> and <italic>NLGN4</italic>, are both on the X chromosome and were among the first few genes to be linked with ASD and intellectual disability (ID). In addition, there is a less studied human neuroligin on the Y chromosome, NLGN4Y, which forms an X-Y pair with NLGN4X. We will discuss recent findings of these neuroligin isoforms regarding function at the synapse in both rodent models and human-derived differentiated neurons, and highlight the exciting challenges moving forward to a better understanding of ASD/ID.</p></abstract><kwd-group><kwd>autism</kwd><kwd>intellectual disabililties</kwd><kwd>NLGN3</kwd><kwd>NLGN4X</kwd><kwd>neuroligin</kwd></kwd-group><counts><fig-count count=\"2\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"99\"/><page-count count=\"10\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder affecting one in 54 children in the United States. ASD is characterized by deficits in communication and social interaction (<xref rid=\"B57\" ref-type=\"bibr\">Miles, 2011</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Fombonne, 2013</xref>). Intellectual disability (ID) is characterized by deficits in intellectual functioning and adaptive behavior thus limiting an individual’s ability to thrive independently (<xref rid=\"B69\" ref-type=\"bibr\">Raymond, 2006</xref>; <xref rid=\"B52\" ref-type=\"bibr\">Lubs et al., 2012</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Ellison et al., 2013</xref>). Interestingly, both ASD and ID are more prevalent in males (<xref rid=\"B30\" ref-type=\"bibr\">Geschwind, 2011</xref>; <xref rid=\"B57\" ref-type=\"bibr\">Miles, 2011</xref>; <xref rid=\"B87\" ref-type=\"bibr\">Werling and Geschwind, 2013</xref>; <xref rid=\"B88\" ref-type=\"bibr\">Werling et al., 2016</xref>), although this strong sex bias in ASD remains unclear. It is notable that a subset of ASD-associated genes are located on the X chromosome indicating that the sex chromosomes may play a role in at least some of the sexual dimorphism in these disorders.</p><p>Autism spectrum disorder is divided into two categories: syndromic and nonsyndromic. Syndromic ASD is defined as a condition in patients who already have an existing neurological disorder. For example, a subset of patients with Fragile-X syndrome, tuberous sclerosis, or Rett syndrome display phenotypes that are attributed to ASD (<xref rid=\"B73\" ref-type=\"bibr\">Singh and Eroglu, 2013</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Geschwind and State, 2015</xref>). Nonsyndromic ASD accounts for ASD cases that are not linked to any neurological disorders. Before the advancement of next-generation sequencing (NGS), genetic researchers focused on finding rare genetic variants in ASD and ID pedigrees to link genes to these disorders, which led to the association of the neuroligins NLGN3 and NLGN4X to ASD/ID (<xref rid=\"B39\" ref-type=\"bibr\">Jamain et al., 2003</xref>; <xref rid=\"B50\" ref-type=\"bibr\">Laumonnier et al., 2004</xref>). Other notable genes identified through rare <italic>de novo</italic> mutations and recessive inheritance mutations include <italic>SHANK3</italic>, <italic>CNTNAP2</italic>, <italic>NRXN1</italic>, <italic>PTEN</italic>, <italic>FMR1</italic>, and <italic>TSC1</italic> (<xref rid=\"B31\" ref-type=\"bibr\">Geschwind and State, 2015</xref>). Although these cases are rare, functional and genetic studies definitively showed their link with ASD and ID. With NGS becoming cheaper and easier to access, genome wide association studies (GWAS) and whole exome sequencing (WES) studies became the major approaches used to identify common and rare variants for ASD/ID. Large cohort studies continue to identify more genes associated with ASD/ID, including genes that are important in chromatin modification, transcriptional regulation, or are FMRP-associated, embryonically expressed, or affect synaptic function (<xref rid=\"B72\" ref-type=\"bibr\">Sanders et al., 2012</xref>; <xref rid=\"B93\" ref-type=\"bibr\">Yu et al., 2013</xref>; <xref rid=\"B22\" ref-type=\"bibr\">De Rubeis et al., 2014</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Iossifov et al., 2014</xref>). Although NGS has dramatically accelerated the identification of new risk genes, it is important to mention that NGS studies often ignore the sex chromosomes due to the limitations for statistical analysis (<xref rid=\"B89\" ref-type=\"bibr\">Wise et al., 2013</xref>; <xref rid=\"B61\" ref-type=\"bibr\">No Author List, 2017</xref>).</p><p>The neuroligin (NLGN) family of postsynaptic cell adhesion molecules have emerged as important factors regulating neuronal development and synaptic transmission. There are five members of the NLGN family in humans and other primates: NLGN1, 2, 3, 4X, and 4Y (<xref rid=\"B6\" ref-type=\"bibr\">Bemben et al., 2015b</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Jeong et al., 2017</xref>; <xref rid=\"B75\" ref-type=\"bibr\">Südhof, 2017</xref>, <xref rid=\"B76\" ref-type=\"bibr\">2018</xref>). However, in rodents, there are only four members: NLGN1, 2, 3, and 4-like (<xref rid=\"B9\" ref-type=\"bibr\">Bolliger et al., 2001</xref>, <xref rid=\"B10\" ref-type=\"bibr\">2008</xref>). NLGNs have an isoform-specific localization: NLGN1 is localized to excitatory synapses, NLGN2 at inhibitory synapses, and NLGN3 is at both (<xref rid=\"B17\" ref-type=\"bibr\">Chih et al., 2005</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Chubykin et al., 2007</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Bemben et al., 2015b</xref>). Interestingly human NLGN4X is localized at excitatory synapses, whereas mouse NLGN4-like is at glycinergic synapses (<xref rid=\"B35\" ref-type=\"bibr\">Hoon et al., 2011</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Bemben et al., 2015a</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Chanda et al., 2016</xref>; <xref rid=\"B53\" ref-type=\"bibr\">Marro et al., 2019</xref>). NLGN4X and NLGN4Y were historically grouped together and assumed to have the same function due to their almost identical sequence identity. However, recent findings show that a single amino acid difference in NLGN4Y results in a trafficking deficit leading to decreased surface expression and synaptic function (<xref rid=\"B60\" ref-type=\"bibr\">Nguyen et al., 2020</xref>).</p><p>Neuroligins are highly dynamic, regulated via posttranslational modifications and protein–protein interactions. NLGN1 is phosphorylated by calcium/calmodulin-dependent protein kinase 2 (CaMKII), protein kinase A (PKA), and tyrosine kinases to regulate its function at excitatory synapses (<xref rid=\"B5\" ref-type=\"bibr\">Bemben et al., 2013</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Giannone et al., 2013</xref>; <xref rid=\"B51\" ref-type=\"bibr\">Letellier et al., 2018</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Jeong et al., 2019</xref>). Furthermore, a recent paper established that NLGN1-mediated synaptogenic properties are mediated by interacting with Kalirin7, a Rho guanine nucleotide exchange factor (GEF) (<xref rid=\"B64\" ref-type=\"bibr\">Paskus et al., 2019</xref>, <xref rid=\"B63\" ref-type=\"bibr\">2020</xref>). Phosphorylation of NLGN2 affects binding with inhibitory scaffolding proteins, thus regulating its function at inhibitory synapses (<xref rid=\"B65\" ref-type=\"bibr\">Poulopoulos et al., 2009</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Antonelli et al., 2014</xref>; <xref rid=\"B59\" ref-type=\"bibr\">Nguyen et al., 2016</xref>). NLGN3 can be cleaved by proteases to reduce its function at synapses (<xref rid=\"B4\" ref-type=\"bibr\">Bemben et al., 2019</xref>). Interestingly, the extracellular cleaved fragment of NLGN3 has been identified as a potent mitogen in brain cancer (<xref rid=\"B82\" ref-type=\"bibr\">Venkatesh et al., 2015</xref>, <xref rid=\"B83\" ref-type=\"bibr\">2017</xref>). Lastly, NLGN4X can be phosphorylated by protein kinase C (PKC) to enhance excitatory synapses (<xref rid=\"B3\" ref-type=\"bibr\">Bemben et al., 2015a</xref>). Together, NLGNs comprise an important class of proteins that are dynamic and have multiple functions at synapses.</p><p>Of the NLGN family, <italic>NLGN3</italic>, <italic>NLGN4X</italic>, and <italic>NLGN4Y</italic> are sex-linked with <italic>NLGN3</italic> and <italic>NLGN4X</italic> on the X-chromosome and <italic>NLGN4Y</italic> on the Y-chromosome. Early genetic studies using a family pedigree of ASD/ID probands associated <italic>NLGN3</italic> and <italic>NLGN4X</italic> with ASD/ID (<xref rid=\"B39\" ref-type=\"bibr\">Jamain et al., 2003</xref>; <xref rid=\"B50\" ref-type=\"bibr\">Laumonnier et al., 2004</xref>) (<xref rid=\"T1\" ref-type=\"table\">Tables 1</xref>, <xref rid=\"T2\" ref-type=\"table\">2</xref>). Interestingly, the majority of cases for NLGN3- and NLGN4X-associated ASD/ID are males. In this review, we provide an overview of the current literature of sex-linked NLGNs functions and their links to ASD/ID.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>ASD-associated NLGN3 variants.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Variants</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Sex</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Inheritance pattern</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Primary Phenotype</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Additional Comments/References</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">P104Qfs42X</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B47\" ref-type=\"bibr\">Kenny et al. (2014)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R195W</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">De novo</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B38\" ref-type=\"bibr\">Iossifov et al. (2014)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">V306M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B44\" ref-type=\"bibr\">Jiang et al. (2013)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">V321A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B93\" ref-type=\"bibr\">Yu et al. (2013)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N390X</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B94\" ref-type=\"bibr\">Yuen et al. (2017)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">G426S</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">De novo</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B90\" ref-type=\"bibr\">Xu et al. (2014)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">W433X</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B56\" ref-type=\"bibr\">McRae et al. (2017)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R451C</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B39\" ref-type=\"bibr\">Jamain et al. (2003)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">P514S</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M × 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B68\" ref-type=\"bibr\">Quartier et al. (2019)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R597W</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M × 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B68\" ref-type=\"bibr\">Quartier et al. (2019)</xref>; <xref rid=\"B70\" ref-type=\"bibr\">Redin et al. (2014)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R617W</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M × 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD/ID</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B70\" ref-type=\"bibr\">Redin et al. (2014)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">T632A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B8\" ref-type=\"bibr\">Blasi et al. (2006)</xref></td></tr></tbody></table></table-wrap><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>ASD-associated NLGN4X variants.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Variants</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Inheritance Pattern</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Sex</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Primary Phenotype</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Additional Comments/References</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">G84R</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Asymptomatic mothers (<xref rid=\"B90\" ref-type=\"bibr\">Xu et al., 2014</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R87W</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">De novo</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B97\" ref-type=\"bibr\">Zhang et al. (2009)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">P94L</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GeneDX submitted on ClinVar with unknown significance</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">G99S</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mother also has learning disability. A brother also has learning disability (<xref rid=\"B92\" ref-type=\"bibr\">Yan et al., 2005</xref>)</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mother also has learning disability. Sibling of above (<xref rid=\"B92\" ref-type=\"bibr\">Yan et al., 2005</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R101Q</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B60\" ref-type=\"bibr\">Nguyen et al. (2020)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">V109L</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ID</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B60\" ref-type=\"bibr\">Nguyen et al. (2020)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Q162K</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">De novo</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B90\" ref-type=\"bibr\">Xu et al. (2014)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">L211X</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Anxiety, ADHD, Cerebral palsy</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B94\" ref-type=\"bibr\">Yuen et al. (2017)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Q274X</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ADHD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B94\" ref-type=\"bibr\">Yuen et al. (2017)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">A283T</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B90\" ref-type=\"bibr\">Xu et al. (2014)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Q329X</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B93\" ref-type=\"bibr\">Yu et al. (2013)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">K378R</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B62\" ref-type=\"bibr\">Pampanos et al. (2009)</xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B92\" ref-type=\"bibr\">Yan et al. (2005)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">396X frameshift 1186t</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 × M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Asperger’s syndrome/ASD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B39\" ref-type=\"bibr\">Jamain et al. (2003)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">V403M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Have both affected and unaffected siblings (<xref rid=\"B90\" ref-type=\"bibr\">Xu et al., 2014</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">429X (nt1253del(AG)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13 × M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD/ID</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B50\" ref-type=\"bibr\">Laumonnier et al. (2004)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">V454_A457X</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">De novo</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ID</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B54\" ref-type=\"bibr\">Martínez et al. (2016)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">V522M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">De novo</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">TD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B86\" ref-type=\"bibr\">Wang et al. (2018)</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R704C</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Unaffected sister (+/−) (<xref rid=\"B92\" ref-type=\"bibr\">Yan et al., 2005</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R766Q</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Maternal</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">M</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ASD</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B93\" ref-type=\"bibr\">Yu et al. (2013)</xref></td></tr></tbody></table></table-wrap></sec><sec id=\"S2\"><title>NLGN3 and ASD</title><p>The first link between ASD and <italic>NLGN3</italic> was revealed from a case study of ASD patients. <xref rid=\"B39\" ref-type=\"bibr\">Jamain et al. (2003)</xref> identified a missense mutation in a Swedish family with two affected brothers, one with ASD and the other with Asperger’s syndrome. They showed that both probands contain a missense mutation in NLGN3 (NLGN3 R451C), which encodes an arginine instead of a cysteine at amino acid 451 within the extracellular domain (ECD) of NLGN3. The NLGN3 R451C mutant displays a decrease in surface expression compared to WT, and is retained in the ER by binding to the chaperone protein BiP (<xref rid=\"B16\" ref-type=\"bibr\">Chih et al., 2004</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Comoletti et al., 2004</xref>; <xref rid=\"B21\" ref-type=\"bibr\">De Jaco et al., 2006</xref>). Unlike the human specific <italic>NLGN4X</italic>, <italic>NLGN3</italic> is highly conserved across mammals, facilitating the development of knock-in (KI) mouse models to study how NLGN3 mutations affect behavior.</p><p>In agreement with molecular studies, the NLGN3 R451C KI mouse displays a significant (∼90%) decrease in protein levels compared to WT. Interestingly, the NLGN3 R451C mutant demonstrated a synaptic transmission gain-of-function phenotype, and these effects are synapse specific. Although the NLGN3 R451C KI mice have reduced protein levels, NLGN3 R451C mice, but not NLGN3 KO mice, display an increase in inhibitory synapses as measured by VGAT and gephyrin immunoreactivity. Furthermore, a concomitant increase in mIPSCs frequency in somatosensory cortex was observed in NLGN3 R451C mice, but not NLGN3 KO mice (<xref rid=\"B77\" ref-type=\"bibr\">Tabuchi et al., 2007</xref>). In addition, NLGN3 R451C leads to impaired inhibitory synaptic transmission in PV-neurons in KI animals, unlike the NLGN3 KO; however, both mouse lines show enhanced inhibitory synaptic transmission in cholecystokinin basket cells (<xref rid=\"B28\" ref-type=\"bibr\">Földy et al., 2013</xref>). <xref rid=\"B36\" ref-type=\"bibr\">Horn and Nicoll (2018)</xref> also provide additional evidence of the synapse-specific function of NLGN3 by showing that knocking down NLGN3 using miRNA specifically affected IPSCs recorded from somatostatin neurons, but not from PV-neurons. In addition, NLGN3 R451C mice, but not NLGN3 KO mice, have a striking phenotype at glutamatergic synapses. In the CA1 region of the hippocampus, NLGN3 R451C mice display an increase in excitatory synaptic transmission (<xref rid=\"B25\" ref-type=\"bibr\">Etherton et al., 2011</xref>). Along with this observation, <xref rid=\"B25\" ref-type=\"bibr\">Etherton et al. (2011)</xref> saw an increase in dendritic complexity and NMDAR protein levels in NLGN3 R451C mice. In contrast, NLGN3 R451C mice display impaired synaptic transmission at the calyx of Held synapses. Furthermore, <xref rid=\"B96\" ref-type=\"bibr\">Zhang et al. (2017)</xref> elegantly demonstrated that the synaptic effect of NLGN3 on the calyx of Held synapses is only observed when NLGN3 is deleted late, but not early, in development. Lastly, NLGN3 R451C KI mice and NLGN3 KO mice share a common synaptic defect at striatal synapses; the deletion or KI of NLGN3 in D1 neurons, but not D2 neurons, results in a decrease in mIPSCs frequency (<xref rid=\"B71\" ref-type=\"bibr\">Rothwell et al., 2014</xref>). Taken together, the NLGN3 R451C mutation differentially alter synaptic function depending on neuron and synapse type.</p><p>Behavioral analyses of NLGN3 R451C KI mice revealed a deficit in social interaction and an enhancement in spatial learning; however, these findings were not reproduced in a separate independent study, likely due to differences in mouse strains or behavioral protocols (<xref rid=\"B77\" ref-type=\"bibr\">Tabuchi et al., 2007</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Chadman et al., 2008</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Jaramillo et al., 2014</xref>; <xref rid=\"B49\" ref-type=\"bibr\">Lakhani et al., 2019</xref>). Another phenotype of ASD is repetitive behavior; and, interestingly, the NLGN3 R451C KI and NLGN3 KO mice share this phenotype despite differences in social interaction and spatial memory paradigms (<xref rid=\"B71\" ref-type=\"bibr\">Rothwell et al., 2014</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Burrows et al., 2015</xref>). Indeed, NLGN3 R451C KI and NLGN3 KO mice both have an enhanced ability to stay on an accelerated rod (<xref rid=\"B14\" ref-type=\"bibr\">Chadman et al., 2008</xref>; <xref rid=\"B71\" ref-type=\"bibr\">Rothwell et al., 2014</xref>). Importantly, the repetitive behavior of NLGN3 mutants is due to dysfunction of D1-dopamine receptor-expressing medium spiny neurons, but not D2 neurons. Taken together, the ASD phenotypes of NLGN3 R451C KI and NLGN3 KO mice are circuit- and neuron-specific. Further investigations into which circuits affect the social interaction, spatial memory, and social memory phenotypes in NLGN3 R451C and NLGN3 KO are needed to better understand the mechanisms driving these behavioral deficits in ASD.</p><p>Studies in NLGN3 R451C KI and NLGN3 KO mice highlighted a need to better understand the physiological function of NLGN3. For example, a striking observation in NLGN3 R451C KI mice is a ∼90% reduction in protein levels, while displaying both gain-of-function and loss-of-function phenotypes depending on the type of synapses. Different synaptic phenotypes induced by the single point mutation, NLGN3 R451C, suggest that WT NLGN3 normally functions in a context-dependent manner. Indeed, context-dependent function of NLGNs has been reported in which excitatory synapses are regulated by the relative expression of NLGN1. For example, NLGN1 KO mice display similar spine density as WT animals, but when NLGN1 KO neurons are co-cultured with WT neurons, the NLGN1 KO neurons show a reduction in spine density (<xref rid=\"B48\" ref-type=\"bibr\">Kwon et al., 2012</xref>). Applying this model of competition to NLGN3 R451C KI mice to explain the gain-of-function observed in this animal is worthy of investigation. It is also important to carefully study NLGN3 function throughout development. <xref rid=\"B96\" ref-type=\"bibr\">Zhang et al. (2017)</xref> demonstrated reduced synaptic transmission at the calyx of Held synapse when NLGN3 is deleted late, but not early, in development. They further showed that when NLGN3 is conditionally knocked out in early development, cerebellin-1 can compensate for the lack of NLGN3.</p></sec><sec id=\"S3\"><title>NLGN4X and Its Link to ASD</title><sec id=\"S3.SS1\"><title>Divergence of NLGN4</title><p>Of the ASD-associated genes identified from human genetic screens, NLGNs are of particular interest due to their important function at synapses. Early genetic studies on the X chromosome indicated that a deletion at Xp22.3 was found in ASD/ID probands (<xref rid=\"B78\" ref-type=\"bibr\">Thomas et al., 1999</xref>; <xref rid=\"B99\" ref-type=\"bibr\">Zinn et al., 2007</xref>). Interestingly, <italic>NLGN4X</italic> is located within this region. Although disease-associated mutations in NLGNs are relatively rare, rigorous genetic studies using probands’ pedigrees have established a causal link between NLGN4X and ASD/ID (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>).</p><p>Because <italic>NLGN4X</italic> is a human-specific gene, the discovery of mouse <italic>NLGN4-like</italic> was exciting because it allowed the study of NLGN4 in rodents to probe its role in ASD/ID. Although, there have been enormous advances in the field regarding the synaptic function of NLGN1-3, there are still many gaps in our understanding of the NLGN4 isoforms, which is complicated due to their unusually rapid divergence in humans and rodents. In humans, NLGN4 is sex-linked, and <italic>NLGN4X</italic> and <italic>NLGN4Y</italic> combine to form an X-Y gene pair. However, in mice, NLGN4 exists as a pseudo-autosomal gene often referred to as NLGN4-like. In addition, <xref rid=\"B55\" ref-type=\"bibr\">Maxeiner et al. (2020)</xref> observed that mouse NLGN4-like undergoes rapid evolution resulting in changes in protein sequence. Sequence alignment of NLGN4X with NLGN4-like shows seven insertions in NLGN4-like across both the ECD and intracellular domain (ICD). Interestingly, NLGN4 from the rodent infra-orders <italic>castorimorpha</italic>, <italic>hystricomorpha</italic>, and <italic>sciuromorpha</italic> retains similarity to human NLGN4X, whereas the rodent infra-order <italic>myomorpha</italic>, which includes mice, do not. Thus far NLGN4 has not been identified in rats (<xref rid=\"B10\" ref-type=\"bibr\">Bolliger et al., 2008</xref>; <xref rid=\"B55\" ref-type=\"bibr\">Maxeiner et al., 2020</xref>). Sequence alignment of mouse NLGN4-like, human NLGN4X, and NLGN4Y shows that NLGN4-like only shares ∼60% sequence identity with NLGN4X/4Y, whereas NLGN4X shares ∼97% sequence identity with NLGN4Y (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). A decade of research later, it is now clear that the human and rodent NLGN4 genes do not share the same function as previously assumed.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Alignment of NLGN4. Alignment of mouse and human NLGN4s and their conservation.</p></caption><graphic xlink:href=\"fnsyn-12-00033-g001\"/></fig></sec><sec id=\"S3.SS2\"><title>Human and Mouse NLGN4</title><p>Human NLGN4X was first cloned almost two decades ago. In the initial studies, NLGN4X was shown to be expressed and processed in a similar fashion to that of NLGN1. NLGN4X, like NLGN1, is glycosylated, traffics to the cell surface, and can bind to PSD-95 (<xref rid=\"B9\" ref-type=\"bibr\">Bolliger et al., 2001</xref>). Furthermore, NLGN4X is found at excitatory synapses. NLGN4X overexpression in mouse hippocampal neurons increases dendritic spine density, but it decreases mEPSCs frequency and amplitude (<xref rid=\"B15\" ref-type=\"bibr\">Chanda et al., 2016</xref>; <xref rid=\"B97\" ref-type=\"bibr\">Zhang et al., 2009</xref>). However, exogenously expressed human NLGN4X in rat hippocampal slices in combination with NLGN1-3 microRNA to knockdown endogenous NLGN1-3 showed an increase in spine density and a concomitant increase in both AMPAR- and NMDAR-mediated EPSCs (<xref rid=\"B3\" ref-type=\"bibr\">Bemben et al., 2015a</xref>). The difference between these two sets of experiments is the presence of endogenous NLGN1-3. It is unclear whether NLGN4X can form heterodimers with NLGN1-3 <italic>in vivo</italic>, although NLGN4X has been shown to form heterodimers with NLGN1 (<xref rid=\"B66\" ref-type=\"bibr\">Poulopoulos et al., 2012</xref>). Further investigation into this subject can provide a better understanding of the function of endogenous NLGN4X at synapses.</p><p>Using differentiated neurons from human induced pluripotent stem cells (iPS cells), NLGN4X was shown to colocalize with VGLUT and PSD-95, revealing NLGN4X localization at excitatory synapses (<xref rid=\"B53\" ref-type=\"bibr\">Marro et al., 2019</xref>). However, in NLGN4X KO differentiated neurons, <xref rid=\"B53\" ref-type=\"bibr\">Marro et al. (2019)</xref> did not observe any changes in either EPSCs or IPSCs. It is important to note that although differentiated human neurons from iPS cells can be useful, these differentiated neurons are not fully mature and are lacking NMDARs, a key component of excitatory synapses (<xref rid=\"B98\" ref-type=\"bibr\">Zhang et al., 2013</xref>; <xref rid=\"B67\" ref-type=\"bibr\">Quadrato et al., 2017</xref>; <xref rid=\"B53\" ref-type=\"bibr\">Marro et al., 2019</xref>).</p><p>In contrast to NLGN4X, mouse NLGN4-like functions at inhibitory synapses. Localization experiments in mice show that NLGN4-like is at glycinergic inhibitory synapses where it colocalizes with glycine receptors and gephyrin, but not PSD-95 in brainstem, spinal cord, and retina. Moreover, NLGN4-like KO mice were shown to have deficits in glycinergic synaptic transmission (<xref rid=\"B40\" ref-type=\"bibr\">Jamain et al., 2008</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Hoon et al., 2011</xref>; <xref rid=\"B95\" ref-type=\"bibr\">Zhang et al., 2018</xref>). In addition, NLGN4-like also functions at GABAergic synapses (<xref rid=\"B33\" ref-type=\"bibr\">Hammer et al., 2015</xref>; <xref rid=\"B81\" ref-type=\"bibr\">Unichenko et al., 2018</xref>). In KO NLGN4-like mice, GABAergic synaptic transmission is impaired in hippocampal CA3 region (<xref rid=\"B33\" ref-type=\"bibr\">Hammer et al., 2015</xref>). Together, NLGN4-like primarily acts at inhibitory synapses, either glycinergic or GABAergic, whereas human NLGN4X acts at excitatory synapses.</p><p>NLGN4-like KO mice were generated over a decade ago and have been characterized extensively. However, the behavioral data have been complicated. For instance, NLGN4-like KO mice were first characterized as having a deficit in social interaction and vocalization (<xref rid=\"B40\" ref-type=\"bibr\">Jamain et al., 2008</xref>; <xref rid=\"B23\" ref-type=\"bibr\">El-Kordi et al., 2013</xref>; <xref rid=\"B45\" ref-type=\"bibr\">Ju et al., 2014</xref>); however, another study using the same NLGN4-like KO mice did not find any deficit in social interaction or vocalization (<xref rid=\"B26\" ref-type=\"bibr\">Ey et al., 2012</xref>). Although NLGN4-like KO mice provide insights into how this protein may function at synapses, because human NLGN4X and mouse NLGN4-like are divergent, there should be caution in linking mouse NLGN4-like studies with NLGN4X-associated neurodevelopmental disorders.</p><p>Lastly, NLGNs are dynamically regulated through posttranslational modifications (<xref rid=\"B6\" ref-type=\"bibr\">Bemben et al., 2015b</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Jeong et al., 2017</xref>). Similar to NLGN1 and NLGN2, posttranslational modifications have an important role in regulating NLGN4X function (<xref rid=\"B6\" ref-type=\"bibr\">Bemben et al., 2015b</xref>; <xref rid=\"B43\" ref-type=\"bibr\">Jeong et al., 2017</xref>). NLGN4X is phosphorylated by PKC at T707 (<xref rid=\"B3\" ref-type=\"bibr\">Bemben et al., 2015a</xref>). Unlike CaMKII phosphorylation of NLGN1, PKC phosphorylation of NLGN4X does not increase its trafficking to the surface. However, phosphorylated NLGN4X T707 does lead to increases in spine density and aggregation of the excitatory synapse markers VGLUT and PSD-95 (<xref rid=\"B5\" ref-type=\"bibr\">Bemben et al., 2013</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Bemben et al., 2015a</xref>). In addition, analyses of the NLGN4X phospho-mimetic mutation, T707D, reveal significant enhancement of both AMPAR and NMDAR EPSCs compared to WT (<xref rid=\"B3\" ref-type=\"bibr\">Bemben et al., 2015a</xref>). How phosphorylated NLGN4X is able to increase excitatory synaptic strength will require additional investigation to reveal the precise mechanisms underlying synaptic potentiation. This topic would benefit from techniques that allow the characterization of spatiotemporal dynamics of PKC phosphorylation of NLGN4X <italic>in vivo</italic>. Furthermore, NLGN4X T707 is conserved in mouse NLGN4-like, but it is unclear whether this residue is phosphorylated in mouse NLGN4-like. Would the phosphorylation of this conserved threonine residue in mouse NLGN4-like enhance synaptic transmission as it does in human NLGN4X? Investigation on the mechanism of phosphorylation and the enhancement of synaptic transmission is a worthy topic to study.</p></sec></sec><sec id=\"S4\"><title>NLGN4X and ASD/ID</title><p><xref rid=\"B39\" ref-type=\"bibr\">Jamain et al. (2003)</xref> first established NLGN4X as causative genes for ASD/ID through screening patients with ASD and Asperger’s syndrome, and identified a frameshift mutation (1186insT) in <italic>NLGN4X</italic>, which leads to a premature stop codon at amino acid 396. Interestingly, in addition to the two probands, their mother also carries the mutation, but does not display any autistic symptoms (<xref rid=\"B39\" ref-type=\"bibr\">Jamain et al., 2003</xref>). The most convincing case for <italic>NLGN4X</italic> as an ASD/ID risk gene is from a study following a French family with a nonsense mutation in <italic>NLGN4X</italic>. <xref rid=\"B50\" ref-type=\"bibr\">Laumonnier et al. (2004)</xref> observed a 2-base-pair deletion in <italic>NLGN4X</italic> that resulted in a stop codon at position 429. By documenting the clinical data from this large family, <xref rid=\"B50\" ref-type=\"bibr\">Laumonnier et al. (2004)</xref> observed that 13 males with the nonsense mutation were diagnosed with ASD, ID, or pervasive neurodevelopmental disorders, whereas female carriers were unaffected. This finding is remarkable in showing that this mutation in <italic>NLGN4X</italic> follows an X-linked recessive pattern. Many subsequent studies have linked <italic>NLGN4X</italic> with neurodevelopmental disorders, and the recurrent theme is that the majority of affected probands are males (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>).</p><p>Along with frameshift and nonsense mutations, many disease-associated missense mutations have been identified in <italic>NLGN4X</italic>. How might these missense mutations affect NLGN4X function? A missense mutation was identified in two ASD probands resulting in a substitution of an arginine residue to tryptophan (NLGN4X R87W). The NLGN4X R87W variant displays a profound deficit in NLGN4X surface expression, which leads to hypofunction of the protein due to decreased synaptogenesis. Furthermore, expression of NLGN4X R87W results in increased synaptic strength when overexpressed in neurons on a WT background (<xref rid=\"B97\" ref-type=\"bibr\">Zhang et al., 2009</xref>). It is puzzling why a variant that failed to induce synaptogenesis on a null background can still enhance synaptic function. Interestingly, a cluster of NLGN4X-associated variants has been identified near the NLGN4X R87W that also display a deficit in surface expression (<xref rid=\"B60\" ref-type=\"bibr\">Nguyen et al., 2020</xref>). Because these NLGN4X-associated variants are in the ECD, it is of interest to investigate their ability to bind to neurexin. Using the solved structure of NLGN4X, it was shown that ASD-associated mutations, such as NLGN4X G99S, are located outside of the neurexin binding domain (<xref rid=\"B27\" ref-type=\"bibr\">Fabrichny et al., 2007</xref>). These data suggest the observed phenotype from the cluster of NLGN4X-associated mutations is due to a deficit in trafficking.</p><p>Another NLGN4X rare variant that has garnered much attention is a substitution in the ICD from arginine to cysteine, NLGN4X R704C (<xref rid=\"B92\" ref-type=\"bibr\">Yan et al., 2005</xref>). As discussed above, NLGN4X is phosphorylated by PKC at T707 resulting in an increase in spine numbers and EPSCs (<xref rid=\"B3\" ref-type=\"bibr\">Bemben et al., 2015a</xref>). Interestingly, there were significant deficits in phosphorylation of NLGN4X T707 in the NLGN4X R704C variant, and the effects mediated by phosphorylation were abolished (<xref rid=\"B3\" ref-type=\"bibr\">Bemben et al., 2015a</xref>). In a separate study, <xref rid=\"B15\" ref-type=\"bibr\">Chanda et al. (2016)</xref> expressed NLGN4X R704C in cultured mouse neurons on a WT background and observed an increase in both NMDAR and AMPAR EPSCs compared to WT. Interestingly, neither study observed a change in surface trafficking. The discrepancy in these studies likely results from differences in experimental design, chiefly whether to include or exclude endogenous NLGN1-3. Taken together, NLGN4X R704C displays profound differences, compared to WT, in regulation of excitatory synapses. Using human differentiated neurons from NLGN4X R704C KI hiPSCs, <xref rid=\"B53\" ref-type=\"bibr\">Marro et al. (2019)</xref> observed an increase in EPSCs compared to WT. In addition, NLGN4X R704C was shown to increase binding with GluA1, but not PSD-95 (<xref rid=\"B53\" ref-type=\"bibr\">Marro et al., 2019</xref>), again revealing that this rare variant has multiple functional effects.</p><p>With the advances in stem cell research, it is now possible to study how different NLGN4X variants function in human neurons. Although studies taking this approach provide attractive new tools to study endogenous NLGN4X and its variants, there are pitfalls that needs to be addressed. Use of differentiated neurons from hiPSCs is still in its infancy and synaptic activity from these neurons does not represent the full endogenous nature of a synapse. For instance, it has been shown that differentiated neurons using single transcription expression models lack NMDA receptors (<xref rid=\"B98\" ref-type=\"bibr\">Zhang et al., 2013</xref>; <xref rid=\"B67\" ref-type=\"bibr\">Quadrato et al., 2017</xref>; <xref rid=\"B85\" ref-type=\"bibr\">Wang et al., 2017</xref>; <xref rid=\"B58\" ref-type=\"bibr\">Nehme et al., 2018</xref>). These neurons can express NMDARs if, and only if, they are allowed to grow for a long period of time (35+ days). Even so, to date, there is little biochemical evidence that NMDARs are present under these differentiation protocols. For the study of neuroligins, this is particularly problematic as they have been shown to directly interact with NMDARs via their ECDs (<xref rid=\"B11\" ref-type=\"bibr\">Budreck et al., 2013</xref>). Thus, although stem cell and differentiation technology are attractive and can be a powerful tool to study human neurons and diseases, a better understanding of the PSD in these neurons is needed before it can be used with great confidence as a model to study synaptic transmission.</p></sec><sec id=\"S5\"><title>NLGN4X and NLGN4Y</title><p>Until recently, the studies on human specific NLGN4s have focused on NLGN4X. However, it is important to explore the function of NLGN4Y as well. NLGN4X and NLGN4Y are remarkably conserved with only 19 amino acid differences between them. Due to this high sequence conservation, the two proteins have been assumed to have the same function (<xref rid=\"B6\" ref-type=\"bibr\">Bemben et al., 2015b</xref>; <xref rid=\"B76\" ref-type=\"bibr\">Südhof, 2018</xref>); however, this hypothesis had not been experimentally examined until recently. Because NLGN4X/Y are sex-linked genes, an important consideration is the sex-bias in the expression of NLGN4X. Outside of the pseudo autosomal regions (PARs), some genes on the X chromosome can escape X-inactivation thus providing an imbalance of gene dosage between males and females (<xref rid=\"B13\" ref-type=\"bibr\">Carrel and Willard, 2005</xref>; <xref rid=\"B74\" ref-type=\"bibr\">Skuse, 2005</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Helena Mangs and Morris, 2007</xref>; <xref rid=\"B80\" ref-type=\"bibr\">Tukiainen et al., 2017</xref>). Interestingly, there are Y-linked genes that are homologs to X-linked genes that escaped X-inactivation in order to balance the gene dosage in males. Furthermore, these X-Y gene pairs have been shown to have an important function in transcription, translation and protein stability (<xref rid=\"B2\" ref-type=\"bibr\">Bellott et al., 2014</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Cortez et al., 2014</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Hughes and Page, 2015</xref>). Together, these studies reveal an important role for genes on the Y chromosome other than sex determining genes. Indeed, comparison of <italic>NLGN4X</italic> and <italic>NLGN4Y</italic> expression in males and females reveals interesting differences. In a large transcriptomic study, <italic>NLGN4Y</italic> was shown to express only in males, as expected; however, <italic>NLGN4X</italic> was shown to express at similar level between males and females (<xref rid=\"B46\" ref-type=\"bibr\">Kang et al., 2011</xref>; <xref rid=\"B79\" ref-type=\"bibr\">Trabzuni et al., 2013</xref>). To complicate the issue further, a separate study reported that incomplete X-inactivation exists in mammals, and <italic>NLGN4X</italic> partially escapes (<xref rid=\"B13\" ref-type=\"bibr\">Carrel and Willard, 2005</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Berletch et al., 2011</xref>). Interestingly, in a study using different tissues to study X-inactivation, <italic>NLGN4X</italic> expression is higher in the cortex in female vs. male (<xref rid=\"B80\" ref-type=\"bibr\">Tukiainen et al., 2017</xref>). Although gene expression of <italic>NLGN4X</italic> and <italic>NLGN4Y</italic> has been compared, research comparing NLGN4X and NLGN4Y protein function has lagged behind.</p><p>Although it was reasonable to hypothesize that NLGN4X and NLGN4Y served the same function due to their high sequence homology (97%), this hypothesis had never been tested. Interestingly, many ASD/ID variants have been identified in NLGN4X (<xref rid=\"B39\" ref-type=\"bibr\">Jamain et al., 2003</xref>; <xref rid=\"B50\" ref-type=\"bibr\">Laumonnier et al., 2004</xref>; <xref rid=\"B92\" ref-type=\"bibr\">Yan et al., 2005</xref>; <xref rid=\"B84\" ref-type=\"bibr\">Volaki et al., 2013</xref>; <xref rid=\"B90\" ref-type=\"bibr\">Xu et al., 2014</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Bemben et al., 2015a</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Chanda et al., 2016</xref>), whereas only one missense mutation has been identified in NLGN4Y (<xref rid=\"B91\" ref-type=\"bibr\">Yan et al., 2008</xref>). Furthermore, ASD/ID-associated mutations in NLGN4X selectively affect more males than females, and the reason for this male bias is unknown. This strong male bias observation in NLGN4X-associated diseases, prompted us to focus on NLGN4Y. If NLGN4Y and NLGN4X are functionally redundant, then there should not be a male bias in NLGN4X-associated diseases.</p><p>To explore the function of NLGN4Y, in a recent study, we directly compared NLGN4X and NLGN4Y and found that NLGN4Y cannot traffic to the surface to induce synapses (<xref rid=\"B60\" ref-type=\"bibr\">Nguyen et al., 2020</xref>). Furthermore, the differential trafficking observed between NLGN4X and NLGN4Y is due to an amino acid difference at position 93, with proline for NLGN4X and serine for NLGN4Y. Indeed, the NLGN4Y S93P mutant was able to efficiently traffic to the surface and induce synapses. Interestingly, there is a cluster of disease-associated NLGN4X variants surrounding the critical amino acid in NLGN4X. Upon analysis, these variants phenocopied the NLGN4Y trafficking deficit and cannot induce synapses (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>NLGN4X and NLGN4Y function. Schematic for differential trafficking of NLGN4X vs NLGN4Y. NLGN4X can traffic to the surface and induce excitatory synapses. Furthermore, phosphorylation of NLGN4X by PKC drastically enhances excitatory postsynaptic currents (EPSCs). In contrast, NLGN4Y cannot traffic to the surface, thus decreasing EPSCs through binding with other NLGNs.</p></caption><graphic xlink:href=\"fnsyn-12-00033-g002\"/></fig><p>What is the function of NLGN4Y if it cannot get to the surface? <xref rid=\"B60\" ref-type=\"bibr\">Nguyen et al. (2020)</xref> demonstrated that NLGN4Y can oligomerize with NLGN1, 2, 3, and 4X and reduce their surface trafficking. In addition, exogenously expressed NLGN4Y on a WT background decreased mEPSCs suggesting NLGN4Y acts to inhibit NLGN1-3 function. However, this study relies on exogenously expressed NLGNs in heterologous cells or rat hippocampal neurons. What the role is for endogenous NLGN4Y in human neurons is an important lingering question.</p></sec><sec id=\"S6\"><title>Conclusion</title><p>With the advances in NGS technologies, a wide variety of genes have been associated with ASD/ID. However, many of these studies have ignored the sex chromosomes due to the additional expense and a lack of statistical power. However, historically many genes on the X-chromosome have been linked to ASD/ID by evaluating proband pedigrees. NLGN3 and NLGN4X, both on the X chromosome, were among the first genes associated with ASD/ID. Although NLGN3 and NLGN4X variants only occur in a small population of ASD/ID cases, studies using NLGN3 and NLGN4 mouse models have provided many insights into how disruptions in NLGN3 and NLGN4 function contribute to ASD/ID phenotypes. With advances in stem cell and neuronal differentiation, it is now possible to study NLGN3 and NLGN4X variants using human iPSCs to explore the causality between disruption in sex-linked NLGNs and ASD/ID by examining endogenous human neuroligins. Although neuronal differentiation is an exciting new technology to further our understanding of the human brain, differentiated neurons from human iPSCs are still relatively immature. Further improvement in the technologies to develop reliable mature neurons will be of paramount importance going forward. In addition, the unexpected revelations from the study of NLGN4X and NLGN4Y highlight the need to investigate the often-ignored Y-chromosome. Although many facets of the sex-linked NLGNs have been characterized, many important questions remain unanswered and provide a fertile topic for future investigation into synaptic regulation and to develop therapeutic treatments.</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>TN and KR wrote the manuscript. AL helped to create table for variants.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This review was supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program (1ZIANS003140-06).</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Antonelli</surname><given-names>R.</given-names></name><name><surname>Pizzarelli</surname><given-names>R.</given-names></name><name><surname>Pedroni</surname><given-names>A.</given-names></name><name><surname>Fritschy</surname><given-names>J.-M.</given-names></name><name><surname>Del Sal</surname><given-names>G.</given-names></name><name><surname>Cherubini</surname><given-names>E.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Pin1-dependent signalling negatively affects GABAergic transmission by modulating neuroligin2/gephyrin interaction.</article-title>\n<source><italic>Nat. Commun.</italic></source>\n<volume>5</volume>:<issue>5066</issue>. <pub-id pub-id-type=\"doi\">10.1038/ncomms6066</pub-id>\n<pub-id pub-id-type=\"pmid\">25297980</pub-id></mixed-citation></ref><ref id=\"B2\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bellott</surname><given-names>D. W.</given-names></name><name><surname>Hughes</surname><given-names>J. F.</given-names></name><name><surname>Skaletsky</surname><given-names>H.</given-names></name><name><surname>Brown</surname><given-names>L. G.</given-names></name><name><surname>Pyntikova</surname><given-names>T.</given-names></name><name><surname>Cho</surname><given-names>T. J.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Mammalian y chromosomes retain widely expressed dosage-sensitive regulators.</article-title>\n<source><italic>Nature</italic></source>\n<volume>508</volume>\n<fpage>494</fpage>–<lpage>499</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature13206</pub-id>\n<pub-id pub-id-type=\"pmid\">24759411</pub-id></mixed-citation></ref><ref id=\"B3\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bemben</surname><given-names>M. A.</given-names></name><name><surname>Nguyen</surname><given-names>Q. A.</given-names></name><name><surname>Wang</surname><given-names>T.</given-names></name><name><surname>Li</surname><given-names>Y.</given-names></name><name><surname>Nicoll</surname><given-names>R. A.</given-names></name><name><surname>Roche</surname><given-names>K. W.</given-names></name></person-group> (<year>2015a</year>). <article-title>Autism-associated mutation inhibits protein kinase C-mediated neuroligin-4X enhancement of excitatory synapses.</article-title>\n<source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source>\n<volume>112</volume>\n<fpage>2551</fpage>–<lpage>2556</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1500501112</pub-id>\n<pub-id pub-id-type=\"pmid\">25675530</pub-id></mixed-citation></ref><ref id=\"B4\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bemben</surname><given-names>M. A.</given-names></name><name><surname>Nguyen</surname><given-names>T. A.</given-names></name><name><surname>Li</surname><given-names>Y.</given-names></name><name><surname>Wang</surname><given-names>T.</given-names></name><name><surname>Nicoll</surname><given-names>R. A.</given-names></name><name><surname>Roche</surname><given-names>K. W.</given-names></name></person-group> (<year>2019</year>). <article-title>Isoform-specific cleavage of neuroligin-3 reduces synapse strength.</article-title>\n<source><italic>Mol. Psychiatry</italic></source>\n<volume>24</volume>\n<fpage>145</fpage>–<lpage>160</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41380-018-0242-y</pub-id>\n<pub-id pub-id-type=\"pmid\">30242227</pub-id></mixed-citation></ref><ref id=\"B5\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bemben</surname><given-names>M. A.</given-names></name><name><surname>Shipman</surname><given-names>S. L.</given-names></name><name><surname>Hirai</surname><given-names>T.</given-names></name><name><surname>Herring</surname><given-names>B. E.</given-names></name><name><surname>Li</surname><given-names>Y.</given-names></name><name><surname>Badger</surname><given-names>J. D.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>CaMKII phosphorylation of neuroligin-1 regulates excitatory synapses.</article-title>\n<source><italic>Nat. Neurosci.</italic></source>\n<volume>17</volume>\n<fpage>56</fpage>–<lpage>64</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nn.3601</pub-id>\n<pub-id pub-id-type=\"pmid\">24336150</pub-id></mixed-citation></ref><ref id=\"B6\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bemben</surname><given-names>M. A.</given-names></name><name><surname>Shipman</surname><given-names>S. L.</given-names></name><name><surname>Nicoll</surname><given-names>R. A.</given-names></name><name><surname>Roche</surname><given-names>K. W.</given-names></name></person-group> (<year>2015b</year>). <article-title>The cellular and molecular landscape of neuroligins.</article-title>\n<source><italic>Trends Neurosci.</italic></source>\n<volume>38</volume>\n<fpage>496</fpage>–<lpage>505</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.tins.2015.06.004</pub-id>\n<pub-id pub-id-type=\"pmid\">26209464</pub-id></mixed-citation></ref><ref id=\"B7\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Berletch</surname><given-names>J. B.</given-names></name><name><surname>Yang</surname><given-names>F.</given-names></name><name><surname>Xu</surname><given-names>J.</given-names></name><name><surname>Carrel</surname><given-names>L.</given-names></name><name><surname>Disteche</surname><given-names>C. M.</given-names></name></person-group> (<year>2011</year>). <article-title>Genes that escape from X inactivation.</article-title>\n<source><italic>Hum. Genet.</italic></source>\n<volume>130</volume>\n<fpage>237</fpage>–<lpage>245</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00439-011-1011-z</pub-id>\n<pub-id pub-id-type=\"pmid\">21614513</pub-id></mixed-citation></ref><ref id=\"B8\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Blasi</surname><given-names>F.</given-names></name><name><surname>Bacchelli</surname><given-names>E.</given-names></name><name><surname>Pesaresi</surname><given-names>G.</given-names></name><name><surname>Carone</surname><given-names>S.</given-names></name><name><surname>Bailey</surname><given-names>A. J.</given-names></name><name><surname>Maestrini</surname><given-names>E.</given-names></name></person-group> (<year>2006</year>). <article-title>Absence of coding mutations in the X-linked genes neuroligin 3 and neuroligin 4 in individuals with autism from the IMGSAC collection.</article-title>\n<source><italic>Am. J. Med. Genet. Neuropsychiatr. Genet.</italic></source>\n<volume>141B</volume>\n<fpage>220</fpage>–<lpage>221</lpage>. <pub-id pub-id-type=\"doi\">10.1002/ajmg.b.30287</pub-id>\n<pub-id pub-id-type=\"pmid\">16508939</pub-id></mixed-citation></ref><ref id=\"B9\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bolliger</surname><given-names>M. F.</given-names></name><name><surname>Frei</surname><given-names>K.</given-names></name><name><surname>Winterhalter</surname><given-names>K. H.</given-names></name><name><surname>Gloor</surname><given-names>S. M.</given-names></name></person-group> (<year>2001</year>). <article-title>Identification of a novel neuroligin in humans which binds to PSD-95 and has a widespread expression.</article-title>\n<source><italic>Biochem. J.</italic></source>\n<volume>356</volume>(Pt 2), <fpage>581</fpage>–<lpage>588</lpage>. <pub-id pub-id-type=\"doi\">10.1042/0264-6021:3560581</pub-id><pub-id pub-id-type=\"pmid\">11368788</pub-id></mixed-citation></ref><ref id=\"B10\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bolliger</surname><given-names>M. F.</given-names></name><name><surname>Pei</surname><given-names>J.</given-names></name><name><surname>Maxeiner</surname><given-names>S.</given-names></name><name><surname>Boucard</surname><given-names>A. A.</given-names></name><name><surname>Grishin</surname><given-names>N. V.</given-names></name><name><surname>Südhof</surname><given-names>T. C.</given-names></name></person-group> (<year>2008</year>). <article-title>Unusually rapid evolution of Neuroligin-4 in mice.</article-title>\n<source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source>\n<volume>105</volume>\n<fpage>6421</fpage>–<lpage>6426</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.0801383105</pub-id>\n<pub-id pub-id-type=\"pmid\">18434543</pub-id></mixed-citation></ref><ref id=\"B11\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Budreck</surname><given-names>E. C.</given-names></name><name><surname>Kwon</surname><given-names>O.-B.</given-names></name><name><surname>Jung</surname><given-names>J. H.</given-names></name><name><surname>Baudouin</surname><given-names>S.</given-names></name><name><surname>Thommen</surname><given-names>A.</given-names></name><name><surname>Kim</surname><given-names>H.-S.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling.</article-title>\n<source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source>\n<volume>110</volume>\n<fpage>725</fpage>–<lpage>730</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1214718110</pub-id>\n<pub-id pub-id-type=\"pmid\">23269831</pub-id></mixed-citation></ref><ref id=\"B12\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Burrows</surname><given-names>E. L.</given-names></name><name><surname>Laskaris</surname><given-names>L.</given-names></name><name><surname>Koyama</surname><given-names>L.</given-names></name><name><surname>Churilov</surname><given-names>L.</given-names></name><name><surname>Bornstein</surname><given-names>J. C.</given-names></name><name><surname>Hill-Yardin</surname><given-names>E. L.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>A neuroligin-3 mutation implicated in autism causes abnormal aggression and increases repetitive behavior in mice.</article-title>\n<source><italic>Mol. Autism</italic></source>\n<volume>6</volume>:<issue>62</issue>. <pub-id pub-id-type=\"doi\">10.1186/s13229-015-0055-7</pub-id>\n<pub-id pub-id-type=\"pmid\">26583067</pub-id></mixed-citation></ref><ref id=\"B13\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carrel</surname><given-names>L.</given-names></name><name><surname>Willard</surname><given-names>H. F.</given-names></name></person-group> (<year>2005</year>). <article-title>X-inactivation profile reveals extensive variability in X-linked gene expression in females.</article-title>\n<source><italic>Nature</italic></source>\n<volume>434</volume>\n<fpage>400</fpage>–<lpage>404</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature03479</pub-id>\n<pub-id pub-id-type=\"pmid\">15772666</pub-id></mixed-citation></ref><ref id=\"B14\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chadman</surname><given-names>K. K.</given-names></name><name><surname>Gong</surname><given-names>S.</given-names></name><name><surname>Scattoni</surname><given-names>M. L.</given-names></name><name><surname>Boltuck</surname><given-names>S. E.</given-names></name><name><surname>Gandhy</surname><given-names>S. U.</given-names></name><name><surname>Heintz</surname><given-names>N.</given-names></name><etal/></person-group> (<year>2008</year>). <article-title>Minimal aberrant behavioral phenotypes of neuroligin-3 R451C knockin mice.</article-title>\n<source><italic>Autism Res.</italic></source>\n<volume>1</volume>\n<fpage>147</fpage>–<lpage>158</lpage>. <pub-id pub-id-type=\"doi\">10.1002/aur.22</pub-id>\n<pub-id pub-id-type=\"pmid\">19360662</pub-id></mixed-citation></ref><ref id=\"B15\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chanda</surname><given-names>S.</given-names></name><name><surname>Aoto</surname><given-names>J.</given-names></name><name><surname>Lee</surname><given-names>S. J.</given-names></name><name><surname>Wernig</surname><given-names>M.</given-names></name><name><surname>Südhof</surname><given-names>T. C.</given-names></name></person-group> (<year>2016</year>). <article-title>Pathogenic mechanism of an autism-associated neuroligin mutation involves altered AMPA-receptor trafficking.</article-title>\n<source><italic>Mol. Psychiatry</italic></source>\n<volume>21</volume>\n<fpage>169</fpage>–<lpage>177</lpage>. <pub-id pub-id-type=\"doi\">10.1038/mp.2015.20</pub-id>\n<pub-id pub-id-type=\"pmid\">25778475</pub-id></mixed-citation></ref><ref id=\"B16\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chih</surname><given-names>B.</given-names></name><name><surname>Afridi</surname><given-names>S. K.</given-names></name><name><surname>Clark</surname><given-names>L.</given-names></name><name><surname>Scheiffele</surname><given-names>P.</given-names></name></person-group> (<year>2004</year>). <article-title>Disorder-associated mutations lead to functional inactivation of neuroligins.</article-title>\n<source><italic>Hum. Mol. Genet.</italic></source>\n<volume>13</volume>\n<fpage>1471</fpage>–<lpage>1477</lpage>. <pub-id pub-id-type=\"doi\">10.1093/hmg/ddh158</pub-id>\n<pub-id pub-id-type=\"pmid\">15150161</pub-id></mixed-citation></ref><ref id=\"B17\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chih</surname><given-names>B.</given-names></name><name><surname>Engelman</surname><given-names>H.</given-names></name><name><surname>Scheiffele</surname><given-names>P.</given-names></name></person-group> (<year>2005</year>). <article-title>Control of excitatory and inhibitory synapse formation by neuroligins.</article-title>\n<source><italic>Science</italic></source>\n<volume>307</volume>\n<fpage>1324</fpage>–<lpage>1328</lpage>. <pub-id pub-id-type=\"doi\">10.1126/science.1107470</pub-id>\n<pub-id pub-id-type=\"pmid\">15681343</pub-id></mixed-citation></ref><ref id=\"B18\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chubykin</surname><given-names>A. A.</given-names></name><name><surname>Atasoy</surname><given-names>D.</given-names></name><name><surname>Etherton</surname><given-names>M. R.</given-names></name><name><surname>Brose</surname><given-names>N.</given-names></name><name><surname>Kavalali</surname><given-names>E. T.</given-names></name><name><surname>Gibson</surname><given-names>J. R.</given-names></name><etal/></person-group> (<year>2007</year>). <article-title>Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2.</article-title>\n<source><italic>Neuron</italic></source>\n<volume>54</volume>\n<fpage>919</fpage>–<lpage>931</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuron.2007.05.029</pub-id>\n<pub-id pub-id-type=\"pmid\">17582332</pub-id></mixed-citation></ref><ref id=\"B19\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Comoletti</surname><given-names>D.</given-names></name><name><surname>De Jaco</surname><given-names>A.</given-names></name><name><surname>Jennings</surname><given-names>L. L.</given-names></name><name><surname>Flynn</surname><given-names>R. E.</given-names></name><name><surname>Gaietta</surname><given-names>G.</given-names></name><name><surname>Tsigelny</surname><given-names>I.</given-names></name><etal/></person-group> (<year>2004</year>). <article-title>The Arg451Cys-neuroligin-3 mutation associated with autism reveals a defect in protein processing.</article-title>\n<source><italic>J. Neurosci.</italic></source>\n<volume>24</volume>\n<fpage>4889</fpage>–<lpage>4893</lpage>. <pub-id pub-id-type=\"doi\">10.1523/JNEUROSCI.0468-04.2004</pub-id>\n<pub-id pub-id-type=\"pmid\">15152050</pub-id></mixed-citation></ref><ref id=\"B20\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cortez</surname><given-names>D.</given-names></name><name><surname>Marin</surname><given-names>R.</given-names></name><name><surname>Toledo-Flores</surname><given-names>D.</given-names></name><name><surname>Froidevaux</surname><given-names>L.</given-names></name><name><surname>Liechti</surname><given-names>A.</given-names></name><name><surname>Waters</surname><given-names>P. D.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Origins and functional evolution of y chromosomes across mammals.</article-title>\n<source><italic>Nature</italic></source>\n<volume>508</volume>\n<fpage>488</fpage>–<lpage>493</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature13151</pub-id>\n<pub-id pub-id-type=\"pmid\">24759410</pub-id></mixed-citation></ref><ref id=\"B21\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Jaco</surname><given-names>A.</given-names></name><name><surname>Comoletti</surname><given-names>D.</given-names></name><name><surname>Kovarik</surname><given-names>Z.</given-names></name><name><surname>Gaietta</surname><given-names>G.</given-names></name><name><surname>Radiæ</surname><given-names>Z.</given-names></name><name><surname>Lockridge</surname><given-names>O.</given-names></name><etal/></person-group> (<year>2006</year>). <article-title>A mutation linked with autism reveals a common mechanism of endoplasmic reticulum retention for theα,β-hydrolase fold protein family.</article-title>\n<source><italic>J. Biol. Chem.</italic></source>\n<volume>281</volume>\n<fpage>9667</fpage>–<lpage>9676</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M510262200</pub-id>\n<pub-id pub-id-type=\"pmid\">16434405</pub-id></mixed-citation></ref><ref id=\"B22\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Rubeis</surname><given-names>S.</given-names></name><name><surname>He</surname><given-names>X.</given-names></name><name><surname>Goldberg</surname><given-names>A. P.</given-names></name><name><surname>Poultney</surname><given-names>C. S.</given-names></name><name><surname>Samocha</surname><given-names>K.</given-names></name><name><surname>Cicek</surname><given-names>A. E.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Synaptic, transcriptional and chromatin genes disrupted in autism.</article-title>\n<source><italic>Nature</italic></source>\n<volume>515</volume>\n<fpage>209</fpage>–<lpage>215</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature13772</pub-id>\n<pub-id pub-id-type=\"pmid\">25363760</pub-id></mixed-citation></ref><ref id=\"B23\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>El-Kordi</surname><given-names>A.</given-names></name><name><surname>Winkler</surname><given-names>D.</given-names></name><name><surname>Hammerschmidt</surname><given-names>K.</given-names></name><name><surname>Kästner</surname><given-names>A.</given-names></name><name><surname>Krueger</surname><given-names>D.</given-names></name><name><surname>Ronnenberg</surname><given-names>A.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>Development of an autism severity score for mice using Nlgn4 null mutants as a construct-valid model of heritable monogenic autism.</article-title>\n<source><italic>Behav. Brain Res.</italic></source>\n<volume>251</volume>\n<fpage>41</fpage>–<lpage>49</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbr.2012.11.016</pub-id>\n<pub-id pub-id-type=\"pmid\">23183221</pub-id></mixed-citation></ref><ref id=\"B24\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ellison</surname><given-names>J. W.</given-names></name><name><surname>Rosenfeld</surname><given-names>J. A.</given-names></name><name><surname>Shaffer</surname><given-names>L. G.</given-names></name></person-group> (<year>2013</year>). <article-title>Genetic Basis of Intellectual Disability.</article-title>\n<source><italic>Annu. Rev. Med.</italic></source>\n<volume>64</volume>\n<fpage>441</fpage>–<lpage>450</lpage>. <pub-id pub-id-type=\"doi\">10.1146/annurev-med-042711-140053</pub-id>\n<pub-id pub-id-type=\"pmid\">23020879</pub-id></mixed-citation></ref><ref id=\"B25\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Etherton</surname><given-names>M.</given-names></name><name><surname>Földy</surname><given-names>C.</given-names></name><name><surname>Sharma</surname><given-names>M.</given-names></name><name><surname>Tabuchi</surname><given-names>K.</given-names></name><name><surname>Liu</surname><given-names>X.</given-names></name><name><surname>Shamloo</surname><given-names>M.</given-names></name></person-group> (<year>2011</year>). <article-title>Autism-linked neuroligin-3 R451C mutation differentially alters hippocampal and cortical synaptic function.</article-title>\n<source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source>\n<volume>108</volume>\n<fpage>13764</fpage>–<lpage>13769</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1111093108</pub-id>\n<pub-id pub-id-type=\"pmid\">21808020</pub-id></mixed-citation></ref><ref id=\"B26\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ey</surname><given-names>E.</given-names></name><name><surname>Yang</surname><given-names>M.</given-names></name><name><surname>Katz</surname><given-names>A. M.</given-names></name><name><surname>Woldeyohannes</surname><given-names>L.</given-names></name><name><surname>Silverman</surname><given-names>J. L.</given-names></name><name><surname>Leblond</surname><given-names>C. S.</given-names></name><etal/></person-group> (<year>2012</year>). <article-title>Absence of deficits in social behaviors and ultrasonic vocalizations in later generations of mice lacking neuroligin4.</article-title>\n<source><italic>Genes Brain Behav.</italic></source>\n<volume>11</volume>\n<fpage>928</fpage>–<lpage>941</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1601-183X.2012.00849.x</pub-id>\n<pub-id pub-id-type=\"pmid\">22989184</pub-id></mixed-citation></ref><ref id=\"B27\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fabrichny</surname><given-names>I. P.</given-names></name><name><surname>Leone</surname><given-names>P.</given-names></name><name><surname>Sulzenbacher</surname><given-names>G.</given-names></name><name><surname>Comoletti</surname><given-names>D.</given-names></name><name><surname>Miller</surname><given-names>M. T.</given-names></name><name><surname>Taylor</surname><given-names>P.</given-names></name><etal/></person-group> (<year>2007</year>). <article-title>Structural analysis of the synaptic protein neuroligin and its β-neurexin complex: determinants for folding and cell adhesion.</article-title>\n<source><italic>Neuron</italic></source>\n<volume>56</volume>\n<fpage>979</fpage>–<lpage>991</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuron.2007.11.013</pub-id>\n<pub-id pub-id-type=\"pmid\">18093521</pub-id></mixed-citation></ref><ref id=\"B28\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Földy</surname><given-names>C.</given-names></name><name><surname>Malenka</surname><given-names>R. C.</given-names></name><name><surname>Südhof</surname><given-names>T. C.</given-names></name></person-group> (<year>2013</year>). <article-title>Autism-associated neuroligin-3 mutations commonly disrupt tonic endocannabinoid signaling.</article-title>\n<source><italic>Neuron</italic></source>\n<volume>78</volume>\n<fpage>498</fpage>–<lpage>509</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuron.2013.02.036</pub-id>\n<pub-id pub-id-type=\"pmid\">23583622</pub-id></mixed-citation></ref><ref id=\"B29\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fombonne</surname><given-names>E.</given-names></name></person-group> (<year>2013</year>). <article-title>The epidemiology of autism: a review.</article-title>\n<source><italic>Sci. Ment. Health</italic></source>\n<volume>2</volume>\n<fpage>65</fpage>–<lpage>82</lpage>. <pub-id pub-id-type=\"doi\">10.1017/S0033291799008508</pub-id>\n<pub-id pub-id-type=\"pmid\">10473304</pub-id></mixed-citation></ref><ref id=\"B30\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Geschwind</surname><given-names>D. H.</given-names></name></person-group> (<year>2011</year>). <article-title>Genetics of autism spectrum disorders.</article-title>\n<source><italic>Trends Cogn. Sci.</italic></source>\n<volume>15</volume>\n<fpage>409</fpage>–<lpage>416</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.tics.2011.07.003</pub-id>\n<pub-id pub-id-type=\"pmid\">21855394</pub-id></mixed-citation></ref><ref id=\"B31\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Geschwind</surname><given-names>D. H.</given-names></name><name><surname>State</surname><given-names>M. W.</given-names></name></person-group> (<year>2015</year>). <article-title>Gene hunting in autism spectrum disorder: on the path to precision medicine.</article-title>\n<source><italic>Lancet Neurol.</italic></source>\n<volume>14</volume>\n<fpage>1109</fpage>–<lpage>1120</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S1474-4422(15)00044-7</pub-id><pub-id pub-id-type=\"pmid\">25891009</pub-id></mixed-citation></ref><ref id=\"B32\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Giannone</surname><given-names>G.</given-names></name><name><surname>Mondin</surname><given-names>M.</given-names></name><name><surname>Grillo-Bosch</surname><given-names>D.</given-names></name><name><surname>Tessier</surname><given-names>B.</given-names></name><name><surname>Saint-Michel</surname><given-names>E.</given-names></name><name><surname>Czöndör</surname><given-names>K.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>Neurexin-1β binding to neuroligin-1 triggers the preferential recruitment of PSD-95 versus gephyrin through tyrosine phosphorylation of neuroligin-1.</article-title>\n<source><italic>Cell Rep.</italic></source>\n<volume>3</volume>\n<fpage>1996</fpage>–<lpage>2007</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.celrep.2013.05.013</pub-id>\n<pub-id pub-id-type=\"pmid\">23770246</pub-id></mixed-citation></ref><ref id=\"B33\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hammer</surname><given-names>M.</given-names></name><name><surname>Krueger-Burg</surname><given-names>D.</given-names></name><name><surname>Tuffy</surname><given-names>L. P.</given-names></name><name><surname>Cooper</surname><given-names>B. H.</given-names></name><name><surname>Taschenberger</surname><given-names>H.</given-names></name><name><surname>Goswami</surname><given-names>S. P.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>Perturbed hippocampal synaptic inhibition and γ-oscillations in a neuroligin-4 knockout mouse model of autism.</article-title>\n<source><italic>Cell Rep.</italic></source>\n<volume>13</volume>\n<fpage>516</fpage>–<lpage>523</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.celrep.2015.09.011</pub-id>\n<pub-id pub-id-type=\"pmid\">26456829</pub-id></mixed-citation></ref><ref id=\"B34\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Helena Mangs</surname><given-names>A.</given-names></name><name><surname>Morris</surname><given-names>B. J.</given-names></name></person-group> (<year>2007</year>). <article-title>The human pseudoautosomal region (PAR): origin, function and future.</article-title>\n<source><italic>Curr. Genom.</italic></source>\n<volume>8</volume>\n<fpage>129</fpage>–<lpage>136</lpage>. <pub-id pub-id-type=\"doi\">10.2174/138920207780368141</pub-id>\n<pub-id pub-id-type=\"pmid\">18660847</pub-id></mixed-citation></ref><ref id=\"B35\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hoon</surname><given-names>M.</given-names></name><name><surname>Soykan</surname><given-names>T.</given-names></name><name><surname>Falkenburger</surname><given-names>B.</given-names></name><name><surname>Hammer</surname><given-names>M.</given-names></name><name><surname>Patrizi</surname><given-names>A.</given-names></name><name><surname>Schmidt</surname><given-names>K.-F. K.-F.</given-names></name><etal/></person-group> (<year>2011</year>). <article-title>Neuroligin-4 is localized to glycinergic postsynapses and regulates inhibition in the retina.</article-title>\n<source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source>\n<volume>108</volume>\n<fpage>3053</fpage>–<lpage>3058</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1006946108</pub-id>\n<pub-id pub-id-type=\"pmid\">21282647</pub-id></mixed-citation></ref><ref id=\"B36\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Horn</surname><given-names>M. E.</given-names></name><name><surname>Nicoll</surname><given-names>R. A.</given-names></name></person-group> (<year>2018</year>). <article-title>Somatostatin and parvalbumin inhibitory synapses onto hippocampal pyramidal neurons are regulated by distinct mechanisms.</article-title>\n<source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source>\n<volume>115</volume>\n<fpage>589</fpage>–<lpage>594</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1719523115</pub-id>\n<pub-id pub-id-type=\"pmid\">29295931</pub-id></mixed-citation></ref><ref id=\"B37\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hughes</surname><given-names>J. F.</given-names></name><name><surname>Page</surname><given-names>D. C.</given-names></name></person-group> (<year>2015</year>). <article-title>The Biology and Evolution of Mammalian Y Chromosomes.</article-title>\n<source><italic>Annu. Rev. Genet.</italic></source>\n<volume>49</volume>\n<fpage>507</fpage>–<lpage>527</lpage>. <pub-id pub-id-type=\"doi\">10.1146/annurev-genet-112414-055311</pub-id>\n<pub-id pub-id-type=\"pmid\">26442847</pub-id></mixed-citation></ref><ref id=\"B38\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Iossifov</surname><given-names>I.</given-names></name><name><surname>O’Roak</surname><given-names>B. J.</given-names></name><name><surname>Sanders</surname><given-names>S. J.</given-names></name><name><surname>Ronemus</surname><given-names>M.</given-names></name><name><surname>Krumm</surname><given-names>N.</given-names></name><name><surname>Levy</surname><given-names>D.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>The contribution of de novo coding mutations to autism spectrum disorder.</article-title>\n<source><italic>Nature</italic></source>\n<volume>515</volume>\n<fpage>216</fpage>–<lpage>221</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature13908</pub-id>\n<pub-id pub-id-type=\"pmid\">25363768</pub-id></mixed-citation></ref><ref id=\"B39\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jamain</surname><given-names>S.</given-names></name><name><surname>Quach</surname><given-names>H.</given-names></name><name><surname>Betancur</surname><given-names>C.</given-names></name><name><surname>Råstam</surname><given-names>M.</given-names></name><name><surname>Colineaux</surname><given-names>C.</given-names></name><name><surname>Gillberg</surname><given-names>I. C.</given-names></name><etal/></person-group> (<year>2003</year>). <article-title>Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism.</article-title>\n<source><italic>Nat. Genet.</italic></source>\n<volume>34</volume>\n<fpage>27</fpage>–<lpage>29</lpage>. <pub-id pub-id-type=\"doi\">10.1038/ng1136</pub-id>\n<pub-id pub-id-type=\"pmid\">12669065</pub-id></mixed-citation></ref><ref id=\"B40\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jamain</surname><given-names>S.</given-names></name><name><surname>Radyushkin</surname><given-names>K.</given-names></name><name><surname>Hammerschmidt</surname><given-names>K.</given-names></name><name><surname>Granon</surname><given-names>S.</given-names></name><name><surname>Boretius</surname><given-names>S.</given-names></name><name><surname>Varoqueaux</surname><given-names>F.</given-names></name><etal/></person-group> (<year>2008</year>). <article-title>Reduced social interaction and ultrasonic communication in a mouse model of monogenic heritable autism.</article-title>\n<source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source>\n<volume>105</volume>\n<fpage>1710</fpage>–<lpage>1715</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.0711555105</pub-id>\n<pub-id pub-id-type=\"pmid\">18227507</pub-id></mixed-citation></ref><ref id=\"B41\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jaramillo</surname><given-names>T. C.</given-names></name><name><surname>Liu</surname><given-names>S.</given-names></name><name><surname>Pettersen</surname><given-names>A.</given-names></name><name><surname>Birnbaum</surname><given-names>S. G.</given-names></name><name><surname>Powell</surname><given-names>C. M.</given-names></name></person-group> (<year>2014</year>). <article-title>Autism-related neuroligin-3 mutation alters social behavior and spatial learning.</article-title>\n<source><italic>Autism Res.</italic></source>\n<volume>7</volume>\n<fpage>264</fpage>–<lpage>272</lpage>. <pub-id pub-id-type=\"doi\">10.1002/aur.1362</pub-id>\n<pub-id pub-id-type=\"pmid\">24619977</pub-id></mixed-citation></ref><ref id=\"B42\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jeong</surname><given-names>J.</given-names></name><name><surname>Pandey</surname><given-names>S.</given-names></name><name><surname>Li</surname><given-names>Y.</given-names></name><name><surname>Badger</surname><given-names>J. D.</given-names></name><name><surname>Lu</surname><given-names>W.</given-names></name><name><surname>Roche</surname><given-names>K. W.</given-names></name></person-group> (<year>2019</year>). <article-title>PSD-95 binding dynamically regulates NLGN1 trafficking and function.</article-title>\n<source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source>\n<volume>116</volume>\n<fpage>12035</fpage>–<lpage>12044</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1821775116</pub-id>\n<pub-id pub-id-type=\"pmid\">31138690</pub-id></mixed-citation></ref><ref id=\"B43\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jeong</surname><given-names>J.</given-names></name><name><surname>Paskus</surname><given-names>J. D.</given-names></name><name><surname>Roche</surname><given-names>K. W.</given-names></name></person-group> (<year>2017</year>). <article-title>Posttranslational modifications of neuroligins regulate neuronal and glial signaling.</article-title>\n<source><italic>Curr. Opin. Neurobiol.</italic></source>\n<volume>45</volume>\n<fpage>130</fpage>–<lpage>138</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.conb.2017.05.017</pub-id>\n<pub-id pub-id-type=\"pmid\">28577430</pub-id></mixed-citation></ref><ref id=\"B44\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jiang</surname><given-names>Y. H.</given-names></name><name><surname>Yuen</surname><given-names>R. K. C.</given-names></name><name><surname>Jin</surname><given-names>X.</given-names></name><name><surname>Wang</surname><given-names>M.</given-names></name><name><surname>Chen</surname><given-names>N.</given-names></name><name><surname>Wu</surname><given-names>X.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>Detection of clinically relevant genetic variants in autism spectrum disorder by whole-genome sequencing.</article-title>\n<source><italic>Am. J. Hum. Genet.</italic></source>\n<volume>93</volume>\n<fpage>249</fpage>–<lpage>263</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ajhg.2013.06.012</pub-id>\n<pub-id pub-id-type=\"pmid\">23849776</pub-id></mixed-citation></ref><ref id=\"B45\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ju</surname><given-names>A.</given-names></name><name><surname>Hammerschmidt</surname><given-names>K.</given-names></name><name><surname>Tantra</surname><given-names>M.</given-names></name><name><surname>Krueger</surname><given-names>D.</given-names></name><name><surname>Brose</surname><given-names>N.</given-names></name><name><surname>Ehrenreich</surname><given-names>H.</given-names></name></person-group> (<year>2014</year>). <article-title>Juvenile manifestation of ultrasound communication deficits in the neuroligin-4 null mutant mouse model of autism.</article-title>\n<source><italic>Behav. Brain Res.</italic></source>\n<volume>270</volume>\n<fpage>159</fpage>–<lpage>164</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbr.2014.05.019</pub-id>\n<pub-id pub-id-type=\"pmid\">24855039</pub-id></mixed-citation></ref><ref id=\"B46\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kang</surname><given-names>H. J.</given-names></name><name><surname>Kawasawa</surname><given-names>Y. I.</given-names></name><name><surname>Cheng</surname><given-names>F.</given-names></name><name><surname>Zhu</surname><given-names>Y.</given-names></name><name><surname>Xu</surname><given-names>X.</given-names></name><name><surname>Li</surname><given-names>M.</given-names></name><etal/></person-group> (<year>2011</year>). <article-title>Spatio-temporal transcriptome of the human brain.</article-title>\n<source><italic>Nature</italic></source>\n<volume>478</volume>\n<fpage>483</fpage>–<lpage>489</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature10523</pub-id>\n<pub-id pub-id-type=\"pmid\">22031440</pub-id></mixed-citation></ref><ref id=\"B47\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kenny</surname><given-names>E. M.</given-names></name><name><surname>Cormican</surname><given-names>P.</given-names></name><name><surname>Furlong</surname><given-names>S.</given-names></name><name><surname>Heron</surname><given-names>E.</given-names></name><name><surname>Kenny</surname><given-names>G.</given-names></name><name><surname>Fahey</surname><given-names>C.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Excess of rare novel loss-of-function variants in synaptic genes in schizophrenia and autism spectrum disorders.</article-title>\n<source><italic>Mol. Psychiatry</italic></source>\n<volume>19</volume>\n<fpage>872</fpage>–<lpage>879</lpage>. <pub-id pub-id-type=\"doi\">10.1038/mp.2013.127</pub-id>\n<pub-id pub-id-type=\"pmid\">24126926</pub-id></mixed-citation></ref><ref id=\"B48\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kwon</surname><given-names>H.-B.</given-names></name><name><surname>Kozorovitskiy</surname><given-names>Y.</given-names></name><name><surname>Oh</surname><given-names>W.-J.</given-names></name><name><surname>Peixoto</surname><given-names>R. T.</given-names></name><name><surname>Akhtar</surname><given-names>N.</given-names></name><name><surname>Saulnier</surname><given-names>J. L.</given-names></name><etal/></person-group> (<year>2012</year>). <article-title>Neuroligin-1-dependent competition regulates cortical synaptogenesis and synapse number.</article-title>\n<source><italic>Nat. Neurosci.</italic></source>\n<volume>15</volume>\n<fpage>1667</fpage>–<lpage>1674</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nn.3256</pub-id>\n<pub-id pub-id-type=\"pmid\">23143522</pub-id></mixed-citation></ref><ref id=\"B49\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lakhani</surname><given-names>C. M.</given-names></name><name><surname>Manrai</surname><given-names>A. K.</given-names></name><name><surname>Yang</surname><given-names>J.</given-names></name><name><surname>Visscher</surname><given-names>P. M.</given-names></name><name><surname>Patel</surname><given-names>C. J.</given-names></name><name><surname>Department</surname><given-names>B. T. T.</given-names></name></person-group> (<year>2019</year>). <article-title>Genetic background effects in neuroligin-3 mutant mice: minimal behavioral abnormalities on C57 background.</article-title>\n<source><italic>Physiol. Behav.</italic></source>\n<volume>176</volume>\n<fpage>139</fpage>–<lpage>148</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.physbeh.2017.03.040</pub-id>\n<pub-id pub-id-type=\"pmid\">28363838</pub-id></mixed-citation></ref><ref id=\"B50\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Laumonnier</surname><given-names>F.</given-names></name><name><surname>Bonnet-Brilhault</surname><given-names>F.</given-names></name><name><surname>Gomot</surname><given-names>M.</given-names></name><name><surname>Blanc</surname><given-names>R.</given-names></name><name><surname>David</surname><given-names>A.</given-names></name><name><surname>Moizard</surname><given-names>M.-P.</given-names></name><etal/></person-group> (<year>2004</year>). <article-title>X-linked mental retardation and autism are associated with a mutation in the NLGN4 gene, a member of the neuroligin family.</article-title>\n<source><italic>Am. J. Hum. Genet.</italic></source>\n<volume>74</volume>\n<fpage>552</fpage>–<lpage>557</lpage>. <pub-id pub-id-type=\"doi\">10.1086/382137</pub-id>\n<pub-id pub-id-type=\"pmid\">14963808</pub-id></mixed-citation></ref><ref id=\"B51\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Letellier</surname><given-names>M.</given-names></name><name><surname>Szíber</surname><given-names>Z.</given-names></name><name><surname>Chamma</surname><given-names>I.</given-names></name><name><surname>Saphy</surname><given-names>C.</given-names></name><name><surname>Papasideri</surname><given-names>I.</given-names></name><name><surname>Tessier</surname><given-names>B.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>A unique intracellular tyrosine in neuroligin-1 regulates AMPA receptor recruitment during synapse differentiation and potentiation.</article-title>\n<source><italic>Nat. Commun.</italic></source>\n<volume>9</volume>:<issue>3979</issue>. <pub-id pub-id-type=\"doi\">10.1038/s41467-018-06220-2</pub-id>\n<pub-id pub-id-type=\"pmid\">30266896</pub-id></mixed-citation></ref><ref id=\"B52\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lubs</surname><given-names>H. A.</given-names></name><name><surname>Stevenson</surname><given-names>R. E.</given-names></name><name><surname>Schwartz</surname><given-names>C. E.</given-names></name></person-group> (<year>2012</year>). <article-title>Fragile X and X-linked intellectual disability: four decades of discovery.</article-title>\n<source><italic>Am. J. Hum. Genet.</italic></source>\n<volume>90</volume>\n<fpage>579</fpage>–<lpage>590</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ajhg.2012.02.018</pub-id>\n<pub-id pub-id-type=\"pmid\">22482801</pub-id></mixed-citation></ref><ref id=\"B53\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Marro</surname><given-names>S. G.</given-names></name><name><surname>Chanda</surname><given-names>S.</given-names></name><name><surname>Yang</surname><given-names>N.</given-names></name><name><surname>Janas</surname><given-names>J. A.</given-names></name><name><surname>Valperga</surname><given-names>G.</given-names></name><name><surname>Trotter</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Neuroligin-4 regulates excitatory synaptic transmission in human neurons.</article-title>\n<source><italic>Neuron</italic></source>\n<volume>103</volume>\n<fpage>617.e6</fpage>–<lpage>626.e6</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuron.2019.05.043</pub-id>\n<pub-id pub-id-type=\"pmid\">31257103</pub-id></mixed-citation></ref><ref id=\"B54\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martínez</surname><given-names>F.</given-names></name><name><surname>Caro-Llopis</surname><given-names>A.</given-names></name><name><surname>Roselló</surname><given-names>M.</given-names></name><name><surname>Oltra</surname><given-names>S.</given-names></name><name><surname>Mayo</surname><given-names>S.</given-names></name><name><surname>Monfort</surname><given-names>S.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>High diagnostic yield of syndromic intellectual disability by targeted next-generation sequencing.</article-title>\n<source><italic>J. Med. Genet.</italic></source>\n<volume>54</volume>\n<fpage>87</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.1136/jmedgenet-2016-103964</pub-id>\n<pub-id pub-id-type=\"pmid\">27620904</pub-id></mixed-citation></ref><ref id=\"B55\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maxeiner</surname><given-names>S.</given-names></name><name><surname>Benseler</surname><given-names>F.</given-names></name><name><surname>Krasteva-Christ</surname><given-names>G.</given-names></name><name><surname>Brose</surname><given-names>N.</given-names></name><name><surname>Südhof</surname><given-names>T. C.</given-names></name></person-group> (<year>2020</year>). <article-title>Evolution of the autism-associated neuroligin-4 gene reveals broad erosion of pseudoautosomal regions in rodents.</article-title>\n<source><italic>Mol. Biol. Evol.</italic></source>\n<volume>37</volume>\n<fpage>1243</fpage>–<lpage>1258</lpage>. <pub-id pub-id-type=\"doi\">10.1093/molbev/msaa014</pub-id>\n<pub-id pub-id-type=\"pmid\">32011705</pub-id></mixed-citation></ref><ref id=\"B56\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McRae</surname><given-names>J. F.</given-names></name><name><surname>Clayton</surname><given-names>S.</given-names></name><name><surname>Fitzgerald</surname><given-names>T. W.</given-names></name><name><surname>Kaplanis</surname><given-names>J.</given-names></name><name><surname>Prigmore</surname><given-names>E.</given-names></name><name><surname>Rajan</surname><given-names>D.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Prevalence and architecture of de novo mutations in developmental disorders.</article-title>\n<source><italic>Nature</italic></source>\n<volume>542</volume>\n<fpage>433</fpage>–<lpage>438</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature21062</pub-id>\n<pub-id pub-id-type=\"pmid\">28135719</pub-id></mixed-citation></ref><ref id=\"B57\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Miles</surname><given-names>J. H.</given-names></name></person-group> (<year>2011</year>). <article-title>Autism spectrum disorders-A genetics review.</article-title>\n<source><italic>Genet. Med.</italic></source>\n<volume>13</volume>\n<fpage>278</fpage>–<lpage>294</lpage>. <pub-id pub-id-type=\"doi\">10.1097/GIM.0b013e3181ff67ba</pub-id>\n<pub-id pub-id-type=\"pmid\">21358411</pub-id></mixed-citation></ref><ref id=\"B58\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nehme</surname><given-names>R.</given-names></name><name><surname>Zuccaro</surname><given-names>E.</given-names></name><name><surname>Ghosh</surname><given-names>S. D.</given-names></name><name><surname>Li</surname><given-names>C.</given-names></name><name><surname>Sherwood</surname><given-names>J. L.</given-names></name><name><surname>Pietilainen</surname><given-names>O.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Combining NGN2 programming with developmental patterning generates human excitatory neurons with NMDAR-mediated synaptic transmission.</article-title>\n<source><italic>Cell Rep.</italic></source>\n<volume>23</volume>\n<fpage>2509</fpage>–<lpage>2523</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.celrep.2018.04.066</pub-id>\n<pub-id pub-id-type=\"pmid\">29791859</pub-id></mixed-citation></ref><ref id=\"B59\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nguyen</surname><given-names>Q. A.</given-names></name><name><surname>Horn</surname><given-names>M. E.</given-names></name><name><surname>Nicoll</surname><given-names>R. A.</given-names></name></person-group> (<year>2016</year>). <article-title>Distinct roles for extracellular and intracellular domains in neuroligin function at inhibitory synapses.</article-title>\n<source><italic>eLife</italic></source>\n<volume>5</volume>\n<fpage>1</fpage>–<lpage>21</lpage>. <pub-id pub-id-type=\"doi\">10.7554/eLife.19236</pub-id>\n<pub-id pub-id-type=\"pmid\">27805570</pub-id></mixed-citation></ref><ref id=\"B60\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nguyen</surname><given-names>T. A.</given-names></name><name><surname>Wu</surname><given-names>K.</given-names></name><name><surname>Pandey</surname><given-names>S.</given-names></name><name><surname>Lehr</surname><given-names>A. W.</given-names></name><name><surname>Li</surname><given-names>Y.</given-names></name><name><surname>Bemben</surname><given-names>M. A.</given-names></name><etal/></person-group> (<year>2020</year>). <article-title>A cluster of autism-associated variants on X-Linked NLGN4X functionally resemble NLGN4Y.</article-title>\n<source><italic>Neuron</italic></source>\n<volume>106</volume>\n<fpage>759.e7</fpage>–<lpage>768.e7</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuron.2020.03.008</pub-id>\n<pub-id pub-id-type=\"pmid\">32243781</pub-id></mixed-citation></ref><ref id=\"B61\"><mixed-citation publication-type=\"journal\"><collab>No Author List</collab> (<year>2017</year>). <article-title>Accounting for sex in the genome.</article-title>\n<source><italic>Nat. Med.</italic></source>\n<volume>23</volume>:<issue>1243</issue>. <pub-id pub-id-type=\"doi\">10.1038/nm.4445</pub-id>\n<pub-id pub-id-type=\"pmid\">29117171</pub-id></mixed-citation></ref><ref id=\"B62\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pampanos</surname><given-names>A.</given-names></name><name><surname>Volaki</surname><given-names>K.</given-names></name><name><surname>Kanavakis</surname><given-names>E.</given-names></name><name><surname>Papandreou</surname><given-names>O.</given-names></name><name><surname>Youroukos</surname><given-names>S.</given-names></name><name><surname>Thomaidis</surname><given-names>L.</given-names></name><etal/></person-group> (<year>2009</year>). <article-title>A substitution involving the NLGN4 gene associated with autistic behavior in the greek population.</article-title>\n<source><italic>Genet. Test. Mol. Biomark.</italic></source>\n<volume>13</volume>\n<fpage>611</fpage>–<lpage>615</lpage>. <pub-id pub-id-type=\"doi\">10.1089/gtmb.2009.0005</pub-id>\n<pub-id pub-id-type=\"pmid\">19645625</pub-id></mixed-citation></ref><ref id=\"B63\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Paskus</surname><given-names>J. D.</given-names></name><name><surname>Herring</surname><given-names>B. E.</given-names></name><name><surname>Roche</surname><given-names>K. W.</given-names></name></person-group> (<year>2020</year>). <article-title>Kalirin and trio: RhoGEFs in synaptic transmission, plasticity, and complex brain disorders.</article-title>\n<source><italic>Trends Neurosci.</italic></source>\n<volume>43</volume>\n<fpage>505</fpage>–<lpage>518</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.tins.2020.05.002</pub-id>\n<pub-id pub-id-type=\"pmid\">32513570</pub-id></mixed-citation></ref><ref id=\"B64\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Paskus</surname><given-names>J. D.</given-names></name><name><surname>Tian</surname><given-names>C.</given-names></name><name><surname>Fingleton</surname><given-names>E.</given-names></name><name><surname>Shen</surname><given-names>C.</given-names></name><name><surname>Chen</surname><given-names>X.</given-names></name><name><surname>Li</surname><given-names>Y.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Synaptic kalirin-7 and trio interactomes reveal a gef protein-dependent neuroligin-1 mechanism of action.</article-title>\n<source><italic>Cell Rep.</italic></source>\n<volume>29</volume>\n<fpage>2944.e5</fpage>–<lpage>2952.e5</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.celrep.2019.10.115</pub-id>\n<pub-id pub-id-type=\"pmid\">31801062</pub-id></mixed-citation></ref><ref id=\"B65\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Poulopoulos</surname><given-names>A.</given-names></name><name><surname>Aramuni</surname><given-names>G.</given-names></name><name><surname>Meyer</surname><given-names>G.</given-names></name><name><surname>Soykan</surname><given-names>T.</given-names></name><name><surname>Hoon</surname><given-names>M.</given-names></name><name><surname>Papadopoulos</surname><given-names>T.</given-names></name><etal/></person-group> (<year>2009</year>). <article-title>Neuroligin 2 drives postsynaptic assembly at perisomatic inhibitory synapses through gephyrin and collybistin.</article-title>\n<source><italic>Neuron</italic></source>\n<volume>63</volume>\n<fpage>628</fpage>–<lpage>642</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuron.2009.08.023</pub-id>\n<pub-id pub-id-type=\"pmid\">19755106</pub-id></mixed-citation></ref><ref id=\"B66\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Poulopoulos</surname><given-names>A.</given-names></name><name><surname>Soykan</surname><given-names>T.</given-names></name><name><surname>Tuffy</surname><given-names>L. P.</given-names></name><name><surname>Hammer</surname><given-names>M.</given-names></name><name><surname>Varoqueaux</surname><given-names>F.</given-names></name><name><surname>Brose</surname><given-names>N.</given-names></name></person-group> (<year>2012</year>). <article-title>Homodimerization and isoform-specific heterodimerization of neuroligins.</article-title>\n<source><italic>Biochem. J.</italic></source>\n<volume>446</volume>\n<fpage>321</fpage>–<lpage>330</lpage>. <pub-id pub-id-type=\"doi\">10.1042/BJ20120808</pub-id>\n<pub-id pub-id-type=\"pmid\">22671294</pub-id></mixed-citation></ref><ref id=\"B67\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quadrato</surname><given-names>G.</given-names></name><name><surname>Nguyen</surname><given-names>T.</given-names></name><name><surname>Macosko</surname><given-names>E. Z.</given-names></name><name><surname>Sherwood</surname><given-names>J. L.</given-names></name><name><surname>Min Yang</surname><given-names>S.</given-names></name><name><surname>Berger</surname><given-names>D. R.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Cell diversity and network dynamics in photosensitive human brain organoids.</article-title>\n<source><italic>Nature</italic></source>\n<volume>545</volume>\n<fpage>48</fpage>–<lpage>53</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature22047</pub-id>\n<pub-id pub-id-type=\"pmid\">28445462</pub-id></mixed-citation></ref><ref id=\"B68\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quartier</surname><given-names>A.</given-names></name><name><surname>Courraud</surname><given-names>J.</given-names></name><name><surname>Thi Ha</surname><given-names>T.</given-names></name><name><surname>McGillivray</surname><given-names>G.</given-names></name><name><surname>Isidor</surname><given-names>B.</given-names></name><name><surname>Rose</surname><given-names>K.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Novel mutations in NLGN3 causing autism spectrum disorder and cognitive impairment.</article-title>\n<source><italic>Hum. Mutat.</italic></source>\n<volume>40</volume>\n<fpage>2021</fpage>–<lpage>2032</lpage>. <pub-id pub-id-type=\"doi\">10.1002/humu.23836</pub-id>\n<pub-id pub-id-type=\"pmid\">31184401</pub-id></mixed-citation></ref><ref id=\"B69\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Raymond</surname><given-names>F. L.</given-names></name></person-group> (<year>2006</year>). <article-title>X linked mental retardation: a clinical guide.</article-title>\n<source><italic>J. Med. Genet.</italic></source>\n<volume>43</volume>\n<fpage>193</fpage>–<lpage>200</lpage>. <pub-id pub-id-type=\"doi\">10.1136/jmg.2005.033043</pub-id>\n<pub-id pub-id-type=\"pmid\">16118346</pub-id></mixed-citation></ref><ref id=\"B70\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Redin</surname><given-names>C.</given-names></name><name><surname>Gérard</surname><given-names>B.</given-names></name><name><surname>Lauer</surname><given-names>J.</given-names></name><name><surname>Herenger</surname><given-names>Y.</given-names></name><name><surname>Muller</surname><given-names>J.</given-names></name><name><surname>Quartier</surname><given-names>A.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Efficient strategy for the molecular diagnosis of intellectual disability using targeted high-throughput sequencing.</article-title>\n<source><italic>J. Med. Genet.</italic></source>\n<volume>51</volume>\n<fpage>724</fpage>–<lpage>736</lpage>. <pub-id pub-id-type=\"doi\">10.1136/jmedgenet-2014-102554</pub-id>\n<pub-id pub-id-type=\"pmid\">25167861</pub-id></mixed-citation></ref><ref id=\"B71\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rothwell</surname><given-names>P. E.</given-names></name><name><surname>Fuccillo</surname><given-names>M. V.</given-names></name><name><surname>Maxeiner</surname><given-names>S.</given-names></name><name><surname>Hayton</surname><given-names>S. J.</given-names></name><name><surname>Gokce</surname><given-names>O.</given-names></name><name><surname>Lim</surname><given-names>B. K.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Autism-associated neuroligin-3 mutations commonly impair striatal circuits to boost repetitive behaviors.</article-title>\n<source><italic>Cell</italic></source>\n<volume>158</volume>\n<fpage>198</fpage>–<lpage>212</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cell.2014.04.045</pub-id>\n<pub-id pub-id-type=\"pmid\">24995986</pub-id></mixed-citation></ref><ref id=\"B72\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sanders</surname><given-names>S. J.</given-names></name><name><surname>Murtha</surname><given-names>M. T.</given-names></name><name><surname>Gupta</surname><given-names>A. R.</given-names></name><name><surname>Murdoch</surname><given-names>J. D.</given-names></name><name><surname>Raubeson</surname><given-names>M. J.</given-names></name><name><surname>Willsey</surname><given-names>A. J.</given-names></name><etal/></person-group> (<year>2012</year>). <article-title>De novo mutations revealed by whole-exome sequencing are strongly associated with autism.</article-title>\n<source><italic>Nature</italic></source>\n<volume>485</volume>\n<fpage>237</fpage>–<lpage>241</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature10945</pub-id>\n<pub-id pub-id-type=\"pmid\">22495306</pub-id></mixed-citation></ref><ref id=\"B73\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Singh</surname><given-names>S. K.</given-names></name><name><surname>Eroglu</surname><given-names>C.</given-names></name></person-group> (<year>2013</year>). <article-title>Neuroligins provide molecular links between syndromic and nonsyndromic autism.</article-title>\n<source><italic>Sci. Signal.</italic></source>\n<volume>6</volume>:<issue>re4</issue>. <pub-id pub-id-type=\"doi\">10.1126/scisignal.2004102</pub-id>\n<pub-id pub-id-type=\"pmid\">23838185</pub-id></mixed-citation></ref><ref id=\"B74\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Skuse</surname><given-names>D. H.</given-names></name></person-group> (<year>2005</year>). <article-title>X-linked genes and mental functioning.</article-title>\n<source><italic>Hum. Mol. Genet.</italic></source>\n<volume>14</volume>\n<fpage>27</fpage>–<lpage>32</lpage>. <pub-id pub-id-type=\"doi\">10.1093/hmg/ddi112</pub-id>\n<pub-id pub-id-type=\"pmid\">15809269</pub-id></mixed-citation></ref><ref id=\"B75\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Südhof</surname><given-names>T. C.</given-names></name></person-group> (<year>2017</year>). <article-title>Synaptic neurexin complexes: a molecular code for the logic of neural circuits.</article-title>\n<source><italic>Cell</italic></source>\n<volume>171</volume>\n<fpage>745</fpage>–<lpage>769</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cell.2017.10.024</pub-id>\n<pub-id pub-id-type=\"pmid\">29100073</pub-id></mixed-citation></ref><ref id=\"B76\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Südhof</surname><given-names>T. C.</given-names></name></person-group> (<year>2018</year>). <article-title>Towards an understanding of synapse formation.</article-title>\n<source><italic>Neuron</italic></source>\n<volume>100</volume>\n<fpage>276</fpage>–<lpage>293</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuron.2018.09.040</pub-id>\n<pub-id pub-id-type=\"pmid\">30359597</pub-id></mixed-citation></ref><ref id=\"B77\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tabuchi</surname><given-names>K.</given-names></name><name><surname>Blundell</surname><given-names>J.</given-names></name><name><surname>Etherton</surname><given-names>M. R.</given-names></name><name><surname>Hammer</surname><given-names>R. E.</given-names></name><name><surname>Liu</surname><given-names>X.</given-names></name><name><surname>Powell</surname><given-names>C. M.</given-names></name><etal/></person-group> (<year>2007</year>). <article-title>A neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice.</article-title>\n<source><italic>Science</italic></source>\n<volume>318</volume>\n<fpage>71</fpage>–<lpage>76</lpage>. <pub-id pub-id-type=\"doi\">10.1126/science.1146221</pub-id>\n<pub-id pub-id-type=\"pmid\">17823315</pub-id></mixed-citation></ref><ref id=\"B78\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thomas</surname><given-names>N. S.</given-names></name><name><surname>Sharp</surname><given-names>A. J.</given-names></name><name><surname>Browne</surname><given-names>C. E.</given-names></name><name><surname>Skuse</surname><given-names>D.</given-names></name><name><surname>Hardie</surname><given-names>C.</given-names></name><name><surname>Dennis</surname><given-names>N. R.</given-names></name></person-group> (<year>1999</year>). <article-title>Xp deletions associated with autism in three females.</article-title>\n<source><italic>Hum. Genet.</italic></source>\n<volume>104</volume>\n<fpage>43</fpage>–<lpage>48</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s004390050908</pub-id>\n<pub-id pub-id-type=\"pmid\">10071191</pub-id></mixed-citation></ref><ref id=\"B79\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Trabzuni</surname><given-names>D.</given-names></name><name><surname>Ramasamy</surname><given-names>A.</given-names></name><name><surname>Imran</surname><given-names>S.</given-names></name><name><surname>Walker</surname><given-names>R.</given-names></name><name><surname>Smith</surname><given-names>C.</given-names></name><name><surname>Weale</surname><given-names>M. E.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>Widespread sex differences in gene expression and splicing in the adult human brain.</article-title>\n<source><italic>Nat. Commun.</italic></source>\n<volume>4</volume>:<issue>2771</issue>. <pub-id pub-id-type=\"doi\">10.1038/ncomms3771</pub-id>\n<pub-id pub-id-type=\"pmid\">24264146</pub-id></mixed-citation></ref><ref id=\"B80\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tukiainen</surname><given-names>T.</given-names></name><name><surname>Villani</surname><given-names>A. C.</given-names></name><name><surname>Yen</surname><given-names>A.</given-names></name><name><surname>Rivas</surname><given-names>M. A.</given-names></name><name><surname>Marshall</surname><given-names>J. L.</given-names></name><name><surname>Satija</surname><given-names>R.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Landscape of X chromosome inactivation across human tissues.</article-title>\n<source><italic>Nature</italic></source>\n<volume>550</volume>\n<fpage>244</fpage>–<lpage>248</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature24265</pub-id>\n<pub-id pub-id-type=\"pmid\">29022598</pub-id></mixed-citation></ref><ref id=\"B81\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Unichenko</surname><given-names>P.</given-names></name><name><surname>Yang</surname><given-names>J. W.</given-names></name><name><surname>Kirischuk</surname><given-names>S.</given-names></name><name><surname>Kolbaev</surname><given-names>S.</given-names></name><name><surname>Kilb</surname><given-names>W.</given-names></name><name><surname>Hammer</surname><given-names>M.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Autism related neuroligin-4 knockout impairs intracortical processing but not sensory inputs in mouse barrel cortex.</article-title>\n<source><italic>Cereb. Cortex</italic></source>\n<volume>28</volume>\n<fpage>2873</fpage>–<lpage>2886</lpage>. <pub-id pub-id-type=\"doi\">10.1093/cercor/bhx165</pub-id>\n<pub-id pub-id-type=\"pmid\">29106499</pub-id></mixed-citation></ref><ref id=\"B82\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Venkatesh</surname><given-names>H. S.</given-names></name><name><surname>Johung</surname><given-names>T. B.</given-names></name><name><surname>Caretti</surname><given-names>V.</given-names></name><name><surname>Noll</surname><given-names>A.</given-names></name><name><surname>Tang</surname><given-names>Y.</given-names></name><name><surname>Nagaraja</surname><given-names>S.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>Neuronal activity promotes glioma growth through neuroligin-3 secretion.</article-title>\n<source><italic>Cell</italic></source>\n<volume>161</volume>\n<fpage>803</fpage>–<lpage>816</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cell.2015.04.012</pub-id>\n<pub-id pub-id-type=\"pmid\">25913192</pub-id></mixed-citation></ref><ref id=\"B83\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Venkatesh</surname><given-names>H. S.</given-names></name><name><surname>Tam</surname><given-names>L. T.</given-names></name><name><surname>Woo</surname><given-names>P. J.</given-names></name><name><surname>Lennon</surname><given-names>J.</given-names></name><name><surname>Nagaraja</surname><given-names>S.</given-names></name><name><surname>Gillespie</surname><given-names>S. M.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Targeting neuronal activity-regulated neuroligin-3 dependency in high-grade glioma.</article-title>\n<source><italic>Nature</italic></source>\n<volume>549</volume>\n<fpage>533</fpage>–<lpage>537</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature24014</pub-id>\n<pub-id pub-id-type=\"pmid\">28959975</pub-id></mixed-citation></ref><ref id=\"B84\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Volaki</surname><given-names>K.</given-names></name><name><surname>Pampanos</surname><given-names>A.</given-names></name><name><surname>Kitsiou-Tzeli</surname><given-names>S.</given-names></name><name><surname>Vrettou</surname><given-names>C.</given-names></name><name><surname>Oikonomakis</surname><given-names>V.</given-names></name><name><surname>Sofocleous</surname><given-names>C.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>Mutation screening in the Greek population and evaluation of NLGN3 and NLGN4X genes causal factors for autism.</article-title>\n<source><italic>Psychiatr. Genet.</italic></source>\n<volume>23</volume>\n<fpage>198</fpage>–<lpage>203</lpage>. <pub-id pub-id-type=\"doi\">10.1097/YPG.0b013e3283643644</pub-id>\n<pub-id pub-id-type=\"pmid\">23851596</pub-id></mixed-citation></ref><ref id=\"B85\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>C.</given-names></name><name><surname>Ward</surname><given-names>M. E.</given-names></name><name><surname>Chen</surname><given-names>R.</given-names></name><name><surname>Liu</surname><given-names>K.</given-names></name><name><surname>Tracy</surname><given-names>T. E.</given-names></name><name><surname>Chen</surname><given-names>X.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Scalable production of iPSC-derived human neurons to identify tau-lowering compounds by high-content screening.</article-title>\n<source><italic>Stem Cell Rep.</italic></source>\n<volume>9</volume>\n<fpage>1221</fpage>–<lpage>1233</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.stemcr.2017.08.019</pub-id>\n<pub-id pub-id-type=\"pmid\">28966121</pub-id></mixed-citation></ref><ref id=\"B86\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>S.</given-names></name><name><surname>Mandell</surname><given-names>J. D.</given-names></name><name><surname>Kumar</surname><given-names>Y.</given-names></name><name><surname>Sun</surname><given-names>N.</given-names></name><name><surname>Morris</surname><given-names>M. T.</given-names></name><name><surname>Arbelaez</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>De novo sequence and copy number variants are strongly associated with tourette disorder and implicate cell polarity in pathogenesis.</article-title>\n<source><italic>Cell Rep.</italic></source>\n<volume>24</volume>\n<fpage>3441.e12</fpage>–<lpage>3454.e12</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.celrep.2018.08.082</pub-id>\n<pub-id pub-id-type=\"pmid\">30257206</pub-id></mixed-citation></ref><ref id=\"B87\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Werling</surname><given-names>D. M.</given-names></name><name><surname>Geschwind</surname><given-names>D. H.</given-names></name></person-group> (<year>2013</year>). <article-title>Understanding sex bias in autism spectrum disorder.</article-title>\n<source><italic>Curr. Opin. Neurol.</italic></source>\n<volume>26</volume>\n<fpage>146</fpage>–<lpage>153</lpage>. <pub-id pub-id-type=\"doi\">10.1097/WCO.0b013e32835ee548</pub-id>\n<pub-id pub-id-type=\"pmid\">23406909</pub-id></mixed-citation></ref><ref id=\"B88\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Werling</surname><given-names>D. M.</given-names></name><name><surname>Parikshak</surname><given-names>N. N.</given-names></name><name><surname>Geschwind</surname><given-names>D. H.</given-names></name></person-group> (<year>2016</year>). <article-title>Gene expression in human brain implicates sexually dimorphic pathways in autism spectrum disorders.</article-title>\n<source><italic>Nat. Commun.</italic></source>\n<volume>7</volume>:<issue>10717</issue>. <pub-id pub-id-type=\"doi\">10.1038/ncomms10717</pub-id>\n<pub-id pub-id-type=\"pmid\">26892004</pub-id></mixed-citation></ref><ref id=\"B89\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wise</surname><given-names>A. L.</given-names></name><name><surname>Gyi</surname><given-names>L.</given-names></name><name><surname>Manolio</surname><given-names>T. A.</given-names></name></person-group> (<year>2013</year>). <article-title>EXclusion: toward integrating the X chromosome in genome-wide association analyses.</article-title>\n<source><italic>Am. J. Hum. Genet.</italic></source>\n<volume>92</volume>\n<fpage>643</fpage>–<lpage>647</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ajhg.2013.03.017</pub-id>\n<pub-id pub-id-type=\"pmid\">23643377</pub-id></mixed-citation></ref><ref id=\"B90\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xu</surname><given-names>X.</given-names></name><name><surname>Xiong</surname><given-names>Z.</given-names></name><name><surname>Zhang</surname><given-names>L.</given-names></name><name><surname>Liu</surname><given-names>Y.</given-names></name><name><surname>Lu</surname><given-names>L.</given-names></name><name><surname>Peng</surname><given-names>Y.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Variations analysis of NLGN3 and NLGN4X gene in Chinese autism patients.</article-title>\n<source><italic>Mol. Biol. Rep.</italic></source>\n<volume>41</volume>\n<fpage>4133</fpage>–<lpage>4140</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s11033-014-3284-5</pub-id>\n<pub-id pub-id-type=\"pmid\">24570023</pub-id></mixed-citation></ref><ref id=\"B91\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yan</surname><given-names>J.</given-names></name><name><surname>Feng</surname><given-names>J.</given-names></name><name><surname>Schroer</surname><given-names>R.</given-names></name><name><surname>Li</surname><given-names>W.</given-names></name><name><surname>Skinner</surname><given-names>C.</given-names></name><name><surname>Schwartz</surname><given-names>C. E.</given-names></name><etal/></person-group> (<year>2008</year>). <article-title>Analysis of the neuroligin 4Y gene in patients with autism.</article-title>\n<source><italic>Psychiatr. Genet.</italic></source>\n<volume>18</volume>\n<fpage>204</fpage>–<lpage>207</lpage>. <pub-id pub-id-type=\"doi\">10.1097/YPG.0b013e3282fb7fe6</pub-id>\n<pub-id pub-id-type=\"pmid\">18628683</pub-id></mixed-citation></ref><ref id=\"B92\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yan</surname><given-names>J.</given-names></name><name><surname>Oliveira</surname><given-names>G.</given-names></name><name><surname>Coutinho</surname><given-names>A.</given-names></name><name><surname>Yang</surname><given-names>C.</given-names></name><name><surname>Feng</surname><given-names>J.</given-names></name><name><surname>Katz</surname><given-names>C.</given-names></name><etal/></person-group> (<year>2005</year>). <article-title>Analysis of the neuroligin 3 and 4 genes in autism and other neuropsychiatric patients.</article-title>\n<source><italic>Mol. Psychiatry</italic></source>\n<volume>10</volume>\n<fpage>329</fpage>–<lpage>332</lpage>. <pub-id pub-id-type=\"doi\">10.1038/sj.mp.4001629</pub-id>\n<pub-id pub-id-type=\"pmid\">15622415</pub-id></mixed-citation></ref><ref id=\"B93\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>T. W.</given-names></name><name><surname>Chahrour</surname><given-names>M. H.</given-names></name><name><surname>Coulter</surname><given-names>M. E.</given-names></name><name><surname>Jiralerspong</surname><given-names>S.</given-names></name><name><surname>Okamura-Ikeda</surname><given-names>K.</given-names></name><name><surname>Ataman</surname><given-names>B.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>Using whole-exome sequencing to identify inherited causes of autism.</article-title>\n<source><italic>Neuron</italic></source>\n<volume>77</volume>\n<fpage>259</fpage>–<lpage>273</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuron.2012.11.002</pub-id>\n<pub-id pub-id-type=\"pmid\">23352163</pub-id></mixed-citation></ref><ref id=\"B94\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yuen</surname><given-names>R. K. C.</given-names></name><name><surname>Merico</surname><given-names>D.</given-names></name><name><surname>Bookman</surname><given-names>M.</given-names></name><name><surname>Howe</surname><given-names>J. L.</given-names></name><name><surname>Thiruvahindrapuram</surname><given-names>B.</given-names></name><name><surname>Patel</surname><given-names>R. V.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Whole genome sequencing resource identifies 18 new candidate genes for autism spectrum disorder.</article-title>\n<source><italic>Nat. Neurosci.</italic></source>\n<volume>20</volume>\n<fpage>602</fpage>–<lpage>611</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nn.4524</pub-id>\n<pub-id pub-id-type=\"pmid\">28263302</pub-id></mixed-citation></ref><ref id=\"B95\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>B.</given-names></name><name><surname>Gokce</surname><given-names>O.</given-names></name><name><surname>Hale</surname><given-names>W. D.</given-names></name><name><surname>Brose</surname><given-names>N.</given-names></name><name><surname>Südhof</surname><given-names>T. C.</given-names></name></person-group> (<year>2018</year>). <article-title>Autism-associated neuroligin-4 mutation selectively impairs glycinergic synaptic transmission in mouse brainstem synapses.</article-title>\n<source><italic>J. Exp. Med.</italic></source>\n<volume>215</volume>\n<fpage>1543</fpage>–<lpage>1553</lpage>. <pub-id pub-id-type=\"doi\">10.1084/jem.20172162</pub-id>\n<pub-id pub-id-type=\"pmid\">29724786</pub-id></mixed-citation></ref><ref id=\"B96\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>B.</given-names></name><name><surname>Seigneur</surname><given-names>E.</given-names></name><name><surname>Wei</surname><given-names>P.</given-names></name><name><surname>Gokce</surname><given-names>O.</given-names></name><name><surname>Morgan</surname><given-names>J.</given-names></name><name><surname>Südhof</surname><given-names>T. C.</given-names></name></person-group> (<year>2017</year>). <article-title>Developmental plasticity shapes synaptic phenotypes of autism-associated neuroligin-3 mutations in the calyx of Held.</article-title>\n<source><italic>Mol. Psychiatry</italic></source>\n<volume>22</volume>\n<fpage>1483</fpage>–<lpage>1491</lpage>. <pub-id pub-id-type=\"doi\">10.1038/mp.2016.157</pub-id>\n<pub-id pub-id-type=\"pmid\">27725662</pub-id></mixed-citation></ref><ref id=\"B97\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>C.</given-names></name><name><surname>Milunsky</surname><given-names>J. M.</given-names></name><name><surname>Newton</surname><given-names>S.</given-names></name><name><surname>Ko</surname><given-names>J.</given-names></name><name><surname>Zhao</surname><given-names>G.</given-names></name><name><surname>Maher</surname><given-names>T. A.</given-names></name><etal/></person-group> (<year>2009</year>). <article-title>A neuroligin-4 missense mutation associated with autism impairs neuroligin-4 folding and endoplasmic reticulum export.</article-title>\n<source><italic>J. Neurosci.</italic></source>\n<volume>29</volume>\n<fpage>10843</fpage>–<lpage>10854</lpage>. <pub-id pub-id-type=\"doi\">10.1523/JNEUROSCI.1248-09.2009</pub-id>\n<pub-id pub-id-type=\"pmid\">19726642</pub-id></mixed-citation></ref><ref id=\"B98\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>Y.</given-names></name><name><surname>Pak</surname><given-names>C. H.</given-names></name><name><surname>Han</surname><given-names>Y.</given-names></name><name><surname>Ahlenius</surname><given-names>H.</given-names></name><name><surname>Zhang</surname><given-names>Z.</given-names></name><name><surname>Chanda</surname><given-names>S.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>Rapid single-step induction of functional neurons from human pluripotent stem cells.</article-title>\n<source><italic>Neuron</italic></source>\n<volume>78</volume>\n<fpage>785</fpage>–<lpage>798</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuron.2013.05.029</pub-id>\n<pub-id pub-id-type=\"pmid\">23764284</pub-id></mixed-citation></ref><ref id=\"B99\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zinn</surname><given-names>A. R.</given-names></name><name><surname>Roeltgen</surname><given-names>D.</given-names></name><name><surname>Stefanatos</surname><given-names>G.</given-names></name><name><surname>Ramos</surname><given-names>P.</given-names></name><name><surname>Elder</surname><given-names>F. F.</given-names></name><name><surname>Kushner</surname><given-names>H.</given-names></name><etal/></person-group> (<year>2007</year>). <article-title>A Turner syndrome neurocognitive phenotype maps to Xp22.3.</article-title>\n<source><italic>Behav. Brain Funct.</italic></source>\n<volume>3</volume>:<issue>24</issue>. <pub-id pub-id-type=\"doi\">10.1186/1744-9081-3-24</pub-id>\n<pub-id pub-id-type=\"pmid\">17517138</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Immunol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Immunol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Immunol.</journal-id><journal-title-group><journal-title>Frontiers in Immunology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-3224</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849644</article-id><article-id pub-id-type=\"pmc\">PMC7431522</article-id><article-id pub-id-type=\"doi\">10.3389/fimmu.2020.01794</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Immunology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>The Generation of an Engineered Interleukin-10 Protein With Improved Stability and Biological Function</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Minshawi</surname><given-names>Faisal</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/944821/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Lanvermann</surname><given-names>Sebastian</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>McKenzie</surname><given-names>Edward</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Jeffery</surname><given-names>Rebecca</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1042313/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Couper</surname><given-names>Kevin</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/176713/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Papoutsopoulou</surname><given-names>Stamatia</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/736852/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Roers</surname><given-names>Axel</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Muller</surname><given-names>Werner</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/31897/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Umm Al-Qura University</institution>, <addr-line>Makkah</addr-line>, <country>Saudi Arabia</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Lydia Becker Institute of Immunology and Inflammation, University of Manchester</institution>, <addr-line>Manchester</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Centre for Translational Medicine, Thomas Jefferson University</institution>, <addr-line>Philadelphia, PA</addr-line>, <country>United States</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Manchester Institute of Biotechnology, Faculty of Science and Engineering, University of Manchester</institution>, <addr-line>Manchester</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Department of Cellular and Molecular Physiology, University of Liverpool</institution>, <addr-line>Liverpool</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff6\"><sup>6</sup><institution>Institute of Immunology, Medical Faculty Carl Gustav Carus, University of Technology Dresden</institution>, <addr-line>Dresden</addr-line>, <country>Germany</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Fulvio D'Acquisto, University of Roehampton London, United Kingdom</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Krishna Rajarathnam, University of Texas Medical Branch at Galveston, United States; Maria Laura Belladonna, University of Perugia, Italy</p></fn><corresp id=\"c001\">*Correspondence: Faisal Minshawi <email>fominshawi@uqu.edu.sa</email></corresp><corresp id=\"c002\">Werner Muller <email>Werner.Muller@manchester.ac.uk</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Inflammation, a section of the journal Frontiers in Immunology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1794</elocation-id><history><date date-type=\"received\"><day>14</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>06</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Minshawi, Lanvermann, McKenzie, Jeffery, Couper, Papoutsopoulou, Roers and Muller.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Minshawi, Lanvermann, McKenzie, Jeffery, Couper, Papoutsopoulou, Roers and Muller</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Interleukin-10 (IL-10) is an immunoregulatory cytokine that plays a pivotal role in modulating inflammation. IL-10 has inhibitory effects on proinflammatory cytokine production and function <italic>in vitro</italic> and <italic>in vivo</italic>; as such, IL-10 is viewed as a potential treatment for various inflammatory diseases. However, a significant drawback of using IL-10 in clinical application is the fact that the biologically active form of IL-10 is an unstable homodimer, which has a short half-life and is easily degraded <italic>in vivo</italic>. Consequently, IL-10 therapy using recombinant native IL-10 has had only limited success in the treatment of human disease. To improve the therapeutic potential of IL-10, we have generated a novel form of IL-10, which consists of two IL-10 monomer subunits linked in a head to tail fashion by a flexible linker. We show that the linker length <italic>per se</italic> did not affect the expression and biological activity of the stable IL-10 molecule, which was more active than natural IL-10, both <italic>in vitro</italic> and <italic>in vivo</italic>. We confirmed that the new form of IL-10 had a much-improved temperature- and pH-dependent biological stability compared to natural IL-10. The IL-10 dimer protein binds to the IL-10 receptor similarly to the natural IL-10 protein, as shown by antibody blocking and through the genetic modifications of one monomer in the IL-10 dimer specifically at the IL-10 receptor binding site. Finally, we showed that stable IL-10 is more effective at suppressing LPS-induced-inflammation <italic>in vivo</italic> compared to the natural IL-10. In conclusion, we have developed a new stable dimer version of the IL-10 protein with improved stability and efficacy to suppress inflammation. We propose that this novel stable IL-10 dimer could serve as the basis for the development of targeted anti-inflammatory drugs.</p></abstract><kwd-group><kwd>interleukin-10</kwd><kwd>immunoregulation</kwd><kwd>inflammation</kwd><kwd>cytokine</kwd><kwd>covalent linker</kwd><kwd>stable dimer</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Medical Research Council<named-content content-type=\"fundref-id\">10.13039/501100000265</named-content></funding-source><award-id rid=\"cn001\">MR/R010099/1</award-id></award-group></funding-group><counts><fig-count count=\"9\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"42\"/><page-count count=\"18\"/><word-count count=\"10509\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>IL-10 is a pleiotropic cytokine that is produced by different cell types, including myeloid cells (dendritic cells, macrophages, eosinophils, neutrophils, and mast cells) and lymphoid cells (NK, B cells, and T cells) with broad anti-inflammatory activity. Macrophages and myeloid dendritic cells express IL-10 upon activation of MyD88 and TRIF-dependent TLR pathways such as TLR3 and TLR4, by stimulation with dsDNA and LPS, respectively (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Moreover, tolerogenic dendritic cells (CD11c<sup>low</sup>CD45RB<sup>high</sup>) produce large amounts of IL-10 in response to LPS, which induces T regulatory cell development (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B3\" ref-type=\"bibr\">3</xref>). Natural regulatory cells (nTreg) produce IL-10 in the response to IL-2, which is vital for immune homeostasis (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>).</p><p>Structurally, IL-10 belongs to the class II cytokine family, which involves IL-19, IL-20, IL-22, IL-24 (Mda-7), IL-26, and interferons (IFN-α, -β, and -γ) (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). The IL-10 is a member of helical cytokines as an IL-10 monomer consists of six α-helices (A-F) (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). Biologically active IL-10 is a non-covalent homodimer, which is described as a three-dimensional (3D) domain swapping protein with a molecular mass of 37 kDa (18.5 kDa for each one) (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>–<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). The IL-10 receptor (IL-10R) is a member of the class II cytokine receptor family and consists of two subunits, IL-10R1 and IL-10R2 (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B11\" ref-type=\"bibr\">11</xref>). IL-10 binds to IL-10R with high affinity; however, it can be species-specific. For example, mouse IL-10 was able to block the binding of human IL-10 to mouse cells but not human cells (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Analysis of the protein crystal formed of IL-10 bound to soluble IL-10R1 revealed that the 3D domain-swapped homodimer IL-10, which consists of helices E and F from one chain inserted into the hydrophobic cleft formed by into helices A–D of the other chain, is essential for receptor-binding (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). IL-10 binding to the IL-10R complex activates a Janus kinase- Signal Transducers and Activation Transcription system (JAK/STAT) signaling pathway. IL-10/IL-10R promotes phosphorylation and activation of the transcription factor STAT3, which is required for the IL-10 immunoregulatory effect (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>).</p><p>The anti-inflammatory activity of IL-10 is in part due to the inhibition of the synthesis of pro-inflammatory cytokines such as tumor necrosis factor (TNF) (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). Furthermore, IL-10 downregulates MHC class II expression and helps to promote wound healing (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>). The IL-10 showed to have an immunoregulatory effect during an infection with <italic>Toxoplasma gondii</italic> (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>), <italic>Mycobacterium</italic> spp. (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>), <italic>Herpes simplex virus</italic> (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>), and malaria (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>) by ameliorating the exaggerated T helper 1 and CD8<sup>+</sup> T cells response including. A defect of IL-10 or the IL-10 receptor has been linked to excessive immune reactions and a disposition to chronic inflammatory disease, such as the early onset of inflammatory bowel disease (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>–<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). Also, changes of the gut microflora could lead to a change in the regulation of the gut-associated immune system, resulting in chronic gut inflammation, which in part could be the result of dysregulated IL-10 expression (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>, <xref rid=\"B24\" ref-type=\"bibr\">24</xref>).</p><p>Here we report the generation of a new form of IL-10 more suitable for therapeutic intervention, as the natural IL-10 has only a short half-life <italic>in vivo</italic>. The stability of the non-covalent IL-10 dimer strongly depends on physical parameters such as temperature and pH. The IL-10 dimer dissociates to a monomeric form at low protein concentrations or at acidic pHs, as typically found in inflamed tissue. Acidic pH has been found, for instance, in fracture-related hematomas (ranging as low as pH 4.7), in cardiac ischemia (pH 5.7) (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Examinations of inflamed skin showed pH values of 5.8–7.2 (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>–<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). It has been demonstrated that ~10 and ~50% of human IL-10 was dissociated (i.e., decayed) when heated to 37 and 55°C, respectively, for 1 h (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). Moreover, to date, the free IL-10 monomer has not been found in the solution. The IL-10 monomer could not exist in solution due to the presence of significant hydrophobic residues, which are shielded by interaction involved in the dimer form (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). Therefore, we reasoned that the generation of a stabilized dimer form of IL-10 might be a promising approach to overcome the inherent IL-10 instability and thereby improve its therapeutic potential.</p><p>One way to improve the stability of the IL-10 structure has been proposed by generating a stable IL-10 dimer using an internal flexible linker (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). Expressing the dimer as a single continuous fusion protein in which the monomers are connected by a flexible linkers (Gly-Ser) may offer a stability advantage and improve biological activity (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). Moreover, Gly-Ser linkers in recombinant proteins could play a general role in overall stability and solubility. A previous study has shown that using a Gly-rich linker to tether the dimeric forms of HIV-1 proteases (HIV-PR) results in a more stable form compared to the natural HIV-PR at pH 7.0 (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Moreover, Foss <italic>et al</italic>. showed that using flexible linkers (Gly-rich linker) in transthyretin, the carrier of the thyroid hormone, is more stable than the natural after urea treatment (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). We now generated a functional IL-10 by linking two monomers using a flexible linker. We examined and compared the biological activity of the stable IL-10 dimer against the natural or non-covalently linked IL-10, both <italic>in vitro and in vivo</italic>. Here, we demonstrate that novel recombinant IL-10 dimer has improved stability and higher biological activity; therefore, it has the potential to be a building block for future IL-10 based immunotherapy treatment regimes.</p></sec><sec sec-type=\"methods\" id=\"s2\"><title>Methods</title><sec><title>Plasmid Design and IL-10 Construct</title><p>The eukaryotic expression vector pCEP V19 was used to express the cDNA of natural mouse IL-10 (Nm) and stable mouse IL-10 (STm). The C-terminus of pCEP V19 includes Factor XA (FXA), human serum albumin (HSA), thrombin. oriP: replication origin of Epstein-Barr virus, EBNA-1: Epstein-Barr virus nuclear antigen-1, ampicillin: ampicillin resistance gene (β-lactamase), pUC ori: the bacterial origin of replication, puromycin: puromycin resistance gene, CMV: Promoter of cytomegalovirus, BM40: a signal sequence of protein BM40, 8 His tag: 8 histidine residues, thrombin: thrombin, NheI, Bsu36I, BamHI: restriction sites, mouse IL-10 cDNA, SV40 pA: polyadenylation signal of SV40 Virus. The expression vector was generated and provided to us by Manuel Koch (Institute of Biochemistry II, University of Cologne). The nine amino acids (-G<sub>3</sub>SG<sub>4</sub>S-) and 13 amino acid linker(-G<sub>3</sub>SG<sub>4</sub>-SG<sub>4</sub>-) were generated by inserting the synthetic insert into the second monomer of mouse IL-10. The stable IL-10 cDNA was digested with the BamHI enzyme before ligating the synthetic inserts containing a nine and 13 amino acid linker with 5′ BamHI restriction site and 3′ BglII restriction sites. The confirmation of the correct insert orientation was carried out using NheI and BamHI restriction digestion. The stable human IL-10 (STh), and mutant cDNAs of mouse IL-10 were synthesized (GeneArt Gene Synthesis: Thermofisher). Oligonucleotides were cloned into pMA-RQ, Col E1 origin: the bacterial origin of replication, ampicillin: ampicillin resistance gene (β-lactamase) (Thermofisher), then subcloned into the eukaryotic expression vector pCEP V19.</p></sec><sec><title>Transfection of HEK<sub>293</sub>EBNA, IL-10 Expression, and Purification</title><p>HEK<sub>293</sub>EBNA (HEK) mammalian cell line was generated from human embryonic kidney cells. The cell line was maintained in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L glucose, 10% fetal bovine serum (FBS) (Life Technologies Ltd), 1% L-Glutamine, and 1% Penicillin-Streptomycin (Pen-Strep) (Sigma). HEK cells carry the Epstein-Barr Virus Nuclear Antigen-1 (EBNA-1) gene and allow for improved protein production since plasmids replicate competently in positive cells. HEK cells were transfected using Lipofectamine 3000 (Invitrogen). HEK cells were first seeded at 1 × 10<sup>6</sup> cells/well in a 6-well plate. Upon reaching a confluence of ~80–90%, 5 μg of the plasmid DNA was mixed with different volumes of lipofectamine 3000 (3.5 and 7.5 μl) in 125 μl of Opti-MEM™ (Thermofisher), and the mixture was incubated for 15 min at room temperature. Afterward, the lipo-DNA mixture was added dropwise onto the cells. After 24 h, the cells were washed once with PBS, and selection media containing puromycin (Gibco) was added (DMEM, 10% FBS, 1% Pen-Strep, 1% of L-Glutamine, (2 μg/mL) and incubated for 3 days. The cells were then washed with PBS before adding fresh medium minus puromycin. The supernatant was harvested and stored at −20°C until further use. The transfected cell supernatant was loaded at 4°C onto a HisTrap HP column (GE Healthcare). The column was then washed with 10 CV of binding buffer, followed by stepwise elution of the protein with increasing imidazole concentration (50, 100, 250, and 500 mM) in binding buffer. Protein content and purity of each fraction were visualized by Coomassie staining. Positive fractions were pooled and dialyzed at 4°C against PBS.</p></sec><sec><title>Animal Models</title><p>Cells derived from mice that were used in the <italic>in vitro</italic> experiments were housed at the University of Manchester Biological Services Facility (BSF) under specific-pathogen-free conditions. They had easy access to food and water on a 12/12-h light cycle. All breeding mice were routinely screened (3 monthly or annually where applicable) according to BSF recommendations. The mouse strains (hTNF.LucBAC and C57BL/6) were bred in this study under a Home Office project license (70/7800) (P8829D3B4) in agreement with the Animal (Scientific Procedures) Act 1986. The C57BL/6 mice were ordered from Charles River (Charles River Laboratories, Inc., Harlow, UK). The <italic>in vivo</italic> experiments were performed at the University of Cologne, Germany, under animal experimental license 24-9168.11-1/2009-22.</p></sec><sec><title>Purification of Bone Marrow-Derived Macrophages</title><p>Mouse bone marrow-derived macrophages cells (BMDMs) were isolated, as described previously (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). Briefly, bone marrow-cells were dissected from femurs and tibiae and plated into a complete media (RPMI 1640 medium (Sigma) supplemented with 10% v/v FBS (Life Technologies Ltd), 100 IU/mL penicillin 100 μg/mL streptomycin (Sigma), 50 ng/mL mouse colony-stimulating factor (MCSF) (Promega), and 50 μM β-mercaptoethanol) (Sigma) at 5 × 10<sup>6</sup> cells per 90 mm bacterial petri dish (Sterilin, UK) for 4 days. On day 4, 10 mL of complete media was added and incubated for 3 days. Adherent cells were then harvested with 5 mL of PBS supplemented with 5% v/v FBS and 2.5 mM EDTA. For splenocyte isolation, the spleen was homogenized and filtered through nylon mesh filters (70 μM; Becton Dickinson, UK) to generate a single-cell suspension. RBCs were lysed with ammonium chloride potassium (ACK) lysis buffer before the cell pellet was resuspended in DMEM medium supplemented with 10% v/v/FBS, 1% w/v/Penicillin/Streptomycin, 1 mM glutamine and 50 μM β-mercaptoethanol.</p></sec><sec><title>Cell-Based Luciferase Reporter Assay</title><p>The cell-based luciferase reporter assay has been previously described (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). BMDMs were plated in 96 well plates (OptiPlate-96, White Opaque 96-well Microplate; Perkin Elmer, UK) at 1 × 10<sup>5</sup>/well in 0.1 mL medium containing 1 mM luciferin (Promega) and left to rest overnight. Cells were stimulated with LPS (<italic>Salmonella enterica</italic> serovar Minnesota R595; Alexis Biochemicals, UK) (10 ng/mL) alone or in the presence of commercial mouse IL-10 mouse (Protech), natural or stable IL-10 proteins. The anti-IL-10R antibody (clone: 1B1.3a) (Biolegend UK Ltd) was used to validate that the alteration in the luciferase response observed was dependent on the IL-10 receptor's engagement with IL-10. Unstimulated cells were used as a negative control. The luciferase activity was measured over time in a CO<sub>2</sub> Lumistar Omega luminometer (BMG Labtech, UK).</p></sec><sec><title>Temperature- and pH-Dependent Stability Study</title><p>Stability experiments were performed as previously described (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>), with the biological activity of IL-10 being monitored by luciferase assay. Briefly, 0.1μg/mL of IL-10 sample was incubated at 55°C in time-course (to 30 min). The pre-heated IL-10 sample was added together with LPS on hTNF.LucBAC BMDMs and the luciferase activity was monitored over time. The pH-effect on the biological stability was determined by first pre-incubating a 0.1 μg/mL sample in different pH buffers (sodium citrate pH 2.5, sodium phosphate pH 5.5–6.5 and TRIS-base pH 8.0–10.0) for 24 h at 4°C followed by buffer exchange with PBS using a spin Desalting column (Thermofisher). Protein was diluted to a final concentration in each experiment of 10 ng/mL before testing for biological activity.</p></sec><sec><title>Enzyme-Linked Immunosorbent Assay (ELISA) for IL-10</title><p>The recombinant fusion mouse and human IL-10 proteins were detected after purification using Ready Set Go ELISA kits (Cat mIL-10 50-112 eBioscience, UK, Cat hIL-10 88-7106) according to the manufacturer instructions. Briefly, 96-well flat-bottom high-affinity ELISA plates were coated overnight at 4°C with the capture antibody. Plates were washed three times with washing buffer (0.05% Tween 20 PBS) before the addition of blocking buffer to each well with 1X ELISA Diluent (supplied) for 1 h. Standards were prepared and added in 2-fold serial dilutions (4,000–31.25 pg/mL) after washing the plate three times as above. The recombinant protein was diluted 100-fold before serial dilution (1/2) were incubated for 2 h at RT. The detection antibody was added and incubated for 1 h after washing three times with washing buffer as above. Plates were further rewashed three times before incubation with streptavidin-horseradish peroxidase (HRP) for 30 min at RT. Plates were then washed five times with substrate solution, Tetramethylbenzidine (TMB). After incubation for another 15–30 min at RT in the dark, stop solution (2N H<sub>2</sub>SO<sub>4</sub>) was added (25 μl) to each well. Optical density was measured using Versa-Max ELISA Microplate Reader with 450 filter.</p></sec><sec><title>Western Blot</title><p>Purified IL-10 was detected by western blot using the anti-mouse IL-10 (JES5-2A5) (eBiosciences). For Splenocytes (5 × 10<sup>6</sup>) were either left unstimulated or were stimulated with either Nm or STm for 24 h After 24 h cells were washed three times with cold PBS and lysed in 0.5 mL of RIPA Buffer (Sigma) containing 5 ul of Protease Inhibitor Cocktail (Sigma) and incubated on ice for 5 min. Samples were then centrifuged at 5,000 × g for 5 min, and the supernatants stored at −20°C. A nanodrop machine was used to determine the concentration of the protein (absorbance at 280 nm) for each sample. The total protein concentration was adjusted in all samples with the addition of a RIPA lysis buffer.</p><p>Protein samples were loaded onto 4–12% BIS-Tris Gels (Invitrogen) using running buffer MED SDS (Invitrogen). 24 μl of sample and 6 μl of SDS Sample Buffer (4X) (Thermofisher) were mixed and heated at 80°C for 5 min. 10 μl of electrophoresis marker (Sigma) was used to determine the molecular size. The gel was run at 100 V until the tracking dye reached the bottom of the gel. The gel was removed from the gel cassettes and placed in the nitrocellulose membrane (Bio-Rad) and blotted using the Trans-Blot Turbo Transfer System (Bio-Rad). The blot was then incubated with blocking solution (5% w/v milk in TBS-Tween) for 90 min at room temperature; before incubation with a primary antibody: anti-rat IL-10 (JES5-2A5) (eBiosciences), anti-mouse total STAT3 (Cell signaling), and anti-rabbit p-STAT3(Cell signaling) in 1:1000 in 5% w/v milk in TBS-Tween overnight at 4°C. After that, the blots were washed four times with TBS-Tween for 20 min; before incubated with the secondary antibody (horseradish-peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit (Cell signaling) at 1:2000 in 5% w/v milk in TBS-Tween for 60 min. The signals were developed using a Western Blot Chemiluminescent Substrate (ECL western blotting substrate (Cell signaling).</p></sec><sec><title><italic>In vivo</italic> Experiments</title><p>Treatment of LPS-induced inflammation of the skin with IL-10: On three consecutive days, 10 μg each of LPS in a volume of 50 μl, with or without varying amounts of Nm or STm, were injected into the flank at the same site. On the 5th day, the tissue surrounding the injection site became removed en bloc and hematoxylin and eosin (H & E) stained. The tissue in 4% formalin was cut to 5 microns thickness using a microtome. After drying the sections onto slides, the deparaffinization and rehydration stages were carried out. The preparations were subsequently stained with Mayer's hemalum solution (Sigma) and eosin (Sigma), dehydrated again, and covered with entellan (Merck). When viewed in the light microsphere, the basic cytoplasm, elastin, and collagen appeared red-orange, the nuclei dark blue, and erythrocytes yellow-orange.</p><p>Treatment of Endotoxin Shock by IL-10: 100 μl PBS containing 25 μg LPS (serotype 055: B5) was injected retro-orbitally (i.v) in C57BL/6 mice (8–12 weeks) to indicate the optimum time for TNF synthesis after LPS treatment. To test the efficacy of IL-10 in TNF suppression, C57BL/6 mice were pre-treated with different amounts of either PBS, Nm or STm (2 μg each) for 30 min before injecting LPS (10 μg) At the indicated time points (1.5, 3, or 6 h), blood was taken retro-orbitally, and ELISA used to determine the TNF serum concentration. IL-10<sup>−/−</sup> mice were given 10 μg LPS together with increasing concentrations of Nm or STm being injected intravenously. After 3 h, blood was withdrawn, and the IL 6 serum concentration determined by ELISA.</p></sec><sec><title>Statistical Analysis</title><p>Results were represented as the mean ± standard error of the mean (SEM). Following assessment for normality and equality of variances, statistical inferences on data were performed using one-way, or two-way analysis of variance (ANOVA) followed by unpaired comparisons of treatment means using Dunnett's <italic>post-hoc</italic> test (LPS-treated or vs. LPS+IL-10) used in the luciferase inhibition assay and (untreated or vs. treated) used in the stability study. Differences were considered statistically significant when <italic>p</italic> < 0.05. Luciferase activity represented as area under the curve (AUC) Statistical analyses were performed using GraphPad Prism-7 Software Statistical Package, La Jolla CA; the USA.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>Generation, Expression, and Purification of a Stable IL-10 Protein Using a Mammalian Expression System</title><p>The crystal structure of the IL-10 dimer shows that the C-terminus of one monomer is in close proximity to the <italic>N</italic> terminus of the second monomer due to 3D domain swapping with the antiparallel association. This suggested that a stabilized dimer could be generated by linking these two termini. A new recombinant stable IL-10 was generated by cloning two copies of the same IL-10 cDNA in tandem as a continuous polypeptide in the same orientation separated by 7 amino acids linkers (-G<sub>3</sub>SG<sub>3</sub>-) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). Because glycine-serine linkers do not form α-helices and have no reactive side chains (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>), they are often used for a flexible and neutral connection of protein domains. Importantly, molecular dynamics simulation (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>) suggested that the linker would not interfere with the secondary structure of the monomers or IL-10 receptor binding site. In this report, we name the natural IL-10 (non-covalently linked) from mouse as Nm and human as Nh; besides, we name the stable IL-10 dimer from the mouse as STm and human as STh.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Expression and ELISA measurement of natural and stable mouse IL-10. <bold>(A)</bold> The cDNA construct of both natural mouse IL-10 (Nm) and stable mouse IL-10 (STm). The cartoon illustration of the expected protein folding of both Nm and STm versions. <bold>(B)</bold> The ribbon plot of the natural IL-10 homo-dimer with <italic>N</italic> and <italic>C</italic> terminus obtained from the previous study (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>) and the molecular dynamics simulation of stable IL-10. The basis for this simulation is the crystal structure of the hIL-10 dimer (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>) (PDB-ID: 2ILK). After about 2 ns of simulated time, the state of the system shown here was found. The peptide linker (-G3SG3-) is colored red. The molecular simulation of stable IL-10 was carried out by Jan-Philip Gehrcke (Biotechnology Center Dresden). <bold>(C)</bold> Both Nm and STm were stained with Coomassie Blue under reducing condition in SDS-page and <bold>(D)</bold> detected by IL-10 antibody in Western Blot. <bold>(E)</bold> Nm (black line) and STm proteins (blue line) were detected ELISA (1/2 dilutions). All data are representative of triplicate wells, and the bars represent standard error of the mean.</p></caption><graphic xlink:href=\"fimmu-11-01794-g0001\"/></fig><p>Both STm and Nm were generated from HEK cells with a His-tagged pCEP V19 expression vector. Recombinant Nm and STm proteins from parental vector-transfected cells were subjected to purification by an N-terminal His-tag purification column and analysis by SDS-PAGE and Western blotting. Under reducing conditions, the Nm migrated as a monomeric band in the region of 23 kDa, while the STm as a stable dimer migrated as a dimer band of ~41 kDa in SDS-PAGE (<xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref>). This result corresponds to the calculated molecular weights of 18.7 for IL-10 monomers and 37.3 kDa for IL-10 dimers, respectively (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). Both proteins were also detected using a specific IL-10 antibody (clone: JES5-2A5) in the western blot with different migration profiles due to the monomeric and dimeric forms (<xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>). Finally, both STm and Nm were detected by IL-10-ELISA to determine the recombinant IL-10 protein concentrations (<xref ref-type=\"fig\" rid=\"F1\">Figure 1E</xref>) with a commercially sourced IL-10 protein (CmIL-10) (PeproTech) being used as an internal standard.</p></sec><sec><title>The Biological Activity of Stabilized Mouse IL-10 Dimer <italic>in vitro</italic></title><p>Different approaches were used to measure the biological activities of the various versions of the IL-10 proteins we generated. First, we determined IL-10 activity by detection of STAT3 phosphorylation in IL-10 treated lymphocytes. To demonstrate proof of concept, spleen cells from wild-type, IL-10<sup>−/−</sup>, and IL-10R1<sup>−/−</sup> mice were prepared and stimulated with the purified IL-10 proteins (5 ng/mL). Phosphorylated STAT3 (p-STAT3) (75 kDa) was detected by western blot analysis of cell lysates. Both STm and Nm induced p-STAT3 in murine wild-type and IL-10<sup>−/−</sup> but not in IL-10R1<sup>−/−</sup> receptor-deficient cells (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>), demonstrating that the STm IL-10 uses the IL-10R for signaling.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Stable mouse IL-10 (STm) is biologically active <italic>in vitro</italic>. <bold>(A)</bold> Specific induction of STAT3 phosphorylation by STm (5 ng/mL) was verified by the use of spleen cells from different mouse lines: Wild type C57BL/6 (WT), IL-10 knockout (IL-10<sup>−/−</sup>) and IL-10 receptor knockout (IL-10R1<sup>−/−</sup>). <bold>(B)</bold> Luciferase activity was monitored as the area under the curve (AUC) from BMDMs reporter mouse; cells were either unstimulated or stimulated with LPS (10 ng/mL) in the presence or absence of IL-10 from HEK<sub>293</sub> EBNA supernatants. A commercial mouse IL-10 (CmIL-10) was used as a positive control, and un-transfected cells supernatants (Medium) served as a negative control. Significant difference considered by comparing to LPS stimulated cells as follows: not significant (ns), ***<italic>p</italic> < 0.001; ANOVA. <bold>(C)</bold> The dose-response effect of IL-10 on LPS-treated BMDMs of the transgenic mouse: the ED<sub>50</sub> of Nm (black line) and STm (blue line) was calculated as 0.17 and 0.04 ng/mL, respectively. The maximum luciferase induction was determined by treating the BMDMs of the transgenic mouse with LPS alone at 10 ng/mL as it showed a black dot line. <bold>(D)</bold> Soluble mouse TNF was measured from the medium 24 h from BMDMs of the transgenic mouse either after LPS stimulation alone or after co-treating with either Nm (black line) or STm (blue line) in a dose-dependent fashion. <bold>(E)</bold> Splenocyte lysates of the C57BL/6 mouse were either unstimulated (∅) or stimulated with IL-10 (Natural type or Stable) in a dose-dependent manner. <bold>(F)</bold> Inhibition of Nm and STm induced STAT3 phosphorylation by a blocking IL-10 antibody (clone JES5-2A5). IL 10 (2.5 ng) was mixed with the indicated amounts of antibody in one volume of 25 μl of medium and preincubated on ice for 30 min before adding this batch to 475 μl of spleen cell suspension. <bold>(G)</bold> The Luciferase activity represented as AUC from LPS-induce cells from transgenic mouse were either unstimulated or treated with 0.2 μg/mL of the antiIL-10 receptor (antiIL-10Ra) antibody (clone 1B1.3a) for 30 min at 37°C before treatment with LPS (10 ng/mL) or LPS (10 ng/mL), Nm (10 ng/mL), or STm (10 ng/mL). Furthermore, a significant difference is also calculated to compered Nm with STm after antiIL-10Ra treatment; ANOVA. <bold>(H)</bold> The dose-response stable IL-10 on LPS-treated BMDMs of transgenic mouse: the ED<sub>50</sub> of Nm (black line) is 1.5 ng/mL and the ED<sub>50</sub> of STm proteins: STm7 (green) STm9 (blue line) and STm13 (red line) is calculated as 0.45 ng/mL and 0.40 and 0.38 ng/mL respectively. All data are representative of three independent experiments, with triplicate cultures per experiment (<italic>N</italic> = 3, <italic>n</italic> = 3), and bars represent standard error of the mean.</p></caption><graphic xlink:href=\"fimmu-11-01794-g0002\"/></fig><p>We compared regulatory activities of STm, Nm, and commercial mouse IL-10 (CmIL-10) using BMDMs isolated from the hTNF.LucBAC reporter mouse reports the activation of the human <italic>TNF gene</italic> promoter, as previously described (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). STm, Nm, and CmIL-10 (all at 10 ng/mL) significantly suppressed the LPS-induced luciferase production by about 60% (<italic>p</italic> < 0.001 ANOVA) (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>) Subsequently, we titrated STm and Nm IL-10 and determined half-maximal suppression of luciferase induction (ED<sub>50</sub>) values of 0.04 and 0.17 ng/mL for STm and Nm, respectively (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>). For validation of this result, we isolated the supernatant of LPS- and IL-10-treated BMDMs after 24 h and measured soluble mouse TNF by ELISA. This alternative readout yielded ED<sub>50</sub> values in the same range as obtained by quantification by the bioluminescence reporter system (<xref ref-type=\"fig\" rid=\"F2\">Figure 2D</xref>). Thus, STm was 4- to 8-fold more active than natural IL-10. Likewise, Western blot analysis of STAT3 phosphorylation induced by titrating amounts of IL-10 showed about 100-fold higher activity of STm compared to natural mouse IL-10 (<xref ref-type=\"fig\" rid=\"F2\">Figure 2E</xref>). As a further test to quantify the biological activity of STm, we determined concentrations of anti-IL-10 mAb required to inhibit STm-induced STAT3 phosphorylation. While the activity of Nm was almost entirely blocked by pre-incubation with 0.05 mg/ml anti-IL-10 antibody, this antibody concentration did not affect STm activity (<xref ref-type=\"fig\" rid=\"F2\">Figure 2F</xref>).</p><p>We believe that the suppression of luciferase is dependent on the IL-10R engagement with IL-10. Therefore, we tested the capability of the IL-10R antibody to block the effect of IL-10 on LPS-BMDMs of h.TNF.LucBAC. Our data showed that the presence of anti-IL-10R1 blocking antibodies completely blocked the biological activity of STm. Our data demonstrate that there is a non-significant change in the luciferase induction of a pre-treated BMDMs with 0.2 μg/mL anti-IL-10 receptors antibody (antiIL-10Ra) compared to the BMDMs treated with LPS only (10 ng/mL) (<xref ref-type=\"fig\" rid=\"F2\">Figure 2G</xref>).</p><p>In order to address whether the length of the flexible linker, which may affect solubility, stability, and function of the fusion protein, we generated dimeric IL-10 molecules with different linker lengths -G<sub>3</sub>SG<sub>3</sub>- (STm7), -G<sub>3</sub>SG<sub>4</sub>S- (STm9), and -G<sub>3</sub>SG<sub>4</sub>-SG<sub>4</sub>- (STm13) as described above. Linker length did not impact on the bioactivity of the stable IL-10 as determined by suppression of LPS-induced TNF reporter expression (<xref ref-type=\"fig\" rid=\"F2\">Figure 2H</xref>). Collectively, stable mouse IL-10 shows significantly more potent bioactivity compared to natural mouse IL-10.</p></sec><sec><title>Temperature- and pH-Dependent Stability of Stable Mouse IL-10 Protein</title><p>The human IL-10 homodimer was shown to rapidly dissociate into inactive monomers at lower pH and higher temperature (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). We compared the effect of temperature and pH on the biological activity of STm and Nm. A 30 min incubation at 37°C did not affect the capacity of Nm or STm to suppress LPS-induced luciferase expression (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>). Nm lost bioactivity already upon 5 min exposure to 55°C, whereas the treatment did not affect STm (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>, <xref ref-type=\"supplementary-material\" rid=\"SM1\">Figure S1A</xref>). ED<sub>50</sub> of commercial IL-10 and Nm were significantly reduced by exposure to 55°C for 10 min while STm was not affected by this treatment (<xref ref-type=\"fig\" rid=\"F3\">Figures 3C–E</xref>). Moreover, we investigate the impact of acidic and basic pH on the biological activity of IL-10 protein. Biological activity of both Nm and STm decreased in acidic and alkaline pH compared to neutral pH (pH 7). However, at an acidic pH of 5, STm was significantly more active over a wide concentration range (<xref ref-type=\"fig\" rid=\"F3\">Figure 3F</xref>). We also addressed the effects of freezing and frozen storage on IL-10 bioactivity. Our data indicate that the ED50 of STm IL-10 was 0.041 ng/mL, and ED<sub>50</sub> Nm was 0.15 ng/mL after storage at −80°C for 6 months (<xref ref-type=\"fig\" rid=\"F3\">Figure 3G</xref>). In summary, we show that stable mouse IL-10 is more resistant to heat and low pH than natural mouse IL-10.</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Biological stability of mouse IL-10 variants upon treatment at different temperatures and pH <italic>in vitro</italic>. <bold>(A)</bold> Area Under the Curve (AUC) of luciferase induction was measured after incubating 100 ng/mL Nm (black line) and STm (blue line) at 37°C in the time course. <bold>(B)</bold> Both Nm (black line) and STm (blue line) were treated at 55°C in time course before luciferase activity was measured as the area under the curve (AUC) after LPS-stimulated BMDMs of reporter mouse. <bold>(C–E)</bold> LPS-induced luciferase inhibition was measured after co-stimulated with either heat-treated at 55°C for 10 min (10 min) or untreated (0 min) of the commercial mouse IL-10 (CmIL-10), Nm and STm. CmIL-10 was used as a control in this experiment. The ED<sub>50</sub> was calculated as follow: <bold>(C)</bold> CmIL-10 heat-treated for (2.24 ng/mL) or untreated (0.137); <bold>(D)</bold> Nm heat-treated for (3.23 ng/mL) or untreated (0.8 ng/mL); <bold>(E)</bold> STm heat-treated for (0.09 ng/mL) or untreated (0.08 ng/mL). <bold>(F)</bold> Both Nm and STm at 100 ng/mL were pre-incubated with different pH buffers at 4°C for 24 h flowed by buffer exchange columns. BMDMs of the transgenic mouse then stimulated with LPS (10 ng/mL), and pH treated IL-10 (Nm and STm) at 10 ng/mL. The percentage of luciferase activities is relative to LPS treatment. The significant difference compared between pH treatments with neutral pH (pH7) of Nm and STm on LPS-stimulated cells ***<italic>p</italic> < 0.001, **<italic>p</italic> < 0.005, *<italic>p</italic> < 0.05; ANOVA. <bold>(G)</bold> To test the effect of the storage of IL-10 protein in −80°C for 6 months, the ED<sub>50</sub> was calculated and compared between Nm (black line) and STm (blue line). All data are representative of three independent experiments, with triplicate cultures per experiment (<italic>N</italic> = 3, <italic>n</italic> = 3), and bars represent standard error of the mean.</p></caption><graphic xlink:href=\"fimmu-11-01794-g0003\"/></fig></sec><sec><title>The Effect of the Site-Specific Mutation on Stable Mouse IL-10 Biology</title><p>We predict that the STm bound to IL-10R like Nm (i.e., natural IL-10). To investigate this, we mutated the IL-10R binding site in the STm dimer. The location of the IL-10R binding site was obtained from the previous study on IL-10/IL-10R interaction (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). For this purpose, we introduced four-point mutations (L23G, R27G, K34G, and Q38G) at the helix A of the second monomer of the STm dimer (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>). This mutated form of IL-10 named IL-10M2<sup>Mu</sup>. The IL-10 ELISA detected the IL-10M2<sup>Mu</sup> as validation of the presence of the recombinant protein in culture supernatants of HEK cells (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>). The luciferase assay, represented as AUC, showed that IL-10M2<sup>Mu</sup> (10 ng/mL) inhibited the luciferase activity by ~15% (<italic>p</italic> < 0.5, ANOVA). However, 50 ng/mL of IL-10M2<sup>Mu</sup> inhibited luciferase activity by ~40% as the Nm and STm (<italic>p</italic> = 0.001, ANOVA) (<xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>). This data shows that maximal suppression could be achieved with 5-fold of IL-10M2<sup>Mu</sup> compared to STm and Nm. This experiment may represent the significance of dimerization in STm to retain the maximum activity by binding to the IL-10R as the natural IL-10.</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Mutations introduced into one monomer of stable mouse IL-10 at the IL-10R binding site (IL-10M2<sup>Mu</sup>). <bold>(A)</bold> The cDNA construct of both un-mutant IL-10 and IL-10M2<sup>Mu</sup> was cloned into the expression vector (pCEP V19) and the cartoon illustration of IL-10M2<sup>Mu</sup> with the location of the 4-points mutations at the IL-10 receptor-binding site on the helix A of IL-10 protein. <bold>(B)</bold> Purified IL-10M2<sup>Mu</sup> detected by ELISA (1/2 dilutions) to evaluate the expression level. <bold>(C)</bold> Assessment of luciferase suppression after LPS (10 ng/mL) treatment and either co-treated with Nm (10 ng/mL), STm (10 ng/mL), or IL-10M2<sup>Mu</sup> at (10 and 50 ng/mL). The significant difference is calculated concerning LPS stimulated cells only and symbolized as ***<italic>p</italic> < 0.001, ns (non-significant). All data are representative of three independent experiments, with triplicate cultures per experiment (N = 3, n = 3), and bars represent standard error of the mean.</p></caption><graphic xlink:href=\"fimmu-11-01794-g0004\"/></fig><p>In previous investigations, we demonstrated that several alanine substitutions in the IL-10<sup>RRCHR</sup> (i.e., natural IL-10) region have an impact on the structural integrity (α-helical structure) of IL-10 (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>), with substitution of RRCHR to ARCHA causing the most significant loss of structure (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). Importantly, we found a correlation between changing the degree of α-helical structure with the reduction in the biological activity of Nm, as presented in ED<sub>50</sub> and confirmed by the p-STAT3 assay (<xref ref-type=\"fig\" rid=\"F5\">Figures 5B,C</xref>). Based on this data, to further investigate the biological properties and the IL-10R binding of the engineered STm, we assessed the corresponding impact of these substitutions on the biological activity of STm. Our data showed that these mutations affected the biological activity of STm to a different extent compared to Nm, as represented in ED<sub>50</sub> and confirmed by p-STAT3 assay (<xref ref-type=\"fig\" rid=\"F5\">Figures 5D,E</xref>). Specifically, whereas the ARCHA mutation significantly affected Nm biological activity by >500 fold, this only led to a 10-fold loss of function in STm. Conversely, the AACHR and RACHR substitutions that did not dramatically modify STm activity led to a similar loss of function in the STm as the ARCHA mutation. Collectively, the inactivation of one monomer of STm led to a weaker binding to IL-10R; moreover, the impact on the biological activity of amino acid substitution at the RRCHR region was higher in Nm than STm.</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>The effect of amino acid substitution at the RRCHR region of mouse IL-10 on biological activities. <bold>(A)</bold> The secondary structure of IL-10 mutants by CD spectroscopy adopted from unpublished data (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). <bold>(B)</bold> The ED50 of natural IL-10 was calculated as flow: Nm<sup>RRCHR</sup> (un-mutant) (black line) (0.1 ng/mL), Nm<sup>RACHR</sup> (green line) (0.28 ng/mL), Nm<sup>AACHR</sup> (blue line) (0.96 ng/mL), and Nm<sup>ARCHA</sup> (red line) (59.28 ng/mL). <bold>(C)</bold> The activation of cellular signaling (p-STAT3) determines by western blot after lysate from spleen cells of the C57BL/6 mouse treated Nm mutants and un-mutant (control) in dose-response and using the total STAT3 as an internal control. <bold>(D)</bold> The ED50 of stable IL-10 was calculated as flow: STm<sup>RRCHR</sup> (un-mutant) (black line) (0.36 ng/mL), STm<sup>RACHR</sup> (green line) (1.6 ng/mL), STm<sup>AACHR</sup> (blue line) (2.55 ng/mL), and ST<sup>ARCHA</sup> (red line) (3.02 ng/mL). <bold>(E)</bold> The activation p-STAT3 determines by western blot from spleen cells lysate of the C57BL/6 mouse treated STm mutants and un-mutant (control) in dose-response and using the total STAT3 as an internal control. The data represented as AUC (Area Under the Curve). All data are representative of two independent experiments, with triplicate cultures per experiment (<italic>N</italic> = 2, <italic>n</italic> = 3), and bars represent standard error of the mean.</p></caption><graphic xlink:href=\"fimmu-11-01794-g0005\"/></fig></sec><sec><title>Stabilized Mouse IL-10 Dimer Is Biologically Active <italic>in vivo</italic></title><p>While the above data demonstrated the improved biological activity and stability of STm <italic>in vitro</italic>, it was essential also to examine the efficacy of STm <italic>in vivo</italic>. The efficacy of STm compared to Nm under physiological conditions was first examined utilizing a local inflammatory reaction in the skin of mice. The skin consists of a multilayer structure of the epidermis, as well as the bluish coloration of the basophilic epithelial cytoplasm (<xref ref-type=\"fig\" rid=\"F6\">Figures 6A,B</xref>). Subcutaneous injection of LPS causes a dose-dependent inflammatory infiltration of all layers of the skin (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>). Our previous data showed that this reaction is much more severe in IL-10<sup>−/−</sup> mice, with considerable necrosis of epidermis, dermis, and panniculus carnosus (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>). This result indicates the importance of IL-10 in modulating the inflammatory response to LPS in the skin, validating the model to assess the <italic>in vivo</italic> effectiveness of STm in controlling inflammation. Different concentrations of the IL-10 proteins were injected subcutaneously (under the panniculus carnosus) together with LPS (10 μg) into IL-10<sup>−/−</sup> mice. On 3 consecutive days, an injection was made in the same place in the flank. On the 5th day, the mice were sacrificed, and the tissue removed from the injection site and examined histologically. This experiment showed that injections of LPS without IL-10 resulted in a massive inflammatory local reaction with massive recruitment of numerous macrophages and neutrophils (<xref ref-type=\"fig\" rid=\"F6\">Figures 6C,D</xref>). Moreover, all layers of the skin in the center of the lesion became necrotic compared to healthy skin. The enlargement view (<xref ref-type=\"fig\" rid=\"F6\">Figure 6D</xref>) showed a necrotic hair follicle and necrotic interfollicular epidermis with condensed nuclei and reddish acidophilic cytoplasm as opposed to the basophilic cytoplasm of healthy keratinocytes. Coadministration of STm along with LPS (<xref ref-type=\"fig\" rid=\"F6\">Figure 6E</xref>) suppressed the inflammatory response in a dose-dependent fashion. In <xref ref-type=\"fig\" rid=\"F6\">Figure 6F</xref>, we summarize the biological activities of STm and Nm in protecting LPS induced inflammation. Both STm and Nm were protective at 2 and 0.2 μg. STm appeared to be more effective than Nm at 0.02 μg, where the biological activity of Nm was waning. At 0.002 μg, both Nm and STm had no protective effect against skin inflammation (<xref ref-type=\"fig\" rid=\"F6\">Figure 6F</xref>). This experiment demonstrated the biological effect of STm <italic>in vivo</italic> but did not allow a quantitative comparison between Nm and STm.</p><fig id=\"F6\" position=\"float\"><label>Figure 6</label><caption><p>Suppression of LPS-induced dermal inflammation by Nm and STm. <bold>(A)</bold> Overview of healthy skin. Mouse skin consists of a 1–2-layer epidermis (Ep), which forms the hair follicles (Hf) and sebaceous glands (Td) by invagination in the dermis (De). The dermis consists of collagenous connective tissue. This is followed by the muscle layer of the Panniculus carnosus (Pc), the fatty tissue (Fg), and the trunk muscles (Rm). <bold>(B)</bold> Enlargement of healthy skin. The epithelium shows bluish cytoplasm and loose chromatin, as well as two intact hair follicles. <bold>(C)</bold> Overview of necrotic skin. In magnification, a large number of neutrophils is recognizable. <bold>(D)</bold> Enlargement of necrotic skin. The epithelial cells show reddish cytoplasm and condensed chromatin. The hair follicle is dead. <bold>(E)</bold> Overview skin section treated STm (2 μg) STm, which LPS was co-injected. <bold>(F)</bold> A table summarizing the effect of IL-10 (Nm/STm) at different concentrations on LPS-treated (10 μg) skin.</p></caption><graphic xlink:href=\"fimmu-11-01794-g0006\"/></fig><p>The activity of STm was also tested using an <italic>in vivo</italic> model of LPS-induced systemic inflammation. First, the time course of TNF concentration in serum after retro-orbital (i.v.) LPS injection was determined in establishment experiments. A high serum concentration of TNF ~3.2 ng/mL was measurable 1.5 h after the administration of LPS (25 μg i.v.). This value was reduced by about 75% after 3 h and had returned close to baseline levels 6 h after LPS injection. Injections of PBS or Nm and STm (2 μg each) alone did not result in the measurable release of TNF, which could exclude contamination of these reagents with pyrogens (<xref ref-type=\"fig\" rid=\"F7\">Figure 7A</xref>). In the next experiment, different amounts of STm i.v. were injected 30 min before LPS (25 μg i.v.). Here, the STm proved to be highly effective, in a dose-dependent manner, in the suppression of TNF release. The administration of 2 μg Nm and STm reduced serum TNF concentration by ~70% relative to the control mice receiving only LPS. Both at either 2 or 20 μg, Nm and STm were equally effective at suppressing TNF production (<xref ref-type=\"fig\" rid=\"F7\">Figure 7B</xref>). Apparently, in this assay, the maximum level of TNF suppression was reached at 2 μg IL-10, as the injection of 20 mg IL-10 failed to suppress TNF production further. Experiments with titrations of Nm- and STm, however, showed large fluctuations in TNF serum concentration and therefore did not allow a quantitative comparison of the effects of Nm- and STm (data not shown). Therefore, we investigated the ability of Nm and STm IL-10 to inhibit the production of IL-6, which is another cytokine integral within the acute phase inflammatory immune response. The serum concentration of IL-6 was determined 3 h after injection by ELISA. It was found that over a wide dose range, the STm reduced IL-6 release more effectively than Nm. By non-linear regression, Nm suppressed the IL-6 response with an ED<sub>50</sub> value of 274.8 ng/mL, whereas STm suppressed IL-6 production with an ED50 of 112 ng/mL. Thus, STm was ~2.5-fold (<xref ref-type=\"fig\" rid=\"F7\">Figure 7C</xref>) more potent than Nm. Altogether, the stable form of IL-10 is biologically active <italic>in vivo</italic>, which controls both the local and systemic inflammatory responses.</p><fig id=\"F7\" position=\"float\"><label>Figure 7</label><caption><p>Detection of the <italic>in vivo</italic> activity of the STm by suppression of LPS-induced cytokine release. <bold>(A,B)</bold> 100 μl PBS containing 25 μg LPS i.v. Injected in C57BL/6 mice (8–12 weeks). At the indicated time points, blood was taken retro-orbitally, and ELISA determined the TNF-a serum concentration, *n.d.: not detectable. <bold>(A)</bold> To establish, LPS, PBS, Nm, or STm (2 μg protein each) were injected one at a time. <bold>(B)</bold> 30 min before LPS administration, the indicated amounts of Nm or STm were i.v. Injected. <bold>(C)</bold> IL-10 -/- mice were given 10 μg LPS together with increasing concentrations of Nm or STm i.v. Injected. After 3 h, blood was drawn, and the IL 6 serum concentration determined by ELISA.</p></caption><graphic xlink:href=\"fimmu-11-01794-g0007\"/></fig></sec><sec><title>The Biological Activity and Stability of Stabilized Human IL-10 Dimer <italic>in vitro</italic></title><p>Our results demonstrated that STm molecules could be generated with potent regulatory activity. Consequently, given the aim of developing an IL-10 based treatment for human disease, we investigated whether it was also possible to create a biologically active and stable version of human IL-10. We first generated and produced a stable human IL-10 using different lengths of the flexible linker (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Figure S2A</xref>). Our data showed that both Nh and STh, with different linker lengths, are biologically active <italic>in vitro</italic> by suppressing LPS-induced luciferase expression in BMDMs from the h.TNF.LucBAC reporter mice (<xref ref-type=\"fig\" rid=\"F8\">Figure 8A</xref>). STh had a 2.5-fold higher biological activity than Nh, as measured by p-STAT3 activation (<xref ref-type=\"fig\" rid=\"F8\">Figure 8B</xref>), which was confirmed by conventional TNF ELISA assay (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Figure S2B</xref>). Like the mouse IL-10, the effect of human IL-10 on luciferase suppression was blocked by using mouse anti-IL-10R1 in a dose-dependent manner. Interestingly, our data demonstrate that increased amounts of anti-IL-10R1 antibody were required to block the effect of STh in suppressing luciferase production than to block the effect of Nh (<xref ref-type=\"fig\" rid=\"F8\">Figure 8C</xref>). This data may indicate the binding of STh to mouse IL-10R is of higher affinity compared to the Nh.</p><fig id=\"F8\" position=\"float\"><label>Figure 8</label><caption><p>Generation and characterization of the stable version of human IL-10. <bold>(A)</bold> The effect of STh (with different linkers length) and Nh on LPS-induce luciferase activity on BMDMs of transgenic mouse: the ED<sub>50</sub> of Nh(black line) is 0.87 ng/mL and the ED<sub>50</sub> of STm proteins: STh7 (green) STh9 (blue), and STh13 (red) is calculated as 0.82, 1, and 1.2 ng/mL, respectively. <bold>(B)</bold> Splenocyte lysates of the C57BL/6 mouse either unstimulated (∅) or stimulated with IL-10 (Natural type or Stable) in a dose-dependent manner. <bold>(C)</bold> The effect mouse antiIL-10 receptor antibody (clone 1B1.3a) on blocking the suppression induced by Nh and STh (10 ng/mL) in a dose-dependent fashion. Furthermore, a significant difference is also calculated to compare Nm with STm after antiIL-10Ra treatment; ANOVA. <bold>(D)</bold> Luciferase activity represented as AUC from LPS-induced BMDMs co-treated with either Nh (black line) and STh (blue line) at 10 ng/mL after incubated at 37°C in the time course. <bold>(E)</bold> Both Nh and STh were incubated at 55°C in time course, and the luciferase activity was calculated after LPS-stimulated and co-treated with either Nm (black line) and STm (blue line) at 10 ng/mL on BMDMs. <bold>(F,G)</bold> LPS-induced Luciferase activity when co-stimulated with either heat-treated at 55°C for 10 min (10 min) or untreated (0 min) of Nh and STh. The ED<sub>50</sub> is calculated as follow: <bold>(F)</bold> Nh heat-treated for (3.74 ng/mL) or untreated (0.17 ng/mL); <bold>(G)</bold> STh heat-treated for (0.10 ng/mL) or untreated (0.12 ng/mL). <bold>(H)</bold> Both Nh and STh at 100 ng/mL were pre-incubated with different pH buffers at 4°C for 24 h flowed by buffer exchange columns. BMDMs of the transgenic mouse then stimulated with LPS (10 ng/mL) and pH-treated IL-10 (Nh and STh) at 10 ng/mL. The percentage of luciferase activities is relative to the maximum LPS induction of luciferase. The significant difference compared between pH treatments with neutral pH (pH7) of Nh and STh on LPS-stimulated cells; ***<italic>p</italic> < 0.001, **<italic>p</italic> < 0.005; ANOVA. All the data above are representative of three independent experiments, with triplicate cultures per experiment (<italic>N</italic> = 3, <italic>n</italic> = 3), and bars represent standard error of the mean.</p></caption><graphic xlink:href=\"fimmu-11-01794-g0008\"/></fig><p>In terms of stability, both STh and Nh were incubated during a time course at 37°C, which showed no significant difference in luciferase inhibition (<italic>p</italic> > 0.5, ANOVA) (<xref ref-type=\"fig\" rid=\"F8\">Figure 8D</xref>), which indicates the both Nh and STh are biologically active at physiological temperature. However, Nh gradually lost biological activity after 5 min of heat treatment at 55°C, whereas STh maintained biological activity as measured by luciferase assay (<xref ref-type=\"fig\" rid=\"F8\">Figure 8E</xref>) and TNF-ELISA (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Figure S1B</xref>). In a dose-response experiment, we also observed that Nh became less potent after heat treatment (55°C for 10 min) (<xref ref-type=\"fig\" rid=\"F8\">Figure 8F</xref>); however, STh maintained suppressive activity (<xref ref-type=\"fig\" rid=\"F8\">Figure 8G</xref>). The pH-dependent stability study showed that STh behave similarly to Nh, but, the STh was more resistant in pH5 and pH2 (<xref ref-type=\"fig\" rid=\"F8\">Figure 8H</xref>). Overall, these data show that the STh is a biologically active protein <italic>in vitro</italic> with higher stability compared to Nh.</p><p>Overall, we generate a stable IL-10 dimer by linking two IL-10 monomers in a head to tail fashion by a flexible linker (<xref ref-type=\"fig\" rid=\"F9\">Figure 9</xref>). We demonstrate that our novel stable IL-10 dimer is more stable in different stress conditions compared to non-covalently linked IL-10 (i.e., natural IL-10). We assume the stable IL-10 dimer folds as natural IL-10. The IL-10 3D domain-swapped dimerization of IL-10 is essential to form the receptor binding site of the cytokine (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Our mutational experiment has indirectly proved that stable IL-10 dimer binds and acts by the IL-10 receptor.</p><fig id=\"F9\" position=\"float\"><label>Figure 9</label><caption><p>Scheme representing the proposed biological activity and stability of stable IL-10 dimer. Both natural and stable IL-10 are biologically active <italic>in vitro</italic> and <italic>in vivo</italic>. We predict that our stable IL-10 is folded like the natural IL-10 in a domain-swapping fashion. Our mutant IL-10 model demonstrates the importance of dimerization to elicit the maximum inhibitory response. We showed that stable IL-10 is more active after treatment with different physiological stress conditions such as high-temperature and pH compared to natural IL-10.</p></caption><graphic xlink:href=\"fimmu-11-01794-g0009\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Soon after the cytokine IL-10 was cloned, we generated a mouse mutant deficient for IL-10 and could show that these mice developed inflammatory bowel disease that depended on the microflora (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). Based on these findings and subsequent preclinical trials, it was shown that while recombinant IL-10 was safe and well-tolerated in healthy individuals (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>), it had limited efficacy in treating inflammatory diseases (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>). In this report, we reach an important milestone for the generation of a recombinant IL-10 with more stable biochemical properties. Syto et al. studied the structural and biological stability of non-covalently linked IL-10 dimer under different conditions. It was observed that incubation of human IL-10 dimer for 1 h at 37 and 55°C resulted in the formation of 2 and 22% monomers, respectively (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). By the covalent fusion of two IL-10 monomers, we generated a more stable and most likely also more biologically active form of IL-10 both for mouse and human (<xref ref-type=\"fig\" rid=\"F2\">Figures 2</xref>, <xref ref-type=\"fig\" rid=\"F8\">8</xref>). The previous study demonstrates that the covalent fusion of dimeric HIV-1 proteases has been shown to improve protein stability compared to natural dimer (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). However, the mechanism of this stability enhancement is not clear. Our possible explanation is that linking the C-terminus of one IL-10 monomer to the N-terminus of the second monomer may increase the stability of the IL-10 3D domain-swapped dimer. The IL-10 3D domain-swapped dimer defined as the exchange of the helices E and F (from the first monomer) into the hydrophobic cleft (helices A-D) of the other monomer, which may not be profoundly affected by the different conditions of stress in stable IL-10 compared to the natural IL-10. This study did not define the potential of refolding stable IL-10 to the known active state after different stress-inducing conditions were applied. We could demonstrate that the new IL-10 dimer binds and acts via the IL-10 receptor, shown that it does not act on IL10R1 deficient lymphocytes (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>), by blocking the biological activity using anti-IL-10 receptor antibodies (<xref ref-type=\"fig\" rid=\"F2\">Figure 2F</xref>) and by introducing point mutations in the covalent linked IL-10 dimer suggested to be involved in IL-10 and IL-10 receptor interaction (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). A previous study suggested that IL-10 dimer formation is vital to generate the receptor-binding domain that activates IL-10R signaling via side to side interaction (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Therefore, it is believed that the correct dimer formation (i.e., 3D domain swapping) in IL-10 is essential to create a receptor-binding domain (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). This hypothesis has been confirmed by generating a stable monomeric form as a model to bind to IL-10R in 1:1 interaction (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>). The next step along this line is to generate crystal structures for the new IL-10 proteins and to compare these to the known IL-10 structure (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>). Our recombinant stable dimeric IL-10 can now be used as a building block for the development of IL-10 based therapeutic. One of the new features of our stable IL-10 is that we can now prepare asymmetric IL-10 variants as we have control over the complete IL-10 dimer. We can modify each IL-10 molecule in the dimer independently and get 100% variants with a defined order of each of the monomers. This allows in the future more insights into the functional analysis of IL-10 with its receptor and fine-tuning of the biological activity of the stable IL-10 protein.</p><p>Using the conditional gene targeting approach, we generated mouse mutants with selective gene inactivation of the IL-10 (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>) and the IL-10R (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). Using these mutants in many different settings, we have clear evidence that IL-10 is a local acting cytokine, and there is an apparent cell-type specificity at both at the level of the IL-10 producing and at the level of the IL-10 responding cell. For example, in the T-cell specific IL-10 deficient mouse mutant (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>), only T cell-specific responses are dysregulated, while innate responses are unaffected. In contrast, in a macrophage-specific IL-10 receptor-deficient mouse mutant, the T cell responses are unaffected while innate responses are disturbed similarly to IL-10<sup>−/−</sup> mice. These experiments suggest that the next step toward an IL-10 based therapeutic recombinant protein will be to make an IL-10 protein that can mimic the local acting property of IL-10.This might be achieved by joining our recombinant stable IL-10 to other protein domains that can bind to local regions and/or to specific cell types. Of note, we have generated a fusion protein of IL-10 with an antibody and could see that both the IL-10 activity and the antibody binding property is present in the fusion protein (work in progress). One of the challenging steps in the engineering is to use a form of IL-10 that is less active compared to the natural IL-10 protein in order to allow IL-10 action only when a high local concentration of IL-10 through the local accumulation of the IL-10 protein through the antibody part. Examples of such IL-10 weakening mutations are presented in this report (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). We presume that we are maybe even able to generate a local acting IL-10 inhibitor, in which one of the two dimers is mutated in a way that the stable IL-10 dimer binds to one of the IL-10 receptor chains and blocking dimerization of the receptor.</p><p>In conclusion, the effect of IL-10 in clinical trials is limited due to pleiotropic properties on different cells, and the rapid dissociation of the homodimer at the site of inflammation. Our stable IL-10 protein could be a potential building block for generating a potent and more effective and selective IL-10-based immunotherapy for treating inflammatory diseases and cancers. For instance, a stable IL-10 dimer has already been proposed as a model for generating a target IL-10 immunotherapy because it has a higher biological activity compared to the natural IL-10 monomer (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>) and the IL-10 dimer proposed here would allow constructing such cell-type-specific local acting IL-10.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>All datasets generated for this study are included in the article/<xref ref-type=\"sec\" rid=\"s9\">Supplementary Material</xref>.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by Home Office project license (70/7800) (P8829D3B4) in agreement with the Animal (Scientific Procedures) Act 1986 and the <italic>in vivo</italic> experiments were performed at the University of Cologne, Germany, under animal experimental license 24-9168.11-1/2009-22.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>FM, SL, AR, and WM: conception, design, and stable IL-10 construction. FM and SL: <italic>in vitro</italic> experiment. SL and AR: <italic>in vivo</italic> experiments. FM, SL, and WM: data acquisition. FM, SL, RJ, and EM: IL-10 expression. FM and SP: luciferase assay. FM, SL, SP, KC, AR, and WM: data analysis and interpretation. FM, KC, and WM: drafting of the article. All co-authors: final approval of the manuscript.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>We would like to express our deep and sincere gratitude to Prof. Dr. Mats Paulsson (University of Cologne) for his contribution to this study by providing the mammalian expression vector and cell lines. We also would like to thank Prof. Mike White (University of Manchester) for using the illuminance measuring instrument. We also acknowledge the support and advice from all members in Transgenic Unit core facility (TgU), Faculty of Biology, Medicine and Health, University of Manchester.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> The work was supported by a Ph. D. scholarship from Umm Al-Qura University, Saudi Arabia (UMU740) to FM and by the MRC (MR/R010099/1) to KC. MR/R010099/1 was jointly funded by the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement and is also part of the EDCTP2 program supported by the European Union.</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s9\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fimmu.2020.01794/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fimmu.2020.01794/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boonstra</surname><given-names>A</given-names></name><name><surname>Rajsbaum</surname><given-names>R</given-names></name><name><surname>Holman</surname><given-names>M</given-names></name><name><surname>Marques</surname><given-names>R</given-names></name><name><surname>Asselin-Paturel</surname><given-names>C</given-names></name><name><surname>Pereira</surname><given-names>JP</given-names></name><etal/></person-group>. <article-title>Macrophages and myeloid dendritic cells, but not plasmacytoid dendritic cells, produce IL-10 in response to MyD88- and TRIF-dependent TLR signals, TLR-independent signals</article-title>. <source>J Immunol.</source> (<year>2006</year>) <volume>177</volume>:<fpage>7551</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.177.11.7551</pub-id><pub-id pub-id-type=\"pmid\">17114424</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fujita</surname><given-names>S</given-names></name><name><surname>Seino</surname><given-names>K</given-names></name><name><surname>Sato</surname><given-names>K</given-names></name><name><surname>Sato</surname><given-names>Y</given-names></name><name><surname>Eizumi</surname><given-names>K</given-names></name><name><surname>Yamashita</surname><given-names>N</given-names></name><etal/></person-group>. <article-title>Regulatory dendritic cells act as regulators of acute lethal systemic inflammatory response</article-title>. <source>Blood.</source> (<year>2006</year>) <volume>107</volume>:<fpage>3656</fpage>–<lpage>64</lpage>. <pub-id pub-id-type=\"doi\">10.1182/blood-2005-10-4190</pub-id><pub-id pub-id-type=\"pmid\">16410444</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Comi</surname><given-names>M</given-names></name><name><surname>Amodio</surname><given-names>G</given-names></name><name><surname>Gregori</surname><given-names>S</given-names></name></person-group>. <article-title>Interleukin-10-producing DC-10 is a unique tool to promote tolerance via antigen-specific T regulatory type 1 cells</article-title>. <source>Front Immunol.</source> (<year>2018</year>) <volume>9</volume>:<fpage>682</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2018.00682</pub-id><pub-id pub-id-type=\"pmid\">29686676</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Barthlott</surname><given-names>T</given-names></name><name><surname>Moncrieffe</surname><given-names>H</given-names></name><name><surname>Veldhoen</surname><given-names>M</given-names></name><name><surname>Atkins</surname><given-names>CJ</given-names></name><name><surname>Christensen</surname><given-names>J</given-names></name><name><surname>O'Garra</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>CD25+ CD4+ T cells compete with naive CD4+ T cells for IL-2 and exploit it for the induction of IL-10 production</article-title>. <source>Int Immunol.</source> (<year>2005</year>) <volume>17</volume>:<fpage>279</fpage>–<lpage>88</lpage>. <pub-id pub-id-type=\"doi\">10.1093/intimm/dxh207</pub-id><pub-id pub-id-type=\"pmid\">15684039</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ouyang</surname><given-names>W</given-names></name><name><surname>O'Garra</surname><given-names>A</given-names></name></person-group>. <article-title>IL-10 family cytokines IL-10 and IL-22: from basic science to clinical translation</article-title>. <source>Immunity.</source> (<year>2019</year>) <volume>50</volume>:<fpage>871</fpage>–<lpage>91</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.immuni.2019.03.020</pub-id><pub-id pub-id-type=\"pmid\">30995504</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zdanov</surname><given-names>A</given-names></name><name><surname>Schalk-Hihi</surname><given-names>C</given-names></name><name><surname>Gustchina</surname><given-names>A</given-names></name><name><surname>Tsang</surname><given-names>M</given-names></name><name><surname>Weatherbee</surname><given-names>J</given-names></name><name><surname>Wlodawer</surname><given-names>A</given-names></name></person-group>. <article-title>Crystal structure of interleukin-10 reveals the functional dimer with an unexpected topological similarity to interferon gamma</article-title>. <source>Structure.</source> (<year>1995</year>) <volume>3</volume>:<fpage>591</fpage>–<lpage>601</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0969-2126(01)00193-9</pub-id><pub-id pub-id-type=\"pmid\">8590020</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Walter</surname><given-names>MR</given-names></name></person-group>. <article-title>The molecular basis of IL-10 function: from receptor structure to the onset of signaling</article-title>. <source>Curr Top Microbiol Immunol.</source> (<year>2014</year>) <volume>380</volume>:<fpage>191</fpage>–<lpage>212</lpage>. <pub-id pub-id-type=\"doi\">10.1007/978-3-662-43492-5_9</pub-id><pub-id pub-id-type=\"pmid\">25004819</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Walter</surname><given-names>MR</given-names></name><name><surname>Nagabhushan</surname><given-names>TL</given-names></name></person-group>. <article-title>Crystal structure of interleukin 10 reveals an interferon gamma-like fold</article-title>. <source>Biochemistry.</source> (<year>1995</year>) <volume>34</volume>:<fpage>12118</fpage>–<lpage>25</lpage>. <pub-id pub-id-type=\"doi\">10.1021/bi00038a004</pub-id><pub-id pub-id-type=\"pmid\">7547951</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Syto</surname><given-names>R</given-names></name><name><surname>Murgolo</surname><given-names>NJ</given-names></name><name><surname>Braswell</surname><given-names>EH</given-names></name><name><surname>Mui</surname><given-names>P</given-names></name><name><surname>Huang</surname><given-names>E</given-names></name><name><surname>Windsor</surname><given-names>WT</given-names></name></person-group>. <article-title>Structural and biological stability of the human interleukin 10 homodimer</article-title>. <source>Biochemistry.</source> (<year>1998</year>) <volume>37</volume>:<fpage>16943</fpage>–<lpage>51</lpage>. <pub-id pub-id-type=\"doi\">10.1021/bi981555y</pub-id><pub-id pub-id-type=\"pmid\">9836587</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>Y</given-names></name><name><surname>Wei</surname><given-names>SH</given-names></name><name><surname>Ho</surname><given-names>AS</given-names></name><name><surname>de Waal Malefyt</surname><given-names>R</given-names></name><name><surname>Moore</surname><given-names>KW</given-names></name></person-group>. <article-title>Expression cloning and characterization of a human IL-10 receptor</article-title>. <source>J Immunol.</source> (<year>1994</year>) <volume>152</volume>:<fpage>1821</fpage>–<lpage>9</lpage>.<pub-id pub-id-type=\"pmid\">8120391</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ozaki</surname><given-names>K</given-names></name><name><surname>Leonard</surname><given-names>WJ</given-names></name></person-group>. <article-title>Cytokine and cytokine receptor pleiotropy and redundancy</article-title>. <source>J Biol Chem.</source> (<year>2002</year>) <volume>277</volume>:<fpage>29355</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.R200003200</pub-id><pub-id pub-id-type=\"pmid\">12072446</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tan</surname><given-names>JC</given-names></name><name><surname>Indelicato</surname><given-names>SR</given-names></name><name><surname>Narula</surname><given-names>SK</given-names></name><name><surname>Zavodny</surname><given-names>PJ</given-names></name><name><surname>Chou</surname><given-names>CC</given-names></name></person-group>. <article-title>Characterization of interleukin-10 receptors on human and mouse cells</article-title>. <source>J Biol Chem.</source> (<year>1993</year>) <volume>268</volume>:<fpage>21053</fpage>–<lpage>9</lpage>.<pub-id pub-id-type=\"pmid\">8407942</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Josephson</surname><given-names>K</given-names></name><name><surname>Logsdon</surname><given-names>NJ</given-names></name><name><surname>Walter</surname><given-names>MR</given-names></name></person-group>. <article-title>Crystal structure of the IL-10/IL-10R1 complex reveals a shared receptor binding site</article-title>. <source>Immunity.</source> (<year>2001</year>) <volume>15</volume>:<fpage>35</fpage>–<lpage>46</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S1074-7613(01)00169-8</pub-id><pub-id pub-id-type=\"pmid\">11485736</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Riley</surname><given-names>JK</given-names></name><name><surname>Takeda</surname><given-names>K</given-names></name><name><surname>Akira</surname><given-names>S</given-names></name><name><surname>Schreiber</surname><given-names>RD</given-names></name></person-group>. <article-title>Interleukin-10 receptor signaling through the JAK-STAT pathway. Requirement for two distinct receptor-derived signals for anti-inflammatory action</article-title>. <source>J Biol Chem.</source> (<year>1999</year>) <volume>274</volume>:<fpage>16513</fpage>–<lpage>21</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.274.23.16513</pub-id><pub-id pub-id-type=\"pmid\">10347215</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Clarke</surname><given-names>CJ</given-names></name><name><surname>Hales</surname><given-names>A</given-names></name><name><surname>Hunt</surname><given-names>A</given-names></name><name><surname>Foxwell</surname><given-names>BM</given-names></name></person-group>. <article-title>IL-10-mediated suppression of TNF-alpha production is independent of its ability to inhibit NF kappa B activity</article-title>. <source>Eur J Immunol.</source> (<year>1998</year>) <volume>28</volume>:<fpage>1719</fpage>–<lpage>26</lpage>. <pub-id pub-id-type=\"doi\">10.1002/(SICI)1521-4141(199805)28:05<1719::AID-IMMU1719>3.0.CO</pub-id><pub-id pub-id-type=\"pmid\">9603479</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Peranteau</surname><given-names>WH</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Muvarak</surname><given-names>N</given-names></name><name><surname>Badillo</surname><given-names>AT</given-names></name><name><surname>Radu</surname><given-names>A</given-names></name><name><surname>Zoltick</surname><given-names>PW</given-names></name><etal/></person-group>. <article-title>IL-10 overexpression decreases inflammatory mediators and promotes regenerative healing in an adult model of scar formation</article-title>. <source>J Invest Dermatol.</source> (<year>2008</year>) <volume>128</volume>:<fpage>1852</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.1038/sj.jid.5701232</pub-id><pub-id pub-id-type=\"pmid\">18200061</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wilson</surname><given-names>EH</given-names></name><name><surname>Wille-Reece</surname><given-names>U</given-names></name><name><surname>Dzierszinski</surname><given-names>F</given-names></name><name><surname>Hunter</surname><given-names>CA</given-names></name></person-group>. <article-title>A critical role for IL-10 in limiting inflammation during toxoplasmic encephalitis</article-title>. <source>J Neuroimmunol.</source> (<year>2005</year>) <volume>165</volume>:<fpage>63</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jneuroim.2005.04.018</pub-id><pub-id pub-id-type=\"pmid\">16005735</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bekker</surname><given-names>LG</given-names></name><name><surname>Maartens</surname><given-names>G</given-names></name><name><surname>Steyn</surname><given-names>L</given-names></name><name><surname>Kaplan</surname><given-names>G</given-names></name></person-group>. <article-title>Selective increase in plasma tumor necrosis factor-alpha and concomitant clinical deterioration after initiating therapy in patients with severe tuberculosis</article-title>. <source>J Infect Dis.</source> (<year>1998</year>) <volume>178</volume>:<fpage>580</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1086/517479</pub-id><pub-id pub-id-type=\"pmid\">9697749</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Suvas</surname><given-names>S</given-names></name><name><surname>Azkur</surname><given-names>AK</given-names></name><name><surname>Kim</surname><given-names>BS</given-names></name><name><surname>Kumaraguru</surname><given-names>U</given-names></name><name><surname>Rouse</surname><given-names>BT</given-names></name></person-group>. <article-title>CD4+CD25+ regulatory T cells control the severity of viral immunoinflammatory lesions</article-title>. <source>J Immunol.</source> (<year>2004</year>) <volume>172</volume>:<fpage>4123</fpage>–<lpage>32</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.172.7.4123</pub-id><pub-id pub-id-type=\"pmid\">15034024</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Couper</surname><given-names>KN</given-names></name><name><surname>Blount</surname><given-names>DG</given-names></name><name><surname>Riley</surname><given-names>EM</given-names></name></person-group>. <article-title>IL-10: the master regulator of immunity to infection</article-title>. <source>J Immunol.</source> (<year>2008</year>) <volume>180</volume>:<fpage>5771</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.180.9.5771</pub-id><pub-id pub-id-type=\"pmid\">18424693</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuhn</surname><given-names>R</given-names></name><name><surname>Lohler</surname><given-names>J</given-names></name><name><surname>Rennick</surname><given-names>D</given-names></name><name><surname>Rajewsky</surname><given-names>K</given-names></name><name><surname>Muller</surname><given-names>W</given-names></name></person-group>. <article-title>Interleukin-10-deficient mice develop chronic enterocolitis</article-title>. <source>Cell.</source> (<year>1993</year>) <volume>75</volume>:<fpage>263</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1016/0092-8674(93)80068-P</pub-id><pub-id pub-id-type=\"pmid\">8402911</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zigmond</surname><given-names>E</given-names></name><name><surname>Bernshtein</surname><given-names>B</given-names></name><name><surname>Friedlander</surname><given-names>G</given-names></name><name><surname>Walker</surname><given-names>CR</given-names></name><name><surname>Yona</surname><given-names>S</given-names></name><name><surname>Kim</surname><given-names>KW</given-names></name><etal/></person-group>. <article-title>Macrophage-restricted interleukin-10 receptor deficiency, but not IL-10 deficiency, causes severe spontaneous colitis</article-title>. <source>Immunity.</source> (<year>2014</year>) <volume>40</volume>:<fpage>720</fpage>–<lpage>33</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.immuni.2014.03.012</pub-id><pub-id pub-id-type=\"pmid\">24792913</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pils</surname><given-names>MC</given-names></name><name><surname>Pisano</surname><given-names>F</given-names></name><name><surname>Fasnacht</surname><given-names>N</given-names></name><name><surname>Heinrich</surname><given-names>JM</given-names></name><name><surname>Groebe</surname><given-names>L</given-names></name><name><surname>Schippers</surname><given-names>A</given-names></name><etal/></person-group>\n<article-title>Monocytes/macrophages and/or neutrophils are the target of IL-10 in the LPS endotoxemia model</article-title>. <source>Eur J Immunol.</source> (<year>2010</year>) <volume>40</volume>:<fpage>443</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1002/eji.200939592</pub-id><pub-id pub-id-type=\"pmid\">19941312</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sellon</surname><given-names>RK</given-names></name><name><surname>Tonkonogy</surname><given-names>S</given-names></name><name><surname>Schultz</surname><given-names>M</given-names></name><name><surname>Dieleman</surname><given-names>LA</given-names></name><name><surname>Grenther</surname><given-names>W</given-names></name><name><surname>Balish</surname><given-names>E</given-names></name><etal/></person-group>. <article-title>Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice</article-title>. <source>Infect Immun.</source> (<year>1998</year>) <volume>66</volume>:<fpage>5224</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.1128/IAI.66.11.5224-5231.1998</pub-id><pub-id pub-id-type=\"pmid\">9784526</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Steen</surname><given-names>KH</given-names></name><name><surname>Steen</surname><given-names>AE</given-names></name><name><surname>Reeh</surname><given-names>PW</given-names></name></person-group>. <article-title>A dominant role of acid pH in inflammatory excitation and sensitization of nociceptors in rat skin, <italic>in vitro</italic></article-title>. <source>J Neurosci.</source> (<year>1995</year>) <volume>15</volume>:<fpage>3982</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1523/JNEUROSCI.15-05-03982.1995</pub-id><pub-id pub-id-type=\"pmid\">7751959</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Punnia-Moorthy</surname><given-names>A</given-names></name></person-group>. <article-title>Evaluation of pH changes in inflammation of the subcutaneous air pouch lining in the rat, induced by carrageenan, dextran and <italic>Staphylococcus aureus</italic></article-title>. <source>J Oral Pathol.</source> (<year>1987</year>) <volume>16</volume>:<fpage>36</fpage>–<lpage>44</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1600-0714.1987.tb00674.x</pub-id><pub-id pub-id-type=\"pmid\">2435877</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Punnia-Moorthy</surname><given-names>A</given-names></name></person-group>. <article-title>Buffering capacity of normal and inflamed tissues following the injection of local anaesthetic solutions</article-title>. <source>Br J Anaesth.</source> (<year>1988</year>) <volume>61</volume>:<fpage>154</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bja/61.2.154</pub-id><pub-id pub-id-type=\"pmid\">3415887</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Roers</surname><given-names>A</given-names></name><name><surname>Lanvermann</surname><given-names>S</given-names></name><name><surname>Müller</surname><given-names>W</given-names></name></person-group>\n<source>Covalently Linked Interleukin-10</source>. Google Patents, US (<year>2013</year>).</mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>X</given-names></name><name><surname>Zaro</surname><given-names>JL</given-names></name><name><surname>Shen</surname><given-names>WC</given-names></name></person-group>. <article-title>Fusion protein linkers: property, design and functionality</article-title>. <source>Adv Drug Deliv Rev.</source> (<year>2013</year>) <volume>65</volume>:<fpage>1357</fpage>–<lpage>69</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.addr.2012.09.039</pub-id><pub-id pub-id-type=\"pmid\">23026637</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cheng</surname><given-names>YS</given-names></name><name><surname>Yin</surname><given-names>FH</given-names></name><name><surname>Foundling</surname><given-names>S</given-names></name><name><surname>Blomstrom</surname><given-names>D</given-names></name><name><surname>Kettner</surname><given-names>CA</given-names></name></person-group>. <article-title>Stability and activity of human immunodeficiency virus protease: comparison of the natural dimer with a homologous, single-chain tethered dimer</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>1990</year>) <volume>87</volume>:<fpage>9660</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.87.24.9660</pub-id><pub-id pub-id-type=\"pmid\">2263618</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Foss</surname><given-names>TR</given-names></name><name><surname>Kelker</surname><given-names>MS</given-names></name><name><surname>Wiseman</surname><given-names>RL</given-names></name><name><surname>Wilson</surname><given-names>IA</given-names></name><name><surname>Kelly</surname><given-names>JW</given-names></name></person-group>. <article-title>Kinetic stabilization of the native state by protein engineering: implications for inhibition of transthyretin amyloidogenesis</article-title>. <source>J Mol Biol.</source> (<year>2005</year>) <volume>347</volume>:<fpage>841</fpage>–<lpage>54</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jmb.2005.01.050</pub-id><pub-id pub-id-type=\"pmid\">15769474</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Minshawi</surname><given-names>F</given-names></name><name><surname>White</surname><given-names>MRH</given-names></name><name><surname>Muller</surname><given-names>W</given-names></name><name><surname>Humphreys</surname><given-names>N</given-names></name><name><surname>Jackson</surname><given-names>D</given-names></name><name><surname>Campbell</surname><given-names>BJ</given-names></name><etal/></person-group>. <article-title>Human TNF-Luc reporter mouse: a new model to quantify inflammatory responses</article-title>. <source>Sci Rep.</source> (<year>2019</year>) <volume>9</volume>:<fpage>193</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-018-36969-x</pub-id><pub-id pub-id-type=\"pmid\">30655563</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zdanov</surname><given-names>A</given-names></name><name><surname>Schalk-Hihi</surname><given-names>C</given-names></name><name><surname>Wlodawer</surname><given-names>A</given-names></name></person-group>. <article-title>Crystal structure of human interleukin-10 at 1.6 A resolution and a model of a complex with its soluble receptor</article-title>. <source>Protein Sci.</source> (<year>1996</year>) <volume>5</volume>:<fpage>1955</fpage>–<lpage>62</lpage>. <pub-id pub-id-type=\"doi\">10.1002/pro.5560051001</pub-id><pub-id pub-id-type=\"pmid\">8897595</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reddy Chichili</surname><given-names>VP</given-names></name><name><surname>Kumar</surname><given-names>V</given-names></name><name><surname>Sivaraman</surname><given-names>J</given-names></name></person-group>. <article-title>Linkers in the structural biology of protein-protein interactions</article-title>. <source>Protein Sci.</source> (<year>2013</year>) <volume>22</volume>:<fpage>153</fpage>–<lpage>67</lpage>. <pub-id pub-id-type=\"doi\">10.1002/pro.2206</pub-id><pub-id pub-id-type=\"pmid\">23225024</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Soderquist</surname><given-names>RG</given-names></name><name><surname>Milligan</surname><given-names>ED</given-names></name><name><surname>Harrison</surname><given-names>JA</given-names></name><name><surname>Chavez</surname><given-names>RA</given-names></name><name><surname>Johnson</surname><given-names>KW</given-names></name><name><surname>Watkins</surname><given-names>LR</given-names></name><etal/></person-group>. <article-title>PEGylation of interleukin-10 for the mitigation of enhanced pain states</article-title>. <source>J Biomed Mater Res A.</source> (<year>2010</year>) <volume>93</volume>:<fpage>1169</fpage>–<lpage>79</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jbm.a.32611</pub-id><pub-id pub-id-type=\"pmid\">19768789</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Lanvermann</surname><given-names>S</given-names></name></person-group>\n<source>Die Bedeutung von Stabilität und Matrixinteraktion des immunsuppressiven Zytokins Interleukin-10 für seine biologischen Effekte</source>, <publisher-loc>Dresden</publisher-loc>: <publisher-name>Der Technischen Universität Dresden</publisher-name> (<year>2014</year>).</mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Siewe</surname><given-names>L</given-names></name><name><surname>Bollati-Fogolin</surname><given-names>M</given-names></name><name><surname>Wickenhauser</surname><given-names>C</given-names></name><name><surname>Krieg</surname><given-names>T</given-names></name><name><surname>Muller</surname><given-names>W</given-names></name><name><surname>Roers</surname><given-names>A</given-names></name></person-group>\n<article-title>Interleukin-10 derived from macrophages and/or neutrophils regulates the inflammatory response to LPS but not the response to CpG DNA</article-title>. <source>Eur J Immunol.</source> (<year>2006</year>) <volume>36</volume>:<fpage>3248</fpage>–<lpage>55</lpage>. <pub-id pub-id-type=\"doi\">10.1002/eji.200636012</pub-id><pub-id pub-id-type=\"pmid\">17111348</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Asadullah</surname><given-names>K</given-names></name><name><surname>Sterry</surname><given-names>W</given-names></name><name><surname>Volk</surname><given-names>HD</given-names></name></person-group>. <article-title>Interleukin-10 therapy–review of a new approach</article-title>. <source>Pharmacol Rev.</source> (<year>2003</year>) <volume>55</volume>:<fpage>241</fpage>–<lpage>69</lpage>. <pub-id pub-id-type=\"doi\">10.1124/pr.55.2.4</pub-id><pub-id pub-id-type=\"pmid\">12773629</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>MC</given-names></name><name><surname>He</surname><given-names>SH</given-names></name></person-group>. <article-title>IL-10 and its related cytokines for treatment of inflammatory bowel disease</article-title>. <source>World J Gastroenterol.</source> (<year>2004</year>) <volume>10</volume>:<fpage>620</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.3748/wjg.v10.i5.620</pub-id><pub-id pub-id-type=\"pmid\">14991925</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Josephson</surname><given-names>K</given-names></name><name><surname>DiGiacomo</surname><given-names>R</given-names></name><name><surname>Indelicato</surname><given-names>SR</given-names></name><name><surname>Iyo</surname><given-names>AH</given-names></name><name><surname>Nagabhushan</surname><given-names>TL</given-names></name><name><surname>Parker</surname><given-names>MH</given-names></name><etal/></person-group>. <article-title>Design and analysis of an engineered human interleukin-10 monomer</article-title>. <source>J Biol Chem.</source> (<year>2000</year>) <volume>275</volume>:<fpage>13552</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.275.18.13552</pub-id><pub-id pub-id-type=\"pmid\">10788470</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Roers</surname><given-names>A</given-names></name><name><surname>Siewe</surname><given-names>L</given-names></name><name><surname>Strittmatter</surname><given-names>E</given-names></name><name><surname>Deckert</surname><given-names>M</given-names></name><name><surname>Schluter</surname><given-names>D</given-names></name><name><surname>Stenzel</surname><given-names>W</given-names></name><etal/></person-group>\n<article-title>T cell-specific inactivation of the interleukin 10 gene in mice results in enhanced T cell responses but normal innate responses to lipopolysaccharide or skin irritation</article-title>. <source>J Exp Med.</source> (<year>2004</year>) <volume>200</volume>:<fpage>1289</fpage>–<lpage>97</lpage>. <pub-id pub-id-type=\"doi\">10.1084/jem.20041789</pub-id><pub-id pub-id-type=\"pmid\">15534372</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Qiao</surname><given-names>J</given-names></name><name><surname>Liu</surname><given-names>Z</given-names></name><name><surname>Dong</surname><given-names>C</given-names></name><name><surname>Luan</surname><given-names>Y</given-names></name><name><surname>Zhang</surname><given-names>A</given-names></name><name><surname>Moore</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>Targeting tumors with IL-10 prevents dendritic cell-mediated CD8<sup>+</sup> T cell apoptosis</article-title>. <source>Cancer Cell.</source> (<year>2019</year>) <volume>35</volume>:<fpage>901</fpage>–<lpage>15</lpage>.e4. <pub-id pub-id-type=\"doi\">10.1016/j.ccell.2019.05.005</pub-id><pub-id pub-id-type=\"pmid\">31185213</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Immunol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Immunol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Immunol.</journal-id><journal-title-group><journal-title>Frontiers in Immunology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-3224</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849641</article-id><article-id pub-id-type=\"pmc\">PMC7431523</article-id><article-id pub-id-type=\"doi\">10.3389/fimmu.2020.01780</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Immunology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Conformationally Altered C-Reactive Protein Capable of Binding to Atherogenic Lipoproteins Reduces Atherosclerosis</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Pathak</surname><given-names>Asmita</given-names></name><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Singh</surname><given-names>Sanjay K.</given-names></name><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1001588/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Thewke</surname><given-names>Douglas P.</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/999599/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Agrawal</surname><given-names>Alok</given-names></name><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/559726/overview\"/></contrib></contrib-group><aff><institution>Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University</institution>, <addr-line>Johnson City, TN</addr-line>, <country>United States</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Kenji Daigo, Nippon Medical School, Japan</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Tomohide Takaya, Shinshu University, Japan; Lawrence Albert Potempa, Roosevelt University, United States</p></fn><corresp id=\"c001\">*Correspondence: Alok Agrawal <email>agrawal@etsu.edu</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†These authors have contributed equally to this work</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1780</elocation-id><history><date date-type=\"received\"><day>21</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>03</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Pathak, Singh, Thewke and Agrawal.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Pathak, Singh, Thewke and Agrawal</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>The aim of this study was to test the hypothesis that C-reactive protein (CRP) protects against the development of atherosclerosis and that a conformational alteration of wild-type CRP is necessary for CRP to do so. Atherosclerosis is an inflammatory cardiovascular disease and CRP is a plasma protein produced by the liver in inflammatory states. The co-localization of CRP and low-density lipoproteins (LDL) at atherosclerotic lesions suggests a possible role of CRP in atherosclerosis. CRP binds to phosphocholine-containing molecules but does not interact with LDL unless the phosphocholine groups in LDL are exposed. However, CRP can bind to LDL, without the exposure of phosphocholine groups, if the native conformation of CRP is altered. Previously, we reported a CRP mutant, F66A/T76Y/E81A, generated by site-directed mutagenesis, that did not bind to phosphocholine. Unexpectedly, this mutant CRP, without any more conformational alteration, was found to bind to atherogenic LDL. We hypothesized that this CRP mutant, unlike wild-type CRP, could be anti-atherosclerotic and, accordingly, the effects of mutant CRP on atherosclerosis in atherosclerosis-prone LDL receptor-deficient mice were evaluated. Administration of mutant CRP into mice every other day for a few weeks slowed the progression of atherosclerosis. The size of atherosclerotic lesions in the aorta of mice treated with mutant CRP for 9 weeks was ~40% smaller than the lesions in the aorta of untreated mice. Thus, mutant CRP conferred protection against atherosclerosis, providing a proof of concept that a local inflammation-induced structural change in wild-type CRP is a prerequisite for CRP to control the development of atherosclerosis.</p></abstract><kwd-group><kwd>atherosclerosis</kwd><kwd>C-reactive protein</kwd><kwd>inflammation</kwd><kwd>low-density lipoprotein</kwd><kwd>phosphocholine</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Institutes of Health<named-content content-type=\"fundref-id\">10.13039/100000002</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"5\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"55\"/><page-count count=\"8\"/><word-count count=\"6216\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Atherosclerosis is a chronic inflammatory disease whose development begins with the dysfunction of the endothelium of the arteries. Endothelial dysfunction leads to infiltration of plasma low-density lipoprotein (LDL) and monocytes in the arterial wall. LDL is subsequently deposited, modified and engulfed by monocyte-derived macrophages. Lipid-laden macrophage foam cells are proinflammatory which enhances the process of the formation of atherosclerotic lesions (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B2\" ref-type=\"bibr\">2</xref>). The inflammatory microenvironment of atherosclerotic lesions is characterized by macrophage activation, hypoxia, and lactate and proton generation, resulting in an acidic extracellular pH (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>–<xref rid=\"B7\" ref-type=\"bibr\">7</xref>).</p><p>C-reactive protein (CRP) is a plasma protein produced by the liver in inflammatory states (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). CRP is also present, co-localized with modified LDL and macrophages, at atherosclerotic lesions in both humans and experimental animals (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>, <xref rid=\"B10\" ref-type=\"bibr\">10</xref>). CRP is composed of five identical subunits arranged in a pentameric symmetry. The molecular weight of each subunit is ~23 kDa and there are 206 amino acid residues in each subunit. CRP binds to molecules with exposed phosphocholine (PCh) groups in a Ca<sup>2+</sup>-dependent manner. There are five PCh-binding sites in the CRP pentamer, one site per subunit (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B12\" ref-type=\"bibr\">12</xref>).</p><p>Oxidized LDL (ox-LDL), enzymatically-modified LDL (E-LDL) and acetylated LDL (ac-LDL) are different forms of modified atherogenic LDL that are used in <italic>in vitro</italic> experiments (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>). In the presence of Ca<sup>2+</sup>, native or recombinant wild-type (WT) CRP interacts with E-LDL due to the exposure of PCh groups on E-LDL, but does not interact with ox-LDL and ac-LDL (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>–<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). In the absence of Ca<sup>2+</sup>, WT CRP does not interact with any form of atherogenic LDL (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>–<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). The native pentameric structure of CRP is altered in response to a variety of experimental conditions (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>–<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). It has been shown that the pentameric structure of WT CRP is subtly altered by acidic pH and by oxidation, and conformationally altered CRP is capable of binding to atherogenic LDL independent of the PCh-binding site (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>–<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Employing E-LDL, it has also been shown that if CRP is bound to atherogenic LDL, it prevents the formation of lipid-laden macrophage foam cells (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). Due to the presence of CRP at atherosclerotic lesions and due to the binding capability of CRP for atherogenic LDL under certain conditions, CRP has been implicated in the development of atherosclerosis (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>).</p><p>We have previously reported a CRP mutant, F66A/T76Y/E81A, in which Phe<sup>66</sup>, Thr<sup>76</sup>, and Glu<sup>81</sup> (three critical amino acid residues in the PCh-binding site) were substituted with Ala, Tyr, and Ala, respectively, to abolish the PCh-binding activity of CRP (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). Mutant CRP did not bind to PCh and was used as a tool to investigate the importance of the PCh-binding site in CRP-mediated protection of mice against pneumococcal infection (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). Biochemical characterization of this mutant CRP, in comparison to that of WT CRP, has been published previously (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). The overall structure of mutant CRP was pentameric and the mutation did not affect the stability of the protein <italic>in vivo</italic>. Mutant CRP circulated freely in the mouse serum and its rate of clearance <italic>in vivo</italic> was similar to that of WT CRP (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>).</p><p>Further analysis of F66A/T76Y/E81A mutant CRP, presented here, revealed that mutant CRP had inadvertently gained the ability to bind to any protein that was immobilized on microtiter plates, including atherogenic forms of LDL, without the need for any further inflammatory milieu-dependent or acidic pH-induced structural change. We, therefore, hypothesized that mutant CRP might show an atheroprotective effect in murine models of atherosclerosis. We reasoned that even if there was no acidic pH around injected mutant CRP in the available murine models, mutant CRP would be able to recognize and bind atherogenic LDL and exert a protective effect on the development of atherosclerosis. Accordingly, in this study, we evaluated the effects of mutant CRP (F66A/T76Y/E81A) on the development of atherosclerosis employing LDL receptor-deficient (<italic>Ldlr</italic><sup>−/−</sup>) mice, an animal model commonly used to investigate molecules involved in human atherosclerosis.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Preparation of F66A/T76Y/E81A Mutant CRP</title><p>The construction of F66A/T76Y/E81A mutant CRP cDNA used in this study has been reported previously (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). Mutant CRP was expressed in CHO cells using the ExpiCHO Expression System (Thermo Fisher Scientific), according to manufacturer's instructions. As described previously (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>), mutant CRP was purified from cell culture supernatant by Ca<sup>2+</sup>-dependent affinity chromatography on a phosphoethanolamine-Sepharose column, since this CRP mutant does not bind to PCh. Mutant CRP was further purified by gel filtration on a Superose12 column. Eluted mutant CRP was immediately dialyzed against 10 mM Tris-HCl, 150 mM NaCl, pH 7.2 (TBS), containing 2 mM CaCl<sub>2</sub>, stored at 4°C, and was used within a week. The purity of CRP was confirmed by using denaturing SDS-PAGE. For <italic>in vivo</italic> experiments, purified mutant CRP was treated with the Detoxi-Gel Endotoxin Removing Gel (Thermo Fisher Scientific) according to manufacturer's instructions. The removal of endotoxin from mutant CRP preparations was confirmed by using the Limulus Amebocyte Lysate kit QCL-1000 (Lonza) according to manufacturer's instructions.</p></sec><sec><title>Solid Phase Ligand-Binding Assay</title><p>The solid phase ligand-binding assay was used to determine the binding of mutant CRP to immobilized proteins, as described earlier (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>–<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Ox-LDL, E-LDL, ac-LDL, factor H (Complement Technology) and amyloid β peptide 1-42 (Bachem, H-1368) were used as protein ligands. Ox-LDL, E-LDL, and ac-LDL were prepared as described previously (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>–<xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B24\" ref-type=\"bibr\">24</xref>). Briefly, microtiter wells were coated with protein ligands (10 μg/ml) diluted in TBS and incubated overnight at 4°C. The unreacted sites in the wells were blocked with TBS containing 0.5% gelatin. Freshly purified mutant CRP was diluted in TBS-Ca (TBS containing 2 mM CaCl<sub>2</sub>, 0.1% gelatin and 0.02% Tween 20), added to the wells, and incubated overnight at 4°C. Bound mutant CRP was detected by using a polyclonal rabbit anti-human CRP antibody (Millipore Sigma, 235752). HRP-conjugated donkey anti-rabbit IgG (GE Healthcare) was used as the secondary antibody. Color was developed using ABTS as the substrate and the OD<sub>405</sub> was read in a plate reader.</p></sec><sec><title>Atherosclerosis Protection Experiments</title><p>Eight-week-old C57BL/6 male <italic>ldlr</italic><sup>−/−</sup> mice (Jackson Lab, 002207) were used in experiments according to protocols approved by and conducted in accordance with the guidelines administered by the Institutional Animal Care and Usage Committee of East Tennessee State University. Sixty mice were fed on a high-fat western-type diet (21% fat, 0.2% cholesterol), purchased from Envigo (TD.88137), for 10 weeks. After 1 week on high-fat diet, mice were divided into two groups of 30 mice in each group: untreated group and mutant CRP-treated group. Mice were injected with either TBS (untreated group) or mutant CRP (50 μg/injection) on alternate days for 9 weeks, via alternating intravenous and intraperitoneal routes (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). Blood, heart and aorta were collected at five different time points (weeks 1, 3, 5, 7, and 9), as described previously (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). Briefly, six mice from each group, at each time point, were sacrificed, blood was collected by cardiac puncture, and the plasma was separated and stored frozen. After collecting blood, the heart was perfused with 10% formalin, followed by removing fat from the entire aorta. Heart was excised from the aorta, was cut into two halves, and the upper half with the aortic root was mounted using OCT medium and stored at −80°C. Following heart excision, aorta was excised and stored in 10% formalin at 4°C. The whole experiment was repeated once more employing another 60 mice and a separate, freshly purified batch of mutant CRP.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Assessment of atherosclerotic lesions in <italic>ldlr</italic><sup>−/−</sup> mice. <bold>(A)</bold> Protocol for administration of mutant CRP and for sacrifice of mice after mutant CRP administration. <bold>(B)</bold> Quantitation of atherosclerotic lesions in the aorta (<italic>en face</italic>). A representative Sudan IV-stained aorta is shown. The red colored areas are the lesions. Scale bar, 5 mm. <bold>(C)</bold> Quantitation of atherosclerotic lesions in the aortic root. A representative Oil Red O-stained aortic root section is shown. The red colored areas are the lesions. Scale bar, 100 μm.</p></caption><graphic xlink:href=\"fimmu-11-01780-g0001\"/></fig></sec><sec><title>Measurement of the Size of Atherosclerotic Lesions</title><p>To measure the size of atherosclerotic lesions in the whole aorta (<italic>en face</italic>), aorta was cut open longitudinally, stained with Sudan IV, and digitally photographed, as described previously (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). A total of 120 aortae (60 mice for untreated group and 60 mice for mutant CRP-treated group; 12 mice per time point) were processed, stained and photographed. A representative Sudan IV-stained aorta is shown in <xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>.</p><p>To measure the size of atherosclerotic lesions in the aortic root, 8 μm cross-sections of frozen OCT-embedded heart were collected from the appearance of the aortic valve leaflets to their disappearance (48–72 sections per heart). Every other cross-section of the entire aortic root was stained with Oil Red O for lipids, counterstained with hematoxylin, and digitally photographed, as described previously (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). A total of 120 hearts (60 mice for untreated group and 60 mice for mutant CRP-treated group; 12 mice per time point) were processed. A representative Oil Red O-stained aortic root section is shown in <xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref>.</p><p>Digital photographs were acquired with an Olympus BX41 microscope equipped with a CCD color camera (QImaging). The stained lesion areas were quantified in digital images using the ImageJ software (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). Quantification of the lesion areas in the photographs was performed by two observers blinded to the experimental protocol. Non-parametric test (Mann-Whitney test) using GraphPad Prism software was employed to calculate the <italic>p</italic>-values.</p></sec><sec><title>Immunostaining of CRP</title><p>Sudan IV-stained aorta was first processed to remove the stain, as described earlier (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). Immunostaining of CRP was performed using Vectastain ABC Elite kit (Vector laboratories, PK-6100) according to manufacturer's instructions. CRP was detected by using a polyclonal rabbit anti-human CRP antibody (Millipore Sigma, 235752). Biotinylated goat-anti rabbit IgG was used as the secondary antibody. Color was developed using DAB (Vector laboratories, ImmPACT DAB, SK-4105) as the substrate, according to manufacturer's instructions.</p></sec><sec><title>Measurement of Lipoproteins in the Plasma</title><p>The concentrations of high-density lipoprotein (HDL) and LDL in the plasma were measured using Cholesterol Assay Kit-HDL and LDL/VLDL (Abcam; ab65390) according to manufacturer's instructions. Lipoprotein levels were measured in the pooled plasma samples collected at weeks 1, 3, 5, 7, and 9 (12 mice per time point). Unpaired student <italic>t</italic>-test was employed to calculate the <italic>p</italic>-values.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><p>All experiments were performed three times, unless otherwise mentioned, and comparable results were obtained each time. Results of a representative experiment are shown in the figures where the raw data (OD<sub>405</sub>) were used to plot the curves.</p><sec><title>Mutant CRP Binds to Atherogenic LDL</title><p>As shown in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>, mutant CRP bound to all three forms of atherogenic LDL immobilized on microtiter wells. The binding occurred at physiological conditions, that is, in the absence of any protein structure-modifying agent, such as acidic pH. Since mutant CRP does not bind to PCh (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>), the binding of mutant CRP to either ox-LDL, E-LDL, or ac-LDL was independent of the PCh groups present in atherogenic LDL. In addition to testing the binding of mutant CRP to atherogenic LDL, we also included two other proteins, factor H and amyloid β peptide, in the binding assay. Mutant CRP also bound to factor H and amyloid β peptide in a concentration-dependent manner, similar to its binding to atherogenic LDL. These results suggest that mutant CRP did not recognize immobilized atherogenic LDL <italic>per se</italic>, but it recognized a pattern, as yet undefined, on immobilized proteins in general.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Binding of F66A/T76Y/E81A mutant CRP to immobilized proteins including atherogenic LDL at physiological pH. Microtiter wells were coated with protein ligands as shown. After blocking the unreacted sites in the wells, mutant CRP diluted in TBS-Ca was added to the wells and incubated for 2 h at 37°C. Bound mutant CRP was detected by using a rabbit anti-human CRP antibody and HRP-conjugated donkey anti-rabbit IgG. Color was developed and the OD was read at 405 nm. A representative of three experiments are shown.</p></caption><graphic xlink:href=\"fimmu-11-01780-g0002\"/></fig></sec><sec><title>Mutant CRP Reduces Atherosclerotic Lesions in the Whole Aorta</title><p>The total size of all lesion areas in the whole aorta (<italic>en face</italic>) was measured. <xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref> shows the combined results of two separate experiments with 12 mice in each group, for each time point. There was no effect of mutant CRP on the <italic>en face</italic> lesions for the first 5 weeks. The effect of mutant CRP on the lesion size was visible once the disease had progressed further. In comparison to untreated mice, the lesion area in mutant CRP-treated mice was 30.9% less after 7 weeks and 42% less after 9 weeks of CRP administration. As shown, in mutant CRP-treated mice, the lesion area did not increase after 5 weeks unlike in the untreated group where the lesion area kept increasing for another 2 weeks. Similar results were seen when the data from each of the two experiments (6 mice/group/time point/experiment) were analyzed separately (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Figure 1</xref>). We did not determine the specific stages of atherosclerosis at any time point.</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Reduction in the size of atherosclerotic lesions in the aorta of mutant CRP-treated mice. The scatter plot shows the total atherosclerotic lesion area in <italic>en face</italic> aorta from untreated and mutant CRP-treated mice. Data were collected at five different time points: 1, 3, 5, 7, and 9 weeks after mutant CRP administration (12 mice per group per time point). Each blue dot represents one mouse from the untreated group and each green dot represents one mouse from the mutant CRP-treated group. Horizontal black lines indicate the median total lesion area for each group. Asterisks denote statistically significant difference between untreated and mutant CRP-treated groups (*<italic>p</italic> ≤ 0.01).</p></caption><graphic xlink:href=\"fimmu-11-01780-g0003\"/></fig></sec><sec><title>The Effects of Mutant CRP Are Not Visible at the Aortic Root Lesions</title><p>The total size of all lesion areas in the aortic root was measured. <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref> shows the combined results of two separate experiments using 12 mice in each group, for each time point. As shown, the disease progressed for the entire duration of 9 weeks in untreated mice. However, the administration of mutant CRP did not affect the lesion size at any time point. There was no statistically significant difference in the lesion size at any time point between untreated and mutant CRP-treated groups of mice. Similar results were seen when the data from each of the two experiments (6 mice/group/time point/experiment) were analyzed separately (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplemental Figure 2</xref>).</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>No reduction in the size of atherosclerotic lesions in the aortic root of mutant CRP-treated mice. The scatter plot shows the total atherosclerotic lesion area in aortic root sections from untreated and mutant CRP-treated mice. Data were collected at five different time points: 1, 3, 5, 7, and 9 weeks after mutant CRP administration (12 mice per group per time point). Each blue dot represents one mouse from the untreated group and each green dot represents one mouse from the mutant CRP-treated group. Horizontal black lines indicate the median total aortic root lesion area for each group.</p></caption><graphic xlink:href=\"fimmu-11-01780-g0004\"/></fig></sec><sec><title>Administered Mutant CRP Is Present in the Aorta</title><p>Immunostaining of CRP in the aorta isolated from mice treated with mutant CRP for seven weeks was performed to confirm the presence of administered mutant CRP at the <italic>en face</italic> atherosclerotic lesions in mutant CRP-treated mice. As shown (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>), the lesions in the aorta of mutant CRP-treated mice were stained while the lesions in the aorta of untreated mice did not stain for CRP. The polyclonal anti-human CRP antibody used in this study does not react with purified murine CRP (data not shown), consistent with the previously published report that anti-human CRP antibodies do not react with murine CRP (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>).</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>Immunostaining of mutant CRP in the aorta. <bold>(A)</bold> A representative aorta from untreated mice stained for mutant CRP. <bold>(B)</bold> A representative aorta from mice treated with mutant CRP for 7 weeks, stained for CRP. 1–4 represent an aorta through different stages of staining. (1) Sudan IV-stained aorta. Red colored areas are the lesions. (2) Aorta post-dehydration to remove Sudan IV. White colored areas are the lesion areas. (3) Anti-human CRP antibody-stained aorta. Brown colored areas reflect the presence of mutant CRP in the lesions. (4) Magnified aortic arch area of the anti-human CRP antibody-stained aorta. Red boxes show the lesion areas.</p></caption><graphic xlink:href=\"fimmu-11-01780-g0005\"/></fig></sec><sec><title>Mutant CRP Does Not Alter the Concentration of Lipoproteins in the Plasma</title><p>Injecting a total of 1.6 mg of mutant CRP into mouse, in 32 injections over a period of 9 weeks, did not alter the concentration of lipoproteins in the circulation. As shown in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, the concentrations of LDL and HDL in the plasma of untreated and mutant CRP-treated mice were comparable. There was no statistically significant difference in the levels of either LDL or HDL in the plasma at any time point between untreated and mutant CRP-treated groups of mice. Administration of mutant CRP did not affect the body weight. Body weights of untreated and mutant CRP-treated mice were similar at each time point (data not shown).</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Concentration of lipoproteins in the plasma.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Week of sacrifice</bold></th><th valign=\"top\" align=\"center\" colspan=\"2\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>HDL (mg/dl)</bold></th><th valign=\"top\" align=\"center\" colspan=\"2\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>LDL (mg/dl)</bold></th></tr><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Untreated</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>CRP-treated</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Untreated</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>CRP-treated</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">61 ± 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">85 ± 19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">864 ± 16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">713 ± 31</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">91 ± 15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">71 ± 19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">838 ± 231</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">786 ± 246</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">78 ± 4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">74 ± 12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">754 ± 118</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">834 ± 185</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">131 ± 1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">107 ± 15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">932 ± 86</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>1, 086</italic>±23</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">105 ± 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>1, 054</italic>±3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>1, 103</italic>±19</td></tr></tbody></table><table-wrap-foot><p><italic>Plasma HDL and LDL levels were analyzed in samples pooled from all 12 mice in each group. Results are expressed as mean ± SEM</italic>.</p></table-wrap-foot></table-wrap></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>In this study, we investigated the effects of mutant CRP F66A/T76Y/E81A, capable of binding to atherogenic LDL but incapable of binding to PCh, on the development of atherosclerosis in male <italic>ldlr</italic><sup>−/−</sup> mice. Our major finding was that the administration of mutant CRP for 7 weeks slowed the progression of atherosclerosis. The total size of atherosclerotic lesions in the whole aorta of mice treated with mutant CRP on alternate days for 7 weeks and beyond was significantly smaller (~40%) than the lesions in the aorta of untreated mice.</p><p>Previously, the role of WT CRP in atherosclerosis had been explored, employing a variety of experimental strategies, and 12 papers have been published using CRP from man, mouse and rabbit in both murine and rabbit models of atherosclerosis (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>–<xref rid=\"B42\" ref-type=\"bibr\">42</xref>). In these studies, both, normal mice and CRP-deficient mice have been employed. Both, normal rabbits and rabbits in which CRP was inhibited by using anti-sense technology have been employed. Both, passively administered CRP and transgenic human CRP have been employed. Three different types of atherosclerosis-prone mice, <italic>apoE</italic><sup>−/−</sup>, <italic>ldlr</italic><sup>−/−</sup>, and <italic>apoB</italic><sup><italic>100/100</italic></sup>\n<italic>ldlr</italic><sup>−/−</sup>, have been used in these studies. Two of the 12 papers indicated that both human and murine CRP might be playing an atheroprotective role: human CRP was shown to slow the development of atherosclerosis in the <italic>apoB</italic><sup><italic>100/100</italic></sup>\n<italic>ldlr</italic><sup>−/−</sup> mice (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>), and the lesion size in CRP-deficient mice on <italic>apoE</italic><sup>−/−</sup> or <italic>ldlr</italic><sup>−/−</sup> background was either equivalent or increased compared to that in normal mice (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>). In the other 10 papers, regardless of the experimental strategy used, WT CRP from all species did not show any effect on the development of atherosclerosis in animals, suggesting that WT CRP is neither pro-atherogenic nor anti-atherogenic (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B43\" ref-type=\"bibr\">43</xref>).</p><p>The most logical explanation for the observations of zero or nominal effects of WT CRP (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>–<xref rid=\"B42\" ref-type=\"bibr\">42</xref>) on atherosclerosis in animal models is that the animal models of atherosclerosis do not possess the required inflammatory microenvironment that is needed by CRP to change its structure and be able to interact with atherogenic LDL (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B25\" ref-type=\"bibr\">25</xref>). WT CRP shows no effect because pH near the lesions may not be acidic in animal models and, therefore, the structure of administered or endogenous WT CRP remains unchanged (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B25\" ref-type=\"bibr\">25</xref>). WT CRP treated with acidic pH <italic>in vitro</italic> was unsuitable for administration into blood circulation of animals since the acidic pH-induced conformational alteration in the pentameric structure of CRP was found to be reversible at physiological pH (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). Therefore, we hypothesized that an <italic>in vitro</italic>-generated mutant CRP, capable of binding to atherogenic LDL without the requirement of any structural modification <italic>in vivo</italic>, would be suitable to investigate the mechanism of action of CRP on the development of the disease in animal models (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Indeed, in one study, monomeric CRP, that is also capable of binding to atherogenic LDL, was employed and the results showed that monomeric CRP was protective against atherosclerosis in <italic>apoE</italic><sup>−/−</sup> mice (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). Thus, our current findings using mutant CRP and previous findings using monomeric CRP, both molecules capable of binding to atherogenic LDL, indicate that structurally altered CRP protects against atherosclerosis; it is just that WT CRP does not exert a protective effect in most animal models (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B44\" ref-type=\"bibr\">44</xref>).</p><p>The effect of mutant CRP on the size of atherosclerotic lesions in the <italic>en face</italic> aorta was obvious since we found that administered mutant CRP had reached the aorta. The staining of aortic roots for the presence of administered mutant CRP provided inconclusive results (data not shown). It is assumed that if mutant CRP reached the aorta, it also reached the aortic root, and if this assumption is correct, then the effect of mutant CRP was site-specific. Site-specific effects of experimental manipulations have been observed in other studies using <italic>ldlr</italic><sup>−/−</sup> mice where the disease developed differently at various lesion-prone sites (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>–<xref rid=\"B50\" ref-type=\"bibr\">50</xref>). Since the effects of mutant CRP were still observed after week 9 in the <italic>en face</italic> aorta, it is unlikely that anti-CRP antibodies were produced in response to intravenous administration of mutant CRP that could have inhibited its functions.</p><p>The topology of the LDL-binding site and the number of LDL-binding sites on mutant CRP remain undefined. Two possible mechanisms have been proposed for the interaction between conformationally altered CRP and atherogenic LDL. The intrinsically disordered region present in CRP has been shown to participate in the binding of monomeric CRP and atherogenic LDL (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>). We proposed that the loosening of the CRP pentamer contributed to the formation of the LDL-binding site (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>–<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Recently, it has been suggested that the pentameric assembly of CRP harbors a pronounced plasticity in inter-subunit interactions, which may form the basis for a reversible activation of CRP in inflammation (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>). However, it is unknown whether the intrinsically disordered region was exposed or the pentamer was loosened in mutant CRP employed in this study. We speculate that there is only one LDL-binding site per CRP pentamer. However, for the protection of mice against atherosclerosis, it is possible that both sites, the PCh-binding site and the LDL-binding site, participate. The search for a new mutant CRP which can bind to both PCh and to atherogenic LDL is in progress. Any possible similarity between mutant CRP used in this study and previously reported conformationally altered pentameric forms of CRP, pCRP<sup>*</sup>, and mCRPm (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>, <xref rid=\"B21\" ref-type=\"bibr\">21</xref>), is unknown.</p><p>Based on the data obtained from a single regimen for mutant CRP treatment, we conclude that one of the functions of CRP is to confer protection against atherosclerosis. We propose, again, that there is no need to stop the biosynthesis of CRP, and that a drug that can lower cholesterol level but not CRP levels could be superior to statins which reduce both CRP and cholesterol levels (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>, <xref rid=\"B54\" ref-type=\"bibr\">54</xref>). These suggestions are supported by the fact that, in rabbits, the inhibition of plasma CRP did not affect the development of atherosclerosis (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>). It has also been previously postulated that the deposition of CRP at the atherosclerotic lesions may be independent of the CRP levels in the circulation and that CRP-mediated lipoprotein removal likely underlies the regression of early lesions which occurs continuously throughout life and that CRP should be considered as an anti-atherosclerotic protein (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>). A long-term goal should be the discovery and design of small-molecule compounds to aid endogenous native CRP in capturing atherogenic LDL, as proposed earlier (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>, <xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Another goal could be to investigate the possible protective effects of mutant CRP used in this study in animal models of other inflammatory diseases.</p><p>Our data also provide a proof of concept that a local inflammation-induced structural change in native CRP is a prerequisite for CRP to control the development of atherosclerosis. An appropriate inflammatory microenvironment at the site of LDL deposition seems to be critical for CRP to prevent atherosclerosis. One function of inflammation could be to change the structure of proteins, including CRP. Inflammation is not a silent killer, perhaps, as has been suggested (<xref rid=\"B55\" ref-type=\"bibr\">55</xref>).</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>All datasets generated for this study are included in the article/<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Material</xref>.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by University Committee on Animal Care, East Tennessee State University.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>AP, SS, and DT performed the experiments. AP and AA analyzed the data. AA conceived and designed the experiments and wrote the paper. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by National Institutes of Health Grant AR068787.</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s9\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fimmu.2020.01780/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fimmu.2020.01780/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Image_1.JPEG\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM2\"><media xlink:href=\"Image_2.JPEG\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Libby</surname><given-names>P</given-names></name><name><surname>Buring</surname><given-names>JE</given-names></name><name><surname>Badimon</surname><given-names>L</given-names></name><name><surname>Hansson</surname><given-names>GK</given-names></name><name><surname>Deanfield</surname><given-names>J</given-names></name><name><surname>Bittencourt</surname><given-names>MS</given-names></name><etal/></person-group>\n<article-title>Atherosclerosis</article-title>. <source>Nat Rev Dis Primers.</source> (<year>2019</year>) <volume>5</volume>:<fpage>56</fpage>\n<pub-id pub-id-type=\"doi\">10.1038/s41572-019-0106-z</pub-id><pub-id pub-id-type=\"pmid\">31420554</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Galkina</surname><given-names>E</given-names></name><name><surname>Ley</surname><given-names>K</given-names></name></person-group>. <article-title>Immune and inflammatory mechanisms of atherosclerosis</article-title>. <source>Annu Rev Immunol.</source> (<year>2009</year>) <volume>27</volume>:<fpage>165</fpage>–<lpage>97</lpage>. <pub-id pub-id-type=\"doi\">10.1146/annurev.immunol.021908.132620</pub-id><pub-id pub-id-type=\"pmid\">19302038</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Leake</surname><given-names>DS</given-names></name></person-group>. <article-title>Does an acidic pH explain why low-density lipoprotein is oxidised in atherosclerotic lesions?</article-title>\n<source>Atherosclerosis.</source> (<year>1997</year>) <volume>129</volume>:<fpage>149</fpage>–<lpage>57</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0021-9150(96)06035-2</pub-id><pub-id pub-id-type=\"pmid\">9105556</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Björnheden</surname><given-names>T</given-names></name><name><surname>Levin</surname><given-names>M</given-names></name><name><surname>Evaldsson</surname><given-names>M</given-names></name><name><surname>Wiklund</surname><given-names>O</given-names></name></person-group>. <article-title>Evidence of hypoxic areas within the arterial wall in vivo</article-title>. <source>Arterioscler Thromb Vasc Biol.</source> (<year>1999</year>) <volume>19</volume>:<fpage>870</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1161/01.ATV.19.4.870</pub-id><pub-id pub-id-type=\"pmid\">10195911</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Naghavi</surname><given-names>M</given-names></name><name><surname>John</surname><given-names>R</given-names></name><name><surname>Naguib</surname><given-names>S</given-names></name><name><surname>Siadaty</surname><given-names>MS</given-names></name><name><surname>Grasu</surname><given-names>R</given-names></name><name><surname>Kurian</surname><given-names>KC</given-names></name><etal/></person-group>. <article-title>pH heterogeneity of human and rabbit atherosclerotic plaques; a new insight into detection of vulnerable plaque</article-title>. <source>Atherosclerosis.</source> (<year>2002</year>) <volume>164</volume>:<fpage>27</fpage>–<lpage>35</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0021-9150(02)00018-7</pub-id><pub-id pub-id-type=\"pmid\">12119190</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sneck</surname><given-names>M</given-names></name><name><surname>Kovanen</surname><given-names>PT</given-names></name><name><surname>Öörni</surname><given-names>K</given-names></name></person-group>. <article-title>Decrease in pH strongly enhances binding of native, proteolyzed, lipolyzed, and oxidized low density lipoprotein particles to human aortic proteoglycans</article-title>. <source>J Biol Chem.</source> (<year>2005</year>) <volume>280</volume>:<fpage>37449</fpage>–<lpage>54</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M508565200</pub-id><pub-id pub-id-type=\"pmid\">16147996</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haka</surname><given-names>AS</given-names></name><name><surname>Grosheva</surname><given-names>I</given-names></name><name><surname>Chiang</surname><given-names>E</given-names></name><name><surname>Buxbaum</surname><given-names>AR</given-names></name><name><surname>Baird</surname><given-names>BA</given-names></name><name><surname>Pierini</surname><given-names>LM</given-names></name><etal/></person-group>. <article-title>Macrophages create an acidic extracellular hydrolytic compartment to digest aggregated lipoproteins</article-title>. <source>Mol Biol Cell.</source> (<year>2009</year>) <volume>20</volume>:<fpage>4932</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1091/mbc.e09-07-0559</pub-id><pub-id pub-id-type=\"pmid\">19812252</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gabay</surname><given-names>C</given-names></name><name><surname>Kushner</surname><given-names>I</given-names></name></person-group>. <article-title>Acute-phase proteins and other systemic responses to inflammation</article-title>. <source>N Engl J Med.</source> (<year>1999</year>) <volume>340</volume>:<fpage>448</fpage>–<lpage>54</lpage>. <pub-id pub-id-type=\"doi\">10.1056/NEJM199902113400607</pub-id><pub-id pub-id-type=\"pmid\">9971870</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sun</surname><given-names>H</given-names></name><name><surname>Koike</surname><given-names>T</given-names></name><name><surname>Ichikawa</surname><given-names>T</given-names></name><name><surname>Hatakeyama</surname><given-names>K</given-names></name><name><surname>Shiomi</surname><given-names>M</given-names></name><name><surname>Zhang</surname><given-names>B</given-names></name><etal/></person-group>. <article-title>C-reactive protein in atherosclerotic lesions: Its origin and pathophysiological significance</article-title>. <source>Am J Pathol.</source> (<year>2005</year>) <volume>167</volume>:<fpage>1139</fpage>–<lpage>48</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0002-9440(10)61202-3</pub-id><pub-id pub-id-type=\"pmid\">16192648</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Agrawal</surname><given-names>A</given-names></name><name><surname>Hammond</surname><given-names>DJ</given-names><suffix>Jr</suffix></name><name><surname>Singh</surname><given-names>SK</given-names></name></person-group>. <article-title>Atherosclerosis-related functions of C-reactive protein</article-title>. <source>Cardiovasc Haematol Disord Drug Targets.</source> (<year>2010</year>) <volume>10</volume>:<fpage>235</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.2174/187152910793743841</pub-id><pub-id pub-id-type=\"pmid\">20932269</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Volanakis</surname><given-names>JE</given-names></name><name><surname>Kaplan</surname><given-names>MH</given-names></name></person-group>. <article-title>Specificity of C-reactive protein for choline phosphate residues of pneumococcal C-polysaccharide</article-title>. <source>Proc Soc Exp Biol Med.</source> (<year>1971</year>) <volume>136</volume>:<fpage>612</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.3181/00379727-136-35323</pub-id><pub-id pub-id-type=\"pmid\">4395924</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thompson</surname><given-names>D</given-names></name><name><surname>Pepys</surname><given-names>MB</given-names></name><name><surname>Wood</surname><given-names>SP</given-names></name></person-group>. <article-title>The physiological structure of human C-reactive protein and its complex with phosphocholine</article-title>. <source>Structure.</source> (<year>1999</year>) <volume>7</volume>:<fpage>169</fpage>–<lpage>77</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0969-2126(99)80023-9</pub-id><pub-id pub-id-type=\"pmid\">10368284</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Torzewski</surname><given-names>M</given-names></name></person-group>. <article-title>Enzymatically modified LDL, atherosclerosis and beyond: paving the way to acceptance</article-title>. <source>Front Biosci.</source> (<year>2018</year>) <volume>23</volume>:<fpage>1257</fpage>–<lpage>71</lpage>. <pub-id pub-id-type=\"doi\">10.2741/4642</pub-id><pub-id pub-id-type=\"pmid\">28930598</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bhakdi</surname><given-names>S</given-names></name><name><surname>Torzewski</surname><given-names>M</given-names></name><name><surname>Klouche</surname><given-names>M</given-names></name><name><surname>Hemmes</surname><given-names>M</given-names></name></person-group>. <article-title>Complement and atherogenesis: binding of CRP to degraded, nonoxidized LDL enhances complement activation</article-title>. <source>Arterioscler Thromb Vasc Biol.</source> (<year>1999</year>) <volume>19</volume>:<fpage>2348</fpage>–<lpage>54</lpage>. <pub-id pub-id-type=\"doi\">10.1161/01.ATV.19.10.2348</pub-id><pub-id pub-id-type=\"pmid\">10521363</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chang</surname><given-names>M-K</given-names></name><name><surname>Binder</surname><given-names>CJ</given-names></name><name><surname>Torzewski</surname><given-names>M</given-names></name><name><surname>Witztum</surname><given-names>JL</given-names></name></person-group>. <article-title>C-reactive protein binds to both oxidized LDL and apoptotic cells through recognition of a common ligand: phosphorylcholine of oxidized phospholipids</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2002</year>) <volume>99</volume>:<fpage>13043</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.192399699</pub-id><pub-id pub-id-type=\"pmid\">12244213</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Agrawal</surname><given-names>A</given-names></name><name><surname>Gang</surname><given-names>TB</given-names></name><name><surname>Rusiñol</surname><given-names>AE</given-names></name></person-group>. <article-title>Recognition functions of pentameric C-reactive protein in cardiovascular disease</article-title>. <source>Mediators Inflamm.</source> (<year>2014</year>) <volume>2014</volume>:<fpage>319215</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2014/319215</pub-id><pub-id pub-id-type=\"pmid\">24948846</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hammond</surname><given-names>DJ</given-names><suffix>Jr</suffix></name><name><surname>Singh</surname><given-names>SK</given-names></name><name><surname>Thompson</surname><given-names>JA</given-names></name><name><surname>Beeler</surname><given-names>BW</given-names></name><name><surname>Rusiñol</surname><given-names>AE</given-names></name><name><surname>Pangburn</surname><given-names>MK</given-names></name><etal/></person-group>. <article-title>Identification of acidic pH-dependent ligands of pentameric C-reactive protein</article-title>. <source>J Biol Chem.</source> (<year>2010</year>) <volume>285</volume>:<fpage>36235</fpage>–<lpage>44</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M110.142026</pub-id><pub-id pub-id-type=\"pmid\">20843812</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Singh</surname><given-names>SK</given-names></name><name><surname>Thirumalai</surname><given-names>A</given-names></name><name><surname>Hammond</surname><given-names>DJ</given-names><suffix>Jr</suffix></name><name><surname>Pangburn</surname><given-names>MK</given-names></name><name><surname>Mishra</surname><given-names>VK</given-names></name><name><surname>Johnson</surname><given-names>DA</given-names></name><etal/></person-group>. <article-title>Exposing a hidden functional site of C-reactive protein by site-directed mutagenesis</article-title>. <source>J Biol Chem.</source> (<year>2012</year>) <volume>287</volume>:<fpage>3550</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M111.310011</pub-id><pub-id pub-id-type=\"pmid\">22158621</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Singh</surname><given-names>SK</given-names></name><name><surname>Thirumalai</surname><given-names>A</given-names></name><name><surname>Pathak</surname><given-names>A</given-names></name><name><surname>Ngwa</surname><given-names>DN</given-names></name><name><surname>Agrawal</surname><given-names>A</given-names></name></person-group>. <article-title>Functional transformation of C-reactive protein by hydrogen peroxide</article-title>. <source>J Biol Chem.</source> (<year>2017</year>) <volume>292</volume>:<fpage>3129</fpage>–<lpage>36</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M116.773176</pub-id><pub-id pub-id-type=\"pmid\">28096464</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ji</surname><given-names>S-R</given-names></name><name><surname>Wu</surname><given-names>Y</given-names></name><name><surname>Zhu</surname><given-names>L</given-names></name><name><surname>Potempa</surname><given-names>LA</given-names></name><name><surname>Sheng</surname><given-names>F-L</given-names></name><name><surname>Lu</surname><given-names>W</given-names></name><etal/></person-group>. <article-title>Cell membranes and liposomes dissociate C-reactive protein (CRP) to form a new, biologically active structural intermediate: mCRPm</article-title>. <source>FASEB J.</source> (<year>2007</year>) <volume>21</volume>:<fpage>284</fpage>–<lpage>94</lpage>. <pub-id pub-id-type=\"doi\">10.1096/fj.06-6722com</pub-id><pub-id pub-id-type=\"pmid\">17116742</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Braig</surname><given-names>D</given-names></name><name><surname>Nero</surname><given-names>TL</given-names></name><name><surname>Koch</surname><given-names>H-G</given-names></name><name><surname>Kaiser</surname><given-names>B</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name><name><surname>Thiele</surname><given-names>JR</given-names></name><etal/></person-group>. <article-title>Transitional changes in the CRP structure lead to the exposure of proinflammatory binding sites</article-title>. <source>Nat Commun.</source> (<year>2017</year>) <volume>8</volume>:<fpage>14188</fpage>. <pub-id pub-id-type=\"doi\">10.1038/ncomms14188</pub-id><pub-id pub-id-type=\"pmid\">28112148</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>S-L</given-names></name><name><surname>Feng</surname><given-names>J-R</given-names></name><name><surname>Zhou</surname><given-names>H-H</given-names></name><name><surname>Zhang</surname><given-names>C-M</given-names></name><name><surname>Lv</surname><given-names>G-B</given-names></name><name><surname>Tan</surname><given-names>Y-B</given-names></name><etal/></person-group>. <article-title>Acidic pH promotes oxidation-induced dissociation of C-reactive protein</article-title>. <source>Mol Immunol.</source> (<year>2018</year>) <volume>104</volume>:<fpage>47</fpage>–<lpage>53</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.molimm.2018.09.021</pub-id><pub-id pub-id-type=\"pmid\">30408622</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McFadyen</surname><given-names>JD</given-names></name><name><surname>Zeller</surname><given-names>J</given-names></name><name><surname>Potempa</surname><given-names>LA</given-names></name><name><surname>Pietersz</surname><given-names>GA</given-names></name><name><surname>Eisenhardt</surname><given-names>SU</given-names></name><name><surname>Peter</surname><given-names>K</given-names></name></person-group>. <article-title>C-reactive protein and its structural isoforms: an evolutionary conserved marker and central player in inflammatory diseases and beyond</article-title>. <source>Subcell Biochem.</source> (<year>2020</year>) <volume>94</volume>:<fpage>499</fpage>–<lpage>520</lpage>. <pub-id pub-id-type=\"doi\">10.1007/978-3-030-41769-7_20</pub-id><pub-id pub-id-type=\"pmid\">32189313</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Singh</surname><given-names>SK</given-names></name><name><surname>Suresh</surname><given-names>MV</given-names></name><name><surname>Prayther</surname><given-names>DC</given-names></name><name><surname>Moorman</surname><given-names>JP</given-names></name><name><surname>Rusiñol</surname><given-names>AE</given-names></name><name><surname>Agrawal</surname><given-names>A</given-names></name></person-group>. <article-title>C-reactive protein-bound enzymatically modified low-density lipoprotein does not transform macrophages into foam cells</article-title>. <source>J Immunol.</source> (<year>2008</year>) <volume>180</volume>:<fpage>4316</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.180.6.4316</pub-id><pub-id pub-id-type=\"pmid\">18322245</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Singh</surname><given-names>SK</given-names></name><name><surname>Agrawal</surname><given-names>A</given-names></name></person-group>\n<article-title>Functionality of C-reactive protein for atheroprotection</article-title>. <source>Front Immunol.</source> (<year>2019</year>) <volume>10</volume>:<fpage>1655</fpage>\n<pub-id pub-id-type=\"doi\">10.3389/fimmu.2019.01655</pub-id><pub-id pub-id-type=\"pmid\">31379851</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gang</surname><given-names>TB</given-names></name><name><surname>Hammond</surname><given-names>DJ</given-names><suffix>Jr</suffix></name><name><surname>Singh</surname><given-names>SK</given-names></name><name><surname>Ferguson</surname><given-names>DA</given-names><suffix>Jr</suffix></name><name><surname>Mishra</surname><given-names>VK</given-names></name><name><surname>Agrawal</surname><given-names>A</given-names></name></person-group>. <article-title>The phosphocholine-binding pocket on C-reactive protein is necessary for initial protection of mice against pneumococcal infection</article-title>. <source>J Biol Chem.</source> (<year>2012</year>) <volume>287</volume>:<fpage>43116</fpage>–<lpage>25</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M112.427310</pub-id><pub-id pub-id-type=\"pmid\">23139417</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Netherland</surname><given-names>CD</given-names></name><name><surname>Pickle</surname><given-names>TG</given-names></name><name><surname>Bales</surname><given-names>A</given-names></name><name><surname>Thewke</surname><given-names>DP</given-names></name></person-group>. <article-title>Cannabinoid receptor type 2 (CB2) deficiency alters atherosclerotic lesion formation in hyperlipidemic Ldlr-null mice</article-title>. <source>Atherosclerosis.</source> (<year>2010</year>) <volume>213</volume>:<fpage>102</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.atherosclerosis.2010.07.060</pub-id><pub-id pub-id-type=\"pmid\">20846652</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schneider</surname><given-names>CA</given-names></name><name><surname>Rasband</surname><given-names>WS</given-names></name><name><surname>Eliceiri</surname><given-names>KW</given-names></name></person-group>. <article-title>NIH image to imageJ: 25 years of image analysis</article-title>. <source>Nat Methods.</source> (<year>2012</year>) <volume>9</volume>:<fpage>671</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nmeth.2089</pub-id><pub-id pub-id-type=\"pmid\">22930834</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fu</surname><given-names>Z</given-names></name><name><surname>Yan</surname><given-names>K</given-names></name><name><surname>Rosenberg</surname><given-names>A</given-names></name><name><surname>Jin</surname><given-names>Z</given-names></name><name><surname>Crain</surname><given-names>B</given-names></name><name><surname>Athas</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Improved protein extraction and protein identification from archival formalin-fixed paraffin-embedded human aortas</article-title>. <source>Proteomics Clin Appl.</source> (<year>2013</year>) <volume>7</volume>:<fpage>217</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1002/prca.201200064</pub-id><pub-id pub-id-type=\"pmid\">23339088</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maudsley</surname><given-names>S</given-names></name><name><surname>Pepys</surname><given-names>MB</given-names></name></person-group>. <article-title>Immunochemical cross-reactions between pentraxins of different species</article-title>. <source>Immunology.</source> (<year>1987</year>) <volume>62</volume>:<fpage>17</fpage>–<lpage>22</lpage>.<pub-id pub-id-type=\"pmid\">3115892</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Paul</surname><given-names>A</given-names></name><name><surname>Ko</surname><given-names>KWS</given-names></name><name><surname>Li</surname><given-names>L</given-names></name><name><surname>Yechoor</surname><given-names>V</given-names></name><name><surname>McCrory</surname><given-names>MA</given-names></name><name><surname>Szalai</surname><given-names>AJ</given-names></name><etal/></person-group>. <article-title>C-reactive protein accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice</article-title>. <source>Circulation.</source> (<year>2004</year>) <volume>109</volume>:<fpage>647</fpage>–<lpage>55</lpage>. <pub-id pub-id-type=\"doi\">10.1161/01.CIR.0000114526.50618.24</pub-id><pub-id pub-id-type=\"pmid\">14744975</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hirschfield</surname><given-names>GM</given-names></name><name><surname>Gallimore</surname><given-names>JR</given-names></name><name><surname>Kahan</surname><given-names>MC</given-names></name><name><surname>Hutchinson</surname><given-names>WL</given-names></name><name><surname>Sabin</surname><given-names>CA</given-names></name><name><surname>Benson</surname><given-names>GM</given-names></name><etal/></person-group>. <article-title>Transgenic human C-reactive protein is not proatherogenic in apolipoprotein E-deficient mice</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2005</year>) <volume>102</volume>:<fpage>8309</fpage>–<lpage>14</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.0503202102</pub-id><pub-id pub-id-type=\"pmid\">15919817</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reifenberg</surname><given-names>K</given-names></name><name><surname>Lehr</surname><given-names>H-A</given-names></name><name><surname>Baskal</surname><given-names>D</given-names></name><name><surname>Wiese</surname><given-names>E</given-names></name><name><surname>Schaefer</surname><given-names>SC</given-names></name><name><surname>Black</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Role of C-reactive protein in atherogenesis: can the apolipoprotein E knockout mouse provide the answer?</article-title>\n<source>Arterioscler Thromb Vasc Biol.</source> (<year>2005</year>) <volume>25</volume>:<fpage>1641</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1161/01.ATV.0000171983.95612.90</pub-id><pub-id pub-id-type=\"pmid\">15920030</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schwedler</surname><given-names>SB</given-names></name><name><surname>Amann</surname><given-names>K</given-names></name><name><surname>Wernicke</surname><given-names>K</given-names></name><name><surname>Krebs</surname><given-names>A</given-names></name><name><surname>Nauck</surname><given-names>M</given-names></name><name><surname>Wanner</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>Native C-reactive protein increases whereas modified C-reactive protein reduces atherosclerosis in apolipoprotein E-knockout mice</article-title>. <source>Circulation.</source> (<year>2005</year>) <volume>112</volume>:<fpage>1016</fpage>–<lpage>23</lpage>. <pub-id pub-id-type=\"doi\">10.1161/CIRCULATIONAHA.105.556530</pub-id><pub-id pub-id-type=\"pmid\">16087790</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Trion</surname><given-names>A</given-names></name><name><surname>de Matt</surname><given-names>MPM</given-names></name><name><surname>Jukema</surname><given-names>JW</given-names></name><name><surname>van der Laarse</surname><given-names>A</given-names></name><name><surname>Mass</surname><given-names>MC</given-names></name><name><surname>Offerman</surname><given-names>EH</given-names></name><etal/></person-group>. <article-title>No effect of C-reactive protein on early atherosclerosis development in apolipoprotein E<sup>*</sup>3-leiden/human C-reactive protein transgenic mice</article-title>. <source>Arterioscler Thromb Vasc Biol.</source> (<year>2005</year>) <volume>25</volume>:<fpage>1635</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1161/01.ATV.0000171992.36710.1e</pub-id><pub-id pub-id-type=\"pmid\">15920036</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kovacs</surname><given-names>A</given-names></name><name><surname>Tornvall</surname><given-names>P</given-names></name><name><surname>Nilsson</surname><given-names>R</given-names></name><name><surname>Tegnér</surname><given-names>J</given-names></name><name><surname>Hamsten</surname><given-names>A</given-names></name><name><surname>Björkegren</surname><given-names>J</given-names></name></person-group>. <article-title>Human C-reactive protein slows atherosclerosis development in a mouse model with human-like hypercholesterolemia</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2007</year>) <volume>104</volume>:<fpage>13768</fpage>–<lpage>73</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.0706027104</pub-id><pub-id pub-id-type=\"pmid\">17702862</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tennent</surname><given-names>GA</given-names></name><name><surname>Hutchinson</surname><given-names>WL</given-names></name><name><surname>Kahan</surname><given-names>MC</given-names></name><name><surname>Hirschfield</surname><given-names>GM</given-names></name><name><surname>Gallimore</surname><given-names>JR</given-names></name><name><surname>Lewin</surname><given-names>J</given-names></name><etal/></person-group>\n<article-title>Transgenic human CRP is not pro-atherogenic, pro-atherothrombotic or pro-inflammatory in apoE-/- mice</article-title>. <source>Atherosclerosis.</source> (<year>2008</year>) <volume>196</volume>:<fpage>248</fpage>–<lpage>55</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.atherosclerosis.2007.05.010</pub-id><pub-id pub-id-type=\"pmid\">17588586</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Torzewski</surname><given-names>M</given-names></name><name><surname>Reifenberg</surname><given-names>K</given-names></name><name><surname>Cheng</surname><given-names>F</given-names></name><name><surname>Wiese</surname><given-names>E</given-names></name><name><surname>Küpper</surname><given-names>I</given-names></name><name><surname>Crain</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>No effect of C-reactive protein on early atherosclerosis in LDLR-/-/human C-reactive protein transgenic mice</article-title>. <source>Thromb Haemost.</source> (<year>2008</year>) <volume>99</volume>:<fpage>196</fpage>–<lpage>201</lpage>. <pub-id pub-id-type=\"doi\">10.1160/TH07-10-0595</pub-id><pub-id pub-id-type=\"pmid\">18217154</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Koike</surname><given-names>T</given-names></name><name><surname>Kitajima</surname><given-names>S</given-names></name><name><surname>Yu</surname><given-names>Y</given-names></name><name><surname>Nishijima</surname><given-names>K</given-names></name><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Ozaki</surname><given-names>Y</given-names></name><etal/></person-group>\n<article-title>Human C-reactive protein does not promote atherosclerosis in transgenic rabbits</article-title>. <source>Circulation.</source> (<year>2009</year>) <volume>120</volume>:<fpage>2088</fpage>–<lpage>94</lpage>. <pub-id pub-id-type=\"doi\">10.1161/CIRCULATIONAHA.109.872796</pub-id><pub-id pub-id-type=\"pmid\">19901190</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ortiz</surname><given-names>MA</given-names></name><name><surname>Campana</surname><given-names>GL</given-names></name><name><surname>Woods</surname><given-names>JR</given-names></name><name><surname>Boguslawski</surname><given-names>G</given-names></name><name><surname>Sosa</surname><given-names>MJ</given-names></name><name><surname>Walker</surname><given-names>CL</given-names></name><etal/></person-group>. <article-title>Continously-infused human C-reactive protein is neither proatherosclerotic nor proinflammatory in apolipoprotein E-deficient mice</article-title>. <source>Exp Biol Med.</source> (<year>2009</year>) <volume>234</volume>:<fpage>624</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.3181/0812-RM-347</pub-id><pub-id pub-id-type=\"pmid\">19359657</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Teupser</surname><given-names>D</given-names></name><name><surname>Weber</surname><given-names>O</given-names></name><name><surname>Rao</surname><given-names>TN</given-names></name><name><surname>Sass</surname><given-names>K</given-names></name><name><surname>Thiery</surname><given-names>J</given-names></name><name><surname>Fehling</surname><given-names>HJ</given-names></name></person-group>. <article-title>No reduction of atherosclerosis in C-reactive protein (CRP)-deficient mice</article-title>. <source>J Biol Chem.</source> (<year>2011</year>) <volume>286</volume>:<fpage>6272</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M110.161414</pub-id><pub-id pub-id-type=\"pmid\">21149301</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>Q</given-names></name><name><surname>Liu</surname><given-names>Z</given-names></name><name><surname>Waqar</surname><given-names>AB</given-names></name><name><surname>Ning</surname><given-names>B</given-names></name><name><surname>Yang</surname><given-names>X</given-names></name><name><surname>Shiomi</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Effects of antisense oligonucleotides against C-reactive protein on the development of atherosclerosis in WHHL rabbits</article-title>. <source>Mediators Inflamm.</source> (<year>2014</year>) <volume>2014</volume>:<fpage>979132</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2014/979132</pub-id><pub-id pub-id-type=\"pmid\">24872601</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Badimon</surname><given-names>L</given-names></name><name><surname>Peña</surname><given-names>E</given-names></name><name><surname>Arderiu</surname><given-names>G</given-names></name><name><surname>Padró</surname><given-names>T</given-names></name><name><surname>Slevin</surname><given-names>M</given-names></name><name><surname>Vilahur</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>C-reactive protein in atherothrombosis and angiogenesis</article-title>. <source>Front Immunol.</source> (<year>2018</year>) <volume>9</volume>:<fpage>430</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2018.00430</pub-id><pub-id pub-id-type=\"pmid\">29552019</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boncler</surname><given-names>M</given-names></name><name><surname>Wu</surname><given-names>Y</given-names></name><name><surname>Watala</surname><given-names>C</given-names></name></person-group>. <article-title>The multiple faces of C-reactive protein - physiological and pathophysiological implications in cardiovascular disease</article-title>. <source>Molecules.</source> (<year>2019</year>) <volume>24</volume>:<fpage>2062</fpage>. <pub-id pub-id-type=\"doi\">10.3390/molecules24112062</pub-id><pub-id pub-id-type=\"pmid\">31151201</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Babaev</surname><given-names>VR</given-names></name><name><surname>Patel</surname><given-names>MB</given-names></name><name><surname>Semenkovich</surname><given-names>CF</given-names></name><name><surname>Fazio</surname><given-names>S</given-names></name><name><surname>Linton</surname><given-names>MF</given-names></name></person-group>. <article-title>Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in low density lipoprotein receptor-deficient mice</article-title>. <source>J Biol Chem.</source> (<year>2000</year>) <volume>275</volume>:<fpage>26293</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M002423200</pub-id><pub-id pub-id-type=\"pmid\">10858435</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schiller</surname><given-names>NK</given-names></name><name><surname>Kubo</surname><given-names>N</given-names></name><name><surname>Boisvert</surname><given-names>WA</given-names></name><name><surname>Curtiss</surname><given-names>LK</given-names></name></person-group>. <article-title>Effect of γ-irradiation and bone marrow transplantation on atherosclerosis in LDL receptor-deficient mice</article-title>. <source>Arterioscler Thromb Vasc Biol.</source> (<year>2001</year>) <volume>21</volume>:<fpage>1674</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.1161/hq1001.096724</pub-id><pub-id pub-id-type=\"pmid\">11597944</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>King</surname><given-names>VL</given-names></name><name><surname>Szilvassy</surname><given-names>SJ</given-names></name><name><surname>Daugherty</surname><given-names>A</given-names></name></person-group>. <article-title>Interleukin-4 deficiency decreases atherosclerotic lesion formation in a site-specific manner in female LDL receptor-/- mice</article-title>. <source>Arterioscler Thromb Vasc Biol.</source> (<year>2002</year>) <volume>22</volume>:<fpage>456</fpage>–<lpage>61</lpage>. <pub-id pub-id-type=\"doi\">10.1161/hq0302.104905</pub-id><pub-id pub-id-type=\"pmid\">11884290</pub-id></mixed-citation></ref><ref id=\"B48\"><label>48.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>VanderLaan</surname><given-names>PA</given-names></name><name><surname>Reardon</surname><given-names>CA</given-names></name><name><surname>Getz</surname><given-names>GS</given-names></name></person-group>. <article-title>Site specificity of atherosclerosis: Site-selective responses to atherosclerotic modulators</article-title>. <source>Arterioscler Thromb Vasc Biol.</source> (<year>2004</year>) <volume>24</volume>:<fpage>12</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.1161/01.ATV.0000105054.43931.f0</pub-id><pub-id pub-id-type=\"pmid\">14604830</pub-id></mixed-citation></ref><ref id=\"B49\"><label>49.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Goel</surname><given-names>R</given-names></name><name><surname>Schrank</surname><given-names>BR</given-names></name><name><surname>Arora</surname><given-names>S</given-names></name><name><surname>Boylan</surname><given-names>B</given-names></name><name><surname>Fleming</surname><given-names>B</given-names></name><name><surname>Miura</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Site-specific effects of PECAM-1 on atherosclerosis in LDL receptor-deficient mice</article-title>. <source>Arterioscler Thromb Vasc Biol.</source> (<year>2008</year>) <volume>28</volume>:<fpage>1996</fpage>–<lpage>2002</lpage>. <pub-id pub-id-type=\"doi\">10.1161/ATVBAHA.108.172270</pub-id><pub-id pub-id-type=\"pmid\">18669884</pub-id></mixed-citation></ref><ref id=\"B50\"><label>50.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Melgar-Lesmes</surname><given-names>P</given-names></name><name><surname>Sánchez-Herrero</surname><given-names>A</given-names></name><name><surname>Lozano-Juan</surname><given-names>F</given-names></name><name><surname>de la Torre Hernández</surname><given-names>JM</given-names></name><name><surname>Montell</surname><given-names>E</given-names></name><name><surname>Jiménez</surname><given-names>W</given-names></name><etal/></person-group>. <article-title>Chondroitin sulphate attenuates atherosclerosis in apoE knockout mice involving cellular regulation of the inflammatory response</article-title>. <source>Thromb Haemost.</source> (<year>2018</year>) <volume>118</volume>:<fpage>1329</fpage>–<lpage>39</lpage>. <pub-id pub-id-type=\"doi\">10.1055/s-0038-1657753</pub-id><pub-id pub-id-type=\"pmid\">29874688</pub-id></mixed-citation></ref><ref id=\"B51\"><label>51.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>H-Y</given-names></name><name><surname>Wang</surname><given-names>J</given-names></name><name><surname>Meng</surname><given-names>F</given-names></name><name><surname>Jia</surname><given-names>Z-K</given-names></name><name><surname>Su</surname><given-names>Y</given-names></name><name><surname>Bai</surname><given-names>Q-F</given-names></name><etal/></person-group>. <article-title>An intrinsically disordered motif mediates diverse actions of monomeric C-reactive protein</article-title>. <source>J Biol Chem.</source> (<year>2016</year>) <volume>291</volume>:<fpage>8795</fpage>–<lpage>804</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M115.695023</pub-id><pub-id pub-id-type=\"pmid\">26907682</pub-id></mixed-citation></ref><ref id=\"B52\"><label>52.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lv</surname><given-names>J-M</given-names></name><name><surname>Chen</surname><given-names>J-Y</given-names></name><name><surname>Liu</surname><given-names>Z-P</given-names></name><name><surname>Yao</surname><given-names>Z-Y</given-names></name><name><surname>Wu</surname><given-names>Y-X</given-names></name><name><surname>Tong</surname><given-names>C-S</given-names></name><etal/></person-group>. <article-title>Cellular folding determinants and conformational plasticity of native C-reactive protein</article-title>. <source>Front Immunol.</source> (<year>2020</year>) <volume>11</volume>:<fpage>583</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2020.00583</pub-id><pub-id pub-id-type=\"pmid\">32296446</pub-id></mixed-citation></ref><ref id=\"B53\"><label>53.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Voleti</surname><given-names>B</given-names></name><name><surname>Agrawal</surname><given-names>A</given-names></name></person-group>. <article-title>Statins and nitric oxide reduce C-reactive protein production while inflammatory conditions persist</article-title>. <source>Mol Immunol.</source> (<year>2006</year>) <volume>43</volume>:<fpage>891</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.molimm.2005.06.045</pub-id><pub-id pub-id-type=\"pmid\">16054696</pub-id></mixed-citation></ref><ref id=\"B54\"><label>54.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Agrawal</surname><given-names>A</given-names></name></person-group>. <article-title>CRP after 2004</article-title>. <source>Mol Immunol.</source> (<year>2005</year>) <volume>42</volume>:<fpage>927</fpage>–<lpage>30</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.molimm.2004.09.028</pub-id><pub-id pub-id-type=\"pmid\">15829284</pub-id></mixed-citation></ref><ref id=\"B55\"><label>55.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McDade</surname><given-names>TW</given-names></name></person-group>. <article-title>Early environments and the ecology of inflammation</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2012</year>) <volume>109</volume>:<fpage>17281</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1202244109</pub-id><pub-id pub-id-type=\"pmid\">23045646</pub-id></mixed-citation></ref></ref-list><glossary><def-list><title>Abbreviations</title><def-item><term>CRP</term><def><p>C-reactive protein</p></def></def-item><def-item><term>LDL</term><def><p>Low-density lipoprotein</p></def></def-item><def-item><term><italic>Ldlr</italic><sup>−/−</sup></term><def><p>LDL receptor-deficient</p></def></def-item><def-item><term>PCh</term><def><p>Phosphocholine</p></def></def-item><def-item><term>WT</term><def><p>wild-type.</p></def></def-item></def-list></glossary></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Neurol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Neurol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Neurol.</journal-id><journal-title-group><journal-title>Frontiers in Neurology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2295</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849252</article-id><article-id pub-id-type=\"pmc\">PMC7431524</article-id><article-id pub-id-type=\"doi\">10.3389/fneur.2020.00826</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Neurology</subject><subj-group><subject>Hypothesis and Theory</subject></subj-group></subj-group></article-categories><title-group><article-title>How Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) Progresses: The Natural History of ME/CFS</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Nacul</surname><given-names>Luis</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/593281/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>O'Boyle</surname><given-names>Shennae</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/624287/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Palla</surname><given-names>Luigi</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/42379/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Nacul</surname><given-names>Flavio E.</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Mudie</surname><given-names>Kathleen</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/623672/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Kingdon</surname><given-names>Caroline C.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/642289/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Cliff</surname><given-names>Jacqueline M.</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/370409/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Clark</surname><given-names>Taane G.</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/642245/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Dockrell</surname><given-names>Hazel M.</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/404313/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Lacerda</surname><given-names>Eliana M.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/638737/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine</institution>, <addr-line>London</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff2\"><sup>2</sup><institution>B.C. Women's Hospital and Health Centre</institution>, <addr-line>Vancouver, BC</addr-line>, <country>Canada</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Medical Statistics, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine</institution>, <addr-line>London</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Global Health, School of Tropical Medicine and Global Health, Nagasaki University</institution>, <addr-line>Nagasaki</addr-line>, <country>Japan</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Pro-Cardiaco Hospital and Federal University of Rio de Janeiro</institution>, <addr-line>Rio de Janeiro</addr-line>, <country>Brazil</country></aff><aff id=\"aff6\"><sup>6</sup><institution>Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine</institution>, <addr-line>London</addr-line>, <country>United Kingdom</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Bernhard Schaller, University of Zurich, Switzerland</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Indre Bileviciute-Ljungar, Karolinska Institutet (KI), Sweden; Lucinda Bateman, Bateman Horne Center, United States</p></fn><corresp id=\"c001\">*Correspondence: Shennae O'Boyle <email>shennae.oboyle@lshtm.ac.uk</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Autonomic Neuroscience, a section of the journal Frontiers in Neurology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>826</elocation-id><history><date date-type=\"received\"><day>18</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>01</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Nacul, O'Boyle, Palla, Nacul, Mudie, Kingdon, Cliff, Clark, Dockrell and Lacerda.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Nacul, O'Boyle, Palla, Nacul, Mudie, Kingdon, Cliff, Clark, Dockrell and Lacerda</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>We propose a framework for understanding and interpreting the pathophysiology of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) that considers wider determinants of health and long-term temporal variation in pathophysiological features and disease phenotype throughout the natural history of the disease. As in other chronic diseases, ME/CFS evolves through different stages, from asymptomatic predisposition, progressing to a prodromal stage, and then to symptomatic disease. Disease incidence depends on genetic makeup and environment factors, the exposure to singular or repeated insults, and the nature of the host response. In people who develop ME/CFS, normal homeostatic processes in response to adverse insults may be replaced by aberrant responses leading to dysfunctional states. Thus, the predominantly neuro-immune manifestations, underlined by a hyper-metabolic state, that characterize early disease, may be followed by various processes leading to multi-systemic abnormalities and related symptoms. This abnormal state and the effects of a range of mediators such as products of oxidative and nitrosamine stress, may lead to progressive cell and metabolic dysfunction culminating in a hypometabolic state with low energy production. These processes do not seem to happen uniformly; although a spiraling of progressive inter-related and self-sustaining abnormalities may ensue, reversion to states of milder abnormalities is possible if the host is able to restate responses to improve homeostatic equilibrium. With time variation in disease presentation, no single ME/CFS case description, set of diagnostic criteria, or molecular feature is currently representative of all patients at different disease stages. While acknowledging its limitations due to the incomplete research evidence, we suggest the proposed framework may support future research design and health care interventions for people with ME/CFS.</p></abstract><kwd-group><kwd>Myalgic Encephalomyelitis/Chronic Fatigue Syndrome</kwd><kwd>Chronic Fatigue Syndrome</kwd><kwd>ME/CFS</kwd><kwd>chronic illness</kwd><kwd>management</kwd><kwd>research</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Institutes of Health<named-content content-type=\"fundref-id\">10.13039/100000002</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"2\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"178\"/><page-count count=\"13\"/><word-count count=\"11514\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>The lack of progress in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) research has been attributed to a range of factors, including the paucity of large, high quality, hypothesis-driven studies, and controversy around diagnosis. Without recognized and validated biomarkers or diagnostic tests, there is an over-reliance on patient history for diagnosis, which is based on criteria with limited sensitivity and specificity (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>) and which ignore disease sub-groups. Furthermore, the lack of consistency in the choice and application of research case definition has led to problems with reliability and comparability of research findings (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). An additional factor complicating diagnosis and case definition for research studies is the time-related variation in phenotype both in the short- (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B4\" ref-type=\"bibr\">4</xref>) and long-term (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>), which has seldom been considered in research studies.</p><p>In addition to often marked variability in disease presentation, severity, progression, and duration among different individuals, the way disease manifests in each individual may change with time. Inter- and intra-individual phenotypic variations lend toward the categorization of different subtype trajectories of ME/CFS that may differ in pathogenesis and prognosis. In some studies, female sex, increased age (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>–<xref rid=\"B8\" ref-type=\"bibr\">8</xref>), and lower socio-economic status (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>) have been found to predict poor prognosis; however, the variable nature of both population sampling and diagnostic criteria has led to ambiguous results and has reinforced the need for ongoing research in this area (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). Further subtypes have been defined on the basis of “minor” symptoms i.e., musculoskeletal, infectious, or neurological (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>), through genetic studies (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>), metabolomics studies (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>), and, duration of disease studies (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>), highlighting the multitude of possible ways ME/CFS patients can be categorized. Other studies have identified variations in symptom profiles as disease progresses; however, such results are often limited by cross-sectional study design (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>), and/or recall bias (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). The breadth of subtype studies available follow a similar model of looking for patterns across patient groups at single time-points; far fewer consider longitudinal subtyping and disease progression of a single patient cohort over time.</p><p>The concept of the natural history of disease is well-understood in public health and medicine: many, if not all, diseases are framed using this construct to formulate how they progress from a pre-illness stage to a final disease outcome, which may vary from full recovery to death. A good understanding of the disease course is vital not only for the design of preventative and intervention studies (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>), but also to assess the timing and type of intervention that minimizes disease risk or optimizes prognosis. Although there is some understanding of the natural history of ME/CFS, this has been limited by problems in case definition (as above) as well as by the paucity of longitudinal studies, and in particular those that follow up individuals' pre-illness. A review of studies on CFS prognosis (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>) suggested recovery rates under 10% in adults, and an improvement rate over 40% for people with fatigue lasting <6 months. The prognosis was worse: when more stringent case definitions were used; in older people; in cases with more severe symptoms; and, in the presence of psychiatric co-morbidity. A subsequent systematic review on prognosis found a median recovery rate of 5%, and median proportion of people improving of 39.5% (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>) with most reporting symptoms still present at follow-up.</p><p>This conceptual paper explores the long-term course of ME/CFS and how presentation and pathophysiological abnormalities may vary with time. The pathophysiological concepts discussed are based on evidence from clinical observations and research, where available, and, as such, are not claimed to be original or indeed conclusive. Instead, they serve to highlight our proposed characterization of ME/CFS's distinct stages within the framework of the natural history of the disease.</p></sec><sec id=\"s2\"><title>Pathophysiological and Cellular Abnormalities Following Host Exposure to “Insults” or “Stressors”</title><p>Prior to exploring the course of ME/CFS, we propose to revisit some concepts related to mechanisms of disease that have been used in the context of life-threatening emergencies and to potential return to homeostasis, such as those occurring in sepsis or poly-trauma. Although very different to ME/CFS, these acute injuries have been extensively studied, and the high intensity and speed of events result in changes that are easily identified and well-described, from potential homeostatic failure to recovery. We present the following models as a paradigm for the understanding of disease mechanisms, based on well-studied examples. They merely serve as a reference for mechanisms that the host may partially engage with in the presence of insults of different severities. Hence, in the following paragraphs, we explore the pathophysiological mechanisms that may be taking place in ME/CFS, which have been related to abnormal homeostasis guided by these established disease descriptions.</p><p>The response to an insult frequently involves multiple body-systems and has components that are independent of the etiology of the insult and, to some extent, its severity. There are many commonalities between the response to sepsis and to poly-trauma: both are acute and severe insults, to which many of the aspects of the host response are indistinguishable. Our proposal is based on the idea that there may be some similar mechanisms at play when individuals predisposed to ME/CFS are faced with a range of “insults” or “stressors.” Needless to say, the hyper-acute changes and co-factors in both sepsis and poly-trauma occur in very rapid sequence, whereas in ME/CFS, physiological changes, even if they resemble those of acute injury in some respects, take place at a much slower pace with less obvious and uniform patterns.</p><sec><title>Non-specific Changes in Response to Severe Acute Injury</title><p>In both sepsis (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>) and poly-trauma, (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>, <xref rid=\"B21\" ref-type=\"bibr\">21</xref>) a state of hyper-inflammation is observed initially as the host responds to the infection or traumatic stress with marked production of pro-inflammatory mediators, e.g., cytokines and polypeptides. A failing circulatory system is associated with activation of the hypothalamic-pituitary-adrenal (HPA) axis and increased sympathetic drive, contributing to metabolic changes and to increased energy expenditure (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>, <xref rid=\"B23\" ref-type=\"bibr\">23</xref>).</p><p>In these conditions, the acute pro-inflammatory state is usually followed by a compensatory anti-inflammatory response, with a different profile of biochemical and molecular mediators. The success of the host in balancing pro- with anti- inflammatory responses alongside injury-related factors, are key to improved long-term outcomes. The direct and indirect effects of immune cells and active products derived from immune, neural, and endocrine systems (some of which cause pathology if present in excess) contribute to a number of physiological changes, including those leading to the formation of reactive oxygen species (ROS, oxidative stress) and reactive nitrogen species (RNS, nitrosative stress). Endothelial and parenchymal (organ) cell damage may result because of a combination of factors, such as polymorphonuclear leukocyte infiltration and the action of reactive oxygen and nitrogen species, cytokines, vasoactive amines, and other products. Endothelial dysfunction results in capillary leakage, accelerated inflammation, platelet aggregation, coagulation, and loss of vascular tone (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). Vascular dysfunction is associated to peripheral vasodilation due to increased nitric oxide and prostacyclin synthesis (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>) and to a decrease in the proportion of perfused vessels and an increase in the heterogeneity of blood flow distribution (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). This results in relative hypovolemia, decreased capillary flow, haemo-concentration, and micro-thrombi formation, and further contributes to reduced exchanges of oxygen and nutrients at the microcirculatory level. The consequent decreased cellular oxygen delivery eventually leads to cytopathic hypoxia. Adenosine triphosphate (ATP) increased consumption and ensuing deficits cascade into a range of metabolic disturbances with systemic effects (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>), and promote changes in membrane permeability that lead to dysfunctional transmembrane ion transport. In acutely and severely ill patients, reperfusion results in further oxidative damage (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>, <xref rid=\"B28\" ref-type=\"bibr\">28</xref>). Additional failures of biological and cell processes lead to multiple dysfunction, to system and organ failure, and to potentially irreversible disease (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>).</p></sec><sec><title>Evidence of Abnormalities in ME/CFS and Loss of Normal Homeostasis</title><p>Concepts that are relevant here are those of homeostasis and allostasis. While homeostasis refers to the “<italic>stability of physiological systems</italic>,” allostasis has been defined as “<italic>the adaptive processes aimed to maintain homeostasis following acute stress, and which contribute to wear and tear on the body and the brain, or allostatic overload</italic>” (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). A central characteristic of individuals with ME/CFS points to a state of homeostatic failure (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>), aggravated by the incidence of, or increase in, levels of new stressors or by the increase in allostatic load (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). Typical stressors include infection [(<xref rid=\"B32\" ref-type=\"bibr\">32</xref>): 17–21], physical exertion and cognitive effort (e.g., reading or solving mental puzzles) triggering post-exertional malaise (PEM) (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>), comorbid conditions (e.g., sleep disturbances) (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>) and a range of environmental and individual factors (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>–<xref rid=\"B40\" ref-type=\"bibr\">40</xref>).</p><p>In those who do not develop ME/CFS or prolonged illness following an insult such as an acute infection, external stressors may initially cause physiological changes accompanied by non-specific symptoms, but the state of homeostatic equilibrium that operated before the insult is quickly restored. Failing re-establishment of this equilibrium, there may be a shift to a state of “aberrant homeostasis,” where physiological processes converge to a new or alternative state of functioning; a state that remains homeostatic in nature, but functions at a less optimum level (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>). While such a state may be adequate for many physiological processes, it will be inadequate or inefficient for a number of other processes and functions and the prolongation of such aberrant functioning will represent another potential source of ongoing stress.</p><p>There is a growing body of evidence on biological abnormalities in ME/CFS that has been reviewed elsewhere (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B42\" ref-type=\"bibr\">42</xref>, <xref rid=\"B43\" ref-type=\"bibr\">43</xref>), and summarized by Komaroff (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>). Of note, many of the abnormalities shown in severe injury have also been identified in ME/CFS such as: immune dysfunction, including pro-inflammatory response (especially at early stages of disease) (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>, <xref rid=\"B46\" ref-type=\"bibr\">46</xref>); autonomic nervous system (<xref rid=\"B47\" ref-type=\"bibr\">47</xref>–<xref rid=\"B49\" ref-type=\"bibr\">49</xref>); HPA axis dysfunction (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>); hypovolemia (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>); nitrosamine and oxidative stress (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>); endothelial dysfunction (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>); metabolic dysfunction (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>–<xref rid=\"B55\" ref-type=\"bibr\">55</xref>); dysfunction of membrane transport (<xref rid=\"B56\" ref-type=\"bibr\">56</xref>); and, tissue hypoxia (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>).</p></sec></sec><sec id=\"s3\"><title>The Stages of ME/CFS</title><p>Other tools widely used in clinical medicine are staging systems. Using sepsis again as an example, such a system was proposed at the International Sepsis Definitions Conference in 2001 to introduce the stratification of patients with sepsis (<xref rid=\"B58\" ref-type=\"bibr\">58</xref>). By applying PIRO (predisposition, infection/insult, response, and organ dysfunction) patients are stratified into appropriate subgroups allowing for more accurate prognostication in emergency medical services (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>). The idea of classifying people with ME or CFS into distinct categories or stages has been explored previously by several theorists. One school of thought proposes categories based on the psychological process of coming to terms with this new and evolving state of health rather than addressing biological differences, and are defined as such by the emotions common to any trauma experience: e.g., denial, fear, frustration, and acceptance (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>, <xref rid=\"B61\" ref-type=\"bibr\">61</xref>). Alternatively, Schweitzer (<xref rid=\"B62\" ref-type=\"bibr\">62</xref>) proposes the different presentations of CFS according to more physical categories (Prodrome, Relapse and Remission, Improvement and Plateau, and Collapse followed by slow worsening with no remission); it is these that we aim to expand on, as follows.</p><p>We show a tentative representation of the key pathophysiological mechanisms operating in each stage of ME/CFS in <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>. As in severe injury or sepsis, the range and order of occurrence of biological processes taking place in ME/CFS may vary, as may their relative significance and impact on each individual. Therefore, it is important to note that although the various abnormalities may occur continuously and often simultaneously, the predominance of specific dysfunctions varies over time and from individual to individual.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Hypothesized key pathophysiological mechanisms for ME/CFS.</p></caption><graphic xlink:href=\"fneur-11-00826-g0001\"/></fig><p>Furthermore, we propose a characterization of disease stages in ME/CFS, based on the natural history of disease framework considering available descriptions from the literature (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>), and the life-stories reported by our own cohort of research participants with ME/CFS (including those with mild/moderate or severe symptoms) (<xref rid=\"B63\" ref-type=\"bibr\">63</xref>). This characterization is summarized in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, which may be used in support of research designs that consider the disease presentation in distinct phases.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Proposed characterization of disease stages in an individual with ME/CFS, within the framework of natural history of diseases.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr style=\"border-bottom: thin solid #000000;\"><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Timing</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>No disease</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Onset</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>0–4 months<xref ref-type=\"table-fn\" rid=\"TN4\"><sup><bold>¶</bold></sup></xref></bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>4–24 months<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>2 years +<sup>†</sup></bold></th></tr><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Stage</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Predisposition</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Trigger and pre-illness</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Prodromal period</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Early disease</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Established disease</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Clinical phenotype</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">No symptoms</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Non-specific or related to triggering “insult”</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fatigue-complex symptoms<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>‡</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fatigue-complex symptoms<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>‡</sup></xref> variable severity and progress</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mild, moderate, severe and complicated disease</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Prevention level<xref ref-type=\"table-fn\" rid=\"TN4\"><sup>¶</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Primary prevention</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Treatment of “insult” and primary prevention</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Secondary prevention</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Treatment and secondary prevention</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Treatment and tertiary prevention</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recovery Potential<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>§</sup></xref></td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Likely</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Possible</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Less likely</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pathophysiology</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Predisposing factors</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Non-specific host response and related to specific trigger factor</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neuro-immune response to insult and fight for homeostasis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neuro-inflammation and systemic consequences; aberrant homeostasis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Systemic disease, aberrant or failed homeostasis</td></tr></tbody></table><table-wrap-foot><fn id=\"TN1\"><label>*</label><p><italic>3–6 months is commonly proposed as the minimum period of symptoms before diagnosis is made in children and adults, respectively (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>)</italic>.</p></fn><p><italic>2 years has been used as a cut off to distinguish between short and long term duration of disease (<xref rid=\"B94\" ref-type=\"bibr\">94</xref>, <xref rid=\"B95\" ref-type=\"bibr\">95</xref>), but its use as defining established disease is variable and depends on a range of factors, including individual response to early disease</italic>.</p><fn id=\"TN2\"><label>‡</label><p><italic>Fatigue-complex symptoms: initially predominantly neuro-immune (prior to early disease), and variable systemic symptoms in established disease</italic>.</p></fn><fn id=\"TN3\"><label>§</label><p><italic>Tentative proportions for recovery are: likely (>75%); possible (<20%); less likely (<5%). “Likely” and “possible” are based on recovery from arboviruses and EBV [(<xref rid=\"B96\" ref-type=\"bibr\">96</xref>); 100]; “less likely” is based on reviews on prognosis (<xref rid=\"B97\" ref-type=\"bibr\">97</xref>)</italic>.</p></fn><fn id=\"TN4\"><label>¶</label><p><italic>The Prevention level will be considered further in a subsequent publication which is being prepared by the authors</italic>.</p></fn></table-wrap-foot></table-wrap><sec><title>Predisposition and Triggering of Disease</title><p>Individuals with a combination of genetic predispositions and exposures to environmental factors may first manifest symptoms of ME/CFS following their encounter with a specific trigger, of which acute infections of various etiologies are the most commonly reported (<xref rid=\"B64\" ref-type=\"bibr\">64</xref>, <xref rid=\"B65\" ref-type=\"bibr\">65</xref>); other patients report a more insidious onset with no obvious initiating factor (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). While it remains unclear exactly which individuals are predisposed to develop ME/CFS and why, some patterns have emerged. For example, gender- and age-specific factors are thought to contribute to the risk of ME/CFS (<xref rid=\"B66\" ref-type=\"bibr\">66</xref>), with epidemiological studies consistently reporting higher rates of the disease in females (<xref rid=\"B67\" ref-type=\"bibr\">67</xref>, <xref rid=\"B68\" ref-type=\"bibr\">68</xref>). Although most cases are endemic, there have been reports of epidemic cases, suggesting an infectious or other environmental cause play a role (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>, <xref rid=\"B69\" ref-type=\"bibr\">69</xref>–<xref rid=\"B72\" ref-type=\"bibr\">72</xref>); although discrepancies in onset patterns and case definitions make these epidemics difficult to compare (<xref rid=\"B72\" ref-type=\"bibr\">72</xref>). Many studies have reported an association between acute viral infection and the development of ME/CFS (<xref rid=\"B73\" ref-type=\"bibr\">73</xref>–<xref rid=\"B76\" ref-type=\"bibr\">76</xref>). Cases are predominantly reported in North America, Europe, and Oceania; however, the occurrence of ME/CFS is thought to be global with evidence of cases in other parts of the world (<xref rid=\"B77\" ref-type=\"bibr\">77</xref>–<xref rid=\"B79\" ref-type=\"bibr\">79</xref>).</p><p>Psychiatric morbidity, experiences of stress and trauma, either physical or emotional have been reported to precipitate the disease (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B80\" ref-type=\"bibr\">80</xref>–<xref rid=\"B82\" ref-type=\"bibr\">82</xref>) and to predict disease progression (<xref rid=\"B83\" ref-type=\"bibr\">83</xref>), under the explanatory biopsychosocial models. However, these models have not been replicated (<xref rid=\"B84\" ref-type=\"bibr\">84</xref>, <xref rid=\"B85\" ref-type=\"bibr\">85</xref>). Furthermore, Chu et al. (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>) found that even when a significant proportion of their research population report stress or a major life event as a precipitating factor for ME/CFS, “<italic>stressful events were rarely chosen as the only precipitant though, endorsed only by 8% of our subjects, and appeared mostly in conjunction with infection or other precipitants.”</italic> We acknowledge that stress may play a role in the development and perpetuation of ME/CFS through its role on the immune system and HPA axis dysfunction (<xref rid=\"B86\" ref-type=\"bibr\">86</xref>), or by aiding transmission or reactivation of viral infections (<xref rid=\"B87\" ref-type=\"bibr\">87</xref>), or as a consequence of the loss of normal functioning experienced by the individual.</p><p>The role of genetic variation has been supported by a number of family-based studies assessing the possibility of a heritable component (<xref rid=\"B88\" ref-type=\"bibr\">88</xref>–<xref rid=\"B90\" ref-type=\"bibr\">90</xref>). Genes underpinning immune system function and inflammatory response may contribute to genetic susceptibility for ME/CFS; some studies suggest associations with human leucocyte antigen class II alleles (<xref rid=\"B91\" ref-type=\"bibr\">91</xref>, <xref rid=\"B92\" ref-type=\"bibr\">92</xref>) and in genes related to the complement cascade, chemokines, cytokine signaling, and toll-like receptor signaling (<xref rid=\"B93\" ref-type=\"bibr\">93</xref>). Small genome-wide association studies (GWAS) have had little overlap in results save for two SNPs in the GRIK2 gene: a gene implicated in a number of neurological conditions such as autism and schizophrenia (<xref rid=\"B98\" ref-type=\"bibr\">98</xref>); in the GRIK3 gene: relating to a pattern recognition receptor capable of binding to a broad range of pathogens; and in the non-coding regions of T-cell receptor loci (<xref rid=\"B99\" ref-type=\"bibr\">99</xref>). A further study reported SNP markers in candidate genes involved in HPA axis function and neurotransmitter systems that distinguished individuals with ME/CFS (<xref rid=\"B100\" ref-type=\"bibr\">100</xref>).</p></sec><sec><title>Prodromal Period</title><p>It is important to preface here that, with the current diagnostic methodology of ME/CFS stipulating the presence of symptoms for more than 6 months (<xref rid=\"B101\" ref-type=\"bibr\">101</xref>, <xref rid=\"B102\" ref-type=\"bibr\">102</xref>) and the absence of a positive validated diagnostic test, the following processes (occurring pre-diagnosis) are difficult to substantiate from existing biomedical research. However, based on the published work on ME/CFS and considering the pathophysiological events happening in sepsis and polytrauma may be similar (though in a much slower pace), we hypothesize that the following may occur.</p><p>In addition to any manifestations specifically related to the acute insult or triggering event, the mechanisms involved in producing the first symptoms of ME/CFS may be similar to what has been described in relation to “sickness behavior” (<xref rid=\"B103\" ref-type=\"bibr\">103</xref>) or in those with severe acute disease, i.e., “systemic inflammatory response syndrome” (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). These result from the interaction of an infective agent or other insult with the host's immune system, as well as their potential effect on the host's central nervous system (CNS). The immune system-nervous system interactions involve bidirectional signals (<xref rid=\"B104\" ref-type=\"bibr\">104</xref>–<xref rid=\"B106\" ref-type=\"bibr\">106</xref>): while immune system activity may interfere with CNS function via various mechanisms, e.g., release and action of pro-inflammatory cytokines and other mediators, various neurotransmitters, neuropeptides, and neuro-hormones may also affect immune function. Additionally, the HPA system and the autonomic nervous system (ANS) are affected, with consequences that may be observed well-beyond the CNS. These effects may vary according to different factors, such as host susceptibility, the nature and persistence (or return to normality) of systemic and local immune dysfunction, altered CNS metabolism, neuro-transmission, brain perfusion changes, and the integrity of the blood-brain barrier (<xref rid=\"B107\" ref-type=\"bibr\">107</xref>–<xref rid=\"B110\" ref-type=\"bibr\">110</xref>).</p><p>Particular characteristics of the specific infectious agent or stressor may also play a role during this prodromal stage, which would explain the different risks of disease development following acute infection. For example, there has long been an interest in the association between ME/CFS and infections such as Epstein-Barr virus (EBV) and other herpesviruses (<xref rid=\"B73\" ref-type=\"bibr\">73</xref>, <xref rid=\"B111\" ref-type=\"bibr\">111</xref>–<xref rid=\"B116\" ref-type=\"bibr\">116</xref>). Herpesviruses tend to be neurotropic and persist following acute infection in a latent state. Similar to EBV infection (<xref rid=\"B117\" ref-type=\"bibr\">117</xref>), the risk of chronic fatigue has been shown to be substantially increased following viral meningitis, a relatively severe infection of the CNS (<xref rid=\"B83\" ref-type=\"bibr\">83</xref>).</p></sec><sec><title>Early Disease</title><p>Early disease represents a continuation of the processes initiated at the prodromal period, when there is a failure of physiological and homeostatic processes to resume previous levels of equilibrium and normality. Fatigue and other symptoms may be largely explained by a combination of the local and systemic effects of pro-inflammatory and other mediators or toxins, CNS metabolic dysfunction (with enhanced excitability and other changes), and a systemic hyper-metabolic state. With higher energy demands for essential biological processes, there will be a reduction in the available energy for less essential tasks, including those demanding increased physical or mental exertion. The increased production and action of anti-inflammatory mediators, as well as their ability to counter-balance pro-inflammatory stimuli, modulate physiological responses, and symptoms and affect disease progression or reversibility. As mentioned previously, without a validated biomarker to diagnose ME/CFS early it is difficult to substantiate the exact mechanisms occurring in the early disease phase. Research into potential diagnostic markers, such as the recent study on impedance signatures (<xref rid=\"B118\" ref-type=\"bibr\">118</xref>), are crucial not only clinically, but to identify these mechanisms as possible targets for early intervention.</p></sec><sec><title>Established ME/CFS</title><p>The persistence of immune and CNS dysfunction with the initial over-production of pro-inflammatory and neurotoxic factors may result in a prolonged state of low-grade neurological and systemic inflammation. In the CNS, a status of glial activation with microglial hypersensitivity to peripheral (<xref rid=\"B119\" ref-type=\"bibr\">119</xref>) and regional stimuli is established (<xref rid=\"B104\" ref-type=\"bibr\">104</xref>, <xref rid=\"B119\" ref-type=\"bibr\">119</xref>–<xref rid=\"B121\" ref-type=\"bibr\">121</xref>), akin to what has been described in chronic pain states (<xref rid=\"B122\" ref-type=\"bibr\">122</xref>). In support of CNS dysfunction, neuroimaging studies have shown various abnormalities in ME/CFS, often associated with symptoms of fatigue and other indications of severity (<xref rid=\"B123\" ref-type=\"bibr\">123</xref>). Glial activation in several areas of the brain has also been demonstrated in positron emission tomography (PET) scans of patients with fibromyalgia (FM), compared to controls, which was correlated to the severity of fatigue (<xref rid=\"B123\" ref-type=\"bibr\">123</xref>, <xref rid=\"B124\" ref-type=\"bibr\">124</xref>). Neuro-glial bidirectional signaling is associated with increased production of neuro-excitatory neurotransmitters and immune-inflammatory mediators (<xref rid=\"B120\" ref-type=\"bibr\">120</xref>).</p><p>Nervous system dysfunction affecting parts of the brain, brain stem, and ANS, could explain not only the encephalopathic or neuro-cognitive type of symptoms, but also those resulting from disruption of key central regulatory mechanisms, such as those involved in endocrine, circulatory, thermoregulation, and respiratory control (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B48\" ref-type=\"bibr\">48</xref>, <xref rid=\"B120\" ref-type=\"bibr\">120</xref>, <xref rid=\"B125\" ref-type=\"bibr\">125</xref>). Examples of these include intolerance to extremes of temperature, chills and temperature variations, intolerance to exertion, hyperventilation or irregular breathing, orthostatic intolerance, with hypotension or postural orthostatic tachycardia, and other symptoms related to autonomic and endocrine control function (<xref rid=\"B102\" ref-type=\"bibr\">102</xref>).</p><p>Among the various by-products produced as a consequence of ongoing abnormalities, are highly ROS and nitric oxide synthase (NOS) or free radicals, which affect cell signaling and cell functioning and structure, particularly when present at high levels. It has been hypothesized that free radicals, and increased levels of nitric oxide and peroxynitrite in particular, play a significant role in ME/CFS (<xref rid=\"B126\" ref-type=\"bibr\">126</xref>, <xref rid=\"B127\" ref-type=\"bibr\">127</xref>); their links to immune and neuro signaling, cell integrity, mitochondrial function, and energy metabolism may play an important part in the long term abnormalities in ME/CFS.</p><p>The nature of neuro-immune and other dysfunctions may change as disease progresses. While a pro-inflammatory state is typical of the early response to insults, immune abnormalities may become less marked (and less pro-inflammatory) with time (<xref rid=\"B128\" ref-type=\"bibr\">128</xref>), and patients with longer periods of illness may show fewer inflammatory immunological abnormalities. In support of this, our preliminary results from the analysis of over 200 ME/CFS patients participating in the UK ME/CFS Biobank (UKMEB), showed that the reported time since disease onset was significantly associated with 2 cytokines, namely SCD40L and IL1RA (manuscript in preparation). These results were found after aliquots of peripheral blood mononuclear cells (PBMC) from participants were stimulated (i.e., subjected to an infection resembling stimulus) and analyzed with MAGPIX® multiplexing system. The statistical analyses were conducted after transforming each cytokine measurement to the logarithm scale to approximate normality; linear regression of these log-transformed values (adapted for truncated outcome variables to account for the assay's limits of detections) was applied to the variables' time since onset, level of severity (mild to moderate vs. severe) and the interaction between severity and time since onset, while also adjusting for age and sex. The results evidenced a decrease of sCD40L—a pro-inflammatory cytokine—and an increase of IL1RA—an anti-inflammatory cytokine—for every additional year since onset of ME.</p></sec><sec><title>Long-Term, Advanced, and Complicated Disease</title><p>As the disease progresses, physiological, and systems abnormalities take their toll and cell dysfunction becomes more pronounced. Endothelial dysfunction may arise as a consequence of a range of factors, including, but not limited to, persistent oxidative and nitrosative stress and circulatory dysfunction (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>, <xref rid=\"B52\" ref-type=\"bibr\">52</xref>, <xref rid=\"B126\" ref-type=\"bibr\">126</xref>, <xref rid=\"B129\" ref-type=\"bibr\">129</xref>, <xref rid=\"B130\" ref-type=\"bibr\">130</xref>). The associated reduced delivery of oxygen and nutrients to the cell leads to a deterioration of cell function and impaired energy metabolism (<xref rid=\"B129\" ref-type=\"bibr\">129</xref>, <xref rid=\"B131\" ref-type=\"bibr\">131</xref>, <xref rid=\"B132\" ref-type=\"bibr\">132</xref>) and a decreased ability of the cell to extract oxygen and produce energy, a condition known as cytopathic hypoxia. As suggested by Naviaux et al. (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>), in cases of ME/CFS with mean duration of symptoms over 17 years, there is a shutting down of various metabolic processes leading to a hypometabolic state, i.e., a move to an energy-saving mode. At this stage, symptoms are likely to be severe, with profound fatigue, intolerance to effort, PEM and other systemic symptoms, which are largely explained by the slowness of physiological and metabolic processes and decreased energy production.</p></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><sec><title>Disease Severity and Reversibility</title><p>It is unknown how the initial host response to a stressor or insult compares in individuals who do or do not develop typical symptoms of ME/CFS. However, the return to good health, which happens to most people following exposure to mild or moderate levels of insult, seems to be impeded in ME/CFS when symptoms persist for longer than 3–6 months; the time interval that is featured in some of the currently used diagnostic criteria (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B101\" ref-type=\"bibr\">101</xref>, <xref rid=\"B102\" ref-type=\"bibr\">102</xref>). This suggests that subsequent mechanisms involved in the host response will differ at some point in those who develop ME/CFS from those who regain full health. Therefore, a key question is what determines full recovery? Or alternatively, what determines the perpetuation and transformation of symptoms?</p><p>While the abnormalities observed in acute disease are general and mostly reversible once the challenge from the stressor ceases, some degree of dysfunction may persist for longer periods. The degree of reversibility of various physiological abnormalities is likely to decrease with time, and some permanent functional, and even structural, damage may occur consequently. This is likely caused by either the persistence or frequent reactivation of the initial stressor (<xref rid=\"B87\" ref-type=\"bibr\">87</xref>, <xref rid=\"B133\" ref-type=\"bibr\">133</xref>), an accumulation of insults, a continuing dysfunctional host-response, or the effects of the numerous psychosocial risk factors that influence disease development and progression (<xref rid=\"B134\" ref-type=\"bibr\">134</xref>), or a combination of all of these.</p><p>Although our framework focuses on the underlying biological mechanisms that may be at play in the development and progression of ME/CFS, it is important to acknowledge the impact of psychosocial and behavioral aspects in the progression of chronic diseases. Stressors such as stressful life events, low satisfaction with social and medical support, and excessive use of coping mechanisms, have been shown to contribute to the neuroendocrine and immune responses by acting through complex pathways that ultimately affect health and health outcomes (<xref rid=\"B134\" ref-type=\"bibr\">134</xref>–<xref rid=\"B136\" ref-type=\"bibr\">136</xref>).</p><p>The interplay between these three dimensions (biological, psychosocial, and behavior) has been noted in the development and the progression of a number of chronic diseases and to influence disease outcomes (<xref rid=\"B136\" ref-type=\"bibr\">136</xref>–<xref rid=\"B139\" ref-type=\"bibr\">139</xref>). The combined effects of stress from work or family life, social deprivation, and depression have been found to contribute to the risk of cardiovascular diseases, including coronary heart disease (<xref rid=\"B140\" ref-type=\"bibr\">140</xref>) and myocardial infarction (<xref rid=\"B141\" ref-type=\"bibr\">141</xref>), and to a worse prognosis (<xref rid=\"B142\" ref-type=\"bibr\">142</xref>) by enhancing cortisol secretion, increasing sympathetic activation, and elevating plasma catecholamine levels (<xref rid=\"B143\" ref-type=\"bibr\">143</xref>). A higher cumulative average number of stressful life events, when coping involves denial, and higher levels of serum cortisol have been found to be associated with a faster progression to AIDS (<xref rid=\"B144\" ref-type=\"bibr\">144</xref>). Correspondingly, low stress levels and low scores of avoidance coping behaviors were shown to be protective against relapse in Crohn's disease patients (<xref rid=\"B135\" ref-type=\"bibr\">135</xref>) in contrast to high levels, which act as mediators, overloading the sympathetic nervous system.</p><p>In the case of ME/CFS, the effect of these dimensions is the same. In fact, one framework has been used to propose a model for managing patients with this disease in which it is considered that genes predispose, life events precipitate, and behaviors perpetuate (<xref rid=\"B145\" ref-type=\"bibr\">145</xref>–<xref rid=\"B147\" ref-type=\"bibr\">147</xref>). However, this model may downplay the important role of the biological mechanisms involved in ME/CFS and overstate the role of psychosocial and behavioral factors (<xref rid=\"B148\" ref-type=\"bibr\">148</xref>).</p><p>The pathophysiological distinction between cases from the milder to the more severe end of the ME/CFS spectrum may relate to near-normal homeostatic regulation in milder cases, and established “aberrant homeostasis” or homeostatic dysregulation with multi-systemic consequences in moderate to severe cases. Alternatively, homeostatic failure, along with variable multi-system physiological failure and increasing degrees of irreversibility, may happen in the most severe cases.</p><p>The early stage of ME/CFS is of variable duration but is usually considered to be between 4 and 6 months to 2 years after the start of prodromal symptoms. Reversibility is possible, but often people will evolve to chronicity or established ME/CFS with either: (a) partial reversal of dysfunctional physiological mechanisms (mild cases with slow improvement over time); (b) persistence of dysfunctions and symptoms (mild or moderate cases with stable symptoms or slow changes over time); or (c) worsening dysfunctions and symptoms (moderate and severe cases) (<xref rid=\"B149\" ref-type=\"bibr\">149</xref>). Note that some cases present early with severe symptoms, which not uncommonly evolve to a milder form (<xref rid=\"B150\" ref-type=\"bibr\">150</xref>). The use of coping mechanisms, such as pacing, can also help improve energy management in people with ME/CFS over time and reduce the risk of relapse into a more severe state; however, there is little evidence that these will lead to a reversibility (<xref rid=\"B151\" ref-type=\"bibr\">151</xref>). There is some indication that rates of resolution are higher in cases of epidemic CFS compared to sporadic cases, although very few of these individuals will recover to their pre-morbid level (<xref rid=\"B152\" ref-type=\"bibr\">152</xref>).</p><p>One way of thinking about these phases is as interconnected spirals, each representing a distinct disease phase. Individuals may either remain for long periods in a single phase with symptoms fluctuating within the “spiral section” or move between phases either upwards (i.e., toward better health status) or downwards (i.e., toward disease deterioration). <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref> represents an illustration of the multi-spiraling disease course suggested for ME/CFS, and shows how patients may move across spirals, with different molecular and system abnormalities.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Hypothetical stages of disease in ME/CFS.</p></caption><graphic xlink:href=\"fneur-11-00826-g0002\"/></fig></sec><sec><title>Common Comorbidities in ME/CFS</title><p>There are a number of comorbid conditions associated with ME/CFS and, as such, these comorbidities can complicate diagnosis, treatment and research of the disease. Comorbidities have been found in up to 97% of people with ME/CFS (PWME) (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B153\" ref-type=\"bibr\">153</xref>) with some developing before, with, or after ME/CFS onset (<xref rid=\"B102\" ref-type=\"bibr\">102</xref>). The complexity of ME/CFS is in part due to the number of different systems affected that contribute to the many and varied symptoms experienced. ME/CFS and FM share a number of overlapping core symptoms that mean the two are commonly experienced together; FM has been reported to co-occur in 12–91% of PWME (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B154\" ref-type=\"bibr\">154</xref>, <xref rid=\"B155\" ref-type=\"bibr\">155</xref>). However, there is evidence to suggest the two conditions differ in their hormone dynamics, genetic/molecular biology, and autonomic function (<xref rid=\"B156\" ref-type=\"bibr\">156</xref>, <xref rid=\"B157\" ref-type=\"bibr\">157</xref>). This is reiterated by the absence of post-exertional malaise in FM (<xref rid=\"B158\" ref-type=\"bibr\">158</xref>, <xref rid=\"B159\" ref-type=\"bibr\">159</xref>), which is one of the key features of ME/CFS (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B101\" ref-type=\"bibr\">101</xref>, <xref rid=\"B102\" ref-type=\"bibr\">102</xref>, <xref rid=\"B160\" ref-type=\"bibr\">160</xref>).</p><p>Sleep disturbances can cause some symptoms that are also present in ME/CFS including fatigue, joint pain, and impaired cognition (<xref rid=\"B161\" ref-type=\"bibr\">161</xref>–<xref rid=\"B165\" ref-type=\"bibr\">165</xref>). Additionally, as part of a bidirectional relationship, comorbid pain conditions may further impact sleep quality (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). Sleep disturbances are also present in a number of neurological diseases (<xref rid=\"B166\" ref-type=\"bibr\">166</xref>), which would explain their presence as an important feature in ME/CFS (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B160\" ref-type=\"bibr\">160</xref>); however, differences in sleep cycle patterns and distinct sleep phenotypes suggest that ME/CFS and primary sleep disorders are, in fact, different entities (<xref rid=\"B167\" ref-type=\"bibr\">167</xref>, <xref rid=\"B168\" ref-type=\"bibr\">168</xref>) with many PWME showing normal sleep study results (<xref rid=\"B169\" ref-type=\"bibr\">169</xref>). Primary sleep disturbances are considered exclusionary for ME/CFS by a number of diagnostic criteria (<xref rid=\"B101\" ref-type=\"bibr\">101</xref>, <xref rid=\"B102\" ref-type=\"bibr\">102</xref>, <xref rid=\"B160\" ref-type=\"bibr\">160</xref>), however, with little evidence that treatment of these disorders improves symptoms of ME/CFS it is argued they are better considered as comorbid conditions (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B34\" ref-type=\"bibr\">34</xref>, <xref rid=\"B170\" ref-type=\"bibr\">170</xref>).</p><p>Also highly prevalent in those with ME/CFS is orthostatic intolerance (OI), a common multifactorial disorder commonly accompanying neurodegenerative, cardiovascular, metabolic, and renal disorders (<xref rid=\"B171\" ref-type=\"bibr\">171</xref>). Disruptions to ANS and reduced blood volume contribute to OI (<xref rid=\"B172\" ref-type=\"bibr\">172</xref>) and the same systemic dysfunctions have been reported in those with ME/CFS (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>); however, not all people with OI disorders have ME/CFS (<xref rid=\"B173\" ref-type=\"bibr\">173</xref>, <xref rid=\"B174\" ref-type=\"bibr\">174</xref>).</p><p>Intestinal dysbiosis thought to be associated with some CNS-related disorders via the gut-brain-axis (<xref rid=\"B175\" ref-type=\"bibr\">175</xref>). IBS is another largely overlapping syndrome with both ME/CFS and FM but metabolic profiles are distinct in ME/CFS and ME/CFS with IBS subgroups (<xref rid=\"B176\" ref-type=\"bibr\">176</xref>). Some authors hypothesize IBS could be considered an initial symptom of ME/CFS, as they reported that 65% of IBS patients followed up developed ME (<xref rid=\"B177\" ref-type=\"bibr\">177</xref>). Authors of a co-twin control study found significant associations between CFS and FM, IBS, chronic pelvic pain, multiple chemical sensitivities, and temporomandibular disorder. After controlling for psychiatric risk factors, they argued that these associations could not be attributed to uniquely psychiatric illness, thus suggesting a “<italic>complex interplay of genes and environmental factors”</italic> to help explain the clinical picture (<xref rid=\"B178\" ref-type=\"bibr\">178</xref>).</p><p>While healthcare costs likely increase following the diagnosis of additional comorbidities (<xref rid=\"B178\" ref-type=\"bibr\">178</xref>), treating comorbidities may improve the quality of life of PWME (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>) not only symptomatically but also in what they might be able to contribute to the economy. We argue that by using the proposed natural history framework, how and when common comorbidities develop in relation to ME/CFS may be highlighted, allowing researchers, and clinicians to better tailor potential interventions according to each phase, thus resulting in a more efficient management of costs.</p></sec><sec><title>Research Implications</title><p>These distinct hypothetical stages may help explain the apparent inconsistency of findings from ME/CFS studies, which likely include cases at distinct stages of disease with potentially diverse systems abnormalities. Hence, we consider that the conceptual approach presented in this paper may help to elucidate pathophysiological mechanisms that may be more prominent at different stages of disease; and consequently, could indicate potential target therapeutic approaches in future. We argue that the different stages patients go through during the course of the disease, their severity, and the presence and degree of complications are key parameters for disease stratification.</p><p>Research leading to an understanding of what is occurring during the first three stages of progression to ME/CFS is greatly needed but requires the recruitment of individuals for research at pre-illness stage. Such research could be invaluable to understanding the biological mechanisms at play before, during and after an insult, and research using proxy disease models for ME/CFS (<xref rid=\"B85\" ref-type=\"bibr\">85</xref>) or follow up of patients after an acute viral infection [e.g., mononucleosis (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>) or more presently COVID-19] could begin to address this knowledge gap. Electronic health records could also be a valuable source of retrospective pre-illness data in people with ME/CFS. Well-designed longitudinal studies, with strict protocols, would help refine this attempted description of the natural course of the ME/CFS, and the interpretation of the findings.</p></sec></sec><sec sec-type=\"conclusions\" id=\"s5\"><title>Conclusions</title><p>The concept of the natural history of disease, common in the field of public health and medicine, serves to frame a disease according to how it progresses from a pre-illness stage to the final disease outcome. Due to the lack of knowledge surrounding the etiology of ME/CFS, the heterogeneous presentation of symptoms and their severity, and the lack of a recognized and validated biomarker to determine diagnosis, the natural history of this disease has been hard to determine. While current research efforts tend to group ME/CFS subtypes according to clusters of symptoms, few studies have considered ME/CFS as a continuum.</p><p>Pathophysiological patterns and changes along and across disease stages result in the expression of different, albeit overlapping phenotypes as seen in the preliminary UKMEB findings related to changes in cytokine levels and symptoms scores with time of disease, reported here. Ignoring phenotype temporal variation in ME/CFS may have an impact on the outputs and the interpretation of research investigating disease mechanisms, pathways, and interventions.</p><p>This paper sought to provide a simple framework, similar to those of other chronic diseases, in an effort to extend the temporal perception of ME/CFS and better incorporate the less defined pre-illness stages of the disease. We believe that by applying this framework to ME/CFS research efforts could better elucidate the pathophysiological mechanisms of the disease and identify potential therapeutic targets at distinct stages.</p></sec><sec sec-type=\"data-availability\" id=\"s6\"><title>Data Availability Statement</title><p>The datasets generated for this study are available on request to the corresponding author.</p></sec><sec id=\"s7\"><title>Ethics Statement</title><p>Ethical approval was granted by the LSHTM Ethics Committee 16 January 2012 (Ref.6123) and the National Research Ethics Service (NRES) London-Bloomsbury Research Ethics Committee 22 December 2011 (REC ref.11/10/1760, IRAS ID: 77765). All biobank participants provided written consent for questionnaire, clinical measurement and laboratory test data, and samples to be made available for ethically approved research, after receiving an extensive information sheet and consent form, which includes an option to withdraw from the study at any time and without any restrictions.</p></sec><sec id=\"s8\"><title>Author Contributions</title><p>LN and EL conceived the paper. LP and EL provided the preliminary findings from data from the UKMEB participants and possible interpretation of them. SO'B contributed to drafting, referencing, and formatting. All authors contributed to drafting and to revising the manuscript and approved the final version to be published.</p></sec><sec id=\"s9\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> The UK ME/CFS Biobank was established with a joint grant from the charities ME Association (including continuing support), ME Research UK and Action for ME, as well as private donors. Research reported in this manuscript was supported by the National Institutes of Health under award number 2R01AI103629. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nacul</surname><given-names>LC</given-names></name><name><surname>Lacerda</surname><given-names>EM</given-names></name><name><surname>Kingdon</surname><given-names>CC</given-names></name><name><surname>Curran</surname><given-names>H</given-names></name><name><surname>Bowman</surname><given-names>EW</given-names></name></person-group>. <article-title>How have selection bias and disease misclassification undermined the validity of myalgic encephalomyelitis/chronic fatigue syndrome studies?</article-title>\n<source>J Health Psychol.</source> (<year>2017</year>) <volume>24</volume>:<fpage>1765</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1177/1359105317695803</pub-id><pub-id pub-id-type=\"pmid\">28810428</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><collab>Institute of Medicine (IOM)</collab></person-group>\n<source>Beyond Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Redefining an Illness</source>. Washington, DC: The National Academies Press (<year>2015</year>). Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.nap.edu/catalog.php?record_id=19012\">http://www.nap.edu/catalog.php?record_id=19012</ext-link></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Twisk</surname><given-names>F</given-names></name></person-group>\n<article-title>Myalgic Encephalomyelitis (ME) or what? An operational definition</article-title>. <source>Diagnostics</source>. (<year>2018</year>) <volume>8</volume>:<fpage>64</fpage>\n<pub-id pub-id-type=\"doi\">10.3390/diagnostics8030064</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Ramsay</surname><given-names>AM</given-names></name></person-group>\n<source>Postviral Fatigue Syndrome—The Saga of Royal Free Disease</source>. <edition>1st ed</edition>\n<publisher-loc>London</publisher-loc>: <publisher-name>Gower Medical Publishing Limited</publisher-name> (<year>1986</year>). Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.meassociation.org.uk/shop/books/saga-of-royal-free-disease/\">https://www.meassociation.org.uk/shop/books/saga-of-royal-free-disease/</ext-link>.</mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reynolds</surname><given-names>GK</given-names></name><name><surname>Lewis</surname><given-names>DP</given-names></name><name><surname>Richardson</surname><given-names>AM</given-names></name><name><surname>Lidbury</surname><given-names>BA</given-names></name></person-group>. <article-title>Comorbidity of postural orthostatic tachycardia syndrome and chronic fatigue syndrome in an Australian cohort</article-title>. <source>J Intern Med.</source> (<year>2014</year>) <volume>275</volume>:<fpage>409</fpage>–<lpage>17</lpage>. <pub-id pub-id-type=\"doi\">10.1111/joim.12161</pub-id><pub-id pub-id-type=\"pmid\">24206536</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Clark</surname><given-names>MR</given-names></name><name><surname>Katon</surname><given-names>W</given-names></name><name><surname>Russo</surname><given-names>J</given-names></name><name><surname>Kith</surname><given-names>P</given-names></name><name><surname>Sintay</surname><given-names>M</given-names></name><name><surname>Buchwald</surname><given-names>D</given-names></name></person-group>. <article-title>Chronic fatigue: risk factors for symptom persistence in a 2 1/2-year follow-up study</article-title>. <source>Am J Med.</source> (<year>1995</year>) <volume>98</volume>:<fpage>187</fpage>–<lpage>95</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0002-9343(99)80403-3</pub-id><pub-id pub-id-type=\"pmid\">7847436</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kroenke</surname><given-names>K</given-names></name><name><surname>Wood</surname><given-names>DR</given-names></name><name><surname>Mangelsdorff</surname><given-names>AD</given-names></name><name><surname>Meier</surname><given-names>NJ</given-names></name><name><surname>Powell</surname><given-names>JB</given-names></name></person-group>. <article-title>Chronic fatigue in primary care: prevalence, patient characteristics, and outcome</article-title>. <source>JAMA.</source> (<year>1988</year>) <volume>260</volume>:<fpage>929</fpage>. <pub-id pub-id-type=\"doi\">10.1001/jama.1988.03410070057028</pub-id><pub-id pub-id-type=\"pmid\">3398197</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Joyce</surname><given-names>J</given-names></name><name><surname>Hotopf</surname><given-names>M</given-names></name><name><surname>Wessely</surname><given-names>S</given-names></name></person-group>. <article-title>The prognosis of chronic fatigue and chronic fatigue syndrome: a systematic review</article-title>. <source>QJM.</source> (<year>1997</year>) <volume>90</volume>:<fpage>223</fpage>–<lpage>33</lpage>. <pub-id pub-id-type=\"doi\">10.1093/qjmed/90.3.223</pub-id><pub-id pub-id-type=\"pmid\">9093600</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bierl</surname><given-names>C</given-names></name><name><surname>Nisenbaum</surname><given-names>R</given-names></name><name><surname>Hoaglin</surname><given-names>DC</given-names></name><name><surname>Randall</surname><given-names>B</given-names></name><name><surname>Jones</surname><given-names>A</given-names></name><name><surname>Unger</surname><given-names>ER</given-names></name><etal/></person-group>. <article-title>Regional distribution of fatiguing illnesses in the United States: a pilot study</article-title>. <source>Population Health Metr</source>. (<year>2004</year>) <volume>2</volume>:<fpage>1</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1478-7954-2-1</pub-id><pub-id pub-id-type=\"pmid\">14761250</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jason</surname><given-names>LA</given-names></name><name><surname>Corradi</surname><given-names>K</given-names></name><name><surname>Torres-Harding</surname><given-names>S</given-names></name><name><surname>Taylor</surname><given-names>RR</given-names></name><name><surname>King</surname><given-names>C</given-names></name></person-group>. <article-title>Chronic fatigue syndrome: the need for subtypes</article-title>. <source>Neuropsychol Rev.</source> (<year>2005</year>) <volume>15</volume>:<fpage>29</fpage>–<lpage>58</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s11065-005-3588-2</pub-id><pub-id pub-id-type=\"pmid\">15929497</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Janal</surname><given-names>MN</given-names></name><name><surname>Ciccone</surname><given-names>DS</given-names></name><name><surname>Natelson</surname><given-names>BH</given-names></name></person-group>. <article-title>Sub-typing CFS patients on the basis of ‘minor' symptoms</article-title>. <source>Biol Psychol.</source> (<year>2006</year>) <volume>73</volume>:<fpage>124</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.biopsycho.2006.01.003</pub-id><pub-id pub-id-type=\"pmid\">16473456</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Gough</surname><given-names>J</given-names></name><name><surname>Christmas</surname><given-names>D</given-names></name><name><surname>Mattey</surname><given-names>DL</given-names></name><name><surname>Richards</surname><given-names>SC</given-names></name><name><surname>Main</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Microbial infections in eight genomic subtypes of chronic fatigue syndrome/myalgic encephalomyelitis</article-title>. <source>J Clin Pathol.</source> (<year>2010</year>) <volume>63</volume>:<fpage>156</fpage>–<lpage>64</lpage>. <pub-id pub-id-type=\"doi\">10.1136/jcp.2009.072561</pub-id><pub-id pub-id-type=\"pmid\">19955554</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kerr</surname><given-names>JR</given-names></name><name><surname>Burke</surname><given-names>B</given-names></name><name><surname>Petty</surname><given-names>R</given-names></name><name><surname>Gough</surname><given-names>J</given-names></name><name><surname>Fear</surname><given-names>D</given-names></name><name><surname>Mattey</surname><given-names>DL</given-names></name><etal/></person-group>. <article-title>Seven genomic subtypes of chronic fatigue syndrome/myalgic encephalomyelitis: a detailed analysis of gene networks and clinical phenotypes</article-title>. <source>J Clin Pathol.</source> (<year>2008</year>) <volume>61</volume>:<fpage>730</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1136/jcp.2007.053553</pub-id><pub-id pub-id-type=\"pmid\">18057078</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nagy-Szakal</surname><given-names>D</given-names></name><name><surname>Barupal</surname><given-names>DK</given-names></name><name><surname>Lee</surname><given-names>B</given-names></name><name><surname>Che</surname><given-names>X</given-names></name><name><surname>Williams</surname><given-names>BL</given-names></name><name><surname>Kahn</surname><given-names>EJR</given-names></name><etal/></person-group>. <article-title>Insights into myalgic encephalomyelitis/chronic fatigue syndrome phenotypes through comprehensive metabolomics</article-title>. <source>Sci Rep.</source> (<year>2018</year>) <volume>8</volume>:<fpage>10056</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-018-28477-9</pub-id><pub-id pub-id-type=\"pmid\">29968805</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Friedberg</surname><given-names>F</given-names></name><name><surname>Dechene</surname><given-names>L</given-names></name><name><surname>McKenzie</surname><given-names>MJ</given-names></name><name><surname>Fontanetta</surname><given-names>R</given-names></name></person-group>. <article-title>Symptom patterns in long-duration chronic fatigue syndrome</article-title>. <source>J Psychosomatic Res.</source> (<year>2000</year>) <volume>48</volume>:<fpage>59</fpage>–<lpage>68</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0022-3999(99)00077-X</pub-id><pub-id pub-id-type=\"pmid\">10750631</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chu</surname><given-names>L</given-names></name><name><surname>Valencia</surname><given-names>IJ</given-names></name><name><surname>Garvert</surname><given-names>DW</given-names></name><name><surname>Montoya</surname><given-names>JG</given-names></name></person-group>. <article-title>Onset patterns and course of myalgic encephalomyelitis/chronic fatigue syndrome</article-title>. <source>Front Pediatrics.</source> (<year>2019</year>) <volume>7</volume>:<fpage>12</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fped.2019.00012</pub-id><pub-id pub-id-type=\"pmid\">30805319</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jewell</surname><given-names>NP</given-names></name></person-group>. <article-title>Natural history of diseases: statistical designs and issues</article-title>. <source>Clin Pharmacol Therap.</source> (<year>2016</year>) <volume>100</volume>:<fpage>353</fpage>–<lpage>61</lpage>. <pub-id pub-id-type=\"doi\">10.1002/cpt.423</pub-id><pub-id pub-id-type=\"pmid\">27393601</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cairns</surname><given-names>R</given-names></name><name><surname>Hotopf</surname><given-names>M</given-names></name></person-group>. <article-title>A systematic review describing the prognosis of chronic fatigue syndrome</article-title>. <source>Occup Med.</source> (<year>2005</year>) <volume>55</volume>:<fpage>20</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.1093/occmed/kqi013</pub-id><pub-id pub-id-type=\"pmid\">15699087</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Mello</surname><given-names>P</given-names></name><name><surname>Gusmao-Flores</surname><given-names>D</given-names></name><name><surname>Dellinger</surname><given-names>RP</given-names></name></person-group>\n<article-title>Sepsis</article-title>. In: O'Donnell JM, Nácul FE, editors. <edition>3rd ed</edition>\n<source>Surgical Intensive Care Medicine.</source>\n<publisher-loc>Cham</publisher-loc>: <publisher-name>Springer International Publishing</publisher-name> (<year>2016</year>). p. <fpage>373</fpage>–<lpage>87</lpage>. <pub-id pub-id-type=\"doi\">10.1007/978-3-319-19668-8_28</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Keel</surname><given-names>M</given-names></name><name><surname>Trentz</surname><given-names>O</given-names></name></person-group>. <article-title>Pathophysiology of polytrauma</article-title>. <source>Injury.</source> (<year>2005</year>) <volume>36</volume>:<fpage>691</fpage>–<lpage>709</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.injury.2004.12.037</pub-id><pub-id pub-id-type=\"pmid\">15910820</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Rosenblatt</surname><given-names>MS</given-names></name><name><surname>Delmonico</surname><given-names>TR</given-names></name></person-group>\n<article-title>Trauma</article-title>. In: O'Donnell JM, Nácul FEM, editors, <edition>3rd ed</edition>\n<source>Surgical Intensive Care Medicine.</source>\n<publisher-loc>Cham</publisher-loc>: <publisher-name>Springer International Publishing</publisher-name> (<year>2016</year>). p. <fpage>599</fpage>–<lpage>610</lpage>. <pub-id pub-id-type=\"doi\">10.1007/978-3-319-19668-8_44</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>O'Donnell</surname><given-names>JM</given-names></name><name><surname>Nacul</surname><given-names>FE</given-names></name></person-group> (editors). <source>Surgical Intensive Care Medicine</source>. <edition>3rd ed</edition>\n<publisher-loc>Basel</publisher-loc>: <publisher-name>Springer International Publishing</publisher-name> (<year>2016</year>).</mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Herman</surname><given-names>JP</given-names></name><name><surname>Mcklveen</surname><given-names>JM</given-names></name><name><surname>Ghosal</surname><given-names>S</given-names></name><name><surname>Kopp</surname><given-names>B</given-names></name><name><surname>Wulsin</surname><given-names>A</given-names></name><name><surname>Makinson</surname><given-names>R</given-names></name><etal/></person-group>. <article-title>Regulation of the hypothalamic-pituitary-adrenocortical stress response</article-title>. <source>Compr Physiol.</source> (<year>2016</year>) <volume>6</volume>:<fpage>603</fpage>–<lpage>21</lpage>. <pub-id pub-id-type=\"doi\">10.1002/cphy.c150015</pub-id><pub-id pub-id-type=\"pmid\">27065163</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martin</surname><given-names>L</given-names></name><name><surname>Koczera</surname><given-names>P</given-names></name><name><surname>Zechendorf</surname><given-names>E</given-names></name><name><surname>Schuerholz</surname><given-names>T</given-names></name></person-group>. <article-title>The endothelial glycocalyx: new diagnostic and therapeutic approaches in sepsis</article-title>. <source>BioMed Res Int.</source> (<year>2016</year>) <volume>2016</volume>:<fpage>3758278</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2016/3758278</pub-id><pub-id pub-id-type=\"pmid\">27699168</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Russell</surname><given-names>JA</given-names></name><name><surname>Rush</surname><given-names>B</given-names></name><name><surname>Boyd</surname><given-names>J</given-names></name></person-group>. <article-title>Pathophysiology of septic shock</article-title>. <source>Crit Care Clin.</source> (<year>2018</year>) <volume>34</volume>:<fpage>43</fpage>–<lpage>61</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ccc.2017.08.005</pub-id><pub-id pub-id-type=\"pmid\">29149941</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pool</surname><given-names>R</given-names></name><name><surname>Gomez</surname><given-names>H</given-names></name><name><surname>Kellum</surname><given-names>JA</given-names></name></person-group>. <article-title>Mechanisms of organ dysfunction in sepsis</article-title>. <source>Crit Care Clin.</source> (<year>2018</year>) <volume>34</volume>:<fpage>63</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ccc.2017.08.003</pub-id><pub-id pub-id-type=\"pmid\">29149942</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Singer</surname><given-names>M</given-names></name></person-group>. <article-title>The role of mitochondrial dysfunction in sepsis-induced multi-organ failure</article-title>. <source>Virulence.</source> (<year>2014</year>) <volume>5</volume>:<fpage>66</fpage>–<lpage>72</lpage>. <pub-id pub-id-type=\"doi\">10.4161/viru.26907</pub-id><pub-id pub-id-type=\"pmid\">24185508</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kalogeris</surname><given-names>T</given-names></name><name><surname>Baines</surname><given-names>CP</given-names></name><name><surname>Krenz</surname><given-names>M</given-names></name><name><surname>Korthuis</surname><given-names>RJ</given-names></name></person-group>. <article-title>Cell biology of ischemia/reperfusion injury</article-title>. <source>Int Rev Cell Mol Biol.</source> (<year>2012</year>) <volume>298</volume>:<fpage>229</fpage>–<lpage>317</lpage>. <pub-id pub-id-type=\"doi\">10.1016/B978-0-12-394309-5.00006-7</pub-id><pub-id pub-id-type=\"pmid\">22878108</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McEwen</surname><given-names>BS</given-names></name></person-group>\n<article-title>Stressed or stressed out: what is the difference?</article-title>\n<source>J Psychiatry Neurosci</source>. (<year>2005</year>) <volume>30</volume>:<fpage>315</fpage>–<lpage>8</lpage>.<pub-id pub-id-type=\"pmid\">16151535</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hatziagelaki</surname><given-names>E</given-names></name><name><surname>Adamaki</surname><given-names>M</given-names></name><name><surname>Tsilioni</surname><given-names>I</given-names></name><name><surname>Dimitriadis</surname><given-names>G</given-names></name><name><surname>Theoharides</surname><given-names>TC</given-names></name></person-group>\n<article-title>Myalgic encephalomyelitis/chronic fatigue syndrome—metabolic disease or disturbed homeostasis due to focal inflammation in the hypothalamus?</article-title>\n<source>J Pharmacol Exp Therap</source>. (<year>2018</year>) <volume>367</volume>:<fpage>155</fpage>–<lpage>67</lpage>. <pub-id pub-id-type=\"doi\">10.1124/jpet.118.250845</pub-id><pub-id pub-id-type=\"pmid\">30076265</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arroll</surname><given-names>MA</given-names></name></person-group>. <article-title>Allostatic overload in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)</article-title>. <source>Med Hypotheses.</source> (<year>2013</year>) <volume>81</volume>:<fpage>506</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.mehy.2013.06.023</pub-id><pub-id pub-id-type=\"pmid\">23850395</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Shepherd</surname><given-names>C</given-names></name><name><surname>Chaudhuri</surname><given-names>A</given-names></name></person-group>\n<source>ME/CFS/PVFS: An Exploration of the Key Clinical Issues</source>. <edition>11th ed</edition>\n<publisher-loc>Gawco</publisher-loc>: <publisher-name>The ME Association</publisher-name> (<year>2019</year>).</mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chu</surname><given-names>L</given-names></name><name><surname>Valencia</surname><given-names>IJ</given-names></name><name><surname>Garvert</surname><given-names>DW</given-names></name><name><surname>Montoya</surname><given-names>JG</given-names></name></person-group>. <article-title>Deconstructing post-exertional malaise in myalgic encephalomyelitis/ chronic fatigue syndrome: a patient-centered, cross-sectional survey</article-title>. <source>PLoS ONE.</source> (<year>2018</year>) <volume>13</volume>:<fpage>e0197811</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0197811</pub-id><pub-id pub-id-type=\"pmid\">29856774</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jackson</surname><given-names>ML</given-names></name><name><surname>Bruck</surname><given-names>D</given-names></name></person-group>. <article-title>Sleep abnormalities in chronic fatigue syndrome/myalgic encephalomyelitis: a review</article-title>. <source>J Clin Sleep Med</source>. (<year>2012</year>) <volume>8</volume>:<fpage>719</fpage>–<lpage>28</lpage>. <pub-id pub-id-type=\"doi\">10.5664/jcsm.2276</pub-id><pub-id pub-id-type=\"pmid\">23243408</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arroll</surname><given-names>MA</given-names></name><name><surname>Attree</surname><given-names>EA</given-names></name><name><surname>O'Leary</surname><given-names>JM</given-names></name><name><surname>Dancey</surname><given-names>CP</given-names></name></person-group>\n<article-title>The delayed fatigue effect in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)</article-title>. <source>Fatigue Biomed Health Behav.</source> (<year>2014</year>) <volume>64</volume>:<fpage>393</fpage>–<lpage>94</lpage>. <pub-id pub-id-type=\"doi\">10.1080/21641846.2014.892755</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arseneau</surname><given-names>L</given-names></name><name><surname>Ko</surname><given-names>G</given-names></name><name><surname>Elgez</surname><given-names>A</given-names></name><name><surname>Romero</surname><given-names>L</given-names></name></person-group>\n<article-title>Environmental exposures as a potential underlying factor in chronic fatigue syndrome; a case report</article-title>. <source>Med Res Arch.</source> (<year>2017</year>) <volume>5</volume>\n<pub-id pub-id-type=\"doi\">10.18103/mra.v5i12.1573</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brown</surname><given-names>AE</given-names></name><name><surname>Jones</surname><given-names>DE</given-names></name><name><surname>Walker</surname><given-names>M</given-names></name><name><surname>Newton</surname><given-names>JL</given-names></name></person-group>. <article-title>Abnormalities of AMPK activation and glucose uptake in cultured skeletal muscle cells from individuals with chronic fatigue syndrome</article-title>. <source>PLoS ONE.</source> (<year>2015</year>) <volume>10</volume>:<fpage>e0122982</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0122982</pub-id><pub-id pub-id-type=\"pmid\">25836975</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hu</surname><given-names>H</given-names></name><name><surname>Baines</surname><given-names>C</given-names></name></person-group>. <article-title>Recent insights into 3 underrecognized conditions: myalgic encephalomyelitis-chronic fatigue syndrome, fibromyalgia, and environmental sensitivities-multiple chemical sensitivity</article-title>. <source>Can Fam Phys.</source> (<year>2018</year>) <volume>64</volume>:<fpage>413</fpage>–<lpage>15</lpage>.<pub-id pub-id-type=\"pmid\">29898928</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Keller</surname><given-names>BA</given-names></name><name><surname>Pryor</surname><given-names>JL</given-names></name><name><surname>Ludovic Giloteaux </surname><given-names>L</given-names></name></person-group>. <article-title>Inability of myalgic encephalomyelitis/chronic fatigue syndrome patients to reproduce VO2peak indicates functional impairment</article-title>. <source>J Transl Med</source>. (<year>2014</year>) <volume>12</volume>:<fpage>104</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1479-5876-12-104</pub-id><pub-id pub-id-type=\"pmid\">24755065</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><collab>Centers for Disease Control and Prevention</collab></person-group>. <source>Genetic And Environmental Factors Impact Chronic Fatigue Syndrome Patients</source>. (<year>2006</year>). Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.sciencedaily.com/releases/2006/04/060421112146.htm\">www.sciencedaily.com/releases/2006/04/060421112146.htm</ext-link> (accessed January 04, 2019).</mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Klimas</surname><given-names>NG</given-names></name><name><surname>Broderick</surname><given-names>G</given-names></name><name><surname>Fletcher</surname><given-names>MA</given-names></name></person-group>. <article-title>Biomarkers for chronic fatigue</article-title>. <source>Brain Behav Immun</source>. (<year>2012</year>) <volume>26</volume>:<fpage>1202</fpage>–<lpage>10</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbi.2012.06.006</pub-id><pub-id pub-id-type=\"pmid\">22732129</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Edwards</surname><given-names>JCW</given-names></name><name><surname>McGrath</surname><given-names>S</given-names></name><name><surname>Baldwin</surname><given-names>A</given-names></name><name><surname>Livingstone</surname><given-names>M</given-names></name><name><surname>Kewley</surname><given-names>A</given-names></name></person-group>. <article-title>The biological challenge of myalgic encephalomyelitis/chronic fatigue syndrome: a solvable problem</article-title>. <source>Fatigue.</source> (<year>2016</year>) <volume>4</volume>:<fpage>63</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1080/21641846.2016.1160598</pub-id><pub-id pub-id-type=\"pmid\">27226928</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Monro</surname><given-names>JA</given-names></name><name><surname>Puri</surname><given-names>BK</given-names></name></person-group>. <article-title>A molecular neurobiological approach to understanding the aetiology of chronic fatigue syndrome (Myalgic Encephalomyelitis or Systemic Exertion Intolerance Disease) with treatment implications</article-title>. <source>Mol Neurobiol.</source> (<year>2018</year>) <volume>55</volume>:<fpage>7377</fpage>–<lpage>88</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s12035-018-0928-9</pub-id><pub-id pub-id-type=\"pmid\">29411266</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Komaroff</surname><given-names>AL</given-names></name></person-group>. <article-title>Advances in understanding the pathophysiology of chronic fatigue syndrome</article-title>. <source>JAMA</source>. (<year>2019</year>) <volume>322</volume>:<fpage>499</fpage>–<lpage>500</lpage>. <pub-id pub-id-type=\"doi\">10.1001/jama.2019.8312</pub-id><pub-id pub-id-type=\"pmid\">31276153</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lord</surname><given-names>JM</given-names></name><name><surname>Midwinter</surname><given-names>MJ</given-names></name><name><surname>Chen</surname><given-names>Y</given-names></name><name><surname>Belli</surname><given-names>A</given-names></name><name><surname>Brohi</surname><given-names>K</given-names></name><name><surname>Kovacs</surname><given-names>EJ</given-names></name><etal/></person-group>. <article-title>The systemic immune response to trauma: an overview of pathophysiology and treatment</article-title>. <source>Lancet.</source> (<year>2014</year>) <volume>384</volume>:<fpage>1455</fpage>–<lpage>65</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0140-6736(14)60687-5</pub-id><pub-id pub-id-type=\"pmid\">25390327</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cliff</surname><given-names>JM</given-names></name><name><surname>King</surname><given-names>EC</given-names></name><name><surname>Lee</surname><given-names>J</given-names></name><name><surname>Sepúlveda</surname><given-names>N</given-names></name><name><surname>Wolf</surname><given-names>A</given-names></name><name><surname>Kingdon</surname><given-names>C</given-names></name><etal/></person-group>\n<article-title>Cellular immune function in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)</article-title>. <source>Front Immunol.</source> (<year>2019</year>) <volume>1</volume>:<fpage>796</fpage>\n<pub-id pub-id-type=\"doi\">10.3389/fimmu.2019.00796</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Esterov</surname><given-names>D</given-names></name><name><surname>Greenwald</surname><given-names>B</given-names></name></person-group>. <article-title>Autonomic dysfunction after mild traumatic brain injury</article-title>. <source>Brain Sci.</source> (<year>2017</year>) <volume>7</volume>:<fpage>100</fpage>. <pub-id pub-id-type=\"doi\">10.3390/brainsci7080100</pub-id><pub-id pub-id-type=\"pmid\">28800081</pub-id></mixed-citation></ref><ref id=\"B48\"><label>48.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cambras</surname><given-names>T</given-names></name><name><surname>Jesú</surname><given-names>Castro-Marrero J</given-names></name><name><surname>Zaragoza</surname><given-names>MC</given-names></name><name><surname>Díez-Noguera</surname><given-names>A</given-names></name><name><surname>Alegre</surname><given-names>J</given-names></name></person-group>. <article-title>Circadian rhythm abnormalities and autonomic dysfunction in patients with chronic fatigue syndrome/myalgic encephalomyelitis</article-title>. <source>PLoS ONE.</source> (<year>2018</year>) <volume>13</volume>:<fpage>e0198106</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0198106</pub-id><pub-id pub-id-type=\"pmid\">29874259</pub-id></mixed-citation></ref><ref id=\"B49\"><label>49.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van Oosterwijck</surname><given-names>J</given-names></name><name><surname>Marusic</surname><given-names>U</given-names></name><name><surname>De Wandele</surname><given-names>I</given-names></name><name><surname>Paul</surname><given-names>L</given-names></name><name><surname>Meeus</surname><given-names>M</given-names></name><name><surname>Moorkens</surname><given-names>G</given-names></name><etal/></person-group>\n<article-title>The role of autonomic function in exercise-induced endogenous analgesia: a case-control study in myalgic encephalomyelitis/chronic fatigue syndrome and healthy people</article-title>. <source>Pain Phys.</source> (<year>2017</year>) <volume>20</volume>:<fpage>E389</fpage>–<lpage>99</lpage>. <pub-id pub-id-type=\"doi\">10.36076/ppj.2017.E399</pub-id></mixed-citation></ref><ref id=\"B50\"><label>50.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tomas</surname><given-names>C</given-names></name><name><surname>Newton</surname><given-names>J</given-names></name><name><surname>Watson</surname><given-names>S</given-names></name></person-group>. <article-title>A review of hypothalamic-pituitary-adrenal axis function in chronic fatigue syndrome</article-title>. <source>ISRN Neurosci.</source> (<year>2013</year>) <volume>2013</volume>:<fpage>784520</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2013/784520</pub-id><pub-id pub-id-type=\"pmid\">24959566</pub-id></mixed-citation></ref><ref id=\"B51\"><label>51.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van Campen</surname><given-names>CLMC</given-names></name><name><surname>Rowe</surname><given-names>PC</given-names></name><name><surname>Visser</surname><given-names>FC</given-names></name></person-group>\n<article-title>Blood volume status in me/cfs correlates with the presence or absence of orthostatic symptoms: preliminary results</article-title>. <source>Front Pediatrics.</source> (<year>2018</year>) <volume>6</volume>:<fpage>352</fpage>\n<pub-id pub-id-type=\"doi\">10.3389/fped.2018.00352</pub-id></mixed-citation></ref><ref id=\"B52\"><label>52.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Newton</surname><given-names>DJ</given-names></name><name><surname>Kennedy</surname><given-names>G</given-names></name><name><surname>Chan</surname><given-names>KKF</given-names></name><name><surname>Lang</surname><given-names>CC</given-names></name><name><surname>Belch</surname><given-names>JJF</given-names></name><name><surname>Khan</surname><given-names>F</given-names></name></person-group>. <article-title>Large and small artery endothelial dysfunction in chronic fatigue syndrome</article-title>. <source>Int J Cardiol.</source> (<year>2012</year>) <volume>154</volume>:<fpage>335</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijcard.2011.10.030</pub-id><pub-id pub-id-type=\"pmid\">22078396</pub-id></mixed-citation></ref><ref id=\"B53\"><label>53.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Germain</surname><given-names>A</given-names></name><name><surname>Ruppert</surname><given-names>D</given-names></name><name><surname>Levine</surname><given-names>S</given-names></name><name><surname>Hanson</surname><given-names>M</given-names></name><name><surname>Germain</surname><given-names>A</given-names></name><name><surname>Ruppert</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Prospective biomarkers from plasma metabolomics of myalgic encephalomyelitis/chronic fatigue syndrome implicate redox imbalance in disease symptomatology</article-title>. <source>Metabolites.</source> (<year>2018</year>) <volume>8</volume>:<fpage>90</fpage>. <pub-id pub-id-type=\"doi\">10.3390/metabo8040090</pub-id><pub-id pub-id-type=\"pmid\">30563204</pub-id></mixed-citation></ref><ref id=\"B54\"><label>54.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Naviaux</surname><given-names>RK</given-names></name><name><surname>Naviaux</surname><given-names>JC</given-names></name><name><surname>Li</surname><given-names>K</given-names></name><name><surname>Bright</surname><given-names>AT</given-names></name><name><surname>Alaynick</surname><given-names>WA</given-names></name><name><surname>Wang</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Metabolic features of chronic fatigue syndrome</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2016</year>) <volume>113</volume>:<fpage>5472</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1607571113</pub-id><pub-id pub-id-type=\"pmid\">27147602</pub-id></mixed-citation></ref><ref id=\"B55\"><label>55.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Missailidis</surname><given-names>D</given-names></name><name><surname>Annesley</surname><given-names>SJ</given-names></name><name><surname>Fisher</surname><given-names>PR</given-names></name></person-group>. <article-title>Pathological mechanisms underlying myalgic encephalomyelitis/chronic fatigue syndrome</article-title>. <source>Diagnostics.</source> (<year>2019</year>) <volume>9</volume>:<fpage>80</fpage>. <pub-id pub-id-type=\"doi\">10.3390/diagnostics9030080</pub-id><pub-id pub-id-type=\"pmid\">31330791</pub-id></mixed-citation></ref><ref id=\"B56\"><label>56.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chaudhuri</surname><given-names>A</given-names></name><name><surname>Watson</surname><given-names>WS</given-names></name><name><surname>Pearn</surname><given-names>J</given-names></name><name><surname>Behan</surname><given-names>PO</given-names></name></person-group>. <article-title>The symptoms of chronic fatigue syndrome are related to abnormal ion channel function</article-title>. <source>Med Hypotheses.</source> (<year>2000</year>) <volume>54</volume>:<fpage>59</fpage>–<lpage>63</lpage>. <pub-id pub-id-type=\"doi\">10.1054/mehy.1998.0822</pub-id><pub-id pub-id-type=\"pmid\">10790725</pub-id></mixed-citation></ref><ref id=\"B57\"><label>57.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vermeulen</surname><given-names>RCW</given-names></name><name><surname>Vermeulen van Eck</surname><given-names>IWG</given-names></name></person-group>. <article-title>Decreased oxygen extraction during cardiopulmonary exercise test in patients with chronic fatigue syndrome</article-title>. <source>J Transl Med.</source> (<year>2014</year>) <volume>12</volume>:<fpage>20</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1479-5876-12-20</pub-id><pub-id pub-id-type=\"pmid\">24456560</pub-id></mixed-citation></ref><ref id=\"B58\"><label>58.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Levy</surname><given-names>MM</given-names></name><name><surname>Fink</surname><given-names>MP</given-names></name><name><surname>Marshall</surname><given-names>JC</given-names></name><name><surname>Abraham</surname><given-names>E</given-names></name><name><surname>Angus</surname><given-names>D</given-names></name><name><surname>Cook</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>2001 SCCM/ESICM/ACCP/ATS/SIS international sepsis definitions conference</article-title>. <source>Crit Care Med.</source> (<year>2003</year>) <volume>31</volume>:<fpage>1250</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1097/01.CCM.0000050454.01978.3B</pub-id><pub-id pub-id-type=\"pmid\">12682500</pub-id></mixed-citation></ref><ref id=\"B59\"><label>59.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rathour</surname><given-names>S</given-names></name><name><surname>Kumar</surname><given-names>S</given-names></name><name><surname>Hadda</surname><given-names>V</given-names></name><name><surname>Bhalla</surname><given-names>A</given-names></name><name><surname>Sharma</surname><given-names>N</given-names></name><name><surname>Varma</surname><given-names>S</given-names></name></person-group>. <article-title>PIRO concept: staging of sepsis</article-title>. <source>J Postgraduate Med.</source> (<year>2015</year>) <volume>61</volume>:<fpage>235</fpage>–<lpage>42</lpage>. <pub-id pub-id-type=\"doi\">10.4103/0022-3859.166511</pub-id><pub-id pub-id-type=\"pmid\">26440393</pub-id></mixed-citation></ref><ref id=\"B60\"><label>60.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jason</surname><given-names>LA</given-names></name><name><surname>Fennell</surname><given-names>PA</given-names></name><name><surname>Klein</surname><given-names>S</given-names></name><name><surname>Fricano</surname><given-names>G</given-names></name><name><surname>Halpert</surname><given-names>J</given-names></name></person-group>\n<article-title>An investigation of the different phases of the CFS illness</article-title>. <source>J Chronic Fatigue Syndrome.</source> (<year>1999</year>) <volume>5</volume>:<fpage>35</fpage>–<lpage>54</lpage>. <pub-id pub-id-type=\"doi\">10.1300/J092v05n03_03</pub-id></mixed-citation></ref><ref id=\"B61\"><label>61.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fennell</surname><given-names>PA</given-names></name></person-group>\n<article-title>The four progressive stages of the CFS experience: a coping tool for patients</article-title>. <source>J Chronic Fatigue Syndromeme</source>. (<year>1995</year>) <volume>1</volume>:<fpage>69</fpage>–<lpage>79</lpage>. <pub-id pub-id-type=\"doi\">10.1300/J092v01n03_11</pub-id></mixed-citation></ref><ref id=\"B62\"><label>62.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><name><surname>Schweitzer</surname><given-names>MM</given-names></name></person-group>\n<source>WECAN Testimony to the Chronic Fatigue Syndrome Coordinating Committee of DHHS</source>. (<year>1999</year>). Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.cfids-me.org/cfscc/cfsccs98.html\">http://www.cfids-me.org/cfscc/cfsccs98.html</ext-link> (accessed January 04, 2019).</mixed-citation></ref><ref id=\"B63\"><label>63.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lacerda</surname><given-names>EM</given-names></name><name><surname>Mudie</surname><given-names>K</given-names></name><name><surname>Kingdon</surname><given-names>CC</given-names></name><name><surname>Butterworth</surname><given-names>JD</given-names></name><name><surname>O'Boyle</surname><given-names>S</given-names></name><name><surname>Nacul</surname><given-names>L</given-names></name></person-group>. <article-title>The UK ME/CFS biobank: a disease-specific biobank for advancing clinical research into myalgic encephalomyelitis/chronic fatigu syndrome</article-title>. <source>Front Neurol.</source> (<year>2018</year>) <volume>9</volume>:<fpage>1026</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fneur.2018.01026</pub-id><pub-id pub-id-type=\"pmid\">30564186</pub-id></mixed-citation></ref><ref id=\"B64\"><label>64.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gow</surname><given-names>JW</given-names></name><name><surname>Hagan</surname><given-names>S</given-names></name><name><surname>Herzyk</surname><given-names>P</given-names></name><name><surname>Cannon</surname><given-names>C</given-names></name><name><surname>Behan</surname><given-names>PO</given-names></name><name><surname>Chaudhuri</surname><given-names>A</given-names></name></person-group>. <article-title>A gene signature for post-infectious chronic fatigue syndrome</article-title>. <source>BMC Med Genomics.</source> (<year>2009</year>) <volume>2</volume>:<fpage>38</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1755-8794-2-38</pub-id><pub-id pub-id-type=\"pmid\">19555476</pub-id></mixed-citation></ref><ref id=\"B65\"><label>65.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kerr</surname><given-names>JR</given-names></name><name><surname>Petty</surname><given-names>R</given-names></name><name><surname>Burke</surname><given-names>B</given-names></name><name><surname>Gough</surname><given-names>J</given-names></name><name><surname>Fear</surname><given-names>D</given-names></name><name><surname>Sinclair</surname><given-names>LI</given-names></name><etal/></person-group>. <article-title>Gene expression subtypes in patients with chronic fatigue syndrome/myalgic encephalomyelitis</article-title>. <source>J Infect Dis.</source> (<year>2008</year>) <volume>197</volume>:<fpage>1171</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.1086/533453</pub-id><pub-id pub-id-type=\"pmid\">18462164</pub-id></mixed-citation></ref><ref id=\"B66\"><label>66.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bakken</surname><given-names>IJ</given-names></name><name><surname>Tveito</surname><given-names>K</given-names></name><name><surname>Gunnes</surname><given-names>N</given-names></name><name><surname>Ghaderi</surname><given-names>S</given-names></name><name><surname>Stoltenberg</surname><given-names>C</given-names></name><name><surname>Trogstad</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Two age peaks in the incidence of chronic fatigue syndrome/myalgic encephalomyelitis: a population-based registry study from Norway 2008-2012</article-title>. <source>BMC Med.</source> (<year>2014</year>) <volume>12</volume>:<fpage>167</fpage>. <pub-id pub-id-type=\"doi\">10.1186/PREACCEPT-1843684980131041</pub-id><pub-id pub-id-type=\"pmid\">25274261</pub-id></mixed-citation></ref><ref id=\"B67\"><label>67.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Prins</surname><given-names>JB</given-names></name><name><surname>van der Meer</surname><given-names>JWM</given-names></name><name><surname>Bleijenberg</surname><given-names>G</given-names></name></person-group>. <article-title>Chronic fatigue syndrome</article-title>. <source>Lancet.</source> (<year>2006</year>) <volume>367</volume>:<fpage>346</fpage>–<lpage>55</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0140-6736(06)68073-2</pub-id><pub-id pub-id-type=\"pmid\">16443043</pub-id></mixed-citation></ref><ref id=\"B68\"><label>68.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nacul</surname><given-names>LC</given-names></name><name><surname>Lacerda</surname><given-names>EM</given-names></name><name><surname>Pheby</surname><given-names>D</given-names></name><name><surname>Campion</surname><given-names>P</given-names></name><name><surname>Molokhia</surname><given-names>M</given-names></name><name><surname>Fayyaz</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Prevalence of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) in three regions of england: a repeated cross-sectional study in primary care</article-title>. <source>BMC Med.</source> (<year>2011</year>) <volume>9</volume>:<fpage>91</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1741-7015-9-91</pub-id><pub-id pub-id-type=\"pmid\">21794183</pub-id></mixed-citation></ref><ref id=\"B69\"><label>69.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Naess</surname><given-names>H</given-names></name><name><surname>Nyland</surname><given-names>M</given-names></name><name><surname>Hausken</surname><given-names>T</given-names></name><name><surname>Follestad</surname><given-names>I</given-names></name><name><surname>Nyland</surname><given-names>HI</given-names></name></person-group>. <article-title>Chronic fatigue syndrome after giardia enteritis: clinical characteristics, disability and long-term sickness absence</article-title>. <source>BMC Gastroenterol.</source> (<year>2012</year>) <volume>12</volume>:<fpage>13</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1471-230X-12-13</pub-id><pub-id pub-id-type=\"pmid\">22316329</pub-id></mixed-citation></ref><ref id=\"B70\"><label>70.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Levine</surname><given-names>PH</given-names></name><name><surname>Jacobson</surname><given-names>S</given-names></name><name><surname>Pocinki</surname><given-names>AG</given-names></name><name><surname>Cheney</surname><given-names>P</given-names></name><name><surname>Peterson</surname><given-names>D</given-names></name><name><surname>Connelly</surname><given-names>RR</given-names></name><etal/></person-group>. <article-title>Clinical, epidemiologic, and virologic studies in four clusters of the chronic fatigue syndrome</article-title>. <source>Arch Intern Med.</source> (<year>1992</year>) <volume>152</volume>:<fpage>1611</fpage>. <pub-id pub-id-type=\"doi\">10.1001/archinte.1992.00400200049009</pub-id><pub-id pub-id-type=\"pmid\">1323246</pub-id></mixed-citation></ref><ref id=\"B71\"><label>71.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Levine</surname><given-names>PH</given-names></name><name><surname>Snow</surname><given-names>PG</given-names></name><name><surname>Ranum</surname><given-names>BA</given-names></name><name><surname>Paul</surname><given-names>C</given-names></name><name><surname>Holmes</surname><given-names>MJ</given-names></name></person-group>. <article-title>Epidemic neuromyasthenia and chronic fatigue syndrome in West Otago, New Zealand</article-title>. <source>Arch Intern Med.</source> (<year>1997</year>) <volume>157</volume>:<fpage>750</fpage>. <pub-id pub-id-type=\"doi\">10.1001/archinte.1997.00440280064005</pub-id><pub-id pub-id-type=\"pmid\">9125006</pub-id></mixed-citation></ref><ref id=\"B72\"><label>72.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Underhill</surname><given-names>RA</given-names></name></person-group>. <article-title>Myalgic encephalomyelitis, chronic fatigue syndrome: an infectious disease</article-title>. <source>Med Hypotheses.</source> (<year>2015</year>) <volume>85</volume>:<fpage>765</fpage>–<lpage>73</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.mehy.2015.10.011</pub-id><pub-id pub-id-type=\"pmid\">26604026</pub-id></mixed-citation></ref><ref id=\"B73\"><label>73.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rasa</surname><given-names>S</given-names></name><name><surname>Nora-Krukle</surname><given-names>Z</given-names></name><name><surname>Henning</surname><given-names>N</given-names></name><name><surname>Eliassen</surname><given-names>E</given-names></name><name><surname>Shikova</surname><given-names>E</given-names></name><name><surname>Harrer</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>Chronic viral infections in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)</article-title>. <source>J Transl Med.</source> (<year>2018</year>) <volume>16</volume>:<fpage>268</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s12967-018-1644-y</pub-id><pub-id pub-id-type=\"pmid\">30285773</pub-id></mixed-citation></ref><ref id=\"B74\"><label>74.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chia</surname><given-names>J</given-names></name><name><surname>Chia</surname><given-names>A</given-names></name><name><surname>Voeller</surname><given-names>M</given-names></name><name><surname>Lee</surname><given-names>T</given-names></name><name><surname>Chang</surname><given-names>R</given-names></name></person-group>. <article-title>Acute enterovirus infection followed by myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and viral persistence</article-title>. <source>J Clin Pathol.</source> (<year>2010</year>) <volume>63</volume>:<fpage>165</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1136/jcp.2009.070466</pub-id><pub-id pub-id-type=\"pmid\">19828908</pub-id></mixed-citation></ref><ref id=\"B75\"><label>75.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hickie</surname><given-names>I</given-names></name><name><surname>Davenport</surname><given-names>T</given-names></name><name><surname>Wakefield</surname><given-names>D</given-names></name><name><surname>Vollmer-Conna</surname><given-names>U</given-names></name><name><surname>Cameron</surname><given-names>B</given-names></name><name><surname>Vernon</surname><given-names>SD</given-names></name><etal/></person-group>. <article-title>Post-infective and chronic fatigue syndromes precipitated by viral and non-viral pathogens: prospective cohort study</article-title>. <source>BMJ.</source> (<year>2006</year>) <volume>333</volume>:<fpage>575</fpage>. <pub-id pub-id-type=\"doi\">10.1136/bmj.38933.585764.AE</pub-id><pub-id pub-id-type=\"pmid\">16950834</pub-id></mixed-citation></ref><ref id=\"B76\"><label>76.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Katz</surname><given-names>BZ</given-names></name><name><surname>Shiraishi</surname><given-names>Y</given-names></name><name><surname>Mears</surname><given-names>CJ</given-names></name><name><surname>Binns</surname><given-names>HJ</given-names></name><name><surname>Taylor</surname><given-names>R</given-names></name></person-group>\n<article-title>Chronic fatigue syndrome following infectious mononucleosis in adolescents: a prospective cohort study</article-title>. <source>Pediatrics.</source> (<year>2009</year>) <volume>124</volume>:<fpage>189</fpage>\n<pub-id pub-id-type=\"doi\">10.1542/peds.2008-1879</pub-id><pub-id pub-id-type=\"pmid\">19564299</pub-id></mixed-citation></ref><ref id=\"B77\"><label>77.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cho</surname><given-names>HJ</given-names></name><name><surname>Menezes</surname><given-names>PR</given-names></name><name><surname>Hotopf</surname><given-names>M</given-names></name><name><surname>Bhugra</surname><given-names>D</given-names></name><name><surname>Wessely</surname><given-names>S</given-names></name></person-group>. <article-title>Comparative epidemiology of chronic fatigue syndrome in Brazilian and British primary care: prevalence and recognition</article-title>. <source>Br J Psychiatry.</source> (<year>2009</year>) <volume>194</volume>:<fpage>117</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.1192/bjp.bp.108.051813</pub-id><pub-id pub-id-type=\"pmid\">19182171</pub-id></mixed-citation></ref><ref id=\"B78\"><label>78.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nacul</surname><given-names>LC</given-names></name><name><surname>Perreira</surname><given-names>FLR</given-names></name><name><surname>Jacques</surname><given-names>GA</given-names></name><name><surname>Ramos</surname><given-names>AG</given-names></name><name><surname>Rodrigues</surname><given-names>CJA</given-names></name><name><surname>Oliva</surname><given-names>IB</given-names></name><etal/></person-group>\n<article-title>What do doctors and health workers of Northeast Brazil know and do about chronic fatigue syndrome?</article-title>\n<source>J Chronic Fatigue Syndrome.</source> (<year>1999</year>) <volume>5</volume>:<fpage>89</fpage>–<lpage>90</lpage>.</mixed-citation></ref><ref id=\"B79\"><label>79.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Njoku</surname><given-names>MG</given-names></name><name><surname>Jason</surname><given-names>LA</given-names></name><name><surname>Torres-Harding</surname><given-names>SR</given-names></name></person-group>. <article-title>The prevalence of chronic fatigue syndrome in Nigeria</article-title>. <source>J Health Psychol.</source> (<year>2007</year>) <volume>12</volume>:<fpage>461</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1177/1359105307076233</pub-id><pub-id pub-id-type=\"pmid\">17439996</pub-id></mixed-citation></ref><ref id=\"B80\"><label>80.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Heim</surname><given-names>C</given-names></name><name><surname>Nater</surname><given-names>UM</given-names></name><name><surname>Maloney</surname><given-names>E</given-names></name><name><surname>Boneva</surname><given-names>R</given-names></name><name><surname>Jones</surname><given-names>JF</given-names></name><name><surname>Reeves</surname><given-names>WC</given-names></name></person-group>. <article-title>Childhood trauma and risk for chronic fatigue syndrome: association with neuroendocrine dysfunction</article-title>. <source>Arch Gen Psychiatry.</source> (<year>2009</year>) <volume>66</volume>:<fpage>72</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.1001/archgenpsychiatry.2008.508</pub-id><pub-id pub-id-type=\"pmid\">19124690</pub-id></mixed-citation></ref><ref id=\"B81\"><label>81.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nater</surname><given-names>UM</given-names></name><name><surname>Maloney</surname><given-names>E</given-names></name><name><surname>Heim</surname><given-names>C</given-names></name><name><surname>Reeves</surname><given-names>WC</given-names></name></person-group>. <article-title>Cumulative life stress in chronic fatigue syndrome</article-title>. <source>Psychiatry Res.</source> (<year>2011</year>) <volume>189</volume>:<fpage>318</fpage>–<lpage>20</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.psychres.2011.07.015</pub-id><pub-id pub-id-type=\"pmid\">21840607</pub-id></mixed-citation></ref><ref id=\"B82\"><label>82.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Harvey</surname><given-names>SB</given-names></name><name><surname>Wadsworth</surname><given-names>M</given-names></name><name><surname>Wessely</surname><given-names>S</given-names></name><name><surname>Hotopf</surname><given-names>M</given-names></name></person-group>. <article-title>The relationship between prior psychiatric disorder and chronic fatigue: evidence from a national birth cohort study</article-title>. <source>Psychol Med.</source> (<year>2008</year>) <volume>38</volume>:<fpage>933</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1017/S0033291707001900</pub-id><pub-id pub-id-type=\"pmid\">17976252</pub-id></mixed-citation></ref><ref id=\"B83\"><label>83.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hotopf</surname><given-names>M</given-names></name><name><surname>Noah</surname><given-names>N</given-names></name><name><surname>Wessely</surname><given-names>S</given-names></name></person-group>. <article-title>Chronic fatigue and minor psychiatric morbidity after viral meningitis: a controlled study</article-title>. <source>J Neurol Neurosurg Psychiatry.</source> (<year>1996</year>) <volume>60</volume>:<fpage>504</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1136/jnnp.60.5.504</pub-id><pub-id pub-id-type=\"pmid\">8778253</pub-id></mixed-citation></ref><ref id=\"B84\"><label>84.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Song</surname><given-names>S</given-names></name><name><surname>Jason</surname><given-names>LA</given-names></name></person-group>\n<article-title>A population-based study of Chronic Fatigue Syndrome (CFS) experienced in differing patient groups: an effort to replicate Vercoulen et Al.'s model of CFS</article-title>. <source>J Mental Health.</source> (<year>2005</year>) <volume>14</volume>:<fpage>277</fpage>–<lpage>89</lpage>. <pub-id pub-id-type=\"doi\">10.1080/09638230500076165</pub-id></mixed-citation></ref><ref id=\"B85\"><label>85.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Russell</surname><given-names>A</given-names></name><name><surname>Hepgul</surname><given-names>N</given-names></name><name><surname>Nikkheslat</surname><given-names>N</given-names></name><name><surname>Borsini</surname><given-names>A</given-names></name><name><surname>Zajkowska</surname><given-names>Z</given-names></name><name><surname>Moll</surname><given-names>N</given-names></name><etal/></person-group>. <article-title>Persistent fatigue induced by interferon-alpha: a novel, inflammation-based, proxy model of chronic fatigue syndrome</article-title>. <source>Psychoneuroendocrinology.</source> (<year>2019</year>) <volume>100</volume>:<fpage>276</fpage>–<lpage>85</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.psyneuen.2018.11.032</pub-id><pub-id pub-id-type=\"pmid\">30567628</pub-id></mixed-citation></ref><ref id=\"B86\"><label>86.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grinde</surname><given-names>B</given-names></name></person-group>\n<article-title>Stress and Chronic Fatigue Syndrome</article-title>. In: Cooper CL, Quick JC, editors. <source>The Handbook of Stress and Health Chichester: John Wiley & Sons, Ltd</source>. (<year>2017</year>). p. <fpage>135</fpage>–<lpage>46</lpage>. <pub-id pub-id-type=\"doi\">10.1002/9781118993811.ch8</pub-id></mixed-citation></ref><ref id=\"B87\"><label>87.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kerr</surname><given-names>JR</given-names></name></person-group>. <article-title>Epstein-barr virus induced Gene-2 upregulation identifies a particular subtype of chronic fatigue syndrome/myalgic encephalomyelitis</article-title>. <source>Front Pediatrics</source>. (<year>2019</year>) <volume>7</volume>:<fpage>59</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fped.2019.00059</pub-id><pub-id pub-id-type=\"pmid\">30918887</pub-id></mixed-citation></ref><ref id=\"B88\"><label>88.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Albright</surname><given-names>F</given-names></name><name><surname>Light</surname><given-names>K</given-names></name><name><surname>Light</surname><given-names>A</given-names></name><name><surname>Bateman</surname><given-names>L</given-names></name><name><surname>Cannon-Albright</surname><given-names>LA</given-names></name></person-group>. <article-title>Evidence for a heritable predisposition to chronic fatigue syndrome</article-title>. <source>BMC Neurol.</source> (<year>2011</year>) <volume>11</volume>:<fpage>62</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1471-2377-11-62</pub-id><pub-id pub-id-type=\"pmid\">21619629</pub-id></mixed-citation></ref><ref id=\"B89\"><label>89.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Walsh</surname><given-names>CM</given-names></name><name><surname>Zainal</surname><given-names>NZ</given-names></name><name><surname>Middleton</surname><given-names>SJ</given-names></name><name><surname>Paykel</surname><given-names>ES</given-names></name></person-group>. <article-title>A family history study of chronic fatigue syndrome</article-title>. <source>Psychiatric Genetics.</source> (<year>2001</year>) <volume>11</volume>:<fpage>123</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1097/00041444-200109000-00003</pub-id><pub-id pub-id-type=\"pmid\">11702053</pub-id></mixed-citation></ref><ref id=\"B90\"><label>90.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Buchwald</surname><given-names>D</given-names></name><name><surname>Herrell</surname><given-names>R</given-names></name><name><surname>Ashton</surname><given-names>S</given-names></name><name><surname>Belcourt</surname><given-names>M</given-names></name><name><surname>Schmaling</surname><given-names>K</given-names></name><name><surname>Sullivan</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>A twin study of chronic fatigue</article-title>. <source>Psychosomatic Med.</source> (<year>2001</year>) <volume>63</volume>:<fpage>936</fpage>–<lpage>43</lpage>. <pub-id pub-id-type=\"doi\">10.1097/00006842-200111000-00012</pub-id><pub-id pub-id-type=\"pmid\">11719632</pub-id></mixed-citation></ref><ref id=\"B91\"><label>91.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Smith</surname><given-names>J</given-names></name><name><surname>Fritz</surname><given-names>EL</given-names></name><name><surname>Kerr</surname><given-names>JR</given-names></name><name><surname>Cleare</surname><given-names>AJ</given-names></name><name><surname>Wessely</surname><given-names>S</given-names></name><name><surname>Mattey</surname><given-names>DL</given-names></name></person-group>. <article-title>Association of chronic fatigue syndrome with human leucocyte antigen Class II alleles</article-title>. <source>J Clin Pathol.</source> (<year>2005</year>) <volume>58</volume>:<fpage>860</fpage>–<lpage>3</lpage>. <pub-id pub-id-type=\"doi\">10.1136/jcp.2004.022681</pub-id><pub-id pub-id-type=\"pmid\">16049290</pub-id></mixed-citation></ref><ref id=\"B92\"><label>92.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Keller</surname><given-names>RH</given-names></name><name><surname>Lane</surname><given-names>JL</given-names></name><name><surname>Klimas</surname><given-names>N</given-names></name><name><surname>Reiter</surname><given-names>WM</given-names></name><name><surname>Fletcher</surname><given-names>MA</given-names></name><name><surname>van Riel</surname><given-names>F</given-names></name><etal/></person-group>. <article-title>Association between HLA Class II antigens and the chronic fatigue immune dysfunction syndrome</article-title>. <source>Clin Infect Dis.</source> (<year>1994</year>) <volume>18</volume>(<issue>Suppl. 1</issue>):<fpage>S154</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1093/clinids/18.Supplement_1.S154</pub-id><pub-id pub-id-type=\"pmid\">8148444</pub-id></mixed-citation></ref><ref id=\"B93\"><label>93.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rajeevan</surname><given-names>MS</given-names></name><name><surname>Dimulescu</surname><given-names>I</given-names></name><name><surname>Murray</surname><given-names>J</given-names></name><name><surname>Falkenberg</surname><given-names>VR</given-names></name><name><surname>Unger</surname><given-names>ER</given-names></name></person-group>. <article-title>Pathway-focused genetic evaluation of immune and inflammation related genes with chronic fatigue syndrome</article-title>. <source>Hum Immunol.</source> (<year>2015</year>) <volume>76</volume>:<fpage>553</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.humimm.2015.06.014</pub-id><pub-id pub-id-type=\"pmid\">26116897</pub-id></mixed-citation></ref><ref id=\"B94\"><label>94.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nisenbaum</surname><given-names>R</given-names></name><name><surname>Jones</surname><given-names>JF</given-names></name><name><surname>Unger</surname><given-names>ER</given-names></name><name><surname>Reyes</surname><given-names>M</given-names></name><name><surname>Reeves</surname><given-names>WC</given-names></name></person-group>. <article-title>A population-based study of the clinical course of chronic fatigue syndrome</article-title>. <source>Health Qual Life Outcomes.</source> (<year>2003</year>) <volume>1</volume>:<fpage>49</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1477-7525-1-49</pub-id><pub-id pub-id-type=\"pmid\">14613572</pub-id></mixed-citation></ref><ref id=\"B95\"><label>95.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Russell</surname><given-names>L</given-names></name><name><surname>Broderick</surname><given-names>G</given-names></name><name><surname>Taylor</surname><given-names>R</given-names></name><name><surname>Fernandes</surname><given-names>H</given-names></name><name><surname>Harvey</surname><given-names>J</given-names></name><name><surname>Barnes</surname><given-names>Z</given-names></name><etal/></person-group>. <article-title>Illness progression in chronic fatigue syndrome: a shifting immune baseline</article-title>. <source>BMC Immunol.</source> (<year>2016</year>) <volume>17</volume>:<fpage>3</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s12865-016-0142-3</pub-id><pub-id pub-id-type=\"pmid\">26965484</pub-id></mixed-citation></ref><ref id=\"B96\"><label>96.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Seet</surname><given-names>RCS</given-names></name><name><surname>Quek</surname><given-names>AML</given-names></name><name><surname>Lim</surname><given-names>ECH</given-names></name></person-group>. <article-title>Post-infectious fatigue syndrome in dengue infection</article-title>. <source>J Clin Virol.</source> (<year>2007</year>) <volume>38</volume>:<fpage>1</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1016/J.JCV.2006.10.011</pub-id><pub-id pub-id-type=\"pmid\">17137834</pub-id></mixed-citation></ref><ref id=\"B97\"><label>97.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wilshire</surname><given-names>C</given-names></name><name><surname>Kindlon</surname><given-names>T</given-names></name><name><surname>Matthees</surname><given-names>A</given-names></name><name><surname>McGrath</surname><given-names>S</given-names></name></person-group> (<year>2017</year>) <article-title>Can patients with chronic fatigue syndrome really recover after graded exercise or cognitive behavioural therapy? A critical commentary and preliminary re-analysis of the PACE trial</article-title>. <source>Fatigue Biomed Health Behav.</source>\n<volume>5</volume>:<fpage>43</fpage>–<lpage>56</lpage>. <pub-id pub-id-type=\"doi\">10.1080/21641846.2017.1259724</pub-id></mixed-citation></ref><ref id=\"B98\"><label>98.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Smith</surname><given-names>AK</given-names></name><name><surname>Fang</surname><given-names>H</given-names></name><name><surname>Whistler</surname><given-names>T</given-names></name><name><surname>Unger</surname><given-names>ER</given-names></name><name><surname>Rajeevan</surname><given-names>MS</given-names></name></person-group>. <article-title>Convergent genomic studies identify association of GRIK2 and NPAS2 with chronic fatigue syndrome</article-title>. <source>Neuropsychobiology.</source> (<year>2011</year>) <volume>64</volume>:<fpage>183</fpage>–<lpage>94</lpage>. <pub-id pub-id-type=\"doi\">10.1159/000326692</pub-id><pub-id pub-id-type=\"pmid\">21912186</pub-id></mixed-citation></ref><ref id=\"B99\"><label>99.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schlauch</surname><given-names>KA</given-names></name><name><surname>Khaiboullina</surname><given-names>SF</given-names></name><name><surname>De Meirleir</surname><given-names>KL</given-names></name><name><surname>Rawat</surname><given-names>S</given-names></name><name><surname>Petereit</surname><given-names>J</given-names></name><name><surname>Rizvanov</surname><given-names>AA</given-names></name><etal/></person-group>. <article-title>Genome-wide association analysis identifies genetic variations in subjects with myalgic encephalomyelitis/chronic fatigue syndrome</article-title>. <source>Transl Psychiatry.</source> (<year>2016</year>) <volume>6</volume>:<fpage>e730</fpage>. <pub-id pub-id-type=\"doi\">10.1038/tp.2015.208</pub-id><pub-id pub-id-type=\"pmid\">26859813</pub-id></mixed-citation></ref><ref id=\"B100\"><label>100.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Smith</surname><given-names>AK</given-names></name><name><surname>White</surname><given-names>PD</given-names></name><name><surname>Aslakson</surname><given-names>E</given-names></name><name><surname>Vollmer-Conna</surname><given-names>U</given-names></name><name><surname>Rajeevan</surname><given-names>MS</given-names></name></person-group>. <article-title>Polymorphisms in genes regulating the HPA axis associated with empirically delineated classes of unexplained chronic fatigue</article-title>. <source>Pharmacogenomics.</source> (<year>2006</year>) <volume>7</volume>:<fpage>387</fpage>–<lpage>94</lpage>. <pub-id pub-id-type=\"doi\">10.2217/14622416.7.3.387</pub-id><pub-id pub-id-type=\"pmid\">16610949</pub-id></mixed-citation></ref><ref id=\"B101\"><label>101.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fukuda</surname><given-names>K</given-names></name><name><surname>Straus</surname><given-names>SE</given-names></name><name><surname>Hickie</surname><given-names>I</given-names></name><name><surname>Sharpe</surname><given-names>MC</given-names></name><name><surname>Dobbins</surname><given-names>JG</given-names></name><name><surname>Komaroff</surname><given-names>A</given-names></name></person-group>\n<article-title>The chronic fatigue syndrome: a comprehensive approach to its definition and study. international chronic fatigue syndrome study group</article-title>. <source>Ann Intern Med.</source> (<year>1994</year>) <volume>121</volume>:<fpage>953</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.7326/0003-4819-121-12-199412150-00009</pub-id><pub-id pub-id-type=\"pmid\">7978722</pub-id></mixed-citation></ref><ref id=\"B102\"><label>102.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carruthers</surname><given-names>BM</given-names></name><name><surname>Jain</surname><given-names>AK</given-names></name><name><surname>De Meirleir</surname><given-names>KL</given-names></name><name><surname>Peterson</surname><given-names>D</given-names></name><name><surname>Klimas</surname><given-names>NG</given-names></name><name><surname>Lerner</surname><given-names>AM</given-names></name><etal/></person-group>\n<article-title>Myalgic encephalomyelitis/chronic fatigue syndrome: clinical working case definition, diagnostic and treatment protocols</article-title>. <source>J Chronic Fatigue Syndrome.</source> (<year>2003</year>) <volume>11</volume>:<fpage>7</fpage>–<lpage>36</lpage>. <pub-id pub-id-type=\"doi\">10.1300/J092v11n01_02</pub-id></mixed-citation></ref><ref id=\"B103\"><label>103.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Low</surname><given-names>CA</given-names></name><name><surname>Kalinski</surname><given-names>P</given-names></name><name><surname>Bovbjerg</surname><given-names>DH</given-names></name></person-group>. <article-title>Neurocognitive impairment as one facet of cancer-related sickness behavior symptoms</article-title>. <source>J Natl Cancer Inst.</source> (<year>2015</year>) <volume>107</volume>:<fpage>176</fpage>. <pub-id pub-id-type=\"doi\">10.1093/jnci/djv176</pub-id><pub-id pub-id-type=\"pmid\">26101332</pub-id></mixed-citation></ref><ref id=\"B104\"><label>104.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Black</surname><given-names>PH</given-names></name></person-group>. <article-title>Immune system-central nervous system interactions: effect and immunomodulatory consequences of immune system mediators on the brain</article-title>. <source>Antimicrobial Agents Chemother.</source> (<year>1994</year>) <volume>38</volume>:<fpage>7</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.1128/AAC.38.1.7</pub-id><pub-id pub-id-type=\"pmid\">8141583</pub-id></mixed-citation></ref><ref id=\"B105\"><label>105.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Prüss</surname><given-names>H</given-names></name><name><surname>Tedeschi</surname><given-names>A</given-names></name><name><surname>Thiriot</surname><given-names>A</given-names></name><name><surname>Lynch</surname><given-names>L</given-names></name><name><surname>Loughhead</surname><given-names>SM</given-names></name><name><surname>Stutte</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Spinal cord injury-induced immunodeficiency is mediated by a sympathetic-neuroendocrine adrenal reflex</article-title>. <source>Nat Neurosci.</source> (<year>2017</year>) <volume>20</volume>:<fpage>1549</fpage>–<lpage>59</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nn.4643</pub-id><pub-id pub-id-type=\"pmid\">28920935</pub-id></mixed-citation></ref><ref id=\"B106\"><label>106.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wrona</surname><given-names>D</given-names></name></person-group>. <article-title>Neural–immune interactions: an integrative view of the bidirectional relationship between the brain and immune systems</article-title>. <source>J Neuroimmunol.</source> (<year>2006</year>) <volume>172</volume>:<fpage>38</fpage>–<lpage>58</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jneuroim.2005.10.017</pub-id><pub-id pub-id-type=\"pmid\">16375977</pub-id></mixed-citation></ref><ref id=\"B107\"><label>107.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Natelson</surname><given-names>BH</given-names></name><name><surname>Weaver</surname><given-names>SA</given-names></name><name><surname>Tseng</surname><given-names>CL</given-names></name><name><surname>Ottenweller</surname><given-names>JE</given-names></name></person-group>. <article-title>Spinal fluid abnormalities in patients with chronic fatigue syndrome</article-title>. <source>Clin Diagn Lab Immunol.</source> (<year>2005</year>) <volume>12</volume>:<fpage>52</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1128/CDLI.12.1.52-55.2005</pub-id><pub-id pub-id-type=\"pmid\">15642984</pub-id></mixed-citation></ref><ref id=\"B108\"><label>108.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schutzer</surname><given-names>SE</given-names></name><name><surname>Angel</surname><given-names>TE</given-names></name><name><surname>Liu</surname><given-names>T</given-names></name><name><surname>Schepmoes</surname><given-names>AA</given-names></name><name><surname>Clauss</surname><given-names>TR</given-names></name><name><surname>Adkins</surname><given-names>JN</given-names></name><etal/></person-group>. <article-title>Distinct cerebrospinal fluid proteomes differentiate post-treatment lyme disease from chronic fatigue syndrome</article-title>. <source>PLoS ONE.</source> (<year>2011</year>) <volume>6</volume>:<fpage>e17287</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0017287</pub-id><pub-id pub-id-type=\"pmid\">21383843</pub-id></mixed-citation></ref><ref id=\"B109\"><label>109.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chaudhuri</surname><given-names>A</given-names></name><name><surname>Behan</surname><given-names>PO</given-names></name></person-group>. <article-title>Fatigue and basal ganglia</article-title>. <source>J Neurol Sci.</source> (<year>2000</year>) <volume>179</volume>:<fpage>34</fpage>–<lpage>42</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0022-510X(00)00411-1</pub-id><pub-id pub-id-type=\"pmid\">11054483</pub-id></mixed-citation></ref><ref id=\"B110\"><label>110.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bested</surname><given-names>AC</given-names></name><name><surname>Saunders</surname><given-names>PR</given-names></name><name><surname>Logan</surname><given-names>AC</given-names></name></person-group>. <article-title>Chronic fatigue syndrome: neurological findings may be related to blood–brain barrier permeability</article-title>. <source>Med Hypotheses.</source> (<year>2001</year>) <volume>57</volume>:<fpage>231</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1054/mehy.2001.1306</pub-id><pub-id pub-id-type=\"pmid\">11461179</pub-id></mixed-citation></ref><ref id=\"B111\"><label>111.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cameron</surname><given-names>B</given-names></name><name><surname>Flamand</surname><given-names>L</given-names></name><name><surname>Juwana</surname><given-names>H</given-names></name><name><surname>Middeldorp</surname><given-names>J</given-names></name><name><surname>Naing</surname><given-names>Z</given-names></name><name><surname>Rawlinson</surname><given-names>W</given-names></name><etal/></person-group>. <article-title>Serological and virological investigation of the role of the herpesviruses EBV, CMV and HHV-6 in post-infective fatigue syndrome</article-title>. <source>J Med Virol.</source> (<year>2010</year>) <volume>82</volume>:<fpage>1684</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jmv.21873</pub-id><pub-id pub-id-type=\"pmid\">20827765</pub-id></mixed-citation></ref><ref id=\"B112\"><label>112.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cameron</surname><given-names>B</given-names></name><name><surname>Galbraith</surname><given-names>S</given-names></name><name><surname>Zhang</surname><given-names>Y</given-names></name><name><surname>Davenport</surname><given-names>T</given-names></name><name><surname>Vollmer-Conna</surname><given-names>U</given-names></name><name><surname>Wakefield</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Gene expression correlates of postinfective fatigue syndrome after infectious mononucleosis</article-title>. <source>J Infect Dis.</source> (<year>2007</year>) <volume>196</volume>:<fpage>56</fpage>–<lpage>66</lpage>. <pub-id pub-id-type=\"doi\">10.1086/518614</pub-id><pub-id pub-id-type=\"pmid\">17538884</pub-id></mixed-citation></ref><ref id=\"B113\"><label>113.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cameron</surname><given-names>B</given-names></name><name><surname>Bharadwaj</surname><given-names>M</given-names></name><name><surname>Burrows</surname><given-names>J</given-names></name><name><surname>Fazou</surname><given-names>C</given-names></name><name><surname>Wakefield</surname><given-names>D</given-names></name><name><surname>Hickie</surname><given-names>I</given-names></name><etal/></person-group>. <article-title>Prolonged illness after infectious mononucleosis is associated with altered immunity but not with increased viral load</article-title>. <source>J Infect Dis.</source> (<year>2006</year>) <volume>193</volume>:<fpage>664</fpage>–<lpage>71</lpage>. <pub-id pub-id-type=\"doi\">10.1086/500248</pub-id><pub-id pub-id-type=\"pmid\">16453261</pub-id></mixed-citation></ref><ref id=\"B114\"><label>114.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vernon</surname><given-names>SD</given-names></name><name><surname>Whistler</surname><given-names>T</given-names></name><name><surname>Cameron</surname><given-names>B</given-names></name><name><surname>Hickie</surname><given-names>IB</given-names></name><name><surname>Reeves</surname><given-names>WC</given-names></name><name><surname>Lloyd</surname><given-names>A</given-names></name></person-group>. <article-title>Preliminary evidence of mitochondrial dysfunction associated with post-infective fatigue after acute infection with epstein barr virus</article-title>. <source>BMC Infect Dis.</source> (<year>2006</year>) <volume>6</volume>:<fpage>15</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1471-2334-6-15</pub-id><pub-id pub-id-type=\"pmid\">16448567</pub-id></mixed-citation></ref><ref id=\"B115\"><label>115.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>White</surname><given-names>PD</given-names></name><name><surname>Thomas</surname><given-names>JM</given-names></name><name><surname>Sullivan</surname><given-names>PF</given-names></name><name><surname>Buchwald</surname><given-names>D</given-names></name></person-group>. <article-title>The nosology of sub-acute and chronic fatigue syndromes that follow infectious mononucleosis</article-title>. <source>Psychol Med.</source> (<year>2004</year>) <volume>34</volume>:<fpage>499</fpage>–<lpage>507</lpage>. <pub-id pub-id-type=\"doi\">10.1017/S0033291703001302</pub-id><pub-id pub-id-type=\"pmid\">15259835</pub-id></mixed-citation></ref><ref id=\"B116\"><label>116.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>White</surname><given-names>PD</given-names></name></person-group>\n<article-title>What causes prolonged fatigue after infectious mononucleosis-and does it tell us anything about chronic fatigue syndrome?</article-title>\n<source>J Infect Dis.</source> (<year>2007</year>) <volume>196</volume>:<fpage>4</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1086/518615</pub-id><pub-id pub-id-type=\"pmid\">17538875</pub-id></mixed-citation></ref><ref id=\"B117\"><label>117.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>White</surname><given-names>PD</given-names></name><name><surname>Thomas</surname><given-names>JM</given-names></name><name><surname>Amess</surname><given-names>J</given-names></name><name><surname>Crawford</surname><given-names>DH</given-names></name><name><surname>Grover</surname><given-names>SA</given-names></name><name><surname>Kangro</surname><given-names>HO</given-names></name><etal/></person-group>. <article-title>Incidence, risk and prognosis of acute and chronic fatigue syndromes and psychiatric disorders after glandular fever</article-title>. <source>Br J Psychiatry.</source> (<year>1998</year>) <volume>173</volume>:<fpage>475</fpage>–<lpage>81</lpage>. <pub-id pub-id-type=\"doi\">10.1192/bjp.173.6.475</pub-id><pub-id pub-id-type=\"pmid\">9926075</pub-id></mixed-citation></ref><ref id=\"B118\"><label>118.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Esfandyarpour</surname><given-names>R</given-names></name><name><surname>Kashi</surname><given-names>A</given-names></name><name><surname>Nemat-Gorgani</surname><given-names>M</given-names></name><name><surname>Wilhelmy</surname><given-names>J</given-names></name><name><surname>Davis</surname><given-names>RW</given-names></name></person-group>. <article-title>A nanoelectronics-blood-based diagnostic biomarker for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2019</year>) <volume>116</volume>:<fpage>10250</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1901274116</pub-id><pub-id pub-id-type=\"pmid\">31036648</pub-id></mixed-citation></ref><ref id=\"B119\"><label>119.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rowe</surname><given-names>PC</given-names></name><name><surname>Fontaine</surname><given-names>KR</given-names></name><name><surname>Lauver</surname><given-names>M</given-names></name><name><surname>Jasion</surname><given-names>SE</given-names></name><name><surname>Marden</surname><given-names>CL</given-names></name><name><surname>Moni</surname><given-names>M</given-names></name></person-group>. <article-title>Neuromuscular strain increases symptom intensity in chronic fatigue syndrome</article-title>. <source>PLoS ONE.</source> (<year>2016</year>) <volume>11</volume>:<fpage>e0159386</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0159386</pub-id><pub-id pub-id-type=\"pmid\">27428358</pub-id></mixed-citation></ref><ref id=\"B120\"><label>120.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Glassford</surname><given-names>JAG</given-names></name></person-group>. <article-title>The neuroinflammatory etiopathology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)</article-title>. <source>Front Physiol.</source> (<year>2017</year>) <volume>8</volume>:<fpage>88</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fphys.2017.00088</pub-id><pub-id pub-id-type=\"pmid\">28261110</pub-id></mixed-citation></ref><ref id=\"B121\"><label>121.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rowe</surname><given-names>PC</given-names></name><name><surname>Fontaine</surname><given-names>KR</given-names></name><name><surname>Violand</surname><given-names>RL</given-names></name></person-group>. <article-title>Neuromuscular strain as a contributor to cognitive and other symptoms in chronic fatigue syndrome: hypothesis and conceptual model</article-title>. <source>Front Physiol.</source> (<year>2013</year>) <volume>4</volume>:<fpage>115</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fphys.2013.00115</pub-id><pub-id pub-id-type=\"pmid\">23720638</pub-id></mixed-citation></ref><ref id=\"B122\"><label>122.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dubin</surname><given-names>AE</given-names></name><name><surname>Patapoutian</surname><given-names>A</given-names></name></person-group>. <article-title>Nociceptors: the sensors of the pain pathway</article-title>. <source>J Clin Invest.</source> (<year>2010</year>) <volume>120</volume>:<fpage>3760</fpage>–<lpage>72</lpage>. <pub-id pub-id-type=\"doi\">10.1172/JCI42843</pub-id><pub-id pub-id-type=\"pmid\">21041958</pub-id></mixed-citation></ref><ref id=\"B123\"><label>123.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morris</surname><given-names>G</given-names></name><name><surname>Berk</surname><given-names>M</given-names></name><name><surname>Puri</surname><given-names>BK</given-names></name></person-group>\n<article-title>A comparison of neuroimaging abnormalities in multiple sclerosis, major depression and chronic fatigue syndrome (myalgic encephalomyelitis): is there a common cause?</article-title>\n<source>Mol Neurobiol.</source> (<year>2018</year>) <volume>55</volume>:<fpage>3592</fpage>–<lpage>609</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s12035-017-0598-z</pub-id><pub-id pub-id-type=\"pmid\">28516431</pub-id></mixed-citation></ref><ref id=\"B124\"><label>124.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Albrecht</surname><given-names>DS</given-names></name><name><surname>Forsberg</surname><given-names>A</given-names></name><name><surname>Sandström</surname><given-names>A</given-names></name><name><surname>Bergan</surname><given-names>C</given-names></name><name><surname>Kadetoff</surname><given-names>D</given-names></name><name><surname>Protsenko</surname><given-names>E</given-names></name><etal/></person-group>. <article-title>Brain glial activation in fibromyalgia – a multi-site positron emission tomography investigation</article-title>. <source>Brain Behav Immun.</source> (<year>2019</year>) <volume>75</volume>:<fpage>72</fpage>–<lpage>83</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbi.2018.09.018</pub-id><pub-id pub-id-type=\"pmid\">30223011</pub-id></mixed-citation></ref><ref id=\"B125\"><label>125.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Beaumont</surname><given-names>A</given-names></name><name><surname>Burton</surname><given-names>AR</given-names></name><name><surname>Lemon</surname><given-names>J</given-names></name><name><surname>Bennett</surname><given-names>BK</given-names></name><name><surname>Lloyd</surname><given-names>A</given-names></name><name><surname>Vollmer-Conna</surname><given-names>U</given-names></name></person-group>. <article-title>Reduced cardiac vagal modulation impacts on cognitive performance in chronic fatigue syndrome</article-title>. <source>PLoS ONE.</source> (<year>2012</year>) <volume>7</volume>:<fpage>e49518</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0049518</pub-id><pub-id pub-id-type=\"pmid\">23166694</pub-id></mixed-citation></ref><ref id=\"B126\"><label>126.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pall</surname><given-names>ML</given-names></name></person-group>. <article-title>Elevated peroxynitrite as the cause of chronic fatigue syndrome</article-title>. <source>J Chronic Fatigue Syndrome.</source> (<year>2000</year>) <volume>7</volume>:<fpage>45</fpage>–<lpage>58</lpage>. <pub-id pub-id-type=\"doi\">10.1300/J092v07n04_05</pub-id><pub-id pub-id-type=\"pmid\">10790736</pub-id></mixed-citation></ref><ref id=\"B127\"><label>127.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pall</surname><given-names>ML</given-names></name></person-group>. <article-title>Elevated nitric oxide/peroxynitrite theory of multiple chemical sensitivity: central role of N-Methyl-D-Aspartate receptors in the sensitivity mechanism</article-title>. <source>Environ Health Perspect.</source> (<year>2003</year>) <volume>111</volume>:<fpage>1461</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1289/ehp.5935</pub-id><pub-id pub-id-type=\"pmid\">12948884</pub-id></mixed-citation></ref><ref id=\"B128\"><label>128.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hornig</surname><given-names>M</given-names></name><name><surname>Montoya</surname><given-names>JG</given-names></name><name><surname>Klimas</surname><given-names>NG</given-names></name><name><surname>Levine</surname><given-names>S</given-names></name><name><surname>Felsenstein</surname><given-names>D</given-names></name><name><surname>Bateman</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Distinct plasma immune signatures in ME/CFS are present early in the course of illness</article-title>. <source>Sci Adv.</source> (<year>2015</year>) <volume>1</volume>:<fpage>1</fpage>–<lpage>10</lpage>. <pub-id pub-id-type=\"doi\">10.1126/sciadv.1400121</pub-id><pub-id pub-id-type=\"pmid\">26079000</pub-id></mixed-citation></ref><ref id=\"B129\"><label>129.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Bell</surname><given-names>DS</given-names></name></person-group>\n<source>Cellular Hypoxia and Neuro-Immune Fatigue</source>. <edition>1st ed</edition>\n<publisher-loc>Livermore</publisher-loc>: <publisher-name>WingSpan Press</publisher-name> (<year>2007</year>).</mixed-citation></ref><ref id=\"B130\"><label>130.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maes</surname><given-names>M</given-names></name><name><surname>Kubera</surname><given-names>M</given-names></name><name><surname>Uytterhoeven</surname><given-names>M</given-names></name><name><surname>Vrydags</surname><given-names>N</given-names></name><name><surname>Bosmans</surname><given-names>E</given-names></name></person-group>. <article-title>Increased plasma peroxides as a marker of oxidative stress in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)</article-title>. <source>Med Sci Monit.</source> (<year>2011</year>) <volume>17</volume>:<fpage>11</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.12659/MSM.881699</pub-id><pub-id pub-id-type=\"pmid\">21455120</pub-id></mixed-citation></ref><ref id=\"B131\"><label>131.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Germain</surname><given-names>A</given-names></name><name><surname>Ruppert</surname><given-names>D</given-names></name><name><surname>Levine</surname><given-names>SM</given-names></name><name><surname>Hanson</surname><given-names>MR</given-names></name></person-group>. <article-title>Metabolic profiling of a myalgic encephalomyelitis/chronic fatigue syndrome discovery cohort reveals disturbances in fatty acid and lipid metabolism</article-title>. <source>Mol BioSyst.</source> (<year>2017</year>) <volume>13</volume>:<fpage>371</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1039/C6MB00600K</pub-id><pub-id pub-id-type=\"pmid\">28059425</pub-id></mixed-citation></ref><ref id=\"B132\"><label>132.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rutherford</surname><given-names>G</given-names></name><name><surname>Manning</surname><given-names>P</given-names></name><name><surname>Newton</surname><given-names>JL</given-names></name></person-group>. <article-title>Understanding muscle dysfunction in chronic fatigue syndrome</article-title>. <source>J Aging Res.</source> (<year>2016</year>) <volume>2016</volume>:<fpage>2497348</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2016/2497348</pub-id><pub-id pub-id-type=\"pmid\">26998359</pub-id></mixed-citation></ref><ref id=\"B133\"><label>133.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ablashi</surname><given-names>DV</given-names></name><name><surname>Eastman</surname><given-names>HB</given-names></name><name><surname>Owen</surname><given-names>CB</given-names></name><name><surname>Roman</surname><given-names>MM</given-names></name><name><surname>Friedman</surname><given-names>J</given-names></name><name><surname>Zabriskie</surname><given-names>JB</given-names></name><etal/></person-group>. <article-title>Frequent HHV-6 reactivation in Multiple Sclerosis (MS) and Chronic Fatigue Syndrome (CFS) patients</article-title>. <source>J Clin Virol.</source> (<year>2000</year>) <volume>16</volume>:<fpage>179</fpage>–<lpage>91</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S1386-6532(99)00079-7</pub-id><pub-id pub-id-type=\"pmid\">10738137</pub-id></mixed-citation></ref><ref id=\"B134\"><label>134.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Bolton</surname><given-names>D</given-names></name><name><surname>Gillett</surname><given-names>G</given-names></name></person-group>\n<article-title>Biopsychosocial conditions of health and disease</article-title>. <source>In: The Biopsychosocial Model of Health and Disease: New Philosophical and Scientific Developments.</source>\n<publisher-loc>Cham</publisher-loc>: <publisher-name>Palgrave Pivot</publisher-name> (<year>2019</year>). p. <fpage>109</fpage>–<lpage>45</lpage>. <pub-id pub-id-type=\"doi\">10.1007/978-3-030-11899-0_4</pub-id></mixed-citation></ref><ref id=\"B135\"><label>135.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bitton</surname><given-names>A</given-names></name><name><surname>Dobkin</surname><given-names>PL</given-names></name><name><surname>Edwardes</surname><given-names>MD</given-names></name><name><surname>Sewitch</surname><given-names>MJ</given-names></name><name><surname>Meddings</surname><given-names>JB</given-names></name><name><surname>Rawal</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Predicting relapse in crohn's disease: a biopsychosocial model</article-title>. <source>Gut.</source> (<year>2008</year>) <volume>57</volume>:<fpage>1386</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.1136/gut.2007.134817</pub-id><pub-id pub-id-type=\"pmid\">18390994</pub-id></mixed-citation></ref><ref id=\"B136\"><label>136.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schneiderman</surname><given-names>N</given-names></name></person-group>\n<article-title>Psychosocial, behavioral, and biological aspects of chronic diseases</article-title>. <source>Curr Direct Psychol Sci.</source> (<year>2004</year>) <volume>13</volume>:<fpage>247</fpage>–<lpage>51</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.0963-7214.2004.00318.x</pub-id></mixed-citation></ref><ref id=\"B137\"><label>137.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Glaser</surname><given-names>R</given-names></name><name><surname>Kiecolt-Glaser</surname><given-names>JK</given-names></name></person-group>. <article-title>Stress-associated immune modulation: relevance to viral infections and chronic fatigue syndrome</article-title>. <source>Am J Med.</source> (<year>1998</year>) <volume>105</volume>:<fpage>35S</fpage>−<lpage>42S</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0002-9343(98)00160-0</pub-id><pub-id pub-id-type=\"pmid\">9790480</pub-id></mixed-citation></ref><ref id=\"B138\"><label>138.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schoenfeld-Smith</surname><given-names>K</given-names></name><name><surname>Petroski</surname><given-names>GF</given-names></name><name><surname>Hewett</surname><given-names>JE</given-names></name><name><surname>Johnson</surname><given-names>JC</given-names></name><name><surname>Wright</surname><given-names>GE</given-names></name><name><surname>Smarr</surname><given-names>KL</given-names></name><etal/></person-group>. <article-title>A biopsychosocial model of disability in rheumatoid arthritis</article-title>. <source>Arthritis Rheumatism.</source> (<year>1996</year>) <volume>9</volume>:<fpage>368</fpage>–<lpage>75</lpage>. <pub-id pub-id-type=\"doi\">10.1002/1529-0131(199610)9:5<368::AID-ANR1790090505>3.0.CO;2-5</pub-id><pub-id pub-id-type=\"pmid\">8997926</pub-id></mixed-citation></ref><ref id=\"B139\"><label>139.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sahle</surname><given-names>BW</given-names></name><name><surname>Chen</surname><given-names>W</given-names></name><name><surname>Melaku</surname><given-names>YA</given-names></name><name><surname>Akombi</surname><given-names>BJ</given-names></name><name><surname>Rawal</surname><given-names>LB</given-names></name><name><surname>Renzaho</surname><given-names>AMN</given-names></name></person-group>. <article-title>Association of psychosocial factors with risk of chronic diseases: a nationwide longitudinal study</article-title>. <source>Am J Preventive Med.</source> (<year>2020</year>) <volume>58</volume>:<fpage>e39</fpage>–<lpage>50</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.amepre.2019.09.007</pub-id><pub-id pub-id-type=\"pmid\">31959325</pub-id></mixed-citation></ref><ref id=\"B140\"><label>140.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pogosova</surname><given-names>N</given-names></name><name><surname>Kotseva</surname><given-names>K</given-names></name><name><surname>De Bacquer</surname><given-names>D</given-names></name><name><surname>Von</surname><given-names>Känel R</given-names></name><name><surname>De Smedt</surname><given-names>D</given-names></name><name><surname>Bruthans</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Psychosocial risk factors in relation to other cardiovascular risk factors in coronary heart disease: results from the EUROASPIRE IV survey. a registry from the European society of cardiology</article-title>. <source>Eur J Preventive Cardiol.</source> (<year>2017</year>) <volume>24</volume>:<fpage>1371</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.1177/2047487317711334</pub-id><pub-id pub-id-type=\"pmid\">28534422</pub-id></mixed-citation></ref><ref id=\"B141\"><label>141.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yusuf</surname><given-names>S</given-names></name><name><surname>Hawken</surname><given-names>S</given-names></name><name><surname>Ôunpuu</surname><given-names>S</given-names></name><name><surname>Dans</surname><given-names>T</given-names></name><name><surname>Avezum</surname><given-names>A</given-names></name><name><surname>Lanas</surname><given-names>F</given-names></name><etal/></person-group>. <article-title>Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART Study): case-control study</article-title>. <source>Lancet.</source> (<year>2004</year>) <volume>364</volume>:<fpage>937</fpage>–<lpage>52</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0140-6736(04)17018-9</pub-id><pub-id pub-id-type=\"pmid\">15364185</pub-id></mixed-citation></ref><ref id=\"B142\"><label>142.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Piepoli</surname><given-names>MF</given-names></name><name><surname>Hoes</surname><given-names>AW</given-names></name><name><surname>Agewall</surname><given-names>S</given-names></name><name><surname>Albus</surname><given-names>C</given-names></name><name><surname>Brotons</surname><given-names>C</given-names></name><name><surname>Catapano</surname><given-names>AL</given-names></name><etal/></person-group>. <article-title>2016 European guidelines on cardiovascular disease prevention in clinical practice</article-title>. <source>Eur Heart J.</source> (<year>2016</year>) <volume>37</volume>:<fpage>2315</fpage>–<lpage>81</lpage>. <pub-id pub-id-type=\"doi\">10.1093/eurheartj/ehw106</pub-id><pub-id pub-id-type=\"pmid\">27222591</pub-id></mixed-citation></ref><ref id=\"B143\"><label>143.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><collab>Wittstein IS</collab></person-group>. <article-title>Depression, anxiety, and platelet reactivity in patients with coronary heart disease</article-title>. <source>Eur Heart J.</source> (<year>2010</year>) <volume>31</volume>:<fpage>1548</fpage>–<lpage>50</lpage>. <pub-id pub-id-type=\"doi\">10.1093/eurheartj/ehq097</pub-id><pub-id pub-id-type=\"pmid\">20413400</pub-id></mixed-citation></ref><ref id=\"B144\"><label>144.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Leserman</surname><given-names>J</given-names></name><name><surname>Petitto</surname><given-names>JM</given-names></name><name><surname>Golden</surname><given-names>RN</given-names></name><name><surname>Gaynes</surname><given-names>BN</given-names></name><name><surname>Gu</surname><given-names>H</given-names></name><name><surname>Perkins</surname><given-names>DO</given-names></name><etal/></person-group>. <article-title>Impact of stressful life events, depression, social support, coping, and cortisol on progression to aids</article-title>. <source>Am J Psychiatry.</source> (<year>2000</year>) <volume>157</volume>:<fpage>1221</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1176/appi.ajp.157.8.1221</pub-id><pub-id pub-id-type=\"pmid\">10910783</pub-id></mixed-citation></ref><ref id=\"B145\"><label>145.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wiborg</surname><given-names>JF</given-names></name><name><surname>Knoop</surname><given-names>H</given-names></name><name><surname>Stulemeijer</surname><given-names>M</given-names></name><name><surname>Prins</surname><given-names>JB</given-names></name><name><surname>Bleijenberg</surname><given-names>G</given-names></name></person-group>. <article-title>How does cognitive behaviour therapy reduce fatigue in patients with chronic fatigue syndrome? The role of physical activity</article-title>. <source>Psychol Med.</source> (<year>2010</year>) <volume>40</volume>:<fpage>1281</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1017/S0033291709992212</pub-id><pub-id pub-id-type=\"pmid\">20047707</pub-id></mixed-citation></ref><ref id=\"B146\"><label>146.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Surawy</surname><given-names>C</given-names></name><name><surname>Hackmann</surname><given-names>A</given-names></name><name><surname>Hawton</surname><given-names>K</given-names></name><name><surname>Sharpe</surname><given-names>M</given-names></name></person-group>. <article-title>Chronic fatigue syndrome: a cognitive approach</article-title>. <source>Behav Res Ther.</source> (<year>1995</year>) <volume>33</volume>:<fpage>535</fpage>–<lpage>44</lpage>. <pub-id pub-id-type=\"doi\">10.1016/0005-7967(94)00077-W</pub-id><pub-id pub-id-type=\"pmid\">7598674</pub-id></mixed-citation></ref><ref id=\"B147\"><label>147.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moss-Morris</surname><given-names>R</given-names></name><name><surname>Deary</surname><given-names>V</given-names></name><name><surname>Castell</surname><given-names>B</given-names></name></person-group>. <article-title>Chronic fatigue syndrome</article-title>. In: Aminoff MJ, Boller F, Swaab DE, editors. <source>Handbook of Clinical Neurology.</source> Elsevier BV. (<year>2013</year>). p. <volume>110</volume>:<fpage>303</fpage>–<lpage>14</lpage>. <pub-id pub-id-type=\"doi\">10.1016/B978-0-444-52901-5.00025-3</pub-id><pub-id pub-id-type=\"pmid\">23312650</pub-id></mixed-citation></ref><ref id=\"B148\"><label>148.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Geraghty</surname><given-names>KJ</given-names></name><name><surname>Blease</surname><given-names>C</given-names></name></person-group>. <article-title>Myalgic encephalomyelitis/chronic fatigue syndrome and the biopsychosocial model: a review of patient harm and distress in the medical encounter</article-title>. <source>Disabil Rehabil</source>. (<year>2019</year>) <volume>41</volume>:<fpage>3092</fpage>–<lpage>102</lpage>. <pub-id pub-id-type=\"doi\">10.1080/09638288.2018.1481149</pub-id><pub-id pub-id-type=\"pmid\">29929450</pub-id></mixed-citation></ref><ref id=\"B149\"><label>149.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><collab>CFS/ME Working Group</collab></person-group>\n<source>Report to the Chief Medical Officer of an Independent Working Group</source>. London (<year>2002</year>). Avaiable online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.meassociation.org.uk/wp-content/uploads/CMO-Report-2002.pdf\">https://www.meassociation.org.uk/wp-content/uploads/CMO-Report-2002.pdf</ext-link> (accessed January 17, 2019).</mixed-citation></ref><ref id=\"B150\"><label>150.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><collab>National Institute of Clinical Excellence</collab></person-group>. <article-title>Chronic fatigue syndrome/myalgic encephalomyelitis (or Encephalopathy): diagnosis and management of CFS/ME in adults and children</article-title>. In: National Institute of Clinical Excellence, editor. <source>NICE Clinical Guidelines</source>. London (<year>2007</year>) <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.nice.org.uk/guidance/cg53\">https://www.nice.org.uk/guidance/cg53</ext-link>.</mixed-citation></ref><ref id=\"B151\"><label>151.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Goudsmit</surname><given-names>EM</given-names></name><name><surname>Jo</surname><given-names>N</given-names></name><name><surname>Leonard</surname><given-names>AJ</given-names></name><name><surname>Karen</surname><given-names>EW</given-names></name></person-group>. <article-title>Pacing as a strategy to improve energy management in myalgic encephalomyelitis/chronic fatigue syndrome: a consensus document</article-title>. <source>Disabil Rehabil</source>. (<year>2012</year>) <volume>34</volume>:<fpage>1140</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.3109/09638288.2011.635746</pub-id><pub-id pub-id-type=\"pmid\">22181560</pub-id></mixed-citation></ref><ref id=\"B152\"><label>152.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Levine</surname><given-names>PH</given-names></name></person-group>. <article-title>Epidemic neuromyasthenia and chronic fatigue syndrome: epidemiological importance of a cluster definition</article-title>. <source>Clin Infect Dis.</source> (<year>1994</year>) <volume>18</volume>(<issue>Suppl. 1</issue>):<fpage>S16</fpage>–<lpage>20</lpage>. <pub-id pub-id-type=\"doi\">10.1093/clinids/18.Supplement_1.S16</pub-id><pub-id pub-id-type=\"pmid\">8148446</pub-id></mixed-citation></ref><ref id=\"B153\"><label>153.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Castro-Marrero</surname><given-names>J</given-names></name><name><surname>Faro</surname><given-names>M</given-names></name><name><surname>Aliste</surname><given-names>L</given-names></name><name><surname>Sáez-Francàs</surname><given-names>N</given-names></name><name><surname>Calvo</surname><given-names>N</given-names></name><name><surname>Martínez-Martínez</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Comorbidity in chronic fatigue syndrome/myalgic encephalomyelitis: a nationwide population-based cohort study</article-title>. <source>Psychosomatics.</source> (<year>2017</year>) <volume>58</volume>:<fpage>533</fpage>–<lpage>43</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.psym.2017.04.010</pub-id><pub-id pub-id-type=\"pmid\">28596045</pub-id></mixed-citation></ref><ref id=\"B154\"><label>154.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aaron</surname><given-names>LA</given-names></name><name><surname>Burke</surname><given-names>MM</given-names></name><name><surname>Buchwald</surname><given-names>D</given-names></name></person-group>. <article-title>Overlapping conditions among patients with chronic fatigue syndrome, fibromyalgia, and temporomandibular disorder</article-title>. <source>Arch Internal Med.</source> (<year>2000</year>) <volume>160</volume>:<fpage>221</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1001/archinte.160.2.221</pub-id><pub-id pub-id-type=\"pmid\">10647761</pub-id></mixed-citation></ref><ref id=\"B155\"><label>155.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bateman</surname><given-names>L</given-names></name><name><surname>Darakjy</surname><given-names>S</given-names></name><name><surname>Klimas</surname><given-names>N</given-names></name><name><surname>Peterson</surname><given-names>D</given-names></name><name><surname>Levine</surname><given-names>SM</given-names></name><name><surname>Allen</surname><given-names>A</given-names></name><etal/></person-group>\n<article-title>Chronic fatigue syndrome and co-morbid and consequent conditions: evidence from a multi-site clinical epidemiology study</article-title>. <source>Fatigue Biomed Health Behav.</source> (<year>2015</year>) <volume>3</volume>:<fpage>1</fpage>–<lpage>15</lpage>. <pub-id pub-id-type=\"doi\">10.1080/21641846.2014.978109</pub-id></mixed-citation></ref><ref id=\"B156\"><label>156.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Natelson</surname><given-names>BH</given-names></name></person-group>. <article-title>Myalgic encephalomyelitis/chronic fatigue syndrome and fibromyalgia: definitions, similarities, and differences</article-title>. <source>Clin Therap.</source> (<year>2019</year>) <volume>41</volume>:<fpage>612</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.clinthera.2018.12.016</pub-id><pub-id pub-id-type=\"pmid\">30795933</pub-id></mixed-citation></ref><ref id=\"B157\"><label>157.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abbi</surname><given-names>B</given-names></name><name><surname>Natelson</surname><given-names>BH</given-names></name></person-group>\n<article-title>Is chronic fatigue syndrome the same illness as fibromyalgia: evaluating the ‘single syndrome' hypothesis | qjm: an international journal of medicine | oxford academic</article-title>. <source>Q J Med.</source> (<year>2013</year>) <volume>106</volume>:<fpage>3</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1093/qjmed/hcs156</pub-id></mixed-citation></ref><ref id=\"B158\"><label>158.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wolfe</surname><given-names>F</given-names></name><name><surname>Clauw</surname><given-names>DJ</given-names></name><name><surname>Fitzcharles</surname><given-names>MA</given-names></name><name><surname>Goldenberg</surname><given-names>DL</given-names></name><name><surname>Katz</surname><given-names>RS</given-names></name><name><surname>Mease</surname><given-names>P</given-names></name><etal/></person-group>\n<article-title>The American college of rheumatology preliminary diagnostic criteria for fibromyalgia and measurement of symptom severity</article-title>. <source>Arthritis Care Res.</source> (<year>2010</year>) <volume>62</volume>:<fpage>600</fpage>–<lpage>10</lpage>. <pub-id pub-id-type=\"doi\">10.1002/acr.20140</pub-id></mixed-citation></ref><ref id=\"B159\"><label>159.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wolfe</surname><given-names>F</given-names></name><name><surname>Clauw</surname><given-names>DJ</given-names></name><name><surname>Fitzcharles</surname><given-names>MA</given-names></name><name><surname>Goldenberg</surname><given-names>DL</given-names></name><name><surname>Häuser</surname><given-names>W</given-names></name><name><surname>Katz</surname><given-names>RS</given-names></name><etal/></person-group>. <article-title>Fibromyalgia criteria and severity scales for clinical and epidemiological studies: a modification of the ACR preliminary diagnostic criteria for fibromyalgia</article-title>. <source>J Rheumatol.</source> (<year>2011</year>) <volume>38</volume>:<fpage>1113</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.3899/jrheum.100594</pub-id><pub-id pub-id-type=\"pmid\">21285161</pub-id></mixed-citation></ref><ref id=\"B160\"><label>160.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carruthers</surname><given-names>BM</given-names></name><name><surname>van de Sande</surname><given-names>MI</given-names></name><name><surname>De Meirleir</surname><given-names>KL</given-names></name><name><surname>Klimas</surname><given-names>NG</given-names></name><name><surname>Broderick</surname><given-names>G</given-names></name><name><surname>Mitchell</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>Myalgic encephalomyelitis: international consensus criteria</article-title>. <source>J Intern Med.</source> (<year>2011</year>) <volume>270</volume>:<fpage>327</fpage>–<lpage>38</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-2796.2011.02428.x</pub-id><pub-id pub-id-type=\"pmid\">21777306</pub-id></mixed-citation></ref><ref id=\"B161\"><label>161.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Unger</surname><given-names>ER</given-names></name><name><surname>Nisenbaum</surname><given-names>R</given-names></name><name><surname>Moldofsky</surname><given-names>H</given-names></name><name><surname>Cesta</surname><given-names>A</given-names></name><name><surname>Sammut</surname><given-names>C</given-names></name><name><surname>Reyes</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Sleep assessment in a population-based study of chronic fatigue syndrome</article-title>. <source>BMC Neurol.</source> (<year>2004</year>) <volume>4</volume>:<fpage>6</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1471-2377-4-6</pub-id><pub-id pub-id-type=\"pmid\">15096280</pub-id></mixed-citation></ref><ref id=\"B162\"><label>162.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cameron</surname><given-names>C</given-names></name></person-group>. <article-title>A theory of fatigue</article-title>. <source>Ergonomics.</source> (<year>1973</year>) <volume>16</volume>:<fpage>633</fpage>–<lpage>48</lpage>. <pub-id pub-id-type=\"doi\">10.1080/00140137308924554</pub-id><pub-id pub-id-type=\"pmid\">4772989</pub-id></mixed-citation></ref><ref id=\"B163\"><label>163.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Horne</surname><given-names>JA</given-names></name></person-group>. <article-title>Sleep loss and ‘divergent' thinking ability</article-title>. <source>Sleep.</source> (<year>1988</year>) <volume>11</volume>:<fpage>528</fpage>–<lpage>36</lpage>. <pub-id pub-id-type=\"doi\">10.1093/sleep/11.6.528</pub-id><pub-id pub-id-type=\"pmid\">3238256</pub-id></mixed-citation></ref><ref id=\"B164\"><label>164.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moldofsky</surname><given-names>H</given-names></name><name><surname>Scarisbrick</surname><given-names>P</given-names></name><name><surname>England</surname><given-names>R</given-names></name><name><surname>Smythe</surname><given-names>H</given-names></name></person-group>. <article-title>Musculoskeletal symptoms and non REM sleep disturbance in patients with ‘fibrositis syndrome' and healthy subjects</article-title>. <source>Psychosomatic Med.</source> (<year>1975</year>) <volume>37</volume>:<fpage>341</fpage>–<lpage>51</lpage>. <pub-id pub-id-type=\"doi\">10.1097/00006842-197507000-00008</pub-id><pub-id pub-id-type=\"pmid\">169541</pub-id></mixed-citation></ref><ref id=\"B165\"><label>165.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Samkoff</surname><given-names>JS</given-names></name><name><surname>Jacques</surname><given-names>CHM</given-names></name></person-group>. <article-title>A review of studies concerning effects of sleep deprivation and fatigue on residents' performance</article-title>. <source>Acad Med.</source> (<year>1991</year>) <volume>66</volume>:<fpage>687</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1097/00001888-199111000-00013</pub-id><pub-id pub-id-type=\"pmid\">1747181</pub-id></mixed-citation></ref><ref id=\"B166\"><label>166.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dyken</surname><given-names>ME</given-names></name><name><surname>Afifi</surname><given-names>AK</given-names></name><name><surname>Lin-Dyken</surname><given-names>DC</given-names></name></person-group>. <article-title>Sleep-related problems in neurologic diseases</article-title>. <source>Chest</source>. (<year>2012</year>) <volume>141</volume>:<fpage>528</fpage>–<lpage>44</lpage>. <pub-id pub-id-type=\"doi\">10.1378/chest.11-0773</pub-id><pub-id pub-id-type=\"pmid\">22315121</pub-id></mixed-citation></ref><ref id=\"B167\"><label>167.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Neu</surname><given-names>D</given-names></name><name><surname>Cappeliez</surname><given-names>B</given-names></name><name><surname>Hoffmann</surname><given-names>G</given-names></name><name><surname>Verbanck</surname><given-names>P</given-names></name><name><surname>Linkowski</surname><given-names>P</given-names></name><name><surname>Le Bon</surname><given-names>O</given-names></name></person-group>. <article-title>High slow-wave sleep and low-light sleep: chronic fatigue syndrome is not likely to be a primary sleep disorder</article-title>. <source>J Clin Neurophysiol.</source> (<year>2009</year>) <volume>26</volume>:<fpage>207</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.1097/WNP.0b013e3181a1841b</pub-id><pub-id pub-id-type=\"pmid\">19424087</pub-id></mixed-citation></ref><ref id=\"B168\"><label>168.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gotts</surname><given-names>ZM</given-names></name><name><surname>Deary</surname><given-names>V</given-names></name><name><surname>Newton</surname><given-names>J</given-names></name><name><surname>Der Van Dussen</surname><given-names>D</given-names></name><name><surname>De Roy</surname><given-names>P</given-names></name><name><surname>Ellis</surname><given-names>JG</given-names></name></person-group>. <article-title>Are there sleep-specific phenotypes in patients with chronic fatigue syndrome? A cross-sectional polysomnography analysis</article-title>. <source>BMJ Open.</source> (<year>2013</year>) <volume>3</volume>:<fpage>e002999</fpage>. <pub-id pub-id-type=\"doi\">10.1136/bmjopen-2013-002999</pub-id><pub-id pub-id-type=\"pmid\">23794547</pub-id></mixed-citation></ref><ref id=\"B169\"><label>169.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reeves</surname><given-names>WC</given-names></name><name><surname>Heim</surname><given-names>C</given-names></name><name><surname>Maloney</surname><given-names>EM</given-names></name><name><surname>Youngblood</surname><given-names>LS</given-names></name><name><surname>Unger</surname><given-names>ER</given-names></name><name><surname>Decker</surname><given-names>MJ</given-names></name><etal/></person-group>. <article-title>Sleep characteristics of persons with chronic fatigue syndrome and non-fatigued controls: results from a population-based study</article-title>. <source>BMC Neurol.</source> (<year>2006</year>) <volume>6</volume>:<fpage>41</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1471-2377-6-41</pub-id><pub-id pub-id-type=\"pmid\">17109739</pub-id></mixed-citation></ref><ref id=\"B170\"><label>170.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Libman</surname><given-names>E</given-names></name><name><surname>Creti</surname><given-names>L</given-names></name><name><surname>Baltzan</surname><given-names>M</given-names></name><name><surname>Rizzo</surname><given-names>D</given-names></name><name><surname>Fichten</surname><given-names>CS</given-names></name><name><surname>Bailes</surname><given-names>S</given-names></name></person-group>. <article-title>Sleep apnea and psychological functioning in chronic fatigue syndrome</article-title>. <source>J Health Psychol.</source> (<year>2009</year>) <volume>14</volume>:<fpage>1251</fpage>–<lpage>67</lpage>. <pub-id pub-id-type=\"doi\">10.1177/1359105309344895</pub-id><pub-id pub-id-type=\"pmid\">19858344</pub-id></mixed-citation></ref><ref id=\"B171\"><label>171.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fedorowski</surname><given-names>A</given-names></name><name><surname>Melander</surname><given-names>O</given-names></name></person-group>. <article-title>Syndromes of orthostatic intolerance: a hidden danger</article-title>. <source>J Internal Med.</source> (<year>2013</year>) <volume>273</volume>:<fpage>322</fpage>–<lpage>35</lpage>. <pub-id pub-id-type=\"doi\">10.1111/joim.12021</pub-id><pub-id pub-id-type=\"pmid\">23216860</pub-id></mixed-citation></ref><ref id=\"B172\"><label>172.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stewart</surname><given-names>JM</given-names></name></person-group>. <article-title>Common syndromes of orthostatic intolerance</article-title>. <source>Pediatrics</source>. (<year>2013</year>) <volume>131</volume>:<fpage>968</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.1542/peds.2012-2610</pub-id><pub-id pub-id-type=\"pmid\">23569093</pub-id></mixed-citation></ref><ref id=\"B173\"><label>173.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Okamoto</surname><given-names>LE</given-names></name><name><surname>Raj</surname><given-names>SR</given-names></name><name><surname>Peltier</surname><given-names>A</given-names></name><name><surname>Gamboa</surname><given-names>A</given-names></name><name><surname>Shibao</surname><given-names>C</given-names></name><name><surname>Diedrich</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Neurohumoral and haemodynamic profile in postural tachycardia and chronic fatigue syndromes</article-title>. <source>Clin Sci.</source> (<year>2012</year>) <volume>122</volume>:<fpage>183</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.1042/CS20110200</pub-id><pub-id pub-id-type=\"pmid\">21906029</pub-id></mixed-citation></ref><ref id=\"B174\"><label>174.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Garland</surname><given-names>EM</given-names></name><name><surname>Celedonio</surname><given-names>JE</given-names></name><name><surname>Raj</surname><given-names>SR</given-names></name></person-group>. <article-title>Postural tachycardia syndrome: beyond orthostatic intolerance</article-title>. <source>Curr Neurol Neurosci Rep</source>. (<year>2015</year>) <volume>15</volume>:<fpage>60</fpage>. <pub-id pub-id-type=\"doi\">10.1007/s11910-015-0583-8</pub-id><pub-id pub-id-type=\"pmid\">26198889</pub-id></mixed-citation></ref><ref id=\"B175\"><label>175.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carding</surname><given-names>S</given-names></name><name><surname>Verbeke</surname><given-names>K</given-names></name><name><surname>Vipond</surname><given-names>DT</given-names></name><name><surname>Corfe</surname><given-names>BM</given-names></name><name><surname>Owen</surname><given-names>LJ</given-names></name></person-group>. <article-title>Dysbiosis of the gut microbiota in disease</article-title>. <source>Microb Ecol Health Dis.</source> (<year>2015</year>) <volume>26</volume>:<fpage>10</fpage>.3402/mehd.v26.26191. <pub-id pub-id-type=\"doi\">10.3402/mehd.v26.26191</pub-id><pub-id pub-id-type=\"pmid\">25651997</pub-id></mixed-citation></ref><ref id=\"B176\"><label>176.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nagy-Szakal</surname><given-names>D</given-names></name><name><surname>Williams</surname><given-names>BL</given-names></name><name><surname>Mishra</surname><given-names>N</given-names></name><name><surname>Che</surname><given-names>X</given-names></name><name><surname>Lee</surname><given-names>B</given-names></name><name><surname>Bateman</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Fecal metagenomic profiles in subgroups of patients with myalgic encephalomyelitis/chronic fatigue syndrome</article-title>. <source>Microbiome.</source> (<year>2017</year>) <volume>5</volume>:<fpage>44</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s40168-017-0261-y</pub-id><pub-id pub-id-type=\"pmid\">28441964</pub-id></mixed-citation></ref><ref id=\"B177\"><label>177.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Berstad</surname><given-names>A</given-names></name><name><surname>Hauso</surname><given-names>O</given-names></name><name><surname>Berstad</surname><given-names>K</given-names></name><name><surname>Berstad</surname><given-names>JER</given-names></name></person-group>. <article-title>From IBS to ME – the dysbiotic march hypothesis</article-title>. <source>Med Hypotheses.</source> (<year>2020</year>) <volume>140</volume>:<fpage>109648</fpage>. <pub-id pub-id-type=\"doi\">10.1016/j.mehy.2020.109648</pub-id><pub-id pub-id-type=\"pmid\">32126475</pub-id></mixed-citation></ref><ref id=\"B178\"><label>178.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aaron</surname><given-names>LA</given-names></name><name><surname>Herrell</surname><given-names>R</given-names></name><name><surname>Ashton</surname><given-names>S</given-names></name><name><surname>Belcourt</surname><given-names>M</given-names></name><name><surname>Schmaling</surname><given-names>K</given-names></name><name><surname>Goldberg</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Comorbid clinical conditions in chronic fatigue. a co-twin control study</article-title>. <source>J General Internal Med.</source> (<year>2001</year>) <volume>16</volume>:<fpage>24</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1525-1497.2001.03419.x</pub-id><pub-id pub-id-type=\"pmid\">11251747</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"brief-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Neth Heart J</journal-id><journal-id journal-id-type=\"iso-abbrev\">Neth Heart J</journal-id><journal-title-group><journal-title>Netherlands Heart Journal</journal-title></journal-title-group><issn pub-type=\"ppub\">1568-5888</issn><issn pub-type=\"epub\">1876-6250</issn><publisher><publisher-name>Bohn Stafleu van Loghum</publisher-name><publisher-loc>Houten</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32193702</article-id><article-id pub-id-type=\"pmc\">PMC7431526</article-id><article-id pub-id-type=\"publisher-id\">1403</article-id><article-id pub-id-type=\"doi\">10.1007/s12471-020-01403-3</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Image Puzzle – Answer</subject></subj-group></article-categories><title-group><article-title>A patient with recurrent palpitations and unusual anatomy</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Lawson</surname><given-names>C. J. M.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><contrib contrib-type=\"author\"><name><surname>Margulescu</surname><given-names>A. D.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Barry</surname><given-names>J.</given-names></name><address><email>james.barry@wales.nhs.uk</email></address><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><aff id=\"Aff1\">Department of Cardiology, Morriston Regional Cardiac Centre, SA6 6NL Swansea, UK </aff></contrib-group><pub-date pub-type=\"epub\"><day>19</day><month>3</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>19</day><month>3</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><month>9</month><year>2020</year></pub-date><volume>28</volume><issue>9</issue><fpage>498</fpage><lpage>499</lpage><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Answer</title><p>Complete atresia of the inferior vena cava (IVC) with cavo-azygos (CA) continuity was diagnosed by contrast venography performed using a long sheath (SL0, Abbott Medical, USA) (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>a). Following the CA vein route, a decapolar and a quadripolar catheter were advanced into the superior vena cava, right atrium, and then into the coronary sinus and right ventricle, respectively (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b). However, the right subclavian vein needed to be used to map the triangle of Koch with the ablation catheter, due to better reach and stability at this region compared with the CA route (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>c).<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Inferior vena cava atresia with cavo-azygos (CA) continuity accounts for the unusual placement of the electrophysiology catheters from the right inguinal region into the heart. <bold>a</bold> LAO 30, left anterior oblique 30 degrees view; <bold>b</bold> RAO 30, right anterior oblique 30 degrees view; <bold>c</bold> Contrast venogram helped deliniate the anatomical structures through which the electrophysiology catheter and sheaths were advanced from the right inguinal region; <bold>d</bold> Schematic representation of normal venous anatomy (<italic>left</italic>) vs. CA continuity (<italic>right</italic>). <italic>CS</italic> coronary sinus, <italic>RA</italic> right atrium <italic>RV</italic> right ventricle <italic>SP</italic> slow pathway <italic>TV</italic> tricuspid valve, <italic>SVC</italic> superior vena cava, <italic>IV</italic> innominate vein, <italic>AV</italic> azygos vein, <italic>HAV</italic> hemiazygos vein, <italic>HV</italic> hepatic veins, <italic>RRV</italic> right renal vein, <italic>LRV</italic> left renal vein</p></caption><graphic xlink:href=\"12471_2020_1403_Fig1_HTML\" id=\"d30e262\"/></fig></p><p>IVC atresia with CA continuity is a rare congenital anomaly that results from lack of interruption of the right cardinal vein at the level of the diaphragm during embryological development [<xref ref-type=\"bibr\" rid=\"CR1\">1</xref>]. As a result, the intrahepatic trajectory of the IVC is not formed, and the hepatic veins will drain separately into the right atrium. Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>d shows a schematic representation of normal venous anatomy vs. CA continuity. CA continuity may be associated with more extensive embryological abnormalities, such as the heterotaxy syndrome (abnormal arrangement of internal organs across the left-right axis of the body) [<xref ref-type=\"bibr\" rid=\"CR2\">2</xref>]. In our patient, chest X‑ray, abdominal ultrasound and echocardiogram revealed normal internal organ arrangement.</p><p>For electrophysiology procedures, IVC atresia with CA continuity can cause significant challenges, especially if left atrial access is required, because the impossibility of performing transseptal puncture through the usual inferior approach [<xref ref-type=\"bibr\" rid=\"CR3\">3</xref>, <xref ref-type=\"bibr\" rid=\"CR4\">4</xref>]. However, right-sided ablations (including ablation of atrioventricular nodal re-entrant tachycardia) can be performed with small variations of standard techniques, by looping the catheters back into the right cardiac chambers through the superior vena cava, as demonstrated in this case.</p></sec></body><back><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Conflict of interest</title><p>C.J.M. Lawson, A.D. Margulescu and J. Barry declare that they have no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hikspoors</surname><given-names>JPJM</given-names></name><name><surname>Soffers</surname><given-names>JHM</given-names></name><name><surname>Mekonen</surname><given-names>HK</given-names></name><etal/></person-group><article-title>Development of the human infrahepatic inferior caval and azygos venous systems</article-title><source>J Anat</source><year>2015</year><volume>226</volume><fpage>113</fpage><lpage>125</lpage><pub-id pub-id-type=\"doi\">10.1111/joa.12266</pub-id><pub-id pub-id-type=\"pmid\">25496171</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>SJ</given-names></name></person-group><article-title>Heterotaxy syndrome</article-title><source>Korean Circ J</source><year>2011</year><volume>41</volume><fpage>227</fpage><lpage>232</lpage><pub-id pub-id-type=\"doi\">10.4070/kcj.2011.41.5.227</pub-id><pub-id pub-id-type=\"pmid\">21731561</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Okajima</surname><given-names>K</given-names></name><name><surname>Nakanishi</surname><given-names>T</given-names></name><name><surname>Ichibori</surname><given-names>H</given-names></name><etal/></person-group><article-title>Trans-aortic pulmonary vein isolation using magnetic navigation system for paroxysmal atrial fibrillation in a patient with dextrocardia, situs inversus, and inferior vena cava continuity with azygos vein</article-title><source>J Arrhythm</source><year>2018</year><volume>34</volume><fpage>583</fpage><lpage>585</lpage><pub-id pub-id-type=\"doi\">10.1002/joa3.12096</pub-id><pub-id pub-id-type=\"pmid\">30327707</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kato</surname><given-names>H</given-names></name><name><surname>Kubota</surname><given-names>S</given-names></name><name><surname>Goto</surname><given-names>T</given-names></name><etal/></person-group><article-title>Transseptal puncture and catheter ablation via the superior vena cava approach for persistent atrial fibrillation in a patient with polysplenia syndrome and interruption of the inferior vena cava: contact force-guided pulmonary vein isolation</article-title><source>Europace</source><year>2017</year><volume>19</volume><fpage>1227</fpage><lpage>1232</lpage><pub-id pub-id-type=\"pmid\">27174901</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Commun Biol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Commun Biol</journal-id><journal-title-group><journal-title>Communications Biology</journal-title></journal-title-group><issn pub-type=\"epub\">2399-3642</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807853</article-id><article-id pub-id-type=\"pmc\">PMC7431527</article-id><article-id pub-id-type=\"publisher-id\">1165</article-id><article-id pub-id-type=\"doi\">10.1038/s42003-020-01165-z</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>2-Nitroimidazoles induce mitochondrial stress and ferroptosis in glioma stem cells residing in a hypoxic niche</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-1952-0252</contrib-id><name><surname>Koike</surname><given-names>Naoyoshi</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kota</surname><given-names>Ryuichi</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Naito</surname><given-names>Yoshiko</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hayakawa</surname><given-names>Noriyo</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Matsuura</surname><given-names>Tomomi</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hishiki</surname><given-names>Takako</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Onishi</surname><given-names>Nobuyuki</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Fukada</surname><given-names>Junichi</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-7165-6336</contrib-id><name><surname>Suematsu</surname><given-names>Makoto</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Shigematsu</surname><given-names>Naoyuki</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Saya</surname><given-names>Hideyuki</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-5783-1049</contrib-id><name><surname>Sampetrean</surname><given-names>Oltea</given-names></name><address><email>oltea@a6.keio.jp</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.26091.3c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9959</institution-id><institution>Division of Gene Regulation, Institute for Advanced Medical Research, </institution><institution>Keio University School of Medicine, </institution></institution-wrap>Tokyo, Japan </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.26091.3c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9959</institution-id><institution>Department of Radiology, </institution><institution>Keio University School of Medicine, </institution></institution-wrap>Tokyo, Japan </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.26091.3c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9959</institution-id><institution>Clinical and Translational Research Center, </institution><institution>Keio University School of Medicine, </institution></institution-wrap>Tokyo, Japan </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.26091.3c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9959</institution-id><institution>Department of Biochemistry, </institution><institution>Keio University School of Medicine, </institution></institution-wrap>Tokyo, Japan </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>3</volume><elocation-id>450</elocation-id><history><date date-type=\"received\"><day>12</day><month>11</month><year>2019</year></date><date date-type=\"accepted\"><day>20</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Under hypoxic conditions, nitroimidazoles can replace oxygen as electron acceptors, thereby enhancing the effects of radiation on malignant cells. These compounds also accumulate in hypoxic cells, where they can act as cytotoxins or imaging agents. However, whether these effects apply to cancer stem cells has not been sufficiently explored. Here we show that the 2-nitroimidazole doranidazole potentiates radiation-induced DNA damage in hypoxic glioma stem cells (GSCs) and confers a significant survival benefit in mice harboring GSC-derived tumors in radiotherapy settings. Furthermore, doranidazole and misonidazole, but not metronidazole, manifested radiation-independent cytotoxicity for hypoxic GSCs that was mediated by ferroptosis induced partially through blockade of mitochondrial complexes I and II and resultant metabolic alterations in oxidative stress responses. Doranidazole also limited the growth of GSC-derived subcutaneous tumors and that of tumors in orthotopic brain slices. Our results thus reveal the theranostic potential of 2-nitroimidazoles as ferroptosis inducers that enable targeting GSCs in their hypoxic niche.</p></abstract><abstract id=\"Abs2\" abstract-type=\"web-summary\"><p id=\"Par2\">Koike et al. show that the 2-nitroimidazole doranidazole increases radiation-induced DNA damage in hypoxic glioma stem cells (GSCs). They further demonstrate that additional radiation-independent cytotoxicity of 2-nitroimidazoles is due to ferroptosis that occurs through blockade of mitochondrial complexes I and II leading to metabolic changes in the oxidative stress response.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Cancer therapy</kwd><kwd>CNS cancer</kwd><kwd>Cancer</kwd><kwd>Cancer metabolism</kwd><kwd>Cancer stem cells</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100002241</institution-id><institution>MEXT | Japan Science and Technology Agency (JST)</institution></institution-wrap></funding-source><award-id>16K07124</award-id><award-id>19K07671</award-id><award-id>18K15602</award-id><principal-award-recipient><name><surname>Sampetrean</surname><given-names>Oltea</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par3\">Intratumoral regions exposed to low contents of molecular oxygen (O<sub>2</sub>) harbor therapy-resistant cancer cells and are a key therapeutic target<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. Although no single drug is able to eradicate all hypoxic malignant cells, two major pharmacological approaches to their specific targeting have been developed: inhibition of proteins that function in the cellular response to hypoxia, and the administration of hypoxia-selective drugs<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>.</p><p id=\"Par4\">Among compounds that are able to exert anticancer effects in the absence of oxygen, nitroimidazoles are considered promising theranostic agents<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup> for several reasons. The electrophilic properties of nitroimidazoles allow them to oxidize the radiation-induced DNA radicals<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>–<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>, thereby enhancing the effects of radiation. Furthermore, on entering cells by diffusion, nitroimidazoles undergo one-electron reduction of the nitro group. In the presence of O<sub>2</sub>, the reduced metabolites of these compounds are immediately reoxidized, resulting in redox cycling of the drugs. However, in the absence of oxygen, the reduced derivatives are stabilized and accumulate within the cell, where they can undergo further serial reduction<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. This characteristic has allowed several 2-nitroimidazoles to serve as exogenous markers of hypoxia. Pimonidazole and etanidazole pentafluoride (EF5) have thus been widely adopted in preclinical studies<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>, whereas misonidazole is applied clinically in the form of [<sup>18</sup>F]fluoromisonidazole as a hypoxia tracer for positron emission tomography<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. In addition, nitroreduction of 2-nitroimidazoles has been reported to result in the production and accumulation of toxic metabolites as well as in the consumption of reducing equivalents under anaerobic conditions, thus, leading to direct, radiation-independent cytotoxic effects on hypoxic cells<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>.</p><p id=\"Par5\">The biological implications of the bioreductive metabolism of nitroimidazoles in cancer have not been fully explored, however, which has limited their therapeutic application. The 5-nitroimidazole nimorazole has been shown to significantly improve the efficacy of radiotherapeutic management in cancer patients<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. In contrast, although they possess a higher electron affinity, 2-nitroimidazoles showed only a moderate effect in this regard<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>, with this inadequacy having been attributed to dose-limiting neurotoxic side effects<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. However, the recent finding that cancer cells with stem-like properties often reside in hypoxic lesions<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup> has suggested that the limited therapeutic effect of 2-nitroimidazoles might also reflect a resistance of cancer stem cells to this class of compounds.</p><p id=\"Par6\">Doranidazole, or 1-(1′,3′,4′-trihydroxy-2′-butoxy)-methyl-2-nitroimidazole (PR-350), was designed to reduce the neurotoxicity of 2-nitroimidazoles by limiting blood–brain barrier permeability<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>–<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. It has shown radiosensitizing effects both in vitro and in vivo in preclinical studies<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>, and its administration before radiotherapy contributed to long-term survival in patients with unresectable pancreatic or locally advanced non-small cell lung cancer<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. It has remained unclear, however, whether doranidazole is able to serve as a radiosensitizer for cancer stem cells and whether it exerts yet unidentified radiation-independent cytotoxic effects on hypoxic cells.</p><p id=\"Par7\">We have now examined these issues with the use of a mouse model of glioblastoma (GBM), a highly hypoxic tumor type<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. We found that doranidazole enhanced radiation-induced DNA damage in hypoxic glioma stem cells (GSCs) and conferred a survival benefit in mice harboring GSC-derived tumors in the radiotherapy setting. Furthermore, doranidazole and misonidazole, but not metronidazole, also induced mitochondrial dysfunction and ROS accumulation in GSCs, resulting in the induction of ferroptosis and a reduction of the hypoxic GSC niche, shedding light on possible theranostic applications of these compounds.</p></sec><sec id=\"Sec2\" sec-type=\"results\"><title>Results</title><sec id=\"Sec3\"><title>Radiosensitizing effect of doranidazole on GSCs</title><p id=\"Par8\">To investigate radiosensitization by and hypoxic cell-specific toxicity of nitroimidazoles, we adopted our previously established syngeneic mouse GBM model<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Glioma-initiating cells (GICs) were established by transduction of <italic>Ink4a/Arf</italic>-null neural stem/progenitor cells (NSCs) with a vector for the oncoprotein H-Ras<sup>V12</sup> (but without a fluorescent marker). The GIC-H cells obtained after selection with hygromycin expressed the stem cell markers Nestin and SOX2, underwent glial differentiation on exposure to fetal bovine serum (FBS), and formed heterogeneous and aggressive tumors resembling human GBMs on orthotopic implantation (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1a–d</xref>). Cells with stem-like properties were isolated from late-stage intracranial tumors that had interacted with the syngeneic environment and formed hypoxic regions. The resulting GSC-H cells showed increased tumor formation potential and radioresistance compared with GIC-H cells (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1c–e</xref>), and they were adopted for subsequent experiments.</p><p id=\"Par9\">We first examined the effect of doranidazole on the radiosensitivity of GSC-H cells. Mice were exposed to 15 Gy of ionizing radiation, with or without prior intraperitoneal (i.p.) administration of doranidazole (200 mg/kg), at 10 days after orthotopic implantation of GSC-H cells. Doranidazole administration combined with radiation resulted in a significant prolongation of survival compared with radiation alone (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>). In contrast, a single dose of doranidazole had no effect on survival in the absence of radiation (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>). Cultures of GSC-H cells maintained in the presence of <0.1% O<sub>2</sub> showed a significant increase in the number of foci positive for histone γH2AX, a marker of DNA double-strand breaks, on exposure to radiation in the presence of doranidazole compared with irradiation alone, whereas doranidazole had no such effect under normoxic conditions (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b, c</xref>) and had no effect in the absence of radiation (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1f</xref>). In a colony formation assay, doranidazole had a significant radiosensitizing effect on GSC-H cells that was more pronounced under severely hypoxic (<0.1% O<sub>2</sub>) than under normoxic conditions (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1d, e</xref>). A 3-h exposure to doranidazole alone tended to reduce the plating efficiency of GSC-H cells in the presence of either 20% or <0.1% O<sub>2</sub> (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1f, g</xref>), but this effect did not achieve statistical significance.<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Radiosensitizing effect of doranidazole on GSCs.</title><p><bold>a</bold> Survival curves for the indicated numbers (<italic>n</italic>) of mice exposed to 0 or 15 Gy of radiation 10 days after orthotopic implantation of 1000 GSC-H cells, with or without administration of doranidazole (200 mg/kg, i.p.) 30 min before irradiation. PBS phosphate-buffered saline. Representative experiment, <italic>n</italic> = 2 independent experiments performed. GSC-H cells cultured under normoxic (<bold>b</bold>) or hypoxic (<bold>c</bold>) conditions were incubated for 30 min in the absence or presence of 3 mM doranidazole, exposed to 2 Gy of radiation, and then subjected to immunofluorescence staining of γH2AX at 30 min after irradiation. Nuclei were stained with Hoechst 33342. The boxed regions of the left images are shown at higher magnification in the right images. Scale bars, 10 µm. The number of γH2AX foci per nucleus was determined for all nuclei from <italic>n</italic> = 3 independent experiments, including a total of >150 nuclei per group. GSC-H cells were incubated for 3 h in the absence or presence of 3 mM doranidazole under normoxic <bold>d</bold>, <bold>f</bold> or hypoxic <bold>e</bold>, <bold>g</bold> conditions, exposed to the indicated doses of radiation <bold>d</bold>, <bold>e</bold>, and then subjected to a colony formation assay for 10 days under normoxic conditions. The surviving fraction <bold>d</bold>, <bold>e</bold> and plating efficiency <bold>f</bold>, <bold>g</bold> determined as the mean ± s.d. from <italic>n</italic> = 3 <bold>d</bold>, <bold>e</bold> or <italic>n</italic> = 4 <bold>f</bold>, <bold>g</bold> independent experiments are shown. Statistical analysis was performed with the log-rank test <bold>a</bold>; the unpaired two-tailed Student’s <italic>t</italic>-test <bold>b</bold>, <bold>c</bold>; two-way ANOVA followed by Sidak’s post hoc test <bold>d</bold>, <bold>e</bold>, with the <italic>P</italic> values being for comparison of plus or minus doranidazole at 10 Gy; or the paired two-tailed Student’s <italic>t</italic>-test <bold>f</bold>, <bold>g</bold>.</p></caption><graphic xlink:href=\"42003_2020_1165_Fig1_HTML\" id=\"d30e620\"/></fig></p><p id=\"Par10\">Together, these results showed that doranidazole mediates radiosensitization of both GSCs and GSC-based tumors.</p></sec><sec id=\"Sec4\"><title>Doranidazole induces GSC death under hypoxic conditions</title><p id=\"Par11\">In addition to regions of severe hypoxia or anoxia (<0.1% O<sub>2</sub>; binding of hypoxia-marker EF5, 30–100%)<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>, which are primary targets of radiosensitization, GBMs contain regions with O<sub>2</sub> levels corresponding to 0.5–2.5% (EF5 binding, 3–10%)<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>, usually considered as “modest” or “mild” hypoxia<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. Given that hypoxic conditions of 1–2% O<sub>2</sub> increase the stem cell fraction and cell proliferation in GBMs<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>, we next asked whether 2-nitroimidazoles possess additional radiation-independent cytotoxic effects on GSCs under mild hypoxia.</p><p id=\"Par12\">Flow cytometric analysis of the cell cycle showed that the proportion of GSC-H cells with a DNA content of between 2N and 4N (corresponding to S phase) declined, whereas that of those with a content of 4N increased, after exposure to doranidazole (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2a, b</xref>). The effects of doranidazole on the cell cycle were similar under both normoxic (20% O<sub>2</sub>) and hypoxic (1% O<sub>2</sub>) conditions, with the exception that the drug also induced an increase in the proportion of cells with a DNA content of <2N (representing dead cells) under the hypoxic condition. Equimolar concentrations of metronidazole and misonidazole did not affect cell cycle distribution (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2b</xref>).</p><p id=\"Par13\">To evaluate the cell cycle profile in more detail, we generated a bicistronic vector encoding mCherry-hCdt1 and EGFP-hGem fusion proteins (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2c</xref>) that was based on the FUCCI (fluorescent ubiquitination-based cell cycle indicator) system<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. The vector was validated in mouse embryonic fibroblasts to confirm that cells in G<sub>1</sub> phase express mCherry, those in S–G<sub>2</sub>–M phases express EGFP (enhanced green fluorescent protein), and contact inhibition induces an accumulation of mCherry-positive cells (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2d</xref>). The construct was then introduced into GSC-H cells, with the resulting cells being designated GSC-F. Time-lapse imaging of GSC-F cells exposed to doranidazole under the normoxic condition revealed a gradual decline in the number of EGFP-positive (S–G<sub>2</sub>–M) cells and an accumulation of mCherry-positive (G<sub>0</sub>–G<sub>1</sub>) cells (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2e</xref>), suggesting that the doranidazole-induced increase in the 4N population of GSC-H cells detected by flow cytometry (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2a</xref>) reflected the appearance of cells that had undergone mitotic slippage and entered the subsequent G<sub>1</sub> phase.</p><p id=\"Par14\">To evaluate cytotoxicity, we quantified the binding of propidium iodide (PI) to DNA in unfixed GSC-H cells. Flow cytometry revealed that doranidazole and misonidazole, but not metronidazole, each induced a significant increase in the proportion of dead cells after drug treatment for 72 h in the presence of 1% O<sub>2</sub> (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a, b</xref>). Of note, the induction of cell death by doranidazole was apparent earlier and was more pronounced in the presence of severe hypoxia (<0.1% O<sub>2</sub>) (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a</xref>, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2f</xref>), consistent with less redox cycling of the drug. Serum-treated NSCs, which differentiate along the astrocytic line (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1b</xref>) underwent significant cell death after exposure to doranidazole in the presence of 0.1% O<sub>2</sub>, but not in the presence of 20% or 1% O<sub>2</sub> (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2g</xref>).<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Cytotoxic effect of doranidazole on GSCs.</title><p><bold>a</bold> Flow cytometric analysis of cell death for GSC-H cells exposed to 0 or 3 mM doranidazole under normoxic or hypoxic conditions for 3 days. Representative profiles and quantification of PI-positive cells are shown. FSC forward scatter. <bold>b</bold> Flow cytometric analysis of cell death for GSC-H cells exposed to 3 mM metronidazole (Metro), 3 mM misonidazole (Miso), or dimethyl sulfoxide (DMSO) vehicle under normoxic or hypoxic conditions for 3 days. <bold>c</bold> Immunofluorescence staining for EF5 (marker of hypoxic cells) in a sphere formed by GSC-H cells. Nuclear staining, Hoechst 33342. <bold>d</bold> GSC-H spheres formed over 7 days were exposed to the indicated concentrations of doranidazole for 12 or 24 h, after which dead cells were identified based on PI uptake. Representative images and quantification of the PI-positive sphere area at 24 h are shown. <bold>e</bold> Evaluation of cell death based on PI uptake for GSC-H spheres incubated for 24 h with the indicated drugs or DMSO vehicle. Quantification of the PI-positive sphere area is shown. <bold>f</bold> Evaluation of cell death based on PI uptake for GSC-H spheres incubated for 24 h with the indicated drugs or DMSO vehicle. Quantification of the PI-positive sphere area is shown. <bold>g</bold> Immunofluorescence staining for EF5 incorporation in spheres formed by U251 and Becker cells over 2 days. Nuclear staining, Hoechst 33342. <bold>h</bold> Evaluation of cell death based on PI uptake for U251 and Becker spheres incubated for 24 h with the indicated concentrations of doranidazole. Representative images and quantification of the PI-positive sphere area are shown. Quantitative data for (<bold>a</bold>, <bold>b</bold>) are means ± s.d. for <italic>n</italic> = 3 independent experiments and were analyzed with the paired two-tailed Student’s <italic>t</italic>-test (<bold>a</bold>) or one-way ANOVA followed by Dunnett’s post hoc test <bold>b</bold>. Data for <bold>d</bold>–<bold>f</bold>, <bold>h</bold> are means ± s.d. of six biologically distinct replicates in a representative experiment (<italic>n</italic> = 3 independent experiments performed) and were analyzed by one-way ANOVA followed by Dunnett’s post hoc test. *<italic>P</italic> < 0.05, ***<italic>P</italic> < 0.001. Scale bars, 100 µm (<bold>c</bold>, <bold>g</bold>) 300 µm (<bold>d</bold>, <bold>h</bold>).</p></caption><graphic xlink:href=\"42003_2020_1165_Fig2_HTML\" id=\"d30e825\"/></fig></p><p id=\"Par15\">We next examined cell death in a model that more closely mimics tumors with regard to the presence of regions with different oxygen tensions. GSC-based spheres possess not only a three-dimensional structure but also a hypoxic core, as revealed by EF5 staining (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2c</xref>). Exposure of GSC-H spheres to doranidazole for 24 h induced cell death (as revealed by PI staining) in the central region of the spheres in a concentration-dependent manner (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2d</xref>). Exposure to 3 mM doranidazole for a period as short as 6 h also induced marked cell death in the central region of the spheres (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2h</xref>). The 2-nitroimidazoles misonidazole and etanidazole, but not the 5-nitroimidazoles metronidazole or nimorazole, also manifested a similar effect at equimolar concentrations (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2e, f</xref>).</p><p id=\"Par16\">We next examined the response to doranidazole in two human glioma cell lines. Under monolayer culture conditions, in which cells are uniformly exposed to the same O<sub>2</sub> level, treatment with 3 mM doranidazole for 24 h induced significant cell death in both cell lines in the presence of <0.1% O<sub>2</sub> (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2i, j</xref>). In sphere culture, the GBM cell line U251 formed spheres with an EF5-positive core (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2g</xref>) and showed significant cell death on exposure to doranidazole for 24 h (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2h</xref>). In contrast, spheres formed by the astrocytoma cell line Becker had a necrotic core with a proliferating outer layer, but with minimal EF5-positive hypoxic cells, and were not sensitive to doranidazole (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2g, h</xref>). Together, these results suggested that doranidazole and misonidazole exert pronounced cytotoxic effects on hypoxic GSCs.</p></sec><sec id=\"Sec5\"><title>Ferroptosis mediates doranidazole-induced hypoxic GSC death</title><p id=\"Par17\">To characterize the mechanism of GSC death induced by doranidazole, we first examined the effects of inhibitors of necroptosis, ferroptosis, and apoptosis. Doranidazole toxicity in GSC-H spheres was attenuated markedly by the ferroptosis inhibitor ferrostatin-1 and to a lesser extent by the necroptosis inhibitor necrostatin-1, whereas the apoptosis inhibitor Z-VAD-FMK had no significant effect (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3a</xref>). The iron-chelator deferoxamine also inhibited doranidazole-induced cell death in a concentration-dependent manner (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b</xref>). These results thus suggested that doranidazole-induced GSC death is mediated, at least in part, by ferroptosis.<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Mechanism of doranidazole-induced GSC death.</title><p><bold>a</bold> Evaluation of cell death based on PI uptake for GSC-H spheres incubated for 24 h with 3 mM doranidazole and either DMSO vehicle or the cell death inhibitors (100 µM) necrostatin-1 (Nec-1), ferrostatin-1 (Fer-1), or Z-VAD-FMK (Z-VAD). Representative images and quantification of the PI-positive sphere area are shown. <bold>b</bold> Evaluation of cell death as in (<bold>a</bold>) for GSC-H spheres incubated for 24 h with 3 mM doranidazole and the indicated concentrations of deferoxamine (DFO). <bold>c</bold> Evaluation of cell death as in (<bold>a</bold>) for GSC-H spheres incubated for 24 h with 3 mM doranidazole and SKQ1 (0 or 10 µM). Flow cytometric analysis of MitoSOX Red staining in GSC-H cells incubated with or without 3 mM doranidazole and 10 µM SKQ1 under normoxic (<bold>d</bold>) or hypoxic (<bold>e</bold>) conditions for 12 h. Representative profiles as well as quantification of the mean fluorescence intensity (MFI) of MitoSOX Red from <italic>n</italic> = 5 (<bold>d</bold>) or <italic>n</italic> = 3 (<bold>e</bold>) independent experiments are shown. <bold>f</bold>, <bold>g</bold> Flow cytometric analysis of BODIPY 581/591 C11 staining in GSC-H cells incubated with or without 3 mM doranidazole and 10 µM SKQ1 under normoxic (<bold>f</bold>) or hypoxic (<bold>g</bold>) conditions for 18 h. Representative profiles and quantification of the MFI of BODIPY 581/591 C11 from <italic>n</italic> = 3 independent experiments are shown. Quantitative data are means ± s.d. from six biologically distinct replicates in a representative experiment, <italic>n</italic> = 3 independent experiments performed <bold>a</bold>–<bold>c</bold> or from the indicated number of independent experiments <bold>d</bold>–<bold>g</bold> and were analyzed by one-way ANOVA followed by Dunnett’s post hoc test <bold>a</bold>, <bold>b</bold>, the unpaired two-tailed Student’s <italic>t</italic>-test (<bold>c</bold>), the paired two-tailed Student’s <italic>t</italic>-test <bold>d</bold>, <bold>f</bold>, or one-way ANOVA followed by Tukey’s post hoc test <bold>e</bold>, <bold>g</bold>. *<italic>P</italic> < 0.05, ***<italic>P</italic> < 0.001. Scale bars, 300 µm (<bold>a</bold>–<bold>c</bold>).</p></caption><graphic xlink:href=\"42003_2020_1165_Fig3_HTML\" id=\"d30e991\"/></fig></p><p id=\"Par18\">An increase in the intracellular amounts of ROS is one trigger of ferroptosis<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. Indeed, gene ontology analysis of gene set enrichment analysis pathways for doranidazole-induced changes in the transcriptome of GSC-H cells revealed that “oxidation reduction process” and “response to oxidative stress” were the most upregulated biological processes common to both normoxic and mildly hypoxic (1% O<sub>2</sub>) conditions (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3b–d</xref>). In addition to genes encoding antioxidant enzymes such as NAD(P)H-dependent quinone oxidoreductase 1 (<italic>Nqo1</italic>) and catalase (<italic>Cat</italic>), genes in the “oxidation reduction process” set whose expression was upregulated by doranidazole included those for enzymes related to the reduction of nitroimidazoles—<italic>Txnrd1</italic><sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>, which encodes thioredoxin reductase 1—or to ferroptosis, including <italic>Steap3</italic> and <italic>Hmox1</italic>, which encode a metalloreductase<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup> and heme oxygenase 1<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>, respectively (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3e</xref>). Consistent with the doranidazole-induced increase in the expression of genes related to “response to oxidative stress,” the antioxidants 10-(6′-plastoquinonyl) decyltriphenylphosphonium (SKQ1)<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref>,<xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup> and <italic>N</italic>-acetyl cysteine (NAC) partially inhibited doranidazole-induced GSC-H death (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3c</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3f</xref>).</p><p id=\"Par19\">Given that SKQ1 targets mitochondria and that mitochondrial ROS play a context-dependent role in ferroptosis<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, we examined the effect of nitroimidazoles on mitochondrial ROS production in GSC-H cells. Staining of mitochondria in these cells with MitoTracker Green revealed that doranidazole or misonidazole, but not metronidazole, induced a change in mitochondrial morphology from tubular to smaller and rounder (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3g</xref>). Staining with MitoSOX Red also showed that doranidazole or misonidazole, but not metronidazole, induced ROS accumulation in mitochondria, with this effect being more pronounced under hypoxic conditions (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3d, e</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3h</xref>). In addition, this effect of doranidazole tended to be inhibited by SKQ1, although not to a statistically significant extent (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3e</xref>). Furthermore, treatment with doranidazole and SKQ1 had similar effects on lipid peroxidation as detected with the fluorescent probe BODIPY C11 (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3f, g</xref>).</p></sec><sec id=\"Sec6\"><title>Doranidazole attenuates mitochondrial complex activity in GSCs</title><p id=\"Par20\">Mitochondrial dysfunction—in particular, dysfunction of mitochondrial complex I and an increase in the NADH/NAD<sup>+</sup> ratio—has been found to be a major cause of elevated mitochondrial ROS levels<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. Assessment of mitochondrial function by extracellular flux analysis showed that the basal oxygen consumption rate (OCR) of GSC-H cells was reduced by incubation with doranidazole under normoxic (20% O<sub>2</sub>) or hypoxic (1% O<sub>2</sub>) conditions for 12 h (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4a, b</xref>). Uncoupling of oxidative phosphorylation with carbonylcyanide-<italic>p</italic>-trifluoromethoxyphenylhydrazone (FCCP) revealed that mitochondrial spare respiratory capacity of GSC-H cells was also reduced by incubation with doranidazole (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4a, b</xref>). Similar effects were apparent with misonidazole, but not with metronidazole (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4a</xref>). Permeabilization of the plasma membrane allows complex specific substrates to access mitochondria. Mitochondrial complex I-dependent respiration, measured after the addition of pyruvate, was reduced by incubation with doranidazole for 12 h (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c</xref>), as was mitochondrial complex II-dependent respiration, measured after the addition of succinate (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c, d</xref>). Mitochondrial complex III- and complex IV-dependent respiration, measured after the addition of duroquinol and <italic>N</italic>,<italic>N</italic>,<italic>N</italic>′,<italic>N</italic>′-tetramethyl-<italic>p</italic>-phenylenediamine (TMPD), respectively, were not substantially affected by doranidazole (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c, d</xref>).<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>Effect of doranidazole on mitochondrial function.</title><p>Extracellular flux analysis of GSC-H cells treated for 12 h with the indicated concentrations of doranidazole under normoxic (<bold>a</bold>) or hypoxic (<bold>b</bold>) conditions. Representative examples of changes in OCR after sequential injection of the indicated inhibitors (Oligo oligomycin, Rtn rotenone, AA antimycin A) as well as quantitative data for basal OCR and spare respiratory capacity (means ± s.d. for <italic>n</italic> = 3 independent experiments) are shown. Mitochondrial complex I-, complex II-, and complex IV-dependent OCR (<bold>c</bold>) and complex II-, complex III-, and complex IV-dependent OCR (<bold>d</bold>) measured in GSC-H cells after exposure to the indicated concentrations of doranidazole for 12 h and permeabilization of the plasma membrane. Suc succinate, Malo malonate, Duro duroquinol. Data are means ± s.d. for at least five biologically distinct replicates in <italic>n</italic> = 1 experiment. <bold>e</bold> Immunoblot analysis of mitochondrial complex (C) proteins in GSC-H cells treated for 24 h with the indicated concentrations of doranidazole. <bold>f</bold> ATP content of GSC-H cells exposed to doranidazole under normoxic or hypoxic conditions for 12 h. Data are expressed relative to the value for normoxia and 0 mM doranidazole and are means ± s.d. from <italic>n</italic> = 3 three independent experiments. Statistical analysis was performed by one-way ANOVA followed by Dunnett’s post hoc test <bold>a</bold>, <bold>b</bold>, or with the two-way ANOVA followed by Sidak’s post hoc test <bold>f</bold>.</p></caption><graphic xlink:href=\"42003_2020_1165_Fig4_HTML\" id=\"d30e1177\"/></fig></p><p id=\"Par21\">To investigate the molecular basis of the dysfunction of the mitochondrial respiratory chain induced by doranidazole, we examined protein expression for the complexes by immunoblot analysis. Doranidazole-induced downregulation of mitochondrial complex I and II proteins in a concentration-dependent manner (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4e</xref>), although similar downregulation of the corresponding mRNAs was not apparent (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4b, c</xref>). Furthermore, the proteasome inhibitor MG132 did not affect the depletion of mitochondrial complex proteins induced by doranidazole (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4d</xref>), suggesting that this depletion was not due to proteolysis. Misonidazole, but not metronidazole, showed effects similar to those of doranidazole on mitochondrial complex protein expression (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4e</xref>). Consistent with the marked decrease in basal OCR, a 12-h doranidazole treatment significantly reduced the ATP content of GSC-H cells under mild hypoxic conditions (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4f</xref>).</p></sec><sec id=\"Sec7\"><title>Doranidazole changes intracellular metabolite levels in GSCs</title><p id=\"Par22\">Both ferroptosis<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup> and mitochondrial function are intimately linked to cellular metabolism. We therefore examined the effects of doranidazole on GSC metabolism by capillary electrophoresis coupled with mass spectrometry. Intermediates of the tricarboxylic acid (TCA) cycle showed the most marked changes in abundance after exposure of GSC-H cells to doranidazole. The intracellular levels of citrate, <italic>cis</italic>-aconitate, and succinate were increased, whereas those of fumarate and malate were decreased, by exposure of the cells to doranidazole for 24 h (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5a</xref>), consistent with changes resulting from a decrease in the activity of mitochondrial complex II. The amounts of glutamine, glutamate, and α-ketoglutarate were also significantly reduced by doranidazole treatment, suggestive of perturbation of glutaminolysis. Of note, the ratio of NADH to NAD<sup>+</sup> was significantly increased by doranidazole treatment (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5b</xref>). Considering that elevated levels of NADH/NAD<sup>+</sup> inhibit dehydrogenases in the TCA cycle, and that increased succinate suggests a blockade of complex II, these alterations in TCA metabolites appear to result from impairment of mitochondrial respiration. Furthermore, while a 12-h treatment with doranidazole was insufficient to induce robust changes in ATP in normoxic conditions (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4f</xref>), a 24-h treatment significantly decreased ATP and increased AMP contents (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5c</xref>). These effects coincided with a significant but modest drop of the adenylate charge (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5d</xref>). In contrast to doranidazole, metronidazole had a negligible effect on metabolites overall (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5a–d</xref>).<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>Effects of doranidazole on intracellular metabolite levels in GSC-H cells.</title><p><bold>a</bold> Metabolome analysis for the TCA cycle in GSC-H cells exposed to 0 or 3 mM doranidazole for 24 h. N.D. not detected. The amounts of NAD<sup>+</sup> and NADH and the NADH/NAD<sup>+</sup> ratio (<bold>b</bold>) as well as ATP and AMP (<bold>c</bold>) and the total adenylate charge (<bold>d</bold>) were also determined. Metabolite levels were normalized by total protein amount, are means ± s.d. from eight biologically distinct replicates, <italic>n</italic> = 1 experiment, and were analyzed by the unpaired two-tailed Student’s <italic>t-</italic>test.</p></caption><graphic xlink:href=\"42003_2020_1165_Fig5_HTML\" id=\"d30e1266\"/></fig></p></sec><sec id=\"Sec8\"><title>Doranidazole limits GSC growth in cultured brain slices</title><p id=\"Par23\">Finally, to assess the effects of doranidazole on GSCs in their niche, we first examined cultured brain slices from mice with orthotopic implants of GSC-F cells. Such brain slices allow the visualization of glioma cells in their native environment, without the limitations imposed by a functional blood–brain barrier<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. Under normoxic conditions, GSC-F cells in brain slices incubated with 3 mM doranidazole for 2 days gradually accumulated in G<sub>1</sub> phase of the cell cycle (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6a</xref>) and tumor growth was inhibited (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6c</xref>). Given that brain slices containing large hypoxic tumors are less amenable to imaging, we next exposed slices with similar small-sized tumors to 1% O<sub>2</sub>. Under this condition, doranidazole induced cell death, as revealed by the binding of 4′,6-diamidino-2-phenylindole (DAPI) to DNA and by morphological changes apparent on hematoxylin-eosin staining (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6b, c</xref>). The effects of doranidazole under both normoxic and hypoxic conditions were concentration dependent (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6a, b</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6a, b</xref>).<fig id=\"Fig6\"><label>Fig. 6</label><caption><title>Effects of doranidazole on cultured brain slices bearing implants of GSC-F cells.</title><p>Sequential images of brain slices prepared from mice bearing GSC-F cell–based orthotopic tumors and incubated with or without 3 mM doranidazole under normoxic (<bold>a</bold>) or hypoxic (<bold>b</bold>) conditions. Overlays of green fluorescence (S–G<sub>2</sub>–M phase), red fluorescence (G<sub>1</sub> phase), and phase-contrast images are shown for days 0, 1, and 2. A DAPI exclusion assay for the same areas as well as hematoxylin-eosin (HE) staining of paraffin-embedded sections of the explants are also presented for day 2. Scale bars, 300 µm (fluorescence images) or 50 µm (hematoxylin-eosin). <bold>c</bold> Quantification of tumor area for explants as in (<bold>a</bold>, <bold>b</bold>) at days 0 and 2. Data are for explants from <italic>n</italic> = 3 mice per condition and were analyzed by two-way ANOVA followed by Sidak’s post hoc test. A.U. arbitrary units. <bold>d</bold> Model for the effects of doranidazole on GSCs in their hypoxic niche. DSB double-strand breaks. The scheme was created with biorender.com.</p></caption><graphic xlink:href=\"42003_2020_1165_Fig6_HTML\" id=\"d30e1336\"/></fig></p><p id=\"Par24\">Next, to reproduce and examine early and pronounced intratumoral hypoxic regions, we injected mice subcutaneously with GSC-H cells. Doranidazole (200 mg/kg, i.p.) administration for 5 days delayed the growth of tumors formed by cells injected 15 days previously (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6c</xref>) as well as reduced the EF5-positive hypoxic tumor area (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6d</xref>), although this latter effect was not statistically significant. Together, these results thus showed that doranidazole induced GSC death in hypoxic intratumoral regions in both the ex vivo and in vivo settings.</p></sec></sec><sec id=\"Sec9\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par25\">We have here shown that 2-nitroimidazole drugs sensitize hypoxic murine GSCs to ionizing radiation in association with an increase in the number of double-strand breaks. Furthermore, under hypoxic conditions, these drugs manifest radiation-independent cytotoxicity for GSCs associated with the induction of mitochondrial dysfunction and ferroptotic cell death. The decrease in the number of metabolically active GSCs induced by these agents is likely to reduce the intratumoral competition for oxygen and thereby ultimately to limit the size of the hypoxic niche.</p><p id=\"Par26\">Nitroimidazoles have previously been shown to potentiate radiation-induced DNA damage and the subsequent death of actively cycling cancer cells in several tumor types<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. However, cancer stem cells often possess an enhanced ability to repair DNA damage and cycle more slowly compared with differentiated tumor cells<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR37\">37</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. We have now shown that doranidazole potentiated the radiation-induced increase in the number of double-strand breaks in mouse GSCs and served as a radiosensitizer for GSC-derived tumors. At least for GBM, these findings exclude the possibility that intrinsic resistance of cancer stem-like cells to 2-nitroimidazoles is a major cause of the limited radiosensitization observed in clinical studies<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref>–<xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup> and suggest the need for an increased focus on theranostic applications.</p><p id=\"Par27\">GBMs are thought to contain substantial hypoxic regions, to experience loss of blood–brain barrier function, and to be highly recalcitrant to the effects of chemo- and radiotherapy. In particular, large tumors presenting with contrast enhancement and compression symptoms as well as recurrent tumors usually contain not only hypoxic regions but also an increased number of GSCs, and such tumors might therefore benefit from doranidazole treatment. The identification of such tumors would be an essential step in any future clinical applications of this drug. A high initial value followed by a gradual decrease in [<sup>18</sup>F]fluoromisonidazole uptake by GBM tumors could indicate treatment efficacy, with such imaging thus representing a companion diagnostic option. In the case of other solid tumors, stratification of patients based on [<sup>18</sup>F]fluoromisonidazole uptake might be necessary for maximal exploitation of the radiosensitizing potential of 2-nitroimidazoles.</p><p id=\"Par28\">Monitoring of hypoxia would also be essential for clinical exploitation of the direct cytotoxicity of doranidazole for GSCs. We found that doranidazole, misonidazole, and etanidazole each induced pronounced radiation- and cell cycle-independent cell death within the oxygen-deprived center of GSC spheres. In spheres with a diameter of >500 µm, which already have a small core of dead cells as a result of oxygen and nutrient limitation, GSC death was apparent as early as 12 h after initiation of drug treatment. Such cell death was markedly, but not completely, attenuated by the ferroptosis inhibitor ferrostatin-1, the iron-chelator deferoxamine, and the mitochondrial ROS scavenger SKQ1, whereas it was not affected by the caspase inhibitor Z-VAD-FMK. These results suggest that the GSC death induced by 2-nitroimidazoles is mediated largely by ferroptosis rather than by apoptosis.</p><p id=\"Par29\">Ferroptosis is a form of iron-dependent oxidative cell death mediated by ROS accumulation and lipid peroxidation<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. The relation between mitochondrial ROS generation and ferroptosis remains poorly understood but seems to be context dependent<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. Partial attenuation of ferroptosis by oligomycin<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>, an inhibitor of mitochondrial complex V, implicates the electron transport chain as a possible initial source of ROS. In contrast, the detection of ferroptosis in cells that lack mitochondrial DNA or mitochondrial function has led to the suggestion that changes in mitochondrial morphology and membrane potential associated with ferroptosis represent the final stages of the death cascade<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. We have now found that doranidazole induced a decrease in mitochondrial complex I and II activity, an increase in the NADH/NAD<sup>+</sup> ratio, and the accumulation of succinate in GSCs. Together with the partial rescue of doranidazole-induced GSC death by SKQ1, these results suggest that the accumulation of mitochondrial ROS, even though due to aberrant function of complexes I and II<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>, takes place during the reversible phase of ferroptosis and thus closer to the initiating rather than the end stage (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6d</xref>). Of note, the decrease in mitochondrial complex activity, the increase in ROS and lipid peroxidation, and the changes in the transcriptome induced by doranidazole were qualitatively similar in normoxic and mildly hypoxic conditions but tended to be more pronounced in the latter. The degree of ferroptosis will likely be maximal under conditions in which oxygen tension is sufficiently low to allow the intracellular accumulation of nitroimidazoles, but sufficiently high to allow cells to rely on mitochondrial and electron transport chain function and to favor a net increase in ROS levels.</p><p id=\"Par30\">The net oxidative damage will also depend on the antioxidant responses triggered by the treatment in a cell-specific manner. In GSCs, doranidazole increased the expression of several antioxidant-related genes, including the aldehyde dehydrogenase gene <italic>Aldh3a1</italic>. Aldh3a1 protects cells by detoxifying lipid peroxidation-derived reactive aldehydes such as 4-hydroxynonenal<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. Aldh3a1 can also release antioxidant nitric oxide (NO) from S-nitrosothiols<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref>–<xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>. Upregulation of Aldh3a1 has been identified as a cause of resistance to ferroptosis induced by sulfasalazine<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>, an inhibitor of the cystine–glutamate transporter subunit xCT, and drug screening for synthetic lethality identified aldehyde dehydrogenase inhibitors that cooperatively enhance the effects of sulfasalazine on cell lines derived from several types of solid tumors as well as on subcutaneous tumors in animal models<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref>,<xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup>. The upregulation of antioxidant proteins might thus be another reason why nitroimidazoles have failed to realize their expected clinical potential. Screening for synthetic lethality under hypoxic conditions might identify aldehyde dehydrogenase inhibitors that specifically enhance the action of nitroimidazoles.</p><p id=\"Par31\">Differences in electron affinity and reduction potential between nitroimidazoles appear to play a crucial role in explaining the effects of doranidazole in ferroptosis, since those characteristics have been found to be correlated with hypoxia selectivity and toxicity<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>. Consistent with these previous findings, we found that equimolar concentrations of 2-nitroimidazoles doranidazole, misonidazole, and etanidazole (electron affinity values of more than −400 mV<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>), but not of 5-nitroimidazoles metronidazole and nimorazole (electron affinity values of <−400 mV), were able to induce hypoxic GSC death. In our previous study using the same GBM model, we reported upregulation of nucleophilic polysulfides in tumors<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>, and further investigation is necessary to determine whether the electrophilic doranidazole breaks the balance between endogenous aldehydes and polysulfides to render GSCs more susceptible to cell death.</p><p id=\"Par32\">In parallel with mitochondrial dysfunction, mobilization of the intracellular labile iron pool is a major determinant of ferroptosis. Doranidazole treatment upregulated <italic>Steap3</italic> and <italic>Hmox1</italic> in GSCs in both normoxic and mildly hypoxic conditions. Metalloreductase Steap3 catalyzes the reduction of ferric iron (Fe<sup>3+</sup>) to ferrous one (Fe<sup>2+</sup>)<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. Heme oxygenase 1 increases the labile iron pool by releasing Fe<sup>2+</sup> from heme<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup> and is instrumental in erastin- and withaferin A-induced ferroptosis<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>. Furthermore, NADH, which was increased after doranidazole treatment, can also contribute to the reduction and mobilization of Fe<sup>2+</sup> from ferritin, especially under anaerobic conditions<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>.</p><p id=\"Par33\">An issue that will require further consideration is the involvement of ferroptosis in 2-nitroimidazole-induced normal tissue toxicity. We found that doranidazole induces a similar extent of cell death in NSCs differentiated along astrocytic lines as it does in the GSCs in conditions of <0.1% O<sub>2</sub>. Given that in the human brain, normal cells are expected to reside at physiological oxygen concentrations of 0.5–7%<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>, massive cell death due to accumulation of the drugs in severely hypoxic normal cells is less likely to occur. However, although not specifically addressed in this study, the doranidazole-induced mitochondrial dysfunction and sublethal damage to oxic GSCs could also occur in non-transformed cells and might partially contribute to the toxicities reported for 2-nitroimidazoles in normal tissues.</p><p id=\"Par34\">In conclusion, we have demonstrated the potential of 2-nitroimidazoles as targeted inducers of ferroptosis in hypoxic GSCs within their niche. Advances in knowledge of cancer cell hierarchies and types of cell death have thus prompted further exploration of 2-nitroimidazole compounds that has led to the uncovering of previously unknown aspects of their reductive metabolism and suggested possible future theranostic applications.</p></sec><sec id=\"Sec10\"><title>Methods</title><sec id=\"Sec11\"><title>Establishment of induced GICs and GSCs and cell culture</title><p id=\"Par35\">Primary <italic>Ink4a/Arf</italic>-null NSCs were isolated from the subventricular zone of 6-week-old mice as previously described<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. NSCs and all derived cells were cultured in neural stem cell medium (NSM), consisting of Dulbecco’s modified Eagle’s medium (DMEM)-F12 (Wako, Osaka, Japan) without serum and containing recombinant human epidermal growth factor and basic fibroblast growth factor (PeproTech, Rocky Hill, NJ) at 20 ng/ml, heparan sulfate (Sigma-Aldrich, St. Louis, MO) at 200 ng/ml, and B27 supplement without vitamin A (Invitrogen, Carlsbad, CA). For establishment of GICs, retrovirus-containing culture supernatants were prepared as previously described<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup> from the pBABE-Hygro retroviral vector containing human <italic>H-Ras</italic><sup><italic>V12</italic></sup> cDNA (kindly provided by P. P. Pandolfi and T. Maeda). NSCs were infected with the retrovirus-containing supernatants, and the infected cells were purified by selection with hygromycin (200 μg/ml) for 14 days. The resulting cells were designated GIC-H<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>. Primary tumors formed by orthotopic implantation of GIC-H cells in syngeneic mice were excised at the onset of tumor-related symptoms. Dissociated tumor cells were subjected to selection with hygromycin (200 μg/ml) for 14 days, and the resulting cells were designated GSC-H and maintained in NSM. The human glioma cell lines U251MG (GBM) and Becker (astrocytoma grade III–IV) were obtained from American Type Culture Collection and Japanese Collection of Research Bioresources Cell Bank, respectively, and they were maintained in DMEM-F12 supplemented with 10% FBS before a switch to NSM for the assays in this study. For induction of cell differentiation, NSCs or GICs were cultured in DMEM-F12 supplemented with 1% or 10% FBS for 72 h. All cells were studied within 20 passages after establishment.</p></sec><sec id=\"Sec12\"><title>Chemicals and reagents</title><p id=\"Par36\">Doranidazole was obtained from POLA Pharma Inc. (Tokyo, Japan) and dissolved in phosphate-buffered saline (PBS). Metronidazole and misonidazole (Sigma-Aldrich) as well as etanidazole (Santa Cruz Biotechnology, Dallas, TX) and nimorazole (Aobious, Gloucester, MA) were dissolved in dimethyl sulfoxide (DMSO). Ferrostatin-1, DAPI, and NAC were obtained from Sigma-Aldrich, necrostatin-1 from Abcam (Cambridge, UK), Z-VAD-FMK from MBL (Nagoya, Japan), deferoxamine mesylate from Tokyo Chemical Industry (Tokyo, Japan), SKQ1 from Cayman Chemical (Ann Arbor, MI), MG132 and EF5 from Merck Millipore (Billerica, MA), and Hoechst 33342 from Thermo Fisher Scientific Inc. (Waltham, MA).</p></sec><sec id=\"Sec13\"><title>Irradiation</title><p id=\"Par37\">X-irradiation was performed with the use of an MBR-1520R-4 system (Hitachi Power Solutions, Ibaraki, Japan) at settings of 150 kV and 20 mA. The dose rate of radiation was 1.45 Gy/min.</p></sec><sec id=\"Sec14\"><title>γH2AX staining and imaging</title><p id=\"Par38\">Cells were incubated with drug under normoxic or hypoxic conditions for 30 min, exposed to 0 or 2 Gy of ionizing radiation, and fixed 30 min later with 4% paraformaldehyde. They were then stained consecutively with rabbit polyclonal antibodies to γH2AX (phospho-S139, Abcam, 1:1000 dilution) and Alexa Fluor 488-conjugated secondary antibodies (Thermo Fisher Scientific Inc.). Nuclei were counterstained with Hoechst 33342. Images were acquired with a Fluoview FV10i confocal microscope and were uniformly processed with Fluoview software v4.2 (Olympus, Tokyo, Japan). The number of immunoreactive foci was counted with the use of ImageJ software and the plugin PzFociEZ<sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>. In brief, a nuclear region of interest was generated from Hoechst 33342 staining, and the number of foci in this region was automatically counted for γH2AX staining. Background subtraction was performed with the rolling ball algorithm. The radius of the rolling ball was set at 50 pixels and Subtract Value set to 5. The number of foci was counted with the use of FindMaxima at a noise tolerance of 7. At least 50 cells were analyzed in each experiment, and three independent experiments were performed.</p></sec><sec id=\"Sec15\"><title>Hypoxia induction</title><p id=\"Par39\">For irradiation under hypoxic culture conditions, a BIONIX-3 hypoxic culture kit (Sugiyama-Gen, Tokyo, Japan) was used to obtain an O<sub>2</sub> concentration of <0.1%. Cells were irradiated 3 h after pouch sealing, and the O<sub>2</sub> concentration was monitored during the experiments. For experiments not including irradiation, cells were cultured under 1% O<sub>2</sub> with nitrogen replacement in a multigas incubator or under <0.1% O<sub>2</sub> after pouch sealing at 37 °C. Experiments were performed under normoxic conditions when not indicated otherwise.</p></sec><sec id=\"Sec16\"><title>Colony formation assay</title><p id=\"Par40\">For evaluation of the surviving fraction after irradiation, cells were seeded in 10-cm dishes. On the basis of preliminary experiments to determine the cell numbers necessary to compensate for plating efficiency, cells were plated at densities of 1 × 10<sup>3</sup>, 2 × 10<sup>3</sup>, 5 × 10<sup>3</sup>, or 1 × 10<sup>5</sup> for the 0, 2, 5, and 10 Gy treatment groups, respectively. They were allowed to settle for 2 h before treatment. Doranidazole was added 3 h before irradiation, and the cells were incubated under normoxic or hypoxic conditions. After irradiation, the medium was replaced with fresh NSM and the cells were cultured under normoxic conditions for the remainder of the experiment. Ten days after plating, the cells were fixed with 4% paraformaldehyde and stained with toluidine blue O (Sigma-Aldrich). Images of individual plates were obtained, and colonies were counted with the use of the Analyze Particles Function of ImageJ software. Three independent experiments were performed.</p></sec><sec id=\"Sec17\"><title>Sphere growth assay</title><p id=\"Par41\">GIC-H or GSC-H cells were manually plated in low-binding 96-well plates (Corning, Corning, NY) at a density of 100 cells per well and were treated with 0, 2, 4, 6, or 8 Gy. Images of the cells were acquired with a Biorevo BZ-9000 inverted microscope (Keyence, Osaka, Japan) at 10 days after plating. Sphere area was quantified with Keyence Analysis Software.</p></sec><sec id=\"Sec18\"><title>Flow cytometric analysis of cell death</title><p id=\"Par42\">Cell death was analyzed by flow cytometry with the use of PI<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>. Cells incubated with drug for 1 or 3 days were collected and exposed to PI (1 μg/ml) before flow cytometric analysis with an Attune flow cytometer (Thermo Fisher Scientific).</p></sec><sec id=\"Sec19\"><title>Visualization of cell death and hypoxic regions in spheres</title><p id=\"Par43\">For evaluation of cell death, cells were manually plated in low-binding 96-well plates (Corning) at a density of 100 cells per well. Drugs, inhibitors, and PI (0.2 μg/ml) were added after spheres had achieved diameters of ~500 μm, and the cells were then incubated for a further 24 h. Images were acquired with a Biorevo BZ-9000 inverted microscope (Keyence), and the PI-positive-area (area containing dead cells) of each sphere was quantified with Keyence Analysis Software. For evaluation of sphere hypoxia, GSC-H cells were manually plated at a density of 100 cells per well and cultured for 5 days. The resulting spheres were incubated in the presence of 200 μM EF5<sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup> for 4 h, fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 μm. The sections were stained with the use of an EF5 Hypoxia Detection Kit (Merck Millipore), and nuclei were counterstained with Hoechst 33342. U251MG and Becker cells were plated at a density of 10,000 cells per well and cultured for 2 days before evaluation of sphere hypoxia in the same manner.</p></sec><sec id=\"Sec20\"><title>Animal experiments</title><p id=\"Par44\">All animal experiments were approved by the Animal Care and Use Committee of Keio University School of Medicine. Orthotopic implantation of cells was performed as described previously<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. In brief, female C57BL/6 J mice (age 6–8 weeks) were anesthetized and placed into a stereotactic apparatus (David Kopf Instruments, Tujunga, CA). One thousand viable GSC-H cells were injected into the right hemisphere at a position 2 mm lateral to the bregma and 3 mm below the brain surface. After 10 days, animals were exposed to 15 Gy (radiation dose rate, 1.45 Gy/min) with or without prior injection of doranidazole (200 mg/kg, i.p.). Radiation was confined to the brain by protection of the body with a lead shield. Animals were monitored for the development of neurological deficits and weight loss. Brain slice explants were established as described previously<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. In brief, 1 × 10<sup>4</sup> viable GSC-F cells were injected into the right hemisphere. After 7 days, the brain was removed and cut into 200-μm-thick coronal slices with the use of a VS1200 vibratome (Leica, Wetzlar, Germany). The explants were cultured on Millicell-CM culture inserts (Merck Millipore) in glass-bottom dishes and were maintained in the presence of drug in NSM under normoxic or hypoxic conditions. Images were acquired with an FV10i confocal microscope (Olympus) and uniformly processed with the Olympus Fluoview Software v4.2b. Tumor area was contoured manually and calculated with the use of ImageJ. For evaluation of the effect of doranidazole on subcutaneous tumors, 5 × 10<sup>5</sup> viable GSC-H cells were injected subcutaneously into the flank of female C57BL/6J mice. After 15 days, doranidazole was injected i.p. at a dose of 200 mg/kg for 5 days. Control animals received vehicle only. At 20 days after cell implantation, 10 mM EF5 (volume in milliliters equal to 1/100 of body weight in grams) was injected i.p., and tumors were excised 4 h later and measured with calipers. Tumor volume was calculated<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup> as (width<sup>2</sup> × length)/2. The tumors were then fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 μm. The sections were depleted of paraffin before staining with Alexa Fluor 488-conjugated antibodies to EF5 (EF5 Hypoxia Detection Kit, Merck Millipore) at a concentration of 75 μg/ml. Nuclei were counterstained with Hoechst 33342. Images were acquired with an FV10i confocal microscope (Olympus) and processed in a uniform manner with the Olympus Fluoview Software v4.2b. EF5 staining is presented in orange. The EF5-positive area was determined as a percentage of the total tumor (Hoechst 33342-positive) area with the use of ImageJ.</p></sec><sec id=\"Sec21\"><title>Measurement of ATP production</title><p id=\"Par45\">Cells incubated with drug for 12 h were isolated to obtain a single-cell suspension in PBS, washed, and plated at a density of 5 × 10<sup>4</sup> per well in 96-well plates in the absence of drug for 30 min. Cellular ATP content was determined with the use of a CellTiter-Glo kit (Promega, Madison, WI) and microplate reader (Perkin Elmer, Waltham, MA).</p></sec><sec id=\"Sec22\"><title>Measurement of OCR</title><p id=\"Par46\">OCR was determined with the use of a Seahorse XF Extracellular Flux Analyzer (Agilent, Santa Clara, CA). In brief, dissociated cells were suspended in NSM containing drug, plated in 24-well plates (Agilent) that had been coated with Matrigel diluted 1:10 in Seahorse XF Assay Medium (Agilent), and incubated under normoxic or hypoxic conditions for 12 h. The cells were then incubated in Seahorse XF Assay Medium supplemented with 17.5 mM glucose, 2 mM pyruvate, and 2.5 mM glutamine for 1 h. For evaluation of mitochondrial function, cells were exposed sequentially to metabolic inhibitors: 1 μM oligomycin (inhibitor of ATP synthase), followed by 4 μM FCCP (uncoupler of mitochondrial oxidative phosphorylation), followed by a combination of 500 nM rotenone (inhibitor of mitochondrial complex I) and 500 nM antimycin A (inhibitor of mitochondrial complex III). Basal OCR and changes induced by the metabolic inhibitors were measured, and values were normalized by cell number at the end of the experiment.</p><p id=\"Par47\">For analysis of mitochondrial complex activity, cells were incubated in NSM with or without doranidazole, after which the medium was replaced with Mitochondria Assay Solution<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>. The plasma membrane was permeabilized to allow nonpermeable substrates to access mitochondria with the use of XF Plasma Membrane Permeabilizer (Agilent). The activity of individual complexes was assayed with combinations of specific substrates and inhibitors<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>: for assay of complex I-dependent respiration, 10 mM pyruvate and 2.5 mM malate as substrates and 2 μM rotenone as an inhibitor; for assay of complex II-dependent respiration, 10 mM succinate as substrate and 2 μM rotenone (complex I activity) followed by 10 mM malonate (complex II activity) as inhibitors; for assay of complex III-dependent respiration, 0.5 mM duroquinol as substrate and 2 μM antimycin A as inhibitor; and for assay of complex IV-dependent respiration, 0.5 mM TMPD and 2 mM ascorbate as substrates.</p></sec><sec id=\"Sec23\"><title>Immunoblot analysis</title><p id=\"Par48\">Cells were passed repeatedly through a 27-gauge needle in radioimmunoprecipitation buffer (Sigma-Aldrich) containing PhosSTOP and cOmplete Mini phosphatase and protease inhibitor cocktails (Sigma). The resulting extracts were fractionated by SDS-polyacrylamide gel electrophoresis, and the separated proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) with the use of a Trans-Blot Turbo Transfer Starter System (Bio-Rad). Immunoblot analysis was performed with the primary antibodies listed in Supplementary Information.</p></sec><sec id=\"Sec24\"><title>Measurement of mitochondrial superoxide production</title><p id=\"Par49\">Mitochondrial superoxide production was measured by flow cytometry with the use of MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fisher Scientific Inc.). Cells were incubated with drug under normoxic or hypoxic conditions for 12 h. After the addition of MitoSOX Red to a final concentration of 5 μM, the cells were incubated for an additional 10 min and then analyzed with an Attune flow cytometer (Thermo Fisher Scientific Inc.). Mean fluorescence intensity (MFI) was calculated with Attune software v2.1.0 (Thermo Fisher Scientific Inc.).</p></sec><sec id=\"Sec25\"><title>Measurement of lipid peroxidation</title><p id=\"Par50\">Lipid peroxidation was measured by flow cytometry with the use of BODIPY 581/591 C11 (Thermo Fisher Scientific). Cells were incubated with drug under normoxic or hypoxic conditions for 18 h. After the addition of BODIPY 581/591 C11 to a final concentration of 5 μM, the cells were incubated for an additional 30 min, isolated by exposure to trypsin, stained for 5 min with PI in DMEM for dead cell exclusion, and then analyzed with an Attune flow cytometer (Thermo Fisher Scientific Inc.) with excitation at 488 nm and a 530/30-nm emission filter to detect oxidized forms of the probe. MFI was calculated with Attune software v2.1.0 (Thermo Fisher Scientific Inc.).</p></sec><sec id=\"Sec26\"><title>Metabolome analysis</title><p id=\"Par51\">Cells were plated at a density of 2 × 10<sup>5</sup> per 10-cm dish and cultured for 2 days, after which the medium was replaced and the cells were incubated for 24 h with drug, washed with 10% mannitol (Wako), and exposed to ice-cold methanol containing internal standards of methionine sulfone and 2-morpholinoethanesulfonic acid. Intracellular metabolites were quantified by capillary electrophoresis–mass spectrometry with an Agilent CE system as described previously<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref>,<xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup>. A total of eight biologically distinct replicates was prepared for each group, and metabolite levels were normalized by total protein amount.</p></sec><sec id=\"Sec27\"><title>Statistics and reproducibility</title><p id=\"Par52\">Statistical analysis was performed with GraphPad Prism software. A <italic>P</italic> value of <0.05 was considered statistically significant. For animal experiments, survival curves were compared with the log-rank test. For experiments using GSCs in culture, quantitative data compare means ± s.d. obtained from the indicated number (<italic>n</italic>) of independent experiments. Within each independent experiment, biologically distinct replicates were used to generate averages for each condition as described in the figure legend. Data were compared with the paired or unpaired two-tailed Student’s <italic>t</italic>-test, by one-way ANOVA followed by Dunnett’s or Tukey’s post hoc tests or by two-way ANOVA followed by Sidak’s post hoc test, as appropriate. Markers are used to distinguish paired data in the graphs.</p></sec><sec id=\"Sec28\"><title>Reporting summary</title><p id=\"Par53\">Further information on research design is available in the Nature Research Reporting Summary linked to this article.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec29\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"42003_2020_1165_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"42003_2020_1165_MOESM2_ESM.pdf\"><caption><p>Description of Additional Supplementary Files</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM3\"><media xlink:href=\"42003_2020_1165_MOESM3_ESM.pdf\"><caption><p>Reporting Summary</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM4\"><media xlink:href=\"42003_2020_1165_MOESM4_ESM.pdf\"><caption><p>Peer Review File</p></caption></media></supplementary-material></sec></sec></body><back><app-group><app id=\"App1\"><sec id=\"Sec30\"><title>Source data</title><p id=\"Par56\"><media position=\"anchor\" xlink:href=\"42003_2020_1165_MOESM5_ESM.xlsx\" id=\"MOESM5\"><caption><p>Source Data</p></caption></media></p></sec></app></app-group><fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary information</bold> is available for this paper at 10.1038/s42003-020-01165-z.</p></sec><ack><title>Acknowledgements</title><p>We thank I. Ishimatsu for preparing the histopathology samples, M. Sato and M. Kobori for help in preparation of the paper, and Collaborative Research Resources of Keio University School of Medicine for technical assistance. Funding: This work was supported by KAKENHI Grants-in Aid for Scientific Research (C) to O.S. (nos. 16K07124 and 19K07671) and a Grant-in Aid for Early Career Scientists to N.K. (no. 18K15602) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>N.K., O.S., and H.S. designed the study. N.K. and O.S. analyzed the data and wrote the paper. N.K. acquired most of the experimental data. Y.N., N.H., T.M., and T.H. performed the metabolome analysis. N.K., R.K. and J.F. performed irradiation experiments. N.K. and N.O. constructed the bicistronic vector. M.S. was the leader of JST ERATO Suematsu Gas Biology until March 2015 who established the infrastructure for metabolomics analysis used in this study. N.S., M.S., and H.S. supervised the project.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>All source data underlying the graphs presented in the main and supplementary figures are provided in the <xref rid=\"MOESM1\" ref-type=\"media\">Supplementary Information</xref> file “Source data”. Uncropped images of the immunoblots and gating information for the flow cytometry analyses are presented in the “<xref rid=\"MOESM1\" ref-type=\"media\">Supplementary Information</xref>” file. The microarray data was deposited in the Gene Expression Omnibus (GEO) database and can be accessed under GSE135858. Other data supporting the findings of this study are available from the corresponding author upon reasonable request.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par54\">H.S. has received commercial research grants from POLA Pharma Inc. and Nihon Noyaku Co. Ltd. O.S. has received research support from Nihon Noyaku Co. Ltd. The findings of this study are the subject of a Japanese patent application as follows: Applicants: Keio University and POLA Pharma Inc. Inventors: H.S., O.S., N.K., and N. Kubota. Application no.: PCT/JP2018/036792. Status: international publication. Specific aspects of the study covered in the application: radiosensitizing effect of doranidazole on GSCs as well as its effects on mitochondrial complexes and ROS. Doranidazole used in this study was provided by POLA Pharma Inc. All other authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wilson</surname><given-names>WR</given-names></name><name><surname>Hay</surname><given-names>MP</given-names></name></person-group><article-title>Targeting hypoxia in cancer therapy</article-title><source>Nat. Rev. Cancer</source><year>2011</year><volume>11</volume><fpage>393</fpage><lpage>410</lpage><pub-id pub-id-type=\"pmid\">21606941</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brown</surname><given-names>JM</given-names></name><name><surname>Wilson</surname><given-names>WR</given-names></name></person-group><article-title>Exploiting tumour hypoxia in cancer treatment</article-title><source>Nat. Rev. Cancer</source><year>2004</year><volume>4</volume><fpage>437</fpage><lpage>447</lpage><pub-id pub-id-type=\"pmid\">15170446</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sharma</surname><given-names>R</given-names></name></person-group><article-title>Nitroimidazole radiopharmaceuticals in hypoxia: Part II cytotoxicity and radiosensitization applications</article-title><source>Curr. Radiopharm.</source><year>2011</year><volume>4</volume><fpage>379</fpage><lpage>393</lpage><pub-id pub-id-type=\"pmid\">22202160</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brown</surname><given-names>JM</given-names></name></person-group><article-title>The hypoxic cell: a target for selective cancer therapy–eighteenth Bruce F. Cain Memorial Award lecture</article-title><source>Cancer Res.</source><year>1999</year><volume>59</volume><fpage>5863</fpage><lpage>5870</lpage><pub-id pub-id-type=\"pmid\">10606224</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Overgaard</surname><given-names>J</given-names></name></person-group><article-title>Hypoxic radiosensitization: adored and ignored</article-title><source>J. Clin. Oncol.</source><year>2007</year><volume>25</volume><fpage>4066</fpage><lpage>4074</lpage><pub-id pub-id-type=\"pmid\">17827455</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wardman</surname><given-names>P</given-names></name></person-group><article-title>Chemical radiosensitizers for use in radiotherapy</article-title><source>Clin. Oncol.</source><year>2007</year><volume>19</volume><fpage>397</fpage><lpage>417</lpage></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martin Brown</surname><given-names>J</given-names></name><name><surname>Yu</surname><given-names>NY</given-names></name><name><surname>Brown</surname><given-names>DM</given-names></name><name><surname>Lee</surname><given-names>WW</given-names></name></person-group><article-title>SR-2508: A 2-nitroimidazole amide which should be superior to misonidazole as a radiosensitizer for clinical use</article-title><source>Int. J. Rad. Oncol. Biol. Phys.</source><year>1981</year><volume>7</volume><fpage>695</fpage><lpage>703</lpage></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rauth</surname><given-names>AM</given-names></name></person-group><article-title>Pharmacology and toxicology of sensitizers: mechanism studies</article-title><source>Int. J. Rad. Oncol. Biol. Phys.</source><year>1984</year><volume>10</volume><fpage>1293</fpage><lpage>1300</lpage></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kizaka-Kondoh</surname><given-names>S</given-names></name><name><surname>Konse-Nagasawa</surname><given-names>H</given-names></name></person-group><article-title>Significance of nitroimidazole compounds and hypoxia-inducible factor-1 for imaging tumor hypoxia</article-title><source>Cancer Sci.</source><year>2009</year><volume>100</volume><fpage>1366</fpage><lpage>1373</lpage><pub-id pub-id-type=\"pmid\">19459851</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rischin</surname><given-names>D</given-names></name><etal/></person-group><article-title>Prognostic significance of [18F]-misonidazole positron emission tomography-detected tumor hypoxia in patients with advanced head and neck cancer randomly assigned to chemoradiation with or without tirapazamine: a substudy of trans-Tasman radiation oncolog</article-title><source>J. Clin. Oncol.</source><year>2006</year><volume>24</volume><fpage>2098</fpage><lpage>2104</lpage><pub-id pub-id-type=\"pmid\">16648512</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Overgaard</surname><given-names>J</given-names></name><etal/></person-group><article-title>A randomized double-blind phase III study of nimorazole as a hypoxic radiosensitizer of primary radiotherapy in supraglottic larynx and pharynx carcinoma. Results of the Danish Head and Neck Cancer Study (DAHANCA) Protocol 5–85</article-title><source>Radiother. Oncol.</source><year>1998</year><volume>46</volume><fpage>135</fpage><lpage>146</lpage><pub-id pub-id-type=\"pmid\">9510041</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eschwège</surname><given-names>F</given-names></name><etal/></person-group><article-title>Results of a European randomized trial of Etanidazole combined with radiotherapy in head and neck carcinomas</article-title><source>Int. J. Rad. Oncol. Biol. Phys.</source><year>1997</year><volume>39</volume><fpage>275</fpage><lpage>281</lpage></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grigsby</surname><given-names>PW</given-names></name><etal/></person-group><article-title>Irradiation with or without misonidazole for patients with stages IIIb and IVa carcinoma of the cervix: final results of RTOG 80-05</article-title><source>Int. J. Rad. Oncol. Biol. Phys.</source><year>1999</year><volume>44</volume><fpage>513</fpage><lpage>517</lpage></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lytle</surname><given-names>NK</given-names></name><name><surname>Barber</surname><given-names>AG</given-names></name><name><surname>Reya</surname><given-names>T</given-names></name></person-group><article-title>Stem cell fate in cancer growth, progression and therapy resistance</article-title><source>Nat. Rev. Cancer</source><year>2018</year><volume>18</volume><fpage>669</fpage><lpage>680</lpage><pub-id pub-id-type=\"pmid\">30228301</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oya</surname><given-names>N</given-names></name><etal/></person-group><article-title>Optical isomers of a new 2-nitroimidazole nucleoside analog (PR-350 series): radiosensitization efficiency and toxicity</article-title><source>Int. J. Rad. Oncol. Biol. Phys.</source><year>1995</year><volume>33</volume><fpage>119</fpage><lpage>127</lpage></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuwabara</surname><given-names>M</given-names></name><etal/></person-group><article-title>Radiation-chemical properties of the hypoxic cell radiosensitizer doranidazole (PR-350)</article-title><source>J. Radiat. Res.</source><year>2002</year><volume>43</volume><fpage>77</fpage><lpage>77</lpage><pub-id pub-id-type=\"pmid\">12056332</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yasui</surname><given-names>H</given-names></name><etal/></person-group><article-title>The prospective application of a hypoxic radiosensitizer, doranidazole to rat intracranial glioblastoma with blood–brain barrier disruption</article-title><source>BMC Cancer</source><year>2013</year><volume>13</volume><fpage>106</fpage><pub-id pub-id-type=\"pmid\">23496909</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yahiro</surname><given-names>T</given-names></name><etal/></person-group><article-title>Effects of hypoxic cell radiosensitizer doranidazole (PR-350) on the radioresponse of murine and human tumor cells in vitro and in vivo</article-title><source>J. Radiat. Res.</source><year>2005</year><volume>46</volume><fpage>363</fpage><lpage>372</lpage><pub-id pub-id-type=\"pmid\">16210793</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Murata</surname><given-names>R</given-names></name><name><surname>Tsujitani</surname><given-names>M</given-names></name><name><surname>Horsman</surname><given-names>MR</given-names></name></person-group><article-title>Enhanced local tumour control after single or fractionated radiation treatment using the hypoxic cell radiosensitizer doranidazole</article-title><source>Radiother. Oncol.</source><year>2008</year><volume>87</volume><fpage>331</fpage><lpage>338</lpage><pub-id pub-id-type=\"pmid\">18375007</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Karasawa</surname><given-names>K</given-names></name><etal/></person-group><article-title>Efficacy of novel hypoxic cell sensitiser doranidazole in the treatment of locally advanced pancreatic cancer: long-term results of a placebo-controlled randomised study</article-title><source>Radiother. Oncol.</source><year>2008</year><volume>87</volume><fpage>326</fpage><lpage>330</lpage><pub-id pub-id-type=\"pmid\">18342968</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nishimura</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Phase I/II Trial of sequential chemoradiotherapy using a novel hypoxic cell radiosensitizer, doranidazole (PR-350), in patients with locally advanced non-small-cell lung cancer (WJTOG-0002)</article-title><source>Int. J. Rad. Oncol. Biol. Phys.</source><year>2007</year><volume>69</volume><fpage>786</fpage><lpage>792</lpage></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Colwell</surname><given-names>N</given-names></name><etal/></person-group><article-title>Hypoxia in the glioblastoma microenvironment: shaping the phenotype of cancer stem-like cells</article-title><source>Neuro-Oncol.</source><year>2017</year><volume>19</volume><fpage>887</fpage><lpage>896</lpage><pub-id pub-id-type=\"pmid\">28339582</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sampetrean</surname><given-names>O</given-names></name><etal/></person-group><article-title>Invasion precedes tumor mass formation in a malignant brain tumor model of genetically modified neural stem cells</article-title><source>Neoplasia</source><year>2011</year><volume>13</volume><fpage>784</fpage><lpage>IN3</lpage><pub-id pub-id-type=\"pmid\">21969812</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Evans</surname><given-names>SM</given-names></name></person-group><article-title>Hypoxia is important in the biology and aggression of human glial brain tumors</article-title><source>Clin. Cancer Res.</source><year>2004</year><volume>10</volume><fpage>8177</fpage><lpage>8184</lpage><pub-id pub-id-type=\"pmid\">15623592</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bar</surname><given-names>EE</given-names></name><name><surname>Lin</surname><given-names>A</given-names></name><name><surname>Mahairaki</surname><given-names>V</given-names></name><name><surname>Matsui</surname><given-names>W</given-names></name><name><surname>Eberhart</surname><given-names>CG</given-names></name></person-group><article-title>Hypoxia increases the expression of stem-cell markers and promotes clonogenicity in glioblastoma neurospheres</article-title><source>Am. J. Pathol.</source><year>2010</year><volume>177</volume><fpage>1491</fpage><lpage>1502</lpage><pub-id pub-id-type=\"pmid\">20671264</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bar</surname><given-names>EE</given-names></name></person-group><article-title>Glioblastoma, cancer stem cells and hypoxia: hypoxia regulates glioma stem cells</article-title><source>Brain Pathol.</source><year>2011</year><volume>21</volume><fpage>119</fpage><lpage>129</lpage><pub-id pub-id-type=\"pmid\">21054626</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sakaue-Sawano</surname><given-names>A</given-names></name><etal/></person-group><article-title>Visualizing spatiotemporal dynamics of multicellular cell-cycle progression</article-title><source>Cell</source><year>2008</year><volume>132</volume><fpage>487</fpage><lpage>498</lpage><pub-id pub-id-type=\"pmid\">18267078</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gao</surname><given-names>M</given-names></name><name><surname>Jiang</surname><given-names>X</given-names></name></person-group><article-title>To eat or not to eat—the metabolic flavor of ferroptosis</article-title><source>Curr. Opin. Cell Biol.</source><year>2018</year><volume>51</volume><fpage>58</fpage><lpage>64</lpage><pub-id pub-id-type=\"pmid\">29175614</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Leitsch</surname><given-names>D</given-names></name><name><surname>Kolarich</surname><given-names>D</given-names></name><name><surname>Wilson</surname><given-names>IBH</given-names></name><name><surname>Altmann</surname><given-names>F</given-names></name><name><surname>Duchêne</surname><given-names>M</given-names></name></person-group><article-title>Nitroimidazole action in Entamoeba histolytica: a central role for thioredoxin reductase</article-title><source>PLoS Biol.</source><year>2007</year><volume>5</volume><fpage>e211</fpage><pub-id pub-id-type=\"pmid\">17676992</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bogdan</surname><given-names>AR</given-names></name><name><surname>Miyazawa</surname><given-names>M</given-names></name><name><surname>Hashimoto</surname><given-names>K</given-names></name><name><surname>Tsuji</surname><given-names>Y</given-names></name></person-group><article-title>Regulators of iron homeostasis: new players in metabolism, cell death, and disease</article-title><source>Trends Biochem. Sci.</source><year>2016</year><volume>41</volume><fpage>274</fpage><lpage>286</lpage><pub-id pub-id-type=\"pmid\">26725301</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kwon</surname><given-names>M-Y</given-names></name><name><surname>Park</surname><given-names>E</given-names></name><name><surname>Lee</surname><given-names>S-J</given-names></name><name><surname>Chung</surname><given-names>SW</given-names></name></person-group><article-title>Heme oxygenase-1 accelerates erastin-induced ferroptotic cell death</article-title><source>Oncotarget</source><year>2015</year><volume>6</volume><fpage>24393</fpage><lpage>24403</lpage><pub-id pub-id-type=\"pmid\">26405158</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Skulachev</surname><given-names>VP</given-names></name><etal/></person-group><article-title>An attempt to prevent senescence: a mitochondrial approach</article-title><source>Biochim. Biophys. Acta</source><year>2009</year><volume>1787</volume><fpage>437</fpage><lpage>461</lpage><pub-id pub-id-type=\"pmid\">19159610</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Skulachev</surname><given-names>VP</given-names></name><etal/></person-group><article-title>Prevention of cardiolipin oxidation and fatty acid cycling as two antioxidant mechanisms of cationic derivatives of plastoquinone (SkQs)</article-title><source>Biochim. Biophys. Acta</source><year>2010</year><volume>1797</volume><fpage>878</fpage><lpage>889</lpage><pub-id pub-id-type=\"pmid\">20307489</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Murphy</surname><given-names>MP</given-names></name></person-group><article-title>How mitochondria produce reactive oxygen species</article-title><source>Biochem. J.</source><year>2009</year><volume>417</volume><fpage>1</fpage><lpage>13</lpage><pub-id pub-id-type=\"pmid\">19061483</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Minami</surname><given-names>N</given-names></name><etal/></person-group><article-title>Organotypic brain explant culture as a drug evaluation system for malignant brain tumors</article-title><source>Cancer Med.</source><year>2017</year><volume>6</volume><fpage>2635</fpage><lpage>2645</lpage><pub-id pub-id-type=\"pmid\">28980419</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Adams</surname><given-names>GE</given-names></name></person-group><article-title>Hypoxia-mediated drugs for radiation and chemotherapy</article-title><source>Cancer</source><year>1981</year><volume>48</volume><fpage>696</fpage><lpage>707</lpage><pub-id pub-id-type=\"pmid\">7018662</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bao</surname><given-names>S</given-names></name><etal/></person-group><article-title>Glioma stem cells promote radioresistance by preferential activation of the DNA damage response</article-title><source>Nature</source><year>2006</year><volume>444</volume><fpage>756</fpage><lpage>760</lpage><pub-id pub-id-type=\"pmid\">17051156</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Blanpain</surname><given-names>C</given-names></name><name><surname>Mohrin</surname><given-names>M</given-names></name><name><surname>Sotiropoulou</surname><given-names>PA</given-names></name><name><surname>Passegué</surname><given-names>E</given-names></name></person-group><article-title>DNA-damage response in tissue-specific and cancer stem cells</article-title><source>Cell Stem Cell</source><year>2011</year><volume>8</volume><fpage>16</fpage><lpage>29</lpage><pub-id pub-id-type=\"pmid\">21211780</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Urtasun</surname><given-names>RC</given-names></name><name><surname>Coleman</surname><given-names>CN</given-names></name><name><surname>Wasserman</surname><given-names>TH</given-names></name><name><surname>Phillips</surname><given-names>TL</given-names></name></person-group><article-title>Clinical trials with hypoxic cell sensitizers: time to retrench or time to push forward?</article-title><source>Int. J. Rad. Oncol. Biol. Phys.</source><year>1984</year><volume>10</volume><fpage>1691</fpage><lpage>1696</lpage></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fulton</surname><given-names>DS</given-names></name><etal/></person-group><article-title>Misonidazole combined with hyperfractionation in the management of malignant glioma</article-title><source>Int. J. Rad. Oncol. Biol. Phys.</source><year>1984</year><volume>10</volume><fpage>1709</fpage><lpage>1712</lpage></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Deutsch</surname><given-names>M</given-names></name><etal/></person-group><article-title>Results of a randomized trial comparing BCNU plus radiotherapy, streptozotocin plus radiotherapy, BCNU plus hyperfractionated radiotherapy, and BCNU following misonidazole plus radiotherapy in the postoperative treatment of malignant glioma</article-title><source>Int. J. Rad. Oncol. Biol. Phys.</source><year>1989</year><volume>16</volume><fpage>1389</fpage><lpage>1396</lpage></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dixon</surname><given-names>SJ</given-names></name><etal/></person-group><article-title>Ferroptosis: an iron-dependent form of nonapoptotic cell death</article-title><source>Cell</source><year>2012</year><volume>149</volume><fpage>1060</fpage><lpage>1072</lpage><pub-id pub-id-type=\"pmid\">22632970</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gao</surname><given-names>M</given-names></name><name><surname>Monian</surname><given-names>P</given-names></name><name><surname>Quadri</surname><given-names>N</given-names></name><name><surname>Ramasamy</surname><given-names>R</given-names></name><name><surname>Jiang</surname><given-names>X</given-names></name></person-group><article-title>Glutaminolysis and transferrin regulate ferroptosis</article-title><source>Mol. Cell</source><year>2015</year><volume>59</volume><fpage>298</fpage><lpage>308</lpage><pub-id pub-id-type=\"pmid\">26166707</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cao</surname><given-names>JY</given-names></name><name><surname>Dixon</surname><given-names>SJ</given-names></name></person-group><article-title>Mechanisms of ferroptosis</article-title><source>Cell. Mol. Life Sci.</source><year>2016</year><volume>73</volume><fpage>2195</fpage><lpage>2209</lpage><pub-id pub-id-type=\"pmid\">27048822</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lin</surname><given-names>S</given-names></name><name><surname>Page</surname><given-names>NA</given-names></name><name><surname>Fung</surname><given-names>SM</given-names></name><name><surname>Fung</surname><given-names>H-L</given-names></name></person-group><article-title>In vitro organic nitrate bioactivation to nitric oxide by recombinant aldehyde dehydrogenase 3A1</article-title><source>Nitric Oxide</source><year>2013</year><volume>35</volume><fpage>137</fpage><lpage>143</lpage><pub-id pub-id-type=\"pmid\">24126018</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Biaglow</surname><given-names>JE</given-names></name><etal/></person-group><article-title>Biochemistry of reduction of nitro heterocycles</article-title><source>Biochem. Pharm.</source><year>1986</year><volume>35</volume><fpage>77</fpage><lpage>70</lpage><pub-id pub-id-type=\"pmid\">2934068</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><mixed-citation publication-type=\"other\">Wardman, P. Nitroimidazoles as hypoxic cell radiosensitizers and hypoxia probes: misonidazole, myths and mistakes. <italic>Br. J. Radiol.</italic> 20170915 10.1259/bjr.20170915 (2018).</mixed-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Okazaki</surname><given-names>S</given-names></name><etal/></person-group><article-title>Synthetic lethality of the ALDH3A1 inhibitor dyclonine and xCT inhibitors in glutathione deficiency-resistant cancer cells</article-title><source>Oncotarget</source><year>2018</year><volume>9</volume><fpage>33832</fpage><lpage>33843</lpage><pub-id pub-id-type=\"pmid\">30333913</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><mixed-citation publication-type=\"other\">Otsuki, Y. et al. Vasodilator oxyfedrine inhibits aldehyde metabolism and thereby sensitizes cancer cells to xCT‐targeted therapy. <italic>Cancer Sci.</italic> 14224, 10.1111/cas.14224 (2019).</mixed-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bonnet</surname><given-names>M</given-names></name><etal/></person-group><article-title>Next-generation hypoxic cell radiosensitizers: nitroimidazole alkylsulfonamides</article-title><source>J. Med. Chem.</source><year>2018</year><volume>61</volume><fpage>1241</fpage><lpage>1254</lpage><pub-id pub-id-type=\"pmid\">29253343</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shiota</surname><given-names>M</given-names></name><etal/></person-group><article-title>Gold-nanofève surface-enhanced Raman spectroscopy visualizes hypotaurine as a robust anti-oxidant consumed in cancer survival</article-title><source>Nat. Commun.</source><year>2018</year><volume>9</volume><fpage>1561</fpage><pub-id pub-id-type=\"pmid\">29674746</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hassannia</surname><given-names>B</given-names></name><name><surname>Vandenabeele</surname><given-names>P</given-names></name><name><surname>Vanden Berghe</surname><given-names>T</given-names></name></person-group><article-title>Targeting ferroptosis to iron out cancer</article-title><source>Cancer Cell</source><year>2019</year><volume>35</volume><fpage>830</fpage><lpage>849</lpage><pub-id pub-id-type=\"pmid\">31105042</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bou-Abdallah</surname><given-names>F</given-names></name><name><surname>Paliakkara</surname><given-names>J</given-names></name><name><surname>Melman</surname><given-names>G</given-names></name><name><surname>Melman</surname><given-names>A</given-names></name></person-group><article-title>Reductive mobilization of iron from intact ferritin: Mechanisms and physiological implication</article-title><source>Pharmaceuticals</source><year>2018</year><volume>11</volume><fpage>120</fpage></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shibao</surname><given-names>S</given-names></name><etal/></person-group><article-title>Metabolic heterogeneity and plasticity of glioma stem cells in a mouse glioblastoma model</article-title><source>Neuro-Oncol.</source><year>2018</year><volume>20</volume><fpage>343</fpage><lpage>354</lpage><pub-id pub-id-type=\"pmid\">29016888</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Znojek</surname><given-names>P</given-names></name><name><surname>Willmore</surname><given-names>E</given-names></name><name><surname>Curtin</surname><given-names>NJ</given-names></name></person-group><article-title>Preferential potentiation of topoisomerase I poison cytotoxicity by PARP inhibition in S phase</article-title><source>Br. J. Cancer</source><year>2014</year><volume>111</volume><fpage>1319</fpage><lpage>1326</lpage><pub-id pub-id-type=\"pmid\">25003660</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Crowley</surname><given-names>LC</given-names></name><etal/></person-group><article-title>Measuring cell death by propidium iodide uptake and flow cytometry</article-title><source>Cold Spring Harb. Prot.</source><year>2016</year><volume>2016</volume><fpage>647</fpage><lpage>651</lpage></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ashton</surname><given-names>TM</given-names></name><etal/></person-group><article-title>The anti-malarial atovaquone increases radiosensitivity by alleviating tumour hypoxia</article-title><source>Nat. Commun.</source><year>2016</year><volume>7</volume><fpage>1</fpage><lpage>13</lpage></element-citation></ref><ref id=\"CR58\"><label>58.</label><mixed-citation publication-type=\"other\">Faustino-Rocha, A. et al. Estimation of rat mammary tumor volume using caliper and ultrasonography measurements. <italic>Lab Anim.</italic> 10.1038/laban.254 (2013).</mixed-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Salabei</surname><given-names>JK</given-names></name><name><surname>Gibb</surname><given-names>AA</given-names></name><name><surname>Hill</surname><given-names>BG</given-names></name></person-group><article-title>Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis</article-title><source>Nat. Protoc.</source><year>2014</year><volume>9</volume><fpage>421</fpage><lpage>438</lpage><pub-id pub-id-type=\"pmid\">24457333</pub-id></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ohmura</surname><given-names>M</given-names></name><etal/></person-group><article-title>Impacts of CD44 knockdown in cancer cells on tumor and host metabolic systems revealed by quantitative imaging mass spectrometry</article-title><source>Nitric Oxide</source><year>2015</year><volume>46</volume><fpage>102</fpage><lpage>113</lpage><pub-id pub-id-type=\"pmid\">25461272</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807830</article-id><article-id pub-id-type=\"pmc\">PMC7431528</article-id><article-id pub-id-type=\"publisher-id\">70914</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70914-1</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Clinical features of irreversible rejection after allogeneic uterus transplantation in cynomolgus macaques</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Kisu</surname><given-names>Iori</given-names></name><address><email>iori71march@a7.keio.jp</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Emoto</surname><given-names>Katsura</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Masugi</surname><given-names>Yohei</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yamada</surname><given-names>Yohei</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Matsubara</surname><given-names>Kentaro</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Obara</surname><given-names>Hideaki</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Matoba</surname><given-names>Yusuke</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Banno</surname><given-names>Kouji</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kato</surname><given-names>Yojiro</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>Saiki</surname><given-names>Yoko</given-names></name><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Itagaki</surname><given-names>Iori</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref><xref ref-type=\"aff\" rid=\"Aff8\">8</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kawamoto</surname><given-names>Ikuo</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><name><surname>Iwatani</surname><given-names>Chizuru</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><name><surname>Murase</surname><given-names>Mitsuru</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><name><surname>Nakagawa</surname><given-names>Takahiro</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><name><surname>Tsuchiya</surname><given-names>Hideaki</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ishigaki</surname><given-names>Hirohito</given-names></name><xref ref-type=\"aff\" rid=\"Aff9\">9</xref></contrib><contrib contrib-type=\"author\"><name><surname>Urano</surname><given-names>Hiroyuki</given-names></name><xref ref-type=\"aff\" rid=\"Aff10\">10</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ema</surname><given-names>Masatsugu</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ogasawara</surname><given-names>Kazumasa</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref><xref ref-type=\"aff\" rid=\"Aff9\">9</xref></contrib><contrib contrib-type=\"author\"><name><surname>Aoki</surname><given-names>Daisuke</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Nakagawa</surname><given-names>Kenshi</given-names></name><xref ref-type=\"aff\" rid=\"Aff10\">10</xref></contrib><contrib contrib-type=\"author\"><name><surname>Shiina</surname><given-names>Takashi</given-names></name><xref ref-type=\"aff\" rid=\"Aff11\">11</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.26091.3c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9959</institution-id><institution>Department of Obstetrics and Gynecology, </institution><institution>Keio University School of Medicine, </institution></institution-wrap>35 Shinanomachi , Shinjuku-ku, Tokyo 1608582 Japan </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.26091.3c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9959</institution-id><institution>Department of Pathology, </institution><institution>Keio University School of Medicine, </institution></institution-wrap>Shinjuku, Tokyo 1608582 Japan </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.26091.3c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9959</institution-id><institution>Department of Pediatric Surgery, </institution><institution>Keio University School of Medicine, </institution></institution-wrap>Shinjuku, Tokyo 1608582 Japan </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.26091.3c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9959</institution-id><institution>Department of Surgery, </institution><institution>Keio University School of Medicine, </institution></institution-wrap>Shinjuku, Tokyo 1608582 Japan </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.410714.7</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 8864 3422</institution-id><institution>Department of Surgery, Division of Gastroenterological and General Surgery, School of Medicine, </institution><institution>Showa University, </institution></institution-wrap>Shinagawa, Tokyo 1428666 Japan </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.416609.c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0642 4752</institution-id><institution>Department of Anesthesiology, </institution><institution>Saiseikai Kanagawaken Hospital, </institution></institution-wrap>Yokohama, Kanagawa 2210821 Japan </aff><aff id=\"Aff7\"><label>7</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.410827.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9747 6806</institution-id><institution>Research Center for Animal Life Science, </institution><institution>Shiga University of Medical Science, </institution></institution-wrap>Ōtsu, Shiga 5202192 Japan </aff><aff id=\"Aff8\"><label>8</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.417584.b</institution-id><institution>The Corporation for Production and Research of Laboratory Primates, </institution></institution-wrap>Tsukuba, Ibaraki 3050003 Japan </aff><aff id=\"Aff9\"><label>9</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.410827.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9747 6806</institution-id><institution>Department of Pathology, </institution><institution>Shiga University of Medical Science, </institution></institution-wrap>Ōtsu, Shiga 5202192 Japan </aff><aff id=\"Aff10\"><label>10</label>Safety Research Center, Ina Research Inc., Ina, Nagano 3994501 Japan </aff><aff id=\"Aff11\"><label>11</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.265061.6</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 1516 6626</institution-id><institution>Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, </institution><institution>Tokai University School of Medicine, </institution></institution-wrap>Hiratsuka, Kanagawa 2591193 Japan </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13910</elocation-id><history><date date-type=\"received\"><day>11</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>22</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Uterus transplantation (UTx) is a potential option for women with uterine factor infertility to have a child. The clinical features indicating irreversible rejection of the uterus are unknown. In our experimental series of allogeneic UTx in cynomolgus macaques, six female macaques were retrospectively examined, which were unresponsive to treatment with immunosuppressants (i.e. irreversible rejection). Clinical features including general condition, hematology, uterine size, indocyanine green (ICG) fluorescence imaging by laparotomy, and histopathological findings of the removed uterus were evaluated. In all cases, general condition was good at the time of diagnosis of irreversible rejection and thereafter. Laboratory evaluation showed temporary increases in white blood cells, lactate dehydrogenase and C-reactive protein, then these levels tended to decrease gradually. In transabdominal ultrasonography, the uterus showed time-dependent shrinkage after transient swelling at the time of diagnosis of irreversible rejection. In laparotomy, a whitish transplanted uterus was observed and enhancement of the transplanted uterus was absent in ICG fluorescence imaging. Histopathological findings in each removed uterus showed hyalinized fibrosis, endometrial deficit, lymphocytic infiltration and vasculitis. These findings suggest that uterine transplantation rejection is not fatal, in contrast to rejection of life-supporting organs. Since the transplanted uterus with irreversible rejection atrophies naturally, hysterectomy may be unnecessary.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Experimental models of disease</kwd><kwd>Reproductive disorders</kwd><kwd>Infertility</kwd></kwd-group><funding-group><award-group><funding-source><institution>the Adaptable and Seamless Technology Transfer Program through Target-Driven Research and Development (A-STEP), AMED</institution></funding-source><award-id>AS2525033N</award-id><principal-award-recipient><name><surname>Kisu</surname><given-names>Iori</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>the Japan Society for the Promotion of Science (JSPS) KAKENHI</institution></funding-source><award-id>17H05099</award-id><principal-award-recipient><name><surname>Kisu</surname><given-names>Iori</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Uterus transplantation (UTx) has become a potential option for women with uterine factor infertility to have a child, following the first successful delivery by Brännström et al. in Sweden in 2014<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. This major achievement attracted considerable attention worldwide, and many centers have recently begun clinical application of UTx, leading to more than 60 procedures and 18 live births<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. Despite this success, UTx is still in an experimental stage and there are many clinical and technical issues to be resolved for its full clinical establishment<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. In particular, rejection remains as a concern in this procedure. There have been several reported cases of rejection after UTx in humans<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>–<xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup> and pathological criteria for uterine rejection have been proposed by the group in Sweden<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. All of these cases overcame rejection with subsequent immunosuppressive therapy and some achieved successful live birth after treatment of the rejection<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>.\n</p><p id=\"Par3\">Organ elimination due to rejection after transplantation of a life-supporting organ generally results in a fatal outcome. However, UTx is not a vital organ transplantation, and organ elimination due to rejection might not lead to life-threatening conditions. However, there are no reports of a uterus that became non-functional or was removed due to irreversible rejection that was resistant to treatment, and the clinical features indicating irreversible rejection of the uterus are unclear.</p><p id=\"Par4\">Our team in Japan launched UTx research in 2009 using approximately 100 cynomolgus macaques, which are anatomically and physiologically similar to humans. Since then, we have accumulated a large archive of results, including examination of uterine blood flow, surgical procedures for autologous and allogeneic UTx, organ perfusion methods in deceased donor models, immunological response and rejection, and ischemia/reperfusion injury<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. In this experimental series, we had cases of hysterectomy after allogeneic UTx due to diagnosis of irreversible rejection that was resistant to immunosuppressants.</p><p id=\"Par5\">These cases arose from difficulty and limitations in postoperative managements and monitoring in cynomolgus macaques<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>, which are likely to develop rejection and have high antigenicity, in contrast to humans<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. These events may be of value in understanding rejection in UTx because similar events have not been observed in humans. Therefore, in this study, we retrospectively examined the clinical features associated with irreversible rejection after allogeneic UTx in cynomolgus macaques.</p></sec><sec id=\"Sec2\"><title>Results</title><sec id=\"Sec3\"><title>Irreversible rejection</title><p id=\"Par6\">In this study, irreversible rejection was defined as progressive rejection in spite of the following treatments. Steroid pulse therapy was administered in all cases and ATG in cases 4 and 5, but the rejections were unresponsive and not overcome by these treatments. Moreover, necrotic change in uterine tissues was often observed histologically. The cases were diagnosed as irreversible rejection on postoperative day (POD) 11, 35, 35, 41, 67 and 206 (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>).<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Summary of the timing of irreversible rejection in cases 1–6.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Case</th><th align=\"left\">POD of irreversible rejection</th><th align=\"left\">Treatment for rejection</th><th align=\"left\">POD of autopsy</th></tr></thead><tbody><tr><td align=\"left\">1</td><td char=\".\" align=\"char\">11</td><td align=\"left\">Steroid pulse</td><td char=\".\" align=\"char\">85</td></tr><tr><td align=\"left\">2</td><td char=\".\" align=\"char\">35</td><td align=\"left\">Steroid pulse</td><td char=\".\" align=\"char\">196</td></tr><tr><td align=\"left\">3</td><td char=\".\" align=\"char\">35</td><td align=\"left\">Steroid pulse</td><td char=\".\" align=\"char\">126</td></tr><tr><td align=\"left\">4</td><td char=\".\" align=\"char\">41</td><td align=\"left\">Steroid pulse + ATG</td><td char=\".\" align=\"char\">104</td></tr><tr><td align=\"left\">5</td><td char=\".\" align=\"char\">67</td><td align=\"left\">Steroid pulse + ATG</td><td char=\".\" align=\"char\">103</td></tr><tr><td align=\"left\">6</td><td char=\".\" align=\"char\">206</td><td align=\"left\">Steroid pulse</td><td char=\".\" align=\"char\">265</td></tr></tbody></table><table-wrap-foot><p><italic>POD</italic> postoperative day, <italic>ATG</italic> antithymocyte globulin.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec4\"><title>Well-being of the animals</title><p id=\"Par7\">In all cases, general condition of the animals (activity, appetite, bowel movement, vomiting, urination) was good at the time of diagnosis of irreversible rejection and thereafter. No animals did not show body weight loss greater than 15%. In cases 2, 5 and 6, continuous vaginal bleeding was observed before and/or after the day of irreversible rejection.</p></sec><sec id=\"Sec5\"><title>Laboratory evaluation</title><p id=\"Par8\">Hematology and blood chemistry in all animals showed temporary increases in white blood cells (WBC), lactate dehydrogenase (LDH), and C-reactive protein (CRP) before and after rejection was diagnosed (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). Thereafter, these levels tended to decrease gradually. There were no marked changes in electrolytes or in liver and renal functions.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Changes in WBC, LDH and CRP before and after irreversible rejection. WBC, LDH and CRP temporarily increased before and after diagnosis of irreversible rejection, and then tended to decrease gradually (day 0 = day of diagnosis of irreversible rejection).</p></caption><graphic xlink:href=\"41598_2020_70914_Fig1_HTML\" id=\"MO1\"/></fig></p></sec><sec id=\"Sec6\"><title>Assessment of the transplanted uterus by ultrasonography</title><p id=\"Par9\">In transabdominal ultrasonography, the uterus was swollen at the time of diagnosis of irreversible rejection (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). The uterus then showed time-dependent shrinkage (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>), which made it difficult to identify blood flow in the uterine artery on duplex Doppler ultrasonography. The endometrium of the uterus was not detected in all cases.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Transabdominal ultrasonography of the long axis of the uterine body in case 5. (<bold>A</bold>) The uterine body (28.4 × 17.6 mm: long axis × anteroposterior diameter) after surgery. (<bold>B</bold>) An enlarged uterine body (60.0 × 28.1 mm: long axis × anteroposterior diameter) without an endometrium was found at the time of diagnosis of irreversible rejection.</p></caption><graphic xlink:href=\"41598_2020_70914_Fig2_HTML\" id=\"MO2\"/></fig><fig id=\"Fig3\"><label>Figure 3</label><caption><p>Changes in the long axis diameter of the uterus before and after irreversible rejection. The uterus was swollen at the time of diagnosis of irreversible rejection, and then showed time-dependent shrinkage (day 0 = day of diagnosis of irreversible rejection).</p></caption><graphic xlink:href=\"41598_2020_70914_Fig3_HTML\" id=\"MO3\"/></fig></p></sec><sec id=\"Sec7\"><title>Gross findings and ICG fluorescence imaging in laparotomy</title><p id=\"Par10\">Autopsy in cases 1–6 was performed on POD 85, 196, 126, 104, 103 and 265, respectively, because of irreversible rejection that was resistant to additional immunosuppressant therapy. Laparotomy showed a white shrinking uterus that was highly adhesive with surrounding tissues, especially to the bladder, the greater omentum and rectum (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>A), in cases other than cases 4 and 5. In cases 4 and 5, a whitish swollen transplanted uterus was observed (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>B). Moreover, the uteri of all cases were hard and sclerotic by gross diagnosis. In ICG fluorescence imaging, enhancement of the transplanted uterus was absent in all cases (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>).<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Macroscopic findings in the pelvis at autopsy in case 3 (<bold>A</bold>) and case 4 (<bold>B</bold>). (<bold>A</bold>) A whitish atrophic uterus (yellow triangles) adhered to the omentum and bladder. (<bold>B</bold>) A swollen uterus that was highly adhesive with surrounding tissues, especially to the omentum and rectum.</p></caption><graphic xlink:href=\"41598_2020_70914_Fig4_HTML\" id=\"MO4\"/></fig><fig id=\"Fig5\"><label>Figure 5</label><caption><p>ICG fluorescence imaging of the transplanted uterus at autopsy in case 4. (<bold>A</bold>) A markedly swollen uterus (yellow triangles). (<bold>B</bold>) Enhancement of the grafted uterus (yellow triangles) was absent in ICG fluorescence imaging.</p></caption><graphic xlink:href=\"41598_2020_70914_Fig5_HTML\" id=\"MO5\"/></fig></p></sec><sec id=\"Sec8\"><title>Pathological findings of the removed uterus</title><p id=\"Par11\">Histologically, compared to the normal uterus (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>A–C), uteri of the six cases showed no endometrium and myometrium which were replaced by sclerotic fibrosis (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>D–F) or coagulative necrosis (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>G–I), which suggested of end stage of the rejection. All cases demonstrated perivascular lymphocytic inflammation, and some cases showed vascular occlusion and/or fibrin thrombi. Inflammation was more severe in perimetrium than in endometrium and myometrium. These histologic findings, loss of endometrial and myometrial structure, indicated that these uteri were no longer functional.<fig id=\"Fig6\"><label>Figure 6</label><caption><p>Histopathological findings of normal uterus in a cynomolgus macaque (<bold>A</bold>–<bold>C</bold>) and the removed uterus at autopsy in case 2 (<bold>D</bold>–<bold>F</bold>) and case 4 (<bold>G</bold>–<bold>I</bold>). (<bold>A</bold>) Normal uterine corpus is composed of endometrium, myometrium and perimetrium. (<bold>B</bold>) High power field of endometrium. (<bold>C</bold>) High power field of myometrium. (<bold>D</bold>) The uterine corpus of case 2 demonstrated fibrous change of the whole wall and inflammation in the perimetrium. (<bold>E</bold>) Endometrium was not seen and replaced by sclerotic fibrosis. (<bold>F</bold>) No smooth muscle cells were observed in the myometrium which was also replaced by sclerotic fibrosis. Mild perivascular inflammation was observed. (<bold>G</bold>) The uterine corpus of case 4 demonstrated necrotic change of the whole wall and inflammation in the perimetrium. (<bold>H</bold>) No endometrium but degenerated fibrotic tissue was seen. (<bold>I</bold>) Myometrium showed coagulative necrosis and vascular occlusion (yellow triangles) which was highlighted by Elastica van Gieason stain in the inset. H&E stain (<bold>A</bold>–<bold>I</bold>). Bar = 4 mm (<bold>A</bold>, <bold>D</bold>), 200 µm (<bold>B</bold>, <bold>C</bold>, <bold>E</bold>, <bold>F</bold>, <bold>H</bold>, <bold>I</bold>), and 6 mm (<bold>G</bold>).</p></caption><graphic xlink:href=\"41598_2020_70914_Fig6_HTML\" id=\"MO6\"/></fig></p></sec></sec><sec id=\"Sec9\"><title>Discussion</title><p id=\"Par12\">An important goal of UTx is improving quality of life (QOL) for women with uterine factor infertility by allowing these women to have a child, in contrast to transplantation of life-supporting organs. UTx has provided great hope to couples with no children due to uterine factor infertility. However, various risks are involved in organ transplantation, and rejection is a particular concern. The antigenicity of a transplanted organ depends on the organ type and is unclear for the uterus. A pregnant uterus allows a non-self organism to develop within, and thus may be presumed to maintain immune tolerance. On the other hand, organs such as skin, small intestine and lungs that are in contact with the external environment have well-developed immune mechanisms that are likely to cause rejection. The uterus is similarly in contact with the external environment via the vagina, and this may explain the strong potential for rejection of a transplanted uterus.</p><p id=\"Par13\">Some cases of human UTx have resulted in uterine rejection<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>–<xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>, but none have required hysterectomy due to refractory rejection. Organ elimination due to rejection after transplantation of a life-supporting organ generally results in a fatal outcome. Since UTx is not life-supporting, but focused on improving QOL, particular attention should be paid to the safety of the healthy recipient. The clinical features of irreversible rejection after UTx are unknown, including the potential mortality that occurs in other organ transplantations, and preparation for unexpected events after UTx is important. Thus, in this study we examined six cynomolgus macaques in which irreversible rejection occurred in our experimental series of allogeneic UTx to clarify the clinical features of irreversible rejection. The main features found were temporary increases in WBC, LDH and CRP before and after irreversible rejection, a swollen uterus at the time of irreversible rejection that then shrinks over time, and a good general condition. In laparotomy, the uterus without uterine blood flow was highly adhesive with surrounding tissues. Pathological findings of the uterus showed hyalinization in the interstitium in all uterine layers, with an endometrial deficit.</p><p id=\"Par14\">The increases in WBC and CRP imply an inflammatory response, and elevated LDH suggests cell breakdown due to rejection. In conventional life-supporting organ transplants, clinical symptoms and biochemical data can be used for monitoring of rejection, instead of biopsy. In contrast, the uterus is not a life-supporting organ and rejection is not immediately life-threatening and is frequently asymptomatic; therefore, no clear findings showing rejection have not been reported in human UTx. The biochemical findings above may be one indicator; however, these findings are often caused by irreversible rejection and it is likely to be difficult to recover uterine function once they occur. Thus, monitoring of rejection by an alternative means to biopsy is more difficult in UTx than in life-supporting organ transplantation.</p><p id=\"Par15\">The swelling of the uterus at the time of rejection shows inflammatory edema due to rejection, and the subsequent shrinkage reflects cell damage, necrotic cell death, and hyalinization and formation of granulation tissues due to ischemia over time, resulting in atrophy. The uteri in cases 4 and 5 were still swollen at autopsy, but it is likely that these uteri will be atrophic thereafter because these cases could not overcome uterine rejection. These processes also occur in life-supporting organs, such as in kidney and liver transplantation.</p><p id=\"Par16\">A major concern in UTx is whether a uterus with irreversible rejection should be removed. Such a uterus is not likely to cause serious problems after rejection and subsequent uterine atrophy and loss of function because the uterus is not a vital organ. This study also showed that general condition remained good in the macaques in which irreversible rejection occurred. Likewise, a good general condition is maintained in macaques with an atrophied uterus after warm ischemia for 8 h, which we used to examine the allowable warm ischemic time and ischemic reperfusion injury of the uterus in cynomolgus macaques<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. Thus, vital organs result in life-threatening conditions if their function is lost, but the uterus is not critical for survival. This difference from other life-supporting organs is an advantage of non-life-supporting organ transplantation.</p><p id=\"Par17\">In kidney transplantation, chronic allograft dysfunction is a chronic, progressive, and irreversible state of a transplanted kidney and one of the leading causes of allograft loss among kidney transplant recipients. In such a case, hemodialysis is introduced as an alternative life-saving treatment. The graft will gradually be atrophied if it is left in the abdomen, and whether an asymptomatic failed renal allograft should be removed before retransplantation is still controversial. The reported rate of surgical allograft nephrectomy after graft failure varies from 20 to 80%, and mainly depends on the center policy<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. Some investigators have advocated against removal of an asymptomatic failed allograft due to the morbidity and mortality associated with transplant nephrectomy<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. In contrast, others have contended that a failed allograft is a source of sepsis or chronic inflammation that may lead to complications, and therefore, should be routinely removed. There is no consensus on the timing and indications for allograft nephrectomy.</p><p id=\"Par18\">Unsurprisingly, continuation of immunosuppression after graft failure results in an increase in infectious complications. Therefore, after early graft failure the graft is often removed to prevent acute rejection and allow rapid reduction or complete withdrawal of immunosuppressive medication. In auxiliary partial orthotopic liver transplantation (APOLT), a partial liver graft is implanted in an orthotopic position after leaving behind part of the native liver, which has a potential advantage of immunosuppression withdrawal in acute liver failure<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. In this technique, withdrawal of immunosuppressants can be gradual, so that the graft undergoes slow rejection and ultimately becomes atrophic and fibrotic, while the native liver continues to regenerate to compensate for the loss in graft volume. However, the atrophic graft may have to be removed after immunosuppression withdrawal if it becomes infected<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>.</p><p id=\"Par19\">Given this background, it is uncertain if an atrophic uterus after irreversible rejection should be left in the pelvis or removed. Continuous rejection under immunosuppressants leads to promote infection because the uterine cavity is in contact with the outside of the body through the vagina, whereas kidney and liver do not contact to the outside directly. In the first Swedish trial of UTx, the patient had repeated intrauterine infection after UTx and the uterus had to be resected<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. We also experienced this situation in cynomolgus macaques that had abscesses temporarily present in the uterine cavity after allogeneic UTx in which continuous rejection occurred<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Since organ transplant recipients receive immunosuppressants, they have higher risks for infection than general patients. Intraabdominal infection is occasionally fatal and hysterectomy should be the best choice to prioritize the safety of the recipient in such a situation.</p><p id=\"Par20\">One concern with hysterectomy is that the rejected uterus is likely to be strongly adhered to the surrounding tissues due to inflammation, which leads to cause more operative complications than in a regular hysterectomy. Furthermore, unless uterine infection occurs, the transplanted graft will atrophy naturally. This natural course supports the opinion that a reoperation for resection of the graft is unnecessary and is likely to lead to risks and burdens for the patient. In reports in humans, post-delivery operations have been routinely performed to allow withdrawal of immunosuppressants to decrease the risk of infection. The question of whether to perform hysterectomy for irreversible uterine rejection, an atrophied uterus, or a transplanted uterus after delivery clearly requires further discussion. Moreover, this study has a limitation that these results in cynomolgus macaques cannot always be extrapolated to humans even if nonhuman primate models have the anatomic and physiologic similarities of their reproductive organs and immune systems to humans.</p><p id=\"Par21\">In conclusion, irreversible rejection after allogeneic UTx in cynomolgus monkeys was associated with increases in WBC, LDH and CRP and uterine shrinkage after transient swelling. General condition was good, even after the uterus failed due to rejection, which suggests that uterine transplantation rejection is not fatal, in contrast to rejection of life-supporting organs.</p></sec><sec id=\"Sec10\"><title>Materials and methods</title><sec id=\"Sec11\"><title>Animals</title><p id=\"Par22\">In our experimental series of allogeneic UTx in cynomolgus macaques, six female recipients of macaques (cases 1–6) (Macaca fascicularis, age 6–13 years; average body weight, 3.85 ± 0.91 kg [mean ± standard deviation]) were retrospectively examined. The six recipients and their donors had compatible ABO blood type and a high degree of polymorphism in the major histocompatibility complex (MHC) gene. All of the recipients were diagnosed with uterine rejection after allogeneic UTx and were unresponsive to treatment with immunosuppressants (i.e. irreversible rejection). The study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council, and was approved by the Animal Care and Use Committee of the Research Center for Animal Life Science, Shiga University of Medical Science, Japan (permit numbers: 2013-4-2, 2016-4-8 and 2019-3-12).</p></sec><sec id=\"Sec12\"><title>Immunosuppressive treatment</title><p id=\"Par23\">The animals received immunosuppressive treatment as shown in Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>. Immunosuppressive protocol varied in these animals. As induction treatment, animals received antithymocyte globulin (ATG) (10 mg/kg; Thymoglobulin, Genzyme, Cambridge, MA, USA) intravenously on postoperative day (POD) 0 (the day of surgery) in cases 2 and 3, and 20 mg/kg of ATG on POD 0 and POD 2 in cases 4–6. Rituximab (2 mg/kg; Rituxan; Genentech, San Francisco, CA, USA) before approximately 3 weeks (within 17–23 days before the surgery) and on POD 0 was also given intravenously on POD 0 and POD 2 in case 5 and 6. Maintenance treatment consisted of tacrolimus (TAC) (Prograf; Astellas Pharma) given orally twice a day in case 1, cyclosporine (CyA) (Sandimmune:Novartis, Basel, Switzerland) given subcutaneously in cases 2 and 3, and TAC (Prograf;Astellas Pharma, Tokyo, Japan) given intramuscularly in cases 4–6. The target trough levels for CyA and TAC up to 1 month after surgery were planned to be in the ranges of 300–400 ng/mL and 15–20 ng/mL, respectively, and thereafter were adjusted as required. As another maintenance treatment,mycophenolate mofetil (MMF) (40–100 mg/kg; Cellcept; Chugai Pharmaceutical, Tokyo, Japan) was administered orally from recovery of appetite after surgery in case 2, 4, 5 and 6. Methylprednisolone (10 mg/kg; Solu-Medrol; Pfizer, NY, USA) was injected intravenously on POD 0 and then injected intramuscularly daily starting on POD 1. The dose of methylprednisolone was gradually tapered. If rejection occurred, steroid pulse therapy of 10 mg/kg methylprednisolone for 2 days was administered and gradually tapered. When the rejection was refractory to conventional steroid pulse treatment, antithymocyte globulin (ATG) were added.<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Immunosuppressive treatment in cases 1–6.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Case</th><th align=\"left\">Induction treatment</th><th align=\"left\">Maintenance treatment</th></tr></thead><tbody><tr><td align=\"left\">1</td><td align=\"left\">None</td><td align=\"left\">Tac + mPSL</td></tr><tr><td align=\"left\">2</td><td align=\"left\">ATG</td><td align=\"left\">CyA + MMF + mPSL</td></tr><tr><td align=\"left\">3</td><td align=\"left\">ATG</td><td align=\"left\">CyA + mPSL</td></tr><tr><td align=\"left\">4</td><td align=\"left\">ATG</td><td align=\"left\">Tac + MMF + mPSL</td></tr><tr><td align=\"left\">5</td><td align=\"left\">Rxm + ATG</td><td align=\"left\">Tac + MMF + mPSL</td></tr><tr><td align=\"left\">6</td><td align=\"left\">Rxm + ATG</td><td align=\"left\">Tac + MMF + mPSL</td></tr></tbody></table><table-wrap-foot><p><italic>ATG</italic> antithymocyte globulin, <italic>CyA</italic> cyclosporine, <italic>MMF</italic> mycophenolate mofetil, <italic>mPSL</italic> methylprednisolone, <italic>Tac</italic> tacrolimus, <italic>Rxm</italic> rituximab.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec13\"><title>Postoperative observation</title><p id=\"Par24\">After allogeneic UTx, these cynomolgus macaques were reared in each cage under the following environmental conditions: temperature of 23–29 °C, humidity of 35–75%, lighting of 12 h/day, and toys to play with all day long. The general condition of the animals (activity, appetite, bowel movement, vomiting, urination) and the laparotomy wound were evaluated daily. Laboratory assessments including hematology, blood chemistry and trough levels of CyA and TAC were performed three times per week for the first two postoperative weeks, twice per week for 2 months, and weekly thereafter. To monitor for potential rejection, transabdominal ultrasonography and transvaginal biopsy of the transplanted uterine tissues of uterine cervix and body by using clamps (Storz 5Fr; Karl Storz) in case 1 and a puncture needle (BARD MAX-CORE; BD [C.R. Bard, Inc.], Tempe, AZ) in case 2–6 were routinely conducted under anesthesia monthly or when considered necessary (whenever graft rejection was suspected). The animals were fed a commercial monkey diet once daily, with supplemental fruits and vegetables seven times weekly before and after the procedure. Lactated Ringer’s solution was administered i.v. if there was a change in the post-operative condition, such as appetite loss or dehydration. Antibiotics and sufficient painkiller were also administered after the invasive procedure. Animals were euthanized if severe weakness, weight loss or abnormal behavior was seen after the procedure or when determined to be necessary by veterinary staff and investigators.</p></sec><sec id=\"Sec14\"><title>Laparotomy and indocyanine green (ICG) fluorescence imaging</title><p id=\"Par25\">When irreversible rejection was diagnosed, laparotomy was performed and ICG fluorescence imaging of the uterus was conducted. ICG (Diagnogreen 0.5%; Daiichi Pharmaceutical, Tokyo, Japan) was injected intravenously and the blood supply to the uterus was monitored in real time<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. An increasing intensity of fluorescence was displayed using the Photodynamic Eye Neo system (Hamamatsu Photonics K.K., Hamamatsu, Japan). Grafted uteri were then removed and evaluated pathologically.</p></sec><sec id=\"Sec15\"><title>Endpoints</title><p id=\"Par26\">Clinical features including general condition, body weight loss, hematology, uterine size by transabdominal ultrasonography, intraabdominal gross findings and ICG fluorescence imaging by laparotomy, and histopathological findings of the removed uterus were evaluated. Day 0 was defined as the day when rejection was diagnosed as irreversible based on the course and pathological findings of biopsy.</p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><ack><title>Acknowledgements</title><p>The authors thank the helpful staff at Research Center for Animal Life Science, Shiga University of Medical Science. This work was supported by the Adaptable and Seamless Technology Transfer Program through Target-Driven Research and Development (A-STEP), AMED (AS2525033N), and the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Number 17H05099).</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>I.K. was involved in the design, execution and analysis of the experimental study and drafting of the manuscript. K.E. revised the manuscript. K.E. and Y.M. contributed to the intellectual input in pathology. Y.Y., K.M., H.O., Y.M., Y.K., Y.S., H.I., H.U. and T.S. were involved in the execution of the experimental study. I.I, I.K., C.I., M.M., T.N., and H.T. were involved in experimental support in animals. K.B., M.E., K.O, D.A., K.K. and T.S. provided intellectual input and supervision in the overall study design. All authors read and approved the final manuscript.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par27\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brannstrom</surname><given-names>M</given-names></name><etal/></person-group><article-title>Livebirth after uterus transplantation</article-title><source>Lancet</source><year>2015</year><volume>385</volume><fpage>607</fpage><lpage>616</lpage><pub-id pub-id-type=\"doi\">10.1016/S0140-6736(14)61728-1</pub-id><pub-id pub-id-type=\"pmid\">25301505</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Farrell</surname><given-names>RM</given-names></name><etal/></person-group><article-title>Evolving ethical issues with advances in uterus transplantation</article-title><source>Am. J. Obstet. Gynecol.</source><year>2020</year><pub-id pub-id-type=\"doi\">10.1016/j.ajog.2020.01.032</pub-id><pub-id pub-id-type=\"pmid\">32446998</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kisu</surname><given-names>I</given-names></name><name><surname>Banno</surname><given-names>K</given-names></name><name><surname>Mihara</surname><given-names>M</given-names></name><name><surname>Suganuma</surname><given-names>N</given-names></name><name><surname>Aoki</surname><given-names>D</given-names></name></person-group><article-title>Current status of uterus transplantation in primates and issues for clinical application</article-title><source>Fertil. Steril.</source><year>2013</year><volume>100</volume><fpage>280</fpage><lpage>294</lpage><pub-id pub-id-type=\"doi\">10.1016/j.fertnstert.2013.03.004</pub-id><pub-id pub-id-type=\"pmid\">23557761</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fageeh</surname><given-names>W</given-names></name><name><surname>Raffa</surname><given-names>H</given-names></name><name><surname>Jabbad</surname><given-names>H</given-names></name><name><surname>Marzouki</surname><given-names>A</given-names></name></person-group><article-title>Transplantation of the human uterus</article-title><source>Int. J. Gynaecol. Obstet.</source><year>2002</year><volume>76</volume><fpage>245</fpage><lpage>251</lpage><pub-id pub-id-type=\"doi\">10.1016/S0020-7292(01)00597-5</pub-id><pub-id pub-id-type=\"pmid\">11880127</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brannstrom</surname><given-names>M</given-names></name><etal/></person-group><article-title>First clinical uterus transplantation trial: a six-month report</article-title><source>Fertil. Steril.</source><year>2014</year><volume>101</volume><fpage>1228</fpage><lpage>1236</lpage><pub-id pub-id-type=\"doi\">10.1016/j.fertnstert.2014.02.024</pub-id><pub-id pub-id-type=\"pmid\">24582522</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Johannesson</surname><given-names>L</given-names></name><etal/></person-group><article-title>Uterus transplantation trial: 1-year outcome</article-title><source>Fertil. Steril.</source><year>2015</year><volume>103</volume><fpage>199</fpage><lpage>204</lpage><pub-id pub-id-type=\"doi\">10.1016/j.fertnstert.2014.09.024</pub-id><pub-id pub-id-type=\"pmid\">25439846</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chmel</surname><given-names>R</given-names></name><etal/></person-group><article-title>Revaluation and lessons learned from the first 9 cases of a Czech uterus transplantation trial: four deceased donor and 5 living donor uterus transplantations</article-title><source>Am. J. Transplant.</source><year>2019</year><volume>19</volume><fpage>855</fpage><lpage>864</lpage><pub-id pub-id-type=\"doi\">10.1111/ajt.15096</pub-id><pub-id pub-id-type=\"pmid\">30151893</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kristek</surname><given-names>J</given-names></name><etal/></person-group><article-title>Acute appendicitis in a patient after a uterus transplant: a case report</article-title><source>World J. Clin. Cases</source><year>2019</year><volume>7</volume><fpage>4270</fpage><lpage>4276</lpage><pub-id pub-id-type=\"doi\">10.12998/wjcc.v7.i24.4270</pub-id><pub-id pub-id-type=\"pmid\">31911907</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Puntambekar</surname><given-names>S</given-names></name><etal/></person-group><article-title>Laparoscopic-assisted uterus retrieval from live organ donors for uterine transplant: our experience of two patients</article-title><source>J. Minim. Invasive Gynecol.</source><year>2018</year><volume>25</volume><fpage>622</fpage><lpage>631</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jmig.2018.01.009</pub-id><pub-id pub-id-type=\"pmid\">29366966</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Molne</surname><given-names>J</given-names></name><etal/></person-group><article-title>Monitoring of human uterus transplantation with cervical biopsies: a provisional scoring system for rejection</article-title><source>Am. J. Transplant.</source><year>2017</year><volume>17</volume><fpage>1628</fpage><lpage>1636</lpage><pub-id pub-id-type=\"doi\">10.1111/ajt.14135</pub-id><pub-id pub-id-type=\"pmid\">27868389</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kisu</surname><given-names>I</given-names></name><name><surname>Banno</surname><given-names>K</given-names></name><name><surname>Matoba</surname><given-names>Y</given-names></name><name><surname>Adachi</surname><given-names>M</given-names></name><name><surname>Aoki</surname><given-names>D</given-names></name></person-group><article-title>Basic research on uterus transplantation in nonhuman primates in Japan</article-title><source>J. Obstet. Gynaecol. Res.</source><year>2018</year><volume>44</volume><fpage>1871</fpage><lpage>1881</lpage><pub-id pub-id-type=\"pmid\">29974599</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tzakis</surname><given-names>AG</given-names></name></person-group><article-title>Nonhuman primates as models for transplantation of the uterus</article-title><source>Fertil. Steril.</source><year>2013</year><volume>100</volume><issue>1</issue><fpage>61</fpage><pub-id pub-id-type=\"doi\">10.1016/j.fertnstert.2013.03.041</pub-id><pub-id pub-id-type=\"pmid\">23651624</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kisu</surname><given-names>I</given-names></name><name><surname>Banno</surname><given-names>K</given-names></name><name><surname>Mihara</surname><given-names>M</given-names></name><name><surname>Aoki</surname><given-names>D</given-names></name></person-group><article-title>Uterus transplantation in nonhuman primates</article-title><source>Fertil. Steril.</source><year>2013</year><volume>100</volume><fpage>e3</fpage><pub-id pub-id-type=\"doi\">10.1016/j.fertnstert.2013.04.011</pub-id><pub-id pub-id-type=\"pmid\">23668993</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kisu</surname><given-names>I</given-names></name><etal/></person-group><article-title>Long-term outcome and rejection after allogeneic uterus transplantation in cynomolgus macaques</article-title><source>J. Clin. Med.</source><year>2019</year><volume>8</volume><fpage>E1572</fpage><pub-id pub-id-type=\"doi\">10.3390/jcm8101572</pub-id><pub-id pub-id-type=\"pmid\">31581534</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kisu</surname><given-names>I</given-names></name><etal/></person-group><article-title>Allowable warm ischemic time and morphological and biochemical changes in uterine ischemia/reperfusion injury in cynomolgus macaque: a basic study for uterus transplantation</article-title><source>Hum. Reprod.</source><year>2017</year><volume>32</volume><fpage>2581</fpage><pub-id pub-id-type=\"doi\">10.1093/humrep/dex325</pub-id><pub-id pub-id-type=\"pmid\">29077872</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ghyselen</surname><given-names>L</given-names></name><name><surname>Naesens</surname><given-names>M</given-names></name></person-group><article-title>Indications, risks and impact of failed allograft nephrectomy</article-title><source>Transplant. Rev. (Orlando).</source><year>2019</year><volume>33</volume><fpage>48</fpage><lpage>54</lpage><pub-id pub-id-type=\"doi\">10.1016/j.trre.2018.08.001</pub-id><pub-id pub-id-type=\"pmid\">30236837</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vavallo</surname><given-names>A</given-names></name><etal/></person-group><article-title>Allograft nephrectomy: what is the best surgical technique?</article-title><source>Transplant. Proc.</source><year>2012</year><volume>44</volume><fpage>1922</fpage><lpage>1925</lpage><pub-id pub-id-type=\"doi\">10.1016/j.transproceed.2012.06.011</pub-id><pub-id pub-id-type=\"pmid\">22974872</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mazzucchi</surname><given-names>E</given-names></name><etal/></person-group><article-title>Surgical complications of graft nephrectomy in the modern transplant era</article-title><source>J. Urol.</source><year>2003</year><volume>170</volume><fpage>734</fpage><lpage>737</lpage><pub-id pub-id-type=\"doi\">10.1097/01.ju.0000080566.42381.94</pub-id><pub-id pub-id-type=\"pmid\">12913685</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rela</surname><given-names>M</given-names></name><name><surname>Kaliamoorthy</surname><given-names>I</given-names></name><name><surname>Reddy</surname><given-names>MS</given-names></name></person-group><article-title>Current status of auxiliary partial orthotopic liver transplantation for acute liver failure</article-title><source>Liver Transpl.</source><year>2016</year><volume>22</volume><fpage>1265</fpage><lpage>1274</lpage><pub-id pub-id-type=\"doi\">10.1002/lt.24509</pub-id><pub-id pub-id-type=\"pmid\">27357489</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weiner</surname><given-names>J</given-names></name><etal/></person-group><article-title>Longterm outcomes of auxiliary partial orthotopic liver transplantation in preadolescent children with fulminant hepatic failure</article-title><source>Liver Transpl.</source><year>2016</year><volume>22</volume><fpage>485</fpage><lpage>494</lpage><pub-id pub-id-type=\"doi\">10.1002/lt.24361</pub-id><pub-id pub-id-type=\"pmid\">26479577</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kisu</surname><given-names>I</given-names></name><etal/></person-group><article-title>Indocyanine green fluorescence imaging for evaluation of uterine blood flow in cynomolgus macaque</article-title><source>PLoS ONE</source><year>2012</year><volume>7</volume><fpage>e35124</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0035124</pub-id><pub-id pub-id-type=\"pmid\">22606213</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"other\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Nat Commun</journal-id><journal-id journal-id-type=\"iso-abbrev\">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type=\"epub\">2041-1723</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807772</article-id><article-id pub-id-type=\"pmc\">PMC7431529</article-id><article-id pub-id-type=\"publisher-id\">17724</article-id><article-id pub-id-type=\"doi\">10.1038/s41467-020-17724-1</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Q&A</subject></subj-group></article-categories><title-group><article-title>Robert Langer and Mark Tibbitt answer questions about additive manufacturing</article-title></title-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>3994</elocation-id><permissions><copyright-statement>© Springer Nature Limited 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\" abstract-type=\"Standfirst\"><p id=\"Par1\">Robert S. Langer is an Institute Professor at the Massachusetts Institute of Technology. Leading one of the largest biomedical engineering labs in the world his research covers many areas of biotechnology including tissue engineering, drug delivery, biofabrication and the development of medical devices. Mark Tibbitt is an Assistant Professor of Macromolecular Engineering at ETH Zürich. His research focuses on combining polymer engineering, synthetic chemistry, mechanical and bioengineering for biofabrication, drug delivery and mechanobiology applications.</p></abstract><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><p id=\"Par2\">Tell us a little bit about you and what sparked your interest in additive manufacturing.</p><p id=\"Par3\"><bold>Bob:</bold> I like to think that our lab at MIT has made pioneering contributions in two cores areas of biomedical engineering: tissue engineering and drug delivery. We are always seeking out emerging technologies, like additive manufacturing, that can advance these paradigms and accelerate our impact on human health. We became interested in additive manufacturing in the context of engineering personalized implants, standardizing microtissues for disease modeling and pharmaceutical development, and fabricating controlled release technologies. This work builds upon paradigms we established in the field and exploits new processing, design, and fabrication opportunities afforded by additive manufacturing.</p><p id=\"Par4\"><bold>Mark:</bold> I joined the Langer Lab as a postdoc in 2013 and at this time the benefit of additive manufacturing for biomedical engineering was becoming more apparent, especially for the design of personalized or precision biomaterials. We first applied additive manufacturing in the lab in a project with Héloise Ragelle, another former postdoc of the Langer Lab, to design and manufacture a multi-material tracheal stent to help cardiothoracic surgeons looking to repair or replace damage tracheal tissue<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. We designed lattice tubes using stereolithography to meet the geometric and mechanical needs of the stent, which would not have been feasible using another manufacturing technique, and developed a process to use surface tension to form a cell-laden hydrogel film around the stent, to introduce biofunctionality to the device. In my lab at ETH Zürich, we are currently designing materials (or inks) for additive manufacturing that can be used to fabricate personalized tissue models and drug delivery systems.</p><p id=\"Par5\">How has additive manufacturing changed your field? Is there anything specific that it has enabled or made possible? Tell us how it impacted your research.</p><p id=\"Par6\">Additive manufacturing is a transformative enabling technology that provides researchers across many domains improved access to design the form and function of the materials they produce. We are already seeing the impact of additive manufacturing in the design and development of commercial products as it transitions from a technology for prototyping to a means of production. In biomedical engineering, additive manufacturing offers specific advantages for both tissue engineering and drug delivery as it enables access to complex structures and detailed geometries that are often not possible (or cost-prohibitive) with traditional subtractive manufacturing. Further, additive manufacturing uses digital design for high precision fabrication, which can enable the production of personalized implants based on medical imaging.</p><p id=\"Par7\">One recent example of the impact of additive manufacturing is the recent demonstration of complex vascularized tissue constructs enabled by advanced stereolithography<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. Vascularization of human-scale constructs remains a major challenge in the field of tissue engineering and additive manufacturing may be one solution that addresses this challenge.</p><p id=\"Par8\">In our lab, additive manufacturing has been used, as Mark mentioned, to design multi-material structures to help surgeons design better tracheal stents. We have also leveraged bioprinting to generate reproducible 3D tissue models to investigate disease processes and for drug testing ex vivo. This includes the design of microscale cardiac valve models, in collaboration with Elena Aikawa at Harvard Medical School, to study calcific valve disease<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. We have also applied the technology to drug delivery and medical device design, Yong Kong and Giovanni Traverso used additive processes in the Langer Lab to design gastric resident devices<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. Kevin McHugh, Thanh Nguyen, and Ana Jaklenec developed a new additive process, StampEd Assembly of polymer Layers or SEAL, to engineer single-injection vaccinations<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>.</p><p id=\"Par9\">What does additive manufacturing have to do to be more widely adopted? Are there any specific hurdles it has to overcome?</p><p id=\"Par10\">While additive manufacturing has already advanced our ability to design complex yet precise biomaterials, there are several open problems that should be addressed to increase its adoption and clinical use. A core area of focus remains on the technology itself. Often additive processes are limited in the scale and speed of production as well as the resolution and complexity of the final part. Technologies like CLIP, commercialized by carbon, volumetric additive manufacturing, and multi-material printing have improved the speed, resolution, and complexity of additively manufactured products<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>–<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. Yet, further developments are needed to realize intricate, multi-cellular tissues of arbitrary complexity, rapid production of patient-specific implants, and the design of personalized pharmaceutical products.</p><p id=\"Par11\">Another current challenge is the range of biomaterials suitable for additive processes. The biomaterials community has made tremendous advances and many GRAS-listed and FDA-approved materials exist for the design of medical devices. Many of these are suitable for additive processes, but not all. We will need to continue working closely with engineers, materials scientists, and clinicians to develop the next-generation of compatible biomaterials that integrate with additive processes.</p><p id=\"Par12\">A further challenge in the broad adoption of additive manufacturing in the biomedical sciences is the pathway to regulatory approval. Regulatory agencies, including the FDA, have provided guidelines for how additive manufacturing will be considered in the regulatory process and already some products have been approved. However, uncertainties remain about how clinical production would operate for additively manufactured biomedical products and whether this would require centralized production facilities, as in standard medical device fabrication, or if technologies will be approved as decentralized production facilities in individual care centers or pharmacies.</p><p id=\"Par13\">Looking forward: where do you see additive manufacturing going next?</p><p id=\"Par14\">We see that additive manufacturing will have the most impact in the field of biomedical engineering where the benefits of digital design, rapid production, and personalized are needed. In the future, this may include the design of complex tissue constructs with spatial resolution at the single cell level containing both vascularization and innervation. Digital design and fabrication based on medical imaging will likely advance the paradigm of precision biomaterials that are built specifically to address individual patients or subsets of patients. Finally, an emerging use of additive manufacturing is on the design of personalized drug delivery systems<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>, and future pharmaceutical development may combine controlled release technologies with additive manufacturing to further enable personalized drug delivery.</p><p id=\"Par15\">For you, is there a difference between additive manufacturing and 3D printing? If they are not equivalent, how do they differ?</p><p id=\"Par16\">The definitions of these terms are not very precise, but one view is that 3D printing describes processes that build 3D structures through successive layer-by-layer formation, like stacking many prints into a 3D object. Whereas additive manufacturing is a more comprehensive term that describes all processes that can be used to fabricate 3D forms by adding, combining, or joining material components, including those that are beyond layer-by-layer, in contrast to traditional subtractive manufacturing where material is removed to produce the final part. Both terms describe methods to produce 3D objects and in practice they have often become synonymous.</p></body><back><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ragelle</surname><given-names>H</given-names></name><etal/></person-group><article-title>Surface tension-assisted additive manufacturing</article-title><source>Nat. Commun.</source><year>2018</year><volume>9</volume><fpage>1184</fpage><pub-id pub-id-type=\"doi\">10.1038/s41467-018-03391-w</pub-id><pub-id pub-id-type=\"pmid\">29567939</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grigoryan</surname><given-names>B</given-names></name><etal/></person-group><article-title>Multivascular networks and functional intravascular topologies within biocompatible hydrogels</article-title><source>Science</source><year>2019</year><volume>364</volume><fpage>458</fpage><lpage>464</lpage><pub-id pub-id-type=\"doi\">10.1126/science.aav9750</pub-id><pub-id pub-id-type=\"pmid\">31048486</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van der Valk</surname><given-names>D</given-names></name><etal/></person-group><article-title>Engineering a 3D-bioprinted model of human heart valve disease using nanoindentation-based biomechanics</article-title><source>Nanomaterials</source><year>2018</year><volume>8</volume><fpage>296</fpage><pub-id pub-id-type=\"doi\">10.3390/nano8050296</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kong</surname><given-names>YL</given-names></name><etal/></person-group><article-title>3D‐printed gastric resident electronics</article-title><source>Adv. Mater. Technol.</source><year>2018</year><volume>4</volume><fpage>1800490</fpage><pub-id pub-id-type=\"doi\">10.1002/admt.201800490</pub-id><pub-id pub-id-type=\"pmid\">32010758</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McHugh</surname><given-names>KJ</given-names></name><etal/></person-group><article-title>Fabrication of fillable microparticles and other complex 3D microstructures</article-title><source>Science</source><year>2017</year><volume>357</volume><fpage>1138</fpage><lpage>1142</lpage><pub-id pub-id-type=\"doi\">10.1126/science.aaf7447</pub-id><pub-id pub-id-type=\"pmid\">28912242</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tumbleston</surname><given-names>JR</given-names></name><etal/></person-group><article-title>Continuous liquid interface production of 3D objects</article-title><source>Science</source><year>2015</year><volume>347</volume><fpage>1349</fpage><lpage>1352</lpage><pub-id pub-id-type=\"doi\">10.1126/science.aaa2397</pub-id><pub-id pub-id-type=\"pmid\">25780246</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kelly</surname><given-names>BE</given-names></name><etal/></person-group><article-title>Volumetric additive manufacturing via tomographic reconstruction</article-title><source>Science</source><year>2019</year><volume>363</volume><fpage>1075</fpage><lpage>1079</lpage><pub-id pub-id-type=\"doi\">10.1126/science.aau7114</pub-id><pub-id pub-id-type=\"pmid\">30705152</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kang</surname><given-names>H-W</given-names></name><etal/></person-group><article-title>A 3D bioprinting system to produce human-scale tissue constructs with structural integrity</article-title><source>Nat. Biotechnol.</source><year>2016</year><volume>34</volume><fpage>312</fpage><lpage>319</lpage><pub-id pub-id-type=\"doi\">10.1038/nbt.3413</pub-id><pub-id pub-id-type=\"pmid\">26878319</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liang</surname><given-names>K</given-names></name><name><surname>Carmone</surname><given-names>S</given-names></name><name><surname>Brambilla</surname><given-names>D</given-names></name><name><surname>Leroux</surname><given-names>J-C</given-names></name></person-group><article-title>3D printing of a wearable personalized oral delivery device: a first-in-human study</article-title><source>Sci. Adv.</source><year>2018</year><volume>4</volume><fpage>eaat2544</fpage><pub-id pub-id-type=\"doi\">10.1126/sciadv.aat2544</pub-id><pub-id pub-id-type=\"pmid\">29750201</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807797</article-id><article-id pub-id-type=\"pmc\">PMC7431530</article-id><article-id pub-id-type=\"publisher-id\">70420</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70420-4</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Oral Phyto-thymol ameliorates the stress induced IBS symptoms</article-title></title-group><contrib-group><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Subramaniyam</surname><given-names>Selvaraj</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Yang</surname><given-names>Shuyou</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Diallo</surname><given-names>Bakary N’tji</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Fanshu</surname><given-names>Xu</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lei</surname><given-names>Luo</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Chong</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-6861-7849</contrib-id><name><surname>Tastan Bishop</surname><given-names>Özlem</given-names></name><address><email>o.tastanbishop@ru.ac.za</email></address><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-8738-7188</contrib-id><name><surname>Bhattacharyya</surname><given-names>Sanjib</given-names></name><address><email>sanjib2017@swu.edu.cn</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.263906.8</institution-id><institution>Department of Pharmaceutical Science and Chinese Traditional Medicine, </institution><institution>Southwest University, </institution></institution-wrap>Beibei, Chongqing, 400715 China </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.91354.3a</institution-id><institution>Research Unit in Bioinformatics (RUBi), Department of Biochemistry and Microbiology, </institution><institution>Rhodes University, </institution></institution-wrap>P.O. Box 94, Grahamstown, 6140 South Africa </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13900</elocation-id><history><date date-type=\"received\"><day>6</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>29</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Physical stressors play a crucial role in the progression of irritable bowel syndrome (IBS). Here we report a heterogeneous physical stress induced IBS rat model which shows depression and subsequent modulation of IBS by oral treatment of thymol. Oral administration of Thymol reduces the stress induced IBS significantly altering the stress induced gastrointestinal hypermotility, prolonged the whole gut transit time, and increased abdominal withdrawal reflex suggesting gastrointestinal hypermotility and visceral discomfort caused the onset of depression. Immunohistochemical analysis in small intestine and colon of rats shows the decreased 5-HT<sub>3A</sub>R expression level while thymol treatment normalized the 5-HT<sub>3A</sub>R expression in the stressed rats. Molecular docking studies showed that thymol competes with endogenous serotonin and an antagonist, Tropisetron and all have similar binding energies to 5-HT<sub>3A</sub>R. Molecular dynamics simulations revealed that thymol and tropisetron might have similar effects on 5-HT<sub>3A</sub>R. Our study suggest that thymol improves IBS symptoms through 5-HT<sub>3A</sub>R, could be useful for the treatment of IBS.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Receptor pharmacology</kwd><kwd>Biophysical chemistry</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">501100006250</institution-id><institution>Southwest University (SWU)</institution></institution-wrap></funding-source><award-id>104290/22300504</award-id></award-group></funding-group><funding-group><award-group><funding-source><institution>DELTAS AFRICA INITIATIVE</institution></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Stress remains an inextricable part of our life throughout the history of civilization, and perhaps changed its course during the modern era in terms of urbanization and lifestyle. Causes and circumstances of stress could vary in different instances, subsequently changing the manifestations of the cause–effect relationship. Stress in life comes from various origins, such as physical trauma, early life events, loss of parents, physical/sexual abuse, and acts as predisposing risk factors for the development of irritable bowel syndrome (IBS), a functional gastrointestinal disorder (FGID). Physical stressors can alter the gut brain axis affecting the visceral events<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. Traumatic events can induce changes in the brain sensory response that modulates the neuroendocrine hypothalamus–pituitary–adrenal (HPA) crosstalk<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. A “fight” response generated due to threat (stressor) activates a feedback mechanism to quench the stress to reinstate the system allostasis<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. However a prolonged stressor can ruin the adaptive system to achieve stress homeostasis, and could subsequently turn into pathogenesis of whole body disorders including gastrointestinal tract (GI) of viscera<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. The consequence of stress episodes and associated anxiety is often compensated in adults at the cost of irritable bowel syndrome (IBS)<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. Hence social stress and relevant maladaptation of life style are often buffered at the expense of IBS. IBS is a complex, polygenic disorder that often includes various symptoms such as abdominal pain and discomfort, visceral hyperalgesia, altered fecal output and GI transit time<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. Visceral pain can arise from wide arrays of disorders such as gallstone, pancreatitis, esophageal reflux and many others<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. Nociceptive pain stems from the central nervous system (CNS) innervating viscera to the site of signal transmission<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. The outcome of visceral pain management has remained unsatisfactory during the last decades including a cost burden of diminished quality of life. However, efforts are ongoing with opioid receptor agonist/antagonist, serotonergic agent, bile acid regulator, which have shown promising results in clinical trials<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. IBS could arise from different scenario of serotonin level giving different phenotypes; such as either diarrhea, or constipation or none of these<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. This variable spectrum of IBS symptoms is the key foundation for developing various serotonin based agonist and antagonist to treat IBS. Recent serotonin transporter knock out animal model study suggests mimicking some spectrum of humanized IBS<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>.</p></sec><sec id=\"Sec2\"><title>Results</title><p id=\"Par3\">Herein we report a physical stressor mediated IBS in rat model that shows alternation of serotonin receptor (5-HT<sub>3</sub>AR) surface presentation in the intestine and colon. We also report that thymol treatment smooths out the IBS symptoms by altering the 5-HT<sub>3</sub>AR level. Thymol, a mono terpenoid phytochemical found in Southeast Asian herbs call Ajwaan, is used as traditional medicine and food component. It is typically used for bowel related complications and digestion problems<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Thymol derivatives have a pharmacologic use as a gastrointestinal modulator, but the biochemical mechanisms are largely unknown. Further studies are required to find out whether thymol could hold a putative potential for pain medication by alleviating the visceral pain associated with IBS and other diseases, modulating the opioid receptor.</p><p id=\"Par4\">We used post-natal stress induced rat model of IBS<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Rats were given a mixture of heterotypic physical stressor (see “<xref rid=\"Sec4\" ref-type=\"sec\">Experimental procedure</xref>” section) by alternating modality to mimic the humanized effects in social milieu for a period of 4–6 weeks. Cotton nest behavioral studies were performed preliminarily in order to see whether application of prolonged stress caused their mood fluctuation in terms of depression. We noticed that rat exposed to various stress factors showed visible signs of depression and mood alteration as evident from their participation to play with the cotton nest to reshuffle the cotton (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>A–C). The tests were scored based on their zeal for participation and performance to play with cotton nest (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>D). At the end of 4 week of stress, we used gastrointestinal transit (GIT) as readout for both the consequence of stress and subsequent healing ability of thymol (for 2-week treatment starting from week 5 and continued in week 6) to ease out stress induced IBS. GIT is a surrogate marker for evaluating drug efficacy to inhibit bowel abnormalities in IBS, also clinically relevant for the assessment of “organic” disorder such as GI motility<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Indeed, drugs that target normalizing GIT such as Lubiprostone, Linaclotide turned out to be useful in relieving abdominal discomfort in Phase III trials<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. We found that oral thymol treatment enhanced the GI transit (similar to untreated control animals) which was higher in stress induced animals possibly due to leaky gut (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A). This could be due to the stress driven change in local intestinal permeability resulting in modulation of mucosal inflammation and gut sensory visceromotor reflex. Since IBS is often associated with visceral hyperalgesia, fecal pellet output has also been used to study the response of stress and drugs in rodent models; this is in some instances homologous to and in some instances contrary to human GIT studies where the patients as well as healthy control subjects were evaluated in absence of acute stressor<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. We noticed that fecal output was higher for stress induced rats (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>B). The thymol treatment significantly decreased the fecal count that was associated with stressed rats (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>B). This result corroborates other rat model studies that showed stress driven anxiety behavior and evaluation of corticotropin-releasing factor 1 (CRF1),\nan antagonist altering the accelerated fecal output in human IBS patients as well<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. Besides leaky gut, stress can change the fecal microbiota that initiated many studies of fecal transplant to donor from IBS patients and subsequent analysis of recipient animal behavior such as visceral hypersensitivity<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. This archetype is used for the study of anxiety associated psychiatry in mouse model by transferring other’s behaviors. Next, abdominal withdrawal reflex (AWR) was used to measure the visceral hypersensitivity which is pre-clinically and clinically used to assess colorectal distension (CRD)<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. This method is widely used in human and other rodent subjects as an index of visceral pain for the evaluation of analgesic compounds. We found that the animal under stress showed extreme abdominal discomfort which is evident from the tolerance to external pain stimuli applied by catheterization of balloon in colorectal cavity (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>C and Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>). The threshold of barostat tolerance was higher for the thymol treated rat upon pain stimuli as evident from the AWRanalysis (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>C).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Exposure to chronic stress reveals altered behavioral response in SD rats. Shredding of a cotton nestlet was measured in rat exposed to (<bold>A</bold>) control, (<bold>B</bold>) Stress, (<bold>C</bold>) Stress with treatment of thymol 50 mg/kg b w, and (<bold>D</bold>) Summary data showing that thymol 50 mg/kg b w treated increased the cotton nestlet shredded in chronic stress induced rat. The amount shredded is shown as percentage of total nestlet area. Data shown as mean ± S.E.M (n = 8). *<italic>P</italic> < 0.05 (one-way ANOVA and TMCT compared with stress).</p></caption><graphic xlink:href=\"41598_2020_70420_Fig1_HTML\" id=\"MO1\"/></fig><fig id=\"Fig2\"><label>Figure 2</label><caption><p>(<bold>A</bold>) Effect of thymol on whole gut transit time (WGTT) in chronic stress induced rat. Summary data shows that thymol 50 mg/kg b w treated prolonged the whole gut transit time in chronic stress induced rat. Data revealed as mean ± SEM (n = 8–10). *<italic>P</italic> < 0.05 (one-way ANOVA and TMCT compared with stress). (<bold>B</bold>) Effect of thymol on abdominal withdrawal reflex (AWR) in chronic stress induced rat. Summary data showing that thymol 50 mg/kg b w treated increased the abdominal withdrawal reflex in chronic induced rat. Data revealed as mean ± SEM (n = 8–10). ***<italic>P</italic> < 0.0001 (one-way ANOVA and TMCT compared with stress). (<bold>C</bold>) Effect of thymol on gastrointestinal hypermotility in chronic stress induced rat. Summary data showing that thymol 50 mg/kg b w treated decreased the gastrointestinal hypermotility in chronic stress induced rat. Data revealed as mean ± SEM (n = 8–10). ***<italic>P</italic> < 0.0001 (one-way ANOVA and TMCT compared with stress).</p></caption><graphic xlink:href=\"41598_2020_70420_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par5\">Next, we wanted to confirm whether the serotonin receptor is the target involved here for the regulation of stress induced IBS and subsequent thymol treatment of rats by oral administration. Serotonin agonist and antagonist are already known to treat IBS related visceral pain<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. We have chosen 5-HT<sub>3</sub>AR among other serotonin receptor homologues, because 5-HT<sub>3</sub>A antagonist compound is capable of managing stress driven IBS defecation. After the rats were sacrificed, the intestine and colon were used for immunohistochemistry by hematoxylin and eosin staining (IHC), and immunohistofluorescence (IHF) analysis of serotonin receptor. IHC analysis clearly showed that visible symptoms of local intestinal tissue atrophy induced by physical stress, while thymol treatment showed the recovery of the intestine tissue architecture possibly by tissue remodeling (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>). Similar tissue damage was observed for the colonic tissue (Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>). It was noticed that stress caused reduction of serotonin receptor (5-HT<sub>3</sub>AR) density presence on the surface membrane of the intestine tissue which was enhanced upon thymol treatment (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>A,B). 5-HT<sub>3</sub>A receptor expression was also upregulated in the case of colonic tissue (Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>). This study indicates that possibly thymol is antagonizing the serotonin receptor to quench the stress mediated IBS that usually results in symptoms of GI hypermotility in rat.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Histopathology of small intestine from chronic stress induced rat. (<bold>A</bold>) Control, (<bold>B</bold>) Stress group: note the crypt flanged with distorted and increasing goblet cells and small intestinal mucosa showing chronic inflammatory changes, epithelial cells of villi damaged and (<bold>C</bold>) Stress induced with treatment of thymol 50 mg/kg b w showing intestinal crypt normal compared to stress.</p></caption><graphic xlink:href=\"41598_2020_70420_Fig3_HTML\" id=\"MO3\"/></fig><fig id=\"Fig4\"><label>Figure 4</label><caption><p>Immunofluorescence detection of 5-HT<sub>3</sub>AR expression in small intestine from chronic stress induced rat. The fluorescent intensity was measured in rat exposed to (<bold>A</bold>) control, (<bold>B</bold>) Stress, (<bold>C</bold>) Stress with treatment of thymol 50 mg/kg b w, and (<bold>D</bold>) Summary data showing that thymol 50 mg/kg b w treated increased the 5-HT<sub>3</sub>AR expression levels in chronic stress induced rat. Data shown as mean ± SEM (n = 8). *<italic>P</italic> < 0. 05 (one-way ANOVA and TMCT compared with stress).</p></caption><graphic xlink:href=\"41598_2020_70420_Fig4_HTML\" id=\"MO4\"/></fig></p><p id=\"Par6\">During IBS, opioid receptors are one of the important regulators of pain sensation among the vast array of other receptors involved in pain perception<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. We observed that physical stressor increased the pain sensation as evident from the overexpression of the mu (µ) opioid receptor that jumped higher upon stress induction (Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S4</xref>). Subsequent thymol treatment of the rats partly adjusts the µ opioid receptor as part of IBS allied visceral pain. However further study is desired to understand how thymol alters the µ opioid receptor axis during pain regulation spurred by IBS. Multi moderators in pain perception are the reason for the complexity to precisely perturb the pain regulator as an IBS related drug target.</p><p id=\"Par7\">A previous study indicated that thymol has a tendency to bind to the transmembrane region of the receptor<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Here, we used molecular docking and MD simulation calculations in order to predict whether thymol also has the ability to compete with serotonin towards 5-HT<sub>3</sub>AR that associates with the nociceptive pain signal in IBS via binding to the extracellular domain (ECD). 5-HT<sub>3</sub>A receptor is a pentamer with five equivalent binding sites; the neurotransmitter site is at the subunit interfaces in the ECD<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Our molecular docking studies against the entire receptor as well as the different conformations of the ECD of the receptor, as explained in the Experimental Procedure section, clearly revealed that serotonin and thymol bind into the same pocket of the 5-HT<sub>3</sub>AR with similar binding energies (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>, Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S5</xref>, Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S6</xref> and Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>, Table <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>). Thymol, in all best poses (most energetically favorable), only, bound to the ECD of the structures. All the other poses of thymol also bound to the same domain in all structures except in 6HIS. In that last case, the 7th and 8th poses bound in the intracellular domains in an extreme region close to the extracellular domain. However, the binding region was still different from the one proposed by Lansdell et al.<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup> The very same pocket is also a binding site for tropisetron, an antagonist for 5-HT<sub>3</sub> receptor, as was shown in crystal structure 6HIS<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Further, residue interaction analysis revealed that these compounds interact with protein residues in a similar manner (Table <xref rid=\"MOESM1\" ref-type=\"media\">S5</xref>, Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S7</xref>). Interestingly, in 6HIS conformation both tropisetron and thymol interacted with identical residues TRP156 (chain A), TYR207 (chain A), ILE44 (chain E), TRP63 (chain E), ARG65 (chain E); while serotonin differed. However, we also observed similar interacting residues when serotonin binds to all other three conformations (6HIQ, 6HIO, 6HIN). Therefore, the difference in the agonist and antagonist may not be linked to the difference in the compounds’ interacting residues.<fig id=\"Fig5\"><label>Figure 5</label><caption><p>Serotonin and thymol docked receptor. (<bold>A</bold>) Cartoon representation of extra cellular domain (ECD) of serotonin-receptor in transition conformation (PDB ID: 6HIS) with docked serotonin (in green) and thymol (in cyan). Docked compounds and crystalized tropisetron (magenta) are superimposed in active site. (<bold>B</bold>) Zoomed in view of serotonin, Tymol and tropisetron. Serotonin interacting residues are indicated in light grey. (<bold>C</bold>) 2D structural presentation of serotonin, tropisetron and thymol.</p></caption><graphic xlink:href=\"41598_2020_70420_Fig5_HTML\" id=\"MO5\"/></fig></p><p id=\"Par8\">To further analyze protein–ligand complexes, MD simulations were performed for the ECD, and root mean square deviation (RMSD), radius of gyration (Rg), root mean square fluctuations (RMSF) and hydrogen bond formation were calculated. RMSD, Rg and hydrogen bond results are presented in Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S8</xref>. According to RMSD results, all the complexes were stable, and no ligand dissociation was observed. The maximum observed ligand RMSD was ~ 1.25 Å for all ligands except tropisetron in 6HIS which increased up to ~ 2 Å. We also observed that tropisetron moves from its original position that was presented in the crystal structure towards the inner part of the binding pocket (Data not shown). Rg also did not show any significant variation. Concerning hydrogen bonding, serotonin formed a higher number of hydrogen bonds than thymol had in all cases (and also more than tropisetron had in 6HIS). Interestingly, in both 6HIO and 6HIQ, Thymol seemed to rearrange and adopt a more stable pose shown here with its more consistent hydrogen bonding toward the end of the simulation. Thymol in 6HIS presented a special case in which the compound does not make any hydrogen bond, but it is mainly stabilized by hydrophobic contacts.</p><p id=\"Par9\">Even though, serotonin, thymol and tropisetron are binding to the same binding pocket of the receptor and showing similar residue interaction pattern, RMSF results revealed that the protein is behaving differently in the presence of serotonin and thymol (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>). In general, residues showed more flexibility when serotonin was bound to protein, contrary to both thymol and tropisetron (6HIS). In the antagonist (tropisetron) bound conformation of the receptor (6HIS), protein residues presented similar behavior in the presence of thymol or tropisetron (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>). These results might be an indication of antagonist behavior of thymol.<fig id=\"Fig6\"><label>Figure 6</label><caption><p>Root mean square fluctuation calculations of extra cellular domain (ECD) of the receptor in compound-bound form for four different conformations. 5-HT<sub>3</sub>A receptor is a pentamer with five equivalent binding sites at the subunit interfaces in the extra cellular domain (ECD). Only RMSF values for residues in chains forming the binding site for the simulated compound are plotted. 6HIQ: intermediate conformation (I2) of 5-HT<sub>3</sub>A receptor with serotonin and TMPPAA (positive allosteric modulator)); 6HIO: intermediate conformation (I1) of the receptor with serotonin; 6HIN: open state of the extracellular domain (ECD) of the receptor with serotonin; 6HIS: transition (T) conformation with tropisetron as antagonist.</p></caption><graphic xlink:href=\"41598_2020_70420_Fig6_HTML\" id=\"MO6\"/></fig></p></sec><sec id=\"Sec3\"><title>Discussion/conclusion</title><p id=\"Par10\">5-HT<sub>3</sub>AR antagonists have been used for IBS treatment modulating the visceral pain situation<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. Thymol has been also used as an essential oil component for various biological benefits<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. In our study, we find that thymol can manage the intestinal hypermotility and ameliorates the visceral sensitivity that is associated with physical stressor mediated IBS (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>). Thus, oral thymol administration could be a potential option to handle the leaky gut with subsequent soothing of the IBS symptoms possibly by regulating the serotonin receptor (5-HT<sub>3</sub>AR). Besides animal model, molecular docking simulation study confirms that thymol competes with native ligand towards the same binding site of 5-HT<sub>3</sub>AR. Whether thymol can intervene in the nociceptive pain triggered by noxious stimuli mediating through pain receptor such as opioid and cannabinoid, could certainly lead to a new direction in developing pain medication related to an array of multiple disorders encompassing viscera and innervated spinal cord to GI tract. Thymol mediated recovery of intestinal tissue architecture that was locally damaged by stress induction also requires further evaluation as to whether thymol has the ability of tissue remodeling during IBS mediated tissue loss. Similarly, we could not find conclusive changes from the CaCO-2 cellular competition experiment to obtain more molecular insight for the thymol mediated 5-HT<sub>3</sub>AR antagonism (data not shown). However, 5-HT<sub>3 </sub>R genetic polymorphism (greater C/C genotype) is associated with the severity of IBS patient symptoms, with enhanced anxiety and amygdala<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. Further investigation is required to understand the precise molecular mechanism of thymol driven 5-HT<sub>3</sub>AR antagonism, even though the 5-HT<sub>3 </sub>R antagonists are viable therapeutic modules to treat IBS and anxiolytic effects.. None the less the non-invasive (oral) validation of thymol as therapeutic target for taming preclinical anxiety associated IBS model in rats warrants further investigation to translate this phytochemical for the application of anxiety associated IBS, other hepatobiliary disease and psycho somatosensory disorders.<fig id=\"Fig7\"><label>Figure 7</label><caption><p>Cartoon presentation of the impact of stress induced IBS during gut brain sensation and subsequent adaptation by producing 5 HT (serotonin). Thymol interferes with the effects of neurotransmitter 5 HT during physical stress mediated IBS and subsequently quench deleterious effects of IBS.</p></caption><graphic xlink:href=\"41598_2020_70420_Fig7_HTML\" id=\"MO7\"/></fig></p><p id=\"Par11\">Comparing ligand structures, thymol does not present the indole ring present in both serotonin and tropisetron. This moiety is very common among bioactive compounds on the 5-HT<sub>3</sub>A receptor (CHEMBL4972). Indeed, except for its aromatic ring which is common among to 5-HT<sub>3</sub>A receptor antagonists, thymol lacks the carbonyl and the basic amine which are considered as key pharmacophoric point for 5-HT<sub>3</sub>A receptor antagonists. However as previously shown, from histological analysis of 5-HT<sub>3</sub>A receptors, and MD simulation analysis of binding similarity as serotonin and tropisetron, thymol interact with the 5-HT<sub>3</sub>A receptor during stress mediated IBS. These interactions are mainly driven by hydrophobic contacts on its aromatic ring and its substituents as evident from the MD simulation. Hence thymol may present a different scaffold for a new class of 5-HT<sub>3</sub>A modulator and may be worthy of additional structure–activity relation (SAR) study.</p></sec><sec id=\"Sec4\"><title>Experimental procedure</title><sec id=\"Sec5\"><title>Materials and methods</title><p id=\"Par12\">\nThymol and serotonin hydrochloride were purchased from Sigma Aldrich (Catalogue No: G8802B, and H9523), Bradford reagent (Sigma Aldrich, Catalogue No: B6916-500ML), Tris base (Geneview, BT350-500G), RAPA lysis buffer (Beyotime Biotechnology, Catalog No: P0013), Paraformaldehyde (Keshi, Catalog No: 30525-89-4), Sucrose (VWR Life science, Catalog No: M117-500G), PVDF membrane (Millipore, Catalog No: IEVH00005), 5HTR3A rabbit polyclonal and β-actin mouse monoclonal antibody were purchased from Proteintech (Catalog No: 10443-1-AP, and Catalog No: 60008-1-Ig), mu Opioid R rabbit polyclonal (Novus Biologicals, Catalog No: NB100-1620). All methods were performed in accordance with the relevant guidelines and regulation.</p></sec><sec id=\"Sec6\"><title>Animals</title><p id=\"Par13\">Adult male Sprague–Dawley (SD) rats at eight weeks old, weighing 222–254 g, were obtained from Chongqing Medical University, Chongqing, China, Animals (n = 30) were transported in a standard cage under similar conditions and were held according to the standards of Animal Ethical Regulations. All methods were performed in accordance with the relevant guidelines and regulation. They were used for the experiment and procured from the animal house of the Southwest University, Chongqing. The animals were maintained in a room with a 12:12 h L:D cycle temperature of 22 ± 2 °C, and Relative humidity of 50–70%. The animals were fed with a balanced commercial diet and water ad libitum. They were allowed for 6-day rest period before the start of the experiment. Experimental protocol approved by International Ethics Committee of Southwest University, Chongqing, China.</p></sec><sec id=\"Sec7\"><title>Induction of stress protocol</title><p id=\"Par14\">The stressor was as follows: (a) no water for 12 h, (b) no food for 12 h, (c) wet/dirty bed for 10 min, (d) press tail for 2 min, (e) day and night reverse, (f) shake the cage for 10 min, (g) tight the legs for 3 min. The animals were exposed to one of the seven various restraint stressors for different time manner. The animals exposed to stressor were returned to the animal house after completion following stressor exposure to minimize the disturbance of the control group, group 1. The sequences of the stressors were exchanged alternatively every week. Rats were kept under rest remaining days of the week. After four weeks of stressors the animals were randomly divided into two groups, groups 2 and 3. Each group had eight to ten animals (n = 8–10). Group 3 was treated with 50 mg thymol/kg body weight (Sigma Aldrich, Catalog No: G8802B). Thymol was dissolved in 0.9% NaCl along with tween 80 (1%) and then orally fed on the fifth and sixth consecutive weeks to the animals of group-3 as indicated. The experiments were conducted for six weeks.</p></sec><sec id=\"Sec8\"><title>Cotton nestle shredding test</title><p id=\"Par15\">Rats were individually placed in a clean cage with a cotton material comprising 5 cm<sup>2</sup> squares of compressed cotton allowed in the cage at 12 h; the percentage of the area of cotton shredded was measured by NIH software (ImageJ).</p></sec><sec id=\"Sec9\"><title>Whole gut transit time (GIT) test</title><p id=\"Par16\">In the morning at 8 a.m. of each experimental animal were transferred into individual empty polyethylene cage and were left to acclimatize to the cage for 1 h. 5% Evan blue and 5% gum Arabic dissolved in 0.9% saline was feed orally to those rats. Rats were returned to their individual cage. The time from the end of the experiment to the notice of the first blue fecal pellet was measured in minutes and constituted the whole gut transit time. During the whole gut transit time the number of fecal pellets in each cage was counted for gastro intestinal hypermotility.</p></sec><sec id=\"Sec10\"><title>Abdominal withdraw reflex (AWR) test</title><p id=\"Par17\">Rats were anaesthetized by ethyl ether and catheter with syringe was inserted (about 8 cm after lubrication with paraffin oil) through anus. When the rats woke up, they were put in a special transparent plastic cage; after the rat adapted to the environment, air was gradually injected into the catheter to expand the intestinal tract, recording the volume of injected air when rats reach to three points. This experiment was repeated three times, with an interval of five minutes.</p></sec><sec id=\"Sec11\"><title>Tissue processing for histology and immunofluorescence</title><sec id=\"Sec12\"><title>Intestine and colon for histology</title><p id=\"Par18\">At the end of the behavioral experimental, animals were autopsied after anesthesia with ether. 2 cm of intestine and colon were removed, placed in 4% paraformaldehyde overnight, and sent for histologic processing. The small intestine and colon were fixed in formalin, dehydrated in grade of alcohols and xylene, embedded in paraffin wax, cut into 5 µm cross section by microtome. A cross section of intestine and colon were attached in pre albumin coated slide to be stained with hematoxylin and eosin. Intestine and colon section slides were imaged using a NIKON Eclipse e100.</p></sec><sec id=\"Sec13\"><title>Intestine and colon for immunofluorescence histochemistry</title><p id=\"Par19\">The intestine and colon were dissected and stored in fixative overnight, after that washed with PBS 3–4 times, and transferred into 30% sucrose (VWR Life Science, Catalog No.: M117-500G), stored at 4 °C. Intestine and colon were sectioned at 20 µm cut on a freezing cryostat. Then the sections were rinsed three times with Tribase saline and 0.1% tween 20 (TBST). Non-specific antibody reactions were blocked with 5% horse serum (v/v) in TBST. Sections were incubated with primary 5-HT<sub>3</sub>AR rabbit polyclonal (Proteintech, Catalog No.: 100443-1-AP, dilution 1:200) and mu Opioid R rabbit polyclonal (Novus Biologicals, Catalog No: NB100-1,620, dilution 1:200) in TBST containing 5% horse serum at 4 °C for overnight. After being washed three times in TBST, sections were incubated with secondary anti rabbit IgG (H + L) F(ab′) fragment conjugate with Alexa Fluor 488 antibody (Cell signaling Technology, Catalog No: 4480S, dilution 1:250) at room temperature for 1 h, keeping in a dark humidified chamber. Finally, sections were cover slipped with antifade mounting medium with DAPI (Vectashield, Catalog No: H-1200). Fluorescent images were captured with Leica dmi8, and processed using the program Leica Application Suite X 3.3.3.16958.</p></sec></sec><sec id=\"Sec14\"><title>Statistical analysis</title><p id=\"Par20\">All data were analyzed using Prism 6 software (GraphPad software). Experiments were analyzed using one-way ANOVA and Tukey corrections for multiple testing between categories. Data is presented with means ± standard error of the mean (SEM) shown as line and sticker.</p></sec><sec id=\"Sec15\"><title>Data retrieval and molecular docking studies</title><p id=\"Par21\">For the entire receptor, which includes intra and extra cellular domains, 4PIR was used<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. For extra cellular domain (ECD) four receptor structures in different conformations were retrieved from Protein Data Bank (PDB). These are 6HIQ in intermediate conformation (I2) of 5-HT<sub>3</sub>A receptor with serotonin and TMPPAA (positive allosteric modulator); 6HIO in intermediate conformation (I1) of the receptor with serotonin; 6HIN in open state of the extracellular domain (ECD) of the receptor with serotonin; 6HIS in transition (T) conformation with tropisetron as antagonist. Thymol structure was obtained from PubChem (CID: 6989). Missing atoms in 6HIS ECD were modelled using Prime version 5.4 (r012)<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>.</p><p id=\"Par22\">The docking validation was, first, done by re-docking serotonin to the 5-HT<sub>3</sub> receptor (6HIQ) using blind docking approach via QuickVina-W<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. The RMSD value between crystalized and docked serotonin was RMSD 2.2 Å (Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S9</xref>). Blind docking experiments were, then, repeated against full length protein (4PIR) as well as against the ECD domain in four different conformations. Thymol, serotonin, tropisetron and NAG (<italic>N</italic>-acetly-<sc>d</sc>-Glucosamine, the co-crystal ligand in 4PIR) were docked in the 5 five structures. The complete docking parameters are in Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref> and Table <xref rid=\"MOESM1\" ref-type=\"media\">S4</xref>. Root mean square deviation (RMSD) values were computed using GROMACS (Version 5.1.2)<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup> without least-squares fitting of the structures.</p></sec><sec id=\"Sec16\"><title>Molecular dynamics simulations and analysis</title><p id=\"Par23\">Nine MD simulations were done using the ECD of the receptor extracted from different conformations. TMPPAA was not included in the simulations. Systems were simulated in a dodecahedron box. The distance between the solute and the box was set to 1.0 nm. TIP3P water model was used with a concentration of 0.15 M Na + (sodium) and Cl− (chloride) ions. Energy minimization was done via steepest descent method with a maximum force set at < 1,000.0 kJ/mol/nm and a maximum number of steps to 50,000. This was followed by equilibration at 300 K and 1 atm with 50 ps of MD simulation in the isothermal-isobaric ensemble and subsequently in the canonical one. A cutoff of 10 Å was used for the Lennard–Jones and short-range electrostatic interactions. The smooth particle mesh Ewald method and a fourth-order interpolation scheme were used for the long-range electrostatic interactions. The leap-frog algorithm was used for integration. Ligand topology files were generated via ACPYPE<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup> using a total charge of zero for all ligands. Simulations were conducted on a remote machine at Center for High Performance Computing (CHPC) with GROMACS version 2016 using the Amber ff99SB-ILDN<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup> force field. The generated trajectories were analyzed with GROMACS modules using RMSD, radius of gyration (Rg) and root mean square fluctuations (RMSF) to assess protein stability. Ligand stability was assessed using the RMSD of the ligand heavy atoms after being least-squares fitted to the protein backbone and also using the number of hydrogen bonds they formed.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec17\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70420_MOESM1_ESM.docx\"><caption><p>Supplementary Information.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Selvaraj Subramaniyam and Shuyou Yang.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70420-4.</p></sec><ack><title>Acknowledgements</title><p>SB sincerely thanks to Southwest University for the financial support (SWU Grant No 104290/22300504) of the experiments. BND is supported through the DELTAS Africa Initiative [DELGEME Grant 107740/Z/15/Z]. The DELTAS Africa Initiative is an independent funding scheme of the African Academy of Sciences (AAS)’s Alliance for Accelerating Excellence in Science in Africa (AESA) and supported by the New Partnership for Africa’s Development Planning and Coordinating Agency (NEPAD Agency) with funding from the Wellcome Trust [DELGEME Grant 107740/Z/15/Z] and the UK government. The views expressed in this publication are those of the author(s) and not necessarily those of AAS, NEPAD Agency, Wellcome Trust or the UK government. Authors acknowledge the use of the Centre for High Performance Computing (CHPC), Cape Town, South Africa for the MD simulations.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>Prof. S.B. conceived and designed the study. Prof. Ö.T.B. further advanced the concept and provided rigorous MD simulation analysis. Dr. S.S., Ms. S.Y., Dr. B.N.D., Mr. X.F., conducted experiments. Dr. S.S. advanced the experimental study. Prof. L.L. and Prof. C.L. provided experimental aid and helping hand to accomplish the experiment. Dr. S.S. and Prof. S.B. interpreted the results and Dr. S.S. carried out statistical analysis. Prof. S.B. wrote the paper which was edited and revised by Prof. Ö.T.B.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par24\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moloney</surname><given-names>RD</given-names></name><name><surname>O'Mahony</surname><given-names>SM</given-names></name><name><surname>Dinan</surname><given-names>TG</given-names></name><name><surname>Cryan</surname><given-names>JF</given-names></name></person-group><article-title>Stress-induced visceral pain: toward animal models of irritable-bowel syndrome and associated comorbidities</article-title><source>Front. Psychiatry</source><year>2015</year><volume>6</volume><fpage>15</fpage><pub-id pub-id-type=\"doi\">10.3389/fpsyt.2015.00015</pub-id><pub-id pub-id-type=\"pmid\">25762939</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Larauche</surname><given-names>M</given-names></name><name><surname>Mulak</surname><given-names>A</given-names></name><name><surname>Tache</surname><given-names>Y</given-names></name></person-group><article-title>Stress-related alterations of visceral sensation: animal models for irritable bowel syndrome study</article-title><source>J. Neurogastroenterol. Motil.</source><year>2011</year><volume>17</volume><fpage>213</fpage><lpage>234</lpage><pub-id pub-id-type=\"doi\">10.5056/jnm.2011.17.3.213</pub-id><pub-id pub-id-type=\"pmid\">21860814</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mayer</surname><given-names>EA</given-names></name><name><surname>Collins</surname><given-names>SM</given-names></name></person-group><article-title>Evolving pathophysiologic models of functional gastrointestinal disorders</article-title><source>Gastroenterology</source><year>2002</year><volume>122</volume><fpage>2032</fpage><lpage>2048</lpage><pub-id pub-id-type=\"doi\">10.1053/gast.2002.33584</pub-id><pub-id pub-id-type=\"pmid\">12055608</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Videlock</surname><given-names>EJ</given-names></name><etal/></person-group><article-title>Childhood trauma is associated with hypothalamic-pituitary-adrenal axis responsiveness in irritable bowel syndrome</article-title><source>Gastroenterology</source><year>2009</year><volume>137</volume><fpage>1954</fpage><lpage>1962</lpage><pub-id pub-id-type=\"doi\">10.1053/j.gastro.2009.08.058</pub-id><pub-id pub-id-type=\"pmid\">19737564</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McEwen</surname><given-names>BS</given-names></name></person-group><article-title>Allostasis and allostatic load: implications for neuropsychopharmacology</article-title><source>Neuropsychopharmacology</source><year>2000</year><volume>22</volume><fpage>108</fpage><lpage>124</lpage><pub-id pub-id-type=\"doi\">10.1016/S0893-133X(99)00129-3</pub-id><pub-id pub-id-type=\"pmid\">10649824</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chrousos</surname><given-names>GP</given-names></name></person-group><article-title>Stress and disorders of the stress system</article-title><source>Nat. Rev. Endocrinol.</source><year>2009</year><volume>5</volume><fpage>374</fpage><lpage>381</lpage><pub-id pub-id-type=\"doi\">10.1038/nrendo.2009.106</pub-id><pub-id pub-id-type=\"pmid\">19488073</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McEwen</surname><given-names>BS</given-names></name></person-group><article-title>Protective and damaging effects of stress mediators</article-title><source>N. Engl. J. Med.</source><year>1998</year><volume>338</volume><fpage>171</fpage><lpage>179</lpage><pub-id pub-id-type=\"doi\">10.1056/NEJM199801153380307</pub-id><pub-id pub-id-type=\"pmid\">9428819</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Holschneider</surname><given-names>DP</given-names></name><name><surname>Bradesi</surname><given-names>S</given-names></name><name><surname>Mayer</surname><given-names>EA</given-names></name></person-group><article-title>The role of experimental models in developing new treatments for irritable bowel syndrome</article-title><source>Expert Rev. Gastroenterol. Hepatol.</source><year>2011</year><volume>5</volume><fpage>43</fpage><lpage>57</lpage><pub-id pub-id-type=\"doi\">10.1586/egh.10.88</pub-id><pub-id pub-id-type=\"pmid\">21309671</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fass</surname><given-names>R</given-names></name><name><surname>Achem</surname><given-names>SR</given-names></name></person-group><article-title>Noncardiac chest pain: epidemiology, natural course and pathogenesis</article-title><source>J. Neurogastroenterol. Motil.</source><year>2011</year><volume>17</volume><fpage>110</fpage><lpage>123</lpage><pub-id pub-id-type=\"doi\">10.5056/jnm.2011.17.2.110</pub-id><pub-id pub-id-type=\"pmid\">21602987</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grundy</surname><given-names>D</given-names></name></person-group><article-title>Neuroanatomy of visceral nociception: vagal and splanchnic afferent</article-title><source>Gut</source><year>2002</year><volume>51</volume><issue>Suppl 1</issue><fpage>i2</fpage><lpage>5</lpage><pub-id pub-id-type=\"doi\">10.1136/gut.51.suppl_1.i2</pub-id><pub-id pub-id-type=\"pmid\">12077054</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Barboza</surname><given-names>JL</given-names></name><name><surname>Talley</surname><given-names>NJ</given-names></name><name><surname>Moshiree</surname><given-names>B</given-names></name></person-group><article-title>Current and emerging pharmacotherapeutic options for irritable bowel syndrome</article-title><source>Drugs</source><year>2014</year><volume>74</volume><fpage>1849</fpage><lpage>1870</lpage><pub-id pub-id-type=\"doi\">10.1007/s40265-014-0292-7</pub-id><pub-id pub-id-type=\"pmid\">25260888</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Camilleri</surname><given-names>M</given-names></name></person-group><article-title>Is there a SERT-ain association with IBS?</article-title><source>Gut</source><year>2004</year><volume>53</volume><fpage>1396</fpage><lpage>1399</lpage><pub-id pub-id-type=\"doi\">10.1136/gut.2004.039826</pub-id><pub-id pub-id-type=\"pmid\">15361483</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bhalerao</surname><given-names>YP</given-names></name><name><surname>Wagh</surname><given-names>S</given-names></name></person-group><article-title>J review on thymol encapsulation and its controlled release through biodegradable polymer shells</article-title><source>Int. J. Pharm. Sci. Res.</source><year>2018</year><volume>9</volume><fpage>4522</fpage><lpage>4532</lpage><pub-id pub-id-type=\"doi\">10.13040/IJPSR.0975-8232.9(11).4522-32</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Camilleri</surname><given-names>M</given-names></name></person-group><article-title>Scintigraphic biomarkers for colonic dysmotility</article-title><source>Clin. Pharmacol. Ther.</source><year>2010</year><volume>87</volume><fpage>748</fpage><lpage>753</lpage><pub-id pub-id-type=\"doi\">10.1038/clpt.2010.23</pub-id><pub-id pub-id-type=\"pmid\">20410880</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>SW</given-names></name><name><surname>Rao</surname><given-names>SS</given-names></name></person-group><article-title>Advances in the management of constipation-predominant irritable bowel syndrome: the role of linaclotide</article-title><source>Therap. Adv. Gastroenterol.</source><year>2014</year><volume>7</volume><fpage>193</fpage><lpage>205</lpage><pub-id pub-id-type=\"doi\">10.1177/1756283X14537882</pub-id><pub-id pub-id-type=\"pmid\">25177366</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Camilleri</surname><given-names>M</given-names></name><etal/></person-group><article-title>Pharmacological and pharmacokinetic aspects of functional gastrointestinal disorders</article-title><source>Gastroenterology</source><year>2006</year><volume>130</volume><fpage>1421</fpage><lpage>1434</lpage><pub-id pub-id-type=\"doi\">10.1053/j.gastro.2005.08.062</pub-id><pub-id pub-id-type=\"pmid\">16678556</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bercik</surname><given-names>P</given-names></name><etal/></person-group><article-title>The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice</article-title><source>Gastroenterology</source><year>2011</year><volume>141</volume><fpage>599</fpage><lpage>609</lpage><pub-id pub-id-type=\"doi\">10.1053/j.gastro.2011.04.052</pub-id><pub-id pub-id-type=\"pmid\">21683077</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ness</surname><given-names>TJ</given-names></name><name><surname>Gebhart</surname><given-names>GF</given-names></name></person-group><article-title>Colorectal distension as a noxious visceral stimulus: physiologic and pharmacologic characterization of pseudaffective reflexes in the rat</article-title><source>Brain Res.</source><year>1988</year><volume>450</volume><fpage>153</fpage><lpage>169</lpage><pub-id pub-id-type=\"doi\">10.1016/0006-8993(88)91555-7</pub-id><pub-id pub-id-type=\"pmid\">3401708</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mayer</surname><given-names>EA</given-names></name><etal/></person-group><article-title>The effect of the 5-HT3 receptor antagonist, alosetron, on brain responses to visceral stimulation in irritable bowel syndrome patients</article-title><source>Aliment. Pharmacol. Ther.</source><year>2002</year><volume>16</volume><fpage>1357</fpage><lpage>1366</lpage><pub-id pub-id-type=\"doi\">10.1046/j.1365-2036.2002.01287.x</pub-id><pub-id pub-id-type=\"pmid\">12144587</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rousseaux</surname><given-names>C</given-names></name><etal/></person-group><article-title>Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors</article-title><source>Nat. Med.</source><year>2007</year><volume>13</volume><fpage>35</fpage><lpage>37</lpage><pub-id pub-id-type=\"doi\">10.1038/nm1521</pub-id><pub-id pub-id-type=\"pmid\">17159985</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Holdgate</surname><given-names>A</given-names></name><name><surname>Pollock</surname><given-names>T</given-names></name></person-group><article-title>Nonsteroidal anti-inflammatory drugs (NSAIDs) versus opioids for acute renal colic</article-title><source>Cochrane Database Syst. Rev.</source><year>2004</year><pub-id pub-id-type=\"doi\">10.1002/14651858.CD004137.pub2</pub-id><pub-id pub-id-type=\"pmid\">14974058</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lansdell</surname><given-names>SJ</given-names></name><name><surname>Sathyaprakash</surname><given-names>C</given-names></name><name><surname>Doward</surname><given-names>A</given-names></name><name><surname>Millar</surname><given-names>NS</given-names></name></person-group><article-title>Activation of human 5-hydroxytryptamine type 3 receptors via an allosteric transmembrane site</article-title><source>Mol Pharmacol</source><year>2015</year><volume>87</volume><fpage>87</fpage><lpage>95</lpage><pub-id pub-id-type=\"doi\">10.1124/mol.114.094540</pub-id><pub-id pub-id-type=\"pmid\">25338672</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Polovinkin</surname><given-names>L</given-names></name><etal/></person-group><article-title>Conformational transitions of the serotonin 5-HT3 receptor</article-title><source>Nature</source><year>2018</year><volume>563</volume><fpage>275</fpage><lpage>279</lpage><pub-id pub-id-type=\"doi\">10.1038/s41586-018-0672-3</pub-id><pub-id pub-id-type=\"pmid\">30401839</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fichna</surname><given-names>J</given-names></name><name><surname>Storr</surname><given-names>M</given-names></name></person-group><article-title>Brain-Gut Interactions in IBS</article-title><source>Front. Pharmacol.</source><year>2012</year><pub-id pub-id-type=\"doi\">10.3389/fphar.2012.00127</pub-id><pub-id pub-id-type=\"pmid\">22783191</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boruga</surname><given-names>O</given-names></name><etal/></person-group><article-title>Thymus vulgaris essential oil: chemical composition and antimicrobial activity</article-title><source>J. Med. Life</source><year>2014</year><volume>7</volume><issue>Spec No. 3</issue><fpage>56</fpage><lpage>60</lpage><pub-id pub-id-type=\"pmid\">25870697</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kilpatrick</surname><given-names>LA</given-names></name><etal/></person-group><article-title>The HTR3A polymorphism c. -42C>T is associated with amygdala responsiveness in patients with irritable bowel syndrome</article-title><source>Gastroenterology</source><year>2011</year><volume>140</volume><fpage>1943</fpage><lpage>1951</lpage><pub-id pub-id-type=\"doi\">10.1053/j.gastro.2011.03.011</pub-id><pub-id pub-id-type=\"pmid\">21420406</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jacobson</surname><given-names>MP</given-names></name><etal/></person-group><article-title>A hierarchical approach to all-atom protein loop prediction</article-title><source>Proteins Struct. Funct. Bioinform.</source><year>2004</year><volume>55</volume><fpage>351</fpage><lpage>367</lpage><pub-id pub-id-type=\"doi\">10.1002/prot.10613</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hassan</surname><given-names>NM</given-names></name><name><surname>Alhossary</surname><given-names>AA</given-names></name><name><surname>Mu</surname><given-names>Y</given-names></name><name><surname>Kwoh</surname><given-names>CK</given-names></name></person-group><article-title>Protein-ligand blind docking using QuickVina-W with inter-process spatio-temporal integration</article-title><source>Sci. Rep.</source><year>2017</year><volume>7</volume><fpage>15451</fpage><pub-id pub-id-type=\"doi\">10.1038/s41598-017-15571-7</pub-id><pub-id pub-id-type=\"pmid\">29133831</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pronk</surname><given-names>S</given-names></name><etal/></person-group><article-title>GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit</article-title><source>Bioinformatics</source><year>2013</year><volume>29</volume><fpage>845</fpage><lpage>854</lpage><pub-id pub-id-type=\"doi\">10.1093/bioinformatics/btt055</pub-id><pub-id pub-id-type=\"pmid\">23407358</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sousa da Silva</surname><given-names>AW</given-names></name><name><surname>Vranken</surname><given-names>WF</given-names></name></person-group><article-title>ACPYPE—AnteChamber PYthon Parser interfacE</article-title><source>BMC Res Notes</source><year>2012</year><volume>5</volume><fpage>367</fpage><pub-id pub-id-type=\"doi\">10.1186/1756-0500-5-367</pub-id><pub-id pub-id-type=\"pmid\">22824207</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lindorff-Larsen</surname><given-names>K</given-names></name><etal/></person-group><article-title>Improved side-chain torsion potentials for the Amber ff99SB protein force field</article-title><source>Proteins</source><year>2010</year><volume>78</volume><fpage>1950</fpage><lpage>1958</lpage><pub-id pub-id-type=\"doi\">10.1002/prot.22711</pub-id><pub-id pub-id-type=\"pmid\">20408171</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807857</article-id><article-id pub-id-type=\"pmc\">PMC7431531</article-id><article-id pub-id-type=\"publisher-id\">70483</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70483-3</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Social isolation, loneliness and physical performance in older-adults: fixed effects analyses of a cohort study</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-9614-3580</contrib-id><name><surname>Philip</surname><given-names>Keir E. J.</given-names></name><address><email>k.philip@imperial.ac.uk</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Polkey</surname><given-names>Michael I.</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hopkinson</surname><given-names>Nicholas S.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Steptoe</surname><given-names>Andrew</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Fancourt</surname><given-names>Daisy</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.7445.2</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2113 8111</institution-id><institution>NHLI Respiratory Muscle Laboratory, </institution><institution>National Heart and Lung Institute, Imperial College London, </institution></institution-wrap>Royal Brompton Campus, Fulham Rd., London, SW3 6NP UK </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.421662.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9216 5443</institution-id><institution>Respiratory Medicine, </institution><institution>Royal Brompton and Harefield NHS Foundation Trust, </institution></institution-wrap>London, UK </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.83440.3b</institution-id><institution-id institution-id-type=\"ISNI\">0000000121901201</institution-id><institution>Department of Behavioural Science and Health, </institution><institution>University College London, </institution></institution-wrap>London, UK </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13908</elocation-id><history><date date-type=\"received\"><day>24</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>14</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Isolation and loneliness are related to various aspects of health. Physical performance is a central component of health. However, its relationship with isolation and loneliness is not well understood. We therefore assessed the relationship between loneliness, different aspects of social isolation, and physical performance over time. 8,780 participants from the English Longitudinal Study of Ageing, assessed three times over 8 years of follow-up, were included. Measures included physical performance (Short Physical Performance Battery), loneliness (modified UCLA Loneliness Scale), and isolation considered in three ways (domestic isolation, social disengagement, low social contact). Fixed effects regression models were used to estimate the relationship between changes in these parameters. Missing data were imputed to account for variable response and ensure a representative sample. Loneliness, domestic isolation and social disengagement were longitudinally associated with poorer physical performance when accounting for both time-invariant and time-variant confounders (loneliness: coef = − 0.06, 95% CI − 0.09 to − 0.02; domestic isolation: coef = − 0.32, 95% CI − 0.46 to − 0.19; social disengagement: coef = − 0.10, 95% CI − 0.12 to − 0.07). Low social contact was not associated with physical performance. These findings suggest social participation and subjectively meaningful interpersonal interactions are related to physical performance, and highlight additional considerations regarding social distancing related to COVID-19 control measures.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Geriatrics</kwd><kwd>Public health</kwd><kwd>Quality of life</kwd></kwd-group><funding-group><award-group><funding-source><institution>National Institute for Health Research</institution></funding-source><award-id>Academic Clinical Fellowship</award-id><principal-award-recipient><name><surname>Philip</surname><given-names>Keir E. J.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/501100000761</institution-id><institution>Imperial College London</institution></institution-wrap></funding-source><award-id>Imperial College Clinician Investigator Scholarship</award-id><principal-award-recipient><name><surname>Philip</surname><given-names>Keir E. J.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>The Wellcome Trust</institution></funding-source><award-id>205407/Z/16/Z</award-id><principal-award-recipient><name><surname>Fancourt</surname><given-names>Daisy</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>The Nuffield Foundation</institution></funding-source><award-id>WEL/FR-000022583</award-id><principal-award-recipient><name><surname>Fancourt</surname><given-names>Daisy</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Social factors including isolation (frequency of social interactions) and loneliness (the subjective quality of social interaction) are related but distinct concepts, both of which have been shown to be associated with morbidity and mortality<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. Much of this literature has focused on mental health, showing associations with higher levels of stress and depression<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>, but there has been increasing interest in effects on physical health. For example, both isolation and loneliness have been linked with systemic inflammation<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>, autonomic dysfunction<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>, neuroendocrine dysregulation<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>, chronically increased allostatic load<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>, stroke and coronary heart disease<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>.\n</p><p id=\"Par3\">However, a less researched area is the relationship between changes in isolation, loneliness and physical performance. Physical performance, based on objective testing, depends on factors including strength, balance and endurance is a key component of physical function, mobility and independence<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Impaired physical performance can be associated with significant personal, societal and economic costs<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Furthermore, understanding the relationship between social isolation, loneliness and physical performance in older-adults has become acutely important in the context of the COVID-19 pandemic. Because older people are particularly at risk from the disease, social distancing measures have been introduced throughout the world to reduce their exposure to SARS-CoV-2, the virus that causes COVID-19. These measures are appropriate in the current context but are likely to have various unintended mental and physical health impacts. Improving our understanding of these impacts can facilitate mitigation of negative sequalae through targeted interventions, such as homebased exercise programmes and online communal activities.</p><p id=\"Par4\">A small number of studies have suggested relationships between social isolation, loneliness and physical performance. For example, loneliness and less frequent engagement in social activities have been associated with reductions in objective markers of motor function in older adults<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup> and walking speed<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. Other studies using subjective measures of function have shown that individuals with more social ties show slower rates of functional decline<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>, and reduced participation in social activities has been linked to functional disability<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. However, these studies have focused on how social factors measured at a single point in time are associated with physical performance in subsequent years. Yet social isolation and loneliness are dynamic states that fluctuate, both according to life circumstances but also as people age. Further, there are a wide range of potential confounding factors that also vary over time and could explain any potential association. Additionally, isolation can, and has been, defined in a variety of ways. People may be isolated because they live alone, because they have few social interactions with friends or family, or because they don’t participate in organisations and community activities. The inclusion or exclusion of various components of isolation may influence the relationship between these variables and physical performance. This is of crucial importance if the research findings are to make useful contributions to planning health and social care interventions and policy.</p><p id=\"Par5\">To address these challenges, we used a statistical approach that enables the tracking of patterns of behaviour over time to explore the relationship between time-varying loneliness and social isolation and time-varying physical performance amongst a representative sample of adults aged 50+ in England, whilst accounting for time-varying confounding demographic and life-style factors. In addition, in this study we also explored three different types of social isolation to help clarify which components of isolation were of greatest importance in relation to physical performance.</p></sec><sec id=\"Sec2\"><title>Methods</title><sec id=\"Sec3\"><title>Study design and participants</title><p id=\"Par6\">We used data from the English Longitudinal Study of Ageing (ELSA), a nationally representative cohort study of community dwelling adults aged over 50 in England. The sample was drawn from households that had previously responded to the Health Survey for England (HSE) in 1998, 1999 or 2001. HSE used a two-stage sampling design consisting of the selection of postcode sectors from a postcode address file and then subsequent selection of postcode addresses from within each postcode sector<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. All core participants from wave 2 of ELSA (2004/05—when physical performance was first measured) were included (n = 8,780), and were followed up in waves 4 (2008/09) and 6 (2012/13). We maintained a sample size of 8,780 participants through the waves by imputation of missing data, as described below. In ELSA, the primary method of data collection includes computer assisted interviews which are completed face-to-face at the participants’ usual place of residence, and a physical assessment conducted by a qualified nurse trained in the specific study protocols. Full documentation of data collection protocols is available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.elsa-project.ac.uk/\">https://www.elsa-project.ac.uk/</ext-link>. ELSA received ethical approval from the National Research Ethics Service and all participants provided written informed consent.</p></sec><sec id=\"Sec4\"><title>Measures</title><sec id=\"Sec5\"><title>Physical performance</title><p id=\"Par7\">Physical performance was assessed using the Short Physical Performance Battery (SPPB), which is rated highly in terms of validity, reliability and responsiveness<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. SPPB assesses three components: five times sit-to-stand, standing balance, and gait-speed<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Sit-to-stand was assessed by timing participants completing five repetitions of standing from a seated position, without using their arms to push themselves up. Balance was assessed through sequentially more difficult balance tasks involving standing first with feet side-by-side, then with feet in semi-tandem, then in full tandem. Participants were asked to try and stay in each position for 10 s without moving their feet or holding any supports, with their ability to complete the task scored according to established categories. Walking speed was assessed by timing participants as they completed an 8ft (2.44 m) walking course at their normal speed, with the fastest speed of two attempts recorded. Walking speed was only measured for those over the age of 60, so for those below we used multiple imputation to impute missing values and then ran sensitivity analyses for individuals above and below the age of 60 to assess whether the results were affected by these imputations. Each component was then categorised into a score ranging from 0 to 4, with the scores for each of these three components then summed to provide a total score from 0 to 12 (with higher scores indicating better function), see Guralnik et al. for further details regarding scorin<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. A detailed standardised protocol for the performance tests is available online at <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.elsa-project.ac.uk/data-and-documentation\">https://www.elsa-project.ac.uk/data-and-documentation</ext-link>.</p></sec><sec id=\"Sec6\"><title>Social isolation</title><p id=\"Par8\">We used three measures of social isolation. Domestic isolation we defined based on whether individuals lived alone or not. Although domestic isolation is highly correlated with marital status, being married does not guarantee living with a spouse, and individuals who are unmarried can live with friends or family. So, living status is a more reliable marker of domestic isolation.</p><p id=\"Par9\">To assess social contact, we used self-reported frequency of social interactions. This included (a) face to face interaction, (b) telephone conversations, or (c) email or written communication with (1) children, (2) other family members or (3) friends. A point was given for use of each mode of communication with each group of people that an individual did not have contact with, providing an overall score from 0 to 9 with higher scores indicating greater social isolation. The index had a Cronbach’s alpha of 0.80.</p><p id=\"Par10\">For social disengagement, we measured frequency of (1) participation in community group activities (including political party, trade union or environmental groups, tenant groups, resident groups, neighbourhood watch groups, church or other religious groups, charitable associations, education, arts or music groups or evening classes, social clubs, sports clubs, exercise classes, or any other organisations, clubs or societies), and (2) engagement with community cultural activities (including going to museums, exhibitions, the theatre, concerts, opera or the cinema). Frequency of community group activities was measured as number in the past 12 months and then recoded as never (score of 4), once or twice a year (score of 3), every few months (score of 2), or monthly or more (score of 1). Frequency of cultural activities was as never (score of 4), less than once a year (score of 3), once or twice a year (score of 2), or every few months or more (score of 1). These scores were summed to provide an overall index of 2–8, with higher scores indicating higher levels of social disengagement. The index had a Cronbach’s alpha of 0.73.</p></sec><sec id=\"Sec7\"><title>Loneliness</title><p id=\"Par11\">Loneliness was measured using an adapted 3-item questionnaire<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup> based on the UCLA loneliness scale, which has strong psychometric properties<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. ELSA respondents were asked how often they (1) felt that they lacked companionship (2) felt left out and (3) felt isolated from the people around them. Frequencies ranged from hardly ever or never (assigned a score of 3) some of the time (assigned a score of 2) and often (assigned a score of 1). The scores for each measure were then summed to give a loneliness score ranging from 3 to 9 where higher scores indicated higher levels of loneliness. The index had a Cronbach’s alpha of 0.83.</p></sec><sec id=\"Sec8\"><title>Covariates</title><p id=\"Par12\">Time variable covariates included age (continuous), marital status (married/cohabiting vs other), employment status (working part-time or full-time vs not currently working), wealth (quintiles). Health related covariates included body mass index (BMI) (continuous), eyesight (very poor/registered blind vs fair/good/very good/excellent); presence of significant physician diagnosed co-morbidities (binary composite variable including arthritis, Parkinson’s disease, congestive heart failure, ‘other heart problem’, Alzheimer’s disease or other dementia, previous stroke with ongoing limb weakness, chronic lung disease such as chronic bronchitis, asthma); chronic pain (no/mild vs moderate/severe); frequency of alcohol consumption (less than once a week vs once to four times per week vs five or more times per week); smoking (never vs ex-smoker vs current); inactive (binary variable—undertakes any kind of sports or activities that are mildly energetic less than weekly vs at least weekly); cognition (using an index of scores from neuropsychiatric batteries testing memory, executive function, processing speed and orientation in time, averaged and standardised); and depression (using the Centre for Epidemiological Studies scale CES-D<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>).</p></sec></sec><sec id=\"Sec9\"><title>Statistical analysis</title><p id=\"Par13\">Analyses were carried out using Stata 14 (StataCorp, College Station, TX). Missing data were imputed using multiple imputation by chained equations to provide 50 imputed datasets using the following predictor variables: physical performance (total SPPB score, five times sit-to-stand score, balance and walking speed), demographic factors (age, sex, marital status, employment status and wealth), and health factors (BMI, eyesight, comorbidities, chronic pain, frequency of alcohol consumption, smoking habits, inactivity, cognition and mental health). Patterns of missing data are shown in the Supplementary Material (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">9</xref>).</p><p id=\"Par14\">Fixed effects regression models were used to estimate the relationship between changes in isolation, loneliness and physical performance. Fixed effects regression is a longitudinal statistical technique that has several strengths: (1) it considers time-varying relationships, which is important when considering states such as isolation and loneliness which are likely to change as people age and be influenced by other time-varying factors such as health or retirement status; (2) in fixed-effects regression, within-person variation is explored with individuals acting as their own reference point over time. So all time-invariant factors (e.g. gender, ethnicity, genetics, personality, educational attainment and socio-economic status) are accounted for even if unobserved<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. This can help to reduce the potential for unobserved confounding leading to spurious results. The basic model for physical performance (SPPB) can be expressed as follows:<disp-formula id=\"Equa\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${SPPB}_{it}={{\\beta }_{0t}+ {\\beta }_{1}S}_{it}+ {\\beta }_{2}{T}_{it}+{\\alpha }_{i}+ {\\varepsilon }_{it}$$\\end{document}</tex-math><mml:math id=\"M2\" display=\"block\"><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"italic\">SPPB</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"italic\">it</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mrow><mml:msub><mml:mi>β</mml:mi><mml:mrow><mml:mn>0</mml:mn><mml:mi>t</mml:mi></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>β</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mi>S</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"italic\">it</mml:mi></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>β</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:msub><mml:mi>T</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">it</mml:mi></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>α</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>ε</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">it</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70483_Article_Equa.gif\" position=\"anchor\"/></alternatives></disp-formula>where SPPB<sub>it</sub> is a measure of individual i’s levels of physical performance at time t, α<sub>i</sub> is unobserved time invariant confounding factors, S is whether an individual was experiencing isolation or loneliness, at time t, and T is measured time-varying confounding factors. Data were strongly balanced. A Hausman test was used to confirm the selection of a fixed effects over a random effects model. The modified Wald test for group-wise heteroscedasticity was significant so sandwich estimators were applied. Coefficients for all years were not jointly equal to zero, so time-fixed effects were included in the model. All other model assumptions were met.</p><p id=\"Par16\">In addition to time-invariant factors already considered in the model and time itself (model 1), model 2 additionally adjusted for time-varying demographic factors (age, marital status, employment status and wealth). Model 3: additionally adjusted for time-varying health factors (BMI, eyesight, comorbidities, chronic pain, frequency of alcohol consumption, smoking habits, inactivity and cognition). Model 4: additionally adjusted for time-varying mental health (depression).</p><p id=\"Par17\">Our main analyses involved entering all exposures simultaneously in the model. But our first sensitivity analysis tested the consistency of results when running separate models for each exposure. Other sensitivity analyses included (1) additional measures of moderate and vigorous physical activity alongside the measure of inactivity to confirm that time-varying engagement in sports or energetic activities did not act as a confounding factor; (2) responses split by gender and age (above and below 60 and 65) to identify if any relationship was more clearly present in some groups than others; (3) a binary outcome variable for SPPB categorising responses into above or below a score of 10, which indicates increased risk of disability<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup> and all-cause mortality<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. All analyses were weighted using cross-sectional sampling weights.</p></sec></sec><sec id=\"Sec10\"><title>Results</title><sec id=\"Sec11\"><title>Descriptive analysis</title><p id=\"Par18\">Of the 8,780 participants, 53.9% were female, the average age was 69.0 years (standard error = 0.07). Of these, 47.0% had either no or only basic qualifications (less than GCSE/O-level/qualification at age 16). At baseline, a quarter of the sample (25.5%) were employed in either full time or part time work. Full details of the sample are provided in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>. Domestic isolation was associated only very slightly with low social contact (r = 0.10, p < 0.001, 1.0% of variance shared) and greater social disengagement (r = 0.09, p < 0.001, 0.8% of variance shared) and showed a small association with higher loneliness (r = 0.27, p < 0.001, 7.2% of variance shared). Low social contact was associated in a very small way with greater social disengagement (r = 0.06, p < 0.001, 0.4% of variance shared) and loneliness (r = 0.08, p < 0.001, 0.6% of variance shared) (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">8</xref>). Social disengagement and loneliness were very slightly associated (r = 0.18, p < 0.001, 3.2% of variance shared). Over time, there was a slight decline in social activities and physical function (see Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>, which also shows within and between variation in the standard deviation of measures).<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Weighted participant characteristics at baseline.</p></caption><table frame=\"hsides\" rules=\"groups\"><tbody><tr><td align=\"left\" colspan=\"3\">Time-invariant characteristics (stated at baseline)<sup>a</sup></td></tr><tr><td align=\"left\">Gender</td><td align=\"left\">% female</td><td align=\"left\">53.9%</td></tr><tr><td align=\"left\">Ethnicity</td><td align=\"left\">White (%)</td><td align=\"left\">97.1%</td></tr><tr><td align=\"left\" rowspan=\"4\">Educational attainment<sup>b</sup></td><td align=\"left\">No qualifications/basic qualifications</td><td align=\"left\">47.0%</td></tr><tr><td align=\"left\">GCSE/O-level/qualification at age 16</td><td align=\"left\">15.9%</td></tr><tr><td align=\"left\">A-levels/higher education/qualification at age 18</td><td align=\"left\">25.9%</td></tr><tr><td align=\"left\">Degree/further higher qualification</td><td align=\"left\">11.2%</td></tr><tr><td align=\"left\" colspan=\"3\">Time-varying characteristics (stated at baseline)</td></tr><tr><td align=\"left\">Age</td><td align=\"left\">Mean (standard error)</td><td align=\"left\">69.0 (0.07)</td></tr><tr><td align=\"left\">Employment</td><td align=\"left\">Working full- or part-time (%)</td><td align=\"left\">25.5%</td></tr><tr><td align=\"left\">Wealth</td><td align=\"left\">Split into quintiles</td><td align=\"left\">–</td></tr><tr><td align=\"left\">BMI</td><td align=\"left\">Mean (standard error)</td><td align=\"left\">26.4 (0.07)</td></tr><tr><td align=\"left\">Eyesight</td><td align=\"left\">% with very poor eyesight/registered blind</td><td align=\"left\">4.0%</td></tr><tr><td align=\"left\">Chronic health conditions</td><td align=\"left\">% with one or more of cancer, chronic lung conditions, arthritis, stroke, diabetes, angina</td><td align=\"left\">45.5%</td></tr><tr><td align=\"left\">Chronic pain</td><td align=\"left\">% with moderate or severe chronic pain</td><td align=\"left\">27.9%</td></tr><tr><td align=\"left\" rowspan=\"3\">Alcohol consumption</td><td align=\"left\">Less than once a week</td><td align=\"left\">41.1%</td></tr><tr><td align=\"left\">Once to four times a week</td><td align=\"left\">36.5%</td></tr><tr><td align=\"left\">5 or more times a week</td><td align=\"left\">22.4%</td></tr><tr><td align=\"left\" rowspan=\"3\">Smoking status</td><td align=\"left\">Never smoked (%)</td><td align=\"left\">36.5%</td></tr><tr><td align=\"left\">Ex-smoker (%)</td><td align=\"left\">53.3%</td></tr><tr><td align=\"left\">Current smoker (%)</td><td align=\"left\">10.2%</td></tr><tr><td align=\"left\">Inactivity</td><td align=\"left\">Undertakes any kind of sports or energetic activities less than weekly (%)</td><td align=\"left\">8.8%</td></tr><tr><td align=\"left\">Cognition</td><td align=\"left\">Standardised</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Depression</td><td align=\"left\">(scored 3 + in CES-D) (%)</td><td align=\"left\">21.3%</td></tr></tbody></table><table-wrap-foot><p><sup>a</sup>Excluded from the analysis as time-invariant factors are automatically included within fixed-effects models, but shown here for descriptive purposes.</p><p><sup>b</sup>Considered to be time-invariant for the purpose of this investigation and therefore automatically included within analyses.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec12\"><title>Social isolation</title><p id=\"Par19\">Domestic isolation and social disengagement were longitudinally associated with poorer physical performance. Although time-varying demographic and health-related factors explained some of this association, results were significant even when accounting for all time-invariant and time-variant confounders (domestic isolation: coef = − 0.32, 95% CI − 0.46 to − 0.19; social disengagement: coef = − 0.10, 95% CI − 0.12 to − 0.07) (see Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>). When exploring the subscales of physical performance, social disengagement was associated with lower ability in sit-to-stand, poorer balance and slower walking speed, but domestic isolation was only associated with poorer balance and walking speed. In terms of effect size, the change from living alone to living with somebody was associated with an increase in average SPPB score from 9.59 (95% CI 9.51–9.67) to 9.94 (95% CI 9.91–9.97); an improvement of 4.0%. The change from being socially completely disengaged to highly engaged, was associated with an increase in average SPPB score from 9.72 (95% CI 9.66–9.77) to 10.05 (95% CI 9.99–10.12); an improvement of (3.5%). Low social contact was not associated with poorer physical performance or any of its subscales.<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Results from fixed effects models showing the relationship between isolation, loneliness and physical performance: all predictors entered simultaneously.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\" colspan=\"2\">Total physical performance</th><th align=\"left\" colspan=\"2\">5 times sit-to-stand</th><th align=\"left\" colspan=\"2\">Balance</th><th align=\"left\" colspan=\"2\">Walking speed</th></tr><tr><th align=\"left\">Coef (95% CI)</th><th align=\"left\">p</th><th align=\"left\">Coef (95% CI)</th><th align=\"left\">p</th><th align=\"left\">Coef (95% CI)</th><th align=\"left\">p</th><th align=\"left\">Coef (95% CI)</th><th align=\"left\">p</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"9\">Domestic isolation</td></tr><tr><td align=\"left\">Model 1</td><td align=\"left\">− 1.454 (− 1.71 to − 1.37)</td><td align=\"left\">< 0.001</td><td align=\"left\"><bold>− 0.47 (− 0.56 to − 0.39)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\">− <bold>0.49 (</bold>− <bold>0.56 to </bold>− <bold>0.43)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.57 (− 0.64 to − 0.50)</bold></td><td align=\"left\"><bold>< 0.001</bold></td></tr><tr><td align=\"left\">Model 2</td><td align=\"left\"><bold>− 0.36 (− 0.50 to − 0.21)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\">− 0.05 (− 0.13 to 0.02)</td><td align=\"left\">0.16</td><td align=\"left\"><bold>− 0.15 (− 0.21 to − 0.08)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.16 (− 0.22 to − 0.09)</bold></td><td align=\"left\"><bold>< 0.001</bold></td></tr><tr><td align=\"left\">Model 3</td><td align=\"left\"><bold>− 0.34 (− 0.48 to − 0.21)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\">− 0.05 (− 0.13 to 0.02)</td><td align=\"left\">0.15</td><td align=\"left\"><bold>− 0.14 (− 0.20 to − 0.08)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.15 (− 0.21 to − 0.09)</bold></td><td align=\"left\"><bold>< 0.001</bold></td></tr><tr><td align=\"left\">Model 4</td><td align=\"left\"><bold>− 0.32 (− 0.46 to − 0.19)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\">− 0.05 (− 0.12 to 0.03)</td><td align=\"left\">0.20</td><td align=\"left\"><bold>− 0.14 (− 0.20 to − 0.07)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.14 (− 0.20 to − 0.08)</bold></td><td align=\"left\"><bold>< 0.001</bold></td></tr><tr><td align=\"left\" colspan=\"9\">Low social contact</td></tr><tr><td align=\"left\">Model 1</td><td align=\"left\">0.009 (− 0.04 to 0.02)</td><td align=\"left\">0.54</td><td align=\"left\">− 0.007 (− 0.02 to 0.01)</td><td align=\"left\">0.39</td><td align=\"left\">− 0.004 (− 0.01 to 0.01)</td><td align=\"left\">0.47</td><td align=\"left\">0.002 (− 0.01 to 0.02)</td><td align=\"left\">0.81</td></tr><tr><td align=\"left\">Model 2</td><td align=\"left\">0.004 (− 0.03 to 0.02)</td><td align=\"left\">0.77</td><td align=\"left\">− 0.005 (− 0.02 to 0.01)</td><td align=\"left\">0.49</td><td align=\"left\">− 0.001 (− 0.01 to 0.01)</td><td align=\"left\">0.80</td><td align=\"left\">0.002 (− 0.01 to − 0.02)</td><td align=\"left\">0.69</td></tr><tr><td align=\"left\">Model 3</td><td align=\"left\">0.002 (− 0.02 to 0.03)</td><td align=\"left\">0.90</td><td align=\"left\">− 0.004 (− 0.02 to 0.01)</td><td align=\"left\">0.55</td><td align=\"left\">0.001 (− 0.01 to 0.01)</td><td align=\"left\">0.88</td><td align=\"left\">0.005 (− 0.007 to 0.02)</td><td align=\"left\">0.39</td></tr><tr><td align=\"left\">Model 4</td><td align=\"left\">0.001 (− 0.02 to 0.03)</td><td align=\"left\">0.94</td><td align=\"left\">− 0.005 (− 0.02 to 0.01)</td><td align=\"left\">0.53</td><td align=\"left\">0.001 (− 0.01 to 0.01)</td><td align=\"left\">0.90</td><td align=\"left\">0.005 (− 0.007 to 0.02)</td><td align=\"left\">0.42</td></tr><tr><td align=\"left\" colspan=\"9\">Social disengagement</td></tr><tr><td align=\"left\">Model 1</td><td align=\"left\"><bold>− 0.27 (− 0.30 to − 0.23)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.08 (− 0.09 to − 0.06)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.08 (− 0.10 to − 0.07)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.11 (− 0.12 to − 0.09)</bold></td><td align=\"left\"><bold>< 0.001</bold></td></tr><tr><td align=\"left\">Model 2</td><td align=\"left\"><bold>− 0.14 (− 0.17 to − 0.11)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.03 (− 0.05 to − 0.02)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.05 (− 0.06 to − 0.03)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.06 (− 0.08 to − 0.05)</bold></td><td align=\"left\"><bold>< 0.001</bold></td></tr><tr><td align=\"left\">Model 3</td><td align=\"left\"><bold>− 0.10 (− 0.13 to − 0.07)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.02 (− 0.04 to − 0.01)</bold></td><td align=\"left\"><bold>0.003</bold></td><td align=\"left\"><bold>− 0.03 (− 0.05 to − 0.02)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.04 (− 0.06 to − 0.03)</bold></td><td align=\"left\"><bold>< 0.001</bold></td></tr><tr><td align=\"left\">Model 4</td><td align=\"left\"><bold>− 0.10 (− 0.12 to − 0.07)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.02 (− 0.04 to − 0.01)</bold></td><td align=\"left\"><bold>0.004</bold></td><td align=\"left\"><bold>− 0.03 (− 0.05 to − 0.02)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.04 (− 0.06 to − 0.03)</bold></td><td align=\"left\"><bold>< 0.001</bold></td></tr><tr><td align=\"left\" colspan=\"9\">Loneliness</td></tr><tr><td align=\"left\">Model 1</td><td align=\"left\"><bold>− 0.16 (− 0.20 to − 0.12)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.06 (− 0.08 to − 0.04)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.03 (− 0.05 to − 0.02)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.07 (− 0.09 to − 0.05)</bold></td><td align=\"left\"><bold>< 0.001</bold></td></tr><tr><td align=\"left\">Model 2</td><td align=\"left\"><bold>− 0.14 (− 0.17 to − 0.11)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.05 (− 0.07 to − 0.03)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.03 (− 0.04 to − 0.02)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.06 (− 0.07 to − 0.05)</bold></td><td align=\"left\"><bold>< 0.001</bold></td></tr><tr><td align=\"left\">Model 3</td><td align=\"left\"><bold>− 0.09 (− 0.12 to − 0.06)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.03 (− 0.05 to − 0.02)</bold></td><td align=\"left\"><bold>< 0.001</bold></td><td align=\"left\"><bold>− 0.02 (− 0.03 to − 0.002)</bold></td><td align=\"left\"><bold>0.02</bold></td><td align=\"left\"><bold>− 0.04 (− 0.05 to − 0.02)</bold></td><td align=\"left\"><bold>< 0.001</bold></td></tr><tr><td align=\"left\">Model 4</td><td align=\"left\"><bold>− 0.06 (− 0.09 to − 0.02)</bold></td><td align=\"left\"><bold>0.001</bold></td><td align=\"left\"><bold>− 0.03 (− 0.04 to − 0.01)</bold></td><td align=\"left\"><bold>0.002</bold></td><td align=\"left\">− 0.01 (− 0.02 to 0.01)</td><td align=\"left\">0.23</td><td align=\"left\"><bold>− 0.02 (− 0.03 to − 0.01)</bold></td><td align=\"left\"><bold>0.002</bold></td></tr></tbody></table><table-wrap-foot><p>N = 8,780, 3 observations per person, total observations 26,340. Model 1: accounting for all time-invariant factors and time. Model 2: additionally adjusted for time-varying demographic factors (age, marital status, employment status and wealth). Model 3: additionally adjusted for time-varying health factors (BMI, eyesight, comorbidities, chronic pain, frequency of alcohol consumption, smoking habits, inactivity and cognition). Model 4: additionally adjusted for time-varying mental health (depression).</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec13\"><title>Loneliness</title><p id=\"Par20\">Higher levels of loneliness were also longitudinally associated with lower physical performance. Although time-varying demographic and health-related factors explained some of this association, results were significant even when accounting for all time-invariant and time-variant confounders (coef = − 0.06, 95% CI − 0.09 to − 0.02) (see Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>). When exploring the subscales of physical performance, there was a significant association with poorer sit-to-stand and slower walking speed but not poorer balance, for which the association was attenuated when accounting for depression (coef = − 0.01, 95% CI − 0.02 to 0.01). If an individual improved from having the highest possible loneliness score to the lowest possible loneliness score, this was associated with an increase in their average SPPB score 9.73 (95% CI 9.62–9.85) to 9.90 (95% CI 9.87–9.93); a 1.7% improvement.</p></sec><sec id=\"Sec14\"><title>Sensitivity analyses</title><p id=\"Par21\">When incorporating our exposures separately into the models so they did not mutually adjust for one another, the pattern of results was completely maintained, with the exception that loneliness was still related to balance when not adjusting for isolation (see Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>). When additionally adjusting for both moderate and vigorous sports or energetic activities, results were materially unaffected (see Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). When splitting results by gender, findings were almost identical (see Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>). When splitting results by age, those over the age of 65 showed consistent results for both loneliness and anxiety. For those under 65, the association between social disengagement and physical performance was still clearly apparent but all other associations were attenuated (see Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>). When restricting the measures for walking speed and total physical performance to adults aged 60+ (who had provided full walking test data), results were entirely maintained (see Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">6</xref>). Finally, when applying the SPPB clinical cut-off of < 10 indicating increased risk of disability<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup> and all-cause mortality <sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>, individuals were 10% more likely to be below this threshold if they were socially disengaged isolated and 5% more likely if they were lonely when accounting for all demographic and physical health-related factors. (See Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">7</xref>).</p></sec></sec><sec id=\"Sec15\"><title>Discussion and conclusions</title><p id=\"Par22\">The main finding of this study is that loneliness and aspects of social isolation, including domestic isolation and social disengagement, are independently associated with poorer physical performance in older age. However, low social contact (i.e. low frequency of social contact with family and friends) was not related to physical performance. These relationships were independent of all time-invariant confounders and all identified demographic, physical and mental health-related time-varying confounders and were also broadly consistent across gender. However, for adults under the age of 65, only the results for social disengagement remained. These results are important given that lower SPPB scores have been associated with adverse clinical outcomes including falls<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, future disability<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>, loss of independence in activities of daily living, rehospitalisation following discharge<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>–<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>, reductions in mobility and function<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, decline in health status<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>, longer hospital inpatient stay<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>, nursing home admission<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>, and death<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>.</p><p id=\"Par23\">As our study was observational rather than interventional, causality cannot be assumed. We have shown a persistent association between social factors and physical performance, and confirmed this is independent of all time-invariant and all identified time-varying confounders. For certain exposures, such as domestic isolation, there is little theoretical rationale for substantial reverse causality (i.e. the causal link between declining physical performance and no longer living with a spouse is less clear than the reverse). However, it is possible that the results for other social factors could be bi-directional, in that lower physical performance may lead to isolation, which may contribute to the findings related to social disengagement. Reductions in physical performance may make social engagement more challenging due to the difficulties experienced getting to the social and cultural events and groups. This may lead to avoidance of these activities resulting in social disengagement. However, it is worth noting that this would still have to be independent of other physical activity as we controlled for time-varying physical activity in our analyses.</p><p id=\"Par24\">These findings are broadly supportive of other studies on this topic, showing that isolation and loneliness are independently associated with physical performance. For example, loneliness and social isolation have previously been shown to be associated with increased rate of motor decline in older adults<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup><sup>,</sup><sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>, and reduced walking speed<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. However, our results extend these findings in three key ways. First, we used a sophisticated statistical approach that identified that the relationship with physical performance is not just present for static social factors but also for time-varying loneliness and isolation. This suggests that changes in loneliness and isolation rather than just absolute scores at a particular moment in time are related to physical performance. Second, we used a more nuanced categorisation of isolation and showing specific associations between domestic isolation and social disengagement but not frequency of social contact. Third, as our analyses automatically accounted for all time-invariant factors, even if unobserved, these results suggest that although factors such as socio-economic status may explain some of the relationship between loneliness, isolation and physical performance, they do not fully explain the association, as the finding persists independent of these factors.</p><p id=\"Par25\">In considering why some aspects of social isolation, but not frequency of social contact, were related to physical performance, the most obvious explanation is to do with physical activity. Social contact (which included telephone, email and writing as well as face-to-face contact) may not lead to increased physical activity as reliably as social engagement or living with somebody. Indeed, social engagement involves active participation in community activities, which has been proposed to constitute a form of physical activity of adequate intensity to prevent, or at least reduce, deconditioning in physical performance, while individuals who live with somebody have been shown to be more physically active<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>–<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. We did also control for inactivity in our analyses, and our sensitivity analyses further controlled for moderate or vigorous sport or physical activity. As the results were not attenuated by the inclusion of such factors, this suggests that while physical activity engaged in as part of social participation may make some contribution to the relationship between social participation and physical performance, it does not fully explain the association. But nonetheless, it is possible that social engagement and living with someone reduces broader sedentary behaviours that are known to lead to increases in physical decline without individuals being consciously aware (and therefore formally reporting) that their activity levels are higher. Alternatively, it is possible that living with somebody and being engaged in community social activities help to provide a sense of purpose greater than that achieved merely through socialising. Purpose and feeling that what one does in life is worthwhile are related, both cross-sectionally and longitudinally, to a wide range of factors that influence physical performance including better mental and physical health (self-rated depressive symptoms and chronic disease), and healthier lifestyles (more physical activity, less smoking, better diet)<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. An additional potential explanation for the findings regarding isolation is that by physically seeing people, health problems that may impact physical performance are more likely to be brought to the attention of healthcare professionals and receive subsequent intervention or management. This may happen more for face-to-face contact than for email or telephone contact. So, our social isolation variable (which combined face-to-face and technology interactions) may have been a less strong factor in encouraging engagement with health services. Future research is recommended that looks in even greater detail into how different types of face-to-face and remote contact are associated with health outcomes.</p><p id=\"Par26\">It is also of note that we found an association between loneliness and physical performance that was independent of social isolation measures. Much of the literature on loneliness has discussed the potential explanatory role of mental health, due to the association between loneliness and depression<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. Notably, the strength of our findings was reduced when accounting for depression, and for balance significance was lost, suggesting that mental health does mediate some of the relationship. However, as the association persisted independently for total performance and for walking speed and sit-to-stand, this suggests that there are other independent paths linking loneliness with physical performance. These may be similar to those linking social isolation to physical performance, such as an individual’s sense of purpose, or they may differ. For example, loneliness has been associated with poorer concentration and memory, which could affect physical performance directly or via reduced confidence<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>.</p><p id=\"Par27\">Amongst other limitations not yet mentioned, it is also possible that unmeasured confounding variables may exist that impact or explain the results presented here. Using fixed effects models for our statistical analysis should have reduced the impact of any such un-measured variables, as all time-invariant factors are automatically accounted for even if unobserved. But time-varying unobserved factors remain a challenge. Finally, there may have been an underestimation of changes in SPPB in individuals with high levels of physical functioning, due to a potentially ceiling effect in SPPB for such people. However, this merely suggests that the association presented here may be an underestimation. Future research could consider in more detail how different sub-groups are affected and whether the interaction of risk factors leads to stronger associations between social factors and physical performance.</p><p id=\"Par28\">In conclusion, we have shown that components of isolation and loneliness are independently associated with poorer physical performance in older adults, including worse sit-to-stand ability, balance and slower walking speed. When considering an outcome of physical performance, an important question is whether findings are clinically meaningful. The minimal clinically important difference (MCID) for total SPPB is 0.5 points for a small change and 1 for a substantial change<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. Therefore, although loneliness and aspects of isolation are associated with poorer physical performance in older age, our findings suggest the degree of this association falls below the MCID. These associations may be of importance on a population scale, but less so on an individual basis. However, the substantially increased risk of falling below the SPPB cut-off of < 10, for socially disengaged or lonely individuals, is of major importance given the implications regarding disability and mortality risk. Arguably, the implication for clinicians and policy makers is that greater attention to social isolation and related factors could be beneficial in designing programmes to improve physical performance. Our findings suggest that societal change, including the increasing reports of isolation and loneliness, may be related to the physical performance of older adults, with further implications for other aspects of health and functioning. Our findings also suggest that physical performance in older adults should be considered in relation to social distancing measures related to the COVID-19 pandemic. These findings do not suggest that social distancing is inappropriate; rather that physical performance interventions that respect the requirements of social distancing measures during this period should be considered. Future intervention studies could explore the impact of social interventions on physical performance to clarify whether this relationship could be causal.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec16\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70483_MOESM1_ESM.pdf\"><caption><p>Supplementary Tables.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p> is available for this paper at 10.1038/s41598-020-70483-3.</p></sec><ack><title>Acknowledgements</title><p>The English Longitudinal Study of Ageing was developed by a team of researchers based at the University College London, NatCen Social Research, the Institute for Fiscal Studies and the University of Manchester. The data were collected by NatCen Social Research. The funding is provided by National Institute of Aging Grant R01AG017644 and a consortium of UK government departments coordinated by the National Institute for Health Research. For this analysis, DF was supported by the Wellcome Trust [205407/Z/16/Z] and KP was supported by National Institute for Health Research Academic Clinical Fellowship award and the Imperial College Clinician Investigator Scholarship. The research was also supported by the Wellcome Trust [221400/Z/20/Z], and the Nuffield Foundation [WEL/FR-000022583], but the views expressed are those of the authors and not necessarily the Foundation. The funders had no say in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. This publication presents independent research. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>All the authors participated in designing the study, writing the manuscript, and making the decision to submit the manuscript for publication. K.P. and D.F. analysed the data and vouch for its accuracy.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>The Data used in this study are available on application from the UK Data Service (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ukdataservice.ac.uk/\">https://www.ukdataservice.ac.uk/</ext-link>).</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par29\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Holt-Lunstad</surname><given-names>J</given-names></name><name><surname>Smith</surname><given-names>TB</given-names></name><name><surname>Baker</surname><given-names>M</given-names></name><name><surname>Harris</surname><given-names>T</given-names></name><name><surname>Stephenson</surname><given-names>D</given-names></name></person-group><article-title>Loneliness and social isolation as risk factors for mortality: A meta-analytic review</article-title><source>Perspect. Psychol. Sci. J. Assoc. Psychol. Sci.</source><year>2015</year><volume>10</volume><fpage>227</fpage><lpage>237</lpage><pub-id pub-id-type=\"doi\">10.1177/1745691614568352</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shor</surname><given-names>E</given-names></name><name><surname>Roelfs</surname><given-names>DJ</given-names></name></person-group><article-title>Social contact frequency and all-cause mortality: A meta-analysis and meta-regression</article-title><source>Soc. Sci. Med.</source><year>2015</year><volume>128</volume><fpage>76</fpage><lpage>86</lpage><pub-id pub-id-type=\"doi\">10.1016/j.socscimed.2015.01.010</pub-id><pub-id pub-id-type=\"pmid\">25594955</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Courtin</surname><given-names>E</given-names></name><name><surname>Knapp</surname><given-names>M</given-names></name></person-group><article-title>Social isolation, loneliness and health in old age: A scoping review</article-title><source>Health Soc. Care Commun.</source><year>2017</year><volume>25</volume><fpage>799</fpage><lpage>812</lpage><pub-id pub-id-type=\"doi\">10.1111/hsc.12311</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Loucks</surname><given-names>EB</given-names></name><name><surname>Berkman</surname><given-names>LF</given-names></name><name><surname>Gruenewald</surname><given-names>TL</given-names></name><name><surname>Seeman</surname><given-names>TE</given-names></name></person-group><article-title>Relation of social integration to inflammatory marker concentrations in men and women 70–79 years</article-title><source>Am. J. Cardiol.</source><year>2006</year><volume>97</volume><fpage>1010</fpage><lpage>1016</lpage><pub-id pub-id-type=\"doi\">10.1016/j.amjcard.2005.10.043</pub-id><pub-id pub-id-type=\"pmid\">16563907</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>YC</given-names></name><etal/></person-group><article-title>Social relationships and physiological determinants of longevity across the human life span</article-title><source>Proc. Natl. Acad. Sci. USA</source><year>2016</year><volume>113</volume><fpage>578</fpage><lpage>583</lpage><pub-id pub-id-type=\"doi\">10.1073/pnas.1511085112</pub-id><pub-id pub-id-type=\"pmid\">26729882</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grippo</surname><given-names>AJ</given-names></name><name><surname>Lamb</surname><given-names>DG</given-names></name><name><surname>Carter</surname><given-names>CS</given-names></name><name><surname>Porges</surname><given-names>SW</given-names></name></person-group><article-title>Social isolation disrupts autonomic regulation of the heart and influences negative affective behaviors</article-title><source>Biol. Psychiat.</source><year>2007</year><volume>62</volume><fpage>1162</fpage><lpage>1170</lpage><pub-id pub-id-type=\"doi\">10.1016/j.biopsych.2007.04.011</pub-id><pub-id pub-id-type=\"pmid\">17658486</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Troxel</surname><given-names>WM</given-names></name><etal/></person-group><article-title>Social integration, social contacts, and blood pressure dipping in African–Americans and whites</article-title><source>J. Hypertens.</source><year>2010</year><volume>28</volume><fpage>265</fpage><lpage>271</lpage><pub-id pub-id-type=\"doi\">10.1097/HJH.0b013e328333ab01</pub-id><pub-id pub-id-type=\"pmid\">20051909</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cacioppo</surname><given-names>JT</given-names></name><name><surname>Cacioppo</surname><given-names>S</given-names></name><name><surname>Capitanio</surname><given-names>JP</given-names></name><name><surname>Cole</surname><given-names>SW</given-names></name></person-group><article-title>The neuroendocrinology of social isolation</article-title><source>Annu. Rev. Psychol.</source><year>2015</year><volume>66</volume><fpage>733</fpage><lpage>767</lpage><pub-id pub-id-type=\"doi\">10.1146/annurev-psych-010814-015240</pub-id><pub-id pub-id-type=\"pmid\">25148851</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Seeman</surname><given-names>TE</given-names></name><name><surname>Singer</surname><given-names>BH</given-names></name><name><surname>Ryff</surname><given-names>CD</given-names></name><name><surname>Love</surname><given-names>GD</given-names></name><name><surname>Levy-Storms</surname><given-names>L</given-names></name></person-group><article-title>Social relationships, gender, and allostatic load across two age cohorts</article-title><source>Psych. Med.</source><year>2002</year><volume>64</volume><fpage>395</fpage><lpage>406</lpage><pub-id pub-id-type=\"doi\">10.1097/00006842-200205000-00004</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Valtorta</surname><given-names>NK</given-names></name><name><surname>Kanaan</surname><given-names>M</given-names></name><name><surname>Gilbody</surname><given-names>S</given-names></name><name><surname>Ronzi</surname><given-names>S</given-names></name><name><surname>Hanratty</surname><given-names>B</given-names></name></person-group><article-title>Loneliness and social isolation as risk factors for coronary heart disease and stroke: Systematic review and meta-analysis of longitudinal observational studies</article-title><source>Heart</source><year>2016</year><volume>102</volume><fpage>1009</fpage><lpage>1016</lpage><pub-id pub-id-type=\"doi\">10.1136/heartjnl-2015-308790</pub-id><pub-id pub-id-type=\"pmid\">27091846</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Beaudart</surname><given-names>C</given-names></name><etal/></person-group><article-title>Assessment of muscle function and physical performance in daily clinical practice: A position paper endorsed by the European Society for Clinical and Economic Aspects of Osteoporosis, Osteoarthritis and Musculoskeletal Diseases (ESCEO)</article-title><source>Calcif. Tissue Int.</source><year>2019</year><pub-id pub-id-type=\"doi\">10.1007/s00223-019-00545-w</pub-id><pub-id pub-id-type=\"pmid\">31729554</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van Lummel</surname><given-names>RC</given-names></name><etal/></person-group><article-title>Physical performance and physical activity in older adults: Associated but separate domains of physical function in old age</article-title><source>PLoS ONE</source><year>2015</year><volume>10</volume><fpage>e0144048</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0144048</pub-id><pub-id pub-id-type=\"pmid\">26630268</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tieland</surname><given-names>M</given-names></name><name><surname>Trouwborst</surname><given-names>I</given-names></name><name><surname>Clark</surname><given-names>BC</given-names></name></person-group><article-title>Skeletal muscle performance and ageing</article-title><source>J. Cachexia Sarcopenia Muscle</source><year>2018</year><volume>9</volume><fpage>3</fpage><lpage>19</lpage><pub-id pub-id-type=\"doi\">10.1002/jcsm.12238</pub-id><pub-id pub-id-type=\"pmid\">29151281</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Buchman</surname><given-names>AS</given-names></name><etal/></person-group><article-title>Loneliness and the rate of motor decline in old age: The Rush Memory and Aging Project, a community-based cohort study</article-title><source>BMC Geriatr.</source><year>2010</year><volume>10</volume><fpage>77</fpage><pub-id pub-id-type=\"doi\">10.1186/1471-2318-10-77</pub-id><pub-id pub-id-type=\"pmid\">20969786</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Buchman</surname><given-names>AS</given-names></name><etal/></person-group><article-title>Association between late-life social activity and motor decline in older adults</article-title><source>Arch. Intern. Med.</source><year>2009</year><volume>169</volume><fpage>1139</fpage><lpage>1146</lpage><pub-id pub-id-type=\"doi\">10.1001/archinternmed.2009.135</pub-id><pub-id pub-id-type=\"pmid\">19546415</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shankar</surname><given-names>A</given-names></name><name><surname>McMunn</surname><given-names>A</given-names></name><name><surname>Demakakos</surname><given-names>P</given-names></name><name><surname>Hamer</surname><given-names>M</given-names></name><name><surname>Steptoe</surname><given-names>A</given-names></name></person-group><article-title>Social isolation and loneliness: Prospective associations with functional status in older adults</article-title><source>Health Psychol. Off. J. Div. Health Psychol. Am. Psychol. Assoc.</source><year>2017</year><volume>36</volume><fpage>179</fpage><lpage>187</lpage><pub-id pub-id-type=\"doi\">10.1037/hea0000437</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Unger</surname><given-names>JB</given-names></name><name><surname>McAvay</surname><given-names>G</given-names></name><name><surname>Bruce</surname><given-names>ML</given-names></name><name><surname>Berkman</surname><given-names>L</given-names></name><name><surname>Seeman</surname><given-names>T</given-names></name></person-group><article-title>Variation in the impact of social network characteristics on physical functioning in elderly persons: MacArthur Studies of Successful Aging</article-title><source>J. Gerontol. Series Psychol. Sci. Social Sci.</source><year>1999</year><volume>54</volume><fpage>S245</fpage><lpage>251</lpage></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kanamori</surname><given-names>S</given-names></name><etal/></person-group><article-title>Social participation and the prevention of functional disability in older Japanese: The JAGES cohort study</article-title><source>PLoS ONE</source><year>2014</year><volume>9</volume><fpage>e99638</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0099638</pub-id><pub-id pub-id-type=\"pmid\">24923270</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Steptoe</surname><given-names>A</given-names></name><name><surname>Breeze</surname><given-names>E</given-names></name><name><surname>Banks</surname><given-names>J</given-names></name><name><surname>Nazroo</surname><given-names>J</given-names></name></person-group><article-title>Cohort profile: The English longitudinal study of ageing</article-title><source>Int. J. Epidemiol.</source><year>2013</year><volume>42</volume><fpage>1640</fpage><lpage>1648</lpage><pub-id pub-id-type=\"doi\">10.1093/ije/dys168</pub-id><pub-id pub-id-type=\"pmid\">23143611</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Freiberger</surname><given-names>E</given-names></name><etal/></person-group><article-title>Performance-based physical function in older community-dwelling persons: A systematic review of instruments</article-title><source>Age Ageing</source><year>2012</year><volume>41</volume><fpage>712</fpage><lpage>721</lpage><pub-id pub-id-type=\"doi\">10.1093/ageing/afs099</pub-id><pub-id pub-id-type=\"pmid\">22885845</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Guralnik</surname><given-names>JM</given-names></name><etal/></person-group><article-title>A short physical performance battery assessing lower extremity function: Association with self-reported disability and prediction of mortality and nursing home admission</article-title><source>J. Gerontol.</source><year>1994</year><volume>49</volume><fpage>M85</fpage><lpage>94</lpage><pub-id pub-id-type=\"doi\">10.1093/geronj/49.2.M85</pub-id><pub-id pub-id-type=\"pmid\">8126356</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hughes</surname><given-names>ME</given-names></name><name><surname>Waite</surname><given-names>LJ</given-names></name><name><surname>Hawkley</surname><given-names>LC</given-names></name><name><surname>Cacioppo</surname><given-names>JT</given-names></name></person-group><article-title>A short scale for measuring loneliness in large surveys: Results from two population-based studies</article-title><source>Res. Aging</source><year>2004</year><volume>26</volume><fpage>655</fpage><lpage>672</lpage><pub-id pub-id-type=\"doi\">10.1177/0164027504268574</pub-id><pub-id pub-id-type=\"pmid\">18504506</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Russell</surname><given-names>D</given-names></name><name><surname>Peplau</surname><given-names>LA</given-names></name><name><surname>Ferguson</surname><given-names>ML</given-names></name></person-group><article-title>Developing a measure of loneliness</article-title><source>J. Pers. Assess.</source><year>1978</year><volume>42</volume><fpage>290</fpage><lpage>294</lpage><pub-id pub-id-type=\"doi\">10.1207/s15327752jpa4203_11</pub-id><pub-id pub-id-type=\"pmid\">660402</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Radloff</surname><given-names>L</given-names></name></person-group><article-title>The CES-D scale: A self-report depression scale for research in the general population</article-title><source>Appl. Psychol. Meas.</source><year>1977</year><volume>1</volume><fpage>385</fpage><lpage>401</lpage><pub-id pub-id-type=\"doi\">10.1177/014662167700100306</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Allison</surname><given-names>P</given-names></name></person-group><source>Fixed Effects Regression Models</source><year>2009</year><publisher-loc>Thousand Oaks</publisher-loc><publisher-name>SAGE publications</publisher-name></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Guralnik</surname><given-names>JM</given-names></name><etal/></person-group><article-title>Lower extremity function and subsequent disability: Consistency across studies, predictive models, and value of gait speed alone compared with the short physical performance battery</article-title><source>J. Gerontol. Series A Biol. Sci. Med. Sci.</source><year>2000</year><volume>55</volume><fpage>M221</fpage><lpage>231</lpage><pub-id pub-id-type=\"doi\">10.1093/gerona/55.4.M221</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pavasini</surname><given-names>R</given-names></name><etal/></person-group><article-title>Short physical performance battery and all-cause mortality: Systematic review and meta-analysis</article-title><source>BMC Med.</source><year>2016</year><volume>14</volume><fpage>215</fpage><pub-id pub-id-type=\"doi\">10.1186/s12916-016-0763-7</pub-id><pub-id pub-id-type=\"pmid\">28003033</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Veronese</surname><given-names>N</given-names></name><etal/></person-group><article-title>Association between short physical performance battery and falls in older people: the Progetto Veneto Anziani Study</article-title><source>Rejuvenation Res.</source><year>2014</year><volume>17</volume><fpage>276</fpage><lpage>284</lpage><pub-id pub-id-type=\"doi\">10.1089/rej.2013.1491</pub-id><pub-id pub-id-type=\"pmid\">24387140</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Guralnik</surname><given-names>JM</given-names></name><name><surname>Ferrucci</surname><given-names>L</given-names></name><name><surname>Simonsick</surname><given-names>EM</given-names></name><name><surname>Salive</surname><given-names>ME</given-names></name><name><surname>Wallace</surname><given-names>RB</given-names></name></person-group><article-title>Lower-extremity function in persons over the age of 70 years as a predictor of subsequent disability</article-title><source>N. Engl. J. Med.</source><year>1995</year><volume>332</volume><fpage>556</fpage><lpage>561</lpage><pub-id pub-id-type=\"doi\">10.1056/NEJM199503023320902</pub-id><pub-id pub-id-type=\"pmid\">7838189</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Volpato</surname><given-names>S</given-names></name><etal/></person-group><article-title>Predictive value of the Short Physical Performance Battery following hospitalization in older patients</article-title><source>J. Gerontol. Series A Biol. Sci. Med. Sci.</source><year>2011</year><volume>66</volume><fpage>89</fpage><lpage>96</lpage><pub-id pub-id-type=\"doi\">10.1093/gerona/glq167</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Studenski</surname><given-names>S</given-names></name><etal/></person-group><article-title>Physical performance measures in the clinical setting</article-title><source>J. Am. Geriatr. Soc.</source><year>2003</year><volume>51</volume><fpage>314</fpage><lpage>322</lpage><pub-id pub-id-type=\"doi\">10.1046/j.1532-5415.2003.51104.x</pub-id><pub-id pub-id-type=\"pmid\">12588574</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Penninx</surname><given-names>BW</given-names></name><etal/></person-group><article-title>Lower extremity performance in nondisabled older persons as a predictor of subsequent hospitalization</article-title><source>J. Gerontol. Series A Biol. Sci. Med. Sci.</source><year>2000</year><volume>55</volume><fpage>M691</fpage><lpage>697</lpage><pub-id pub-id-type=\"doi\">10.1093/gerona/55.11.M691</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vasunilashorn</surname><given-names>S</given-names></name><etal/></person-group><article-title>Use of the Short Physical Performance Battery Score to predict loss of ability to walk 400 meters: Analysis from the InCHIANTI study</article-title><source>J. Gerontol. Series A Biol. Sci. Med. Sci.</source><year>2009</year><volume>64</volume><fpage>223</fpage><lpage>229</lpage><pub-id pub-id-type=\"doi\">10.1093/gerona/gln022</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fisher</surname><given-names>S</given-names></name><name><surname>Ottenbacher</surname><given-names>KJ</given-names></name><name><surname>Goodwin</surname><given-names>JS</given-names></name><name><surname>Graham</surname><given-names>JE</given-names></name><name><surname>Ostir</surname><given-names>GV</given-names></name></person-group><article-title>Short physical performance battery in hospitalized older adults</article-title><source>Aging Clin. Exp. Res.</source><year>2009</year><volume>21</volume><fpage>445</fpage><lpage>452</lpage><pub-id pub-id-type=\"doi\">10.1007/BF03327444</pub-id><pub-id pub-id-type=\"pmid\">20154514</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schrempft</surname><given-names>S</given-names></name><name><surname>Jackowska</surname><given-names>M</given-names></name><name><surname>Hamer</surname><given-names>M</given-names></name><name><surname>Steptoe</surname><given-names>A</given-names></name></person-group><article-title>Associations between social isolation, loneliness, and objective physical activity in older men and women</article-title><source>BMC Public Health</source><year>2019</year><volume>19</volume><fpage>74</fpage><pub-id pub-id-type=\"doi\">10.1186/s12889-019-6424-y</pub-id><pub-id pub-id-type=\"pmid\">30651092</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kobayashi</surname><given-names>LC</given-names></name><name><surname>Steptoe</surname><given-names>A</given-names></name></person-group><article-title>Social isolation, loneliness, and health behaviors at older ages: Longitudinal cohort study</article-title><source>Ann. Behav. Med. Publ. Soc. Behav. Med.</source><year>2018</year><volume>52</volume><fpage>582</fpage><lpage>593</lpage><pub-id pub-id-type=\"doi\">10.1093/abm/kax033</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pettee</surname><given-names>KK</given-names></name><etal/></person-group><article-title>Influence of marital status on physical activity levels among older adults</article-title><source>Med. Sci. Sports Exerc.</source><year>2006</year><volume>38</volume><fpage>541</fpage><lpage>546</lpage><pub-id pub-id-type=\"doi\">10.1249/01.mss.0000191346.95244.f7</pub-id><pub-id pub-id-type=\"pmid\">16540843</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Steptoe</surname><given-names>A</given-names></name><name><surname>Fancourt</surname><given-names>D</given-names></name></person-group><article-title>Leading a meaningful life at older ages and its relationship with social engagement, prosperity, health, biology, and time use</article-title><source>Proc. Natl. Acad. Sci. USA</source><year>2019</year><volume>116</volume><fpage>1207</fpage><lpage>1212</lpage><pub-id pub-id-type=\"doi\">10.1073/pnas.1814723116</pub-id><pub-id pub-id-type=\"pmid\">30617082</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Holvast</surname><given-names>F</given-names></name><etal/></person-group><article-title>Loneliness is associated with poor prognosis in late-life depression: Longitudinal analysis of the Netherlands study of depression in older persons</article-title><source>J. Affect. Disord.</source><year>2015</year><volume>185</volume><fpage>1</fpage><lpage>7</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jad.2015.06.036</pub-id><pub-id pub-id-type=\"pmid\">26142687</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boss</surname><given-names>L</given-names></name><name><surname>Kang</surname><given-names>DH</given-names></name><name><surname>Branson</surname><given-names>S</given-names></name></person-group><article-title>Loneliness and cognitive function in the older adult: A systematic review</article-title><source>Int. Psychogeriatr.</source><year>2015</year><volume>27</volume><fpage>541</fpage><lpage>553</lpage><pub-id pub-id-type=\"doi\">10.1017/S1041610214002749</pub-id><pub-id pub-id-type=\"pmid\">25554219</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Perera</surname><given-names>S</given-names></name><name><surname>Mody</surname><given-names>SH</given-names></name><name><surname>Woodman</surname><given-names>RC</given-names></name><name><surname>Studenski</surname><given-names>SA</given-names></name></person-group><article-title>Meaningful change and responsiveness in common physical performance measures in older adults</article-title><source>J. Am. Geriatr. Soc.</source><year>2006</year><volume>54</volume><fpage>743</fpage><lpage>749</lpage><pub-id pub-id-type=\"doi\">10.1111/j.1532-5415.2006.00701.x</pub-id><pub-id pub-id-type=\"pmid\">16696738</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807849</article-id><article-id pub-id-type=\"pmc\">PMC7431532</article-id><article-id pub-id-type=\"publisher-id\">70888</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70888-0</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Dietary ceramide 2-aminoethylphosphonate, a marine sphingophosphonolipid, improves skin barrier function in hairless mice</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Tomonaga</surname><given-names>Nami</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Manabe</surname><given-names>Yuki</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Aida</surname><given-names>Kazuhiko</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Sugawara</surname><given-names>Tatsuya</given-names></name><address><email>sugawara@kais.kyoto-u.ac.jp</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.258799.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0372 2033</institution-id><institution>Laboratory of Technology of Marine Bioproducts, Division of Applied Biosciences, Graduate School of Agriculture, </institution><institution>Kyoto University, </institution></institution-wrap>Kitashirakawaoiwakecho, Sakyo-ku, Kyoto, 606-8502 Japan </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.471412.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1763 6304</institution-id><institution>Innovation Center, </institution><institution>Nippon Flour Mills Co., Ltd, </institution></institution-wrap>5-1-3 Midorigaoka, Atsugi, Kanagawa 243-0041 Japan </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13891</elocation-id><history><date date-type=\"received\"><day>8</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>3</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Sphingolipids are one of the major components of cell membranes and are ubiquitous in eukaryotic organisms. Ceramide 2-aminoethylphosphonate (CAEP) of marine origin is a unique and abundant sphingophosphonolipid with a C-P bond. Although molluscs such as squids and bivalves, containing CAEP, are consumed globally, the dietary efficacy of CAEP is not understood. We investigated the efficacy of marine sphingophosphonolipids by studying the effect of dietary CAEP on the improvement of the skin barrier function in hairless mice fed a diet that induces severely dry-skin condition. The disrupted skin barrier functions such as an increase in the transepidermal water loss (TEWL), a decrease in the skin hydration index, and epidermal hyperplasia were restored by CEAP dietary supplementation. Correspondingly, dietary CAEP significantly increased the content of covalently bound ω-hydroxyceramide, and the expression of its biosynthesis-related genes in the skin. These effects of dietary CAEP mimic those of dietary plant glucosylceramide. The novel observations from this study show an enhancement in the skin barrier function by dietary CAEP and the effects could be contributed by the upregulation of covalently bound ω-hydroxyceramide synthesis in the skin.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Nutrition</kwd><kwd>Lipids</kwd></kwd-group><funding-group><award-group><funding-source><institution>JSPS KAKENHI</institution></funding-source><award-id>JP15J01143</award-id><award-id>JP16H04923</award-id><principal-award-recipient><name><surname>Tomonaga</surname><given-names>Nami</given-names></name><name><surname>Sugawara</surname><given-names>Tatsuya</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">The mammalian skin barrier is in the stratum corneum, the outermost layers of the epidermis, which protects against excessive transepidermal water loss (TEWL) and to block of irritants. In this study, we focused on the function to retain water in the epidermis as the skin barrier and the epidermal structures which conducive to the barrier. Lipid lamellae in the extracellular space of corneocytes play a vital role in the barrier function and maintain a hydrophobic environment. These lipids, consisting of 50% ceramides, 25% cholesterol, and 15% fatty acids (on a total lipid mass basis), contribute to the water-holding properties and prevent desiccation by TEWL<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">3</xref></sup>. Ceramide formation occurs by binding of a fatty acid to an amide group of the sphingoid base. The molecular structures of ceramides are various. Ceramides are essential for the skin barrier function since changes in ceramide profile of the lipid lamellae have been associated with impaired barrier function<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>–<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>.\n</p><p id=\"Par3\">The structure formed by the binding of ω-hydroxyceramides to cornified envelope proteins is important for the skin barrier<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. The cornified envelope is a rigid structure with an outer lipid layer and an inner protein, which is produced by the crosslinking of precursor proteins such as involucrin and loricrin<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Ultra-long-chain ceramide participates in the formation of covalently bound ω-hydroxyceramides<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. The amount of covalently bound ω-hydroxyceramides correlates with skin hydration and skin barrier function<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>.</p><p id=\"Par4\">Sphingoid base is a common structure of sphingolipids which are one of the major families of lipids. Since sphingolipids are components of cell membranes, they are ubiquitous in eukaryotic organisms<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. However, the polar head groups and ceramide structure of sphingolipids vary among biological species<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. For example, sphingomyelin, which has a phosphocholine as a polar head, is a major mammalian sphingophospholipid, and is present in foods such as meat and milk. Glucosylceramide (GluCer) has a glucose as a head group and is a major glycosphingolipid frequently found in not only animals but also higher plants (cereals, beans, and vegetables). A certain quantity of sphingolipids is ingested daily from meals<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">23</xref></sup>. Recent reports show a protective effect on the skin barrier function by dietary intake of sphingomyelin and GluCer<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">27</xref></sup>. Dietary sphingomyelin and GluCer enhanced mRNA expression of epidermal ceramide synthases (CERS), contributing to ultra-long-chain ceramide synthesis in the dry-skin hairless mouse model<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. Sphingoid bases from dietary sphingolipids might participate in the upregulation of epidermal ultra-long-chain ceramide synthesis because sphingoid bases increase the expression of these CERS genes in normal human foreskin keratinocytes<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. Earlier studies reported that dietary GluCer is digested intestinally and absorbed as sphingoid bases into the lymph<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">29</xref></sup>. Additionally, dietary milk phospholipids (consisted mainly of phosphatidylcholine and sphingomyelin) increased epidermal covalently bound ω-hydroxyceramides, and improved skin barrier function in hairless mice<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>.</p><p id=\"Par5\">In contrast, general marine sphingolipid, ceramide 2-aminoethylphosphonate (CAEP), frequently contains unique structures<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. Unlike the C-O-P linkage encountered in the polar head of major sphingophospholipids such as sphingomyelin, the phosphorus atom of 2-aminoethylphosphonate, the polar head of CAEP is directly bound to a carbon atom (C-P bond)<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. Sphingolipids with C-P bonds, including CAEP, are sphingophosphonolipids<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. CAEP consists of not only sphingosine (d18:1) and hexadeca-4-sphingenine (d16:1), often found in mammals, but also a unique triene-type, odd-numbered carbon chain sphingoid base, 2-amino-9-methyl-4,8,10-octadecatriene-1,3-diol (d19:3), which differ from those in mammals<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref>–<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. The nomenclature for sphingoid bases is to indicate the number of hydroxyl groups (d for di- and t for tri-) followed by the chain length and number of double bonds. CAEP is widely present in marine invertebrates<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>, including molluscs such as squids<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup> and bivalves<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>, consumed globally. Previously, we showed digestion of dietary CAEP to sphingoid bases and absorption of these sphingoid bases, including unique d19:3 into the lymph in experimental animal models<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. Therefore, dietary CAEP has the potential to improve the skin barrier function via modulation of ceramide synthesis. In the present study, we evaluated the effect of dietary CAEP in comparison with GluCer on the skin barrier function to elucidate its mechanism of action by focusing on the synthesis of covalently bound ω-hydroxyceramides.</p></sec><sec id=\"Sec2\"><title>Results</title><sec id=\"Sec3\"><title>Effect on skin properties</title><p id=\"Par6\">After feeding mice HR-AD diet for 11 weeks, severe dry-skin and systemic erythema manifested. A HR-AD diet is used to induce skin damages with dry-skin condition and is especially characterized by a deficiency of magnesium<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref>,<xref ref-type=\"bibr\" rid=\"CR44\">45</xref></sup>. The dry-skin showed an increase in the TEWL to 18.7 ± 0.9 g/m<sup>2</sup>/h with a decrease in the hydration index to 24.2 ± 1.3 (arbitrary unit) at the end of HR-AD diet feeding period. The body weight of HR-AD group at the end of HR-AD diet feeding period (day 0 of the recovery treatment period) was 23.1 ± 1.0 g. Daily food intake and body weight were not significantly different among each group during the recovery treatment period (data not shown). After feeding the experimental diet for seven days, tissue weights (liver, spleen, and kidney) were not significantly different among different groups (data not shown). During the recovery treatment period, the dry-skin improved dramatically in all groups. Compared to the control group, TEWL levels on day 3 and 6 of recovery treatment period were reduced significantly by dietary CAEP (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>A). The hydration index in CAEP and GluCer groups were significantly increased compared to the control group on day 6 of the recovery treatment period (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Effect of dietary sphingolipids on TEWL levels (<bold>A</bold>) and the hydration index (<bold>B</bold>) in the HR-AD induced barrier perturbation model. The dorsal skin measurements performed every three days during the recovery period. Data reported as means ± standard errors (HR-AD and control groups, n = 5; CAEP and GluCer groups, n = 6). Bars with different letters at each time point are significantly different from each other by one-way ANOVA, followed by Tukey–Kramer tests (<italic>P</italic> < 0.05).</p></caption><graphic xlink:href=\"41598_2020_70888_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par7\">Epidermal hyperplasia was caused by disruption of the skin barrier in mice fed HR-AD diet for 11 weeks. CEAP and GluCer treatment reduced the thickness of epidermal hyperplasia and improved the skin appearance compared to control mice (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). Deep wrinkles developed by feeding the HR-AD diet (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>A). In comparison with the control group, dietary CAEP significantly decreased the number of wrinkles by day 3 of the recovery period (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>B). In addition, the average depth of wrinkles, wrinkle area ratio, and wrinkle volume ratio were reduced significantly by ingestion of CAEP and GluCer compared to control diet on day 3 of the recovery period (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>C – E). These results showed accelerated recovery of the skin barrier function and condition by dietary CAEP, in the dry-skin induced by HR-AD diets, comparable to dietary GluCer.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>The thickness of the epidermis in mice fed different diets (HR-AD and control groups, n = 5; CAEP and GluCer groups, n = 6). Photographs of mice dorsal skin sections stained with H&E (<bold>A</bold>). The thickness of each specimen was measured using a microscope (<bold>B</bold>). Values reported as means ± standard error. Data analysed by one-way ANOVA, followed by Tukey–Kramer tests. Bars with different letters are significantly different with <italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"41598_2020_70888_Fig2_HTML\" id=\"MO2\"/></fig><fig id=\"Fig3\"><label>Figure 3</label><caption><p>Representative photographs of replicas taken from mice dorsal skin (<bold>A</bold>). The images show skin wrinkles in each group. The number (<bold>B</bold>), the average depth of wrinkles (<bold>C</bold>), wrinkle area ratio (<bold>D</bold>), and volume ratio (<bold>E</bold>) analysed by an imaging analyser. Data reported as means ± standard errors. Bars with different letters at each time point are significantly different from each other by one-way ANOVA, followed by Tukey–Kramer tests (<italic>P</italic> < 0.05).</p></caption><graphic xlink:href=\"41598_2020_70888_Fig3_HTML\" id=\"MO3\"/></fig></p><p id=\"Par8\">Immunohistochemical staining of skin sections showed involucrin localized to keratinocytes (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>A). In semi-quantitative assay using immunostaining pictures, the average of percentage areas positive for involucrin immunostaining in adequate area (800–900μm<sup>2</sup>) of epidermis were 4.9% for HR-AD group, 8.5% for Control group, 16.9% for CAEP group, and 11.6% for GluCer group (n = 2). In CAEP and GluCer groups, the number of keratinocytes was comparable to the control group. In the stratum corneum, the staining intensity of filaggrin was not different among control, CAEP, and GluCer groups (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>B).<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Immunohistochemistry of mice dorsal skin after fed different diets. Photographs of immunohistochemically stained sections with an antibody against involucrin (<bold>A</bold>) and filaggrin (<bold>B</bold>).</p></caption><graphic xlink:href=\"41598_2020_70888_Fig4_HTML\" id=\"MO4\"/></fig></p></sec><sec id=\"Sec4\"><title>Effect on covalently bound ω-hydroxyceramide synthesis in the skin</title><p id=\"Par9\">Each ceramide molecules structure is shown as “sphingoid base/fatty acid” (“h” placed at numerical symbols of fatty acid means hydroxyl group) with reference to the nomenclature<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>. The covalently bound ω-hydroxyceramides levels were elevated during the recovery treatment period (Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>, Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>). Especially, d17:1/32:0 h, d17:1/32:1 h, d17:1/34:1 h, d18:1/32:1 h, and d18:1/34:1 h increased significantly by dietary CAEP and GluCer compared with the HR-AD group. These ω-hydroxyceramide molecules excluding d17:1/32:0 h increased in CAEP and GluCer groups, and d17:1/34:1 h showed a particularly significant change in CAEP group compared with the control group.<fig id=\"Fig5\"><label>Figure 5</label><caption><p>Levels of covalently bound ω-hydroxyceramides in mice epidermis fed different diets (HR-AD and control groups, n = 5; CAEP and GluCer groups, n = 6). The relative peak area per epidermal protein content presented as the fold change relative to ω-hydroxyceramide standard (d18:1/30:0 h, 1.0 pmol). Data reported as means ± standard errors and were analysed by one-way ANOVA, followed by Tukey–Kramer tests. Bars with different letters in each molecular species are significantly different with <italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"41598_2020_70888_Fig5_HTML\" id=\"MO5\"/></fig></p><p id=\"Par10\">Multiple enzymes contribute to the synthesis of covalently bound ω-hydroxyceramide. In this study, dietary CAEP increased mRNA expression of fatty acid elongase (ELOVL4) and ceramide synthases (CERS2 and 3) in the dorsal skin of mice (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>B–D), especially CERS2 and 3 mRNA expression levels in CAEP group increased significantly than in the HR-AD and control groups (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>C and D). Additionally, patatin-like phospholipase domain-containing protein 1 (PNPLA1) mRNA expression in CAEP group was significantly higher compared with the HR-AD group (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>E). PNPLA1 catalyses the synthesis of acylceramide, which is a precursor of covalently bound ω-hydroxyceramide<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref>,<xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>. Dietary GluCer significantly upregulated mRNA expression of ELOVL1 and CERS3 in the skin, compared with the HR-AD diet (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>A and D). These results suggested that dietary CAEP and GluCer promoted the synthesis of covalently bound ω-hydroxyceramides.<fig id=\"Fig6\"><label>Figure 6</label><caption><p>Effect of dietary sphingolipids on the expression of genes related to the synthesis of covalently bound ω-hydroxyceramides in the dorsal epidermis of the AD-like murine model. The quantification of relative expression of ELOVL1 (<bold>A</bold>), ELOVL4 (<bold>B</bold>), CERS2 (<bold>C</bold>), CERS3 (<bold>D</bold>), and PNPLA1 (<bold>E</bold>) mRNAs was by real-time RT-PCR. The expression of ACTB mRNA was used as an internal control. Values presented as means ± standard errors (HR-AD and control groups, n = 5; CAEP and GluCer groups, n = 6). Data analysed by one-way ANOVA, followed by Tukey–Kramer tests. Bars with different letters are significantly different with <italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"41598_2020_70888_Fig6_HTML\" id=\"MO6\"/></fig></p></sec></sec><sec id=\"Sec5\"><title>Discussion</title><p id=\"Par11\">Our results show an improvement of skin barrier function by dietary CAEP, and these effects were similar to that of dietary GluCer. The content of CAEP from squids is about 0.2–2% (weight percent of total lipids)<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>, thus supplementation of CAEP would be useful to achieve the functions. In the present study, the dry-skin condition was produced by feeding HR-AD diet as described earlier<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref>,<xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>. Dietary CAEP enhanced the reduction of TEWL and the increment of the skin hydration index during the recovery treatment period in this model. The epidermal hyperplasia is associated with skin dryness and indicated by the disordered skin barrier<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref>,<xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>. The epidermal thickness in CAEP group from this study closely matches that in normal mature hairless mice HR-1 (approximately 10–15 μm <sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>). Wrinkles frequently appear due to the dry-skin, such as photodamaged and aged skin, which is closely related to the skin barrier disruption<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref>,<xref ref-type=\"bibr\" rid=\"CR52\">54</xref></sup>. In these dry-skin conditions, abnormal terminal differentiation of keratinocytes induces the disorder of functional stratum corneum formation that contributes to the skin barrier<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref>,<xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>. The decrease in wrinkles by dietary CAEP might be associated with an improvement in the skin barrier function because dryness influences the skin wrinkles.</p><p id=\"Par12\">Involucrin is present in the granular and upper spinous layers, and earlier clinical reports showed reduced involucrin expression, while premature expression after barrier disruption was observed in lower spinous layer<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref>,<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>. In this study, the relatively low staining intensity of involucrin tended to be more broadly in the epidermis of HR-AD group compared to the other groups. Since involucrin participates in the maintenance of rigid cornified envelope, involucrin level might contribute to improvement in the skin barrier function<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Additionally, Jensen et al. suggested that decreased involucrin expression may cause a reduction in ω-hydroxyceramide levels in atopic dermatitis (AD) by failing to provide enough substrate for the binding of ceramides<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>. The immune-stained involucrin in the stratum corneum of CAEP group appeared to be more intense than in other groups, but it was not remarkable. The formation of stratum corneum is related to the upward migration and terminal differentiation of the keratinocytes in the epidermis <sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR50\">50</xref>,<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>. To elucidate the effects of dietary sphingolipids, further histological evaluation in detail is required. On the other hand, filaggrin expression was similar among CAEP, GluCer, and control groups. Filaggrin is a specific epidermal protein which is the precursor of the natural moisturizing factors and involved in the stratum corneum hydration<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>. In the dry-skin, filaggrin expression is downregulated<sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup>; however, filaggrin knock-down did not affect lipid composition of stratum corneum<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup>. Hence, suggesting that a shortage of filaggrin participates in dry-skin, although filaggrin knock-down alone does not necessarily affect the barrier function<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup>. Furthermore, Danso et al. reported that ELOVL1 and 6 expressions in the AD patients with filaggrin mutations were comparable to those in the wild-type AD patients<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>. Therefore, the effect of dietary CAEP on skin barrier function might not involve filaggrin expression changes.</p><p id=\"Par13\">Earlier reports showed a correlation between dry-skin and decrease in covalently bound ω-hydroxyceramide levels<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. Covalently bound ω-hydroxyceramides are one of the major components of the cornified lipid envelope<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref>,<xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup>. The impairment of covalently bound ω-hydroxyceramides production pathway causes ichthyosis<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref>,<xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup>. Previous reports mentioned that covalently bound ω-hydroxyceramides are thought to play an important role in stabilizing lamellar structure as compared with other species<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup>. In this study, administration of CAEP and GluCer increased epidermal covalently bound ω-hydroxyceramide levels and its synthesis-related gene expression in the skin compared to the control group. We found major covalently bound ω-hydroxyceramides in mice, although the minor molecule species (dihydrosphingosine-type, 4,14-sphingadiene-type and sphingosine-type involving odd-numbered carbon chain fatty acid) indicated by Kawana et al.<sup><xref ref-type=\"bibr\" rid=\"CR62\">62</xref></sup> were not detected. Dietary CAEP and GluCer intake effectively restore the skin condition in this mouse model, because ingestion of CAEP and GluCer normalizes the levels of epidermal ω-hydroxyceramide in mice compared to normal, hairless mice (data not shown). Interestingly, dietary CAEP and GluCer increased levels of covalently bound ω-hydroxyceramides having unsaturated fatty acids than those having saturated fatty acids. Previous studies reported that covalently bound ω-hydroxyceramides containing unsaturated fatty acid were less sensitive to aging, seasonal variation, and AD-like damage compared to those containing saturated fatty acid<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup>. The presence of an unsaturated acyl chain is necessary for the formation of the lipid lamellar structure<sup><xref ref-type=\"bibr\" rid=\"CR63\">63</xref></sup>. Importance of covalently bound ω-hydroxyceramides containing unsaturated fatty acid in maintenance and strengthening of epidermal lamellar structures are shown earlier<sup><xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup>. In other previous reports, changes in not only covalently bound ω-hydroxyceramides but also epidermal ceramide profile or other lipids content are associated with skin barrier function<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR7\">10</xref></sup>. Although this study evaluated ω-hydroxyceramides which are significantly associated with dry skin as the first step for elucidation of the effects of dietary CAEP on skin barrier, further studies to evaluate epidermal lipids profiles are needed to elucidate the more detailed mechanism of the effects. Macheleidt et al. reported that epidermal covalently bound ω-hydroxyceramides decreased in atopic dermatitis skin. In earlier reports, it has been shown that sphingomyelin rich diet and dietary GluCer exerted anti-inflammatory effects in the dry-skin hairless mouse model and a mouse model of oxazolone-induced chronic irritant contact dermatitis (ICD), respectively<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR64\">64</xref></sup>. Therefore, dietary CAEP and GluCer may exert the improving effect on skin barrier function which may be contributed by covalently bound ω-hydroxyceramides synthesis and anti-inflammatory effects also in atopic dermatitis model mice, while this study has not focused on atopic dermatitis cytokines.</p><p id=\"Par14\">In this study, we showed an increase in the level of covalently bound ω-hydroxyceramide in the epidermis by dietary CAEP and GluCer comparable to milk sphingomyelin as reported earlier<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. Fatty acids are elongated by the enzymes (ELOVL1-7) which have substrate specificities after conversion to acyl CoA<sup><xref ref-type=\"bibr\" rid=\"CR65\">65</xref>–<xref ref-type=\"bibr\" rid=\"CR67\">67</xref></sup>. In this study, dietary CAEP increased mRNA expression of ELOVL4, involved in the elongation of ultra-long-chain fatty acids (C26 <), highly expressed in the skin, and essential for skin barrier formation<sup><xref ref-type=\"bibr\" rid=\"CR68\">68</xref>–<xref ref-type=\"bibr\" rid=\"CR71\">71</xref></sup>. In ELOVL4 mutant mice, perinatal death by skin barrier disruption was caused by the deficiency of ultra-long-chain ceramides and acylceramides<sup><xref ref-type=\"bibr\" rid=\"CR72\">72</xref>,<xref ref-type=\"bibr\" rid=\"CR73\">73</xref></sup>. On the other hand, dietary GluCer upregulated mRNA expression of ELOVL1 in this study. ELOVL1 is involved in the elongation of very-long-chain fatty acids (especially C20-26) <sup><xref ref-type=\"bibr\" rid=\"CR74\">74</xref></sup>. Reports show reduced ELOVL1 mRNA levels in AD lesioned skin<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>. Sassa et al. showed early death in ELOVL1 knockout mice due to epidermal barrier defects<sup><xref ref-type=\"bibr\" rid=\"CR75\">75</xref></sup>. Additionally, since C24 < very-long-chain acyl CoA is a substrate for ELOVL4, the upregulation of ELOVL1 might facilitate ultra-long-chain fatty acid synthesis<sup><xref ref-type=\"bibr\" rid=\"CR67\">67</xref></sup>.</p><p id=\"Par15\">We also showed that dietary CAEP upregulated CERS2 and 3 mRNA expression compared to HR-AD and control groups. CERS3 synthesizes ultra-long-chain ceramide (C26 <), and its deficiency indicates skin barrier defect<sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref>,<xref ref-type=\"bibr\" rid=\"CR76\">77</xref></sup>. CERS2 synthesizes very-long-chain ceramides (C20 <)<sup><xref ref-type=\"bibr\" rid=\"CR77\">77</xref>,<xref ref-type=\"bibr\" rid=\"CR78\">78</xref></sup>. Moreover, Jennemann et al. reported that CERS3 uses ultra- but also very-long-chain acyl-CoAs as substrates. Thus, increased expression of ELOVL1 and CERS3 mRNA by dietary GluCer might also contribute to very-long-chain ceramide synthesis.</p><p id=\"Par16\">PNPLA1 plays an essential role in skin barrier function by catalysing the transacylation of the linoleic acid to ultra-long-chain ω-hydroxyceramide for acylceramide production<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR43\">43</xref>,<xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>. Hydrolysis of the ester bond of oxidized linoleic acid residue of acylceramide produce ω-hydroxyceramide<sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup>. Covalently bound ω-hydroxyceramide is formed by crosslinking the exposed ω-OH groups of ω-hydroxyceramide with cornified envelope protein<sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup>. Thus, acylceramide is a precursor of covalently bound ω-hydroxyceramide. The enhancement of PNPLA1 mRNA expression by dietary CAEP shown in this study could contribute to improved efficacy of dietary CAEP on skin barrier function. On the other hand, TEWL and wrinkles were improved at day 3, so the expression of mRNAs may increase before the day 3.</p><p id=\"Par17\">An earlier report showed that each sphingoid base such as sphinganine (d18:0), d18:1 and 4,8-sphingadienine (d18:2) similarly enhanced mRNA expression of CERS2-4 in human foreskin keratinocytes<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. Sphingatrienine (d18:3) and d19:3, purified from sea star, enhanced the de novo ceramide synthesis and mRNA expression of CERSs and ELOVLs in undifferentiated keratinocytes<sup><xref ref-type=\"bibr\" rid=\"CR79\">79</xref></sup>. Thus, sphingoid bases derived from CAEP may have a similar effect on skin barrier function. Further investigation is needed to elucidate whether dietary CAEP enhances de novo synthesis of sphingoid bases which can contribute to the improving effect on skin barrier function. Additionally, sphingoid bases (d18:2 and t18:1 (4-hydroxy-8-sphinganine)) facilitated the expression of PPARβ/δ and PPARγ mRNA and acted as a ligand for PPARγ<sup><xref ref-type=\"bibr\" rid=\"CR80\">80</xref></sup>. Recent studies have shown that activators of PPARα, PPARβ/δ, and PPARγ improved skin barrier function due to increased expression of epidermal ceramide synthesis-related genes and regulation of keratinocyte differentiation<sup><xref ref-type=\"bibr\" rid=\"CR81\">81</xref>–<xref ref-type=\"bibr\" rid=\"CR83\">83</xref></sup>. Thus, sphingoid bases might induce ceramide synthesis via PPAR activation.</p><p id=\"Par18\">Due to low dietary absorption of sphingoid bases, as shown earlier<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>, likely, sphingoid bases from dietary sources are hardly reutilized in the skin. However, even a modest delivery of sphingoid bases from dietary CAEP to skin might affect the ceramide synthesis because a low dose of sphingoid bases upregulated CERSs mRNA expression in keratinocytes<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. CAEP used in this experiment is composed not only d18:1, which is a principal sphingoid base in mammals, but also unique sphingoid bases d19:3 and d16:1. d19:3 and d16:1 from CAEP were undetectable and d18:1 did not increase in the epidermis after CAEP administration in this study (data not shown). Therefore, suggesting that sphingoid bases derived from dietary sphingolipids are hardly reutilized to form the sphingolipids in the epidermis; however, they or their metabolites might play a role as triggers or signalling molecules in epidermal ceramide synthesis.</p><p id=\"Par19\">In conclusion, the present study shows a novel role of dietary CAEP on the improvement of skin barrier function and its involvement in the synthesis of epidermal covalently bound ω-hydroxyceramide constituted by ultra-long-chain ceramides. Moreover, the upregulation of ultra- and very-long-chain ceramide synthesis by dietary CAEP could contribute to the cornified lipid envelope formation. These effects of dietary CAEP were comparable to the dietary GluCer. The potential of dietary CAEP might be due to its easy digestion and absorption compared to other sphingolipids as described earlier<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. Sphingoid base composition of maize GluCer was 66.1% d18:2, 13.6% t18:1, and others as described previously<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. Structural difference among sphingolipids may affect their degradation and absorption. The findings in this study provide novel insights into the efficacy of dietary marine sphingophosphonolipid.</p></sec><sec id=\"Sec6\"><title>Materials and methods</title><sec id=\"Sec7\"><title>Preparation of sphingolipids</title><p id=\"Par20\">CAEP was purified from crude lipids extracted from the skin of jumbo flying squid, <italic>Dosidicus gigas</italic>, kindly donated by Dr. Saito (Ishikawa Prefectural University, Japan) using previously described methods<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. Constituent sphingoid base of the CAEP (purity 98%) were 41.4% d19:3, 28.6% d16:1, 12.9% d18:1, with smaller fractions of others as shown previously<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. GluCer prepared from maize was kindly donated by Nippon Flour Mills Co. Ltd. (Atsugi, Japan). The purity of the GluCer was 92%.</p></sec><sec id=\"Sec8\"><title>Animals and diets</title><p id=\"Par21\">Female hairless mice (Hos: HR-1, 4-week-old) were purchased from Hoshino Laboratory Animals, Inc (Ibaragi, Japan) and maintained following the Guide for the Care and Use of Laboratory Animals (Animal Care Committee, Kyoto University). Approval for all protocols used in this study was from the Kyoto University animal committee (no. 27–37). The mice were individually housed in plastic cages at 24 ± 1 °C with a 12-h light/dark cycle and free access to diet and distilled water. All mice were fed MF standard chow (Oriental Yeast, Tokyo, Japan) for a week, acclimated before the start of the experiments, and were subjected to the experimental protocols as described earlier<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. For developing the dry-skin condition (i.e., perturbations in the skin barrier), mice were fed magnesium-deficient diet (HR-AD chow, Nosan Corp., Yokohama, Japan) for 11 weeks<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref>,<xref ref-type=\"bibr\" rid=\"CR84\">84</xref></sup>. Subsequently, mice were randomly divided into four groups. The mice sacrificed immediately after the HR-AD feeding period were designated as HR-AD group (n = 5). After the HR-AD feeding period, mice in the control group were fed AIN-93G for seven days (n = 5). Mice in the CAEP and GluCer group received the AIN-93G diet containing 0.1% CAEP and 0.1% GluCer, respectively (n = 6 in each group) (Table S2). Body weights and dietary intake of each mouse were measured daily. At the end of each treatment, mice were sacrificed under isoflurane anaesthesia, and the dorsal skin specimens collected immediately. Pieces of dorsal skin were fixed in 10% neutral buffered formalin solution for morphological analysis. To analyse the mRNA expression, part of the skin specimens was stored in RNAlater (Qiagen, Valencia, CA) at − 80 °C until use. The skin specimens for lipid analysis were frozen immediately at − 80 °C.</p></sec><sec id=\"Sec9\"><title>Measurement of skin barrier functions</title><p id=\"Par22\">TEWL and the dorsal skin hydration index of the mice were measured on day-0, -3, and -6 after switching the HR-AD diet to the experimental diets. The measurements of TEWL and the hydration index by Tewameter TM 300 and Corneometer CM 825 (Courage Khazaka electronic GmbH, Cologne, Germany), respectively, were continued until the TEWL recovered to the normal level (below 10 g/m<sup>2</sup>/h).</p></sec><sec id=\"Sec10\"><title>Evaluation of the wrinkles in the skin</title><p id=\"Par23\">On day 0, 3, and 6 of the recovery treatment, the skin surface replicas were collected from the dorsal skin, using a silicone product (Asch Japan Co., Ltd, Hachioji, Japan) under isoflurane anaesthesia. For evaluating the wrinkling degree, parameters (number of wrinkles, the average depth of wrinkles, wrinkle area ratio, and wrinkle volume ratio) of the skin replica were measured using skin wrinkle analysis software (Asch Japan Co., Ltd)<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref>,<xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>.</p></sec><sec id=\"Sec11\"><title>Histological analysis of epidermis</title><p id=\"Par24\">For evaluating the morphological changes, pieces of dorsal skin were sectioned and stained with haematoxylin and eosin (H&E) by Biopathology Institute (Oita, Japan). Immunohistochemical staining with antibody against involucrin and filaggrin was performed for typical two animals in each group at Biopathology Institute. The thickness of the epidermis and the area of immune-stained involucrin were measured using a Biorevo all-in-one microscope (BZ-9000, Keyence Co., Osaka, Japan) and Image J software (Wayne Rasband, National Institutes of Health, Bethesda, Maryland, USA), respectively. The average value of 10 random determinations was considered as the representative value for the individual animal.</p></sec><sec id=\"Sec12\"><title>HPLC analysis of covalently bound ceramides in the epidermis</title><p id=\"Par25\">Covalently bound ceramides were extracted with a slight modification of earlier methods<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. The skin epidermis was separated from the dermis at the basement membrane by overnight incubation at 4 °C with 2.5 U/mL Dispase II (neutral protease, grade II, Roche Diagnostics GmbH Mannheim, Germany) in Hanks' balanced salt solution + (HBSS( +), Nacalai Tesque). After removal of the epidermal lipids using chloroform/methanol (2:1, v/v), ω-hydroxyceramides bound to the stratum corneum by ester bonds were released by overnight incubation in 1 M KOH in 95% methanol at room temperature. Subsequent extraction of ω-hydroxyceramides was with chloroform/methanol (2:1, v/v) after neutralization with acetic acid. The protein level in the residue quantified using a DC Protein Assay kit (Bio-Rad Laboratories, CA, USA). Analysis of ω-hydroxyceramides was done using HPLC system coupled with an ion trap time-of-flight mass-spectrometer (LCMS-IT-TOF; Shimadzu Co., Kyoto, Japan), equipped with atmospheric pressure chemical ionisation (APCI) or electrospray ionisation (ESI) interface (Shimadzu) using an earlier method with some modifications<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. Mobile phase A consisted of 1 mM ammonium acetate in methanol and 2 mM ammonium acetate in water (80:20, v/v). Mobile phase B consisted of 1 mM ammonium acetate in methanol and 2 mM ammonium acetate in water (99:1, v/v). The TSKgel ODS-100Z column (2.0 mm × 50 mm, i.d., 3 μm, Tosoh, Tokyo, Japan) was eluted using the following binary gradients: 0–5 min, 50–100% B; 5–35 min, 100% B; 35–40 min, 100–50% B. The MS (range, m/z 400 to 1,200) and MS/MS (range, m/z 200 to 350) spectra were acquired in the positive scan mode. For structural analysis of ω-hydroxyceramides, [M + H – H<sub>2</sub>O]<sup>+</sup> was used to obtain product ions by MS/MS analysis following earlier method<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. In Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>, typical signals of d17:1 [M + H – 2H<sub>2</sub>O]<sup>+</sup> (m/z 250.3) and d18:1 [M + H – 2H<sub>2</sub>O]<sup>+</sup> (m/z 264.3), characteristic sphingoid bases present in the mouse skin<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup> were observed as product ions using auto MS/MS detection mode<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. Pairs of structurally-specific product ions of sphingoid bases and their precursor-ions were used to identify ω-hydroxyceramide molecules. The precursor-ions as [M + H – H<sub>2</sub>O]<sup>+</sup> were used to semi-quantify each molecule of ω-hydroxyceramide by LCMS-2010 eV (Shimadzu) equipped with APCI probe. The analytical conditions were as described before with some modifications<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. HPLC conditions were like that of LCMS-IT-TOF analysis described above. The relative peak area of ω-hydroxyceramide was calculated using an authentic standard (N-<italic>omega</italic>-hydroxy-C30:0-<sub>D</sub>-<italic>erythro</italic>-ceramide, Matreya LLC, Inc., Pleasant Gap, PA, USA).</p></sec><sec id=\"Sec13\"><title>RNA preparation and real-time qRT-PCR</title><p id=\"Par26\">Total RNA extraction, cDNA synthesis, and real-time quantitative qPCR were performed as described before with some modifications<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref>,<xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. Total RNA was extracted from the skin samples in RNAlater by using Sepasol reagent (Nacalai Tesque) following the manufacturer's instructions. cDNAs were synthesized using SuperScript RNase II reverse transcriptase (Invitrogen, CA, USA) with random hexamers. For RT-PCR, 3 μL diluted cDNA was mixed with 7 μL iQ SYBR Green Supermix (Bio-Rad Laboratories, CA, USA) containing 2 μL PCR primer (5 μM, primer sequences are shown in Table S3). Real-time qRT-PCR performed by using a DNA Engine Option system (Bio-Rad Laboratories). The thermal cycling conditions were 3 min at 95 °C for 1 cycle, followed by amplification for 40 cycles with melting for 15 s at 95 °C, and annealing and extension for 30 s at 60 °C. The expression level of each gene was normalized using β-actin (ACTB) mRNA as an internal control.</p></sec><sec id=\"Sec14\"><title>Statistical analysis</title><p id=\"Par27\">Data are reported as means ± standard errors. Statistical analysis was by using Stat View software (SAS Institute, NC, USA). Two-way repeated ANOVA analysed body weight and food intake. Analysis of other measurements was by one-way ANOVA and Tukey–Kramer tests as a post hoc test. Data were considered statistically significant with <italic>P</italic>-values < 0.05.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec15\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70888_MOESM1_ESM.docx\"><caption><p>Supplementary information</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70888-0.</p></sec><ack><title>Acknowledgements</title><p>We thank Dr. Saito and Dr. Itonori for providing valuable materials. This work was supported by JSPS KAKENHI [Grant Numbers JP15J01143, JP16H04923 and JP20H02931].</p></ack><notes notes-type=\"author-contribution\"><title>Author Contributions</title><p>T.S. and N.T. designed the study. N.T. carried out the experiments. N.T. analysed the data. N.T. and T.S. wrote the main manuscript text. Y.M. and K.A. contributed to the interpretation of the results and discussion. All authors reviewed the manuscript.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par28\">Nippon Flour Mills Corporation funded this study and provided support for the author T.S. The other authors state no conflict of interest.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Breiden</surname><given-names>B</given-names></name><name><surname>Sandhoff</surname><given-names>K</given-names></name></person-group><article-title>The role of sphingolipid metabolism in cutaneous permeability barrier formation</article-title><source>Biochim. Biophys. Acta.</source><year>2014</year><volume>1841</volume><fpage>441</fpage><lpage>452</lpage><pub-id pub-id-type=\"pmid\">23954553</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Feingold</surname><given-names>KR</given-names></name></person-group><article-title>Thematic review series: skin lipids. The role of epidermal lipids in cutaneous permeability barrier homeostasis</article-title><source>J. Lipid Res.</source><year>2007</year><volume>48</volume><fpage>2531</fpage><lpage>46</lpage><pub-id pub-id-type=\"pmid\">17872588</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gray</surname><given-names>GM</given-names></name><name><surname>White</surname><given-names>RJ</given-names></name><name><surname>Williams</surname><given-names>RH</given-names></name><name><surname>Yardley</surname><given-names>HJ</given-names></name></person-group><article-title>Lipid composition of the superficial stratum corneum cells of pig epidermis</article-title><source>Br. J. Dermatol.</source><year>1982</year><volume>106</volume><fpage>59</fpage><lpage>63</lpage><pub-id pub-id-type=\"pmid\">7059505</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Motta</surname><given-names>S</given-names></name><etal/></person-group><article-title>Ceramide composition of the psoriatic scale</article-title><source>Biochim. Biophys. Acta.</source><year>1993</year><volume>1182</volume><fpage>147</fpage><lpage>151</lpage><pub-id pub-id-type=\"pmid\">8357845</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Motta</surname><given-names>S</given-names></name><etal/></person-group><article-title>Abnormality of water barrier function in psoriasis Role of ceramide fractions</article-title><source>Arch. Dermatol.</source><year>1994</year><volume>130</volume><fpage>52</fpage><lpage>56</lpage></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yoon</surname><given-names>JS</given-names></name><etal/></person-group><article-title>Alteration of stratum corneum ceramide profiles in spontaneous canine model of atopic dermatitis</article-title><source>Exp. Dermatol.</source><year>2011</year><volume>20</volume><fpage>732</fpage><lpage>736</lpage><pub-id pub-id-type=\"pmid\">21649737</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Franco</surname><given-names>J</given-names></name><name><surname>Ferreira</surname><given-names>C</given-names></name><name><surname>Paschoal Sobreira</surname><given-names>TJ</given-names></name><name><surname>Sundberg</surname><given-names>JP</given-names></name><name><surname>HogenEsch</surname><given-names>H</given-names></name></person-group><article-title>Profiling of epidermal lipids in a mouse model of dermatitis: Identification of potential biomarkers</article-title><source>PLoS ONE</source><year>2018</year><volume>13</volume><fpage>e0196595</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0196595</pub-id><pub-id pub-id-type=\"pmid\">29698466</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Imokawa</surname><given-names>G</given-names></name><etal/></person-group><article-title>Decreased level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry skin?</article-title><source>J. Invest. Dermatol.</source><year>1991</year><volume>96</volume><fpage>523</fpage><lpage>526</lpage><pub-id pub-id-type=\"pmid\">2007790</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Di Nardo</surname><given-names>A</given-names></name><name><surname>Wertz</surname><given-names>P</given-names></name><name><surname>Giannetti</surname><given-names>A</given-names></name><name><surname>Seidenari</surname><given-names>S</given-names></name></person-group><article-title>Ceramide and cholesterol composition of the skin of patients with atopic dermatitis</article-title><source>Acta. Derm Venereol.</source><year>1998</year><volume>78</volume><fpage>27</fpage><lpage>30</lpage><pub-id pub-id-type=\"pmid\">9498022</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Joo</surname><given-names>KM</given-names></name><etal/></person-group><article-title>Relationship between cutaneous barrier function and ceramide species in human stratum corneum</article-title><source>J. Dermatol. Sci.</source><year>2010</year><volume>60</volume><fpage>47</fpage><lpage>50</lpage><pub-id pub-id-type=\"pmid\">20837392</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Meguro</surname><given-names>S</given-names></name><name><surname>Arai</surname><given-names>Y</given-names></name><name><surname>Masukawa</surname><given-names>Y</given-names></name><name><surname>Uie</surname><given-names>K</given-names></name><name><surname>Tokimitsu</surname><given-names>I</given-names></name></person-group><article-title>Relationship between covalently bound ceramides and transepidermal water loss (TEWL)</article-title><source>Arch. Dermatol. Res.</source><year>2000</year><volume>292</volume><fpage>463</fpage><lpage>468</lpage><pub-id pub-id-type=\"pmid\">11000290</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Macheleidt</surname><given-names>O</given-names></name><name><surname>Kaiser</surname><given-names>HW</given-names></name><name><surname>Sandhoff</surname><given-names>K</given-names></name></person-group><article-title>Deficiency of epidermal protein-bound omega-hydroxyceramides in atopic dermatitis</article-title><source>J. Invest. Dermatol.</source><year>2002</year><volume>119</volume><fpage>166</fpage><lpage>173</lpage><pub-id pub-id-type=\"pmid\">12164940</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kypriotou</surname><given-names>M</given-names></name><name><surname>Huber</surname><given-names>M</given-names></name><name><surname>Hohl</surname><given-names>D</given-names></name></person-group><article-title>The human epidermal differentiation complex: cornified envelope precursors, S100 proteins and the 'fused genes' family</article-title><source>Exp. Dermatol.</source><year>2012</year><volume>21</volume><fpage>643</fpage><lpage>649</lpage><pub-id pub-id-type=\"pmid\">22507538</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Muñoz-Garcia</surname><given-names>A</given-names></name><name><surname>Thomas</surname><given-names>CP</given-names></name><name><surname>Keeney</surname><given-names>DS</given-names></name><name><surname>Zheng</surname><given-names>Y</given-names></name><name><surname>Brash</surname><given-names>AR</given-names></name></person-group><article-title>The importance of the lipoxygenase-hepoxilin pathway in the mammalian epidermal barrier</article-title><source>Biochim. Biophys. Acta.</source><year>2014</year><volume>1841</volume><fpage>401</fpage><lpage>408</lpage><pub-id pub-id-type=\"pmid\">24021977</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ohno</surname><given-names>Y</given-names></name><name><surname>Kamiyama</surname><given-names>N</given-names></name><name><surname>Nakamichi</surname><given-names>S</given-names></name><name><surname>Kihara</surname><given-names>A</given-names></name></person-group><article-title>PNPLA1 is a transacylase essential for the generation of the skin barrier lipid ω-O-acylceramide</article-title><source>Nat. Commun.</source><year>2017</year><volume>8</volume><fpage>14610</fpage><pub-id pub-id-type=\"doi\">10.1038/ncomms14610</pub-id><pub-id pub-id-type=\"pmid\">28248318</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morifuji</surname><given-names>M</given-names></name><etal/></person-group><article-title>A novel mechanism for improvement of dry skin by dietary milk phospholipids: effect on epidermal covalently bound ceramides and skin inflammation in hairless mice</article-title><source>J. Dermatol. Sci.</source><year>2015</year><volume>78</volume><fpage>224</fpage><lpage>231</lpage><pub-id pub-id-type=\"pmid\">25816721</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Karlsson</surname><given-names>KA</given-names></name></person-group><article-title>Sphingolipid long chain bases</article-title><source>Lipids</source><year>1970</year><volume>5</volume><fpage>878</fpage><lpage>891</lpage><pub-id pub-id-type=\"pmid\">4921900</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vesper</surname><given-names>H</given-names></name><etal/></person-group><article-title>Critical Review Sphingolipids in food and the emerging importance of sphingolipids to nutrition</article-title><source>J. Nutr.</source><year>1999</year><volume>129</volume><fpage>1239</fpage><lpage>1250</lpage><pub-id pub-id-type=\"pmid\">10395583</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yunoki</surname><given-names>K</given-names></name><etal/></person-group><article-title>Analysis of sphingolipid classes and their contents in meals</article-title><source>Biosci. Biotechnol. Biochem.</source><year>2008</year><volume>72</volume><fpage>222</fpage><lpage>225</lpage><pub-id pub-id-type=\"pmid\">18175909</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fischbeck</surname><given-names>A</given-names></name><name><surname>Krüger</surname><given-names>M</given-names></name><name><surname>Blaas</surname><given-names>N</given-names></name><name><surname>Humpf</surname><given-names>HU</given-names></name></person-group><article-title>Analysis of sphingomyelin in meat based on hydrophilic interaction liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (HILIC-HPLC-ESI-MS/MS)</article-title><source>J. Agric. Food Chem.</source><year>2009</year><volume>57</volume><fpage>9469</fpage><lpage>9474</lpage><pub-id pub-id-type=\"pmid\">20175585</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zeisel</surname><given-names>SH</given-names></name><name><surname>Char</surname><given-names>D</given-names></name><name><surname>Sheard</surname><given-names>NF</given-names></name></person-group><article-title>Choline, phosphatidylcholine and sphingomyelin in human and bovine milk and infant formulas</article-title><source>J. Nutr.</source><year>1986</year><volume>116</volume><fpage>50</fpage><lpage>58</lpage><pub-id pub-id-type=\"pmid\">3944656</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Newburg</surname><given-names>DS</given-names></name><name><surname>Chaturvedi</surname><given-names>P</given-names></name></person-group><article-title>Neutral glycolipids of human and bovine milk</article-title><source>Lipids</source><year>1992</year><volume>27</volume><fpage>923</fpage><lpage>927</lpage><pub-id pub-id-type=\"pmid\">1491612</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sugawara</surname><given-names>T</given-names></name><name><surname>Miyazawa</surname><given-names>T</given-names></name></person-group><article-title>Separation and determination of glycolipids from edible plant sources by high-performance liquid chromatography and evaporative light-scattering detection</article-title><source>Lipids</source><year>1999</year><volume>34</volume><fpage>1231</fpage><lpage>1237</lpage><pub-id pub-id-type=\"pmid\">10606047</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tsuji</surname><given-names>K</given-names></name><etal/></person-group><article-title>Dietary glucosylceramide improves skin barrier function in hairless mice</article-title><source>J. Dermatol. Sci.</source><year>2006</year><volume>44</volume><fpage>101</fpage><lpage>107</lpage><pub-id pub-id-type=\"pmid\">17000082</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haruta</surname><given-names>Y</given-names></name><name><surname>Kato</surname><given-names>K</given-names></name><name><surname>Yoshioka</surname><given-names>T</given-names></name></person-group><article-title>Dietary phospholipid concentrate from bovine milk improves epidermal function in hairless mice</article-title><source>Biosci. Biotechnol. Biochem.</source><year>2008</year><volume>72</volume><fpage>2151</fpage><lpage>2157</lpage><pub-id pub-id-type=\"pmid\">18685188</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Duan</surname><given-names>J</given-names></name><etal/></person-group><article-title>Dietary sphingolipids improve skin barrier functions via the upregulation of ceramide synthases in the epidermis</article-title><source>Exp. Dermatol.</source><year>2012</year><volume>21</volume><fpage>448</fpage><lpage>452</lpage><pub-id pub-id-type=\"pmid\">22621186</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Duan</surname><given-names>J</given-names></name><etal/></person-group><article-title>Dietary cerebroside from sea cucumber (<italic>Stichopus japonicus</italic>): absorption and effects on skin barrier and cecal short-chain fatty acids</article-title><source>J. Agric. Food Chem.</source><year>2016</year><volume>64</volume><fpage>7014</fpage><lpage>7021</lpage><pub-id pub-id-type=\"pmid\">27585906</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sugawara</surname><given-names>T</given-names></name><name><surname>Kinoshita</surname><given-names>M</given-names></name><name><surname>Ohnishi</surname><given-names>M</given-names></name><name><surname>Nagata</surname><given-names>J</given-names></name><name><surname>Saito</surname><given-names>M</given-names></name></person-group><article-title>Digestion of maize sphingolipids in rats and uptake of sphingadienine by Caco-2 cells</article-title><source>J. Nutr.</source><year>2003</year><volume>133</volume><fpage>2777</fpage><lpage>2782</lpage><pub-id pub-id-type=\"pmid\">12949364</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sugawara</surname><given-names>T</given-names></name><etal/></person-group><article-title>Intestinal absorption of dietary maize glucosylceramide in lymphatic duct cannulated rats</article-title><source>J. Lipid Res.</source><year>2010</year><volume>51</volume><fpage>1761</fpage><lpage>1769</lpage><pub-id pub-id-type=\"pmid\">20211933</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Simon</surname><given-names>G</given-names></name><name><surname>Rouser</surname><given-names>G</given-names></name></person-group><article-title>Phospholipids of the sea anemone: quantitative distribution; absence of carbon-phosphorus linkages in glycerol phospholipids; structural elucidation of ceramide aminoethylphosphonate</article-title><source>Lipids</source><year>1967</year><volume>2</volume><fpage>55</fpage><lpage>59</lpage><pub-id pub-id-type=\"pmid\">17805722</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moschidis</surname><given-names>MC</given-names></name></person-group><article-title>Phosphonolipids</article-title><source>Prog. Lipid Res.</source><year>1984</year><volume>23</volume><fpage>223</fpage><lpage>246</lpage><pub-id pub-id-type=\"pmid\">6400665</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Joseph</surname><given-names>JD</given-names></name></person-group><article-title>Lipid composition of marine and estuarine invertebrates Part II: mollusca</article-title><source>Prog. Lipid Res.</source><year>1982</year><volume>21</volume><fpage>109</fpage><lpage>153</lpage><pub-id pub-id-type=\"pmid\">7051043</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Irie</surname><given-names>A</given-names></name><name><surname>Kubo</surname><given-names>H</given-names></name><name><surname>Hoshi</surname><given-names>M</given-names></name></person-group><article-title>Glucosylceramide having a novel tri-unsaturated long-chain base from the spermatozoa of the starfish Asterias amurensis</article-title><source>J. Biochem.</source><year>1990</year><volume>107</volume><fpage>578</fpage><lpage>586</lpage><pub-id pub-id-type=\"pmid\">2358431</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jin</surname><given-names>W</given-names></name><name><surname>Rinehart</surname><given-names>KL</given-names></name><name><surname>Jares-Erijman</surname><given-names>EA</given-names></name></person-group><article-title>Ophidiacerebrosides: cytotoxic glycosphingolipids containing a novel sphingosine from a sea star</article-title><source>J. Org. Chem.</source><year>1994</year><volume>59</volume><fpage>144</fpage><lpage>147</lpage></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ohashi</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Squid nerve sphingomyelin containing an unusual sphingoid base</article-title><source>J. Lipid Res.</source><year>2000</year><volume>41</volume><fpage>1118</fpage><lpage>1124</lpage><pub-id pub-id-type=\"pmid\">10884294</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sugawara</surname><given-names>T</given-names></name><name><surname>Aida</surname><given-names>K</given-names></name><name><surname>Duan</surname><given-names>J</given-names></name><name><surname>Hirata</surname><given-names>T</given-names></name></person-group><article-title>Analysis of glucosylceramides from various sources by liquid chromatography-ion trap mass spectrometry</article-title><source>J. Oleo. Sci.</source><year>2010</year><volume>59</volume><fpage>387</fpage><lpage>394</lpage><pub-id pub-id-type=\"pmid\">20513973</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Facchini</surname><given-names>L</given-names></name><name><surname>Losito</surname><given-names>I</given-names></name><name><surname>Cataldi</surname><given-names>TR</given-names></name><name><surname>Palmisano</surname><given-names>F</given-names></name></person-group><article-title>Ceramide lipids in alive and thermally stressed mussels: an investigation by hydrophilic interaction liquid chromatography-electrospray ionization fourier transform mass spectrometry</article-title><source>J. Mass Spectrom.</source><year>2016</year><volume>51</volume><fpage>768</fpage><lpage>781</lpage><pub-id pub-id-type=\"pmid\">27479706</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mukhamedova</surname><given-names>KhS</given-names></name><name><surname>Glushenkova</surname><given-names>AI</given-names></name></person-group><article-title>Natural phosphonolipids</article-title><source>Chem. Nat. Comp.</source><year>2000</year><volume>36</volume><fpage>329</fpage><lpage>341</lpage></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Saito</surname><given-names>H</given-names></name><name><surname>Ishikawa</surname><given-names>S</given-names></name></person-group><article-title>Characteristic of lipids and fatty acid compositions of the neon flying squid <italic>Ommastrephes bartramii</italic></article-title><source>J. Oleo. Sci.</source><year>2012</year><volume>61</volume><fpage>547</fpage><lpage>564</lpage><pub-id pub-id-type=\"pmid\">23018852</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hori</surname><given-names>T</given-names></name><name><surname>Arakawa</surname><given-names>I</given-names></name><name><surname>Sugita</surname><given-names>M</given-names></name></person-group><article-title>Distribution of ceramide 2-aminoethylphosphonate and ceramide aminoethylphosphate (sphingoethanolamine) in some aquatic animals</article-title><source>J. Biochem.</source><year>1967</year><volume>62</volume><fpage>67</fpage><lpage>70</lpage><pub-id pub-id-type=\"pmid\">6073176</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tomonaga</surname><given-names>N</given-names></name><name><surname>Manabe</surname><given-names>Y</given-names></name><name><surname>Sugawara</surname><given-names>T</given-names></name></person-group><article-title>Digestion of ceramide 2-aminoethylphosphonate, a sphingolipid from the jumbo flying squid <italic>Dosidicus gigas</italic>, in mice</article-title><source>Lipids</source><year>2017</year><volume>52</volume><fpage>353</fpage><lpage>362</lpage><pub-id pub-id-type=\"pmid\">28243820</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tomonaga</surname><given-names>N</given-names></name><name><surname>Tsuduki</surname><given-names>T</given-names></name><name><surname>Manabe</surname><given-names>Y</given-names></name><name><surname>Sugawara</surname><given-names>T</given-names></name></person-group><article-title>Sphingoid bases of dietary ceramide 2-aminoethylphosphonate, a marine sphingolipid, absorb into lymph in rats</article-title><source>J. Lipid Res.</source><year>2019</year><volume>60</volume><fpage>333</fpage><lpage>340</lpage><pub-id pub-id-type=\"pmid\">30552287</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fujii</surname><given-names>M</given-names></name><etal/></person-group><article-title>Atopic dermatitis-like pruritic skin inflammation caused by feeding a special diet to HR-1 hairless mice</article-title><source>Exp. Dermatol.</source><year>2005</year><volume>14</volume><fpage>460</fpage><lpage>468</lpage><pub-id pub-id-type=\"pmid\">15885082</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Makiura</surname><given-names>M</given-names></name><etal/></person-group><article-title>Atopic dermatitis-like symptoms in HR-1 hairless mice fed a diet low in magnesium and zinc</article-title><source>J. Int. Med. Res.</source><year>2004</year><volume>32</volume><fpage>392</fpage><lpage>399</lpage><pub-id pub-id-type=\"pmid\">15303770</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><mixed-citation publication-type=\"other\">National Research Council (US). Subcommittee on Laboratory Animal Nutrition. <italic>Nutrient requirements of laboratory animals</italic>, National Academy of Sciences, Washington, D.C., (1995)</mixed-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liebisch</surname><given-names>G</given-names></name><etal/></person-group><article-title>Shorthand notation for lipid structures derived from mass spectrometry</article-title><source>J. Lipid Res.</source><year>2013</year><volume>54</volume><fpage>1523</fpage><lpage>1530</lpage><pub-id pub-id-type=\"pmid\">23549332</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Elias</surname><given-names>PM</given-names></name><etal/></person-group><article-title>Formation and functions of the corneocyte lipid envelope (CLE)</article-title><source>Biochim. Biophys. Acta.</source><year>2014</year><volume>1841</volume><fpage>314</fpage><lpage>318</lpage><pub-id pub-id-type=\"pmid\">24076475</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hirabayashi</surname><given-names>T</given-names></name><etal/></person-group><article-title>PNPLA1 has a crucial role in skin barrier function by directing acylceramide biosynthesis</article-title><source>Nat. Commun.</source><year>2017</year><volume>8</volume><fpage>14609</fpage><pub-id pub-id-type=\"doi\">10.1038/ncomms14609</pub-id><pub-id pub-id-type=\"pmid\">28248300</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Denda</surname><given-names>M</given-names></name><name><surname>Sato</surname><given-names>J</given-names></name><name><surname>Tsuchiya</surname><given-names>T</given-names></name><name><surname>Elias</surname><given-names>PM</given-names></name><name><surname>Feingold</surname><given-names>KR</given-names></name></person-group><article-title>Low humidity stimulates epidermal DNA synthesis and amplifies the hyperproliferative response to barrier disruption: implication for seasonal exacerbations of inflammatory dermatoses</article-title><source>J. Invest. Dermatol.</source><year>1998</year><volume>111</volume><fpage>873</fpage><lpage>878</lpage><pub-id pub-id-type=\"pmid\">9804352</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jensen</surname><given-names>JM</given-names></name><etal/></person-group><article-title>Impaired sphingomyelinase activity and epidermal differentiation in atopic dermatitis</article-title><source>J. Invest Dermatol.</source><year>2004</year><volume>122</volume><fpage>1423</fpage><lpage>1431</lpage><pub-id pub-id-type=\"pmid\">15175033</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hashizume</surname><given-names>H</given-names></name></person-group><article-title>Skin aging and dry skin</article-title><source>J. Dermatol.</source><year>2004</year><volume>31</volume><fpage>603</fpage><lpage>609</lpage><pub-id pub-id-type=\"pmid\">15492432</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Urikura</surname><given-names>I</given-names></name><name><surname>Sugawara</surname><given-names>T</given-names></name><name><surname>Hirata</surname><given-names>T</given-names></name></person-group><article-title>Protective effect of Fucoxanthin against UVB-induced skin photoaging in hairless mice</article-title><source>Biosci. Biotechnol. Biochem.</source><year>2011</year><volume>75</volume><fpage>757</fpage><lpage>760</lpage><pub-id pub-id-type=\"pmid\">21512228</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Komatsu</surname><given-names>T</given-names></name><name><surname>Sasaki</surname><given-names>S</given-names></name><name><surname>Manabe</surname><given-names>Y</given-names></name><name><surname>Hirata</surname><given-names>T</given-names></name><name><surname>Sugawara</surname><given-names>T</given-names></name></person-group><article-title>Preventive effect of dietary astaxanthin on UVA-induced skin photoaging in hairless mice</article-title><source>PLoS ONE</source><year>2017</year><volume>12</volume><fpage>e0171178</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0171178</pub-id><pub-id pub-id-type=\"pmid\">28170435</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shimada</surname><given-names>E</given-names></name><name><surname>Aida</surname><given-names>K</given-names></name><name><surname>Sugawara</surname><given-names>T</given-names></name><name><surname>Hirata</surname><given-names>T</given-names></name></person-group><article-title>Inhibitory effect of topical maize glucosylceramide on skin photoaging in UVA-irradiated hairless mice</article-title><source>J. Oleo. Sci.</source><year>2011</year><volume>60</volume><fpage>321</fpage><lpage>325</lpage><pub-id pub-id-type=\"pmid\">21606620</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ekanayake-Mudiyanselage</surname><given-names>S</given-names></name><etal/></person-group><article-title>Expression of epidermal keratins and the cornified envelope protein involucrin is influenced by permeability barrier disruption</article-title><source>J. Invest. Dermatol.</source><year>1998</year><volume>111</volume><fpage>517</fpage><lpage>523</lpage><pub-id pub-id-type=\"pmid\">9740250</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scott</surname><given-names>IR</given-names></name><name><surname>Harding</surname><given-names>CR</given-names></name></person-group><article-title>Filaggrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment</article-title><source>Dev. Biol.</source><year>1986</year><volume>115</volume><fpage>84</fpage><lpage>92</lpage><pub-id pub-id-type=\"pmid\">3516761</pub-id></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Seguchi</surname><given-names>T</given-names></name><etal/></person-group><article-title>Decreased expression of filaggrin in atopic skin</article-title><source>Arch. Dermatol. Res..</source><year>1996</year><volume>288</volume><fpage>442</fpage><lpage>446</lpage><pub-id pub-id-type=\"pmid\">8844122</pub-id></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van Drongelen</surname><given-names>V</given-names></name><etal/></person-group><article-title>Knock-down of filaggrin does not affect lipid organization and composition in stratum corneum of reconstructed human skin equivalents</article-title><source>Exp. Dermatol.</source><year>2013</year><volume>22</volume><fpage>807</fpage><lpage>712</lpage><pub-id pub-id-type=\"pmid\">24164439</pub-id></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Danso</surname><given-names>M</given-names></name><etal/></person-group><article-title>Altered expression of epidermal lipid bio-synthesis enzymes in atopic dermatitis skin is accompanied by changes in stratum corneum lipid composition</article-title><source>J. Dermatol. Sci.</source><year>2017</year><volume>88</volume><fpage>57</fpage><lpage>66</lpage><pub-id pub-id-type=\"pmid\">28571749</pub-id></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kihara</surname><given-names>A</given-names></name></person-group><article-title>Synthesis and degradation pathways, functions, and pathology of ceramides and epidermal acylceramides</article-title><source>Prog. Lipid Res.</source><year>2016</year><volume>63</volume><fpage>50</fpage><lpage>69</lpage><pub-id pub-id-type=\"pmid\">27107674</pub-id></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fujiwara</surname><given-names>A</given-names></name><etal/></person-group><article-title>Age-related and seasonal changes in covalently bound ceramide content in forearm stratum corneum of Japanese subjects: determination of molecular species of ceramides</article-title><source>Arch. Dermatol. Res.</source><year>2018</year><volume>310</volume><fpage>729</fpage><lpage>735</lpage><pub-id pub-id-type=\"pmid\">30182275</pub-id></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kawana</surname><given-names>M</given-names></name><name><surname>Miyamoto</surname><given-names>M</given-names></name><name><surname>Ohno</surname><given-names>Y</given-names></name><name><surname>Kihara</surname><given-names>A</given-names></name></person-group><article-title>Comparative profiling and comprehensive quantification of stratum corneum ceramides in humans and mice by LC/MS/MS</article-title><source>J. Lipid Res.</source><year>2020</year><volume>61</volume><fpage>884</fpage><lpage>895</lpage><pub-id pub-id-type=\"pmid\">32265320</pub-id></element-citation></ref><ref id=\"CR63\"><label>63.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bouwstra</surname><given-names>JA</given-names></name><name><surname>Gooris</surname><given-names>GS</given-names></name><name><surname>Dubbelaar</surname><given-names>FE</given-names></name><name><surname>Ponec</surname><given-names>M</given-names></name></person-group><article-title>Phase behavior of stratum corneum lipid mixtures based on human ceramides: the role of natural and synthetic ceramide 1</article-title><source>J. Invest. Dermatol.</source><year>2002</year><volume>118</volume><fpage>606</fpage><lpage>617</lpage><pub-id pub-id-type=\"pmid\">11918706</pub-id></element-citation></ref><ref id=\"CR64\"><label>64.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yeom</surname></name><etal/></person-group><article-title>Oral administration of glucosylceramide ameliorates inflammatory dry-skin condition in chronic oxazolone-induced irritant contact dermatitis in the mouse ear</article-title><source>J. Dermatol. Sci.</source><year>2012</year><volume>67</volume><fpage>101</fpage><lpage>110</lpage><pub-id pub-id-type=\"pmid\">22726258</pub-id></element-citation></ref><ref id=\"CR65\"><label>65.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nugteren</surname><given-names>DH</given-names></name></person-group><article-title>The enzymic chain elongation of fatty acids by rat-liver microsomes</article-title><source>Biochim. Biophys. Acta.</source><year>1965</year><volume>106</volume><fpage>280</fpage><lpage>290</lpage><pub-id pub-id-type=\"pmid\">4379659</pub-id></element-citation></ref><ref id=\"CR66\"><label>66.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jakobsson</surname><given-names>A</given-names></name><name><surname>Westerberg</surname><given-names>R</given-names></name><name><surname>Jacobsson</surname><given-names>A</given-names></name></person-group><article-title>Fatty acid elongases in mammals: their regulation and roles in metabolism</article-title><source>Prog. Lipid Res.</source><year>2006</year><volume>45</volume><fpage>237</fpage><lpage>249</lpage><pub-id pub-id-type=\"pmid\">16564093</pub-id></element-citation></ref><ref id=\"CR67\"><label>67.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ohno</surname><given-names>Y</given-names></name><etal/></person-group><article-title>ELOVL1 production of C24 acyl-CoAs is linked to C24 sphingolipid synthesis</article-title><source>Proc. Natl. Acad. Sci. USA</source><year>2010</year><volume>107</volume><fpage>18439</fpage><lpage>18444</lpage><pub-id pub-id-type=\"pmid\">20937905</pub-id></element-citation></ref><ref id=\"CR68\"><label>68.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Agbaga</surname><given-names>MP</given-names></name><etal/></person-group><article-title>Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids</article-title><source>Proc. Natl. Acad. Sci. USA</source><year>2008</year><volume>105</volume><fpage>12843</fpage><lpage>12848</lpage><pub-id pub-id-type=\"pmid\">18728184</pub-id></element-citation></ref><ref id=\"CR69\"><label>69.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sassa</surname><given-names>T</given-names></name><name><surname>Kihara</surname><given-names>A</given-names></name></person-group><article-title>Metabolism of very long-chain Fatty acids: genes and pathophysiology</article-title><source>Biomol. Ther. (Seoul)</source><year>2008</year><volume>22</volume><fpage>83</fpage><lpage>92</lpage></element-citation></ref><ref id=\"CR70\"><label>70.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mandal</surname><given-names>MN</given-names></name><etal/></person-group><article-title>Characterization of mouse orthologue of ELOVL4: genomic organization and spatial and temporal expression</article-title><source>Genomics</source><year>2004</year><volume>83</volume><fpage>626</fpage><lpage>635</lpage><pub-id pub-id-type=\"pmid\">15028285</pub-id></element-citation></ref><ref id=\"CR71\"><label>71.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McMahon</surname><given-names>A</given-names></name><name><surname>Butovich</surname><given-names>IA</given-names></name><name><surname>Kedzierski</surname><given-names>W</given-names></name></person-group><article-title>Epidermal expression of an Elovl4 transgene rescues neonatal lethality of homozygous Stargardt disease-3 mice</article-title><source>J. Lipid Res.</source><year>2011</year><volume>52</volume><fpage>1128</fpage><lpage>1138</lpage><pub-id pub-id-type=\"pmid\">21429867</pub-id></element-citation></ref><ref id=\"CR72\"><label>72.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cameron</surname><given-names>DJ</given-names></name><etal/></person-group><article-title>Essential role of Elovl4 in very long chain fatty acid synthesis, skin permeability barrier function, and neonatal survival</article-title><source>Int. J. Biol. Sci.</source><year>2007</year><volume>3</volume><fpage>111</fpage><lpage>119</lpage><pub-id pub-id-type=\"pmid\">17304340</pub-id></element-citation></ref><ref id=\"CR73\"><label>73.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vasireddy</surname><given-names>V</given-names></name><etal/></person-group><article-title>Loss of functional ELOVL4 depletes very long-chain fatty acids (≥C28) and the unique omega-O-acylceramides in skin leading to neonatal death</article-title><source>Hum. Mol. Genet.</source><year>2007</year><volume>16</volume><fpage>471</fpage><lpage>482</lpage><pub-id pub-id-type=\"pmid\">17208947</pub-id></element-citation></ref><ref id=\"CR74\"><label>74.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Uchida</surname><given-names>Y</given-names></name></person-group><article-title>The role of fatty acid elongation in epidermal structure and function</article-title><source>Dermatoendocrinol</source><year>2011</year><volume>3</volume><fpage>65</fpage><lpage>69</lpage><pub-id pub-id-type=\"pmid\">21695014</pub-id></element-citation></ref><ref id=\"CR75\"><label>75.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sassa</surname><given-names>T</given-names></name><etal/></person-group><article-title>Impaired epidermal permeability barrier in mice lacking elovl1, the gene responsible for very-long-chain fatty acid production</article-title><source>Mol. Cell. Biol.</source><year>2013</year><volume>33</volume><fpage>2787</fpage><lpage>2796</lpage><pub-id pub-id-type=\"pmid\">23689133</pub-id></element-citation></ref><ref id=\"CR76\"><label>76.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jennemann</surname><given-names>R</given-names></name><etal/></person-group><article-title>Loss of ceramide synthase 3 causes lethal skin barrier disruption</article-title><source>Hum. Mol. Genet.</source><year>2012</year><volume>21</volume><fpage>586</fpage><lpage>608</lpage><pub-id pub-id-type=\"pmid\">22038835</pub-id></element-citation></ref><ref id=\"CR77\"><label>77.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sassa</surname><given-names>T</given-names></name><name><surname>Hirayama</surname><given-names>T</given-names></name><name><surname>Kihara</surname><given-names>A</given-names></name></person-group><article-title>Enzyme activities of the ceramide synthases CERS2-6 are regulated by phosphorylation in the c-terminal region</article-title><source>J. Biol. Chem.</source><year>2016</year><volume>291</volume><fpage>7477</fpage><lpage>7487</lpage><pub-id pub-id-type=\"pmid\">26887952</pub-id></element-citation></ref><ref id=\"CR78\"><label>78.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Laviad</surname><given-names>EL</given-names></name><etal/></person-group><article-title>Characterization of ceramide synthase 2: tissue distribution, substrate specificity, and inhibition by sphingosine 1-phosphate</article-title><source>J. Biol. Chem.</source><year>2008</year><volume>283</volume><fpage>5677</fpage><lpage>5684</lpage><pub-id pub-id-type=\"pmid\">18165233</pub-id></element-citation></ref><ref id=\"CR79\"><label>79.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mikami</surname><given-names>D</given-names></name><name><surname>Sakai</surname><given-names>S</given-names></name><name><surname>Sasaki</surname><given-names>S</given-names></name><name><surname>Igarashi</surname><given-names>Y</given-names></name></person-group><article-title>Effects of Asterias amurensis-derived Sphingoid Bases on the <italic>de novo</italic> ceramide synthesis in cultured normal human epidermal keratinocytes</article-title><source>J. Oleo. Sci.</source><year>2016</year><volume>65</volume><fpage>671</fpage><lpage>680</lpage><pub-id pub-id-type=\"pmid\">27430385</pub-id></element-citation></ref><ref id=\"CR80\"><label>80.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shirakura</surname><given-names>Y</given-names></name><etal/></person-group><article-title>4,8-Sphingadienine and 4-hydroxy-8-sphingenine activate ceramide production in the skin</article-title><source>Lipids Health Dis.</source><year>2012</year><volume>11</volume><fpage>108</fpage><pub-id pub-id-type=\"pmid\">22937840</pub-id></element-citation></ref><ref id=\"CR81\"><label>81.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rivier</surname><given-names>M</given-names></name><name><surname>Castiel</surname><given-names>I</given-names></name><name><surname>Safonova</surname><given-names>I</given-names></name><name><surname>Ailhaud</surname><given-names>G</given-names></name><name><surname>Michel</surname><given-names>S</given-names></name></person-group><article-title>Peroxisome proliferator-activated receptor-alpha enhances lipid metabolism in a skin equivalent model</article-title><source>J. Invest. Dermatol.</source><year>2000</year><volume>114</volume><fpage>681</fpage><lpage>687</lpage><pub-id pub-id-type=\"pmid\">10733673</pub-id></element-citation></ref><ref id=\"CR82\"><label>82.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kömüves</surname><given-names>LG</given-names></name><etal/></person-group><article-title>Stimulation of PPARalpha promotes epidermal keratinocyte differentiation in vivo</article-title><source>J. Invest. Dermatol.</source><year>2000</year><volume>115</volume><fpage>353</fpage><lpage>360</lpage><pub-id pub-id-type=\"pmid\">10951268</pub-id></element-citation></ref><ref id=\"CR83\"><label>83.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Man</surname><given-names>MQ</given-names></name><etal/></person-group><article-title>Basis for improved permeability barrier homeostasis induced by PPAR and LXR activators: liposensors stimulate lipid synthesis, lamellar body secretion, and post-secretory lipid processing</article-title><source>J. Invest. Dermatol.</source><year>2006</year><volume>126</volume><fpage>386</fpage><lpage>392</lpage><pub-id pub-id-type=\"pmid\">16374473</pub-id></element-citation></ref><ref id=\"CR84\"><label>84.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ishina</surname><given-names>K</given-names></name><etal/></person-group><article-title>Latent essential fatty acid deficiency in a special diet deteriorates skin barrier</article-title><source>Osaka City Med. J.</source><year>2008</year><volume>54</volume><fpage>63</fpage><lpage>74</lpage></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807842</article-id><article-id pub-id-type=\"pmc\">PMC7431533</article-id><article-id pub-id-type=\"publisher-id\">70969</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70969-0</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>High levels of ubidecarenone (oxidized CoQ<sub>10</sub>) delivered using a drug-lipid conjugate nanodispersion (BPM31510) differentially affect redox status and growth in malignant glioma versus non-tumor cells</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Sun</surname><given-names>Jiaxin</given-names></name><address><email>JIAXSUN@STANFORD.EDU</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Patel</surname><given-names>Chirag B.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Jang</surname><given-names>Taichang</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Merchant</surname><given-names>Milton</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Chen</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kazerounian</surname><given-names>Shiva</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Diers</surname><given-names>Anne R.</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kiebish</surname><given-names>Michael A.</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Vishnudas</surname><given-names>Vivek K.</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Gesta</surname><given-names>Stephane</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sarangarajan</surname><given-names>Rangaprasad</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Narain</surname><given-names>Niven R.</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Nagpal</surname><given-names>Seema</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Recht</surname><given-names>Lawrence</given-names></name><address><email>LRECHT@STANFORD.EDU</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.168010.e</institution-id><institution-id institution-id-type=\"ISNI\">0000000419368956</institution-id><institution>Department of Neurology and Clinical Neurosciences, </institution><institution>Stanford University, </institution></institution-wrap>Palo Alto, CA 94305 USA </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.168010.e</institution-id><institution-id institution-id-type=\"ISNI\">0000000419368956</institution-id><institution>Molecular Imaging Program at Stanford (MIPS), Department of Radiology, </institution><institution>Stanford University School of Medicine, </institution></institution-wrap>Stanford, CA 94305 USA </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.168010.e</institution-id><institution-id institution-id-type=\"ISNI\">0000000419368956</institution-id><institution>Department of Otolaryngology, </institution><institution>Stanford University, </institution></institution-wrap>Palo Alto, CA 94305 USA </aff><aff id=\"Aff4\"><label>4</label>BERG LLC, Framingham, MA 01701 USA </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13899</elocation-id><history><date date-type=\"received\"><day>18</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>4</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Metabolic reprogramming in cancer cells, vs. non-cancer cells, elevates levels of reactive oxygen species (ROS) leading to higher oxidative stress. The elevated ROS levels suggest a vulnerability to excess prooxidant loads leading to selective cell death, a therapeutically exploitable difference. Co-enzyme Q<sub>10</sub> (CoQ<sub>10</sub>) an endogenous mitochondrial resident molecule, plays an important role in mitochondrial redox homeostasis, membrane integrity, and energy production. BPM31510 is a lipid-drug conjugate nanodispersion specifically formulated for delivery of supraphysiological concentrations of ubidecarenone (oxidized CoQ<sub>10</sub>) to the cell and mitochondria, in both in vitro and in vivo model systems. In this study, we sought to investigate the therapeutic potential of ubidecarenone in the highly treatment-refractory glioblastoma. Rodent (C6) and human (U251) glioma cell lines, and non-tumor human astrocytes (HA) and rodent NIH3T3 fibroblast cell lines were utilized for experiments. Tumor cell lines exhibited a marked increase in sensitivity to ubidecarenone vs. non-tumor cell lines. Further, elevated mitochondrial superoxide production was noted in tumor cells vs. non-tumor cells hours before any changes in proliferation or the cell cycle could be detected. In vitro co-culture experiments show ubidecarenone differentially affecting tumor cells vs. non-tumor cells, resulting in an equilibrated culture. In vivo activity in a highly aggressive orthotopic C6 glioma model demonstrated a greater than 25% long-term survival rate. Based on these findings we conclude that high levels of ubidecarenone delivered using BPM31510 provide an effective therapeutic modality targeting cancer-specific modulation of redox mechanisms for anti-cancer effects.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Cancer metabolism</kwd><kwd>CNS cancer</kwd></kwd-group><funding-group><award-group><funding-source><institution>Eric Sun and Karen Law at Brain Tumor Research fund</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>Berg LLC Research Support</institution></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">The Warburg effect was originally described a century ago as an aspect of metabolic rewiring in cancer cells<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>, and is now considered a distinctive hallmark of cancer, emerging in recent years as an important concept in the field of cancer biology<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. Further, recent studies reveal the Warburg phenotype as more than the simple overutilization of glycolysis vs. oxidative metabolism; rather, it reflects a complex re-circuitry of the metabolic machinery, culminating in the facilitation of a hyper-proliferative state<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. The study of metabolic reprogramming in cancer highlights, as a potential vulnerability, the increased levels of steady state reactive oxygen species (ROS) relative to normal tissue<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>–<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>.\n</p><p id=\"Par3\">ROS, which include H<sub>2</sub>O<sub>2</sub>, superoxide anions (O<sub>2</sub><sup>−</sup>), and hydroxyl radicals (OH<sup>•</sup>), are byproducts of aerobic metabolism, previously considered detrimental to cellular health, but now recognized as important signal transducers with optimal cellular function ranges, which if exceeded, induce pathology due to the increased oxidative stress that damages lipids, proteins, and DNA<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. Metabolic reprogramming in cancer cells results in the generation of higher than normal levels of ROS from mitochondria and cytoplasmic NADPH oxidases<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>, which require counterbalancing through antioxidant activity<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Consequently, the elevated levels of ROS in cancer cells create a potential vulnerability to prooxidants, rendering them susceptible to oxidative-stress-induced cell death<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Conventional anti-cancer agents such as doxorubicin are in fact prooxidants that drive ROS levels above a death-inducing threshold in cancer cells<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>–<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>; however, due to toxicity, there are limits on dosing, emphasizing the need for less toxic agents with similar functions based on inducing selective ROS production. CoQ<sub>10</sub> (ubidecarenone) is a lipophilic antioxidant with the potential to serve as the basis for the aforementioned strategy.</p><p id=\"Par4\">CoQ<sub>10</sub> is hydrophobic due to its side chains, and thus resides in membranous fractions such as mitochondria and plasma membranes<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>, naturally serving as an electron carrier, exploiting the redox profile of the p-benzoquinone ring moiety<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Within the inner mitochondrial membrane, the activity of CoQ<sub>10</sub> is dependent on its redox state<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup> of which there are three: oxidized (ubiquinone, also known as ubidecarenone or CoQ<sub>10</sub>), a free-radical intermediate (semiquinone, CoQ<sub>10</sub>H<sup>•</sup>), and the most abundant reduced form (ubiquinol, CoQ<sub>10</sub>H<sub>2</sub>)<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>.</p><p id=\"Par5\">In its reduced form, CoQ<sub>10</sub>H<sub>2</sub> serves as a potent endogenous antioxidant that prevents lipid peroxidation, protein carbonylation, and oxidative damage to DNA<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. The aforementioned antioxidant function is sub-served via two types of reducing quinone-related oxidoreductases. NADPH dehydrogenase (quinone) 1 catalyzes the two-electron reduction of quinones, producing stable quinols<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>–<xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. In contrast, enzymes such as NADPH-cytochrome P450 reductase catalyze the reduction to a semiquinone radical in the presence of a suitable electron donor such as NADPH<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>, which because of its own lability and high reactivity easily donates an electron to a neighboring oxygen molecule, resulting in the production of an O<sub>2</sub><sup>−</sup> anion. Multiple such reactions result in an overabundance of O<sub>2</sub><sup>−</sup> anions and consequently cell toxicity. Considering the increased levels of oxidative stress within cancer cells, exposure to the optimum amount of CoQ<sub>10</sub> could potentially exclusively affect cancer cells, thus providing a potentially well-tolerated and effective anti-cancer therapy.</p><p id=\"Par6\">The aforementioned approach is however limited by CoQ<sub>10</sub>′s insolubility, which restricts the amount that can be delivered to cells, and to date, only modest anti-cancer efficacy has been reported<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. Furthermore, since oxidative stress can be cell supportive when therapeutic agents fail to raise ROS levels beyond toxic thresholds<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, the potential for a cancer therapeutic agent to work will depend on its markedly increased delivery to cancer tissues.</p><p id=\"Par7\">To address the aforementioned challenge, an oxidized form of CoQ<sub>10</sub> (ubidecarenone) formulated as a drug-lipid conjugate nanodispersion (BPM31510) and optimized for stability and delivery was developed<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup> to investigate the therapeutic potential of delivering supraphysiological concentrations of ubidecarenone to tumors. Given that CoQ<sub>10</sub> is known to cross the blood brain barrier<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>, we investigated its efficacy in the treatment-refractory malignant glioma. Using co-cultures of glioma cells and non-tumor cells, we demonstrate that BPM31510 treatment differentially and rapidly raises intramitochondrial O<sub>2</sub><sup>−</sup> anion levels in glioma cells relative to non-tumor cells, an effect that precedes any changes in proliferation or cell cycle status. Importantly, we demonstrate unique in vivo activity using an orthotopic glioblastoma model. These findings suggest a selective therapeutic potential for BPM31510 in the highly aggressive glioma cancer cells with minimal impact on non-tumor cells.</p></sec><sec id=\"Sec2\"><title>Results</title><sec id=\"Sec3\"><title>Differential effects of ubidecarenone on glioblastoma and non-tumor cell lines</title><p id=\"Par8\">CoQ<sub>10</sub> is a highly lipophilic molecule with limited water solubility that requires dissolving in highly toxic organic solvents such as ethanol (0.3 mg/ml) or dimethyl formamide (DMF, 10 mg/ml) prior to use. Based on preliminary studies, we determined that a limit of 0.5% DMF in cell cultures prevented solvent toxicity, thus limiting the concentration of CoQ<sub>10</sub> to a maximum of 10 µM (Supplementary Figure <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>). At this dose, neither native ubidecarenone nor BPM31510-delivered ubidecarenone had an effect on cell proliferation in either the rat C6 or human U251 glioma cells (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>A).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Differential effects of oxidized CoQ<sub>10</sub> on glioblastoma and non-tumor cell lines. (<bold>A</bold>) Relative cell viability of rodent C6 glioma and human U251 glioma cells after exposure to 0.1% DMF, 10 µM native CoQ10 (in 0.1% DMF), or 10 µM of ubidecarenone using BPM31510 for 72 h. Values are normalized to control. No significant effect on cell growth is noted under any condition. (<bold>B</bold>) Dose response curves for rat C6 glioma and mouse NIH3T3 fibroblast cells (left panel) and human U251 glioma and HA cells (right panel) after incubation for 72 h with BPM31510. Note that in each case, the tumor line was more sensitive than the control. (<bold>C</bold>) Cell viability analysis of each cell line over time after incubation with increasing doses of ubidecarenone. The number of live cells is converted from a pre-established standard curve with known cells numbers. All data presented as Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to control (no BPM31510) counts on the same day. Each color corresponds to the BPM31510 dose.</p></caption><graphic xlink:href=\"41598_2020_70969_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par9\">The lipid formulation of BPM31510 enables the achievement of supraphysiological ubidecarenone levels allowing for assessment of much higher dosing. Figure <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B demonstrates the effects of higher ubidecarenone dosing on the cell viability of two glioma cell lines, rat C6 and human U251, and two non-tumor cell lines, mouse NIH3T3 fibroblast cells (immortalized but not neoplastic) and normal human astrocytes (HA). After 72 h of incubation, rat C6 glioma cell growth was inhibited to a significantly higher degree than mouse NIH3T3 cell growth (IC<sub>50</sub> C6: 230 μM vs. IC<sub>50</sub> NIH3T3: > 460 μM). Interestingly, a more dramatic effect was observed in the human U251 glioma and HA cells (IC<sub>50</sub> U251: 230 µM vs. IC<sub>50</sub> HA: 1,840 µM), suggesting that the non-tumor HA cells were essentially unaffected by the high concentrations of ubidecarenone (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B).</p><p id=\"Par10\">To assess growth inhibitory effects of ubidecarenone on glioma and non-tumor cells, timed proliferation assays were performed as a function of drug dose; differential effects were noted over time (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>C). Notably, a cytostatic effect of ubidecarenone was noted at the lower doses (230 µM and 460 µM) upon treatment of rat C6 glioma cells, while a cytocidal effect was noted at the highest dose (1,840 µM). Similar but more pronounced results were noted for human U251 glioma cells, where both 460 µM and 1,840 µM doses were cytocidal. In contrast, only cytostatic effects were noted for mouse NIH3T3 fibroblast cells, and the highest dose of BPM31510 had minimal effect on the non-tumor HA cells. In fact, a sixfold increase in HA cell numbers was observed after a 72-h exposure to 1,840 µM of Ubidecarenone, suggesting maintenance of proliferative responses for these non-tumor cells.</p></sec><sec id=\"Sec4\"><title>Ubidecarenone induces G<sub>2</sub>/M cell cycle arrest of glioblastoma but not non-tumor cell lines</title><p id=\"Par11\">To define the mechanistic underpinnings of ubidecarenone’s growth inhibitory effects, we conducted cell cycle analysis. As demonstrated in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A, treatment of human U251 glioma cells with 460 µM ubidecarenone for 48 h resulted in significant accumulation of cells in the G2/M phase. We next performed cell cycle analyses for both glioma and non-tumor cell lines at various doses (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>B). In the glioma cells, a significant dose-dependent relationship with regards to G2/M phase arrest was noted, while in the non-tumor cells, the differences noted at all doses were not statistically significant. Of note, the G2/M delay was significant in both glioma cell lines at approximately the IC<sub>50</sub> dose (between 230 µM and 460 µM ubidecarenone).<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Ubidecarenone induces G<sub>2</sub>/M cell-cycle arrest of glioblastoma but not non-cancerous cell lines. (<bold>A</bold>) Cell cycle analysis and quantification of human U251 glioma cells treated with 0, 75, 115, 230, 345, or 460 µM ubidecarenone. The percentage of cells in each phase (G<sub>0/1</sub>, S or G<sub>2</sub> + M) is estimated from the frequency histograms. The percentage of cells in G<sub>2</sub> + M phase is highlighted. (<bold>B</bold>) Quantification of human U251 glioma, rat C6 glioma, HA, or mouse NIH3T3 fibroblast cells in each cell phase. All data presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to control (no drug exposure) at the same cell cycle phase.</p></caption><graphic xlink:href=\"41598_2020_70969_Fig2_HTML\" id=\"MO2\"/></fig></p></sec><sec id=\"Sec5\"><title>Differential redox vulnerabilities to ubidecarenone exposure between glioma and non-tumor cells</title><p id=\"Par12\">Given that most ROS is produced within the mitochondria<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>, we next assessed differential effects of ubidecarenone on mitochondria-derived ROS by measuring O<sub>2</sub><sup>−</sup> production using a dye that specifically localizes within mitochondria. A 48-h incubation of human U251 glioma or HA cells with ubidecarenone concentrations between 0–460 µM resulted in a dose-dependent accumulation of O<sub>2</sub><sup>−</sup> that was markedly increased in the glioma cells as compared to HA cells (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>A,B). Specifically, we noted that while O<sub>2</sub><sup>−</sup> levels for HA cells were elevated approximately 1.8-fold when exposed to 115 µM ubidecarenone, higher doses did not result in additional increased ROS production. In contrast, a dose-dependent increase in O<sub>2</sub><sup>−</sup> production was noted in human U251 glioma cells, with the highest dose producing over a fourfold elevation in O<sub>2</sub><sup>−</sup> (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>B; Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>).<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Differential redox vulnerabilities to ubidecarenone exposure between non-tumor and glioblastoma cells. (<bold>A</bold>) Flow cytometry analysis of O<sub>2</sub><sup>−</sup> in HA or human U251 glioma cells treated with 0, 115, or 460 µM ubidecarenone. O<sub>2</sub><sup>-</sup> intensity in each cell is demonstrated in the frequency histograms. (<bold>B</bold>) Quantification of mean O<sub>2</sub><sup>−</sup> levels in HA and human U251 glioma cells. A modest increase in O<sub>2</sub><sup>−</sup> is noted in HA in the presence of ubidecarenone, although this increase is not dose-dependent. In contrast, there is a fourfold O<sub>2</sub><sup>−</sup> increase in rat C6 glioma cells in a dose-dependent manner. (<bold>C</bold>) Flow cytometry analysis (scatter plot) of O<sub>2</sub><sup>−</sup> and DAPI in human U251 glioma and HA cells. Notably, there is minimal change in O<sub>2</sub><sup>−</sup> intensity with increasing ubidecarenone dose in HA cells, while there is a marked increase in both O<sub>2</sub><sup>−</sup> and DAPI in human U251 glioma cells after drug exposure. (<bold>D</bold>) Quantification of relative cell populations of O<sub>2</sub><sup>−</sup><sub>low</sub>/DAPI<sub>low</sub>, O<sub>2</sub><sup>−</sup><sub>high</sub>/DAPI<sub>low</sub>, and O<sub>2</sub><sup>−</sup><sub>high</sub>/DAPI<sub>high</sub> in human U251 glioma cells demonstrating increasing numbers of high O<sub>2</sub><sup>-</sup> and DAPI labeled cells with increasing ubidecarenone dose. <bold>(E</bold>) Flow cytometry analysis of O<sub>2</sub><sup>−</sup> for human U251 glioma cells treated with 230 µM ubidecarenone for 0, 2, 6, or 24 h. Relative mean values of O<sub>2</sub><sup>−</sup> were quantified and normalized to control at 0 h. (<bold>F</bold>) Cell cycle analysis by flow cytometry for human U251 glioma cells treated with 230 µM ubidecarenone for 0, 2, 6, or 24 h. The percentage of human U251 glioma cells in each cell cycle phase (G<sub>0/1</sub>, S or G<sub>2</sub> + M) was quantified. All data presented as Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant, compared to control (0 µM) at the same cell cycle phase.</p></caption><graphic xlink:href=\"41598_2020_70969_Fig3_HTML\" id=\"MO3\"/></fig></p><p id=\"Par13\">Additionally, DAPI co-staining was utilized to determine the flow cytometry profiles of human U251 glioma and HA cells. Human U251 glioma cells exhibited three distinct populations after exposure to BPM31510: O<sub>2</sub><sup>−</sup><sub>low</sub>/DAPI<sub>low</sub>, O<sub>2</sub><sup>−</sup><sub>high</sub>/DAPI<sub>low</sub>, and O<sub>2</sub><sup>−</sup><sub>high</sub>/DAPI<sub>high</sub> (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>C and Supplementary Figure <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>). In Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>D, a dose-dependent accumulation of cells in both O<sub>2</sub><sup>−</sup><sub>high</sub> and DAPI<sub>high</sub> populations is graphically depicted. As shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>C (bottom panels) HA cell populations were indistinguishable even at the highest ubidecarenone dose. These data support the existence of differential redox vulnerabilities between glioma and non-tumor cells. Similar results were noted with the rat C6 glioma and mouse NIH3T3 cells, where images demonstrate the active superoxide (red) being highly correlated with the C6 (GFP) population, while essentially no fluorescent signal was noted until its occasional appearance with these nonfluorescent cells was noted at the highest doses examined (345 μM or 460 μM) (indicated by white arrows in Supplementary Figure <xref rid=\"MOESM1\" ref-type=\"media\">S4</xref>). These observations affirm the differential induction of mitochondrial superoxide by BPM31510 in non-cancer vs. neoplastic cells.</p></sec><sec id=\"Sec6\"><title>Ubidecarenone induces an early-onset and increased accumulation of O<sub>2</sub><sup>−</sup> that precedes cell cycle arrest</title><p id=\"Par14\">Given the noted increase in superoxide production and the decrease in proliferation after 48 h, we wanted to determine the sequence of the onset of cell cycle changes and ROS production in glioma and non-tumor cells. Consequently, we assessed both O<sub>2</sub><sup>−</sup> levels and cell cycle state in human U251 glioma cells incubated with 230 µM BPM31510 for 0, 2, 6, and 24 h. O<sub>2</sub><sup>−</sup> levels were significantly elevated after 6 h and continued to increase over the 24-h course of the experiment (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>E). In contrast, no significant changes were detected in cell cycle state even at 24 h (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>F). This supports the contention that elevated mitochondrial O<sub>2</sub><sup>−</sup> levels represent an early event in the cascade leading to slowed proliferation and death of cancer cells.</p></sec><sec id=\"Sec7\"><title>Ubidecarenone differentially affects glioma and non-tumor cell growth in co-culture experiments</title><p id=\"Par15\">Given the higher levels of oxidative stress in cancer cells relative to non-cancer cells, a potential exists for capitalizing on these differences by differentially stressing the cancer cells through exposure to prooxidants. Therefore, we next assessed the effects of ubidecarenone on viability and redox homeostasis in both rodent and human co-cultures of glioma and non-tumor cells (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>).<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Strategy of human and rodent co-culture experiments using glioma and non-tumor cells.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Co-culture</th><th align=\"left\" colspan=\"2\">Human</th><th align=\"left\" colspan=\"2\">Rodent</th></tr></thead><tbody><tr><td align=\"left\">Predominant type of CoQ</td><td align=\"left\" colspan=\"2\">CoQ10</td><td align=\"left\" colspan=\"2\">CoQ9</td></tr></tbody></table><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Cell type</th><th align=\"left\">Astrocyte</th><th align=\"left\">Glioma U251</th><th align=\"left\">Fibroblast NIH3T3</th><th align=\"left\">Glioma C6</th></tr></thead><tbody><tr><td align=\"left\">Label</td><td align=\"left\">None</td><td align=\"left\">GFP</td><td align=\"left\">None</td><td align=\"left\">GFP</td></tr><tr><td align=\"left\">Starting ratio in population</td><td align=\"left\">60%</td><td align=\"left\">40%</td><td align=\"left\">50%</td><td align=\"left\">50%</td></tr></tbody></table><table-wrap-foot><p>In the human cells model, HA (non-labeled) and U251 glioma (GFP-labeled) cells are co-cultured at designated cell densities. In the rodent cells model, NIH3T3 fibroblast (non-labeled) and C6 glioma (GFP-labeled) cells are co-cultured at designated cell densities.</p></table-wrap-foot></table-wrap></p><p id=\"Par16\">GFP-labeled rat C6 glioma cells (C6<sub>GFP</sub>) form clusters and surround non-labeled mouse NIH3T3 fibroblast cells after 72 h in co-culture under basal conditions (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>A). Co-culture incubation with 230 or 460 µM ubidecarenone induced a dose-dependent decrease in the distribution of C6<sub>GFP</sub> cells in comparison to non-labeled NIH3T3 cells, which were less impacted by ubidecarenone (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>A). Graphically illustrated in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>B, 72 h after equal numbers of C6<sub>GFP</sub> and non-labeled NIH3T3 cells were initially plated, 75% of the cell population was GFP labeled (C6<sub>GFP</sub>) in the absence of ubidecarenone compared to only 35% after incubation in 230 µM ubidecarenone. Of note, the inhibitory effects on the cancer cell population persisted. In cultures maintained up to 12 days without ubidecarenone, C6<sub>GFP</sub> cells represented the entire cell population. In contrast, cultures with ubidecarenone doses above 115 µM were equilibrated and persisted for 12 days (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>C). Utilizing a similar co-culture strategy with GFP-labeled human glioma U251 and non-labeled HA cells, a robust response wherein increasing ubidecarenone doses differentially depleted GFP-labeled human glioma U251 cells in comparison to non-labeled HA cells was noted (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>A).<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Ubidecarenone induces differential effects on cell growth and redox vulnerabilities between non-tumor and glioblastoma cells in co-culture. (<bold>A</bold>) Phase and fluorescent images of GFP-labeled rat C6 glioma cells and non-labeled mouse NIH3T3 fibroblast, co-cultured and treated with 0, 230, or 460 µM ubidecarenone, demonstrates a dose-dependent decrease in glioma cells with a relative sparing of HA cells. <bold>(B</bold>) Flow cytometry analysis (scatter plot) of GFP and O<sub>2</sub><sup>−</sup> in GFP-labeled rat C6 glioma cells and non-labeled mouse NIH3T3 co-culture. Cell populations are characterized based on GFP intensity. Note the increase in the GFP<sub>neg</sub> population relative to GFP<sub>high</sub> population in the presence of ubidecarenone. (<bold>C</bold>) Flow cytometry analysis of GFP-labeled rat C6 glioma cells and non-labeled mouse NIH3T3 fibroblasts co-cultures treated with ubidecarenone for up to 12 days. Cells are characterized based on their GFP intensity (GFP<sub>neg</sub> or GFP<sub>pos</sub>). Results are grouped based on ubidecarenone dose and are shown as a percentage of the entire cell population. While glioma cells (GFP<sub>pos</sub>) represent the entire cell population by day 9 in co-cultures without ubidecarenone, there are essentially equal numbers of glioma and non-tumor cell populations at doses ≥ 115 µM ubidecarenone.</p></caption><graphic xlink:href=\"41598_2020_70969_Fig4_HTML\" id=\"MO4\"/></fig><fig id=\"Fig5\"><label>Figure 5</label><caption><p>Ubidecarenone induces differential effects on cell growth and redox vulnerabilities between non-tumor and glioblastoma cells in co-culture experiments. (<bold>A</bold>) Phase and fluorescent images of GFP-labeled human U251 glioma cells and non-labeled HA cells co-cultured and treated with 0, 230, or 460 µM ubidecarenone demonstrate a dose-dependent decrease in glioma cells with a relative sparing of HA. <bold>(B</bold>) Flow cytometry analysis (scatter plot) of GFP and O<sub>2</sub><sup>-</sup> in GFP-labeled human U251 glioma cells and non-labeled HA cells in co-culture. Cell populations are characterized based on GFP intensity (GFP<sub>neg</sub>, GFP<sub>low</sub>, and GFP<sub>high</sub>). Note the dose-dependent increase in the GFP<sub>neg</sub> population relative to the GFP<sub>high</sub> population in the presence of ubidecarenone. Quantification of flow cytometry cell populations illustrates a dose-dependent increase in GFP<sub>low</sub> cell numbers. (<bold>C</bold>) Flow cytometry analysis of O<sub>2</sub><sup>−</sup> and DAPI in GFP-labeled human U251 glioma cells and non-labeled HA cells, co-cultured and treated with 0, 230, or 460 µM ubidecarenone. Note the increase in O<sub>2</sub><sup>−</sup> values for both GFP<sub>low</sub> and GFP<sub>high</sub> cells, with insignificant changes noted in the GFP<sub>neg</sub> (HA cells) population. (<bold>D</bold>) Graphical depiction of flow cytometry analysis of O<sub>2</sub><sup>−</sup> and DAPI in GFP-labeled human U251 glioma cells and non-labeled HA cells, co-cultured and treated with 0, 230, or 460 µM ubidecarenone. In contrast to the GFP<sub>neg</sub> population, BPM exposure results in a marked increase in superoxide production in both the low and high GFP fractions. (<bold>E</bold>) DAPI levels are significantly elevated only in the GFP<sub>low</sub> cell population, consistent with this population representing dying cells.</p></caption><graphic xlink:href=\"41598_2020_70969_Fig5_HTML\" id=\"MO5\"/></fig></p><p id=\"Par17\">Next, flow cytometry was utilized to assess the differential effects on O<sub>2</sub><sup>−</sup> and DAPI staining when ubidecarenone doses are increased. Three distinct cell populations, GFP-negative, GFP-low, and GFP-high were noted (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>B). Given the unlikely event that HA cells acquired GFP, the GFP<sub>low</sub> population is thus interpreted to represent human glioma U251 cells. Consistent with this, a dose-dependent accumulation of GFP<sub>low</sub> human glioma U251 cells that correlated with a significant reduction in the GFP<sub>high</sub> population was noted (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>B), implicating ubidecarenone exposure in this transition.</p></sec><sec id=\"Sec8\"><title>Ubidecarenone exploits differential redox vulnerabilities between non-cancerous and glioblastoma cells</title><p id=\"Par18\">We next assessed each of the three cell populations (GFP<sub>neg</sub>, GFP<sub>low</sub>, and GFP<sub>high</sub>) to compare changes in O<sub>2</sub><sup>−</sup> and DAPI intensity, which occur after exposure to ubidecarenone (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>C). First, consistent with the known increase in basal oxidative stress, we noted that while O<sub>2</sub><sup>−</sup> levels for human glioma U251 cells were 1.5-fold higher than HA cells under basal conditions, the difference increased over fourfold after treatment with BPM31510 (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>D). These findings are consistent with previous observations in experiments conducted with independent cell lines (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>D) and support the contention that ubidecarenone exploits differential redox vulnerabilities between human glioma U251 cells and HA to mediate its anti-cancer activity. Consistent with knowledge regarding the Warburg effect, our findings further suggest that our system mimics reality.</p><p id=\"Par19\">Noteworthy, ubidecarenone did not induce significant changes in either O<sub>2</sub><sup>−</sup> production or DAPI staining in the non-labeled HA cells (GFP<sub>neg</sub>). In contrast, the GFP<sub>low</sub> population exhibited a notable increase in both O<sub>2</sub><sup>−</sup> production and DAPI staining, while the GFP<sub>high</sub> population exhibited a significant increase in O<sub>2</sub><sup>−</sup> levels only (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>C–E). Consistent with the cell cycle analysis, these findings suggest that the GFP<sub>low</sub> population represents the actively dying fraction of tumor cells.</p></sec><sec id=\"Sec9\"><title>Ubidecarenone exerts efficacy in an orthotopic model of glioblastoma</title><p id=\"Par20\">To address whether ubidecarenone exerts in vivo efficacy, we assessed its effects in an orthotopic model using rat C6 glioma cells. After inoculation of 10<sup>6</sup> cells into the right striatum, control rats (n = 32) all died within 16 days of implantation. In contrast, nine of 31 (29%) rats, treated with BPM31510 50 mg/kg i.p. twice per day beginning either 4 or 8 days after implantation, and continuing through post-implantation days 35–42 were long-term survivors (P < 0.02, log rank statistic; Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>A).<fig id=\"Fig6\"><label>Figure 6</label><caption><p>Ubidecarenone demonstrates efficacy in an orthotopic glioma model. Wistar rats received either saline or 50 mg/kg BPM31510 IP, twice daily, five days per week, starting at either 4 (n = 12) or 8 (n = 19) days after implantation of 10<sup>6</sup> C6 glioma cells into the right striatum. (<bold>A</bold>) Survival of rats treated either with saline (n = 32) or BPM31510. Over 25% of BPM31510 treated rats survived to the end of experiment (at least 75 days) compared to 0% of PBS treated controls (P < 0.01, log rank statistic). No significant difference was noted between the BPM31510 treated groups starting at 4- or 8-days. (<bold>B</bold>) Serial MRI of a long-term survivor (Day 27, Day 34, and Day 102 post-implantation) demonstrating persistent effects even after treatment was withdrawn. Lower right panel is a coronal plane H&E stained section of the same long-term survivor demonstrating a cystic cavity with no obvious tumor present.</p></caption><graphic xlink:href=\"41598_2020_70969_Fig6_HTML\" id=\"MO6\"/></fig></p><p id=\"Par21\">While median survival was increased modestly (median 12.0 vs. 13.0, saline vs. BPM31510, P < 0.01, log rank statistic), there was a marked increase in the number of rats surviving greater than 16 days (0% vs. 29%, p < 0.001, Fisher’s Exact test). Furthermore, gradual involution was noted over time in long-term survivors with the appearance of ex vacuo changes pathologically characterized as cystic cavities containing no macroscopic tumor over six weeks after drug cessation (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>B). No significant differences were noted relative to the start of treatment relative to tumor implantation.</p></sec></sec><sec id=\"Sec10\"><title>Discussion</title><p id=\"Par22\">Ubidecarenone delivered using BPM31510 at levels equivalent to those achieved using native Ubidecarenone has no appreciable effects on glioma cells; however, the increased solubility in the lipid nanodispersion formulation allows for increased exposure (> 200 fold). Utilizing cellular proliferation assays, cell cycle analysis, and measurements of mitochondrial O<sub>2</sub><sup>−</sup> production, even at ubidecarenone doses well below the maximum levels achievable, differential effects were observed in tumor cells relative to non-tumor cells. Furthermore, the aforementioned differential effects were maintained in co-culture experiments, with prolonged drug exposure resulting in an equilibrated cultures where neither tumor nor non-tumor cells dominated over time.</p><p id=\"Par23\">Malignant gliomas possess several features that make it a prototypical tumor type for novel metabolic approaches, including the presence of extensive metabolic reprogramming, a high level of oxidative stress, and its development within an environment that is at once relatively inaccessible and very sensitive to normal tissue damage. This was the impetus for the investigation into ubidecarenone’s efficacy both in vitro and in an orthotopic glioma model. In vitro, our results indicate that administration of BPM31510 results in a marked differential elevation in mitochondrial O<sub>2</sub><sup>−</sup> species in two established glioma cell lines compared to two non-tumor lines derived from both human and rat. This elevation in mitochondrial O<sub>2</sub><sup>−</sup> species preceded the onset of slowed growth and G2/M cell cycle arrest.</p><p id=\"Par24\">The noted slowed growth is of particular interest when comparing the effects of ubidecarenone in an immortalized 3T3 murine line and HA, the latter being almost completely insensitive to growth delay, and exhibiting markedly diminished elevations in O<sub>2</sub><sup>−</sup> production, even at high doses. Whether the marked resistance of HA relative to NIH3T3 cells to the effects of ubidecarenone reflects the fact that human cells express only CoQ<sub>10,</sub> compared to the relative abundance of CoQ<sub>9</sub> in rodents<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>, rather than reflecting a difference in sensitivity related to immortalized vs. non-immortalized cells, remains to be determined.</p><p id=\"Par25\">The co-culture studies are especially illuminating in how this therapy might result in a different, but equally efficacious, outcome compared to conventional cytotoxic approaches. The observation of equilibrated cultures which persisted over time suggest that modulating the redox status “leveled the playing field” between cancer and non-cancer cells (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>). Translating this to potential in situ observations, one might expect that optimal dosing redox balance could be achieved long term, resulting in a period of extended control without a marked impact on conventional anatomical imaging.</p><p id=\"Par26\">The idea of creating equilibrium through exerting differential redox toxicity is also of interest in light of the in vivo experimental results. The observation of an essentially “all or none” response characterized by either rapid death or a slow involution of established tumors is unusual and raises the question of why this agent does not result in a cure in all rats. While one could posit that variable penetration of drug to tumor tissue might underlie this variation, it is also very possible that ubidecarenone’s effectiveness is dependent on maintaining an optimal redox balance. Generally supporting this contention is accumulating evidence that raising ROS levels can selectively induce cancer cell death by disabling antioxidants<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref>,<xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup> only if ROS is sufficiently elevated to preferentially disable cancer cells relative to normal ones. Therefore, methods in which this effect can be measured in vivo are required if this strategy is to succeed in the clinic.</p><p id=\"Par27\">While a standardized in vivo measure of oxidation status in cancer tissues does not yet exist, a number of potential imaging methods might prove useful in this regard. For instance, measuring compensatory endogenous antioxidants such as glutathione could be envisioned as a readout, wherein the ratio of the oxidized and reduced forms would reflect redox status. Alternatively, PET tracers such as hydroascorbate, which can also provide key information about redox status<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>, are currently being studied.</p><p id=\"Par28\">In summary, we demonstrate that exposure to high levels of ubidecarenone produce differential changes in glioma cells relative to non-glioma cells. This effect correlates with the production of intramitochondrial O<sub>2</sub><sup>−</sup>, an increase that is noted well before changes in proliferation or the cell cycle can be measured. Considering that non-tumor cells appear resistant to its growth inhibiting effects, including O<sub>2</sub><sup>−</sup> production, BPM31510 possesses attributes warranting further evaluation as a redox biologic agent.</p></sec><sec id=\"Sec11\"><title>Methods</title><sec id=\"Sec12\"><title>Orthotopic C6 glioma model</title><p id=\"Par29\">1 × 10<sup>6</sup> rat C6 glioma cells were injected intracranially into 6–8 week-old Wistar female rats as previously described<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup> with approval of the Stanford University School of Medicine IACUC (Protocol #11396). In brief, Wistar adult rats weighing 150 to 200 g were anesthetized initially with isofluane (3–4% followed by maintenance 1–2%) and placed in a stereotactic head holder. A burr hole approximately 3 mm lateral and posterior to the bregma using a 19-gauge dental drill was then performed. Suspensions of 10<sup>6</sup> exponentially growing C6 glioma cells in 30 µl of MEM were injected over a 5-min period through a Hamilton syringe placed 3 mm lateral and 3 mm posterior to the bregma and 0.6 mm deep to the dura. Four and eight-days post-implantation, rats were randomly allocated to receive either isotonic PBS (n = 32) or 50 mg/kg bid BPM 31,510 starting either 4 (n = 12) or 8 days post-tumor implantation (n = 19). I.P. injections were continued for 5 days per week until rats met requirements for euthanasia (i.e., eye rings lethargy, motor incoordination) or day 35 post-implantation.</p></sec><sec id=\"Sec13\"><title>Reagents</title><p id=\"Par31\">CoQ<sub>10</sub> (C9538) and dimethyl formamide (DMF, 33120) were purchased from SIGMA-ALDRICH (St. Louis, MO). BPM31510 was prepared as previously described<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup> and provided by BERG LLC (Framingham, MA).</p></sec><sec id=\"Sec14\"><title>Cell culture</title><p id=\"Par32\">GP2-293 cells (Catalog #. 631458) was obtained from CLONTECH, now TAKARA BIO USA (Mountain View, CA). Rat C6 GBM (Catalog# CCL-107), and mouse NIH3T3 fibroblasts (Catalog# CRL-1658) were obtained from ATCC (Manassas, VA). Human U251 GBM (Catalog# 03063001) was from SIGMA-ALDRICH (St. Louis, MO). All cells were maintained following manufacturer’s protocols. Normal human astrocytes (HA, Catalog# CC-2565) were purchased from LONZA (Basel, Switzerland) and grown in HA growth medium kit (Catalog# 821–500) from APPLICATION INC. (San Diego, CA). For the co-culture system experiments, labeled tumor and unlabeled non-tumor cells were seeded in 6-well plates at designated densities and treated with BPM31510 or vehicle, for 24–72 h. Rat C6 GBM and NIH3T3 fibroblast cells were co-cultured in DMEM medium supplemented with 10% FBS, and human U251 GBM and HA cells were co-cultured in HA growth medium.</p></sec><sec id=\"Sec15\"><title>Retroviral production and establishment of stable GBM cell lines</title><p id=\"Par33\">GP2-293 cells were grown in a T75 flask dish to 85% confluence and transfected with pQCXIP-EGFP and pVSVG vector (Gifts from Dr. Nan Gao, Rutgers-Newark) using Lipofectamine 3,000 (Catalog# L3000015) from LIFE TECHNOLOGIES (Carlsbad, CA) following manufacturer’s protocol. The plasmid information was described at a previous paper<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. Virus collection and cell infection steps were modified from a previous protocol<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. Briefly, the medium containing the retroviruses was collected 48 h post-transfection, centrifuged at 3,000 rpm to remove cell debris, and the supernatant passed through a 0.45 µM filter. Glioma cells (80% confluence) were then incubated with viral-DMEM medium containing 40% filtered supernatant and 60% fresh DMEM with 10% FBS supplement for 6 h, followed by culture in DMEM medium with 10% FBS supplement for 48 h. Stable clones were selected by culturing with puromycin (1 μg/ml) for 7 days, followed by flow cytometry to purify the GFP-positive population.</p></sec><sec id=\"Sec16\"><title>Cell viability assay and cell counts</title><p id=\"Par34\">Cell viability was evaluated by measuring the fluorescence signal generated from the cell viability reagent PrestoBlue following manufacturer’s instructions (Catalog# A13261) from THERMOFISHER (Waltham, MA). In brief, cells were plated in cell culture medium in a 96-well plate in triplicate at 5,000 cells/well and incubated with indicated concentration of compounds. Cell viability are assessed after 24, 48 or 72 h of treatment by fluorometer analysis (Excitation/ Emission (nm) is 560/595) using Prestoblue assay. All data is presented as Mean ± SEM of three replicated experiments.</p><p id=\"Par35\">Cell numbers for human U251 GBM, rat C6 GBM, mouse NIH3T3 fibroblasts, and normal human astrocytes were calculated based on individual standard curves generated from known numbers (ranged from 5,000 to 600,000 cells per well) of each cell line.</p></sec><sec id=\"Sec17\"><title>Cell cycle analysis</title><p id=\"Par36\">10<sup>6</sup> cells were fixed and permeabilized with 70% ethanol for 30 min at − 20 °C. After permeabilization, cells were washed twice with cold PBS and pelleted. Cells were then resuspended in 300–500 µL FxCycle PI/RNase staining solution (Catalog# F10797) from INVITROGEN (Carlsbad, CA). Cells were subjected to flow cytometry after incubation for 30 min in the dark. The percentage of cells in each phase (G<sub>0/1</sub>, S, or G<sub>2</sub> + M) was then estimated from the frequency histograms.</p></sec><sec id=\"Sec18\"><title>MitoSOX-based O<sub>2</sub><sup>−</sup> staining</title><p id=\"Par37\">Cells were cultured in 6-well plates with designated reagents and incubated according to experimental design. For flow cytometry measurements, cells were gently twice-washed with 37 °C pre-warmed Hank’s Balanced Salt Solution (HBSS, Catalog# 14175079) from THERMOFISHER (Waltham, MA) and then incubated in the dark with 5 µM MitoSOX working solution (Catalog# M36008) from THERMOFISHER (Waltham, MA) for 10 min at 37 °C. Cells were then harvested from the plates and washed twice with 37 °C pre-warmed HBSS. A final concentration of 3 µM DAPI dye (Catalog# 62248) from THERMOFISHER (Waltham, MA) was then added into each sample to distinguish live/dead cells. The cell samples were then analyzed by flow cytometry to determine the mean fluorescence intensity and percentage of stained cells.</p></sec><sec id=\"Sec19\"><title>Flow cytometry</title><p id=\"Par38\">Flow cytometry data were obtained from a Scanford instrument and the transfected GFP-positive glioma cells were sorted using a Megatron instrument in the Shared FACS Facility at Stanford University.</p></sec><sec id=\"Sec20\"><title>Microscopic analysis</title><p id=\"Par39\">Microscopic images were obtained using a Leica CTR5000 microscope and MetaMorph software program. Images were processed using ImageJ.</p></sec><sec id=\"Sec21\"><title>Quantification and statistical analysis</title><p id=\"Par40\">All data were analyzed from indicated independent experiments. Cell viability or fold change in cell numbers were averaged from six independent wells in each independent experiment, and each experimental condition was repeated three or more times. Data were plotted as mean ± SEM. FACS experiments were independently repeated in triplicate and analyzed using Flowjo. Statistical analysis was performed using two-way ANOVAs on the basis of experimental setups for cell cycle analysis and percentage of cell populations, or one-way ANOVA on the basis of experimental setups for cell viability, fold change in cell numbers, and mean of O<sub>2</sub><sup>-</sup> or DAPI. Graphs were constructed with GraphPad Prism 5 and results represented graphically as *P < 0.05; **P < 0.01; ***P < 0.001 or ****P < 0.0001.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec22\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70969_MOESM1_ESM.pdf\"><caption><p>Supplementary file1</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70969-0.</p></sec><ack><title>Acknowledgements</title><p>Eric Sun and Karen Law at Brain Tumor Research fund. Berg LLC for providing drug and research support. Rina Kara at Dissertation-Editor.com for providing academic editing.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>Conceptualization: J.S., A.D., S.G., R.S., N.N., L.R.; Methodology: J.S., C.P., T.J., M.M., C.C., L.R.; Software: J.S.; Validation: J.S., L.R.; Formal analysis: J.S., C.P.; Investigation: J.S., C.P., T.J., M.M., C.C., L.R.; Resources: L.R.; Data curation: J.S.; Writing-original draft: J.S., L.R.; Writing-review and editing: J.S., C.P, S.K. A.D., S.G., R.S., N.N., S.N., V.V., M.K., L.R.; Supervision: N.N., L.R.; Project administration: J.S., L.R.; Funding acquisition: L.R.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>All data supporting the findings of this study are available with the article and can also be obtained from the authors.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par41\">Jiaxin Sun, Taichang Jang, Milton Merchant, Seema Nagpal and Lawrence Recht received research support from both Brain Tumor Research fund and BERG.LLC. Shiva Kazerounian, Anne R. Diers, Michael A. Kiebish, Vivek K. Vishnudas, Stephane Gesta, Rangaprasad Sarangarajan and Niven R. Narain are all employed by BERG.LLC. Chirag B. Patel and Chen Chen have no competing interest to declare.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Warburg</surname><given-names>O</given-names></name></person-group><source>The metabolism of tumours</source><year>1930</year><publisher-loc>London</publisher-loc><publisher-name>Arnold Constable</publisher-name></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Warburg</surname><given-names>O</given-names></name><name><surname>Wind</surname><given-names>F</given-names></name><name><surname>Negelein</surname><given-names>E</given-names></name></person-group><article-title>The metabolism of tumors in the body</article-title><source>J. Gen. Physiol.</source><year>1927</year><volume>8</volume><fpage>519</fpage><lpage>530</lpage><pub-id pub-id-type=\"pmid\">19872213</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hanahan</surname><given-names>D</given-names></name><name><surname>Weinberg</surname><given-names>RA</given-names></name></person-group><article-title>Hallmarks of cancer: The next generation</article-title><source>Cell</source><year>2011</year><volume>144</volume><issue>5</issue><fpage>646</fpage><lpage>674</lpage><pub-id pub-id-type=\"pmid\">21376230</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vander Heiden</surname><given-names>MG</given-names></name><name><surname>Cantley</surname><given-names>LC</given-names></name><name><surname>Thompson</surname><given-names>CB</given-names></name></person-group><article-title>Understanding the Warburg effect: The metabolic requirements of cell proliferation</article-title><source>Science</source><year>2009</year><volume>324</volume><fpage>1029</fpage><lpage>1034</lpage><pub-id pub-id-type=\"pmid\">19460998</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ward</surname><given-names>PS</given-names></name><name><surname>Thompson</surname><given-names>CB</given-names></name></person-group><article-title>Metabolic reprogramming: A cancer hallmark even Warburg did not anticipate</article-title><source>Cancer Cell</source><year>2012</year><volume>21</volume><fpage>297</fpage><lpage>308</lpage><pub-id pub-id-type=\"pmid\">22439925</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Park</surname><given-names>SJ</given-names></name><name><surname>Kim</surname><given-names>YT</given-names></name><name><surname>Jeon</surname><given-names>YJ</given-names></name></person-group><article-title>Antioxidant dieckol downregulates the Rac1/ROS signaling pathway and inhibits Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein 2 (WAVE2)-mediated invasive migration of B16 mouse melanoma cells</article-title><source>Mol. Cells</source><year>2012</year><volume>33</volume><issue>4</issue><fpage>363</fpage><lpage>369</lpage><pub-id pub-id-type=\"pmid\">22441674</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Laurent</surname><given-names>A</given-names></name><etal/></person-group><article-title>Controlling tumor growth by modulating endogenous production of reactive oxygen species</article-title><source>Cancer Res.</source><year>2005</year><volume>65</volume><issue>3</issue><fpage>948</fpage><lpage>956</lpage><pub-id pub-id-type=\"pmid\">15705895</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>P</given-names></name><etal/></person-group><article-title>NAC selectively inhibit cancer telomerase activity: A higher redox homeostasis threshold exists in cancer cells</article-title><source>Redox Biol.</source><year>2016</year><volume>8</volume><fpage>91</fpage><lpage>97</lpage><pub-id pub-id-type=\"pmid\">26771767</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cross</surname><given-names>CE</given-names></name><etal/></person-group><article-title>Oxygen radicals and human disease</article-title><source>Ann. Intern. Med.</source><year>1987</year><volume>107</volume><issue>4</issue><fpage>526</fpage><lpage>545</lpage><pub-id pub-id-type=\"pmid\">3307585</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cairns</surname><given-names>R</given-names></name><name><surname>Harris</surname><given-names>IS</given-names></name><name><surname>Mak</surname><given-names>TW</given-names></name></person-group><article-title>Regulation of cancer cell metabolism</article-title><source>Nat. Rev. Cancer</source><year>2011</year><volume>11</volume><fpage>85</fpage><lpage>95</lpage><pub-id pub-id-type=\"pmid\">21258394</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Szatrowski</surname><given-names>TP</given-names></name><name><surname>Nathan</surname><given-names>CF</given-names></name></person-group><article-title>Production of large amounts of hydrogen peroxide by human tumor cells</article-title><source>Cancer Res</source><year>1991</year><volume>51</volume><issue>3</issue><fpage>794</fpage><lpage>798</lpage><pub-id pub-id-type=\"pmid\">1846317</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gorrini</surname><given-names>C</given-names></name><name><surname>Harris</surname><given-names>IS</given-names></name><name><surname>Mak</surname><given-names>TW</given-names></name></person-group><article-title>Modulation of oxidative stress as an anticancer strategy</article-title><source>Nat. Rev. Drug Discov.</source><year>2013</year><volume>12</volume><issue>12</issue><fpage>931</fpage><lpage>947</lpage><pub-id pub-id-type=\"pmid\">24287781</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Trachootham</surname><given-names>D</given-names></name><etal/></person-group><article-title>Selective killing of oncogenically transformed cells through a ROS-mediated mechanism by beta-phenylethyl isothiocyanate</article-title><source>Cancer Cell</source><year>2006</year><volume>10</volume><issue>3</issue><fpage>241</fpage><lpage>252</lpage><pub-id pub-id-type=\"pmid\">16959615</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nogueira</surname><given-names>V</given-names></name><etal/></person-group><article-title>Akt determines replicative senescence and oxidative or oncogenic premature senescence and sensitizes cells to oxidative apoptosis</article-title><source>Cancer Cell</source><year>2008</year><volume>14</volume><issue>6</issue><fpage>458</fpage><lpage>470</lpage><pub-id pub-id-type=\"pmid\">19061837</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Raza</surname><given-names>MH</given-names></name><etal/></person-group><article-title>ROS-modulated therapeutic approaches in cancer treatment</article-title><source>J. Cancer Res. Clin. Oncol.</source><year>2017</year><volume>143</volume><issue>9</issue><fpage>1789</fpage><lpage>1809</lpage><pub-id pub-id-type=\"pmid\">28647857</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Galadari</surname><given-names>S</given-names></name><etal/></person-group><article-title>Reactive oxygen species and cancer paradox: To promote or to suppress?</article-title><source>Free Radic. Biol. Med.</source><year>2017</year><volume>104</volume><fpage>144</fpage><lpage>164</lpage><pub-id pub-id-type=\"pmid\">28088622</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Milkovic</surname><given-names>L</given-names></name><etal/></person-group><article-title>Oxidative stress and antioxidants in carcinogenesis and integrative therapy of cancer</article-title><source>Curr. Pharm. Des.</source><year>2014</year><volume>20</volume><issue>42</issue><fpage>6529</fpage><lpage>6542</lpage><pub-id pub-id-type=\"pmid\">25341930</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cabello</surname><given-names>CM</given-names></name><name><surname>Bair</surname><given-names>WB</given-names><suffix>3rd</suffix></name><name><surname>Wondrak</surname><given-names>GT</given-names></name></person-group><article-title>Experimental therapeutics: Targeting the redox Achilles heel of cancer</article-title><source>Curr. Opin. Investig. Drugs</source><year>2007</year><volume>8</volume><issue>12</issue><fpage>1022</fpage><lpage>1037</lpage><pub-id pub-id-type=\"pmid\">18058573</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aberg</surname><given-names>F</given-names></name><etal/></person-group><article-title>Distribution and redox state of ubiquinones in rat and human tissues</article-title><source>Arch. Biochem. Biophys.</source><year>1992</year><volume>295</volume><issue>2</issue><fpage>230</fpage><lpage>234</lpage><pub-id pub-id-type=\"pmid\">1586151</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Takahashi</surname><given-names>T</given-names></name><etal/></person-group><article-title>Distribution of ubiquinone and ubiquinol homologues in rat tissues and subcellular fractions</article-title><source>Lipids</source><year>1993</year><volume>28</volume><issue>9</issue><fpage>803</fpage><lpage>809</lpage><pub-id pub-id-type=\"pmid\">8231656</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lenaz</surname><given-names>G</given-names></name><name><surname>Genova</surname><given-names>ML</given-names></name></person-group><article-title>Mobility and function of coenzyme Q (ubiquinone) in the mitochondrial respiratory chain</article-title><source>Biochim. Biophys. Acta</source><year>2009</year><volume>1787</volume><issue>6</issue><fpage>563</fpage><lpage>573</lpage><pub-id pub-id-type=\"pmid\">19268424</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sun</surname><given-names>IL</given-names></name><etal/></person-group><article-title>Requirement for coenzyme Q in plasma membrane electron transport</article-title><source>Proc. Natl. Acad. Sci. USA</source><year>1992</year><volume>89</volume><issue>23</issue><fpage>11126</fpage><lpage>11130</lpage><pub-id pub-id-type=\"pmid\">1454789</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gant</surname><given-names>TW</given-names></name><etal/></person-group><article-title>Redox cycling and sulphydryl arylation; their relative importance in the mechanism of quinone cytotoxicity to isolated hepatocytes</article-title><source>Chem. Biol. Interact</source><year>1988</year><volume>65</volume><issue>2</issue><fpage>157</fpage><lpage>173</lpage><pub-id pub-id-type=\"pmid\">2835188</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>Y</given-names></name><name><surname>Hekimi</surname><given-names>S</given-names></name></person-group><article-title>Understanding ubiquinone</article-title><source>Trends Cell Biol.</source><year>2016</year><volume>26</volume><issue>5</issue><fpage>367</fpage><lpage>378</lpage><pub-id pub-id-type=\"pmid\">26827090</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quinzii</surname><given-names>CM</given-names></name><name><surname>Hirano</surname><given-names>M</given-names></name></person-group><article-title>Coenzyme Q and mitochondrial disease</article-title><source>Dev. Disabil. Res. Rev.</source><year>2010</year><volume>16</volume><issue>2</issue><fpage>183</fpage><lpage>188</lpage><pub-id pub-id-type=\"pmid\">20818733</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bentinger</surname><given-names>M</given-names></name><name><surname>Tekle</surname><given-names>M</given-names></name><name><surname>Dallner</surname><given-names>G</given-names></name></person-group><article-title>Coenzyme Q–biosynthesis and functions</article-title><source>Biochem. Biophys. Res. Commun.</source><year>2010</year><volume>396</volume><issue>1</issue><fpage>74</fpage><lpage>79</lpage><pub-id pub-id-type=\"pmid\">20494114</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lind</surname><given-names>C</given-names></name><name><surname>Hochstein</surname><given-names>P</given-names></name><name><surname>Ernster</surname><given-names>L</given-names></name></person-group><article-title>DT-diaphorase as a quinone reductase: A cellular control device against semiquinone and superoxide radical formation</article-title><source>Arch. Biochem. Biophys.</source><year>1982</year><volume>216</volume><issue>1</issue><fpage>178</fpage><lpage>185</lpage><pub-id pub-id-type=\"pmid\">6285828</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ernster</surname><given-names>L</given-names></name><name><surname>Danielson</surname><given-names>L</given-names></name><name><surname>Ljunggren</surname><given-names>M</given-names></name></person-group><article-title>DT diaphorase. I. Purification from the soluble fraction of rat-liver cytoplasm, and properties</article-title><source>Biochim. Biophys. Acta</source><year>1962</year><volume>58</volume><fpage>171</fpage><lpage>188</lpage><pub-id pub-id-type=\"pmid\">13890666</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Siegel</surname><given-names>D</given-names></name><etal/></person-group><article-title>NAD(P)H:quinone oxidoreductase 1: Role as a superoxide scavenger</article-title><source>Mol. Pharmacol.</source><year>2004</year><volume>65</volume><issue>5</issue><fpage>1238</fpage><lpage>1247</lpage><pub-id pub-id-type=\"pmid\">15102952</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kappus</surname><given-names>H</given-names></name><name><surname>Sies</surname><given-names>H</given-names></name></person-group><article-title>Toxic drug effects associated with oxygen metabolism: Redox cycling and lipid peroxidation</article-title><source>Experientia</source><year>1981</year><volume>37</volume><issue>12</issue><fpage>1233</fpage><lpage>1241</lpage><pub-id pub-id-type=\"pmid\">7035210</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Powis</surname><given-names>G</given-names></name><name><surname>Svingen</surname><given-names>BA</given-names></name><name><surname>Appel</surname><given-names>P</given-names></name></person-group><article-title>Quinone-stimulated superoxide formation by subcellular fractions, isolated hepatocytes, and other cells</article-title><source>Mol. Pharmacol.</source><year>1981</year><volume>20</volume><issue>2</issue><fpage>387</fpage><lpage>394</lpage><pub-id pub-id-type=\"pmid\">6272094</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Frontinan-Rubio</surname><given-names>J</given-names></name><etal/></person-group><article-title>Regulation of the oxidative balance with coenzyme Q10 sensitizes human glioblastoma cells to radiation and temozolomide</article-title><source>Radiother. Oncol.</source><year>2018</year><volume>128</volume><fpage>236</fpage><lpage>244</lpage><pub-id pub-id-type=\"pmid\">29784452</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gupta</surname><given-names>SC</given-names></name><etal/></person-group><article-title>Upsides and downsides of reactive oxygen species for cancer: The roles of reactive oxygen species in tumorigenesis, prevention, and therapy</article-title><source>Antioxid. Redox. Signal</source><year>2012</year><volume>16</volume><issue>11</issue><fpage>1295</fpage><lpage>1322</lpage><pub-id pub-id-type=\"pmid\">22117137</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Garg</surname><given-names>S</given-names></name><etal/></person-group><article-title>CoQ10 selective miscibility and penetration into lipid monolayers with lower lateral packing density</article-title><source>Biochim. Biophys. Acta Biomembr.</source><year>2017</year><volume>1859</volume><issue>7</issue><fpage>1173</fpage><lpage>1179</lpage><pub-id pub-id-type=\"pmid\">28366515</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mitsui</surname><given-names>J</given-names></name><etal/></person-group><article-title>Three-year follow-up of high-dose ubiquinol supplementation in a case of familial multiple system atrophy with compound heterozygous COQ2 mutations</article-title><source>Cerebellum</source><year>2017</year><volume>16</volume><issue>3</issue><fpage>664</fpage><lpage>672</lpage><pub-id pub-id-type=\"pmid\">28150130</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fernandez-Ayala</surname><given-names>DJ</given-names></name><etal/></person-group><article-title>Coenzyme Q distribution in HL-60 human cells depends on the endomembrane system</article-title><source>Biochim. Biophys. Acta</source><year>2005</year><volume>1713</volume><issue>2</issue><fpage>129</fpage><lpage>137</lpage><pub-id pub-id-type=\"pmid\">15993380</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bae</surname><given-names>YS</given-names></name><etal/></person-group><article-title>Regulation of reactive oxygen species generation in cell signaling</article-title><source>Mol. Cells</source><year>2011</year><volume>32</volume><issue>6</issue><fpage>491</fpage><lpage>509</lpage><pub-id pub-id-type=\"pmid\">22207195</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kawamukai</surname><given-names>M</given-names></name></person-group><article-title>Biosynthesis of coenzyme Q in eukaryotes</article-title><source>Biosci. Biotechnol. Biochem.</source><year>2016</year><volume>80</volume><issue>1</issue><fpage>23</fpage><lpage>33</lpage><pub-id pub-id-type=\"pmid\">26183239</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Glasauer</surname><given-names>A</given-names></name><etal/></person-group><article-title>Targeting SOD1 reduces experimental non-small-cell lung cancer</article-title><source>J. Clin. Invest</source><year>2014</year><volume>124</volume><issue>1</issue><fpage>117</fpage><lpage>128</lpage><pub-id pub-id-type=\"pmid\">24292713</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shaw</surname><given-names>AT</given-names></name><etal/></person-group><article-title>Selective killing of K-ras mutant cancer cells by small molecule inducers of oxidative stress</article-title><source>Proc. Natl. Acad. Sci. USA</source><year>2011</year><volume>108</volume><issue>21</issue><fpage>8773</fpage><lpage>8778</lpage><pub-id pub-id-type=\"pmid\">21555567</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carroll</surname><given-names>VN</given-names></name><etal/></person-group><article-title>[(11)C]Ascorbic and [(11)C]dehydroascorbic acid, an endogenous redox pair for sensing reactive oxygen species using positron emission tomography</article-title><source>Chem. Commun. (Camb)</source><year>2016</year><volume>52</volume><issue>27</issue><fpage>4888</fpage><lpage>4890</lpage><pub-id pub-id-type=\"pmid\">26963495</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tsujikawa</surname><given-names>T</given-names></name><etal/></person-group><article-title>Assessment of the tumor redox status in head and neck cancer by 62Cu-ATSM PET</article-title><source>PLoS ONE</source><year>2016</year><volume>11</volume><issue>5</issue><fpage>e0155635</fpage><pub-id pub-id-type=\"pmid\">27187778</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kubiatowski</surname><given-names>T</given-names></name><etal/></person-group><article-title>Association of increased phosphatidylinositol 3-kinase signaling with increased invasiveness and gelatinase activity in malignant gliomas</article-title><source>J. Neurosurg.</source><year>2001</year><volume>95</volume><fpage>480</fpage><lpage>488</lpage><pub-id pub-id-type=\"pmid\">11565871</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sun</surname><given-names>J</given-names></name><etal/></person-group><article-title>A Wntless-SEC12 complex on the ER membrane regulates early Wnt secretory vesicle assembly and mature ligand export</article-title><source>J. Cell Sci.</source><year>2017</year><volume>130</volume><issue>13</issue><fpage>2159</fpage><lpage>2171</lpage><pub-id pub-id-type=\"pmid\">28515233</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807822</article-id><article-id pub-id-type=\"pmc\">PMC7431534</article-id><article-id pub-id-type=\"publisher-id\">70887</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70887-1</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Tanshinone II A attenuates vascular remodeling through klf4 mediated smooth muscle cell phenotypic switching</article-title></title-group><contrib-group><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Lou</surname><given-names>Guanhua</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Hu</surname><given-names>Wangming</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Wu</surname><given-names>Ziqiang</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Xu</surname><given-names>Huan</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yao</surname><given-names>Huan</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Yang</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Huang</surname><given-names>Qinwan</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Baojia</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wen</surname><given-names>Li</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Gong</surname><given-names>Daoying</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Xiongbing</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Shi</surname><given-names>Yaping</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yang</surname><given-names>Lan</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Xu</surname><given-names>Yiming</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Wang</surname><given-names>Yong</given-names></name><address><email>yongwang1008@hotmail.com</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.411304.3</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0376 205X</institution-id><institution>Chengdu University of Traditional Chinese Medicine, </institution><institution>College of Basic Medicine, </institution></institution-wrap>Chengdu, China </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.411304.3</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0376 205X</institution-id><institution>Chengdu University of Traditional Chinese Medicine, College Pharmacy, </institution></institution-wrap>Chengdu, China </aff><aff id=\"Aff3\"><label>3</label>Chengdu University of Traditional Chinese Medicine, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, China </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.410737.6</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 8653 1072</institution-id><institution>Guangzhou Medical University, School of Basic Medical Sciences, </institution></institution-wrap>Guangzhou, China </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13858</elocation-id><history><date date-type=\"received\"><day>26</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>5</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">The aim of this study is to investigate the therapeutic role of Tanshinone II A, a key integrant from salvia miltiorrhiza, against pathological vascular remodeling. Completed ligation of mouse left common carotid arteries animal model and rat smooth muscle cells used to investigate the role of Tanshinone II A in regulating pathological vascular remodeling through hematoxylin and eosin staining, immunohistochemistry staining, immunofluorescence staining, adenovirus infection, real time PCR and western blotting. Our data demonstrated that Tanshinone II A treatment suppresses vascular injury-induced neointima formation. In vitro studies on rat smooth muscle cell indicated that Tanshinone II A treatment attenuates PDGF-BB induced cell growth, and promotes smooth muscle cell differentiated marker genes expression that induced by rapamycin treatment. Tanshinone II A treatment significant inhibits rat smooth muscle cell proliferation and migration. Tanshinone II A promotes KLF4 expression during smooth muscle phenotypic switching. Overexpression of KLF4 exacerbates Tanshinone II A mediated smooth muscle cell growth inhibition. Tanshinone II A plays a pivotal role in regulating pathological vascular remodeling through KLF4 mediated smooth muscle cell phenotypic switching. This study demonstrated that Tanshinone II A is a potential therapeutic agent for vascular diseases.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Drug discovery</kwd><kwd>Molecular biology</kwd><kwd>Cardiology</kwd><kwd>Medical research</kwd><kwd>Molecular medicine</kwd></kwd-group><funding-group><award-group><funding-source><institution>National Natural Science Foundation of China</institution></funding-source><award-id>81741007</award-id><award-id>81870363</award-id><principal-award-recipient><name><surname>Wang</surname><given-names>Yong</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>Cheng Du University of Traditional Chinese Medicine</institution></funding-source><award-id>008066</award-id><award-id>030038199</award-id><award-id>030041023</award-id><award-id>030041224</award-id><award-id>242030016</award-id><principal-award-recipient><name><surname>Wang</surname><given-names>Yong</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>science & technology departments of Sichuan province</institution></funding-source><award-id>2020JDTD0025</award-id><principal-award-recipient><name><surname>Wang</surname><given-names>Yong</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par18\">Vascular smooth muscle cells phenotypic switching contributes to development of variety vascular diseases, including post angioplasty restenosis, aneurysm, atherosclerosis and pulmonary hypertension<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. Maturated smooth muscle cell exhibits dramatically plasticity. The principal function of maturated smooth muscle cells is contraction regulating. However, smooth muscle cell can undergo phenotypic switching from differentiated stage to dedifferentiated stage, which characterized by reduced expression of smooth muscle specific genes, such as smooth muscle myosin heave chain, smooth muscle light chains, smooth muscle α-actin, SM22α, smooth muscle α-tropomyosin, smoothelin, h1-calponin, h-calponin, h-caldesmon, β-vinculin, metavinculin, telokin and desmin, whereas enhanced proliferation and migration<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. Emerging evidence indicated that Traditional Chinese Medicine is effective in treatment variety cardiovascular diseases. Salvia miltiorrhiza is a traditional Chinese medicine and widely used for treatment of cardiovascular diseases. Tanshinone II A is a key ingredient separated from salvia miltiorrhiza. Previously reports demonstrated that Tanshinone II A attenuates angiotensin II induced cardiac hypertrophy and cardiac fibroblast proliferation through MEK/ERK pathway<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. Tanshinone II A inhibits inflammatory response induced by myocardial infarction<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. Tanshinone II A suppresses cells growth on human hepatocellular carcinoma cells<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>, human gastric carcinoma cells. Tanshinone II A prevents apoptosis in PC12 induced by serum starvation<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. Tanshinone II A inhibits HCC cell invasion through suppressing the activity of MMPs<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. In treatment of vascular diseases, Tanshinone II A attenuates atherosclerosis calcification through suppressing oxidative stress<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Tanshinone II A inhibits proliferation of pulmonary artery smooth muscle cells induced by hypoxia<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. Tanshinone II A induces pulmonary artery smooth muscle cell apoptosis through suppressing JAK1/STATS signaling pathway<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. And inhibits vascular smooth muscle migration through suppressing ERK1/2 MAPK signaling pathway<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. However, whether Tanshinone II A plays a critical role during pathological vascular remodeling is largely unknown. Our preliminary data shown that Tanshinone II A significant promotes KLF4 expression. This study aimed to test hypothesis that Tanshinone II A regulates pathological vascular remodeling through KLF4 mediated smooth muscle cell phenotypic switching.</p></sec><sec id=\"Sec2\"><title>Material and methods</title><sec id=\"Sec3\"><title>Mouse common carotid artery complete ligation injury animal model</title><p id=\"Par19\">Completed ligation of mouse left common carotid artery induced vascular remodeling was performed as previously described<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Briefly, Mouse were pretreated with Tanshinone II A (5 mg/kg) for 3 consecutive days and anesthetized with ketamine (80 mg/kg) and xylazine (5 mg/kg) by intraperitoneal injection<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. Exposed the left common carotid arteries and completely ligated at bifurcation with 6-0 silk. The right common carotid artery was going the same process but not ligated. After Tanshinone II A consecutive treatment for 3 weeks and collected sections (5 µm) between 0.2 to 2.0 mm proximal to the ligation site. Morphological analysis based on HE-staining. The quantification of neointima areas and media layer area measured using Image J software.</p></sec><sec id=\"Sec4\"><title>Rat aortic smooth muscle separation</title><p id=\"Par20\">Smooth muscle cells culture from thoracic artery of Sprague–Dawley rats separated as our previous report<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. Briefly, the thoracic arteries for Sprague–Dawley rats harvested after euthanized and removed adhering periadventitial and endothelium under microscope. The adventitial layer removed after Blend enzyme III solution (Roche, 0.5 U/ml) digested for 5–15 min at 37 °C. The smooth muscle medial layer further digested with Blend enzyme III for 2 h at 37 °C. Collected the cells and suspended in DMEM medium contained 10% FBS.</p></sec><sec id=\"Sec5\"><title>WST-1 cell proliferation assay</title><p id=\"Par21\">3 × 10<sup>3</sup> rat smooth muscle cells (each well) were seeded in 96-well culture plate. Treated the cells with Tanshinone II A (1 μM) for 24 h<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>, and measured the absorbance at 450 nm using WST-1 proliferation assay.</p></sec><sec id=\"Sec6\"><title>Scratch wound healing assay</title><p id=\"Par22\">Rat smooth muscle cells were seeded into 6-well culture plate with a density of 1 × 10<sup>6</sup> cells/well. Treated the cells with Tanshinone II A (1 μM). A scratch across the center of the well gently and slowly made with a 10 µl pipette tip. The relative distance of the gaps monitored at different time points after crystal violet staining.</p></sec><sec id=\"Sec7\"><title>Boyden chamber migration assay</title><p id=\"Par23\">Rat smooth muscle cells were treated with 1 μM Tanshinone II A for 24 h. Trypsinized and suspended 5 × 10<sup>4</sup> cells in 100 μl serum-free medium, and seeded in Boyden chambers (353093, BD Biosciences, San Jose, CA, USA). Settle down the Boyden chambers into a 12-well culture plate, which contained 600 µl full culture growth medium contained 50 ng/ml Tanshinone II A and incubated at 37 °C for 4–24 h. The non-migrating cells on the upper side removed. The migrated cells on the bottom side fixed with 4% formaldehyde at room temperature for 20 min, and visualized after 0.1% Crystal violet staining. Migrated cells were counted manually in five random microscopic fields.</p></sec><sec id=\"Sec8\"><title>BRDU incorporation assay</title><p id=\"Par24\">Rat smooth muscle cells treated with 1 μM Tanshinone II A overnight, following treatment with BRDU labeling reagent for 24 h. Fixed the cells with 4% PFA for 20 min at room temperature. Permeabilized with PBS contained 0.20% Triton-X-100 for 30 min, treated with 2 N HCl at room temperature for 30 min, blocked with 10% goat serum at room temperature for 1 h and incubated with BRDU antibody (Invitrogen).</p></sec><sec id=\"Sec9\"><title>Quantitative real time PCR analysis</title><p id=\"Par25\">Total RNA from cells was extracted using TRIzol reagent. 600 ng RNA were using as template for reverse transcription with random hexamer primers using iScript cDNA synthesis kit<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. Real time PCR performed duplicated on Bio-Rad real time PCR system with specific genes primers listed in table (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>). Relative gene expression level was analysis using the 2<sup>−∆∆ct</sup> method against RPLPO.<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>List of primer sequences used in the study primers used for quantitative RT-PCR.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Gene name</th><th align=\"left\">Species</th><th align=\"left\">Sequence</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"2\">SM 22a</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-TGACATGTTCCAGACTGTTGACCTCT-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-CTTCATAAACCAGTTGGGATCTCCAC-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">SM-α-actin</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-ATGCTCCCAGGGCTGTTTTCCCAT-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-GTGGTGCCAGATCTTTTCCATGTCG-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">MHC</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-CAGTTGGACACTATGTCAGGGAAA-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-ATGGAGACAAATGCTAATCAGCC-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">Calponin</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-AACTGGCACCAGCTGGAGAACATAG-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-GAGTAGACTGAACTTGTGTATGATTGG-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">Myocardin</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-GTTCAGCTACCCTGGGATGCACCAA-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-GGCCTGGTTTGAGAGAAGAAACACC-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">SRF</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-GATGGAGTTCATCGACAACAAGCTG-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-CCCTGTCAGCGTGGACAGCTCATA- 3′</td></tr><tr><td align=\"left\" rowspan=\"2\">CDKN1A</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-ATGACTGAGTATAAACTTGTGG-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-TCACATGACTATACACCTTGTC-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">CDKN1B</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-GTCTCAGGCAAACTCTGAG-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-GTTTACGTCTGGCGTCGAAG-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">CCND1</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-AATGGAACTGCTTCTGGTGAACA-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-CGGATGATCTGCTTGTTCTCATC-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">pCNA</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-ACGTCTCCTTAGTGCAGCTTACTCT-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-TAATGATGTCTTCATTACCAGCACAT-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">PTEN</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-GCACAAGAGGCCCTGGATT-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-TGAAACAACAGTGCCACTGG-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">c-fos</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-GGGACAGCCTTTCCTACTACC-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-AGATCTGCGCAAAAGTCCTG-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">Gadd45</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-ATGACTTTGGAGGAATTCTCGG-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-TCACCGTTCGGGGAGATTAATC-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">KLF2</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-ACTTGCAGCTACACCAACTG-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-CTGTGACCCGTGTGCTTG-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">KLF3</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-TCATGTACACCAGCCACCTG-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-TAGTCAGTCCTCTGTGGTTC-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">KLF4</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-CGGGAAGGGAGAAGACACTGC-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-GCTAGCTGGGGAAGACGAGGA-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">KLF5</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-AGCTCACCTGAGGACTCATA-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-GTGCGCAGTGCTCAGTTCT-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">KLF15</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-GATGAGTTGTCACGGCACC-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-CACTGCGCTCAGTTGATGG-3′</td></tr><tr><td align=\"left\" rowspan=\"2\">RPLP0</td><td align=\"left\">Rat</td><td align=\"left\">F: 5′-GGACCCGAGAAGACCTCCTT-3′</td></tr><tr><td align=\"left\">Rat</td><td align=\"left\">R: 5′-TGCTGCCGTTGTCAAACACC-3′</td></tr></tbody></table></table-wrap></p></sec><sec id=\"Sec10\"><title>Protein extraction and Western blotting</title><p id=\"Par26\">Total protein from rat smooth muscle cells lysed with RIPA buffer. Protein concentration determined using BCA kit. 20–40 μg protein from each groups denatured for SDS-PAGE. After blocked with 5% nonfat milk for 1 h, and incubated with specific antibodies at 4 ℃ for immunoblot analyses<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>.</p></sec><sec id=\"Sec11\"><title>Hematoxylin and eosin (H&E) stain, immunohistochemistry (IHC) and immunofluorescence staining (IF)</title><p id=\"Par27\">The mouse carotid arteries fixed with 4% paraformaldehyde overnight at 4 °C and following paraffin embedded process, 5-μm thickness of slides collected and deparaffinized. Hematoxylin/eosin (H&E) staining performed as previously described<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. For IHC staining, the deparaffinized slides were treated with citric acid and antigenic unmasked at 98 °C for 5 min, incubated with primary antibodies overnight at 4 °C, followed by incubation with biotinylated secondary antibody at room temperature for 1 h (Vector Laboratories, 1:200), and ABC solution (Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. Expression level of the targets visualized after DAB solution incubation<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. For IF staining for test BRDU incorporation, the deparaffinized slides, permeabilized with PBS contained 0.2% Triton-X-100, treated with 2 N HCl and blocked with 10% goat serum, incubated with primary antibodies overnight at 4 °C, washed with PBST and incubated with Alexa 594-conjugated secondary antibody at room temperature for 1 h. Nuclei visualized with 4′,6′-diamidino-2-phenylindole (DAPI) staining<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Images were collected using confocal microscopy (LS510, Zeiss).</p></sec><sec id=\"Sec12\"><title>Statistics</title><p id=\"Par28\">Quantitative data presented as mean ± SEM. Comparisons between two groups were analysis by unpaired student’s <italic>t</italic> test using GraphPad prism software<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>–<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. A value of P < 0.05 was considered statistically significant.</p></sec><sec id=\"Sec13\"><title>Ethical approval</title><p id=\"Par29\">The use of mice and rat approved by the Experimental Animal Ethics Committee at Chengdu University of Traditional Chinese Medicine. Ethical approval number: 2019–04.</p></sec><sec id=\"Sec23\"><title>Consent for publication</title><p id=\"Par46\">Yes.</p></sec></sec><sec id=\"Sec14\"><title>Results</title><sec id=\"Sec15\"><title>Tanshinone II A attenuates vascular injury induced neointimal hyperplasia</title><p id=\"Par30\">Tanshinone II A reported to suppress proliferation of smooth muscle cells. To determine whether Tanshinone II A plays a critical role in regulating smooth muscle cell phenotypic switching, we pretreated C57BL/6 mice with 5 mg/kg Tanshinone II A by intraperitoneal injection for 3 consecutive days and following left common carotid artery ligation to induce vascular injury (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>A). Following three weeks of consecutive treatment with Tanshinone II A, harvested the arteries and undergoing paraffin embedded. We performed H&E staining to visualize vascular morphological change induced by vascular injury. Our results indicated that treatment with Tanshinone II A dramatically suppresses neointima formation (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B). We analyzed neointima areas using Image J software from different locations far away from the ligation site. Our data shown the neointima areas from 100 to 700 μm were significant decreased (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>C). We compared the ratios of neointima areas to the medium layer areas, which were significant decreased (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>D). Those data indicated that Tanshinone II A involves in regulating vascular remodeling induced by vascular injury.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Tanshinone II A attenuates vascular neointimal hyperplasia in left common carotid artery ligated mice. (<bold>A</bold>) Schematic diagram for common left carotid artery ligation. (<bold>B</bold>) The representative images of H&E staining of the arteries. Mouse were pretreated with Tanshinone II A (5 mg/kg) for 3 consecutive days by intraperitoneal injection and following common left carotid artery ligation. After 3 consecutive weeks treatment with of Tanshinone II A, the arteries harvested and following paraffin embedded. (<bold>C</bold>) Neointimal area measured using Image J software (n = 6 mice per group). and the ratio of neointima area to the medium layer area shown in (<bold>D</bold>) (n = 6 mice per group). Data represented as mean ± SEM. *P < 0.05.</p></caption><graphic xlink:href=\"41598_2020_70887_Fig1_HTML\" id=\"MO1\"/></fig></p></sec><sec id=\"Sec16\"><title>Tanshinone II A regulates smooth muscle phenotypic switching</title><p id=\"Par31\">Smooth muscle cells phenotypic switching is critical for Pathological vascular remodeling. To determine whether Tanshinone II A contributes to smooth muscle cell phenotypic switching in vitro, we treated rat aortic smooth muscle cells with tanshinone IIA (1 μM) for 30 h, and real time PCR performed to evaluate the expression of SMC differentiated genes and cell growth-regulating genes. Our data indicated that Tanshinone II A treatment significant promotes expression of smooth muscle specific genes, including MHC, calponin, SM α-actin, myocardin and SRF, whereas dramatically suppresses Cyclin D1 expression (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A). We further treated rat smooth muscle cell with PDGF-BB to induce cell growth (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>A,B). The data shown that tanshinone II A treatment attenuates PDGF-BB induced cell growth and expression of Cyclin D1, whereas enhances the expression of MHC, Calponin, SM 22α, myocardin (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>B). We next induced rat smooth muscle cell differentiation by rapamycin treatment (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>A,B). Tanshinone II A treatment promotes the expression of smooth muscle differentiated marker genes, including SM 22α, MHC, Calponin, myocardin (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). Those data suggested that Tanshinone II A modulates smooth muscle phenotypic switching.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Tanshinone II A regulates rat aortic smooth muscle cell phenotypic switching. (<bold>A</bold>) Rat SMCs were treated with Tanshinone II A (1 μM) for 36 h and real time PCR performed to detect expression of cell growth related genes, Cyclin D1, CDKN1A, CDKN1B and smooth muscle specific genes (n = 6 independent experiments). (<bold>B</bold>) Growth of rat smooth muscle cells induced by PDGF-BB treatment (20 ng/mL) and real time PCR performed to observe the expression of smooth muscle differentiated genes and cell growth related genes (n = 6 independent experiments). Data represented presented as mean ± SEM. *P < 0.05.</p></caption><graphic xlink:href=\"41598_2020_70887_Fig2_HTML\" id=\"MO2\"/></fig></p></sec><sec id=\"Sec17\"><title>Tanshinone II A suppresses migration of smooth muscle cells</title><p id=\"Par32\">Migration of VSMCs contributes to neointimal hyperplasia after vascular injury<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. To determine the function role of Tanshinone II A in regulating smooth muscle cell migration, we treated rat smooth muscle cells with Tanshinone II A (1 μΜ) and wound scratch healing assay was performed to monitor the scratching gap at different time point. No difference exhibited at 12 h. However, the distance of scratching gap is larger after Tanshinone II A treatment at 24 h (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>A,B). Similar results were obtained using Boyden chamber migration assay (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>C,D). Those data demonstrated that Tanshinone II A treatment dramatically suppresses migration of rat aortic smooth muscle cells.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Tanshinone II A suppresses rat aortic smooth muscle cell migration. (<bold>A</bold>) Rat smooth muscle cells treated with 1 μM Tanshinone II A for 24 h and Wound scratch experiment performed. The relative gap distance shown in (<bold>B</bold>) (n = 6 independent experiments). (<bold>C</bold>) Rat smooth muscle cells treated with Tanshinone II A (1 μM) and Boyden Chamber Migration Assay was performed, the quantification of migrated cells were exhibited in (<bold>D</bold>) (n = 4 independent experiments). Data represented means ± SEM. *P < 0.05.</p></caption><graphic xlink:href=\"41598_2020_70887_Fig3_HTML\" id=\"MO3\"/></fig></p></sec><sec id=\"Sec18\"><title>Tanshinone II A suppresses proliferation of smooth muscle cells</title><p id=\"Par33\">Smooth muscle cell phenotypic switching is characterized extremely reduced expression of differentiated markers genes and enhanced proliferation. To determine the function role of Tanshinone II A in regulation smooth muscle cell proliferation, immunohistochemistry staining against PCNA was performed on slides from completed ligation of mouse left common carotid arteries (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>A). The percentage of PCNA positive smooth muscle cells within neointima area was extensive decreased (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>B). We next sought to determine whether Tanshinone II A treatment could suppress rat aortic smooth muscle cells growth. After Tanshinone II A treatment, we monitored the proliferation and viability of smooth muscle cells using WST-1 assay. The results indicated that absorbance (OD) at 450 nm is remarkable decreased after transhinone II A treatment (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>C). Seminar results were obtained from cell number counting experiment, Tanshinone II A treatment extremely decreases the cell numbers (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>D). Our Real time PCR data shown that Tanshinone II A treatment suppresses Cyclin D1 expression and promotes expression of cell cycle negative regulated genes, CDKN1A and CDKN1B (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>E). We further confirmed the results by BRDU incorporation assay. We treated rat smooth muscle cells with Tanshinone II A and following treated with BRDU labeling reagent, immunofluorescent staing was performed against BRDU antibody, the percentage of BRDU positive cell was quantified. Our data shown that Tanshinone II A treatment dramatically suppressed BRDU incorporation (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>F,G). These results above demonstrated that Tanshinone II A treatment significant suppresses rat aortic smooth muscle cell proliferation.<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Tanshinone II A suppresses rat aortic smooth muscle cells proliferation. (<bold>A</bold>) Representative Images of IHC staining from left common carotid artery complete ligation injury animal model against with PCNA antibody. (<bold>B</bold>) Quantification of PCNA positive smooth muscle cells in the neointima areas from (<bold>A</bold>) (n = 5 mice per group). (<bold>C</bold>) Rat smooth muscle cells treated with 1 μM Tanshinone II A for 24 h and proliferation and viability were detected by WST-1 assay (n = 5 independent experiments). (<bold>D</bold>) Rat smooth muscle cells treated with 1 μM Tanshinone II A and cell number counted at different time point (n = 5 independent experiments). (<bold>E</bold>) Tanshinone II A treated rat smooth muscle cells for 30 h and real time PCR performed to investigate the expression of cell cycles related genes (n = 6 independent experiments). (<bold>F</bold>) Representative images of IHC staining against BRDU antibody from rat smooth muscle cells. Rat smooth muscle cells treated with 1 μM Tanshinone II A overnight, following the treatment with BRDU labeling reagent for 24 h and following IHC staining, the nuclei visualized using DAPI staining. The BRDU positive cells from (<bold>F</bold>) quantified in (<bold>G</bold>) (n = 5 independent experiments). The quantify data represented as means ± SEM. *P < 0.05.</p></caption><graphic xlink:href=\"41598_2020_70887_Fig4_HTML\" id=\"MO4\"/></fig></p></sec><sec id=\"Sec19\"><title>Tanshinone II A promotes KLF4 expression during smooth muscle phenotypic switching</title><p id=\"Par34\">Tanshinone II A plays a critical role in regulating smooth muscle cell phenotypic switching. However, the underlie mechanism is poorly understood. We used rat aortic smooth muscle cells and treated with 1 μM Tanshinone II A for 30 h, real time PCR performed to screen multiple signaling pathways. Interesting, we observed that Tanshinone II A treatment promotes the expression of Pten, c-Fos, Gadd45, KLF4 and KLF1 expression (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>). Since KLF4 plays an pivotal role during smooth muscle cell phenotypic switching<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>, we confirmed the expression of KLF4 after Tanshinone II A treatment. Our data demonstrated that Tanshinone II A treatment obviously enhances the expression level of KLF4 at both transcription level and protein level (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>A–C). This was consistent with our in vitro study. We performed immunohistochemistry staining of KLF4 in common carotid arteries. The expression of KLF4 within neointima area dramatically decreased after Tanshinone II A treatment (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>D,E). Our results suggested that Tanshinone II A promotes KLF4 expression in rat aortic smooth muscle cell.<fig id=\"Fig5\"><label>Figure 5</label><caption><p>Tanshinone II A treatment promotes KLF4 expression. (<bold>A</bold>) Rat smooth muscle cells treated with Tanshinone II A (1 μM) for 30 h and real time PCR performed to detect KLF4 transcription level (n = 6 independent experiments). (<bold>B</bold>) Rat smooth muscle cells treated with Tanshinone II A (1 μM) for 3 h, 20 h and 30 h. The expression of KLF4 investigated by western blotting and the quantification data showed in (<bold>C</bold>) (n = 5 independent experiments). (<bold>D</bold>) Representative Images of IHC staining from left common carotid artery complete ligation injury animal model against with KLF4 antibody. (<bold>E</bold>) Relative expression level of KLF4 in (<bold>D</bold>) was quantified by integrated optical density (IOD) using Image J software (n = 5 mice per group). Data represented as means ± SEM. *P < 0.05.</p></caption><graphic xlink:href=\"41598_2020_70887_Fig5_HTML\" id=\"MO5\"/></fig></p></sec><sec id=\"Sec20\"><title>Interruption KLF4 expression contributes to Tanshinone II A in regulating pathological vascular remodeling</title><p id=\"Par35\">To determine whether Tanshinone II A regulates smooth muscle cell phenotypic switching through mediated KLF4 expression, we first overexpressed of KLF4 in rat aortic smooth muscle cells by adenovirus infection and the expression efficiency validated by real time PCR (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>). We next sought to determine whether overexpression of KLF4 in smooth muscle cell could interrupt Tanshinone II A in regulating smooth muscle cell migration. We infected rat aortic smooth muscle cells with adenoviral KLF4 to generate KLF4 overexpression smooth muscle cells, and treated with 1 μM Tanshinone II A. The migration was monitored scratch wound healing assay. Our results indicated that overexpression of KLF4 suppresses migration of rat aortic smooth muscle cells (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>A,B). We further detected whether overexpression of KLF4 exacerbates Tanshinone II A in suppression smooth muscle cell proliferation using WST-1 cell proliferation assay. Both Tanshinone II A treatment and adenovirus mediated KLF4 overexpression can inhibit rat aortic smooth muscle cell proliferation. However, after adenovirus infection and following Tanshinone II A treatment, the cells exhibited much lower OD compared to the control group (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>C). We finally determined whether overexpression of KLF4 interrupts Tanshinone II A in regulating the expression of smooth muscle differentiated marker genes using real time PCR. Overexpression of KLF4 in rat aortic smooth muscle cells promotes the expression of SM 22α, MHC, calponin, SRF and Myocardin (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>D).<fig id=\"Fig6\"><label>Figure 6</label><caption><p>Tanshinone II A regulates smooth muscle phenotypic switching through enhances KLF4 expression. (<bold>A</bold>) Wound healing assay was performed to detect migration of KLF4 overexpress VSMCs treated with Tanshinone II A in 3 different time periods (0 h, 12 h, 24 h). The quantification of relative gap distance from (<bold>A</bold>) displayed in (<bold>B</bold>) (n = 6 independent experiments). (<bold>C</bold>) WST-1 proliferation assay was used to assess proliferation of rat smooth muscle cells with different treatment (n = 5 independent experiments). (<bold>D</bold>) Real Time PCR performed to detect transcription level of smooth muscle cell phenotypic switching regulating genes (n = 6 independent experiments). (<bold>E</bold>) Schematic diagram indicates that Tanshinone II A attenuates vascular remodeling through klf4 mediated smooth muscle cell phenotypic switching.</p></caption><graphic xlink:href=\"41598_2020_70887_Fig6_HTML\" id=\"MO6\"/></fig></p><p id=\"Par36\">Taken together, our data in this study demonstrated that Tanshinone II A attenuates smooth muscle cell phenotypic switching induced by vascular injure partially through regulating KLF4 expression (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>E).</p></sec></sec><sec id=\"Sec21\"><title>Discussion</title><p id=\"Par37\">This study for the first time provides the evidence that Tanshinone II A administration suppresses vascular remodeling through KLF4 mediated smooth muscle cell phenotypic switching. We found that Tanshinone II A intraperitoneal injection attenuates complete carotid artery ligation induced neointima formation. Tanshinone II A attenuates rat aortic smooth muscle cell growth induced by PDGF-BB treatment, and promotes smooth muscle cell differentiated marker genes expression induced by Rapamycin. The critical role of Tanshinone II A in regulating smooth muscle cell phenotypic switching exhibited in dramatically promoting the expression of smooth muscle cell differentiated marker genes, as well as impaired cell proliferation.</p><p id=\"Par38\">The Traditional Chinses Medicine, salvia miltiorrhiza, is traditional used in treatment of cardiovascular diseases. At least 108 kinds of different integrants have been identified from salvia miltiorrhiza. However, the biofunction of most integrants are undefined. In our study, we only focused on a key integrant, Tanshinone II A, and observed its function in regulating smooth muscle cell phenotypic switching.</p><p id=\"Par39\">In this study we reported a valuable mechanism that Tanshinone II A regulates smooth muscle phenotypic switching through enhanced KLF4 expression to suppress smooth muscle cells proliferation, whereas induce differentiate, and eventually regulates pathological vascular remodeling.</p><p id=\"Par40\">KLF4, a member of Krüppel-like family of transcription factors (KLFs), plays a crucial role in various vascular diseases. Expression level of KFL4 is extremely low in normal condition, whereas the expression level dramatically increased in response to injury stresses. We observed that Tanshinone II A treatment promotes the expression of Pten, c-Fos, Gadd45 and Krüppel-like family of transcription family (KLFs). We detected the other members of Krüppel-like family of transcription family, including KLF2, KLF3, KLF5 and KLF15, the expression of KLF4 is remarkable increased compared to the other members (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). We tried to knockout KLF4 in rat smooth muscle cells using adenovirus infection. However, the knockdown efficiency of KLF4 virus was not high enough. While forced expression of KLF4 in rat smooth muscle cell aggravated the function of Tanshinone II A in suppressing proliferation and promoting expression of muscle differentiated genes (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>). Those results suggested Tanshinone II A regulates smooth muscle phenotypic switching, at least partially, through active KLF4 signaling pathway.</p><p id=\"Par41\">In this study we did not adopted multiple biological methods to detect the interaction between Tanshinone II A and KLF4, as endogenously genes which involved in signaling pathway transmission.</p><p id=\"Par42\">Tanshinone II A treatment suppresses rat aortic smooth muscle growth. Our real time PCR data indicated that Tanshinone II A treatment dramatically enhances the expression of CDKN1A and CDKN1B, which are cell cycle negative control genes. The results suggested that Tanshinone II A, at least partially, inhibits cell cycle to regulate cell growth.</p><p id=\"Par43\">Rapamycin induced smooth muscle cell differentiation<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. After Rapamycin and Tanshinone II A treatment, our real time PCR data shown that Tanshinone II A treatment can not significant promote smooth muscle differentiated genes expression (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). The optimal explanation is that both control and Tanshinone II A treatment smooth muscle cell have been treated with Rapamycin. We treated smooth muscle cell with Tanshinone II A alone dramatically promotes differentiated genes expression (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>).</p><p id=\"Par44\">In summary, our study not only demonstrate that Tanshinone II A is critical in regulating smooth muscle cell pathological phenotypic switching, but also identify a target for vascular diseases treatment.</p></sec><sec id=\"Sec22\"><title>Conclusion</title><p id=\"Par45\">Tanshinone II A plays a pivotal role in regulating pathological vascular remodeling through KLF4 mediated smooth muscle cell phenotypic switching. This study demonstrated that Tanshinone II A is a potential therapeutic agent for vascular diseases.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec24\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70887_MOESM1_ESM.pdf\"><caption><p>Supplementary Legends.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41598_2020_70887_MOESM2_ESM.tif\"><caption><p>Supplementary Figure 1.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM3\"><media xlink:href=\"41598_2020_70887_MOESM3_ESM.tif\"><caption><p>Supplementary Figure 2.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM4\"><media xlink:href=\"41598_2020_70887_MOESM4_ESM.tif\"><caption><p>Supplementary Figure 3.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM5\"><media xlink:href=\"41598_2020_70887_MOESM5_ESM.tif\"><caption><p>Supplementary Figure 4.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM6\"><media xlink:href=\"41598_2020_70887_MOESM6_ESM.tif\"><caption><p>Supplementary Figure 5.</p></caption></media></supplementary-material></p></sec></sec></body><back><glossary><title>Abbreviations</title><def-list><def-item><term>Tans</term><def><p id=\"Par2\">Tanshinone II A</p></def></def-item><def-item><term>KLF4</term><def><p id=\"Par3\">Krüppel-like factor 4</p></def></def-item><def-item><term>HCC</term><def><p id=\"Par4\">Hepatic cell carcinoma</p></def></def-item><def-item><term>PDGF-BB</term><def><p id=\"Par5\">Platelet-derived growth factor BB</p></def></def-item><def-item><term>PCR</term><def><p id=\"Par6\">Polymerase chain reaction</p></def></def-item><def-item><term>ERK</term><def><p id=\"Par7\">Extracellular signal regulated protein kinase</p></def></def-item><def-item><term>JAK1</term><def><p id=\"Par8\">Janus kinase 1</p></def></def-item><def-item><term>STAT1</term><def><p id=\"Par9\">Signal transduction and activation of transcription-1</p></def></def-item><def-item><term>H&E</term><def><p id=\"Par10\">Hematoxylin and eosin stain</p></def></def-item><def-item><term>IHC</term><def><p id=\"Par11\">Immunohistochemistry</p></def></def-item><def-item><term>IF</term><def><p id=\"Par12\">Immunofluorescence staining</p></def></def-item><def-item><term>BRDU</term><def><p id=\"Par13\">5-Bromo-2-deoxyUridine</p></def></def-item><def-item><term>DAPI</term><def><p id=\"Par14\">4′,6′-Diamidino-2-phenylindole</p></def></def-item><def-item><term>PCNA</term><def><p id=\"Par15\">Proliferating cell nuclear antigen</p></def></def-item><def-item><term>SM22α</term><def><p id=\"Par16\">Smooth muscle protein 22α</p></def></def-item><def-item><term>MHC</term><def><p id=\"Par17\">Myosin heavy chain</p></def></def-item></def-list></glossary><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Guanhua Lou, Wangming Hu and Ziqiang Wu.</p></fn></fn-group><sec><title>Supplementary information</title><p> is available for this paper at 10.1038/s41598-020-70887-1.</p></sec><ack><title>Acknowledgements</title><p>We thank Dr Yuqing Huo from Augusta University and Dr Quansheng Feng from Chengdu University of Traditional Chinese Medicine for design the study and manuscript preparation. This study supported by Grant (81741007, 81870363) from National Natural Science Foundation of China; Grant 008066, 030038199, 030041023, 030041224 and 242030016 from Cheng Du University of Traditional Chinese Medicine; Grant from 2020JDTD0025 from science & technology departments of Sichuan province.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>Y.W., Y.X. designed research; G.L., Z.W., B.W. and L.W. performed experiments; H.X., H.Y., Y.W., W.H. and D.G. analyzed data; Y.W., Z.W., G.L. wrote and revised the manuscript; Q.H., X.C., Y.S., L.Y. participated in discussing experiments.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>The authors state that all relevant data are available within the article and the online Supplementary material or are available from the corresponding authors upon reasonable request.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par47\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alexander</surname><given-names>MR</given-names></name><name><surname>Owens</surname><given-names>GK</given-names></name></person-group><article-title>Epigenetic control of smooth muscle cell differentiation and phenotypic switching in vascular development and disease</article-title><source>Annu. Rev. Physiol.</source><year>2012</year><volume>74</volume><fpage>13</fpage><lpage>40</lpage><pub-id pub-id-type=\"doi\">10.1146/annurev-physiol-012110-142315</pub-id><pub-id pub-id-type=\"pmid\">22017177</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gomez</surname><given-names>D</given-names></name><name><surname>Owens</surname><given-names>GK</given-names></name></person-group><article-title>Smooth muscle cell phenotypic switching in atherosclerosis</article-title><source>Cardiovasc. Res.</source><year>2012</year><volume>95</volume><fpage>156</fpage><lpage>164</lpage><pub-id pub-id-type=\"doi\">10.1093/cvr/cvs115</pub-id><pub-id pub-id-type=\"pmid\">22406749</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Owens</surname><given-names>GK</given-names></name><name><surname>Kumar</surname><given-names>MS</given-names></name><name><surname>Wamhoff</surname><given-names>BR</given-names></name></person-group><article-title>Molecular regulation of vascular smooth muscle cell differentiation in development and disease</article-title><source>Physiol. Rev.</source><year>2004</year><volume>84</volume><fpage>767</fpage><lpage>801</lpage><pub-id pub-id-type=\"doi\">10.1152/physrev.00041.2003</pub-id><pub-id pub-id-type=\"pmid\">15269336</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Takahashi</surname><given-names>K</given-names></name><etal/></person-group><article-title>Sodium tanshinone IIA sulfonate derived from Danshen (<italic>Salvia miltiorrhiza</italic>) attenuates hypertrophy induced by angiotensin II in cultured neonatal rat cardiac cells</article-title><source>Biochem. Pharmacol.</source><year>2002</year><volume>64</volume><fpage>745</fpage><lpage>749</lpage><pub-id pub-id-type=\"doi\">10.1016/S0006-2952(02)01250-9</pub-id><pub-id pub-id-type=\"pmid\">12167494</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>L</given-names></name><etal/></person-group><article-title>Sodium tanshinone IIA sulfonate depresses angiotensin II-induced cardiomyocyte hypertrophy through MEK/ERK pathway</article-title><source>Exp. Mol. Med.</source><year>2007</year><volume>39</volume><fpage>65</fpage><lpage>73</lpage><pub-id pub-id-type=\"doi\">10.1038/emm.2007.8</pub-id><pub-id pub-id-type=\"pmid\">17334230</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ren</surname><given-names>ZH</given-names></name><name><surname>Tong</surname><given-names>YH</given-names></name><name><surname>Xu</surname><given-names>W</given-names></name><name><surname>Ma</surname><given-names>J</given-names></name><name><surname>Chen</surname><given-names>Y</given-names></name></person-group><article-title>Tanshinone II A attenuates inflammatory responses of rats with myocardial infarction by reducing MCP-1 expression</article-title><source>Phytomed. Int. J. Phytother. Phytopharmacol.</source><year>2010</year><volume>17</volume><fpage>212</fpage><lpage>218</lpage></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yuan</surname><given-names>SL</given-names></name><etal/></person-group><article-title>Growth inhibition and apoptosis induction of tanshinone II-A on human hepatocellular carcinoma cells</article-title><source>World J. Gastroenterol.</source><year>2004</year><volume>10</volume><fpage>2024</fpage><lpage>2028</lpage><pub-id pub-id-type=\"doi\">10.3748/wjg.v10.i14.2024</pub-id><pub-id pub-id-type=\"pmid\">15237427</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ji</surname><given-names>ZN</given-names></name><name><surname>Liu</surname><given-names>GQ</given-names></name></person-group><article-title>Inhibition of serum deprivation-induced PC12 cell apoptosis by tanshinone II A</article-title><source>Acta Pharmacol. Sin.</source><year>2001</year><volume>22</volume><fpage>459</fpage><lpage>462</lpage><pub-id pub-id-type=\"pmid\">11743897</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yuxian</surname><given-names>X</given-names></name><name><surname>Feng</surname><given-names>T</given-names></name><name><surname>Ren</surname><given-names>L</given-names></name><name><surname>Zhengcai</surname><given-names>L</given-names></name></person-group><article-title>Tanshinone II-A inhibits invasion and metastasis of human hepatocellular carcinoma cells in vitro and in vivo</article-title><source>Tumori</source><year>2009</year><volume>95</volume><fpage>789</fpage><lpage>795</lpage><pub-id pub-id-type=\"doi\">10.1177/030089160909500623</pub-id><pub-id pub-id-type=\"pmid\">20210245</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tang</surname><given-names>F</given-names></name><etal/></person-group><article-title>Tanshinone II A attenuates atherosclerotic calcification in rat model by inhibition of oxidative stress</article-title><source>Vascul. Pharmacol.</source><year>2007</year><volume>46</volume><fpage>427</fpage><lpage>438</lpage><pub-id pub-id-type=\"doi\">10.1016/j.vph.2007.01.001</pub-id><pub-id pub-id-type=\"pmid\">17337361</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luo</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Tanshinone IIA inhibits hypoxia-induced pulmonary artery smooth muscle cell proliferation via Akt/Skp2/p27-associated pathway</article-title><source>PLoS ONE</source><year>2013</year><volume>8</volume><fpage>e56774</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0056774</pub-id><pub-id pub-id-type=\"pmid\">23437233</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>M</given-names></name><etal/></person-group><article-title>Tanshinone IIA promotes pulmonary artery smooth muscle cell apoptosis in vitro by inhibiting the JAK2/STAT3 signaling pathway</article-title><source>Cell. Physiol. Biochem. Int. J. Exp. Cell. Physiol. Biochem. Pharmacol.</source><year>2014</year><volume>33</volume><fpage>1130</fpage><lpage>1138</lpage><pub-id pub-id-type=\"doi\">10.1159/000358682</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lu</surname><given-names>M</given-names></name><name><surname>Luo</surname><given-names>Y</given-names></name><name><surname>Hu</surname><given-names>P</given-names></name><name><surname>Dou</surname><given-names>L</given-names></name><name><surname>Huang</surname><given-names>S</given-names></name></person-group><article-title>Tanshinone IIA inhibits AGEs-induced proliferation and migration of cultured vascular smooth muscle cells by suppressing ERK1/2 MAPK signaling</article-title><source>Iranian journal of basic medical sciences</source><year>2018</year><volume>21</volume><fpage>83</fpage><lpage>88</lpage><pub-id pub-id-type=\"pmid\">29372041</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kumar</surname><given-names>A</given-names></name><name><surname>Lindner</surname><given-names>V</given-names></name></person-group><article-title>Remodeling with neointima formation in the mouse carotid artery after cessation of blood flow</article-title><source>Arterioscler. Thromb. Vasc. Biol.</source><year>1997</year><volume>17</volume><fpage>2238</fpage><lpage>2244</lpage><pub-id pub-id-type=\"doi\">10.1161/01.ATV.17.10.2238</pub-id><pub-id pub-id-type=\"pmid\">9351395</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>SB</given-names></name><name><surname>Guo</surname><given-names>XF</given-names></name><name><surname>Weng</surname><given-names>B</given-names></name><name><surname>Tang</surname><given-names>SP</given-names></name><name><surname>Zhang</surname><given-names>HJ</given-names></name></person-group><article-title>Tanshinone IIA attenuates ovalbumin-induced airway inflammation and hyperresponsiveness in a murine model of asthma</article-title><source>Iran. J. Basic Med. Sci.</source><year>2019</year><volume>22</volume><fpage>160</fpage><lpage>165</lpage><pub-id pub-id-type=\"pmid\">30834081</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xu</surname><given-names>S</given-names></name><etal/></person-group><article-title>Development of an optimized protocol for primary culture of smooth muscle cells from rat thoracic aortas</article-title><source>Cytotechnology</source><year>2009</year><volume>61</volume><fpage>65</fpage><lpage>72</lpage><pub-id pub-id-type=\"doi\">10.1007/s10616-009-9236-6</pub-id><pub-id pub-id-type=\"pmid\">19898948</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>X</given-names></name><etal/></person-group><article-title>The induction of yes-associated protein expression after arterial injury is crucial for smooth muscle phenotypic modulation and neointima formation</article-title><source>Arterioscler. Thromb. Vasc. Biol.</source><year>2012</year><volume>32</volume><fpage>2662</fpage><lpage>2669</lpage><pub-id pub-id-type=\"doi\">10.1161/ATVBAHA.112.254730</pub-id><pub-id pub-id-type=\"pmid\">22922963</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cheng</surname><given-names>W</given-names></name><name><surname>Xiang</surname><given-names>W</given-names></name><name><surname>Wang</surname><given-names>S</given-names></name></person-group><article-title>Tanshinone IIA ameliorates oxaliplatin-induced neurotoxicity via mitochondrial protection and autophagy promotion</article-title><source>Am. J. Transl. Res.</source><year>2019</year><volume>11</volume><fpage>3140</fpage><lpage>3149</lpage><pub-id pub-id-type=\"pmid\">31217883</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Deletion of yes-associated protein (YAP) specifically in cardiac and vascular smooth muscle cells reveals a crucial role for YAP in mouse cardiovascular development</article-title><source>Circ. Res.</source><year>2014</year><volume>114</volume><fpage>957</fpage><lpage>965</lpage><pub-id pub-id-type=\"doi\">10.1161/CIRCRESAHA.114.303411</pub-id><pub-id pub-id-type=\"pmid\">24478334</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xu</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Regulation of endothelial intracellular adenosine via adenosine kinase epigenetically modulates vascular inflammation</article-title><source>Nat. Commun.</source><year>2017</year><volume>8</volume><fpage>943</fpage><pub-id pub-id-type=\"doi\">10.1038/s41467-017-00986-7</pub-id><pub-id pub-id-type=\"pmid\">29038540</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xu</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Intracellular adenosine regulates epigenetic programming in endothelial cells to promote angiogenesis</article-title><source>EMBO Mol. Med.</source><year>2017</year><volume>9</volume><fpage>1263</fpage><lpage>1278</lpage><pub-id pub-id-type=\"doi\">10.15252/emmm.201607066</pub-id><pub-id pub-id-type=\"pmid\">28751580</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yao</surname><given-names>H</given-names></name><etal/></person-group><article-title>Andrographolide attenuates imbalance of gastric vascular homeostasis induced by ethanol through glycolysis pathway</article-title><source>Sci. Rep.</source><year>2019</year><volume>9</volume><fpage>4968</fpage><pub-id pub-id-type=\"doi\">10.1038/s41598-019-41417-5</pub-id><pub-id pub-id-type=\"pmid\">30899067</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kallenbach</surname><given-names>K</given-names></name><etal/></person-group><article-title>Inhibition of smooth muscle cell migration and neointima formation in vein grafts by overexpression of matrix metalloproteinase-3</article-title><source>J. Vasc. Surg.</source><year>2009</year><volume>49</volume><fpage>750</fpage><lpage>758</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jvs.2008.11.001</pub-id><pub-id pub-id-type=\"pmid\">19268777</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zheng</surname><given-names>B</given-names></name><name><surname>Han</surname><given-names>M</given-names></name><name><surname>Wen</surname><given-names>JK</given-names></name></person-group><article-title>Role of Kruppel-like factor 4 in phenotypic switching and proliferation of vascular smooth muscle cells</article-title><source>IUBMB Life</source><year>2010</year><volume>62</volume><fpage>132</fpage><lpage>139</lpage><pub-id pub-id-type=\"pmid\">20073036</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martin</surname><given-names>KA</given-names></name><etal/></person-group><article-title>Rapamycin promotes vascular smooth muscle cell differentiation through insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt2 feedback signaling</article-title><source>J. Biol. Chem.</source><year>2007</year><volume>282</volume><fpage>36112</fpage><lpage>36120</lpage><pub-id pub-id-type=\"doi\">10.1074/jbc.M703914200</pub-id><pub-id pub-id-type=\"pmid\">17908691</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807805</article-id><article-id pub-id-type=\"pmc\">PMC7431535</article-id><article-id pub-id-type=\"publisher-id\">70623</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70623-9</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Salt coatings functionalize inert membranes into high-performing filters against infectious respiratory diseases</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Rubino</surname><given-names>Ilaria</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Oh</surname><given-names>Euna</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Han</surname><given-names>Sumin</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kaleem</surname><given-names>Sana</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hornig</surname><given-names>Alex</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lee</surname><given-names>Su-Hwa</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kang</surname><given-names>Hae-Ji</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lee</surname><given-names>Dong-Hun</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Chu</surname><given-names>Ki-Back</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kumaran</surname><given-names>Surjith</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Armstrong</surname><given-names>Sarah</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lalani</surname><given-names>Romani</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Choudhry</surname><given-names>Shivanjali</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kim</surname><given-names>Chun Il</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Quan</surname><given-names>Fu-Shi</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>Jeon</surname><given-names>Byeonghwa</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Choi</surname><given-names>Hyo-Jick</given-names></name><address><email>hyojick@ualberta.ca</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.17089.37</institution-id><institution>Department of Chemical and Materials Engineering, </institution><institution>University of Alberta, </institution></institution-wrap>Edmonton, AB T6G 1H9 Canada </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.17089.37</institution-id><institution>School of Public Health, </institution><institution>University of Alberta, </institution></institution-wrap>Edmonton, AB T6G 1H9 Canada </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.289247.2</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2171 7818</institution-id><institution>Department of Biomedical Science, Graduate School, </institution><institution>Kyung Hee University, </institution></institution-wrap>Seoul, 130-701 South Korea </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.17089.37</institution-id><institution>Department of Mechanical Engineering, </institution><institution>University of Alberta, </institution></institution-wrap>Edmonton, AB T6G 1H9 Canada </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.289247.2</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2171 7818</institution-id><institution>Department of Medical Zoology, </institution><institution>Kyung Hee University School of Medicine, </institution></institution-wrap>Seoul, 130-701 South Korea </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.17635.36</institution-id><institution-id institution-id-type=\"ISNI\">0000000419368657</institution-id><institution>Environmental Health Sciences, School of Public Health, </institution><institution>University of Minnesota, </institution></institution-wrap>Saint Paul, MN 55108 USA </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13875</elocation-id><history><date date-type=\"received\"><day>17</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>30</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Respiratory protection is key in infection prevention of airborne diseases, as highlighted by the COVID-19 pandemic for instance. Conventional technologies have several drawbacks (i.e., cross-infection risk, filtration efficiency improvements limited by difficulty in breathing, and no safe reusability), which have yet to be addressed in a single device. Here, we report the development of a filter overcoming the major technical challenges of respiratory protective devices. Large-pore membranes, offering high breathability but low bacteria capture, were functionalized to have a uniform salt layer on the fibers. The salt-functionalized membranes achieved high filtration efficiency as opposed to the bare membrane, with differences of up to 48%, while maintaining high breathability (> 60% increase compared to commercial surgical masks even for the thickest salt filters tested). The salt-functionalized filters quickly killed Gram-positive and Gram-negative bacteria aerosols in vitro, with CFU reductions observed as early as within 5 min, and in vivo by causing structural damage due to salt recrystallization. The salt coatings retained the pathogen inactivation capability at harsh environmental conditions (37 °C and a relative humidity of 70%, 80% and 90%). Combination of these properties in one filter will lead to the production of an effective device, comprehensibly mitigating infection transmission globally.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Disease prevention</kwd><kwd>Public health</kwd><kwd>Infectious diseases</kwd><kwd>Bacterial infection</kwd><kwd>Biomaterials</kwd><kwd>Respiratory tract diseases</kwd><kwd>Nanobiotechnology</kwd><kwd>Design, synthesis and processing</kwd></kwd-group><funding-group><award-group><funding-source><institution>Natural Sciences and Engineering Research Council of Canada</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>Mitacs</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>University of Alberta</institution></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Airborne pathogens, including bacteria and viruses, transmit in the environment in the form of droplets (> 5 µm) or aerosols (< 5 µm)<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. Due to long travelling distance and respirability of aerosols, airborne transmission can occur very easily<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. As such, respiratory protection measures are essential first lines of defense in health care settings, congregate settings (e.g., correctional facilities, military barracks, homeless shelters, refugee camps, dormitories, and nursing homes), households (including family members and caregivers), and in the event of pandemic or epidemic outbreaks. As the World Health Organization (WHO) and scientific community highlight the urgency in stopping infectious diseases and preparing for the next disease outbreaks<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>, and new pandemic strains such as COVID-19 emerge, development of effective, readily available infection control measures is recognized as a primary challenge in health care.</p><p id=\"Par3\">Specifically, in health care facilities, bacteria including <italic>Klebsiella pneumoniae </italic>(<italic>K. pneumoniae</italic>), <italic>Staphylococcus aureus</italic> (<italic>S. aureus</italic>), <italic>Pseudomonas aeruginosa </italic>(<italic>P. aeruginosa</italic>), <italic>Streptococcus pyogenes</italic> (<italic>S. pyogenes</italic>) and <italic>Escherichia coli </italic>(<italic>E. coli</italic>) are the major causes of nosocomial (hospital-associated) infections<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. Bacteria can transmit infections through the air in locations such as operating theatres, corridors, waste containers as well as intensive care, burn and orthopedic units<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>–<xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. Nosocomial <italic>K. pneumoniae</italic> infections have mortality rates as high as 50%; additionally, the WHO has reported global resistance to third-generation cephalosporins and carbapenem in 30–60% and up to 50% of cases, respectively<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Methicillin-resistant <italic>S. aureus</italic> (MRSA), also transmissible through the air<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>, constitutes another major pathogen in nosocomial infections, causing 20% to above 80% of <italic>S. aureus</italic> nosocomial infections worldwide<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>.</p><p id=\"Par4\">The most commonly used respiratory protective device in health care is the N95 respirator, which is designed to capture aerosols. Surgical masks, commonly used to block large droplets during surgeries, were historically utilized against bioaerosols. Surgical masks have seen a continued use for this purpose in the general public during more recent outbreaks, in spite of the improper application<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>–<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. This is due to their high availability, affordability and comfort. Although respirators and masks play a critical role in the protection against bioaerosols, they are limited by four major technical issues of the filters: (i) cross-infection, (ii) filtration efficiency, (iii) breathability, and (iv) recyclability<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. When respirators/masks capture bioaerosols, they become contaminated, since pathogens survive on the surface of the filters. This presents a threat to the wearer and people they come in contact with. As the devices can easily become a source of infection, commercial respirators/masks are limited to a single use. Additionally, in the traditional technologies, it is well-known that increasing the filtration efficiency leads to filters that cause higher pressure drop across the masks, with consequent difficulty in breathing. Some efforts have been directed towards production of filters that can overcome the burden of decreased breathability<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. However, these methods cannot address the risks arising from the survival of pathogens captured on the filters.</p><p id=\"Par5\">We envisioned the development of a technology that could solve all major technical challenges simultaneously, which cannot be addressed by conventional respiratory protective devices. The mechanism we developed consists of functionalizing the polypropylene (PP) fibers of large-pore (~ 60 μm) membranes with salt. Large-pore membranes have high breathability compared to commercial respirators/masks, but no pathogen filtration activity. We hypothesized that the salt coating would: (i) increase the filtration efficiency of the breathable membranes, turning them into an active filtration unit, and (ii) inactivate bacteria. Our previous report showed that coating the filters of surgical masks with sodium chloride (NaCl) salt kills multiple strains of influenza virus; additionally, a significant increase in the filtration efficiency of viral aerosols on the NaCl-coated filters compared to the bare surgical mask filters was observed<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. In this work, non-functional large-pore membranes were coated with three types of salt [NaCl, potassium sulfate (K<sub>2</sub>SO<sub>4</sub>) and potassium chloride (KCl)]. We demonstrate that salt functionalization greatly increases the filtration efficiency of large-pore membranes, while at the same time exhibiting no significant increase in pressure drop. The proposed filtration mechanism is non-mechanical in nature, which is evidenced by the insensitivity of filtration efficiency with respect to increasing layer numbers and the high filtration efficiency of the top layer (the first layer interacting with aerosols) of salt-functionalized filters even with presence of large-pore fiber mesh. Additionally, the salt recrystallization upon exposure of the salt-functionalized filters to infectious aerosols caused the physical damage of the bacteria independent of strain (<italic>K. pneumoniae,</italic> MRSA<italic>, P. aeruginosa, S. pyogenes</italic> and <italic>E. coli</italic>), leading to their inactivation. As such, we report a diverse respiratory protection system that is manufactured without extensive engineering efforts, and achieves quick universal pathogen inactivation, high filtration efficiency, high breathability, and safe recyclability, all in one platform.</p></sec><sec id=\"Sec2\"><title>Results and discussion</title><sec id=\"Sec3\"><title>Production and characterization of salt-coated filters</title><p id=\"Par6\">Development of environment-resistant filters is key to their universal application. Thus, when designing the salt-functionalized filters, fine-tuning of the properties of the pathogen-inactivating mask to satisfy different temperature/humidity conditions during use and storage was considered. To this end, safe and inexpensive salt types with different critical relative humidity (RH) were selected. At a given temperature, if a salt is exposed to an RH above its critical RH, it will take up water from the atmosphere. NaCl, KCl and K<sub>2</sub>SO<sub>4</sub> have critical RH of 74.7%, 81.2% and 95.7% at 40 °C, respectively<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>, and were investigated in this study. The salt coatings were applied onto the PP microfibers of large-pore membranes, which were not designed to provide protection against bacteria aerosols. The membranes (bare membranes) were dried in different volumes of coating solution (V<sub>salt</sub>; NaCl, K<sub>2</sub>SO<sub>4</sub> or KCl), to produce filters coated with different amounts of salts per unit area (W<sub>salt</sub>). The linear relationship between W<sub>salt</sub> and V<sub>salt</sub> for each salt type at different thicknesses can be found in Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>a–c (Pearson test, <italic>P</italic> values on graphs).</p><p id=\"Par7\">By stacking different numbers of membrane layers during drying, the overall thickness was varied. This allows for control over the final design of the salt-functionalized filters based on application needs. The formation of NaCl, K<sub>2</sub>SO<sub>4</sub> or KCl salt coatings was analyzed by scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) mapping. Homogenous coating formation on the surface of the fibers and throughout the cross section of multi-layer filters was observed (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>a and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>d). These results show the successful fabrication of filters uniformly functionalized with NaCl, K<sub>2</sub>SO<sub>4</sub> or KCl salts at controlled thicknesses.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Characterization and performance of salt-coated filters. (<bold>a</bold>) EDX mapping images of NaCl (left—combination of Na (red) and Cl (green) mapping images), K<sub>2</sub>SO<sub>4</sub> [center—combination of K (red) and S (green)], and KCl [right—combination of K (red) and Cl (green)] filters, showing formation of NaCl, K<sub>2</sub>SO<sub>4</sub>, and KCl coatings, respectively. Top: plain view, bottom: cross-sectional view. (<bold>b</bold>) Filtration efficiency of Bare, NaCl<sub>600</sub>, K<sub>2</sub>SO<sub>4</sub> <sub>600</sub>, and KCl<sub>600</sub> with 1, 3 and 5 stacked layers with no air flow [<italic>n</italic> = 7–20, mean ± standard deviation (SD)]. (<bold>c</bold>) Filtration efficiency of Bare × 1, and NaCl × 1, K<sub>2</sub>SO<sub>4</sub> × 1, and KCl × 1 coated with 3, 6 and 7 mg salt/cm<sup>2</sup> with no air flow (<italic>n</italic> = 7–15, mean ± SD). (<bold>d</bold>) Filtration efficiency of Bare, NaCl<sub>600</sub>, K<sub>2</sub>SO<sub>4</sub> <sub>600</sub>, and KCl<sub>600</sub> with 1, 3 and 5 stacked layers at an air flow rate of 15 Lpm (<italic>n</italic> = 10, mean ± SD). (<bold>e</bold>) Pressure drop of Bare × 3, and NaCl × 3, K<sub>2</sub>SO<sub>4</sub> × 3, and KCl × 3 coated with different amount of salt (<italic>n</italic> = 26–45, mean ± SD). No star: ns. (<bold>f</bold>) Pressure drop of Bare, NaCl<sub>600</sub>, K<sub>2</sub>SO<sub>4</sub> <sub>600</sub>, and KCl<sub>600</sub> with 1, 3 and 5 stacked layers (<italic>n</italic> = 27–65, mean ± SD). No star: ns. Dotted line: average pressure drop of commercial surgical mask. For all panels: ns: <italic>P</italic> > 0.05; *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01; ***<italic>P</italic> < 0.001; ****<italic>P</italic> < 0.0001, by ANOVA.</p></caption><graphic xlink:href=\"41598_2020_70623_Fig1_HTML\" id=\"MO1\"/></fig></p></sec><sec id=\"Sec4\"><title>Filtration efficiency against bacteria aerosols</title><p id=\"Par8\">The bacteria filtration performance of the salt-coated filters was probed against <italic>K. pneumoniae</italic> aerosols at 0 and 15 Lpm air flow rates. Overall, at 0 Lpm air flow, salt-coated filters showed significantly higher filtration efficiency than bare membranes at all conditions of salt type (NaCl, K<sub>2</sub>SO<sub>4</sub> and KCl), thickness (1, 3 or 5 layers), and salt amount (3, 6 or 7 mg/cm<sup>2</sup>) (two-way ANOVA, <italic>P</italic> values on graphs) (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b,c). In particular, the NaCl, K<sub>2</sub>SO<sub>4</sub> and KCl filters exhibited an increase in filtration efficiency from 50 to 79%, 98% and 81%, respectively, as compared to bare membranes (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b). Even when the <italic>K. pneumoniae</italic> aerosols were passed through the filters at 15 Lpm, NaCl and KCl salt filters still captured significantly more bacteria than bare membranes; 5-layered K<sub>2</sub>SO<sub>4</sub> filters showed a significant increase in filtration efficiency compared to bare membranes as well (two-way ANOVA, <italic>P</italic> values on graphs) (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>d). Interestingly, with the sole exception of K<sub>2</sub>SO<sub>4</sub> filters tested at 15 Lpm air flow, the filtration efficiency of the salt-coated filters did not depend on the filter thickness (two-way ANOVA, <italic>P</italic> > 0.05) (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b,d). This led us to hypothesize that the top layer (first layer to interact with the aerosols) is mainly responsible for the increased filtration efficiency observed in salt-coated filters. Considering the relatively large size of pores against aerosols, the collection of aerosols possibly occurs via the absorption mechanisms of coated dry-salt agents (mainly those applied on the top layer), as opposed to the conventional filtration mechanism where the collection efficiency is governed by the fine mesh size and the number of added layers.</p></sec><sec id=\"Sec5\"><title>Pressure drop across the salt-coated filters</title><p id=\"Par9\">As traditional technologies that enhance capture of pathogens/particles negatively affect the breathability of the filters, the pressure drop of the salt-coated filters at an air flow rate of 8 Lpm was measured next. Pressure drop levels evaluate the perceived resistance breathing through the filters. Notably, the pressure drop across NaCl, K<sub>2</sub>SO<sub>4</sub> and KCl filters did not significantly change as compared to bare membranes, irrespective of the amount of coated salt (one-way ANOVA, <italic>P</italic> > 0.05) (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>e) and the thickness of the filters (two-way ANOVA, <italic>P</italic> > 0.05) (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>f). Since the pore size of the fiber mesh remains sufficiently large after the salt coating treatment (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>e and Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>), the above results further suggest that the proposed filtration mechanism is non-mechanical in nature, ensuring maximum filtration efficiency with high breathability.</p><p id=\"Par10\">To represent the overall filter performance, the quality factors (QF) of the salt-coated filters were compared with those of the bare membranes at 15 Lpm. The QF values are reported in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref> and represent the ratio between the filtration efficiency and the pressure drop of filters<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. The QF of NaCl, K<sub>2</sub>SO<sub>4</sub> and KCl filters increased by 107%, 26% and 103%, respectively, compared with bare membranes at 1-layer thickness, 16%, 23% and 57% for 3 layers and 23%, 66% and 51% for 5 layers. The increase in QF of the salt-functionalized filters compared to bare membranes indicates an overall enhancement in performance. It is also important to note that, in theory, the QF is independent of the filter thickness. However, NaCl and KCl filters show differences in QF at different thicknesses. This is related to the fact that the top layer constitutes the main capture medium responsible for increased filtration efficiency of the salt-coated filters compared to bare membranes, as mentioned. Overall, these performance test results indicate that functionalizing large-pore membranes with salts leads to a substantial increase of the amount of captured bacteria, without increasing the resistance to air flow, yielding improved filter quality.</p></sec><sec id=\"Sec6\"><title>Inactivation of <italic>K. pneumoniae</italic> on the salt-coated filters and protective efficacy in vivo</title><p id=\"Par11\">The pathogen inactivation on NaCl, K<sub>2</sub>SO<sub>4</sub> and KCl filters was investigated by exposing them to <italic>K. pneumoniae</italic> bacterial aerosols. Time-dependent inactivation of bacteria incubated on filters coated with all salt types was observed (General Linear Model, <italic>P</italic> < 0.001) (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>a). In particular, the KCl-coated filters exhibited significant colony forming units (CFU) reduction compared to bare membranes even within 5 min from aerosol exposure (two-way ANOVA, <italic>P</italic> ≤ 0.0001). As shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>b and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>a, in contrast to intact bacteria in the control and on bare membranes, bacteria recovered from the salt-coated filters were found to be severely damaged due to the salt growth during the evaporation process. This explains the destabilization of the bacteria measured by CFU. Furthermore, the effect of different amounts of NaCl coated on the filters on the stability of <italic>K. pneumoniae</italic> aerosols was investigated. NaCl-coated filters showed rapid time-dependent bacteria inactivation (General Linear Model, <italic>P</italic> < 0.001) (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>c). Notably, NaCl × 3<sub>1200</sub> caused a 4 log CFU reduction within 30 min from aerosol exposure. The TEM analysis revealed the rapture, structural damage and morphological changes incurred by the bacteria due to the salt recrystallization (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>b). Similarly, when <italic>K. pneumoniae</italic> aerosols were exposed to the K<sub>2</sub>SO<sub>4</sub> and KCl filters prepared to contain a lower amount of salt (K<sub>2</sub>SO<sub>4</sub> × 3<sub>0</sub> and KCl × 3<sub>0</sub>), quick time-dependent CFU reduction was still measured (General Linear Model, <italic>P</italic> < 0.001) (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>a,b)<sub>.</sub> The bacteria inactivation on K<sub>2</sub>SO<sub>4</sub> × 3<sub>0</sub> and KCl × 3<sub>0</sub> was also confirmed in the TEM analysis (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>c). In general, it is worth noting that the actual infectious aerosols will have significantly lower levels of bacterial concentration than the exposure aerosols used in the tests; as such, it is expected that further pathogen inactivation will be observed in real-life scenarios. Overall, the bacteria stability results indicate that the NaCl, K<sub>2</sub>SO<sub>4</sub> and KCl coatings rapidly neutralize bacteria by physical damage induced during the salt recrystallization process.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Pathogen inactivation on salt-coated filters due to salt recrystallization. (<bold>a</bold>) CFU change showing the effect of incubation time on <italic>K. pneumoniae</italic> exposed to bare membranes and NaCl, K<sub>2</sub>SO<sub>4</sub>, and KCl filters (<italic>n</italic> = 5–38, mean ± SD). (<bold>b</bold>) TEM images of <italic>K. pneumoniae</italic> incubated on Bare × 3, NaCl × 3<sub>600</sub>, K<sub>2</sub>SO<sub>4</sub> × 3<sub>600</sub>, and KCl × 3<sub>600</sub> for 5 and 30 min (top), and of <italic>K. pneumoniae</italic> suspension as control (bottom). (<bold>c</bold>) CFU change showing the effect of incubation time on <italic>K. pneumoniae</italic> exposed to NaCl filters coated with different amount of salt (<italic>n</italic> = 5–38, mean ± SD). (<bold>d</bold>–<bold>g</bold>) Mice body weight change after infection with bacteria incubated on bare membranes, and NaCl × 3<sub>600</sub> (<bold>d</bold>), K<sub>2</sub>SO<sub>4</sub> × 3<sub>600</sub> (<bold>e</bold>), and KCl × 3<sub>600</sub> (<bold>f</bold>) for 5 and 30 min (<italic>n</italic> = 3–8, mean ± SD), and OD<sub>600</sub> of lungs (<italic>n</italic> = 3–8, mean ± SD; §: below detection limit) (<bold>g</bold>). **<italic>P</italic> < 0.01; ****<italic>P</italic> < 0.0001, by one-way ANOVA. (<bold>h–k</bold>) Mice body weight change after infection with bacteria incubated on bare membranes, NaCl × 3<sub>0</sub>, and K<sub>2</sub>SO<sub>4</sub> × 3<sub>0</sub> for 5 min (<bold>h</bold>), 15 min (<bold>i</bold>), and 30 min (<bold>j</bold>) (<italic>n</italic> = 4–8, mean ± SD), and OD<sub>600</sub> of lungs (<italic>n</italic> = 3–4, mean ± SD; §: below detection limit) (<bold>k</bold>).</p></caption><graphic xlink:href=\"41598_2020_70623_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par12\">The bacteria inactivation findings were confirmed in vivo. Mice were infected with <italic>K. pneumoniae</italic> recovered from NaCl × 3<sub>600</sub>, K<sub>2</sub>SO<sub>4</sub> × 3<sub>600</sub> and KCl × 3<sub>600</sub> at 5 and 30 min incubation. At 30-min incubation, the bare membrane group reached a body weight loss that was significantly higher than that of NaCl, K<sub>2</sub>SO<sub>4</sub> and KCl filter groups by 10% (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>d–f) (two-way ANOVA, <italic>P</italic> ≤ 0.05). As shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>g, OD<sub>600</sub> measured from the lungs of salt filter group mice were significantly lower than those of the control groups (bare membranes) (one-way ANOVA, <italic>P</italic> values on graph). Furthermore, the OD<sub>600</sub> levels decreased with incubation time (General Linear Model, <italic>P</italic> < 0.001). After 2–4 days, all salt filter mice groups showed a significant rapid recovery in body weight as opposed to the bacteria aerosol control group (two-way ANOVA, <italic>P</italic> ≤ 0.05), unlike bare membrane groups (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>d–f). Additionally, the in vivo response to <italic>K. pneumoniae</italic> was investigated after incubation on the NaCl × 3<sub>0</sub> and K<sub>2</sub>SO<sub>4</sub> × 3<sub>0</sub> for 5, 15 and 30 min. At day 2 post-infection, the salt filter groups exhibited a significantly lower body weight loss than the bacteria aerosol control group (two-way ANOVA, <italic>P</italic> ≤ 0.05), unlike the bare membrane group (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>h–j). The lung OD<sub>600</sub> measurements further supported these results by showing significantly lower levels in NaCl × 3<sub>0</sub> and K<sub>2</sub>SO<sub>4</sub> × 3<sub>0</sub> mice groups than bare membrane groups (one-way ANOVA, <italic>P</italic> ≤ 0.0001) (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>k). In general, a decrease in lung OD<sub>600</sub> was also observed with the increase in incubation time on the filters (General Linear Model, <italic>P</italic> < 0.001). Thus, these studies support the in vitro results, indicating that protection of the mice was obtained by the NaCl, K<sub>2</sub>SO<sub>4</sub> and KCl salt coatings due to inactivation of the aerosolized bacteria.</p></sec><sec id=\"Sec7\"><title>Strain-independent inactivation of bacteria on salt-coated filters</title><p id=\"Par13\">Strain-nonspecific bacteria inactivation on NaCl, K<sub>2</sub>SO<sub>4</sub> and KCl coated filters was tested by exposure to four further bacteria strains (MRSA<italic>, P. aeruginosa, S. pyogenes</italic> and <italic>E. coli</italic>). Similar to <italic>K. pneumoniae</italic>, the salt filters exhibited inactivation capabilities irrespective of pathogen strain (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a–d). In particular, in the case of <italic>E. coli, P. aeruginosa</italic> and <italic>S. pyogenes,</italic> the bacteria showed significant decrease in CFU on all salt filters compared to bare membranes, even within 5 min (General Linear Model, <italic>P</italic> < 0.001 for <italic>E. coli</italic>, <italic>P</italic> < 0.001 for <italic>P. aeruginosa</italic>, <italic>P</italic> < 0.05 for <italic>S. pyogenes</italic>) (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a–c). NaCl-coated filters showed effective time-dependent MRSA inactivation (General Linear Model, <italic>P</italic> < 0.001) (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>d). Therefore, these data indicate that, due to exploitation of a physical mechanism to inactivate the pathogens (i.e., evaporation-induced salt recrystallization), the salt-coated filters offer a universal infection prevention unit.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Strain-nonspecific protective efficacy and environmental stability. (<bold>a</bold>–<bold>d</bold>) CFU change showing the effect of incubation time on <italic>E. coli</italic> (<bold>a</bold>), <italic>S. pyogenes</italic> (<bold>b</bold>), <italic>P. aeruginosa</italic> (<bold>c</bold>), and MRSA (<bold>d</bold>) exposed to bare membranes and NaCl, K<sub>2</sub>SO<sub>4</sub>, and KCl filters (<italic>n</italic> = 4–19 for (<bold>a</bold>), <italic>n</italic> = 3–19 for (<bold>b</bold>), <italic>n</italic> = 3–25 for (<bold>c</bold>), <italic>n</italic> = 2–10 for (<bold>d</bold>), mean ± SD). (<bold>e</bold>) SEM images of NaCl × 3<sub>600</sub>, K<sub>2</sub>SO<sub>4</sub> × 3<sub>600</sub>, and KCl × 3<sub>600</sub> at ambient condition (top), and following 5-days storage at 37 °C and 70%, 80% and 90% RH. (<bold>f</bold>) CFU change showing the effect of temperature and humidity (5-days storage) on bare membranes and NaCl, K<sub>2</sub>SO<sub>4</sub>, and KCl filters in inactivating <italic>K. pneumoniae</italic> after 30-min incubation (<italic>n</italic> = 3–10, mean ± SD). §: below detection limit. ns: <italic>P</italic> > 0.05; *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01; ****<italic>P</italic> < 0.0001, by <italic>t</italic> test.</p></caption><graphic xlink:href=\"41598_2020_70623_Fig3_HTML\" id=\"MO3\"/></fig></p></sec><sec id=\"Sec8\"><title>Exposure of salt coatings to harsh environmental conditions</title><p id=\"Par14\">Next, the environmental stability of the salt-functionalized filters was investigated. The filters were stored at 37 °C and 70%, 80% and 90% RH for 5 days. The SEM analysis showed that all salt types remained coated on the surface of the filter fibers (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>e). Additionally, the salt coatings showed morphological changes and increased roughness due to recrystallization at the conditions above the respective critical RH (i.e., at 80% and 90% RH for NaCl, and at 90% RH for KCl). The salt-functionalized filters retained their ability to inactivate <italic>K. pneumoniae</italic> (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>f). Interestingly, the salt-coated filters showed a significant increase in inactivation properties after exposure to the humid environment, and no bacteria were detected from the filters after storage at 90% RH (<italic>t</italic> test, <italic>P</italic> values on graph). This phenomenon is due to the morphological change of the salt coatings after storage, leading to increased surface roughness which affects viability of bacteria<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Additionally, bare membranes showed the opposite trend, where the bacteria recovered from filters stored at higher RH levels yielded significantly higher CFU counts as compared to the ambient condition (<italic>t</italic> test, <italic>P</italic> values on graph). This is due to remaining humidity trapped in the membranes, protecting the bacteria. Altogether, these findings indicate that prolonged exposure of the salt-coated filters to harsh environmental conditions does not compromise the stability of the coating, while inducing further antimicrobial activity. Notably, two important conclusions come from these findings: (i) the salt-based coatings can be safely used at different environmental conditions of use and storage, and (ii) the salt-coated filters will remain highly effective against pathogens after exposure to the humidity levels generated while breathing, ensuring reusability of the filters.</p></sec></sec><sec id=\"Sec9\"><title>Conclusion</title><p id=\"Par15\">In summary, we developed an antimicrobial respiratory protective system with high filtering performance and improved breathability levels compared to regular masks, based on the salt functionalization technology of large-pore membranes. The filters coated with different salt types are able to capture more pathogens than the bare membranes, converting a non-functional system into an active respiratory protection unit. Since the infectious aerosols tested in this work have a small particle size (2.5–4 µm), high filtration efficiency can also be expected against bigger particles responsible for disease transmission through large droplets. Additionally, commercial mask filters are produced by melt-blowing; by converting the more inexpensive spunbond large-pore PP membranes into functional filters by salt coating and controlling the final mask design, the production cost of masks will be reduced. Simultaneously, the enhancement in filtration efficiency does not cause any decrease in the breathability of the large-pore membranes, which is not achievable by traditional technologies. Due to the natural salt recrystallization process physically neutralizing both Gram-positive and Gram-negative bacteria within a short time, the salt-coated filters offer broad-spectrum protection. This results in no risk of cross infection, safe reusability of the device without further processing, reduced amounts of biohazardous waste and no risk of shortage of respirators during outbreaks. Additionally, the salt coatings remained stable on the filters following prolonged exposure to high temperature and humidity. This offers complete protection at varying environmental conditions of use or storage as well as ensures the recyclability of the filters. Overall, the results indicate that our technology can be used to fabricate respiratory protective devices with high filtration and breathability performance, which achieve strain-nonspecific protective efficacy. Notably, the simple and cost-effective functionalization process with salt addresses all major technical challenges of respiratory protection devices in a single system and could be readily applied to other devices that are already in use, such as filters in buildings and hospitals. This comprehensive technology could lead to an enhanced and timely response to epidemics and pandemics, such as the case of COVID-19, and provides a safe and effective solution to prevent diseases globally.</p></sec><sec id=\"Sec10\"><title>Methods</title><sec id=\"Sec11\"><title>Filters preparation</title><p id=\"Par16\">Large-pore membranes were obtained from three-ply surgical masks (Fisherbrand Facemasks; Fisher Scientific, Pittsburgh, PA, USA). The middle membrane (active filtration unit) and outer protective membrane were discarded; the innermost PP membrane (typically used against the wearer’s face for mechanical protection of the middle filter; ~ 25 g/m<sup>2</sup>) was used to produce the salt filter samples. As the innermost mask membrane has higher porosity than the middle filter, it was selected for the study; by stacking different numbers of layers, the performance of the salt filter samples was controlled by varying the thickness. Circular samples (3-cm radius) of the mask innermost large-pore membrane were cut (bare membranes, labelled Bare × # where # is the number of stacked layers). The membranes were coated with different salt types to obtain the salt-functionalized filters: sodium chloride (NaCl; Sigma Aldrich, St. Louis, MO, USA), potassium sulfate (K<sub>2</sub>SO<sub>4</sub>; Sigma Aldrich), and potassium chloride (KCl; Sigma Aldrich). To prepare the coating solutions, the salts were dissolved in filtered (0.22 µm pore size; Corning, Tewksbury, MA, USA) DI water under stirring at 400 rpm and 90 °C for NaCl, and 400 rpm and room temperature (RT) for K<sub>2</sub>SO<sub>4</sub> and KCl (final salt concentrations: 29.03 w/v%, 9.72 w/v% and 26.31 w/v% for NaCl, K<sub>2</sub>SO<sub>4</sub> and KCl, respectively). Surfactant (Tween 20, Fisher Scientific) was added at 1 v/v%. The salt filters were prepared by completely pre-wetting the membrane samples with ~ 350 μL of a given coating solution (pre-wet membranes). The amount of coated salt was controlled by varying the volume of coating solution (0, 300, 600, or 1,200 µL) in which the pre-wet membranes were deposited in 60 × 15 mm petri dishes; more layers of pre-wet membrane samples were added on top based on the desired total number of layers (1, 3 or 5), and any air bubbles were carefully removed. The salt filter samples were dried overnight in an incubator (Thermolyne 42000; Thermolyne, Dubuque, IA, USA) at 45 °C. The salt-coated filter samples were labelled Salt × #<sub>vol</sub>, where Salt is the salt type (NaCl, K<sub>2</sub>SO<sub>4</sub>, or KCl), # the number of stacked layers (1, 3 or 5), and <sub>vol</sub> the volume of coating solution in which the filters were dried (0, 300, 600, or 1,200 µL).</p></sec><sec id=\"Sec12\"><title>Bacteria cultures</title><p id=\"Par17\"><italic>Klebsiella pneumoniae</italic> (ATCC BAA-1705), methicillin-resistant <italic>Staphylococcus aureus</italic> (ATCC 33593), <italic>Escherichia coli</italic> (ATCC 25922), <italic>Pseudomonas aeruginosa</italic> (ATCC 10145), and <italic>Streptococcus pyogenes</italic> (ATCC 19615) were streaked and grown in appropriate agars and growth media, respectively. The bacteria cultures were grown following standard practice. The detailed procedures are in the <xref rid=\"MOESM1\" ref-type=\"media\">Supplementary Methods</xref>. The cultures were washed 3 times in phosphate buffered saline (PBS) before experiments.</p></sec><sec id=\"Sec13\"><title>Filtration efficiency tests</title><p id=\"Par18\">Filtration efficiency tests were designed by adapting the ASTM F2101-14/19 standard tests<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup> recommended by the Food and Drug Administration (FDA)<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. The detailed operation of the filtration efficiency test apparatus is in the <xref rid=\"MOESM1\" ref-type=\"media\">Supplementary Methods</xref>. Briefly, the filter samples (4.9 cm<sup>2</sup> exposed area) were exposed to 60 µL of aerosolized (diameter = 2.5–4 μm) <italic>K. pneumoniae</italic> DI water suspension (OD<sub>600</sub> = 10), under an air flow rate of 0 or 15 Lpm.</p><p id=\"Par19\">The bacteria were reconstituted from the filter samples as follows. The filter samples were incubated in PBS for 30 s–1 min. After vortexing, the filters were centrifuged (6,000 rpm, RT, 1 min) in a new tube to collect any remaining bacteria. The recovered bacteria were centrifuged (14,000 rpm, 15 min, 4 °C) to discard any salt/surfactant dissolved from the filters, and then resuspended in 1.2 mL of fresh PBS, eliminating any interference with assays.</p><p id=\"Par20\">The total amount of bacteria contained in the exposure aerosols was determined by aerosolizing 60 µL of <italic>K. pneumoniae</italic> suspension into a 15-mL tube containing 1.5 mL of PBS for 30 s, followed by 1-min aerosolization of DI water to avoid drying of the bacteria condensed against the tube wall. After vortexing, the bacteria were centrifuged (14,000 rpm, 15 min, 4 °C) and resuspended in 1.2 mL of fresh PBS.</p><p id=\"Par21\">The filtration efficiency was calculated as the ratio of the amount of bacteria recovered from the filter sample to the total amount of bacteria contained in the exposure aerosols. The amount of bacteria was determined as the protein concentration measured with bicinchoninic acid assay (Micro BCA protein assay kit; Thermo Fischer Scientific, Waltham, IL, USA), with bovine serum albumin (BSA) standard.</p></sec><sec id=\"Sec14\"><title>Pressure drop tests</title><p id=\"Par22\">Pressure drop tests were designed by adapting the MIL-M-36945C standard test<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>, recommended by the FDA<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. The detailed operation of the pressure drop test apparatus is in the <xref rid=\"MOESM1\" ref-type=\"media\">Supplementary Methods</xref>. Differential pressure measurements were conducted at an air flow rate of 8 Lpm (breathing condition<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>) for 15 s, with (P<sub>1</sub>) and without (P<sub>0</sub>) loaded filter samples. The final pressure drop was calculated as ΔP = (P<sub>1</sub> – P<sub>0</sub>)/A, where A is the area of filter sample exposed to the air flow (6.6 cm<sup>2</sup>). The quality factors (QF) of the bare membranes and salt-coated filters were calculated at 15 Lpm (QF = – ln(1 – F))/(P<sub>1</sub> – P<sub>0</sub>), where F is the fraction of captured bacteria<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>); notably, filtration efficiency and pressure drop tests were conducted separately.</p></sec><sec id=\"Sec15\"><title>Test of bacteria stability change on filters in vitro</title><p id=\"Par23\">Aerosols of multiple bacteria were exposed to the bare membranes (3 layers) and salt-functionalized filters (3 layers; NaCl, K<sub>2</sub>SO<sub>4</sub>, KCl). After washing, the bacteria were resuspended in DI water to an OD<sub>600</sub> of 12.5 (<italic>K. pneumoniae, P. aeruginosa</italic> and <italic>E. coli</italic>) or 100 (MRSA and <italic>S. pyogenes</italic>). Highly concentrated bacteria stocks were used to ensure that the bacteria recovered from the filters were detectable, due to the low filtration efficiency of the bare membranes and the inactivation of the bacteria occurring on the salt filters. For reference, the <italic>K. pneumoniae</italic> stock used is 5 × 10<sup>8</sup> CFU/mL, as opposed to the 3 × 10<sup>0</sup>–5 × 10<sup>1</sup> CFU/mL of bacteria detected in hospitals (assuming 50% RH indoors)<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>.</p><p id=\"Par24\">The nebulizer unit was placed on top of the filter samples (radius = 1.2 cm), which were loaded on a porous support, and 20 μL of bacteria suspension were aerosolized (30 s). After 3, 5, 15 or 30 min incubation on the filter samples, the bacteria were reconstituted as described above. After removing the supernatant containing salt/surfactant, the bacteria were resuspended in 100 μL of PBS. The amount of bacteria contained in the exposure aerosols (0 min incubation on the filters) was determined by aerosolizing 40 µL of bacteria suspension into a 15-mL tube, similar to above; the bacteria were resuspended in 100 μL of PBS. CFU measurements were obtained by incubating 5 μL of 10 × dilutions onto MH II agar plates (<italic>K. pneumoniae,</italic> MRSA and <italic>E. coli</italic>), TSA plates (<italic>P. aeruginosa</italic>) or BHI agar plates (<italic>S. pyogenes</italic>) at 37 °C overnight. CFU were divided by the amount of bacteria recovered from the filters (determined as protein concentration). The measurements were expressed relative to the CFU at 0 min incubation on the filters (Relative CFU = CFU<sub>sample</sub>/CFU<sub>0 min</sub>).</p></sec><sec id=\"Sec16\"><title>Test of bacteria stability change on filters in vivo</title><p id=\"Par25\">20 μL of <italic>K. pneumoniae</italic> DI water suspension (OD<sub>600</sub> = 10.5) were aerosolized on the filter samples and incubated for 5, 15 and 30 min. A lethal dose of bacteria reconstituted from the filters was used to infect 8 7-week old BALB/c mice (KOATECH, Pyeongtaek, Republic of Korea) per group by the intranasal route. As negative controls, two mice groups were infected with the bacteria before and after aerosolization, respectively. The body weight change was measured daily for 10 days. The mice were euthanized if the body weight reached below 75% of the starting weight. Kyung Hee University (KHU) Institutional Animal Care and Use Committee (IACUC), which operates under National Veterinary Research and Quarantine Service (NVRQS) and animal welfare law and regulations of the WOAH-OIE (World organization for animal health), provided approval for all animal protocols (KHUASP(SE)-18-085). The approved protocols and guidelines of KHU IACUC were followed for all animal experiments and husbandry related to this study. At day 3 post-infection, 4 mice per group were sacrificed to collect the lung tissues. The lung supernatants from the homogenizing process were used to measure OD<sub>600</sub> (NanoDrop One C; Thermo Fisher Scientific).</p></sec><sec id=\"Sec17\"><title>Performance of filters at different environmental conditions</title><p id=\"Par26\">Bare membranes (Bare × 3) and salt-functionalized filters (NaCl × 3<sub>600</sub>, K<sub>2</sub>SO<sub>4</sub> × 3<sub>600</sub>, KCl × 3<sub>600</sub>) were stored in an environmental chamber (Memmert HPP260; Memmert, Buchenbach, Germany) at 37 °C, and 70%, 80% and 90% RH. The bacteria stability change on filters in vitro was tested against <italic>K. pneumoniae</italic> aerosols at day 5 of storage, by measuring the CFU after 30 min incubation of the bacteria on the filters, as described above.</p></sec><sec id=\"Sec18\"><title>Electron microscopy analysis</title><p id=\"Par27\">SEM analysis (secondary electron mode at 20 kV, Hitachi S-3000N; Hitachi, Toronto, Canada) was performed on the salt filters (NaCl, K<sub>2</sub>SO<sub>4</sub>, KCl) coated with a gold layer (thickness = 10 nm), and an EDX detector (Oxford Instruments, Concord, MA, USA) was used for the EDX analysis. The pore size of bare membranes and salt filters was determined from SEM images.</p><p id=\"Par28\">TEM analysis (200 kV, JEOL JEM 2100; JEOL, Peabody, MA, USA) was performed on <italic>K. pneumoniae</italic> reconstituted after incubation on the bare membranes and salt-functionalized filters, as described above. The bacteria were deposited on copper grids (Electron Microscopy Sciences, Hatfield, PA, USA) and negatively stained with tungsten using a solution of 1.5 w/v% phosphotungstic acid hydrate (pH = 7.0) (Sigma Aldrich).</p></sec><sec id=\"Sec19\"><title>Statistical analysis</title><p id=\"Par29\">Pearson correlation coefficients basis of correlations was conducted between levels of salt weight and volume. The statistical analysis was performed by using one-way analysis of variance (ANOVA), two-way ANOVA, General Linear Model, <italic>t</italic> test, and chi-square analysis for multiple comparisons. SPSS ver. 21 (IBM, Armonk, NY, USA) and Minitab (Minitab, State College, PA) were used. The significance of multiple comparisons was considered by <italic>P</italic> value; <italic>P</italic> value of less than 0.05 was considered significant.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec20\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70623_MOESM1_ESM.pdf\"><caption><p>Supplementary Information.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70623-9.</p></sec><ack><title>Acknowledgements</title><p>The authors wish to thank Mr. Ankit Kumar at the University of Alberta for technical assistance on the SEM/EDX, and Ms. Sally Fung, Ms. Iryna Roever and Ms. Miyoung Park at the University of Alberta for assistance in the sample preparation. This research was financially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant, University of Alberta Faculty of Engineering, and Mitacs Globalink Research Award.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>H.J.C. conceived the experiments. H.J.C. and I.R. designed the experiments. I.R., E.O., S.H., S.Kaleem, A.H., S.H.L., H.J.K., D.H.L., K.B.C., S.Kumaran, S.A., R.L., S.C., F.S.Q., B.H.J., and H.J.C. performed the experiments. I.R., E.O., S.H., S.H.L., H.J.K., D.H.L., K.B.C., C.I.K, F.S.Q., B.H.J., and H.J.C. analyzed the data. I.R. and H.J.C. wrote the manuscript. E.O., S.H., S.Kaleem, A.H., S.H.L., H.J.K., D.H.L., K.B.C., S.Kumaran, S.A., R.L., S.C., C.I.K., F.S.Q., and B.H.J. edited the manuscript.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>All data generated or analysed during this study are included in this published article (and its Supplementary Information files).</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par30\">The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: H.J.C is an inventor of the salt-coated pathogen deactivation technology and the patent application has been filed.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Hinds</surname><given-names>WC</given-names></name></person-group><source>Aerosol Technology: Properties, Behavior, and Measurement of Airborne Particles</source><year>1999</year><publisher-loc>New York</publisher-loc><publisher-name>Wiley</publisher-name></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weber</surname><given-names>TP</given-names></name><name><surname>Stilianakis</surname><given-names>NI</given-names></name></person-group><article-title>Inactivation of influenza A viruses in the environment and modes of transmission: a critical review</article-title><source>J. Infect.</source><year>2008</year><volume>57</volume><fpage>361</fpage><lpage>373</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jinf.2008.08.013</pub-id><pub-id pub-id-type=\"pmid\">18848358</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><mixed-citation publication-type=\"other\">World Health Organization. <italic>Urgent Health Challenges for the Next Decade</italic>. <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.who.int/news-room/photo-story/photo-story-detail/urgent-health-challenges-for-the-next-decade\">https://www.who.int/news-room/photo-story/photo-story-detail/urgent-health-challenges-for-the-next-decade</ext-link> (2020).</mixed-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xia</surname><given-names>J</given-names></name><name><surname>Gao</surname><given-names>J</given-names></name><name><surname>Tang</surname><given-names>W</given-names></name></person-group><article-title>Nosocomial infection and its molecular mechanisms of antibiotic resistance</article-title><source>Biosci. Trends</source><year>2016</year><volume>10</volume><fpage>14</fpage><lpage>21</lpage><pub-id pub-id-type=\"doi\">10.5582/bst.2016.01020</pub-id><pub-id pub-id-type=\"pmid\">26877142</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schaal</surname><given-names>KP</given-names></name></person-group><article-title>Medical and microbiological problems arising from airborne infection in hospitals</article-title><source>J. Hosp. Infect.</source><year>1991</year><volume>18</volume><fpage>451</fpage><lpage>459</lpage><pub-id pub-id-type=\"doi\">10.1016/0195-6701(91)90056-E</pub-id><pub-id pub-id-type=\"pmid\">1679815</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kelsen</surname><given-names>SG</given-names></name><name><surname>McGuckin</surname><given-names>M</given-names></name></person-group><article-title>The role of airborne bacteria in the contamination of fine particle nebulizers and the development of nosocomial pneumonia</article-title><source>Ann. N. Y. Acad. Sci.</source><year>1980</year><volume>353</volume><fpage>218</fpage><lpage>229</lpage><pub-id pub-id-type=\"doi\">10.1111/j.1749-6632.1980.tb18925.x</pub-id><pub-id pub-id-type=\"pmid\">6939387</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kibbler</surname><given-names>CC</given-names></name><name><surname>Quick</surname><given-names>A</given-names></name><name><surname>O'Neill</surname><given-names>AM</given-names></name></person-group><article-title>The effect of increased bed numbers on MRSA transmission in acute medical wards</article-title><source>J. Hosp. Infect.</source><year>1998</year><volume>39</volume><fpage>213</fpage><lpage>219</lpage><pub-id pub-id-type=\"doi\">10.1016/S0195-6701(98)90260-2</pub-id><pub-id pub-id-type=\"pmid\">9699141</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grieble</surname><given-names>HG</given-names></name><name><surname>Bird</surname><given-names>TJ</given-names></name><name><surname>Nidea</surname><given-names>HM</given-names></name><name><surname>Miller</surname><given-names>CA</given-names></name></person-group><article-title>Chute-hydropulping waste disposal system: a reservoir of enteric bacilli and pseudomonas in a modern hospital</article-title><source>J. Infect. Dis.</source><year>1974</year><volume>130</volume><fpage>602</fpage><lpage>607</lpage><pub-id pub-id-type=\"doi\">10.1093/infdis/130.6.602</pub-id><pub-id pub-id-type=\"pmid\">4214881</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Beggs</surname><given-names>CB</given-names></name></person-group><article-title>The airborne transmission of infection in hospital buildings: fact or fiction?</article-title><source>Indoor Built Environ.</source><year>2003</year><volume>12</volume><fpage>9</fpage><lpage>18</lpage><pub-id pub-id-type=\"doi\">10.1177/1420326X03012001002</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Vani</surname><given-names>WH</given-names></name></person-group><source>Antimicrobial Resistance: Global Report on Surveillance</source><year>2014</year><publisher-loc>Geneva</publisher-loc><publisher-name>World Health Organization</publisher-name></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shiomori</surname><given-names>T</given-names></name><name><surname>Miyamoto</surname><given-names>H</given-names></name><name><surname>Makishima</surname><given-names>K</given-names></name></person-group><article-title>Significance of airborne transmission of methicillin-resistant <italic>Staphylococcus aureus</italic> in an otolaryngology–head and neck surgery unit</article-title><source>Arch. Otolaryngol. Head Neck Surg.</source><year>2001</year><volume>127</volume><fpage>644</fpage><lpage>648</lpage><pub-id pub-id-type=\"doi\">10.1001/archotol.127.6.644</pub-id><pub-id pub-id-type=\"pmid\">11405862</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Seto</surname><given-names>WH</given-names></name><etal/></person-group><article-title>Effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (SARS)</article-title><source>Lancet</source><year>2003</year><volume>361</volume><fpage>1519</fpage><lpage>1520</lpage><pub-id pub-id-type=\"doi\">10.1016/S0140-6736(03)13168-6</pub-id><pub-id pub-id-type=\"pmid\">12737864</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Balazy</surname><given-names>A</given-names></name><etal/></person-group><article-title>Do N95 respirators provide 95% protection level against airborne viruses, and how adequate are surgical masks?</article-title><source>Am. J. Infect. Control</source><year>2006</year><volume>34</volume><fpage>51</fpage><lpage>57</lpage><pub-id pub-id-type=\"doi\">10.1016/j.ajic.2005.08.018</pub-id><pub-id pub-id-type=\"pmid\">16490606</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Loeb</surname><given-names>M</given-names></name><etal/></person-group><article-title>Surgical mask vs N95 respirator for preventing influenza among health care workers: a randomized trial</article-title><source>JAMA</source><year>2009</year><volume>302</volume><fpage>1865</fpage><lpage>1871</lpage><pub-id pub-id-type=\"doi\">10.1001/jama.2009.1466</pub-id><pub-id pub-id-type=\"pmid\">19797474</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bunyan</surname><given-names>D</given-names></name><name><surname>Ritchie</surname><given-names>L</given-names></name><name><surname>Jenkins</surname><given-names>D</given-names></name><name><surname>Coia</surname><given-names>JE</given-names></name></person-group><article-title>Respiratory and facial protection: a critical review of recent literature</article-title><source>J. Hosp. Infect.</source><year>2013</year><volume>85</volume><fpage>165</fpage><lpage>169</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jhin.2013.07.011</pub-id><pub-id pub-id-type=\"pmid\">24035256</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rubino</surname><given-names>I</given-names></name><name><surname>Choi</surname><given-names>HJ</given-names></name></person-group><article-title>Respiratory protection against pandemic and epidemic diseases</article-title><source>Trends Biotechnol.</source><year>2017</year><volume>35</volume><fpage>907</fpage><lpage>910</lpage><pub-id pub-id-type=\"doi\">10.1016/j.tibtech.2017.06.005</pub-id><pub-id pub-id-type=\"pmid\">28733078</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Urbaniak-Domagala</surname><given-names>W</given-names></name><name><surname>Wrzosek</surname><given-names>H</given-names></name><name><surname>Szymanowski</surname><given-names>H</given-names></name><name><surname>Majchrzycka</surname><given-names>K</given-names></name><name><surname>Brochocka</surname><given-names>A</given-names></name></person-group><article-title>Plasma modification of filter nonwovens used for the protection of respiratory tracts</article-title><source>Fibres Text. East. Eur.</source><year>2010</year><volume>18</volume><fpage>94</fpage><lpage>99</lpage></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tiliket</surname><given-names>G</given-names></name><etal/></person-group><article-title>A new material for airborne virus filtration</article-title><source>Chem. Eng. J.</source><year>2011</year><volume>173</volume><fpage>341</fpage><lpage>351</lpage><pub-id pub-id-type=\"doi\">10.1016/j.cej.2011.07.059</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quan</surname><given-names>FS</given-names></name><name><surname>Rubino</surname><given-names>I</given-names></name><name><surname>Lee</surname><given-names>SH</given-names></name><name><surname>Koch</surname><given-names>B</given-names></name><name><surname>Choi</surname><given-names>HJ</given-names></name></person-group><article-title>Universal and reusable virus deactivation system for respiratory protection</article-title><source>Sci. Rep.</source><year>2017</year><volume>7</volume><fpage>39956</fpage><pub-id pub-id-type=\"doi\">10.1038/srep39956</pub-id><pub-id pub-id-type=\"pmid\">28051158</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Adams</surname><given-names>JR</given-names></name><name><surname>Merz</surname><given-names>AR</given-names></name></person-group><article-title>Hygroscopicity of fertilizer materials and mixtures</article-title><source>Ind. Eng. Chem. Res.</source><year>1929</year><volume>21</volume><fpage>305</fpage><lpage>307</lpage><pub-id pub-id-type=\"doi\">10.1021/ie50232a003</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hasan</surname><given-names>J</given-names></name><etal/></person-group><article-title>Multi-scale surface topography to minimize adherence and viability of nosocomial drug-resistant bacteria</article-title><source>Mater. Des.</source><year>2018</year><volume>140</volume><fpage>332</fpage><lpage>344</lpage><pub-id pub-id-type=\"doi\">10.1016/j.matdes.2017.11.074</pub-id><pub-id pub-id-type=\"pmid\">29391661</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><mixed-citation publication-type=\"other\">ASTM International. <italic>ASTM F2101-14 Standard Test Method for Evaluating the Bacterial Filtration Efficiency </italic>(<italic>BFE</italic>)<italic> of Medical Face Mask Materials, Using a Biological Aerosol of Staphylococcus aureus</italic> (2014).</mixed-citation></ref><ref id=\"CR23\"><label>23.</label><mixed-citation publication-type=\"other\">ASTM International. <italic>ASTM F2101-19, Standard Test Method for Evaluating the Bacterial Filtration Efficiency </italic>(<italic>BFE</italic>)<italic> of Medical Face Mask Materials, Using a Biological Aerosol of Staphylococcus aureus</italic> (2019).</mixed-citation></ref><ref id=\"CR24\"><label>24.</label><mixed-citation publication-type=\"other\">FDA. <italic>Guidance for Industry and FDA Staff: Surgical Masks—Premarket Notification </italic>[<italic>510</italic>(<italic>k</italic>)] <italic>Submissions</italic> (2004).</mixed-citation></ref><ref id=\"CR25\"><label>25.</label><mixed-citation publication-type=\"other\">Military Specification. <italic>MIL-M-36945C 4.4.1.1.1, Method 1 Military Specifications: Surgical Mask, Disposable</italic> (1975).</mixed-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Cotes</surname><given-names>JE</given-names></name></person-group><source>Lung Function: Assessment and Application in Medicine</source><year>1965</year><publisher-loc>New York</publisher-loc><publisher-name>Blackwell Publishing</publisher-name></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luksamijarulkul</surname><given-names>P</given-names></name><name><surname>Aiempradit</surname><given-names>N</given-names></name><name><surname>Vatanasomboon</surname><given-names>P</given-names></name></person-group><article-title>Microbial contamination on used surgical masks among hospital personnel and microbial air quality in their working wards: a hospital in Bangkok</article-title><source>Oman Med. J.</source><year>2014</year><volume>29</volume><fpage>346</fpage><lpage>350</lpage><pub-id pub-id-type=\"doi\">10.5001/omj.2014.92</pub-id><pub-id pub-id-type=\"pmid\">25337311</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807915</article-id><article-id pub-id-type=\"pmc\">PMC7431536</article-id><article-id pub-id-type=\"publisher-id\">70895</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70895-1</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Amygdalin based G-6-P synthase inhibitors as novel preservatives for food and pharmaceutical products</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Lather</surname><given-names>Amit</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sharma</surname><given-names>Sunil</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-0856-3620</contrib-id><name><surname>Khatkar</surname><given-names>Anurag</given-names></name><address><email>dranuragkhatkarmdurtk@gmail.com</email></address><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.411524.7</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1790 2262</institution-id><institution>Research Scholar, Faculty of Pharmaceutical Sciences, </institution><institution>Maharshi Dayanand University, </institution></institution-wrap>Rohtak, Haryana India </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.411892.7</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0500 4297</institution-id><institution>Department of Pharmaceutical Sciences, </institution><institution>G.J.U.S.&T, </institution></institution-wrap>Hisar, India </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.411524.7</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1790 2262</institution-id><institution>Laboratory for Preservation Technology and Enzyme Inhibition Studies, Faculty of Pharmaceutical Sciences, </institution><institution>Maharshi Dayanand University, </institution></institution-wrap>Rohtak, Haryana India </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13903</elocation-id><history><date date-type=\"received\"><day>27</day><month>1</month><year>2020</year></date><date date-type=\"accepted\"><day>6</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">G-6-P synthase enzyme has been involved in the synthesis of the microbial cell wall, and its inhibition may lead to the antimicrobial effect. In the present study, we designed a library of amygdalin derivatives, and two most active derivatives selected on the basis of various parameters viz<italic>.</italic> dock score, binding energy, and ADMET data using molecular docking software (Schrodinger’s Maestro). The selected derivatives were synthesized and evaluated for their antioxidant and antimicrobial potential against several Gram (+ ve), Gram (−ve), as well as fungal strains. The results indicated that synthesized compounds exhibited good antioxidant, antimicrobial, and better preservative efficacy in food preparation as compared to the standard compounds. No significant differences were observed in different parameters as confirmed by Kruskal–Wallis test (p < 0.05). Docking results have been found in good correlation with experimental wet-lab data. Moreover, the mechanistic insight into the docking poses has also been explored by binding interactions of amygdalin derivative inside the dynamic site of G-6-P synthase.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Screening</kwd><kwd>Antimicrobial resistance</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Nowadays, several antimicrobial and antioxidant based preservatives such as p-hydroxybenzoates, parabens, benzalkonium chloride, dibromodicyanobutane, dimethyl dithiocarbamate, dimethoxy dimethyl hydantoin, formaldehyde, etc. are available in the market<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. However, the existing preservatives have been associated with severe side effects such as estrogenic effect, breast cancer, contact eczema, endocrine disruptors and many other type cancers, etc. Hence, the researchers have been compelled to search for new, better, and safe preservatives<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>–<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>.</p><p id=\"Par3\">To attain this, the researchers have focused on discovery of novel mechanisms and target sites in addition to the reported mechanisms responsible for antimicrobial action. The different target site for the action of antimicrobials includes inhibition of the cell membrane synthesis, metabolic pathways (folic acid synthesis inhibition), protein synthesis, leakage from cell membrane, and interference with DNA and RNA replication, etc. The microbial cell wall provides mechanical support to microbes and regulates the diffusion process<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. Hence, for pathogenic bacteria the inhibition of microbial cell wall synthesis may be used as a vital target to produce the antimicrobial effect. The probable target site for the inhibition of microbial cell wall synthesis includes PBPs (transpeptidases), β-lactamase, terminal D-Ala-D-Ala in Lipid II, and Glucosamine-6-Phosphate synthase (G-6-P synthase), etc.<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>.</p><p id=\"Par4\">G-6-P synthase is a complex enzyme involved in the formation of Uridine diphosphate acetylglucosamine (UDP-GlcNAc) and catalyzes the first step in hexosamine biosynthesis. It converts Fru-6-P into Glucosamine-6-Phosphate (GlcN-6-P) using glutamine as the source of ammonia. GlcN-6-P is a precursor of Uridine diphosphate N-acetylglucosamine (UDP-NAG) from which other amino sugar-containing molecules are derived. One of these products, N-acetyl glucosamine, is an essential constituent of the peptidoglycan layer of bacterial and fungal cell wall.</p><p id=\"Par5\">The molecular docking can screen thousands of molecules for their affinity towards a particular target site using various softwares<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. The availability of a three-dimensional crystal structure of G-6-P synthase (protein data bank id 1moq) shall be used to explore, and evaluate a large number of molecules to find out better inhibitors of G-6-P synthase.</p><p id=\"Par6\">A large number of plant-based extracts and phytoconstituents are available, which possess excellent antimicrobial activity; however, there is a lack of data for their preservative effectiveness if compared to the commercially available preservatives<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Amygdalin, a cyanogenic glycoside and is present in variable amounts in seeds of fruits like apricot, peach, plum, etc. and fruits like nectarine, chokeberry, christmas berry, barley, brown rice, buckwheat groats, cherry, etc.<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>.</p><p id=\"Par7\">The pharmacological potential of amygdalin includes antitussive, antiasthmatic, digestive, anti-atherogenic, inhibition of renal interstitial fibrosis, prevention of pulmonary fibrosis, lung injury due to hypoxia, immune system regulation, antitumor, anti-inflammatory, keratoconjunctivitis sicca, emphysema, leprosy, vitiligo, antimicrobial and antiulcer etc.<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>–<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>.</p><p id=\"Par8\">Some literature data also concluded that several natural moieties like gallic acid derivatives, ferulic acid, p-coumaric acid, ε-Polylysine, etc. have been evaluated for their preservative effectiveness<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>–<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. Hence, based on the available data, it was planned to explore the amygdalin derivatives for their G-6-P synthase inhibitory potential along with their antioxidant, antimicrobial, and preservative efficacy potential in food preparation.</p></sec><sec id=\"Sec2\"><title>Experimental</title><sec id=\"Sec3\"><title>Material and methods</title><p id=\"Par9\">All the chemicals required for experimental work were of analytical grade and were purchased from LobaChemie, SRL, and Sigma Aldrich. Nutrient agar, nutrient broth, sabouraud dextrose agar, and sabouraud dextrose broth required for antimicrobial and preservative efficacy were obtained from Hi-media Laboratories. Streptomycin, ciprofloxacin, ampicillin and fluconazole were obtained as a gift sample from Belco Pharma, Bahadurgarh. Microbial strains <italic>S. aureus MTCC</italic> 3,160, <italic>P. aeruginosa MTCC</italic> 1934, <italic>E. coli MTCC</italic> 45, <italic>C. albicans MTCC</italic> 183, and <italic>A. niger MTCC</italic> 282 were purchased from MTCC, Chandigarh. Chemical reactions were monitored by TLC on silica gel plates in iodine and UV chamber. The Sonar melting point apparatus in open capillary tube was used for the recording of melting points. <sup>1</sup>H NMR and <sup>13</sup>C NMR spectra were confirmed in DMSO and deuterated CDCl<sub>3</sub> on Bruker Avance II 400 NMR spectrometer at a frequency of 400 MHz downfield to tetramethylsilane standard. The FTIR spectra were recorded on Perkin Elmer FTIR spectrophotometer with the help of the KBr pellets technique. Waters Micromass Q-ToF Micro instrument was used for Mass spectrum recording, and elemental analysis was done by Perkin Elmer 2,400 elemental analyzer.</p></sec><sec id=\"Sec4\"><title>In silico molecular docking studies</title><p id=\"Par10\">The Schrodinger, Inc. (New York, USA) software Maestro 11 was used for the computational calculations and docking studies. Laboratory for Enzyme Inhibition Studies, Department of Pharmaceutical Sciences, M.D. University, Rohtak, INDIA, was used for the computational work. The receptor-grid files were generated by grid-receptor generation program Glide, version 6.6, 2015. Grid-based ligand docking was used, which utilized the hierarchical sequence of filters to produce possible conformations of the ligand in the active-site region of the protein receptor. At this stage, raw score values and geometric filters were prepared out unlikely binding modes. The next filter phase involves a grid-based force field evaluation and refinement of docking experiments, including torsional and rigid-body movements of the ligand<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. The remained docking evaluations were subjected to a Monte Carlo procedure to minimize the energy score.</p><p id=\"Par11\">The energy differences were calculated using the equation:<disp-formula id=\"Equa\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\Delta E = \\, E_{complex} - \\, E_{ligand} - \\, E_{protein} $$\\end{document}</tex-math><mml:math id=\"M2\" display=\"block\"><mml:mrow><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:mi>E</mml:mi><mml:mo>=</mml:mo><mml:mspace width=\"0.166667em\"/><mml:msub><mml:mi>E</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">complex</mml:mi></mml:mrow></mml:msub><mml:mo>-</mml:mo><mml:mspace width=\"0.166667em\"/><mml:msub><mml:mi>E</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">ligand</mml:mi></mml:mrow></mml:msub><mml:mo>-</mml:mo><mml:mspace width=\"0.166667em\"/><mml:msub><mml:mi>E</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">protein</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70895_Article_Equa.gif\" position=\"anchor\"/></alternatives></disp-formula></p></sec><sec id=\"Sec5\"><title>Protein preparation</title><p id=\"Par12\">The X-ray protein structure coordinates of G-6-P synthase were downloaded from Protein Data Bank and were prepared with the help of the Schrödinger protein preparation wizard ‘Prepwiz’<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Pdb id 1moq, having resolution 1.57 Å was selected based on the lowest resolution and availability. All the water molecules except metals coordinate and present between the ligand and protein were removed. The energy-restrained structure of the protein G-6-P synthase was constructed with the help of the Optimized Potentials for Liquid Simulations -2005 (OPLS-2005) force field.</p></sec><sec id=\"Sec6\"><title>Ligand preparation</title><p id=\"Par13\">The three-dimensional structural library was prepared using the Chemdraw software and preceded for energy minimization using the LigPrep tool for the correction of coordinates, ionization, stereochemistry, and tautomeric structure to gain the appropriate conformation through the addition or removal of hydrogen bonds. The partial charges were computed according to the OPLS-2005 force field (32 stereoisomer, tautomers, and ionization) at biological pH and used for molecular docking studies.</p></sec><sec id=\"Sec7\"><title>General procedure for the synthesis of amygdalin derivatives</title><p id=\"Par14\">The amygdalin derivatives were synthesized by using the procedure outlined in Scheme 1 by enzyme catalysis. Here, 40 ml of acetone containing 0.1 mol/L amygdalin and 0.3 mol/L of sinapic and syringic acid in 1 g of Novozyme 435 were taken in incubator shaker at 200 rpm at 45 °C (48 h). The filtration of the mixture terminated the reaction. Both the compounds in the series were synthesized according to the standard procedures as outlined in Scheme 1 (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). The completion of the reaction was confirmed by single spot TLC. After the completion of reaction the concentrated reaction mixture was concentrated and the formed precipitated were filtered off desiccated. The crude products were recrystallized using alcohol yielded compound 1–2. The confirmation of the final compounds was made by physicochemical and spectral methods like FTIR, 1H NMR, 13C NMR spectra, CHN and mass analysis.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Design strategy and Scheme used for the synthesis of amygdalin derivatives.</p></caption><graphic xlink:href=\"41598_2020_70895_Fig1_HTML\" id=\"MO1\"/></fig></p></sec><sec id=\"Sec8\"><title>Spectral data</title><sec id=\"Sec9\"><title>(E)-(6-((6-(cyano(phenyl)methoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2yl)methoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-yl)methyl3-(4-hydroxy-3,5 dimethoxyphenyl) acrylate</title><p id=\"Par15\">mp: 230–232 °C; TLC (Ethyl Acetate: Methanol, 4:1 v/v): R<sub>f</sub> = 0.60; Yield = 65.50%; M.Wt. = 663.62; <sup>1</sup>H NMR (400 MHz, CDCL<sub>3</sub>): δ 8.69 (s, 1H), 7.50–7.43 (m, 2H), 7.41–7.32 (m, 3H), 7.32–7.24 (m, 1H), 6.82 (s, 2H), 6.28 (d, J = 15.1 Hz, 1H), 5.80 (s, 1H), 5.00 (d, J = 6.9 Hz, 1H), 4.76 (d, J = 8.8 Hz, 1H), 4.71 (dd, J = 8.8, 2.7 Hz, 3H), 4.59 (d, J = 7.0 Hz, 1H), 4.40–4.30 (m, 2H), 4.33–4.25 (m, 1H), 4.12 (dd, J = 12.5, 7.0 Hz, 1H), 3.90 (dd, J = 12.3, 7.0 Hz, 1H), 3.81 (s, 5H), 3.70–3.56 (m, 2H), 3.56–3.47 (m, 1H), 3.45–3.24 (m, 6H), 3.16 (dt, J = 8.9, 7.0 Hz, 1H); <sup>13</sup>C NMR (400 MHz, CDCL<sub>3</sub>) δ 168.20, 149.08, 146.99, 138.98, 134.91, 130.91, 130.11, 128.91, 126.93, 119.31, 115.50, 106.56, 104.04, 100.85, 76.13, 74.19, 74.10, 74.00, 73.12, 72.60, 71.10, 70.23, 68.91, 68.25, 63.67, 56.59; IR (KBr pellets): 1,029 cm<sup>−1</sup> (–C–O–C), 1,074 cm<sup>−1</sup> (–C–C–), 1,449 cm<sup>−1</sup> (–C=C–), 1642 cm<sup>−1</sup> (–C=N–), 2,875 cm<sup>−1</sup> (–C–H–), 3,371 (–OH–); MS ES + (ToF): m/z 663.22 [M<sup>+</sup> + 2]; CHNS: Calc (C<sub>31</sub>H<sub>37</sub>NO<sub>15</sub>): C, 56.11; H, 5.62; N, 2.11; O, 36.16; Found C, 56.14; H, 5.64; N, 2.10; O, 36.18.</p></sec><sec id=\"Sec10\"><title>(6-((6-(cyano(phenyl)methoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-yl)methoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-yl)methyl-4-hydroxy-3,5 dimethoxy benzoate</title><p id=\"Par16\">mp: 239–241 °C; TLC (Ethyl Acetate: Methanol, 4:1 v/v): R<sub>f</sub> = 0.67; Yield = 69.55%; M.Wt. = 622.55; <sup>1</sup>H NMR (400 MHz, CDCL<sub>3</sub>): δ 8.69 (s, 1H), 7.50–7.43 (m, 2H), 7.41–7.32 (m, 2H), 7.32–7.24 (m, 1H), 7.18 (s, 2H), 5.80 (s, 1H), 5.00 (d, J = 6.9 Hz, 1H), 4.76 (d, J = 8.8 Hz, 1H), 4.71 (dd, J = 8.8, 2.6 Hz, 3H), 4.59 (d, J = 7.0 Hz, 1H), 4.37 (d, J = 8.0 Hz, 1H), 4.33–4.25 (m, 1H), 4.24 (dd, J = 12.4, 7.0 Hz, 1H), 4.01 (dd, J = 12.4, 7.0 Hz, 1H), 3.90 (dd, J = 12.3, 7.0 Hz, 1H), 3.81 (s, 5H), 3.65 (dd, J = 12.3, 6.9 Hz, 1H), 3.57–3.27 (m, 7H), 3.16 (dt, J = 8.9, 6.9 Hz, 1H)); <sup>13</sup>C NMR (400 MHz, CDCL<sub>3</sub>): δ 166.71, 147.85, 141.19, 134.88, 130.91, 130.11, 128.91, 120.59, 119.27, 106.79, 102.61, 101.58, 76.55, 75.99, 75.68, 75.49, 74.47, 73.99, 71.60, 70.81, 69.52, 68.85, 64.07, 56.35; IR (KBr pellets): 1,029 cm<sup>−1</sup> (–C–O–C), 1,074 cm<sup>−1</sup> (–C–C–), 1,449 cm<sup>−1</sup> (–C=C–), 1642 cm<sup>−1</sup> (–C=N–), 3,029 cm<sup>−1</sup> (–C–H–), 3,371 cm<sup>−1</sup> (–OH–); MS ES + (ToF): m/z 622.18 [M<sup>+</sup> + 2]; CHNS: Calc (C<sub>28</sub>H<sub>32</sub>NO<sub>15</sub>): C, 54.02; H, 5.18; N, 2.25; O, 38.55; Found C, 54.05; H, 5.15; N, 2.27; O, 38.56.</p></sec></sec><sec id=\"Sec11\"><title>Antioxidant activity</title><sec id=\"Sec12\"><title>2,2-Diphenyl-1-pycrilhydrazil hydrate (DPPH) radical scavenging assay</title><p id=\"Par17\">Antioxidant activity of the synthesized was evaluated photocolorimetric assay by using DPPH free radical scavenging method. Briefly, 0.1 mM solution of DPPH in methyl alcohol was prepared, and 1 mL of this solution was added to 3 mL of sample or standard. Discolorations were measured at 517 nm after incubation for 30 min at 30 °C in the dark. Lower absorbance of the reaction mixture indicates higher free radical scavenging activity. The test was performed in triplicate and the % inhibition values of given samples was calculated by using the formula:<disp-formula id=\"Equb\"><alternatives><tex-math id=\"M3\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\% {\\text{ Inhibition }} = \\, \\left( {{\\text{A}}_{{\\text{c}}} - {\\text{A}}_{{\\text{s}}} } \\right) \\, \\times { 1}00/{\\text{A}}_{{\\text{c}}} $$\\end{document}</tex-math><mml:math id=\"M4\" display=\"block\"><mml:mrow><mml:mo>%</mml:mo><mml:mrow><mml:mspace width=\"0.333333em\"/><mml:mtext>Inhibition</mml:mtext><mml:mspace width=\"0.333333em\"/></mml:mrow><mml:mo>=</mml:mo><mml:mspace width=\"0.166667em\"/><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:msub><mml:mtext>A</mml:mtext><mml:mtext>c</mml:mtext></mml:msub><mml:mo>-</mml:mo><mml:msub><mml:mtext>A</mml:mtext><mml:mtext>s</mml:mtext></mml:msub></mml:mrow></mml:mfenced><mml:mspace width=\"0.166667em\"/><mml:mo>×</mml:mo><mml:mn>100</mml:mn><mml:mo stretchy=\"false\">/</mml:mo><mml:msub><mml:mtext>A</mml:mtext><mml:mtext>c</mml:mtext></mml:msub></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70895_Article_Equb.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p id=\"Par18\">Here, A<sub>c</sub> was the absorbance of the control, and A<sub>s</sub> was the absorbance of the sample<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>.</p></sec></sec><sec id=\"Sec13\"><title>Antimicrobial activity</title><sec id=\"Sec14\"><title>Minimum inhibitory concentration (MIC)</title><p id=\"Par19\">The antimicrobial activity of the synthesized compounds was determined against <italic>S. aureus MTCC</italic> 3,160, <italic>P. aeruginosa MTCC</italic> 1934, <italic>E. coli MTCC</italic> 45, <italic>P. mirabilis MTCC</italic> 3,310<italic>, C. albicans MTCC</italic> 183, and <italic>A. niger MTCC</italic> 282 by using the tube dilution method. Dilutions of test and standard compounds were prepared in double strength nutrient broth I.P. (bacteria) or sabouraud dextrose broth I.P. (fungi)<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. The slants of <italic>E. coli</italic>, <italic>P. aeruginosa, P. mirabilis,</italic> and <italic>S. aureus</italic> were incubated at the 30–35 °C for 24 h. The slants of <italic>C. albicans</italic> were incubated at 20–25 °C for 48 h, whereas; the slants of <italic>A. niger</italic> were incubated at 20–25 °C for 5 days. After the incubation period sterilized 0.9% NaCl solution was used to harvest the bacterial, and fungal cultures from agar slant through proper shaking and then the suspensions of microorganisms were diluted with the sterile 0.9% NaCl solution to Colony Forming Unit (CFU) count was adjusted by adjusting the density of microorganism suspension to that of 0.5 McFarland standards by adding distilled water. The number of CFU was determined by dilution pour-plate method. A serial dilution of 50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, 3.12 µg/mL and 1.62 µg/mL was used for determination of MIC. The samples tubes were incubated at 37 °C for 24 h (bacteria), at 25 °C for 7 days (<italic>A. niger</italic>), and at 37 °C for 48 h (<italic>C. albicans</italic>), and the results were recorded in pMIC<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>.</p></sec><sec id=\"Sec15\"><title>Preservative effectiveness study</title><p id=\"Par20\">The selected most active antioxidant/antimicrobial compounds were further evaluated for their preservative potential in the cosmetic product, White lotion USP and food products; such as fresh aloe vera juice the cosmetic product as per the procedure mentioned in USP 2004<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>.</p></sec><sec id=\"Sec16\"><title>Preparation of aloe vera juice</title><p id=\"Par21\">Aloe vera leaves were cleaned with distilled water and cleaned with 0.5% chlorine water. The leaf base and tip were chopped 1.5 inches and 3 inches, respectively. Margins of leaves were removed with the help of a stainless steel knife. The pulp was washed 2–3 times with distilled water to remove the exudates and homogenized with the help of a blender, and then filtered through an autoclave muslin cloth. The aloe vera juice thus obtained was used for the testing of food preservative efficacy<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>.</p></sec><sec id=\"Sec17\"><title>Preparation of white lotion USP</title><p id=\"Par22\">Ingredients: Zinc sulfate 40 gm, sulfurated potash 40 gm and purified water q.s. to 1,000 mL. Firstly, zinc sulfate and sulfurated potash were dissolved in 450 mL of water separately and filtered. Then, sulfurated potash solution was added to zinc sulfate with stirring. At last, the required amount of water was added and mixed thoroughly and sterilized. For preservative efficacy testing, the White lotion USP was prepared using the equimolar amount of compounds <bold>1</bold>–<bold>2</bold> as novel preservatives by replacing sodium benzoate, methyl paraben and propyl paraben from the formula<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>.</p></sec><sec id=\"Sec18\"><title>Challenge microorganism</title><p id=\"Par23\"><italic>S. aureus MTCC</italic> 3,160, <italic>P. aeruginosa MTCC</italic> 1934, <italic>E. coli MTCC</italic> 45, <italic>C. albicans MTCC</italic> 183, and <italic>A. niger MTCC</italic> 282 were used as common contaminants in the study as prescribed in USP for preservative efficacy testing in the pharmaceutical preparations<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>.</p></sec><sec id=\"Sec19\"><title>Preparation of inoculums</title><p id=\"Par24\">The slants of <italic>E. coli</italic>, <italic>P. aeruginosa,</italic> and <italic>S. aureus</italic> were incubated at the 37 °C for 24 h. The slants of <italic>C. albicans</italic> were incubated at 37 °C for 48 h, whereas; the slants of <italic>A. niger</italic> were incubated at 25 °C for 7 days<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>.</p></sec></sec><sec id=\"Sec20\"><title>Preservative efficacy procedure</title><sec id=\"Sec21\"><title>Aloe vera juice</title><p id=\"Par25\">Preservative efficacy of the selected compound 1 and sodium benzoate (standard) was estimated in freshly prepared aloe vera juice as per the method with minor modifications as described by Ahlawat et al<italic>.</italic><sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. A concentration of 600 mg/kg or 600 ppm of sodium benzoate in aloe vera juice was taken as per food safety and standard guidelines. Equimolar quantity (0.0004 mol) of selected compound was taken as a preservative system in test samples. Challenged microbial cell suspension was inoculated the juice preparation with inoculum size never exceed more than 1%. After inoculation with microbes juice was incubated at room temperature for consecutive 28 days, and samples were collected on the 14th and 28th day of the experiment. The viable count of microorganisms was performed on nutrient agar (bacteria), and sabouraud dextrose agar (fungi) plates<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. Each experiment was done in triplicate and log cfu/ml of juice was determined with comparison to standard.</p></sec><sec id=\"Sec22\"><title>White lotions USP</title><p id=\"Par26\">White lotions USP was added in final containers and were used in the challenge test. The preparation was inoculated with a 0.5–1% volume of microbial inoculum having a concentration of 1 × 10<sup>5</sup>–1 × 10<sup>6</sup> CFU/mL<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. Inoculated samples were mixed thoroughly to ensure homogeneous microorganism distribution and incubated. The CFU/mL of the product was determined at an interval of 0 days, 7 days, 14 days, 21 days, and 28 days in agar plates. Log CFU/mL of white lotion USP was calculated as not as not less than 2.0 log reductions from initial count on 14th day of incubation and no increase in CFU from 14th day count to 28th day in case of bacteria and no increase from the initial calculated count on 14th day and 28th day in case of fungi<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>.</p></sec><sec id=\"Sec23\"><title>Stability study</title><p id=\"Par27\">Compound 1 was selected for stability study in their final container containing the formulation of Aole vera gel and White Lotion USP. Formulation having different preservatives i.e., standard and compound <bold>1</bold> were stored at 40° ± 2 °C at 75% RH ± 5% RH (as per ICH guidelines) and were analyzed for the pH and cfu/ml of the product at 0, 1, 2, 3, 4, 5, and 6 months.</p></sec></sec><sec id=\"Sec24\"><title>Statistical analyses</title><p id=\"Par28\">Data are expressed as mean values ± standard deviation or standard error as described in the legend of the figures and tables. Analysis of results was done by Kruskal–Wallis test as appropriate. Differences were considered to be statistically significant at P < 0.05. Statistical analyses were performed using MS excel data statistics analysis tool.</p></sec></sec><sec id=\"Sec25\"><title>Results and discussion</title><sec id=\"Sec26\"><title>Molecular docking study</title><p id=\"Par29\">The selection of amygdalin for further evaluation as preservative has been made on the basis of our prior evaluation in docking and absorption, distribution, metabolism and excretion (ADMET) study data<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. Then the proposed library of amygdalin derivatives was again evaluated for their molecular docking behavior with the help of Schrodinger’s Maestro docking software<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. Molecules were docked with the help of the G-6-P synthase crystallographic complex having pdb id 1moq. The Induced Fit docking (IFD) method of docking was utilized for the same. The predicted binding pattern revealed that synthesized ligand binds within the catalytic cavity of G-6-P synthase firmly via hydrogen bond formation, pi-pi stacking, and hydrophobic interactions. Two compounds 1 and 2, were selected via docking score, binding energy, and ADMET study parameters. Compound 1 showed the hydrogen bonding with Asp 354, Cys 300, and hydrophobic interactions with Val 605, Leu 601, while compound 2 showed hydrogen bonding with Asp 354, Ala 602, Ser 349, Ser 347, Thr 352, and hydrophobic interactions Leu 484, Cys 300. The synthesized compounds 1 and 2 possessed excellent dock score − 9.65, − 6.97, respectively and binding energy − 1.40 kJ/mol, − 51.31 kJ/mol, respectively as compared to standard drugs ciprofloxacin, ampicillin and fluconazole, dock score − 5.18, − 5.06, − 5.12, and binding energy − 37.16 kJ/mol, − 25.41 kJ/mol and − 23.15 kJ/mol, respectively. Hence, it is cleared that both the compounds 1 and 2 behave as G-6-P synthase inhibitor. Here, the inhibition of G-6-P synthase enzyme further evaluated by the outcomes of the inhibition likes antimicrobial activity. This further made the clearance behind the inhibition of G-6-P synthase enzyme by different proposed molecules. The molecular docking of the proposed amygdalin derivatives with the target site of G-6-P synthase (PDB ID 1MOQ) showed that all the selected compounds exhibited better binding affinity with different amino acid residues in active pocket of the enzyme. The results of molecular docking for different ligands within G-6-P synthase pocket and their interaction with different amino acid residues have been shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>. Molecular docking results of proposed amygdalin derivatives have been shown in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Interaction patterns of ligands within the G-6-P synthase pocket.</p></caption><graphic xlink:href=\"41598_2020_70895_Fig2_HTML\" id=\"MO2\"/></fig><table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Docking parameters, ADMET profile and pMIC value of selected amygdalin derivatives.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Compound(s)</th><th align=\"left\" colspan=\"2\">G-6-P synthase binding affinity</th><th align=\"left\" colspan=\"7\">ADMET profile</th><th align=\"left\" colspan=\"7\">pMIC values in μM</th></tr><tr><th align=\"left\">Docking score</th><th align=\"left\">Energy</th><th align=\"left\">No. of rotatable bond</th><th align=\"left\">DonorHB</th><th align=\"left\">AcceptHB</th><th align=\"left\">QplogPo/w</th><th align=\"left\">QplogBB</th><th align=\"left\">QPPMDCK</th><th align=\"left\">QPPCaco</th><th align=\"left\"><italic>K.pneumoniae</italic></th><th align=\"left\"><italic>P. mirabilis</italic></th><th align=\"left\"><italic>P. aeruginosa</italic></th><th align=\"left\"><italic>S. aureus</italic></th><th align=\"left\"><italic>E. coli</italic></th><th align=\"left\"><italic>C.albicans</italic></th><th align=\"left\"><italic>A. niger</italic></th></tr></thead><tbody><tr><td align=\"left\">Compound 1</td><td char=\".\" align=\"char\">− 9.65</td><td char=\".\" align=\"char\">− 71.40</td><td char=\".\" align=\"char\">8</td><td char=\".\" align=\"char\">6</td><td char=\".\" align=\"char\">20</td><td char=\".\" align=\"char\">− 2.01</td><td char=\".\" align=\"char\">− 4.41</td><td char=\".\" align=\"char\">0.84</td><td align=\"left\">2.70</td><td align=\"left\">2.02</td><td align=\"left\">1.72</td><td align=\"left\">1.42</td><td align=\"left\">1.42</td><td align=\"left\">2.02</td><td align=\"left\">1.72</td><td align=\"left\">2.02</td></tr><tr><td align=\"left\">Compound 2</td><td char=\".\" align=\"char\">− 6.97</td><td char=\".\" align=\"char\">− 51.31</td><td char=\".\" align=\"char\">10</td><td char=\".\" align=\"char\">7</td><td char=\".\" align=\"char\">22</td><td char=\".\" align=\"char\">− 1.97</td><td char=\".\" align=\"char\">− 4.34</td><td char=\".\" align=\"char\">0.92</td><td align=\"left\">3.0</td><td align=\"left\">1.39</td><td align=\"left\">1.39</td><td align=\"left\">1.69</td><td align=\"left\">1.39</td><td align=\"left\">1.69</td><td align=\"left\">1.39</td><td align=\"left\">1.39</td></tr><tr><td align=\"left\">Amygdalin</td><td char=\".\" align=\"char\">− 6.60</td><td char=\".\" align=\"char\">− 57.22</td><td char=\".\" align=\"char\">6</td><td char=\".\" align=\"char\">5</td><td char=\".\" align=\"char\">18</td><td char=\".\" align=\"char\">− 1.02</td><td char=\".\" align=\"char\">− 3.35</td><td char=\".\" align=\"char\">0.98</td><td align=\"left\">4</td><td align=\"left\">0.86</td><td align=\"left\">1.06</td><td align=\"left\">1.76</td><td align=\"left\">1.06</td><td align=\"left\">0.96</td><td align=\"left\">0.76</td><td align=\"left\">0.86</td></tr><tr><td align=\"left\">Streptomycin</td><td char=\".\" align=\"char\">− 5.44</td><td char=\".\" align=\"char\">− 40.20</td><td char=\".\" align=\"char\">9</td><td char=\".\" align=\"char\">12</td><td char=\".\" align=\"char\">15</td><td char=\".\" align=\"char\">− 2.06</td><td char=\".\" align=\"char\">− 4.20</td><td char=\".\" align=\"char\">0.78</td><td align=\"left\">3</td><td align=\"left\">1.96</td><td align=\"left\">1.06</td><td align=\"left\">1.36</td><td align=\"left\">1.06</td><td align=\"left\">1.96</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Ciprofloxacin</td><td char=\".\" align=\"char\">− 5.18</td><td char=\".\" align=\"char\">− 37.16</td><td char=\".\" align=\"char\">3</td><td char=\".\" align=\"char\">2</td><td char=\".\" align=\"char\">6</td><td char=\".\" align=\"char\">− 1.02</td><td char=\".\" align=\"char\">2.23</td><td char=\".\" align=\"char\">0.80</td><td align=\"left\">4</td><td align=\"left\">2.02</td><td align=\"left\">1.12</td><td align=\"left\">1.42</td><td align=\"left\">1.12</td><td align=\"left\">1.42</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Ampicillin</td><td char=\".\" align=\"char\">− 5.06</td><td char=\".\" align=\"char\">− 25.41</td><td char=\".\" align=\"char\">4</td><td char=\".\" align=\"char\">3</td><td char=\".\" align=\"char\">5</td><td char=\".\" align=\"char\">− 1.35</td><td char=\".\" align=\"char\">0.99</td><td char=\".\" align=\"char\">0.90</td><td align=\"left\">0.89</td><td align=\"left\">2.04</td><td align=\"left\">1.14</td><td align=\"left\">0.84</td><td align=\"left\">0.84</td><td align=\"left\">1.74</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Fluconazole</td><td char=\".\" align=\"char\">− 5.12</td><td char=\".\" align=\"char\">− 23.15</td><td char=\".\" align=\"char\">5</td><td char=\".\" align=\"char\">1</td><td char=\".\" align=\"char\">5</td><td char=\".\" align=\"char\">− 2.32</td><td char=\".\" align=\"char\">0.88</td><td char=\".\" align=\"char\">0.87</td><td align=\"left\">0.93</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">1.08</td><td align=\"left\">1.38</td></tr></tbody></table></table-wrap></p></sec><sec id=\"Sec27\"><title>ADMET study</title><p id=\"Par30\">The compounds selected from docking have been further evaluated for their ADME parameters so that the selection of final preservative becomes easy. The evaluation of different ADMET parameters of selected amygdalin derivatives has been represented in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>. All the synthesized compounds fulfilled the standard Rule of Five<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. All the synthesized compounds qualified the conditions for various descriptors like lipophilicity (LogP), hydrogen bond acceptor (HBA), hydrogen bond donor (HBD) and moleculat weight (MW). All parameters were in a suitable range for drug-like characteristics. In addition, according to Veber et al<italic>.</italic> (2002) for better bioavailability, rotatable bonds should be ≤ 10 as the rotatable bonds in ligand impart elasticity<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. The values of QPlogBB should be > 1.0 CNS active compounds, and value < 1.0 CNS inactive compounds. QPPCaco cell permeability should be in a range from 4–70<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. All the synthesized compounds exhibited a suitable drug-like profile and could be used for further evaluation as a novel preservative for food and pharmaceutical preparations.</p></sec><sec id=\"Sec28\"><title>Chemistry</title><p id=\"Par31\">The amygdalin derivatives selected on the basis of docking and ADMET parameters were synthesized <bold>(</bold>derivatives 1–2) by the reaction according to Vemula et al<italic>.</italic> (2006), outlined in Scheme 1<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. The chemical structural parts of all the synthesized compounds were confirmed by FTIR, <sup>1</sup>H NMR, <sup>13</sup>C NMR, mass spectroscopy, and elemental analysis, which were in full agreement with their structures. The synthesis of amygdalin esters was completed by enzyme catalysis. In general, 40 ml of acetone containing 0.1 mol/L amygdalin and 0.3 mol/L vinyl ester was added in 1 g of Novozyme 435. The reaction mixtures were placed it in an incubator shaker at 200 rpm on 45 °C for 48 h. The filtration of reaction mixtures terminated the reaction.</p><p id=\"Par32\">Both of the compounds were synthesized according to the standard procedures as outlined in Scheme 1. The completion of the reaction was confirmed by TLC under UV lamp and FTIR. Formation of compounds 1 and 2 was further confirmed by peak shifted from 2,730 cm<sup>−1</sup> (–OH) and appearance of a peak at 1,730 cm<sup>−1</sup> and 1744 cm<sup>−1</sup> (–C=O), respectively for compound 1 and compound 2. The change in chemical shift value, coupling constant and multiplicities were analyzed by <sup>1</sup>HNMR and <sup>13</sup>C NMR signals of synthesized compounds. The FTIR, <sup>1</sup>H NMR and <sup>13</sup>C NMR data confirmed the chemical structures of synthesized amygdalin derivatives. Final confirmation of the synthesized compounds was done by analyzing the mass spectrum of synthesized derivatives for molecular weight determination, and the Q-ToF Micro instrument was used as ion source. Most of the derivatives showed M<sup>+</sup> (molecular ion peak), (M<sup>++1</sup>), (M<sup>++2</sup>) in positive chemical ionization, and (M<sup>1+</sup>), (M<sup>2+</sup>), M<sup>+</sup> during negative chemical ionization mode. Finally, establishment of synthesis of amygdalin derivatives was done by elemental analysis where C, H, and N in percent were found within acceptable limits.</p></sec><sec id=\"Sec29\"><title>Antioxidant activity</title><sec id=\"Sec30\"><title>DPPH radical scavenging activity</title><p id=\"Par33\">The plant-based antioxidants can be used in food and pharmaceuticals to enhance their shelf life against oxidation<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. In the present study, DPPH radical scavenging assay method was utilized for the evaluation of the antioxidant profile of the synthesized compounds<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. In this screening, compound 1 was observed as the most potent antioxidant compound (IC<sub>50</sub> values 5.54 ± 0.03 µM) as compared to reference standard L-ascorbic acid (IC<sub>50</sub> values 8.11 ± 0.0.69 µM). However, compound 2 showed moderate antioxidant activity (IC<sub>50</sub> value 6.51 ± 0.04 µM). The antioxidant activity of the amygdalin was found 7.72 ± 0.03 µM<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. Here, the better antioxidant property of amygdalin derivative shall be useful in the preservation of food, cosmetics, and pharmaceuticals<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. All the results were expressed as mean ± standard deviation (n = 5) and results were found significant with Krukal-Wallis test (p < 0.05).</p></sec></sec><sec id=\"Sec31\"><title>Antimicrobial activity</title><sec id=\"Sec32\"><title>MIC</title><p id=\"Par34\">Newly synthesized amygdalin derivatives were evaluated for their in vitro antimicrobial activity against standard MTCC strains of <italic>K. reparati, P. mirabilis, P.aeruginosa, S. aureus, E. coli, C. albicans,</italic> and <italic>A. niger</italic>. Antimicrobial activity of the test compounds revealed that the compound <bold>1</bold> was found to be the most potent compound ((pMIC 2.02, 1.72, 1.42, 1.42, 2.02, 1.72 and 2.02 µM/ml against <italic>P. mirabilis, P. aeruginosa</italic>, <italic>S. aureus, E. coli, C. albicans,</italic> and <italic>A. niger</italic> respectively) as compared to the standard drugs streptomycin (pMIC 1.06, 1.36, 1.06, and 1.96 μM for <italic>P. mirabilis, P. aeruginosa, S. aureus,</italic> and <italic>E. coli</italic> respectively), ciprofloxacin (pMIC 1.12, 1.42, 1.12, and 1.42 μM for <italic>P. mirabilis, P. aeruginosa, S. aureus,</italic> and <italic>E. coli</italic> respectively), ampicillin (pMIC 1.14, 0.84, 0.84, and 1.74 μM for <italic>P. mirabilis, P. aeruginosa, S. aureus,</italic> and <italic>E. coli</italic> respectively) and fluconazole (pMIC 1.08 and 1.38 μM for <italic>C. albicans,</italic> and <italic>A. niger</italic> respectively) using tube dilution method. Here, the results of MIC studies (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>) revealed that the synthesized compounds have better antimicrobial potential as compared to standard ciprofloxacin, ampecillin, and fluconazole. The probable mechanism of antimicrobial activity of amygdalin derivatives may be due to the better inhibition of G-6-P synthase.</p></sec><sec id=\"Sec33\"><title>Preservative efficacy</title><p id=\"Par35\">The results of preservative efficacy study of the aloe vera juice and White lotion USP were performed and were reported as mean ± standard deviation. Results of microbial growth at 14th day and 28th day were found to be significant with p < 0.05 as confirmed by Kruskal–Wallis test.</p></sec><sec id=\"Sec34\"><title>Aloe vera juice</title><p id=\"Par36\">The results of preservative effectiveness have been summarized in Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>. The log CFU/ml for compound 1 revealed that the values were within the prescribed limit as per USP criteria. The selected compound 1 reduced the growth of microbes on the 14th day from the initial count and found to be effective on the 28th day and results were also comparable to sodium benzoate. The preservative efficacy of the amygdalin compound 1 has been represented for number of days vs. degree of microbial log reduction and has been shown graphically in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>.<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Log CFU/ml values of the selected compound <bold>1</bold> in Aloe vera juice and White lotion USP.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" colspan=\"2\">Compound</th><th align=\"left\" colspan=\"2\"><italic>E.coli</italic></th><th align=\"left\" colspan=\"2\"><italic>P.aeruginosa</italic></th><th align=\"left\" colspan=\"2\"><italic>S.aureus</italic></th><th align=\"left\" colspan=\"2\"><italic>C.albicans</italic></th><th align=\"left\" colspan=\"2\"><italic>A.niger</italic></th></tr><tr><th align=\"left\" colspan=\"2\">Cfu/ml after days</th><th align=\"left\">14 days</th><th align=\"left\">28 days</th><th align=\"left\">14 days</th><th align=\"left\">28 days</th><th align=\"left\">14 days</th><th align=\"left\">28 days</th><th align=\"left\">14 days</th><th align=\"left\">28 days</th><th align=\"left\">14 days</th><th align=\"left\">28 days</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"2\">Compound 1</td><td align=\"left\">#</td><td align=\"left\">2.25 ± 0.10<sup>a</sup></td><td align=\"left\">2.19 ± 0.14<sup>b</sup></td><td align=\"left\">2.31 ± 0.12<sup>c</sup></td><td align=\"left\">2.19 ± 0.12<sup>d</sup></td><td align=\"left\">2.4 ± 0.14<sup>e</sup></td><td align=\"left\">2.28 ± 0.15f.</td><td align=\"left\">2.16 ± 0.15<sup>g</sup></td><td align=\"left\">2.3 ± 0.11<sup>h</sup></td><td align=\"left\">2.13 ± 0.12<sup>i</sup></td><td align=\"left\">2.12 ± 0.17<sup>j</sup></td></tr><tr><td align=\"left\">@</td><td align=\"left\">3.22 ± 0.12<sup>a</sup></td><td align=\"left\">3.20 ± 0.13<sup>b</sup></td><td align=\"left\">3.31 ± 0.12<sup>c</sup></td><td align=\"left\">3.22 ± 0.22<sup>d</sup></td><td align=\"left\">3.11 ± 0.14<sup>e</sup></td><td align=\"left\">3.62 ± 0.13f.</td><td align=\"left\">3.22 ± 0.16<sup>g</sup></td><td align=\"left\">3.12 ± 0.21<sup>h</sup></td><td align=\"left\">3.14 ± 0.22<sup>i</sup></td><td align=\"left\">3.64 ± 0.12<sup>j</sup></td></tr><tr><td align=\"left\" rowspan=\"2\">Sodium Benzoate</td><td align=\"left\">#</td><td align=\"left\">2.26 ± 0.11<sup>a</sup></td><td align=\"left\">2.19 ± 0.12<sup>b</sup></td><td align=\"left\">2.32 ± 0.13<sup>c</sup></td><td align=\"left\">2.27 ± 0.22<sup>d</sup></td><td align=\"left\">2.35 ± 0.23<sup>e</sup></td><td align=\"left\">2.3 ± 0.21f.</td><td align=\"left\">2.18 ± 0.16<sup>g</sup></td><td align=\"left\">2.09 ± 0.18<sup>h</sup></td><td align=\"left\">2.19 ± 0.17<sup>i</sup></td><td align=\"left\">2.12 ± 0.15<sup>j</sup></td></tr><tr><td align=\"left\">@</td><td align=\"left\">3.13 ± 0.21<sup>a</sup></td><td align=\"left\">3.23 ± 0.22<sup>b</sup></td><td align=\"left\">3.21 ± 0.16<sup>c</sup></td><td align=\"left\">3.22 ± 0.13<sup>d</sup></td><td align=\"left\">3.54 ± 0.24<sup>e</sup></td><td align=\"left\">3.26 ± 0.12f.</td><td align=\"left\">3.17 ± 0.08<sup>g</sup></td><td align=\"left\">2.22 ± 0.28<sup>h</sup></td><td align=\"left\">3.17 ± 0.13<sup>i</sup></td><td align=\"left\">3.13 ± 0.12<sup>j</sup></td></tr><tr><td align=\"left\" rowspan=\"2\">Propyl Paraben</td><td align=\"left\">#</td><td align=\"left\">2.19 ± 0.15<sup>a</sup></td><td align=\"left\">2.24 ± 0.16<sup>b</sup></td><td align=\"left\">2.24 ± 0.2<sup>c</sup></td><td align=\"left\">2.19 ± 0.15<sup>d</sup></td><td align=\"left\">2.66 ± 0.14<sup>e</sup></td><td align=\"left\">2.41 ± 0.15f.</td><td align=\"left\">2.4 ± 0.16<sup>g</sup></td><td align=\"left\">2.22 ± 0.16<sup>h</sup></td><td align=\"left\">2.11 ± 0.18<sup>i</sup></td><td align=\"left\">2.02 ± 0.18<sup>j</sup></td></tr><tr><td align=\"left\">@</td><td align=\"left\">3.22 ± 0.51<sup>a</sup></td><td align=\"left\">3.22 ± 0.26<sup>b</sup></td><td align=\"left\">3.32 ± 0.34<sup>c</sup></td><td align=\"left\">3.33 ± 0.16<sup>d</sup></td><td align=\"left\">3.23 ± 0.22<sup>e</sup></td><td align=\"left\">3.22 ± 0.23f.</td><td align=\"left\">3.29 ± 0.13<sup>g</sup></td><td align=\"left\">3.22 ± 0.21<sup>h</sup></td><td align=\"left\">3.19 ± 0.16<sup>i</sup></td><td align=\"left\">3.10 ± 0.18<sup>j</sup></td></tr><tr><td align=\"left\" rowspan=\"2\">Ethyl Paraben</td><td align=\"left\">#</td><td align=\"left\">2.2 ± 0.18<sup>a</sup></td><td align=\"left\">2.09 ± 0.18<sup>b</sup></td><td align=\"left\">2.24 ± 0.16<sup>c</sup></td><td align=\"left\">2.22 ± 0.14<sup>d</sup></td><td align=\"left\">2.16 ± 0.15<sup>e</sup></td><td align=\"left\">2.11.18f.</td><td align=\"left\">2.56 ± 0.2<sup>g</sup></td><td align=\"left\">2.29 ± 0.19<sup>h</sup></td><td align=\"left\">2.04 ± 0.17<sup>i</sup></td><td align=\"left\">2.02 ± 0.12<sup>j</sup></td></tr><tr><td align=\"left\">@</td><td align=\"left\">3.39 ± 0.12<sup>a</sup></td><td align=\"left\">3.10 ± 0.17<sup>b</sup></td><td align=\"left\">3.23 ± 0.36<sup>c</sup></td><td align=\"left\">3.39 ± 0.12<sup>d</sup></td><td align=\"left\">3.12 ± 0.13<sup>e</sup></td><td align=\"left\">3.19 ± 0.12f.</td><td align=\"left\">3.50 ± 0.21<sup>g</sup></td><td align=\"left\">3.20 ± 0.41<sup>h</sup></td><td align=\"left\">3.17 ± 0.54<sup>i</sup></td><td align=\"left\">3.09 ± 0.28<sup>j</sup></td></tr><tr><td align=\"left\">Control</td><td align=\"left\" colspan=\"11\"><bold>5 ± 0.20</bold></td></tr></tbody></table><table-wrap-foot><p><sup>#</sup> Aloe vera juice; <sup>@</sup>White lotion USP.</p><p><italic>CFU</italic> Colony forming unit, all experiments were conducted in triplicate (n = 5) and the mean values are presented. Different letters mean p < 0.05 in each line by Kruskal–Wallis test.</p></table-wrap-foot></table-wrap><fig id=\"Fig3\"><label>Figure 3</label><caption><p>Preservative efficacy results of compound 1 in Aloe vera juice and degree of microbial log reduction. Data are means of five replicates; standard deviation is shown as error bars. Chart indicates statistically significant differences between groups (p < 0.05).</p></caption><graphic xlink:href=\"41598_2020_70895_Fig3_HTML\" id=\"MO3\"/></fig></p></sec><sec id=\"Sec35\"><title>White lotion USP</title><p id=\"Par37\">The highly active antimicrobial compound 1 was selected for evaluation of its preservative efficacy. Result showed a less than 2.0 log reductions from initial count on 14th day and number of CFU/ml in some samples increased on the 14th day to 28th day as compared to that of the standard preservatives sodium benzoate, propyl paraben and ethyl paraben. The log CFU/ml (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>) for compound 1 revealed that the values were within the prescribed limit as per USP criteria<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. The graphical representation of preservative efficacy of amygdalin compound 1 in white lotion USP has been presented between the numbers of days <italic>vs</italic>. degree of microbial log reduction and graphically same has been represented in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>.<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Preservative efficacy results of compound 1 in White Lotion USP and degree of microbial log reduction. Data are means of five replicates; standard deviation is shown as error bars. Chart indicates statistically significant differences between groups (p < 0.05).</p></caption><graphic xlink:href=\"41598_2020_70895_Fig4_HTML\" id=\"MO4\"/></fig></p></sec><sec id=\"Sec36\"><title>Stability study</title><p id=\"Par38\">The results of stability testing were performed in triplicate, and were reported as mean values in Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref>. Results revealed that the pH of Aloe vera juice and White lotion USP samples were in range of 7.6–8.0, which indicated the stability of compound 1 ((E)-(6-((6-(cyano(phenyl)methoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2yl)methoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-yl)methyl3-(4-hydroxy-3,5 dimethoxyphenyl) acrylate) as preservative over, the six months period as compared to that of the standard preservatives sodium benzoate, propyl paraben, and methyl paraben. The results of the microbial study indicated that no microbial growth was observed in samples containing compound <bold>1,</bold> over 6 months period. These results indicated that the products were found stable over 6 months with added preservatives. Results for microbial growth and pH changes also found to be significant at p < 0.05.<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Stability studies of compound <bold>1 </bold>in Aloe vera juice and White Lotion USP for pH.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\" colspan=\"2\">Compound(s)</th><th align=\"left\" colspan=\"7\">Change in pH with time</th></tr><tr><th align=\"left\">0 month</th><th align=\"left\">1 month</th><th align=\"left\">2 month</th><th align=\"left\">3 month</th><th align=\"left\">4 month</th><th align=\"left\">5 month</th><th align=\"left\">6 month</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"2\">Compound 1</td><td align=\"left\">#</td><td align=\"left\">7.9 ± 0.32<sup>a</sup></td><td align=\"left\">8.2 ± 0.34<sup>b</sup></td><td align=\"left\">7.4 ± 0.14<sup>c</sup></td><td align=\"left\">7.8 ± 0.24<sup>d</sup></td><td align=\"left\">7.3 ± 0.30<sup>e</sup></td><td align=\"left\">7.8 ± 0.22f.</td><td align=\"left\">7.6 ± 0.34<sup>g</sup></td></tr><tr><td align=\"left\">@</td><td align=\"left\">7.8 ± 0.25<sup>a</sup></td><td align=\"left\">7.8 ± 0.22<sup>b</sup></td><td align=\"left\">7.9 ± 0.33<sup>c</sup></td><td align=\"left\">7.7 ± 0.33<sup>d</sup></td><td align=\"left\">7.6 ± 0.32<sup>e</sup></td><td align=\"left\">7.7 ± 0.22f.</td><td align=\"left\">8.1 ± 0.23<sup>g</sup></td></tr><tr><td align=\"left\" rowspan=\"2\">Sodium benzoate</td><td align=\"left\">#</td><td align=\"left\">8.8 ± 0.14<sup>a</sup></td><td align=\"left\">9.2 ± 0.39<sup>b</sup></td><td align=\"left\">9.2 ± 0.21<sup>c</sup></td><td align=\"left\">9.4 ± 0.39<sup>d</sup></td><td align=\"left\">9.1 ± 0.42<sup>e</sup></td><td align=\"left\">9.2 ± 0.22f.</td><td align=\"left\">9.2 ± 0.56<sup>g</sup></td></tr><tr><td align=\"left\">@</td><td align=\"left\">9.2 ± 0.54<sup>a</sup></td><td align=\"left\">9.2 ± 0.84<sup>b</sup></td><td align=\"left\">9.4 ± 0.33<sup>c</sup></td><td align=\"left\">9.7 ± 0.43<sup>d</sup></td><td align=\"left\">9.2 ± 0.50<sup>e</sup></td><td align=\"left\">9.1 ± 0.94f.</td><td align=\"left\">9.2 ± 0.17<sup>g</sup></td></tr><tr><td align=\"left\" rowspan=\"2\">Propyl paraben</td><td align=\"left\">#</td><td align=\"left\">7.3 ± 0.21<sup>a</sup></td><td align=\"left\">7.5 ± 0.25<sup>b</sup></td><td align=\"left\">7.5 ± 0.25<sup>c</sup></td><td align=\"left\">7.5 ± 0.25<sup>d</sup></td><td align=\"left\">7.8 ± 0.28<sup>e</sup></td><td align=\"left\">7.3 ± 0.33f.</td><td align=\"left\">7.2 ± 0.54<sup>g</sup></td></tr><tr><td align=\"left\">@</td><td align=\"left\">8.2 ± 0.04<sup>a</sup></td><td align=\"left\">8.5 ± 0.69<sup>b</sup></td><td align=\"left\">8.8 ± 0.68<sup>c</sup></td><td align=\"left\">8.7 ± 0.76<sup>d</sup></td><td align=\"left\">8.5 ± 0.32<sup>e</sup></td><td align=\"left\">8.3 ± 0.39f.</td><td align=\"left\">8.7 ± 0.26<sup>g</sup></td></tr><tr><td align=\"left\" rowspan=\"2\">Ethyl paraben</td><td align=\"left\">#</td><td align=\"left\">8.2 ± 0.02<sup>a</sup></td><td align=\"left\">8.4 ± 0.44<sup>b</sup></td><td align=\"left\">8.4 ± 0.26<sup>c</sup></td><td align=\"left\">8.5 ± 0.24<sup>d</sup></td><td align=\"left\">8.4 ± 0.21<sup>e</sup></td><td align=\"left\">8.3 ± 0.49f.</td><td align=\"left\">8.4 ± 0.28<sup>g</sup></td></tr><tr><td align=\"left\">@</td><td align=\"left\">8.4 ± 0.35<sup>a</sup></td><td align=\"left\">8.6 ± 0.36<sup>b</sup></td><td align=\"left\">8.0 ± 0.66<sup>c</sup></td><td align=\"left\">8.2 ± 0.18<sup>d</sup></td><td align=\"left\">8.1 ± 0.14<sup>e</sup></td><td align=\"left\">8.1 ± 0.69f.</td><td align=\"left\">8.4 ± 0.32<sup>g</sup></td></tr><tr><td align=\"left\">Control</td><td align=\"left\" colspan=\"8\"># 8.0 ± 23; @ 8.2 ± 0.08</td></tr></tbody></table><table-wrap-foot><p><sup>#</sup>Aloe vera juice; <sup>@</sup>White lotion USP.</p><p>All pH values were recorded in triplicate (n = 5) and the mean values are presented. Different letters mean p < 0.05 in each line by Kruskal–Wallis test.</p></table-wrap-foot></table-wrap></p></sec></sec></sec><sec id=\"Sec37\"><title>Conclusion</title><p id=\"Par39\">It has also been reported in our previous study that amygdalin can act as an active inhibitor of G-6-P synthase enzyme based upon the results of molecular docking and ADMET data<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. In current study the amygdalin derivatives were found active G-6-P synthase inhibitors, and molecular docking study, provided a new insight of mechanism for the inhibition with visual binding interactions. The derivatives of amygdalin ((E)-(6-((6-(cyano(phenyl)methoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2yl)methoxy)-3,4,5-trihydroxy-tetrahydro-2H-pyran-2-yl)methyl3-(4-hydroxy-3,5 dimethoxyphenyl) acrylate) showed antioxidant, antimicrobial, better preservative efficacy and prevent the change in pH as well microbial count of formulation for food as well as pharmaceutical products, which were in agreement with the results of molecular docking and highlight the mechanism of their preservative activity. Therefore, the synthesized amygdalin derivatives can be used as novel food and pharmaceutical preservatives to prevent them from microbial degradation.</p></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><ack><title>Acknowledgements</title><p>Authors are grateful to the Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak, Haryana (India) for providing the necessary facilities to carry out this research work.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>The authors A.L., S.S. and A.K. have designed, synthesized and carried out the work in equal contribution. All authors have read and approved the final manuscript.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par40\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Black</surname><given-names>MT</given-names></name></person-group><article-title>Evidence that the catalytic activity of prokaryote leader peptidase depends upon the operation of a serine-lysine catalytic dyad</article-title><source>J. Bacteriol.</source><year>1993</year><volume>175</volume><fpage>4957</fpage><lpage>4961</lpage><pub-id pub-id-type=\"doi\">10.1128/jb.175.16.4957-4961.1993</pub-id><pub-id pub-id-type=\"pmid\">8394311</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bockstael</surname><given-names>K</given-names></name><name><surname>Aerschot</surname><given-names>AV</given-names></name></person-group><article-title>Antimicrobial resistance in bacteria</article-title><source>Central Eur. J. Med.</source><year>2009</year><volume>4</volume><fpage>141</fpage><lpage>155</lpage><pub-id pub-id-type=\"doi\">10.2478/s11536-008-0088-9</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Darbre</surname><given-names>PD</given-names></name><name><surname>Harvey</surname><given-names>PW</given-names></name></person-group><article-title>Paraben esters: Review of recent studies of endocrine toxicity, absorption, esterase and human exposure, and discussion of potential human health risks</article-title><source>J. Appl. Toxicol.</source><year>2008</year><volume>28</volume><fpage>561</fpage><lpage>578</lpage><pub-id pub-id-type=\"doi\">10.1002/jat.1358</pub-id><pub-id pub-id-type=\"pmid\">18484575</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tavares</surname><given-names>RS</given-names></name><name><surname>Martins</surname><given-names>FC</given-names></name><name><surname>Oliveira</surname><given-names>PJ</given-names></name><name><surname>Ramalho-Santos</surname><given-names>J</given-names></name><name><surname>Peixoto</surname><given-names>FP</given-names></name></person-group><article-title>Parabens in male infertility is there a mitochondrial connection</article-title><source>Reprod. Toxicol.</source><year>2009</year><volume>27</volume><fpage>1</fpage><lpage>7</lpage><pub-id pub-id-type=\"doi\">10.1016/j.reprotox.2008.10.002</pub-id><pub-id pub-id-type=\"pmid\">19007877</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lundov</surname><given-names>MD</given-names></name><name><surname>Moesby</surname><given-names>L</given-names></name><name><surname>Zachariae</surname><given-names>C</given-names></name><name><surname>Johansen</surname><given-names>JD</given-names></name></person-group><article-title>Contamination versus preservation of cosmetics: A review on legislation, usage, infections, and contact allergy</article-title><source>Contact Dermatol.</source><year>2009</year><volume>60</volume><fpage>70</fpage><lpage>80</lpage><pub-id pub-id-type=\"doi\">10.1111/j.1600-0536.2008.01501.x</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kahne</surname><given-names>D</given-names></name><name><surname>Leimkuhler</surname><given-names>C</given-names></name><name><surname>Lu</surname><given-names>W</given-names></name><name><surname>Walsh</surname><given-names>C</given-names></name></person-group><article-title>Glycopeptide and lipo glycopeptides antibiotics</article-title><source>Chem. Rev.</source><year>2005</year><volume>105</volume><fpage>425</fpage><lpage>448</lpage><pub-id pub-id-type=\"doi\">10.1021/cr030103a</pub-id><pub-id pub-id-type=\"pmid\">15700951</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lange</surname><given-names>RP</given-names></name><name><surname>Locher</surname><given-names>HH</given-names></name><name><surname>Wyss</surname><given-names>PC</given-names></name><name><surname>Then</surname><given-names>RL</given-names></name></person-group><article-title>The targets of currently used antibacterial agents: lessons for drug discovery</article-title><source>C. P. D.</source><year>2007</year><volume>13</volume><fpage>3140</fpage><lpage>3154</lpage><pub-id pub-id-type=\"doi\">10.1101/cshperspect.a030239</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Satyendra</surname><given-names>RV</given-names></name><etal/></person-group><article-title>In vitro antimicrobial and molecular docking of dichloro substituted benzoxazole derivatives</article-title><source>Med. Chem. Res.</source><year>2012</year><volume>21</volume><fpage>4193</fpage><lpage>4199</lpage><pub-id pub-id-type=\"doi\">10.1186/s13065-017-0347-4</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dhiman</surname><given-names>P</given-names></name><name><surname>Malik</surname><given-names>N</given-names></name><name><surname>Khatkar</surname><given-names>A</given-names></name></person-group><article-title>Docking related survey on natural product based new monoamine oxidase inhibitors and their therapeutic potential</article-title><source>C. C. H. T. S.</source><year>2017</year><volume>20</volume><fpage>474</fpage><lpage>491</lpage><pub-id pub-id-type=\"doi\">10.2174/1386207320666170414102814</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Koehn</surname><given-names>FE</given-names></name><name><surname>Carter</surname><given-names>GT</given-names></name></person-group><article-title>The evolving role of natural products in drug discovery</article-title><source>Nat. Revol. Drug Dis.</source><year>2005</year><volume>4</volume><fpage>206</fpage><lpage>209</lpage><pub-id pub-id-type=\"doi\">10.1038/nrd1657</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Santos</surname><given-names>PLP</given-names></name><name><surname>Schilthuizen</surname><given-names>M</given-names></name><name><surname>Verpoorte</surname><given-names>R</given-names></name><name><surname>Choi</surname><given-names>YH</given-names></name></person-group><article-title>Quantitative analysis of amygdalin and prunasin in <italic>Prunusserotina Ehrh</italic>. Using (1) H-NMR spectroscopy</article-title><source>Phytochem. Anal.</source><year>2014</year><volume>25</volume><fpage>122</fpage><lpage>126</lpage><pub-id pub-id-type=\"doi\">10.1002/pca.2476</pub-id><pub-id pub-id-type=\"pmid\">24115144</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Enculescu</surname><given-names>M</given-names></name></person-group><article-title>Vitamin B17/laetrile/amygdalin (a review)</article-title><source>Bull. UASVM Anim. Sci. Biotechnol.</source><year>2009</year><volume>66</volume><fpage>1</fpage><lpage>2</lpage><pub-id pub-id-type=\"doi\">10.15835/buasvmcn-asb:66:1-2:3316</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Du</surname><given-names>HK</given-names></name><name><surname>Song</surname><given-names>FC</given-names></name><name><surname>Zhou</surname><given-names>X</given-names></name><name><surname>Li</surname><given-names>H</given-names></name><name><surname>Zhang</surname><given-names>JP</given-names></name></person-group><article-title>Effect of amygdalin on serum proteinic biomarker in pulmonary fibrosis of bleomycin-induced rat</article-title><source>Chin. J. Ind. Hyg. Occup. Dis.</source><year>2010</year><volume>28</volume><fpage>260</fpage><lpage>263</lpage></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mirmiranpour</surname><given-names>H</given-names></name><etal/></person-group><article-title>Amygdalin inhibits angiogenesis in the cultured endothelial cells of diabetic rats</article-title><source>Indian J. Patho. Microbiol.</source><year>2012</year><volume>55</volume><fpage>211</fpage><lpage>214</lpage><pub-id pub-id-type=\"doi\">10.4103/0377-4929.97874</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hyun</surname><given-names>SW</given-names></name><etal/></person-group><article-title>Apricot kernel extract and amygdalin inhibit urban particulate matter induced kerato conjunctivitis sicca</article-title><source>Molecules</source><year>2019</year><volume>24</volume><fpage>1</fpage><lpage>12</lpage><pub-id pub-id-type=\"doi\">10.3390/molecules24030650</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><mixed-citation publication-type=\"other\">Al-Bakri, S. A., Nima, Z. A., Jabri, R. R. & Ajeel, E. A. Antibacterial activity of Apricot kernel extracts containing amygdalin. <italic>Iraqi J. Sci.</italic><bold>51</bold>, 571–576; <ext-link ext-link-type=\"uri\" xlink:href=\"https://pdfs.semanticscholar.org/9859/192679896c46a4546c63feba49b90bb\">https://pdfs.semanticscholar.org/9859/192679896c46a4546c63feba49b90bb</ext-link> 14ea7.pdf (2010).</mixed-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bhattacharya</surname><given-names>S</given-names></name><name><surname>Bhayani</surname><given-names>K</given-names></name><name><surname>Ghosh</surname><given-names>T</given-names></name><name><surname>Bajaj</surname><given-names>S</given-names></name><name><surname>Trivedi</surname><given-names>N</given-names></name></person-group><article-title>Stability of phycobiliproteins using natural preservative ε-polylysine (ε-PL)</article-title><source>Ferment. Technol.</source><year>2018</year><volume>7</volume><fpage>149</fpage><pub-id pub-id-type=\"doi\">10.4172/2167-7972.1000149</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Proestos</surname><given-names>C</given-names></name><name><surname>Boziaris</surname><given-names>IS</given-names></name><name><surname>Nychas</surname><given-names>GJE</given-names></name><name><surname>Komaitis</surname><given-names>M</given-names></name></person-group><article-title>Analysis of flavonoids and phenolic acids in greek aromatic plants: Investigation of their antioxidant capacity and antimicrobial activity</article-title><source>Food Chem.</source><year>2006</year><volume>95</volume><fpage>664</fpage><lpage>671</lpage><pub-id pub-id-type=\"doi\">10.1016/j.foodchem.2005.01.049</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ou</surname><given-names>S</given-names></name><name><surname>Kwok</surname><given-names>KC</given-names></name></person-group><article-title>Ferulic acid: Pharmaceutical functions, preparation and applications in foods</article-title><source>J. Sci. Food Agric.</source><year>2004</year><volume>84</volume><fpage>1261</fpage><lpage>1269</lpage><pub-id pub-id-type=\"doi\">10.1002/jsfa.1873</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Khatkar</surname><given-names>A</given-names></name><name><surname>Nanda</surname><given-names>A</given-names></name><name><surname>Narasimhan</surname><given-names>B</given-names></name></person-group><article-title>Stability studies of the selected derivatives of ferulic acid, gallic acid and p-coumaric acid as per the ICH guidelines</article-title><source>HYGEIA J. Drugs Res.</source><year>2013</year><volume>5</volume><fpage>34</fpage><lpage>42</lpage><pub-id pub-id-type=\"doi\">10.1021/jf011348w</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Friesner</surname><given-names>RA</given-names></name><etal/></person-group><article-title>Extra precision glide: Docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes</article-title><source>J. Med. Chem.</source><year>2006</year><volume>49</volume><fpage>6177</fpage><lpage>6196</lpage><pub-id pub-id-type=\"doi\">10.1021/jm051256o</pub-id><pub-id pub-id-type=\"pmid\">17034125</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><mixed-citation publication-type=\"other\">Kriza, A., Ignat, I., Stanica, N. & Draghici, C. Synthesis and characterization of Cu(II), Co(II) and Ni(II) complexes with Schiff bases derived from isatin. <italic>Revista Chimie</italic>, <bold>62</bold>, 696–701; <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.orientjchem.org/?p=23200\">https://www.orientjchem.org/?p=23200</ext-link> (2011).</mixed-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Blois</surname><given-names>MS</given-names></name></person-group><article-title>Antioxidant determinations by the use of a stable free radical</article-title><source>Nature</source><year>1958</year><volume>181</volume><fpage>1199</fpage><lpage>1200</lpage><pub-id pub-id-type=\"doi\">10.1038/1811199a0</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><mixed-citation publication-type=\"other\">Indian Pharmacopoeia Vol-I. Indian Pharmacopoeia Commission. The Controller of Publications, New Delhi, 37; <ext-link ext-link-type=\"uri\" xlink:href=\"http://pharmacentral.in/wp-content/uploads/2018/05/INDIAN%20PHARMACOPOEIA%202007.pdf\">https://pharmacentral.in/wp-content/uploads/2018/05/INDIAN%20PHARMACOPOEIA%202007.pdf</ext-link> (2007).</mixed-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Andrews</surname><given-names>JM</given-names></name></person-group><article-title>Determination of minimum inhibitory concentration</article-title><source>J. Antimicrob. Chem.</source><year>2001</year><volume>48</volume><fpage>5</fpage><lpage>16</lpage><pub-id pub-id-type=\"doi\">10.1093/jac/48.suppl_1.5</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><mixed-citation publication-type=\"other\">Maughan, R. G., Poore, R. A. & Phan, B. V. Product and procedure for stabilizing Aloe vera gel, US Patent, 0211182A1; <ext-link ext-link-type=\"uri\" xlink:href=\"https://patents.google.com/patent/US7033620B2/en\">https://patents.google.com/patent/US7033620B2/en</ext-link> (2006).</mixed-citation></ref><ref id=\"CR27\"><label>27.</label><mixed-citation publication-type=\"other\">Talib, M. I., Wayal, R. R. & Parate, V. R. Development of Aloevera based Ready to Serve Soft drink. <italic>Int. Conference on Global Trends in Engineering, Tech. Mgt.</italic> 216–233; <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.ijettjournal.org/Special%20issue/ICGTETM-2016/ICGTETM_2016_paper_69.pdf\">https://www.ijettjournal.org/Special%20issue/ICGTETM-2016/ICGTETM_2016_paper_69.pdf</ext-link> (2016).</mixed-citation></ref><ref id=\"CR28\"><label>28.</label><mixed-citation publication-type=\"other\">Narang, R., Narasimhan, B., Judge, V., Ohlan, S. & Ohlan, R. Evaluaton of reparation effectiveness in an official antacid reparation. <italic>Acta Pharmaceutica Sciencia</italic>, <bold>51</bold>, 2259; <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.actapharmsci.com/abstract.php?id=17\">https://www.actapharmsci.com/abstract.php?id=17</ext-link> (2009).</mixed-citation></ref><ref id=\"CR29\"><label>29.</label><mixed-citation publication-type=\"other\">Indian Pharmacopoeia. Indian Pharmacopoeia Commission, Ghaziabad, India, 27–28; <ext-link ext-link-type=\"uri\" xlink:href=\"https://igmpiindia.Org/ipc.pdf\">https://igmpiindia.Org/ipc.pdf</ext-link> (2010).</mixed-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ahlawat</surname><given-names>KS</given-names></name><name><surname>Khatkar</surname><given-names>BS</given-names></name></person-group><article-title>Processing, food applications and safety of aloe vera products: A review</article-title><source>J. Food Sci. Technol.</source><year>2011</year><volume>48</volume><fpage>525</fpage><lpage>533</lpage><pub-id pub-id-type=\"doi\">10.1007/s13197-011-0229-z</pub-id><pub-id pub-id-type=\"pmid\">23572784</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Khatkar</surname><given-names>A</given-names></name><name><surname>Nanda</surname><given-names>A</given-names></name><name><surname>Kumar</surname><given-names>P</given-names></name><name><surname>Narasimhan</surname><given-names>B</given-names></name></person-group><article-title>Synthesis, antimicrobial evaluation and QSAR studies of p-coumaric acid derivatives</article-title><source>Arab. J. Chem.</source><year>2017</year><volume>10</volume><fpage>S3804</fpage><lpage>S3815</lpage><pub-id pub-id-type=\"doi\">10.1016/j.arabjc.2014.05.018</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dafale</surname><given-names>NA</given-names></name><name><surname>Semwal</surname><given-names>UP</given-names></name><name><surname>Agarwal</surname><given-names>PK</given-names></name><name><surname>Sharma</surname><given-names>P</given-names></name><name><surname>Singh</surname><given-names>GN</given-names></name></person-group><article-title>Evaluation of preservative effectiveness in antacid, cough syrup and ophthalmic solution by microbial challenge test</article-title><source>Int. J. Pharmacy</source><year>2014</year><volume>1</volume><fpage>193</fpage><lpage>199</lpage><pub-id pub-id-type=\"doi\">10.1111/j.1365-2710.2012.01354.x</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><mixed-citation publication-type=\"other\">The United States Pharmacopoeia. Antimicrobial effectiveness testing. Rockville: <italic>United States Pharmacopoeial Conventon Inc</italic>., 214850; <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.drugfuture.com/Pharmacopoeia/usp35/PDF/00520054%20%5B51%5D\">https://www.drugfuture.com/Pharmacopoeia/usp35/PDF/00520054%20%5B51%5D</ext-link>% 20ANTIMICROBIA%20EFFECTIVENESS%20TESTING.pdf (2004).</mixed-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lather</surname><given-names>A</given-names></name><name><surname>Sharma</surname><given-names>S</given-names></name><name><surname>Khatkar</surname><given-names>A</given-names></name></person-group><article-title>Virtual screening of novel glucosamine-6-phosphate synthase inhibitors</article-title><source>C. C. H. T. S.</source><year>2018</year><volume>21</volume><fpage>1</fpage><lpage>12</lpage><pub-id pub-id-type=\"doi\">10.2174/1386207321666180330114457</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Friesner</surname><given-names>RA</given-names></name><etal/></person-group><article-title>Glide: A new approach for rapid, accurate docking and scoring method and assessment of docking accuracy</article-title><source>J. Med. Chem.</source><year>2004</year><volume>47</volume><fpage>1739</fpage><lpage>1749</lpage><pub-id pub-id-type=\"doi\">10.1021/jm0306430</pub-id><pub-id pub-id-type=\"pmid\">15027865</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hopkins</surname><given-names>AL</given-names></name><name><surname>Groom</surname><given-names>CR</given-names></name></person-group><article-title>The drug gable genome</article-title><source>Nat. Revol. Drug Dis.</source><year>2002</year><volume>1</volume><fpage>727</fpage><lpage>733</lpage><pub-id pub-id-type=\"doi\">10.1038/nrd892</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Veber</surname><given-names>DF</given-names></name><etal/></person-group><article-title>Molecular properties that influence the oral bioavailability of drug candidates</article-title><source>J. Med. Chem.</source><year>2002</year><volume>45</volume><fpage>2615</fpage><lpage>2623</lpage><pub-id pub-id-type=\"doi\">10.1021/jm020017n</pub-id><pub-id pub-id-type=\"pmid\">12036371</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Irvine</surname><given-names>JD</given-names></name><etal/></person-group><article-title>MDCK (Madin Darby Canine Kidney) cells: A tool for membrane permeability screening</article-title><source>J. Pharmaceut. Sci.</source><year>1999</year><volume>88</volume><fpage>28</fpage><lpage>33</lpage><pub-id pub-id-type=\"doi\">10.1021/js9803205</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vemula</surname><given-names>PK</given-names></name><name><surname>Li</surname><given-names>J</given-names></name><name><surname>John</surname><given-names>G</given-names></name></person-group><article-title>Enzyme catalysis: Tool to make and break amygdalin hydrogelators from renewable resources: a delivery model for hydrophobic drugs</article-title><source>J. Am. Chem. Soc.</source><year>2006</year><volume>128</volume><fpage>8932</fpage><lpage>8938</lpage><pub-id pub-id-type=\"doi\">10.1021/ja062650u</pub-id><pub-id pub-id-type=\"pmid\">16819889</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grzesik</surname><given-names>M</given-names></name><name><surname>Naparło</surname><given-names>K</given-names></name><name><surname>Bartosz</surname><given-names>G</given-names></name><name><surname>Sadowska</surname><given-names>BI</given-names></name></person-group><article-title>Antioxidant properties of catechins: Comparison with other antioxidants</article-title><source>Food Chem.</source><year>2018</year><volume>241</volume><fpage>480</fpage><lpage>492</lpage><pub-id pub-id-type=\"doi\">10.1016/j.foodchem.2017.08.117</pub-id><pub-id pub-id-type=\"pmid\">28958556</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manzocco</surname><given-names>L</given-names></name><name><surname>Anese</surname><given-names>M</given-names></name><name><surname>Nicoli</surname><given-names>MC</given-names></name></person-group><article-title>Antioxidant propertiesof tea extracts as affected by processing</article-title><source>Lebens-mittel-Wissenschaft Und-Technologie</source><year>1998</year><volume>31</volume><fpage>694</fpage><lpage>698</lpage><pub-id pub-id-type=\"doi\">10.1006/fstl.1998.0491</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Piao</surname><given-names>XL</given-names></name><name><surname>Park</surname><given-names>IH</given-names></name><name><surname>Baek</surname><given-names>SH</given-names></name><name><surname>Kim</surname><given-names>HY</given-names></name><name><surname>Park</surname><given-names>MK</given-names></name><name><surname>Park</surname><given-names>JH</given-names></name></person-group><article-title>Antioxidative activity of furanocoumarins isolated from <italic>Angelica edahuricae</italic></article-title><source>J. Ethnopharmacol.</source><year>2004</year><volume>93</volume><fpage>243</fpage><lpage>246</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jep.2004.03.054</pub-id><pub-id pub-id-type=\"pmid\">15234759</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Basanta</surname><given-names>MF</given-names></name><etal/></person-group><article-title>Antioxidant Japanese plum (<italic>Prunussalicina</italic>) microparticles with potential for food preservation</article-title><source>J. Funct. Foods.</source><year>2016</year><volume>24</volume><fpage>287</fpage><lpage>296</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jff.2016.04.015</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Nat Commun</journal-id><journal-id journal-id-type=\"iso-abbrev\">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type=\"epub\">2041-1723</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807781</article-id><article-id pub-id-type=\"pmc\">PMC7431537</article-id><article-id pub-id-type=\"publisher-id\">17952</article-id><article-id pub-id-type=\"doi\">10.1038/s41467-020-17952-5</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-3737-2468</contrib-id><name><surname>Coelho</surname><given-names>Matthew A.</given-names></name><address><email>matthew.coelho@sanger.ac.uk</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>De Braekeleer</surname><given-names>Etienne</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Firth</surname><given-names>Mike</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Bista</surname><given-names>Michal</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lukasiak</surname><given-names>Sebastian</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-6429-2290</contrib-id><name><surname>Cuomo</surname><given-names>Maria Emanuela</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-6101-3786</contrib-id><name><surname>Taylor</surname><given-names>Benjamin J. M.</given-names></name><address><email>benjamin.taylor@astrazeneca.com</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.417815.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 5929 4381</institution-id><institution>Discovery Sciences, R&D, AstraZeneca, </institution></institution-wrap>Cambridge, UK </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.417815.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 5929 4381</institution-id><institution>Oncology R&D, AstraZeneca, </institution></institution-wrap>Cambridge, UK </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.10306.34</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0606 5382</institution-id><institution>Present Address: Wellcome Sanger Institute, </institution></institution-wrap>Hinxton, Cambridge CB10 1RQ UK </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>4132</elocation-id><history><date date-type=\"received\"><day>23</day><month>5</month><year>2019</year></date><date date-type=\"accepted\"><day>23</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Precise genome editing using CRISPR-Cas9 is a promising therapeutic avenue for genetic diseases, although off-target editing remains a significant safety concern. Guide RNAs shorter than 16 nucleotides in length effectively recruit Cas9 to complementary sites in the genome but do not permit Cas9 nuclease activity. Here we describe CRISPR <underline>Gu</underline>ide RNA <underline>A</underline>ssisted <underline>R</underline>eduction of <underline>D</underline>amage (CRISPR GUARD) as a method for protecting off-targets sites by co-delivery of short guide RNAs directed against off-target loci by competition with the on-target guide RNA. CRISPR GUARD reduces off-target mutagenesis while retaining on-target editing efficiencies with Cas9 and base editor. However, we discover that short guide RNAs can also support base editing if they contain cytosines within the deaminase activity window. We explore design rules and the universality of this method through in vitro studies and high-throughput screening, revealing CRISPR GUARD as a rapidly implementable strategy to improve the specificity of genome editing for most genomic loci. Finally, we create an online tool for CRISPR GUARD design.</p></abstract><abstract id=\"Abs2\" abstract-type=\"web-summary\"><p id=\"Par2\">Off-target editing remains a concern for therapeutic applications of CRISPR-Cas9. Here the authors present CRISPR GUARD, which uses very short non-cleaving gRNAs to prevent editing at off-target sites.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Mutagenesis</kwd><kwd>Targeted gene repair</kwd><kwd>CRISPR-Cas9 genome editing</kwd><kwd>CRISPR-Cas9 genome editing</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par3\">A major limitation of gene editing with CRISPR-Cas9 systems is off-target mutagenesis through guide RNA (gRNA) directed binding at closely matched sequences in the genome<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Although gRNA design algorithms are under continual refinement<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>, off-target activity can be unavoidable when the gRNA window is restricted to a narrow genomic location, such as for therapeutic correction of disease causing mutations<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Protein engineering strategies have resulted in higher-fidelity Cas9 variants that reduce, but do not eliminate, off-target mutations; such variants can also show impaired on-target activity<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. While off-target mutations remain a persistent problem, they can be readily detected using a variety of methodologies<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref>–<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. A system that is not restricted to a Cas9 variant and further reduces or eliminates particularly detrimental off-target mutations whilst retaining on-target activity would be invaluable for therapeutic strategies. Here, we develop CRISPR GUARD, a methodology that aims to block mismatched gRNAs from binding off-target sites through competition with an inactive Cas9 complex. The inactive complexes are generated by Cas9 binding short gRNAs, or GUARD RNAs, with perfect complementary to the off-target site; gRNAs shorter than 16 nucleotides (nt) in length can direct Cas9 binding but do not support nuclease activity<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>–<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. This method can be adapted to use catalytically inactive Cas9 (dCas9), related RNA-guided nucleases, base editors or other sequence-specific DNA binding proteins to form inert complexes occupying off-target loci to render them inaccessible (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>).<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>CRISPR GUARD protection of Cas9 off-target sites.</title><p>Schematic showing how short gRNAs (orange) forming catalytically inactive complexes with Cas9 can occupy specific off-target sites in the genome and compete with the mismatched nuclease competent gRNA (black), thereby providing protection from Cas9-mediated DNA cleavage. Mismatches in the gRNA are shown in red. RNA bases overlapping the gRNA PAM are shown in bold.</p></caption><graphic xlink:href=\"41467_2020_17952_Fig1_HTML\" id=\"d30e377\"/></fig></p></sec><sec id=\"Sec2\" sec-type=\"results\"><title>Results</title><sec id=\"Sec3\"><title>In vitro protection of Cas9 off-targets with CRISPR GUARD</title><p id=\"Par4\">We tested the concept of CRISPR GUARD by measuring the binding kinetics of a perfectly complementary 15-nt GUARD RNA versus a mismatched gRNA to an immobilised DNA off-target template for a gRNA targeting <italic>VEGFA</italic> with four mismatches<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>–<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Bio-layer interferometry (BLI) revealed comparable off-target association kinetics for catalytically inactive Cas9 complexed with GUARD RNA or mismatched gRNA, with 29 ± 4% slower binding for the GUARD RNA (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a</xref>), suggesting that competition at off-target loci is feasible. To test the possibility of DNA protection by CRISPR GUARD, we selected an array of potential GUARD RNA designs for in vitro Cas9 DNA cleavage assays. We considered GUARD RNA lengths of 14-nt or 15-nt, or a full length 20-nt design with only 15-nt of target complementarity (15-nt+ spacer). GUARD RNAs were designed as competitive molecules that are truncated versions of the on-target gRNA but incorporate mismatches found at the off-target site (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2b</xref>). Alternatively, we designed proximal GUARD RNAs that bind flanking regions of the off-target site, speculating that the reduced sequence homology would reduce competition at the on-target site, but still block the protospacer and protospacer adjacent motif (PAM). We termed these designs as competitive and non-competitive GUARD RNAs, respectively (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2b</xref>). Firstly, we confirmed that GUARD RNAs cannot direct nuclease activity by in vitro assessment of on-target and off-target cleavage using short purified DNA targets. None of the GUARD RNAs directed DNA cleavage when complexed with Cas9 alone (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2c</xref>). Cas9 complexed with <italic>VEGFA</italic> gRNA showed robust cleavage of both on-target, and off-target <italic>CAVIN4</italic> DNA. However, addition of non-competitive GUARD RNAs failed to protect the off-target site. Significant protection was only observed using a 15-nt competitive GUARD RNA, but on-target cleavage was also impaired suggesting competition at both sites (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2c</xref>). Competition with the on-target gRNA may be particularly pertinent when there are very few mismatches, or when the mismatches of the gRNA cannot be incorporated in the truncated GUARD RNA design.<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>GUARD RNAs reduce Cas9 off-target cleavage activity without affecting on-target editing.</title><p><bold>a</bold> Bio-layer interferometry (BLI) analysis of binding kinetics for dead Cas9 ribonucleoprotein complex to an immobilised biotinylated DNA substrate. Cas9 was precomplexed with tracrRNA and (NT) non-targeting control gRNA; (gRNA) 20-nt <italic>VEGFA</italic> gRNA with 4 mismatches; (GUARD RNA) a 15-nt GUARD RNA targeting the off-target site or no guide RNA. <bold>b</bold> CRISPR GUARD design. Schematic showing GUARD RNAs designs. Variation in length (14-nt versus 15-nt) and protospacer positioning likely influence binding energy and competition with the on-target gRNA (i.e., competitive/overlapping, non-competitive/proximal). Mismatches in the gRNA are shown in red. RNA bases overlapping the gRNA PAM are shown in bold. <bold>c</bold> Competition between on-target gRNA and GUARD RNA can be reduced by proximal positioning. On-target (<italic>VEGFA</italic>) and off-target (<italic>CAVIN4</italic>) DNA abundance was measured by qPCR following an in vitro Cas9 DNA cleavage assay. The corresponding GUARD RNA was added at a molar ratio of 5:1 to the <italic>VEGFA</italic> on-target gRNA (100 nM to 20 nM). <bold>d</bold> GUARD RNAs are more effective at blocking Cas9-mediated DNA cutting with pre-incubation in vitro. On-target (<italic>VEGFA</italic>) and off-target (<italic>CAVIN4</italic>) DNA abundance was measured by qPCR following an in vitro Cas9 DNA cleavage assay, with a 30 min pre-incubation with GUARD RNAs and Cas9, before addition of the on-target gRNA and Cas9. Increasing molar ratios of GUARD RNA to the <italic>VEGFA</italic> on-target gRNA were used (10:10, 20:10, 50:10, 100:10 nM). <bold>e</bold> GUARD RNAs are effective in blocking Cas9-mediated DNA cleavage of off-target sites within a 10 bp window of the gRNA PAM. Synthetic DNA fragments containing the <italic>VEGFA</italic> gRNA off-target site <italic>CAVIN4</italic> positioned progressively further away from the GUARD RNA binding site were quantified by qPCR following an in vitro Cas9 DNA cleavage assay (50:10 nM GUARD RNA to gRNA ratio). <bold>f</bold> CRISPR GUARD is effective at blocking off-target editing in cells. Indel rates from NGS of amplicons from Cas9-expressing HEK293 cells transfected with <italic>VEGFA</italic> gRNA and multiple GUARD RNA designs for protection of the <italic>CAVIN4</italic> proximal off-target site (25:25 nM GUARD RNA to gRNA ratio). Data represent the mean of two (<bold>f</bold>) independent experiments, or the mean ± SD of three (<bold>c, e</bold>) or five (<bold>d</bold>) independent experiments with symbols representing each replicate. For <bold>a</bold>, data are representative of two independent experiments. NT non-targeting gRNA, UTC untransfected control. Unpaired, two-tailed student’s <italic>t</italic>-test. For <bold>c</bold>, *<italic>P</italic> = 0.0208, **<italic>P</italic> = 0.0083. For <bold>d</bold>, *<italic>P</italic> = 0.0164, **<italic>P</italic> = 0.0021 (<italic>VEGFA</italic>) and **<italic>P</italic> = 0.007 (<italic>CAVIN4</italic>), ***<italic>P</italic> = 0.0003, ***<italic>P</italic> < 0.0001. For <bold>e</bold>, **<italic>P</italic> = 0.0041 and ***<italic>P</italic> = 0.0001. Source data are provided as a Source Data file.</p></caption><graphic xlink:href=\"41467_2020_17952_Fig2_HTML\" id=\"d30e540\"/></fig></p><p id=\"Par5\">Cas9-gRNA complexes show rapid target binding and cleavage in vitro<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. The failure of GUARD RNAs to protect off-target sites could therefore be due to these rapid kinetics. In line with this, a 30-min pre-incubation of Cas9 and a 14-nt non-competitive GUARD RNA with the DNA substrate resulted in robust protection of the off-target DNA, even at a 1:1 ratio of GUARD RNA to gRNA (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2d</xref>). Further increasing the ratio of GUARD RNA to a five-fold excess led to complete protection from cleavage. In addition, on-target cleavage could be completely prevented using a competitive 14-nt GUARD RNA against <italic>VEGFA</italic>, demonstrating that GUARD RNAs can compete against perfectly complementary gRNAs (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2d</xref>). On-target protection of <italic>VEGFA</italic> required a higher ratio of GUARD RNA to gRNA, presumably due to the higher binding affinity of the on-target gRNA.</p><p id=\"Par6\">To better understand GUARD RNA positioning rules, we designed an in vitro Cas9 DNA cleavage assay for the <italic>VEGFA</italic> off-target site <italic>CAVIN4</italic>, whereby the same non-competitive GUARD RNA binding site was positioned incrementally further away within a synthetic DNA fragment (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>). After incubation with Cas9 complexes, the remaining uncleaved DNA was quantified by qPCR. In this way, we could determine the optimal distance between the GUARD RNA and the off-target region for protection from Cas9 nuclease. As expected, the 14-nt non-competitive GUARD RNA against the <italic>CAVIN4</italic> off-target site provided significant protection when it was overlapping the off-target site, but also proved similarly effective when placed 10 bp away from the gRNA PAM (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2e</xref>). However, GUARD RNAs positioned 25 and 50 bp distal to the off-target gRNA PAM on either the 5’ or 3’ flank provided no protection (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2e</xref>), implying that GUARD RNAs are effective in rendering an off-target region inaccessible for editing if positioned ≤10 bp away from the gRNA PAM.</p></sec><sec id=\"Sec4\"><title>Reduced off-target editing without affecting on-target editing</title><p id=\"Par7\">Next, we tested the cellular activity of CRISPR GUARD. Using a Cas9-expressing HEK293 cell line<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>, we co-transfected a <italic>VEGFA</italic> gRNA and multiple GUARD RNA designs (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2b</xref>) at equimolar ratios for protection of the <italic>CAVIN4</italic> proximal off-target site and performed next-generation sequencing (NGS) of amplicons to detect insertion and deletions (indels). Strikingly, all tested GUARD RNA designs significantly protected the off-target site (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2f</xref>). Notably, none of the GUARD RNA designs significantly interfered with on-target cutting efficiency, including the competitive GUARD RNA. Furthermore, unlike the in vitro scenario, phased delivery of the GUARD RNA before the gRNA provided no additional benefit (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>). We reasoned that, in contrast to the in vitro setting, the kinetics of Cas9 binding to the off-target region is slower due to scanning of the mammalian genome, thus concomitant delivery of GUARD RNA is sufficient to allow for effective competition at off-target sites and may also reduce the effects of direct competition at the on-target locus with competitive GUARD RNAs. Due to the apparent efficacy of the 14-nt and 15-nt GUARDs in vitro and in cells, we disregarded the 15-nt GUARD RNA with a 5-nt mismatched spacer sequence (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2b</xref>), as this provided the least protection (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2f</xref>) and could potentially give rise to a catalytically active Cas9 complex if the GUARD RNA binds to a DNA site where the mismatched spacer has >2-nt of base pairing.</p><p id=\"Par8\">Next, we investigated the optimal dosing for CRISPR GUARD. We co-transfected a gRNA against <italic>VEGFA</italic> and increasing concentrations of GUARD RNA to protect the <italic>CAVIN4</italic> proximal off-target site. Although an equimolar ratio of GUARD RNA to gRNA was effective in reducing off-target indel rates (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2f</xref>), higher concentrations of GUARD RNA provided additional reduction of off-target editing (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref>). Notably, the highest concentration of GUARD RNA (10:1 ratio) reduced off-target indel rates to background levels (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref>). We noted that indel rates in untransfected control cells for the <italic>CAVIN4</italic> proximal region are approximately 0.3%, owing to intrinsic error in the NGS of a polymeric cytosine tract at this locus. We assume that Cas9 concentration is in excess in the cell as increasing the ratio of GUARD RNA to gRNA did not have a negative impact on on-target editing, so in this context we recommend routinely adopting a 5:1 ratio of GUARD RNA to gRNA.<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Protection of multiple, endogenous off-target sites with CRISPR GUARD.</title><p><bold>a</bold> Increasing GUARD RNA concentration leads to complete protection of Cas9 off-target sites in cells. Indel rates from NGS of amplicons from Cas9-expressing HEK293 cells transfected with <italic>VEGFA</italic> gRNA and increasing concentrations of 14-nt GUARD RNA for protection of the <italic>CAVIN4</italic> off-target site (10:10, 20:10, 50:10, 100:10 nM). The total concentration of delivered RNA in each case was kept constant by co-delivery of NT gRNA (first condition is 100 nM NT). <bold>b</bold> Indel rates from amplicons sequencing of Cas9-expressing HEK293 cells transfected with <italic>EMX1</italic> or <italic>VEGFA</italic> on-target gRNAs, and a single GUARD RNA (25:25 nM ratio). GUARD RNA positioning relative to the off-target protospacer sequence is shown schematically in each panel. Data represent the mean of two independent experiments with symbols representing each replicate. Source data are provided as a Source Data file.</p></caption><graphic xlink:href=\"41467_2020_17952_Fig3_HTML\" id=\"d30e659\"/></fig></p><p id=\"Par9\">To determine if CRISPR GUARD was also effective in the therapeutically relevant setting of Cas9 ribonucleoprotein (RNP) delivery, we separately precomplexed catalytically inactive Cas9 (dCas9) protein with a GUARD RNA against <italic>VEGFA</italic> off-target site <italic>TENT4A</italic>, and wild-type Cas9 protein with the <italic>VEGFA</italic> on-target guide RNA, to co-deliver these RNPs for protection and cutting, respectively. In principle, it is an option to use a full-length 20-nt gRNA for protection with dCas9. However, we continued to use 14-nt GUARD RNAs in this scenario in order to prevent guide swapping in the cell<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>, whereby wild-type Cas9 acquires full-length gRNA against the off-target site. Encouragingly, CRISPR GUARD using RNPs successfully protected <italic>VEGFA</italic> off-target <italic>TENT4A</italic>, and there was no detectable impact of protection of the <italic>TENT4A</italic> off-target site on the other known off-target sites by redistribution of active Cas9 (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>).</p><p id=\"Par10\">To begin to understand GUARD RNA design rules, we assessed the activity of an array of GUARD RNAs protecting known endogenous off-target sites of <italic>EMX1</italic> and <italic>VEGFA</italic> gRNAs<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>–<xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>, assessing nucleotide composition and positioning. We used equimolar GUARD RNA and gRNA in order discern off-target protection that would be masked at higher GUARD RNA concentrations (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref>). Notably, both <italic>MYC</italic> and <italic>CAVIN4</italic> GUARDs worked exceptionally well, reducing indel rates from 2.6 ± 0.3% to 0.08 ± 0.08%, and 19 ± 1.9% to 3.3 ± 0.4%, respectively (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b</xref>). All GUARD RNAs tested reduced off-target indel rates except for one (<italic>VEGFA</italic> off-target <italic>HDLBP</italic>). Upon inspection of the non-functional <italic>HDLBP</italic> GUARD RNA, we noted that it had relatively low GC-content compared to the cognate on-target gRNA, potentially leading to low binding affinity and reduced ability to compete at the off-target locus. Introduction of a revised GUARD RNA with higher GC-content, length and increased overlap of the gRNA seed region, achieved only modestly improved protection (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>). We only observed protection at a 5:1 molar ratio of GUARD RNA to gRNA, and no protection at a 1:1 ratio. Taken together, these data suggest that some off-target loci are more amenable to protection by CRISPR GUARD than others, indicating more systematic investigation is warranted. This is likely contingent on the relative affinities of the GUARD RNA and the cognate gRNA for the off-target region.</p><p id=\"Par11\">As protection of one off-target with CRISPR GUARD apparently does not increase editing at other off-target sites (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>), we attempted multiplexing of three GUARD RNAs together to assess if it was possible to protect multiple off-target sites simultaneously. We performed multiplexed GUARD RNA experiments by transfecting Cas9 expressing HEK293 cells with on-target gRNA and three independent GUARD RNAs all at equimolar concentrations. For both <italic>EMX1</italic> and <italic>VEGFA</italic>, multiplex GUARD RNA delivery was generally effective in protecting multiple off-target sites from indel formation (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>). Consistent with the in vitro data (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2b</xref>), the non-competitive GUARD RNAs used in these experiments showed no effect at the on-target site (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>), thus allowing robust protection of several off-targets whilst maintaining efficient on-target editing.</p><p id=\"Par12\">To further validate the safety of CRISPR GUARD, we transfected various functional GUARDs (14-nt and 15-nt in length) at the highest effective concentration used in this study (125 nM) and assessed if they could support Cas9-mediated DNA cleavage in cells by deep sequencing of amplicons. In concordance with our in vitro data (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2b, c</xref>), none of the GUARD RNAs could generate Cas9-induced indels in isolation, in contrast to a 20-nt control gRNA at the same concentration (~82% indel formation), affirming that GUARD RNAs form nuclease-dead complexes with Cas9 in the cell (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>).</p></sec><sec id=\"Sec5\"><title>CRISPR GUARD for base editing</title><p id=\"Par13\">Next, we applied CRISPR GUARD to base editing. Due to the strict positioning requirements for base editing activity, gRNA design possibilities are more limited and therefore off-targets are harder to avoid<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. We reasoned that CRISPR GUARD could reduce Base Editor 3 (BE3) activity at off-target sites, since the activity window of BE3 is mostly absent from GUARD RNAs (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6a</xref>), and optimal activity of BE3 is dependent on nickase Cas9 nuclease acitivity<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, which is compromised with short gRNAs<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>–<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. Using a HEK293 cell line expressing BE3, we tested GUARD RNA designs for protection of <italic>EMX1</italic> and <italic>VEGFA</italic> off-target sites (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6b</xref>). We demonstrated significant protection of the <italic>EMX1</italic> off-target cytosines proximal to <italic>MYC</italic> (a reduction from 6.03 ± 0.6% to 0.98 ± 0.09% editing), and the <italic>VEGFA</italic> off-target cytosines proximal to <italic>CAVIN4</italic> (a reduction of 23.33 ± 0.45% to 11.37 ± 2.72%; Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4a</xref>). These GUARD RNAs also performed well in the CRISPR/Cas9 system (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b</xref>). As with Cas9, introduction of GUARD RNAs did not compromise on-target editing efficiencies (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4a</xref>).<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>GUARD RNAs can reduce off-target base editing if designed to avoid cytosine exposure.</title><p><bold>a</bold> Protection from off-target cytosine deamination with CRISPR GUARD. Base editing rates from amplicon sequencing of BE3-expressing HEK293 cells transfected with <italic>EMX1</italic> or <italic>VEGFA</italic> on-target gRNA (25 nM) and the indicated GUARD RNA (125 nM). Shown is the cumulative editing rate for the cytosines highlighted. The predicted cytosine deamination activity window is indicated. <bold>b</bold> Increased base editing frequencies with GUARD RNAs that expose cytosines. Base editing rates from amplicon sequencing of BE3-expressing HEK293 cells transfected with <italic>EMX1</italic> (left) or <italic>VEGFA</italic> (right) on-target gRNA (25 nM) and the indicated GUARD RNA (125 nM). <bold>c</bold> GUARD RNAs can facilitate base editing without full-length on-target gRNAs. Base editing rates from amplicon sequencing of BE3-expressing HEK293 cells transfected with <italic>MFAP1</italic> GUARD RNA only (125 nM). Data represent the mean of two independent experiments with symbols representing each replicate. Source data are provided as a Source Data file.</p></caption><graphic xlink:href=\"41467_2020_17952_Fig4_HTML\" id=\"d30e852\"/></fig></p><p id=\"Par14\">To date, the activity of short gRNAs for base editing applications has not been systematically investigated. The <italic>HDBLP</italic> and <italic>MFAP1</italic> GUARD RNAs are predicted to expose cytosines as single-stranded DNA near the BE3 deaminase activity window (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6a</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">6b</xref>). Interestingly, when BE3 expressing cells were transfected with both on-target gRNA and GUARD RNA, an overall increase in deamination rates was observed (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4b</xref>). Strikingly, the <italic>HDBLP</italic> GUARD RNA introduced two distinct G-to-A mutations that were not detected in the absence of GUARD RNA (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4b</xref>). These mutations were found both linked and unlinked to those generated by the on-target gRNA, suggesting they could be occurring in the same editing event (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6c</xref>). Moreover, when we transfected the <italic>MFAP1</italic> GUARD RNA alone (without <italic>EMX1</italic> on-target gRNA), we could detect a low but significant number of C-to-T deamination events (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c</xref>; 3.44 ± 0.40%), suggesting that short GUARD RNAs are sufficient to independently support base editing in cells. It is likely that the low level of base editing observed with short GUARD RNAs alone is due to the lack of nickase activity, which is analogous to editing with base editor 2 versions<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>.</p></sec><sec id=\"Sec6\"><title>A high-throughput screen identifies functional GUARD RNAs</title><p id=\"Par15\">Thus far, we have analysed the performance of a relatively small number of GUARD RNAs targeting endogenous loci. To systematically screen all possible Cas9 or BE3 GUARD RNAs for a given target, we adapted a high-throughput approach<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup> using a library of lentiviral constructs that express both gRNA and GUARD RNA and harbour the off-target sequence within 79 bp of its genomic context. Using this system, we screened the performance of ~600 GUARD RNAs including NT GUARD RNA controls, obviating amplification of individual endogenous loci to analyse editing events (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5a</xref>). We tested four gRNAs based on their therapeutic relevance (<italic>HBB;</italic> a therapeutic target for correcting sickle-cell disease<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>) or because of an extensive knowledge of experimentally validated off-target sites (<italic>EMX1</italic>, <italic>FANCF,</italic> and <italic>HEKsite1</italic>). Each gRNA expression vector was linked to one of 30 experimentally validated off-target sites<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. For each of the off-target sites, we tested all possible 15-nt GUARD RNAs within a 10 bp window either side of the mismatched gRNA protospacer with an NRG PAM (where R is a purine). We introduced the lentiviral pooled library into HEK293 cells expressing doxycycline-inducible Cas9 or BE3 and measured the number of indels or base edits for each GUARD RNA by NGS (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5a</xref>).<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>High-throughput screening identifies functional GUARD RNAs.</title><p><bold>a</bold> Schematic of the lentiviral pooled screening approach to analyse off-target editing frequencies in cells expressing different GUARD RNA species. Numbers indicate the length of the plasmid segment in base pairs. <bold>b</bold> Scatter plots of indels (Cas9) or SNPs (base edits) for two independent replicate experiments with or without induction of Cas9 or BE3 expression with doxycycline for 48 h. <bold>c</bold> Box and whiskers plot of off-target indel or base editing frequency for coding and non-targeting (NT) GUARD RNAs in the Cas9 or base editor screen, respectively. NB: some outliers fall outside of the <italic>y</italic>-axis limit. <bold>d</bold> GUARD RNAs binding to the same DNA strand as the gRNA are more effective at blocking Cas9 off-target editing but can increase base editing efficiency. Box and whiskers plot of off-target indel or SNP frequencies in the screens. NB: some outliers fall outside of the <italic>y</italic>-axis limit. Box and whiskers plot: centre line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. Data were compared using unpaired, two-tailed student’s <italic>t</italic>-tests.</p></caption><graphic xlink:href=\"41467_2020_17952_Fig5_HTML\" id=\"d30e964\"/></fig></p><p id=\"Par16\">As expected, we observed a significant induction of editing at off-target sites by Cas9 and BE3 in the presence of doxycycline with a high correlation of editing frequencies between biological replicates (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5b</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">7</xref>). Editing profiles were compared to those derived from deep sequencing of the original plasmid library, such that we focussed on mutations caused by Cas9 or BE3 expression in the cell in downstream analyses. Low frequency editing was observed without doxycycline, likely reflecting a degree of leakiness in the inducible system. To further validate the screen, we compared the Cas9-driven indel rates in the screen with those detected with GUIDE-seq methodologies on endogenous genomic loci<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup> and found that the observed mutation rates correlated well (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">8</xref>), verifying the physiological relevance of the system<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>.</p><p id=\"Par17\">Comparing the abundance of Cas9-driven off-target indels, we observed that expression of GUARD RNAs tended to reduce off-target mutations compared to cells expressing NT GUARD molecules (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5c</xref>). It was also clear that many of the screened GUARD RNAs have minimal effects, which is perhaps to be expected as we did not pre-select GUARD RNAs based on sequence features and many of these GUARD molecules may perform better at higher concentrations relative to the gRNA (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref>).</p><p id=\"Par18\">Conversely, we discovered that GUARD RNAs significantly increased base editing at off-target sites on average when compared to NT GUARD RNA controls (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5c</xref>), consistent with some GUARD RNAs being able to support base editing (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>). BE3 had a higher rate of off-target editing than Cas9 (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5c</xref>), with most SNPs detected being C->T and G->A mutations, consistent with cytosine deamination (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">9</xref>). Notably, base editing with GUARD RNAs was promoted by binding to the same DNA strand as the gRNA (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5d</xref>), presumably because the full-length gRNA-BE3 complex nicks the opposite strand, thus encouraging repair of the unedited strand and retention of the mutation<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. In contrast, Cas9 GUARD RNAs binding on the same DNA strand as the gRNA significantly reduced indel rates (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5d</xref>), implying that this may be sterically more obstructive.</p></sec><sec id=\"Sec7\"><title>Sequence features of functional GUARD RNAs</title><p id=\"Par19\">To delineate features of protective GUARD RNAs, we designated GUARD RNAs that reduced off-target editing by more than two standard deviations from the mean of the NT GUARD RNA controls as functional molecules (Figs. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>a, <xref rid=\"Fig6\" ref-type=\"fig\">6b</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">10</xref>). We selected a pair of reproducibly functional and non-functional Cas9 GUARD RNAs and performed validation experiments on the endogenous genomic locus. Results from these experiments were in direct concordance with the screening data supporting the validity of this categorisation (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6c</xref>).<fig id=\"Fig6\"><label>Fig. 6</label><caption><title>GUARD RNA features that significantly reduce off-target editing.</title><p><bold>a</bold> GUARD RNA performance for Cas9 and, (<bold>b</bold>) BE3. Base editing rates for BE (% of NGS reads with SNPs) or indel rates for Cas9 (% of NGS reads with indels) for a subset of off-target sites. Off-targets are labelled with an identifier, gene name (if genic), gRNA name, and the number of mismatches between the gRNA and the off-target site. Data are pooled from two independent experiments from cells treated with doxycycline. Each point represents a GUARD RNA and are compared to three non-targeting (NT) control RNAs. GUARD RNAs are classed as functional if they reduce off-target editing by at least two standard deviations from the mean of the NT controls. Data shown here is a subset of that presented in Supplementary Fig. 10. <bold>c</bold> Lentiviral plasmid screening reveals GUARD RNAs that are effective at endogenous genomic loci. (Left panel) indel rates from GUARD RNA screening of <italic>EMX1</italic> gRNA off-target site <italic>MFAP1</italic>, highlighting non-functional GUARD RNA (v1) and functional GUARD RNA (v2). Screening data shown here is a subset of that presented in Supplementary Fig. 10. (Right panels) indel rates from amplicon sequencing of on-target and off-target endogenous loci from Cas9-expressing HEK293 cells transfected with <italic>EMX1</italic> gRNA and GUARD RNA v1, GUARD RNA v2 or a NT control (all at 25 nM). Data represent mean of two independent experiments with symbols representing each replicate. <bold>d</bold> GUARD RNAs can mediate base editing at position 1 and 2 within the 15-nt GUARD RNA. GUARD RNAs were grouped according to whether they had a cytosine at each nucleotide position or not, and <italic>P</italic> values were generated by comparing the % of NGS reads with SNPs between GUARD RNA groups with an unpaired, two-tailed student’s <italic>t</italic>-tests. <bold>e</bold> Functional GUARD RNAs predominantly have NGG PAMs. The proportion of functional GUARD RNAs from the Cas9 screen that had NGG PAMs versus NAG PAMs was compared using a two-sided Fisher’s exact test. <bold>f</bold> Model depicting important parameters for GUARD RNA design. For GUARD RNAs that reduce Cas9 off-target editing, reducing distance from the gRNA, having an NGG PAM, binding to the same DNA strand as the gRNA, increased GC-content and high concentration in the cell will all increase the effectiveness of CRISPR GUARD. For BE3, the presence of cytosines at position 1 or 2 is to be avoided for 15-nt GUARD RNAs to prevent editing. Source data are provided as a Source Data file.</p></caption><graphic xlink:href=\"41467_2020_17952_Fig6_HTML\" id=\"d30e1080\"/></fig></p><p id=\"Par20\">Half of the analysed off-target sites (15 of 30) had functional GUARD RNAs for Cas9, and 80% (24 of 30) had functional GUARD RNAs for BE3 (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">10</xref>). Functional GUARD RNAs were evenly distributed between off-targets with low and high mutation rates and for off-targets with different numbers of gRNA mismatches (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6a</xref>). Of the 510 targeting GUARD RNA designs tested, 18 ± 1% showed functional protection from Cas9 off-target activity, with 23 ± 0.3% of these reducing off-target editing to below 0.5%. For BE3, fewer GUARD RNAs showed functionality, with 8 ± 1% showing reproducible protection of off-target sites (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6b</xref>). Only one GUARD RNA was able to reduce BE3 editing to below 0.5%, consistent with the finding that some GUARD RNAs can also support base editing. Specifically, the presence of a cytosine within the first 2-nt of the 15-nt GUARD RNA gave rise to significantly more base editing events (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6d</xref>). Concordant with GUARD RNAs with high affinity having superior protective effects, NGG PAMs were significantly enriched in functional GUARD RNAs over NAG (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6e</xref>). Selecting only Cas9 GUARD RNAs with an NGG PAM significantly increased the percentage of functional Cas9 GUARD RNAs to 26 ± 1%. Moreover, the proportion of functional GUARD RNAs tended to increase with higher GC-content (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">11a</xref>). Finally, GUARD RNAs with a higher degree of spatial overlap with the gRNA, especially at the seed and PAM region, led to superior off-target protection (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">11b</xref>). Taken together, these data support a model of off-target protection by CRISPR GUARD through direct competition with the mismatched gRNA and highlight important parameters for GUARD RNA design (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6f</xref>).</p><p id=\"Par21\">Finally, we generated a publicly available tool for automated GUARD RNA design called CRISPR GUARD Finder (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.sanger.ac.uk/tool/crispr-guard-finder/\">https://www.sanger.ac.uk/tool/crispr-guard-finder/</ext-link> and <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/MatthewACoelho/CRISPRGUARDFinder\">https://github.com/MatthewACoelho/CRISPRGUARDFinder</ext-link>), which predicts potential off-target sites for a given gRNA, and generates a list of possible GUARD RNAs with relevant sequence features. An example is given in Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">12</xref>.</p></sec></sec><sec id=\"Sec8\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par22\">In this report, we reveal CRISPR GUARD as a rapidly implementable tool to reduce off-target editing by Cas9 without reducing on-target editing efficiency. Importantly, on-target editing is maintained even when GUARD RNAs are multiplexed to protect multiple off-target sites simultaneously (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>). Using in vitro assays, in-cell assays and screening hundreds of GUARD RNAs, we show that the majority of off-target sites can be protected by GUARD RNA molecules and identify key parameters for GUARD RNA design for Cas9 and BE, including distance from the off-target, GC-content, PAM, DNA strand, concentration and cytosine positioning (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6f</xref>). In addition, GUARD RNAs with extensive homology to the on-target site may interfere with on-target editing in some cases (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2c</xref>).</p><p id=\"Par23\">GUARD RNA design rules for BE is complicated by the ability of short gRNAs to direct cytosine deamination in isolation. Many features of Cas9 GUARD RNA design were not significantly enriched in functional GUARD RNAs for base editing, presumably because features promoting strong GUARD RNA binding also promote base editing. This property could be exploited to reduce bystander cytosine editing at the 5’ of the gRNA, or may be used in tandem with full-length gRNAs to broaden the editing window when multiple editing events are desired. Nevertheless, as long as cytosines are avoided within the base editing window, functional BE GUARD RNAs can be identified that significantly reduce off-target base editing (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6b</xref>).</p><p id=\"Par24\">Although the minority of GUARD RNAs were protective in the Cas9 lentiviral screen where we cannot directly control the molar ratio between gRNA and GUARD RNA, it is clear that higher doses of GUARD RNAs are often required to compete with the full-length gRNA. Moreover, GUARD RNAs that satisfy sequence features identified here are more likely to be successful in blocking off-target mutation. At high effective concentrations, we verify that GUARD RNAs cannot support nuclease activity in vitro or in cells. However, it is possible that GUARD RNA-Cas9 complexes might transiently interfere with transcription<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. Thus, more work is required to refine GUARD RNA design computationally to minimise the number of potential binding sites within the genome (intrinsically high for short polynucleotides), and to optimise binding energies relative to the cognate mismatched gRNA. For example, locked nucleic acid bases could further improve the specificity and binding energy of GUARD RNAs to favour displacement of the mismatched gRNA. In addition, the CRISPR GUARD Finder tool will be useful in allowing researchers to rapidly assess the optimal CRISPR GUARD designs for gene editing experiments. Despite our demonstration that multiplexing is feasible for CRISPR GUARD, there may be instances where a particular gRNA is predicted to have many off-target sites and screening all promising GUARD RNA designs may not be feasible. In such situations, we suggest that the user considers refining gRNA design, and failing this, prioritising off-targets that are experimentally validated, bioinformatically scored as most probable, or likely to be particularly detrimental (e.g., genic).</p><p id=\"Par25\">We envisage that CRISPR GUARD could be employed for therapeutic applications that require high editing efficiency and where high-fidelity Cas9 variants cannot fully supress persistent off-target editing<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. We demonstrate CRISPR GUARD is effective with RNP delivery, where editing efficiencies may be maximised by complexing GUARD RNAs with dCas9, and the gRNA with active Cas9. However, in situations where this becomes less practical (e.g., multiplexing GUARD RNAs), co-delivery with active Cas9 will be more appropriate. Many of our experiments involved co-transfection of gRNA and GUARD RNAs in Cas9-expressing cells lines, with no apparent consequences of guide-swapping<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. The design principles of CRISPR GUARD could be easily adapted for use with Cas9 orthologues, TALENs and zinc-finger nucleases, while Cas9 variants that utilise distinct PAMs will increase the number of suitable GUARD RNAs for each off-target. By demonstrating that we can also block editing at perfectly complementary on-target sites (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2d</xref>), we open the possibility for controlling the kinetics of gene editing by introducing GUARD RNAs as an off-switch. In summary, CRISPR GUARD has exciting potential for improving the specificity and safety of genome editing. During review, a similar technology report was published with complementary and supportive data<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>.</p></sec><sec id=\"Sec9\"><title>Methods</title><sec id=\"Sec10\"><title>CRISPR-GUARD RNAs</title><p id=\"Par26\">GUARD RNA and full-length gRNA sequences are listed in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">2</xref> and are AltR modified IDT crRNAs.</p></sec><sec id=\"Sec11\"><title>Bio-layer interferometry (BLI)</title><p id=\"Par27\">DNA substrate was generated from duplexing a biotin-conjugated oligo with an unlabelled oligo coding for the <italic>CAVIN4</italic> off-target site of the <italic>VEGFA</italic> gRNA. Labelled strand (AGCCACAACCCTGTTGGACGTCCTGAGGCGGGGTGGGGGGGTGTGCAAGGGAACTCTCC), unlabelled strand (GGAGAGTTCCCTTGCACACCCCCCCACCCCGCCTCAGGACGTCCAACAGGGTTGTGGCT). GUARD RNA (CCCCCACCCCGCCTC), gRNA (GACCCCCTCCACCCCGCCTC). BLI measurements were performed at 25 °C using an Octet Red96 instrument (ForteBio). The measurement buffer consisted of PBS supplemented with 0.005% Tween-20, 1 mM MgCl<sub>2</sub> and 50 μg ml<sup>−1</sup> heparin. SA tips were used to immobilise ca. 0.1 unit (nm) of 5′-biotinylated duplex DNA oligo. Subsequently, tips were typically dipped in the measurement buffer for 30 s and transferred to precomplexed dCas9:tracrRNA:guideRNA (formed at 100 nM:200 nM:200 nM, respectively) for a 1000 s association step.</p></sec><sec id=\"Sec12\"><title>In vitro Cas9 cleavage assays</title><p id=\"Par28\">Cas9 DNA cleavage reactions were made up in a final reaction volume of 10 µL and incubated at 37 °C for 1 h. Reactions consisted of: <italic>S. pyrogenes</italic> Cas9 (120 nM; NEB), 1× NEB Cas9 reaction buffer, on-target gRNA (10 nM), a range of concentrations of GUARD RNA (10–100 nM), 120 nM tracrRNA, a purified PCR amplicon containing the gRNA protospacer sequence (0.5 nM) or a purified PCR amplicon containing the mismatched off-target protospacer sequence (0.5 nM), as indicated in the figures. In each case, the non-targeting gRNA (NT) was used at the highest concentration (100 nM) as a negative control and used to back-calculate the final concentration of GUARD RNA in the titration experiments such that the total concentration of RNA in each condition was equivalent. For control reactions with GUARD RNA only, GUARD RNA was used at the highest concentration (100 nM). For pre-incubation with GUARD RNAs, the reactions were set up as above, except for a 30 min preincubation of Cas9 (120 nM in 5 µL) with GUARD RNA and the PCR amplicon, before addition of the gRNA and Cas9 (120 nM in 5 µL) for a further 1 h. For Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1e</xref>, gblocks (IDT) were synthesised containing the GUARD RNA binding region in the following positions: overlapping, 10 bp away, 25 bp away or 50 bp away from the off-target protospacer. These gblocks were used as input in Cas9 DNA cleavage assays as above (0.5 nM input DNA). Reactions were stopped by the addition of an equal volume of 100 mM EDTA and heating for 5 min to 65 °C and subsequently diluted 10-fold with water before analysis of cutting efficiency by qPCR. The non-targeting guide RNA was also from IDT and has the protospacer sequence: GCCCCGCCGCCCTCCCCTCC<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>.</p></sec><sec id=\"Sec13\"><title>qPCR</title><p id=\"Par29\">qPCR primers used are listed in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>. An ABI 7900 (ThermoFisher) and Fast SYBR Green Master Mix (ThermoFisher) were used to quantify cutting efficiency using the no Cas9 condition as a control for input, where % input = 100<sup>2</sup>(Ct input − Ct experimental).</p></sec><sec id=\"Sec14\"><title>CRISPR GUARD in cells</title><p id=\"Par30\">HEK293 (ATCC) Cas9-expressing cells and BE3-expressing cells were cultured in RPMI medium (Sigma-Aldrich) supplemented with 10% FCS (ThermoFisher), 1% GlutaMAX (ThermoFisher) in a 37 °C, 5% CO2, 95% air incubator. HEK293 Cas9-expressing cells and BE3-expressing cells were generated using ObLiGaRe-mediated incorporation of an expression cassette into the AAVS1 safe-harbour locus by co-transfection of plasmids encoding AAVS1-targeting zinc-finger nucleases and the expression plasmid<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref>,<xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>, followed by selection in G418. Parental HEK293 cells were acquired from ATCC and all cell lines used were STR profiled and verified as mycoplasma-free.</p><p id=\"Par31\">For CRISPR GUARD experiments in cells, 200,000 cells were seeded in a 24 well tissue culture plate in 500 µL medium. The following day, cells were co-transfected. Briefly, each gRNA (crRNA AltR modified; IDT) was separately precomplexed with tracrRNA (IDT) at a 1:1 molar ratio in IDT duplex buffer by heating to 95 °C for 5 min. After duplexing with tracrRNA, on-target gRNA, non-targeting gRNA, or GUARD RNAs were diluted in Opti-MEM (ThermoFisher) and co-transfected with RNAiMAX (ThermoFisher) according to manufacturer’s instructions. Forty-eight hours later, DNA was extracted using DNeasy Blood and Tissue kit (Qiagen) and used for amplicon sequencing.</p><p id=\"Par32\">For RNP experiments, Cas9 RNPs were delivered using the Neon electroporation system (ThermoFisher) using protocol 20. Briefly, 500,000 HEK293 cells were electroporated with 1 µg of Cas9 precomplexed with gRNA and 1 µg of dCas9 precomplexed with GUARD RNA, with 0.1 µg of AltR electroporation enhancer (IDT).</p></sec><sec id=\"Sec15\"><title>Amplicon sequencing and analysis</title><p id=\"Par33\">Primers used for amplicon sequencing are listed in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>. PCR reactions were performed using Phusion Flash Hi-Fidelity PCR Master Mix (ThermoFisher) using an optimised number of PCR cycles ranging from 22 to 25. The NGS indexing PCR was 10 cycles, using 1 ng of purified product from PCR1 as input. PCR fragments were purified using SPRI beads (MAGBIO), size verified using the QIAXcel (Qiagen), quantified with the Qubit (ThermoFisher) and finally pooled and sequenced on a NextSeq 500 or MiSeq (Illumina). Bioinformatics analysis of NGS data was performed essentially as described<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>, using Fast Length Adjustment of Short reads (FLASH v1.2.11) for paired reads, BWA-MEM for alignment to the human genome, and Samtools to generate indexed BAM files and variant calling, with >0.001 allele frequency and >1000 read cut-off.</p></sec><sec id=\"Sec16\"><title>CRISPR GUARD screening plasmid construction</title><p id=\"Par34\">Using a scaffoldless version of the pKLV2 lentiviral base vector, we integrated a gBlock (IDT) containing H1 promoter-gRNA-scaffold into the Apa1-Mlu1 site using Gibson assembly (NEB). This produced four distinct plasmids containing either the <italic>EMX1</italic>, <italic>FANCF</italic>, <italic>HEKsite1</italic>, or <italic>HBB</italic> gRNA. We used the improved scaffold (encoding the tracrRNA) for gRNA and GUARD RNAs. Next, we designed all possible GUARD 15-nt sequences for each off-target site using our custom tool (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.sanger.ac.uk/tool/crispr-guard-finder/\">https://www.sanger.ac.uk/tool/crispr-guard-finder/</ext-link> and <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/MatthewACoelho/CRISPRGUARDFinder\">https://github.com/MatthewACoelho/CRISPRGUARDFinder</ext-link>) and designed an oligo library for each gRNA where each of the GUARD sequences are linked to the corresponding off-target embedded between 28 bp of flanking genomic context on either side. For each off-target sequence, we also included three non-targeting GUARD RNA controls, which were truncated versions of published Avana NT control guide RNAs<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>, verified to have minimal complementarity to the human genome (sequences are listed in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>). Single stranded oligo pools were obtained from IDT and were PCR amplified (10 PCR cycles) to make them double-stranded and to append adaptors containing short homology arms for Gibson assembly (NEB). Double-stranded oligo-pools were SPRI bead purified (MAGBIO) and subsequently inserted into the BbsI site of one of the four corresponding lentiviral plasmids to generate vectors expressing the gRNA and GUARD RNA. We pooled at least four transformations by electroporation of NEB Stable bacteria (NEB) to maintain library diversity on Amp agar plates and then harvested the DNA from a 3-h Amp liquid culture derived from a plate-scrape of colonies. Because the length of oligo synthesis was limiting, we designed the oligos to contain an internal AvrII site, which we then used to complete the tracrRNA scaffold sequence with a short PCR fragment using Gibson assembly (NEB). We harvested plasmid DNA as above and the resulting four completed libraries were combined into one pool in a ratio reflecting the total number of unique constructs in each sub-pool. The single pool of plasmids was sequence verified by NGS using primers designed to capture the GUARD and the off-target sequence.</p></sec><sec id=\"Sec17\"><title>CRISPR GUARD screen execution</title><p id=\"Par35\">We generated lentiviral particles in HEK293T cells with psPAX2 and pMD2.G packaging plasmids using FuGeneHD transfection reagent (Promega). We empirically determined the titre required to infect 40% of cells in the presence of 8 µg/ml Polybrene using BFP fluorescence detected by flow cytometry. The screen was performed in two independent infections treated as biological replicates for HEK293-Cas9 or HEK293-BE3 cells. The day after infection, HEK293 cells were selected for three days with puromycin (1 µg/ml), and surviving cells were induced with doxycycline (0.1 µg/ml) to induce expression of Cas9 or BE3. Control cells were treated with medium alone. After 48 h, DNA was harvest and purified using DNeasy Blood and Tissue kit (Qiagen). To maintain sample complexity, we pooled 24 × 20 µl PCR reactions, each using 500 ng of genomic DNA as input. For PCR1 we used 22 cycles and for the subsequent indexing PCR2 we used 10 cycles with 5 ng of purified PCR1 as input.</p></sec><sec id=\"Sec18\"><title>CRISPR GUARD screen analysis</title><p id=\"Par36\">NGS was performed on a NextSeq or MiSeq (Illumina) and fastq files were grouped into pairs as above using Fast Length Adjustment of Short reads (FLASH v1.2.11). A custom Perl script (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.sanger.ac.uk/tool/crispr-guard-finder/\">https://www.sanger.ac.uk/tool/crispr-guard-finder/</ext-link> and <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/MatthewACoelho/CRISPRGUARDFinder\">https://github.com/MatthewACoelho/CRISPRGUARDFinder</ext-link>) was used to process the data. Firstly, we scanned for a GUARD sequence within the first 30 bp of the paired reads in the specific U6-GUARD-scaffold context. We searched for exact matches except for the three non-targeting GUARDs where we allowed a single mismatch as this would be without functional consequence. For reads with matching GUARD sequences, we aligned these to the oligo reference library and checked whether the correct corresponding context-off-target-context (79 bp) was aligned. For correctly assigned reads, we used a dpAlign from BioPerl for alignment and quantification of SNPs and indel frequency. Mutations occurring in the plasmid library were removed from the experimental data, as they are likely caused by NGS and cloning artefacts and not due to Cas9 or BE. All further processing and graphing of the data was performed in R and can be found here: (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.sanger.ac.uk/tool/crispr-guard-finder/\">https://www.sanger.ac.uk/tool/crispr-guard-finder/</ext-link> and <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/MatthewACoelho/CRISPRGUARDFinder\">https://github.com/MatthewACoelho/CRISPRGUARDFinder</ext-link>). In downstream analyses, we only considered GUARD RNA constructs with a minimum of 250 reads. Thus, coverage ranged between 250 and approximately 16,000 reads per GUARD RNA construct for ~600 unique constructs.</p></sec><sec id=\"Sec19\"><title>CRISPR GUARD finder tool</title><p id=\"Par37\">For each guide (Gon) we identified off-target sites with up to 5 mismatches using our own implementation of the method employed in the Sanger WGE website<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>(<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.sanger.ac.uk/htgt/wge/\">https://www.sanger.ac.uk/htgt/wge/</ext-link>), enhanced with the calculation of the probability of the off-target<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. We used version GRCh38 (hg38) of the human genome, and GRCm38 (mm10) of the mouse genome, and gene annotation obtained from Ensembl. For each off-target locus (G<sub>off</sub>) with a probability above a threshold value, we scanned a user-defined proximal region of x bp for GUARD sequences (G<sub>Guard</sub>) of a user-defined length (e.g., 14-nt or 15-nt) adjacent to an NRG PAM on either strand. The code is available from (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.sanger.ac.uk/tool/crispr-guard-finder/\">https://www.sanger.ac.uk/tool/crispr-guard-finder/</ext-link> and <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/MatthewACoelho/CRISPRGUARDFinder\">https://github.com/MatthewACoelho/CRISPRGUARDFinder</ext-link>).</p></sec><sec id=\"Sec20\"><title>Reporting summary</title><p id=\"Par38\">Further information on research design is available in the <xref rid=\"MOESM3\" ref-type=\"media\">Nature Research Reporting Summary</xref> linked to this article.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec21\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41467_2020_17952_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41467_2020_17952_MOESM2_ESM.pdf\"><caption><p>Peer Review File</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM3\"><media xlink:href=\"41467_2020_17952_MOESM3_ESM.pdf\"><caption><p>Reporting Summary</p></caption></media></supplementary-material></sec></sec></body><back><app-group><app id=\"App1\"><sec id=\"Sec22\"><title>Source data</title><p id=\"Par41\"><media position=\"anchor\" xlink:href=\"41467_2020_17952_MOESM4_ESM.xls\" id=\"MOESM4\"><caption><p>Source Data</p></caption></media></p></sec></app></app-group><fn-group><fn><p><bold>Peer review information</bold>\n<italic>Nature Communications</italic> thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary information</bold> is available for this paper at 10.1038/s41467-020-17952-5.</p></sec><ack><title>Acknowledgements</title><p>Thanks to Discovery Biology AstraZeneca for helpful discussion, Marcello Maresca for conceptual advice and Ian Barrett and Stephanie Ashenden for bioinformatic support. We thank Euan Gordon for Cas9 protein production. We thank Ohad Yogev and Ardi Liaunardy for critical reading of the manuscript. MC is a fellow of the AstraZeneca postdoc programme. This work was funded by AstraZeneca plc. We thank the Wellcome Sanger Institute for hosting the CRISPR GUARD Finder tool.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>M.A.C. and B.J.M.T. designed the study. M.A.C. carried out the experiments, analysed and interpreted the data. E.D.B. performed experiments for paper revisions. M.B. performed B.L.I. experiments. M.F. provided bioinformatics support for NGS data analysis and wrote the CRISPR GUARD Finder tool. M.A.C. wrote the CRISPR GUARD Finder Shiny app. S.L., M.E.C., M.M. and B.J.M.T. provided conceptual advice. M.A.C. and B.J.M.T. wrote the manuscript. All authors contributed to manuscript revision and review.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>All data generated or analysed during this study are included in this published article, its supplementary information files, and publicly available repositories. Sequencing data is available from the NCBI Sequence Read Archive database, accession SRP252950, BioProject accession <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/bioproject/PRJNA612602/\">PRJNA612602</ext-link>. Source data are provided with this paper. Any additional relevant data are available upon reasonable request. Source data are provided with this paper.</p></notes><notes notes-type=\"data-availability\"><title>Code availability</title><p>Code for the CRISPR GUARD Finder tool is freely available from: <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/MatthewACoelho/CRISPRGUARDFinder\">https://github.com/MatthewACoelho/CRISPRGUARDFinder</ext-link>. Code and analyses used to analyse sequencing data are available from: <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/MatthewACoelho/CRISPRGUARDFinder\">https://github.com/MatthewACoelho/CRISPRGUARDFinder</ext-link>.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par39\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><mixed-citation publication-type=\"other\">Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. <italic>Nat. Biotechnol.</italic>10.1038/nbt.2623 (2013).</mixed-citation></ref><ref id=\"CR2\"><label>2.</label><mixed-citation publication-type=\"other\">Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. <italic>Nat. Biotechnol.</italic>10.1038/nbt.3117 (2015).</mixed-citation></ref><ref id=\"CR3\"><label>3.</label><mixed-citation publication-type=\"other\">Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. <italic>Nat. Biotechnol.</italic>10.1038/nbt.2647 (2013).</mixed-citation></ref><ref id=\"CR4\"><label>4.</label><mixed-citation publication-type=\"other\">Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. <italic>Nat. Biotechnol.</italic>10.1038/nbt.2673 (2013).</mixed-citation></ref><ref id=\"CR5\"><label>5.</label><mixed-citation publication-type=\"other\">Cradick, T. J., Fine, E. J., Antico, C. J. & Bao, G. CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. <italic>Nucleic Acids Res.</italic>10.1093/nar/gkt714 (2013).</mixed-citation></ref><ref id=\"CR6\"><label>6.</label><mixed-citation publication-type=\"other\">Wang, X. et al. Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. <italic>Nat. Biotechnol.</italic>10.1038/nbt.3127 (2015).</mixed-citation></ref><ref id=\"CR7\"><label>7.</label><mixed-citation publication-type=\"other\">Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. <italic>Nat. Methods</italic>10.1038/nmeth.3284 (2015).</mixed-citation></ref><ref id=\"CR8\"><label>8.</label><mixed-citation publication-type=\"other\">Lin, Y. et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. <italic>Nucleic Acids Res.</italic>10.1093/nar/gku402 (2014).</mixed-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cho</surname><given-names>SW</given-names></name><etal/></person-group><article-title>Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases</article-title><source>Genome Res.</source><year>2014</year><volume>24</volume><fpage>132</fpage><lpage>141</lpage><pub-id pub-id-type=\"doi\">10.1101/gr.162339.113</pub-id><pub-id pub-id-type=\"pmid\">24253446</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Frock</surname><given-names>RL</given-names></name><etal/></person-group><article-title>Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases</article-title><source>Nat. Biotechnol.</source><year>2015</year><volume>33</volume><fpage>179</fpage><lpage>186</lpage><pub-id pub-id-type=\"doi\">10.1038/nbt.3101</pub-id><pub-id pub-id-type=\"pmid\">25503383</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><mixed-citation publication-type=\"other\">Doench, J. G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. <italic>Nat. Biotechnol.</italic>10.1038/nbt.3437 (2016).</mixed-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Doench</surname><given-names>JGAm</given-names></name></person-group><article-title>I ready for CRISPR? A user’s guide to genetic screens</article-title><source>Nat. Rev. Genet.</source><year>2018</year><volume>19</volume><fpage>67</fpage><lpage>80</lpage><pub-id pub-id-type=\"doi\">10.1038/nrg.2017.97</pub-id><pub-id pub-id-type=\"pmid\">29199283</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dever</surname><given-names>DP</given-names></name><etal/></person-group><article-title>CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells</article-title><source>Nature</source><year>2016</year><volume>539</volume><fpage>384</fpage><lpage>389</lpage><pub-id pub-id-type=\"doi\">10.1038/nature20134</pub-id><pub-id pub-id-type=\"pmid\">27820943</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><mixed-citation publication-type=\"other\">Vakulskas, C. A. et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. <italic>Nat. Med.</italic><bold>24</bold>, 1216 (2018).</mixed-citation></ref><ref id=\"CR15\"><label>15.</label><mixed-citation publication-type=\"other\">Tsai, S. Q. et al. CIRCLE-seq: A highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. <italic>Nat. Meth.</italic>10.1038/nmeth.4278 (2017).</mixed-citation></ref><ref id=\"CR16\"><label>16.</label><mixed-citation publication-type=\"other\">Wienert, B. et al. Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq. <italic>Science</italic>10.1126/science.aav9023 (2019).</mixed-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Akcakaya</surname><given-names>P</given-names></name><etal/></person-group><article-title>In vivo CRISPR editing with no detectable genome-wide off-target mutations</article-title><source>Nature</source><year>2018</year><volume>561</volume><fpage>416</fpage><lpage>419</lpage><pub-id pub-id-type=\"doi\">10.1038/s41586-018-0500-9</pub-id><pub-id pub-id-type=\"pmid\">30209390</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kiani</surname><given-names>S</given-names></name><etal/></person-group><article-title>Cas9 gRNA engineering for genome editing, activation and repression</article-title><source>Nat. Methods</source><year>2015</year><volume>12</volume><fpage>1051</fpage><lpage>1054</lpage><pub-id pub-id-type=\"doi\">10.1038/nmeth.3580</pub-id><pub-id pub-id-type=\"pmid\">26344044</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><mixed-citation publication-type=\"other\">Dagdas, Y. S., Chen, J. S., Sternberg, S. H., Doudna, J. A. & Yildiz, A. A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9. <italic>Sci. Adv.</italic>10.1126/sciadv.aao0027 (2017).</mixed-citation></ref><ref id=\"CR20\"><label>20.</label><mixed-citation publication-type=\"other\">Dahlman, J. E. et al. Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease. <italic>Nat. Biotechnol.</italic>10.1038/nbt.3390 (2015).</mixed-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coelho</surname><given-names>MA</given-names></name><etal/></person-group><article-title>BE-FLARE: a fluorescent reporter of base editing activity reveals editing characteristics of APOBEC3A and APOBEC3B</article-title><source>BMC Biol.</source><year>2018</year><volume>16</volume><fpage>150</fpage><pub-id pub-id-type=\"doi\">10.1186/s12915-018-0617-1</pub-id><pub-id pub-id-type=\"pmid\">30593278</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><mixed-citation publication-type=\"other\">Rees, H. A. et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. <italic>Nat. Commun.</italic><bold>8</bold>, 10.1038/ncomms15790 (2017).</mixed-citation></ref><ref id=\"CR23\"><label>23.</label><mixed-citation publication-type=\"other\">Gehrke, J. M. et al. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. <italic>Nat. Biotechnol.</italic>10.1038/nbt.4199 (2018).</mixed-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rueda</surname><given-names>FO</given-names></name><etal/></person-group><article-title>Mapping the sugar dependency for rational generation of a DNA-RNA hybrid-guided Cas9 endonuclease</article-title><source>Nat. Commun.</source><year>2017</year><volume>8</volume><fpage>1610</fpage><pub-id pub-id-type=\"doi\">10.1038/s41467-017-01732-9</pub-id><pub-id pub-id-type=\"pmid\">29151576</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><mixed-citation publication-type=\"other\">Taheri-Ghahfarokhi, A. et al. Decoding non-random mutational signatures at Cas9 targeted sites. <italic>Nucleic Acids Res.</italic>10.1093/nar/gky653 (2018).</mixed-citation></ref><ref id=\"CR26\"><label>26.</label><mixed-citation publication-type=\"other\">Ting, P. Y. et al. Guide Swap enables genome-scale pooled CRISPR–Cas9 screening in human primary cells. <italic>Nat. Methods</italic>10.1038/s41592-018-0149-1 (2018).</mixed-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Slaymaker</surname><given-names>IM</given-names></name><etal/></person-group><article-title>Rationally engineered Cas9 nucleases with improved specificity</article-title><source>Science</source><year>2016</year><volume>351</volume><fpage>84</fpage><lpage>88</lpage><pub-id pub-id-type=\"doi\">10.1126/science.aad5227</pub-id><pub-id pub-id-type=\"pmid\">26628643</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Komor</surname><given-names>AC</given-names></name><name><surname>Kim</surname><given-names>YB</given-names></name><name><surname>Packer</surname><given-names>MS</given-names></name><name><surname>Zuris</surname><given-names>JA</given-names></name><name><surname>Liu</surname><given-names>DR</given-names></name></person-group><article-title>Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage</article-title><source>Nature</source><year>2016</year><volume>533</volume><fpage>420</fpage><lpage>424</lpage><pub-id pub-id-type=\"doi\">10.1038/nature17946</pub-id><pub-id pub-id-type=\"pmid\">27096365</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><mixed-citation publication-type=\"other\">Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. <italic>Science</italic><bold>353</bold>, 10.1126/science.aaf8729 (2016).</mixed-citation></ref><ref id=\"CR30\"><label>30.</label><mixed-citation publication-type=\"other\">Allen, F. et al. Predicting the mutations generated by repair of Cas9-induced double-strand breaks. <italic>Nat. Biotechnol.</italic>10.1038/nbt.4317 (2019).</mixed-citation></ref><ref id=\"CR31\"><label>31.</label><mixed-citation publication-type=\"other\">Shen, M. W. et al. Predictable and precise template-free CRISPR editing of pathogenic variants. <italic>Nature</italic>10.1038/s41586-018-0686-x (2018).</mixed-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gilbert</surname><given-names>LA</given-names></name><etal/></person-group><article-title>CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes</article-title><source>Cell</source><year>2013</year><volume>154</volume><fpage>442</fpage><lpage>451</lpage><pub-id pub-id-type=\"doi\">10.1016/j.cell.2013.06.044</pub-id><pub-id pub-id-type=\"pmid\">23849981</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rose</surname><given-names>JC</given-names></name><etal/></person-group><article-title>Suppression of unwanted CRISPR-Cas9 editing by co-administration of catalytically inactivating truncated guide RNAs</article-title><source>Nat. Commun.</source><year>2020</year><volume>11</volume><fpage>2697</fpage><pub-id pub-id-type=\"doi\">10.1038/s41467-020-16542-9</pub-id><pub-id pub-id-type=\"pmid\">32483117</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maresca</surname><given-names>M</given-names></name><name><surname>Lin</surname><given-names>VG</given-names></name><name><surname>Guo</surname><given-names>N</given-names></name><name><surname>Yang</surname><given-names>Y</given-names></name></person-group><article-title>Obligate ligation-gated recombination (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining</article-title><source>Genome Res.</source><year>2013</year><volume>23</volume><fpage>539</fpage><lpage>546</lpage><pub-id pub-id-type=\"doi\">10.1101/gr.145441.112</pub-id><pub-id pub-id-type=\"pmid\">23152450</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><mixed-citation publication-type=\"other\">Hodgkins, A. et al. WGE: a CRISPR database for genome engineering. <italic>Bioinformatics</italic>10.1093/bioinformatics/btv308 (2015).</mixed-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Qi</surname><given-names>LS</given-names></name><etal/></person-group><article-title>Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression</article-title><source>Cell</source><year>2013</year><volume>152</volume><fpage>1173</fpage><lpage>1183</lpage><pub-id pub-id-type=\"doi\">10.1016/j.cell.2013.02.022</pub-id><pub-id pub-id-type=\"pmid\">23452860</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807802</article-id><article-id pub-id-type=\"pmc\">PMC7431538</article-id><article-id pub-id-type=\"publisher-id\">70808</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70808-2</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Socio-economic, built environment, and mobility conditions associated with crime: a study of multiple cities</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-8466-3933</contrib-id><name><surname>De Nadai</surname><given-names>Marco</given-names></name><address><email>denadai@fbk.eu</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-5429-3177</contrib-id><name><surname>Xu</surname><given-names>Yanyan</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>Letouzé</surname><given-names>Emmanuel</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>González</surname><given-names>Marta C.</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lepri</surname><given-names>Bruno</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.11696.39</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1937 0351</institution-id><institution>Department of Information Engineering and Computer Science, </institution><institution>University of Trento, </institution></institution-wrap>Via Sommarive, 9I, 38123 Povo, TN Italy </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.11469.3b</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9780 0901</institution-id><institution>Mobs Lab, Fondazione Bruno Kessler, </institution></institution-wrap>Via Sommarive 18, 38123 Povo, TN Italy </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.47840.3f</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2181 7878</institution-id><institution>Department of City and Regional Planning and Department of Civil and Environmental Engineering, </institution><institution>University of California Berkeley, </institution></institution-wrap>230 Wurster Hall #1820, Berkeley, CA 94720–1820 USA </aff><aff id=\"Aff4\"><label>4</label>Data-pop Alliance, 99 Madison Avenue, New York, NY 10016 USA </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.116068.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2341 2786</institution-id><institution>Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, </institution></institution-wrap>77 Massachusetts Ave, Cambridge, MA 02139 USA </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13871</elocation-id><history><date date-type=\"received\"><day>15</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>31</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Nowadays, 23% of the world population lives in multi-million cities. In these metropolises, criminal activity is much higher and violent than in either small cities or rural areas. Thus, understanding what factors influence urban crime in big cities is a pressing need. Seminal studies analyse crime records through historical panel data or analysis of historical patterns combined with ecological factor and exploratory mapping. More recently, machine learning methods have provided informed crime prediction over time. However, previous studies have focused on a single city at a time, considering only a limited number of factors (such as socio-economical characteristics) and often at large in a single city. Hence, our understanding of the factors influencing crime across cultures and cities is very limited. Here we propose a Bayesian model to explore how violent and property crimes are related not only to socio-economic factors but also to the built environmental (e.g. land use) and mobility characteristics of neighbourhoods. To that end, we analyse crime at small areas and integrate multiple open data sources with mobile phone traces to compare how the different factors correlate with crime in diverse cities, namely Boston, Bogotá, Los Angeles and Chicago. We find that the combined use of socio-economic conditions, mobility information and physical characteristics of the neighbourhood effectively explain the emergence of crime, and improve the performance of the traditional approaches. However, we show that the socio-ecological factors of neighbourhoods relate to crime very differently from one city to another. Thus there is clearly no “one fits all” model.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Computational science</kwd><kwd>Statistics</kwd><kwd>Computer science</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">501100011061</institution-id><institution>Agence Française de Développement (French Development Agency)</institution></institution-wrap></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">100004421</institution-id><institution>World Bank Group (World Bank)</institution></institution-wrap></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">501100011061</institution-id><institution>Agence Française de Développement (French Development Agency)</institution></institution-wrap></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">100004421</institution-id><institution>World Bank Group (World Bank)</institution></institution-wrap></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">100006978</institution-id><institution>University of California Berkeley (University of California, Berkeley)</institution></institution-wrap></funding-source><award-id>DeepDrive</award-id><award-id>ITS Berkeley 2018 2018-19 SB1</award-id><principal-award-recipient><name><surname>González</surname><given-names>Marta C.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">501100011061</institution-id><institution>Agence Française de Développement (French Development Agency)</institution></institution-wrap></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">100004421</institution-id><institution>World Bank Group (World Bank)</institution></institution-wrap></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Criminology widely recognizes the importance of places<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>: crime occurs in small areas such as street segments, buildings or parks, and it is spatially stable over time<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. However, theoretical and empirical research showed that crime is also a consequence of socio-economic contextual characteristics, usually referred to as the “neighbourhood effect”<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. In criminology, cooperation, as opposed to disorganization of neighbours, is indeed believed to create the mechanisms by which residents themselves achieve guardianship and public order<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>, solve common problems, and reduce violence<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>–<xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. This mechanism also finds its roots in urban planning, where the relationship between specific aspects of urban architecture<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> and urban physical characteristics<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup> are related to security. Places and neighbourhoods are not to be considered islands unto themselves, as they are embedded in a city-wide system of social interactions. On a daily basis, people’s routine exposes residents to different conditions, possibilities<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>, and this routine may favour crime<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Nevertheless, many empirical studies focus on just a subset of static factors at a time such as socio-economic factors without considering the contextual built environment<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref>–<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>, or ignoring mobility<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>, and often only drawing results in a single city (e.g. Chicago)<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref>–<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>.\n</p><p id=\"Par3\">Studies on small areas and neighbourhoods roughly come from two streams of literature. The first stream focuses on the routine activity and crime pattern theories<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup> at places. These studies suggest that crime occurs when an offender, its suitable target, and the absence of any deterrence system, such as police or even ordinary citizens<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>, converge at a place. The presence of people influence the number of offenders and targets, but the daily routine of residents exposes homes and people to predatory crimes<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. The built environment was also found to affect criminal activities, as physical disorder and specific locations (e.g. bar, taverns) attract offenders and suitable targets<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref>–<xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>. The second stream of literature builds on the social context upon which the place of the crime is embedded. A notable example is the Social Disorganization theory<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>, which found high crime concentration in socially and economically disadvantaged neighbourhoods. In it, the structural predictors are often seen through the concentrated disadvantage, ethnic diversity, residential instability of neighbourhoods<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup> While most of these studies use census data as primary data source, recent years have witnessed a growing interest in alternative data. For example, scholars exploited synthetic social ties to simulate neighbourhood cohesion<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>, and mobility flows to indicate crime opportunities and connections between neighbourhoods<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Others leveraged crowd-sourced Point of Interests (POIs), taxi flows<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>, and dynamic population mapping from satellite imagery<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>,<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup> and mobile phone activity<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup> to assess the presence of people. Altogether, these results highlight the tight relation between the socio-economic, the built environment and mobility conditions, and their impact on criminal activities. Although the two streams of the theory are often seen as competing, we argue that they can complement each other. However, very limited work has integrated socio-economic, built environment and mobility conditions together in multiple cities and in small areas. Moreover, while crime theories are not limited to specific cities<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>, and several cross-disciplinary results suggest common and universal patterns in mobility<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>, urban environment<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup> and aggregated crime<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup> in urban systems, our comparative knowledge base is limited<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. These limitations result in a fragmented and incomplete picture<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup> of how the numerous factors influence crime in the urban context and limit the impact of the conclusions.</p><p id=\"Par4\">Here, we seek to shed light on the diverse set of factors at play with urban crime exploring how violent and property crimes are related, at the same time, to the Social Disorganisation, to the built environment characteristics and to human mobility. Specifically, we analyse crime at the level of group of blocks (measuring on average 0.378 square kilometers), considering both the local features of the group and its surrounding context, represented by all the blocks within a half-mile. The contribution of this paper is twofold. First, we address the need for a comprehensive study that explores crime patterns at fine grained resolution across multiple cities of the world, analysing Bogotá, Boston, Los Angeles and Chicago. Secondly, we show that taking into account the complex interplay between crime, people, places, and human mobility significantly improves the performance of the crime inference. We make use of massive and ubiquitous data sources such as mobile phone records and geographical data, implying that the resulting framework can be replicated at scale. Our generated insights can help recommend effective policies and interventions that improve urban security.</p></sec><sec id=\"Sec2\"><title>Results</title><p id=\"Par5\">We study criminal activity in Bogotá (Colombia), Boston (USA), Chicago (USA) and Los Angeles (USA), four very different cities with respect to cultural, urban and socio-economic conditions.</p><p id=\"Par6\">Our approach follows the aforementioned two streams of literature of place and neighborhood, assuming the existence of a social process named neighborhood effect, namely the relation of crime patterns with small places characteristics and mobility. To account for all these factors we analyse criminal activity and small places characteristics at census block group, the smallest geographical unit for which the U.S. census publishes data, and measuring on average 0.378 square kilometers. Each block group, here called <italic>core</italic>, is exposed to a surrounding context, named <italic>corehood</italic>, which is the set of all the surrounding block groups within a half mile from the core (see Figure <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). As the context of nearby cores is similar, corehoods might overlap. The idea of using overlapping units is not new<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR44\">44</xref>,<xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>, and it is focused on creating an ego-centric neighborhood for each core (see Supplementary Information (SI) Note <xref rid=\"MOESM1\" ref-type=\"media\">11</xref> for a technical discussion). We describe the characteristics of the place where crime happens through specific features of the <italic>core</italic>, while we describe the context at which it is embedded through the features at the <italic>corehood</italic>. As neighborhoods in literature are loosely defined, we tested different sizes of the corehood, finding the half mile distance as the best to describe the neighborhood effect (see the SI Note 11).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>For each block group (the core), we consider the block groups within a half mile as its corehood. Blocks that are near each other share most of their corehood. In this example, we show two cores in Bogotá and their corresponding corehood. We focus on three aspects of the core and the corehood: the Social Disorganization (SD), the Built Environment (BE), and the Mobility (M). The core, where crime is predicted, measures on average 0.378 square kilometers.</p></caption><graphic xlink:href=\"41598_2020_70808_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par7\">Criminal activity is provided by police agencies, which record through police reports the geographic location (i.e. latitude and longitude), date, time of day and category of each crime event. For all the cities we map each category of crime into the US Uniform Crime Reporting (UCR) categories<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup> and analyse crime belonging to two broad categories: violent and property crimes. They include homicides, sexual and non-sexual aggravated assaults, robbery, motor vehicle thefts and arson. We assign each crime to a corehood through its geographical position.</p><p id=\"Par8\">We describe cores through the features that were previously found to attract potential offenders and targets<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>, such as the census <italic>residential population</italic> and the number of <italic>nightlife</italic>, <italic>shops</italic> and <italic>food</italic> POIs inside each core, which are extracted from web data (more details in the Methods Section). Then, we describe corehoods through the environmental (neighbourhood) characteristics found to influence crime<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. The corehood features are estimated from all the block groups surrounding the core. We group them in Social Disorganization (SD), Built Environment (BE) and Mobility (M) features. The SD characteristics include some of the standard Social Disorganization theory features, namely concentrated <italic>disadvantage</italic>, <italic>instability</italic> and <italic>ethnic diversity</italic>. Consistently with the literature<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>, <italic>disadvantage</italic> and <italic>instability</italic> are composite variables built from the two largest principal components of: (i) unemployment rate, (ii) poverty rate, defined as people living below the poverty line, and (iii) residential mobility rate, defined as the percentage of people who recently changed residency. Again, in accordance with the literature<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref>,<xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>, <italic>ethnic diversity</italic> is computed as the Hirschman-Herfindahl index across six population groups (e.g. hispanic, black, white people, etc.). Additional details are present in the Methods Section. Note that we excluded all race-specific variables that are usually employed (e.g. percentage of black people in<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>) to build an evidence-based and race-neutral model.</p><p id=\"Par9\">The BE corehood features are based on the Jane Jacobs theory<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>, which states that the presence of people and a vibrant neighborhood life form a virtuous loop controlling local crime. From her own words “a well-used city street is apt to be a safe street and a deserted city street is apt to be unsafe”<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. Four conditions have to be valid to ensure this virtuous loop. First, a district should serve at least two or more functions to have streets continuously used by residents and strangers. Second, street blocks should be small and short to ensure both high <italic>walkability</italic> and frequent meeting of people at street intersections. Third, diverse buildings make it possible to have low- and high-rent spaces, and thus a mixture of people and enterprises. The fourth condition is about dense concentration, which ensures a sufficient presence of people and enterprises to attract dwellers from different neighbourhoods continuously. Thus, in accordance with the literature<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup> we operationalize through census and geographical data the four conditions in: i) <italic>land-use mix</italic>; ii) <italic>block size</italic> iii) <italic>building age diversity</italic>; iv) <italic>population density</italic> and <italic>walkability</italic>, which promotes social relations<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup> and is connected to local cohesion of neighbors. The details, data sources, and formula for these metrics are available in the Methods Section.</p><p id=\"Par10\">The M features are built upon recent mobility and criminology literature, which found mobility to be tightly coupled with criminal activity in space and time<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. People at risk in urban areas can be essentially measured through residential and floating population. The first one measures the number of people who resides in an area, while the second one measures the average number of people that can be expected in an area at any given time<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup> (e.g. average number of people at a mall). We measure floating population through the average number of people for each core, named <italic>ambient population</italic><sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>, and the <italic>attractiveness</italic> of the corehood, measured through the number of people movements to the corehood for reasons different than travelling to work or home. We extract this valuable information from passively and anonimized mobile phone data, collected by mobile phone operators for billing reasons. From mobile phone data, we fit the mobility model TimeGeo<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup> and simulate realistic urban traces that are used to extract the <italic>ambient population</italic> and <italic>attractiveness</italic> features. We do not include M features for Chicago, as we do not have mobile phone traces. Even if mobility is not available, Chicago is considered by many the testbed for empirical crime analysis, thus we include it to allow readers to do useful comparisons for socio-economic and urban environment characteristics.</p><p id=\"Par11\">Crime patterns have been observed to be highly concentrated in the space, overdispersed<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>, and positively spatial correlated. Thus, we model and predict crime through a spatially filtered Bayesian Negative Binomial, which is specifically tailored for discrete data, accounts for the overdispersion of crime events, models uncertainty and avoids the biased parameters of non-spatial models<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref>,<xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>. Through this model, criminal activity at cores is described by a linear combination of an intercept, the fixed effects (i.e. the aforementioned core and corehood features), and some random effects that represent the latent and unexplained variance that emerge from the spatial-autocorrelation of neighboring areas. Our model accounts for the high spatial correlation in crime events, and we did not find any significant spatial auto-correlation in the model residuals (see Note 4 in the SI).\nThe reader can refer to the Methods section for additional details about the model and its formulation.<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Quantitative results of crime description and predictions in Bogotá, Boston, Los Angeles and Chicago. The model including Social Disorganization, Built Environment and Mobility features achieves the highest descriptive (<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_m$$\\end{document}</tex-math><mml:math id=\"M2\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq1.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq2\"><alternatives><tex-math id=\"M3\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_c$$\\end{document}</tex-math><mml:math id=\"M4\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>c</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq2.gif\"/></alternatives></inline-formula>) and predictive (LOO) performance. Here, we can see that contextual features of the neighborhood significantly increase our model’s performance against the model considering only the core features. The LOO metric is calculated through the Pareto smoothed importance sampling Leave-One-Out cross-validation.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Model</th><th align=\"left\" colspan=\"2\">Bogotá</th><th align=\"left\" colspan=\"2\">Boston</th><th align=\"left\" colspan=\"2\">Los Angeles</th><th align=\"left\" colspan=\"2\">Chicago</th></tr><tr><th align=\"left\"><inline-formula id=\"IEq3\"><alternatives><tex-math id=\"M5\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_m$$\\end{document}</tex-math><mml:math id=\"M6\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq3.gif\"/></alternatives></inline-formula> (<inline-formula id=\"IEq4\"><alternatives><tex-math id=\"M7\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_c$$\\end{document}</tex-math><mml:math id=\"M8\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>c</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq4.gif\"/></alternatives></inline-formula>)</th><th align=\"left\">LOO</th><th align=\"left\"><inline-formula id=\"IEq5\"><alternatives><tex-math id=\"M9\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_m$$\\end{document}</tex-math><mml:math id=\"M10\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq5.gif\"/></alternatives></inline-formula> (<inline-formula id=\"IEq6\"><alternatives><tex-math id=\"M11\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_c$$\\end{document}</tex-math><mml:math id=\"M12\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>c</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq6.gif\"/></alternatives></inline-formula>)</th><th align=\"left\">LOO</th><th align=\"left\"><inline-formula id=\"IEq7\"><alternatives><tex-math id=\"M13\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_m$$\\end{document}</tex-math><mml:math id=\"M14\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq7.gif\"/></alternatives></inline-formula> (<inline-formula id=\"IEq8\"><alternatives><tex-math id=\"M15\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_c$$\\end{document}</tex-math><mml:math id=\"M16\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>c</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq8.gif\"/></alternatives></inline-formula>)</th><th align=\"left\">LOO</th><th align=\"left\"><inline-formula id=\"IEq9\"><alternatives><tex-math id=\"M17\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_m$$\\end{document}</tex-math><mml:math id=\"M18\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq9.gif\"/></alternatives></inline-formula> (<inline-formula id=\"IEq10\"><alternatives><tex-math id=\"M19\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_c$$\\end{document}</tex-math><mml:math id=\"M20\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>c</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq10.gif\"/></alternatives></inline-formula>)</th><th align=\"left\">LOO</th></tr></thead><tbody><tr><td align=\"left\">Core</td><td align=\"left\">0.54 (0.75)</td><td align=\"left\">−3897</td><td align=\"left\">0.21 (0.64)</td><td align=\"left\">−2035</td><td align=\"left\">0.18 (0.68)</td><td align=\"left\">−9665</td><td align=\"left\">0.09 (0.68)</td><td align=\"left\">−8415</td></tr><tr><td align=\"left\">Social-disorganization (SD)</td><td align=\"left\">0.57 (0.75)</td><td align=\"left\">−3891</td><td align=\"left\">0.55 (0.68)</td><td align=\"left\">−2019</td><td align=\"left\">0.53 (0.72)</td><td align=\"left\">−9529</td><td align=\"left\">0.66 (0.78)</td><td align=\"left\">−8019</td></tr><tr><td align=\"left\">Built environment (BE)</td><td align=\"left\">0.61 (0.76)</td><td align=\"left\">−3881</td><td align=\"left\">0.36 (0.68)</td><td align=\"left\">−2014</td><td align=\"left\">0.27 (0.69)</td><td align=\"left\">−9629</td><td align=\"left\">0.21 (0.69)</td><td align=\"left\">−8371</td></tr><tr><td align=\"left\">Mobility (M)</td><td align=\"left\">0.64 (0.80)</td><td align=\"left\">−3804</td><td align=\"left\">0.42 (0.70)</td><td align=\"left\">−2001</td><td align=\"left\">0.25 (0.70)</td><td align=\"left\">−9570</td><td align=\"left\">-</td><td align=\"left\">-</td></tr><tr><td align=\"left\">SD+BE</td><td align=\"left\">0.64 (0.76)</td><td align=\"left\">−3881</td><td align=\"left\">0.65 (0.72)</td><td align=\"left\">−1987</td><td align=\"left\">0.56 (0.72)</td><td align=\"left\">−9508</td><td align=\"left\"><inline-formula id=\"IEq11\"><alternatives><tex-math id=\"M21\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\mathbf{0.67\\ (0.79) }$$\\end{document}</tex-math><mml:math id=\"M22\"><mml:mrow><mml:mn mathvariant=\"bold\">0.67</mml:mn><mml:mspace width=\"4pt\"/><mml:mo stretchy=\"false\">(</mml:mo><mml:mn mathvariant=\"bold\">0.79</mml:mn><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq11.gif\"/></alternatives></inline-formula></td><td align=\"left\"><inline-formula id=\"IEq12\"><alternatives><tex-math id=\"M23\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-\\mathbf{8003 }$$\\end{document}</tex-math><mml:math id=\"M24\"><mml:mrow><mml:mo>-</mml:mo><mml:mn mathvariant=\"bold\">8003</mml:mn></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq12.gif\"/></alternatives></inline-formula></td></tr><tr><td align=\"left\">SD+M</td><td align=\"left\">0.66 (0.81)</td><td align=\"left\"><inline-formula id=\"IEq13\"><alternatives><tex-math id=\"M25\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-\\mathbf{3795 }$$\\end{document}</tex-math><mml:math id=\"M26\"><mml:mrow><mml:mo>-</mml:mo><mml:mn mathvariant=\"bold\">3795</mml:mn></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq13.gif\"/></alternatives></inline-formula></td><td align=\"left\">0.67 (0.73)</td><td align=\"left\">−1973</td><td align=\"left\">0.55 (0.73)</td><td align=\"left\">−9467</td><td align=\"left\">-</td><td align=\"left\">-</td></tr><tr><td align=\"left\">BE+M</td><td align=\"left\">0.68 (0.80)</td><td align=\"left\">−3819</td><td align=\"left\">0.50 (0.72)</td><td align=\"left\">−1989</td><td align=\"left\">0.30 (0.70)</td><td align=\"left\">−9585</td><td align=\"left\">-</td><td align=\"left\">-</td></tr><tr><td align=\"left\">SD+BE+M (Full)</td><td align=\"left\"><inline-formula id=\"IEq14\"><alternatives><tex-math id=\"M27\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\mathbf{0.70\\, (0.80) }$$\\end{document}</tex-math><mml:math id=\"M28\"><mml:mrow><mml:mn mathvariant=\"bold\">0.70</mml:mn><mml:mspace width=\"0.166667em\"/><mml:mo stretchy=\"false\">(</mml:mo><mml:mn mathvariant=\"bold\">0.80</mml:mn><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq14.gif\"/></alternatives></inline-formula></td><td align=\"left\">−3808</td><td align=\"left\"><inline-formula id=\"IEq15\"><alternatives><tex-math id=\"M29\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\mathbf{0.70\\, (0.75) }$$\\end{document}</tex-math><mml:math id=\"M30\"><mml:mrow><mml:mn mathvariant=\"bold\">0.70</mml:mn><mml:mspace width=\"0.166667em\"/><mml:mo stretchy=\"false\">(</mml:mo><mml:mn mathvariant=\"bold\">0.75</mml:mn><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq15.gif\"/></alternatives></inline-formula></td><td align=\"left\"><inline-formula id=\"IEq16\"><alternatives><tex-math id=\"M31\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-\\mathbf{1957 }$$\\end{document}</tex-math><mml:math id=\"M32\"><mml:mrow><mml:mo>-</mml:mo><mml:mn mathvariant=\"bold\">1957</mml:mn></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq16.gif\"/></alternatives></inline-formula></td><td align=\"left\"><inline-formula id=\"IEq17\"><alternatives><tex-math id=\"M33\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\mathbf{0.56\\, (0.74) }$$\\end{document}</tex-math><mml:math id=\"M34\"><mml:mrow><mml:mn mathvariant=\"bold\">0.56</mml:mn><mml:mspace width=\"0.166667em\"/><mml:mo stretchy=\"false\">(</mml:mo><mml:mn mathvariant=\"bold\">0.74</mml:mn><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq17.gif\"/></alternatives></inline-formula></td><td align=\"left\"><inline-formula id=\"IEq18\"><alternatives><tex-math id=\"M35\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-\\mathbf{9454 }$$\\end{document}</tex-math><mml:math id=\"M36\"><mml:mrow><mml:mo>-</mml:mo><mml:mn mathvariant=\"bold\">9454</mml:mn></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq18.gif\"/></alternatives></inline-formula></td><td align=\"left\">-</td><td align=\"left\">-</td></tr></tbody></table><table-wrap-foot><p>The best performance is highlighted in bold.</p></table-wrap-foot></table-wrap></p><sec id=\"Sec3\"><title>Description and prediction of crime</title><p id=\"Par12\">We begin by presenting the aggregated performance of our model predicting crime in the four analysed cities. We evaluate our model under various feature combinations to assess the contribution of each group of features. We measure the capability of the model to describe crime through the marginal <inline-formula id=\"IEq19\"><alternatives><tex-math id=\"M37\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_m$$\\end{document}</tex-math><mml:math id=\"M38\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq19.gif\"/></alternatives></inline-formula><sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup> and the conditional <inline-formula id=\"IEq20\"><alternatives><tex-math id=\"M39\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_c$$\\end{document}</tex-math><mml:math id=\"M40\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>c</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq20.gif\"/></alternatives></inline-formula><sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup> (the higher the better). The marginal <inline-formula id=\"IEq21\"><alternatives><tex-math id=\"M41\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_m$$\\end{document}</tex-math><mml:math id=\"M42\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq21.gif\"/></alternatives></inline-formula> measures the proportion of variance explained by the fixed effects (i.e. the input features), while the conditional <inline-formula id=\"IEq22\"><alternatives><tex-math id=\"M43\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_c$$\\end{document}</tex-math><mml:math id=\"M44\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>c</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq22.gif\"/></alternatives></inline-formula><sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup> takes also into account the variance explained by the auto-correlation but not the input features (absorbed by the random effects). The difference between the two can be used to find clustering effects and missing variables. To assess the point-wise out-of-sample prediction accuracy we use the Pareto-smoothed importance sampling Leave-One-Out cross-validation (here called LOO for simplicity)<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup> (the higher, the better).</p><p id=\"Par13\">First, we evaluate the baseline model that includes only the core variables, namely the residential population and the number of nightlife, shops and food POIs. Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> shows that the core-only model performs poorly in Chicago, Los Angeles and Boston, while it has high <inline-formula id=\"IEq23\"><alternatives><tex-math id=\"M45\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_m$$\\end{document}</tex-math><mml:math id=\"M46\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq23.gif\"/></alternatives></inline-formula> in Bogotá. We observe high difference between <inline-formula id=\"IEq24\"><alternatives><tex-math id=\"M47\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_m$$\\end{document}</tex-math><mml:math id=\"M48\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq24.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq25\"><alternatives><tex-math id=\"M49\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_c$$\\end{document}</tex-math><mml:math id=\"M50\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>c</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq25.gif\"/></alternatives></inline-formula>, which means that there is a significant unexplained variance that is not explained by the core features.</p><p id=\"Par14\">The SD, BE and M features significantly increase the explanatory power of our model. Particularly, in US cities, the <inline-formula id=\"IEq26\"><alternatives><tex-math id=\"M51\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_m$$\\end{document}</tex-math><mml:math id=\"M52\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq26.gif\"/></alternatives></inline-formula> increases up to 161%, 194% and 633% in Boston, Los Angeles and Chicago. Notably, and not surprisingly, the SD features are very important, especially in Chicago, where the “Chicago school”<sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup> forged the Social Disorganization theory and further elaborated the role of collective efficacy on dealing with crime. Differently, the increase in Bogotá is less pronounced, suggesting that the neighbourhood impact on crime is limited. Turning to M and BE features, we find that they describe the crime, but they are often as not meaningful as the SD features for crime prediction. However, the importance of mobility confirms the importance of floating population at describing micro-dynamic behaviour of criminal activity<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. We observe that in all cities the conditional <inline-formula id=\"IEq27\"><alternatives><tex-math id=\"M53\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_c$$\\end{document}</tex-math><mml:math id=\"M54\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>c</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq27.gif\"/></alternatives></inline-formula> increases when adding the SD, BE and M features, revealing that the included variables also help explain the variance of crime.</p><p id=\"Par15\">Overall, Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> shows that considering together SD, BE and M variables result in the highest descriptive (<inline-formula id=\"IEq28\"><alternatives><tex-math id=\"M55\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_m$$\\end{document}</tex-math><mml:math id=\"M56\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq28.gif\"/></alternatives></inline-formula>) and predictive (LOO) performance. This result means that, in order to model crime, one needs to account for multiple aspects of urban life, including Social Disorganization, the physical characteristics of the neighbourhoods, and mobility. This result holds also against different combinations of the features (i.e. SD+BE, SD+M and BE+M). Nonetheless, some of the SD+BE and SD+M models are very competitive and might be considered when all data-sources are available. Particularly, the ambient population (i.e. the average number of people who stop at the core) is one of the most important variables in the model and allows to better assess the number of people at risk, as suggested by previous works on aggregated mobility<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>, satellite imagery<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>, Twitter<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup> and census data<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup>. The <inline-formula id=\"IEq29\"><alternatives><tex-math id=\"M57\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_m$$\\end{document}</tex-math><mml:math id=\"M58\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq29.gif\"/></alternatives></inline-formula> improvements also indicate that the model relies less on the random effects and it is better at explaining crime from the input features. However, we found that it might generate large errors due to places that are outliers of mobility in densely populated areas or hotspots of activity (see Figure S16 and Figure S17 in the SI).<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Maps of the estimated number of crime for each neighborhood in Bogotá for the A) Social-disorganization, B) Built environment, C) Full model. D) shows the Full model’s prediction. E) shows the ground truth crime count.</p></caption><graphic xlink:href=\"41598_2020_70808_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par16\">Figure <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> shows the spatial gain in performance from the baseline in Bogotá. First, it reveals that our Full model prediction resembles the ground truth data (Figure <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> D-E), as confirmed by the high value of <inline-formula id=\"IEq30\"><alternatives><tex-math id=\"M59\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_c = 0.80$$\\end{document}</tex-math><mml:math id=\"M60\"><mml:mrow><mml:msubsup><mml:mi>R</mml:mi><mml:mi>c</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mo>=</mml:mo><mml:mn>0.80</mml:mn></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq30.gif\"/></alternatives></inline-formula>. Second, it shows that, while the SD and BE models achieve localized improvements (Figure <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> A-B), the Full model improves the prediction almost everywhere. However, the Full model performs quite poorly in a specific area of Bogotá (see Figure <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> C), part of the Engativá neighbourhood. By inspecting the coefficients of the model, we find that this area is an outlier as it is densely populated, thus causing an inflated prediction of crime, due to the high importance of residential and ambient population in the Bogotá model. Note, however, that our prediction is at the block level and the city-wide goodness of fit is <inline-formula id=\"IEq31\"><alternatives><tex-math id=\"M61\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_c = 0.80$$\\end{document}</tex-math><mml:math id=\"M62\"><mml:mrow><mml:msubsup><mml:mi>R</mml:mi><mml:mi>c</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mo>=</mml:mo><mml:mn>0.80</mml:mn></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq31.gif\"/></alternatives></inline-formula>.</p><p id=\"Par17\">The difference between <inline-formula id=\"IEq32\"><alternatives><tex-math id=\"M63\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_c$$\\end{document}</tex-math><mml:math id=\"M64\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>c</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq32.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq33\"><alternatives><tex-math id=\"M65\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2_m$$\\end{document}</tex-math><mml:math id=\"M66\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq33.gif\"/></alternatives></inline-formula> represents the unexplained variance due to spatial auto-correlation, which might suggest missing effects and variables. In Bogotá, our model points out that the touristic and dangerous neighbourhood La Candelaria, and the populous district of Engativá have significant unexplained variance that our input features cannot capture (see Figure S13 in the SI). In Boston, the area near the Franklin park indicates missing local factors (see Figure S12 in SI). In Los Angeles, unexplained variance seems to be tied to places with a large number of people, namely the international airport and the UCLA campus (see Figure S14 in SI). Again, in Chicago, missing variables are suggested near the prison and the southern area (see Figure S15 in SI). Altogether, these signals could help policymakers on including the best factors for each city and enacting policies that prevent crime.</p><p id=\"Par18\">Previous results suggested that human movements between different regions might help describing crime<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref>,<xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>. Thus, we test our model against this hypothesis by using the people trips between areas to model the auto-correlation between corehoods. This connectivity is not only influenced by distance but also by geographical barriers, roads, traffic, and public transportation. Moreover, it could be interpreted as a proxy of spatial mismatch and isolation, which was empirically found to be connected with crime<sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup>. To build the connectivity matrix we use the TimeGeo model, which simulates a reliable Origin-Destination matrix between regions and it is validated towards transportation surveys (see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">3.1</xref>). However, we find that mobility flows alone do not have good predictive power in LA and Boston. The interested reader can find more information on the definition and results of this connectivity matrix in Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">6</xref>.</p><p id=\"Par19\">While the effects of urban environment characteristics, socio-economic conditions, and mobility have been empirically tested separately<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR49\">49</xref>,<xref ref-type=\"bibr\" rid=\"CR60\">60</xref>–<xref ref-type=\"bibr\" rid=\"CR62\">62</xref></sup>, to the best of our knowledge, this is the first study to support with large-scale data the association of crime with socio-economic conditions, the built environment, and mobility. However, we find that these aspects do not play the same role across cities, and only some of them contribute to the crime prediction model.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Generalized Linear Model’s <inline-formula id=\"IEq34\"><alternatives><tex-math id=\"M67\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\beta $$\\end{document}</tex-math><mml:math id=\"M68\"><mml:mi>β</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq34.gif\"/></alternatives></inline-formula> coefficients showing that Social Disorganization, Built Environment and Mobility features do not play the same role in all cities. We highlight in blue the minimum and maximum coefficient for each feature. Overall, this figure shows that there is no universal theory of crime for spatial predictions.</p></caption><graphic xlink:href=\"41598_2020_70808_Fig3_HTML\" id=\"MO3\"/></fig></p></sec><sec id=\"Sec4\"><title>Neighborhood variables across cities</title><p id=\"Par20\">By comparing how features play different roles in different cities, we can understand how far can we push previous theoretical and empirical studies. In this section, we turn our attention to the standardized <inline-formula id=\"IEq35\"><alternatives><tex-math id=\"M69\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\beta }$$\\end{document}</tex-math><mml:math id=\"M70\"><mml:mi>β</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq35.gif\"/></alternatives></inline-formula> coefficients that reveal how features correlate with criminal activity.</p><p id=\"Par21\">First, we focus on the coefficients of the Full model, which combines socio-economic features with the characteristics of the built environment and human mobility. Note that here Chicago is excluded for lack of data. Figure <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> pictures that the <inline-formula id=\"IEq36\"><alternatives><tex-math id=\"M71\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\beta }$$\\end{document}</tex-math><mml:math id=\"M72\"><mml:mi>β</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq36.gif\"/></alternatives></inline-formula> coefficients vary greatly across cities. For example, land-use mix correlates negatively with criminal activity in Bogotá and Los Angeles, but positively in Boston. Similarly, higher population building age diversity is present in low-crime areas in Boston and Los Angeles, but in high-crime areas in Bogotá. Social Disorganization variables are no less different, as corehood instability is correlated with crime activity only in Bogotá, differently from what expected from the theory<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR63\">63</xref></sup>.</p><p id=\"Par22\">The discrepancies between cities could be explained by the different spatial and socio-economic processes at play. When we look at the bivariate correlations across features, we observe interesting patterns. For example, in Los Angeles and Boston, <italic>walkability</italic> is strongly positively correlated with population density and neighbourhood attractiveness, as expected<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR63\">63</xref></sup>, and slightly correlated with advantaged neighbourhoods. Differently, walkable areas in Bogotá have low population density and are highly advantaged, while the attractiveness is slightly correlated (see Figure S20 in SI). A possible reason for the <inline-formula id=\"IEq37\"><alternatives><tex-math id=\"M73\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\beta }$$\\end{document}</tex-math><mml:math id=\"M74\"><mml:mi>β</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq37.gif\"/></alternatives></inline-formula> coefficients disagreement lies on the multi-collinearity of the input features. Although we use the QR decomposition and Ridge penalty to shrink down the variables that are not necessary, the difference between the coefficients is present also in simpler models (e.g. core-only).</p><p id=\"Par23\">The difference between the results across cities also suggests that crime correlates differently with space and people. For example, we observe that in Bogotá high crime areas relate to advantaged neighbourhoods, while in Boston and Los Angeles higher crime seem to be linked to disadvantaged neighbourhoods, according to the theory<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR63\">63</xref></sup>. A possible explanation might be related to under-reporting and police disrespecting, which seems to be a problem particularly in Bogotá<sup><xref ref-type=\"bibr\" rid=\"CR64\">64</xref></sup>. However, literature has shown how neighbourhood cultural codes, informal local control, and problematic policing are also related to violent criminal activities<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>.</p><p id=\"Par24\">We also found some commonalities in all the cities. We find that corehoods with high disadvantage and ethnic diversity but, surprisingly, smaller blocks have higher crime activity. While in the core we find that the presence of Shops, Food POIs, and population (both residential and ambient) correlates positively with criminal activity. These results resonate with literature showing that the presence of POIs and ambient population increase crime due to a higher number of potential targets and offenders in an area. Additionally, we find that corehood attractiveness has a strong connection with crimes, suggesting that the presence of people that do not live nor work in the area might influence crime. This result is in contrast with literature based on Jacobs’ theory<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>, but resonate with Oscar Newman’s one arguing that a high number of visitors results in higher anonymity and, thus, crime<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Additionally, a recent empirical study from survey data<sup><xref ref-type=\"bibr\" rid=\"CR65\">65</xref></sup> agrees with our result, obtained instead with large-scale and passively collected information. In the supplementary materials (SI), we compare all the cities in detail (see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>-<xref rid=\"MOESM1\" ref-type=\"media\">11</xref>).</p><p id=\"Par25\">We acknowledge the big difference between crime types. In this paper, we analysed serious crimes, which comprise heterogeneous crime types such as rape and robberies. Thus, we also test our model by disentangling criminal activity into two main categories: property and violent crimes. We found that the Full model still outperforms the others, and that precise patterns can be extracted from the <inline-formula id=\"IEq38\"><alternatives><tex-math id=\"M75\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\beta $$\\end{document}</tex-math><mml:math id=\"M76\"><mml:mi>β</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq38.gif\"/></alternatives></inline-formula> coefficients analysis. For example, in Bogota <italic>walkability</italic> is much more important in describing property crime than violent crime, while in Los Angeles, higher <italic>walkability</italic> seems to suggest a lower presence of property crimes. However, we observe that the multifaceted picture found in the aggregated crimes still holds for the disentangled models.</p><p id=\"Par26\">We also tested the alternative assumption where all corehood features are computed at the core, and found that the models with features computed at the corehood perform better than the models using SD, BE and M features only at the core, which highlights the validity of the corehood (and neighbourhood) assumptions (see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">11</xref>).</p><p id=\"Par27\">Previous research have found universal common patterns even in highly heterogeneous data and behaviour. Literature has shown the existence of common mathematical models describing mobility<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>, cities<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup> and aggregated crime at the city level<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. To test the possibility of having a universal model that predicts crime in small areas, we test a model that uses only the features that behave in the same direction in all the cities. This model consistently performs worse than the Full model (see Note 10 in SI), showing that at this moment, no model is convenient to be easily applied to all cities. We also studied at what extent a model trained in one city can be tested to another city. We found that US cities are, as expected, more similar to each other than Bogotá, and that Los Angeles behave similarly to Chicago.</p></sec></sec><sec id=\"Sec5\"><title>Discussion</title><p id=\"Par28\">In this paper, we modelled the presence of crime across four cities, widely different with respect to cultural, economic, historical and geographical aspects. We found that the variability of the dynamics and history of each city poses a challenge to the existence of a model that “fits it all”, able to learn from one city and to predict on another one. Instead, we presented a model that could describe and disentangle the role of diverse factors in urban crime and draw some theoretical and practical implications.</p><p id=\"Par29\">The goal of this research goes beyond crime prediction in time (i.e. forecasting). Offences are concentrated in a small number of places<sup><xref ref-type=\"bibr\" rid=\"CR66\">66</xref></sup>, and are tightly coupled with places, stable over time<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. Thus, the easiest way to predict crime is modelling those few places with the highest number of crimes, also known as <italic>hotspots</italic><sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR67\">67</xref></sup>. On the contrary, we seek to shed light on the diverse set of factors at play with urban crime and do predictions for those areas without crime statistics (i.e. nowcasting).</p><p id=\"Par30\">Our cumulative results show little evidence in support of the Jane Jacobs’ theory, arguing that specific urban features and people on the street generate higher security. On the contrary, we often found that Jacobs’ features and urban vibrancy increase people’s vulnerability to crime, suggesting that further work has to be done in this direction.</p><p id=\"Par31\">We found that different theories often seen as competing can complement each other in models that take into account the socio-economic, built environment and mobility conditions together. The importance of mobility and built environment characteristics showed that competitive descriptive and predictive models can be built from data available at large scale without the necessity of costly in-field survey studies. However, we found that aspects related to the Social Disorganisation are important for crime description and prediction. Therefore, it is crucial to consider alternative sources of data to infer social cohesion and interactions and overcome the use of census information, which is costly to collect and rarely updated. There have been multiple attempts at inferring social interactions<sup><xref ref-type=\"bibr\" rid=\"CR68\">68</xref></sup>, poverty<sup><xref ref-type=\"bibr\" rid=\"CR69\">69</xref></sup>, well-being<sup><xref ref-type=\"bibr\" rid=\"CR70\">70</xref></sup> and unemployment<sup><xref ref-type=\"bibr\" rid=\"CR71\">71</xref></sup> but so far very little work has been done at small areas.</p><p id=\"Par32\">Comparing multiple cities in different countries do not come without limitations. First, our analysis ignore temporal variation such as opening times of POIs or temporal variation in mobility. Second, due to lack of consistent data, we did not account for variables such as political and housing policies, security perception, community participation, and social ties within family and within neighbourhoods that were previously found to be related to crime<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR72\">72</xref>,<xref ref-type=\"bibr\" rid=\"CR73\">73</xref></sup>. Finally, official crime data do not come without errors, given that not all crimes are reported nor recorded<sup><xref ref-type=\"bibr\" rid=\"CR74\">74</xref></sup>, and there is no “ground truth” data to gauge any bias in police records. We use official police records similarly to recent literature in the field<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>.</p><p id=\"Par33\">Our work seeks to make headway on the previous limitation of a single site of study. While recent works have started the use of street units and blocks to study criminal activity<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR75\">75</xref>,<xref ref-type=\"bibr\" rid=\"CR76\">76</xref></sup>, they often relied on a small subset of variables and one city. Analysing multiple cities together exposed criminology theories to discrepancies and differences, and answers to the call of a framework to compare crime in different cities<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. Descriptive and comparative modelling can help policymakers to see common patterns between cities, understand the use of urban space and deploy future investments and resources thoughtfully. Moreover, from the scientific perspective, descriptive modelling can provide insights for strong predictors, and potentially for explanatory variables, to be further investigated by explanatory modelling and experiments<sup><xref ref-type=\"bibr\" rid=\"CR77\">77</xref></sup>. Thus, we hope that additional research keeps exploring multi-dimensional aspects related to crime, to clarify potential crime causes and design better cities.</p></sec><sec id=\"Sec6\"><title>Methods</title><p id=\"Par34\">The socio-economical and Jane Jacobs’ urban theories are dependent upon the actions and activities at work in communities. Thus, we identified corehoods as social and geographical units of analysis. Then, we obtained and aggregated the data for each corehood of Bogotá, Boston, Los Angeles and Chicago.</p><sec id=\"Sec7\"><title>Crime data</title><p id=\"Par35\">Our crime data is obtained directly from police departments. Crime records are collected by the police, which annotates in the report the crime event at point locations (latitude and longitude) along with the category of crime and the time it happened.</p><p id=\"Par36\">Through its category, we associate each event to the Uniform Crime Reporting (UCR)<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup> categorization. The UCR program is a US statistical effort to make crime reports uniform across the country. The UCR divides crime in two main groups: Part 1 and Part 2 offences. The former is composed by violent crimes (aggravated assault, forcible rape, robbery and murder) and property crimes (larceny-theft, motor vehicle theft, burglary and arson), while the latter are considered less serious and they include offences such as simple assaults and nuisance crimes.</p><p id=\"Par37\">We filter out those crimes not belonging to Part 1 of UCR, similarly to most of the criminology literature. For Bogotá we mapped crime categories consistently with UCR categories, and we released the mapping for future research and comparisons. We also filtered out larceny crime events, which include among others thefts of bicycles, shoplifting, pick-pocketing, or the stealing of any property or article that is not taken by force and violence or by fraud. We consider larceny-thefts (except motor vehicle theft) as sometimes noisy and we expect the neighborhood effect to have a negligible impact on larceny-thefts (e.g. social cohesion with pick-pocketing in a shop). We geo-reference crimes to cores and, when a crime event happens in a street segment shared between cores, we evenly assign the event to both cores. Due to the limit in accuracy of GPS positioning, we create a buffer of 30 meters for each crime, which is the distance usually employed for stop location detection algorithms<sup><xref ref-type=\"bibr\" rid=\"CR78\">78</xref></sup> and criminology literature at micro-places<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR44\">44</xref>,<xref ref-type=\"bibr\" rid=\"CR76\">76</xref></sup>. We have no reason to suspect that the effect of the crime events stops at distances lower than 30 meters (e.g. robberies on the other side of the street are likely to affect residents on both sides). On the contrary, crime risk at hotspots has been observed to spread to distances up to 2000 meters<sup><xref ref-type=\"bibr\" rid=\"CR67\">67</xref></sup> spatially. Moreover, we note that the median area of cores are 0.378 square kilometers, which roughly means that each core has a median side of 615 meters (see Figure S11 in the SI).</p><p id=\"Par38\">More details are presented in the SI. We summed crime events over one year to minimize seasonal fluctuations.</p></sec><sec id=\"Sec8\"><title>Mobile phone data</title><p id=\"Par39\">We computed the ambient population and the OD matrices for Bogotá, Boston and Los Angeles from Call Detail Records (CDRs) of millions of individuals in the three cities. Mobile phone activity includes received and made calls and SMS activity. Each time a call or SMS is made/received, a CDR is generated. It includes some metadata such as the time and the tower at which the phone was connected when the activity was collected. Due to the inherent noise of CDRs<sup><xref ref-type=\"bibr\" rid=\"CR79\">79</xref></sup>, which are collected only for billing purposes, we follow seminal literature<sup><xref ref-type=\"bibr\" rid=\"CR78\">78</xref>,<xref ref-type=\"bibr\" rid=\"CR80\">80</xref>,<xref ref-type=\"bibr\" rid=\"CR81\">81</xref></sup> and apply a stop location algorithm to classify the geo-located points where people <italic>stay</italic> or <italic>pass-by</italic>. Then, we simulate reliable human mobility traces through the TimeGeo modelling framework<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>, which generates traces that well describe the real mobility of people. To be consistent with the travel surveys of each city it simulates the time, duration, direction and type of travels within the city. The types of travels are classified as Home-Based from/to Work (HBW), Home-Based from/to Other type of locations (HBO) and Non-Home-based from/to Other type of locations (NHB).</p><p id=\"Par40\">We fitted the model starting from aggregated and anonymized Call Detailed Records (CDRs) collected from 12-01-2013 to 05-31-2014, 6 weeks in 2010, and 10-15, 2012 to 11-24, 2012 for Bogotá, Boston and Los Angeles respectively. We validated the model with the National Household Travel Survey (NHTS)<sup><xref ref-type=\"bibr\" rid=\"CR82\">82</xref></sup> and California Household Travel Survey (CHTS)<sup><xref ref-type=\"bibr\" rid=\"CR83\">83</xref></sup> datasets. We refer to the SI for the validation of TimeGeo.</p><p id=\"Par41\">To build the <italic>ambient population</italic> we counted the number of people who stops at a specific location for at least one hour. Since TimeGeo is validated and peer reviewed with HBW, HBO and NHB types of trips, we define the corehood <italic>attractiveness</italic> counting the number of NHB trips with the corehood as destination. We did not use HBW trips, as we cannot differentiate the origin from the destination and thus attractiveness could correlate with residential places. For the same reason, we excluded HBO trips from the <italic>attractiveness</italic> definition.</p><p id=\"Par42\">The anonymized data for the three cities was collected for billing purposes by two mobile operators, who also kindly provided to us the data for the present research.</p></sec><sec id=\"Sec9\"><title>Spatial and census data</title><p id=\"Par43\">Census blocks, population, employment and poverty for US cities were drawn from the American Community Survey (ACS) (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.census.gov/programs-surveys/acs\">https://www.census.gov/programs-surveys/acs</ext-link>). The census data of Bogotá was obtained by the Departmento Administrativo Nacional de Estadística (DANE), which organized the 2005 general census for the city (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.dane.gov.co\">http://www.dane.gov.co</ext-link>). The poverty data of Bogotá was extracted from the Sisbén in the Identification System III of 2014. We also use the US Tiger dataset, OpenStreetMap (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.openstreetmap.org\">http://www.openstreetmap.org</ext-link>) geographical data and the POIs extracted from Foursquare (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.foursquare.com\">http://www.foursquare.com</ext-link>). The detailed description of datasets and related source URLs are listed in the SI.</p></sec><sec id=\"Sec10\"><title>Built environment features</title><p id=\"Par44\">We operationalize the Jane Jacobs conditions through some state of the art metrics defined in literature<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup> in all the corehoods. The land-use mix is computed as the average entropy among land uses: <inline-formula id=\"IEq39\"><alternatives><tex-math id=\"M77\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\text {LUM}_{L,i} = - \\sum _{j \\in L} \\frac{P_{i,j} \\log (P_{i,j})}{\\log (|L|)}$$\\end{document}</tex-math><mml:math id=\"M78\"><mml:mrow><mml:msub><mml:mtext>LUM</mml:mtext><mml:mrow><mml:mi>L</mml:mi><mml:mo>,</mml:mo><mml:mi>i</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mo>-</mml:mo><mml:msub><mml:mo>∑</mml:mo><mml:mrow><mml:mi>j</mml:mi><mml:mo>∈</mml:mo><mml:mi>L</mml:mi></mml:mrow></mml:msub><mml:mfrac><mml:mrow><mml:msub><mml:mi>P</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mo>,</mml:mo><mml:mi>j</mml:mi></mml:mrow></mml:msub><mml:mo>log</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mi>P</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mo>,</mml:mo><mml:mi>j</mml:mi></mml:mrow></mml:msub><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:mo>log</mml:mo><mml:mo stretchy=\"false\">(</mml:mo><mml:mo stretchy=\"false\">|</mml:mo><mml:mi>L</mml:mi><mml:mo stretchy=\"false\">|</mml:mo><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mfrac></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq39.gif\"/></alternatives></inline-formula>, where <inline-formula id=\"IEq40\"><alternatives><tex-math id=\"M79\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$P_{i,j}$$\\end{document}</tex-math><mml:math id=\"M80\"><mml:msub><mml:mi>P</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mo>,</mml:mo><mml:mi>j</mml:mi></mml:mrow></mml:msub></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq40.gif\"/></alternatives></inline-formula> is the percentage of square meters having land use <italic>j</italic> in unit <italic>i</italic>, and <inline-formula id=\"IEq41\"><alternatives><tex-math id=\"M81\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$L = \\{\\text {residential}, \\text {commercial and institutional},$$\\end{document}</tex-math><mml:math id=\"M82\"><mml:mrow><mml:mi>L</mml:mi><mml:mo>=</mml:mo><mml:mo stretchy=\"false\">{</mml:mo><mml:mtext>residential</mml:mtext><mml:mo>,</mml:mo><mml:mtext>commercial and institutional</mml:mtext><mml:mo>,</mml:mo></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq41.gif\"/></alternatives></inline-formula><inline-formula id=\"IEq42\"><alternatives><tex-math id=\"M83\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\text {park and recreational}\\}$$\\end{document}</tex-math><mml:math id=\"M84\"><mml:mrow><mml:mtext>park and recreational</mml:mtext><mml:mo stretchy=\"false\">}</mml:mo></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq42.gif\"/></alternatives></inline-formula> represents the considered land uses in the metric. The LUM ranges between 0, wherein the unit is composed by only one land use (e.g. residential), and 1, wherein developed area is equally shared among the <italic>n</italic> land-uses.</p><p id=\"Par45\">Then, for each corehood we determine the <italic>walkability</italic> through the accessibility of the core to the nearest point of interests (e.g. convenience stores, restaurants, sport facilities). Consistently with literature<sup><xref ref-type=\"bibr\" rid=\"CR84\">84</xref></sup>, we define the weighted <italic>walkability</italic> score as: <inline-formula id=\"IEq43\"><alternatives><tex-math id=\"M85\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\text {walk}_i = \\frac{1}{|B_i|} \\sum _{c \\in C} \\sum _{b \\in B_i} {{\\,{\\mathrm{wdist}}\\,}}(b, {{\\,{\\mathrm{closest}}\\,}}(b, \\text {POI}_c))$$\\end{document}</tex-math><mml:math id=\"M86\"><mml:mrow><mml:msub><mml:mtext>walk</mml:mtext><mml:mi>i</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:mrow><mml:mo stretchy=\"false\">|</mml:mo></mml:mrow><mml:msub><mml:mi>B</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mrow><mml:mo stretchy=\"false\">|</mml:mo></mml:mrow></mml:mrow></mml:mfrac><mml:msub><mml:mo>∑</mml:mo><mml:mrow><mml:mi>c</mml:mi><mml:mo>∈</mml:mo><mml:mi>C</mml:mi></mml:mrow></mml:msub><mml:msub><mml:mo>∑</mml:mo><mml:mrow><mml:mi>b</mml:mi><mml:mo>∈</mml:mo><mml:msub><mml:mi>B</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mrow></mml:msub><mml:mrow><mml:mspace width=\"0.166667em\"/><mml:mi mathvariant=\"normal\">wdist</mml:mi><mml:mspace width=\"0.166667em\"/></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>b</mml:mi><mml:mo>,</mml:mo><mml:mrow><mml:mspace width=\"0.166667em\"/><mml:mi mathvariant=\"normal\">closest</mml:mi><mml:mspace width=\"0.166667em\"/></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>b</mml:mi><mml:mo>,</mml:mo><mml:msub><mml:mtext>POI</mml:mtext><mml:mi>c</mml:mi></mml:msub><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq43.gif\"/></alternatives></inline-formula>, where <italic>C</italic> is the set of categories (i.e. Food, Shops, Grocery, Schools, Entertainment, Parks and outside, Coffee, Banks, Books), <inline-formula id=\"IEq44\"><alternatives><tex-math id=\"M87\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${{\\,{\\mathrm{wdist}}\\,}}$$\\end{document}</tex-math><mml:math id=\"M88\"><mml:mrow><mml:mspace width=\"0.166667em\"/><mml:mi mathvariant=\"normal\">wdist</mml:mi><mml:mspace width=\"0.166667em\"/></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq44.gif\"/></alternatives></inline-formula> is the street-network distance decay function, and <inline-formula id=\"IEq45\"><alternatives><tex-math id=\"M89\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\text {POI}_c$$\\end{document}</tex-math><mml:math id=\"M90\"><mml:msub><mml:mtext>POI</mml:mtext><mml:mi>c</mml:mi></mml:msub></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq45.gif\"/></alternatives></inline-formula> is the set of POIs of category <italic>c</italic>. The distance decay function gives a weight (importance) to each POI reachable from a starting point. Additional information about the <italic>walkability</italic> score can be find in the SI.</p><p id=\"Par46\">We then compute the average block area among the set <inline-formula id=\"IEq46\"><alternatives><tex-math id=\"M91\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$B_i$$\\end{document}</tex-math><mml:math id=\"M92\"><mml:msub><mml:mi>B</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq46.gif\"/></alternatives></inline-formula> of blocks in unit <italic>i</italic> as <inline-formula id=\"IEq47\"><alternatives><tex-math id=\"M93\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\text {Blocks area}_i = \\frac{1}{|B_i|} \\sum _{b \\in B_i} {{\\,{\\mathrm{area}}\\,}}(b)$$\\end{document}</tex-math><mml:math id=\"M94\"><mml:mrow><mml:mtext>Blocks</mml:mtext><mml:mspace width=\"0.333333em\"/><mml:msub><mml:mtext>area</mml:mtext><mml:mi>i</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:mrow><mml:mo stretchy=\"false\">|</mml:mo></mml:mrow><mml:msub><mml:mi>B</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mrow><mml:mo stretchy=\"false\">|</mml:mo></mml:mrow></mml:mrow></mml:mfrac><mml:msub><mml:mo>∑</mml:mo><mml:mrow><mml:mi>b</mml:mi><mml:mo>∈</mml:mo><mml:msub><mml:mi>B</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mrow></mml:msub><mml:mrow><mml:mspace width=\"0.166667em\"/><mml:mi mathvariant=\"normal\">area</mml:mi><mml:mspace width=\"0.166667em\"/></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>b</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq47.gif\"/></alternatives></inline-formula>, and the building age diversity as the standard deviation of building ages in the corehood.</p><p id=\"Par47\">Finally, we operationalize Jacobs’ density condition with the dwelling units density, computed from census data. Additional details are described in the SI.</p></sec><sec id=\"Sec11\"><title>Social Disorganization</title><p id=\"Par48\">We create the feature <italic>disadvantage</italic> and <italic>instability</italic><sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup> through the two largest PCA principal components of: (i) unemployment rate, (ii) poverty rate, defined as people living below the poverty line, and (iii) residential mobility rate, defined as the percentage of people who recently changed residency (one year for US cities and fiver years for Bogotá). From the loadings of the PCA linear combination we verified that disadvantage is mainly a linear combination of poverty rate and unemployment, while instability is mainly about residential mobility rate.</p><p id=\"Par49\">In the Social Disorganization variables we do not include any ethnic-specific variables (e.g. percentage of black people) other than diversity because they might be present only in some places and not in others (e.g. native Americans in Bogotá), and to avoid any ethnic-specific bias. Ethnic diversity represents the difficulties of a community to communicate and collaborate for a common goal. Accordingly to the literature<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref>,<xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>, it is computed as the Hirschman-Herfindahl diversity index of six population groups <inline-formula id=\"IEq48\"><alternatives><tex-math id=\"M95\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$H = 1- \\sum _{i=1}^N s_i^2$$\\end{document}</tex-math><mml:math id=\"M96\"><mml:mrow><mml:mi>H</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn><mml:mo>-</mml:mo><mml:msubsup><mml:mo>∑</mml:mo><mml:mrow><mml:mi>i</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>N</mml:mi></mml:msubsup><mml:msubsup><mml:mi>s</mml:mi><mml:mi>i</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq48.gif\"/></alternatives></inline-formula>, where <inline-formula id=\"IEq49\"><alternatives><tex-math id=\"M97\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$s_i$$\\end{document}</tex-math><mml:math id=\"M98\"><mml:msub><mml:mi>s</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq49.gif\"/></alternatives></inline-formula> is the proportion of people belonging to the ethnicity <italic>i</italic>, and <italic>N</italic> is the number of ethnicities. Consistently with the literature we include for US cities: Hispanics, non-Hispanic Blacks, Whites, Asians, Native Hawaiians - Pacific Islanders and others. For Bogotá we include: Indigenous, Rom, Islanders (San Andrés), Palenquero, Black and others.</p></sec><sec id=\"Sec12\"><title>Bayesian model</title><p id=\"Par50\">Let <inline-formula id=\"IEq50\"><alternatives><tex-math id=\"M99\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$y_i$$\\end{document}</tex-math><mml:math id=\"M100\"><mml:msub><mml:mi>y</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq50.gif\"/></alternatives></inline-formula> be the discrete number of crimes for a set of spatial regions <inline-formula id=\"IEq51\"><alternatives><tex-math id=\"M101\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$i= 1 ,\\ldots , N$$\\end{document}</tex-math><mml:math id=\"M102\"><mml:mrow><mml:mi>i</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn><mml:mo>,</mml:mo><mml:mo>…</mml:mo><mml:mo>,</mml:mo><mml:mi>N</mml:mi></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq51.gif\"/></alternatives></inline-formula>. We approximate the relation between crimes and spatial features through a Negative Binomial approach that models the non-negative nature of the crime-counts in a city, but also the overdispersion found in the data (Note 4 in the SI). Specifically, <inline-formula id=\"IEq52\"><alternatives><tex-math id=\"M103\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\ln ({{\\mathbb {E}}}(Y)) = {\\mathbf {X}}\\beta + {\\mathbf {b}}$$\\end{document}</tex-math><mml:math id=\"M104\"><mml:mrow><mml:mo>ln</mml:mo><mml:mo stretchy=\"false\">(</mml:mo><mml:mi mathvariant=\"double-struck\">E</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>Y</mml:mi><mml:mo stretchy=\"false\">)</mml:mo><mml:mo stretchy=\"false\">)</mml:mo><mml:mo>=</mml:mo><mml:mi mathvariant=\"bold\">X</mml:mi><mml:mi>β</mml:mi><mml:mo>+</mml:mo><mml:mi mathvariant=\"bold\">b</mml:mi></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq52.gif\"/></alternatives></inline-formula> where <inline-formula id=\"IEq53\"><alternatives><tex-math id=\"M105\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathbf {X}}$$\\end{document}</tex-math><mml:math id=\"M106\"><mml:mi mathvariant=\"bold\">X</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq53.gif\"/></alternatives></inline-formula> is the input data and <inline-formula id=\"IEq54\"><alternatives><tex-math id=\"M107\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\beta }$$\\end{document}</tex-math><mml:math id=\"M108\"><mml:mi>β</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq54.gif\"/></alternatives></inline-formula> the coefficients of the model. <inline-formula id=\"IEq55\"><alternatives><tex-math id=\"M109\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathbf {b}}$$\\end{document}</tex-math><mml:math id=\"M110\"><mml:mi mathvariant=\"bold\">b</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq55.gif\"/></alternatives></inline-formula> are the random effects that accounts for the unexplained variability of crime (i.e. the spatial-autocorrelation). In this paper, we account the spatial auto-correlation with the Bayesian Spatial Filtering (BSF)<sup><xref ref-type=\"bibr\" rid=\"CR85\">85</xref></sup> that defines <inline-formula id=\"IEq56\"><alternatives><tex-math id=\"M111\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathbf {b}} = {\\mathbf {E}}{\\gamma }$$\\end{document}</tex-math><mml:math id=\"M112\"><mml:mrow><mml:mi mathvariant=\"bold\">b</mml:mi><mml:mo>=</mml:mo><mml:mi mathvariant=\"bold\">E</mml:mi><mml:mi>γ</mml:mi></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq56.gif\"/></alternatives></inline-formula> where <inline-formula id=\"IEq57\"><alternatives><tex-math id=\"M113\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\gamma }$$\\end{document}</tex-math><mml:math id=\"M114\"><mml:mi>γ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq57.gif\"/></alternatives></inline-formula> are coefficients to be found. <inline-formula id=\"IEq58\"><alternatives><tex-math id=\"M115\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathbf {E}}$$\\end{document}</tex-math><mml:math id=\"M116\"><mml:mi mathvariant=\"bold\">E</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq58.gif\"/></alternatives></inline-formula> is instead defined as the first principal components of <inline-formula id=\"IEq59\"><alternatives><tex-math id=\"M117\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathbf {E}}_{\\mathrm{full}} = \\mathbf {MCM}$$\\end{document}</tex-math><mml:math id=\"M118\"><mml:mrow><mml:msub><mml:mi mathvariant=\"bold\">E</mml:mi><mml:mi mathvariant=\"normal\">full</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mi mathvariant=\"bold\">MCM</mml:mi></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq59.gif\"/></alternatives></inline-formula>, where <inline-formula id=\"IEq60\"><alternatives><tex-math id=\"M119\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathbf {C}}$$\\end{document}</tex-math><mml:math id=\"M120\"><mml:mi mathvariant=\"bold\">C</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq60.gif\"/></alternatives></inline-formula> is a spatial matrix that describes the graph between spatial locations, while <inline-formula id=\"IEq61\"><alternatives><tex-math id=\"M121\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathbf {M}} = {\\mathbf {I}} - {\\mathbf {X}}({\\mathbf {X}}'{\\mathbf {X}})-{\\mathbf {X}}'$$\\end{document}</tex-math><mml:math id=\"M122\"><mml:mrow><mml:mi mathvariant=\"bold\">M</mml:mi><mml:mo>=</mml:mo><mml:mi mathvariant=\"bold\">I</mml:mi><mml:mo>-</mml:mo><mml:mi mathvariant=\"bold\">X</mml:mi><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:msup><mml:mrow><mml:mi mathvariant=\"bold\">X</mml:mi></mml:mrow><mml:mo>′</mml:mo></mml:msup><mml:mi mathvariant=\"bold\">X</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mo>-</mml:mo><mml:msup><mml:mrow><mml:mi mathvariant=\"bold\">X</mml:mi></mml:mrow><mml:mo>′</mml:mo></mml:msup></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq61.gif\"/></alternatives></inline-formula>, which is an approximation of the spatial error model<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>. We tested for the presence of spatial auto-correlation on the residuals of all the models without finding significant auto-correlation. As the results might change with different definitions of <inline-formula id=\"IEq62\"><alternatives><tex-math id=\"M123\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathbf {C}}$$\\end{document}</tex-math><mml:math id=\"M124\"><mml:mi mathvariant=\"bold\">C</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq62.gif\"/></alternatives></inline-formula>, we tested all the models for three definitions: i) <inline-formula id=\"IEq63\"><alternatives><tex-math id=\"M125\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathbf {C}}$$\\end{document}</tex-math><mml:math id=\"M126\"><mml:mi mathvariant=\"bold\">C</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq63.gif\"/></alternatives></inline-formula> is a binary adjacency matrix identifying whether a corehood overlaps another corehood, ii) <inline-formula id=\"IEq64\"><alternatives><tex-math id=\"M127\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathbf {C}}$$\\end{document}</tex-math><mml:math id=\"M128\"><mml:mi mathvariant=\"bold\">C</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq64.gif\"/></alternatives></inline-formula> is a inverse distance matrix between corehoods, iii) <inline-formula id=\"IEq65\"><alternatives><tex-math id=\"M129\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathbf {C}}$$\\end{document}</tex-math><mml:math id=\"M130\"><mml:mi mathvariant=\"bold\">C</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq65.gif\"/></alternatives></inline-formula> describes the flow of people between corehoods, which is extracted from mobile phone data. We found that the binary matrix consistently outperforms other definitions. Additional details of the presented models, definition of <inline-formula id=\"IEq66\"><alternatives><tex-math id=\"M131\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathbf {C}}$$\\end{document}</tex-math><mml:math id=\"M132\"><mml:mi mathvariant=\"bold\">C</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq66.gif\"/></alternatives></inline-formula>, and other competitive models tested are present in the SI.</p><p id=\"Par51\">As we have to account for collinearity, we employ a Ridge penalty to all fixed effects.</p></sec><sec id=\"Sec13\"><title>Model calibration ed evaluation</title><p id=\"Par52\">Model calibration is carried out by means of Markov Chain Monte Carlo (MCMC) approach. We run the MCMC method for 20,000 iterations and chose as burn-in the first 15,000 iterations to ensure that the remaining 5,000 iterations are in the high-probability region. Convergence for all the models was assured by the Gelman-Rubin convergence statistics<sup><xref ref-type=\"bibr\" rid=\"CR86\">86</xref></sup> and visual inspection of the traces.</p><p id=\"Par53\">We assess how well the models describe crime through the conditional <inline-formula id=\"IEq67\"><alternatives><tex-math id=\"M133\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2$$\\end{document}</tex-math><mml:math id=\"M134\"><mml:msup><mml:mi>R</mml:mi><mml:mn>2</mml:mn></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq67.gif\"/></alternatives></inline-formula> and the marginal <inline-formula id=\"IEq68\"><alternatives><tex-math id=\"M135\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$R^2$$\\end{document}</tex-math><mml:math id=\"M136\"><mml:msup><mml:mi>R</mml:mi><mml:mn>2</mml:mn></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq68.gif\"/></alternatives></inline-formula><sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>, which adapt the popular coefficient of determination to the generalized linear mixed-effects models. They are defined as:<disp-formula id=\"Equ2\"><alternatives><tex-math id=\"M137\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\begin{aligned} R^2_m&= \\frac{\\sigma _f^2}{\\sigma _f^2 + \\sigma _r^2 + \\sigma _{\\epsilon }^2}\\\\ R^2_c&= \\frac{\\sigma _f^2 + \\sigma _r^2}{\\sigma _f^2 + \\sigma _r^2 + \\sigma _{\\epsilon }^2} \\end{aligned}$$\\end{document}</tex-math><mml:math id=\"M138\" display=\"block\"><mml:mrow><mml:mtable><mml:mtr><mml:mtd columnalign=\"right\"><mml:msubsup><mml:mi>R</mml:mi><mml:mi>m</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:mtd><mml:mtd columnalign=\"left\"><mml:mrow><mml:mo>=</mml:mo><mml:mfrac><mml:msubsup><mml:mi>σ</mml:mi><mml:mi>f</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mrow><mml:msubsup><mml:mi>σ</mml:mi><mml:mi>f</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mo>+</mml:mo><mml:msubsup><mml:mi>σ</mml:mi><mml:mi>r</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mo>+</mml:mo><mml:msubsup><mml:mi>σ</mml:mi><mml:mrow><mml:mi>ϵ</mml:mi></mml:mrow><mml:mn>2</mml:mn></mml:msubsup></mml:mrow></mml:mfrac></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd columnalign=\"right\"><mml:mrow><mml:mrow/><mml:msubsup><mml:mi>R</mml:mi><mml:mi>c</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:mrow></mml:mtd><mml:mtd columnalign=\"left\"><mml:mrow><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msubsup><mml:mi>σ</mml:mi><mml:mi>f</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mo>+</mml:mo><mml:msubsup><mml:mi>σ</mml:mi><mml:mi>r</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:mrow><mml:mrow><mml:msubsup><mml:mi>σ</mml:mi><mml:mi>f</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mo>+</mml:mo><mml:msubsup><mml:mi>σ</mml:mi><mml:mi>r</mml:mi><mml:mn>2</mml:mn></mml:msubsup><mml:mo>+</mml:mo><mml:msubsup><mml:mi>σ</mml:mi><mml:mrow><mml:mi>ϵ</mml:mi></mml:mrow><mml:mn>2</mml:mn></mml:msubsup></mml:mrow></mml:mfrac></mml:mrow></mml:mtd></mml:mtr></mml:mtable></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70808_Article_Equ2.gif\" position=\"anchor\"/></alternatives></disp-formula>where <inline-formula id=\"IEq69\"><alternatives><tex-math id=\"M139\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sigma _f^2$$\\end{document}</tex-math><mml:math id=\"M140\"><mml:msubsup><mml:mi>σ</mml:mi><mml:mi>f</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq69.gif\"/></alternatives></inline-formula> is the variance explained by the fixed effects, <inline-formula id=\"IEq70\"><alternatives><tex-math id=\"M141\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sigma _r^2$$\\end{document}</tex-math><mml:math id=\"M142\"><mml:msubsup><mml:mi>σ</mml:mi><mml:mi>r</mml:mi><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq70.gif\"/></alternatives></inline-formula> is the variance explained by the random effects, and <inline-formula id=\"IEq71\"><alternatives><tex-math id=\"M143\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sigma _{\\epsilon }^2$$\\end{document}</tex-math><mml:math id=\"M144\"><mml:msubsup><mml:mi>σ</mml:mi><mml:mrow><mml:mi>ϵ</mml:mi></mml:mrow><mml:mn>2</mml:mn></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq71.gif\"/></alternatives></inline-formula> is the variance of the residuals. Specifically, <inline-formula id=\"IEq72\"><alternatives><tex-math id=\"M145\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$f= {\\mathbf {X}}\\beta $$\\end{document}</tex-math><mml:math id=\"M146\"><mml:mrow><mml:mi>f</mml:mi><mml:mo>=</mml:mo><mml:mi mathvariant=\"bold\">X</mml:mi><mml:mi>β</mml:mi></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq72.gif\"/></alternatives></inline-formula>, <inline-formula id=\"IEq73\"><alternatives><tex-math id=\"M147\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$r= {\\mathbf {E}}\\gamma $$\\end{document}</tex-math><mml:math id=\"M148\"><mml:mrow><mml:mi>r</mml:mi><mml:mo>=</mml:mo><mml:mi mathvariant=\"bold\">E</mml:mi><mml:mi>γ</mml:mi></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq73.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq74\"><alternatives><tex-math id=\"M149\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\epsilon $$\\end{document}</tex-math><mml:math id=\"M150\"><mml:mi>ϵ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq74.gif\"/></alternatives></inline-formula> is specific to the Negative Binomial and defined<sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup> as <inline-formula id=\"IEq75\"><alternatives><tex-math id=\"M151\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\epsilon = \\ln {(1+1/\\mu +1/\\phi )}$$\\end{document}</tex-math><mml:math id=\"M152\"><mml:mrow><mml:mi>ϵ</mml:mi><mml:mo>=</mml:mo><mml:mo>ln</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mn>1</mml:mn><mml:mo>+</mml:mo><mml:mn>1</mml:mn><mml:mo stretchy=\"false\">/</mml:mo><mml:mi>μ</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn><mml:mo stretchy=\"false\">/</mml:mo><mml:mi>ϕ</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq75.gif\"/></alternatives></inline-formula>, with <inline-formula id=\"IEq76\"><alternatives><tex-math id=\"M153\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\mu = \\frac{1}{N} \\sum _i^N y_i$$\\end{document}</tex-math><mml:math id=\"M154\"><mml:mrow><mml:mi>μ</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mi>N</mml:mi></mml:mfrac><mml:msubsup><mml:mo>∑</mml:mo><mml:mi>i</mml:mi><mml:mi>N</mml:mi></mml:msubsup><mml:msub><mml:mi>y</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq76.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq77\"><alternatives><tex-math id=\"M155\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\phi $$\\end{document}</tex-math><mml:math id=\"M156\"><mml:mi>ϕ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq77.gif\"/></alternatives></inline-formula> is the shape parameter of the Negative Binomial distribution.</p><p id=\"Par54\">We assess the out of sample predictive accuracy through the Pareto-smoothed importance sampling Leave-One-Out cross-validation (PSIS-LOO, here simply referred as LOO)<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup> and the Deviance Information Criterion (DIC)<sup><xref ref-type=\"bibr\" rid=\"CR87\">87</xref></sup>. Even though DIC has been used extensively for practical model comparison in many disciplines, recent literature on Bayesian models evaluation strongly discourage the use of DIC due to its numerous disadvantages including the fact that it works well only if the posterior is close to a Gaussian, its lack of consistency and the fact that is not a proper predictive criterion<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref>,<xref ref-type=\"bibr\" rid=\"CR88\">88</xref></sup>. Since LOO overcome the DIC issues, it has rapidly become the state of the art for evaluating Bayesian models. We employ the LOO in the main paper, while we present the DIC results in the supplementary. The LOO is defined in the log score as:<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M157\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\begin{aligned} \\text {LOO} = \\sum _{i=1}^n \\ln \\left( \\frac{\\sum _{s=1}^S w_i^s p(y_i|\\theta ^s)}{\\sum _{s=1}^S w_i^s}\\right) . \\end{aligned}$$\\end{document}</tex-math><mml:math id=\"M158\" display=\"block\"><mml:mrow><mml:mtable><mml:mtr><mml:mtd columnalign=\"right\"><mml:mrow><mml:mtext>LOO</mml:mtext><mml:mo>=</mml:mo><mml:munderover><mml:mo>∑</mml:mo><mml:mrow><mml:mi>i</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>n</mml:mi></mml:munderover><mml:mo>ln</mml:mo><mml:mfenced close=\")\" open=\"(\"><mml:mfrac><mml:mrow><mml:msubsup><mml:mo>∑</mml:mo><mml:mrow><mml:mi>s</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>S</mml:mi></mml:msubsup><mml:msubsup><mml:mi>w</mml:mi><mml:mi>i</mml:mi><mml:mi>s</mml:mi></mml:msubsup><mml:mi>p</mml:mi><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mi>y</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mo stretchy=\"false\">|</mml:mo><mml:msup><mml:mi>θ</mml:mi><mml:mi>s</mml:mi></mml:msup><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:msubsup><mml:mo>∑</mml:mo><mml:mrow><mml:mi>s</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>S</mml:mi></mml:msubsup><mml:msubsup><mml:mi>w</mml:mi><mml:mi>i</mml:mi><mml:mi>s</mml:mi></mml:msubsup></mml:mrow></mml:mfrac></mml:mfenced><mml:mo>.</mml:mo></mml:mrow></mml:mtd></mml:mtr></mml:mtable></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70808_Article_Equ1.gif\" position=\"anchor\"/></alternatives></disp-formula>where <italic>n</italic> is the number of data points, <inline-formula id=\"IEq78\"><alternatives><tex-math id=\"M159\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\theta ^s$$\\end{document}</tex-math><mml:math id=\"M160\"><mml:msup><mml:mi>θ</mml:mi><mml:mi>s</mml:mi></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq78.gif\"/></alternatives></inline-formula> are draws from the full posterior <inline-formula id=\"IEq79\"><alternatives><tex-math id=\"M161\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$p(\\theta |y)$$\\end{document}</tex-math><mml:math id=\"M162\"><mml:mrow><mml:mi>p</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>θ</mml:mi><mml:mo stretchy=\"false\">|</mml:mo><mml:mi>y</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq79.gif\"/></alternatives></inline-formula>, <inline-formula id=\"IEq80\"><alternatives><tex-math id=\"M163\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$s=1,\\dots ,S$$\\end{document}</tex-math><mml:math id=\"M164\"><mml:mrow><mml:mi>s</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn><mml:mo>,</mml:mo><mml:mo>⋯</mml:mo><mml:mo>,</mml:mo><mml:mi>S</mml:mi></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq80.gif\"/></alternatives></inline-formula> represent the <italic>S</italic> draws, and <inline-formula id=\"IEq81\"><alternatives><tex-math id=\"M165\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$w_i^s$$\\end{document}</tex-math><mml:math id=\"M166\"><mml:msubsup><mml:mi>w</mml:mi><mml:mi>i</mml:mi><mml:mi>s</mml:mi></mml:msubsup></mml:math><inline-graphic xlink:href=\"41598_2020_70808_Article_IEq81.gif\"/></alternatives></inline-formula> is a vector of weights that are the Pareto Smoothed importance ratios built through an algorithm described in the LOO original paper<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>. The best model is associated with the smallest LOO value.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec14\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70808_MOESM1_ESM.pdf\"><caption><p>Supplementary information.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70808-2.</p></sec><ack><title>Acknowledgements</title><p>We thank Paolo Bosetti and Junpeng Lao for the helpful comments. We especially thank Andrés Clavijo for his support on the data, we all hope that this work could make Bogotá better. This work was supported by the Berkeley DeepDrive and the ITS Berkeley 2018-19 SB1 Research Grant (to M.C.G.); the French Development Agency and the World Bank (to M.D.N., B.L. and E.L.).</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>M.D.N, E.L., M.C.G. and B.L. designed research and experiments; M.D.N, Y.X., M.C.G. and B.L. performed research and experiments; M.D.N, M.C.G. and B.L. contributed new analytic tools; M.D.N, and Y.X. analysed the data; and M.D.N, M.C.G. and B.L. wrote the paper. All authors read, reviewed and approved the final manuscript.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>We are pleased to make available the source-code and datasets accompanying this research. The projects files are available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/denadai2/bayesian-crime-multiple-cities/\">https://github.com/denadai2/bayesian-crime-multiple-cities/</ext-link>.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par58\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Weisburd</surname><given-names>D</given-names></name><name><surname>Groff</surname><given-names>ER</given-names></name><name><surname>Yang</surname><given-names>S-M</given-names></name></person-group><source>The Criminology of Place: Street Segments and Our Understanding of the Crime Problem</source><year>2012</year><publisher-loc>Oxford</publisher-loc><publisher-name>Oxford University Press</publisher-name></element-citation></ref><ref id=\"CR2\"><label>2.</label><mixed-citation publication-type=\"other\">Tita, G. E. & Greenbaum, R. T. Crime, neighborhoods, and units of analysis: putting space in its place. In <italic>Putting Crime in Its Place</italic>, 145–170 (Springer, Berlin, 2009).</mixed-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Spelman</surname><given-names>W</given-names></name></person-group><article-title>Criminal careers of public places</article-title><source>Crime Place</source><year>1995</year><volume>4</volume><fpage>115</fpage><lpage>144</lpage></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weisburd</surname><given-names>D</given-names></name><name><surname>Bushway</surname><given-names>S</given-names></name><name><surname>Lum</surname><given-names>C</given-names></name><name><surname>Yang</surname><given-names>S-M</given-names></name></person-group><article-title>Trajectories of crime at places: a longitudinal study of street segments in the city of seattle</article-title><source>Criminology</source><year>2004</year><volume>42</volume><fpage>283</fpage><lpage>322</lpage></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Sampson</surname><given-names>RJ</given-names></name></person-group><source>Great American City: Chicago and the Enduring Neighborhood Effect</source><year>2012</year><publisher-loc>Chicago</publisher-loc><publisher-name>University of Chicago Press</publisher-name></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Graif</surname><given-names>C</given-names></name><name><surname>Gladfelter</surname><given-names>AS</given-names></name><name><surname>Matthews</surname><given-names>SA</given-names></name></person-group><article-title>Urban poverty and neighborhood effects on crime: incorporating spatial and network perspectives</article-title><source>Soc. Compass</source><year>2014</year><volume>8</volume><fpage>1140</fpage><lpage>1155</lpage></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sampson</surname><given-names>RJ</given-names></name><name><surname>Groves</surname><given-names>WB</given-names></name></person-group><article-title>Community structure and crime: testing social-disorganization theory</article-title><source>Am. J. Sociol.</source><year>1989</year><volume>94</volume><fpage>774</fpage><lpage>802</lpage></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Graif</surname><given-names>C</given-names></name><name><surname>Sampson</surname><given-names>RJ</given-names></name></person-group><article-title>Spatial heterogeneity in the effects of immigration and diversity on neighborhood homicide rates</article-title><source>Homicide Stud.</source><year>2009</year><volume>13</volume><fpage>242</fpage><lpage>260</lpage><pub-id pub-id-type=\"doi\">10.1177/1088767909336728</pub-id><pub-id pub-id-type=\"pmid\">20671811</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sampson</surname><given-names>RJ</given-names></name></person-group><article-title>Neighborhoods and violent crime: a multilevel study of collective efficacy</article-title><source>Science</source><year>1997</year><volume>277</volume><fpage>918</fpage><lpage>924</lpage><pub-id pub-id-type=\"doi\">10.1126/science.277.5328.918</pub-id><pub-id pub-id-type=\"pmid\">9252316</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Newman</surname><given-names>O</given-names></name></person-group><source>Defensible Space</source><year>1972</year><publisher-loc>New York</publisher-loc><publisher-name>Macmillan</publisher-name></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Jacobs</surname><given-names>J</given-names></name></person-group><source>The Death and Life of Great American Cities</source><year>1961</year><publisher-loc>New York</publisher-loc><publisher-name>Vintage</publisher-name></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>Q</given-names></name><name><surname>Phillips</surname><given-names>NE</given-names></name><name><surname>Small</surname><given-names>ML</given-names></name><name><surname>Sampson</surname><given-names>RJ</given-names></name></person-group><article-title>Urban mobility and neighborhood isolation in Americas 50 largest cities</article-title><source>PNAS</source><year>2018</year><volume>115</volume><fpage>7735</fpage><lpage>7740</lpage><pub-id pub-id-type=\"pmid\">29987019</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cohen</surname><given-names>LE</given-names></name><name><surname>Felson</surname><given-names>M</given-names></name></person-group><article-title>Social change and crime rate trends: a routine activity approach</article-title><source>Am. Soc. Rev.</source><year>1979</year><volume>44</volume><fpage>588</fpage><lpage>608</lpage></element-citation></ref><ref id=\"CR14\"><label>14.</label><mixed-citation publication-type=\"other\">Bogomolov, A. <italic>et al.</italic> Once upon a crime: towards crime prediction from demographics and mobile data. In <italic>ICMI</italic>, 427–434 (ACM, 2014).</mixed-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kubrin</surname><given-names>CE</given-names></name><name><surname>Weitzer</surname><given-names>R</given-names></name></person-group><article-title>Retaliatory homicide: concentrated disadvantage and neighborhood culture</article-title><source>Soc. Probl.</source><year>2003</year><volume>50</volume><fpage>157</fpage><lpage>180</lpage></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hipp</surname><given-names>JR</given-names></name><name><surname>Boessen</surname><given-names>A</given-names></name></person-group><article-title>Egohoods as waves washing across the city: a new measure of neighborhoods</article-title><source>Criminology</source><year>2013</year><volume>51</volume><fpage>287</fpage><lpage>327</lpage></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Andresen</surname><given-names>MA</given-names></name></person-group><article-title>The ambient population and crime analysis</article-title><source>Prof. Geogr.</source><year>2011</year><volume>63</volume><fpage>193</fpage><lpage>212</lpage></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jones</surname><given-names>RW</given-names></name><name><surname>Pridemore</surname><given-names>WA</given-names></name></person-group><article-title>Toward an integrated multilevel theory of crime at place: routine activities, social disorganization, and the law of crime concentration</article-title><source>J. Quant. Criminol.</source><year>2019</year><volume>35</volume><fpage>543</fpage><lpage>572</lpage></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Contreras</surname><given-names>C</given-names></name></person-group><article-title>A block-level analysis of medical marijuana dispensaries and crime in the city of los angeles</article-title><source>Justice Q.</source><year>2017</year><volume>34</volume><fpage>1069</fpage><lpage>1095</lpage></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Malleson</surname><given-names>N</given-names></name><name><surname>Andresen</surname><given-names>MA</given-names></name></person-group><article-title>Spatio-temporal crime hotspots and the ambient population</article-title><source>Crime Sci.</source><year>2015</year><volume>4</volume><fpage>10</fpage></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hipp</surname><given-names>JR</given-names></name><name><surname>Kim</surname><given-names>Y-A</given-names></name><name><surname>Kane</surname><given-names>K</given-names></name></person-group><article-title>The effect of the physical environment on crime rates: capturing housing age and housing type at varying spatial scales</article-title><source>Crime Delinq.</source><year>2019</year><volume>65</volume><fpage>1570</fpage><lpage>1595</lpage></element-citation></ref><ref id=\"CR22\"><label>22.</label><mixed-citation publication-type=\"other\">Traunmueller, M., Quattrone, G. & Capra, L. Mining mobile phone data to investigate urban crime theories at scale. In <italic>International Conference on Social Informatics</italic>, 396–411 (Springer, 2014).</mixed-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Song</surname><given-names>G</given-names></name><etal/></person-group><article-title>Crime feeds on legal activities: daily mobility flows help to explain thieves target location choices</article-title><source>J. Quant. Criminol.</source><year>2019</year><volume>35</volume><fpage>831</fpage><lpage>854</lpage></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sohn</surname><given-names>D-W</given-names></name></person-group><article-title>Residential crimes and neighbourhood built environment: assessing the effectiveness of crime prevention through environmental design (CPTED)</article-title><source>Cities</source><year>2016</year><volume>52</volume><fpage>86</fpage><lpage>93</lpage></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kadar</surname><given-names>C</given-names></name><name><surname>Pletikosa</surname><given-names>I</given-names></name></person-group><article-title>Mining large-scale human mobility data for long-term crime prediction</article-title><source>EPJ Data Sci.</source><year>2018</year><volume>7</volume><fpage>26</fpage></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sampson</surname><given-names>RJ</given-names></name><name><surname>Morenoff</surname><given-names>JD</given-names></name><name><surname>Earls</surname><given-names>F</given-names></name></person-group><article-title>Beyond social capital: spatial dynamics of collective efficacy for children</article-title><source>Am. Soc. Rev.</source><year>1999</year><volume>64</volume><fpage>633</fpage><lpage>660</lpage></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Felson</surname><given-names>M</given-names></name><name><surname>Clarke</surname><given-names>RV</given-names></name></person-group><article-title>Opportunity makes the thief</article-title><source>Police Res. Ser. Paper</source><year>1998</year><volume>98</volume><fpage>1</fpage><lpage>36</lpage></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brantingham</surname><given-names>PL</given-names></name><name><surname>Brantingham</surname><given-names>PJ</given-names></name></person-group><article-title>Nodes, paths and edges: considerations on the complexity of crime and the physical environment</article-title><source>J. Environ. Psychol.</source><year>1993</year><volume>13</volume><fpage>3</fpage><lpage>28</lpage></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Felson</surname><given-names>M</given-names></name><name><surname>Boba</surname><given-names>RL</given-names></name></person-group><source>Crime and Everyday Life</source><year>2010</year><publisher-loc>Thousand Oaks</publisher-loc><publisher-name>Sage</publisher-name></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Hindelang</surname><given-names>MJ</given-names></name><name><surname>Gottfredson</surname><given-names>MR</given-names></name><name><surname>Garofalo</surname><given-names>J</given-names></name></person-group><source>Victims of Personal Crime: An Empirical Foundation for a Theory of Personal Victimization</source><year>1978</year><publisher-loc>Cambridge, MA</publisher-loc><publisher-name>Ballinger</publisher-name></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>OBrien</surname><given-names>DT</given-names></name><name><surname>Sampson</surname><given-names>RJ</given-names></name></person-group><article-title>Public and private spheres of neighborhood disorder: assessing pathways to violence using large-scale digital records</article-title><source>J. Res. Crime Delinq.</source><year>2015</year><volume>52</volume><fpage>486</fpage><lpage>510</lpage></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Murray</surname><given-names>RK</given-names></name><name><surname>Roncek</surname><given-names>DW</given-names></name></person-group><article-title>Measuring diffusion of assaults around bars through radius and adjacency techniques</article-title><source>Criminal Justice Rev.</source><year>2008</year><volume>33</volume><fpage>199</fpage><lpage>220</lpage></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Salesses</surname><given-names>P</given-names></name><name><surname>Schechtner</surname><given-names>K</given-names></name><name><surname>Hidalgo</surname><given-names>CA</given-names></name></person-group><article-title>The collaborative image of the city: mapping the inequality of urban perception</article-title><source>PloS ONE</source><year>2013</year><volume>8</volume><fpage>e0119352</fpage></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sampson</surname><given-names>RJ</given-names></name></person-group><article-title>Neighborhood and crime: the structural determinants of personal victimization</article-title><source>J. Res. Crime Delinq.</source><year>1985</year><volume>22</volume><fpage>7</fpage><lpage>40</lpage><pub-id pub-id-type=\"doi\">10.1177/0022427885022001002</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hipp</surname><given-names>JR</given-names></name><name><surname>Butts</surname><given-names>CT</given-names></name><name><surname>Acton</surname><given-names>R</given-names></name><name><surname>Nagle</surname><given-names>NN</given-names></name><name><surname>Boessen</surname><given-names>A</given-names></name></person-group><article-title>Extrapolative simulation of neighborhood networks based on population spatial distribution: do they predict crime?</article-title><source>Soc. Netw.</source><year>2013</year><volume>35</volume><fpage>614</fpage><lpage>625</lpage></element-citation></ref><ref id=\"CR36\"><label>36.</label><mixed-citation publication-type=\"other\">Wang, H., Kifer, D., Graif, C. & Li, Z. Crime rate inference with big data. In <italic>ACM SIGKDD</italic>, KDD’16, 635–644, 10.1145/2939672.2939736 (ACM, New York, NY, USA, 2016).</mixed-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Andresen</surname><given-names>MA</given-names></name></person-group><article-title>Crime measures and the spatial analysis of criminal activity</article-title><source>Br. J. Criminol.</source><year>2006</year><volume>46</volume><fpage>258</fpage><lpage>285</lpage></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gonzalez</surname><given-names>MC</given-names></name><name><surname>Hidalgo</surname><given-names>CA</given-names></name><name><surname>Barabasi</surname><given-names>A-L</given-names></name></person-group><article-title>Understanding individual human mobility patterns</article-title><source>Nature</source><year>2008</year><volume>453</volume><fpage>779</fpage><lpage>782</lpage><pub-id pub-id-type=\"pmid\">18528393</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Barbosa</surname><given-names>H</given-names></name><etal/></person-group><article-title>Human mobility: models and applications</article-title><source>Phys. Rep.</source><year>2018</year><volume>734</volume><fpage>1</fpage><lpage>74</lpage></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Louail</surname><given-names>T</given-names></name><etal/></person-group><article-title>From mobile phone data to the spatial structure of cities</article-title><source>Sci. Rep.</source><year>2014</year><volume>4</volume><fpage>5276</fpage><pub-id pub-id-type=\"pmid\">24923248</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><mixed-citation publication-type=\"other\">Gomez-Lievano, A., Patterson-Lomba, O. & Hausmann, R. Explaining the prevalence, scaling and variance of urban phenomena. <italic>Nat. Energy</italic> 1–9, (2018).</mixed-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Caminha</surname><given-names>C</given-names></name><etal/></person-group><article-title>Human mobility in large cities as a proxy for crime</article-title><source>PLoS ONE</source><year>2017</year><volume>12</volume><fpage>e0171609</fpage><pub-id pub-id-type=\"pmid\">28158268</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Ojo</surname><given-names>A</given-names></name><name><surname>Ojewale</surname><given-names>O</given-names></name><etal/></person-group><source>Urbanisation and Crime in Nigeria</source><year>2019</year><publisher-loc>Berlin</publisher-loc><publisher-name>Springer</publisher-name></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>I</given-names></name><name><surname>Jung</surname><given-names>S</given-names></name><name><surname>Lee</surname><given-names>J</given-names></name><name><surname>Macdonald</surname><given-names>E</given-names></name></person-group><article-title>Street crime prediction model based on the physical characteristics of a streetscape: analysis of streets in low-rise housing areas in South Korea</article-title><source>Environ. Plan. B Urban Anal. City Sci.</source><year>2019</year><volume>46</volume><fpage>862</fpage><lpage>879</lpage></element-citation></ref><ref id=\"CR45\"><label>45.</label><mixed-citation publication-type=\"other\">De Nadai, M. & Lepri, B. The economic value of neighborhoods: predicting real estate prices from the urban environment. In <italic>2018 IEEE 5th International Conference on Data Science and Advanced Analytics (DSAA)</italic>, 323–330 (IEEE, 2018).</mixed-citation></ref><ref id=\"CR46\"><label>46.</label><mixed-citation publication-type=\"other\">FBI. Uniform crime reporting (UCR) program. <ext-link ext-link-type=\"uri\" xlink:href=\"https://ucr.fbi.gov/\">https://ucr.fbi.gov/</ext-link>. Accessed 21 June 2020.</mixed-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sampson</surname><given-names>RJ</given-names></name></person-group><article-title>The place of context: a theory and strategy for criminologys hard problems</article-title><source>Criminology</source><year>2013</year><volume>51</volume><fpage>1</fpage><lpage>31</lpage></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sampson</surname><given-names>RJ</given-names></name><name><surname>Graif</surname><given-names>C</given-names></name></person-group><article-title>Neighborhood social capital as differential social organization: resident and leadership dimensions</article-title><source>Am. Behav. Sci.</source><year>2009</year><volume>52</volume><fpage>1579</fpage><lpage>1605</lpage></element-citation></ref><ref id=\"CR49\"><label>49.</label><mixed-citation publication-type=\"other\">De Nadai, M. <italic>et al.</italic> The death and life of great Italian cities: a mobile phone data perspective. In <italic>WWW</italic>, 413–423, 10.1145/2872427.2883084 (International World Wide Web Conferences Steering Committee, Republic and Canton of Geneva, Switzerland, 2016).</mixed-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Leyden</surname><given-names>KM</given-names></name></person-group><article-title>Social capital and the built environment: the importance of walkable neighborhoods</article-title><source>Am. J. Public Health</source><year>2003</year><volume>93</volume><fpage>1546</fpage><lpage>1551</lpage><pub-id pub-id-type=\"pmid\">12948978</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jiang</surname><given-names>S</given-names></name><etal/></person-group><article-title>The TimeGeo modeling framework for urban mobility without travel surveys</article-title><source>PNAS</source><year>2016</year><volume>113</volume><fpage>E5370</fpage><lpage>E5378</lpage><pub-id pub-id-type=\"doi\">10.1073/pnas.1524261113</pub-id><pub-id pub-id-type=\"pmid\">27573826</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Osgood</surname><given-names>DW</given-names></name></person-group><article-title>Poisson-based regression analysis of aggregate crime rates</article-title><source>J. Quant. Criminol.</source><year>2000</year><volume>16</volume><fpage>21</fpage><lpage>43</lpage></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Griffith</surname><given-names>DA</given-names></name><name><surname>Peres-Neto</surname><given-names>PR</given-names></name></person-group><article-title>Spatial modeling in ecology: the flexibility of eigenfunction spatial analyses</article-title><source>Ecology</source><year>2006</year><volume>87</volume><fpage>2603</fpage><lpage>2613</lpage><pub-id pub-id-type=\"pmid\">17089668</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tiefelsdorf</surname><given-names>M</given-names></name><name><surname>Griffith</surname><given-names>DA</given-names></name></person-group><article-title>Semiparametric filtering of spatial autocorrelation: the eigenvector approach</article-title><source>Environ. Plan. A</source><year>2007</year><volume>39</volume><fpage>1193</fpage><lpage>1221</lpage></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nakagawa</surname><given-names>S</given-names></name><name><surname>Johnson</surname><given-names>PC</given-names></name><name><surname>Schielzeth</surname><given-names>H</given-names></name></person-group><article-title>The coefficient of determination r 2 and intra-class correlation coefficient from generalized linear mixed-effects models revisited and expanded</article-title><source>J. R. Soc. Interface</source><year>2017</year><volume>14</volume><fpage>20170213</fpage><pub-id pub-id-type=\"pmid\">28904005</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vehtari</surname><given-names>A</given-names></name><name><surname>Gelman</surname><given-names>A</given-names></name><name><surname>Gabry</surname><given-names>J</given-names></name></person-group><article-title>Practical bayesian model evaluation using leave-one-out cross-validation and waic</article-title><source>Stat. Comput.</source><year>2017</year><volume>27</volume><fpage>1413</fpage><lpage>1432</lpage></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lutters</surname><given-names>WG</given-names></name><name><surname>Ackerman</surname><given-names>MS</given-names></name></person-group><article-title>An introduction to the chicago school of sociology</article-title><source>Interval Res. Propr.</source><year>1996</year><volume>2</volume><fpage>1</fpage><lpage>25</lpage></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mburu</surname><given-names>LW</given-names></name><name><surname>Helbich</surname><given-names>M</given-names></name></person-group><article-title>Crime risk estimation with a commuter-harmonized ambient population</article-title><source>Ann. Am. Assoc. Geogr.</source><year>2016</year><volume>106</volume><fpage>804</fpage><lpage>818</lpage><pub-id pub-id-type=\"doi\">10.1080/24694452.2016.1163252</pub-id></element-citation></ref><ref id=\"CR59\"><label>59.</label><mixed-citation publication-type=\"other\">Wang, H. & Li, Z. Region representation learning via mobility flow. In <italic>Proceedings of the 2017 ACM on Conference on Information and Knowledge Management</italic>, 237–246 (2017).</mixed-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Graif</surname><given-names>C</given-names></name><name><surname>Lungeanu</surname><given-names>A</given-names></name><name><surname>Yetter</surname><given-names>AM</given-names></name></person-group><article-title>Neighborhood isolation in chicago: violent crime effects on structural isolation and homophily in inter-neighborhood commuting networks</article-title><source>Soc. Netw.</source><year>2017</year><volume>51</volume><fpage>40</fpage><lpage>59</lpage></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>S</given-names></name><name><surname>Yoo</surname><given-names>C</given-names></name><name><surname>Ha</surname><given-names>J</given-names></name><name><surname>Seo</surname><given-names>J</given-names></name></person-group><article-title>Are perceived neighbourhood built environments associated with social capital? Evidence from the Seoul survey in South Korea</article-title><source>Int. J. Urban Sci.</source><year>2012</year><pub-id pub-id-type=\"doi\">10.1080/12265934.2017.1396909</pub-id></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sung</surname><given-names>H</given-names></name><name><surname>Lee</surname><given-names>S</given-names></name></person-group><article-title>Residential built environment and walking activity: empirical evidence of Jane Jacobs Urban Vitality</article-title><source>Transp. Res. D Transp. Environ.</source><year>2015</year><volume>41</volume><fpage>318</fpage><lpage>329</lpage></element-citation></ref><ref id=\"CR63\"><label>63.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Shaw</surname><given-names>CR</given-names></name><name><surname>McKay</surname><given-names>HD</given-names></name></person-group><source>Juvenile Delinquency and Urban Areas</source><year>1942</year><publisher-loc>Chicago</publisher-loc><publisher-name>University of Chicago Press</publisher-name></element-citation></ref><ref id=\"CR64\"><label>64.</label><mixed-citation publication-type=\"other\">Godoy, J. F., Rodriguez, C. & Zuleta, H. Security and sustainable development in bogota, colombia. <italic>Geneva: DCAF</italic> (2018).</mixed-citation></ref><ref id=\"CR65\"><label>65.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boivin</surname><given-names>R</given-names></name><name><surname>Felson</surname><given-names>M</given-names></name></person-group><article-title>Crimes by visitors versus crimes by residents: the influence of visitor inflows</article-title><source>J. Quant. Criminol.</source><year>2018</year><volume>34</volume><fpage>465</fpage><lpage>480</lpage></element-citation></ref><ref id=\"CR66\"><label>66.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>Y</given-names></name><name><surname>Eck</surname><given-names>JE</given-names></name><name><surname>Soo</surname><given-names>Hyun O</given-names></name><name><surname>Martinez</surname><given-names>NN</given-names></name></person-group><article-title>How concentrated is crime at places? a systematic review from 1970 to 2015</article-title><source>Crime Sci.</source><year>2017</year><volume>6</volume><fpage>6</fpage><pub-id pub-id-type=\"doi\">10.1186/s40163-017-0069-x</pub-id></element-citation></ref><ref id=\"CR67\"><label>67.</label><mixed-citation publication-type=\"other\">Short, M. B., Brantingham, P. J., Bertozzi, A. L. & Tita, G. E. Dissipation and displacement of hotspots in reaction-diffusion models of crime. <italic>PNAS</italic> (2010).</mixed-citation></ref><ref id=\"CR68\"><label>68.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eagle</surname><given-names>N</given-names></name><name><surname>Pentland</surname><given-names>AS</given-names></name><name><surname>Lazer</surname><given-names>D</given-names></name></person-group><article-title>Inferring friendship network structure by using mobile phone data</article-title><source>PNAS</source><year>2009</year><volume>106</volume><fpage>15274</fpage><lpage>15278</lpage><pub-id pub-id-type=\"pmid\">19706491</pub-id></element-citation></ref><ref id=\"CR69\"><label>69.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Blumenstock</surname><given-names>J</given-names></name><name><surname>Cadamuro</surname><given-names>G</given-names></name><name><surname>On</surname><given-names>R</given-names></name></person-group><article-title>Predicting poverty and wealth from mobile phone metadata</article-title><source>Science</source><year>2015</year><volume>350</volume><fpage>1073</fpage><lpage>1076</lpage><pub-id pub-id-type=\"doi\">10.1126/science.aac4420</pub-id><pub-id pub-id-type=\"pmid\">26612950</pub-id></element-citation></ref><ref id=\"CR70\"><label>70.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pappalardo</surname><given-names>L</given-names></name><etal/></person-group><article-title>An analytical framework to nowcast well-being using mobile phone data</article-title><source>Int. J. Data Sci. Anal.</source><year>2016</year><volume>2</volume><fpage>75</fpage><lpage>92</lpage><pub-id pub-id-type=\"doi\">10.1007/s41060-016-0013-2</pub-id></element-citation></ref><ref id=\"CR71\"><label>71.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Toole</surname><given-names>JL</given-names></name><etal/></person-group><article-title>Tracking employment shocks using mobile phone data</article-title><source>J. R. Soc. Interface</source><year>2015</year><volume>12</volume><fpage>20150185</fpage><pub-id pub-id-type=\"doi\">10.1098/rsif.2015.0185</pub-id><pub-id pub-id-type=\"pmid\">26018965</pub-id></element-citation></ref><ref id=\"CR72\"><label>72.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Faust</surname><given-names>K</given-names></name><name><surname>Tita</surname><given-names>GE</given-names></name></person-group><article-title>Social networks and crime: pitfalls and promises for advancing the field</article-title><source>Ann. Rev. Criminol.</source><year>2019</year><volume>2</volume><fpage>99</fpage><lpage>122</lpage></element-citation></ref><ref id=\"CR73\"><label>73.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tran</surname><given-names>VC</given-names></name><name><surname>Graif</surname><given-names>C</given-names></name><name><surname>Jones</surname><given-names>AD</given-names></name><name><surname>Small</surname><given-names>ML</given-names></name><name><surname>Winship</surname><given-names>C</given-names></name></person-group><article-title>Participation in context: neighborhood diversity and organizational involvement in boston</article-title><source>City Commun.</source><year>2013</year><volume>12</volume><fpage>187</fpage><lpage>210</lpage></element-citation></ref><ref id=\"CR74\"><label>74.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Small</surname><given-names>ML</given-names></name></person-group><article-title>Understanding when people will report crimes to the police</article-title><source>Proc. Nat. Acad. Sci.</source><year>2018</year><volume>115</volume><fpage>8057</fpage><lpage>8059</lpage><pub-id pub-id-type=\"pmid\">30026194</pub-id></element-citation></ref><ref id=\"CR75\"><label>75.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>Y-A</given-names></name><name><surname>Hipp</surname><given-names>JR</given-names></name></person-group><article-title>Street egohood: an alternative perspective of measuring neighborhood and spatial patterns of crime</article-title><source>J. Quant. Criminol.</source><year>2020</year><volume>36</volume><fpage>29</fpage><lpage>66</lpage></element-citation></ref><ref id=\"CR76\"><label>76.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rosser</surname><given-names>G</given-names></name><name><surname>Davies</surname><given-names>T</given-names></name><name><surname>Bowers</surname><given-names>KJ</given-names></name><name><surname>Johnson</surname><given-names>SD</given-names></name><name><surname>Cheng</surname><given-names>T</given-names></name></person-group><article-title>Predictive crime mapping: arbitrary grids or street networks?</article-title><source>J. Quant. Criminol.</source><year>2017</year><volume>33</volume><fpage>569</fpage><lpage>594</lpage><pub-id pub-id-type=\"pmid\">32025086</pub-id></element-citation></ref><ref id=\"CR77\"><label>77.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kenett</surname><given-names>RS</given-names></name><name><surname>Pfeffermann</surname><given-names>D</given-names></name><name><surname>Steinberg</surname><given-names>DM</given-names></name></person-group><article-title>Election polls—a survey, a critique, and proposals</article-title><source>Ann. Rev. Stat. Appl.</source><year>2018</year><pub-id pub-id-type=\"doi\">10.1146/annurev-statistics-031017-100204</pub-id></element-citation></ref><ref id=\"CR78\"><label>78.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Nadai</surname><given-names>M</given-names></name><name><surname>Cardoso</surname><given-names>A</given-names></name><name><surname>Lima</surname><given-names>A</given-names></name><name><surname>Lepri</surname><given-names>B</given-names></name><name><surname>Oliver</surname><given-names>N</given-names></name></person-group><article-title>Strategies and limitations in app usage and human mobility</article-title><source>Sci. Rep.</source><year>2019</year><volume>9</volume><fpage>1</fpage><lpage>9</lpage><pub-id pub-id-type=\"pmid\">30626917</pub-id></element-citation></ref><ref id=\"CR79\"><label>79.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xu</surname><given-names>Y</given-names></name><name><surname>Çolak</surname><given-names>S</given-names></name><name><surname>Kara</surname><given-names>EC</given-names></name><name><surname>Moura</surname><given-names>SJ</given-names></name><name><surname>González</surname><given-names>MC</given-names></name></person-group><article-title>Planning for electric vehicle needs by coupling charging profiles with urban mobility</article-title><source>Nat. Energy</source><year>2018</year><volume>3</volume><fpage>484</fpage><lpage>493</lpage></element-citation></ref><ref id=\"CR80\"><label>80.</label><mixed-citation publication-type=\"other\">Jiang, S. <italic>et al.</italic> A review of urban computing for mobile phone traces: current methods, challenges and opportunities. In <italic>Proceedings of the 2nd ACM SIGKDD International Workshop on Urban Computing</italic>, 1–9 (2013).</mixed-citation></ref><ref id=\"CR81\"><label>81.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zheng</surname><given-names>Y</given-names></name><name><surname>Xie</surname><given-names>X</given-names></name></person-group><article-title>Learning travel recommendations from user-generated gps traces</article-title><source>ACM Trans. Intell. Syst. Technol. (TIST)</source><year>2011</year><volume>2</volume><fpage>1</fpage><lpage>29</lpage></element-citation></ref><ref id=\"CR82\"><label>82.</label><mixed-citation publication-type=\"other\">US Department of Transportation, F. H. A. National household travel survey. <ext-link ext-link-type=\"uri\" xlink:href=\"http://nhts.ornl.gov\">http://nhts.ornl.gov</ext-link>. Accessed 19 June (2020).</mixed-citation></ref><ref id=\"CR83\"><label>83.</label><mixed-citation publication-type=\"other\">California Department of Transportation (Caltrans). California household travel survey (CHTS). <ext-link ext-link-type=\"uri\" xlink:href=\"https://dot.ca.gov/programs/transportation-planning/economics-data-management/transportation-economics/ca-household-travel-survey\">https://dot.ca.gov/programs/transportation-planning/economics-data-management/transportation-economics/ca-household-travel-survey</ext-link>. Accessed 19 June (2020).</mixed-citation></ref><ref id=\"CR84\"><label>84.</label><mixed-citation publication-type=\"other\">Front seat walk score methodology. Tech. Rep. Available online at <ext-link ext-link-type=\"uri\" xlink:href=\"http://pubs.cedeus.cl/omeka/files/original/b6fa690993d59007784a7a26804d42be.pdf\">http://pubs.cedeus.cl/omeka/files/original/b6fa690993d59007784a7a26804d42be.pdf</ext-link>. Accessed on 3 January 2020, (Accessed February 20, 2020).</mixed-citation></ref><ref id=\"CR85\"><label>85.</label><mixed-citation publication-type=\"other\">Hughes, J. Spatial regression and the bayesian filter. <italic>arXiv preprint</italic><ext-link ext-link-type=\"uri\" xlink:href=\"http://arxiv.org/abs/1706.04651\">arXiv:1706.04651</ext-link> (2017).</mixed-citation></ref><ref id=\"CR86\"><label>86.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brooks</surname><given-names>SP</given-names></name><name><surname>Gelman</surname><given-names>A</given-names></name></person-group><article-title>General methods for monitoring convergence of iterative simulations</article-title><source>J. Comput. Graph. Stat.</source><year>1998</year><volume>7</volume><fpage>434</fpage><lpage>455</lpage></element-citation></ref><ref id=\"CR87\"><label>87.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Spiegelhalter</surname><given-names>DJ</given-names></name><name><surname>Best</surname><given-names>NG</given-names></name><name><surname>Carlin</surname><given-names>BP</given-names></name><name><surname>Van Der Linde</surname><given-names>A</given-names></name></person-group><article-title>Bayesian measures of model complexity and fit</article-title><source>J. R. Stat. Soc. Ser. B (Stat. Methodol.)</source><year>2002</year><volume>64</volume><fpage>583</fpage><lpage>639</lpage><pub-id pub-id-type=\"doi\">10.1111/1467-9868.00353</pub-id></element-citation></ref><ref id=\"CR88\"><label>88.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Spiegelhalter</surname><given-names>DJ</given-names></name><name><surname>Best</surname><given-names>NG</given-names></name><name><surname>Carlin</surname><given-names>BP</given-names></name><name><surname>Van der Linde</surname><given-names>A</given-names></name></person-group><article-title>The deviance information criterion: 12 years on</article-title><source>J. R. Stat. Soc. Ser. B (Stat. Methodol.)</source><year>2014</year><volume>76</volume><fpage>485</fpage><lpage>493</lpage></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Transl Psychiatry</journal-id><journal-id journal-id-type=\"iso-abbrev\">Transl Psychiatry</journal-id><journal-title-group><journal-title>Translational Psychiatry</journal-title></journal-title-group><issn pub-type=\"epub\">2158-3188</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807774</article-id><article-id pub-id-type=\"pmc\">PMC7431539</article-id><article-id pub-id-type=\"publisher-id\">951</article-id><article-id pub-id-type=\"doi\">10.1038/s41398-020-00951-x</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Clustering by phenotype and genome-wide association study in autism</article-title></title-group><contrib-group><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Narita</surname><given-names>Akira</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-0818-3158</contrib-id><name><surname>Nagai</surname><given-names>Masato</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Mizuno</surname><given-names>Satoshi</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ogishima</surname><given-names>Soichi</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Tamiya</surname><given-names>Gen</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ueki</surname><given-names>Masao</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sakurai</surname><given-names>Rieko</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Makino</surname><given-names>Satoshi</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Obara</surname><given-names>Taku</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ishikuro</surname><given-names>Mami</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yamanaka</surname><given-names>Chizuru</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Matsubara</surname><given-names>Hiroko</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kuniyoshi</surname><given-names>Yasutaka</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Murakami</surname><given-names>Keiko</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ueno</surname><given-names>Fumihiko</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Noda</surname><given-names>Aoi</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kobayashi</surname><given-names>Tomoko</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kobayashi</surname><given-names>Mika</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Usuzaki</surname><given-names>Takuma</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ohseto</surname><given-names>Hisashi</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hozawa</surname><given-names>Atsushi</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kikuya</surname><given-names>Masahiro</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>Metoki</surname><given-names>Hirohito</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kure</surname><given-names>Shigeo</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-6445-0911</contrib-id><name><surname>Kuriyama</surname><given-names>Shinichi</given-names></name><address><email>kuriyama@med.tohoku.ac.jp</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.69566.3a</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2248 6943</institution-id><institution>Tohoku Medical Megabank Organization, </institution><institution>Tohoku University, </institution></institution-wrap>Sendai, Japan </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.69566.3a</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2248 6943</institution-id><institution>Graduate School of Medicine, </institution><institution>Tohoku University, </institution></institution-wrap>Sendai, Japan </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.7597.c</institution-id><institution-id institution-id-type=\"ISNI\">0000000094465255</institution-id><institution>RIKEN Center for Advanced Intelligence Project, </institution></institution-wrap>Tokyo, Japan </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.69566.3a</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2248 6943</institution-id><institution>Tohoku University Hospital, </institution><institution>Tohoku University, </institution></institution-wrap>Sendai, Japan </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.264706.1</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9239 9995</institution-id><institution>School of Medicine, </institution><institution>Teikyo University, </institution></institution-wrap>Tokyo, Japan </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.412755.0</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2166 7427</institution-id><institution>School of Medicine, </institution><institution>Tohoku Medical and Pharmaceutical University, </institution></institution-wrap>Sendai, Japan </aff><aff id=\"Aff7\"><label>7</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.69566.3a</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2248 6943</institution-id><institution>International Research Institute of Disaster Science, </institution><institution>Tohoku University, </institution></institution-wrap>Sendai, Miyagi Japan </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>290</elocation-id><history><date date-type=\"received\"><day>14</day><month>4</month><year>2020</year></date><date date-type=\"rev-recd\"><day>15</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>22</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Autism spectrum disorder (ASD) has phenotypically and genetically heterogeneous characteristics. A simulation study demonstrated that attempts to categorize patients with a complex disease into more homogeneous subgroups could have more power to elucidate hidden heritability. We conducted cluster analyses using the k-means algorithm with a cluster number of 15 based on phenotypic variables from the Simons Simplex Collection (SSC). As a preliminary study, we conducted a conventional genome-wide association study (GWAS) with a data set of 597 ASD cases and 370 controls. In the second step, we divided cases based on the clustering results and conducted GWAS in each of the subgroups vs controls (cluster-based GWAS). We also conducted cluster-based GWAS on another SSC data set of 712 probands and 354 controls in the replication stage. In the preliminary study, which was conducted in conventional GWAS design, we observed no significant associations. In the second step of cluster-based GWASs, we identified 65 chromosomal loci, which included 30 intragenic loci located in 21 genes and 35 intergenic loci that satisfied the threshold of <italic>P</italic> < 5.0 × 10<sup>−8</sup>. Some of these loci were located within or near previously reported candidate genes for ASD: <italic>CDH5</italic>, <italic>CNTN5, CNTNAP5, DNAH17, DPP10, DSCAM</italic>, <italic>FOXK1</italic>, <italic>GABBR2, GRIN2A</italic>5, <italic>ITPR1, NTM, SDK1, SNCA</italic>, and <italic>SRRM4</italic>. Of these 65 significant chromosomal loci, rs11064685 located within the <italic>SRRM4</italic> gene had a significantly different distribution in the cases vs controls in the replication cohort. These findings suggest that clustering may successfully identify subgroups with relatively homogeneous disease etiologies. Further cluster validation and replication studies are warranted in larger cohorts.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Molecular neuroscience</kwd><kwd>Autism spectrum disorders</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100002241</institution-id><institution>MEXT | Japan Science and Technology Agency (JST)</institution></institution-wrap></funding-source><award-id>19390171</award-id><award-id>16H05242</award-id><principal-award-recipient><name><surname>Kuriyama</surname><given-names>Shinichi</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par2\">Autism spectrum disorder (ASD) has heterogeneous characteristics in terms of both phenotypic features and genetics. ASD is mainly characterized by difficulties in communication and repetitive behaviors, but ASD also shows many other symptoms<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. Regarding genetics, previous studies have not consistently identified genetic variants that are associated with an increased risk of ASD<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>, although several lines of evidence suggest that genetic factors strongly contribute to the increased risk of ASD. Monozygotic twins have higher concordance rates of ASD (92%) than dizygotic twins (10%)<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. The recurrence risk ratio is 22 for ASD among siblings<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. The Human Gene module of the Simons Foundation Autism Research Initiative (SFARI) gene provides a comprehensive reference for suggested human ASD-related genes in an up-to-date manner<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> and currently demonstrates ~1000 genes that may have links to ASD, potentially indicating the heterogeneity of ASD. In addition to phenotype and genotype heterogeneities, ASD shows heterogeneous responses to interventions. Several kinds of pharmacological treatments are suggested, but the effects of these treatments are controversial<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>.</p><p id=\"Par3\">If the heterogeneous phenotypes and responses to treatment in some way correspond to differences in genotype, grouping persons with ASD according to phenotype and responses to treatment variables may increase the chances of identifying genetic susceptibility factors. Traylor and colleagues<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup> demonstrated that attempts to categorize patients with a complex disease into more homogeneous subgroups could have more power to elucidate the hidden heritability in a simulation study. Several studies on Alzheimer’s disease, neuroticism, or asthma indicated that items or symptoms were to some degree more useful for identifying high-impact genetic factors than broadly defined diagnoses<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>–<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>, although a study of ASD demonstrated modest effects of two-way stratification by individual symptoms<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. In addition, medical researchers have begun to use machine learning methods, which is an artificial intelligence technique that can reveal masked patterns of data sets. In view of the abovementioned circumstances, clustering algorithms of machine learning and subsequent genome-wide association studies (GWASs) could be hypothesized to reveal novel and more genetically homogeneous clusters, but a combinatorial approach of cluster analysis and GWASs, to the best of our knowledge, has not been applied to any diseases including ASD.</p><p id=\"Par4\">We therefore explored whether grouping persons with ASD using a clustering algorithm with phenotype and responses to treatment variables can be used to discriminate more genetically homogeneous persons with ASD. In the present study, we conducted cluster-based GWASs (named cluster-based GWASs) using real data based on the concept of a previous simulation study<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup> adopting a machine learning k-means<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup> algorithm for cluster analysis.</p></sec><sec id=\"Sec2\"><title>Subjects and methods</title><p id=\"Par5\">We conducted the present study in accordance with the guidelines of the Declaration of Helsinki<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup> and all other applicable guidelines. The protocol was reviewed and approved by the institutional review board of Tohoku University Graduate School of Medicine, and written informed consent was obtained from all participants over the age of 18 by the SFARI<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. For participants under the age of 18, informed consent was obtained from a parent and/or legal guardian. In addition, for participants 10–17 years of age, informed assent was obtained from the individuals.</p><sec id=\"Sec3\"><title>data sets</title><p id=\"Par6\">We used phenotypic variables, history of treatment, and genotypic data from the Simons Simplex Collection (SSC)<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. The SSC establishes a repository of phenotypic data and genetic data/samples from mainly simplex families.</p><p id=\"Par7\">The SSC data were publicly released in October 2007 and are directly available from the SFARI. From the SSC data set, we used data from 614 affected white male probands who had no missing information regarding Autism Diagnostic Interview-Revised (ADI-R) scores<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup> and vitamin treatment<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup> and 391 unaffected brothers for whom genotype data, generated by the Illumina Human Omni2.5 (Omni2.5) array, were available for subsequent clustering and genetic analyses. We excluded participants whose ancestries were estimated to be different from the other participants using principal component analyses (PCAs) performed by EIGENSOFT version 7.2.1<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup> for the genotype data. Based on the PCAs, we excluded data beyond four standard deviations of principal components 1 or 2 (Supplementary Fig. <xref rid=\"MOESM2\" ref-type=\"media\">1</xref>). Therefore, we used data from 597 probands and 370 unaffected brothers.</p><p id=\"Par8\">In the replication study, we used another SSC data set genotyped using the Illumina 1Mv3 (1Mv3) array. In the data set, data from 735 affected male probands with no missing information regarding ADI-R scores or vitamin treatment and 387 unaffected brothers were available. After conducting PCA, we excluded data beyond four standard deviations of principal components 1 or 2 as outliers. In this way, we used data from 712 probands and 354 unaffected brothers in the replication study.</p></sec><sec id=\"Sec4\"><title>Clustering</title><p id=\"Par9\">We conducted cluster analyses using phenotypic variables of ADI-R scores and history of vitamin treatment<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. We chose these variables because the ADI-R is one of the most reliable estimates of ASD and has the ability to evaluate substructure domains of ASD<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. Among the ADI-R scores, “the total score for the Verbal Communication Domain of the ADI-R minus the total score for the Nonverbal Communication Domain of the ADI-R”, “the total score for the Nonverbal Communication Domain of the ADI-R”, “the total score for the Restricted, Repetitive, and Stereotyped Patterns of Behavior Domain of the ADI-R”, and “the total score for the Reciprocal Social Interaction Domain of the ADI-R” were included in the preprocessed data set.</p><p id=\"Par10\">Among the treatments, we selected the variable of history of vitamin treatment because we recently found that a cluster of persons with ASD is associated with potential responsiveness to vitamin B6 treatment<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. The history of treatment is not always compatible with responsiveness, but we considered that continuous treatment indicates responsiveness to some degree. The SSC data set includes history of treatment but not variables of responsiveness.</p><p id=\"Par11\">We applied the machine learning k-means<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup> algorithm to conduct a cluster analysis to divide the data set obtained from ASD persons into subgroups using phenotypic variables and history of treatment. The k-means algorithm requires a cluster number (k) determined by researchers. We set a priori k of 5, 10, 15, and 20 under the hypothesis that ASD consists of hundreds of subgroups<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup> and considering statistical power by sample size calculations<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. We performed the analyses using the scikit-learn toolkit in Python 2.7 (Supplementary Information <xref rid=\"MOESM6\" ref-type=\"media\">1</xref>).</p><p id=\"Par12\">Clustering is an exploratory data analysis technique, and the validity of the clustering results may be judged by external knowledge, such as the purpose of the segmentation<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. Several methods have proposed to prespecify a cluster number of k, such as visual examination of the data, and likelihood and error-based approaches; however, these methods do not necessarily provide results that are consistent with each other<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Although there are measures for evaluating the quality of the clusters<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>, the number of clusters should still be determined according to the research purposes. We regarded the inflation factor (λ) of quantile-quantile (Q–Q) plots of the logarithm of the <italic>P</italic> value to base 10 (−log<sub>10</sub><italic>P</italic>) as one of the indicators of successful clustering in the present study. We calculated λ for each cluster number.</p><p id=\"Par13\">When conducting clustering, we combined the two data sets of male probands, one genotyped using the Omni2.5 array and the other genotyped using the 1Mv3 array. After clustering, we redivided the new data set according to the SNP arrays used. In the discovery stage, we used the Omni2.5 data set and the 1Mv3 data set in the replication stage.</p></sec><sec id=\"Sec5\"><title>Genotype data and quality control</title><p id=\"Par14\">We used the SSC data set, in which probands and unaffected brothers had already been genotyped in other previous studies<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. In the discovery stage, we used the data set genotyped by the Omni2.5 array, which has 2,383,385 probes. We excluded SNPs with a minor allele frequency < 0.01, call rate < 0.95, and Hardy–Weinberg equilibrium test <italic>P</italic> < 0.000001.</p><p id=\"Par15\">In the replication study, where we used the data set genotyped using the 1Mv3 array, we applied the same cutoff values for quality control as those used in the discovery stage. The 1Mv3 array includes 1,147,689 SNPs. The Omni2.5 array and the 1Mv3 array shared 675,923 SNPs.</p></sec><sec id=\"Sec6\"><title>Statistical analysis</title><p id=\"Par16\">As a preliminary study, we conducted a conventional GWAS in the whole Omni2.5 data set, with a total of 597 male probands and 370 unaffected brothers. Here, we used the brothers of the cases as controls, in contrast to many previous studies in which genetically unrelated controls were used. We thus adopted the sib transmission disequilibrium test (sib-TDT)<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>, a family-based association test, to take into account familial relationships among the participants. In the second step, in the discovery stage, we conducted cluster-based GWAS in each subgroup of the cases, which had been divided using the k-means<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup> algorithm, and the controls. As mentioned above, the controls were the brothers of the cases, and we then excluded the unaffected brothers of the cases belonging to the subgroup being analyzed. Details of the study design are shown in Supplementary Fig. <xref rid=\"MOESM3\" ref-type=\"media\">2</xref>. We applied the Cochran–Armitage trend test<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>, which examines the risk of disease in those who do not have the allele of interest, those who have a single copy, and those who are homozygous.</p><p id=\"Par17\">We further tested the significantly associated loci found in the discovery studies in the replication stage. The level of significance for association was set as <italic>P</italic> < 0.05 in the replication studies.</p><p id=\"Par18\">Association analyses were performed with the PLINK software package<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. The detected SNPs were subsequently annotated using ANNOVAR<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. Manhattan plots and Q–Q plots were generated using the ‘qqman’ package in R version 3.0.2.</p></sec></sec><sec id=\"Sec7\" sec-type=\"results\"><title>Results</title><sec id=\"Sec8\"><title>Cluster-based GWAS</title><p id=\"Par19\">As a preliminary study, we conducted a conventional GWAS with the Omni2.5 data set using the sib-TDT. We observed no significant associations (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). Although we adopted the sib-TDT here because we used the brothers of the cases as controls, we also used the Cochran–Armitage trend test and found that the −log<sub>10</sub><italic>P</italic> values were distributed downward compared with the expected values, as shown in Supplementary Fig. <xref rid=\"MOESM4\" ref-type=\"media\">3</xref>.<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Manhattan plot and corresponding quantile-quantile plot in GWAS for all male probands vs their unaffected brothers.</title><p>Manhattan plot <bold>a</bold> and corresponding quantile-quantile plot <bold>b</bold> in GWAS for all male probands vs their unaffected brothers. We conducted a GWAS in the Simons Simplex Collection data set of 597 male probands and 370 unaffected brothers genotyped by the Illumina Human Omni2.5 array using the sib transmission/disequilibrium test (sib-TDT). We observed no significant associations in this GWAS with the genome-wide threshold of <italic>P</italic> = 5.0 × 10<sup>−8</sup>. The blue horizontal line indicates the genome-wide suggestive threshold of <italic>p</italic> = 1.0 × 10<sup>−5</sup>.</p></caption><graphic xlink:href=\"41398_2020_951_Fig1_HTML\" id=\"d30e736\"/></fig></p><p id=\"Par20\">We also applied the sib-TDT to cluster 1, which was obtained by dividing all the cases using k-means with <italic>k</italic> of 15, and all the controls and found that the observed −log<italic>P</italic> values were lower than expected, as shown in Supplementary Fig. <xref rid=\"MOESM4\" ref-type=\"media\">3</xref>. As the sib-TDT may efficiently work in a population consisting of a substantial number of sibs, a limited number of brothers of the probands among all the controls probably contributed to a substantial loss of power. Thus, we excluded the brothers of the probands in each subset from the controls so that each subset of probands has no genetic relations with the rest of the controls and conducted the Cochran–Armitage trend test, as in many other studies. In the present study, therefore, we applied the sib-TDT to the GWAS of the whole data set, whereas in the cluster-based GWAS, we excluded in turn the unaffected brothers of the cases belonging to the subgroup being analyzed and used the Cochran–Armitage trend test to account for the relationships between participants.</p><p id=\"Par21\">The average inflation factor <italic>λ</italic> for the cluster-based GWAS with k of 5, 10, 15, and 20 were 1.021, 1.024, 1.038, and 1.053, respectively. Several lines of evidence suggest that regarding an appropriate threshold of <italic>λ</italic>, empirically, a value <1.050 is deemed safe for avoiding false positives<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. Under the hypothesis that ASD consists of hundreds of subgroups<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>, we compared <italic>λ</italic> values giving larger numbers of clusters as priority. We therefore considered the cluster-based GWAS using k-means cluster analysis with <italic>k</italic> of 15 to be the most appropriate approach to the present data set. The characteristics of each cluster are presented in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>.<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Characteristics of each of 15 k-means clusters in the Omni2.5 data set.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"2\">Cluster no.</th><th rowspan=\"2\"><italic>n</italic></th><th colspan=\"4\">Verbal score from ADI-R</th><th colspan=\"4\">Nonverbal score from ADI-R</th><th colspan=\"4\">Restricted and repetitive patterns of behavior score from ADI-R</th><th colspan=\"4\">Social score from ADI-R</th><th rowspan=\"2\">Vitamin B6 treatment (%)</th></tr><tr><th>Mean (SD)</th><th>Median (p25–p75)</th><th>Min</th><th>Max</th><th>Mean (SD)</th><th>Median (p25–p75)</th><th>Min</th><th>Max</th><th>Mean (SD)</th><th>Median (p25–p75)</th><th>Min</th><th>Max</th><th>Mean (SD)</th><th>Median (p25–p75)</th><th>Min</th><th>Max</th></tr></thead><tbody><tr><td>All</td><td>597</td><td>7.7 (2.1)</td><td>8.0 (6.0–9.0)</td><td>0</td><td>12</td><td>8.9 (3.3)</td><td>9.0 (6.0–12.0)</td><td>0</td><td>14</td><td>6.8 (2.5)</td><td>7.0 (5.0–8.0)</td><td>1</td><td>12</td><td>19.8 (5.3)</td><td>20.0 (16.0–24.0)</td><td>8</td><td>30</td><td>59.6</td></tr><tr><td>1</td><td>33</td><td>7.4 (2.2)</td><td>7.0 (6.0–10.0)</td><td>3</td><td>11</td><td>4.4 (1.6)</td><td>4.0 (3.0–6.0)</td><td>1</td><td>7</td><td>8.5 (1.6)</td><td>8.0 (7.0–10.0)</td><td>6</td><td>12</td><td>14.0 (1.5)</td><td>14.0 (13.0–15.0)</td><td>11</td><td>17</td><td>60.6</td></tr><tr><td>2</td><td>49</td><td>8.9 (1.3)</td><td>9.0 (8.0–10.0)</td><td>6</td><td>12</td><td>12.3 (1.5)</td><td>12.0 (11.0–14.0)</td><td>9</td><td>14</td><td>6.2 (1.3)</td><td>6.0 (6.0–7.0)</td><td>3</td><td>8</td><td>27.1 (1.3)</td><td>27.0 (26.0–28.0)</td><td>24</td><td>30</td><td>79.6</td></tr><tr><td>3</td><td>45</td><td>6.0 (1.9)</td><td>6.0 (5.0–7.0)</td><td>2</td><td>10</td><td>8.8 (1.5)</td><td>9.0 (8.0–10.0)</td><td>6</td><td>12</td><td>5.0 (1.5)</td><td>5.0 (4.0–6.0)</td><td>2</td><td>7</td><td>16.8 (1.1)</td><td>17.0 (16.0–18.0)</td><td>15</td><td>19</td><td>64.4</td></tr><tr><td>4</td><td>59</td><td>9.0 (1.5)</td><td>9.0 (8.0–10.0)</td><td>6</td><td>12</td><td>8.1 (1.5)</td><td>8.0 (7.0–9.0)</td><td>4</td><td>10</td><td>8.8 (1.9)</td><td>8.0 (8.0–10.0)</td><td>5</td><td>12</td><td>23.8 (1.4)</td><td>24.0 (23.0–25.0)</td><td>21</td><td>27</td><td>57.6</td></tr><tr><td>5</td><td>28</td><td>7.3 (1.1)</td><td>7.0 (6.5–8.0)</td><td>5</td><td>9</td><td>9.1 (1.7)</td><td>9.0 (8.0–10.0)</td><td>7</td><td>13</td><td>6.1 (2.3)</td><td>6.0 (5.0–7.0)</td><td>1</td><td>12</td><td>12.7 (1.7)</td><td>13.0 (12.0–14.0)</td><td>9</td><td>15</td><td>60.7</td></tr><tr><td>6</td><td>29</td><td>7.7 (1.9)</td><td>8.0 (7.0–9.0)</td><td>2</td><td>12</td><td>4.6 (1.8)</td><td>5.0 (4.0–6.0)</td><td>0</td><td>8</td><td>4.0 (1.1)</td><td>4.0 (3.0–5.0)</td><td>2</td><td>6</td><td>15.8 (1.4)</td><td>16.0 (15.0–17.0)</td><td>14</td><td>19</td><td>44.8</td></tr><tr><td>7</td><td>37</td><td>6.5 (1.8)</td><td>6.0 (5.0–8.0)</td><td>3</td><td>11</td><td>12.5 (1.3)</td><td>12.0 (12.0–14.0)</td><td>10</td><td>14</td><td>5.6 (1.4)</td><td>6.0 (5.0–7.0)</td><td>3</td><td>8</td><td>19.4 (1.8)</td><td>20.0 (18.0–21.0)</td><td>15</td><td>22</td><td>56.8</td></tr><tr><td>8</td><td>23</td><td>8.3 (1.6)</td><td>8.0 (7.0–10.0)</td><td>5</td><td>11</td><td>4.2 (2.1)</td><td>4.0 (3.0–6.0)</td><td>0</td><td>8</td><td>5.9 (1.9)</td><td>6.0 (4.0–8.0)</td><td>3</td><td>10</td><td>9.7 (1.1)</td><td>10.0 (9.0–11.0)</td><td>8</td><td>12</td><td>60.9</td></tr><tr><td>9</td><td>46</td><td>9.0 (1.3)</td><td>9.0 (8.0–10.0)</td><td>5</td><td>12</td><td>12.4 (1.3)</td><td>13.0 (11.0–13.0)</td><td>10</td><td>14</td><td>9.2 (1.8)</td><td>9.0 (8.0–10.0)</td><td>6</td><td>12</td><td>22.7 (1.4)</td><td>22.5 (22.0–24.0)</td><td>20</td><td>25</td><td>69.6</td></tr><tr><td>10</td><td>43</td><td>6.6 (1.4)</td><td>7.0 (6.0–7.0)</td><td>4</td><td>9</td><td>11.7 (1.5)</td><td>12.0 (10.0–13.0)</td><td>9</td><td>14</td><td>5.0 (1.5)</td><td>5.0 (4.0–6.0)</td><td>2</td><td>8</td><td>24.1 (1.3)</td><td>24.0 (23.0–25.0)</td><td>22</td><td>26</td><td>55.8</td></tr><tr><td>11</td><td>34</td><td>4.4 (1.6)</td><td>5.0 (3.0–6.0)</td><td>0</td><td>7</td><td>4.9 (1.8)</td><td>5.0 (4.0–6.0)</td><td>1</td><td>9</td><td>4.1 (1.7)</td><td>4.0 (3.0–5.0)</td><td>1</td><td>9</td><td>10.9 (1.9)</td><td>10.5 (9.0–13.0)</td><td>8</td><td>14</td><td>55.9</td></tr><tr><td>12</td><td>38</td><td>8.8 (1.6)</td><td>9.0 (8.0–10.0)</td><td>5</td><td>12</td><td>9.7 (1.5)</td><td>9.0 (8.0–11.0)</td><td>8</td><td>13</td><td>9.2 (1.3)</td><td>9.0 (8.0–10.0)</td><td>7</td><td>12</td><td>18.1 (1.3)</td><td>18.0 (17.0–19.0)</td><td>15</td><td>20</td><td>65.8</td></tr><tr><td>13</td><td>52</td><td>7.1 (2.0)</td><td>7.0 (5.5–8.5)</td><td>3</td><td>12</td><td>7.4 (1.5)</td><td>7.0 (6.0–9.0)</td><td>4</td><td>10</td><td>4.6 (1.5)</td><td>4.5 (3.5–6.0)</td><td>1</td><td>7</td><td>22.0 (1.7)</td><td>22.0 (21.0–23.0)</td><td>19</td><td>27</td><td>44.2</td></tr><tr><td>14</td><td>46</td><td>7.9 (1.5)</td><td>8.0 (7.0–9.0)</td><td>4</td><td>11</td><td>6.2 (1.6)</td><td>6.0 (5.0–7.0)</td><td>1</td><td>9</td><td>8.4 (1.7)</td><td>8.0 (7.0–10.0)</td><td>5</td><td>12</td><td>19.4 (1.4)</td><td>19.0 (18.0–20.0)</td><td>17</td><td>22</td><td>58.7</td></tr><tr><td>15</td><td>35</td><td>9.5 (1.4)</td><td>9.0 (8.0–11.0)</td><td>7</td><td>12</td><td>12.7 (1.4)</td><td>13.0 (12.0–14.0)</td><td>9</td><td>14</td><td>9.6 (1.1)</td><td>10.0 (9.0–10.0)</td><td>8</td><td>12</td><td>27.5 (1.5)</td><td>27.0 (26.0–29.0)</td><td>25</td><td>30</td><td>54.3</td></tr></tbody></table><table-wrap-foot><p><italic>ADI-R</italic> autism diagnostic interview-revised, <italic>SD</italic> standard deviation.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec9\"><title>Gene interpretation</title><p id=\"Par22\">We observed 65 chromosomal loci that satisfied the threshold of <italic>P</italic> < 5.0 × 10<sup>−8</sup> (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>); 30 out of the 65 loci were located within 21 genes, and the remaining 35 loci were intergenic (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>). Among them, eight loci were located within or near the genes associated with the Human Gene module of the SFARI Gene scoring system<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>; <italic>GABBR2</italic> (score 4, Rare Single Gene Mutation, Syndromic, Functional) in Cluster 1; <italic>CNTNAP5</italic> (score 4, Rare Single Gene Mutation, Genetic Association) in Cluster 3; <italic>ITPR1</italic> (score 4, Rare Single Gene Mutation) in Cluster 5; <italic>DNAH17</italic> (score 4, Rare Single Gene Mutation) in Cluster 7; <italic>SDK1</italic> (score none, Rare Single Gene Mutation, Genetic Association) in Cluster 13; <italic>SRRM4</italic> (score 5, Rare Single Gene Mutation, Functional) in Cluster 13; <italic>CNTN5</italic> (score 3, Rare Single Gene Mutation, Genetic Association) in Cluster 14; and <italic>DPP10</italic> (score 3, Rare Single Gene Mutation) in Cluster 15.<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Manhattan plots and corresponding quantile-quantile plots in cluster-based GWASs.</title><p>Manhattan plots <bold>a</bold> and corresponding quantile-quantile plots <bold>b</bold> in cluster-based GWASs with a cluster number of 15. We performed cluster analysis using k-means with a cluster number of 15 and conducted cluster-based GWAS. Among 15 clusters, significant associations were observed in 14 clusters. In total, we observed 65 chromosomal loci, labeled in the figure, that satisfied the threshold of <italic>P</italic> = 5.0 × 10<sup>−8</sup>. The red horizontal lines indicate the threshold for genome-wide significance (<italic>P</italic> = 5.0 × 10<sup>−8</sup>) and the blue horizontal lines indicate the genome-wide suggestive threshold (<italic>P</italic> = 1.0 × 10<sup>−5</sup>). The names of the suggested genes where the excerpted and circled SNPs are located are typed in Manhattan plots.</p></caption><graphic xlink:href=\"41398_2020_951_Fig2_HTML\" id=\"d30e1912\"/></fig><table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Association table of the cluster-based GWAS with 15 k-means clusters in the Omni2.5 data set.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Cluster no.</th><th>ID</th><th>Chr</th><th>hg19</th><th>Minor/major</th><th>MAF (%)</th><th>OR</th><th>95% CI</th><th><italic>P</italic></th><th>GENESYMBOL</th><th>Function</th><th>Power</th></tr></thead><tbody><tr><td>1</td><td>rs111629286</td><td>11</td><td>130,152,136</td><td>A/G</td><td>1.80</td><td>13.42</td><td>4.38–41.17</td><td>1.36 × 10<sup>−8</sup></td><td>ZBTB44</td><td>Intronic</td><td>0.997</td></tr><tr><td>1</td><td>rs115140946</td><td>6</td><td>37,891,923</td><td>C/A</td><td>1.03</td><td>21.07</td><td>4.79–92.77</td><td>2.87 × 10<sup>−8</sup></td><td>ZFAND3</td><td>Intronic</td><td>0.878</td></tr><tr><td>1</td><td>rs9462391</td><td>6</td><td>38,123,030</td><td>A/G</td><td>1.03</td><td>21.07</td><td>4.79–92.77</td><td>2.87 × 10<sup>−8</sup></td><td>ZFAND3</td><td>Downstream</td><td>0.878</td></tr><tr><td>1</td><td>rs10217283</td><td>9</td><td>101,423,675</td><td>A/G</td><td>1.42</td><td>15.51</td><td>4.45–54.12</td><td>2.95 × 10<sup>−8</sup></td><td>GABBR2</td><td>Intronic</td><td>0.976</td></tr><tr><td>1</td><td>rs114109395</td><td>6</td><td>38,005,546</td><td>A/G</td><td>1.03</td><td>21.01</td><td>4.77–92.51</td><td>3.02 × 10<sup>−8</sup></td><td>ZFAND3</td><td>Intronic</td><td>0.877</td></tr><tr><td>2</td><td>rs115621412</td><td>9</td><td>74,366,033</td><td>C/A</td><td>7.89</td><td>4.42</td><td>2.48–7.87</td><td>8.13 × 10<sup>−9</sup></td><td>CEMIP2</td><td>Intronic</td><td>1.000</td></tr><tr><td>3</td><td>rs77507687</td><td>2</td><td>26,939,229</td><td>G/A</td><td>2.00</td><td>12.43</td><td>4.37–35.36</td><td>6.10 × 10<sup>−9</sup></td><td>KCNK3</td><td>Intronic</td><td>1.000</td></tr><tr><td>3</td><td>rs76880969</td><td>1</td><td>227,711,506</td><td>G/A</td><td>1.00</td><td>27.15</td><td>5.30–139.20</td><td>8.20 × 10<sup>−9</sup></td><td>CDC42BPA, ZNF678</td><td>Intergenic</td><td>0.865</td></tr><tr><td>3</td><td>rs115483919</td><td>2</td><td>125,010,267</td><td>A/G</td><td>1.00</td><td>27.15</td><td>5.30–139.20</td><td>8.20 × 10<sup>−9</sup></td><td>CNTNAP5</td><td>Intronic</td><td>0.865</td></tr><tr><td>5</td><td>rs16965293</td><td>16</td><td>9,551,490</td><td>A/G</td><td>2.31</td><td>14.04</td><td>5.00–39.45</td><td>3.83 × 10<sup>−10</sup></td><td>LINC01195, GRIN2A</td><td>Intergenic</td><td>1.000</td></tr><tr><td>5</td><td>rs77489014</td><td>9</td><td>106,962,281</td><td>A/G</td><td>1.41</td><td>19.47</td><td>5.51–68.82</td><td>6.69 × 10<sup>−10</sup></td><td>SMC2, LOC105376194</td><td>Intergenic</td><td>0.991</td></tr><tr><td>5</td><td>rs117473168</td><td>9</td><td>106,848,270</td><td>A/G</td><td>1.55</td><td>16.90</td><td>5.02–56.93</td><td>2.64 × 10<sup>−9</sup></td><td>SMC2</td><td>ncRNA exonic</td><td>0.991</td></tr><tr><td>5</td><td>rs7199670</td><td>16</td><td>22,875,238</td><td>A/G</td><td>11.28</td><td>5.28</td><td>2.76–10.10</td><td>4.98 × 10<sup>−9</sup></td><td>HS3ST2</td><td>Intronic</td><td>1.000</td></tr><tr><td>5</td><td>rs73142209</td><td>12</td><td>77,859,299</td><td>G/A</td><td>1.54</td><td>16.18</td><td>4.82–54.31</td><td>5.33 × 10<sup>−9</sup></td><td>E2F7, NAV3</td><td>Intergenic</td><td>0.989</td></tr><tr><td>5</td><td>rs118167078</td><td>15</td><td>65,723,796</td><td>A/G</td><td>1.54</td><td>16.18</td><td>4.82–54.31</td><td>5.33 × 10<sup>−9</sup></td><td>IGDCC4, DPP8</td><td>Intergenic</td><td>0.989</td></tr><tr><td>5</td><td>rs11919513</td><td>3</td><td>4,841,384</td><td>G/A</td><td>3.22</td><td>10.18</td><td>3.99–26.03</td><td>8.92 × 10<sup>−9</sup></td><td>ITPR1</td><td>Intronic</td><td>1.000</td></tr><tr><td>5</td><td>rs13332627</td><td>16</td><td>22,874,928</td><td>G/A</td><td>9.23</td><td>6.10</td><td>2.96–12.57</td><td>1.22 × 10<sup>−8</sup></td><td>HS3ST2</td><td>Intronic</td><td>1.000</td></tr><tr><td>5</td><td>rs111920363</td><td>7</td><td>143,656,906</td><td>A/G</td><td>1.15</td><td>19.46</td><td>4.89–77.39</td><td>1.29 × 10<sup>−8</sup></td><td>OR2F1</td><td>Upstream</td><td>0.933</td></tr><tr><td>5</td><td>rs9939816</td><td>16</td><td>22,876,408</td><td>A/C</td><td>9.25</td><td>6.08</td><td>2.95–12.53</td><td>1.30 × 10<sup>−8</sup></td><td>HS3ST2</td><td>Intronic</td><td>1.000</td></tr><tr><td>5</td><td>rs76096239</td><td>14</td><td>97,193,704</td><td>A/G</td><td>1.67</td><td>13.79</td><td>4.27–44.54</td><td>3.25 × 10<sup>−8</sup></td><td>PAPOLA, LINC02299</td><td>Intergenic</td><td>0.986</td></tr><tr><td>5</td><td>rs1054028</td><td>16</td><td>22,927,214</td><td>G/A</td><td>14.36</td><td>5.02</td><td>2.62–9.61</td><td>3.32 × 10<sup>−8</sup></td><td>HS3ST2</td><td>UTR3</td><td>1.000</td></tr><tr><td>5</td><td>rs78486970</td><td>7</td><td>106,127,612</td><td>G/A</td><td>6.87</td><td>5.46</td><td>2.67–11.18</td><td>3.68 × 10<sup>−8</sup></td><td>NAMPT, CCDC71L</td><td>Intergenic</td><td>1.000</td></tr><tr><td>6</td><td>rs148617803</td><td>1</td><td>76,136,228</td><td>G/A</td><td>1.32</td><td>22.57</td><td>5.95–85.64</td><td>2.77 × 10<sup>−10</sup></td><td>SLC44A5, ACADM</td><td>Intergenic</td><td>0.988</td></tr><tr><td>6</td><td>rs55985845</td><td>10</td><td>25,163,664</td><td>T/A</td><td>2.51</td><td>11.71</td><td>4.26–32.20</td><td>7.18 × 10<sup>−9</sup></td><td>PRTFDC1</td><td>Intronic</td><td>1.000</td></tr><tr><td>6</td><td>rs73094424</td><td>12</td><td>39,840,397</td><td>A/G</td><td>2.11</td><td>12.09</td><td>4.12–35.52</td><td>2.70 × 10<sup>−8</sup></td><td>KIF21A, ABCD2</td><td>Intergenic</td><td>0.998</td></tr><tr><td>6</td><td>rs58845693</td><td>3</td><td>122,804,247</td><td>G/A</td><td>1.18</td><td>18.07</td><td>4.55–71.72</td><td>4.24 × 10<sup>−8</sup></td><td>PDIA5</td><td>Intronic</td><td>0.915</td></tr><tr><td>6</td><td>rs11709496</td><td>3</td><td>122,809,400</td><td>G/A</td><td>1.18</td><td>18.07</td><td>4.55–71.72</td><td>4.24 × 10<sup>−8</sup></td><td>PDIA5</td><td>Intronic</td><td>0.915</td></tr><tr><td>6</td><td>rs199531954</td><td>12</td><td>95,064,359</td><td>C/A</td><td>1.19</td><td>17.92</td><td>4.52–71.10</td><td>4.92 × 10<sup>−8</sup></td><td>TMCC3, MIR492</td><td>Intergenic</td><td>0.913</td></tr><tr><td>7</td><td>rs79033134</td><td>17</td><td>76,473,288</td><td>A/G</td><td>1.53</td><td>16.29</td><td>4.87–54.44</td><td>4.24 × 10<sup>−9</sup></td><td>DNAH17</td><td>Intronic</td><td>0.995</td></tr><tr><td>7</td><td>rs57127555</td><td>17</td><td>76,475,811</td><td>C/A</td><td>1.54</td><td>16.24</td><td>4.86–54.28</td><td>4.49 × 10<sup>−9</sup></td><td>DNAH17</td><td>Intronic</td><td>0.995</td></tr><tr><td>7</td><td>rs75382702</td><td>11</td><td>81,149,755</td><td>A/G</td><td>1.28</td><td>16.94</td><td>4.54–63.23</td><td>3.18 × 10<sup>−8</sup></td><td>LINC02720, MIR4300HG</td><td>Intergenic</td><td>0.961</td></tr><tr><td>8</td><td>rs73149247</td><td>3</td><td>100,864,047</td><td>G/A</td><td>2.21</td><td>11.41</td><td>3.88–33.54</td><td>5.80 × 10<sup>−11</sup></td><td>ABI3BP, IMPG2</td><td>Intergenic</td><td>1.000</td></tr><tr><td>8</td><td>rs12418400</td><td>11</td><td>131,263,123</td><td>G/A</td><td>1.56</td><td>20.88</td><td>6.09–71.57</td><td>6.68 × 10<sup>−11</sup></td><td>NTM</td><td>Intronic</td><td>0.996</td></tr><tr><td>8</td><td>rs78323783</td><td>10</td><td>45,084,432</td><td>A/G</td><td>1.17</td><td>24.79</td><td>6.13–100.30</td><td>2.28 × 10<sup>−10</sup></td><td>CXCL12, TMEM72</td><td>Intergenic</td><td>0.997</td></tr><tr><td>8</td><td>rs72991663</td><td>6</td><td>130,143,713</td><td>A/G</td><td>2.85</td><td>13.30</td><td>4.84–36.53</td><td>5.51 × 10<sup>−10</sup></td><td>ARHGAP18, TMEM244</td><td>Intergenic</td><td>1.000</td></tr><tr><td>8</td><td>rs74922057</td><td>21</td><td>41,595,011</td><td>A/G</td><td>1.31</td><td>19.67</td><td>5.22–74.14</td><td>3.13 × 10<sup>−9</sup></td><td>DSCAM</td><td>Intronic</td><td>0.962</td></tr><tr><td>8</td><td>rs115035406</td><td>21</td><td>41,580,474</td><td>G/A</td><td>1.42</td><td>16.53</td><td>4.61–59.31</td><td>1.97 × 10<sup>−8</sup></td><td>DSCAM</td><td>Intronic</td><td>0.957</td></tr><tr><td>8</td><td>rs114994877</td><td>4</td><td>136,731,494</td><td>A/G</td><td>1.42</td><td>16.53</td><td>4.61–59.31</td><td>1.97 × 10<sup>−8</sup></td><td>LINC02485, LINC00613</td><td>Intergenic</td><td>0.957</td></tr><tr><td>8</td><td>rs117008682</td><td>9</td><td>103,245,053</td><td>G/A</td><td>1.43</td><td>16.48</td><td>4.59–59.15</td><td>2.08 × 10<sup>−8</sup></td><td>MSANTD3</td><td>Intronic</td><td>0.957</td></tr><tr><td>8</td><td>rs117772706</td><td>9</td><td>81,338,445</td><td>G/A</td><td>1.43</td><td>16.44</td><td>4.58–58.98</td><td>2.19 × 10<sup>−8</sup></td><td>PSAT1, LOC101927450</td><td>Intergenic</td><td>0.957</td></tr><tr><td>9</td><td>rs4885429</td><td>13</td><td>77,400,673</td><td>G/A</td><td>2.14</td><td>13.69</td><td>4.91–38.16</td><td>4.67 × 10<sup>−10</sup></td><td>LMO7DN, KCTD12</td><td>Intergenic</td><td>1.000</td></tr><tr><td>9</td><td>rs45618836</td><td>7</td><td>73,480,258</td><td>G/A</td><td>2.26</td><td>11.94</td><td>4.43–32.18</td><td>2.30 × 10<sup>−9</sup></td><td>ELN</td><td>Intronic</td><td>1.000</td></tr><tr><td>9</td><td>rs7299395</td><td>12</td><td>41,714,602</td><td>A/G</td><td>3.27</td><td>8.52</td><td>3.65–19.89</td><td>1.15 × 10<sup>−8</sup></td><td>PDZRN4</td><td>Intronic</td><td>1.000</td></tr><tr><td>9</td><td>rs55772967</td><td>7</td><td>73,448,499</td><td>G/A</td><td>2.89</td><td>8.91</td><td>3.66–21.66</td><td>2.09 × 10<sup>−8</sup></td><td>ELN</td><td>Intronic</td><td>1.000</td></tr><tr><td>10</td><td>rs72799348</td><td>2</td><td>22,637,443</td><td>A/G</td><td>2.31</td><td>12.84</td><td>4.74–34.77</td><td>6.57 × 10<sup>−10</sup></td><td>LINC01822, LINC01884</td><td>Intergenic</td><td>1.000</td></tr><tr><td>10</td><td>rs76159464</td><td>5</td><td>169,446,509</td><td>A/G</td><td>1.02</td><td>28.05</td><td>5.47–144.00</td><td>5.03 × 10<sup>−9</sup></td><td>DOCK2</td><td>Intronic</td><td>0.877</td></tr><tr><td>10</td><td>rs12483301</td><td>21</td><td>28,070,591</td><td>G/A</td><td>1.92</td><td>11.89</td><td>3.94–35.92</td><td>6.74 × 10<sup>−9</sup></td><td>CYYR1, ADAMTS1</td><td>Intergenic</td><td>1.000</td></tr><tr><td>10</td><td>rs72883714</td><td>18</td><td>23,987,552</td><td>A/G</td><td>2.17</td><td>11.25</td><td>4.08–31.06</td><td>1.59 × 10<sup>−8</sup></td><td>TAF4B, LINC01543</td><td>Intergenic</td><td>1.000</td></tr><tr><td>10</td><td>rs1876769</td><td>2</td><td>22,678,191</td><td>A/G</td><td>2.17</td><td>11.25</td><td>4.08–31.06</td><td>1.59 × 10<sup>−8</sup></td><td>LINC01822, LINC01884</td><td>Intergenic</td><td>1.000</td></tr><tr><td>10</td><td>rs17043765</td><td>2</td><td>22,656,804</td><td>A/G</td><td>2.17</td><td>11.25</td><td>4.08–31.06</td><td>1.59 × 10<sup>−8</sup></td><td>LINC01822, LINC01884</td><td>Intergenic</td><td>1.000</td></tr><tr><td>11</td><td>rs74645195</td><td>4</td><td>48,330,367</td><td>G/A</td><td>2.71</td><td>10.26</td><td>3.95–26.66</td><td>1.34 × 10<sup>−8</sup></td><td>TEC, SLAIN2</td><td>Intergenic</td><td>1.000</td></tr><tr><td>11</td><td>rs78513244</td><td>1</td><td>2,360,342</td><td>A/G</td><td>3.25</td><td>9.33</td><td>3.79–22.98</td><td>1.35 × 10<sup>−8</sup></td><td>PEX10, PLCH2</td><td>Intergenic</td><td>1.000</td></tr><tr><td>11</td><td>rs10027938</td><td>4</td><td>90,242,059</td><td>A/G</td><td>16.93</td><td>4.48</td><td>2.49–8.05</td><td>2.29 × 10<sup>−8</sup></td><td>GPRIN3, SNCA</td><td>Intergenic</td><td>1.000</td></tr><tr><td>12</td><td>rs117647850</td><td>8</td><td>79,156,756</td><td>A/G</td><td>3.08</td><td>10.88</td><td>4.44–26.68</td><td>5.10 × 10<sup>−11</sup></td><td>LOC102724874, PKIA</td><td>Intergenic</td><td>1.000</td></tr><tr><td>12</td><td>rs4131532</td><td>1</td><td>3,540,256</td><td>A/G</td><td>1.54</td><td>15.63</td><td>4.68–52.14</td><td>8.61 × 10<sup>−9</sup></td><td>MEGF6, TPRG1L</td><td>Intergenic</td><td>0.994</td></tr><tr><td>12</td><td>rs77964987</td><td>4</td><td>183,685,432</td><td>G/A</td><td>4.77</td><td>7.06</td><td>3.21–15.53</td><td>4.97 × 10<sup>−8</sup></td><td>TENM3</td><td>Intronic</td><td>1.000</td></tr><tr><td>13</td><td>rs117954350</td><td>7</td><td>4,440,757</td><td>A/G</td><td>1.02</td><td>52.73</td><td>6.34–438.60</td><td>4.00 × 10<sup>−10</sup></td><td>SDK1, FOXK1</td><td>Intergenic</td><td>0.635</td></tr><tr><td>13</td><td>rs11064685</td><td>12</td><td>119,590,881</td><td>G/A</td><td>6.14</td><td>5.15</td><td>2.66–9.97</td><td>4.46 × 10<sup>−8</sup></td><td>SRRM4</td><td>Intronic</td><td>1.000</td></tr><tr><td>14</td><td>rs77983358</td><td>12</td><td>82,393,237</td><td>G/A</td><td>1.52</td><td>21.71</td><td>5.50–85.76</td><td>1.29 × 10<sup>−10</sup></td><td>LINC02426, CCDC59</td><td>Intergenic</td><td>0.999</td></tr><tr><td>14</td><td>rs7118821</td><td>11</td><td>96,876,267</td><td>C/A</td><td>1.01</td><td>26.18</td><td>5.11–134.00</td><td>1.50 × 10<sup>−8</sup></td><td>LINC02737</td><td>Intergenic</td><td>0.847</td></tr><tr><td>14</td><td>rs7122015</td><td>11</td><td>96,950,548</td><td>G/A</td><td>1.01</td><td>26.18</td><td>5.11–134.00</td><td>1.50 × 10<sup>−8</sup></td><td>LINC02737, CNTN5</td><td>Intergenic</td><td>0.847</td></tr><tr><td>14</td><td>rs7106102</td><td>11</td><td>96,885,969</td><td>A/G</td><td>1.01</td><td>26.10</td><td>5.10–133.70</td><td>1.58 × 10<sup>−8</sup></td><td>LINC02737</td><td>Intergenic</td><td>0.845</td></tr><tr><td>14</td><td>rs7189512</td><td>16</td><td>66,324,048</td><td>A/G</td><td>3.28</td><td>7.26</td><td>3.13–16.88</td><td>4.62 × 10<sup>−8</sup></td><td>LINC00922, CDH5</td><td>Intergenic</td><td>1.000</td></tr><tr><td>15</td><td>rs77311527</td><td>2</td><td>5,516,750</td><td>G/A</td><td>2.45</td><td>11.87</td><td>4.44–31.79</td><td>2.19 × 10<sup>−9</sup></td><td>LINC01249, LINC01248</td><td>Intergenic</td><td>1.000</td></tr><tr><td>15</td><td>rs276833</td><td>2</td><td>114,769,078</td><td>A/G</td><td>1.29</td><td>18.00</td><td>4.81–67.43</td><td>1.25 × 10<sup>−8</sup></td><td>LINC01191, DPP10</td><td>Intergenic</td><td>0.970</td></tr></tbody></table><table-wrap-foot><p><italic>Chr</italic> chromosome, <italic>OR</italic> odds ratio, <italic>CI</italic> confidence interval.</p><p>Powers were calculated using the method based on the results in Nam’s study<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>.</p></table-wrap-foot></table-wrap></p><p id=\"Par23\">The SFARI Gene scoring system ranges from “Category 1”, which indicates “high confidence”, through “Category 6”, which denotes “evidence does not support a role”. Genes of a syndromic disorder (e.g., fragile X syndrome) related to ASD are categorized in a different category. Rare single-gene variants, disruptions/mutations, and submicroscopic deletions/duplications related to ASD are categorized as “Rare Single Gene Mutation”.</p><p id=\"Par24\">In addition to genes in the Human Gene module of the SFARI Gene, several important genes associated with ASD or other related disorders<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup> from previous reports were included in our findings as follows: <italic>CDH5</italic> in Cluster 14, <italic>DSCAM</italic> in Cluster 8, <italic>FOXK1</italic> in Cluster 13, <italic>GRIN2A</italic> in Cluster 5, <italic>NTM</italic> in Cluster 8, and <italic>SNCA</italic> in Cluster 11 previously reported with ASD<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>–<xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>; <italic>PLCH2</italic> in Cluster 11 previously reported with mental retardation<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>; <italic>ARHGAP18</italic> in Cluster 18, <italic>CDC42BPA</italic> in Cluster 3, <italic>CXCL12</italic> in Cluster 8, and <italic>HS3ST2</italic> in Cluster 5 previously reported with schizophrenia<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref>–<xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>; <italic>KCTD12</italic> in Cluster 9 and <italic>PSAT1</italic> in Cluster 8 previously reported with depressive disorder<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>; and <italic>ADAMTS1</italic> in Cluster 10, <italic>DOCK2</italic> in Cluster 10, <italic>HS3ST2</italic> in Cluster 5, <italic>NAMPT</italic> in Cluster 5, and <italic>NAV</italic> in Cluster 5 previously reported with Alzheimer’s disease<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref>–<xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>.</p></sec><sec id=\"Sec10\"><title>Replication study</title><p id=\"Par25\">We conducted replication studies with another independent data set that included a total of 712 male probands and 354 unaffected brothers and had been genotyped using the 1Mv3 array. As mentioned before, we had previously carried out cluster analyses in the combined data set genotyped with either Omni2.5 or 1Mv3 and then redivided it according to the SNP arrays used. The characteristics of each of the 15 clusters in the 1Mv3 data set are presented in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>.</p><p id=\"Par26\">Among the 65 genome-wide significant chromosomal loci found in the discovery study, seven chromosomal loci were included in the 1Mv3 array. Of these loci, rs11064685, within <italic>SRRM4</italic> in Cluster 13, had a significantly different distribution (<italic>p</italic> = 0.03) in cases vs controls in the replication cohort (Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref>).<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Results of replication studies in the 1Mv3 data set for statistically significant chromosomal loci in the discovery studies.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Cluster no.</th><th>ID</th><th>Chr</th><th>hg19</th><th>Minor/major</th><th>MAF (%)</th><th>OR</th><th>95% CI</th><th><italic>P</italic></th><th>GENESYMBOL</th><th>Function</th></tr></thead><tbody><tr><td>5</td><td>rs13332627</td><td>16</td><td>22,874,928</td><td>G/A</td><td>10.0</td><td/><td>0.50</td><td/><td>0.18–1.45</td><td rowspan=\"1\">0.195</td></tr><tr><td>5</td><td>rs7199670</td><td>16</td><td>22,875,238</td><td>A/G</td><td>12.2</td><td>0.51</td><td>0.20–1.33</td><td>0.1629</td><td>HS3ST2</td><td>Intronic</td></tr><tr><td>5</td><td>rs1054028</td><td>16</td><td>22,927,214</td><td>G/A</td><td>15.0</td><td>0.51</td><td>0.22–1.21</td><td>0.121</td><td>HS3ST2</td><td>UTR3</td></tr><tr><td>10</td><td>rs1876769</td><td>2</td><td>22,678,191</td><td>A/G</td><td>1.4</td><td>NA</td><td>–</td><td>0.1822</td><td>LINC01822, LINC01884</td><td>Intergenic</td></tr><tr><td>13</td><td>rs11064685</td><td>12</td><td>119,590,881</td><td>G/A</td><td>8.2</td><td>1.89</td><td>1.06–3.37</td><td>0.02858</td><td>SRRM4</td><td>Intronic</td></tr><tr><td>14</td><td>rs7189512</td><td>16</td><td>66,324,048</td><td>A/G</td><td>3.5</td><td>2.16</td><td>0.83–5.67</td><td>0.1085</td><td>LINC00922, CDH5</td><td>Intergenic</td></tr><tr><td>15</td><td>rs276833</td><td>2</td><td>114,769,078</td><td>A/G</td><td>1.3</td><td>0.71</td><td>0.09–5.75</td><td>0.75</td><td>LINC01191, DPP10</td><td>Intergenic</td></tr></tbody></table><table-wrap-foot><p><italic>Chr</italic> chromosome, <italic>OR</italic> odds ratio, <italic>CI</italic> confidence interval.</p></table-wrap-foot></table-wrap></p></sec></sec><sec id=\"Sec11\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par27\">One of the most important findings of our study was that reasonably decreasing the sample size could increase the statistical power. A plausible explanation is that our clustering may have successfully identified subgroups that are etiologically more homogeneous. At least two reasons could reduce the possibility of false positives of the present results of statistically significant SNPs in cluster-based GWAS. First, the present study validated the usefulness and feasibility of the concept of a previous simulation study<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>, which indicated that homogeneous case subgroups increase power in genetic association studies by Traylor and colleagues, using measurement data in the real world. Second, a substantial number of statistically significant SNPs in cluster-based GWAS observed in the present study were located within or near previously reported candidate genes for ASD<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR30\">30</xref>–<xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>.</p><p id=\"Par28\">We observed many statistically significant SNPs in cluster-based GWAS: <italic>CDH5</italic>, <italic>CNTN5, CNTNAP5, DNAH17, DPP10, DSCAM</italic>, <italic>FOXK1</italic>, <italic>GABBR2, GRIN2A</italic>5, <italic>ITPR1, NTM, SDK1, SNCA</italic>, and <italic>SRRM4</italic>. In particular, loci within the <italic>SRRM4</italic> gene had significantly different distributions in the cases vs controls in the replication cohort. Previous studies indicate that <italic>SRRM4</italic> is strongly associated with ASD, indicating that our results may be valid to some degree. The gene regulates neural microexons. In the brains of individuals with ASD, these microexons are frequently dysregulated<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>. In addition, nSR100/SRRM4 haploinsufficiency in mice induced autistic features such as sensory hypersensitivity and altered social behavior and impaired synaptic transmission and excitability<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup>.</p><p id=\"Par29\">In addition to <italic>SRRM4</italic>, we observed several genes located within or near previously reported candidate genes for ASD. The relatively high correspondence between our results in part and the SFARI Gene scoring system<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> indicates that the statistically significant loci we found may be associated with ASD subgroups (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). We also observed several important genes associated with ASD and other related disorders<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup> from previous reports. These findings suggest that the statistically significant SNPs might explain autistic symptoms because these diseases are suggested to have shared etiology, even in part, with ASD<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. Associations at the remaining significant loci that were not in the SFARI module or described above have not been previously reported, and to the best of our knowledge, some of them might be novel findings. These results might suggest that novel genetic loci of ASD could be found by identifying better defined subgroups, although further confirmation is needed in future cohorts with larger sample sizes.</p><p id=\"Par30\">Previous studies regarding Alzheimer’s disease, neuroticism, or asthma found that items or symptoms showed, to some degree, increased ORs between the case loci and control loci compared with those from previous studies using broadly defined disease diagnoses<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>–<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. These findings may indicate that GWAS based on a symptom or an item could identify genetically more homogeneous subgroups and let us hypothesize that a relatively reasonable combination of symptoms or items could identify more genetically homogeneous subgroups. In contrast, Chaste and colleagues showed that stratifying children with ASD based on the phenotype only modestly increased power in GWAS<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. The discrepancy between their findings and ours might be explained by usage of phenotype variables. Chaste and colleagues used one item or symptom alone with limited number of subgroups, whereas we used combinations of them with a machine learning method with a potentially sufficient number of clusters. DeMichele-Sweet and colleagues reported that subgrouping only by having psychosis could lead to the identification of limited loci that had small effects<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>, but Mukherjee and colleagues found a substantial number of suggestive loci that had extreme ORs after categorizing persons with Alzheimer’s disease based on relative performance across cognitive domains by modern psychometric approaches<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>.</p><p id=\"Par31\">Validation of clusters is essential. In the present study, we selected the k-means algorithm, focused on ADI-R items and treatment as variables, and determined cluster numbers based on the <italic>λ</italic> of the Q–Q plots. Although we believe this approach is one of the relevant ways, selection of variables, selection of algorithms and selection of cluster numbers still remain to be considered in future mathematical and biological cluster validation studies because controversies surrounding evaluation of the quality of the clusters are important issues and are still ongoing and because validated clusters may lead to elucidate the genetic architectures of ASD<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>.</p><p id=\"Par32\">The present study has a limitation to be noted. Substantial differences in the two genotyping platforms may have affected the results of the replication study. The Omni2.5 array includes 2,383,385 autosomal SNPs, whereas the 1Mv3 array includes 1,147,689 SNPs, with 675,923 shared SNPs between the two. Of the 65 statistically significant chromosomal loci in the discovery data, only seven chromosomal loci were shared between the two arrays.</p><p id=\"Par33\">Our study demonstrated that if the data set consists of multiple heterogeneous subgroups, even a subgroup that includes a much smaller number of homogeneous individuals could detect high-impact genetic factors. Hypothetical examples of the concept of cluster-based GWAS are shown in Supplementary Fig. <xref rid=\"MOESM5\" ref-type=\"media\">4</xref>. As shown in Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>, only 30 etiologically homogeneous probands and 300 controls can have a statistical power of ~1.00, calculated using the method based on the results in Nam’s study<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. Although the integral model, which assumes many genetic variants have a small effect, may contribute to the formation of some subgroups of ASD, our results indicate that clustering by specific phenotypic variables may provide a candidate example for identifying etiologically similar cases of ASD.</p><p id=\"Par34\">Our data indicate the relevance of cluster-based GWAS as a means to identify more homogeneous subgroups of ASD than broadly defined subgroups. Future investigation of cluster validation and replication with a larger sample size is therefore warranted. Such studies will provide clues to elucidate the genetic structures and etiologies of ASD and facilitate the development of precision medicine for ASD.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec12\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41398_2020_951_MOESM1_ESM.xlsx\"><caption><p>Supplementary Table 1</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41398_2020_951_MOESM2_ESM.pptx\"><caption><p>Supplementary Fig. 1</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM3\"><media xlink:href=\"41398_2020_951_MOESM3_ESM.tif\"><caption><p>Supplementary Fig. 2</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM4\"><media xlink:href=\"41398_2020_951_MOESM4_ESM.tif\"><caption><p>Supplementary Fig. 3</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM5\"><media xlink:href=\"41398_2020_951_MOESM5_ESM.tif\"><caption><p>Supplementary Fig. 4</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM6\"><media xlink:href=\"41398_2020_951_MOESM6_ESM.docx\"><caption><p>Supplementary Information 1</p></caption></media></supplementary-material></sec></sec></body><back><fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Akira Narita, Masato Nagai, Satoshi Mizuno</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary Information</bold> accompanies this paper at (10.1038/s41398-020-00951-x).</p></sec><ack><title>Acknowledgements</title><p>We are grateful to all of the families at the participating SSC sites, as well as the staff at the Simons Foundation Autism Research Initiative (SFARI). The present study was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) KAKENHI grant numbers 19390171, 16H05242 and 19H03894. MEXT had no role in the design or execution of the study.</p></ack><notes notes-type=\"data-availability\"><title>Data availability</title><p>All data used in the study are available only to those granted access by the Simons Foundation.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Conflict of interest</title><p id=\"Par35\">The authors declare that they have no conflict of interest.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><mixed-citation publication-type=\"other\">American Psychological Association (2013): Diagnostic and Statistical Manual of Mental Disorders (DSM–5). Washington: American Psychological Association.</mixed-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Geschwind</surname><given-names>DH</given-names></name><name><surname>State</surname><given-names>MW</given-names></name></person-group><article-title>Gene hunting in autism spectrum disorder: on the path to precision medicine</article-title><source>Lancet Neurol.</source><year>2015</year><volume>14</volume><fpage>1109</fpage><lpage>1120</lpage><pub-id pub-id-type=\"pmid\">25891009</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bailey</surname><given-names>A</given-names></name><etal/></person-group><article-title>Autism as a strongly genetic disorder: evidence from a British twin study</article-title><source>Psychol. Med.</source><year>1995</year><volume>25</volume><fpage>63</fpage><lpage>77</lpage><pub-id pub-id-type=\"pmid\">7792363</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lauritsen</surname><given-names>MB</given-names></name><name><surname>Pedersen</surname><given-names>CB</given-names></name><name><surname>Mortensen</surname><given-names>PB</given-names></name></person-group><article-title>Effects of familial risk factors and place of birth on the risk of autism: a nationwide register-based study</article-title><source>J. Child Psychol. Psychiatry</source><year>2005</year><volume>46</volume><fpage>963</fpage><lpage>971</lpage><pub-id pub-id-type=\"pmid\">16108999</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><mixed-citation publication-type=\"other\">Gene, S. Gene scoring. 2008. <ext-link ext-link-type=\"uri\" xlink:href=\"https://gene.sfari.org/database/gene-scoring/\">https://gene.sfari.org/database/gene-scoring/</ext-link>.</mixed-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eissa</surname><given-names>N</given-names></name><etal/></person-group><article-title>Current enlightenment about etiology and pharmacological treatment of autism spectrum disorder</article-title><source>Front. Neurosci.</source><year>2018</year><volume>12</volume><fpage>304</fpage><pub-id pub-id-type=\"pmid\">29867317</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Traylor</surname><given-names>M</given-names></name><name><surname>Markus</surname><given-names>H</given-names></name><name><surname>Lewis</surname><given-names>CM</given-names></name></person-group><article-title>Homogeneous case subgroups increase power in genetic association studies</article-title><source>Eur. J. Hum. Genet.</source><year>2015</year><volume>23</volume><fpage>863</fpage><lpage>869</lpage><pub-id pub-id-type=\"pmid\">25271086</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><mixed-citation publication-type=\"other\">Mukherjee, S. et al. Genetic data and cognitively defined late-onset Alzheimer’s disease subgroups. <italic>Mol. Psychiatry</italic>. 2018. 10.1038/s41380-018-0298-8.</mixed-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nagel</surname><given-names>M</given-names></name><name><surname>Watanabe</surname><given-names>K</given-names></name><name><surname>Stringer</surname><given-names>S</given-names></name><name><surname>Posthuma</surname><given-names>D</given-names></name><name><surname>van der Sluis</surname><given-names>S</given-names></name></person-group><article-title>Item-level analyses reveal genetic heterogeneity in neuroticism</article-title><source>Nat. Commun.</source><year>2018</year><volume>9</volume><fpage>905</fpage><pub-id pub-id-type=\"pmid\">29500382</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lavoie-Charland</surname><given-names>E</given-names></name><name><surname>Berube</surname><given-names>JC</given-names></name><name><surname>Boulet</surname><given-names>LP</given-names></name><name><surname>Bosse</surname><given-names>Y</given-names></name></person-group><article-title>Asthma susceptibility variants are more strongly associated with clinically similar subgroups</article-title><source>J. Asthma</source><year>2016</year><volume>53</volume><fpage>907</fpage><lpage>913</lpage><pub-id pub-id-type=\"pmid\">27058054</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chaste</surname><given-names>P</given-names></name><etal/></person-group><article-title>A genome-wide association study of autism using the Simons simplex collection: does reducing phenotypic heterogeneity in autism increase genetic homogeneity?</article-title><source>Biol. Psychiatry</source><year>2015</year><volume>77</volume><fpage>775</fpage><lpage>784</lpage><pub-id pub-id-type=\"pmid\">25534755</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><mixed-citation publication-type=\"other\">MacQueen, J. <italic>Some methods for classification and analysis of multivariate observations.</italic> In: Fifth Berkeley Symposium on Mathematical Statistics and Probability. Berkeley: University of California Press, 1967, pp 281–297.</mixed-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><collab>World Medical Association.</collab></person-group><article-title>World medical association Declaration of Helsinki: ethical principles for medical research involving human subjects</article-title><source>JAMA</source><year>2013</year><volume>310</volume><fpage>2191</fpage><lpage>2194</lpage><pub-id pub-id-type=\"pmid\">24141714</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fischbach</surname><given-names>GD</given-names></name><name><surname>Lord</surname><given-names>C</given-names></name></person-group><article-title>The simons simplex collection: a resource for identification of autism genetic risk factors</article-title><source>Neuron</source><year>2010</year><volume>68</volume><fpage>192</fpage><lpage>195</lpage><pub-id pub-id-type=\"pmid\">20955926</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Beggiato</surname><given-names>A</given-names></name><etal/></person-group><article-title>Gender differences in autism spectrum disorders: divergence among specific core symptoms</article-title><source>Autism Res.</source><year>2017</year><volume>10</volume><fpage>680</fpage><lpage>689</lpage><pub-id pub-id-type=\"pmid\">27809408</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuriyama</surname><given-names>S</given-names></name><etal/></person-group><article-title>Pyridoxine treatment in a subgroup of children with pervasive developmental disorders</article-title><source>Dev. Med. Child Neurol.</source><year>2002</year><volume>44</volume><fpage>284</fpage><lpage>286</lpage><pub-id pub-id-type=\"pmid\">11995900</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Obara</surname><given-names>T</given-names></name><etal/></person-group><article-title>Potential identification of vitamin B6 responsiveness in autism spectrum disorder utilizing phenotype variables and machine learning methods</article-title><source>Sci. Rep.</source><year>2018</year><volume>8</volume><fpage>14840</fpage><pub-id pub-id-type=\"pmid\">30287864</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Patterson</surname><given-names>N</given-names></name><name><surname>Price</surname><given-names>AL</given-names></name><name><surname>Reich</surname><given-names>D</given-names></name></person-group><article-title>Population structure and eigenanalysis</article-title><source>PLoS Genet.</source><year>2006</year><volume>2</volume><fpage>e190</fpage><pub-id pub-id-type=\"pmid\">17194218</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nam</surname><given-names>JM</given-names></name></person-group><article-title>Simple approximation for calculating sample sizes for detecting linear trend in proportions</article-title><source>Biometrics</source><year>1987</year><volume>43</volume><fpage>701</fpage><lpage>705</lpage><pub-id pub-id-type=\"pmid\">3663825</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><mixed-citation publication-type=\"other\">Cutting, D. R., Karger, D. R., Pedersen J. O. & Tukey J. W. Scatter/gather: a cluster-based approach to browsing large document collections. In: Proceedings of the 15th Annual ACM SIGIR Conference on Research and Development in Information Retrieval. 318–329 (New York: Association for Computing Machinery (ACM), 1992).</mixed-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Raykov</surname><given-names>YP</given-names></name><name><surname>Boukouvalas</surname><given-names>A</given-names></name><name><surname>Baig</surname><given-names>F</given-names></name><name><surname>Little</surname><given-names>MA</given-names></name></person-group><article-title>What to do when K-means clustering fails: a simple yet principled alternative algorithm</article-title><source>PLoS ONE</source><year>2011</year><volume>11</volume><fpage>e0162259</fpage></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Guo</surname><given-names>G</given-names></name><name><surname>Chen</surname><given-names>L</given-names></name><name><surname>Ye</surname><given-names>Y</given-names></name><name><surname>Jiang</surname><given-names>Q</given-names></name></person-group><article-title>Cluster validation method for determining the number of clusters in categorical sequences</article-title><source>IEEE Trans. Neural Netw. Learn Syst.</source><year>2017</year><volume>28</volume><fpage>2936</fpage><lpage>2948</lpage><pub-id pub-id-type=\"pmid\">28114078</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sanders</surname><given-names>SJ</given-names></name><etal/></person-group><article-title>Multiple recurrent de novo CNVs, including duplications of the 7q11.23 Williams syndrome region, are strongly associated with autism</article-title><source>Neuron</source><year>2011</year><volume>70</volume><fpage>863</fpage><lpage>885</lpage><pub-id pub-id-type=\"pmid\">21658581</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Spielman</surname><given-names>RS</given-names></name><name><surname>Ewens</surname><given-names>WJ</given-names></name></person-group><article-title>A sibship test for linkage in the presence of association: the sib transmission/disequilibrium test</article-title><source>Am. J. Hum. Genet</source><year>1998</year><volume>62</volume><fpage>450</fpage><lpage>458</lpage><pub-id pub-id-type=\"pmid\">9463321</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Freidlin</surname><given-names>B</given-names></name><name><surname>Zheng</surname><given-names>G</given-names></name><name><surname>Li</surname><given-names>Z</given-names></name><name><surname>Gastwirth</surname><given-names>JL</given-names></name></person-group><article-title>Trend tests for case-control studies of genetic markers: power, sample size and robustness</article-title><source>Hum. Hered.</source><year>2002</year><volume>53</volume><fpage>146</fpage><lpage>152</lpage><pub-id pub-id-type=\"pmid\">12145550</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Purcell</surname><given-names>S</given-names></name><etal/></person-group><article-title>PLINK: a tool set for whole-genome association and population-based linkage analyses</article-title><source>Am. J. Hum. Genet.</source><year>2002</year><volume>81</volume><fpage>559</fpage><lpage>575</lpage></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>K</given-names></name><name><surname>Li</surname><given-names>M</given-names></name><name><surname>Hakonarson</surname><given-names>H</given-names></name></person-group><article-title>ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data</article-title><source>Nucleic Acids Res.</source><year>2010</year><volume>38</volume><fpage>e164</fpage><pub-id pub-id-type=\"pmid\">20601685</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Genome-wide association study of piglet uniformity and farrowing interval</article-title><source>Front. Genet.</source><year>2017</year><volume>8</volume><fpage>194</fpage><pub-id pub-id-type=\"pmid\">29234349</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Anttila</surname><given-names>V</given-names></name><etal/></person-group><article-title>Analysis of shared heritability in common disorders of the brain</article-title><source>Science</source><year>2018</year><volume>360</volume><fpage>eaap8757</fpage><pub-id pub-id-type=\"pmid\">29930110</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Redies</surname><given-names>C</given-names></name><name><surname>Hertel</surname><given-names>N</given-names></name><name><surname>Hubner</surname><given-names>CA</given-names></name></person-group><article-title>Cadherins and neuropsychiatric disorders</article-title><source>Brain Res.</source><year>2012</year><volume>1470</volume><fpage>130</fpage><lpage>144</lpage><pub-id pub-id-type=\"pmid\">22765916</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Varghese</surname><given-names>M</given-names></name><etal/></person-group><article-title>Autism spectrum disorder: neuropathology and animal models</article-title><source>Acta Neuropathol.</source><year>2017</year><volume>134</volume><fpage>537</fpage><lpage>566</lpage><pub-id pub-id-type=\"pmid\">28584888</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Atsem</surname><given-names>S</given-names></name><etal/></person-group><article-title>Paternal age effects on sperm FOXK1 and KCNA7 methylation and transmission into the next generation</article-title><source>Hum. Mol. Genet.</source><year>2016</year><volume>25</volume><fpage>4996</fpage><lpage>5005</lpage><pub-id pub-id-type=\"pmid\">28171595</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Barnby</surname><given-names>G</given-names></name><etal/></person-group><article-title>Candidate-gene screening and association analysis at the autism-susceptibility locus on chromosome 16p: evidence of association at GRIN2A and ABAT</article-title><source>Am. J. Hum. Genet.</source><year>2005</year><volume>76</volume><fpage>950</fpage><lpage>966</lpage><pub-id pub-id-type=\"pmid\">15830322</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Minhas</surname><given-names>HM</given-names></name><etal/></person-group><article-title>An unbalanced translocation involving loss of 10q26.2 and gain of 11q25 in a pedigree with autism spectrum disorder and cerebellar juvenile pilocytic astrocytoma</article-title><source>Am. J. Med. Genet. A</source><year>2013</year><volume>161a</volume><fpage>787</fpage><lpage>791</lpage><pub-id pub-id-type=\"pmid\">23495067</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abou-Donia</surname><given-names>MB</given-names></name><name><surname>Suliman</surname><given-names>HB</given-names></name><name><surname>Siniscalco</surname><given-names>D</given-names></name><name><surname>Antonucci</surname><given-names>N</given-names></name><name><surname>ElKafrawy</surname><given-names>P</given-names></name></person-group><article-title>De novo blood biomarkers in autism: autoantibodies against neuronal and glial proteins</article-title><source>Behav. Sci. (Basel)</source><year>2019</year><volume>9</volume><fpage>E47</fpage><pub-id pub-id-type=\"pmid\">31035713</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lo Vasco</surname><given-names>VR</given-names></name></person-group><article-title>Role of phosphoinositide-specific phospholipase C η2 in isolated and syndromic mental retardation</article-title><source>Eur. Neurol.</source><year>2011</year><volume>65</volume><fpage>264</fpage><lpage>269</lpage><pub-id pub-id-type=\"pmid\">21474938</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Potkin</surname><given-names>SG</given-names></name><etal/></person-group><article-title>Gene discovery through imaging genetics: identification of two novel genes associated with schizophrenia</article-title><source>Mol. Psychiatry</source><year>2009</year><volume>14</volume><fpage>416</fpage><lpage>428</lpage><pub-id pub-id-type=\"pmid\">19065146</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Konopaske</surname><given-names>GT</given-names></name><etal/></person-group><article-title>Dysbindin-1 contributes to prefrontal cortical dendritic arbor pathology in schizophrenia</article-title><source>Schizophr. Res.</source><year>2018</year><volume>201</volume><fpage>270</fpage><lpage>277</lpage><pub-id pub-id-type=\"pmid\">29759351</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Openshaw</surname><given-names>RL</given-names></name><etal/></person-group><article-title>JNK signalling mediates aspects of maternal immune activation: importance of maternal genotype in relation to schizophrenia risk</article-title><source>J. Neuroinflamm.</source><year>2019</year><volume>16</volume><fpage>18</fpage></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ikeda</surname><given-names>M</given-names></name><etal/></person-group><article-title>Identification of novel candidate genes for treatment response to risperidone and susceptibility for schizophrenia: integrated analysis among pharmacogenomics, mouse expression, and genetic case-control association approaches</article-title><source>Biol. Psychiatry</source><year>2010</year><volume>67</volume><fpage>263</fpage><lpage>269</lpage><pub-id pub-id-type=\"pmid\">19850283</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Teng</surname><given-names>X</given-names></name><etal/></person-group><article-title>KCTD: a new gene family involved in neurodevelopmental and neuropsychiatric disorders</article-title><source>CNS Neurosci. Ther.</source><year>2019</year><volume>25</volume><fpage>887</fpage><lpage>902</lpage><pub-id pub-id-type=\"pmid\">31197948</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lin</surname><given-names>CH</given-names></name><name><surname>Huang</surname><given-names>MW</given-names></name><name><surname>Lin</surname><given-names>CH</given-names></name><name><surname>Huang</surname><given-names>CH</given-names></name><name><surname>Lane</surname><given-names>HY</given-names></name></person-group><article-title>Altered mRNA expressions for N-methyl-D-aspartate receptor-related genes in WBC of patients with major depressive disorder</article-title><source>J. Affect. Disord.</source><year>2019</year><volume>245</volume><fpage>1119</fpage><lpage>1125</lpage><pub-id pub-id-type=\"pmid\">30699855</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kunkle</surname><given-names>BW</given-names></name><etal/></person-group><article-title>Genetic meta-analysis of diagnosed Alzheimer’s disease identifies new risk loci and implicates Abeta, tau, immunity and lipid processing</article-title><source>Nat. Genet.</source><year>2019</year><volume>51</volume><fpage>414</fpage><lpage>430</lpage><pub-id pub-id-type=\"pmid\">30820047</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cimino</surname><given-names>PJ</given-names></name><name><surname>Sokal</surname><given-names>I</given-names></name><name><surname>Leverenz</surname><given-names>J</given-names></name><name><surname>Fukui</surname><given-names>Y</given-names></name><name><surname>Montine</surname><given-names>TJ</given-names></name></person-group><article-title>DOCK2 is a microglial specific regulator of central nervous system innate immunity found in normal and Alzheimer’s disease brain</article-title><source>Am. J. Pathol.</source><year>2009</year><volume>175</volume><fpage>1622</fpage><lpage>1630</lpage><pub-id pub-id-type=\"pmid\">19729484</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sepulveda-Diaz</surname><given-names>JE</given-names></name><etal/></person-group><article-title>HS3ST2 expression is critical for the abnormal phosphorylation of tau in Alzheimer’s disease-related tau pathology</article-title><source>Brain</source><year>2015</year><volume>138</volume><fpage>1339</fpage><lpage>1354</lpage><pub-id pub-id-type=\"pmid\">25842390</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ghosh</surname><given-names>D</given-names></name><name><surname>Levault</surname><given-names>KR</given-names></name><name><surname>Brewer</surname><given-names>GJ</given-names></name></person-group><article-title>Relative importance of redox buffers GSH and NAD(P)H in age-related neurodegeneration and Alzheimer disease-like mouse neurons</article-title><source>Aging Cell</source><year>2014</year><volume>13</volume><fpage>631</fpage><lpage>640</lpage><pub-id pub-id-type=\"pmid\">24655393</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zong</surname><given-names>Y</given-names></name><etal/></person-group><article-title>miR-29c regulates NAV3 protein expression in a transgenic mouse model of Alzheimer’s disease</article-title><source>Brain Res.</source><year>2015</year><volume>1624</volume><fpage>95</fpage><lpage>102</lpage><pub-id pub-id-type=\"pmid\">26212654</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Irimia</surname><given-names>M</given-names></name><etal/></person-group><article-title>A highly conserved program of neuronal microexons is misregulated in autistic brains</article-title><source>Cell</source><year>2014</year><volume>159</volume><fpage>1511</fpage><lpage>1523</lpage><pub-id pub-id-type=\"pmid\">25525873</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quesnel-Vallieres</surname><given-names>M</given-names></name><etal/></person-group><article-title>Misregulation of an activity-dependent splicing network as a common mechanism underlying autism spectrum disorders</article-title><source>Mol. Cell</source><year>2016</year><volume>64</volume><fpage>1023</fpage><lpage>1034</lpage><pub-id pub-id-type=\"pmid\">27984743</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>DeMichele-Sweet</surname><given-names>MAA</given-names></name><etal/></person-group><article-title>Genetic risk for schizophrenia and psychosis in Alzheimer disease</article-title><source>Mol. Psychiatry</source><year>2018</year><volume>23</volume><fpage>963</fpage><lpage>972</lpage><pub-id pub-id-type=\"pmid\">28461698</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807792</article-id><article-id pub-id-type=\"pmc\">PMC7431540</article-id><article-id pub-id-type=\"publisher-id\">69287</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-69287-2</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Radiocarbon offsets and old world chronology as relevant to Mesopotamia, Egypt, Anatolia and Thera (Santorini)</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Manning</surname><given-names>Sturt W.</given-names></name><address><email>sm456@cornell.edu</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wacker</surname><given-names>Lukas</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Büntgen</surname><given-names>Ulf</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Bronk Ramsey</surname><given-names>Christopher</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><name><surname>Dee</surname><given-names>Michael W.</given-names></name><xref ref-type=\"aff\" rid=\"Aff8\">8</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kromer</surname><given-names>Bernd</given-names></name><xref ref-type=\"aff\" rid=\"Aff9\">9</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lorentzen</surname><given-names>Brita</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Tegel</surname><given-names>Willy</given-names></name><xref ref-type=\"aff\" rid=\"Aff10\">10</xref><xref ref-type=\"aff\" rid=\"Aff11\">11</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5386.8</institution-id><institution-id institution-id-type=\"ISNI\">000000041936877X</institution-id><institution>Cornell Tree Ring Laboratory, Department of Classics, </institution><institution>Cornell University, </institution></institution-wrap>Ithaca, NY 14853 USA </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5801.c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2156 2780</institution-id><institution>Laboratory for Ion Beam Physics, </institution><institution>Swiss Federal Institute of Technology in Zurich, </institution></institution-wrap>8093 Zurich, Switzerland </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5335.0</institution-id><institution-id institution-id-type=\"ISNI\">0000000121885934</institution-id><institution>Department of Geography, </institution><institution>University of Cambridge, </institution></institution-wrap>Cambridge, CB2 3EN UK </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.419754.a</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2259 5533</institution-id><institution>Swiss Federal Research Institute WSL, </institution></institution-wrap>8903 Birmensdorf, Switzerland </aff><aff id=\"Aff5\"><label>5</label>Global Change Research Institute CAS, 603 00 Brno, Czech Republic </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.10267.32</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2194 0956</institution-id><institution>Department of Geography, Faculty of Science, </institution><institution>Masaryk University, </institution></institution-wrap>611 37 Brno, Czech Republic </aff><aff id=\"Aff7\"><label>7</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.4991.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 8948</institution-id><institution>Research Laboratory for Archaeology, School of Archaeology, </institution><institution>University of Oxford, </institution></institution-wrap>Oxford, OX1 3TG UK </aff><aff id=\"Aff8\"><label>8</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.4830.f</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0407 1981</institution-id><institution>Centre for Isotope Research, Faculty of Science and Engineering, </institution><institution>University of Groningen, </institution></institution-wrap>Nijenborgh 6, 9747 AG Groningen, The Netherlands </aff><aff id=\"Aff9\"><label>9</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.7700.0</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2190 4373</institution-id><institution>Institute of Environmental Physics, </institution><institution>University of Heidelberg, </institution></institution-wrap>69120 Heidelberg, Germany </aff><aff id=\"Aff10\"><label>10</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5963.9</institution-id><institution>Chair of Forest Growth and Dendroecology, Institute of Forest Sciences, </institution><institution>University of Freiburg, </institution></institution-wrap>Freiburg, Germany </aff><aff id=\"Aff11\"><label>11</label>Archaeological Service Kanton Thurgau (AATG), 8510 Frauenfeld, Switzerland </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13785</elocation-id><history><date date-type=\"received\"><day>6</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>8</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">The new IntCal20 radiocarbon record continues decades of successful practice by employing one calibration curve as an approximation for different regions across the hemisphere. Here we investigate three radiocarbon time-series of archaeological and historical importance from the Mediterranean-Anatolian region, which indicate, or may include, offsets from IntCal20 (~0–22 <sup>14</sup>C years). While modest, these differences are critical for our precise understanding of historical and environmental events across the Mediterranean Basin and Near East. Offsets towards older radiocarbon ages in Mediterranean-Anatolian wood can be explained by a divergence between high-resolution radiocarbon dates from the recent generation of accelerator mass spectrometry (AMS) versus dates from previous technologies, such as low-level gas proportional counting (LLGPC) and liquid scintillation spectrometry (LSS). However, another reason is likely differing growing season lengths and timings, which would affect the seasonal cycle of atmospheric radiocarbon concentrations recorded in different geographic zones. Understanding and correcting these offsets is key to the well-defined calendar placement of a Middle Bronze Age tree-ring chronology. This in turn resolves long-standing debate over Mesopotamian chronology in the earlier second millennium BCE. Last but not least, accurate dating is needed for any further assessment of the societal and environmental impact of the Thera/Santorini volcanic eruption.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Climate sciences</kwd><kwd>Environmental sciences</kwd><kwd>Environmental social sciences</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/100000001</institution-id><institution>National Science Foundation</institution></institution-wrap></funding-source><award-id>BCS 1219315</award-id><principal-award-recipient><name><surname>Manning</surname><given-names>Sturt W.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/501100000155</institution-id><institution>Social Sciences and Humanities Research Council of Canada</institution></institution-wrap></funding-source><award-id>895-2011-1026</award-id><principal-award-recipient><name><surname>Manning</surname><given-names>Sturt W.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/501100000781</institution-id><institution>European Research Council</institution></institution-wrap></funding-source><award-id>714679</award-id><award-id>ECHOES</award-id><principal-award-recipient><name><surname>Dee</surname><given-names>Michael W.</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">The 2020 International Northern Hemisphere (NH) Radiocarbon (<sup>14</sup>C) Calibration curve, IntCal20, forms the current basis to calendar ages for many scientific fields from 0 to 55 kyr ago<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. IntCal20 continues the long-standing assumption that a single <sup>14</sup>C calibration curve is applicable to the mid-latitudes of the NH<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. However, there are indications of small, fluctuating, <sup>14</sup>C offsets which, at high-resolution, may affect accurate <sup>14</sup>C-based chronology in some mid-latitude regions<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>–<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Part of such differences may result from inter-laboratory offsets (see Supplementary Discussion 1), or derive from differences between recent AMS <sup>14</sup>C measurements versus those from previous <sup>14</sup>C dating technologies. Another part is inferred as a representation of the differing parts of the intra-annual atmospheric <sup>14</sup>C cycle, recorded because of different plant growth seasons or contexts. An example of the latter is the difference between the growth period of tree rings in central and northern Europe and northern America that comprise the Holocene IntCal record (spring through summer), versus those of many plants in the Mediterranean and Near East (winter to early summer)<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>–<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. The topic is noted, but is not addressed, in IntCal20<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. Finally, there are latitude-based variations in <sup>14</sup>C levels, but these are regarded as minimal within the mid-latitudes<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. Here we show the presence of small, but varying, <sup>14</sup>C offsets versus IntCal20—from one or a combination of the above potential sources—in the east Mediterranean-Anatolia region across the second millennium BCE. These need to be addressed to achieve accurate high-resolution <sup>14</sup>C-based chronology (and revise and clarify indications from initial comparisons with earlier versions of IntCal<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>–<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>). While small, the impact of these <sup>14</sup>C offsets can be substantial for Mediterranean and Near Eastern archaeology because of the intricate and densely integrated timeframes involved and the small margins of tolerance<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Moreover, where present, apparent seasonal <sup>14</sup>C offsets fluctuate over time, and appear associated with changes in <sup>14</sup>C production and thus likely with variations in solar activity and climate (and ocean systems), and potentially also, therefore, changes in percentage contributions of early and late wood to given tree-rings<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>–<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. These circumstances complicate the elegant hypothesis of a single NH calibration curve, with any variation assumed as effectively comparable with (or incorporated within) error terms<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. However, as we illustrate for Old Assyrian/Old Babylonian chronology, it opens the way for more accurate and precise dating through recognition of offsets and by tying sequences to specific appropriate <sup>14</sup>C records.</p><p id=\"Par3\">Among explanations for offsets between <sup>14</sup>C measurements, the least recognized is the role of the intra-annual cycle of atmospheric <sup>14</sup>C levels, with an NH winter low and a summer high<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>–<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. The Holocene part of the NH IntCal20 <sup>14</sup>C calibration curve, constructed mainly from tree-rings from central and northern Europe and northern America, reflects photosynthesis in the spring through summer period<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. In contrast, many plants in lower elevation contexts in the Mediterranean and Near East grow primarily in winter to spring<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>, or exhibit plasticity allowing climate and growth environment to modulate the boundaries of their growing season from year to year<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. Hence, there is a potential for different aspects of the annual <sup>14</sup>C cycle to be represented, especially as measurement of <sup>14</sup>C increases in accuracy and precision<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>–<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Despite a few observations of regional differences<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>, the topic really only became visible and relevant a decade ago in a large-scale study addressing ancient Egypt<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. This demonstrated that <sup>14</sup>C-based dating could achieve accuracy and precision at the level of the approximate historically derived chronology of Egypt. However, the data indicated it was necessary to make allowance for an Egyptian offset in local <sup>14</sup>C levels<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>, <xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. This offset was associated with the different (near opposite) growing season for plant matter in pre-modern Egypt (winter–spring) versus the growing season for the tree-rings used to inform the NH IntCal calibration record (spring–summer). Other work has identified instances of small offsets for the Mediterranean-Near East region, but also indications that they fluctuate<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>.</p><p id=\"Par4\">Whereas Libby employed samples from Old World archaeology to help supply a ‘curve of knowns’ to initially validate <sup>14</sup>C dating<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>, we now employ data from archaeo-historic cases with tight constraints to explore the issue of <sup>14</sup>C offsets, including any Mediterranean-Near East <sup>14</sup>C offset. Based on existing observations, <sup>14</sup>C offsets are typically evident only over certain periods, and become visible in the context of longer high-resolution rigid or near-rigid time-series<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>–<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Here we report comparison and analysis of three high-resolution <sup>14</sup>C time-series from archaeological material from the Mediterranean-Anatolia region against the IntCal20 dataset to identify and quantify <sup>14</sup>C offsets and to discuss sources. Historical chronologies provide constraints; in turn, they are better dated.</p></sec><sec id=\"Sec2\"><title>Results</title><sec id=\"Sec3\"><title>Anatolian Middle Bronze Age tree ring radiocarbon time series versus IntCal20</title><p id=\"Par5\">The first <sup>14</sup>C time-series comprises samples from a Middle Bronze Age (MBA) juniper (<italic>Juniperus</italic> sp.) tree-ring chronology constructed from three archaeological sites in Anatolia (Acemhöyük, ACM, Karahöyük, KBK, and Kültepe, KUL), archaeologically associated with Old Assyrian/Old Babylonian history through texts naming rulers and officials from the earlier second millennium BCE<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. This confluence of evidence enables potential resolution of the long-running debate over Mesopotamian chronology, where text and astronomical data have offered possibilities but not definitive solutions<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. Previous work indicated a likely solution<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. New data improving and extending the MBA <sup>14</sup>C time series, and the availability of the revised IntCal20 <sup>14</sup>C calibration dataset for comparison, provide the context to revisit in order to establish a high-resolution placement. We use the existing data<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup> and incorporate 25 new ETH measurements (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>). Since the wood samples from each site crossdate to form a single secure annual tree-ring chronology<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>, the tree-ring sequenced series of <sup>14</sup>C data (n = 76) over a 200-year period should offer close comparison with the NH <sup>14</sup>C calibration curve. We compare and fit (‘wiggle-match’) the data using the known tree-ring spacing after removing four initial outliers using the OxCal software<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>–<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> (see “<xref rid=\"Sec10\" ref-type=\"sec\">Methods</xref>”).</p><p id=\"Par6\">However, the fit is poor, failing an overall χ<sup>2</sup> test and yielding poor OxCal agreement indices (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>a). An OxCal ΔR test<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>, to assess whether there is systematic difference between the MBA time series and the calibration curve using a neutral prior (0 ± 10 <sup>14</sup>C years), indicates in many cases a bimodal finding (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>). The data are offset on average either (and most likely) about 22 ± 5 <sup>14</sup>C years, or, alternatively about − 32 ± 8 <sup>14</sup>C years. Let us quantify what these differences mean in calendar terms for a specific point in the MBA tree ring series, Relative Year (RY) 701 (the latest dated element), in order to appreciate the scale of the problem. The mid-point of the 68.3% highest posterior density (hpd) range for RY701 with no ΔR is ~ 1,851 BCE, with ΔR 22 ± 5 <sup>14</sup>C years it is ~ 1,803 BCE and with ΔR − 32 ± 8 <sup>14</sup>C years it is ~ 1,883 BCE—a total range of ~ 81 calendar years. Such a large discrepancy is incompatible with high-resolution chronology. It is therefore important to resolve such ambiguity and imprecision. To investigate towards the likely solution, we tried wiggle-matches incorporating an offset effect of 22 ± 5 <sup>14</sup>C years or − 32 ± 8 <sup>14</sup>C years. Runs of the latter model yield poor OxCal agreement indices (A<sub>model</sub> and A<sub>overall</sub> below 30, well below the satisfactory threshold value of 60), the posterior density for the ΔR offers poor OxCal agreement with the prior (< 60), and there is a poor visual fit (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>). We thus exclude this option as not viable. In contrast, the model incorporating an offset effect ΔR of 22 ± 5 <sup>14</sup>C years offers a good visual fit with IntCal20 (A<sub>model</sub> and A<sub>overall</sub> around 60) (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b) and the observed ΔR corresponds successfully with this prior estimate (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>c). In particular, although offset to slightly older <sup>14</sup>C ages, we note how the MBA series as placed in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b closely describes the wiggle ~ 1,850 to 1,810 BCE in the IntCal20 calibration curve (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>d, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>). This provides a specific and secure chronological placement for the later part of the time series, versus a lack of clarity in this region with a smaller dataset and previous calibration curves<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Thus, by identifying, quantifying and then exploiting the relevant offset in this case we can obtain a unique high-resolution chronology.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Fit of the MBA crossdated tree-ring <sup>14</sup>C time series from Acemhöyük (ACM), Karahöyük (KBK) and Kültepe (KUL)<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup> against IntCal20<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. (<bold>a</bold>) Wiggle-match with OxCal<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup> 4.4.1 of the MBA <sup>14</sup>C time series against IntCal20 with no offset allowed for and curve resolution of 1 year (the previous IntCal13 calibration curve<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup> is shown for comparison). The OxCal A<sub>model</sub> and A<sub>overall</sub> values are poor and 33% of the data achieve unsatisfactory individual OxCal Agreement values (< 60). Visual inspection shows most data are placed too old, so they are either below the calibration curve or do not offer good correspondence—especially the set of Acemhöyük dates (black) which show structure, but do not correspond with the calibration curve at this calendar position. (<bold>b</bold>) Fit with an offset of 22 ± 5 <sup>14</sup>C years. 72 data, 39 elements. (<bold>c</bold>) Modelled posterior density (dark histogram) versus the prior of 22 ± 5 <sup>14</sup>C years illustrating good agreement (see Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>). (<bold>d</bold>) Close and specific fit of the ACM <sup>14</sup>C data (black) around the wiggle in IntCal20 between 1,850 and 1,810 BCE. Data ~ 1,890 to 1,850 BCE, during a reversal in atmospheric <sup>14</sup>C levels, indicate a likely (positive) regional or measurement <sup>14</sup>C offset.</p></caption><graphic xlink:href=\"41598_2020_69287_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par7\">The incompatibility (older <sup>14</sup>C values) of the four KUL + ACM elements ~ 1,883 to 1,853 BCE with IntCal20 is conspicuous. To investigate, we measured new ETH data on known-age single-year oak tree-ring samples from Erstein, France, from part of this period (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. These data also do not replicate the strong dip and reversal in IntCal ~ 1,860 to 1,840 BCE (~ 3,809 to 3,789 Cal BP). Instead, they indicate values that are older than IntCal20 and more in the range of those from the MBA time series. Collectively, these new data suggest that IntCal itself needs some revision in this period (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S4</xref>). Hence, while some portion of the visible offset in this case might, as in cases of other reversals in the <sup>14</sup>C record<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>, comprise a manifestation of a regional <sup>14</sup>C offset, in this instance the actual existence of the strong reversal in the IntCal dataset is open to question. We re-run the wiggle-match of the MBA time series excluding this currently problematic interval to check that it is not being unduly influenced by this issue. We thus exclude the five offset data points for RY621, RY631, RY641, RY646 and RY651 (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>d). Over 10 runs with a neutral prior of 0 ± 10 <sup>14</sup>C years, the remaining MBA time series nonetheless consistently finds the same approximate best fit range as in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b (in 5 of 10 runs, or 1 year older, in 4 of 10 runs, or 2 years older, in 1 of 10 runs). Further, within 95.4% probability limits, the reduced time series now avoids the bi-modal probability issue noted above (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>)—we may therefore view the previous ambiguity as caused by the problematic dip in the current IntCal dataset. With the edited time series, the OxCal ΔR offset observed is reduced a little—but nevertheless remains present. The average 68.3% hpd ΔR offset range is 17.0 ± 4.1 <sup>14</sup>C years. If the series is then run with a ΔR of 17 ± 4 <sup>14</sup>C years, it consistently finds a very similar but slightly better defined best fit placement compared to that shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b. The mean placement of the elements is just 0–1 year later and the standard deviation on this mean is 1 year smaller (2 versus 3). The last dated RY701 element is placed 1,805–1,800 BCE (68.3% hpd) and 1,807–1,798 BCE (95.4% hpd), compared with 1,806–1,801 BCE (68.3% hpd) and 1,809–1,797 BCE (95.4% hpd) in the Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b fit using ΔR 22 ± 5 <sup>14</sup>C years. We therefore regard the placement shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b as robust within about 1 year, pending revision of this whole period of the IntCal dataset (we note that this portion of IntCal20 remains largely based on legacy data from IntCal13<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>, and before).</p></sec><sec id=\"Sec4\"><title>Mesopotamian Old Assyrian/Old Babylonian chronology</title><p id=\"Par8\">The MBA wiggle-match in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b places likely earliest use (RY673) of the Waršama Palace at Kültepe<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup> ~ 1,837 to 1,826 BCE (95.4% hpd) and the earliest use (RY732) of the Sarıkaya Palace at Acemhöyük<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup> ~ 1,778 to 1,767 BCE (95.4% hpd). (The alternative reduced dataset 95.4% hpd ranges are almost the same: 1,835–1,826 BCE and 1,776–1,767 BCE.) A rich set of historical associations linked with the Old Assyrian Revised Eponym List (REL) should fit as respectively before, around, and following these dates<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup> (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). For example, the lower town Kültepe Ib period is regarded as commencing around the start of the Waršama Palace and multiple documents link the Assyrian ruler Šamši-Adad I with both Kültepe Ib and the Sarıkaya Palace. The only Mesopotamian chronological schemes<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup> potentially compatible with the wiggle-match are the High Middle or (especially) Low Middle Chronologies (which are only 8 calendar years apart)<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref>–<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. Recent studies assessing the textual and astronomical data have also offered strong support for this solution<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>–<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>.The tree ring sequenced <sup>14</sup>C placement and necessary set of relationships contradict the other candidates (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). Our findings here, with additional data and the new IntCal20 calibration curve, confirm the resolution of Old Assyrian/Old Babylonian chronology around the Middle Chronology range and end a long-running debate.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Comparisons of sequenced <sup>14</sup>C datasets and their historical associations. (<bold>a</bold>) Earliest use dates for the Waršama Palace at Kültepe and the Sarıkaya Palace at Acemhöyük (arrows indicate approximate minimum use periods based on dendrochronologically dated repairs/additions<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>) from the dendro-<sup>14</sup>C wiggle-match (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b). These are compared with historical associations expressed in terms of Revised Eponym List (REL) dates from text records, placed according to the five main rival Mesopotamian chronologies<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>–<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>.</p></caption><graphic xlink:href=\"41598_2020_69287_Fig2_HTML\" id=\"MO2\"/></fig></p></sec><sec id=\"Sec5\"><title>Egyptian New Kingdom radiocarbon time series versus IntCal20</title><p id=\"Par9\">The second <sup>14</sup>C time series comprises the Egyptian New Kingdom (NK) dataset<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. This indicated a seasonal <sup>14</sup>C offset of ~ 19 ± 5 <sup>14</sup>C years against IntCal04<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. Re-run against IntCal20, the offset reduces slightly, but remains present at ~ 16 ± 4 <sup>14</sup>C years (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a,b, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S5</xref>). The revised Egyptian NK model with a neutral prior seasonal offset test of 0 ± 10 <sup>14</sup>C years (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>b), or models running with a ΔR of 16 ± 5 <sup>14</sup>C years, produce modelled ages for the NK rulers with IntCal20 that vary only very slightly, downwards, compared with the ages determined previously<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. However, there are indications that the <sup>14</sup>C offset likely fluctuates. We find that an alternative NK model<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup> which employs some revised reign lengths and the plausible longest reigns for the 18th Dynasty (ultra-high model)<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref>–<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>, and so has a slightly different placement of the constituent groups of <sup>14</sup>C data versus the calibration curve, offers a different (and much smaller) ΔR of ~ 6 ± 6 <sup>14</sup>C years (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S5</xref>). This better fit, and recent review of the historical and astronomical evidence, may favour a longer/higher NK Egyptian historical chronology<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Comparisons of IntCal20<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>, Hd GOR<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> and Egyptian NK<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup> datasets. (<bold>a</bold>) IntCal20 and Hd GOR records (± 1σ) and NK Egyptian time series. (<bold>b</bold>) Seasonal offset of the NK time series with IntCal20. (<bold>c</bold>) <sup>14</sup>C offsets between Hd GOR and IntCal20 overall interpolated. (<bold>d</bold>) Posterior density placement of the GOR felling date RY1,764 versus IntCal20 using the Hd GOR data series minus outliers, placing the overall GOR chronology (RY737–1,764) ~ 3,724 to 2,697 Cal BP/1,775–748 BCE.<bold> (e)</bold> Comparisons of AA IrO, AA GOR (and 10 point adjacent average) and ETH IrO versus each other and IntCal20, <bold>(f)</bold> Differences AA GOR versus AA IrO and AA GOR versus IntCal20 according to placement of GOR chronology last ring (RY1,764).</p></caption><graphic xlink:href=\"41598_2020_69287_Fig3_HTML\" id=\"MO3\"/></fig></p></sec><sec id=\"Sec6\"><title>Gordion tree ring chronology versus IntCal20</title><p id=\"Par10\">The third long time series comprises <sup>14</sup>C measurements on a tree-ring chronology from the Midas Mound Tumulus at Gordion (GOR) in central Anatolia<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR37\">37</xref>–<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. There are two versions: a LLGPC Heidelberg (Hd) series<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup> and a AMS <sup>14</sup>C Arizona (AA) series<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. Wiggle-matched versus IntCal20 (see “<xref rid=\"Sec10\" ref-type=\"sec\">Methods</xref>”, Supplementary Discussion 2) (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a,c,d), the Hd GOR series (GOR RY737–1,764, ~ 1775 to 748 BCE/3,724–2,697 Cal BP) has a weighted average offset of 2.3 ± 2.1 <sup>14</sup>C years (n = 117), with periods of fluctuating offsets in each direction. As observed in other cases, the positive offsets correspond generally with periods around reversals and plateaus in the <sup>14</sup>C calibration curve<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. It is evident, for certain periods, and in particular when there is a marked positive Hd GOR to IntCal20 offset (e.g. ~ 1,360 to 1,330 BCE), that the Egyptian NK time series corresponds better with the Hd GOR data than IntCal20 (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a). An exception is around 1,470 BCE. Here the few and decadal Hd GOR data do not pick up the wiggle and apparent larger offset exhibited by the Egyptian samples.</p><p id=\"Par11\">The AA GOR series is much shorter in overall length (186 years), but comprises annual resolution data<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. Wiggle-matched against IntCal20, they are placed (μ ± σ) 1,678 ± 1 BCE (GOR RY 834) to 1,493 ± 1 BCE (GOR RY 1,019) (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>e, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S6</xref>, extrapolated the 95.4% range for GOR RY1,764 is 751–746 BCE, μ ± σ = 748 ± 1 BCE). This is identical with the Hd GOR fit in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a,d. The publication advocated chronological positioning from a χ<sup>2</sup> fit<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. We consider two approaches<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref>,<xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup> against both the IntCal20 modelled curve<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup> and a weighted average<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup> of recently published Irish Oak (IrO) and bristlecone pine (BCP) datasets<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup> (see Supplementary Discussion 2). These find the best (minimum) fit for the last ring and felling date, GOR RY1,764, 749–747 BCE (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S7</xref>), very similar to the OxCal results (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>e, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S7</xref>). Agreement on the approximate absolute calendar placement of the GOR time series suggests a robust fit (and we use the ~ 748 BCE fit).</p><p id=\"Par12\">However, there is a clear difference comparing the <sup>14</sup>C ages from Hd GOR versus AA GOR versus IntCal20 (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S8</xref>). In contrast to the Hd GOR time series, where the weighted average offset against IntCal20 is calculated as − 2.3 ± 2.1 <sup>14</sup>C years (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S8</xref>), the AA GOR time series (over a much shorter period) and with considerable noise exhibits a much larger weighted average offset of 11.2 ± 1.9 <sup>14</sup>C years (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>e,f). This tendency to an average positive offset is visible in Supplementary S6, where 69% of the AA GOR <sup>14</sup>C data are older than the corresponding IntCal20 value. Latitude is suggested as an explanation<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>, but a previous <sup>14</sup>C time series on Anatolian wood does not illustrate such systematically offset data<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S9</xref>). The Noceto (NOC) series from Italy also exhibits only a small average offset, as does the Miletos series from western Turkey, or data from Bcharre in Lebanon (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>a, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S9</xref>). Since measurements on the same IrO between AA and ETH indicate that AA is on average 6.2 ± 1.8 <sup>14</sup>C years older<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup> (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>f), we might instead consider adjusting the AA GOR offset, perhaps by a similar amount (e.g. to ~ 5.0 ± 2.6 <sup>14</sup>C years). This would then also be a typically small or negligible average offset (with variation, as evident from Hd GOR: Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>c), and not far from the Hd GOR record (see below).<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Instances of the differing <sup>14</sup>C offsets between the Mediterranean-Near East and IntCal20 (± 1σ) at various periods. (<bold>a</bold>) The 1,700–1,500 BCE period, where IntCal20 is informed predominantly by many new AMS <sup>14</sup>C dates<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>, shows little offset (contrary to the previous IntCal13<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>) and low elevation Mediterranean time series from Miletos, western Turkey, and Noceto (NOC), northern Italy, fit the calibration curve closely and show a negligible offset (the ~ 1,487 BCE NOC date may be an interesting exception, see text). Difference NOC versus IntCal20: 1.7 ± 6.1 <sup>14</sup>C years; difference Miletos versus IntCal20: 1.2 ± 10.0 <sup>14</sup>C years. Combined OxCal<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> ΔR with neutral prior of 0 ± 10 <sup>14</sup>C years gives μ ± σ of 2.1 ± 5.3 <sup>14</sup>C years. (<bold>b</bold>) Small positive <sup>14</sup>C offset during the Amarna period in Egypt contemporary with a reversal in the <sup>14</sup>C calibration curve, especially at time of the death of Tutankhamun, when it reaches ~ 19 <sup>14</sup>C years (but IntCal20 in this period is largely based on legacy <sup>14</sup>C data—thus the offset observed may reduce once IntCal20 is updated with modern AMS <sup>14</sup>C data for this interval).</p></caption><graphic xlink:href=\"41598_2020_69287_Fig4_HTML\" id=\"MO4\"/></fig></p></sec></sec><sec id=\"Sec7\"><title>Discussion</title><sec id=\"Sec8\"><title>Radiocarbon offsets and their causes</title><p id=\"Par13\">The three sets of comparisons indicate two key outcomes. First, across the second and early first millennia BCE, there is repeated evidence for the operation and effect of small offsets that impact the high-resolution dating of these Mediterranean-Near Eastern <sup>14</sup>C datasets, even with the latest NH international <sup>14</sup>C calibration curve (IntCal20). Second, such offsets are not constant, but appear to fluctuate over time. This suggests it would be misleading to apply a constant offset factor for individual dating cases that might, or might not, be relevant.</p><p id=\"Par14\">Evidently one key factor relevant to determining the nature and source of the offsets observed is the composition of the <sup>14</sup>C calibration curve at particular periods. Much of the calibration curve record up until IntCal20 derives from laboratories using LLGPC or LSS<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>, and, except for the period ~ 1,700 to 1,500 BCE, most of the second to early first millennia BCE still does<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. As noted, in several instances including this one, detailed new measurements of time intervals with AMS <sup>14</sup>C have indicated slightly older <sup>14</sup>C ages<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref>,<xref ref-type=\"bibr\" rid=\"CR43\">43</xref>–<xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>. The MBA (87%) and Egyptian NK (100%) time series consist of AMS <sup>14</sup>C dates. It is thus unclear how much of the scale of the observed <sup>14</sup>C offsets may in fact be a difference between measurement techniques and technologies—versus an expected small but varying intra-annual seasonal <sup>14</sup>C offset component<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>–<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. For example, Mediterranean-Near Eastern <sup>14</sup>C offsets within the period 1,600–1,900 CE observed comparing AMS <sup>14</sup>C data with the previous LLGPC and LSS IntCal datasets<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup> remain, but are reduced, when compared with the new IntCal20 curve containing many new AMS <sup>14</sup>C data for this period<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. For example, the original Egyptian 18th–19th century CE average offset<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup> reduces from 19 ± 5 to 12 ± 5 <sup>14</sup>C years (and the NK period offset may reduce with revisions to the historical intervals: see above), while the comparisons of the Oxford and AA Jordan juniper datasets<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup> similarly reduce from the reported average OxCal ΔR <sup>14</sup>C year offsets of 19 ± 3 and 21 ± 5 to 12 ± 3 and 12 ± 5 <sup>14</sup>C years. Egypt and the southern Levant represent almost maximally offset mid-latitude NH growth season timings versus central and northern Europe and North America<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. This suggests the scale of a likely real average maximum seasonal offset factor, if the entire calibration curve comprised similar AMS <sup>14</sup>C data, more of the order of ~ 12 ± 5 <sup>14</sup>C years (~ 1 to 2‰). At about half the maximum intra-annual variation observed from atmospheric measurements<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>, this appears plausible. We accordingly revise previous estimates of typical seasonal <sup>14</sup>C offsets<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> downwards to this approximate range. In practice, the additional issue of inter-laboratory differences (see above), evident even among high-precision calibration laboratories, adds a further error component<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup> (Supplementary Discussion 1). Any average <sup>14</sup>C offset in the Aegean–Anatolia region should be rather smaller, since the growing seasons are substantially less offset versus IntCal20 source trees<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>.</p><p id=\"Par15\">Two issues apply particularly to the 1,700–1,480 BCE interval (Supplementary Discussion 1). First, BCP tends to produce <sup>14</sup>C ages older than contemporary IrO or IntCal20 by around 7–9 <sup>14</sup>C years<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref>,<xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup>. Second, AA <sup>14</sup>C data overall for this period<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref>,<xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup> are older than the consensus (IntCal20) or in direct comparisons with ETH by around ~ 6 to 7 <sup>14</sup>C years<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. Thus the incorporation of several hundred AA BCP and IrO ages into IntCal20 1,700–1,480 BCE overly raises <sup>14</sup>C ages in this section of the calibration curve. This AA-effect likely partly incorporates (or hides) any typical positive Mediterranean growing season offset, when relevant (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>c, Supplementary Figs. <xref rid=\"MOESM1\" ref-type=\"media\">S6</xref>, <xref rid=\"MOESM1\" ref-type=\"media\">S9</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. The Egyptian NK data support such a view. Ruling out two extreme outliers, it is noticeable that the 7 <sup>14</sup>C elements of the Egyptian NK time series<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup> in the sixteenth century BCE are either around, or in fact below, IntCal20 (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S5</xref>).</p><p id=\"Par16\">For unknown reasons it is apparent that the Hd German Oak (GeO) data for the period ~ 1,660 to 1,540 BCE are too recent<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR43\">43</xref>,<xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>. Despite good comparisons in other periods<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S9</xref>), there was a problem in this interval. The Hd GeO data 3,629–3,449 Cal BP (1,680–1,500 BCE) are − 15.6 ± 2.4 <sup>14</sup>C years versus IntCal20, n = 57. But as noted, IntCal20 is a little old in this period. The Hd GeO series, when compared versus ETH IrO<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup> (weighted averages) for this period (common data available 3,625–3,431 Cal BP/1,676–1,482 BCE), are – 11.8 ± 2.8 <sup>14</sup>C years, n = 49. In particular, Hd data on Knetzgau 40<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> have been shown to be − 12.9 ± 3.1 <sup>14</sup>C years more recent than measurements by three other laboratories on this tree<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>. Thus a previously observed offset between Hd GeO and Hd GOR in the earlier sixteenth century BCE<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> is likely largely erased (Supplementary Discussion 1). Are the Hd GOR data similarly too recent? We argue no. As published, the Hd GOR data offer reasonable comparison with IntCal20, as would be anticipated given (1) the relevant growing seasons are not markedly offset (contrast Egypt or the southern Levant<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>), but with some periods of small offset when the difference was exaggerated<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>, and (2) the AA–IrO and especially BCP inflation of IntCal20 in this period likely already covers some to all of any typical Aegean–Anatolian offset. For example, were even the smaller of the offsets evident for the Hd GeO (just noted) also applied to Hd GOR, then there would be a large average offset, e.g. + 14.2 ± 2.8 <sup>14</sup>C years versus IntCal20. But, as just discussed, revision and comparison of comparable datasets indicates maximum mid-latitude NH growing season offsets ~ 12 ± 5 <sup>14</sup>C years, and the Gordion context should be substantially less offset. The same criticism of too large an average offset applies to the AA GOR data<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. Since it is evident from a large set of parallel measurements of IrO by both AA and ETH (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>e) that the AA data are ~ 6.2 ± 2.8 <sup>14</sup>C years older<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>, it seems likely these AA GOR data are on average too old also. If they were adjusted by around the ETH to AA IrO factor, as suggested above, then they too would offer a more plausible relationship with IntCal20.</p></sec><sec id=\"Sec9\"><title>Radiocarbon offsets and Mediterranean chronology</title><p id=\"Par17\">The values for possible <sup>14</sup>C offsets mentioned above are averages, and there will be variation around these (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>c,f)<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Such episodes could be important for high-resolution chronology. The historically well-dated Amarna period in Egypt offers a test case for a larger offset during the second millennium BCE, since it lies around the time of an apparent offset in Mediterranean <sup>14</sup>C levels ~ 1,360 to 1,330 BCE from the Hd GOR dataset (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a,c). A model combining the available <sup>14</sup>C dates and the historical constraints<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>, <xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup> (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>) indicates a maximum possible offset around the time of the burial of Tutankhamun of ~ 19 <sup>14</sup>C years versus IntCal20 (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>b). However, since this part of IntCal20 comprises legacy data, we might anticipate this offset reducing a little in the future (compare our MBA case above).</p><p id=\"Par18\">Even small changes in  <sup>14</sup>C ages can make large calendar differences during reversals and plateaus in the calibration record. There is a narrow distinction between a late seventeenth and earlier-mid sixteenth century BCE date range with IntCal20. Yet this determines the much-debated date of the Thera/Santorini volcanic eruption<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR43\">43</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref>,<xref ref-type=\"bibr\" rid=\"CR48\">48</xref>,<xref ref-type=\"bibr\" rid=\"CR51\">51</xref>–<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup> (Supplementary Discussion 3). Analysis with IntCal20 using (1) weighted average <sup>14</sup>C ages<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>, (2) a published dataset and alternative appropriate method<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref>,<xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>, or (3) the series of <sup>14</sup>C dates on an olive branch found buried by the Santorini/Thera eruption<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR53\">53</xref>,<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>, all indicate a most likely late seventeenth century BCE date, but include varying probability in the earlier-mid sixteenth century BCE (Supplementary Figs. <xref rid=\"MOESM1\" ref-type=\"media\">S10</xref>a,b, <xref rid=\"MOESM1\" ref-type=\"media\">S11</xref>a,b, Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>a). However, if the eruption was coeval with a small positive offset—for example of up to ~ 8 <sup>14</sup>C years (1‰) (see above, Supplementary Discussions 1, 3, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S6</xref>)—this moves substantial or majority probability from the later 17th to the earlier-mid sixteenth centuries BCE in (1) and (3) (Supplementary Figs. <xref rid=\"MOESM1\" ref-type=\"media\">S10</xref>c,d, <xref rid=\"MOESM1\" ref-type=\"media\">S11</xref>c, Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>b).<fig id=\"Fig5\"><label>Figure 5</label><caption><p>Calendar dating probabilities and ranges for the Santorini/Thera volcanic eruption following published dataset (with one subsequent addition: see Supplementary Discussion 3, Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>) and an appropriate method<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref>,<xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. (<bold>a</bold>) With IntCal20<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>, resolution = 1 year, and assuming no substantive Mediterranean <sup>14</sup>C offset at this time beyond that covered already by IntCal20 for this period (as indicated in Figs. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a,c, <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>a, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S9</xref>). (<bold>b</bold>) As (<bold>a</bold>), but applying a hypothetical positive Aegean-region ~ 8 <sup>14</sup>C years offset (OxCal ΔR of + 8 <sup>14</sup>Cyears) (Supplementary Discussion 1, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S6</xref>).</p></caption><graphic xlink:href=\"41598_2020_69287_Fig5_HTML\" id=\"MO5\"/></fig></p><p id=\"Par19\">In the Thera case, it was suggested recently that “to gain more precise insight into the timing using <sup>14</sup>C, modelling of multiple <sup>14</sup>C dates will likely be needed”<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. We revise and up-date a Bayesian model<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup> (see Supplementary Discussion 3, Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>, Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S12</xref>) incorporating 147 <sup>14</sup>C dates and archaeological information from Thera and the southern Aegean for the periods before, contemporary with, and after the Thera eruption. The modelled dating probability for the Thera eruption, using the median OxCal A<sub>model</sub> result from 11 model runs (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S13</xref>) is shown in Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>a. Across the 11 runs the total dating window at 95.4% hpd is 1,619–1,543 BCE and the most likely 68.3% hpd regions overall are ~ 1,617 to 1,601 BCE (average 62.8% hpd) and ~ 1,570 to 1,562 BCE (average 5.4% hpd) (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S13</xref>). Did any additional <sup>14</sup>C offset apply beyond that already incorporated in IntCal20 (see above)? If, for example, even an 8 <sup>14</sup>C year offset applied, then the dating probability in the Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>a model largely switches to the earlier-mid sixteenth century BCE (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>b). Contrary to previous advertisements<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>, a date for the Thera eruption after ~ 1,543/1,538 BCE remains improbable (end 95.4% hpd, multiple runs Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref> models), ruling out the conventional ‘low’ chronology range ~ 1,530 to 1,500 BCE<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>, but final placement depends on clarification of the reality (or not) of a small additional positive <sup>14</sup>C offset. While, at first glance, this is perhaps suggested by the AA GOR data (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S6</xref>), it is contradicted by other available data (see above), and is likely not supported even by the AA GOR data series once the evident inter-laboratory offset and excessive noise is removed (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S14</xref>, Supplementary Discussion 1). The better fit of a longer/higher Egyptian NK chronology versus IntCal20 noted above (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S5</xref>b) is potentially important. Such revision brings the time range of the Thera eruption (either Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>a or b) much closer to the start of the New Kingdom. This could minimize a time difference previously viewed as problematic<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>, and might start to permit discussion of suggested possible associations between these episodes<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>.<fig id=\"Fig6\"><label>Figure 6</label><caption><p>Modelled dating probabilities for the Thera eruption from the southern Aegean model (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S12</xref>). (<bold>a</bold>) Modelled Thera eruption boundary (age estimate) including <sup>14</sup>C data from Thera—median A<sub>model</sub> result from 11 runs (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S13</xref>). Arrows indicate major volcanic signals in recently re-dated Greenland ice-core records<sup><xref ref-type=\"bibr\" rid=\"CR67\">67</xref></sup>, along with some published tree-ring growth anomalies suggested potentially to be associated with major volcanic eruptions<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR67\">67</xref>–<xref ref-type=\"bibr\" rid=\"CR69\">69</xref></sup> (see Supplementary Discussion 3). (<bold>b</bold>) As (<bold>a</bold>) but applying a hypothetical additional positive Aegean-region ~ 8 <sup>14</sup>C years offset (OxCal ΔR of + 8 <sup>14</sup>C years) (Supplementary Discussion 1, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S6</xref>). Of the definite volcanic signals represented in the Greenland ice, either (higher option) 1,610 BCE, or (lower option) 1,560 BCE appear respectively plausible and most likely. OxCal<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>–<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> models in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref> and described in Supplementary Discussion 3, using IntCal20<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>, with resolution = 1 year.</p></caption><graphic xlink:href=\"41598_2020_69287_Fig6_HTML\" id=\"MO6\"/></fig></p><p id=\"Par20\">Thera is a well-known case, but there are many other instances of high-resolution <sup>14</sup>C chronologies key to Mediterranean and Near Eastern pre- and proto-history<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR57\">57</xref>–<xref ref-type=\"bibr\" rid=\"CR63\">63</xref></sup>. Our examples highlight the need to determine a high-resolution Mediterranean-Near Eastern <sup>14</sup>C record in order to clarify the question of fluctuating small offsets as relevant to regional <sup>14</sup>C levels over time. At present, a basic problem is that comparisons for many periods (where extensive new annual resolution AMS <sup>14</sup>C data are not yet available) merge two separate issues: (1) differences between older LLGPC and LSS <sup>14</sup>C calibration data versus newer AMS <sup>14</sup>C data, as well as (2) an apparent modest seasonal <sup>14</sup>C component. Any general approximation is an unsatisfactory solution since offsets appear to vary over time (likely associated with varying <sup>14</sup>C production, climate and plant physiology processes<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>–<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>). Especially at times of reversals and plateaus in the <sup>14</sup>C calibration curve, even modest variations may have great import for high-resolution chronology in the Mediterranean and Near East, and could affect a number of long-running debates. For those periods of IntCal20 still primarily based on LLGPC and LSS data, we have shown that such offsets affect accurate high-resolution chronology using AMS <sup>14</sup>C dates. Resolution requires deconvolution of the now mixed IntCal record. Ideally, AMS <sup>14</sup>C dates should be calibrated against an AMS <sup>14</sup>C derived calibration record, and LLGPC and LSS dates against a LLGPC and LSS derived <sup>14</sup>C calibration curve. Remaining offsets and variations would then have other causes, such as seasonal effects.</p></sec></sec><sec id=\"Sec10\"><title>Methods</title><sec id=\"Sec11\"><title>Radiocarbon dates and samples</title><p id=\"Par21\"><sup>14</sup>C dates employed combine sets of dates previously published with methods and full information<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR37\">37</xref>–<xref ref-type=\"bibr\" rid=\"CR39\">39</xref>,<xref ref-type=\"bibr\" rid=\"CR43\">43</xref>,<xref ref-type=\"bibr\" rid=\"CR50\">50</xref>,<xref ref-type=\"bibr\" rid=\"CR51\">51</xref>,<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup> (Supplementary Discussion 3) and sets of new dates run at the Eidgenössische Technische Hochschule (ETH) Zürich <sup>14</sup>C laboratory (Supplementary Tables <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>, <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>). The dendrochronology of the <italic>Juniperus</italic> sp. time series from Acemhöyük, Karahöyük and Kültepe is published<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. Timbers from the Waršama Palace at Kültepe include bark. Hence the felling date and likely primary construction 0–1 year later is placed RY670–672, and so suggest earliest building use likely ~ RY673<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. Timbers from the Sarıkaya Palace at Acemhöyük include bark (felling date and likely primary construction 0–1 year later) at RY730–731, and so suggest earliest building use likely ~ RY732<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. The <sup>14</sup>C dates and dendrochronology of the Gordion time series employed is published<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR37\">37</xref>–<xref ref-type=\"bibr\" rid=\"CR39\">39</xref>,<xref ref-type=\"bibr\" rid=\"CR64\">64</xref></sup>. The Erstein (ERST) <sup>14</sup>C measurements are on oak (<italic>Quercus</italic> sp.) samples from a tree-ring chronology built from preserved timbers recovered as part of archaeological excavations undertaken before the development of the Parc d’Activités du Pays d’Erstein, Erstein, France (48.4269N, 7.6386E)<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. Dendrochronological crossdating places the sample used, ERST 5964-GBS-218-37, at 2,010-1,764 BCE. The Egyptian NK data and the OxCal CQL2 code have been published<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. The revision of this OxCal model, adjusted to incorporate subsequent studies on historical Egyptian chronology and the reign lengths of kings<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref>–<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup> using the ultra-high version for the earlier NK<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup> is also published<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>. Details on the Miletos and Noceto tree-ring samples and <sup>14</sup>C dates are published<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>. Where plotted in the figures, <sup>14</sup>C dates (or weighted averages) are shown with 1σ errors (Y axis). In Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>a–c, <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>a and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref> the X axis width of the plotted date, or weighted average age, indicates the 68.3% hpd wiggle-match range. In Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>b and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S5</xref> the <sup>14</sup>C dates or weighted averages show the <sup>14</sup>C value (age or mean) on the Y axis and the mean ± σ values of the modelled posterior density distributions on the X axis. Pretreatment and processing of samples and their AMS <sup>14</sup>C dating at the ETH laboratory followed methods previously described for similar wood/charcoal samples<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR44\">44</xref>,<xref ref-type=\"bibr\" rid=\"CR65\">65</xref>,<xref ref-type=\"bibr\" rid=\"CR66\">66</xref></sup>.</p></sec><sec id=\"Sec12\"><title>Radiocarbon modelling</title><p id=\"Par22\">We employed OxCal<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>–<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> using versions 4.1.7, 4.3.2 and 4.4.1 with the IntCal20 NH <sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>C calibration curve<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup> (curve resolution set at 1 year). Where <sup>14</sup>C dates comprised the same (cross-dated) tree-rings or mid-points, and so represent estimates of (approximately) the same <sup>14</sup>C date/calendar age relationship, we combined these into weighted averages<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup> using the R_Combine function in OxCal. Where sets of tree-rings comprise the sample we regard the date as the mid-point (e.g. for Relative Years, RY, 1–5 this would be RY3). Where a sample comprised an even number of tree-rings, e.g. RY1–10, then the mid-point is treated as RY5.5 (after RY5 and before RY6). Where applicable, individual outliers were identified and down-weighted using the OxCal SSimple Outlier model<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. The SSimple Outlier model was also used to assess weighted averages against the model. The tree-ring time series were analyzed (‘wiggle-matched’) using the D_Sequence function of OxCal<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>.</p><p id=\"Par23\">The MBA time series comprises 76 <sup>14</sup>C dates. After combining dates with the same mid-points the time series contains 40 elements. However, three of the weighted averages fail a χ<sup>2</sup> test for representing the same age (mid-points RY651, 659 and 691)<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. In each case the OxCal SSimple Outlier model identifies one date as the clear outlier and so we removed three dates: ETH-78942.1.1 (outlier probability ~ 53%), OxA-29963 (outlier probability ~ 65%) and ETH-78947.1.1 (outlier probability ~ 84%) (see Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>). One other date (OxA-30907) had a large offset between the δ<sup>13</sup>C value measured by the AMS versus the stable isotope MS (suggesting fractionation at the level of 1.1%). Sometimes this indicates an issue with a sample and an unexplained age offset, making this sample and date suspect. We thus excluded it on this ground—the date was also an outlier at ~ 20% probability. The remaining time series contains 72 dates and 39 elements. The OxCal runfile is in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>. The dataset does not provide a good visual fit with the calibration curve (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>a)—many data are placed below or away from the calibration curve—failing an overall χ<sup>2</sup> test (T = 65.4 > 52.6 df38 at 5%) and delivering poor OxCal Agreement indices (A<sub>comb</sub> = 10.3 < A<sub>n</sub> = 11.3%, A<sub>model</sub> and A<sub>overall</sub> ≤ 10, well below the satisfactory value of 60). It appears likely there is a systematic offset between the data measured and the calibration curve. To investigate we used the Delta_R (ΔR) function in OxCal<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. This allows investigation of whether a data set exhibits a systematic shift relative to the calibration curve. We employed a neutral prior ΔR value of 0 ± 10 <sup>14</sup>C years. For a number of model runs Convergence values are poor (< 95). The reason is that the ΔR model in these cases produces a bi-modal result. The possible offsets are on average (usually more likely) ~ 22 ± 5 <sup>14</sup>C years or the very different − 32 ± 8 <sup>14</sup>C years. Only in some runs did the model converge successfully (all elements with Convergence, C, values ≥ 95) and in these cases usually a single ΔR range of ~ 22 ± 4 <sup>14</sup>C years was found and occasionally the alternative -32 ± 8 <sup>14</sup>C years range (substantially increasing the kIterations value, and so run time, usually resolved the low C values, but retained the ambiguity). The ΔR posterior densities from ten example runs (six bi-modal, three with about a 22 <sup>14</sup>C years offset, and one with a − 32 <sup>14</sup>C years offset) are illustrated in Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>. It is evident there is an offset. We tried models with a ΔR of 22 ± 5 <sup>14</sup>C years, which appears the likely solution based on the model runs for Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>. We also tried runs with the alternative ΔR − 32 ± 8 <sup>14</sup>C years. The ΔR of 22 ± 5 <sup>14</sup>C years yields a satisfactory visual solution (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b). Without consideration of any further outliers, the OxCal diagnostic values, A<sub>model</sub> and A<sub>overall</sub> are typically ≥ 60 (~ 60 and ~ 65 respectively). At this point there is then one major outlier date, OxA-30908, with an outlier probability of ~ 64% (no other outlier probability is above ~ 25/26%, and in all only 7 values are ≥ 10% from multiple runs). If we exclude OxA-30908 and re-run the model, the placement is identical and the A<sub>model</sub> and A<sub>overall</sub> values exceed the satisfactory threshold value of 60 at ~ 76 and ~ 80. Thus we use the fit and placement shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b. The ΔR posterior density offers good agreement with the prior of 22 ± 5 <sup>14</sup>C years (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>c). In particular, the set of ACM values offer a good and specific fit around the wiggle in the calibration curve ~ 1,850 to 1,810 BCE (contrary the notably poor fit in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>a with the earlier placement). In contrast, model runs with the alternative (earlier) fit with a ΔR of − 32 ± 8 <sup>14</sup>C years achieve unsatisfactory OxCal A<sub>model</sub> and A<sub>overall</sub> values, all < 30, well below the satisfactory threshold value of 60. The ΔR posterior density also offers poor OxCal agreement values (< 60) with the prior. The visual fit is poor with most data not matching the calibration curve, and instead placed below the curve (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>). Thus we exclude this fit range as viable. (We note that the older alternative option, about 81 calendar years earlier than the fit shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b, is in fact likely too early to correspond with the High Mesopotamian Chronology<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>, which is only ~ 56 years earlier than the Middle Chronology. Even at the limits of 68.3% hpd and 95.4% hpd, the difference is at least 72 and 62 calendar years respectively, leaving any correspondence as unlikely. Moreover, regardless, the older solution is clearly unlikely on the basis of the <sup>14</sup>C wiggle-match data just discussed. This instead offers a good correspondence only with the High Middle Chronology or Low Middle Chronology, see text and Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>.)</p><p id=\"Par24\">The Egyptian NK models are used as published<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>. The wiggle-match calendar placement of the Hd GOR time series<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup> uses the placement with satisfactory OxCal agreement indices after removing the 13 or 14 largest outliers (SSimple outlier model<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> applied to individual dates, dates in weighted averages, and the weighted averages<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>). The minimum almost satisfactory case removes 13 individual outliers and achieves Amodel ~ 58 and Aoverall ~ 61, while removing 14 individual outliers achieves Amodel ~ 72 and Aoverall ~ 74 (dates removed are indicated in the OxCal runfile in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>). This places the last year of the chronology RY1,764, with bark (felling date) ~ 748BCE (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>d). This fit is 2 years later than the OxCal best fit using all data (against IntCal20 or IntCal04<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>) but with poor OxCal agreement indices. The OxCal wiggle-match of the AA GOR dataset uses IntCal20 with no outlier model following the publication<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup> (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S6</xref>). The χ<sup>2</sup> least squares and χ<sup>2</sup> fitting of the AA GOR data uses published methodologies<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref>,<xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup> (see Supplementary Discussion 2, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S7</xref>).</p><p id=\"Par25\">Comparisons of <sup>14</sup>C datasets were made using the quoted data, or via 1-year linear interpolations of the multi-year Hd GOR and GeO datasets (e.g. Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a,c, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S6</xref>). Weighted average<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup> comparisons are cited for the relevant pairs of data, 10-year block mid-points were rounded by 0.5 years.</p><p id=\"Par26\">The Miletos and Noceto wiggle-match data were used as published<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>. For details on the Thera/Santorini case and the data analysis, see Supplementary Discussion 3 and Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>. Since it has been suggested in the past that <sup>14</sup>C dates on samples from Thera could have been affected by volcanic CO<sub>2</sub> (despite no positive evidence as regards any archaeological sample)<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR51\">51</xref>,<xref ref-type=\"bibr\" rid=\"CR53\">53</xref>,<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>, we also consider models excluding all <sup>14</sup>C data from Thera (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S15</xref>). These offer similar but slightly less constrained results.</p><p id=\"Par27\">The OxCal CQL2 runfiles, with annotations indicating outliers not used and some other details, are provided in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>. It should be noted that each run of such Bayesian models is different and small variations occur. In well-constrained data sets where there is a single best fit location or Sequence solution, these tend to be small and in the range of, e.g., 0–2 years. It is important to observe that—except where noted (6 cases in Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>)—we only employed data where the model run achieved satisfactory Convergence, C, values ≥ 95. We report typical examples from multiple model runs.</p></sec><sec id=\"Sec13\"><title>Historical and archaeological associations</title><p id=\"Par28\">The archaeological associations between the contexts of the MBA tree ring time series and the sites of Kültepe and Acemhöyük are as previously outlined<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. The construction of the Egyptian NK model and the historical priors included are as published<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>. The Amarna model is explained in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>. The Aegean model, revising a previous model<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>, is explained in Supplementary Discussion 3 and in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec14\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_69287_MOESM1_ESM.docx\"><caption><p>Supplementary information.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-69287-2.</p></sec><ack><title>Acknowledgements</title><p>Funding support for this work was received from the National Science Foundation, Award BCS 1219315 (awarded to S.W.M.); the Social Science and Humanities Research Council, Canada, via the CRANE project, University of Toronto, Award 895-2011-1026, sub-contract to Cornell University (awarded to S.W.M.); and the College of Arts & Sciences and the Department of Classics, Cornell University. M.W.D.’s research is supported by an ERC Grant (714679, ECHOES). We thank Cynthia Kocik for dendrochronological work on the ACM samples. Thanks to Fikri Kulakoğlu for hospitality and collaboration at Kültepe; and thanks also to Pinar Ertepinar and Nuretdin Kaymakci.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>S.W.M. designed the study; L.W. carried out the new <sup>14</sup>C dating; W.T. and U.B. organized and supplied the Erstein tree ring samples; B.L. and S.W.M. worked on Anatolian tree ring samples; C.B.R., M.W.D. and B.K. carried out previous <sup>14</sup>C dating; S.W.M. drafted the manuscript with input from all authors.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>All data generated or analyzed during this study are included in this published article (and its Supplementary Information files), or are previously published. The newly published raw <sup>14</sup>C determinations are in Supplementary Tables <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>. All other <sup>14</sup>C dates have previously been published and are available from the relevant publications<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR37\">37</xref>–<xref ref-type=\"bibr\" rid=\"CR40\">40</xref>,<xref ref-type=\"bibr\" rid=\"CR43\">43</xref>,<xref ref-type=\"bibr\" rid=\"CR50\">50</xref>,<xref ref-type=\"bibr\" rid=\"CR51\">51</xref>,<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup> (and see Supplementary Discussion 3 and Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>). The IntCal20 dataset<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup> is available from <ext-link ext-link-type=\"uri\" xlink:href=\"https://intcal.org/\">https://intcal.org/</ext-link>.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par29\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><mixed-citation publication-type=\"other\">Reimer, P. J. <italic>et al.</italic> The IntCal20 Northern Hemisphere radiocarbon age calibration curve (0–55 kcal BP). <italic>Radiocarbon</italic><bold>62</bold>, 10.1017/RDC.2020.41 (2020).</mixed-citation></ref><ref id=\"CR2\"><label>2.</label><mixed-citation publication-type=\"other\">van der Plicht, J., Bronk Ramsey, C., Heaton, T. J., Scott, E. M. & Talamo, S. Recent developments in calibration for archaeological and environmental samples. <italic>Radiocarbon</italic><bold>62</bold>, 10.1017/RDC.2020.22 (2020).</mixed-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Braziunas</surname><given-names>TF</given-names></name><name><surname>Fung</surname><given-names>IY</given-names></name><name><surname>Stuiver</surname><given-names>M</given-names></name></person-group><article-title>The preindustrial atmospheric <sup>14</sup>CO<sub>2</sub> latitudinal gradient as related to exchanges among atmospheric, oceanic, and terrestrial reservoirs</article-title><source>Glob. Biogeochem. Cycles</source><year>1995</year><volume>9</volume><fpage>565</fpage><lpage>584</lpage></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Büntgen</surname><given-names>U</given-names></name><etal/></person-group><article-title>Tree rings reveal globally coherent signature of cosmogenic radiocarbon events in 774 and 993 CE</article-title><source>Nat. Commun.</source><year>2018</year><volume>9</volume><fpage>3605</fpage><pub-id pub-id-type=\"pmid\">30190505</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kromer</surname><given-names>B</given-names></name></person-group><article-title>Regional <sup>14</sup>CO<sub>2</sub> offsets in the troposphere: Magnitude, mechanisms, and consequences</article-title><source>Science</source><year>2001</year><volume>294</volume><fpage>2529</fpage><lpage>2532</lpage><pub-id pub-id-type=\"pmid\">11743160</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dee</surname><given-names>MW</given-names></name><etal/></person-group><article-title>Investigating the likelihood of a reservoir offset in the radiocarbon record for ancient Egypt</article-title><source>J. Archaeol. Sci.</source><year>2010</year><volume>37</volume><fpage>687</fpage><lpage>693</lpage></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bronk</surname><given-names>RC</given-names></name></person-group><article-title>Radiocarbon-based chronology for dynastic Egypt</article-title><source>Science</source><year>2010</year><volume>328</volume><fpage>1554</fpage><lpage>1557</lpage><pub-id pub-id-type=\"pmid\">20558717</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manning</surname><given-names>SW</given-names></name><etal/></person-group><article-title>Fluctuating radiocarbon offsets observed in the southern Levant and implications for archaeological chronology debates</article-title><source>Proc. Natl Acad. Sci. USA</source><year>2018</year><volume>115</volume><fpage>6141</fpage><lpage>6146</lpage><pub-id pub-id-type=\"pmid\">29844183</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McDonald</surname><given-names>L</given-names></name><name><surname>Chivall</surname><given-names>D</given-names></name><name><surname>Miles</surname><given-names>D</given-names></name><name><surname>Bronk Ramsey</surname><given-names>C</given-names></name></person-group><article-title>Seasonal variations in the <sup>14</sup>C content of tree rings: Influences on radiocarbon calibration and single-year curve construction</article-title><source>Radiocarbon</source><year>2019</year><volume>61</volume><fpage>185</fpage><lpage>194</lpage></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manning</surname><given-names>SW</given-names></name><etal/></person-group><article-title>Mediterranean radiocarbon offsets and calendar dates for prehistory</article-title><source>Sci. Adv.</source><year>2020</year><volume>6</volume><fpage>eaaz1096</fpage><pub-id pub-id-type=\"pmid\">32206721</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stuiver</surname><given-names>M</given-names></name><name><surname>Braziunas</surname><given-names>TF</given-names></name></person-group><article-title>1998 GRL anthropogenic and solar components of hemispheric <sup>14</sup>C</article-title><source>Geophys. Res. Lett.</source><year>1998</year><volume>25</volume><fpage>329</fpage><lpage>332</lpage></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Bietak</surname><given-names>M</given-names></name></person-group><source>The Synchronisation of Civilisations in the Eastern Mediterranean in the Second Millennium BC</source><year>2000</year><publisher-loc>Vienna</publisher-loc><publisher-name>Verlag der Österreichischen Akademie der Wissenschaften</publisher-name></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manning</surname><given-names>SW</given-names></name><etal/></person-group><article-title>High-precision dendro-<sup>14</sup>C dating of two cedar wood sequences from First Intermediate Period and Middle Kingdom Egypt and a small regional climate-related <sup>14</sup>C divergence</article-title><source>J. Archaeol. Sci.</source><year>2014</year><volume>46</volume><fpage>401</fpage><lpage>416</lpage></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Levin</surname><given-names>I</given-names></name><etal/></person-group><article-title>Observations and modelling of the global distribution and long-term trend of atmospheric <sup>14</sup>CO<sub>2</sub></article-title><source>Tellus B</source><year>2010</year><volume>62</volume><fpage>26</fpage><lpage>46</lpage></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Flexas</surname><given-names>J</given-names></name><etal/></person-group><article-title>Photosynthetic limitations in Mediterranean plants: A review</article-title><source>Environ. Exp. Bot.</source><year>2014</year><volume>103</volume><fpage>12</fpage><lpage>23</lpage></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McCormac</surname><given-names>FG</given-names></name><name><surname>Baillie</surname><given-names>MGL</given-names></name><name><surname>Pilcher</surname><given-names>JR</given-names></name><name><surname>Kalin</surname><given-names>RM</given-names></name></person-group><article-title>Location-dependent differences in the <sup>14</sup>C content of wood</article-title><source>Radiocarbon</source><year>1995</year><volume>37</volume><fpage>395</fpage><lpage>407</lpage></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Libby</surname><given-names>WF</given-names></name></person-group><article-title>Radiocarbon dating</article-title><source>Science</source><year>1961</year><volume>133</volume><fpage>621</fpage><lpage>629</lpage><pub-id pub-id-type=\"pmid\">13761865</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manning</surname><given-names>SW</given-names></name><etal/></person-group><article-title>Integrated tree-ring-radiocarbon high-resolution timeframe to resolve earlier second millennium BCE Mesopotamian chronology</article-title><source>PLoS One</source><year>2016</year><volume>11</volume><issue>7</issue><fpage>e0157144</fpage><pub-id pub-id-type=\"pmid\">27409585</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Barjamovic</surname><given-names>G</given-names></name><name><surname>Hertel</surname><given-names>T</given-names></name><name><surname>Larsen</surname><given-names>MT</given-names></name></person-group><source>Ups and Downs at Kanesh-Observations on Chronology, History and Society in the Old Assyrian period</source><year>2012</year><publisher-loc>Leiden</publisher-loc><publisher-name>Nederlands Instituut voor het Nabije Oosten</publisher-name></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Pruzsinszky</surname><given-names>R</given-names></name></person-group><source>Mesopotamian Chronology of the 2nd Millennium BC. An introduction to the Textual Evidence and Related Chronological Issues</source><year>2009</year><publisher-loc>Vienna</publisher-loc><publisher-name>Verlag der Österreichischen Akademie der Wissenschaften</publisher-name></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manning</surname><given-names>SW</given-names></name><name><surname>Barjamovic</surname><given-names>G</given-names></name><name><surname>Lorentzen</surname><given-names>B</given-names></name></person-group><article-title>The course of <sup>14</sup>C dating does not run smooth: Tree-rings, radiocarbon, and potential impacts of a calibration curve wiggle on dating Mesopotamian chronology</article-title><source>J. Ancient Egypt. Interconnect.</source><year>2017</year><volume>13</volume><fpage>70</fpage><lpage>81</lpage></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bronk Ramsey</surname><given-names>C</given-names></name><name><surname>van der Plicht</surname><given-names>J</given-names></name><name><surname>Weninger</surname><given-names>B</given-names></name></person-group><article-title>‘Wiggle matching’ radiocarbon dates</article-title><source>Radiocarbon</source><year>2001</year><volume>43</volume><fpage>381</fpage><lpage>389</lpage></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bronk Ramsey</surname><given-names>C</given-names></name></person-group><article-title>Bayesian analysis of radiocarbon dates</article-title><source>Radiocarbon</source><year>2009</year><volume>51</volume><fpage>337</fpage><lpage>360</lpage></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bronk Ramsey</surname><given-names>C</given-names></name></person-group><article-title>Dealing with outliers and offsets in radiocarbon dating</article-title><source>Radiocarbon</source><year>2009</year><volume>51</volume><fpage>1023</fpage><lpage>1045</lpage></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Croutsch</surname><given-names>C</given-names></name><name><surname>Tegel</surname><given-names>W</given-names></name><name><surname>Rault</surname><given-names>E</given-names></name></person-group><article-title>Les puits de l’âge du Bronze du Parc d’Activités du Pays d’Erstein (Bas-Rhin, Alsace): Des analyses dendroarchéologiques à l’étude de l’occupation du sol</article-title><source>Bull. Soc. Préhistor. Franç.</source><year>2019</year><volume>116</volume><fpage>743</fpage><lpage>774</lpage></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Croutsch</surname><given-names>C</given-names></name><name><surname>Tegel</surname><given-names>W</given-names></name><name><surname>Rault</surname><given-names>E</given-names></name><name><surname>Pierrevelcin</surname><given-names>G</given-names></name></person-group><person-group person-group-type=\"editor\"><name><surname>Montoya</surname><given-names>C</given-names></name><name><surname>Fagnart</surname><given-names>J-L</given-names></name><name><surname>Locht</surname><given-names>J-L</given-names></name></person-group><article-title>Des sites de la fin du Néolithique et de l’âge du Bronze ancien: Nouvelles données sur la chronologie absolue en Alsace (2500–1450 av.J.-C.)</article-title><source>Préhistoire de l'Europe du Nord-Ouest: Mobilité, Climats et Identités Culturelles, Volume 3: Néolithique—Âge du Bronze</source><year>2019</year><publisher-loc>Paris</publisher-loc><publisher-name>Société Préhistorique Française</publisher-name><fpage>475</fpage><lpage>481</lpage></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reimer</surname><given-names>PJ</given-names></name><etal/></person-group><article-title>IntCal13 and Marine13 radiocarbon age calibration curves 0–50,000 years Cal BP</article-title><source>Radiocarbon</source><year>2013</year><volume>55</volume><fpage>1869</fpage><lpage>1887</lpage></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Roaf</surname><given-names>M</given-names></name></person-group><article-title>The Fall of Babylon in 1499 NC or 1595 MC</article-title><source>Akkadica</source><year>2012</year><volume>133</volume><fpage>147</fpage><lpage>174</lpage></element-citation></ref><ref id=\"CR29\"><label>29.</label><mixed-citation publication-type=\"other\">de Jong, T. Astronomical fine-tuning of the chronology of the Hammurabi age. <italic>Jaarbericht van het Vooraziatisch-Egyptisch Genootschap “Ex Oriente Lux”</italic><bold>44</bold>, 147–167 (2013).</mixed-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nahm</surname><given-names>W</given-names></name></person-group><article-title>The case of the lower middle chronology</article-title><source>Altorient. Forsch.</source><year>2013</year><volume>40</volume><fpage>350</fpage><lpage>372</lpage></element-citation></ref><ref id=\"CR31\"><label>31.</label><mixed-citation publication-type=\"other\">de Jong, T. Further astronomical fine-tuning of the old Assyrian and Old Babylonian chronologies. <italic>Jaarbericht van het Vooraziatisch-Egyptisch Genootschap “Ex Oriente Lux”</italic><bold>46</bold>, 127–143 (2016–2017).</mixed-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reimer</surname><given-names>PJ</given-names></name><etal/></person-group><article-title>IntCal04 terrestrial radiocarbon age calibration, 0–26 Cal KYR BP</article-title><source>Radiocarbon</source><year>2004</year><volume>46</volume><fpage>1029</fpage><lpage>1058</lpage></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Manning</surname><given-names>SW</given-names></name></person-group><source>A Test of Time and A Test of Time Revisited. The Volcano of Thera and the Chronology and History of the Aegean and East Mediterranean in the Mid-second Millennium BC</source><year>2014</year><publisher-loc>Oxford</publisher-loc><publisher-name>Oxbow Books</publisher-name></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aston</surname><given-names>D</given-names></name></person-group><article-title>Radiocarbon, Wine Jars and New Kingdom chronology</article-title><source>Ägypt. Levante</source><year>2013</year><volume>22–23</volume><fpage>289</fpage><lpage>315</lpage></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schneider</surname><given-names>T</given-names></name></person-group><article-title>Contributions to the chronology of the New Kingdom and the Third Intermediate Period</article-title><source>Ägypt. Levante</source><year>2010</year><volume>20</volume><fpage>373</fpage><lpage>403</lpage></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Aston</surname><given-names>DA</given-names></name></person-group><person-group person-group-type=\"editor\"><name><surname>Forstner-Müller</surname><given-names>I</given-names></name><name><surname>Moeller</surname><given-names>N</given-names></name></person-group><article-title>How early (and how late) Can Khyan really be: An essay based on “conventional archaeological methods”</article-title><source>The Hyksos Ruler Khyan and the Early Second Intermediate Period in Egypt: Problems and Priorities of Current Research</source><year>2018</year><publisher-loc>Vienna</publisher-loc><publisher-name>Österreichische Archäologisches Institut</publisher-name><fpage>15</fpage><lpage>56</lpage></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manning</surname><given-names>SW</given-names></name><etal/></person-group><article-title><sup>14</sup>C Record and wiggle-match placement for the Anatolian (Gordion Area) Juniper Tree-Ring Chronology ~1729 to 751 Cal BC, and typical Aegean/Anatolian (growing season related) regional <sup>14</sup>C offset assessment</article-title><source>Radiocarbon</source><year>2010</year><volume>52</volume><fpage>1571</fpage><lpage>1597</lpage></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pearson</surname><given-names>C</given-names></name><etal/></person-group><article-title>Securing timelines in the ancient Mediterranean using multiproxy annual tree-ring data</article-title><source>Proc. Natl. Acad. Sci. U.S.A.</source><year>2020</year><volume>117</volume><fpage>8410</fpage><lpage>8415</lpage><pub-id pub-id-type=\"pmid\">32229554</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><mixed-citation publication-type=\"other\">Pearson, C. <italic>et al.</italic> Annual variation in atmospheric <sup>14</sup>C between 1700 BC and 1480 BC. <italic>Radiocarbon</italic><bold>62</bold>, 10.1017/RDC.2020.14 (2020).</mixed-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pearson</surname><given-names>GW</given-names></name></person-group><article-title>Precise calendrical dating of known growth-period samples using a ‘curve fitting’ technique</article-title><source>Radiocarbon</source><year>1986</year><volume>28</volume><fpage>292</fpage><lpage>299</lpage></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wacker</surname><given-names>L</given-names></name><name><surname>Güttler</surname><given-names>D</given-names></name><name><surname>Goll</surname><given-names>J</given-names></name><name><surname>Hurni</surname><given-names>JP</given-names></name><name><surname>Synal</surname><given-names>H-A</given-names></name><name><surname>Walti</surname><given-names>N</given-names></name></person-group><article-title>Radiocarbon dating to a single year by means of rapid atmospheric <sup>14</sup>C changes</article-title><source>Radiocarbon</source><year>2014</year><volume>56</volume><fpage>573</fpage><lpage>579</lpage></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ward</surname><given-names>GK</given-names></name><name><surname>Wilson</surname><given-names>SR</given-names></name></person-group><article-title>Procedures for comparing and combining radiocarbon age determinations: A critique</article-title><source>Archaeometry</source><year>1978</year><volume>20</volume><fpage>19</fpage><lpage>31</lpage></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pearson</surname><given-names>CL</given-names></name><etal/></person-group><article-title>Annual radiocarbon record indicates 16th century BCE date for the Thera eruption</article-title><source>Sci. Adv.</source><year>2018</year><volume>4</volume><fpage>eaar8241</fpage><pub-id pub-id-type=\"pmid\">30116779</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Güttler</surname><given-names>D</given-names></name><name><surname>Wacker</surname><given-names>L</given-names></name><name><surname>Kromer</surname><given-names>B</given-names></name><name><surname>Friedrich</surname><given-names>M</given-names></name><name><surname>Synal</surname><given-names>H-A</given-names></name></person-group><article-title>Evidence of 11-year solar cycles in tree rings from 1010 to 1110 AD—progress on high precision AMS measurements</article-title><source>Nucl. Instrum. Methods Phys. Res. B</source><year>2013</year><volume>294</volume><fpage>459</fpage><lpage>463</lpage></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hogg</surname><given-names>A</given-names></name><etal/></person-group><article-title>The New Zealand Kauri (<italic>Agathis australis</italic>) research project: A radiocarbon dating intercomparison of Younger Dryas Wood and implications for IntCal13</article-title><source>Radiocarbon</source><year>2013</year><volume>55</volume><fpage>2035</fpage><lpage>2048</lpage></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Friedrich</surname><given-names>R</given-names></name><etal/></person-group><article-title>Annual <sup>14</sup>C tree-ring data around 400 AD: Mid- and high-latitude records</article-title><source>Radiocarbon</source><year>2019</year><volume>61</volume><fpage>1305</fpage><lpage>1316</lpage></element-citation></ref><ref id=\"CR47\"><label>47.</label><mixed-citation publication-type=\"other\">Kuitems, M., van der Plicht, J. & Jansma, E. Wood from The Netherlands around the time of the Santorini eruption dated by dendrochronology and radiocarbon. <italic>Radiocarbon</italic><bold>62</bold>, 10.1017/RDC.2020.23 (2020).</mixed-citation></ref><ref id=\"CR48\"><label>48.</label><mixed-citation publication-type=\"other\">Friedrich, R. <italic>et al.</italic> A new annual <sup>14</sup>C dataset for calibrating the Thera eruption. <italic>Radiocarbon</italic><bold>62</bold>, 10.1017/RDC.2020.33 (2020).</mixed-citation></ref><ref id=\"CR49\"><label>49.</label><mixed-citation publication-type=\"other\">Fahrni, S. <italic>et al.</italic> Single-year German oak and Californian bristlecone pine <sup>14</sup>C data at the beginning of the Hallstatt Plateau from 865 BC to 626 BC. <italic>Radiocarbon </italic><bold>62</bold>, 10.1017/RDC.2020.16 (2020).</mixed-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Manning</surname><given-names>SW</given-names></name><name><surname>Kromer</surname><given-names>B</given-names></name><name><surname>Dee</surname><given-names>MW</given-names></name><name><surname>Friedrich</surname><given-names>M</given-names></name><name><surname>Higham</surname><given-names>TFG</given-names></name><name><surname>Bronk Ramsey</surname><given-names>C</given-names></name></person-group><person-group person-group-type=\"editor\"><name><surname>Shortland</surname><given-names>AJ</given-names></name><name><surname>Bronk Ramsey</surname><given-names>C</given-names></name></person-group><article-title>Radiocarbon calibration in the mid to later 14th century BC and radiocarbon dating Tell el-Amarna, Egypt</article-title><source>Radiocarbon and the Chronologies of Ancient Egypt</source><year>2013</year><publisher-loc>Oxford</publisher-loc><publisher-name>Oxbow Books</publisher-name><fpage>121</fpage><lpage>145</lpage></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manning</surname><given-names>SW</given-names></name><etal/></person-group><article-title>Chronology for the Aegean Late Bronze Age</article-title><source>Science</source><year>2006</year><volume>312</volume><fpage>565</fpage><lpage>569</lpage><pub-id pub-id-type=\"pmid\">16645092</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Höflmayer</surname><given-names>F</given-names></name></person-group><article-title>The date of the Minoan Santorini eruption: Quantifying the “offset”</article-title><source>Radiocarbon</source><year>2012</year><volume>54</volume><fpage>435</fpage><lpage>448</lpage></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manning</surname><given-names>SW</given-names></name><etal/></person-group><article-title>Dating the Thera (Santorini) eruption: Archaeological and scientific evidence supporting a high chronology</article-title><source>Antiquity</source><year>2014</year><volume>88</volume><fpage>1164</fpage><lpage>1179</lpage></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cherubini</surname><given-names>P</given-names></name><etal/></person-group><article-title>Olive tree-ring problematic dating: A comparative analysis on Santorini (Greece)</article-title><source>PLoS One</source><year>2013</year><volume>8</volume><issue>1</issue><fpage>e54730</fpage><pub-id pub-id-type=\"pmid\">23382949</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Friedrich</surname><given-names>WL</given-names></name><etal/></person-group><article-title>Santorini eruption radiocarbon dated to 1627–1600 BC</article-title><source>Science</source><year>2006</year><volume>312</volume><fpage>548</fpage><pub-id pub-id-type=\"pmid\">16645088</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ritner</surname><given-names>RK</given-names></name><name><surname>Moeller</surname><given-names>N</given-names></name></person-group><article-title>The Ahmose ‘Tempest Stela’, Thera and comparative chronology</article-title><source>J. Near Eastern Stud.</source><year>2014</year><volume>73</volume><fpage>1</fpage><lpage>19</lpage></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bruins</surname><given-names>HJ</given-names></name><name><surname>van der Plicht</surname><given-names>J</given-names></name><name><surname>Mazar</surname><given-names>A</given-names></name></person-group><article-title><sup>14</sup>C dates from Tel Rehov: Iron-Age chronology, pharaohs, and Hebrew kings</article-title><source>Science</source><year>2003</year><volume>300</volume><fpage>315</fpage><lpage>318</lpage><pub-id pub-id-type=\"pmid\">12690196</pub-id></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Weiss</surname><given-names>H</given-names></name><etal/></person-group><person-group person-group-type=\"editor\"><name><surname>Weiss</surname><given-names>H</given-names></name><etal/></person-group><article-title>Tell Leilan Akkadian imperialization, collapse and short-lived reoccupation defined by high-resolution radiocarbon dating</article-title><source>Seven Generations Since the Fall of Akkad, Studia Chaburensia 3</source><year>2012</year><publisher-loc>Wiesbaden</publisher-loc><publisher-name>Harrassowitz Verlag</publisher-name><fpage>163</fpage><lpage>192</lpage></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Regev</surname><given-names>J</given-names></name><etal/></person-group><article-title>Chronology of the Early Bronze Age in the Southern Levant: New analysis for a high chronology</article-title><source>Radiocarbon</source><year>2012</year><volume>54</volume><fpage>525</fpage><lpage>566</lpage></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kutschera</surname><given-names>W</given-names></name><etal/></person-group><article-title>The chronology of Tell el-Dab‘a: A crucial meeting point of <sup>14</sup>C dating, archaeology, and Egyptology in the 2nd millennium BC</article-title><source>Radiocarbon</source><year>2012</year><volume>54</volume><fpage>407</fpage><lpage>422</lpage></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Toffolo</surname><given-names>MB</given-names></name><etal/></person-group><article-title>Towards an absolute chronology for the Aegean Iron Age: New radiocarbon dates from Lefkandi, Kalapodi and Corinth</article-title><source>PLoS One</source><year>2013</year><volume>8</volume><issue>12</issue><fpage>e83117</fpage><pub-id pub-id-type=\"pmid\">24386150</pub-id></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Toffolo</surname><given-names>MB</given-names></name><name><surname>Arie</surname><given-names>E</given-names></name><name><surname>Martin</surname><given-names>MAS</given-names></name><name><surname>Boaretto</surname><given-names>E</given-names></name><name><surname>Finkelstein</surname><given-names>I</given-names></name></person-group><article-title>Absolute chronology of Megiddo, Israel, in the Late Bronze Age and Iron Ages: High-resolution radiocarbon dating</article-title><source>Radiocarbon</source><year>2014</year><volume>56</volume><fpage>221</fpage><lpage>244</lpage></element-citation></ref><ref id=\"CR63\"><label>63.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Höflmayer</surname><given-names>F</given-names></name><etal/></person-group><article-title>New evidence for Middle Bronze Age chronology and synchronisms in the levant: Radiocarbon dates from Tell el-Burak, Tell el-Dab‘a, and Tel Ifshar Compared</article-title><source>Bull. Am. Sch. Orient. Res.</source><year>2016</year><volume>375</volume><fpage>53</fpage><lpage>76</lpage></element-citation></ref><ref id=\"CR64\"><label>64.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Kuniholm</surname><given-names>PI</given-names></name><name><surname>Newton</surname><given-names>MW</given-names></name><name><surname>Liebhart</surname><given-names>RF</given-names></name></person-group><person-group person-group-type=\"editor\"><name><surname>Rose</surname><given-names>CB</given-names></name><name><surname>Darbyshire</surname><given-names>G</given-names></name></person-group><article-title>Dendrochronology at Gordion</article-title><source>The New Chronology of Iron Age Gordion</source><year>2011</year><publisher-loc>Philadelphia</publisher-loc><publisher-name>University of Pennsylvania Museum of Archaeology and Anthropology</publisher-name><fpage>79</fpage><lpage>122</lpage></element-citation></ref><ref id=\"CR65\"><label>65.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Němec</surname><given-names>N</given-names></name><name><surname>Wacker</surname><given-names>L</given-names></name><name><surname>Hajdas</surname><given-names>I</given-names></name><name><surname>Gäggeler</surname><given-names>H</given-names></name></person-group><article-title>Alternative methods for cellulose preparation for AMS measurement</article-title><source>Radiocarbon</source><year>2010</year><volume>52</volume><fpage>1358</fpage><lpage>1370</lpage></element-citation></ref><ref id=\"CR66\"><label>66.</label><mixed-citation publication-type=\"other\">Sookdeo, A. <italic>et al.</italic> Quality dating: A well-defined protocol implemented at ETH for high-precision<sup>14</sup>C-dates tested on late glacial wood. <italic>Radiocarbon</italic><bold>62</bold>, 10.1017/rdc.2019.132 (2020).</mixed-citation></ref><ref id=\"CR67\"><label>67.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McAneney</surname><given-names>J</given-names></name><name><surname>Baillie</surname><given-names>M</given-names></name></person-group><article-title>Absolute tree-ring dates for the Late Bronze Age eruptions of Aniakchak and Thera in light of a proposed revision of ice-core chronologies</article-title><source>Antiquity</source><year>2019</year><volume>93</volume><fpage>99</fpage><lpage>112</lpage></element-citation></ref><ref id=\"CR68\"><label>68.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Salzer</surname><given-names>MW</given-names></name><name><surname>Hughes</surname><given-names>MK</given-names></name></person-group><article-title>Bristlecone pine tree rings and volcanic eruptions over the last 5000 yr</article-title><source>Quatern. Res.</source><year>2007</year><volume>67</volume><fpage>57</fpage><lpage>68</lpage></element-citation></ref><ref id=\"CR69\"><label>69.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Helama</surname><given-names>S</given-names></name><etal/></person-group><article-title>A chronology of climatic downturns through the mid- and late-Holocene: Tracing the distant effects of explosive eruptions from palaeoclimatic and historical evidence in northern Europe</article-title><source>Polar Res.</source><year>2013</year><volume>32</volume><fpage>15866</fpage></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Commun Biol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Commun Biol</journal-id><journal-title-group><journal-title>Communications Biology</journal-title></journal-title-group><issn pub-type=\"epub\">2399-3642</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807870</article-id><article-id pub-id-type=\"pmc\">PMC7431541</article-id><article-id pub-id-type=\"publisher-id\">1183</article-id><article-id pub-id-type=\"doi\">10.1038/s42003-020-01183-x</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Pheromone components affect motivation and induce persistent modulation of associative learning and memory in honey bees</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Baracchi</surname><given-names>David</given-names></name><address><email>david.baracchi@unifi.it</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Cabirol</surname><given-names>Amélie</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Devaud</surname><given-names>Jean-Marc</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Haase</surname><given-names>Albrecht</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>d’Ettorre</surname><given-names>Patrizia</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\" equal-contrib=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-7173-769X</contrib-id><name><surname>Giurfa</surname><given-names>Martin</given-names></name><address><email>martin.giurfa@univ-tlse3.fr</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff7\">7</xref><xref ref-type=\"aff\" rid=\"Aff8\">8</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.11417.32</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2353 1689</institution-id><institution>Research Centre on Animal Cognition, Center for Integrative Biology, CNRS, </institution><institution>University of Toulouse, </institution></institution-wrap>118 route de Narbonne, F-31062 Toulouse, Cedex 09 France </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.8404.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1757 2304</institution-id><institution>Department of Biology, </institution><institution>University of Florence, </institution></institution-wrap>Via Madonna del Piano, 6, 50019 Sesto Fiorentino, Italy </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.11696.39</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1937 0351</institution-id><institution>Center for Mind/Brain Sciences (CIMeC), </institution><institution>University of Trento, </institution></institution-wrap>Piazza Manifattura 1, I-38068 Rovereto, Italy </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.11696.39</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1937 0351</institution-id><institution>Department of Physics, </institution><institution>University of Trento, </institution></institution-wrap>Via Sommarive 14, I-38123 Povo, Italy </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.11318.3a</institution-id><institution-id institution-id-type=\"ISNI\">0000000121496883</institution-id><institution>Laboratory of Experimental and Comparative Ethology, </institution><institution>University of Paris 13, </institution></institution-wrap>F-93430 Sorbonne Paris Cité, France </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.440891.0</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 1931 4817</institution-id><institution>Institut Universitaire de France (IUF), </institution></institution-wrap>Paris, France </aff><aff id=\"Aff7\"><label>7</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.440891.0</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 1931 4817</institution-id><institution>Institut Universitaire de France (IUF), </institution></institution-wrap>Toulouse, France </aff><aff id=\"Aff8\"><label>8</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.256111.0</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1760 2876</institution-id><institution>College of Animal Science (College of Bee Science), </institution><institution>Fujian Agriculture and Forestry University, </institution></institution-wrap>Fuzhou, 350002 China </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>3</volume><elocation-id>447</elocation-id><history><date date-type=\"received\"><day>11</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>30</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Since their discovery in insects, pheromones are considered as ubiquitous and stereotyped chemical messengers acting in intraspecific animal communication. Here we studied the effect of pheromones in a different context as we investigated their capacity to induce persistent modulations of associative learning and memory. We used honey bees, <italic>Apis mellifera</italic>, and combined olfactory conditioning and pheromone preexposure with disruption of neural activity and two-photon imaging of olfactory brain circuits, to characterize the effect of pheromones on olfactory learning and memory. Geraniol, an attractive pheromone component, and 2-heptanone, an aversive pheromone, improved and impaired, respectively, olfactory learning and memory via a durable modulation of appetitive motivation, which left odor processing unaffected. Consistently, interfering with aminergic circuits mediating appetitive motivation rescued or diminished the cognitive effects induced by pheromone components. We thus show that these chemical messengers act as important modulators of motivational processes and influence thereby animal cognition.</p></abstract><abstract id=\"Abs2\" abstract-type=\"web-summary\"><p id=\"Par2\">David Baracchi et al. show that honey bees exposed to geraniol, a component of an attractant pheromone, exhibit improved olfactory learning and memory while the opposite effect was seen in honey bees exposed to the aversive pheromone, 2- heptanone. These changes occurred through a modulation of appetitive motivation without affecting odor processing, suggesting an important role for pheromones as modulators of motivational and learning processes.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Learning and memory</kwd><kwd>Animal behaviour</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100001665</institution-id><institution>Agence Nationale de la Recherche (French National Research Agency)</institution></institution-wrap></funding-source><award-id>ANR-14-CE18-0003</award-id><award-id>ANR-14-CE18-0003</award-id><principal-award-recipient><name><surname>d’Ettorre</surname><given-names>Patrizia</given-names></name><name><surname>Giurfa</surname><given-names>Martin</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100004795</institution-id><institution>Institut Universitaire de France (IUF)</institution></institution-wrap></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par3\">Pheromones are chemical signals released by a sender into the environment to convey specific messages to members of the same species, in which they release stereotyped and adaptive responses<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. Since their definition, proposed six decades ago, pheromones have been confined to an intraspecific communication scenario, so that less is known about their possible consequences in contexts different from that of information exchange<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. In particular, if exposure to pheromones affects in a durable way subsequent associative learning and memory remains unknown. Research on rodents<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup> and rabbits<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> has shown that some pheromone components can replace reinforcing stimuli <italic>during</italic> associative conditioning and mediate the learning of odor or contextual cues. In contrast to these works, we asked if <italic>prior</italic> exposure to pheromone components induces subsequent and persistent motivational changes affecting the way in which animals learn and memorize when the pheromone signals are no longer present. Motivation is central to animal and human behavior as it affects decision-making processes, stimulus searching or avoidance, and consequently, what individuals learn and remember<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>–<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. If besides providing specific messages to conspecifics, pheromones change an animal’s motivation according to their message, they will exert important consequences on its capacity to learn and memorize.</p><p id=\"Par4\">A relevant species to address this hypothesis is the domestic honey bee <italic>Apis mellifera</italic>, which represents a pinnacle of sociality among insects<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. Social cohesion and colony efficiency rely largely on pheromones, which signal diverse events in a variety of behavioral contexts<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. Furthermore, individual worker bees exhibit impressive learning and memory abilities<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>, which can be studied in the laboratory using controlled conditioning protocols. One of these protocols is the olfactory conditioning of the proboscis extension reflex (PER), in which harnessed bees learn the association between an odorant (the conditioned stimulus or CS) and a reward of sucrose solution (the unconditioned stimulus or US), so that they exhibit the appetitive PER to the odorant that anticipates the food<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. We took advantage of this protocol to determine if prior pheromone exposure modifies subsequent associative learning and memory when pheromone components were no longer present. To disentangle the effect of these components on memory formation and retrieval, which may engage different processes and neural circuits<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>, we exposed honey bees either before conditioning, thereby affecting ongoing learning and memory formation, or after learning and before a retention test, affecting exclusively the process of retrieval. We studied the impact of two pheromonal signals of different “valence”: 2-heptanone (2H), a deterrent pheromone-signaling aversive situation<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>, and geraniol (GER), the major component of the attractive pheromone of the Nasonov gland<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. We preexposed bees to these pheromone components to determine if they affect subsequent olfactory learning and memory formation. Using in vivo two-photon calcium imaging, we analyzed the impact of this preexposure on odor-evoked neuronal activity in the antennal lobes (ALs), the primary olfactory centers of the insect brain. In parallel, we analyzed how these pheromonal signals affect aminergic circuits mediating appetitive motivation and responsiveness in the bee brain<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>–<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Our results show that GER and 2H do not modify olfactory processing, but induce long-lasting changes in motivation, which affect subsequent learning and memory formation. Thus, besides acting as chemical messengers, pheromonal signals contribute to behavioral plasticity by preparing individuals to learn and memorize according to pheromonal valence.</p></sec><sec id=\"Sec2\" sec-type=\"results\"><title>Results</title><sec id=\"Sec3\"><title>Modulation of olfactory learning and memory by pheromones</title><p id=\"Par5\">We first studied if pheromone components modify learning performances using the olfactory conditioning of the PER<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. We preexposed bees to either Geraniol (GER), an attractive pheromone component<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>, or 2-Heptanone (2H), a deterrent pheromone-signaling aversive event<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>—during 15 min and waited for additional 15 min before starting conditioning (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>). Control bees were preexposed to mineral oil, a standard control solvent in studies on olfaction, which was used to dilute pheromone components. During conditioning and in subsequent tests, an air extractor ensured that no pheromone leftovers remained at the experimental site. The three groups of bees were conditioned to discriminate the floral odors limonene and eugenol in the absence of pheromonal stimulation during ten trials spaced by 12 min. Thus, 123 min elapsed since pheromone-component exposure and the end of conditioning. Conditioning consisted of five rewarded presentations of one of the two odorants with sucrose solution (CS+) and five unrewarded presentations of the other odorant (CS−). Within each group, the role of limonene and eugenol as CS+ and CS− was counterbalanced, and the sequence of odorants was pseudorandomized<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>.<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Pheromone components modulate associative olfactory learning and memory retention in honey bees.</title><p><bold>a</bold>–<bold>c</bold> Pheromone components modulate associative olfactory learning and memory according to their valence. <bold>a</bold> Experimental protocol used. <bold>b</bold> Associative olfactory conditioning of PER in honey bees exposed to geraniol (GER), 2-heptanone (2H), or mineral oil 15 min before training. Proportion of bees showing a conditioned response (PER) to the rewarded (solid lines) and unrewarded (dotted lines) odorants during successive conditioning trials. GER-preexposed bees (<italic>n</italic> = 75 independent bees) performed better than control bees exposed to mineral oil (<italic>n</italic> = 129 independent bees), while 2H preexposed bees (<italic>n</italic> = 73 independent bees) performed worse than controls. (*) <italic>p</italic> < 0.05; (**) <italic>p</italic> < 0.001. <bold>c</bold> Retention tests performed 2, 24, or 72 h post conditioning. Preexposure to GER (blue), 2H (orange), or mineral oil (gray) was performed prior to conditioning. The figure shows the proportions of bees showing a specific memory (i.e., responding to the CS+ and not to the CS−) in the retention tests. The sample size is reported above each bar and refers to independent bees. Retention was better in bees exposed to GER compared with control bees. The opposite was observed in bees exposed to 2H. (*) <italic>p</italic> < 0.05; (**) <italic>p</italic> < 0.01; (***) <italic>p</italic> < 0.001.</p></caption><graphic xlink:href=\"42003_2020_1183_Fig1_HTML\" id=\"d30e565\"/></fig></p><p id=\"Par6\">Bees in all three groups learned to discriminate the CS+ from the CS− (generalized linear mixed model, GLMM, trial: <italic>χ</italic><sup>2</sup> = 70.93, d<italic>f</italic> = 1, <italic>p</italic> < 0.0001) and responded differently to both odorants at the end of training (GLMM, CS: <italic>χ</italic><sup>2</sup> = 44.87, d<italic>f</italic> = 1, <italic>p</italic> < 0.0001) (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref>). Bees exposed to GER performed better than controls, while the opposite occurred in bees exposed to 2H (treatment × CS interaction: <italic>χ</italic><sup>2</sup> = 30.48, d<italic>f</italic> = 2, <italic>p</italic> < 0.0001). Differences were based on responses to the CS+ as GER-exposed bees responded more to the CS+ than control bees (GLMM, Tukey’s post hoc test; CS+: <italic>p</italic> = 0.0007; CS−: <italic>p</italic> = 0.80), while 2H-exposed bees responded less to the CS+ than controls (<italic>p</italic> = 0.018). No differences were found in the case of CS− responses (<italic>p</italic> = 0.98). Thus, preexposure to GER facilitated subsequent appetitive olfactory learning, while preexposure to 2H impaired it. Similar results were obtained upon evaluation of individual acquisition scores (ACQS), calculated as the sum of responses to the five CS+ presentations for every preexposed bee (see Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>).</p><p id=\"Par7\">The same three groups previously preexposed and conditioned were further tested with the CS+ and the CS− 2, 24, and 72 h after conditioning (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>). In this way, besides evaluating how preexosure to pheromonal components affected their learning, we evaluated how it affected memory retention after conditioning. As a robust proxy of memory retention, we quantified the percentage of bees exhibiting specific memory, i.e., responding only to the CS+ and not to the CS−<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Preexposure to pheromone components of different valence induced opposite modulation of specific memory (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1c</xref>, GLMM, treatment: <italic>χ</italic><sup>2</sup> = 27.80, d<italic>f</italic> = 2, <italic>p</italic> < 0.0001). The time elapsed between training and test also affected specific memory (testing time: <italic>χ</italic><sup>2</sup> = 6.03, d<italic>f</italic> = 2, <italic>p</italic> < 0.049). The interaction was not significant (<italic>χ</italic><sup>2</sup> = 6.69, d<italic>f</italic> = 4, <italic>p</italic> = 0.153). Overall, a lower percentage of bees with specific memory was observed after 2H preexposure with respect to controls (Tukey’s post hoc test: <italic>p</italic> < 0.001), while a higher proportion was observed after GER preexposure (Tukey’s post hoc test: <italic>p</italic> < 0.001).</p><p id=\"Par8\">We next asked if preexposure to GER and 2H affects memory retrieval, irrespective of the differences in learning induced by preexposure. We trained unexposed bees to discriminate limonene and eugenol, and selected the bees exhibiting correct discrimination (responding to the CS+ and not to the CS−) in the last conditioning trial. Thus, all bees had the same acquisition level at the end of training. They were then exposed to GER, 2H, or mineral oil during 15 min, prior to the memory tests performed 2, 24, or 72 h after conditioning (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2A</xref>). Different groups of bees were used for each treatment and test. Retrieval performances were similar between groups at any testing time, and irrespective of the pheromone component exposed (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2B</xref>, logistic regression, 2 h: treatment: <italic>χ</italic><sup>2</sup> = 0.27, d<italic>f</italic> = 2, <italic>p</italic> = 0.9, 24 h: treatment: <italic>χ</italic><sup>2</sup> = 0.31, d<italic>f</italic> = 2, <italic>p</italic> = 0.9, 72 h: treatment: <italic>χ</italic><sup>2</sup> = 1.48, d<italic>f</italic> = 2, <italic>p</italic> = 0.5). Thus, preexposure to pheromone components differing in valence did not affect memory retrieval.</p><p id=\"Par9\">As olfactory PER conditioning is a case of Pavlovian learning involving repeated exposures to an odorant and sucrose solution<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>, we next studied the effect of GER and 2H preexposure on the processing of odorants and on appetitive motivation evaluated through sucrose responsiveness.</p></sec><sec id=\"Sec4\"><title>Effect of pheromone components on odor processing</title><p id=\"Par10\">We first analyzed the effect of GER and 2H preexposure on odorant processing by recording odor-evoked neural activity in the ALs, the primary olfactory center in the bee brain, following pheromone-component preexposure. ALs are constituted by glomeruli, which are interaction sites between afferent olfactory receptor neurons located on the antennae, local interneurons, and projection neurons (PNs) conveying the olfactory information to higher-order brain centers<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. Odorants are encoded in the ALs as odor-specific glomerular maps, which can be visualized using in vivo calcium imaging<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. Using a fluorescent calcium-sensitive dye, we recorded PN activity at the level of the ALs by means of two-photon fluorescence microscopy. Bees prepared for imaging were preexposed to GER, 2H, or mineral oil. PN responses to limonene and eugenol, the conditioned odorants, were recorded 15 min before pheromone exposure (baseline), as well as 15 min and 2 h after preexposure, in the absence of training (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a</xref>). The latter recording time corresponds to the end of conditioning in trained animals, when odorant discrimination was achieved. In this way, we determined if pheromones affected per se (i.e., in the absence of training) perceptual distances between odorants, thus modulating their discrimination. Fluorescence was recorded along a scanline crossing multiple glomeruli (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2b, c</xref>) upon alternate presentations of limonene and eugenol, lasting 4 s each. The normalized fluorescence-intensity change (−Δ<italic>F</italic>/<italic>F</italic>) provides a measure of the neuronal firing rate (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2d</xref>).<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Effect of pheromone components on neural odor processing.</title><p><bold>a</bold>–<bold>f</bold> Pheromone components do not affect olfactory coding and odorant similarity in the antennal lobe. <bold>a</bold> Experimental protocol used. <bold>b</bold> Two-photon microscopy image of the left antennal lobe (AL) stained with a fluorescent calcium-sensitive dye injected in the projection neurons (PN) of the medial and the lateral antennal protocerebral tracts. Activity in PN dendrites in AL glomeruli can be visualized in this way. <bold>c</bold> Signal intensity was recorded along a line crossing ten glomeruli identified using the antennal lobe atlas of the honey bee<sup><xref ref-type=\"bibr\" rid=\"CR62\">62</xref></sup> (AN antennal nerve, v ventral, l lateral, m medial, d dorsal. Numbers refer to identified glomeruli) and averaged between the identified borders of each glomerulus (white circles). Scale bars = 200 µm. <bold>d</bold> Signal intensity of each identified glomerulus over time after background subtraction and normalization (−Δ<italic>F</italic>/<italic>F</italic>). Red lines represent the onset and offset of limonene (Lim, above) and eugenol (Eug, below) presentation to the bee. <bold>e</bold> Change in normalized fluorescence during the first 600 ms of odor stimulation with Lim and Eug 15 min before (“−15 min”) mineral oil (“Oil”; <italic>n</italic> = 8 independent bees) or pheromone- component (GER, 2H; <italic>n</italic> = 8 independent bees for both components) exposure, as well as 15 min (“+15 min”) and 2 h (“+2 h”) after exposure. No significant variation in activity was found for each odorant over time for both pheromonal treatments. <bold>f</bold> Euclidian distance—a measure of odor distinguishability—in the odor-coding space defined by the activity recorded for the ten identified glomeruli between the odor representations of Lim and Eug 15 min before, as well as 15 min and 2 h after the exposure to Oil, GER, and 2H. The circles constitute individual data, and the horizontal bars represent the medians of each distribution. Odor discrimination did not change over time after exposure to pheromone components.</p></caption><graphic xlink:href=\"42003_2020_1183_Fig2_HTML\" id=\"d30e806\"/></fig></p><p id=\"Par11\">Preexposure to mineral oil, GER, and 2H affected neither limonene nor eugenol encoding in the ALs. The neural activation averaged over all glomeruli in response to odor stimulation was not different along recording times after exposure to pheromone components or mineral oil (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2e</xref>; Friedman test, GER<sub>Limonene</sub>: <italic>χ</italic><sup>2</sup> = 1.75, d<italic>f</italic> = 2, <italic>p</italic> = 0.42; GER<sub>Eugenol</sub>: <italic>χ</italic><sup>2</sup> = 3.25, d<italic>f</italic> = 2, <italic>p</italic> = 0.2; 2H<sub>Limonene</sub>: <italic>χ</italic><sup>2</sup> = 1.75, d<italic>f</italic> = 2, <italic>p</italic> = 0.42; 2H<sub>Eugenol</sub>: <italic>χ</italic><sup>2</sup> = 5.25, d<italic>f</italic> = 2, <italic>p</italic> = 0.07; Mineral Oil<sub>Limonene</sub>: <italic>χ</italic><sup>2</sup> = 2.25, d<italic>f</italic> = 2, <italic>p</italic> = 0.32; Mineral Oil<sub>Eugenol</sub>: <italic>χ</italic><sup>2</sup> = 3.25, d<italic>f</italic> = 2, <italic>p</italic> = 0.2). Similarly, the Euclidian distance (ED) between the glomerular-activation patterns of limonene and eugenol, which constitutes a measure of their discriminability<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>, did not change following pheromone-component or mineral-oil preexposure (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2f</xref>; Friedman test, GER: <italic>χ</italic><sup>2</sup> = 2.25, d<italic>f</italic> = 2, <italic>p</italic> = 0.32; 2H: <italic>χ</italic><sup>2</sup> = 3.25, d<italic>f</italic> = 2, <italic>p</italic> = 0.2; Mineral Oil: <italic>χ</italic><sup>2</sup> = 4, d<italic>f</italic> = 2, <italic>p</italic> = 0.14). Thus, the modulation of odor learning and memory induced by pheromone components does not occur via olfactory (CS) circuits.</p></sec><sec id=\"Sec5\"><title>Effect of pheromone components on appetitive motivation</title><p id=\"Par12\">We reasoned that pheromonal signals might affect appetitive motivation, i.e., the subjective evaluation of sucrose reward, thereby determining different levels of appetitive learning (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref>). To test this hypothesis, we studied if preexposure to GER and 2H induces opposite modulations of sucrose responsiveness, using a standard test in which bees are stimulated with six increasing concentrations of sucrose solution delivered to the antennae (0.1, 0.3, 1, 3, 10, and 30% w/w)<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>.</p><p id=\"Par13\">For each bee responding at least to the highest sucrose concentration (30%), we quantified an individual sucrose-responsiveness score (SRS) as the number of sucrose concentrations that elicited a PER. Higher SRSs reflect a higher appetitive motivation. Preexposure to GER and 2H modified sucrose responsiveness (Kruskal–Wallis test, <italic>H</italic> = 71.17, d<italic>f</italic> = 2, <italic>p</italic> < 0.001, Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>); specifically, GER-preexposed bees responded more to sucrose than controls, i.e., had higher SRSs (Dunn’s post hoc test, <italic>p</italic> = 0.0007), while 2H preexposed bees responded less, i.e., had lower SRSs (Dunn’s post hoc test, <italic>p</italic> < 0.0001), consistently with prior findings<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. A population analysis confirmed that sucrose responsiveness increased with sucrose concentration (GLMM, sucrose concentration: <italic>χ</italic><sup>2</sup> = 408.9, d<italic>f</italic> = 1, <italic>p</italic> < 0.0001), and was higher in GER-preexposed bees and lower in 2H preexposed bees (treatment: <italic>χ</italic><sup>2</sup> = 360.6, d<italic>f</italic> = 2, <italic>p</italic> < 0.0001, Tukey’s post hoc test, <italic>p</italic> < 0.0001 in both cases). SRSs are tightly related to learning success as higher SRSs correspond to better acquisition performances<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. We thus studied if pheromone components modify this relationship. As expected, learning success measured through individual acquisition scores (ACQS, see above) correlated with SRSs (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3A, B</xref>): bees with low sucrose responsiveness (SRSs 1–2) had lower ACQSs, while bees with high sucrose responsiveness (SRSs 5–6) had higher ACQSs; intermediate SRSs (3–4) corresponded to intermediate ACQSs (Kruskal–Wallis test, <italic>χ</italic><sup>2</sup> = 35.69, d<italic>f</italic> = 2, <italic>p</italic> < 0.001). Yet, pheromone preexposure modified this relationship for bees with low (1–2) and high (5–6) SRSs (Kruskal–Wallis test, SRS<sub>1–2</sub>: <italic>p</italic> < 0.009, SRS<sub>3–4</sub>: <italic>p</italic> = 0.17, SRS<sub>5–6</sub>: <italic>p</italic> = 0.05), showing that pheromone components modulate associative learning via their effect on appetitive responsiveness.<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Pheromone components modulate sucrose responsiveness according to their valence.</title><p>Bees were preexposed to geraniol (GER, <italic>n</italic> = 112 independent bees), 2-heptanone (2H, <italic>n</italic> = 156 independent bees), or mineral oil (<italic>n</italic> = 172 independent bees). They were then stimulated with a series of six increasing concentrations of sucrose solution (0.1, 0.3, 1, 3, 10, and 30, w/w). For each bee that responded at least to the highest sucrose concentration (30%), we calculated an individual sucrose-responsiveness score (SRS) as the number of sucrose concentrations to which a bee responded. The figure shows the median, quartiles, and max and min (upper and lower whiskers) SRS values of bees preexposed to GER, 2H, or oil, and retained in the analyses. Individual bees are indicated by the dots. Preexposure to GER and to 2H induced a significant increase and decrease of SRS, respectively, with respect to bees exposed to mineral oil. (*) <italic>p</italic> = 0.0007; (**) <italic>p</italic> < 0.0001.</p></caption><graphic xlink:href=\"42003_2020_1183_Fig3_HTML\" id=\"d30e1075\"/></fig></p></sec><sec id=\"Sec6\"><title>Effect of pheromone components on aminergic signaling</title><p id=\"Par14\">We next focused on neural signaling by octopamine (OA) and dopamine (DA) because these biogenic amines mediate appetitive<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup> and aversive responsiveness<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, respectively, in the bee brain. We injected two doses of OA, DA, epinastine (OA-receptor antagonist), or flupentixol (DA-receptor antagonist) into the brain of bees that were preexposed to GER, 2H, or mineral oil prior to olfactory conditioning and retention tests (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4a</xref>). Phosphate-buffered saline (PBS) was injected into control bees. After preexposure to GER, which facilitates olfactory learning and memory, bees injected with epinastine (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4b</xref>) and with flupentixol (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c</xref>) exhibited impaired learning and specific memory with respect to control bees (epinastine: acquisition: <italic>χ</italic><sup>2</sup> = 12.46, d<italic>f</italic> = 2, <italic>p</italic> = 0.002; memory: <italic>χ</italic><sup>2</sup> = 8.21, d<italic>f</italic> = 2, <italic>p</italic> = 0.016; flupentixol: acquisition: <italic>χ</italic><sup>2</sup> = 7.74, d<italic>f</italic> = 2, <italic>p</italic> = 0.02; memory: <italic>χ</italic><sup>2</sup> = 8.35, d<italic>f</italic> = 2, <italic>p</italic> < 0.015). GER-preexposed bees injected with OA or DA exhibited similar learning and specific-memory performances as control bees (see Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4A–C</xref>), consistent with a ceiling effect. These results indicate that the enhancing effect of GER preexposure on learning and memory was mediated by both octopaminergic and dopaminergic signaling.<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>Effect of pheromone components on aminergic signaling in the bee brain.</title><p><bold>a</bold>–<bold>e</bold> Pharmacological treatment with agonists/antagonists of the dopaminergic and octopaminergic system of honey bees exposed to either GER or 2H counteracted totally or partially the effects of pheromone components on learning and memory. <bold>a</bold> Experimental protocol used. <bold>b</bold> Proportion of conditioned responses (PER) to the rewarded (solid lines) and nonrewarded odors (dotted lines) during five CS+ and CS− trials, and proportion of bees with specific memory (i.e., the proportion of bees responding to the CS+ and not to the CS−) in retention tests performed 2, 24, or 72 h after conditioning (bar diagram) in the case of bees injected either with PBS (<italic>n</italic> = 43 independent bees, controls) or epinastine (OA-receptor antagonist) [0.4 µM (<italic>n</italic> = 42 independent bees), 4 mM (<italic>n</italic> = 45 independent bees)] and preexposed to geraniol (GER). (*) <italic>p</italic> < 0.05; (**) <italic>p</italic> ≤ 0.001. <bold>c</bold> Same as in <bold>b</bold> but for bees injected either with PBS (<italic>n</italic> = 45 independent bees) or flupentixol (DA-receptor antagonist) [0.2 µM (<italic>n</italic> = 40 independent bees), 2 mM (<italic>n</italic> = 43 independent bees)] and exposed to geraniol (GER). Both the OA and the DA-receptor antagonist counteracted the enhancing effect of GER on learning and memory formation. (*) <italic>p</italic> < 0.05. <bold>d</bold> Same as in <bold>b</bold> but for bees injected either with PBS (<italic>n</italic> = 53 independent bees) or with octopamine [20 µM (<italic>n</italic> = 53 independent bees) and 2 mM (<italic>n</italic> = 49 independent bees)] and preexposed to 2-heptanone (2H). OA treatment counteracted the decrement of learning and memory induced by 2H. (*) <italic>p</italic> < 0.05; (**) <italic>p</italic> ≤ 0.001. <bold>e</bold> Same as in <bold>b</bold> but for bees injected either with PBS (<italic>n</italic> = 55 independent bees) or with flupentixol (DA-receptor antagonist) [0.2 µM (<italic>n</italic> = 61 independent bees) and 2 mM (<italic>n</italic> = 62 independent bees)] and exposed to 2-heptanone (2H). The DA-receptor antagonist had no effect on learning but rescued memory retention. (**) <italic>p</italic> ≤ 0.001.</p></caption><graphic xlink:href=\"42003_2020_1183_Fig4_HTML\" id=\"d30e1245\"/></fig></p><p id=\"Par15\">After preexposure to 2H, which impairs olfactory learning and memory, injection of OA (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4d</xref>) rescued both learning (inj:trial: <italic>χ</italic><sup>2</sup> = 18.20, d<italic>f</italic> = 2, <italic>p</italic> < 0.0001) and specific memory (<italic>χ</italic><sup>2</sup> = 145.6, d<italic>f</italic> = 2, <italic>p</italic> < 0.001) with respect to control bees. Accordingly, epinastine did neither affect learning (<italic>χ</italic><sup>2</sup> = 3.99, d<italic>f</italic> = 2, <italic>p</italic> = 0.14) nor memory (<italic>χ</italic><sup>2</sup> = 0.032, d<italic>f</italic> = 2, <italic>p</italic> = 0.98) with respect to control bees (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4D</xref>), consistent with a floor effect. Injection of DA (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4E</xref>) did not affect olfactory learning in PBS-injected bees (<italic>χ</italic><sup>2</sup> = 2.72, d<italic>f</italic> = 2, <italic>p</italic> < 0.26). Yet, DA enhanced specific memory (inj:test: <italic>χ</italic><sup>2</sup> = 12.86, d<italic>f</italic> = 2, <italic>p</italic> = 0.01), similarly to OA. Injection of flupentixol (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4e</xref>) rescued partially the effects of 2H exposure. No effect on learning was detected (<italic>χ</italic><sup>2</sup> = 0.11, d<italic>f</italic> = 2, <italic>p</italic> = 0.94, Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4e</xref>), likely because 2H did not induce a clear effect in the PBS group. Yet, flupentixol improved significantly specific memory (<italic>χ</italic><sup>2</sup> = 159.1, d<italic>f</italic> = 2, <italic>p</italic> < 0.0001), irrespective of the test and the dose used (Tukey’s post hoc test, 2 mM vs. PBS: <italic>p</italic> < 0.0001; 0.2 µM vs. PBS: <italic>p</italic> < 0.0001). These results indicate that the depressing effect of 2H was in part mediated by dopaminergic signaling.</p></sec></sec><sec id=\"Sec7\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par16\">Our results reveal that pheromone components induce a persistent, valence-dependent modulation of learning and memory. They do not affect odor processing but modulate appetitive motivation via aminergic circuits in the bee brain, thereby changing subsequent appetitive learning and memory formation. If bees in an appetitive-search mood are exposed to GER, which is the major component of the pheromone of the Nasonov gland used to attract individuals to sites of interest<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>, they will learn more efficiently the features of that site. On the contrary, if they are exposed to 2H, which signals aversive events in various contexts<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR17\">17</xref>,<xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>, they may no longer be predisposed to learn about appetitive food cues<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>, but rather to avoid or respond defensively to these events. The resulting scenario is adaptive as it improves foraging and orientation, and tunes responses toward relevant stimuli, even when the pheromone perceived is no longer present.</p><p id=\"Par17\">The effect of pheromone components on sucrose responsiveness<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup> relied on aminergic modulation of appetitive motivation (see Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref> for a summary). GER acted on both octopaminergic and dopaminergic pathways. While the participation of OA in the enhancement of appetitive responses was predictable based on prior results<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref>,<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>, the finding that DA has a similar effect represents a novelty in the case of the honey bee. It suggests that appetitive signaling may also recruit the dopaminergic pathway, similarly to the case of the fruit fly, <italic>Drosophila melanogaster</italic><sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>, where sucrose signaling occurs via a specific cluster of dopaminergic neurons. Evidence for a similar neural representation of sucrose has remained elusive in the honey bee until now. Yet, the strict separation between octopaminergic and dopaminergic signaling as mediating mechanisms of appetitive and aversive responsiveness in bees, respectively, needs both to be reconsidered and clarified. On the one hand, DA has been shown to impair appetitive memory consolidation in olfactory PER conditioning, while blockade of DA receptors enhances olfactory memory<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. On the other hand, in experiments using a visual version of PER conditioning<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>, DA-receptor blockade impaired appetitive visual learning and memory, while DA administration improved them. In our experiments, the impairment of learning and memory induced by 2H was counteracted by OA but not by epinastine. Inhibiting DA signaling via flupentixol left acquisition intact but improved memory retention, irrespective of the dose used, while the lower dose of DA also enhanced memory retention, thus showing that the effect of 2H on dopaminergic signaling requires further clarification.</p><p id=\"Par18\">Other molecular actors differing from biogenic amines may participate in the modulation of learning and memory induced by pheromones. For instance, isoamyl acetate (IAA), the main component of the sting alarm pheromone of honey bees, activates the equivalent of an opioid system, and decreases aversive responsiveness in a process that resembles stress-induced analgesia<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref>,<xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. Exposure to both the sting alarm pheromone and to IAA alone impairs appetitive olfactory PER conditioning in a dose-dependent manner, but leaves sucrose responsiveness intact<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. Learning impairment is mediated probably by allatostatins, in particular by the C-type allatostatin (ASTCC), which might be therefore responsible for the stress-induced analgesia<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. If and how allatostatins interact with aminergic pathways to modulate learning and memory remains to be determined. Irrespective of the specific pathway involved, the result is consistent with our scenario: exposure to an aversive pheromone component decreases significantly appetitive learning.</p><p id=\"Par19\">Our findings differ from prior works suggesting that in newborn rabbits<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> and in adult rats<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>, pheromone components can substitute unconditioned stimuli and mediate learning when paired with conditioned odorants or context. In our work, pheromonal components were neither present during learning, nor did we show that GER or 2H replace sucrose. We showed instead that preexposure to these substances changes appetitive motivation, and that this change affects subsequent appetitive learning in the absence of the pheromonal signal. If our pheromone components would simply replace reinforcement, their preexposure should retard olfactory conditioning according to the known US-preexposure effect<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. Clearly, this was not observed upon GER preexposure.</p><p id=\"Par20\">In the context of queen dominance, homovanillyl alcohol, a main component of the queen mandibular pheromone, impaired aversive but not appetitive learning of young bees<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>, an effect that was considered specific of the queen–nurse interaction<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>. Also, isoamyl acetate, the main component of the sting alarm pheromone, impaired appetitive learning, which was considered specific of the sting alarm pheromone<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. Our findings show that these cases were not specific but reflect a generalized effect on learning that extends across ages and behavioral contexts.</p><p id=\"Par21\">These examples highlight the common use of single, major pheromone components to study the effects of pheromones that integrate more compounds in their blends. We used GER, which is the major component of the attractive pheromone of the Nasonov gland, and has an attractive effect per se<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. GER is also part of some floral scents, as is the case of many chemicals that are shared between plants and pheromones<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>. Yet, in contrast to the minute quantities of GER available in plant scents (expressed in ppm)<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup>, the amount of GER used in the present work constituted a massive signal corresponding to several bees recruiting for an appetitive event. In the case of 2H, the amount used corresponded to 1–3 mandibular glands of foragers<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>. Although the value of 2H as alarm signal in collective defensive responses is debated<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>, its use to signal negative events has been verified<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Thus, the use of single pheromone components differing in valence is a valid strategy to estimate how pheromones affect a receiver’s behavior according to their valence.</p><p id=\"Par22\">Pheromones evolved to act as chemical signals, which are effective because the receiver’s response evolved accordingly. Our results indicate that besides the primary effect of conveying an intraspecific message, pheromones have a secondary effect in the receiver, which may have evolved with the primary one. This effect is the adaptive modulation of learning and memory in a way consistent with the valence of the pheromone perceived. We thus suggest that the definition of pheromone should incorporate the capacity of these substances to act as behavioral modulators of both motivational and cognitive processes.</p></sec><sec id=\"Sec8\"><title>Methods</title><sec id=\"Sec9\"><title>Experimental animals and subject preparation</title><p id=\"Par23\">Honey bee workers (<italic>Apis mellifera</italic>) were reared in outdoor hives at the experimental apiary of the CRCA situated in the campus of the University Paul Sabatier. In all cases, honey bee foragers (2–3 weeks old) were used. Foragers were collected each day at a feeder and immediately brought to the laboratory where they were cold-anesthetized for 5 min and gently restrained within a metal holder. Adhesive tape was used to block the bees. Their heads were fastened to the holder with a drop of low-temperature melting wax so that only antennae and mouthparts could be moved<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Harnessed bees were either fed with 5 μl of sucrose solution (50% w/w) or fed to satiation depending on the experiment (see below). After feeding, bees were kept resting in a dark and humid place (ca. 60%) at 25 ± 1 °C until the start of the experiments. Through this procedure, we aimed at equalizing the hunger level across individuals, and at keeping bees with sufficient appetitive motivation and endurance<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>.</p></sec><sec id=\"Sec10\"><title>Pheromone-component exposure</title><p id=\"Par24\">All pheromone substances were diluted to 24% in mineral oil (6-μl pheromone + 19-μl mineral oil), following a standard procedure<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. Hence, the dissolvent, mineral oil, served as a control when presented alone. Restrained bees were confined within a 35-ml glass vial containing a filter paper (1 × 5 cm) soaked with either 25 μl of mineral oil or the diluted pheromone component (Geraniol, GER, or 2-Heptanone, 2H). Doses of pheromone components were based on previous studies showing modulation of stimulus responsiveness<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>, and correspond to the natural situation of several bees signaling a target. The exposure time (15 min) was shown to be sufficient to trigger different physiological and behavioral changes<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup> so that we hypothesized that it could also translate into changes in learning and memory. Pheromone exposure was followed by a rest period that lasted 15 min. All chemicals were purchased from Sigma-Aldrich (France).</p></sec><sec id=\"Sec11\"><title>Sucrose-responsiveness assay</title><p id=\"Par25\">Harnessed bees collected at 5:00 p.m., fed to satiation, and kept resting overnight, were used the day after to quantify their sucrose responsiveness by recording PER in response to increasing concentrations of sucrose, following a standard protocol<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. To this end, early in the morning of the testing day, bees received an additional 5 µl of sucrose (50% w/w) and were kept resting for 1 h. After resting and before mineral oil or pheromone-component exposure, bees were allowed to drink water ad libitum in order to ensure that they would respond only to the sucrose contained in the solutions assayed. Fifteen minutes after the end of exposure, both antennae of each bee were stimulated by means of a toothpick with six sucrose solutions of increasing concentration: 0.1, 0.3, 1, 3, 10, and 30% (w/w). Sucrose of analytical grade (Sigma-Aldrich, France) diluted in deionized water (Milli-Q system, Millipore, Bedford, USA) was used to prepare the solutions. Antennal stimulations with deionized water were interspersed between successive sucrose stimulations to avoid sucrose sensitization. The interstimulus interval was 2 min. Bees that did not respond to any sucrose concentration, that responded to water, or that exhibited inconsistent responses to sucrose (i.e., responding to lower but not to higher sucrose concentrations) were discarded<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. An individual sucrose-responsiveness score (SRS) was obtained for each bee based on the number of responses (PER) to the six sucrose concentrations assayed. A bee with a SRS of 1 only responded to the highest concentration (30%) but not to the lower ones. A bee with a SRS of 6 responded to all six sucrose concentrations, including the most diluted ones.</p><p id=\"Par26\">Within each group, only a small percentage of bees (3.8–9.8%) were discarded as they responded to water or exhibited inconsistent responses to successive sucrose concentrations. The proportion of discarded bees did not differ between groups (<italic>χ</italic><sup>2</sup> < 1.56, d<italic>f</italic> = 1, <italic>p</italic> > 0.20 for all pairwise comparisons). Among the remaining bees, some failed to respond to any sucrose concentration, and they did so more often following preexposure to 2H (34%) than to mineral oil (3.5%) (2H vs. oil: <italic>χ</italic><sup>2</sup> = 51.54, d<italic>f</italic> = 1, <italic>p</italic> < 0.0001). GER had no effect (4.5%) on the absence of responsiveness (GER vs. oil: <italic>χ</italic><sup>2</sup> = 0.17, d<italic>f</italic> = 1, <italic>p</italic> = 0.67). The bees used for assessing the effect of pheromone components on sucrose responsiveness were afterward used for determining the effect of the same components on olfactory learning and retention.</p></sec><sec id=\"Sec12\"><title>Olfactory conditioning and retention tests</title><p id=\"Par27\">Bees tested for sucrose responsiveness were kept in a dark and humid place for one additional hour, and then exposed for a second time to the same pheromone component (or to mineral oil) during 15 min as described above. Two hours elapsed between the two exposures. Previous results showed that the modulatory effects of pheromone components on sucrose responsiveness were consistent and nonadditive over successive exposures<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. Fifteen minutes after the end of the exposure, bees were subjected to olfactory PER conditioning in the form of a differential conditioning<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Bees were trained to discriminate a rewarding odorant (CS+) from a nonrewarding odorant (CS−) during ten trials (5 CS+ trials and 5 CS− trials) presented in a pseudorandom sequence so that the same stimulus (CS+ or CS−) was never presented more than twice consecutively. A 12-min intertrial interval was used. Sucrose solution (30%, w/w) delivered by a toothpick to the bees’ antennae and proboscis was used as appetitive US. Limonene and eugenol (Sigma-Aldrich, France) were used as conditioned odorants (CSs). Both odors were used either as CS+ or CS− in a counterbalanced design. Four microliters of the pure odors were added each to a filter paper (0.4 × 4 cm) placed into a 1-ml syringe connected with a computer-controlled odor-stimulation device, which allowed an efficient temporal control of the odor stimulation. Each acquisition trial lasted 30 s. It consisted of a 13-s familiarization phase with the automated odor releaser and the experimental context, a 6-s forward-paired presentation of the CS and the US in the case of CS+ trials (odorant and sucrose presentations lasted 4 s and 3 s, respectively, with a 1-s overlap), and a 11-s resting phase in the setup. CS− trials followed the same sequence, but no sucrose was delivered upon odorant presentation. Memory tests were performed 2, 24, and 72 h after the end of the conditioning experiments. The tests consisted in CS+ and CS− presentations as in the training phase, but in the absence of US. Each odor was presented during 4 s with the same timing used for the conditioning trials. Odor presentations were separated by an intertrial interval of 12 min. The order of presentation of the CS+ and CS− was randomized between bees. Bees were fed to satiation and kept resting in a dark and humid place (ca. 60%) at 25 ± 1 °C at least 30 min after the end of the 2-h retention test, and once each other day. Although testing the same bees more than once can induce memory extinction, we adopted this procedure in the experiment in which exposure was performed before conditioning. We assumed that this phenomenon, if any, might affect both the experimental and the control groups, which were run in parallel. Bees that did not respond to the CS− or to the CS+ during the tests were stimulated with sucrose to check the integrity of the unconditioned response. Bees not responding to sucrose at the end of the retention tests were discarded<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>.</p><p id=\"Par28\">To study specifically the effect of pheromone components on memory retrieval, a process different from memory formation, we exposed bees to pheromone/mineral oil <italic>after</italic> conditioning and before the memory tests performed 2, 24, and 72 h after conditioning. Retention was assessed in groups of bees that had reached equal learning levels prior to exposure and established an associative memory. Independent groups of bees were used for each memory test so that each bee was assessed only once. An identical experiment was repeated in different groups of bees exposed twice to either pheromone component (GER or 2H) or mineral oil as control to ensure that the number of exposures prior to the memory test did not affect memory retrieval (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5A, B</xref>).</p></sec><sec id=\"Sec13\"><title>Calcium imaging of olfactory coding in the ALs</title><p id=\"Par29\">Animal preparation and in vivo calcium imaging of projection-neuron (PN) activity at the level of the ALs upon stimulation with limonene and eugenol were performed as described previously<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. Foragers were collected at 5:00 p.m. and harnessed in Plexiglas stages designed for calcium imaging experiments. After feeding them with 10 μl of sucrose solution (50% w/w), a small window was opened in the head cuticle. Glands and trachea covering the injection site were gently removed. A fluorescent calcium-sensitive dye (Fura-2 conjugated with dextran, Thermo-Fisher Scientific) was injected into the medial and lateral antenno-protocerebral tracts of PNs. To this end, the dye, crystallized at the tip of a pulled borosilicate glass needle, was manually inserted between the MB calyces, below the alpha lobe<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. The left and right ALs were prepared alternately to avoid potential biases due to lateralization. The cuticle was replaced on top of the head and sealed with n-eicosane to avoid brain desiccation. Bees were fed to satiation and left in a dark humid chamber overnight. On the following morning, after the dye had diffused retrogradely toward the PN dendrites, bees received 5 μl of sucrose solution (50% w/w), the head capsule was reopened, and the glands and trachea were removed to expose the AL. The brain was covered with a transparent two-component silicon (Kwik-Sil, WPI). Imaging of the AL responses was performed under a two-photon microscope (Ultima IV, Bruker) equipped with a 20× water-immersion objective (NA 1.0, Olympus)<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. Fluorescence intensity was recorded along a custom scanline of interest, recording all glomeruli in a given focal plane. Bees were exposed to three alternating stimulations of limonene and eugenol for 4 s each, with an interstimulus interval of 13 s (corresponding to the familiarization time used in the conditioning experiment). Fifteen minutes after this first imaging session, bees were exposed to mineral oil or to one of the two pheromones (GER, 2H) for 15 min. Two additional imaging sessions were performed 15 min and 2 h after pheromone/mineral-oil exposures. The latter period corresponds to the end of conditioning in trained animals. The potential impact of pheromone exposure on odorant coding and differentiation was evaluated by comparing the overall activation signal averaged over all observed glomeruli, and the distinguishability of the odor-coding patterns quantified in terms of the Euclidean distance. Data analyses were performed with MATLAB (R2018, MathWorks). Glomerular response signals were deduced from relative changes in fluorescence, with respect to a background signal: −Δ<italic>F</italic>(<italic>t</italic>)/<italic>F</italic>. The background fluorescence <italic>F</italic> was obtained in a dynamic way by applying a moving average filter with a long time constant (7 s) to the fluorescence time series. Ten glomeruli were identified for each bee. Glomerular responses were averaged over the first 600 ms of olfactory stimulation and over the three repeated stimulations for each odorant.</p><p id=\"Par30\">The ED between the response patterns of two odors <italic>x</italic> and <italic>y</italic> was calculated by the following formula:<disp-formula id=\"Equa\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\rm{ED}}_{x,y} = \\sqrt {\\mathop {\\sum }\\limits_{i = 1}^n (x_i - y_i)^2},$$\\end{document}</tex-math><mml:math id=\"M2\"><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">ED</mml:mi></mml:mrow><mml:mrow><mml:mi>x</mml:mi><mml:mo>,</mml:mo><mml:mi>y</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msqrt><mml:mrow><mml:munderover accent=\"false\" accentunder=\"false\"><mml:mrow><mml:mo>∑</mml:mo></mml:mrow><mml:mrow><mml:mi>i</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:munderover><mml:msup><mml:mrow><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>x</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>y</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:msqrt><mml:mo>,</mml:mo></mml:math><graphic xlink:href=\"42003_2020_1183_Article_Equa.gif\" position=\"anchor\"/></alternatives></disp-formula>where <italic>x</italic><sub><italic>i</italic></sub> and <italic>y</italic><sub><italic>i</italic></sub> are the average responses of the single glomerulus <italic>i</italic> to <italic>x</italic> and <italic>y</italic>, which are summed over all <italic>n</italic> glomeruli observed in a single bee.</p></sec><sec id=\"Sec14\"><title>Neuropharmacological experiments</title><p id=\"Par31\">Bees used in the neuropharmacological experiments were collected each day at 9.00 a.m. (see above in the “Subject Preparation” section for detailed information) and subjected to drug administration, pheromone exposure, and olfactory conditioning of PER during the same day. Octopamine (OA) and dopamine (DA) and their respective receptor antagonists epinastine hydrochloride (epinastine)<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup> and cis-(Z)-flupentixol dihydrochloride (flupentixol)<sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup> (Sigma-Aldrich, France) were injected into the bee brain via the ocellar tract<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. Injections were performed after gentle removal of the median ocellus with a scalpel. This procedure allows the drug to reach and dissolve uniformly into the protocerebrum within few minutes. Injections were done using a WPI 26 gauge needle of a NanoFil 10-µl syringe controlled by a micromanipulator (M3301R, WPI) under a binocular stereomicroscope (Leica)<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. To test for dose–response effects, each substance was used at a lower and a higher dose (OA: 20 µM and 2 mM; DA: 20 µM and 2 mM; epinastine: 0.4 µM and 4 mM; flupentixol: 0.2 µM and 2 mM). Drugs were dissolved in PBS, which was injected (200 nl) alone into control bees. For each exposure treatment (GER, 2H, or mineral oil) and each drug injected (agonist or antagonist), low-dose, high-dose, and PBS-injected groups were run in parallel. Bees losing hemolymph after surgery or not showing drug penetration after a couple of minutes were discarded. Each day, before and after injections, syringes and needles were washed using ethanol and distilled water. Injections were performed 15 min before pheromone exposure (see above) and 30 min before olfactory PER conditioning. Previous studies showed that 30 min are required for these biogenic amines and their antagonists to be effective<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>. Protocols for exposure, conditioning, and memory-retention tests were the same as those previously described.</p></sec><sec id=\"Sec15\"><title>Statistics and reproducibility</title><p id=\"Par32\">Sample sizes are provided in each figure caption for the different types of experiments. The behavioral readout used throughout was the PER (1 or 0) to the US (sucrose-responsiveness experiment) or to the CS− and CS+ (learning and memory experiments). The percentage of bees responding to the US or the CSs was calculated and represented as a population response. Individual scores based on the number of PER to a given number of stimulations (either US or CS) were also computed. A SRS was calculated for each bee as the number of sucrose concentrations eliciting PER. Differences between the SRSs of different groups of bees were analyzed using a Kruskal–Wallis test followed by Dunn’s pairwise test for multiple comparisons. Repeated-measure ANOVA was used to analyze behavioral performances in sucrose-responsiveness assays and in olfactory conditioning of bees exposed to pheromone components and/or bees treated with drugs. Independent models were used for the acquisition phase and the memory test for bees exposed either before or after conditioning. Individual conditioned responses were examined using GLMMs with a binomial error structure—logit-link function—<italic>glmer</italic> function of R package <italic>lme4</italic><sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup>. When necessary, models were optimized with the iterative algorithm BOBYQA<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup>. In the sucrose-responsiveness model, “bee response” was entered as a dependent variable, “treatment” (GER, 2H, or mineral oil) as a fixed factor, and “sucrose concentrations” as covariates. In the learning models, “bee response” was entered as a dependent variable, “treatment” (GER, 2H, or mineral-oil exposure) and “CS” as fixed factors, and “conditioning trial” as covariate. In the memory-retention models for bees exposed before conditioning, “bee response” was entered as a dependent variable, and “treatment” and “testing time” (2, 24, or 72 h) as fixed factors. In all cases, “individual identity” (<italic>IDs</italic>) was considered as a random factor to allow for repeated-measurement analysis.</p><p id=\"Par33\">An acquisition score (ACQS) was calculated for each bee as the sum of responses to the five CS+ presentations during conditioning. We analyzed if pheromone-component exposure changed the ACQS of bees pertaining to different SRS categories. To this end, bees were grouped into three groups based on their SRSs (SRS<sub>1–2</sub>, SRS<sub>3–4</sub>, or SRS<sub>5–6</sub>), and their ACQSs were compared using a Kruskal–Wallis test followed by Dunn’s pairwise test for multiple comparisons. Memory retrieval at 2, 24, and 72 h of independent groups of bees exposed after conditioning was analyzed using a Binomial Logistic Regression test. In the model, “bee response” was entered as a dependent variable and “treatment” (GER, 2H, or mineral-oil exposure) as a fixed factor. For the neuropharmacological experiments, independent models were carried out either for the acquisition phase and the memory test or for each exposure treatment. These GLM models were designed as described above with the exception that the fixed factor “treatment” (exposure) was replaced by the fixed factor “pharmacological treatment”. In all the GLM models, “individual identity” (IDs) was considered as a random factor to allow for repeated-measurement analysis. In many analyses, several models were run and compared to identify significant interactions between fixed factors and/or covariates, and the significant model with the highest explanatory power (i.e., the lowest Akaike information criterion (AIC) value) was retained. When AIC values were very similar, the most significant model was retained. Interactions, wherever significant, are indicated in the text. Tukey’s post hoc tests were used to detect differences between the different groups (<italic>glht</italic> function from R package <italic>multcomp</italic><sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>). All statistical analyses were performed with R 3.4.2<sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup>.</p></sec><sec id=\"Sec16\"><title>Reporting summary</title><p id=\"Par34\">Further information on research design is available in the <xref rid=\"MOESM2\" ref-type=\"media\">Nature Research Reporting Summary</xref> linked to this article.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec17\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"42003_2020_1183_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"42003_2020_1183_MOESM2_ESM.pdf\"><caption><p>Reporting Summary</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM3\"><media xlink:href=\"42003_2020_1183_MOESM3_ESM.pdf\"><caption><p>Peer Review File</p></caption></media></supplementary-material></sec></sec></body><back><fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Patrizia d’Ettorre, Martin Giurfa.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary information</bold> is available for this paper at 10.1038/s42003-020-01183-x.</p></sec><ack><title>Acknowledgements</title><p>We thank Alex Kacelnik for useful discussions on the role of pheromones. This work was supported by the French National Research Agency (Project PHEROMOD, ANR-14-CE18-0003), the Institut Universitaire de France, and the IDEX Program “Excellence Chairs” of the Université Fédérale Toulouse Midi-Pyrénées. A.C. and A.H. acknowledge funding by the Autonomous Province of Bolzano (Project B26J16000310003). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>D.B. performed the behavioral and the pharmacological experiments, and analyzed the corresponding data. A.C. performed the calcium imaging experiments and analyzed the data. M.G. and P.d.’E. supervised the behavioral and the pharmacological experiments. A.H. supervised the calcium imaging experiments. All authors (D.B., A.C., J.-M.D., A.H., P.d.’E., and M.G.) helped design the experiments. D.B. and M.G. wrote the paper. All authors participated in the editing of the paper. M.G. and P.d.’E. obtained the principal funding for the research.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>The datasets generated during this study are available at figshare.com with the following accession ID: 10.6084/m9.figshare.12029526.v1<sup><xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup>.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par35\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Karlson</surname><given-names>P</given-names></name><name><surname>Luscher</surname><given-names>M</given-names></name></person-group><article-title>‘Pheromones’: a new term for a class of biologically active substances</article-title><source>Nature</source><year>1959</year><volume>183</volume><fpage>55</fpage><lpage>56</lpage><pub-id pub-id-type=\"pmid\">13622694</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stowers</surname><given-names>L</given-names></name><name><surname>Marton</surname><given-names>TF</given-names></name></person-group><article-title>What is a pheromone? Mammalian pheromones reconsidered</article-title><source>Neuron</source><year>2005</year><volume>46</volume><fpage>699</fpage><lpage>702</lpage><pub-id pub-id-type=\"pmid\">15924856</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carew</surname><given-names>SJ</given-names></name><etal/></person-group><article-title>Pheromone-induced odor associative fear learning in rats</article-title><source>Sci. Rep.</source><year>2018</year><volume>8</volume><fpage>17701</fpage><pub-id pub-id-type=\"pmid\">30532054</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fortes-Marco</surname><given-names>L</given-names></name><name><surname>Lanuza</surname><given-names>E</given-names></name><name><surname>Martinez-Garcia</surname><given-names>F</given-names></name><name><surname>Agustin-Pavon</surname><given-names>C</given-names></name></person-group><article-title>Avoidance and contextual learning induced by a kairomone, a pheromone and a common odorant in female CD1 mice</article-title><source>Front. Neurosci.</source><year>2015</year><volume>9</volume><fpage>336</fpage><pub-id pub-id-type=\"pmid\">26500474</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coureaud</surname><given-names>G</given-names></name><etal/></person-group><article-title>A pheromone that rapidly promotes learning in the newborn</article-title><source>Curr. Biol.</source><year>2006</year><volume>16</volume><fpage>1956</fpage><lpage>1961</lpage><pub-id pub-id-type=\"pmid\">17027493</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><mixed-citation publication-type=\"other\">Murty, V. P. & Dickerson, K. C. in <italic>Recent Developments in Neuroscience Research on Human Motivation: Advances in Motivation and Achievement</italic> Vol. 19 (eds S.-I. Kim, J. D. Reeve & M. Bong) 203–227 (2016).</mixed-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wise</surname><given-names>RA</given-names></name></person-group><article-title>Dopamine, learning and motivation</article-title><source>Nat. Rev. Neurosci.</source><year>2004</year><volume>5</volume><fpage>483</fpage><lpage>494</lpage><pub-id pub-id-type=\"pmid\">15152198</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dickinson</surname><given-names>A</given-names></name><name><surname>Balleine</surname><given-names>B</given-names></name></person-group><article-title>Motivational control of goal-directed action</article-title><source>Anim. Learn. Behav.</source><year>1994</year><volume>22</volume><fpage>1</fpage><lpage>18</lpage></element-citation></ref><ref id=\"CR9\"><label>9.</label><mixed-citation publication-type=\"other\">Wilson, E. O. <italic>The Insect Societies</italic>. (Harvard University Press, 1971).</mixed-citation></ref><ref id=\"CR10\"><label>10.</label><mixed-citation publication-type=\"other\">Free, J. B. <italic>Pheromones of Social Bees</italic>. (Comstock Publishing Associates, 1987).</mixed-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sandoz</surname><given-names>JC</given-names></name><name><surname>Deisig</surname><given-names>N</given-names></name><name><surname>de Brito Sanchez</surname><given-names>MG</given-names></name><name><surname>Giurfa</surname><given-names>M</given-names></name></person-group><article-title>Understanding the logics of pheromone processing in the honeybee brain: from labeled-lines to across-fiber patterns</article-title><source>Front. Behav. Neurosci.</source><year>2007</year><volume>1</volume><fpage>5</fpage><pub-id pub-id-type=\"pmid\">18958187</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Giurfa</surname><given-names>M</given-names></name></person-group><article-title>Behavioral and neural analysis of associative learning in the honeybee: a taste from the magic well</article-title><source>J. Comp. Physiol. A</source><year>2007</year><volume>193</volume><fpage>801</fpage><lpage>824</lpage></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Menzel</surname><given-names>R</given-names></name></person-group><article-title>Memory dynamics in the honeybee</article-title><source>J. Comp. Physiol. A</source><year>1999</year><volume>185</volume><fpage>323</fpage><lpage>340</lpage></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bitterman</surname><given-names>ME</given-names></name><name><surname>Menzel</surname><given-names>R</given-names></name><name><surname>Fietz</surname><given-names>A</given-names></name><name><surname>Schäfer</surname><given-names>S</given-names></name></person-group><article-title>Classical conditioning of proboscis extension in honeybees (<italic>Apis mellifera</italic>)</article-title><source>J. Comp. Psychol.</source><year>1983</year><volume>97</volume><fpage>107</fpage><lpage>119</lpage><pub-id pub-id-type=\"pmid\">6872507</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Giurfa</surname><given-names>M</given-names></name><name><surname>Sandoz</surname><given-names>JC</given-names></name></person-group><article-title>Invertebrate learning and memory: fifty years of olfactory conditioning of the proboscis extension response in honeybees</article-title><source>Learn. Mem.</source><year>2012</year><volume>19</volume><fpage>54</fpage><lpage>66</lpage><pub-id pub-id-type=\"pmid\">22251890</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><mixed-citation publication-type=\"other\">Rudy, J. W. <italic>The Neurobiology of Learning and Memory</italic>. (Sinauer Associates, 2008).</mixed-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shearer</surname><given-names>DA</given-names></name><name><surname>Boch</surname><given-names>R</given-names></name></person-group><article-title>2-Heptanone in the mandibular gland secretion of the honey-bee</article-title><source>Nature</source><year>1965</year><volume>206</volume><fpage>530</fpage><pub-id pub-id-type=\"pmid\">5831852</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boch</surname><given-names>R</given-names></name><name><surname>Shearer</surname><given-names>DA</given-names></name></person-group><article-title>Identification of geraniol as the active component of the Nassanoff pheromone in the honey bee</article-title><source>Nature</source><year>1962</year><volume>194</volume><fpage>704</fpage><lpage>706</lpage></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scheiner</surname><given-names>R</given-names></name><name><surname>Baumann</surname><given-names>A</given-names></name><name><surname>Blenau</surname><given-names>W</given-names></name></person-group><article-title>Aminergic control and modulation of honeybee behaviour</article-title><source>Curr. Neuropharmacol.</source><year>2006</year><volume>4</volume><fpage>259</fpage><lpage>276</lpage><pub-id pub-id-type=\"pmid\">18654639</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><mixed-citation publication-type=\"other\">Tedjakumala, S. R., Aimable, M. & Giurfa, M. Pharmacological modulation of aversive responsiveness in honey bees. <italic>Front. Behav. Neurosci</italic>. <bold>7</bold> 221 (2014).</mixed-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Giurfa</surname><given-names>M</given-names></name></person-group><article-title>Associative learning: the instructive function of biogenic amines</article-title><source>Curr. Biol.</source><year>2006</year><volume>16</volume><fpage>R892</fpage><lpage>R895</lpage><pub-id pub-id-type=\"pmid\">17055975</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Matsumoto</surname><given-names>Y</given-names></name><name><surname>Menzel</surname><given-names>R</given-names></name><name><surname>Sandoz</surname><given-names>JC</given-names></name><name><surname>Giurfa</surname><given-names>M</given-names></name></person-group><article-title>Revisiting olfactory classical conditioning of the proboscis extension response in honey bees: a step toward standardized procedures</article-title><source>J. Neurosci. Methods</source><year>2012</year><volume>211</volume><fpage>159</fpage><lpage>167</lpage><pub-id pub-id-type=\"pmid\">22960052</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Villar</surname><given-names>ME</given-names></name><name><surname>Marchal</surname><given-names>P</given-names></name><name><surname>Viola</surname><given-names>H</given-names></name><name><surname>Giurfa</surname><given-names>M</given-names></name></person-group><article-title>Redefining single-trial memories in the honey bee</article-title><source>Cell Rep.</source><year>2020</year><volume>30</volume><fpage>2603</fpage><lpage>2613</lpage><pub-id pub-id-type=\"pmid\">32101739</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Galizia</surname><given-names>CG</given-names></name><name><surname>Rössler</surname><given-names>W</given-names></name></person-group><article-title>Parallel olfactory systems in insects: anatomy and function</article-title><source>Annu. Rev. Entomol.</source><year>2010</year><volume>55</volume><fpage>399</fpage><lpage>420</lpage><pub-id pub-id-type=\"pmid\">19737085</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Joerges</surname><given-names>J</given-names></name><name><surname>Küttner</surname><given-names>A</given-names></name><name><surname>Galizia</surname><given-names>CG</given-names></name><name><surname>Menzel</surname><given-names>R</given-names></name></person-group><article-title>Representation of odours and odour mixtures visualized in the honeybee brain</article-title><source>Nature</source><year>1997</year><volume>387</volume><fpage>285</fpage><lpage>288</lpage></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Deisig</surname><given-names>N</given-names></name><name><surname>Giurfa</surname><given-names>M</given-names></name><name><surname>Lachnit</surname><given-names>H</given-names></name><name><surname>Sandoz</surname><given-names>JC</given-names></name></person-group><article-title>Neural representation of olfactory mixtures in the honeybee antennal lobe</article-title><source>Eur. J. Neurosci.</source><year>2006</year><volume>24</volume><fpage>1161</fpage><lpage>1174</lpage><pub-id pub-id-type=\"pmid\">16930442</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Deisig</surname><given-names>N</given-names></name><name><surname>Giurfa</surname><given-names>M</given-names></name><name><surname>Sandoz</surname><given-names>JC</given-names></name></person-group><article-title>Antennal lobe processing increases separability of odor mixture representations in the honeybee</article-title><source>J. Neurophysiol.</source><year>2010</year><volume>103</volume><fpage>2185</fpage><lpage>2194</lpage><pub-id pub-id-type=\"pmid\">20181736</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pankiw</surname><given-names>T</given-names></name><name><surname>Page</surname><given-names>RE</given-names><suffix>Jr.</suffix></name></person-group><article-title>The effect of genotype, age, sex, and caste on response thresholds to sucrose and foraging behavior of honey bees (<italic>Apis mellifera</italic> L.)</article-title><source>J. Comp. Physiol.</source><year>1999</year><volume>185</volume><fpage>207</fpage><lpage>213</lpage><pub-id pub-id-type=\"pmid\">10488557</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scheiner</surname><given-names>R</given-names></name><name><surname>Page</surname><given-names>RE</given-names></name><name><surname>Erber</surname><given-names>J</given-names></name></person-group><article-title>Sucrose responsiveness and behavioral plasticity in honey bees (<italic>Apis mellifera</italic>)</article-title><source>Apidologie</source><year>2004</year><volume>35</volume><fpage>133</fpage><lpage>142</lpage></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Baracchi</surname><given-names>D</given-names></name><name><surname>Devaud</surname><given-names>JM</given-names></name><name><surname>d’Ettorre</surname><given-names>P</given-names></name><name><surname>Giurfa</surname><given-names>M</given-names></name></person-group><article-title>Pheromones modulate reward responsiveness and non-associative learning in honey bees</article-title><source>Sci. Rep.</source><year>2017</year><volume>7</volume><fpage>9875</fpage><pub-id pub-id-type=\"pmid\">28852036</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scheiner</surname><given-names>R</given-names></name><name><surname>Erber</surname><given-names>J</given-names></name><name><surname>Page</surname><given-names>RE</given-names><suffix>Jr.</suffix></name></person-group><article-title>Tactile learning and the individual evaluation of the reward in honey bees (<italic>Apis mellifera</italic> L.)</article-title><source>J. Comp. Physiol. A</source><year>1999</year><volume>185</volume><fpage>1</fpage><lpage>10</lpage><pub-id pub-id-type=\"pmid\">10450609</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scheiner</surname><given-names>R</given-names></name><name><surname>Pluckhahn</surname><given-names>S</given-names></name><name><surname>Oney</surname><given-names>B</given-names></name><name><surname>Blenau</surname><given-names>W</given-names></name><name><surname>Erber</surname><given-names>J</given-names></name></person-group><article-title>Behavioural pharmacology of octopamine, tyramine and dopamine in honey bees</article-title><source>Behav. Brain Res.</source><year>2002</year><volume>136</volume><fpage>545</fpage><lpage>553</lpage><pub-id pub-id-type=\"pmid\">12429417</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tedjakumala</surname><given-names>SR</given-names></name><name><surname>Aimable</surname><given-names>M</given-names></name><name><surname>Giurfa</surname><given-names>M</given-names></name></person-group><article-title>Pharmacological modulation of aversive responsiveness in honey bees</article-title><source>Front Behav. Neurosci.</source><year>2013</year><volume>7</volume><fpage>221</fpage><pub-id pub-id-type=\"pmid\">24431993</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Papachristoforou</surname><given-names>A</given-names></name><etal/></person-group><article-title>The bite of the honeybee: 2-heptanone secreted from honeybee mandibles during a bite acts as a local anaesthetic in insects and mammals</article-title><source>PLoS ONE</source><year>2012</year><volume>7</volume><fpage>e47432</fpage><pub-id pub-id-type=\"pmid\">23091624</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><mixed-citation publication-type=\"other\">Urlacher, E., Frances, B., Giurfa, M. & Devaud, J. M. An alarm pheromone modulates appetitive olfactory learning in the honeybee (<italic>Apis mellifera</italic>). <italic>Front. Behav. Neurosci</italic>. <bold>4</bold> 157 (2010).</mixed-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hammer</surname><given-names>M</given-names></name><name><surname>Menzel</surname><given-names>R</given-names></name></person-group><article-title>Multiple sites of associative odor learning as revealed by local brain microinjections of octopamine in honeybees</article-title><source>Learn. Mem.</source><year>1998</year><volume>5</volume><fpage>146</fpage><lpage>156</lpage><pub-id pub-id-type=\"pmid\">10454379</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Farooqui</surname><given-names>T</given-names></name><name><surname>Robinson</surname><given-names>K</given-names></name><name><surname>Vaessin</surname><given-names>H</given-names></name><name><surname>Smith</surname><given-names>BH</given-names></name></person-group><article-title>Modulation of early olfactory processing by an octopaminergic reinforcement pathway in the honeybee</article-title><source>J. Neurosci.</source><year>2003</year><volume>23</volume><fpage>5370</fpage><lpage>5380</lpage><pub-id pub-id-type=\"pmid\">12832563</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Burke</surname><given-names>CJ</given-names></name><etal/></person-group><article-title>Layered reward signalling through octopamine and dopamine in</article-title><source>Drosoph. Nat.</source><year>2012</year><volume>492</volume><fpage>433</fpage><lpage>437</lpage></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>C</given-names></name><etal/></person-group><article-title>A subset of dopamine neurons signals reward for odour memory in</article-title><source>Drosoph. Nat.</source><year>2012</year><volume>488</volume><fpage>512</fpage><lpage>516</lpage></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Klappenbach</surname><given-names>M</given-names></name><name><surname>Kaczer</surname><given-names>L</given-names></name><name><surname>Locatelli</surname><given-names>F</given-names></name></person-group><article-title>Dopamine interferes with appetitive long-term memory formation in honey bees</article-title><source>Neurobiol. Learn. Mem.</source><year>2013</year><volume>106</volume><fpage>230</fpage><lpage>237</lpage><pub-id pub-id-type=\"pmid\">24076013</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><mixed-citation publication-type=\"other\">Mancini, N., Giurfa, M., Sandoz, J. C. & Avargues-Weber, A. Aminergic neuromodulation of associative visual learning in harnessed honey bees. <italic>Neurobiol. Learn. Mem</italic>. <bold>155</bold>, 556–567 (2018).</mixed-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Núñez</surname><given-names>J</given-names></name><name><surname>Almeida</surname><given-names>L</given-names></name><name><surname>Balderrama</surname><given-names>N</given-names></name><name><surname>Giurfa</surname><given-names>M</given-names></name></person-group><article-title>Alarm pheromone induces stress analgesia via an opioid system in the honeybee</article-title><source>Physiol. Behav.</source><year>1997</year><volume>63</volume><fpage>75</fpage><lpage>80</lpage><pub-id pub-id-type=\"pmid\">9402618</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Balderrama</surname><given-names>N</given-names></name><name><surname>Núñez</surname><given-names>JA</given-names></name><name><surname>Guerrieri</surname><given-names>F</given-names></name><name><surname>Giurfa</surname><given-names>M</given-names></name></person-group><article-title>Different functions of two alarm substances in the honeybee</article-title><source>J. Comp. Physiol. A</source><year>2002</year><volume>188</volume><fpage>485</fpage><lpage>491</lpage></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Urlacher</surname><given-names>E</given-names></name><name><surname>Devaud</surname><given-names>J-M</given-names></name><name><surname>Mercer</surname><given-names>AR</given-names></name></person-group><article-title>C-type allatostatins mimic stress-related effects of alarm pheromone on honey bee learning and memory recall</article-title><source>PLoS ONE</source><year>2017</year><volume>12</volume><fpage>e0174321</fpage><pub-id pub-id-type=\"pmid\">28323874</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Randich</surname><given-names>A</given-names></name><name><surname>LoLordo</surname><given-names>VM</given-names></name></person-group><article-title>Associative and nonassociative theories of the UCS preexposure phenomenon: implications for Pavlovian conditioning</article-title><source>Psychol. Bull.</source><year>1979</year><volume>86</volume><fpage>523</fpage><lpage>548</lpage><pub-id pub-id-type=\"pmid\">377357</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vergoz</surname><given-names>V</given-names></name><name><surname>Schreurs</surname><given-names>HA</given-names></name><name><surname>Mercer</surname><given-names>AR</given-names></name></person-group><article-title>Queen pheromone blocks aversive learning in young worker bees</article-title><source>Science</source><year>2007</year><volume>317</volume><fpage>384</fpage><lpage>386</lpage><pub-id pub-id-type=\"pmid\">17641204</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Galizia</surname><given-names>CG</given-names></name></person-group><article-title>Neuroscience. Brainwashing, honeybee style</article-title><source>Science</source><year>2007</year><volume>317</volume><fpage>326</fpage><lpage>327</lpage><pub-id pub-id-type=\"pmid\">17641186</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reddy</surname><given-names>GVP</given-names></name><name><surname>Guerrero</surname><given-names>A</given-names></name></person-group><article-title>Interactions of insect pheromones and plant semiochemicals</article-title><source>Trends Plant Sci.</source><year>2004</year><volume>9</volume><fpage>253</fpage><lpage>261</lpage><pub-id pub-id-type=\"pmid\">15130551</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><mixed-citation publication-type=\"other\">Geraniol. in <italic>PubChem Database</italic>. Vol. CID=637566 <ext-link ext-link-type=\"uri\" xlink:href=\"https://pubchem.ncbi.nlm.nih.gov/compound/Geraniol\">https://pubchem.ncbi.nlm.nih.gov/compound/Geraniol</ext-link> (National Center for Biotechnology Information, 2020).</mixed-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vallet</surname><given-names>A</given-names></name><name><surname>Cassier</surname><given-names>P</given-names></name><name><surname>Lensky</surname><given-names>Y</given-names></name></person-group><article-title>Ontogeny of the fine-structure of the mandibular glands of the honeybee (<italic>Apis-Mellifera</italic> L) workers and the pheromonal activity of 2-heptanone</article-title><source>J. Insect Physiol.</source><year>1991</year><volume>37</volume><fpage>789</fpage><lpage>804</lpage></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nouvian</surname><given-names>M</given-names></name><name><surname>Reinhard</surname><given-names>J</given-names></name><name><surname>Giurfa</surname><given-names>M</given-names></name></person-group><article-title>The defensive response of the honeybee <italic>Apis mellifera</italic></article-title><source>J. Exp. Biol.</source><year>2016</year><volume>219</volume><fpage>3505</fpage><lpage>3517</lpage><pub-id pub-id-type=\"pmid\">27852760</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><mixed-citation publication-type=\"other\">Rossi, N., d’Ettorre, P. & Giurfa, M. Pheromones modulate responsiveness to a noxious stimulus in honey bees. <italic>J Exp Biol</italic><bold>221</bold>, jeb172270 (2018).</mixed-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Paoli</surname><given-names>M</given-names></name><name><surname>Haase</surname><given-names>A</given-names></name></person-group><article-title>In vivo two-photon imaging of the olfactory system in insects</article-title><source>Methods Mol. Biol.</source><year>2018</year><volume>1820</volume><fpage>179</fpage><lpage>219</lpage><pub-id pub-id-type=\"pmid\">29884947</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Roeder</surname><given-names>T</given-names></name><name><surname>Degen</surname><given-names>J</given-names></name><name><surname>Gewecke</surname><given-names>M</given-names></name></person-group><article-title>Epinastine, a highly specific antagonist of insect neuronal octopamine receptors</article-title><source>Eur. J. Pharm.</source><year>1998</year><volume>349</volume><fpage>171</fpage><lpage>177</lpage></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Blenau</surname><given-names>W</given-names></name><name><surname>Erber</surname><given-names>J</given-names></name></person-group><article-title>Behavioural pharmacology of dopamine, serotonin and putative aminergic ligands in the mushroom bodies of the honeybee (<italic>Apis mellifera</italic>)</article-title><source>Behav. Brain Res.</source><year>1998</year><volume>96</volume><fpage>115</fpage><lpage>124</lpage><pub-id pub-id-type=\"pmid\">9821548</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vergoz</surname><given-names>V</given-names></name><name><surname>Roussel</surname><given-names>E</given-names></name><name><surname>Sandoz</surname><given-names>JC</given-names></name><name><surname>Giurfa</surname><given-names>M</given-names></name></person-group><article-title>Aversive learning in honeybees revealed by the olfactory conditioning of the sting extension reflex</article-title><source>PLoS ONE</source><year>2007</year><volume>2</volume><fpage>e288</fpage><pub-id pub-id-type=\"pmid\">17372627</pub-id></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bates</surname><given-names>D</given-names></name><name><surname>Mächler</surname><given-names>M</given-names></name><name><surname>Bolker</surname><given-names>B</given-names></name><name><surname>Walker</surname><given-names>S</given-names></name></person-group><article-title>Fitting linear mixed-effects models using lme4</article-title><source>J. Stat. Softw.</source><year>2015</year><volume>67</volume><fpage>1</fpage><lpage>48</lpage></element-citation></ref><ref id=\"CR58\"><label>58.</label><mixed-citation publication-type=\"other\">Powell, M. J. <italic>Cambridge NA Report</italic>. 26–46.</mixed-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hothorn</surname><given-names>T</given-names></name><name><surname>Bretz</surname><given-names>F</given-names></name><name><surname>Westfall</surname><given-names>P</given-names></name></person-group><article-title>Simultaneous inference in general parametric models</article-title><source>Biometr. J.</source><year>2008</year><volume>50</volume><fpage>346</fpage><lpage>363</lpage></element-citation></ref><ref id=\"CR60\"><label>60.</label><mixed-citation publication-type=\"other\">R Development Core Team. <italic>R: A Language and Environment for Statistical Computing</italic>. (The R Foundation for Statistical Computing, 2016).</mixed-citation></ref><ref id=\"CR61\"><label>61.</label><mixed-citation publication-type=\"other\">Baracchi, D. et al. 10.6084/m9.figshare.12029526.v1 figshare.com. (2020).</mixed-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brandt</surname><given-names>R</given-names></name><etal/></person-group><article-title>Three-dimensional average-shape atlas of the honeybee brain and its applications</article-title><source>J. Comp. Neurol.</source><year>2005</year><volume>492</volume><fpage>1</fpage><lpage>19</lpage><pub-id pub-id-type=\"pmid\">16175557</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Transl Psychiatry</journal-id><journal-id journal-id-type=\"iso-abbrev\">Transl Psychiatry</journal-id><journal-title-group><journal-title>Translational Psychiatry</journal-title></journal-title-group><issn pub-type=\"epub\">2158-3188</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807767</article-id><article-id pub-id-type=\"pmc\">PMC7431542</article-id><article-id pub-id-type=\"publisher-id\">978</article-id><article-id pub-id-type=\"doi\">10.1038/s41398-020-00978-0</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Developmental exposure to near roadway pollution produces behavioral phenotypes relevant to neurodevelopmental disorders in juvenile rats</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-6413-2561</contrib-id><name><surname>Berg</surname><given-names>Elizabeth L.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Pedersen</surname><given-names>Lauren R.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Pride</surname><given-names>Michael C.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Petkova</surname><given-names>Stela P.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Patten</surname><given-names>Kelley T.</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Valenzuela</surname><given-names>Anthony E.</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wallis</surname><given-names>Christopher</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Bein</surname><given-names>Keith J.</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-1565-814X</contrib-id><name><surname>Wexler</surname><given-names>Anthony</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-7665-7584</contrib-id><name><surname>Lein</surname><given-names>Pamela J.</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-9357-5476</contrib-id><name><surname>Silverman</surname><given-names>Jill L.</given-names></name><address><email>jsilverman@ucdavis.edu</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.27860.3b</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9684</institution-id><institution>MIND Institute and Department of Psychiatry and Behavioral Sciences, </institution><institution>University of California Davis School of Medicine, </institution></institution-wrap>Sacramento, CA USA </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.27860.3b</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9684</institution-id><institution>Department of Molecular Biosciences, </institution><institution>University of California Davis School of Veterinary Medicine, </institution></institution-wrap>Davis, CA USA </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.27860.3b</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9684</institution-id><institution>Air Quality Research Center, </institution><institution>University of California Davis, </institution></institution-wrap>Davis, CA USA </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>289</elocation-id><history><date date-type=\"received\"><day>10</day><month>1</month><year>2020</year></date><date date-type=\"rev-recd\"><day>7</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>15</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Epidemiological studies consistently implicate traffic-related air pollution (TRAP) and/or proximity to heavily trafficked roads as risk factors for developmental delays and neurodevelopmental disorders (NDDs); however, there are limited preclinical data demonstrating a causal relationship. To test the effects of TRAP, pregnant rat dams were transported to a vivarium adjacent to a major freeway tunnel system in northern California where they were exposed to TRAP drawn directly from the face of the tunnel or filtered air (FA). Offspring remained housed under the exposure condition into which they were born and were tested in a variety of behavioral assays between postnatal day 4 and 50. To assess the effects of near roadway exposure, offspring of dams housed in a standard research vivarium were tested at the laboratory. An additional group of dams was transported halfway to the facility and then back to the laboratory to control for the effect of potential transport stress. Near roadway exposure delayed growth and development of psychomotor reflexes and elicited abnormal activity in open field locomotion. Near roadway exposure also reduced isolation-induced 40-kHz pup ultrasonic vocalizations, with the TRAP group having the lowest number of call emissions. TRAP affected some components of social communication, evidenced by reduced neonatal pup ultrasonic calling and altered juvenile reciprocal social interactions. These findings confirm that living in close proximity to highly trafficked roadways during early life alters neurodevelopment.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Psychiatric disorders</kwd><kwd>Learning and memory</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/100000066</institution-id><institution>U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)</institution></institution-wrap></funding-source><award-id>R21 ES025570</award-id><award-id>R21 ES026515</award-id><award-id>T32 ES007059</award-id><award-id>R21 ES025570</award-id><award-id>R21 ES026515</award-id><award-id>P30 ES023513</award-id><award-id>R21 ES025570</award-id><award-id>R21 ES026515</award-id><award-id>P30 ES023513</award-id><award-id>R21 ES025570</award-id><award-id>R21 ES026515</award-id><award-id>P30 ES023513</award-id><principal-award-recipient><name><surname>Pedersen</surname><given-names>Lauren R.</given-names></name><name><surname>Bein</surname><given-names>Keith J.</given-names></name><name><surname>Wexler</surname><given-names>Anthony</given-names></name><name><surname>Lein</surname><given-names>Pamela J.</given-names></name><name><surname>Silverman</surname><given-names>Jill L.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/100000025</institution-id><institution>U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)</institution></institution-wrap></funding-source><award-id>T32 MH112507</award-id><principal-award-recipient><name><surname>Pedersen</surname><given-names>Lauren R.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/100009633</institution-id><institution>U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)</institution></institution-wrap></funding-source><award-id>U54 HD079125</award-id><award-id>U54 HD087011</award-id><principal-award-recipient><name><surname>Lein</surname><given-names>Pamela J.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)</institution></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par2\">Neurodevelopmental disorders (NDDs) result from abnormal brain development and include a wide range of conditions, such as intellectual disability, attention deficit hyperactivity disorder (ADHD), and autism spectrum disorder (ASD). Symptoms present in early childhood and persist throughout life, significantly affecting social, cognitive, and behavioral functioning. ASD and ADHD, which affect ~1 and 5% of children respectively, are among the most common and well-studied NDDs<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. The disorders often co-occur with 30–50% of ASD patients presenting symptoms of ADHD, and their prevalence is on the rise<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. According to the U.S. Centers for Disease Control and Prevention, ASD is currently estimated to affect about 1 in 59 children, which represents a dramatic increase from their previously reported 1 in 68 estimate<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. These increased prevalence rates highlight the crucial need to develop a better understanding of the etiology of these neurological disorders since these conditions already incur immense societal and economic costs.</p><p id=\"Par3\">While there is compelling evidence that susceptibility to NDDs, as well as symptom severity and treatment outcomes, are influenced by the interaction of genetic and environmental risk factors, the underlying mechanisms remain to be elucidated<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>–<xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. Environmental factors also contribute to these conditions—although researchers disagree on the relative contributions of genes and environment. Furthermore, studies suggest that more than 50% of new ASD cases are due to factors other than diagnostic drift<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>–<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>.</p><p id=\"Par4\">Identifying and understanding the environmental risk factors contributing to the rising prevalence rates is crucial and important since they can be modified and/or avoided, unlike genetic risk factors, which are, for the most part, not currently modifiable risk variables. Mounting epidemiological data using independent samples, models, and methods from a variety of geographical locations have implicated exposure to traffic-related air pollution (TRAP) as one of these factors. Human exposure to TRAP and/or proximity to roadways, especially during the late gestational period and/or early life, has been significantly associated with an NDD diagnosis<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>–<xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>.</p><p id=\"Par5\">These studies of humans, however, fall short of establishing a causal relationship between TRAP exposure and NDD development, due to an array of confounding factors and a lack of data quantifying individual exposures to complex environmental mixtures. Animal models, therefore, offer a unique benefit and can be used to fill this knowledge gap and directly test the hypothesis that exposure to TRAP impairs behaviors related to NDDs (e.g., developmental delays, social interaction, learning and memory). While there has been some concentrated research in preclinical models, many of the commonly employed exposure methods are limited in their translational relevance to the human condition due to reasons such as repeated anesthesia and failure to recapitulate the complexity and/or relative concentrations of traffic-related emissions in the real world<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref>–<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>.</p><p id=\"Par6\">In order to fully understand the behavioral consequences of near roadway exposures during early life, we leveraged an innovative real-time rodent exposure facility to expose developing rats. Since composition, dose, duration, intensity, mixtures, and timing of air pollution exposures can influence biological outcomes<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref>–<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>, we designed our study to be translationally relevant by representing human TRAP exposure and combined real-world composition of pollutants and dosing in an animal model. The detailed components of the exposure can be found in our Supplementary Information and are reported in comprehensive detail in Bein et al. (under review)<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. In brief, air from a traffic tunnel in northern California was diverted to a nearby exposure facility housing a large rat colony, with half the colony receiving polluted tunnel air and half the colony receiving filtered air. Using rats, which possess a larger and more sophisticated repertoire of social behaviors compared to mice, allowed for an extensive and nuanced examination of NDD-relevant outcomes. After delivering real-world polluted air to pregnant rats and their offspring, we sought to determine whether the gestational and early life near roadway exposure affected physical growth, neonatal reflexes, communication, social interaction, and/or learning and memory, using a battery of validated behavioral assays.</p><p id=\"Par7\">Numerous epidemiological studies have associated near roadway exposure to a range of diseases, but it is difficult in such studies to disentangle confounders, such as socioeconomic status, smoking, and diet. The Childhood Autism Risks from Genetics and Environment (CHARGE) study examined the link between autism and living near freeways using a distribution of distances: closest 10% (<309 m), next 15% (309–647 m), the next 25% (647–1419 m), and farthest from freeways (>1419 m)<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. In each trimester of pregnancy, living closest (<309 m) to the freeway was associated with autism, with the odds ratio reaching the highest significance during the third trimester, informing the timing of our exposure period. In order to generate toxicological data that complements the epidemiological data, the exposure facility that we employed in this study was designed to model near roadway exposures of air pollution, noise, and vibration, the same stressors experienced by people living in this environment. Epidemiological studies differ on the distance from the roadway that is “safe”, as this distance is partially determined by how much the air pollution from vehicles dilutes and how much the noise and vibration dissipates before the near roadway population is exposed. We drew air from the eastern face of the tunnel, not from inside the tunnel itself, so that the air pollution was somewhat diluted already, we insulated the building to reduce noise, and we installed vibration isolators on the feet of the exposure chambers to reduce vibration. The goal of these measures was to expose the rodents to conditions that well model human exposures.</p><p id=\"Par8\">Our investigation led to the discovery that gestational and early life exposure to TRAP affects some components of social communication. Importantly, we also discovered that both roadside-reared groups, TRAP and filtered air (FA), with exposure to the same noise and vibrational stress, had significantly delayed growth and development of psychomotor reflexes, displayed altered social interactions, and exhibited abnormal motor activity. Histological outcomes from these exposures are described in our companion manuscript<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. Further, we found no evidence for an effect, due to stress or otherwise, of the pregnant dams’ transport to the roadside facility on offspring behavior. This is the first report of functional outcomes of this exposure model, and the first report that illustrates behavioral deficits resulting from near roadway exposure alone.</p></sec><sec id=\"Sec2\"><title>Methods</title><sec id=\"Sec3\"><title>Subjects</title><p id=\"Par9\">All animals were housed in a temperature-controlled vivarium maintained on a 12:12 light–dark cycle. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California Davis (UC Davis) and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. To identify rats, pups were labeled with paw tattoos on postnatal day (PND) 2 using non-toxic animal tattoo ink (Ketchum Manufacturing Inc., Brockville, ON, Canada). Ink was delivered into the center of the paw with a 23-gauge hypodermic needle tip. Rats were also tail marked with non-toxic permanent marker at weaning to allow for additional identification. The tattoo and tail marks for each subject were coded to allow investigators to carry out testing and scoring blind to treatment group.</p></sec><sec id=\"Sec4\"><title>Order of behavioral testing and description of cohorts</title><p id=\"Par10\">Male and female Sprague-Dawley rat breeders (PND 80–90) were paired for two weeks before females were singly housed at approximately gestational day (GD) 14. A group of dams was transported to the roadside exposure facility adjacent to a major freeway tunnel system in the Bay Area of Northern California. Dams were randomly assigned to one of two exposure conditions within the same facility: traffic-related air pollution (TRAP) or filtered air (FA). Two male and two female offspring from each of 20 dams were tested as follows: (1) developmental milestones at PND 4, 6, 7, 9, 10, and 12, (2) pup USV at PND 5, (3) reciprocal social interaction at PND 32–34, (4) open-field behavior at PND 39–41, (5) novel object recognition at PND 40–42, and (6) fear conditioning at PND 44–48.</p><p id=\"Par11\">A second group of dams remained housed at a UC Davis vivarium, constituting the laboratory control group. Two male and two female offspring from each of seven litters were tested as follows: (1) developmental milestones at PND 4, 6, 7, 9, 10, and 12, (2) pup USV at PND 5, (3) reciprocal social interaction at PND 34–36, and (5) open field behavior at PND 42–43.</p><p id=\"Par12\">At a later timepoint, a third group of dams was employed as a control for the approximately 1.5-h vehicular transport required to move the prior groups of dams to the roadside exposure facility. Two weeks after being paired with a male breeder, all dams were singly-housed and half of the group was driven halfway to the roadside tunnel site (~45 min drive) and then back to UC Davis. The other half of the group of dams remained unmoved at the UC Davis vivarium, constituting the control group for the transported group. All of the dams and their offspring remained housed at the UC Davis vivarium for the duration of the study. Two male and two female offspring from each of 11 dams were tested as follows: (1) developmental milestones at PND 4, 6, 7, 9, 10, and 12, (2) pup USV at PND 5, and (3) open field behavior at PND 38–41.</p><p id=\"Par13\">All offspring remained in the location and exposure condition into which they were born. Behavioral testing was conducted in testing rooms adjacent to each vivarium. Two male and two female offspring from each litter were tested. To minimize carry-over effects from repeated testing, assays were performed in order from least to most stressful and at least 48 h elapsed between tests.</p><p id=\"Par14\">At separate timepoints, two additional cohorts of male and female Sprague-Dawley rats were used to collect laboratory control data for the learning and memory paradigms. These data were collected at the UC Davis vivarium prior to the testing equipment being relocated to the roadside exposure facility. Both groups remained housed at the UC Davis vivarium for the duration of the study, were well-handled prior to testing, and were offspring of Sprague-Dawley breeders who remained housed at the UC Davis vivarium. One cohort of rats was sampled from five litters and tested in the novel object recognition test at PND 45–53 and a second cohort was sampled from seven litters and tested in the fear conditioning assay at PND 42–44.</p></sec><sec id=\"Sec5\"><title>Roadside exposure facility</title><p id=\"Par15\">Data from the CHARGE study found residential proximity to freeways to be a risk factor for NDDs when maternal residence was <309 m from a major roadway<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. In order to generate toxicological data that complements the epidemiological data, the exposure facility that we employed in this study was designed to model near roadway exposures of air pollution, noise, and vibration, the same stressors experienced by people living in this environment. We drew air from the eastern face of the tunnel, not from within the tunnel itself, so that the air pollution was somewhat diluted already. Additionally, we installed vibration isolators on the feet of the exposure chambers to reduce vibration and insulated the building to reduce noise below the IACUC-mandated maximal tolerated limit of 85 decibels. Such measures were unnecessary for the UC Davis vivarium and adjacent laboratory testing rooms, which have ambient noise levels of only 64 and 43–47 decibels, respectively.</p><p id=\"Par16\">The dual housing and exposure facility, located adjacent to a major freeway tunnel system in the Bay Area of northern California, was composed of three rooms: one containing equipment for adjusting air temperature and flow, and measuring air pollutant concentrations; a second room for the two exposure chambers; and a third room for behavior testing. Each exposure chamber was 12.8 ft l × 3 ft w × 7.8 ft h and capable of accommodating 108 cages with filter tops removed. In order to minimize noise stress, all pumps and blowers were housed outside the facility and plumbed through walls. The room containing the exposure chambers and the behavioral testing suite were also additionally insulated to block noise.</p><p id=\"Par17\">Air supplied to the TRAP-exposed animals was drawn directly from the face of the tunnel. Flexible ducting carried air from the exit of the tunnel’s two eastbound bores to the exposure facility where rats were exposed to the tunnel air. Air supplied to the filtered air group was drawn from the outside of the exposure facility, where pollutant concentrations were expected to be much lower than at the tunnel face. This air was subjected to several emissions control technologies coupled together in series prior to being plumbed to the exposure chamber. These included (a) a pre-filter for removing large debris and coarse particulate matter (PM), (b) inline activated carbon filters for removing gas-phase volatile and semi-volatile organic compounds, (c) barium oxide-based catalytic converters for removing NO<sub>x</sub> and (d) ultrahigh efficiency Teflon-bound glass microfiber filters for removing fine and ultrafine PM. Flow rate control and temperature conditioning were also included in compliance with IACUC specifications. Pressure within each exposure chamber was monitored constantly and blowers were programmed to maintain a small negative pressure in each chamber, with the TRAP chamber drawing in air from the tunnel and the filtered air chamber drawing in air from the outside via the filtration system.</p></sec><sec id=\"Sec6\"><title>Behavioral testing</title><p id=\"Par18\">Two males and two females from each litter were randomly selected as behavioral test subjects and were tested on all behavioral assays with the exception of 16 animals who were only tested as pups and not as juveniles. This was to carefully control for the effect of the litter, previously described as being the most influential factor in developmental toxicological exposure studies<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>. Rats at the roadside exposure facility were removed from the home exposure chambers for testing and then immediately returned to the chamber following the completion of each test. For behavioral tests involving bedding, the same type of bedding as present in home cages was used.</p><sec id=\"Sec7\"><title>Developmental milestones</title><p id=\"Par19\">Pup developmental milestones were assessed at PND 4, 6, 7, 9, 10, and 12 similarly to methods described previously<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref>–<xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>. Body length (cm; nose to tail base) and body weight (grams) were measured. Rooting reflex was measured as a turn of the head to whisker stimulation. Forelimb grasping was measured as grasping of a bar being moved upward along both front paws.</p></sec><sec id=\"Sec8\"><title>Isolation-induced pup ultrasonic vocalizations</title><p id=\"Par20\">During the first 2 weeks of life, rodent pups will emit ultrasonic vocalizations (USV) upon separation from their mothers and littermates<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref>–<xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. On PND 5, isolation-induced USVs were collected from each pup for three min. A pup was randomly selected from the nest, placed in a small, open top container with bedding, and emitted USV were collected using Avisoft-RECORDER (Avisoft Bioacoustics, Glienicke, Germany) as described previously<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref>,<xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>. The container was cleaned with 70% ethanol and new clean bedding was added between each animal. USV were displayed as spectrograms and counted by a trained observer blinded to group using Avisoft-SASLab Pro (Avisoft Bioacoustics, Glienicke, Germany).</p></sec><sec id=\"Sec9\"><title>Juvenile reciprocal social interaction</title><p id=\"Par21\">Each rat was paired with an unfamiliar strain-, age-, and sex-matched stimulus rat and allowed to freely interact for 10 min in a clean, empty test arena (41.3 cm l × 41.3 cm w × 29 cm h) containing bedding. Behaviors were video recorded through the arena’s transparent front wall and later scored by a trained observer blinded to group as described previously<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>. Both subject and stimulus animals were isolated for 30 min prior to the test session. Subject and stimulus animals were always from different litters and stimuli rats used at the roadside facility were housed in filtered air. All behaviors scored were those of the subject animal. Behaviors scored for duration were: (1) exploring, (2) following or chasing, (3) social sniffing, (4) anogenital sniffing, and (6) self-grooming. The testing room was illuminated to ~30 lux.</p></sec><sec id=\"Sec10\"><title>Open field exploration</title><p id=\"Par22\">In order to control for the potentially confounding effects of hypo- or hyperactivity on the other behavioral assays, exploratory activity in a novel open arena was evaluated over a 30 min session. Rats were placed in the center of the arena at the start of the testing session. Using methods similar to those previously described<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref>,<xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>, total distance traveled and time spent in the center of the arena were measured using one of two comparable automated systems: an opaque matte black arena (54.1 cm l × 54.1 cm w × 34.3 cm h) equipped with video tracking software (EthoVision XT 12; Noldus Information Technology, Wageningen, Netherlands) or the fully automated Digiscan Animal Activity Monitors with Integra software (Omnitech Electronics, Columbus, OH, USA). The testing room was illuminated to ~30 lux.</p></sec><sec id=\"Sec11\"><title>Novel object recognition</title><p id=\"Par23\">Novel object recognition was assayed using methods similar to those described previously<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref>,<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>. Using an opaque matte black box (54.1 cm l × 54.1 cm w × 34.3 cm h), each animal was habituated to the empty arena for 30 min on the day prior to the test. On the day of the test, each subject was again habituated to the arena for 30 min before two identical objects were placed gently in the arena with the animal. After a 10 min familiarization session, the animal was isolated in a clean holding cage with bedding for 60 min. During this time, the arena and the objects were cleaned with 70% ethanol and one clean familiar object and one clean novel object were placed in the original positions of the two identical objects during familiarization. Both the identity and location of the novel object within the arena were counterbalanced to address potential inherent object preferences or side biases. Our protocol has been published as standard by the Intellectual and Developmental Disability’s Behavior Cores<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>. Upon being returned to the arena for the recognition test, the subject was allowed 5 min to interact with the familiar and novel objects. Time spent sniffing each object during each phase of testing was automatically measured via video tracking software (EthoVision XT 10 and 12; Noldus Information Technology, Wageningen, Netherlands). Objects used were orange plastic cones (8.5 cm l × 8.5 cm w × 9.5 cm h) and glass bell jars (7.5 cm d × 10.3 cm h). The testing room was illuminated to ~30 lux.</p></sec><sec id=\"Sec12\"><title>Contextual and cued fear conditioning</title><p id=\"Par24\">Contextual and cued fear conditioning was carried out using an automated fear conditioning chamber (Med Associates, Inc., Fairfax, VT, USA) similar to methods described previously<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref>,<xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup>. During training on day one, rats were exposed to a series of three noise-shock (CS-US) pairings in a testing chamber with specific visual, odor, and tactile cues. The training environment was brightly lit (~100 lux), contained a metal wire floor, and included 0.3 mL of vanilla odor cue (1:100 dilution of McCormick Vanilla Extract). White noise (80 dB) was played for 30 s and a foot shock (0.7 mA) occurred during the final two sec of the noise cue. A two min period for exploration preceded the first noise-shock pairing and elapsed between each noise-shock pairing. A 30 s exploration period followed the final noise-shock pairing and the entire training session was eight min in duration. On day two of testing, the subject was placed back inside the training environment for five min. The chamber contained identical contextual cues as the training session, but no white noise or foot shock occurred. On day three of testing, the subject was placed back inside the training environment for 6 min, but the chamber context was altered. The overhead lighting was turned off and the chamber contained a novel smooth plastic floor, novel black angled walls, and a novel lemon scent (1:100 dilution of McCormick Lemon Extract). An initial three min exploration period was followed by a three min presentation of the white noise conditioned stimulus. Time spent freezing during each test phase was automatically measured by the VideoFreeze software (version 2.7; Med Associates).</p></sec></sec><sec id=\"Sec13\"><title>Statistical analysis</title><p id=\"Par25\">Particulate matter concentrations were compared using paired <italic>t</italic>-test since measurements occurred on the same days in both groups. Vocalizations were analyzed via unpaired (Student’s) <italic>t</italic>-test for two groups or via one-way ANOVA with Tukey’s multiple comparisons post hoc test for three groups. Developmental metrics and open field parameters were analyzed via two-way repeated measures ANOVA with exposure as the between-group factor and time as the within-group factor. Significant ANOVAs were followed by Tukey’s post hoc testing. Log-Rank (Mantel-Cox) test was used to compare the percentage of animals achieving developmental milestones. Social interaction parameters were compared with one-way ANOVA followed by Tukey’s post hoc testing. Comparisons between sniff times of objects were made within each exposure group via paired <italic>t</italic>-test and comparisons between freezing times were compared within test day with repeated measures ANOVA (for training and cued freezing) or unpaired (Student’s) <italic>t</italic>-test (for contextual freezing). Group sizes were chosen based on past experience and power analyses<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup>, and data were analyzed with GraphPad Prism. Behavioral data passed distribution normality tests, were collected using continuous variables, and thus were analyzed via parametric tests. Variances were similar between groups and data points within two standard deviations from the mean were included in analyses. All significance levels were set at <italic>p</italic> < 0.05 and all <italic>t</italic>-tests were two-tailed. Multiple comparisons were corrected for via post hoc testing via Tukey’s multiple comparisons test. Data are presented as mean ± standard error of the mean.</p></sec></sec><sec id=\"Sec14\" sec-type=\"results\"><title>Results</title><sec id=\"Sec15\"><title>Reproductive success</title><p id=\"Par26\">Two of three groups of pregnant female rats were transported to the roadside exposure facility at approximately gestational day (GD) 14, while the third group remained in the laboratory at UC Davis. Dams of the roadside cohort were randomly assigned to be housed in either the TRAP or filtered air (FA) exposure chamber. In the laboratory control setting, 10 of 11 dams gave birth. One litter was cannibalized and did not survive to PND 2. We assayed a final litter count of 9. In the FA-exposed group at the roadside facility, 17 of 18 dams gave birth. One litter was cannibalized and did not survive to PND 2, so we assayed a final litter count of 16. In the TRAP-exposed group at the roadside facility, 10 of 10 dams gave birth. The average number of days between arrival at the roadside vivarium and birth was 10 days for both exposure groups, and there was no effect of group on litter size nor male to female ratio (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>). Figure <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref> illustrates our experimental design described in the methods.<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Timeline and quantification of roadside TRAP exposure.</title><p><bold>a</bold> Pregnant female rats were transported to the roadside exposure facility at approximately gestational day (GD) 14 and were randomly assigned to be housed in either the TRAP or filtered air (FA) exposure chambers. Offspring, remained in the exposure condition into which they were born, were tested on a variety of developmental milestone assays between four days after birth (postnatal day (PND) 4) and weaning at PND 21 and then a battery of standardized behavioral assays between PND 21 and 50. <bold>b</bold>, <bold>c</bold> Particulate matter (PM) concentrations of the TRAP air and FA were quantified on 19 days. Both <bold>b</bold> PM<sub>2.5</sub> and <bold>c</bold> PM<sub>10</sub> concentrations of the TRAP air were significantly higher than those of the FA. *<italic>p</italic> < 0.05, paired <italic>t</italic>-test.</p></caption><graphic xlink:href=\"41398_2020_978_Fig1_HTML\" id=\"d30e684\"/></fig></p></sec><sec id=\"Sec16\"><title>Particulate concentrations in TRAP and FA exposures</title><p id=\"Par27\">Twenty-four-hour PM<sub>2.5</sub> and total suspended particulate mass concentrations measured immediately upstream of the FA and TRAP exposure chambers at the roadside tunnel facility for the study duration are described with extensive detail in Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref> and Bein et al. (under review)<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. A unique and defining characteristic of our design is that it captured significant diurnal and day-to-day variations in exposure concentrations that cannot be readily recreated in the laboratory. These variations were easily seen in the size distribution of particle number concentrations (Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>) and described comprehensively in Bein et al. (under review)<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. Figure <xref rid=\"Fig1\" ref-type=\"fig\">1b, c</xref> illustrate the clearly increased PM<sub>2.5</sub> and PM<sub>10</sub>, respectively, in the tunnel-sampled air (TRAP) compared to filtered air (FA), thereby validating our exposure system (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref><italic>t</italic><sub>(1, 18)</sub> = 4.562, <italic>p</italic> < 0.001 and Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1c</xref><italic>t</italic><sub>(1, 18)</sub> = 4.923, <italic>p</italic> < 0.001).</p></sec><sec id=\"Sec17\"><title>Reduced isolation-induced pup ultrasonic vocalizations (USV)</title><p id=\"Par28\">Isolation-induced USV were collected for 3 min as social communication signals in rat pups on PND 5, as previously described<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref>,<xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>. In male offspring, a significant effect of exposure on USV was discovered (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a</xref><italic>F</italic><sub>(2, 50)</sub> = 4.287, <italic>p</italic> < 0.02). TRAP-exposed pups emitted the fewest USV calls (<italic>p</italic> = 0.014 versus laboratory controls) and, interestingly, the FA-exposed group also trended to emit lower calls compared to the laboratory control group (<italic>p</italic> = 0.154). Raw values show the phenomenon that TRAP had the lowest number of calls: non-significant but noteworthy effects on USV by exposure group using mean ± SD showed that in male laboratory controls USV were 412 ± 132.8, FA USV were 327 ± 99.90, and TRAP USV were 280 ± 145.3. Given that TRAP-exposed did not differ from FA-exposed by Tukey’s multiple comparisons post hoc analysis (<italic>p</italic> = 0.475), we cannot conclude that the air quality alone caused the lower numbers of USV. Although, the SD of the raw values allows us to see the high variability in call numbers by group.<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Reduced isolation-induced ultrasonic vocalizations (USV) of TRAP-exposed pups at PND 5.</title><p><bold>a</bold> Male pups exposed to TRAP emitted significantly fewer USV during the three min isolation compared to lab controls. <bold>b</bold> Exposure did not affect USV emission in females, although the trend indicated reduced numbers of calls in the TRAP group compared to lab controls. *<italic>p</italic> < 0.05, one-way ANOVA followed by Tukey’s multiple comparisons test.</p></caption><graphic xlink:href=\"41398_2020_978_Fig2_HTML\" id=\"d30e788\"/></fig></p><p id=\"Par29\">A similar pattern was illustrated in the female offspring (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2b</xref><italic>F</italic><sub>(2, 52)</sub> = 3.069, <italic>p</italic> = 0.055) albeit statistical significance in the overall ANOVA was not <0.05. Non-significant but noteworthy effects on USV by exposure group using mean ± SD showed that in female laboratory controls USV were 407 ± 154.60, FA USV were 331 ± 155.7, and TRAP USV were 275 ± 162.20. However, as the overall ANOVA was not under <italic>p</italic> = 0.05, we did not run post hoc analyses. Given the effect of the roadside exposure (TRAP and FA) in males and trend in females, we were unable to extract a sound statistical finding on calls that resulted from our intermittent, intensity varying, mixture of real-world pollution in the TRAP group. Trends, raw values, and high SD allow us to see the high variability in call numbers by group.</p><p id=\"Par30\">Body weight and temperature were also collected since body temperature is known to alter pup USV emission<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref>,<xref ref-type=\"bibr\" rid=\"CR59\">59</xref>–<xref ref-type=\"bibr\" rid=\"CR62\">62</xref></sup>. Weights and temperature did not differ by roadside air exposure (weight TRAP versus FA <italic>t</italic><sub>(1, 38)</sub> = 0.753, <italic>ns</italic> and temperature TRAP versus FA <italic>t</italic><sub>(1, 38)</sub> = 1.375, <italic>ns</italic>). On PND 5, males of both roadside exposed groups weighed less than laboratory controls (TRAP versus lab <italic>t</italic><sub>(1, 31)</sub> = 2.603, <italic>p</italic> < 0.02; FA versus lab <italic>t</italic><sub>(1, 31)</sub> = 2.388, <italic>p</italic> < 0.03).</p></sec><sec id=\"Sec18\"><title>Delayed growth and milestone achievement of both TRAP and FA-exposed pups</title><p id=\"Par31\">Figure <xref rid=\"Fig3\" ref-type=\"fig\">3a–h</xref> shows delayed early physical development and neurological reflexes in TRAP- and FA-exposed offspring compared to laboratory controls. All male and female subjects gained weight and grew in length over time (males Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref><sub>length</sub>\n<italic>F</italic><sub>(5, 255)</sub> = 390.8, <italic>p</italic> < 0.0001; Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b</xref><sub>weight</sub>\n<italic>F</italic><sub>(5, 255)</sub> = 1186, <italic>p</italic> < 0.001 and females Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3e</xref><sub>length</sub>\n<italic>F</italic><sub>(5, 270)</sub> = 322.1, <italic>p</italic> < 0.0001; Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3f</xref><sub>weight</sub>\n<italic>F</italic><sub>(5, 255)</sub> = 1092, <italic>p</italic> < 0.001). Significant effects of exposure on body length and weight were discovered in both sexes (males Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref><sub>length</sub>\n<italic>F</italic><sub>(2, 51)</sub> = 12.66, <italic>p</italic> < 0.001; Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b</xref><sub>weight</sub>\n<italic>F</italic><sub>(2, 51)</sub> = 10.04, <italic>p</italic> < 0.001 and females Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3e</xref><sub>length</sub>\n<italic>F</italic><sub>(2, 54)</sub> = 13.05, <italic>p</italic> < 0.001; Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3f</xref><sub>weight</sub>\n<italic>F</italic><sub>(2, 54)</sub> = 6.312, <italic>p</italic> < 0.004). TRAP-exposed (males <italic>p</italic> < 0.001 and females <italic>p</italic> < 0.006) and FA-exposed (males <italic>p</italic> < 0.001 and females <italic>p</italic> < 0.02) offspring differed from laboratory controls in both length and weight. Interestingly, no differences were observed between TRAP- and FA-exposure for length (males <italic>ns</italic> and females <italic>ns</italic>) or weight (males <italic>ns</italic> and females <italic>ns</italic>). The rooting and grasping reflexes were delayed in both the TRAP- and FA-exposed offspring compared to laboratory controls in both males (TRAP Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3c</xref><sub>rooting</sub> Log-rank <italic>χ</italic><sup>2</sup><sub>(1)</sub> = 8.35, <italic>p</italic> < 0.005; FA Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3c</xref><sub>rooting</sub> Log-rank <italic>χ</italic><sup>2</sup><sub>(1)</sub> = 7.18, <italic>p</italic> < 0.01; TRAP Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3d</xref><sub>grasping</sub> Log-rank <italic>χ</italic><sup>2</sup><sub>(1)</sub> <italic>=</italic> 11.05, <italic>p</italic> < 0.001; FA Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3d</xref><sub>grasping</sub> Log-rank <italic>χ</italic><sup>2</sup><sub>(1)</sub> <italic>=</italic> 14.30, <italic>p</italic> < 0.001) and females (TRAP Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3g</xref><sub>rooting</sub> Log-rank <italic>χ</italic><sup>2</sup><sub>(1)</sub> <italic>=</italic> 13.98, <italic>p</italic> < 0.001; FA Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3g</xref><sub>rooting</sub> Log-rank <italic>χ</italic><sup>2</sup><sub>(1)</sub> <italic>=</italic> 13.98, <italic>p</italic> < 0.001; TRAP Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3h</xref><sub>grasping</sub> Log-rank <italic>χ</italic><sup>2</sup><sub>(1)</sub> <italic>=</italic> 5.92, <italic>p</italic> < 0.05; FA Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3h</xref><sub>grasping</sub> Log-rank <italic>χ</italic><sup>2</sup><sub>(1)</sub> <italic>=</italic> 18.63, <italic>p</italic> < 0.001). Additional developmental milestones are shown in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>.<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Delayed growth and milestone achievement of roadside TRAP- and FA-exposed pups.</title><p><bold>a</bold> Male pups exposed to TRAP or FA had significantly reduced body length and <bold>b</bold> body weight throughout early development compared to lab controls. Males of both roadside groups exhibited a significant delay in the development of <bold>c</bold> rooting and <bold>d</bold> forelimb grasping reflexes. <bold>e</bold> Female pups exposed to TRAP or FA also had reduced body length and <bold>f</bold> body weight and developed <bold>g</bold> rooting and <bold>h</bold> forelimb grasping reflexes later than lab controls. <bold>a</bold>, <bold>b</bold>, <bold>e</bold>, <bold>f</bold> *<italic>p</italic> < 0.05, repeated measures ANOVA followed by Tukey’s multiple comparisons test. <bold>c</bold>, <bold>d</bold>, <bold>g</bold>, <bold>h</bold> *<italic>p</italic> < 0.05, Log-Rank (Mantel-Cox) test.</p></caption><graphic xlink:href=\"41398_2020_978_Fig3_HTML\" id=\"d30e1206\"/></fig></p></sec><sec id=\"Sec19\"><title>Juvenile reciprocal dyad social interactions (social play)</title><p id=\"Par32\">Male and female subject exploration did not differ between exposure groups and neither exposure group differed from laboratory controls (males Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4a</xref><italic>F</italic><sub>(2, 42)</sub> = 2.065, <italic>ns</italic> and females Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4f</xref><italic>F</italic><sub>(2, 44)</sub> = 0.2467, <italic>ns</italic>). This key information suggests that any differences in social behavior are not confounded by motor abilities, or hypo-, or hyper-exploration of the arena. Levels of this parameter were comparable and consistent with earlier findings using Sprague-Dawley rats at this age<sup><xref ref-type=\"bibr\" rid=\"CR63\">63</xref>–<xref ref-type=\"bibr\" rid=\"CR68\">68</xref></sup> and with our transported laboratory-tested control group (Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>).<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>Roadside TRAP- and FA-exposed rats differed from lab controls during juvenile reciprocal social interactions on several key parameters.</title><p><bold>a</bold> Roadside exposures did not affect levels of exploration during the social interaction assay for males, however <bold>b</bold> TRAP-exposed males spent significantly more time following or chasing the stimulus animal than did FA-exposed or lab controls. <bold>c</bold> Roadside-reared males showed typical levels of social sniffing but <bold>d</bold> there was a significant effect of group on anogenital sniffing, with post hoc trends suggesting that both roadside exposure groups spent more time anogenital sniffing compared to lab controls. <bold>e</bold> Both TRAP- and FA-exposed males spent more time self-grooming than lab controls. <bold>f</bold> Females of all groups exhibited comparable levels of exploration, but <bold>g</bold> TRAP-exposed females spent more time following or chasing than lab controls. <bold>h</bold> FA-exposed females spent significantly less time social sniffing relative to lab controls and <bold>i</bold> both roadside groups had elevated levels of anogenital sniffing. <bold>j</bold> Females of all groups displayed similar levels of self-grooming. *<italic>p</italic> < 0.05, one-way ANOVA followed by Tukey’s multiple comparisons test.</p></caption><graphic xlink:href=\"41398_2020_978_Fig4_HTML\" id=\"d30e1286\"/></fig></p><p id=\"Par33\">Social deficits, by an unusually high amount of time on the play parameter of following/chasing, were observed in both sexes (males Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4b</xref><italic>F</italic><sub>(2, 42)</sub> = 11.61, <italic>p</italic> < 0.001 and females Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4f</xref><italic>F</italic><sub>(2, 44)</sub> = 5.944, <italic>p</italic> < 0.006). Specifically, TRAP-exposed males spent more time following/chasing compared to FA-exposed males (<italic>p</italic> < 0.05) and laboratory controls (<italic>p</italic> < 0.001). FA-exposed males also trended to spend more time following/chasing compared to laboratory controls (<italic>p</italic> = 0.06). TRAP-exposed females exhibited a strong trend to spend more time following/chasing compared to FA-exposed females (<italic>p</italic> = 0.10) and TRAP-exposed females spent more time following/chasing compared to laboratory controls (<italic>p</italic> < 0.004). FA-exposed females did not differ on time spent following/chasing compared to laboratory controls (<italic>p</italic> = 0.356).</p><p id=\"Par34\">In females, exposure had a significant effect on time engaged in the key interaction metric of social sniffing, which includes nose-to-nose sniffing, neck and body sniffing, and other bouts of contact sniffing with the partner stimulus rat (females Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4g</xref><italic>F</italic><sub>(2, 44)</sub> = 3.264, <italic>p</italic> < 0.05). The TRAP and FA-exposed groups did not differ from one another (<italic>p</italic> = 0.650). Interestingly, FA-exposed (<italic>p</italic> = 0.040) females spent less time engaged in social sniffing compared to laboratory controls but the TRAP-exposed female group did not differ from laboratory controls (<italic>ns</italic>). In contrast, only a trending difference between groups was observed in the key metric of social sniffing in males (males Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c</xref><italic>F</italic><sub>(2, 42)</sub> = 2.622, <italic>p</italic> = 0.085).</p><p id=\"Par35\">Nose-to-anogenital sniffing time, when initiated by the subject rat, was significantly affected in both sexes (males Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4d</xref><italic>F</italic><sub>(2, 42)</sub> = 3.492, <italic>p</italic> = 0.040 and females Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4i</xref><italic>F</italic><sub>(2, 44)</sub> = 5.944, <italic>p</italic> < 0.006). TRAP and FA-exposed groups did not differ from one another (<italic>ns</italic>). Although neither the FA-exposed males (<italic>p</italic> = 0.079) nor the TRAP exposed males (<italic>p</italic> = 0.056) significantly differed compared to laboratory controls in anogenital sniffing upon post hoc analyses, trending differences were discovered. Whereas this parameter did not differ in the transport control group (Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>) suggesting the cause was the roadside exposure conditions and not the transport during gestation.</p><p id=\"Par36\">Time spent engaged in self-grooming differed between exposure groups in males (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4e</xref><italic>F</italic><sub>(2, 42)</sub> = 6.870, <italic>p</italic> < 0.004) but not females (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4j</xref><italic>F</italic><sub>(2, 44)</sub> = 0.6994, <italic>ns</italic>). Tukey’s multiple comparisons post hoc analysis revealed that both the TRAP (<italic>p</italic> = 0.049) and FA-exposed (<italic>p</italic> = 0.002) male groups exhibited higher self-grooming scores compared to laboratory controls. Social interaction metrics that did not differ between the transported group and laboratory control offspring are illustrated in Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref> and additional play metrics that did not differ between groups are summarized in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>.</p></sec><sec id=\"Sec20\"><title>Normal exploratory locomotor behavior in an open field arena</title><p id=\"Par37\">Motor abilities were tested in an open field assay, assessing cm of distance traveled using beam breaks and time spent in the center of the arena. FA- and TRAP-exposed juvenile male rats, as well as a cohort of male laboratory controls, exhibited no group differences in total activity (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5a</xref><italic>F</italic><sub>(2, 42)</sub> = 3.042, <italic>ns</italic>). As expected, all groups decreased activity over time (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5a</xref><italic>F</italic><sub>(4, 151)</sub> = 220.2, <italic>p</italic> < 0.0001). No treatment group differences were detected in center time measures in males (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5b</xref><italic>F</italic><sub>(2, 42)</sub> = 1.367, <italic>ns</italic>). Group effects were observed in FA- and TRAP-exposed juvenile female rats, as well as a cohort of female laboratory controls, in total activity (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5c</xref><italic>F</italic><sub>(2, 44)</sub> = 4.690, <italic>p</italic> < 0.02). There was not a significant difference in performance between TRAP- and FA-exposed rats, except at a single timepoint (20–25 min: <italic>p</italic> = 0.019). TRAP did not differ from the laboratory controls (<italic>ns</italic>) at any timepoint, while the FA-exposed group and lab controls differed at four timepoints upon post hoc analyses in females (5–10 min: <italic>p</italic> = 0.009; 10–15 min: <italic>p</italic> = 0.011; 15–20 min: <italic>p</italic> = 0.049; 20–25 min: <italic>p</italic> = 0.009). Group differences were detected in center time measures in females (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5d</xref><italic>F</italic><sub>(2, 44)</sub> = 10.39, <italic>p</italic> < 0.001). As expected, all groups decreased center time across the 30-min testing session (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5a</xref><italic>F</italic><sub>(3, 140)</sub> = 8.70, <italic>p</italic> < 0.0001). TRAP-exposed females exhibited lower time in the center compared to FA-exposed rats (<italic>p</italic> = 0.007). Both TRAP (0–5 min: <italic>p</italic> = 0.001; 10–15 min: <italic>p</italic> = 0.007) and FA-exposed (0–5 min: <italic>p</italic> = 0.030) females groups differed by lower center times compared to the laboratory controls upon post hoc analyses.<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>Atypical exploratory activity in a novel open field in rats exposed to roadside TRAP and FA.</title><p><bold>a</bold> Roadside exposures did not affect males’ gross locomotion or <bold>b</bold> time spent in the center during a 30-min exploration of a novel arena. <bold>c</bold> In females, there was a significant effect of group on distance moved, with trends suggesting that FA-exposed females covered more distance during the assay compared to lab controls. <bold>d</bold> TRAP-exposed females spent less time in the center than did FA-exposed females, and both TRAP- and FA-exposed females displayed significantly reduced center time relative to lab controls. *<italic>p</italic> < 0.05, repeated measures ANOVA followed by Tukey’s multiple comparisons test.</p></caption><graphic xlink:href=\"41398_2020_978_Fig5_HTML\" id=\"d30e1554\"/></fig></p></sec><sec id=\"Sec21\"><title>Intact object recognition and Pavlovian conditioning learning and memory behavior</title><p id=\"Par38\">Manual and automated scoring indicated both male TRAP- and FA-exposure groups spent more time investigating the novel object versus the familiar object, thereby exhibiting typical novel object preference (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6a</xref> TRAP-exposed, <italic>t</italic><sub>(1, 15)</sub> = 3.269, <italic>p</italic> < 0.006 and FA-exposed, <italic>t</italic><sub>(1, 15)</sub> = 3.081, <italic>p</italic> < 0.008). Times spent exploring the objects during the familiarization component were similar for both groups using mean ± SEM in that FA-exposed sniffing investigation times were 135.5 ± 15.2 s, and TRAP-exposed sniffing investigation times were 110.3 ± 12.1 s. Similarly, both female exposure groups spent more time investigating the novel object versus the familiar object, exhibiting typical novel object preference (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6c</xref> TRAP-exposed, <italic>t</italic><sub>(1, 12)</sub> = 4.316, <italic>p</italic> < 0.001 and FA-exposed, (<italic>t</italic><sub>(1, 12)</sub> = 3.720, <italic>p</italic> < 0.003). Thus, roadway exposure did not adversely affect object learning or short-term memory recall. This negative finding was not the result of a lack of participation or object investigation as times spent exploring the objects during the familiar exposure component, in females, were similar for both groups using mean ± SEM in that FA-exposed sniffing investigation times were 138.6 ± 6.8 s, and TRAP exposed sniffing investigation times were 117.0 ± 9.4 s. Object sniff times observed 60 min following familiarization with one object type in laboratory control subjects (males Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3a</xref><italic>t</italic>\n<sub>(1, 15)</sub> = 3.997, <italic>p</italic> = 0.001 and females Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3b</xref><italic>t</italic><sub>(1, 14)</sub> = 2.788, <italic>p</italic> = 0.015) illustrated typical novel object preference in groups run in our rat behavioral core when given the opportunity to investigate a novel and a familiar object.<fig id=\"Fig6\"><label>Fig. 6</label><caption><title>Learning and memory in roadside exposed rats.</title><p><bold>a</bold> Males exposed to TRAP or FA displayed intact novel object recognition as evidenced by spending significantly more time sniffing the novel object than the familiar object. <bold>b</bold> Exposure to TRAP did not affect contextual or cued fear memory in males and both TRAP and FA groups displayed high levels freezing day 1 post-training and to the cue presentation on day 3. <bold>c</bold> Both groups of roadside exposed females spent significantly more time investigating the novel object compared to the familiar object and <bold>d</bold> no group differences were observed in percent time freezing during the test of contextual and cued memory. <bold>a</bold>, <bold>c</bold> *<italic>p</italic> < 0.05, paired <italic>t</italic>-test, familiar vs. novel. <bold>b</bold>, <bold>d</bold> *<italic>p</italic> < 0.05, Day 1, 3: repeated measures ANOVA; Day 2: Student’s <italic>t</italic>-test, TRAP vs. FA.</p></caption><graphic xlink:href=\"41398_2020_978_Fig6_HTML\" id=\"d30e1668\"/></fig></p><p id=\"Par39\">Learning and memory were further evaluated using two measures of Pavlovian fear conditioning with a 24-h contextual component and a 48-h auditory cued fear conditioning. Significant main effects of time (males Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>b <italic>F</italic><sub>(1, 30)</sub> = 61.06, <italic>p</italic> < 0.0001 and females Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>d <italic>F</italic><sub>(1, 30)</sub> = 31.27, <italic>p</italic> < 0.0001) but not exposure group (males Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>b <italic>F</italic><sub>(1, 30)</sub> = 0.0213, <italic>ns</italic> and females Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>d <italic>F</italic><sub>(1, 30)</sub> = 0.3597, <italic>ns</italic>) or interaction (males Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>b <italic>F</italic><sub>(1, 30)</sub> = 0.0061, <italic>ns</italic> and females Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>d <italic>F</italic><sub>(1, 30)</sub> = 0.3785, <italic>ns</italic>) indicated that high levels of freezing were observed in both groups subsequent to the conditioned stimulus (CS)—unconditioned stimulus (UCS) pairings on the training day. Elevated post-training freezing in both exposure groups with no group difference in training freeze scores in males or females indicates no confounds and no deficits in the learning of the associations between the context stimuli and auditory cues. No difference in freezing scores was observed 24 h following CS-UCS training between TRAP- and FA-exposed subjects freezing scores in males (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6b</xref><italic>t</italic><sub>(1, 30)</sub> = 1.510, <italic>ns</italic>) or females (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6d</xref><italic>t</italic><sub>(1, 30)</sub> = 0.8535, <italic>ns</italic>) when placed in the context chamber from conditioning training with identical stimulus cues. Levels of freezing, between the pre- and post-cue presentation 48 h after training, revealed significant main effects of cue presentation (males Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>b <italic>F</italic><sub>(1, 30)</sub> = 112.1, <italic>p</italic> < 0.0001 and females Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>d <italic>F</italic><sub>(1, 30)</sub> = 47.86, <italic>p</italic> < 0.0001) but not exposure group (males Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>b <italic>F</italic><sub>(1, 30)</sub> = 0.0006, <italic>ns</italic> and females Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>d <italic>F</italic><sub>(1, 30)</sub> = 0.0484, <italic>ns</italic>) or interaction (males Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>b <italic>F</italic><sub>(1, 30)</sub> = 0.0370, <italic>ns</italic> and females Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>d <italic>F</italic><sub>(1, 30)</sub> = 0.0694, <italic>ns</italic>). Therefore, no group difference was found in freezing in response to the auditory cue between TRAP- and FA-exposed subjects when placed in the novel chamber with unique contextual cues (olfactory, visual, and textural). Freezing scores in laboratory control subjects observed 24 h following CS-UCS training compared to pre-training scores (males Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S4a</xref><italic>t</italic><sub>(1, 25)</sub> = 5.722, <italic>p</italic> < 0.0001 and females Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S4b</xref><italic>t</italic><sub>(1, 22)</sub> = 4.486, <italic>p</italic> < 0.001) illustrated typical fear responses in groups run in our rat behavioral core when placed in the context chamber from conditioning training with identical stimulus cues.</p></sec><sec id=\"Sec22\"><title>Transport stress does not cause the observed behavioral phenotypes</title><p id=\"Par40\">The effect of potential stress on the pregnant dam during the transport to the roadside exposure facility was ruled out as a causal mechanism for these physical and behavioral changes. Figure <xref rid=\"Fig7\" ref-type=\"fig\">7a–d</xref> show no delay in early physical development and neurological reflexes. This figure combines sexes as no sex difference was observed throughout the developmental outcomes (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). In transported and control offspring, all male and female subjects gained weight and grew in length over time (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7a</xref><sub>length</sub>\n<italic>F</italic><sub>(5, 173)</sub> = 846.2, <italic>p</italic> < 0.001; Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7b</xref><sub>weight</sub>\n<italic>F</italic><sub>(5, 255)</sub> = 1186, <italic>p</italic> < 0.001). There was a trend but no statistically significant effect of transport on body length (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7a</xref><italic>F</italic><sub>(1, 42)</sub> = 3.278, <italic>p</italic> = 0.077) or body weight (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7b</xref><italic>F</italic><sub>(1, 42)</sub> = 1.764, <italic>ns</italic>). Neurological reflexes, including the rooting and grasping reflexes, were normal in transported and control offspring (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7c</xref><sub>rooting</sub> Log-rank <italic>χ</italic><sup>2</sup><sub>(1)</sub> = 0.1716, <italic>ns</italic>; Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7d</xref><sub>grasping</sub> Log-rank <italic>χ</italic><sup>2</sup><sub>(1)</sub> <italic>=</italic> 00.00, <italic>ns</italic>). Figure <xref rid=\"Fig7\" ref-type=\"fig\">7e</xref> shows that there were no differences in PND 5 pup USV emissions across the transported and control offspring (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7e</xref><italic>t</italic><sub>(1, 38)</sub> = 0.4814, <italic>ns</italic>). No differences in total activity (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7f</xref><italic>F</italic><sub>(1, 42)</sub> = 0.0049, <italic>ns</italic>) or time spent in the center of the open field arena (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7g</xref><italic>F</italic><sub>(1, 42)</sub> = 0.4879, <italic>ns</italic>) were observed between the transported offspring compared to control group. Overall, no effect of transport alone was observed on offspring development. Pups born to dams that experienced a transport at approximately GD 14 exhibited no physical or behavioral abnormalities (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>) and exhibited no differences in social investigative events such as exploring, social sniffing, anogenital sniffing, following/chasing, or the repetitive behavior of self-grooming (Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>). Pups born to dams that experienced a transport at approximately GD 14 also exhibited no differences in social play point events such as pouncing, pinning, and pushing under or crawling over (Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>).<fig id=\"Fig7\"><label>Fig. 7</label><caption><title>No effect of gestational transport alone on offspring development and behavior.</title><p>Pups born to dams that experienced a transport event at approximately GD 14 exhibited no physical or behavioral abnormalities. <bold>a</bold> Body length and <bold>b</bold> body weight were typical throughout early life, as was the timing of the development of <bold>c</bold> rooting and <bold>d</bold> forelimb grasping reflexes. <bold>e</bold> Gestational transport did not affect the number of isolation-induced pup ultrasonic vocalizations at PND 5 and <bold>f</bold> juveniles exhibited similar exploratory activity in a novel open field as indicated by total distance moved and <bold>g</bold> time spent in the center. <bold>a</bold>, <bold>b</bold>, <bold>f</bold>, <bold>g</bold> *<italic>p</italic> < 0.05, repeated measures ANOVA. <bold>c</bold>, <bold>d</bold> *<italic>p</italic> < 0.05, Log-Rank (Mantel-Cox) test. <bold>e</bold> *<italic>p</italic> < 0.05, Student’s <italic>t</italic>-test.</p></caption><graphic xlink:href=\"41398_2020_978_Fig7_HTML\" id=\"d30e2058\"/></fig></p></sec></sec><sec id=\"Sec23\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par41\">Our goal was to corroborate human studies that have linked increased risk of NDDs to near roadway TRAP exposures. To do this, we developed an innovative exposure model that quantifies and delivers TRAP collected from a traffic tunnel to rats during both in utero and post-natal development. This design avoided limitations of single exposure paradigms, including requiring anesthesia and difficulties mimicking real-world mixtures of TRAP, while simultaneously leveraging earlier literature to yield consensus. Both roadside exposure groups had significantly delayed growth and development of psychomotor reflexes, displayed altered social interactions, and exhibited abnormal activity in an open field compared to lab controls. This is the first report that used carefully controlled subgroups to illustrate that developmental exposure to realistic near roadway exposures caused subtle but significant changes in developmental endpoints and functional outcomes (i.e., behavior). This confirms the theory suggested by epidemiological studies that in addition to TRAP, noise, vibration, and proximity to highways may be additional risk factors for NDDs in combination with genetic susceptibility or independently<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR69\">69</xref>–<xref ref-type=\"bibr\" rid=\"CR73\">73</xref></sup>. Our work presented herein is also a novel, important addition because, to our knowledge, this is the first nonclinical study that did not use high levels of particulate matter (PM), concentrated ambient ultrafine particles (CAPS), and/or diesel exhaust and discovered subtle but reportable behavioral outcomes. These findings support the need for further research delineating causal link(s) between exposure to TRAP and behavioral outcomes relevant to NDDs and adding to our understanding of the risks posed by air pollution to the developing nervous system.</p><p id=\"Par42\">Recently, a few well recognized laboratories have used reductionist experimental designs to investigate the effects of diesel exhaust<sup><xref ref-type=\"bibr\" rid=\"CR69\">69</xref>,<xref ref-type=\"bibr\" rid=\"CR70\">70</xref>,<xref ref-type=\"bibr\" rid=\"CR74\">74</xref>,<xref ref-type=\"bibr\" rid=\"CR75\">75</xref></sup> and particulate matter (sizeable and ultrafine)<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR76\">76</xref>–<xref ref-type=\"bibr\" rid=\"CR81\">81</xref></sup>, the components most implicated in mediating the neurotoxic effects of TRAP. We extended this published research with our innovative real-world exposure to a dynamic, complex mixture of components, noise, and vibration. Polluted tunnel air was delivered to subjects in the nearby exposure facility while control animals received thoroughly filtered air from a tunnel-adjacent area. Because behavioral outcomes vary by sex, time of year, vendor, and numerous additional variables, we ran, in parallel, a laboratory control group<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref>,<xref ref-type=\"bibr\" rid=\"CR82\">82</xref></sup>.</p><p id=\"Par43\">An important finding was that we observed no significant difference in litter size between TRAP, FA, and laboratory groups, which eliminated litter size as a potential explanatory variable for effects of near roadway exposure on pup growth and development. Yet, both roadside groups had significantly delayed growth and development of psychomotor reflexes, altered social interactions, and abnormal activity in an open field compared to laboratory controls. A potential explanation was that transport stress confounded our observations. However, we showed a complete absence of behavioral phenotypes resulting from the transport alone, strongly suggesting that adverse functional outcomes observed in the TRAP and FA groups were attributable to near roadway exposures. In both sexes of FA- and TRAP-exposed groups, we observed reduced isolation-induced 40-kHz pup ultrasonic vocalizations. Other atypical behaviors included juvenile social play behavior by the critical investigative parameter of anogenital sniffing and social play behavior of following/chasing. These data have direct translational implications as epidemiological studies directed at investigating ASD and NDDs have reported high levels of physical and developmental effects on health associated with the proximity of residence to heavily trafficked roads, using unique data sets from differing regional areas<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>–<xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR75\">75</xref>,<xref ref-type=\"bibr\" rid=\"CR83\">83</xref></sup>. Follow-up studies will need to delineate the effects of noise and vibration during pregnancy from those of TRAP on offspring development and behavior. These findings add to our understanding of the risks posed to the developing nervous system by living in close proximity to roadways and support the need for further research delineating causal link(s) between exposure to TRAP and behavioral outcomes relevant to NDDs.</p><p id=\"Par44\">We observed a strong trend toward reduced overall social sniffing in the TRAP- and FA-exposed groups. Social sniffing is a key investigative behavior that initiates numerous types of juvenile social play events such as following/chasing, pinning, pouncing, and rough and tumble play. The TRAP- and FA-exposed juveniles exhibited these deficits without confounding motor deficits. In addition, we observed a trend to elevated self-grooming in males, a well-reported, standardized restricted, repetitive behavior in rodents<sup><xref ref-type=\"bibr\" rid=\"CR84\">84</xref></sup>. Less social sniffing in dyadic interactions and elevated self-grooming are likely indicators of stress in both exposure groups.</p><p id=\"Par45\">In addition, others observed elevated self-grooming, repetitive behavior in mice. Similar findings of reduced reciprocal interactions following diesel exhaust exposure from prenatal embryonic day 0 to postnatal day 21 were recently reported in male mice<sup><xref ref-type=\"bibr\" rid=\"CR70\">70</xref></sup>. Chang et al. also reported their diesel exhaust exposure caused increased repeated entries in the T-maze test of spontaneous alternation, a learning and memory assay with the embedded ability to capture restricted behavior. We observed high levels of repetitive motor behavior in the TRAP-exposed group, using a low-order motor stereotypy measure of grooming. Spontaneous alternation in T or Y maze, which may be mimicking higher order restricted, repetitive behaviors, are easily observable in mice<sup><xref ref-type=\"bibr\" rid=\"CR85\">85</xref>–<xref ref-type=\"bibr\" rid=\"CR88\">88</xref></sup>.</p><p id=\"Par46\">Surprisingly, we did not observe deficits on our two standard assays of learning and memory, contextual and cued fear conditioning and novel object recognition, due to TRAP exposure. We hypothesized this behavioral domain to have a robust phenotype given earlier literature. Early postnatal life exposure to concentrated ambient ultrafine particles (CAPS) increased preference for immediate reward, a more complex type of cognition that assesses impulsivity using a fixed-ratio waiting-for-reward paradigm, in mice<sup><xref ref-type=\"bibr\" rid=\"CR80\">80</xref>,<xref ref-type=\"bibr\" rid=\"CR89\">89</xref></sup>. The discounting of delayed rewards in preclinical models is considered to be analogous to impulsivity and delay of gratification in humans and is relevant to ADHD. Follow-up investigations revealed that early postnatal exposures to CAPS caused sexually dimorphic impairments in fixed interval performance on an operant training task, with greater sensitivity in males, while adult exposures caused deficits in females, indicating dysfunctional learning and reduced behavioral flexibility in CAPS-exposed mice. CAPS exposure also impaired short-term memory on the novel object recognition memory task in both sexes<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR80\">80</xref></sup>. Collectively, these observations indicate dysfunctional learning and reduced behavioral flexibility in CAPS-exposed mice. The different results observed in the CAPS study versus our study may be due to (a) our tasks being limited to fear conditioning and novel object recognition because of limitations on the type of equipment that could be housed at the exposure facility, (b) that there is >20 million years of evolution that separate mouse and rat and there is likely species differences<sup><xref ref-type=\"bibr\" rid=\"CR90\">90</xref>,<xref ref-type=\"bibr\" rid=\"CR91\">91</xref></sup>, and/or (c) the variable intensities and concentrations of the exposures. Another limitation of our learning and memory data was that the laboratory control data were collected from cohorts other than those under study herein, thereby precluding direct comparisons of performance scores. We were, however, able to make observational assessments with the knowledge that the cohorts were all Sprague-Dawley rats of similar ages tested by the same experimenter using the same equipment following the standard experimental protocol and the laboratory controls were within our standard validation scores. In future studies, we plan on employing operant touchscreen testing, as performed by our laboratories in mice and rats<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref>,<xref ref-type=\"bibr\" rid=\"CR92\">92</xref>,<xref ref-type=\"bibr\" rid=\"CR93\">93</xref></sup>, which will allow for more direct comparisons of impulsivity via five choice serial reaction and continuous performance assays<sup><xref ref-type=\"bibr\" rid=\"CR94\">94</xref>–<xref ref-type=\"bibr\" rid=\"CR98\">98</xref></sup>. Other groups exposed rats in a highly trafficked location in Portugal to non-filtered air (NFA) during gestation and early life and found a significant decrease in object discrimination when compared to the group exposed to filtered air (FA), suggesting that the exposure to TRAP during the combined pre- and post-natal periods impaired short-term discriminative memory. Animals exposed during only pre- or post-natal period did not show impairment on this assay<sup><xref ref-type=\"bibr\" rid=\"CR99\">99</xref></sup>, similar to our findings. Another group found that ambient concentrated PM<sub>2.5</sub> exposure resulted in robust impairments in adult mice tested in the Barnes Maze, a hippocampal dependent spatial learning task. The PM<sub>2.5</sub>-exposed mice made more errors during training and took longer to reach the target during training trials and the memory retention test, indicating that chronic exposure to airborne fine particulate matter impaired hippocampal related learning and memory<sup><xref ref-type=\"bibr\" rid=\"CR100\">100</xref></sup>.</p><p id=\"Par47\">Multiple groups have reported strong associations between prenatal exposure to TRAP and developmental delays and/or NDDs. Since epidemiology studies are associative, rigorous experiments that test preclinical models in highly controlled environments are warranted. This is particularly pertinent for studies of TRAP, since for decades research has focused on the detrimental effects of tobacco and asthma/allergy-related illnesses. In conclusion, we developed and functionally validated an innovative preclinical model that recapitulated human studies that have linked developmental exposure to TRAP, or proximity to TRAP, and increased risk of NDDs. This confirmation of TRAP as an environmental risk factor for NDDs provides a rationale for controlling and minimizing exposures during critical periods of neurodevelopment thereby reducing the incidence of NDDs and/or decreasing the severity of symptoms. This study sets the stage for future mechanistic investigations to determine the mechanisms by which this risk factor interacts with NDD genes of susceptibility. It will also inform our understanding of the molecular pathophysiology of NDDs, which will be useful for identifying developmental windows of vulnerability and possible novel intervention and/or therapeutic strategies.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec24\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41398_2020_978_MOESM1_ESM.docx\"><caption><p>Berg et al. Supplementary Information</p></caption></media></supplementary-material></sec></sec></body><back><fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary Information</bold> accompanies this paper at (10.1038/s41398-020-00978-0).</p></sec><ack><title>Acknowledgements</title><p>This work was supported by the National Institutes of Health (R21 ES025570, R21 ES026515, and P30 ES023513). This project used core facilities supported by the UC Davis MIND Institute Intellectual and Developmental Disabilities Research Center (IDDRC; U54 HD079125).</p></ack><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Conflict of interest</title><p id=\"Par48\">The authors declare that they have no conflict of interest.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dall’Aglio</surname><given-names>L</given-names></name><etal/></person-group><article-title>The role of epigenetic modifications in neurodevelopmental disorders: a systematic review</article-title><source>Neurosci. Biobehav. Rev.</source><year>2018</year><volume>94</volume><fpage>17</fpage><lpage>30</lpage><pub-id pub-id-type=\"pmid\">30067938</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dalley</surname><given-names>JW</given-names></name><name><surname>Everitt</surname><given-names>BJ</given-names></name><name><surname>Robbins</surname><given-names>TW</given-names></name></person-group><article-title>Impulsivity, compulsivity, and top-down cognitive control</article-title><source>Neuron</source><year>2011</year><volume>69</volume><fpage>680</fpage><lpage>694</lpage><pub-id pub-id-type=\"pmid\">21338879</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Baio</surname><given-names>J</given-names></name><etal/></person-group><article-title>Prevalence of autism spectrum disorder among children aged 8 years—autism and developmental disabilities monitoring network, 11 sites, United States, 2014</article-title><source>MMWR Surveill. Summ.</source><year>2018</year><volume>67</volume><fpage>1</fpage><lpage>23</lpage><pub-id pub-id-type=\"pmid\">29701730</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sahin</surname><given-names>M</given-names></name><name><surname>Sur</surname><given-names>M</given-names></name></person-group><article-title>Genes, circuits, and precision therapies for autism and related neurodevelopmental disorders</article-title><source>Science</source><year>2015</year><volume>350</volume><fpage>aab3897</fpage><pub-id pub-id-type=\"pmid\">26472761</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hallmayer</surname><given-names>J</given-names></name><etal/></person-group><article-title>Genetic heritability and shared environmental factors among twin pairs with autism</article-title><source>Arch. Gen. Psychiatry</source><year>2011</year><volume>68</volume><fpage>1095</fpage><lpage>1102</lpage><pub-id pub-id-type=\"pmid\">21727249</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zuk</surname><given-names>O</given-names></name><name><surname>Hechter</surname><given-names>E</given-names></name><name><surname>Sunyaev</surname><given-names>SR</given-names></name><name><surname>Lander</surname><given-names>ES</given-names></name></person-group><article-title>The mystery of missing heritability: genetic interactions create phantom heritability</article-title><source>Proc. Natl Acad. Sci. USA</source><year>2012</year><volume>109</volume><fpage>1193</fpage><lpage>1198</lpage><pub-id pub-id-type=\"pmid\">22223662</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stamou</surname><given-names>M</given-names></name><name><surname>Streifel</surname><given-names>KM</given-names></name><name><surname>Goines</surname><given-names>PE</given-names></name><name><surname>Lein</surname><given-names>PJ</given-names></name></person-group><article-title>Neuronal connectivity as a convergent target of gene x environment interactions that confer risk for Autism Spectrum Disorders</article-title><source>Neurotoxicology Teratol.</source><year>2012</year><volume>36</volume><fpage>3</fpage><lpage>16</lpage></element-citation></ref><ref id=\"CR8\"><label>8.</label><mixed-citation publication-type=\"other\">Pessah, I. N. & Lein P. J. in: <italic>Autism: Current Theories and Evidence</italic>. (edsZimmerman A.), 409–428 (Humana Press, Totowa, NJ, 2008).</mixed-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hertz-Picciotto</surname><given-names>I</given-names></name><name><surname>Delwiche</surname><given-names>L</given-names></name></person-group><article-title>The rise in autism and the role of age at diagnosis</article-title><source>Epidemiology</source><year>2009</year><volume>20</volume><fpage>84</fpage><lpage>90</lpage><pub-id pub-id-type=\"pmid\">19234401</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grether</surname><given-names>JK</given-names></name><name><surname>Rosen</surname><given-names>NJ</given-names></name><name><surname>Smith</surname><given-names>KS</given-names></name><name><surname>Croen</surname><given-names>LA</given-names></name></person-group><article-title>Investigation of shifts in autism reporting in the California Department of Developmental Services</article-title><source>J. Autism Dev. Disord.</source><year>2009</year><volume>39</volume><fpage>1412</fpage><lpage>1419</lpage><pub-id pub-id-type=\"pmid\">19479197</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>King</surname><given-names>M</given-names></name><name><surname>Bearman</surname><given-names>P</given-names></name></person-group><article-title>Diagnostic change and the increased prevalence of autism</article-title><source>Int. J. Epidemiol.</source><year>2009</year><volume>38</volume><fpage>1224</fpage><lpage>1234</lpage><pub-id pub-id-type=\"pmid\">19737791</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Keil</surname><given-names>KP</given-names></name><name><surname>Lein</surname><given-names>PJ</given-names></name></person-group><article-title>DNA methylation: a mechanism linking environmental chemical exposures to risk of autism spectrum disorders?</article-title><source>Environ. Epigenet</source><year>2016</year><volume>2</volume><fpage>1</fpage><lpage>15</lpage></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kalkbrenner</surname><given-names>AE</given-names></name><etal/></person-group><article-title>Perinatal exposure to hazardous air pollutants and autism spectrum disorders at age 8</article-title><source>Epidemiology</source><year>2010</year><volume>21</volume><fpage>631</fpage><lpage>641</lpage><pub-id pub-id-type=\"pmid\">20562626</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kalkbrenner</surname><given-names>AE</given-names></name><etal/></person-group><article-title>Particulate matter exposure, prenatal and postnatal windows of susceptibility, and autism spectrum disorders</article-title><source>Epidemiology</source><year>2015</year><volume>26</volume><fpage>30</fpage><lpage>42</lpage><pub-id pub-id-type=\"pmid\">25286049</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kalkbrenner</surname><given-names>AE</given-names></name><etal/></person-group><article-title>Air Toxics in relation to autism diagnosis, phenotype, and severity in a U.S. family-based study</article-title><source>Environ. Health Perspect.</source><year>2018</year><volume>126</volume><fpage>037004</fpage><pub-id pub-id-type=\"pmid\">29553459</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McGuinn</surname><given-names>LA</given-names></name><etal/></person-group><article-title>Early life exposure to air pollution and autism spectrum disorder: findings from a multisite case-control study</article-title><source>Epidemiology</source><year>2020</year><volume>31</volume><fpage>103</fpage><lpage>114</lpage><pub-id pub-id-type=\"pmid\">31592868</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thygesen</surname><given-names>M</given-names></name><etal/></person-group><article-title>Exposure to air pollution in early childhood and the association with attention-deficit hyperactivity disorder</article-title><source>Environ. Res.</source><year>2020</year><volume>183</volume><fpage>108930</fpage><pub-id pub-id-type=\"pmid\">31810593</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ladd-Acosta</surname><given-names>C</given-names></name><etal/></person-group><article-title>Epigenetic marks of prenatal air pollution exposure found in multiple tissues relevant for child health</article-title><source>Environ. Int</source><year>2019</year><volume>126</volume><fpage>363</fpage><lpage>376</lpage><pub-id pub-id-type=\"pmid\">30826615</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Volk</surname><given-names>HE</given-names></name><etal/></person-group><article-title>Autism spectrum disorder: interaction of air pollution with the MET receptor tyrosine kinase gene</article-title><source>Epidemiology</source><year>2014</year><volume>25</volume><fpage>44</fpage><lpage>47</lpage><pub-id pub-id-type=\"pmid\">24240654</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Volk</surname><given-names>HE</given-names></name><name><surname>Lurmann</surname><given-names>F</given-names></name><name><surname>Penfold</surname><given-names>B</given-names></name><name><surname>Hertz-Picciotto</surname><given-names>I</given-names></name><name><surname>McConnell</surname><given-names>R</given-names></name></person-group><article-title>Traffic-related air pollution, particulate matter, and autism</article-title><source>JAMA Psychiatry</source><year>2013</year><volume>70</volume><fpage>71</fpage><lpage>77</lpage><pub-id pub-id-type=\"pmid\">23404082</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Becerra</surname><given-names>TA</given-names></name><name><surname>Wilhelm</surname><given-names>M</given-names></name><name><surname>Olsen</surname><given-names>J</given-names></name><name><surname>Cockburn</surname><given-names>M</given-names></name><name><surname>Ritz</surname><given-names>B</given-names></name></person-group><article-title>Ambient air pollution and autism in Los Angeles county, California</article-title><source>Environ. Health Perspect.</source><year>2013</year><volume>121</volume><fpage>380</fpage><lpage>386</lpage><pub-id pub-id-type=\"pmid\">23249813</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jung</surname><given-names>CR</given-names></name><name><surname>Lin</surname><given-names>YT</given-names></name><name><surname>Hwang</surname><given-names>BF</given-names></name></person-group><article-title>Air pollution and newly diagnostic autism spectrum disorders: a population-based cohort study in Taiwan</article-title><source>PLoS ONE</source><year>2013</year><volume>8</volume><fpage>e75510</fpage><pub-id pub-id-type=\"pmid\">24086549</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>von Ehrenstein</surname><given-names>OS</given-names></name><name><surname>Aralis</surname><given-names>H</given-names></name><name><surname>Cockburn</surname><given-names>M</given-names></name><name><surname>Ritz</surname><given-names>B</given-names></name></person-group><article-title>In utero exposure to toxic air pollutants and risk of childhood autism</article-title><source>Epidemiology</source><year>2014</year><volume>25</volume><fpage>851</fpage><lpage>858</lpage><pub-id pub-id-type=\"pmid\">25051312</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Windham</surname><given-names>GC</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Gunier</surname><given-names>R</given-names></name><name><surname>Croen</surname><given-names>LA</given-names></name><name><surname>Grether</surname><given-names>JK</given-names></name></person-group><article-title>Autism spectrum disorders in relation to distribution of hazardous air pollutants in the San Francisco bay area</article-title><source>Environ. Health Perspect.</source><year>2006</year><volume>114</volume><fpage>1438</fpage><lpage>1444</lpage><pub-id pub-id-type=\"pmid\">16966102</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Herr</surname><given-names>CE</given-names></name><etal/></person-group><article-title>Exposure to air pollution in critical prenatal time windows and IgE levels in newborns</article-title><source>Pediatr. Allergy Immunol.</source><year>2011</year><volume>22</volume><issue>1 Pt 1</issue><fpage>75</fpage><lpage>84</lpage><pub-id pub-id-type=\"pmid\">20609135</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kerin</surname><given-names>T</given-names></name><etal/></person-group><article-title>Association between air pollution exposure, cognitive and adaptive function, and ASD severity among children with autism spectrum disorder</article-title><source>J. Autism Dev. Disord.</source><year>2018</year><volume>48</volume><fpage>137</fpage><lpage>150</lpage><pub-id pub-id-type=\"pmid\">28921105</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Payne-Sturges</surname><given-names>DC</given-names></name><etal/></person-group><article-title>Healthy air, healthy brains: advancing air pollution policy to protect children’s health</article-title><source>Am. J. Public Health</source><year>2019</year><volume>109</volume><fpage>550</fpage><lpage>554</lpage><pub-id pub-id-type=\"pmid\">30789769</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Volk</surname><given-names>HE</given-names></name><name><surname>Hertz-Picciotto</surname><given-names>I</given-names></name><name><surname>Delwiche</surname><given-names>L</given-names></name><name><surname>Lurmann</surname><given-names>F</given-names></name><name><surname>McConnell</surname><given-names>R</given-names></name></person-group><article-title>Residential proximity to freeways and autism in the CHARGE study</article-title><source>Environ. Health Perspect.</source><year>2011</year><volume>119</volume><fpage>873</fpage><lpage>877</lpage><pub-id pub-id-type=\"pmid\">21156395</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chun</surname><given-names>H</given-names></name><name><surname>Leung</surname><given-names>C</given-names></name><name><surname>Wen</surname><given-names>SW</given-names></name><name><surname>McDonald</surname><given-names>J</given-names></name><name><surname>Shin</surname><given-names>HH</given-names></name></person-group><article-title>Maternal exposure to air pollution and risk of autism in children: a systematic review and meta-analysis</article-title><source>Environ. Pollut.</source><year>2020</year><volume>256</volume><fpage>113307</fpage><pub-id pub-id-type=\"pmid\">31733973</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Goodrich</surname><given-names>AJ</given-names></name><etal/></person-group><article-title>Joint effects of prenatal air pollutant exposure and maternal folic acid supplementation on risk of autism spectrum disorder</article-title><source>Autism Res.</source><year>2018</year><volume>11</volume><fpage>69</fpage><lpage>80</lpage><pub-id pub-id-type=\"pmid\">29120534</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chandrakumar</surname><given-names>A</given-names></name><name><surname>Willem’t Jong</surname><given-names>G</given-names></name></person-group><article-title>Maternal exposure to air pollution during pregnancy and autism spectrum disorder in offspring</article-title><source>JAMA Pediatr.</source><year>2019</year><volume>173</volume><fpage>697</fpage><lpage>698</lpage><pub-id pub-id-type=\"pmid\">31058952</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pagalan</surname><given-names>L</given-names></name><etal/></person-group><article-title>Association of prenatal exposure to air pollution with autism spectrum disorder</article-title><source>JAMA Pediatr.</source><year>2019</year><volume>173</volume><fpage>86</fpage><lpage>92</lpage><pub-id pub-id-type=\"pmid\">30452514</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pagalan</surname><given-names>L</given-names></name><name><surname>Brauer</surname><given-names>M</given-names></name><name><surname>Lanphear</surname><given-names>B</given-names></name></person-group><article-title>Maternal exposure to air pollution during pregnancy and autism spectrum disorder in offspring-reply</article-title><source>JAMA Pediatr.</source><year>2019</year><volume>173</volume><fpage>698</fpage><pub-id pub-id-type=\"pmid\">31058920</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bein</surname><given-names>KJ</given-names></name><name><surname>Wexler</surname><given-names>AS</given-names></name></person-group><article-title>A high-efficiency, low-biased method for extracting particulate matter from filter and impactor substrates</article-title><source>Atmos. Environ.</source><year>2014</year><volume>90</volume><fpage>87</fpage><lpage>95</lpage></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Plummer</surname><given-names>L</given-names></name><name><surname>Ham</surname><given-names>W</given-names></name><name><surname>Kleeman</surname><given-names>M</given-names></name><name><surname>Wexler</surname><given-names>A</given-names></name><name><surname>Pinkerton</surname><given-names>K</given-names></name></person-group><article-title>Influence of season and location on pulmonary response to California’s San Joaquin Valley airborne particulate matter</article-title><source>J. Toxicol. Environ. Health Part A</source><year>2012</year><volume>75</volume><fpage>253</fpage><lpage>271</lpage><pub-id pub-id-type=\"pmid\">22409489</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>KM</given-names></name><name><surname>Wexler</surname><given-names>AS</given-names></name></person-group><article-title>Evolution of particle number distribution near roadways. Part I: analysis of aerosol dynamics and its implications for engine emission measurement</article-title><source>Atmos. Environ.</source><year>2004</year><volume>38</volume><fpage>6643</fpage><lpage>6653</lpage></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>KM</given-names></name><etal/></person-group><article-title>Evolution of particle number distribution near roadways. Part III: Traffic analysis and on-road size resolved particulate emission factors</article-title><source>Atmos. Environ.</source><year>2005</year><volume>39</volume><fpage>4155</fpage><lpage>4166</lpage></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cory-Slechta</surname><given-names>DA</given-names></name><name><surname>Allen</surname><given-names>JL</given-names></name><name><surname>Conrad</surname><given-names>K</given-names></name><name><surname>Marvin</surname><given-names>E</given-names></name><name><surname>Sobolewski</surname><given-names>M</given-names></name></person-group><article-title>Developmental exposure to low level ambient ultrafine particle air pollution and cognitive dysfunction</article-title><source>Neurotoxicology</source><year>2018</year><volume>69</volume><fpage>217</fpage><lpage>231</lpage><pub-id pub-id-type=\"pmid\">29247674</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sobolewski</surname><given-names>M</given-names></name><etal/></person-group><article-title>Developmental exposures to ultrafine particle air pollution reduces early testosterone levels and adult male social novelty preference: Risk for children’s sex-biased neurobehavioral disorders</article-title><source>Neurotoxicology</source><year>2018</year><volume>68</volume><fpage>203</fpage><lpage>211</lpage><pub-id pub-id-type=\"pmid\">30144459</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carosino</surname><given-names>CM</given-names></name><etal/></person-group><article-title>Allergic airway inflammation is differentially exacerbated by daytime and nighttime ultrafine and submicron fine ambient particles: heme oxygenase-1 as an indicator of PM-mediated allergic inflammation</article-title><source>J. Toxicol. Environ. Health, Part A</source><year>2015</year><volume>78</volume><fpage>254</fpage><lpage>266</lpage><pub-id pub-id-type=\"pmid\">25679046</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Plummer</surname><given-names>LE</given-names></name><etal/></person-group><article-title>Pulmonary inflammatory effects of source-oriented particulate matter from California’s San Joaquin Valley</article-title><source>Atmos. Environ.</source><year>2015</year><volume>119</volume><fpage>174</fpage><lpage>181</lpage></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Papapostolou</surname><given-names>V</given-names></name><etal/></person-group><article-title>Development and characterization of an exposure generation system to investigate the health effects of particles from fresh and aged traffic emissions</article-title><source>Air Qual., Atmosphere Health</source><year>2012</year><volume>6</volume><fpage>419</fpage><lpage>429</lpage></element-citation></ref><ref id=\"CR43\"><label>43.</label><mixed-citation publication-type=\"other\">Bein K. J. et al. Roadway tunnels as real-time exposure systems: a case study. <italic>Environ Sci. Technol.</italic> (2020).</mixed-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hertz-Picciotto</surname><given-names>I</given-names></name><etal/></person-group><article-title>The CHARGE study: an epidemiologic investigation of genetic and environmental factors contributing to autism</article-title><source>Environ. health Perspect.</source><year>2006</year><volume>114</volume><fpage>1119</fpage><lpage>1125</lpage><pub-id pub-id-type=\"pmid\">16835068</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Patten</surname><given-names>KT</given-names></name><etal/></person-group><article-title>Effects of early life exposure to traffic-related air pollution on brain development in juvenile Sprague-Dawley rats</article-title><source>Transl. psychiatry</source><year>2020</year><volume>10</volume><fpage>1</fpage><lpage>12</lpage><pub-id pub-id-type=\"pmid\">32066695</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><mixed-citation publication-type=\"other\">Hood R. D. <italic>Handbook of Developmental Toxicology</italic> (CRC Press, USA, 1996).</mixed-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lazic</surname><given-names>SE</given-names></name><name><surname>Essioux</surname><given-names>L</given-names></name></person-group><article-title>Improving basic and translational science by accounting for litter-to-litter variation in animal models</article-title><source>BMC Neurosci.</source><year>2013</year><volume>14</volume><fpage>37</fpage><pub-id pub-id-type=\"pmid\">23522086</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Berg</surname><given-names>EL</given-names></name><etal/></person-group><article-title>Translational outcomes in a full gene deletion of ubiquitin protein ligase E3A rat model of Angelman syndrome</article-title><source>Transl. Psychiatry</source><year>2020</year><volume>10</volume><fpage>1</fpage><lpage>6</lpage><pub-id pub-id-type=\"pmid\">32066695</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Adhikari</surname><given-names>A</given-names></name><etal/></person-group><article-title>Cognitive deficits in the Snord116 deletion mouse model for Prader-Willi syndrome</article-title><source>Neurobiol. Learn. Mem.</source><year>2019</year><volume>165</volume><fpage>106874</fpage><pub-id pub-id-type=\"pmid\">29800646</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fox</surname><given-names>WM</given-names></name></person-group><article-title>Reflex-ontogeny and behavioural development of the mouse</article-title><source>Anim. Behav.</source><year>1965</year><volume>13</volume><fpage>234</fpage><lpage>241</lpage><pub-id pub-id-type=\"pmid\">5835840</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><mixed-citation publication-type=\"other\">Hofer, M. A., Shair, H. N. & Brunelli S. A. Ultrasonic vocalizations in rat and mouse pups. Curr. Protoc. Neurosci. <bold>Chapter 8:</bold> Unit 8.14 (2002).</mixed-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wohr</surname><given-names>M</given-names></name><name><surname>Schwarting</surname><given-names>RK</given-names></name></person-group><article-title>Maternal care, isolation-induced infant ultrasonic calling, and their relations to adult anxiety-related behavior in the rat</article-title><source>Behav. Neurosci.</source><year>2008</year><volume>122</volume><fpage>310</fpage><lpage>330</lpage><pub-id pub-id-type=\"pmid\">18410171</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><mixed-citation publication-type=\"other\">Brudzynski, S. <italic>Handbook of Ultrasonic Vocalization: A Window Into the Emotional Brain</italic>, vol. 25 (Academic Press, 2018).</mixed-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Berg</surname><given-names>EL</given-names></name><etal/></person-group><article-title>Developmental social communication deficits in the Shank3 rat model of Phelan-Mcdermid syndrome and autism spectrum disorder</article-title><source>Autism Res.</source><year>2018</year><volume>11</volume><fpage>587</fpage><lpage>601</lpage><pub-id pub-id-type=\"pmid\">29377611</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Copping</surname><given-names>NA</given-names></name><etal/></person-group><article-title>Neuronal overexpression of Ube3a isoform 2 causes behavioral impairments and neuroanatomical pathology relevant to 15q11.2-q13.3 duplication syndrome</article-title><source>Hum. Mol. Genet.</source><year>2017</year><volume>26</volume><fpage>3995</fpage><lpage>4010</lpage><pub-id pub-id-type=\"pmid\">29016856</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gulinello</surname><given-names>T</given-names></name><etal/></person-group><article-title>Rigor and reproducibility in rodent behavioral research</article-title><source>Neurobiol. Learn. Mem.</source><year>2019</year><volume>165</volume><fpage>106780</fpage><pub-id pub-id-type=\"pmid\">29307548</pub-id></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gompers</surname><given-names>AL</given-names></name><etal/></person-group><article-title>Germline Chd8 haploinsufficiency alters brain development in mouse</article-title><source>Nat. Neurosci.</source><year>2017</year><volume>20</volume><fpage>1062</fpage><lpage>1073</lpage><pub-id pub-id-type=\"pmid\">28671691</pub-id></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sukoff Rizzo</surname><given-names>SJ</given-names></name><name><surname>Silverman</surname><given-names>JL</given-names></name></person-group><article-title>Methodological considerations for optimizing and validating behavioral assays</article-title><source>Curr. Protoc. Mouse Biol.</source><year>2016</year><volume>6</volume><fpage>364</fpage><lpage>379</lpage><pub-id pub-id-type=\"pmid\">27906464</pub-id></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hofer</surname><given-names>MA</given-names></name><name><surname>Shair</surname><given-names>HN</given-names></name></person-group><article-title>Ultrasonic vocalization by rat pups during recovery from deep hypothermia</article-title><source>Dev. Psychobiol.</source><year>1992</year><volume>25</volume><fpage>511</fpage><lpage>528</lpage><pub-id pub-id-type=\"pmid\">1459345</pub-id></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shair</surname><given-names>HN</given-names></name><name><surname>Brunelli</surname><given-names>SA</given-names></name><name><surname>Masmela</surname><given-names>JR</given-names></name><name><surname>Boone</surname><given-names>E</given-names></name><name><surname>Hofer</surname><given-names>MA</given-names></name></person-group><article-title>Social, thermal, and temporal influences on isolation-induced and maternally potentiated ultrasonic vocalizations of rat pups</article-title><source>Dev. Psychobiol.</source><year>2003</year><volume>42</volume><fpage>206</fpage><lpage>222</lpage><pub-id pub-id-type=\"pmid\">12555284</pub-id></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Allin</surname><given-names>JT</given-names></name><name><surname>Banks</surname><given-names>EM</given-names></name></person-group><article-title>Effects of temperature on ultrasound production by infant albino rats</article-title><source>Dev. Psychobiol.</source><year>1971</year><volume>4</volume><fpage>149</fpage><lpage>156</lpage><pub-id pub-id-type=\"pmid\">5162545</pub-id></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oswalt</surname><given-names>GL</given-names></name><name><surname>Meier</surname><given-names>GW</given-names></name></person-group><article-title>Olfactory, thermal, and tactual influences on infantile ultrasonic vocalization in rats</article-title><source>Dev. Psychobiol.</source><year>1975</year><volume>8</volume><fpage>129</fpage><lpage>135</lpage><pub-id pub-id-type=\"pmid\">1225689</pub-id></element-citation></ref><ref id=\"CR63\"><label>63.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Raza</surname><given-names>S</given-names></name><etal/></person-group><article-title>Effects of prenatal exposure to valproic acid on the development of juvenile-typical social play in rats</article-title><source>Behav. Pharm.</source><year>2015</year><volume>26</volume><fpage>707</fpage><lpage>719</lpage></element-citation></ref><ref id=\"CR64\"><label>64.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thor</surname><given-names>DH</given-names></name><name><surname>Holloway</surname><given-names>WR</given-names><suffix>Jr.</suffix></name></person-group><article-title>Social play in juvenile rats: a decade of methodological and experimental research</article-title><source>Neurosci. Biobehav. Rev.</source><year>1984</year><volume>8</volume><fpage>455</fpage><lpage>464</lpage><pub-id pub-id-type=\"pmid\">6514252</pub-id></element-citation></ref><ref id=\"CR65\"><label>65.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vanderschuren</surname><given-names>LJ</given-names></name><name><surname>Niesink</surname><given-names>RJ</given-names></name><name><surname>Van Ree</surname><given-names>JM</given-names></name></person-group><article-title>The neurobiology of social play behavior in rats</article-title><source>Neurosci. Biobehav. Rev.</source><year>1997</year><volume>21</volume><fpage>309</fpage><lpage>326</lpage><pub-id pub-id-type=\"pmid\">9168267</pub-id></element-citation></ref><ref id=\"CR66\"><label>66.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ku</surname><given-names>KM</given-names></name><name><surname>Weir</surname><given-names>RK</given-names></name><name><surname>Silverman</surname><given-names>JL</given-names></name><name><surname>Berman</surname><given-names>RF</given-names></name><name><surname>Bauman</surname><given-names>MD</given-names></name></person-group><article-title>Behavioral phenotyping of juvenile long-evans and Sprague-Dawley rats: implications for preclinical models of autism spectrum disorders</article-title><source>PLoS ONE</source><year>2016</year><volume>11</volume><fpage>e0158150</fpage><pub-id pub-id-type=\"pmid\">27351457</pub-id></element-citation></ref><ref id=\"CR67\"><label>67.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Panksepp</surname><given-names>J</given-names></name></person-group><article-title>The ontogeny of play in rats</article-title><source>Dev. Psychobiol.</source><year>1981</year><volume>14</volume><fpage>327</fpage><lpage>332</lpage><pub-id pub-id-type=\"pmid\">7250521</pub-id></element-citation></ref><ref id=\"CR68\"><label>68.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Panksepp</surname><given-names>J</given-names></name><name><surname>Beatty</surname><given-names>WW</given-names></name></person-group><article-title>Social deprivation and play in rats</article-title><source>Behav. Neural Biol.</source><year>1980</year><volume>30</volume><fpage>197</fpage><lpage>206</lpage><pub-id pub-id-type=\"pmid\">7447871</pub-id></element-citation></ref><ref id=\"CR69\"><label>69.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Costa</surname><given-names>LG</given-names></name><name><surname>Chang</surname><given-names>YC</given-names></name><name><surname>Cole</surname><given-names>TB</given-names></name></person-group><article-title>Developmental neurotoxicity of traffic-related air pollution: focus on autism</article-title><source>Curr. Environ. Health Rep.</source><year>2017</year><volume>4</volume><fpage>156</fpage><lpage>165</lpage><pub-id pub-id-type=\"pmid\">28417440</pub-id></element-citation></ref><ref id=\"CR70\"><label>70.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chang</surname><given-names>YC</given-names></name><name><surname>Cole</surname><given-names>TB</given-names></name><name><surname>Costa</surname><given-names>LG</given-names></name></person-group><article-title>Prenatal and early-life diesel exhaust exposure causes autism-like behavioral changes in mice</article-title><source>Part Fibre Toxicol.</source><year>2018</year><volume>15</volume><fpage>18</fpage><pub-id pub-id-type=\"pmid\">29678176</pub-id></element-citation></ref><ref id=\"CR71\"><label>71.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lyall</surname><given-names>K</given-names></name><etal/></person-group><article-title>The changing epidemiology of autism spectrum disorders</article-title><source>Annu. Rev. Public Health</source><year>2017</year><volume>38</volume><fpage>81</fpage><lpage>102</lpage><pub-id pub-id-type=\"pmid\">28068486</pub-id></element-citation></ref><ref id=\"CR72\"><label>72.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Costa</surname><given-names>LG</given-names></name><etal/></person-group><article-title>Neurotoxicity of traffic-related air pollution</article-title><source>Neurotoxicology</source><year>2017</year><volume>59</volume><fpage>133</fpage><lpage>139</lpage><pub-id pub-id-type=\"pmid\">26610921</pub-id></element-citation></ref><ref id=\"CR73\"><label>73.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>D</given-names></name><etal/></person-group><article-title>The joint effect of air pollution exposure and copy number variation on risk for autism</article-title><source>Autism Res.</source><year>2017</year><volume>10</volume><fpage>1470</fpage><lpage>1480</lpage><pub-id pub-id-type=\"pmid\">28448694</pub-id></element-citation></ref><ref id=\"CR74\"><label>74.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Block</surname><given-names>ML</given-names></name><etal/></person-group><article-title>The outdoor air pollution and brain health workshop</article-title><source>Neurotoxicology</source><year>2012</year><volume>33</volume><fpage>972</fpage><lpage>984</lpage><pub-id pub-id-type=\"pmid\">22981845</pub-id></element-citation></ref><ref id=\"CR75\"><label>75.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hansen</surname><given-names>CA</given-names></name><name><surname>Barnett</surname><given-names>AG</given-names></name><name><surname>Pritchard</surname><given-names>G</given-names></name></person-group><article-title>The effect of ambient air pollution during early pregnancy on fetal ultrasonic measurements during mid-pregnancy</article-title><source>Environ. Health Perspect.</source><year>2008</year><volume>116</volume><fpage>362</fpage><lpage>369</lpage><pub-id pub-id-type=\"pmid\">18335104</pub-id></element-citation></ref><ref id=\"CR76\"><label>76.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Allen</surname><given-names>JL</given-names></name><etal/></person-group><article-title>Early postnatal exposure to ultrafine particulate matter air pollution: persistent ventriculomegaly, neurochemical disruption, and glial activation preferentially in male mice</article-title><source>Environ. Health Perspect.</source><year>2014</year><volume>122</volume><fpage>939</fpage><lpage>945</lpage><pub-id pub-id-type=\"pmid\">24901756</pub-id></element-citation></ref><ref id=\"CR77\"><label>77.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Allen</surname><given-names>JL</given-names></name><etal/></person-group><article-title>Developmental exposure to concentrated ambient ultrafine particulate matter air pollution in mice results in persistent and sex-dependent behavioral neurotoxicity and glial activation</article-title><source>Toxicol. Sci.</source><year>2014</year><volume>140</volume><fpage>160</fpage><lpage>178</lpage><pub-id pub-id-type=\"pmid\">24690596</pub-id></element-citation></ref><ref id=\"CR78\"><label>78.</label><mixed-citation publication-type=\"other\">Zhao Y. et al. Field evaluation of the versatile aerosol concentration enrichment system (VACES) particle concentrator coupled to the rapid single-particle mass spectrometer (RSMS-3). <italic>J. Geophys. Res. Atmos.</italic>10.1029/2004JD004644 (2005).</mixed-citation></ref><ref id=\"CR79\"><label>79.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Breysse</surname><given-names>PN</given-names></name><etal/></person-group><article-title>US EPA particulate matter research centers: summary of research results for 2005–2011</article-title><source>Air Qual. Atmos. Health</source><year>2013</year><volume>6</volume><fpage>333</fpage><lpage>355</lpage></element-citation></ref><ref id=\"CR80\"><label>80.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Allen</surname><given-names>JL</given-names></name><etal/></person-group><article-title>Developmental neurotoxicity of inhaled ambient ultrafine particle air pollution: Parallels with neuropathological and behavioral features of autism and other neurodevelopmental disorders</article-title><source>Neurotoxicology</source><year>2017</year><volume>59</volume><fpage>140</fpage><lpage>154</lpage><pub-id pub-id-type=\"pmid\">26721665</pub-id></element-citation></ref><ref id=\"CR81\"><label>81.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Allen</surname><given-names>JL</given-names></name><etal/></person-group><article-title>Consequences of developmental exposure to concentrated ambient ultrafine particle air pollution combined with the adult paraquat and maneb model of the Parkinson’s disease phenotype in male mice</article-title><source>Neurotoxicology</source><year>2014</year><volume>41</volume><fpage>80</fpage><lpage>88</lpage><pub-id pub-id-type=\"pmid\">24486957</pub-id></element-citation></ref><ref id=\"CR82\"><label>82.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Silverman</surname><given-names>JL</given-names></name><name><surname>Yang</surname><given-names>M</given-names></name><name><surname>Lord</surname><given-names>C</given-names></name><name><surname>Crawley</surname><given-names>JN</given-names></name></person-group><article-title>Behavioural phenotyping assays for mouse models of autism</article-title><source>Nat. Rev. Neurosci.</source><year>2010</year><volume>11</volume><fpage>490</fpage><lpage>502</lpage><pub-id pub-id-type=\"pmid\">20559336</pub-id></element-citation></ref><ref id=\"CR83\"><label>83.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Currie</surname><given-names>J</given-names></name><name><surname>Neidell</surname><given-names>M</given-names></name><name><surname>Schmieder</surname><given-names>JF</given-names></name></person-group><article-title>Air pollution and infant health: Lessons from New Jersey</article-title><source>J. Health Econ.</source><year>2009</year><volume>28</volume><fpage>688</fpage><lpage>703</lpage><pub-id pub-id-type=\"pmid\">19328569</pub-id></element-citation></ref><ref id=\"CR84\"><label>84.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kalueff</surname><given-names>AV</given-names></name><etal/></person-group><article-title>Neurobiology of rodent self-grooming and its value for translational neuroscience</article-title><source>Nat. Rev. Neurosci.</source><year>2016</year><volume>17</volume><fpage>45</fpage><lpage>59</lpage><pub-id pub-id-type=\"pmid\">26675822</pub-id></element-citation></ref><ref id=\"CR85\"><label>85.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sukoff Rizzo</surname><given-names>SJ</given-names></name><etal/></person-group><article-title>Assessing healthspan and lifespan measures in aging mice: optimization of testing protocols, replicability, and rater reliability</article-title><source>Curr. Protoc. Mouse Biol.</source><year>2018</year><volume>8</volume><fpage>e45</fpage><pub-id pub-id-type=\"pmid\">29924918</pub-id></element-citation></ref><ref id=\"CR86\"><label>86.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sukoff Rizzo</surname><given-names>SJ</given-names></name><name><surname>Crawley</surname><given-names>JN</given-names></name></person-group><article-title>Behavioral phenotyping assays for genetic mouse models of neurodevelopmental, neurodegenerative, and psychiatric disorders</article-title><source>Annu Rev. Anim. Biosci.</source><year>2017</year><volume>5</volume><fpage>371</fpage><lpage>389</lpage><pub-id pub-id-type=\"pmid\">28199172</pub-id></element-citation></ref><ref id=\"CR87\"><label>87.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sukoff Rizzo</surname><given-names>SJ</given-names></name><etal/></person-group><article-title>Behavioral characterization of striatal-enriched protein tyrosine phosphatase (STEP) knockout mice</article-title><source>Genes Brain Behav.</source><year>2014</year><volume>13</volume><fpage>643</fpage><lpage>652</lpage><pub-id pub-id-type=\"pmid\">25103464</pub-id></element-citation></ref><ref id=\"CR88\"><label>88.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sukoff Rizzo</surname><given-names>SJ</given-names></name><name><surname>McTighe</surname><given-names>S</given-names></name><name><surname>McKinzie</surname><given-names>DL</given-names></name></person-group><article-title>Genetic background and sex: impact on generalizability of research findings in pharmacology studies</article-title><source>Handb. Exp. Pharm.</source><year>2019</year><volume>257</volume><fpage>147</fpage><lpage>162</lpage></element-citation></ref><ref id=\"CR89\"><label>89.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Allen</surname><given-names>JL</given-names></name><etal/></person-group><article-title>Developmental exposure to concentrated ambient particles and preference for immediate reward in mice</article-title><source>Environ. Health Perspect.</source><year>2013</year><volume>121</volume><fpage>32</fpage><lpage>38</lpage><pub-id pub-id-type=\"pmid\">23063827</pub-id></element-citation></ref><ref id=\"CR90\"><label>90.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Iannaccone</surname><given-names>PM</given-names></name><name><surname>Jacob</surname><given-names>HJ</given-names></name></person-group><article-title>Rats!</article-title><source>Dis. Model. Mech.</source><year>2009</year><volume>2</volume><fpage>206</fpage><lpage>210</lpage><pub-id pub-id-type=\"pmid\">19407324</pub-id></element-citation></ref><ref id=\"CR91\"><label>91.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parker</surname><given-names>CC</given-names></name><etal/></person-group><article-title>Rats are the smart choice: rationale for a renewed focus on rats in behavioral genetics</article-title><source>Neuropharmacology</source><year>2014</year><volume>76 Pt B</volume><fpage>250</fpage><lpage>258</lpage><pub-id pub-id-type=\"pmid\">23791960</pub-id></element-citation></ref><ref id=\"CR92\"><label>92.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Copping</surname><given-names>NA</given-names></name><etal/></person-group><article-title>Touchscreen learning deficits and normal social approach behavior in the Shank3B model of Phelan-McDermid Syndrome and autism</article-title><source>Neuroscience</source><year>2017</year><volume>345</volume><fpage>155</fpage><lpage>165</lpage><pub-id pub-id-type=\"pmid\">27189882</pub-id></element-citation></ref><ref id=\"CR93\"><label>93.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Petkova</surname><given-names>SP</given-names></name><etal/></person-group><article-title>Cyclin D2-knock-out mice with attenuated dentate gyrus neurogenesis have robust deficits in long-term memory formation</article-title><source>Sci. Rep.</source><year>2020</year><volume>10</volume><fpage>1</fpage><lpage>16</lpage><pub-id pub-id-type=\"pmid\">31913322</pub-id></element-citation></ref><ref id=\"CR94\"><label>94.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bhakta</surname><given-names>SG</given-names></name><name><surname>Young</surname><given-names>JW</given-names></name></person-group><article-title>The 5 choice continuous performance test (5C-CPT): a novel tool to assess cognitive control across species</article-title><source>J. Neurosci. methods</source><year>2017</year><volume>292</volume><fpage>53</fpage><lpage>60</lpage><pub-id pub-id-type=\"pmid\">28754432</pub-id></element-citation></ref><ref id=\"CR95\"><label>95.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cope</surname><given-names>ZA</given-names></name><name><surname>Young</surname><given-names>JW</given-names></name></person-group><article-title>The five-choice continuous performance task (5C-CPT): a cross-species relevant paradigm for assessment of vigilance and response inhibition in rodents</article-title><source>Curr. Protoc. Neurosci.</source><year>2017</year><volume>78</volume><fpage>9</fpage><lpage>56</lpage><pub-id pub-id-type=\"pmid\">28046200</pub-id></element-citation></ref><ref id=\"CR96\"><label>96.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lustig</surname><given-names>C</given-names></name><name><surname>Kozak</surname><given-names>R</given-names></name><name><surname>Sarter</surname><given-names>M</given-names></name><name><surname>Young</surname><given-names>JW</given-names></name><name><surname>Robbins</surname><given-names>TW</given-names></name></person-group><article-title>CNTRICS final animal model task selection: control of attention</article-title><source>Neurosci. Biobehav. Rev.</source><year>2013</year><volume>37</volume><issue>9 Pt B</issue><fpage>2099</fpage><lpage>2110</lpage><pub-id pub-id-type=\"pmid\">22683929</pub-id></element-citation></ref><ref id=\"CR97\"><label>97.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McKenna</surname><given-names>BS</given-names></name><name><surname>Young</surname><given-names>JW</given-names></name><name><surname>Dawes</surname><given-names>SE</given-names></name><name><surname>Asgaard</surname><given-names>GL</given-names></name><name><surname>Eyler</surname><given-names>LT</given-names></name></person-group><article-title>Bridging the bench to bedside gap: validation of a reverse-translated rodent continuous performance test using functional magnetic resonance imaging</article-title><source>Psychiatry Res</source><year>2013</year><volume>212</volume><fpage>183</fpage><lpage>191</lpage><pub-id pub-id-type=\"pmid\">23570915</pub-id></element-citation></ref><ref id=\"CR98\"><label>98.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cope</surname><given-names>ZA</given-names></name><etal/></person-group><article-title>Premature responses in the five-choice serial reaction time task reflect rodents’ temporal strategies: evidence from no-light and pharmacological challenges</article-title><source>Psychopharmacology</source><year>2016</year><volume>233</volume><fpage>3513</fpage><lpage>3525</lpage><pub-id pub-id-type=\"pmid\">27534540</pub-id></element-citation></ref><ref id=\"CR99\"><label>99.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zanchi</surname><given-names>AC</given-names></name><etal/></person-group><article-title>Pre and post-natal exposure to ambient level of air pollution impairs memory of rats: the role of oxidative stress</article-title><source>Inhal. Toxicol.</source><year>2010</year><volume>22</volume><fpage>910</fpage><lpage>918</lpage><pub-id pub-id-type=\"pmid\">20569119</pub-id></element-citation></ref><ref id=\"CR100\"><label>100.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fonken</surname><given-names>LK</given-names></name><etal/></person-group><article-title>Air pollution impairs cognition, provokes depressive-like behaviors and alters hippocampal cytokine expression and morphology</article-title><source>Mol. Psychiatry</source><year>2011</year><volume>16</volume><fpage>987</fpage><lpage>995</lpage><pub-id pub-id-type=\"pmid\">21727897</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807889</article-id><article-id pub-id-type=\"pmc\">PMC7431543</article-id><article-id pub-id-type=\"publisher-id\">70866</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70866-6</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Sleep disturbances as risk factors for suicidal thoughts and behaviours: a meta-analysis of longitudinal studies</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Harris</surname><given-names>Lauren M.</given-names></name><address><email>harris@psy.fsu.edu</email></address><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><contrib contrib-type=\"author\"><name><surname>Huang</surname><given-names>Xieyining</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><contrib contrib-type=\"author\"><name><surname>Linthicum</surname><given-names>Kathryn P.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><contrib contrib-type=\"author\"><name><surname>Bryen</surname><given-names>Chloe P.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ribeiro</surname><given-names>Jessica D.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><aff id=\"Aff1\"><institution-wrap><institution-id institution-id-type=\"GRID\">grid.255986.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0472 0419</institution-id><institution>Department of Psychology, </institution><institution>Florida State University, </institution></institution-wrap>1107 W. Call St., Tallahassee, FL 32306-4301 USA </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13888</elocation-id><history><date date-type=\"received\"><day>1</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>30</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">In recent years, there has been a growing interest in understanding the relationship between sleep and suicide. Although sleep disturbances are commonly cited as critical risk factors for suicidal thoughts and behaviours, it is unclear to what degree sleep disturbances confer risk for suicide. The aim of this meta-analysis was to clarify the extent to which sleep disturbances serve as risk factors (i.e., longitudinal correlates) for suicidal thoughts and behaviours. Our analyses included 156 total effects drawn from 42 studies published between 1982 and 2019. We used a random effects model to analyse the overall effects of sleep disturbances on suicidal ideation, attempts, and death. We additionally explored potential moderators of these associations. Our results indicated that sleep disturbances are statistically significant, yet weak, risk factors for suicidal thoughts and behaviours. The strongest associations were found for insomnia, which significantly predicted suicide ideation (OR 2.10 [95% CI 1.83–2.41]), and nightmares, which significantly predicted suicide attempt (OR 1.81 [95% CI 1.12–2.92]). Given the low base rate of suicidal behaviours, our findings raise questions about the practicality of relying on sleep disturbances as warning signs for imminent suicide risk. Future research is necessary to uncover the causal mechanisms underlying the relationship between sleep disturbances and suicide.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Psychology</kwd><kwd>Risk factors</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Sleep is fundamental to survival<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. A single poor night of sleep can result in mood changes<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>, worsening executive function<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>, and memory impairment<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. Chronic sleep disturbances have been linked to increased risk for depression<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>, bipolar disorder<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>, and anxiety<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. A growing body of research has also uncovered a link between sleep disturbances and suicide.</p><p id=\"Par3\">The notion that sleep disturbances contribute to suicide risk is gaining momentum. Sleep disturbance is commonly considered a warning sign for suicide<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>, and associations between sleep disturbances and suicidal thoughts and behaviours are consistently detected<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>–<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. These associations are notable for several reasons. First, sleep problems are highly prevalent<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>–<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. Approximately one-third of adults experience insomnia symptoms, with 6–10% meeting criteria for insomnia disorder<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. To accurately assess risk, it is critical to determine the magnitude of the association between sleep disturbances and suicide. Second, sleep disturbances, unlike many other suicide risk factors, are modifiable. Sleep is already an intervention target in many mainstream therapeutic approaches, and treatments for sleep disturbances have been firmly established<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. If sleep disturbances are reliably shown to be risk factors for suicide, sleep interventions could be leveraged as suicidality interventions. Third, sleep is an intervention target with relatively low stigma<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>, especially compared to suicidality<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Addressing suicidality by targeting sleep may increase the likelihood that at-risk individuals will seek treatment.</p><p id=\"Par4\">Until recently, research on the relationship between sleep and suicide has been predominantly cross-sectional<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. Because risk factors must <italic>precede</italic> outcomes of interest<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>, cross-sectional evidence is insufficient to conclude that disturbed sleep is a risk factor for suicide. While longitudinal studies are filling this critical gap in the literature, results are mixed. Some studies have found large effects of sleep disturbances on suicide risk<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref>–<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, but others have found smaller<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref>–<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup> or nonsignificant effects<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref>–<xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. These discrepancies raise questions about the extent to which sleep disturbances confer risk for suicidal thoughts and behaviours. Moreover, these studies cannot provide information about causal mechanisms. Although risk factors are typically assumed to play a causal role in the outcome of interest, other variables may be responsible for observed longitudinal associations. For sleep disturbances to be considered <italic>causal</italic> risk factors for suicide, studies must examine whether manipulating sleep leads to systematic differences in suicide-related outcomes.</p><p id=\"Par5\">It is also unclear whether certain sleep disturbances are stronger predictors of suicidal thoughts and behaviours. Many studies focus on insomnia <sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref>,<xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>; others examine nightmares<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>, daytime sleepiness<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, total sleep time<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>, and nonspecific or undifferentiated categories like “sleep problems”<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. Even among studies that examine the same category of sleep disturbance, effect sizes range widely. Perhaps as a result of these inconsistent findings, the existing clinical guidelines are relatively nonspecific. Clinicians must rely on indicators like “unable to sleep” or “sleeping all the time” as warning signs for suicidal behaviours<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Given the seriousness of managing suicide risk, it is critical that clinical guidelines are clear so clinicians can make informed decisions about how to maintain their patients’ safety.</p><p id=\"Par6\">Measurement of sleep disturbances also varies across studies. Some studies use self-report scales<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>; others use clinical interviews<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>, and recent studies have begun to evaluate objective sleep parameters using actigraphy<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup> and polysomnography<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. While novel methodologies refine the measurement of sleep disturbances, the comparative utility of objective versus subjective measures is unclear. Some evidence suggests that objective and subjective sleep measures are highly correlated<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>, whereas other studies find that individuals who present with subjective sleep complaints may not demonstrate objective evidence of disturbed sleep<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. Therefore, their associations with suicide risk may be discrepant. Studies examining the relationship between objective sleep measures and suicide risk remain rare, but recent evidence indicates that both subjective and objective measures significantly predict risk, with similar effect sizes<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. Given the cost and inconvenience of continuous sleep monitoring<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>, scalable subjective self-report measures may be preferable for routine monitoring of sleep disturbances.</p><p id=\"Par7\">Follow-up intervals also vary, ranging from one day<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup> to ten years<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup> for suicidal ideation, one month<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup> to eight years<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup> for suicide attempt, and one week<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup> to up to 50 years<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup> for suicide death. As clinicians are often tasked with identifying risk in the very short term, the most useful risk factors would accurately indicate imminent risk. It is critical to examine whether the effect of sleep disturbances on suicide risk varies with respect to time interval.</p><p id=\"Par8\">In short, the existing literature raises questions about the extent to which sleep disturbances serve as risk factors for future suicidal thoughts and behaviours. Although some have endeavoured to provide quantitative summaries of this literature<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>, these efforts have focused predominately on synthesizing cross-sectional research. Accordingly, it remains unclear whether sleep disturbances confer risk for suicidal thoughts and behaviours or whether they simply represent a correlate of those experiences. Advancing knowledge toward this end is critical to improving suicide prediction and prevention efforts.</p><p id=\"Par9\">The objective of this study is to substantively advance our understanding of the link between sleep disturbance and suicidal thoughts and behaviours. Using meta-analytic methods, the present study advances our knowledge in five ways. First, we will summarize the longitudinal literature on the relationship between sleep disturbances and suicide. Second, we will meta-analyse categories of sleep disturbances to determine whether certain categories (e.g., insomnia, nightmares, sleep quality) are stronger risk factors for suicide-related outcomes. Third, we will examine whether effects vary across outcomes (i.e., suicide ideation, attempts, or death). Fourth, we will evaluate the influence of potential moderators including study publication date, follow-up length, sample severity, and sleep measure type. Given recent methodological advances in the measurement of acute sleep disturbances, we hypothesized that more recent studies, particularly those which objectively measure sleep disturbances, would provide the most robust prediction. Although relatively little research has examined short-term prediction of suicide<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>, recent evidence indicates that shorter follow-up lengths may improve predictive accuracy<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>; therefore, we expected to detect stronger effects over shorter follow-up periods. Fifth, we will evaluate whether sleep disturbances serve as clinically useful predictors of suicidal thoughts and behaviours by contextualizing our findings in terms of the absolute risk of suicide-related outcomes.</p></sec><sec id=\"Sec2\"><title>Methods</title><sec id=\"Sec3\"><title>Literature search</title><p id=\"Par10\">Our literature search was conducted as part of a larger meta-analytic effort<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. Using identical methods and search terms, we updated the comprehensive literature search conducted by Franklin and colleagues<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup> to include articles published through October 31, 2019. Databases used were PubMed, PsycINFO, and Google Scholar. Search terms included variants of the words “longitudinal” (i.e., “longitudinal,” “longitudinally,” “predict,” “predicts,” “prospective,” “prospectively,” “future,” “later,”) and “suicide” (i.e., “self-injury,” “self-injurious,” “self-injurer,” “suicide,” “suicidal,” “suicidality,” “self-harm,” “NSSI,” “DSH,” “self-cutting,” “self-burning,” “self-poisoning”). Because many studies include measures of sleep disturbances even when they are not central to the study (e.g., a study about the effects of mood disorders on suicide may include information about insomnia), we intentionally did not constrain our search based on sleep-specific key words. We reasoned that this more comprehensive approach accordingly increased the likelihood that all potentially relevant articles would be captured.</p></sec><sec id=\"Sec4\"><title>Inclusion and exclusion criteria</title><p id=\"Par11\">All articles were required to include at least one longitudinal analysis in which a sleep-related variable (i.e., any measure designed to assess sleep or sleep-related symptoms) predicted suicide ideation, attempt, or death. We focused on these outcomes for two reasons. First, we were interested in effects on suicidal thoughts and behaviours, which are self-directed and involve a nonzero intent to die. This excludes behaviours unrelated to suicidality, such as nonsuicidal self-injury, or mixed terms capturing both suicidal and nonsuicidal behaviours, such as deliberate self-harm. Second, we were interested in understanding specific effects on discrete suicide-related outcomes. Therefore, studies that collapsed these variables into a single measure (e.g., subsuming suicidal ideation and attempts under a “suicidality” item) were excluded. All articles were required to be peer-reviewed published articles with an English language version available. We chose to include only published studies because we were interested in publicly available data that clinicians may use to make decisions.</p><p id=\"Par12\">Treatment studies were excluded, as treatment effects may influence risk factor effects. Systematic reviews and meta-analyses were also excluded. Studies which did not provide necessary statistical information were also excluded (i.e., insufficient data to calculate an odds ratio and its variance).</p></sec><sec id=\"Sec5\"><title>Study selection</title><p id=\"Par13\">Our initial search yielded 5,091 unique articles published between 1965 and 2019. We screened in 743 papers for a full-text review. We retained 42 studies for our final analyses based on our inclusion criteria. Across studies, there were 156 unique effect sizes (see Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> for PRISMA flowchart; see <xref rid=\"MOESM1\" ref-type=\"media\">Supplementary Materials</xref> for a full reference list of included studies).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>PRISMA flowchart<sup><xref ref-type=\"bibr\" rid=\"CR84\">84</xref></sup>.</p></caption><graphic xlink:href=\"41598_2020_70866_Fig1_HTML\" id=\"MO1\"/></fig></p></sec><sec id=\"Sec6\"><title>Data extraction and coding</title><p id=\"Par14\">The following data were extracted from each study: author, publication year, follow-up length in months, sample size, sample type (i.e., general population, clinical population, or participants recruited for a history of self-injurious or suicidal behaviours), sample age (i.e., “child/adolescent” if the study included only participants under 18 years of age at the start of the study, “mixed” if the study included both participants under and over 18 at the start of the study, or “adult” if all participants were over 18 years old), predictor (i.e., type of sleep disturbance), outcome (i.e., ideation, attempt, or death), sleep measure type (i.e., self-report, interview, actigraphy, or polysomnography), and relevant statistics. Any statistical test in which a sleep-related variable predicted suicide ideation, attempt, or death was retained as a “prediction case.”</p><p id=\"Par15\">Predictor variables varied across studies. To improve interpretability, we coded specific predictor variables into secondary broad predictor categories; see <xref rid=\"MOESM1\" ref-type=\"media\">Supplementary Materials</xref> for a complete list. Initial codes for each article were determined by the first author. Each code was subsequently examined by two additional authors (CPB and KPL). All discrepancies were discussed until consensus and resolved. Outcomes were coded as suicide ideation, attempt, or death. One study<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup> included interrupted and aborted suicide attempts; these were coded as suicide attempts. Our pattern of findings remained unchanged when this study was excluded from analyses.</p><p id=\"Par16\">Meta-analyses often code for study quality, especially when the included studies contain a high degree of methodological variability. Compared to other meta-analyses, however, the present set of studies was relatively uniform, as they all shared a common core design (i.e., longitudinal prediction of a discrete suicide-relevant outcome). Because there are no objective criteria to assess study quality in this particular literature, we conducted moderator analyses of methodological differences (e.g., length of follow-up, predictor type, sample type, etc.) to examine the impact of how certain methodological differences may influence risk factor magnitude. This approach was consistent with the methods used in prior meta-analyses of suicide risk factor research<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref>–<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>.</p></sec><sec id=\"Sec7\"><title>Statistical analyses</title><p id=\"Par17\">All analyses were conducted using R<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>. Meta-analytic procedures were conducted using the metafor package<sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup>. We used odds ratios, which represent the odds of an event in one group compared to another, as our effect sizes. When odds ratios were not available, they were calculated from given data and summary statistics (e.g., 2 × 2 contingency tables, independent group means, risk ratios). If insufficient data were reported to compute an odds ratio and its variance (e.g., beta weights with no additional information, hazard ratios), that prediction case was excluded.</p><p id=\"Par18\">We used random effects models for all meta-analyses. Random effects models do not assume that a single, true effect exists, but that there will be a distribution of effects across studies. Due to the variability in predictors, outcomes, populations, and methodology, significant between-study heterogeneity was expected. Random effects models account for heterogeneity by relying on unconditional variance, which takes into account both sample size and variance between studies and weights effect sizes accordingly. Between-study heterogeneity was quantified using <italic>I</italic><sup>2</sup> tests. To improve the reliability of obtained estimates, only models including at least three effect sizes were run.</p><p id=\"Par19\">Publication bias was examined in several ways. We visually inspected funnel plots, which tend to be asymmetrical when publication bias is present and symmetrical when it is absent. Because visual inspection can be subjective, we also calculated Egger’s regression test as an objective index of funnel plot symmetry and used Duval and Tweedie’s trim and fill method to determine how many studies would be needed to make the funnel plot symmetrical. Classic and Orwin’s failsafe N analyses were conducted to estimate the robustness of observed effects.</p><p id=\"Par20\">Moderator analyses were conducted through a series of metaregressions using a random-effects model with unrestricted maximum likelihood estimation. Moderators included publication date, follow-up length, sample severity, and sleep measure type.</p></sec></sec><sec id=\"Sec8\"><title>Results</title><sec id=\"Sec9\"><title>Descriptive summary</title><p id=\"Par21\">Publication dates ranged from 1982 to 2019. The number of longitudinal studies examining the relationship between sleep disturbances and suicide has increased over time; most studies (N = 22; 52.38%) were published between 2015 and 2019 (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). Suicide ideation was the most common outcome (k = 85; 54.49%), followed by attempt (k = 39; 25%) and death (k = 32; 20.51%).<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Number of studies and effect sizes over time.</p></caption><graphic xlink:href=\"41598_2020_70866_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par22\">Nearly half of prediction cases (k = 75; 48.07%) were drawn from community samples; 19.23% (k = 30) were drawn from clinical samples, and 32.69% (k = 51) were drawn from samples recruited for a history of self-injurious thoughts and behaviours. Most samples comprised only adults (k = 106; 67.95%); 29.49% of samples included only participants under 18 at the start of the study, and 2.56% (k = 4) included both participants under and over 18 at the start of the study.</p><p id=\"Par23\">Follow-up lengths ranged from 1 day to 50 years, with an average of 61.31 months (<italic>SD</italic> = 100.37, <italic>Mdn</italic> = 12). The follow-up lengths for suicide death cases (<italic>M</italic> = 197.40 months, range = 0.25–600, <italic>SD</italic> = 148.04, <italic>Mdn</italic> = 132) were longer on average than those for ideation (<italic>M</italic> = 23.61 months, range = 0.03–120, <italic>SD</italic> = 34.47, <italic>Mdn</italic> = 12) or attempt (<italic>M</italic> = 31.60 months, range = 0.03–100, <italic>SD</italic> = 30.44, <italic>Mdn</italic> = 12).</p><p id=\"Par24\">Insomnia was the most common predictor (k = 55; 35.26%), followed by unspecified “sleep problems” (k = 27; 17.31%), sleep duration (k = 16; 10.26%), and nightmares (k = 13; 8.33%). The majority of cases (k = 110; 70.51%) measured sleep disturbances via self-report, with the remainder using interviews (k = 29; 18.59%), actigraphy (k = 10; 6.41%), or polysomnography (k = 7; 4.49%).</p></sec><sec id=\"Sec10\"><title>Overall prediction and publication bias</title><p id=\"Par25\">Overall prediction estimates reflect the pooled effects of all predictors on the outcome of interest. Odds ratio analyses included 156 prediction cases. The overall weighted odds ratio for all outcomes was 1.59 (95% CI 1.46–1.73). Between-study heterogeneity was high (<italic>I</italic><sup>2</sup> = 83.40%). See Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> for all random-effects results. Significant evidence of publication bias was not detected (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>).\n<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Random-effects results for each broad predictor category.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\" colspan=\"2\">Suicide ideation</th><th align=\"left\" colspan=\"2\">Suicide attempt</th><th align=\"left\" colspan=\"2\">Suicide death</th></tr><tr><th align=\"left\">n</th><th align=\"left\">OR [95% CI]</th><th align=\"left\">n</th><th align=\"left\">OR [95% CI]</th><th align=\"left\">n</th><th align=\"left\">OR [95% CI]</th></tr></thead><tbody><tr><td align=\"left\">Insomnia</td><td align=\"left\">31</td><td align=\"left\">2.10 [1.83–2.41]</td><td align=\"left\">16</td><td align=\"left\">1.78 [1.38–2.29]</td><td align=\"left\">8</td><td align=\"left\">1.54 [1.04–2.29]</td></tr><tr><td align=\"left\">Nightmares</td><td align=\"left\">4</td><td align=\"left\">1.08 [0.61–1.91]</td><td align=\"left\">5</td><td align=\"left\">1.81 [1.12–2.92]</td><td align=\"left\">4</td><td align=\"left\">1.31 [0.83–2.06]</td></tr><tr><td align=\"left\">Sleep disturbances</td><td align=\"left\">6</td><td align=\"left\">1.61 [0.60–2.69]</td><td align=\"left\">3</td><td align=\"left\">1.85 [0.98–3.48]</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Sleep duration</td><td align=\"left\">12</td><td align=\"left\">0.95 [0.70–1.29]</td><td align=\"left\">4</td><td align=\"left\">1.10 [0.75–1.68]</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Sleep efficiency</td><td align=\"left\">4</td><td align=\"left\">1.26 [0.56–2.81]</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Sleep problems</td><td align=\"left\">9</td><td align=\"left\">1.80 [1.30–2.50]</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">16</td><td align=\"left\">1.27 [0.97–1.66]</td></tr><tr><td align=\"left\">Sleep quality</td><td align=\"left\">6</td><td align=\"left\">1.74 [1.13–2.67]</td><td align=\"left\">3</td><td align=\"left\">1.35 [0.84–2.15]</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Sleep onset latency</td><td align=\"left\">6</td><td align=\"left\">1.34 [0.75–2.42]</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Tiredness</td><td align=\"left\">5</td><td align=\"left\">1.81 [1.00–3.27]</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td></tr></tbody></table></table-wrap><table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Publication bias.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\" colspan=\"2\">Fail-safe N</th><th align=\"left\" rowspan=\"2\">Egger’s test of the intercept, z-value (<italic>p</italic>)</th><th align=\"left\" colspan=\"2\">Duval and Tweedie’s trim and fill</th></tr><tr><th align=\"left\">Classic</th><th align=\"left\">Orwin’s</th><th align=\"left\">Missing cases</th><th align=\"left\">Adjusted OR</th></tr></thead><tbody><tr><td align=\"left\">All outcomes</td><td align=\"left\">32,255</td><td align=\"left\">149</td><td align=\"left\"> − 1.31 (0.19)</td><td align=\"left\">24</td><td align=\"left\">1.79</td></tr><tr><td align=\"left\">Suicidal ideation</td><td align=\"left\">13,011</td><td align=\"left\">81</td><td align=\"left\"> − 2.00 (0.05)</td><td align=\"left\">17</td><td align=\"left\">1.92</td></tr><tr><td align=\"left\">Suicide plan</td><td align=\"left\">96</td><td align=\"left\">6</td><td align=\"left\"> − 0.05 (0.96)</td><td align=\"left\">0</td><td align=\"left\">1.63</td></tr><tr><td align=\"left\">Suicide attempt</td><td align=\"left\">2021</td><td align=\"left\">36</td><td align=\"left\">0.83 (0.41)</td><td align=\"left\">1</td><td align=\"left\">1.54</td></tr><tr><td align=\"left\">Suicide death</td><td align=\"left\">378</td><td align=\"left\">32</td><td align=\"left\">0.02 (0.99)</td><td align=\"left\">6</td><td align=\"left\">1.18</td></tr></tbody></table></table-wrap></p><sec id=\"Sec11\"><title>Suicidal ideation</title><p id=\"Par26\">Odds ratio analyses for suicidal ideation included 84 prediction cases. Heterogeneity was high between studies (<italic>I</italic><sup>2</sup> = 83.91%). The overall weighted odds ratio was 1.73 (95% CI 1.54–1.94). While failsafe N analyses indicated that this was a robust effect, evidence of publication bias was detected via visual inspection of the funnel plot and Egger’s test of the intercept (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a; Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>).<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Funnel plots. (<bold>a</bold>) Suicide ideation, (<bold>b</bold>) suicide attempt, and (<bold>c</bold>) suicide death. Filled circles represent observed effects, and open circles represent imputed effects.</p></caption><graphic xlink:href=\"41598_2020_70866_Fig3_HTML\" id=\"MO3\"/></fig></p></sec><sec id=\"Sec12\"><title>Suicide attempt</title><p id=\"Par27\">Odds ratio analyses for suicide attempt included 36 prediction cases. The overall weighted odds ratio was 1.54 (95% CI 1.32–1.81). Between-study heterogeneity was high (<italic>I</italic><sup>2</sup> = 83.16%). While visual inspection of the funnel plot indicated possible publication bias, significant evidence for publication bias was not detected via Egger’s test of the intercept, and failsafe N analyses indicated this was a robust effect (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>b; Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>).</p></sec><sec id=\"Sec13\"><title>Suicide death</title><p id=\"Par28\">Odds ratio analyses for suicide death included 32 prediction cases. The overall weighted odds ratio was 1.33 (95% CI 1.11–1.60). Between-study heterogeneity was high (<italic>I</italic><sup>2</sup> = 73.94%). Although visual inspection of the funnel plot indicated possible publication bias, no significant evidence for publication bias was detected via Egger’s test of the intercept, and failsafe N analyses indicated this was a robust effect (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>c; Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>).</p></sec></sec><sec id=\"Sec14\"><title>Prediction by risk factor category</title><sec id=\"Sec15\"><title>Insomnia</title><p id=\"Par29\">Insomnia significantly predicted suicidal ideation (OR 2.10 [95% CI 1.83–2.41], k = 31). Point estimates were smaller predicting suicide attempt (OR 1.78 [95% CI 1.38–2.29], k = 16) and suicide death (OR 1.54 [95% CI 1.04–2.29], k = 8).</p></sec><sec id=\"Sec16\"><title>Nightmares</title><p id=\"Par30\">Nightmares significantly predicted suicide attempt (OR 1.81 [95% CI 1.12–2.92], k = 5), but not suicide ideation (OR 1.08 [95% CI 0.61–1.91], k = 4) or death (OR 1.31 [95% CI 0.83–2.06], k = 4).</p></sec><sec id=\"Sec17\"><title>Sleep disturbances</title><p id=\"Par31\">Sleep disturbances did not significantly predict suicide ideation (OR 1.61 [95% CI 0.60–2.69], k = 6) or attempt (OR 1.85 [95% CI 0.98–3.48], k = 3). An insufficient number of cases were available to examine effects on suicide death.</p></sec><sec id=\"Sec18\"><title>Sleep duration</title><p id=\"Par32\">Sleep duration did not significantly predict suicide ideation (OR 0.95 [95% CI 0.70–1.29], k = 12), or attempt (OR 1.12 [95% CI 0.75–1.68], k = 4). An insufficient number of cases were available to examine effects on suicide death.</p></sec><sec id=\"Sec19\"><title>Sleep efficiency</title><p id=\"Par33\">Sleep efficiency did not significantly predict suicidal ideation (OR 1.26 [95% CI 0.56–2.81], k = 4). An insufficient number of cases were available to examine effects on suicide attempt or death.</p></sec><sec id=\"Sec20\"><title>Sleep problems</title><p id=\"Par34\">Sleep problems significantly predicted suicidal ideation (OR 1.80 [95% CI 1.30–2.50], k = 9). Effects were not significant for suicide death (OR 1.27 [95% CI 0.97–1.66], k = 16). An insufficient number of cases were available to examine effects on suicide attempt.</p></sec><sec id=\"Sec21\"><title>Sleep quality</title><p id=\"Par35\">Sleep quality significantly predicted suicidal ideation (OR 1.74 [95% CI 1.13–2.67], k = 6). Effects were not significant for suicide attempt (OR 1.35 [95% CI 0.84–2.15], k = 3). An insufficient number of cases were available to examine effects on suicide death.</p></sec><sec id=\"Sec22\"><title>Sleep-onset latency</title><p id=\"Par36\">Sleep-onset latency did not significantly predict suicidal ideation (OR 1.34 [95% CI 0.75–2.42], k = 6). An insufficient number of cases were available to examine effects on suicide attempt or death.</p></sec><sec id=\"Sec250\"><title>Tiredness</title><p id=\"Par37\">Tiredness did not significantly predict suicidal ideation (OR 1.81 [95% CI 1.00–3.27], k = 5). An insufficient number of cases were available to examine effects on suicide attempt or death.</p></sec></sec><sec id=\"Sec231\"><title>Moderator analyses</title><p id=\"Par38\">We report metaregression results in terms of Q<sub>M</sub>, the model sum of squares, which is a test of whether any of the regression coefficients in the model are significantly different from zero.</p><sec id=\"Sec251\"><title>Publication date</title><p id=\"Par39\">Metaregression results indicated no significant effects of publication date on suicide ideation (Q<sub>M</sub> [df = 2] = 0.31, <italic>p</italic> = 0.85), attempt (Q<sub>M</sub>[df = 2] = 1.15, <italic>p</italic> = 0.56), or death (Q<sub>M</sub>[df = 5] = 1.23, <italic>p</italic> = 0.94), indicating that effect sizes have remained stable over time.</p></sec><sec id=\"Sec26\"><title>Follow-up length</title><p id=\"Par40\">There was a significant effect of follow-up length for suicide ideation (Q<sub>M</sub>[df = 5] = 160.62, <italic>p</italic> < 0.0001), attempt (Q<sub>M</sub>[df = 5] = 39.71, <italic>p</italic> < 0.0001), and death (Q<sub>M</sub>[df = 5] = 13.29, <italic>p</italic> = 0.02). Effects were strongest when the follow-up length was 6 months or less for all outcomes (ideation OR 2.30 [95% CI 2.00–2.65]; attempt OR 2.48 [95% CI 1.33–4.62]; death OR 2.19 [95% CI 1.08–4.43]).</p></sec><sec id=\"Sec27\"><title>Sample severity</title><p id=\"Par41\">Moderation analyses revealed a significant effect of sample severity on suicide ideation (Q<sub>M</sub>[df = 2] = 21.61, <italic>p</italic> < 0.001), but not attempt (Q<sub>M</sub>[df = 2] = 0.20, <italic>p</italic> = 0.90) or death (Q<sub>M</sub>[df = 2] = 2.65, <italic>p</italic> = 0.27). Predicting ideation, effects were slightly stronger in community samples (OR 1.52 [95% CI 1.30–1.79]) and samples recruited for a history of self-injurious thoughts and behaviours (OR 1.45 [95% CI 1.17–1.80]) compared to other clinical populations (OR 0.78 [95% CI 0.57–1.05]).</p></sec><sec id=\"Sec28\"><title>Sleep measure type</title><p id=\"Par42\">Metaregression results indicated no significant effects of sleep measure type (i.e., self-report, interview, actigraphy, or polysomnography) on suicide ideation (Q<sub>M</sub>[df = 3] = 4.55, <italic>p</italic> = 0.21), attempt (Q<sub>M</sub>[df = 2] = 3.62, <italic>p</italic> = 0.16), or death (Q<sub>M</sub>[df = 1] = 0.25, <italic>p</italic> = 0.62). We also conducted post-hoc moderator analysis using each <italic>specific</italic> measure (e.g., specific self-report measures, interviews, and actigraphy/polysomnography measures) as a moderator. Our analyses revealed a significant effect of specific measure for suicide ideation (Q<sub>M</sub>[df = 13 ] = 54.50 , <italic>p</italic> < 0.001), but not attempt (Q<sub>M</sub>[df = 10] = 16.73, <italic>p</italic> = 0.08), or death (Q<sub>M</sub>[df = 12] = 11.88, <italic>p</italic> = 0. 46). For suicide ideation, effects were strongest when sleep disturbances were measured with the Insomnia Severity Index (OR = 1.55 [95% CI 1.07–2.23]) and the Adolescent Health Questionnaire (OR 1.63 [95% CI 1.10–2.42]).</p></sec></sec></sec><sec id=\"Sec29\"><title>Discussion</title><p id=\"Par43\">Our findings indicate that sleep disturbances are statistically significant predictors of suicide ideation, attempt, and death. However, these effects were weak, at least as examined within the methodological constraints of the literature. Our results are consistent with a growing body of evidence which demonstrates that most commonly cited risk factors only weakly predict suicide<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref>,<xref ref-type=\"bibr\" rid=\"CR58\">58</xref>,<xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>. Odds ratios for each outcome ranged from 1.33 to 1.73, and effects were consistent regardless of study publication date and type of sleep measure used (i.e., self-report, clinical interview, actigraphy, or polysomnography).</p><p id=\"Par44\">The literature on the longitudinal relationship between sleep and suicide has grown exponentially in recent years. Because the most recent meta-analysis of this literature was published nearly a decade ago and focused primarily on cross-sectional studies, the present study represents a critical step toward advancing our knowledge of the extent to which disturbed sleep confers risk for future suicidal thoughts and behaviours. Indeed, over half of the studies we uncovered in our review of the literature were published within the last 5 years (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). Results indicated, however, that this increase in research has not corresponded with improved predictive accuracy, though it may have contributed to improved reliability of detected effects. We found that most studies examined the effects of disturbed sleep on suicide ideation, rather than suicidal behaviours (i.e., attempts or death). Moreover, very few studies examined proximal risk; the average follow-up length was over 5 years, and follow-up intervals were much longer on average for suicidal behaviours compared to suicidal ideation.</p><p id=\"Par45\">Effects varied depending on follow-up length, with slightly stronger effects observed over shorter follow-up periods (≤ 6 months); however, these effects remained weak. Prior evidence is mixed regarding the effects of follow-up length on risk factor strength. In a meta-analysis of hundreds of risk factors, no consistent patterns of predictive ability over different follow-up intervals were detected<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. Given the fluctuating nature of sleep disturbances<sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref>,<xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup> and suicidality<sup><xref ref-type=\"bibr\" rid=\"CR62\">62</xref>,<xref ref-type=\"bibr\" rid=\"CR63\">63</xref></sup>, it is possible that short-term follow-up windows yielded slightly stronger effects by producing more reliable measurement. Although these results indicate that it is sensible to focus on short-term prediction, studies with brief follow-up periods remain rare. Because suicide is fortunately a low base-rate event, it is difficult to detect statistically meaningful effects over brief intervals. Recent studies have used novel techniques to overcome this challenge, such as leveraging large, severe samples recruited online<sup><xref ref-type=\"bibr\" rid=\"CR64\">64</xref>,<xref ref-type=\"bibr\" rid=\"CR65\">65</xref></sup>. Online studies yield faster recruitment than in-person data collection and produce comparable results to in-person studies<sup><xref ref-type=\"bibr\" rid=\"CR66\">66</xref>,<xref ref-type=\"bibr\" rid=\"CR67\">67</xref></sup>. These methods may provide a fruitful path for future research.</p><p id=\"Par46\">Slightly stronger effects were detected in less severe samples. These findings are likely a methodological artefact. When studies rely on homogenous samples, the range of severity is restricted, making it difficult to detect significant differences from the reference group; in contrast, samples with high levels of heterogeneity capture both extremes of severity, making it easier to detect significant effects. Studies in clinical and self-injurious samples are also likely to control for additional risk factors, which may further reduce effect sizes. Other meta-analyses have found similar moderating effects of sample severity<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref>,<xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup>. Despite statistically significant moderation, effects detected in less severe samples remained weak.</p><p id=\"Par47\">In addition to overall prediction, we examined the effects of specific sleep disturbances. The strongest effects were found for insomnia, which significantly predicted suicide ideation (OR 2.10 [95% CI 1.83–2.41]), and nightmares, which significantly predicted suicide attempts (OR 1.81 [95% CI 1.12–2.92]). Only insomnia significantly predicted suicide death (OR 1.54 [95% CI 1.04–2.29]). This pattern of findings is consistent with other meta-analytic evidence that the strongest predictive effects are typically observed for suicide ideation, followed by attempt and death (e.g., anxiety symptoms<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup>; depression and hopelessness<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>); however, the evidence reliably demonstrates that even the strongest predictors are weak in absolute terms<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. The majority of effects (76%) were nonsignificant, and for several predictors, there were too few cases to provide reliable estimates. Although additional cases may have allowed us to detect more reliable effects, it is unlikely that stronger effects would be detected, as overall estimates all achieved statistical significance yet remained weak.</p><p id=\"Par48\">This meta-analysis cannot provide direct insight into the relationship between sleep disturbances and suicide risk; it can only reflect the value of this relationship as examined within the methodological constraints of the literature. It is therefore important to consider limitations when interpreting these findings. First, although self-report was the most common way to assess sleep disturbances, nearly every study relied on different measures. The exceptions were three studies<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref>,<xref ref-type=\"bibr\" rid=\"CR65\">65</xref>,<xref ref-type=\"bibr\" rid=\"CR68\">68</xref></sup> which used the Insomnia Severity Index (ISI), and two studies<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR69\">69</xref></sup> which used the Women’s Health Initiative Insomnia Rating Scale (WHIIRS). Other validated self-report measures included the Pittsburgh Sleep Quality Index<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>, the Adolescent Health Questionnaire<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, the Youth Self Report questionnaire<sup><xref ref-type=\"bibr\" rid=\"CR70\">70</xref></sup>, the Beck Depression Inventory<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>, and the Uppsala Sleep Inventory<sup><xref ref-type=\"bibr\" rid=\"CR71\">71</xref></sup>. Multiple studies relied on single-item measures rather than validated questionnaires<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR72\">72</xref>,<xref ref-type=\"bibr\" rid=\"CR73\">73</xref></sup>. Although our moderation analyses did not reveal a significant effect of sleep measure <italic>type</italic> (i.e., self-report, interview, actigraphy, or polysomnography) on effect size, our post-hoc moderator analyses revealed that <italic>specific</italic> measures were statistically significant moderators for suicide ideation, with the strongest effects observed for the Insomnia Severity Index and the Adolescent Health Questionnaire. However, these effects remained weak (ORs 1.55 and 1.63, respectively). These two measures accounted for the largest proportion of effect sizes in our analyses; therefore, the significant moderation effect may reflect improved reliability as a consequence of their frequency of use. No significant effects were detected for suicide attempt or death.</p><p id=\"Par49\">Second, several sleep disorders were not represented in the existing literature, including sleep apnoea, restless leg syndrome, and narcolepsy. In fact, prior diagnoses of sleep disorders were sometimes used as exclusion criteria for study participation (e.g.,<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>). Although sleep disturbances may indicate the presence of a sleep disorder, it remains unknown whether their associations with suicide risk are distinct. Due to the lack of studies examining the longitudinal relationship between diagnosed sleep disorders and suicidal thoughts and behaviours, determining the extent to which they may confer risk for suicide is beyond the scope of the present meta-analysis; however, given prior meta-analytic evidence that diagnoses of particular disorders are limited in their ability to accurately predict suicide<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>, we reason that our pattern of results would have been comparable even with the addition of studies examining these disorders. Nevertheless, this represents an important area for future research.</p><p id=\"Par50\">Third, assessment windows varied substantially. Whereas the BDI assesses sleep disturbances over the last week, the ISI assesses the last two weeks, and the WHIIRS assesses the last four weeks. Some studies assessed sleep disturbances over the last year<sup><xref ref-type=\"bibr\" rid=\"CR72\">72</xref>,<xref ref-type=\"bibr\" rid=\"CR74\">74</xref></sup>. Consistent with a priori hypotheses, our moderator analyses demonstrated that effects were strongest over shorter intervals. Although relying on retrospective self-reported symptoms is common, doing so over longer periods of time is likely to be less valid and reliable than short-term assessment, as these methods may be affected by recall biases, especially for symptoms that occurred less recently<sup><xref ref-type=\"bibr\" rid=\"CR75\">75</xref></sup>. These measurement issues may have contributed to the detection of less robust effects; however, the strongest short-term effect we detected (i.e., pooled effect of all sleep disturbances on suicide attempt over a follow-up period of 0–6 months) was still weak (OR = 2.48).</p><p id=\"Par51\">Fourth, although objective sleep measures address several limitations of subjective measures<sup><xref ref-type=\"bibr\" rid=\"CR76\">76</xref></sup>, only three studies <sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref>,<xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup> used these methods, accounting for approximately 10% of effect sizes. It is possible that our failure to detect a significant effect of sleep measure type was due to the small number of studies using objective measures that fit our inclusion criteria. The included studies may not represent the effects of objective measures in general; sleep disturbances may emerge as stronger risk factors as additional studies are published. Because the effects of univariate predictors of self-injurious and suicidal behaviours are weak<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref>,<xref ref-type=\"bibr\" rid=\"CR77\">77</xref></sup>, however, we reason that our results would be similar even with the inclusion of additional studies.</p><p id=\"Par52\">Our findings must also be evaluated with regards to clinical utility. According to the Centers for Disease Control and Prevention, the rate of suicide death in the United States is approximately 14.5 per 100,000. The strongest predictor of suicide death in this study was insomnia, which approximately doubles the risk of suicide death (OR 2.10). This increases the odds to 0.0003, representing a marginal improvement in predictive ability. Although sleep disturbances play a statistically significant role in predicting suicide, these results raise questions about the usefulness of designating sleep disturbances as suicide “warning signs,” at least when considered in isolation.</p><p id=\"Par53\">It is possible that methodological constraints account for the weak prediction estimates found in this study. However, our moderator analyses indicate this is unlikely. Even statistically significant influences on effects due to variations in follow-up length, sleep measure type, and sample severity did not meaningfully improve prediction. We accordingly reason that the most significant methodological issue is the focus on univariate-level prediction.</p><p id=\"Par54\">Our first recommendation for future research is to advance beyond examining the effects of sleep disturbances in isolation, and instead consider their function in the context of complex associations with other biopsychosocial factors. Studies which focus on individual sleep disturbances as predictors of suicide implicitly assume that this relationship is simple. Simple theories are cognitively manageable, but they stand in contrast to evidence demonstrating that it may be necessary to consider many biopsychosocial factors, combined in complex ways, to accurately predict suicide<sup><xref ref-type=\"bibr\" rid=\"CR65\">65</xref>,<xref ref-type=\"bibr\" rid=\"CR78\">78</xref>,<xref ref-type=\"bibr\" rid=\"CR79\">79</xref></sup>. Results of the present study are consistent with complexity. Whereas no individual sleep disturbance was found to be particularly relevant to suicide, several were weak predictors. Although this does not mean that sleep disturbances are inconsequential for suicide risk, it indicates that the relationships between sleep disturbances and suicide are likely to be small, individual, and highly variable. Sleep disturbances are not necessary or sufficient in isolation for suicidality to arise. Continuing to examine <italic>which</italic> sleep disturbances contribute to suicide risk is unlikely to improve suicide prediction and prevention. Instead, a more promising path may be identifying <italic>how</italic> sleep disturbances can be incorporated into complex conceptualizations of suicide risk.</p><p id=\"Par55\">The aim of suicide science is not only to predict risk, but to uncover the causal processes underlying suicide and disrupt them. Our second recommendation is for future research to clarify causal mechanisms underlying the relationship between sleep and suicide. Longitudinal studies can establish risk factors, but they are unable to test causal hypotheses<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. Experimental designs, in contrast, isolate the direct influence of risk factors; only experiments can be used to draw causal conclusions. Experiments are rare within the field of suicide research, but technological advances make it possible to safely and validly test causal hypotheses about suicide<sup><xref ref-type=\"bibr\" rid=\"CR80\">80</xref></sup>. Experiments are more common in sleep research. The effects of experimentally induced sleep deprivation have been examined in the context of working memory<sup><xref ref-type=\"bibr\" rid=\"CR81\">81</xref></sup>, pain perception<sup><xref ref-type=\"bibr\" rid=\"CR82\">82</xref></sup>, and inflammation<sup><xref ref-type=\"bibr\" rid=\"CR83\">83</xref></sup>. To our knowledge, no experiments to date have examined the influence of sleep disturbances on suicide. Future studies which leverage sleep deprivation paradigms in tandem with experimental approaches to studying suicide would advance our knowledge of whether sleep disturbances represent <italic>causal</italic> risk factors for suicide. This is a critical step toward designing interventions that directly target the causes of suicidal behaviour.</p><p id=\"Par56\">In sum, sleep disturbances increase risk for future suicide ideation, attempt, and death; however, these effects are weak in magnitude. The existing literature is methodologically constrained, but even with methodological advances, sleep disturbances are unlikely to emerge as strong univariate predictors of suicide. Although our results cast doubt on the utility of relying on sleep disturbances in isolation as warning signs for suicide, we look forward to future research examining the complex contributions of sleep disturbances to suicide risk. Future experimental studies are also needed to uncover potential causal mechanisms underlying the relationship between sleep disturbances and suicide.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec30\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70866_MOESM1_ESM.docx\"><caption><p>Supplementary information.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70866-6.</p></sec><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>LMH and JDR devised the present project. LMH took the lead in writing the manuscript, in consultation with all other authors. LMH, XH, CPB, and KPL coded data for analyses. LMH analysed the data. JDR supervised the project.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>All relevant data are available upon reasonable request to the corresponding author.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par57\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Czeisler</surname><given-names>CA</given-names></name><name><surname>Klerman</surname><given-names>EB</given-names></name></person-group><article-title>Circadian and sleep-dependent regulation of hormone release in humans</article-title><source>Recent Prog. Hormdiscussion 130–2. Res.</source><year>1999</year><volume>54</volume><fpage>97</fpage><lpage>130</lpage></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dattilo</surname><given-names>M</given-names></name><etal/></person-group><article-title>Sleep and muscle recovery: Endocrinological and molecular basis for a new and promising hypothesis</article-title><source>Med. Hypotheses</source><year>2011</year><volume>77</volume><fpage>220</fpage><lpage>222</lpage><pub-id pub-id-type=\"pmid\">21550729</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stickgold</surname><given-names>R</given-names></name></person-group><article-title>Sleep-dependent memory consolidation</article-title><source>Nature</source><year>2005</year><volume>437</volume><fpage>1272</fpage><lpage>1278</lpage><pub-id pub-id-type=\"pmid\">16251952</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gerner</surname><given-names>RH</given-names></name><name><surname>Post</surname><given-names>RM</given-names></name><name><surname>Gillin</surname><given-names>C</given-names></name><name><surname>Bunney</surname><given-names>WE</given-names></name></person-group><article-title>Biological and behavioral effects of one night’s sleep deprivation in depressed patients and normals</article-title><source>J. Psychiatry Res.</source><year>1979</year><volume>15</volume><fpage>21</fpage><lpage>40</lpage></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nilsson</surname><given-names>JP</given-names></name><etal/></person-group><article-title>Less effective executive functioning after one night’s sleep deprivation</article-title><source>J. Sleep Res.</source><year>2005</year><volume>14</volume><fpage>1</fpage><lpage>6</lpage><pub-id pub-id-type=\"pmid\">15743327</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Williams</surname><given-names>HL</given-names></name><name><surname>Gieseking</surname><given-names>CF</given-names></name><name><surname>Lubin</surname><given-names>A</given-names></name></person-group><article-title>Some effects of sleep loss on memory</article-title><source>Percept. Mot. Skills</source><year>1966</year><volume>23</volume><fpage>1287</fpage><lpage>1293</lpage><pub-id pub-id-type=\"pmid\">5972935</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tsuno</surname><given-names>N</given-names></name><name><surname>Besset</surname><given-names>A</given-names></name><name><surname>Ritchie</surname><given-names>K</given-names></name></person-group><article-title>Sleep and depression</article-title><source>J. Clin. Psychiatry</source><year>2005</year><volume>66</volume><fpage>1254</fpage><lpage>1269</lpage><pub-id pub-id-type=\"pmid\">16259539</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Plante</surname><given-names>DT</given-names></name><name><surname>Winkelman</surname><given-names>JW</given-names></name></person-group><article-title>Sleep disturbance in bipolar disorder: Therapeutic implications</article-title><source>Am. J. Psychiatry</source><year>2008</year><volume>165</volume><fpage>830</fpage><lpage>843</lpage><pub-id pub-id-type=\"pmid\">18483132</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Babson</surname><given-names>KA</given-names></name><name><surname>Trainor</surname><given-names>CD</given-names></name><name><surname>Feldner</surname><given-names>MT</given-names></name><name><surname>Blumenthal</surname><given-names>H</given-names></name></person-group><article-title>A test of the effects of acute sleep deprivation on general and specific self-reported anxiety and depressive symptoms: An experimental extension</article-title><source>J. Behav. Ther. Exp. Psychiatry</source><year>2010</year><volume>41</volume><fpage>297</fpage><lpage>303</lpage><pub-id pub-id-type=\"pmid\">20231014</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rudd</surname><given-names>MD</given-names></name><etal/></person-group><article-title>Warning signs for suicide: Theory, research, and clinical applications</article-title><source>Suicide Life. Threat. Behav.</source><year>2006</year><volume>36</volume><fpage>255</fpage><lpage>262</lpage><pub-id pub-id-type=\"pmid\">16805653</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Agargun</surname><given-names>MY</given-names></name><name><surname>Cartwright</surname><given-names>R</given-names></name></person-group><article-title>REM sleep, dream variables and suicidality in depressed patients</article-title><source>Psychiatry Res.</source><year>2003</year><volume>119</volume><fpage>33</fpage><lpage>39</lpage><pub-id pub-id-type=\"pmid\">12860358</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ağargün</surname><given-names>MY</given-names></name><name><surname>Kara</surname><given-names>H</given-names></name><name><surname>Solmaz</surname><given-names>M</given-names></name></person-group><article-title>Sleep disturbances and suicidal behavior in patients with major depression</article-title><source>J. Clin. Psychiatry</source><year>1997</year><volume>58</volume><fpage>249</fpage><lpage>251</lpage><pub-id pub-id-type=\"pmid\">9228889</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krakow</surname><given-names>B</given-names></name><name><surname>Ribeiro</surname><given-names>JD</given-names></name><name><surname>Ulibarri</surname><given-names>VA</given-names></name><name><surname>Krakow</surname><given-names>J</given-names></name><name><surname>Joiner</surname><given-names>TE</given-names></name></person-group><article-title>Sleep disturbances and suicidal ideation in sleep medical center patients</article-title><source>J. Affect. Disord.</source><year>2011</year><volume>131</volume><fpage>422</fpage><lpage>427</lpage><pub-id pub-id-type=\"pmid\">21211850</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>YJ</given-names></name><name><surname>Cho</surname><given-names>S-J</given-names></name><name><surname>Cho</surname><given-names>IH</given-names></name><name><surname>Kim</surname><given-names>SJ</given-names></name></person-group><article-title>Insufficient sleep and suicidality in adolescents</article-title><source>Sleep</source><year>2012</year><volume>35</volume><fpage>455</fpage><lpage>460</lpage><pub-id pub-id-type=\"pmid\">22467982</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Woosley</surname><given-names>JA</given-names></name><name><surname>Lichstein</surname><given-names>KL</given-names></name><name><surname>Taylor</surname><given-names>DJ</given-names></name><name><surname>Riedel</surname><given-names>BW</given-names></name><name><surname>Bush</surname><given-names>AJ</given-names></name></person-group><article-title>Insomnia complaint versus sleep diary parameters: Predictions of suicidal ideation</article-title><source>Suicide Life. Threat. Behav.</source><year>2016</year><volume>46</volume><fpage>88</fpage><lpage>95</lpage><pub-id pub-id-type=\"pmid\">26052862</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Roth</surname><given-names>T</given-names></name><etal/></person-group><article-title>Sleep problems, comorbid mental disorders, and role functioning in the National Comorbidity Survey Replication (NCS-R)</article-title><source>Biol. Psychiatry</source><year>2006</year><volume>60</volume><fpage>1364</fpage><lpage>1371</lpage><pub-id pub-id-type=\"pmid\">16952333</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sivertsen</surname><given-names>B</given-names></name><name><surname>Krokstad</surname><given-names>S</given-names></name><name><surname>Øverland</surname><given-names>S</given-names></name><name><surname>Mykletun</surname><given-names>A</given-names></name></person-group><article-title>The epidemiology of insomnia: Associations with physical and mental health</article-title><source>J. Psychosom. Res.</source><year>2009</year><volume>67</volume><fpage>109</fpage><lpage>116</lpage><pub-id pub-id-type=\"pmid\">19616137</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Taylor</surname><given-names>DJ</given-names></name><name><surname>Lichstein</surname><given-names>KL</given-names></name><name><surname>Durrence</surname><given-names>HH</given-names></name><name><surname>Reidel</surname><given-names>BW</given-names></name><name><surname>Bush</surname><given-names>AJ</given-names></name></person-group><article-title>Epidemiology of insomnia, depression, and anxiety</article-title><source>Sleep</source><year>2005</year><volume>28</volume><fpage>1457</fpage><lpage>1464</lpage><pub-id pub-id-type=\"pmid\">16335332</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><mixed-citation publication-type=\"other\"><italic>Diagnostic and statistical manual of mental disorders: DSM-5</italic> (American Psychiatric Association, 2013).</mixed-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morin</surname><given-names>CM</given-names></name><name><surname>Colecchi</surname><given-names>C</given-names></name><name><surname>Stone</surname><given-names>J</given-names></name><name><surname>Sood</surname><given-names>R</given-names></name><name><surname>Brink</surname><given-names>D</given-names></name></person-group><article-title>Behavioral and pharmacological therapies for late-life insomnia: A randomized controlled trial</article-title><source>JAMA</source><year>1999</year><volume>281</volume><fpage>991</fpage><lpage>999</lpage><pub-id pub-id-type=\"pmid\">10086433</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Taylor</surname><given-names>DJ</given-names></name><name><surname>Pruiksma</surname><given-names>KE</given-names></name></person-group><article-title>Cognitive and behavioural therapy for insomnia (CBT-I) in psychiatric populations: A systematic review</article-title><source>Int. Rev. Psychiatry</source><year>2014</year><volume>26</volume><fpage>205</fpage><lpage>213</lpage><pub-id pub-id-type=\"pmid\">24892895</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stinson</surname><given-names>K</given-names></name><name><surname>Tang</surname><given-names>NKY</given-names></name><name><surname>Harvey</surname><given-names>AG</given-names></name></person-group><article-title>Barriers to treatment seeking in primary insomnia in the United Kingdom: A cross-sectional perspective</article-title><source>Sleep</source><year>2006</year><volume>29</volume><fpage>1643</fpage><lpage>1646</lpage><pub-id pub-id-type=\"pmid\">17252896</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sudak</surname><given-names>H</given-names></name><name><surname>Maxim</surname><given-names>K</given-names></name><name><surname>Carpenter</surname><given-names>M</given-names></name></person-group><article-title>Suicide and stigma: A review of the literature and personal reflections</article-title><source>Acad. Psychiatry</source><year>2008</year><volume>32</volume><fpage>136</fpage><lpage>142</lpage><pub-id pub-id-type=\"pmid\">18349334</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pigeon</surname><given-names>WR</given-names></name><name><surname>Bishop</surname><given-names>TM</given-names></name><name><surname>Titus</surname><given-names>CE</given-names></name></person-group><article-title>The relationship between sleep disturbance, suicidal ideation, suicide attempts, and suicide among adults: A systematic review</article-title><source>Psychiatry Ann.</source><year>2016</year><volume>46</volume><fpage>177</fpage><lpage>186</lpage></element-citation></ref><ref id=\"CR25\"><label>25.</label><mixed-citation publication-type=\"other\">Kraemer, H. <italic>et al.</italic> Coming to term with the terms of risk. <italic>Arch. Gen. Psychiatry</italic><bold>54</bold>, 337–343 (1997).</mixed-citation></ref><ref id=\"CR26\"><label>26.</label><mixed-citation publication-type=\"other\">Blumenthal, S., Bell, V., Neumann, N. U., Schuttler, R. & Vogel, R. Mortality and rate of suicide of first admission psychiatric patients: A 5-year follow-up of a prospective longitudinal study. <italic>Psychopathology</italic> 50–56 (1989).</mixed-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>SX</given-names></name><name><surname>Lam</surname><given-names>SP</given-names></name><name><surname>Yu</surname><given-names>MWM</given-names></name><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Wing</surname><given-names>Y-K</given-names></name></person-group><article-title>Nocturnal sleep disturbances as a predictor of suicide attempts among psychiatric outpatients: A clinical, epidemiologic Prospective Study</article-title><source>J. Clin. Psychiatry</source><year>2010</year><volume>71</volume><fpage>1440</fpage><lpage>1446</lpage><pub-id pub-id-type=\"pmid\">21114949</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pien</surname><given-names>F-C</given-names></name><etal/></person-group><article-title>Changes in quality of life after a suicide attempt</article-title><source>West. J. Nurs. Res.</source><year>2016</year><volume>38</volume><fpage>721</fpage><lpage>737</lpage><pub-id pub-id-type=\"pmid\">26681669</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bernert</surname><given-names>RA</given-names></name><name><surname>Turvey</surname><given-names>CL</given-names></name><name><surname>Conwell</surname><given-names>Y</given-names></name><name><surname>Joiner</surname><given-names>TE</given-names></name></person-group><article-title>Association of poor subjective sleep quality with risk for death by suicide during a 10-year period: A longitudinal, population-based study of late life</article-title><source>JAMA Psychiatry</source><year>2014</year><volume>71</volume><fpage>1129</fpage><pub-id pub-id-type=\"pmid\">25133759</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bjørngaard</surname><given-names>JH</given-names></name><name><surname>Bjerkeset</surname><given-names>O</given-names></name><name><surname>Romundstad</surname><given-names>P</given-names></name><name><surname>Gunnell</surname><given-names>D</given-names></name></person-group><article-title>Sleeping problems and suicide in 75,000 Norwegian Adults: A 20 year follow-up of the HUNT I Study</article-title><source>Sleep</source><year>2011</year><volume>34</volume><fpage>1155</fpage><lpage>1159</lpage><pub-id pub-id-type=\"pmid\">21886352</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mars</surname><given-names>B</given-names></name><etal/></person-group><article-title>Predictors of future suicide attempt among adolescents with suicidal thoughts or non-suicidal self-harm: A population-based birth cohort study</article-title><source>Lancet Psychiatry</source><year>2019</year><volume>6</volume><fpage>327</fpage><lpage>337</lpage><pub-id pub-id-type=\"pmid\">30879972</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Allan</surname><given-names>NP</given-names></name><etal/></person-group><article-title>Insomnia and suicidal ideation and behaviors in former and current U.S. service members: Does depression mediate the relations?</article-title><source>Psychiatry Res.</source><year>2017</year><volume>252</volume><fpage>296</fpage><lpage>302</lpage><pub-id pub-id-type=\"pmid\">28301827</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><mixed-citation publication-type=\"other\">Liu, X. <italic>et al.</italic> Daytime sleepiness predicts future suicidal behavior: A longitudinal study of adolescents. <italic>Sleep</italic><bold>42</bold>, zsy225 (2019). 10.1093/sleep/zsy225.</mixed-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wakefield</surname><given-names>JC</given-names></name><name><surname>Schmitz</surname><given-names>MF</given-names></name></person-group><article-title>Feelings of worthlessness during a single complicated major depressive episode predict postremission suicide attempt</article-title><source>Acta Psychiatry Scand.</source><year>2016</year><volume>133</volume><fpage>257</fpage><lpage>265</lpage></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eikelenboom</surname><given-names>M</given-names></name><name><surname>Beekman</surname><given-names>ATF</given-names></name><name><surname>Penninx</surname><given-names>BWJH</given-names></name><name><surname>Smit</surname><given-names>JH</given-names></name></person-group><article-title>A 6-year longitudinal study of predictors for suicide attempts in major depressive disorder</article-title><source>Psychol. Med.</source><year>2019</year><volume>49</volume><fpage>911</fpage><lpage>921</lpage><pub-id pub-id-type=\"pmid\">29897037</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hung</surname><given-names>GC-L</given-names></name><name><surname>Kwok</surname><given-names>C-L</given-names></name><name><surname>Yip</surname><given-names>PS</given-names></name><name><surname>Gunnell</surname><given-names>D</given-names></name><name><surname>Chen</surname><given-names>Y-Y</given-names></name></person-group><article-title>Predicting suicide in older adults—A community-based cohort study in Taipei City, Taiwan</article-title><source>J. Affect. Disord.</source><year>2015</year><volume>172</volume><fpage>165</fpage><lpage>170</lpage><pub-id pub-id-type=\"pmid\">25451412</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sandman</surname><given-names>N</given-names></name><etal/></person-group><article-title>Nightmares as predictors of suicide: An extension study including war veterans</article-title><source>Sci. Rep.</source><year>2017</year><volume>7</volume><fpage>44756</fpage><pub-id pub-id-type=\"pmid\">28294195</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>SY</given-names></name><name><surname>Sim</surname><given-names>S</given-names></name><name><surname>Choi</surname><given-names>HG</given-names></name></person-group><article-title>High stress, lack of sleep, low school performance, and suicide attempts are associated with high energy drink intake in adolescents</article-title><source>PLoS ONE</source><year>2017</year><volume>12</volume><fpage>e0187759</fpage><pub-id pub-id-type=\"pmid\">29135989</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schneider</surname><given-names>B</given-names></name><etal/></person-group><article-title>Living alone, obesity, and smoking increase risk for suicide independently of depressive mood findings from the population-based MONICA/KORA Augsburg cohort study</article-title><source>J. Affect. Disord.</source><year>2014</year><volume>152–154</volume><fpage>416</fpage><lpage>421</lpage><pub-id pub-id-type=\"pmid\">24238953</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Suh</surname><given-names>S</given-names></name><etal/></person-group><article-title>Longitudinal course of depression scores with and without insomnia in non-depressed individuals: A 6-year follow-up longitudinal study in a Korean Cohort</article-title><source>Sleep</source><year>2013</year><volume>36</volume><fpage>369</fpage><lpage>376</lpage><pub-id pub-id-type=\"pmid\">23449814</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bernert</surname><given-names>RA</given-names></name><name><surname>Hom</surname><given-names>MA</given-names></name><name><surname>Iwata</surname><given-names>NG</given-names></name><name><surname>Joiner</surname><given-names>TE</given-names></name></person-group><article-title>Objectively assessed sleep variability as an acute warning sign of suicidal ideation in a longitudinal evaluation of young adults at high suicide risk</article-title><source>J. Clin. Psychiatry</source><year>2017</year><volume>78</volume><fpage>e678</fpage><lpage>e687</lpage><pub-id pub-id-type=\"pmid\">28682534</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Littlewood</surname><given-names>DL</given-names></name><etal/></person-group><article-title>Short sleep duration and poor sleep quality predict next-day suicidal ideation: An ecological momentary assessment study</article-title><source>Psychol. Med.</source><year>2018</year><volume>49</volume><fpage>403</fpage><lpage>411</lpage><pub-id pub-id-type=\"pmid\">29697037</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Armitage</surname><given-names>R</given-names></name><name><surname>Trivedi</surname><given-names>M</given-names></name><name><surname>Hoffmann</surname><given-names>RA</given-names></name><name><surname>John</surname><given-names>R</given-names></name></person-group><article-title>Relationship between objective and subjective sleep measures in depressed patients and healthy controls</article-title><source>Depress. Anxiety</source><year>1997</year><volume>5</volume><fpage>97</fpage><lpage>102</lpage><pub-id pub-id-type=\"pmid\">9262940</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bertocci</surname><given-names>MA</given-names></name><etal/></person-group><article-title>Subjective sleep complaints in pediatric depression: A controlled study and comparison with EEG measures of sleep and waking</article-title><source>J. Am. Acad. Child Adolesc. Psychiatry</source><year>2005</year><volume>44</volume><fpage>1158</fpage><lpage>1166</lpage><pub-id pub-id-type=\"pmid\">16239865</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ancoli-Israel</surname><given-names>S</given-names></name><etal/></person-group><article-title>The role of actigraphy in the study of sleep and circadian rhythms</article-title><source>Sleep</source><year>2003</year><volume>26</volume><fpage>342</fpage><lpage>392</lpage><pub-id pub-id-type=\"pmid\">12749557</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ballard</surname><given-names>ED</given-names></name><etal/></person-group><article-title>Nocturnal wakefulness is associated with next-day suicidal ideation in major depressive disorder and bipolar disorder</article-title><source>J. Clin. Psychiatry</source><year>2016</year><volume>77</volume><fpage>825</fpage><lpage>831</lpage><pub-id pub-id-type=\"pmid\">27337418</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ribeiro</surname><given-names>JD</given-names></name><etal/></person-group><article-title>Sleep problems outperform depression and hopelessness as cross-sectional and longitudinal predictors of suicidal ideation and behavior in young adults in the military</article-title><source>J. Affect. Disord.</source><year>2012</year><volume>136</volume><fpage>743</fpage><lpage>750</lpage><pub-id pub-id-type=\"pmid\">22032872</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Britton</surname><given-names>PC</given-names></name><name><surname>Ilgen</surname><given-names>MA</given-names></name><name><surname>Rudd</surname><given-names>MD</given-names></name><name><surname>Conner</surname><given-names>KR</given-names></name></person-group><article-title>Warning signs for suicide within a week of healthcare contact in Veteran decedents</article-title><source>Psychiatry Res.</source><year>2012</year><volume>200</volume><fpage>395</fpage><lpage>399</lpage><pub-id pub-id-type=\"pmid\">22796102</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ribeiro</surname><given-names>JD</given-names></name><name><surname>Yen</surname><given-names>S</given-names></name><name><surname>Joiner</surname><given-names>T</given-names></name><name><surname>Siegler</surname><given-names>IC</given-names></name></person-group><article-title>Capability for suicide interacts with states of heightened arousal to predict death by suicide beyond the effects of depression and hopelessness</article-title><source>J. Affect. Disord.</source><year>2015</year><volume>188</volume><fpage>53</fpage><lpage>59</lpage><pub-id pub-id-type=\"pmid\">26342889</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pigeon</surname><given-names>WR</given-names></name><name><surname>Pinquart</surname><given-names>M</given-names></name><name><surname>Conner</surname><given-names>K</given-names></name></person-group><article-title>Meta-analysis of sleep disturbance and suicidal thoughts and behaviors</article-title><source>J. Clin. Psychiatry</source><year>2012</year><volume>73</volume><fpage>e1160</fpage><lpage>e1167</lpage><pub-id pub-id-type=\"pmid\">23059158</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Glenn</surname><given-names>CR</given-names></name><name><surname>Nock</surname><given-names>MK</given-names></name></person-group><article-title>Improving the short-term prediction of suicidal behavior</article-title><source>Am. J. Prev. Med.</source><year>2014</year><volume>47</volume><fpage>S176</fpage><lpage>S180</lpage><pub-id pub-id-type=\"pmid\">25145736</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Walsh</surname><given-names>CG</given-names></name><name><surname>Ribeiro</surname><given-names>JD</given-names></name><name><surname>Franklin</surname><given-names>JC</given-names></name></person-group><article-title>Predicting risk of suicide attempts over time through machine learning</article-title><source>Clin. Psychol. Sci.</source><year>2017</year><volume>5</volume><fpage>457</fpage><lpage>469</lpage></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Franklin</surname><given-names>JC</given-names></name><etal/></person-group><article-title>Risk factors for suicidal thoughts and behaviors: A meta-analysis of 50 years of research</article-title><source>Psychol. Bull.</source><year>2017</year><volume>143</volume><fpage>187</fpage><lpage>232</lpage><pub-id pub-id-type=\"pmid\">27841450</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ribeiro</surname><given-names>JD</given-names></name><name><surname>Huang</surname><given-names>X</given-names></name><name><surname>Fox</surname><given-names>KR</given-names></name><name><surname>Franklin</surname><given-names>JC</given-names></name></person-group><article-title>Depression and hopelessness as risk factors for suicide ideation, attempts and death: Meta-analysis of longitudinal studies</article-title><source>Br. J. Psychiatry</source><year>2018</year><volume>212</volume><fpage>279</fpage><lpage>286</lpage><pub-id pub-id-type=\"pmid\">29587888</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ribeiro</surname><given-names>JD</given-names></name><etal/></person-group><article-title>Self-injurious thoughts and behaviors as risk factors for future suicide ideation, attempts, and death: A meta-analysis of longitudinal studies</article-title><source>Psychol. Med.</source><year>2016</year><volume>46</volume><fpage>225</fpage><lpage>236</lpage><pub-id pub-id-type=\"pmid\">26370729</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><mixed-citation publication-type=\"other\">R Core Team. <italic>R: A Language and Environment for Statistical Computing</italic>. (R Foundation for Statistical Computing, 2019).</mixed-citation></ref><ref id=\"CR57\"><label>57.</label><mixed-citation publication-type=\"other\">Viechtbauer, W. Conducting Meta-Analyses in <italic>R</italic> with the <bold>metafor</bold> Package (Version 2.4). <italic>J. Stat. Softw.</italic><bold>36</bold>, (2010). <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.jstatsoft.org/v36/i03/\">https://www.jstatsoft.org/v36/i03/</ext-link>.</mixed-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bentley</surname><given-names>KH</given-names></name><etal/></person-group><article-title>Anxiety and its disorders as risk factors for suicidal thoughts and behaviors: A meta-analytic review</article-title><source>Clin. Psychol. Rev.</source><year>2016</year><volume>43</volume><fpage>30</fpage><lpage>46</lpage><pub-id pub-id-type=\"pmid\">26688478</pub-id></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Witte</surname><given-names>TK</given-names></name><name><surname>Gauthier</surname><given-names>JM</given-names></name><name><surname>Huang</surname><given-names>X</given-names></name><name><surname>Ribeiro</surname><given-names>JD</given-names></name><name><surname>Franklin</surname><given-names>JC</given-names></name></person-group><article-title>Is externalizing psychopathology a robust risk factor for suicidal thoughts and behaviors? A meta-analysis of longitudinal studies</article-title><source>J. Clin. Psychol.</source><year>2018</year><volume>74</volume><fpage>1607</fpage><lpage>1625</lpage><pub-id pub-id-type=\"pmid\">29687442</pub-id></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lemola</surname><given-names>S</given-names></name><name><surname>Ledermann</surname><given-names>T</given-names></name><name><surname>Friedman</surname><given-names>EM</given-names></name></person-group><article-title>Variability of sleep duration is related to subjective sleep quality and subjective well-being: An actigraphy study</article-title><source>PLOS ONE</source><year>2013</year><volume>8</volume><fpage>e71292</fpage><pub-id pub-id-type=\"pmid\">23967186</pub-id></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chung</surname><given-names>S</given-names></name><etal/></person-group><article-title>Environmental noise and sleep disturbance: Night-to-night variability of sleep/wake pattern</article-title><source>Sleep Med. Res.</source><year>2016</year><volume>7</volume><fpage>78</fpage><lpage>81</lpage></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cukrowicz</surname><given-names>KC</given-names></name><etal/></person-group><article-title>Course of suicide ideation and predictors of change in depressed older adults</article-title><source>J. Affect. Disord.</source><year>2009</year><volume>113</volume><fpage>30</fpage><lpage>36</lpage><pub-id pub-id-type=\"pmid\">18617271</pub-id></element-citation></ref><ref id=\"CR63\"><label>63.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Glenn</surname><given-names>CR</given-names></name><etal/></person-group><article-title>Implicit identification with death predicts change in suicide ideation during psychiatric treatment in adolescents</article-title><source>J. Child Psychol. Psychiatry</source><year>2017</year><volume>58</volume><fpage>1319</fpage><lpage>1329</lpage><pub-id pub-id-type=\"pmid\">28675456</pub-id></element-citation></ref><ref id=\"CR64\"><label>64.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Franklin</surname><given-names>JC</given-names></name><etal/></person-group><article-title>A brief mobile app reduces nonsuicidal and suicidal self-injury: Evidence from three randomized controlled trials</article-title><source>J. Consult. Clin. Psychol.</source><year>2016</year><volume>84</volume><fpage>544</fpage><lpage>557</lpage><pub-id pub-id-type=\"pmid\">27018530</pub-id></element-citation></ref><ref id=\"CR65\"><label>65.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ribeiro</surname><given-names>JD</given-names></name><name><surname>Huang</surname><given-names>X</given-names></name><name><surname>Fox</surname><given-names>KR</given-names></name><name><surname>Walsh</surname><given-names>CG</given-names></name><name><surname>Linthicum</surname><given-names>KP</given-names></name></person-group><article-title>Predicting imminent suicidal thoughts and nonfatal attempts: The role of complexity</article-title><source>Clin. Psychol. Sci.</source><year>2019</year><volume>7</volume><fpage>941</fpage><lpage>957</lpage></element-citation></ref><ref id=\"CR66\"><label>66.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Casler</surname><given-names>K</given-names></name><name><surname>Bickel</surname><given-names>L</given-names></name><name><surname>Hackett</surname><given-names>E</given-names></name></person-group><article-title>Separate but equal? A comparison of participants and data gathered via Amazon’s MTurk, social media, and face-to-face behavioral testing</article-title><source>Comput. Hum. Behav.</source><year>2013</year><volume>29</volume><fpage>2156</fpage><lpage>2160</lpage></element-citation></ref><ref id=\"CR67\"><label>67.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hauser</surname><given-names>DJ</given-names></name><name><surname>Schwarz</surname><given-names>N</given-names></name></person-group><article-title>Attentive Turkers: MTurk participants perform better on online attention checks than do subject pool participants</article-title><source>Behav. Res. Methods</source><year>2016</year><volume>48</volume><fpage>400</fpage><lpage>407</lpage><pub-id pub-id-type=\"pmid\">25761395</pub-id></element-citation></ref><ref id=\"CR68\"><label>68.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zuromski</surname><given-names>KL</given-names></name><name><surname>Cero</surname><given-names>I</given-names></name><name><surname>Witte</surname><given-names>TK</given-names></name></person-group><article-title>Insomnia symptoms drive changes in suicide ideation: A latent difference score model of community adults over a brief interval</article-title><source>J. Abnorm. Psychol.</source><year>2017</year><volume>126</volume><fpage>739</fpage><lpage>749</lpage><pub-id pub-id-type=\"pmid\">28557509</pub-id></element-citation></ref><ref id=\"CR69\"><label>69.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kivelä</surname><given-names>L</given-names></name><etal/></person-group><article-title>Longitudinal course of suicidal ideation and predictors of its persistence—A NESDA study</article-title><source>J. Affect. Disord.</source><year>2019</year><volume>257</volume><fpage>365</fpage><lpage>375</lpage><pub-id pub-id-type=\"pmid\">31302526</pub-id></element-citation></ref><ref id=\"CR70\"><label>70.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wong</surname><given-names>MM</given-names></name><name><surname>Brower</surname><given-names>KJ</given-names></name><name><surname>Zucker</surname><given-names>RA</given-names></name></person-group><article-title>Sleep problems, suicidal ideation, and self-harm behaviors in adolescence</article-title><source>J. Psychiatry Res.</source><year>2011</year><volume>45</volume><fpage>505</fpage><lpage>511</lpage></element-citation></ref><ref id=\"CR71\"><label>71.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sjöström</surname><given-names>N</given-names></name><name><surname>Hetta</surname><given-names>J</given-names></name><name><surname>Waern</surname><given-names>M</given-names></name></person-group><article-title>Persistent nightmares are associated with repeat suicide attempt</article-title><source>Psychiatry Res.</source><year>2009</year><volume>170</volume><fpage>208</fpage><lpage>211</lpage><pub-id pub-id-type=\"pmid\">19900715</pub-id></element-citation></ref><ref id=\"CR72\"><label>72.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Roane</surname><given-names>BM</given-names></name><name><surname>Taylor</surname><given-names>DJ</given-names></name></person-group><article-title>Adolescent insomnia as a risk factor for early adult depression and substance abuse</article-title><source>Sleep</source><year>2008</year><volume>31</volume><fpage>1351</fpage><lpage>1356</lpage><pub-id pub-id-type=\"pmid\">18853932</pub-id></element-citation></ref><ref id=\"CR73\"><label>73.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tanskanen</surname><given-names>A</given-names></name><etal/></person-group><article-title>Nightmares as predictors of suicide</article-title><source>Sleep</source><year>2001</year><volume>24</volume><fpage>845</fpage><lpage>848</lpage></element-citation></ref><ref id=\"CR74\"><label>74.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>SX</given-names></name><etal/></person-group><article-title>Sleep disturbances and suicide risk in an 8-year longitudinal study of schizophrenia-spectrum disorders</article-title><source>Sleep</source><year>2016</year><volume>39</volume><fpage>1275</fpage><lpage>1282</lpage><pub-id pub-id-type=\"pmid\">27091530</pub-id></element-citation></ref><ref id=\"CR75\"><label>75.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shiffman</surname><given-names>S</given-names></name><etal/></person-group><article-title>Remember that? A comparison of real-time versus retrospective recall of smoking lapses</article-title><source>J. Consult. Clin. Psychol.</source><year>1997</year><volume>65</volume><fpage>292</fpage><pub-id pub-id-type=\"pmid\">9086693</pub-id></element-citation></ref><ref id=\"CR76\"><label>76.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Littlewood</surname><given-names>D</given-names></name><name><surname>Kyle</surname><given-names>SD</given-names></name><name><surname>Pratt</surname><given-names>D</given-names></name><name><surname>Peters</surname><given-names>S</given-names></name><name><surname>Gooding</surname><given-names>P</given-names></name></person-group><article-title>Examining the role of psychological factors in the relationship between sleep problems and suicide</article-title><source>Clin. Psychol. Rev.</source><year>2017</year><volume>54</volume><fpage>1</fpage><lpage>16</lpage><pub-id pub-id-type=\"pmid\">28371648</pub-id></element-citation></ref><ref id=\"CR77\"><label>77.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fox</surname><given-names>KR</given-names></name><etal/></person-group><article-title>Meta-analysis of risk factors for nonsuicidal self-injury</article-title><source>Clin. Psychol. Rev.</source><year>2015</year><volume>42</volume><fpage>156</fpage><lpage>167</lpage><pub-id pub-id-type=\"pmid\">26416295</pub-id></element-citation></ref><ref id=\"CR78\"><label>78.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fox</surname><given-names>KR</given-names></name><etal/></person-group><article-title>Model complexity improves the prediction of nonsuicidal self-injury</article-title><source>J. Consult. Clin. Psychol.</source><year>2019</year><volume>87</volume><fpage>684</fpage><pub-id pub-id-type=\"pmid\">31219275</pub-id></element-citation></ref><ref id=\"CR79\"><label>79.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ribeiro</surname><given-names>JD</given-names></name><etal/></person-group><article-title>Letter to the Editor: Suicide as a complex classification problem: machine learning and related techniques can advance suicide prediction—A reply to Roaldset (2016)</article-title><source>Psychol. Med.</source><year>2016</year><volume>46</volume><fpage>2009</fpage><lpage>2010</lpage><pub-id pub-id-type=\"pmid\">27091309</pub-id></element-citation></ref><ref id=\"CR80\"><label>80.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Franklin</surname><given-names>JC</given-names></name><name><surname>Huang</surname><given-names>X</given-names></name><name><surname>Bastidas</surname><given-names>D</given-names></name></person-group><article-title>Virtual reality suicide: Development of a translational approach for studying suicide causes</article-title><source>Behav. Res. Ther.</source><year>2018</year><pub-id pub-id-type=\"doi\">10.1016/j.brat.2018.12.013</pub-id><pub-id pub-id-type=\"pmid\">30616833</pub-id></element-citation></ref><ref id=\"CR81\"><label>81.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lim</surname><given-names>J</given-names></name><name><surname>Dinges</surname><given-names>DF</given-names></name></person-group><article-title>A meta-analysis of the impact of short-term sleep deprivation on cognitive variables</article-title><source>Psychol. Bull.</source><year>2010</year><volume>136</volume><fpage>375</fpage><lpage>389</lpage><pub-id pub-id-type=\"pmid\">20438143</pub-id></element-citation></ref><ref id=\"CR82\"><label>82.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schrimpf</surname><given-names>M</given-names></name><etal/></person-group><article-title>The effect of sleep deprivation on pain perception in healthy subjects: A meta-analysis</article-title><source>Sleep Med.</source><year>2015</year><volume>16</volume><fpage>1313</fpage><lpage>1320</lpage><pub-id pub-id-type=\"pmid\">26498229</pub-id></element-citation></ref><ref id=\"CR83\"><label>83.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Irwin</surname><given-names>MR</given-names></name><name><surname>Olmstead</surname><given-names>R</given-names></name><name><surname>Carroll</surname><given-names>JE</given-names></name></person-group><article-title>Sleep disturbance, sleep duration, and inflammation: A systematic review and meta-analysis of cohort studies and experimental sleep deprivation</article-title><source>Biol. Psychiatry</source><year>2016</year><volume>80</volume><fpage>40</fpage><lpage>52</lpage><pub-id pub-id-type=\"pmid\">26140821</pub-id></element-citation></ref><ref id=\"CR84\"><label>84.</label><mixed-citation publication-type=\"other\">Moher, D., Liberati, A., Tetzlaff, J., Altman, D. G. & The PRISMA Group. Preferred reporting items for systematic reviews and meta-analyses: The PRISMA statement. <italic>PLoS Med</italic><bold>6</bold>, e1000097 (2009).</mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cell Death Dis</journal-id><journal-id journal-id-type=\"iso-abbrev\">Cell Death Dis</journal-id><journal-title-group><journal-title>Cell Death & Disease</journal-title></journal-title-group><issn pub-type=\"epub\">2041-4889</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807788</article-id><article-id pub-id-type=\"pmc\">PMC7431544</article-id><article-id pub-id-type=\"publisher-id\">2819</article-id><article-id pub-id-type=\"doi\">10.1038/s41419-020-02819-w</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Farnesoid X receptor antagonizes Wnt/β-catenin signaling in colorectal tumorigenesis</article-title></title-group><contrib-group><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Yu</surname><given-names>Junhui</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Li</surname><given-names>Shan</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Guo</surname><given-names>Jing</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Xu</surname><given-names>Zhengshui</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Zheng</surname><given-names>Jianbao</given-names></name><address><email>bobzjb@126.com</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Sun</surname><given-names>Xuejun</given-names></name><address><email>sunxy@mail.xjtu.edu.cn</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.452438.c</institution-id><institution>Department of General Surgery, </institution><institution>First Affiliated Hospital of Xi’an Jiaotong University, </institution></institution-wrap>Xi’an, 710061 Shaanxi Province PR China </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.452438.c</institution-id><institution>Department of Reproductive Medicine, </institution><institution>First Affiliated Hospital of Xi’an Jiaotong University, </institution></institution-wrap>Xi’an, 710061 Shaanxi Province PR China </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><month>8</month><year>2020</year></pub-date><volume>11</volume><issue>8</issue><elocation-id>640</elocation-id><history><date date-type=\"received\"><day>28</day><month>2</month><year>2020</year></date><date date-type=\"rev-recd\"><day>16</day><month>7</month><year>2020</year></date><date date-type=\"accepted\"><day>16</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Farnesoid X receptor (FXR, encoded by NR1H4), a critical regulator of bile acid homeostasis, is widely implicated in human tumorigenesis. However, the functional role of FXR in colorectal cancer (CRC) and the precise molecular mechanism remain unclear. In this study, we demonstrated that FXR expression was downregulated in colon cancer tissues and decreased expression of FXR predicted a poor prognosis. Knockdown of FXR promoted colon cancer cell growth and invasion in vitro, and facilitated xenograft tumor formation and distant metastasis in vivo, whereas ectopic expression of FXR had the reserved change. Mechanistic studies indicated that FXR exerted its tumor suppressor functions by antagonizing Wnt/β-catenin signaling. Furthermore, we identified an FXR/β-catenin interaction in colon cancer cells. The FXR/β-catenin interaction impaired β-catenin/TCF4 complex formation. In addition, our study suggested a reciprocal relationship between FXR and β-catenin, since loss of β-catenin increased the transcriptional activation of SHP by FXR. Altogether, these data indicated that FXR functions a tumor-suppressor role in CRC by antagonizing Wnt/β-catenin signaling.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Colon cancer</kwd><kwd>Oncogenes</kwd><kwd>Diagnostic markers</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100001809</institution-id><institution>National Natural Science Foundation of China (National Science Foundation of China)</institution></institution-wrap></funding-source><award-id>81972720</award-id><principal-award-recipient><name><surname>Sun</surname><given-names>Xuejun</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>the Coordinative and Innovative Plan Projects of the Science and Technology Program in Shaanxi Province (Grant Serial Numbers: 2013KTCQ03-08)</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>the Science and Technology Project of Shaanxi Province (Grant serial number: 2016SF-015, 2019SF-065), the Fundamental Research Funds for the Central Universities (Grant serial number: xjj2018123)</institution></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par2\">Colorectal cancer (CRC) ranks the second leading cause of cancer-related death<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. Globally, ~1,800,000 new cases are diagnosed as CRC every year. With the change in lifestyle, including high-fat diets (HFDs), tobacco use and less or lack of exercise, the incidence of CRC has increased rapidly in developing countries<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. Although great progress has been achieved in multimodality therapy of CRC, the prognosis of late-stage CRC is still unsatisfactory due to distant metastasis and relapse<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. The molecular pathogenesis of CRC is considered a multistep and consecutive process with the accumulation of various aberrant genetic and epigenetic variations<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. Dissecting the precise mechanisms of colorectal tumorigenesis is crucial for developing better prognostic and therapeutic strategies.</p><p id=\"Par3\">Canonical Wnt/β-catenin signaling plays essential roles in embryonic development and maintaining gut homeostasis<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. Persistent activation of Wnt signaling featured by nuclear accumulation of β-catenin is an early event of colorectal tumorigenesis<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. Wnt-related targets, including c-Myc, cyclin D1, MMP-7, and VEGF, are critical contributors to tumor cell proliferation, invasion, and migratory potential<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>–<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Epithelial-mesenchymal transition (EMT) is a biological process that involves the malignant transformation of epithelial cells with a loss of an epithelial and gain of a mesenchyme-like phenotype<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. EMT plays a significant role in tumor progression via endowing tumor cells with the potential for invasive and metastatic growth. In invasive regions, tumor cells undergoing EMT exhibit a strong accumulation of nuclear β-catenin<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Recent study further demonstrates a direct link between β-catenin and EMT by identifying slug, a strong inducer of EMT, as the target of β-catenin<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. These data strongly indicate that Wnt signaling participated in EMT process.</p><p id=\"Par4\">Bile acids are widely involved in the pathogenesis of human malignancies, including hepatocellular carcinoma (HCC)<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>, gastric cancer<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>, esophageal cancer<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>, and pancreatic cancer<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Exposure to elevated fecal bile acids is associated with the occurrence of colon cancer<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. The farnesoid X receptor (FXR, encoded by NR1H4), a nuclear receptor of bile acids, is widely present in the gastrointestinal tract and liver<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. In addition to its essential role in regulating bile acid homeostasis<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>, accumulating evidence supports a critical role of FXR in human tumorigenesis<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. Reduced FXR at the mRNA level is found in colon polyps and is even more remarkable in CRC<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. Restoration of FXR has been shown to suppress abnormal intestinal cell growth and curtail CRC progression<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. However, the functional role of FXR in CRC and the precise molecular mechanism remain to be further elucidated.</p><p id=\"Par5\">In this study, we aim to investigate the correlation between FXR and Wnt/β-catenin signaling during colorectal tumorigenesis</p></sec><sec id=\"Sec2\" sec-type=\"materials|methods\"><title>Materials and methods</title><sec id=\"Sec3\"><title>Clinical samples and cell cultures</title><p id=\"Par6\">One hundred and twenty-three human colon cancer tissues were obtained from patient diagnosed with colon cancer and received surgery at the First Affiliated Hospital of Xi’an Jiaotong University from January 2011 to May 2013. No patient had received preoperative chemotherapy or radiotherapy. The procedure of this study was approved by the institutional review board of the First Affiliated Hospital of Xi’an Jiaotong University. Informed consent was obtained by all participants.</p><p id=\"Par7\">Human colon cancer cells HT-29, Caco-2, HCT116, RKO, SW480, and Lovo were maintained in DMEM medium (Corning, New York, NY, USA) supplemented with 10% FBS (Hyclone, Logan, UT, USA) at 5% CO<sup>2</sup> at 37 °C.</p></sec><sec id=\"Sec4\"><title>Lentiviral vectors and transfection</title><p id=\"Par8\">The phU6-EGFP-shRNA-FXR, β-catenin, and SHP lentiviral vectors and their control vectors were used to inhibit FXR, β-catenin, and SHP expression, while the pUbi-EGFP- FXR, β-catenin, and SHP lentiviral vectors and their control vectors were used to increase FXR, β-catenin, and SHP expression. All transfections were conducted in accordance with the manufacturer’s protocol. All the lentiviral vectors were constructed and prepared by GeneChem Co., Ltd. (Shanghai, China)</p></sec><sec id=\"Sec5\"><title>CCK8, colony formation and cell cycle assays</title><p id=\"Par9\">CCK8 assays were performed as described previously<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. For colony formation assay, three hundred cells were seeded and cultured for 14 days. Colonies (≥50 cells/colony) were counted. Cell cycle distributions were evaluated by flow cytometry as previously described<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. Each experiment was performed in triplicate.</p></sec><sec id=\"Sec6\"><title>Wound-healing assays</title><p id=\"Par10\">Cells were cultured in six-well plates until confluent. Then, three artificial vertical lines were created with pipette tips (10 µL) in each well. The wells were washed with phosphate-buffered saline (PBS) to remove cell debris. The cells were then cultured for an additional 48 h. The scratch lines were imaged under a microscope, and the scratch distances were measured. Each experiment was performed in triplicate.</p></sec><sec id=\"Sec7\"><title>Transwell assays</title><p id=\"Par11\">Cell migration and invasion were measured by using Transwell plates (Corning, New York, NY, USA) with or without Matrigel (BD, Franklin Lakes, NJ, USA). In both assays, the lower chamber was filled with 600 μl of DMEM medium containing 20% FBS. The upper chamber filters were pre-coated with 50 µL of Matrigel and plated at 10 × 10<sup>4</sup> cells per upper chamber. The cells were incubated at 37 °C for 48 h. After incubation, non-migratory cells on the upper surface of the Transwell inserts were removed by washing with fresh PBS. The invading cells on the underside of the membrane were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The number of cells was counted in three randomly selected fields of fixed cells under an inverted microscope. Each experiment was performed in triplicate.</p></sec><sec id=\"Sec8\"><title>Nude mouse xenograft and lung metastasis models</title><p id=\"Par12\">All animal experiments were performed in accordance with the institutional guidelines, and were approved by the Laboratory Animal Center of Xi’an Jiaotong University. The 5-week-old female BALB/c-nude mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The mice were injected with 5 × 10<sup>6</sup> cells into the right flanks to establish xenograft tumor model. Tumor size was were monitored using callipers every 3 days, and the tumor volume was calculated according to the formula (length × width<sup>2</sup> × 0.5). At the end of the experiment, the mice were killed and the xenograft tumors were isolated and weighted. Lung metastasis models were established via tail vein injections into each nude mouse. The weight of the nude mice was monitored every 3 days. At 60 days post-injection, the mice were killed by cervical dislocation, the lungs were excised, and the number of tumor nodules in the lung was recorded.</p></sec><sec id=\"Sec9\"><title>RNA isolation and real-time PCR</title><p id=\"Par13\">RNA isolation, complementary DNA (cDNA) synthesized, and real-time PCR were performed as described previously<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. The sequences of primers were summarized in Supplementary Table <xref rid=\"MOESM2\" ref-type=\"media\">1</xref>. Each experiment was performed in triplicate.</p></sec><sec id=\"Sec10\"><title>Immunohistochemistry</title><p id=\"Par14\">The protocol was performed as previously described<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. The extent of Ki67- and active caspase-3-stained cells was divided into 4 score ranks: 0–5% (0), 6–25% (1), 26–50% (2), 51–75% (3), and 76–100% (4). The staining intensity was divided into 4 score ranks: negative (0), light brown (1), brown (2), and dark brown (3). The immunoreactivity scores (IRSs) = extent score × intensity score. An IRS of ≤3 was defined as negative, and a score of >3 was defined as positive.</p></sec><sec id=\"Sec11\"><title>Preparation of nuclear extracts</title><p id=\"Par15\">Nuclear extract was prepared with the protocol in the the Nuclear Extraction Kit (abcam, Cambridge, MA, USA). The nuclear protein was quantified and used for downstream applications.</p></sec><sec id=\"Sec12\"><title>Total protein extraction and western blotting analysis</title><p id=\"Par16\">The detailed protocol was performed as described previously<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. The antibody information was presented in Supplementary Table <xref rid=\"MOESM2\" ref-type=\"media\">2</xref>. Each experiment was performed in triplicate.</p></sec><sec id=\"Sec13\"><title>Immunofluorescence (IF)</title><p id=\"Par17\">The cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.2% Triton X-100 for 10 min. After blocking with 5% bovine serum albumin (BSA) for 30 min at room temperature, the cells were incubated at 4 °C overnight with primary antibodies against E-cadherin and vimentin (1:100 dilution). The dishes were washed three times with PBS for 10 min each and then incubated with Alexa Fluor 594-conjugated secondary antibodies (1:400 dilution, Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. The nuclei were stained with DAPI (10 mg/ml) for 10 min. The samples were examined via microscopy (Leica Microsystems, Heidelberg, Germany) to analyze the expression of E-cadherin and vimentin.</p></sec><sec id=\"Sec14\"><title>Luciferase reporter assay</title><p id=\"Par18\">Fragments of the SHP 5′-flanking sequence were amplified by PCR using special primers (Table <xref rid=\"MOESM2\" ref-type=\"media\">S1</xref>) and cloned into the luciferase reporter vector pGL3.0-Basic (Promega, Madison, WI, USA) to generate SHP promoter reporter constructs (wild-type SHP promoter, SHP-WT promoter). Mutagenesis of FXR binding site in SHP promoter (SHP-mFXR promoter) was performed using a site-directed mutagenesis kit (Takara Biotechnology Co., Ltd.). The detailed protocol was performed as described previously<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. Each experiment was performed in triplicate.</p></sec><sec id=\"Sec15\"><title>Co-immunoprecipitation assay</title><p id=\"Par19\">The cells were washed three times with ice-cold PBS and harvested at 4 °C in immunoprecipitation lysis buffer. Co-immunoprecipitation (co-IP) assay was then performed as described previously<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>. Each experiment was performed in triplicate.</p></sec><sec id=\"Sec16\"><title>Quantitative chromatin immunoprecipitation</title><p id=\"Par20\">Cells were subjected to ChIP using the EZ-ChIP Kit (Millipore, Bedford, MA, USA)。</p><p id=\"Par21\">The detailed protocol was performed as described previously<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. Real-time PCR was conducted to amplify the regions of DNA fragments by using special primers (Supplementary Table <xref rid=\"MOESM2\" ref-type=\"media\">1</xref>). Each experiment was performed in triplicate.</p></sec><sec id=\"Sec17\"><title>Microarrays and gene expression analysis</title><p id=\"Par22\">Three HT-29-shCtrl cells and three HT-29-shFXR cells were used for microarrays. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). An Affymetrix PrimeView Human Gene Expression Array was used to investigate the changes in transcriptional profiles. The experiment was performed based on the manufacturer’s standard protocols. Genes with ≥1.5-fold change between two groups were identified as differentially expressed genes.</p></sec><sec id=\"Sec18\"><title>Statistical analysis</title><p id=\"Par23\">Each experiment was repeated three times. Data are presented as the mean ± SD. The Student’s <italic>t</italic> test, one-way ANOVA or <italic>χ</italic><sup>2</sup> test was conducted to compare the differences among the groups. Correlations were analyzed by using Pearson linear-regression analysis. OS and RFS rates were plotted using the Kaplan–Meier method and compared with log-rank test. Multivariate statistical analysis was performed using a Cox regression model. Statistical analyses were performed with SPSS 18.0 software (SPSS Inc., Chicago, IL, USA). <italic>P</italic> < 0.05 was considered statistically significant.</p></sec></sec><sec id=\"Sec19\" sec-type=\"results\"><title>Results</title><sec id=\"Sec20\"><title>FXR is expressed at low levels in colon cancer clinical samples and correlates with poor prognosis</title><p id=\"Par24\">To determine whether FXR correlates with CRC development and progression, we first employed immunohistochemistry (IHC) assay to detect the expression of FXR in 123 colon cancer tissues and paired normal tissues. FXR immunostaining was seen in the nuclei of colonic cells (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>). The rate of positive FXR staining was decreased from 67.5% (83/123) in normal tissues to 32.5% (40/123) in colon cancer tissues (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref>). Moreover, colon cancer tissues displayed a low immunoreactivity score (IRS) of FXR staining in relative to normal tissues. The correlation between FXR and patient clinicopathological characteristics was further analyzed. Our study found that FXR expression was negatively correlated with tumor size, T stages, lymph node metastasis, and TNM stages (Supplementary Table <xref rid=\"MOESM2\" ref-type=\"media\">3</xref>). Kaplan–Meier analysis showed that patients with low FXR-expressed tumors had shorter overall survival (OS) times and shorter recurrence-free survival (RFS) times than those with high FXR-expressed tumors (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1d</xref>). Univariate analyses indicated that FXR expression was negatively correlated with OS and RFS, although FXR was not validated as an independent predictor of OS and RFS by multivariate analyses (Supplementary Tables <xref rid=\"MOESM2\" ref-type=\"media\">4</xref> and <xref rid=\"MOESM2\" ref-type=\"media\">5</xref>). Analyses of colon cancer data from the TCGA database also supported a strong relationship between diminished FXR and poor overall survival in patients with colon cancer (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1e</xref>).<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>The expression of FXR in colon cancer tissue samples and normal tissue samples.</title><p><bold>a</bold> The expression of FXR in normal tissue samples and colon cancer tissue samples by immunohistochemistry (IHC) staining. <bold>b</bold> The positivity of FXR staining in normal tissue samples and colon cancer tissue samples. <bold>c</bold> The immunoreactivity score (IRS) of FXR staining in normal tissue samples and colon cancer tissue samples. <bold>d</bold> Kaplan–Meier representation of the overall survival and recurrence-free survival of the two groups of patients with high (<italic>n</italic> = 40, red line) or low (<italic>n</italic> = 83, blue line) FXR expression in colon cancer tissues. <bold>e</bold> Data in TCGA database showed the overall survival of the two groups of patients with high (red line) or low (blue line) FXR expression in colon cancer tissues. <bold>f</bold> Western blotting bands for FXR expression in normal tissue samples and colon cancer tissue samples. <bold>g</bold> Western blotting bands for FXR expression in six colon cancer cells. All data are the mean ± SD of three independent experiments. *<italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"41419_2020_2819_Fig1_HTML\" id=\"d30e641\"/></fig></p><p id=\"Par25\">Next, western blotting analysis was conducted to evaluate the expression of FXR in eight colon cancer tissues and paired normal tissues (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1f</xref>). The results showed that tumor tissues exhibited reduced FXR expression levels compared with normal tissues (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1g</xref>). Finally, the expression of FXR in six colon cancer cell lines was investigated (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1h</xref>). FXR was highly expressed in highly or moderately differentiated HT-29 and Caco-2 cells. However, in poorly differentiated (HCT116 and RKO) or undifferentiated (SW480 and Lovo) colon cancer cells, the levels of FXR were at a low level or not expressed. Taken together, we conclude that FXR expression is reduced in colon cancer tissues and decreased FXR expression correlates with poor prognosis.</p></sec><sec id=\"Sec21\"><title>FXR inhibits tumorigenic properties of colon cancer cells</title><p id=\"Par26\">To further gain insight into the impact of FXR in colorectal tumorigenesis, a series of in vitro experiments were performed in colon cancer cells with gain-of-function and loss-of-function of FXR. Knockdown of FXR in HT-29 and Caco-2 cells, and ectopic expression of FXR in SW480 and HCT116 cells were validated by western blotting analysis (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a, b</xref>).<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>FXR inhibits tumorigenic properties of colon cancer cells.</title><p><bold>a</bold>, <bold>b</bold> FXR expression in FXR-knockdown HT-29 and Caco-2 cells (<bold>a</bold>) or FXR-overexpressing SW480 and HCT116 cells (<bold>b</bold>) detected by western blotting analysis. <bold>c</bold>, <bold>d</bold> The effect of FXR knockdown (<bold>c</bold>) or overexpression (<bold>d</bold>) on the viability of colon cancer cells detected by CCK8 assays. <bold>e</bold>, <bold>f</bold> The effect of FXR knockdown (<bold>e</bold>) or overexpression (<bold>f</bold>) on colony formation of colon cancer cells. <bold>g</bold>, <bold>h</bold> The effect of FXR knockdown (<bold>g</bold>) or overexpression (<bold>h</bold>) on cell cycle distribution of colon cancer cells. All data are the mean ± SD of three independent experiments. *<italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"41419_2020_2819_Fig2_HTML\" id=\"d30e723\"/></fig></p><p id=\"Par27\">CCK8 assays were employed to assess the effect of modulating FXR expression on the viability of colon cancer cells. Our data indicated that knockdown of FXR in HT-29 and Caco-2 cells resulted in an enhanced cell viability (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2c</xref>), whereas ectopic expression of FXR in SW480 and HCT116 cells had the reserved change (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2d</xref>). The inhibitory effect of FXR on tumor cell growth was further verified by colony-formation assay, in which knockdown or ectopic expression of FXR promoted or inhibited the colony-formation ability of colon cancer cells (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2e, f</xref>), respectively. As cell proliferation regulation was observed after modulation of FXR, cell cycle distribution was detected by flow cytometry assay. Knockdown of FXR resulted in a marked decrease of cells in the G0/G1 phase with an accumulation of cells the S phase (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2g</xref>). FXR overexpression experiment had the opposite change (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2h</xref>). However, modulation of FXR expression had no significant impact on the accumulation of cells in the G2 phase.</p><p id=\"Par28\">We next conducted xenograft mouse model to assess in vivo tumor-suppressor role of FXR. The xenograft tumors in the FXR-knockdown group showed a decline in growth rate in relative to the control group (Supplementary Fig. <xref rid=\"MOESM3\" ref-type=\"media\">1a, b</xref>). Moreover, mean weight of xenograft tumors in the FXR-knockdown group is lighter than that in the control group (Supplementary Fig. <xref rid=\"MOESM3\" ref-type=\"media\">1a, b</xref>). FXR-overexpressing group displayed the opposite change (Supplementary Fig. <xref rid=\"MOESM3\" ref-type=\"media\">1c, d</xref>). Ki67 was a well-known maker evaluating cellular proliferation. Thus, we detected Ki67 expression in xenograft tumors by using IHC staining. Enhanced Ki67 staining of the xenograft tumors was observed in the FXR-knockdown group in relative to the control group (Supplementary Fig. <xref rid=\"MOESM3\" ref-type=\"media\">1e</xref>). Conversely, the xenograft tumors in the FXR-overexpressing group had the opposite change (Supplementary Fig. <xref rid=\"MOESM3\" ref-type=\"media\">1f</xref>). Collectively, these data supported a tumor suppressor role of FXR in colon cancer cells.</p></sec><sec id=\"Sec22\"><title>FXR inhibits colon cancer cell invasion and metastasis in vitro and vivo</title><p id=\"Par29\">Considering that tumor metastasis is the leading cause of cancer-related death in CRC, we thus aimed to evaluate the impact of FXR on the invasive and migratory abilities of colon cancer cells. Wound-healing scratch assays showed that knockdown of FXR in HT-29 and Caco-2 cells led to an increase in the percentage of wound healing (Supplementary Fig. <xref rid=\"MOESM4\" ref-type=\"media\">2a, b</xref>). Conversely, ectopic expression of FXR in SW480 and HCT116 cells had the reserved change (Supplementary Fig. <xref rid=\"MOESM4\" ref-type=\"media\">2c, d</xref>). The Transwell assays showed that the number of invasive HT-29-shFXR and Caco-2-shFXR cells was greater than the number of invasive control cells (Supplementary Fig. <xref rid=\"MOESM4\" ref-type=\"media\">2e, g</xref>), whereas the number of invasive SW480-FXR and HCT116-FXR cells was less than the number of invasive control cells (Supplementary Fig. <xref rid=\"MOESM4\" ref-type=\"media\">2f, h</xref>).</p><p id=\"Par30\">To determine whether FXR affects colon cancer cell metastasis in vivo, colon cancer lung metastasis models via tail vein injection were generated in BALB/c-nude mice using HT-29 and Caco-2 cells with stably FXR knockdown. The metastatic tumor nodules of the FXR-knockdown group and the control group were counted under microscopy by H&E staining (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a, c</xref>). The average number of tumor nodules in lung metastasis in HT-29-shFXR and Caco-2-shFXR groups was greater than that in the control groups (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b, d</xref>). Altogether, these data suggested that FXR inhibits colon cancer cells invasion and metastasis.<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Knockdown of FXR promotes the lung metastasis of colon cancer cells in vivo.</title><p>Lung metastasis models of colon cancer were generated in BALB/c-nude mice with FXR-knockdown HT-29 and Caco-2 cells via tail vein injection. <bold>a</bold>, <bold>c</bold> The metastases in lung were shown for HT-29-shFXR (<bold>a</bold>) and Caco-2-shFXR (<bold>c</bold>) group and their control group identified by H&E staining. <bold>b</bold>, <bold>d</bold> Average number of tumor nodules in lung metastasis in the HT-29-shFXR (<bold>b</bold>) and Caco-2-shFXR (<bold>d</bold>) group and their control group. *<italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"41419_2020_2819_Fig3_HTML\" id=\"d30e821\"/></fig></p></sec><sec id=\"Sec23\"><title>FXR inhibits EMT in colon cancer cells</title><p id=\"Par31\">The presence of tumor cells that have undergone EMT is considered to be an important event during the early stage of cancer metastasis<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. Whether EMT is involved in the mechanism of FXR-mediated inhibition of colorectal tumorigenesis was investigated. Our study indicated that knockdown of FXR in HT-29 and Caco-2 cells resulted in a promotion in the levels of slug, snail, vimentin, fibronectin, and MMP-9 with a reduction in the levels of E-cadherin and ZO-1 (Supplementary Fig. <xref rid=\"MOESM5\" ref-type=\"media\">3a, c</xref>). Conversely, ectopic expression of FXR had the opposite effect (Supplementary Fig. <xref rid=\"MOESM5\" ref-type=\"media\">3b, d</xref>).</p><p id=\"Par32\">Furthermore, the results from IF assay indicated that HT-29-shFXR and Caco-2-shFXR cells showed a decrease in E-cadherin staining (Supplementary Fig. <xref rid=\"MOESM6\" ref-type=\"media\">4a, b</xref>) and an increase vimentin staining (Supplementary Fig. <xref rid=\"MOESM6\" ref-type=\"media\">4c, d</xref>) in relative to the control cells. IHC assay showed the lung metastatic tumor tissues formed in the FXR-knockdown group demonstrated a weaker E-cadherin staining and a much stronger vimentin-staining than those formed in the control group (Supplementary Fig. <xref rid=\"MOESM6\" ref-type=\"media\">4e, f</xref>). Altogether, these findings supported a suppressive effect of FXR on EMT in CRC.</p></sec><sec id=\"Sec24\"><title>FXR inhibits colorectal tumorigenesis by antagonizing Wnt/β-catenin signaling</title><p id=\"Par33\">We further explore the mechanism of FXR-mediated tumorigenicity and EMT inhibition by using microarray analyses of HT-29-shFXR cells and the control cells. Hierarchical clustering analysis showed that knockdown of FXR led to a difference in gene expression profile with the upregulation of 310 genes and the downregulation of 206 genes (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4a</xref>). Pathway analysis suggested that multiple signaling pathway might participate in the tumor-promoting mechanism of FXR knockdown (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4b</xref>). Among them, Wnt/β-catenin pathway aroused our interest, as it plays a critical role in promoting EMT, stemness properties and tumorigenicity<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. In addition, previous studies revealed that FXR deficiency in mice led to increased colon and liver cell proliferation, accompanied by an upregulation of β-catenin<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref>,<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. Analysis of Wnt/β-catenin signaling indicated that c-Myc and cyclin D1, two direct target genes, were upregulated in HT-29-shFXR cells in relative to HT-29-shCtrl cells (data not shown). Hence, Wnt/β–catenin signaling was chosen for further research.<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>FXR suppresses the activity of Wnt/β-catenin signaling in colorectal tumorigenesis.</title><p><bold>a</bold> Hierarchical clustering of genes that were significantly and differentially expressed in HT-29-shFXR cells and control cells. Data were log2 normalized. <bold>b</bold> Pathway analysis of genes that were significantly and differentially expressed in HT-29-shFXR cells and control cells using KEGG database. <bold>c</bold> The effect of FXR knockdown or overexpression on the luciferase activities of TOP/FOP-Flash reporter plasmid. <bold>d</bold> The effect of FXR knockdown or overexpression on the mRNA levels of cyclin D1 and c-Myc in colon cancer cells detected by real-time PCR. <bold>e</bold>, <bold>f</bold> The effect of FXR knockdown (<bold>e</bold>) or overexpression (<bold>f</bold>) on the protein levels of cyclin D1 and c-Myc in colon cancer cells detected by western blotting analysis. All data are the mean ± SD of three independent experiments. *<italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"41419_2020_2819_Fig4_HTML\" id=\"d30e907\"/></fig></p><p id=\"Par34\">To further validate the impact of FXR on the activity of Wnt/β-catenin signaling in colon cancer cells, we first performed a TOP/FOP-Flash luciferase reporter assay. Knockdown of FXR in HT-29 and SW480 cells elevated the luciferase intensities compared with the control (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c</xref>). Moreover, the expression of c-Myc and cyclin D1 at both the mRNA and protein levels was elevated in response to FXR knockdown (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4d, e</xref>). FXR overexpression experiment had the opposite change (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c, d, f</xref>).</p><p id=\"Par35\">Furthermore, XAV-939, a selective inhibitor of Wnt signaling, was adopted to block activated Wnt signaling in FXR-knockdown cells. As expected, XAV-939 treatment attenuated the proliferative and invasive abilities inhibited by FXR knockdown (Supplementary Fig. <xref rid=\"MOESM7\" ref-type=\"media\">5a–f</xref>). Consistent with the observations above, XAV-939 treatment reduced the protein levels of vimentin, fibronectin, MMP-9, snail, slug, cyclin D1, and c-Myc but increased the level of E-cadherin and ZO-1 (Supplementary Fig. <xref rid=\"MOESM8\" ref-type=\"media\">6a–d</xref>). These data demonstrate that FXR inhibits colorectal tumorigenesis and EMT induction, which may be attributed to its suppression of Wnt signaling.</p></sec><sec id=\"Sec25\"><title>FXR functions as a repressor of Wnt/β-catenin signaling by interacting with β-catenin in colon cancer cells</title><p id=\"Par36\">In canonical Wnt signaling, elevated nuclear β-catenin is always observed<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. However, our western blotting analysis showed no significant difference in nuclear β-catenin level between modified-FXR cells and the control cells (data not shown). Since FXR is a mainly located in the nucleus, we hypothesized that the FXR might affect the β-catenin/TCF complex<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. First, the co-IP assay showed that FXR did not bind with TCF4 in the nucleus of HEK293 cells (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5a</xref>). Then, we questioned whether FXR could form a complex with β-catenin. FXR co-immunoprecipitated with β-catenin in HEK293 cells co-transfected with FXR and Flag-β-catenin (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5b</xref>). Moreover, reciprocal co-IP experiments were performed by co-transfecting Myc-FXR and β-catenin into HEK293 cells, which further support the interaction between FXR and β-catenin (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5b</xref>). An interaction between endogenous FXR and β-catenin was also observed in HT-29 and Caco-2 cells (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5c</xref>). Next, we attempted to assess that whether FXR abolished the stability of the β-catenin/TCF4 complex by forming a complex with β-catenin. The binding between exogenous β-catenin and exogenous TCF4 was impaired in HEK293 cells upon FXR overexpression (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5d</xref>). Moreover, knockdown of FXR in HT-29 cells retarded the binding between endogenous β-catenin and endogenous TCF4 (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5e</xref>), whereas ectopic expression of FXR in SW480 cells had the opposite effect (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5f</xref>). Altogether, these data indicated that FXR functions as a repressor of Wnt/β-catenin signaling by interacting with β-catenin. This interaction impaired β-catenin/TCF4 complex and subsequent transcriptional activity of Wnt-related target genes.<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>FXR functions as a repressor of Wnt/β-catenin signaling by interacting with β-catenin in colon cancer cells.</title><p><bold>a</bold> Co-immunoprecipitation showed the physical interaction between FXR and TCF4 in HEK293 cells co-expressing HA-tagged TCF4 and Myc-tagged FXR. The cell lysates were subjected to IP with an anti-HA or Myc antibody. <bold>b</bold> Co-immunoprecipitation showed the physical interaction between FXR and β-catenin in HEK293 cells co-expressing Flag-β-catenin and Myc-tagged FXR. The cell lysates were subjected to IP with an anti-Flag or Myc antibody. <bold>c</bold> Co-immunoprecipitation showed the physical interaction between endogenous FXR and β-catenin was also observed in HT-29 and Caco-2 cells. The cell lysates were subjected to IP with an anti-FXR. <bold>d</bold> Ectopic expression of FXR impaired the interaction of β-catenin and TCF4. Plasmids of HA-tagged-TCF4, Flag-tagged-β-catenin, and FXR-shRNA lentivirus were co-transfected into HEK293 cells. The cell lysates were subjected to IP with an anti-Flag antibody. <bold>e</bold> Knockdown of FXR enhanced the interaction of β-catenin and TCF4. HT-29 cells were infected with FXR-shRNA lentivirus or control lentivirus. The nuclear fractions were incubated with an anti-TCF4 antibody for the IP experiment. IgG was used as a negative control. <bold>f</bold> Ectopic expression of FXR enhanced the interaction of β-catenin and TCF4. SW480 cells were infected with FXR lentivirus or control lentivirus. The nuclear fractions were incubated with an anti-TCF4 antibody for the IP experiment.</p></caption><graphic xlink:href=\"41419_2020_2819_Fig5_HTML\" id=\"d30e989\"/></fig></p></sec><sec id=\"Sec26\"><title>FXR inhibits colorectal tumorigenesis by regulating SHP expression</title><p id=\"Par37\">Small heterodimer partner (SHP), the well-known target gene of FXR<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>, retards tumorigenesis by regulating cyclin D1 expression<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. Microarray analyses revealed that SHP is downregulated in HT-29 cells upon FXR knockdown, which was validated by the results of real-time PCR and western blotting analysis (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6a–d</xref>). To further validate the involvement of SHP in FXR-mediated inhibition of colorectal tumorigenesis, we ectopically expressed or knocked down SHP in colon cancer cells. Ectopic expression of SHP attenuated the proliferative and invasive abilities of colon cancer cells enhanced by FXR knockdown (Supplementary Fig. <xref rid=\"MOESM9\" ref-type=\"media\">7a, c, e</xref>). Western blotting analysis indicated that the levels of cell cycle- and EMT-related proteins in HT-29-shFXR and Caco-2-shFXR cells was reversed upon ectopic expression of SHP (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6e</xref>). Conversely, knockdown of SHP significantly restored the tumor-inhibitory effect of FXR on SW480 and HCT116 cells (Supplementary Fig. <xref rid=\"MOESM9\" ref-type=\"media\">7b, d, f</xref>) as well as the levels of cell cycle- and EMT-related proteins (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6f</xref>). These findings further confirm that FXR-mediated colorectal tumorigenesis inhibition might be partly related to its transcriptional activation of SHP.<fig id=\"Fig6\"><label>Fig. 6</label><caption><title>FXR inhibits colorectal tumorigenesis by regulating SHP expression.</title><p>SHP expression in FXR-knockdown HT-29 and Caco-2 cells (<bold>a</bold>) or FXR-overexpressing SW480 and HCT116 cells (<bold>b</bold>) detected by western blotting analysis. <bold>c</bold> Quantitative analysis of SHP expression in FXR-knockdown HT-29 and Caco-2 cells or FXR-overexpressing SW480 and HCT116 cells. <bold>d</bold> SHP expression in FXR-knockdown HT-29 and Caco-2 cells or FXR-overexpressing SW480 and HCT116 cells detected by real-time PCR. <bold>e</bold> The effect of SHP overexpression on cell cycle- and EMT-related protein levels of FXR-knockdown HT-29 and Caco-2 cells detected by western blotting analysis. <bold>f</bold> The effect of SHP knockdown on cell cycle- and EMT-related protein levels of FXR-overexpressing SW480 and HCT116 cells detected by western blotting analysis. All data are the mean ± SD of three independent experiments. *<italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"41419_2020_2819_Fig6_HTML\" id=\"d30e1051\"/></fig></p></sec><sec id=\"Sec27\"><title>Modulation of β-catenin impacts FXR transcriptional activation of SHP expression in colon cancer cells</title><p id=\"Par38\">To determine whether the FXR/β-catenin interaction affects FXR transcriptional activation of SHP expression, we first assessed the impact of modulating β-catenin on SHP expression. Knockdown of β-catenin in SW480-FXR and HCT116-FXR cells elevated the expression of SHP both at the mRNA and protein levels compared with the control (Supplementary Fig. <xref rid=\"MOESM10\" ref-type=\"media\">8a, c</xref>). Conversely, ectopic expression of β-catenin had the opposite effect (Supplementary Fig. <xref rid=\"MOESM10\" ref-type=\"media\">8b, d</xref>). FXR often regulate target genes transcription as a heterodimer with the retinoid X receptor (RXR)<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. We then questioned whether modulation of β-catenin affected the FXR/RXRα complex. The co-IP assay showed that knockdown of β-catenin in SW480-FXR and HCT116-FXR cells resulted in a reduction in the FXR/β-catenin complex but a promotion in the FXR/RXRα complex (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7a, b</xref>), whereas ectopic expression of β-catenin had the reserved effect (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7c, d</xref>). Furthermore, knockdown of β-catenin in SW480-FXR and HCT116-FXR cells increased the luciferase activities of the SHP-WT promoter but not the luciferase activities of the SHP-mFXR promoter. Conversely, ectopic expression of β-catenin decreased the luciferase activities of the SHP-WT promoter but not the SHP-mFXR promoter (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7e</xref>).<fig id=\"Fig7\"><label>Fig. 7</label><caption><title>Modulating β-catenin expression affected the FXR/RXRα interaction in colon cancer cells.</title><p><bold>a</bold>, <bold>b</bold> Knockdown of β-catenin enhanced the FXR/RXRα interaction. SW480-FXR (<bold>a</bold>) and HCT116-FXR (<bold>b</bold>) cells and their control cells were infected with β-catenin-shRNA lentivirus or control lentivirus as indicated above for 48 h. The nuclear fractions were incubated with an anti-FXR antibody for the IP experiment. <bold>c</bold>, <bold>d</bold> Ectopic expression of β-catenin impaired the FXR/RXRα interaction. SW480-FXR (<bold>c</bold>) and HCT116-FXR (<bold>d</bold>) cells and their control cells were infected with β-catenin-overexpressing lentivirus or control lentivirus as indicated above for 48 h. The nuclear fractions were incubated with an anti-FXR antibody for the IP experiment. <bold>e</bold> Modulating β-catenin expression affected the binding of RXRα to SHP promoter. Wild type SHP promoter (SHP-WT) or a mutated SHP loss of FXR binding site (SHP-mFXR) and pRL-TK plasmid were transfected into HEK293 cells for 24 h. Luciferase activity was measured using cell lysates 24 h after transfection. <bold>f</bold> The effect of modulating β-catenin expression on the binding of FXR or RXRα protein to the SHP promoter detected by the qChIP assay. All data are the mean ± SD of three independent experiments. *<italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"41419_2020_2819_Fig7_HTML\" id=\"d30e1120\"/></fig></p><p id=\"Par39\">Finally, a quantitative chromatin immunoprecipitation (qChIP) assay was employed to determine whether modulation of β-catenin affected the occupancy of FXR on the SHP promoter in vivo. Our data revealed that knockdown of β-catenin led to an enhancement of FXR binding to the SHP promoter in SW480-FXR and HCT116-FXR cells (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7f</xref>). Conversely, ectopic expression of β-catenin exerted an opposite effect. Interestingly, RXRα occupancy of the SHP promoter tended to mirror that of FXR, which is likely due to the occupancy of its binding partner FXR and not because of any direct association with β-catenin. These data indicate that the FXR/β-catenin complex antagonizes the FXR/RXRα complex and its transcriptional activity.</p></sec><sec id=\"Sec28\"><title>Correlations among FXR and cell cycle- and EMT-related proteins in colon cancer tissues</title><p id=\"Par40\">Correlations among FXR and cell cycle- and EMT-related proteins were further explored in 30 colon cancer tissues by IHC assay (Supplementary Fig. <xref rid=\"MOESM11\" ref-type=\"media\">9a</xref>). Our study showed that the level of FXR positively correlated with that of SHP (Supplementary Fig. <xref rid=\"MOESM11\" ref-type=\"media\">9b</xref>) and E-cadherin (Supplementary Fig. <xref rid=\"MOESM11\" ref-type=\"media\">9c</xref>) and negatively correlated with that of vimentin, cyclin D1, and c-Myc expression (Supplementary Fig. <xref rid=\"MOESM11\" ref-type=\"media\">9d–f</xref>), further supporting the notion that FXR is a negative regulator of colorectal tumorigenesis.</p></sec></sec><sec id=\"Sec29\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par41\">Mounting epidemiological evidence indicates that HFDs, rich in carbohydrates and saturated fatty acids, are an acknowledged risk factor for CRC<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. HFDs lead to commensurate increases in fecal bile acids, particularly lithocholic and deoxycholic acids, which are potent inducers of CRC<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. FXR, a bile acid-activated nuclear receptor, plays an important role on oncogenic transformation. In this study, we demonstrated that FXR expression was significantly downregulated in colon cancer tissues and decreased FXR expression was negatively related to the location of tumor, lymph node metastasis, and TNM stage. High FXR expression is a strong and independent prognostic indicator in invasive breast carcinoma<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. In colon cancer, low FXR expression was correlated with worse clinical outcome<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. Our date concluded that reduced FXR expression was significantly associated with worse OS and RFS of patients with CRC, although FXR was not validated as an independent predictor of OS and RFS by multivariate analyses.</p><p id=\"Par42\">In vitro and in vivo assays further supported a tumor-suppressor role of FXR in CRC. Knockdown of FXR promoted colon cancer cells cell growth and invasion in vitro, and facilitated tumor formation and distant metastasis in vivo. These changes were the opposite of those seen in the FXR overexpression experiment and further validated the role of FXR in CRC progression. Consistent with our study, FXR-deficient mice exhibited an enhancement in intestinal cell proliferation with upregulation of cyclin D1 expression<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. Moreover, our study demonstrated that knockdown of FXR in colon cancer cells induced EMT, accompanied by upregulation of Snail, Slug, vimentin, fibronectin, and MMP-9, and downregulation of E-cadherin and ZO-1, whereas ectopic expression of FXR had reversed change. In non-small-cell lung cancer, FXR functions as a proto-oncogene, promoting cell proliferation by directly transactivating cyclin D1<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>. In pancreatic cancer, increased FXR promotes cell invasive and migratory ability<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>. These studies indicate the tumor-specific contributions of FXR to the pathogenesis of different cancer types.</p><p id=\"Par43\">Previous study indicated that in FXR-deficient mice, increased activation of Wnt signaling was observed in spontaneous HCC<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. The present study revealed that knockdown of FXR activated Wnt signaling, as evidenced by the high luciferase activity of the TOP/FOP-Flash reporter and the upregulation of Wnt signaling target genes. These changes were opposite to FXR overexpression experiment. Moreover, blockage of Wnt signaling by XAV-939 attenuated the tumor-suppressive effect of FXR knockdown on colon cancer cells. Activation of Wnt/β-catenin signaling is a strong inducer of EMT, as evidenced by the identification of β-catenin-regulated genes, such as fibronectin<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>, slug<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>, MMP-7<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup>, and VEGF<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>. These genes, directly or indirectly involved in EMT, code for direct effectors of CRC progression. These data indicated that FXR-inhibited tumorigenicity and EMT in CRC could be attributed to the inactivation of Wnt/β-catenin signaling. In addition, activation of FXR abolished colon cancer cell growth by inhibiting EGFR/ERK signaling<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>. Intriguingly, a recent study revealed that FXR directly regulated MMP-7 expression by acting as a transcriptional repressor<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>. These data indicate that FXR exerts its tumor suppressor functions via distinct signaling pathways.</p><p id=\"Par44\">FXR has been widely considered a transcriptional factor functioning in cancer cells<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. In the present study, the expression of SHP, a well-known target gene of FXR, was decreased or elevated upon FXR knockdown or FXR overexpression. SHP has also been shown to suppress tumor cell proliferation and invasion via transcriptional repression of cyclin D1 and Ccl2 expression<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref>,<xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>. Our study demonstrated that the tumor-suppressive effects of FXR could be partially attributed to FXR-mediated transcriptional activation of SHP, as ectopic expression of SHP impaired the proliferative and invasive potential of colon cancer cells.</p><p id=\"Par45\">The crosstalk between Wnt signaling and the nuclear receptors (NRs) has been highlighted in the field of cancer research<sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>. Analyses of NR interactions with canonical Wnt signaling reveal two broad themes: Wnt/β-catenin modulation of NRs (theme I) and NR inhibition of the Wnt/β-catenin/TCF cascade (theme II). Glucocorticoid receptor (GR) could inhibit tumor proliferation by repressing cyclin D1 expression via targeting of the β-catenin/TCF complex<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup> (theme II); and β-catenin acts as a coactivator of androgen receptor (AR) transcription and promotes cell proliferation and prostate pathogenesis<sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup> (theme I). Our study revealed that a nontranscriptional mechanism of FXR that FXR forms a complex with β-catenin and subsequently disturbs the transcriptional activity of the β-catenin/TCF complex in colon cancer lines (theme II). FXR appears to regulate β-catenin activity without affecting β-catenin localization and protein stability, since modulation of FXR expression did not affect the translocation of β-catenin (data not shown). On the other hand, our study found that modulation of β-catenin affected the transcriptional activation of SHP by FXR, indicating that β-catenin negatively regulates FXR activity through direct binding (theme I). Based on these observations, we propose a model in which an event initiated in tumor cells activates Wnt signaling at the early phase of colorectal tumorigenesis, thus elevating the levels of nuclear β-catenin, forming β-catenin/FXR complex and subsequently impairing the tumor-suppressor effect of FXR. Furthermore, due to DNA hypermethylation<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>, miRNA regulation<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup>, or transcription factor regulation<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>, loss of FXR enhances the β-catenin/FXR complex and leads to persistent activation of Wnt signaling to further promote tumorigenesis (Supplementary Fig. <xref rid=\"MOESM12\" ref-type=\"media\">10</xref>). Intriguingly, Selmin et al.<sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup> reported that APC mutations, which result in Wnt signaling activation, cause silencing of FXR expression through CpG hypermethylation, although the underlying mechanism remains unclear. Further investigation into the reciprocal relationship between FXR and β-catenin is urgent.</p><p id=\"Par46\">In summary, we found that FXR is downregulated in colon cancer and is negatively associated with poor prognosis. Functional studies indicated that FXR exerts a tumor-suppressive function in CRC. Mechanistically, FXR suppresses the activity of Wnt/β-catenin signaling via interaction with β-catenin. Furthermore, the FXR/β-catenin interaction retards FXR-mediated transcriptional activation of its target gene SHP. These novel findings have identified a heretofore unrecognized relationship between FXR and β-catenin in tumorigenesis, thus providing a novel interventional opportunity.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec30\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41419_2020_2819_MOESM1_ESM.docx\"><caption><p>Supplementary Figures legend</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41419_2020_2819_MOESM2_ESM.docx\"><caption><p>Supplementary Tables</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM3\"><media xlink:href=\"41419_2020_2819_MOESM3_ESM.tif\"><caption><p>Supplementary Figure 1</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM4\"><media xlink:href=\"41419_2020_2819_MOESM4_ESM.tif\"><caption><p>Supplementary Figure 2</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM5\"><media xlink:href=\"41419_2020_2819_MOESM5_ESM.tif\"><caption><p>Supplementary Figure 3</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM6\"><media xlink:href=\"41419_2020_2819_MOESM6_ESM.tif\"><caption><p>Supplementary Figure 4</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM7\"><media xlink:href=\"41419_2020_2819_MOESM7_ESM.tif\"><caption><p>Supplementary Figure 5</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM8\"><media xlink:href=\"41419_2020_2819_MOESM8_ESM.tif\"><caption><p>Supplementary Figure 6</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM9\"><media xlink:href=\"41419_2020_2819_MOESM9_ESM.tif\"><caption><p>Supplementary Figure 7</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM10\"><media xlink:href=\"41419_2020_2819_MOESM10_ESM.tif\"><caption><p>Supplementary Figure 8</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM11\"><media xlink:href=\"41419_2020_2819_MOESM11_ESM.tif\"><caption><p>Supplementary Figure 9</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM12\"><media xlink:href=\"41419_2020_2819_MOESM12_ESM.tif\"><caption><p>Supplementary Figure 10</p></caption></media></supplementary-material></sec></sec></body><back><fn-group><fn><p>Edited by M. Agostini</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Junhui Yu, Shan Li</p></fn><fn><p>These authors jointly supervised this work: Junhui Yu, Shan Li</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary Information</bold> accompanies this paper at (10.1038/s41419-020-02819-w).</p></sec><ack><title>Acknowledgements</title><p>This work was funded by a grant from the National Natural Science Foundation of China (Grant Serial Numbers: 81972720, 81101874, and 81172362), the Coordinative and Innovative Plan Projects of the Science and Technology Program in Shaanxi Province (Grant Serial Numbers: 2013KTCQ03-08), the Science and Technology Project of Shaanxi Province (Grant serial number: 2016SF-015 and 2019SF-065), and the Fundamental Research Funds for the Central Universities (Grant serial number: xjj2018123).</p></ack><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Conflict of interest</title><p id=\"Par47\">The authors declare that they have no conflict of interest.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bray</surname><given-names>F</given-names></name><etal/></person-group><article-title>Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries</article-title><source>Cancer J. Clin.</source><year>2018</year><volume>68</volume><fpage>394</fpage><lpage>424</lpage></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Raskov</surname><given-names>H</given-names></name><name><surname>Pommergaard</surname><given-names>HC</given-names></name><name><surname>Burcharth</surname><given-names>J</given-names></name><name><surname>Rosenberg</surname><given-names>J</given-names></name></person-group><article-title>Colorectal carcinogenesis–update and perspectives</article-title><source>World J. Gastroenterol.</source><year>2014</year><volume>20</volume><fpage>18151</fpage><lpage>18164</lpage><pub-id pub-id-type=\"pmid\">25561783</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aleksandrova</surname><given-names>K</given-names></name><etal/></person-group><article-title>Combined impact of healthy lifestyle factors on colorectal cancer: a large European cohort study</article-title><source>BMC Med.</source><year>2014</year><volume>12</volume><fpage>168</fpage><pub-id pub-id-type=\"pmid\">25319089</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coppede</surname><given-names>F</given-names></name><name><surname>Lopomo</surname><given-names>A</given-names></name><name><surname>Spisni</surname><given-names>R</given-names></name><name><surname>Migliore</surname><given-names>L</given-names></name></person-group><article-title>Genetic and epigenetic biomarkers for diagnosis, prognosis and treatment of colorectal cancer</article-title><source>World J. Gastroenterol.</source><year>2014</year><volume>20</volume><fpage>943</fpage><lpage>956</lpage><pub-id pub-id-type=\"pmid\">24574767</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mei</surname><given-names>Z</given-names></name><etal/></person-group><article-title>Tumour-infiltrating inflammation and prognosis in colorectal cancer: systematic review and meta-analysis</article-title><source>Br. J. Cancer</source><year>2014</year><volume>110</volume><fpage>1595</fpage><lpage>1605</lpage><pub-id pub-id-type=\"pmid\">24504370</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carethers</surname><given-names>JM</given-names></name><name><surname>Jung</surname><given-names>BH</given-names></name></person-group><article-title>Genetics and genetic biomarkers in sporadic colorectal cancer</article-title><source>Gastroenterology</source><year>2015</year><volume>149</volume><fpage>1177</fpage><lpage>1190.e1173</lpage><pub-id pub-id-type=\"pmid\">26216840</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>HS</given-names></name><etal/></person-group><article-title>Molecular testing for gastrointestinal cancer</article-title><source>J. Pathol. Transl. Med.</source><year>2017</year><volume>51</volume><fpage>103</fpage><lpage>121</lpage><pub-id pub-id-type=\"pmid\">28219002</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oittinen</surname><given-names>M</given-names></name><etal/></person-group><article-title>Polycomb Repressive Complex 2 enacts wnt signaling in intestinal homeostasis and contributes to the instigation of stemness in diseases entailing epithelial hyperplasia or neoplasia</article-title><source>Stem Cells</source><year>2017</year><volume>35</volume><fpage>445</fpage><lpage>457</lpage><pub-id pub-id-type=\"pmid\">27570105</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rahmani</surname><given-names>F</given-names></name><name><surname>Avan</surname><given-names>A</given-names></name></person-group><article-title>Role of Wnt/beta-catenin signaling regulatory microRNAs in the pathogenesis of colorectal cancer</article-title><source>J. Cell. Physiol</source><year>2018</year><volume>233</volume><fpage>811</fpage><lpage>817</lpage><pub-id pub-id-type=\"pmid\">28266708</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>He</surname><given-names>TC</given-names></name><etal/></person-group><article-title>Identification of c-MYC as a target of the APC pathway</article-title><source>Science</source><year>1998</year><volume>281</volume><fpage>1509</fpage><lpage>1512</lpage><pub-id pub-id-type=\"pmid\">9727977</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tetsu</surname><given-names>O</given-names></name><name><surname>McCormick</surname><given-names>F</given-names></name></person-group><article-title>Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells</article-title><source>Nature</source><year>1999</year><volume>398</volume><fpage>422</fpage><lpage>426</lpage><pub-id pub-id-type=\"pmid\">10201372</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Neth</surname><given-names>P</given-names></name><etal/></person-group><article-title>Wnt signaling regulates the invasion capacity of human mesenchymal stem cells</article-title><source>Stem Cells</source><year>2006</year><volume>24</volume><fpage>1892</fpage><lpage>1903</lpage><pub-id pub-id-type=\"pmid\">16690780</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moustakas</surname><given-names>A</given-names></name><name><surname>de Herreros</surname><given-names>AG</given-names></name></person-group><article-title>Epithelial-mesenchymal transition in cancer</article-title><source>Mol. Oncol.</source><year>2017</year><volume>11</volume><fpage>715</fpage><lpage>717</lpage><pub-id pub-id-type=\"pmid\">28677253</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brabletz</surname><given-names>T</given-names></name><etal/></person-group><article-title>Down-regulation of the homeodomain factor Cdx2 in colorectal cancer by collagen type I: an active role for the tumor environment in malignant tumor progression</article-title><source>Cancer Res.</source><year>2004</year><volume>64</volume><fpage>6973</fpage><lpage>6977</lpage><pub-id pub-id-type=\"pmid\">15466189</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Conacci-Sorrell</surname><given-names>M</given-names></name><etal/></person-group><article-title>Autoregulation of E-cadherin expression by cadherin-cadherin interactions: the roles of beta-catenin signaling, Slug, and MAPK</article-title><source>J. Cell Biol.</source><year>2003</year><volume>163</volume><fpage>847</fpage><lpage>857</lpage><pub-id pub-id-type=\"pmid\">14623871</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>W</given-names></name><etal/></person-group><article-title>A weighted relative difference accumulation algorithm for dynamic metabolomics data: long-term elevated bile acids are risk factors for hepatocellular carcinoma</article-title><source>Sci. Rep.</source><year>2015</year><volume>5</volume><fpage>8984</fpage><pub-id pub-id-type=\"pmid\">25757957</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stanimirov</surname><given-names>B</given-names></name><name><surname>Stankov</surname><given-names>K</given-names></name><name><surname>Mikov</surname><given-names>M</given-names></name></person-group><article-title>Bile acid signaling through farnesoid X and TGR5 receptors in hepatobiliary and intestinal diseases</article-title><source>Hepatobiliary Pancreat. Dis. Int.</source><year>2015</year><volume>14</volume><fpage>18</fpage><lpage>33</lpage><pub-id pub-id-type=\"pmid\">25655287</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carino</surname><given-names>A</given-names></name><etal/></person-group><article-title>The bile acid receptor GPBAR1 (TGR5) is expressed in human gastric cancers and promotes epithelial-mesenchymal transition in gastric cancer cell lines</article-title><source>Oncotarget</source><year>2016</year><volume>7</volume><fpage>61021</fpage><lpage>61035</lpage><pub-id pub-id-type=\"pmid\">27409173</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Matsuzaki</surname><given-names>J</given-names></name><etal/></person-group><article-title>Bile acids increase levels of microRNAs 221 and 222, leading to degradation of CDX2 during esophageal carcinogenesis</article-title><source>Gastroenterology</source><year>2013</year><volume>145</volume><fpage>1300</fpage><lpage>1311</lpage><pub-id pub-id-type=\"pmid\">23933602</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abdel-Latif</surname><given-names>MM</given-names></name><name><surname>Inoue</surname><given-names>H</given-names></name><name><surname>Kelleher</surname><given-names>D</given-names></name><name><surname>Reynolds</surname><given-names>JV</given-names></name></person-group><article-title>Factors regulating nuclear factor-kappa B activation in esophageal cancer cells: Role of bile acids and acid</article-title><source>J. Cancer Res. Ther.</source><year>2016</year><volume>12</volume><fpage>364</fpage><lpage>373</lpage><pub-id pub-id-type=\"pmid\">27072265</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Joshi</surname><given-names>S</given-names></name><etal/></person-group><article-title>Bile acids-mediated overexpression of MUC4 via FAK-dependent c-Jun activation in pancreatic cancer</article-title><source>Mol. Oncol.</source><year>2016</year><volume>10</volume><fpage>1063</fpage><lpage>1077</lpage><pub-id pub-id-type=\"pmid\">27185392</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Feng</surname><given-names>HY</given-names></name><name><surname>Chen</surname><given-names>YC</given-names></name></person-group><article-title>Role of bile acids in carcinogenesis of pancreatic cancer: an old topic with new perspective</article-title><source>World J. Gastroenterol.</source><year>2016</year><volume>22</volume><fpage>7463</fpage><lpage>7477</lpage><pub-id pub-id-type=\"pmid\">27672269</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Raufman</surname><given-names>JP</given-names></name><etal/></person-group><article-title>Slc10a2-null mice uncover colon cancer-promoting actions of endogenous fecal bile acids</article-title><source>Carcinogenesis</source><year>2015</year><volume>36</volume><fpage>1193</fpage><lpage>1200</lpage><pub-id pub-id-type=\"pmid\">26210740</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Degirolamo</surname><given-names>C</given-names></name><name><surname>Modica</surname><given-names>S</given-names></name><name><surname>Palasciano</surname><given-names>G</given-names></name><name><surname>Moschetta</surname><given-names>A</given-names></name></person-group><article-title>Bile acids and colon cancer: Solving the puzzle with nuclear receptors</article-title><source>Trends Mol. Med.</source><year>2011</year><volume>17</volume><fpage>564</fpage><lpage>572</lpage><pub-id pub-id-type=\"pmid\">21724466</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vaquero</surname><given-names>J</given-names></name><name><surname>Monte</surname><given-names>MJ</given-names></name><name><surname>Dominguez</surname><given-names>M</given-names></name><name><surname>Muntane</surname><given-names>J</given-names></name><name><surname>Marin</surname><given-names>JJ</given-names></name></person-group><article-title>Differential activation of the human farnesoid X receptor depends on the pattern of expressed isoforms and the bile acid pool composition</article-title><source>Biochem. Pharmacol.</source><year>2013</year><volume>86</volume><fpage>926</fpage><lpage>939</lpage><pub-id pub-id-type=\"pmid\">23928191</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Miyazaki-Anzai</surname><given-names>S</given-names></name><name><surname>Masuda</surname><given-names>M</given-names></name><name><surname>Levi</surname><given-names>M</given-names></name><name><surname>Keenan</surname><given-names>AL</given-names></name><name><surname>Miyazaki</surname><given-names>M</given-names></name></person-group><article-title>Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis</article-title><source>PLoS ONE</source><year>2014</year><volume>9</volume><fpage>e108270</fpage><pub-id pub-id-type=\"pmid\">25237811</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chiang</surname><given-names>JY</given-names></name></person-group><article-title>Recent advances in understanding bile acid homeostasis</article-title><source>F1000Research</source><year>2017</year><volume>6</volume><fpage>2029</fpage><pub-id pub-id-type=\"pmid\">29188025</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gadaleta</surname><given-names>RM</given-names></name><name><surname>Cariello</surname><given-names>M</given-names></name><name><surname>Sabba</surname><given-names>C</given-names></name><name><surname>Moschetta</surname><given-names>A</given-names></name></person-group><article-title>Tissue-specific actions of FXR in metabolism and cancer</article-title><source>Biochim. Biophys. Acta</source><year>2015</year><volume>1851</volume><fpage>30</fpage><lpage>39</lpage><pub-id pub-id-type=\"pmid\">25139561</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ding</surname><given-names>L</given-names></name><name><surname>Yang</surname><given-names>L</given-names></name><name><surname>Wang</surname><given-names>Z</given-names></name><name><surname>Huang</surname><given-names>W</given-names></name></person-group><article-title>Bile acid nuclear receptor FXR and digestive system diseases</article-title><source>Acta Pharm. Sin. B</source><year>2015</year><volume>5</volume><fpage>135</fpage><lpage>144</lpage><pub-id pub-id-type=\"pmid\">26579439</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bailey</surname><given-names>AM</given-names></name><etal/></person-group><article-title>FXR silencing in human colon cancer by DNA methylation and KRAS signaling</article-title><source>Am. J. Physiol. Gastrointest. Liver Physiol.</source><year>2014</year><volume>306</volume><fpage>G48</fpage><lpage>G58</lpage><pub-id pub-id-type=\"pmid\">24177031</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fu</surname><given-names>T</given-names></name><etal/></person-group><article-title>FXR regulates intestinal cancer stem cell proliferation</article-title><source>Cell</source><year>2019</year><volume>176</volume><fpage>1098</fpage><lpage>1112.e1018</lpage><pub-id pub-id-type=\"pmid\">30794774</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>J</given-names></name><etal/></person-group><article-title>CDX2 inhibits the proliferation and tumor formation of colon cancer cells by suppressing Wnt/beta-catenin signaling via transactivation of GSK-3beta and Axin2 expression</article-title><source>Cell Death Dis.</source><year>2019</year><volume>10</volume><fpage>26</fpage><pub-id pub-id-type=\"pmid\">30631044</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>S</given-names></name><name><surname>Ma</surname><given-names>YM</given-names></name><name><surname>Zheng</surname><given-names>PS</given-names></name><name><surname>Zhang</surname><given-names>P</given-names></name></person-group><article-title>GDF15 promotes the proliferation of cervical cancer cells by phosphorylating AKT1 and Erk1/2 through the receptor ErbB2</article-title><source>J. Exp. Clin. Cancer Res.</source><year>2018</year><volume>37</volume><fpage>80</fpage><pub-id pub-id-type=\"pmid\">29636108</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Smith</surname><given-names>BN</given-names></name><name><surname>Bhowmick</surname><given-names>NA</given-names></name></person-group><article-title>Role of EMT in metastasis and therapy resistance</article-title><source>J. Clin. Med.</source><year>2016</year><volume>5</volume><fpage>17</fpage></element-citation></ref><ref id=\"CR35\"><label>35.</label><mixed-citation publication-type=\"other\">Basu, S., Cheriyamundath, S. & Ben-Ze’ev, A. Cell-cell adhesion: linking Wnt/beta-catenin signaling with partial EMT and stemness traits in tumorigenesis. <italic>F1000Research</italic><bold>7</bold>, F1000 Faculty Rev–1488 (2018).</mixed-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maran</surname><given-names>RR</given-names></name><etal/></person-group><article-title>Farnesoid X receptor deficiency in mice leads to increased intestinal epithelial cell proliferation and tumor development</article-title><source>J. Pharmacol. Exp. Ther.</source><year>2009</year><volume>328</volume><fpage>469</fpage><lpage>477</lpage><pub-id pub-id-type=\"pmid\">18981289</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wolfe</surname><given-names>A</given-names></name><etal/></person-group><article-title>Increased activation of the Wnt/beta-catenin pathway in spontaneous hepatocellular carcinoma observed in farnesoid X receptor knockout mice</article-title><source>J. Pharmacol. Exp. Ther.</source><year>2011</year><volume>338</volume><fpage>12</fpage><lpage>21</lpage><pub-id pub-id-type=\"pmid\">21430080</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nusse</surname><given-names>R</given-names></name><name><surname>Clevers</surname><given-names>H</given-names></name></person-group><article-title>Wnt/beta-catenin signaling, disease, and emerging therapeutic modalities</article-title><source>Cell</source><year>2017</year><volume>169</volume><fpage>985</fpage><lpage>999</lpage><pub-id pub-id-type=\"pmid\">28575679</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cariello</surname><given-names>M</given-names></name><etal/></person-group><article-title>LXRs, SHP, and FXR in prostate cancer: enemies or menage a quatre with AR?</article-title><source>Nucl. Recept. Signal.</source><year>2018</year><volume>15</volume><fpage>1550762918801070</fpage><pub-id pub-id-type=\"pmid\">30718981</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Orphan receptor small heterodimer partner suppresses tumorigenesis by modulating cyclin D1 expression and cellular proliferation</article-title><source>Hepatology</source><year>2008</year><volume>48</volume><fpage>289</fpage><lpage>298</lpage><pub-id pub-id-type=\"pmid\">18537191</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maloney</surname><given-names>PR</given-names></name><etal/></person-group><article-title>Identification of a chemical tool for the orphan nuclear receptor FXR</article-title><source>J. Med. Chem.</source><year>2000</year><volume>43</volume><fpage>2971</fpage><lpage>2974</lpage><pub-id pub-id-type=\"pmid\">10956205</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>D</given-names></name><etal/></person-group><article-title>Colonic lysine homocysteinylation induced by high-fat diet suppresses DNA damage repair</article-title><source>Cell Rep.</source><year>2018</year><volume>25</volume><fpage>398</fpage><lpage>412.e396</lpage><pub-id pub-id-type=\"pmid\">30304680</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coats</surname><given-names>M</given-names></name><name><surname>Shimi</surname><given-names>SM</given-names></name></person-group><article-title>Cholecystectomy and the risk of alimentary tract cancers: a systematic review</article-title><source>World J. Gastroenterol.</source><year>2015</year><volume>21</volume><fpage>3679</fpage><lpage>3693</lpage><pub-id pub-id-type=\"pmid\">25834337</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Giaginis</surname><given-names>C</given-names></name><etal/></person-group><article-title>High Farnesoid X Receptor (FXR) expression is a strong and independent prognosticator in invasive breast carcinoma</article-title><source>Neoplasma</source><year>2017</year><volume>64</volume><fpage>633</fpage><lpage>639</lpage><pub-id pub-id-type=\"pmid\">28485172</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lax</surname><given-names>S</given-names></name><etal/></person-group><article-title>Expression of the nuclear bile acid receptor/farnesoid X receptor is reduced in human colon carcinoma compared to nonneoplastic mucosa independent from site and may be associated with adverse prognosis</article-title><source>Int. J. Cancer</source><year>2012</year><volume>130</volume><fpage>2232</fpage><lpage>2239</lpage><pub-id pub-id-type=\"pmid\">21780109</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>You</surname><given-names>W</given-names></name><etal/></person-group><article-title>Farnesoid X receptor, a novel proto-oncogene in non-small cell lung cancer, promotes tumor growth via directly transactivating CCND1</article-title><source>Sci. Rep.</source><year>2017</year><volume>7</volume><fpage>591</fpage><pub-id pub-id-type=\"pmid\">28377627</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>JY</given-names></name><etal/></person-group><article-title>Farnesoid X receptor, overexpressed in pancreatic cancer with lymph node metastasis promotes cell migration and invasion</article-title><source>Br. J. Cancer</source><year>2011</year><volume>104</volume><fpage>1027</fpage><lpage>1037</lpage><pub-id pub-id-type=\"pmid\">21364590</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cai</surname><given-names>T</given-names></name><etal/></person-group><article-title>FHL2 promotes tubular epithelial-to-mesenchymal transition through modulating beta-catenin signalling</article-title><source>J. Cell. Mol. Med.</source><year>2018</year><volume>22</volume><fpage>1684</fpage><lpage>1695</lpage><pub-id pub-id-type=\"pmid\">29193729</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>X</given-names></name><etal/></person-group><article-title>SULT2B1b promotes epithelial-mesenchymal transition through activation of the beta-catenin/MMP7 pathway in hepatocytes</article-title><source>Biochem. Biophys. Res. Commun.</source><year>2019</year><volume>510</volume><fpage>495</fpage><lpage>500</lpage><pub-id pub-id-type=\"pmid\">30658852</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liang</surname><given-names>X</given-names></name><etal/></person-group><article-title>PAX3 promotes proliferation of human glioma cells by WNT/beta-catenin signaling pathways</article-title><source>J. Mol. Neurosci.</source><year>2019</year><volume>68</volume><fpage>66</fpage><lpage>77</lpage><pub-id pub-id-type=\"pmid\">30826985</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Peng</surname><given-names>Z</given-names></name><name><surname>Raufman</surname><given-names>JP</given-names></name><name><surname>Xie</surname><given-names>G</given-names></name></person-group><article-title>Src-mediated cross-talk between farnesoid X and epidermal growth factor receptors inhibits human intestinal cell proliferation and tumorigenesis</article-title><source>PLoS ONE</source><year>2012</year><volume>7</volume><fpage>e48461</fpage><pub-id pub-id-type=\"pmid\">23119029</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Peng</surname><given-names>Z</given-names></name><name><surname>Chen</surname><given-names>J</given-names></name><name><surname>Drachenberg</surname><given-names>CB</given-names></name></person-group><article-title>Farnesoid X receptor represses matrix metalloproteinase 7 expression, revealing this regulatory axis as a promising therapeutic target in colon cancer</article-title><source>J. Biol. Chem.</source><year>2019</year><volume>294</volume><fpage>8529</fpage><lpage>8542</lpage><pub-id pub-id-type=\"pmid\">30967475</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>XF</given-names></name><name><surname>Zhao</surname><given-names>WY</given-names></name><name><surname>Huang</surname><given-names>WD</given-names></name></person-group><article-title>FXR and liver carcinogenesis</article-title><source>Acta Pharmacol. Sin.</source><year>2015</year><volume>36</volume><fpage>37</fpage><lpage>43</lpage><pub-id pub-id-type=\"pmid\">25500874</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>Z</given-names></name><name><surname>Koehler</surname><given-names>AN</given-names></name><name><surname>Wang</surname><given-names>L</given-names></name></person-group><article-title>A novel small molecule activator of nuclear receptor SHP inhibits HCC cell migration via suppressing Ccl2</article-title><source>Mol. Cancer Ther.</source><year>2016</year><volume>15</volume><fpage>2294</fpage><lpage>2301</lpage><pub-id pub-id-type=\"pmid\">27486225</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mulholland</surname><given-names>DJ</given-names></name><name><surname>Dedhar</surname><given-names>S</given-names></name><name><surname>Coetzee</surname><given-names>GA</given-names></name><name><surname>Nelson</surname><given-names>CC</given-names></name></person-group><article-title>Interaction of nuclear receptors with the Wnt/beta-catenin/Tcf signaling axis: Wnt you like to know?</article-title><source>Endocr. Rev.</source><year>2005</year><volume>26</volume><fpage>898</fpage><lpage>915</lpage><pub-id pub-id-type=\"pmid\">16126938</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Takayama</surname><given-names>S</given-names></name><name><surname>Rogatsky</surname><given-names>I</given-names></name><name><surname>Schwarcz</surname><given-names>LE</given-names></name><name><surname>Darimont</surname><given-names>BD</given-names></name></person-group><article-title>The glucocorticoid receptor represses cyclin D1 by targeting the Tcf-beta-catenin complex</article-title><source>J. Biol. Chem.</source><year>2006</year><volume>281</volume><fpage>17856</fpage><lpage>17863</lpage><pub-id pub-id-type=\"pmid\">16644723</pub-id></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Truica</surname><given-names>CI</given-names></name><name><surname>Byers</surname><given-names>S</given-names></name><name><surname>Gelmann</surname><given-names>EP</given-names></name></person-group><article-title>Beta-catenin affects androgen receptor transcriptional activity and ligand specificity</article-title><source>Cancer Res.</source><year>2000</year><volume>60</volume><fpage>4709</fpage><lpage>4713</lpage><pub-id pub-id-type=\"pmid\">10987273</pub-id></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krattinger</surname><given-names>R</given-names></name><etal/></person-group><article-title>microRNA-192 suppresses the expression of the farnesoid X receptor</article-title><source>Am. J. Physiol. Gastrointest. Liver Physiol.</source><year>2016</year><volume>310</volume><fpage>G1044</fpage><lpage>G1051</lpage><pub-id pub-id-type=\"pmid\">27079614</pub-id></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Modica</surname><given-names>S</given-names></name><etal/></person-group><article-title>Transcriptional regulation of the intestinal nuclear bile acid farnesoid X receptor (FXR) by the caudal-related homeobox 2 (CDX2)</article-title><source>J. Biol. Chem.</source><year>2014</year><volume>289</volume><fpage>28421</fpage><lpage>28432</lpage><pub-id pub-id-type=\"pmid\">25138215</pub-id></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Selmin</surname><given-names>OI</given-names></name><etal/></person-group><article-title>Inactivation of adenomatous polyposis coli reduces bile acid/farnesoid X receptor expression through Fxr gene CpG methylation in mouse colon tumors and human colon cancer cells</article-title><source>J. Nutr.</source><year>2016</year><volume>146</volume><fpage>236</fpage><lpage>242</lpage><pub-id pub-id-type=\"pmid\">26609171</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Nat Commun</journal-id><journal-id journal-id-type=\"iso-abbrev\">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type=\"epub\">2041-1723</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807807</article-id><article-id pub-id-type=\"pmc\">PMC7431545</article-id><article-id pub-id-type=\"publisher-id\">17994</article-id><article-id pub-id-type=\"doi\">10.1038/s41467-020-17994-9</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Deployable CRISPR-Cas13a diagnostic tools to detect and report Ebola and Lassa virus cases in real-time</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\" equal-contrib=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-8291-4388</contrib-id><name><surname>Barnes</surname><given-names>Kayla G.</given-names></name><address><email>kbarnes@broadinstitute.org</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\" equal-contrib=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-2930-9541</contrib-id><name><surname>Lachenauer</surname><given-names>Anna E.</given-names></name><address><email>annalach@stanford.edu</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-5679-838X</contrib-id><name><surname>Nitido</surname><given-names>Adam</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Siddiqui</surname><given-names>Sameed</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><name><surname>Gross</surname><given-names>Robin</given-names></name><xref ref-type=\"aff\" rid=\"Aff8\">8</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-3798-6411</contrib-id><name><surname>Beitzel</surname><given-names>Brett</given-names></name><xref ref-type=\"aff\" rid=\"Aff9\">9</xref></contrib><contrib contrib-type=\"author\"><name><surname>Siddle</surname><given-names>Katherine J.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff10\">10</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-1939-3102</contrib-id><name><surname>Freije</surname><given-names>Catherine A.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Dighero-Kemp</surname><given-names>Bonnie</given-names></name><xref ref-type=\"aff\" rid=\"Aff8\">8</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mehta</surname><given-names>Samar B.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff11\">11</xref></contrib><contrib contrib-type=\"author\"><name><surname>Carter</surname><given-names>Amber</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Uwanibe</surname><given-names>Jessica</given-names></name><xref ref-type=\"aff\" rid=\"Aff12\">12</xref><xref ref-type=\"aff\" rid=\"Aff13\">13</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ajogbasile</surname><given-names>Fehintola</given-names></name><xref ref-type=\"aff\" rid=\"Aff12\">12</xref><xref ref-type=\"aff\" rid=\"Aff13\">13</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-5046-153X</contrib-id><name><surname>Olumade</surname><given-names>Testimony</given-names></name><xref ref-type=\"aff\" rid=\"Aff12\">12</xref><xref ref-type=\"aff\" rid=\"Aff13\">13</xref></contrib><contrib contrib-type=\"author\"><name><surname>Odia</surname><given-names>Ikponmwosa</given-names></name><xref ref-type=\"aff\" rid=\"Aff14\">14</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sandi</surname><given-names>John Demby</given-names></name><xref ref-type=\"aff\" rid=\"Aff12\">12</xref><xref ref-type=\"aff\" rid=\"Aff15\">15</xref></contrib><contrib contrib-type=\"author\"><name><surname>Momoh</surname><given-names>Mambu</given-names></name><xref ref-type=\"aff\" rid=\"Aff12\">12</xref><xref ref-type=\"aff\" rid=\"Aff15\">15</xref></contrib><contrib contrib-type=\"author\"><name><surname>Metsky</surname><given-names>Hayden C.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff16\">16</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-6136-3257</contrib-id><name><surname>Boehm</surname><given-names>Chloe K.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-7400-4125</contrib-id><name><surname>Lin</surname><given-names>Aaron E.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kemball</surname><given-names>Molly</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff10\">10</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-7226-7781</contrib-id><name><surname>Park</surname><given-names>Daniel J.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Branco</surname><given-names>Luis</given-names></name><xref ref-type=\"aff\" rid=\"Aff17\">17</xref></contrib><contrib contrib-type=\"author\"><name><surname>Boisen</surname><given-names>Matt</given-names></name><xref ref-type=\"aff\" rid=\"Aff17\">17</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-4277-6037</contrib-id><name><surname>Sullivan</surname><given-names>Brian</given-names></name><xref ref-type=\"aff\" rid=\"Aff18\">18</xref></contrib><contrib contrib-type=\"author\"><name><surname>Amare</surname><given-names>Mihret F.</given-names></name><xref ref-type=\"aff\" rid=\"Aff19\">19</xref><xref ref-type=\"aff\" rid=\"Aff20\">20</xref></contrib><contrib contrib-type=\"author\"><name><surname>Tiamiyu</surname><given-names>Abdulwasiu B.</given-names></name><xref ref-type=\"aff\" rid=\"Aff19\">19</xref><xref ref-type=\"aff\" rid=\"Aff21\">21</xref></contrib><contrib contrib-type=\"author\"><name><surname>Parker</surname><given-names>Zahra F.</given-names></name><xref ref-type=\"aff\" rid=\"Aff19\">19</xref><xref ref-type=\"aff\" rid=\"Aff20\">20</xref></contrib><contrib contrib-type=\"author\"><name><surname>Iroezindu</surname><given-names>Michael</given-names></name><xref ref-type=\"aff\" rid=\"Aff19\">19</xref><xref ref-type=\"aff\" rid=\"Aff21\">21</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-4329-0795</contrib-id><name><surname>Grant</surname><given-names>Donald S.</given-names></name><xref ref-type=\"aff\" rid=\"Aff15\">15</xref><xref ref-type=\"aff\" rid=\"Aff22\">22</xref></contrib><contrib contrib-type=\"author\"><name><surname>Modjarrad</surname><given-names>Kayvon</given-names></name><xref ref-type=\"aff\" rid=\"Aff19\">19</xref></contrib><contrib contrib-type=\"author\"><name><surname>Myhrvold</surname><given-names>Cameron</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff10\">10</xref></contrib><contrib contrib-type=\"author\"><name><surname>Garry</surname><given-names>Robert F.</given-names></name><xref ref-type=\"aff\" rid=\"Aff17\">17</xref><xref ref-type=\"aff\" rid=\"Aff23\">23</xref></contrib><contrib contrib-type=\"author\"><name><surname>Palacios</surname><given-names>Gustavo</given-names></name><xref ref-type=\"aff\" rid=\"Aff9\">9</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hensley</surname><given-names>Lisa E.</given-names></name><xref ref-type=\"aff\" rid=\"Aff8\">8</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-6699-3568</contrib-id><name><surname>Schaffner</surname><given-names>Stephen F.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff10\">10</xref></contrib><contrib contrib-type=\"author\"><name><surname>Happi</surname><given-names>Christian T.</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff12\">12</xref><xref ref-type=\"aff\" rid=\"Aff13\">13</xref><xref ref-type=\"aff\" rid=\"Aff14\">14</xref></contrib><contrib contrib-type=\"author\"><name><surname>Colubri</surname><given-names>Andres</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff10\">10</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sabeti</surname><given-names>Pardis C.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff10\">10</xref><xref ref-type=\"aff\" rid=\"Aff24\">24</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.66859.34</institution-id><institution>Broad Institute of MIT and Harvard, </institution></institution-wrap>Cambridge, Massachusetts USA </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.38142.3c</institution-id><institution-id institution-id-type=\"ISNI\">000000041936754X</institution-id><institution>Department of Immunology and Infectious Diseases, </institution><institution>Harvard T.H. Chan School of Public Health, Harvard University, </institution></institution-wrap>Boston, Massachusetts USA </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.301713.7</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0393 3981</institution-id><institution>MRC-University of Glasgow Centre for Virus Research, </institution></institution-wrap>Glasgow, UK </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.168010.e</institution-id><institution-id institution-id-type=\"ISNI\">0000000419368956</institution-id><institution>Stanford University School of Medicine, </institution></institution-wrap>Stanford, California USA </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.461656.6</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0489 3491</institution-id><institution>Ragon Institute of MGH, MIT, and Harvard, </institution></institution-wrap>Cambridge, Massachusetts 02139 USA </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.38142.3c</institution-id><institution-id institution-id-type=\"ISNI\">000000041936754X</institution-id><institution>Ph.D. Program in Virology, Division of Medical Sciences, </institution><institution>Harvard Medical School, </institution></institution-wrap>Boston, Massachusetts 02115 USA </aff><aff id=\"Aff7\"><label>7</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.116068.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2341 2786</institution-id><institution>Computational and Systems Biology, </institution><institution>Massachusetts Institute of Technology, </institution></institution-wrap>Cambridge, Massachusetts USA </aff><aff id=\"Aff8\"><label>8</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.419681.3</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2164 9667</institution-id><institution>Integrated Research Facility, Division of Clinical Research, </institution><institution>National Institute of Allergy and Infectious Diseases, National Institutes of Health, </institution></institution-wrap>Frederick, Maryland USA </aff><aff id=\"Aff9\"><label>9</label>Center for Genome Sciences, The United States Army Medical Research Institute for Infectious Disease, 1425 Porter Street, Fort Detrick, Maryland 21702 USA </aff><aff id=\"Aff10\"><label>10</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.38142.3c</institution-id><institution-id institution-id-type=\"ISNI\">000000041936754X</institution-id><institution>Center for Systems Biology, Department of Organismic and Evolutionary Biology, </institution><institution>Harvard University, </institution></institution-wrap>Cambridge, Massachusetts USA </aff><aff id=\"Aff11\"><label>11</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.239395.7</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9011 8547</institution-id><institution>Division of Infectious Diseases, </institution><institution>Beth Israel Deaconess Medical Center, </institution></institution-wrap>Boston, Massachusetts USA </aff><aff id=\"Aff12\"><label>12</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.442553.1</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0622 6369</institution-id><institution>African Center of Excellence for Genomics of Infectious Disease (ACEGID), </institution><institution>Redeemer’s University, </institution></institution-wrap>Ede, Osun State Nigeria </aff><aff id=\"Aff13\"><label>13</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.442553.1</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0622 6369</institution-id><institution>Department of Biological Sciences, </institution><institution>College of Natural Sciences, Redeemer’s University, </institution></institution-wrap>Ede, Osun State Nigeria </aff><aff id=\"Aff14\"><label>14</label>Institute of Lassa Fever Research and Control, Irrua Specialist Teaching Hospital, Irrua, Edo State Nigeria </aff><aff id=\"Aff15\"><label>15</label>Viral Hemorrhagic Fever Program, Kenema Government Hospital, Kenema, Sierra Leone </aff><aff id=\"Aff16\"><label>16</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.116068.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2341 2786</institution-id><institution>Department of Electrical Engineering and Computer Science, </institution><institution>MIT, </institution></institution-wrap>Cambridge, Massachusetts 02139 USA </aff><aff id=\"Aff17\"><label>17</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.505518.c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 5901 1919</institution-id><institution>Zalgen Labs, </institution></institution-wrap>Germantown, Maryland USA </aff><aff id=\"Aff18\"><label>18</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.214007.0</institution-id><institution-id institution-id-type=\"ISNI\">0000000122199231</institution-id><institution>Department of Immunology and Microbial Science, </institution><institution>The Scripps Research Institute, </institution></institution-wrap>La Jolla, California USA </aff><aff id=\"Aff19\"><label>19</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.507680.c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2230 3166</institution-id><institution>Emerging Infectious Diseases Branch, </institution><institution>Walter Reed Army Institute of Research, </institution></institution-wrap>Silver Spring, Maryland USA </aff><aff id=\"Aff20\"><label>20</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.201075.1</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0614 9826</institution-id><institution>Henry M. Jackson Foundation for the Advancement of Military Medicine, </institution></institution-wrap>Bethesda, Maryland USA </aff><aff id=\"Aff21\"><label>21</label>Henry M. Jackson Foundation Medical Research International, Abuja, Nigeria </aff><aff id=\"Aff22\"><label>22</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.463455.5</institution-id><institution>Ministry of Health and Sanitation, </institution></institution-wrap>Freetown, Sierra Leone </aff><aff id=\"Aff23\"><label>23</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.265219.b</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2217 8588</institution-id><institution>Tulane University School of Medicine, </institution></institution-wrap>New Orleans, Los Angeles 70112 USA </aff><aff id=\"Aff24\"><label>24</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.413575.1</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2167 1581</institution-id><institution>Howard Hughes Medical Institute, </institution></institution-wrap>Chevy Chase, Maryland USA </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>4131</elocation-id><history><date date-type=\"received\"><day>20</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>28</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Recent outbreaks of viral hemorrhagic fevers (VHFs), including Ebola virus disease (EVD) and Lassa fever (LF), highlight the urgent need for sensitive, deployable tests to diagnose these devastating human diseases. Here we develop CRISPR-Cas13a-based (SHERLOCK) diagnostics targeting Ebola virus (EBOV) and Lassa virus (LASV), with both fluorescent and lateral flow readouts. We demonstrate on laboratory and clinical samples the sensitivity of these assays and the capacity of the SHERLOCK platform to handle virus-specific diagnostic challenges. We perform safety testing to demonstrate the efficacy of our HUDSON protocol in heat-inactivating VHF viruses before SHERLOCK testing, eliminating the need for an extraction. We develop a user-friendly protocol and mobile application (HandLens) to report results, facilitating SHERLOCK’s use in endemic regions. Finally, we successfully deploy our tests in Sierra Leone and Nigeria in response to recent outbreaks.</p></abstract><abstract id=\"Abs2\" abstract-type=\"web-summary\"><p id=\"Par2\">Outbreaks of viral hemorrhagic fevers highlight the need for sensitive, field-deployable diagnostics. Here the authors present a CRISPR-based SHERLOCK platform with field protocol and mobile app for Ebola and Lassa fever outbreaks.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>CRISPR-Cas systems</kwd><kwd>Virology</kwd><kwd>Viral infection</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par3\">Ebola virus (EBOV) and Lassa virus (LASV) pose immediate, severe threats to human life and public health, as demonstrated by ongoing outbreaks of EBOV disease (EVD) in the Democratic Republic of the Congo (DRC) and Lassa fever (LF) in Nigeria. Despite their high morbidity and mortality, EVD and LF are difficult to diagnose, because early symptoms, including fever, vomiting, and aches, are often indistinguishable from those of more common tropical diseases<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. Rapid point-of-care diagnostics are vital for facilitating timely clinical care and proper containment<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>.</p><p id=\"Par4\">Despite the critical need for rapid point-of-care diagnostics for these viruses, current gold standards lack the logistical feasibility to effectively diagnose cases in endemic regions with limited infrastructure. PCR-based diagnostics are sensitive and can be rapidly developed for emerging or mutating viruses but they are not practical as a point-of-care test, as they require advanced laboratory infrastructure, a cold chain, and expensive reagents. Rapid antigen- and antibody-based tests are deployable but are less sensitive than PCR and take longer to develop; they can also be ineffective in the early/acute stage of infection<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>–<xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>, a critical period for supportive care and to contain human-to-human spread.</p><p id=\"Par5\">EBOV and LASV both present distinct diagnostic challenges. The live attenuated rVSV∆G-ZEBOV-GP EBOV vaccine (Merck), currently being deployed to combat the DRC outbreak, produces EBOV GP RNA that can yield false-positive tests by glycoprotein (GP)-targeting assays, including the commonly used GeneXpert reverse-transcriptase quantitative PCR (RT-qPCR)<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>–<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>; similar false positives will be a concern whenever new live attenuated vaccines are introduced for any virus. In the case of LASV, high genetic diversity in the virus through western Africa hinders the development of diagnostic tools sensitive to all viral strains. The most widely used diagnostic for LF viral detection, a RT-qPCR developed against Josiah strains derived from Sierra Leone (clade IV), has had false-positive and false-negative results when tested against recent clade II samples from Nigeria<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>.</p><p id=\"Par6\">The recently developed CRISPR-based SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) platform provides a promising approach for rapidly adaptable, deployable diagnostics. SHERLOCK utilizes the RNA-targeting protein Cas13a for sensitive and specific detection of viral nucleic acid<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. It pairs isothermal recombinase polymerase amplification (RPA) with crRNA-guided Cas13a detection, which enables specific pairing of Cas13a with the target sequence and signal amplification via Cas13’s collateral cleavage activity<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. Both amplification and Cas13a-based detection are isothermal, requiring only a low-energy, single-temperature heat block and basic pipette and tips, compatible with point-of-care detection. SHERLOCK can be combined with HUDSON (Heating Unextracted Diagnostic Samples to Obliterate Nucleases), which inactivates pathogens and releases nucleic acid through a combined heat and chemical denaturation, eliminating the need for a column- or bead-based nucleic acid extraction<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Our recent work has shown the high sensitivity of SHERLOCK and HUDSON in detecting Zika virus and dengue virus directly from bodily fluids<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>, allowing for a fully point-of-care diagnostic. Utilizing this system, we develop a diagnostic test for EBOV and LASV that can be deployed in any setting, requires minimal processing of infectious materials, and accurately reports test results in a user-friendly format.</p></sec><sec id=\"Sec2\" sec-type=\"results\"><title>Results</title><sec id=\"Sec3\"><title>CRISPR-Cas13a diagnostic development and validation for VHFs</title><p id=\"Par7\">Motivated by the increased severity and frequency of EBOV and LASV outbreaks, we describe here the development and validation of SHERLOCK assays to detect these viruses. The assays can be detected by two readout methods, either fluorescence or lateral flow. The more sensitive fluorescence-based system allowed us to perform extensive validation during the development of our assay, determine the length of amplification time needed for viral detection, and determine the limit of detection (LOD). The lateral flow readout, which we then validated further, utilizes a commercially available detection strip to provide semi-quantitative point-of-care detection of the virus.</p><p id=\"Par8\">We developed a SHERLOCK EBOV assay to target the <italic>L</italic> gene of the EBOV Zaire strain, which accounts for the majority of known clinical cases of EBOV infections, including the two largest and most recent EVD epidemics<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. We used primer design applications (CATCH<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>) to identify an optimal target within a conserved region of the <italic>L</italic> gene, thus avoiding potential false-positive results caused by the rVSV∆G-ZEBOV-GP EBOV vaccine (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref> and Supplementary Tables <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>–<xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). Our assay detected synthetic DNA at concentrations as low as 10 copies/µl using either fluorescent or lateral flow readout (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b, c</xref>). As EBOV, LASV, and Marburg virus (MARV) infections present with similar symptoms and have been known to co-circulate, we tested for cross-reactivity using seedstock and synthetic DNA of each virus; our assay showed no cross-reactivity to either LASV or MARV (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1d</xref>).<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Detection of EBOV.</title><p><bold>a</bold> Schematic of the SHERLOCK EBOV assay. <bold>b</bold>, <bold>c</bold> Detection of a serial dilution of EBOV synthetic DNA using (<bold>b</bold>) mean fluorescence of three technical replicates and (<bold>c</bold>) lateral flow readouts. Error bars indicate ±1 SD for three technical replicates. <bold>d</bold> Test of cross-reactivity using MARV, EBOV, and LASV viral seedstock cDNA. Heat map is measured in Fluorescence (a.u.). <bold>e</bold> SHERLOCK testing of cDNA extracted from 12 confirmed EBOV-positive and 4 confirmed EBOV-negative samples collected from suspected EVD patients during the 2014 outbreak in Sierra Leone. Error bars indicate 95% confidence interval. <bold>f</bold> Four of the samples from <bold>e</bold> were also tested by collaborators using lateral flow detection. <bold>g</bold>, <bold>h</bold> Detection of serial dilution of synthetic RNA from Ituri, DRC and Makona, Sierra Leone using (<bold>g</bold>) fluorescence where error bars indicate ±1 SD for three technical replicates and (<bold>h</bold>) lateral flow readouts carried out at USAMRIID. Source data are in the Source Data file.</p></caption><graphic xlink:href=\"41467_2020_17994_Fig1_HTML\" id=\"d30e975\"/></fig></p><p id=\"Par9\">We validated the SHERLOCK EBOV assay at the Broad Institute using 16 clinical samples taken from suspected EVD patients in Sierra Leone during the 2014–2016 West Africa outbreak. For safety reasons, we tested complementary DNA (which is not infectious) and benchmarked the results against previously generated sequencing data<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup> (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1e</xref>). Of the 16 samples, 12 were positive for EBOV by sequencing, all 12 of which were positive by SHERLOCK. The four sequencing-negative samples were negative by SHERLOCK (100% sensitivity, 100% concordance).</p><p id=\"Par10\">We also developed and validated SHERLOCK assays for LASV, a challenging target because of the virus’s extreme genetic diversity, both within and especially between clades<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Currently, two clades—clade II, localized in Nigeria, and clade IV, localized in Sierra Leone<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>—account for over 90% of known clinical infections<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Given this extreme genetic diversity, we designed two LASV SHERLOCK assays (Supplementary Tables <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>–<xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). The first assay (LASV-II) targets clade II. To ensure detection of all known genomes in this highly divergent clade, the assay contains two multiplexed crRNAs (LASV-IIA and LASV-IIB) (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a</xref>). When we compared the two LASV-II crRNAs to an alternative assay with only one crRNA, the former detected LASV more quickly and identified an additional positive sample (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2b</xref>). The second assay (LASV-IV) targets clade IV; in this clade, we found a more conserved region that enabled us to use a single crRNA. The LASV-II assay was sensitive down to 10 copies/µl with a fluorescent readout (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1a</xref>) and 100 copies/µl using lateral flow strips (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1c</xref>); the LOD for LASV-IV was 100 copies/µl for the fluorescence-based assay (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1b</xref>) and 1000 copies/µl for the lateral flow assay (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1d</xref>). Neither the LASV-II nor the LASV-IV assay cross-reacted with synthetic DNA from the other clade or with known positive patient samples from the other clade’s geographic region, nor did they cross-react with EBOV or Marburg seedstock cDNA (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2c, d</xref>). The LASV SHERLOCK assays are thus both species and clade specific, and therefore region specific, which can help distinguish possible imported cases from local transmission.<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Detection of LASV clade II and IV.</title><p><bold>a</bold> Schematic of LASV SHERLOCK assays targeting the two most common clades of LASV: clades II (LASV-II assay) and IV (LASV-IV assay). For the LASV-II assay, three crRNAs were designed and tested. Two crRNAs are multiplexed to encompass the clade’s genetic diversity (IIA/IIB or IIA/IIC). Each crRNA was tested using three technical replicates. <bold>b</bold>–<bold>d</bold> Heat maps are measured in fluorescence (a.u.). <bold>b</bold> Detection of LASV RNA from suspected LF clinical samples using crRNAs IIA, IIB, IIC, or a combination of crRNAs. <bold>c</bold> Test of cross-reactivity between different viral species using MARV, EBOV, and LASV viral seedstock cDNA. The LASV-II and LASV-IV assays do not cross-react with MARV or EBOV seed stocks. <bold>d</bold> Test of cross-reactivity between LASV clade-specific assays using clinical samples from recent outbreaks in Nigeria and Sierra Leone. The LASV-II and LASV-IV assays provide clade-specific detection. <bold>e</bold> SHERLOCK testing using the LASV-II assay of RNA extracted from seven confirmed LASV-positive and three confirmed LASV-negative samples collected from suspected LF patients in Nigeria during the 2018 outbreak. Error bar indicates 95% confidence interval. <bold>f</bold> Results from <bold>e</bold> were compared head-to-head to those from the gold standard Nikisins RT-qPCR assay, next-generation sequencing (genome assembled), and lateral flow detection. <bold>g</bold> SHERLOCK testing using the LASV-IV assay of RNA extracted from seven confirmed LASV-positive and three confirmed LASV-negative samples collected from suspected LF patients in Sierra Leone. Error bar indicates 95% confidence interval. <bold>h</bold> Results from <bold>g</bold> were compared head-to-head to those from the gold standard Nikisins RT-qPCR assay, a second Broad RT-qPCR, NGS, and lateral flow detection. Source dare are in the Source Data file.</p></caption><graphic xlink:href=\"41467_2020_17994_Fig2_HTML\" id=\"d30e1080\"/></fig></p></sec><sec id=\"Sec4\"><title>Deployment of CRISPR-Cas13a diagnostics</title><p id=\"Par11\">Next, we deployed the EBOV assay to our collaborators in Sierra Leone to test patient samples stored from the 2014–2016 outbreak using the point-of-care lateral flow assay. This allowed us to validate and assess the practicality of the SHERLOCK assay in a setting with previously circulating EBOV and limited infrastructure. As a head-to-head comparison, we identified 4 whole blood (WB) in trizol aliquots from the same patients tested in our first panel of 16 samples. These samples were stored at the Kenema Government Hospital (KGH) biobank under variable temperature conditions (−20 °C with multiple power cuts). Using the same protocol as for the panel of 16 (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>), all 4 samples were positive by SHERLOCK, consistent with the results obtained at the Broad Institute (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1f</xref>), despite multiple years of an imperfect cold chain.</p><p id=\"Par12\">The SHERLOCK EBOV assay was also highly efficient at detecting a more recent EBOV variant from the DRC. We tested a synthetic version of a 2018 Ebola isolate from Ituri Province (Ituri isolate 18FHV089), DRC (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1g, h</xref>), and, as validation, a 2014 Makona isolate from Sierra Leone that underwent the same synthetic generation process (see “Methods”)<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. Utilizing both the SHERLOCK fluorescence-based and lateral flow-based assays, the Makona and Ituri isolates were both detected at levels down to 10 copies/µl. The DRC isolate was genetically distinct from the 2014 isolate but maintained the key conserved stretch on the L gene that the SHERLOCK assay targets, which remains conserved on all available genomes from the ongoing DRC outbreak.</p><p id=\"Par13\">We also tested our LASV assay in Sierra Leone and Nigeria using clinical samples. We evaluated the sensitivity on a panel of ten RNA and cDNA samples per clade, derived from suspected LF patients (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2e, g</xref>). We compared SHERLOCK results using both fluorescent and lateral flow readouts head-to-head with the Nikisin RT-qPCR assay<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> and benchmarked both results against sequencing data (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2f, h</xref>). The LASV-II fluorescent readout was positive for all seven sequencing-positive samples and negative for all three sequencing-negative samples (100% sensitivity, 100% concordance), as was the Nikisin RT-qPCR. The LASV-II lateral flow readout failed to detect one sequencing-positive sample. For the LASV-IV assay, SHERLOCK performed significantly better than RT-qPCR. SHERLOCK was again positive for all seven sequencing-positive samples (100% sensitivity), whereas the Nikisin assay was only 40% sensitive and our in-house Broad RT-qPCR assay (Supplementary Tables <xref rid=\"MOESM1\" ref-type=\"media\">4</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>), developed on all recent LASV genomes, was only 50% sensitive. The three sequencing-negative samples were negative by SHERLOCK (100% concordance). The low detection rate of clade IV by RT-qPCRs is likely due to multiple mismatched base pairs where the primers anneal; despite this, Nikisin continues to be a primary diagnostic.</p></sec><sec id=\"Sec5\"><title>Safety analysis and efficacy of heat inactivation</title><p id=\"Par14\">Reducing exposure to viral hemorrhagic fevers (VHFs) among healthcare workers is critical for the safe and effective use of diagnostic tests. For diagnostic test design, this requires ensuring that the sample is fully inactivated and, where possible, using non-invasive sample types. To this end, we combined the SHERLOCK assay with the HUDSON technique, which integrates heat inactivation with TCEP : EDTA to denature RNAses and release nucleic acid from viral particles, thus eliminating the need for RNA extraction. Furthermore, as LASV and EBOV are secreted in saliva and urine<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>, HUDSON enables disease diagnosis without the need for an invasive blood draw or specialized equipment, resulting in a faster end-to-end processing time. To confirm HUDSON’s efficacy for viral inactivation and to determine the most sensitive HUDSON protocol for SHERLOCK use, we carried out tests at the BSL4 laboratory facility at the NIH Integrated Research Facilities.</p><p id=\"Par15\">We first showed that HUDSON successfully rendered viruses inactive in three sample types. We spiked human WB, urine, and saliva with the live EBOV Mayinga variant and confirmed that samples had viral activity using an initial plaque assay (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref>). Samples underwent serial dilution to mimic variation in viral load and were then heat and chemical treated using HUDSON. We performed HUDSON using two conditions, either 95 °C for 10 min or 70 °C for 30 min, to determine the most effective heat-inactivation protocol. We used a standard plaque assay, two passages in Vero cells, to determine presence or absence of replication-competent virus. After HUDSON treatment, no viable virus was detected at either temperature, showing complete inactivation at all concentrations and confirming the safety of the HUDSON-SHERLOCK platform. To ensure safety, clinical and laboratory staff should use appropriate personal protective equipment or a glove box until a sample is fully inactivated.<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>HUDSON safety testing.</title><p><bold>a</bold> Schematic overview of the HUDSON, SHERLOCK inactivation validation. Viral inactivation includes dilution with EDTA : TCEP and a 20 min 37 °C inactivation of nucleases. All final results were determined using lateral flow due to the inability to carry out appropriate fluorescent analysis in the BSL4 facility. <bold>b</bold> Lateral flow detection of spiked blood, urine, and saliva inactivated at either 70 °C or 95 °C. Serial dilution shown are PFU/mL. All assays were carried out in the BSL4 facility.</p></caption><graphic xlink:href=\"41467_2020_17994_Fig3_HTML\" id=\"d30e1154\"/></fig></p><p id=\"Par16\">We then performed SHERLOCK on the HUDSON-inactivated samples to establish how HUDSON temperature conditions affect SHERLOCK’s performance. The serially diluted EBOV samples were tested by SHERLOCK using the lateral flow readout (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b</xref>) and the results were compared to the GeneXpert diagnostic. Both heat-inactivation conditions performed equally. Using our combined HUDSON-SHERLOCK method, we detected virus down to 1.1E + 05 PFU/mL in WB, 1.2E + 05 PFU/mL in urine, and 9.4E + 03 PFU/mL in saliva (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">6</xref>). When considering cycle threshold ≤ 36 as definitive positives<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, GeneXpert was more sensitive than SHERLOCK for WB (4.4E + 02 PFU/mL) and urine (1.1E + 02 PFU/mL), but comparable for saliva (1.1E + 03 PFU/mL but for only NP detection, 9.4E + 03 PFU/mL for GP detection). Ultimately, our HUDSON testing highlights the potential to safely and sensitively test saliva in suspected patients, minimizing the need for more invasive blood draws and increasing safety for healthcare workers.</p></sec><sec id=\"Sec6\"><title>HandLens: a mobile application for diagnostic analysis</title><p id=\"Par17\">The lateral flow readout of the current SHERLOCK protocol can be difficult to interpret for low concentration samples due to the correlation of band darkness with viral load. Critical for a deployable rapid diagnostic and surveillance tool is an easy-to-use interface that produces and reports a consistent readout free of operator bias. In addition, lateral flow band strength has the potential to generate a semi-quantitative result. To exploit that potential and to facilitate accurate readout, we developed a mobile phone application called HandLens that captures (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4a, b</xref>) and analyzes (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c–e</xref>) an image of one or more lateral flow strips to quantify test results and resolve ambiguous readouts (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>). Using a prototype version of the HandLens app, we tested a dilution series (ranging from 10<sup>5</sup> to 10 copies/µl) from four EBOV samples and compared our results to RT-qPCR. This yielded estimates of 93% accuracy, 91% sensitivity, and 100% specificity from a total of 21 strips (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3a</xref>). This app can be adapted for use on any smartphone or tablet, allowing a clear, unbiased diagnostic readout.<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>Quantification of SHERLOCK lateral flow strips using HandLens, an Android app prototype.</title><p>Internal image analysis pipeline of the SHERLOCK detector app (HandLens). <bold>a</bold> Images of two positive sample lateral flow strips are imported to the app. <bold>b</bold> The relevant signal regions of the lateral flow strips are detected and demarcated by red bounding boxes. <bold>c</bold> Bilateral filtering is used to extract and smoothen the signal regions from the raw input image. <bold>d</bold> Contrast within the image is increased by applying contrast limited adaptive histogram equalization (CLAHE). <bold>e</bold> The signal is linearized for downstream signal processing; the red curves indicate the signal extracted after applying CLAHE, whereas the blue curves indicate the signal levels if the CLAHE step is skipped. <bold>f</bold> The strip reader app works by allowing the user to take a picture of the test strips where a rectangle can be used to select the control strip on the leftmost side. The raw image data is sent to a backend server that runs the signal detection algorithm and returns the binary and semi-quantitative predictions for each strip.</p></caption><graphic xlink:href=\"41467_2020_17994_Fig4_HTML\" id=\"d30e1217\"/></fig></p></sec></sec><sec id=\"Sec7\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par18\">The recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the need for rapid deployable diagnostics that decrease healthcare worker exposure. Although there has been great improvement with platforms like Abbot and GeneXpert, these RT-qPCRs still require expensive machines and laboratory infrastructure. Furthermore, this pandemic and other recent viral outbreaks have demonstrated the lag time it takes to create a sensitive antibody- or antigen-based rapid diagnostic test (RDT). SHERLOCK provides an alternative viral diagnostic method that addresses these shortcomings. In summary, we have developed sensitive, specific, point-of-care CRISPR-based diagnostics for EBOV and LASV, two hemorrhagic fever viruses that pose immediate global threats. We have validated these diagnostics on laboratory and patient samples, including deployment for testing in partner laboratories in Sierra Leone and Nigeria. In addition, we have shown that the HUDSON protocol not only removes the need for extraction but also inactivates EBOV to allow for a safe low-tech test, and we demonstrated that non-invasive samples including saliva and urine can be used for rapid detection, eliminating the need for a blood draw and increasing safety for clinical staff testing for suspected VHF. We provide a user-friendly readout that can be documented using a mobile device to allow for greater reproducibility and immediate reporting. The HUDSON-SHERLOCK assay minimizes testing time and handling of infectious samples, reduces the cost to less than $1 USD<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup> per sample, and can be run with minimal equipment using only solar power or a small generator to allow for quick diagnostics in any environment, showcasing the growing capabilities of CRISPR-based diagnostics for viral detection.</p></sec><sec id=\"Sec8\"><title>Methods</title><sec id=\"Sec9\"><title>Ethical approval for the use of clinical samples</title><p id=\"Par19\">All patient samples used for this study were de-identified and were obtained through studies that were evaluated and approved by the institutional review boards at the Irrua Specialist Teaching Hospital (Irrua, Nigeria), Redeemer’s University (Nigeria), KGH (Sierra Leone), Sierra Leone Ministry of Health, Ministry of Health of the DRC, and Harvard University (Cambridge, Massachusetts).</p><p id=\"Par20\">LF patients were recruited for this study using protocols approved by human subjects committees at Harvard University, Broad Institute, Irrua Specialist Teaching Hospital, KGH, Oyo State Ministry of Health, Ibadan, Nigeria, and Sierra Leone Ministry of Health. All patients were treated with a similar standard of care and were offered the drug Ribavirin, whether or not they decided to participate in the study.</p><p id=\"Par21\">Due to the severe outbreak for EVD, patients could not be consented through our standard protocols. Instead use of clinical excess samples from EVD patients was evaluated and approved by Institutional Review Boards in Sierra Leone and at Harvard University. The Office of the Sierra Leone Ethics and Scientific Review Committee, the Sierra Leone Ministry of Health and Sanitation, and the Harvard Committee on the Use of Human Subjects have granted a waiver of consent to use de-identified samples collected from all suspected EVD patients receiving care during the outbreak response. The Sierra Leone Ministry of Health and Sanitation also approved shipments of non-infectious non-biological samples from Sierra Leone to the Broad Institute and Harvard University for genomic studies of outbreak samples.</p><p id=\"Par22\">Protocol Title: Genomic Characterization and Surveillance of Microbial Threats in West Africa</p><p id=\"Par23\">Principal Investigator: Pardis C. Sabeti</p><p id=\"Par24\">Protocol #: IRB19-0023</p><p id=\"Par25\">Funding Source: The Broad Institute-5700161-5500000755 (Active), NIH; Military HIV Research Program and Henry M. Jackson Foundation, NIH; Bill and Melinda Gates Foundation</p><p id=\"Par26\">Protocol Title: Sierra Leone Lassa and Ebola Case Control (includes Ebola Clinical Excess from Deceased Patients), Nigeria Lassa Case Control</p><p id=\"Par27\">Protocol #: CUHS 21288 and ORSP 2202</p><p id=\"Par28\">Harvard University</p><p id=\"Par29\">Funding Source: The Broad Institute-5700161-5500000755 (Active), NIH; Military HIV Research Program and Henry M. Jackson Foundation, NIH; Bill and Melinda Gates Foundation</p></sec><sec id=\"Sec10\"><title>Sample preparation</title><p id=\"Par30\">Patient samples were taken by clinical staff using appropriate personal protective equipment. Inactivation of samples occurred in their country of origin. Samples were then shipped to the Broad Institute or tested at the local center (Nigeria, Sierra Leone). Samples were inactivated in AVL buffer (Qiagen) or TRIzol (Life Technologies) following standard operating procedures. Samples were stored in liquid nitrogen or at −20 °C. RNA was isolated at the clinical site using the QIAamp Viral RNA Minikit (Qiagen) according to the manufacturer’s protocol. Poly(rA) and host rRNA were depleted using RNase H selective depletion, using 616 ng oligo (dT) (40 nt long) and/or 1000 ng DNA probes complementary to human rRNA. Samples then underwent RNase-free DNase using a kit (Qiagen) according to the manufacturer’s protocol. AMPure RNA clean beads (Beckman Coulter Genomics) were used to clean and concentrate samples. cDNA synthesis was performed using the Superscript III kit (Thermo Fischer) plus dNTPs, random primers, and SUPERASE-IN for first-strand synthesis. Then, the 10× second-strand buffer kit (New England Biolabs), plus <italic>Escherichia coli</italic> DNA ligase, <italic>E. coli</italic> DNA polymerase, Rnase H, and dNTPs were used for second-strand synthesis. Samples then underwent a final AMpure DNA beads clean-up<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>.</p></sec><sec id=\"Sec11\"><title>SHERLOCK assay design</title><p id=\"Par31\">To design RPA primers and crRNAs, we identified conserved regions of the EBOV and LASV genomes. For the EBOV assay, we used an alignment based on all published sequences. The highly conserved areas of the EBOV genome allowed us to design numerous efficient crRNAs, with two targeting the NP-gene and two targeting the <italic>L</italic> gene (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4a</xref>), neither of which is expected to cross-react with the live attenuated vaccine. We developed an EBOV assay using the most efficient crRNA based on peak fluorescence and minimum time required for SHERLOCK detected to reach saturation (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4b</xref>). For the LASV-IV assay, we used an alignment based on all published LASV clade IV sequences (Sierra Leone)<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. For the LASV-II assay, we used an alignment based on all published LASV clade II sequences (Nigeria)<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. We then used a 50 bp sliding window to identify flanking conserved areas. We identified 21–29 bp primers and a 29 bp crRNA for the LASV-IV assay (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a</xref>). Due to the high diversity in clade II, one crRNA, even with up to six degenerate bases, did not encompass all known genomes. We identified three crRNAs within the same 200 bp region and tested these in tandem (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2b</xref>).</p></sec><sec id=\"Sec12\"><title>RPA reactions</title><p id=\"Par32\">All RPA reactions were carried out using the Twist-Dx RT-RPA kit according to the manufacturer’s instructions. All reactions were run for 20 min. Primer concentrations were 480 nM. For reactions with RNA input, Murine RNase inhibitor (NEB M3014L) was added at a concentration of 2 units/µl. For a complete list of RPA primer names and sequences, see Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>.</p></sec><sec id=\"Sec13\"><title>Production of LwaCas13a and crRNAs</title><p id=\"Par33\">LwaCas13a was purified by Genscript. The crRNAs were determined by aligning all known genomes and using our CATCH method<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup> to identify conserved areas on a sliding scale and were synthesized by Integrated DNA Technologies.</p></sec><sec id=\"Sec14\"><title>CAS13a detection reactions</title><p id=\"Par34\">For detection reaction, refer to our one-page user-friendly protocol (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>). Detection assays were performed for either plate reader (fluorescent) or lateral flow detection. Broadly, Cas13a, crRNas, T7 polymerase (New England Biolabs), RNasae inhibitors (New England Biolabs), buffer (CB—40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl<sub>2</sub>, pH 7.3), MgCl<sub>2</sub> (rNTPs (New England Biolabs), and either a fluorescent substrate reporter (RNase alert v2) or LP probe (Tewist-Dx) were combined. Detection mix was combined with the RPA reaction and incubator for 1 h at 37 °C<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. For multiplexed crRNAs in LASV-II assays, the total volume of crRNA was doubled. Reactions were run on a Biotek Cytation 5 multi-mode reader. All reactions were run in triplicate alongside a no-template control. Fluorescence kinetics were measured via a monochrometer with excitation at 485 nm and emission at 520 nm, with a reading every 5 min. EBOV assays were run for 1 h and LASV assays were run for 3 h. Reported fluorescence values are specified as background-subtracted or template-specific. For EBOV detection, the crRNA EBOVA was used unless otherwise noted. For detection of LASV clade IV, the crRNA LASV-IVA was used. For detection of LASV-II, an equal mix of crRNAs LASV-IIA and LASV-IIB was used. For a complete list of crRNA names and sequences, see Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>.</p></sec><sec id=\"Sec15\"><title>Lateral flow detection reactions</title><p id=\"Par35\">Lateral flow detection reactions were performed as described using commercially available detection strips according to manufacturer’s instructions (Milenia Hybridetect 1, Twist-Dx, Cambridge, UK).</p></sec><sec id=\"Sec16\"><title>Data analysis</title><p id=\"Par36\">For all fluorescence values, background-subtracted fluorescence was calculated by subtracting the minimum fluorescence value, which occurred between 0–20 min, from the final fluorescence value. For all fluorescence values reported for patient samples, found in Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1e, g</xref> and <xref rid=\"Fig2\" ref-type=\"fig\">2b, e, h</xref>, target-specific fluorescence was calculated by subtracting the mean background-subtracted fluorescence of the no template control from the mean background-subtracted fluorescence of a given target with the same crRNA at the same time point.</p></sec><sec id=\"Sec17\"><title>LOD experiments</title><p id=\"Par37\">To determine the sensitivity of SHERLOCK assays, assay-specific synthetically derived DNA templates were derived from clade- and virus-specific alignments<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR17\">17</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref>–<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Synthetically derived DNA (≤500 bp and non-replicant competent) templates were used as input into the RPA reaction at concentrations from 10<sup>4</sup> copies/µl to 1 copy/µl with a 1 : 10 dilution series. Each crRNA was also tested on a no-input negative control. Reactions were run twice, using both the fluorescent readout and the lateral flow readout. All reactions were run in triplicate for the fluorescent readout.</p></sec><sec id=\"Sec18\"><title>Cross-reactivity experiments</title><p id=\"Par38\">To assess the cross-reactivity of assays with other viruses known to cause hemorrhagic symptoms, all assays were tested on LASV (Josiah), EBOV (Makona), and MARV (Angola) viral cDNA seed stocks. Each assay was also tested on a positive control containing an assay-specific synthetically derived DNA template at a concentration of 10<sup>4</sup> copies/µl and on no-input negative control. Synthetically derived DNA were short fragments that encompassed the primers and around 20 base pairs on both the 5′- and 3′-end. All fragments were non-replicant competent. All reactions were run in triplicate. The LASV assays were also assessed for clade-specific detection. The LASV-IV assay was tested on three RNA patient samples from clade II and the LASV-II assay was tested on three RNA patient samples from clade IV. Each assay was also tested on a positive control containing an assay-specific synthetically derived DNA template at a concentration of 10<sup>4</sup> copies/µl and on no-input negative control. All reactions were run in triplicate.</p></sec><sec id=\"Sec19\"><title>Validation on patient samples</title><p id=\"Par39\">The LASV-IV assay was validated on a panel of 12 RNA samples collected from suspected LF patients in Sierra Leone<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Seven of these samples were confirmed LASV positive by antigen-based RDT, enzyme-linked immunosorbent assay (ELISA) IgM, and RT-qPCR<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>; four were confirmed LASV negative by RDT, ELISA, and RT-qPCR; one did not have the full panel of tests. The assay was also tested on a positive control containing synthetically derived cDNA and a no-input control. All reactions were run in triplicate. A subset of these samples was tested using the lateral flow readout. Results were compared to RT-qPCR results and sequencing results. The LASV-II assay was validated on a panel of 12 cDNA samples collected from suspected LF patients in Nigeria during the 2018 outbreak<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Nine of these samples were confirmed LASV positive and three were confirmed LASV negative. The assay was also tested on a positive control containing synthetically derived cDNA at a concentration of 10<sup>4</sup> cp/µl and a no-input control. All reactions were run in triplicate. A subset of these samples was also tested using the lateral flow readout. Results were compared to RT-qPCR results and sequencing results.</p><p id=\"Par40\">The EBOV assay was validated on a panel of 16 cDNA and RNA samples collected from suspected EVD patients in Sierra Leone during the 2014 outbreak<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. Twelve of these samples were confirmed EBOV positive and four were confirmed EBOV negative. The assay was also tested on a positive control containing synthetically derived cDNA at a concentration of 10<sup>4</sup> cp/µl and a no-input control. All reactions were run in triplicate. To validate the lateral flow readout, the EBOV assay was tested on four EBOV-positive cDNA samples, alongside a positive and a no-input control.</p></sec><sec id=\"Sec20\"><title>RT-qPCR experiments</title><p id=\"Par41\">RT-qPCR for LASV detection was performed using the Power SYBR Green RNA-to-Ct 1-step RT-qPCR kit (Thermo Fisher) according to the manufacturer’s instructions. Reactions were performed on a LightCycler 96 machine (Roche). For detection of LASV clade II, the primers Nikisins_F and Nikisins_R were used<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. For detection of LASV clade IV, the in-house assay, including primers Broad_F and Broad_R, was also used with the TaqMan RNA-to-CT 1-Step Kit (Applied Biosystems). For a complete list of primer names and sequences, see Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>; for a complete list of probe names and sequences, see Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>.</p></sec><sec id=\"Sec21\"><title>EBOV DRC experiments</title><p id=\"Par42\">All samples tested at USAMRIID underwent SHERLOCK as described above with the exception of the fluorescent readout which was conducted on a BioRad CFX96. The genomes of both the Makona and Ituri isolates were sequenced with Illumina technology (MiSeq for Makona and iSeq for Ituri) in-country as described in ref. <sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. Briefly, once the genome sequences were obtained, sub-genomic fragments were commercially synthesized and then assembled into plasmids encoding full genomes at USAMRIID. The RNAs were in vitro transcribed from the full genome plasmids. All SHERLOCK work on these isolates was carried out at USAMRIID.</p></sec><sec id=\"Sec22\"><title>Safety testing of HUDSON and SHERLOCK</title><p id=\"Par43\">Before inactivation all samples should be treated using BSL4 safety conditions. All clinical and laboratory staff should wear full PPE and/or use a glove box to acquire and handle patient samples. We also recommend that all equipment should be decontaminated before and after each use. Samples remain highly infectious until the full heat inactivation protocol can be carried out.</p><p id=\"Par44\">Viral titers for each sample were determined by plaque assay; a six-well plate with a confluent monolayer of VeroE6 cells was infected with a predetermined volume of sample. The wells are then overlaid with a medium to ensure the monolayer health and incubated for a set amount of time. If there is live virus in the sample, this virus will infect and kill a cell, and spread cell-to-cell creating a plaque, or a clearing of cells. Plaques are then visualized by a crystal violet stain and counted to determine viral titer. Samples underwent a serial dilution and then were heat-inactivated using methods described in<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Briefly, a 1 : 100 solution of 0.5 M EDTA to TCEP (Thermo) was used to decrease RNase degradation. The EDTA : TCEP was added to spiked samples at a ratio of one part EDTA : TCEP to four parts samples. First, samples were heated to 37 °C for 20 min to inactivate nucleases. The samples were then heated to 95 °C for 10 min or 70 °C for 30 min as described, followed by the SHERLOCK assay as described above. Only Eppendorph Safe-Lock tubes or cryovials with a screw top should be used for heat inactivation and the outside of the tubes should be decontaminated before and after heat inactivation.</p><p id=\"Par45\">The samples then underwent primary passaging. Samples were added to a T25 flask, incubated for 1 h at 37 °C at 5% CO<sub>2</sub>, then replenished with media and incubated for 7 days. Seven days post infection (dpi) pictures were taken of each flask to assess for cytopathic effect. Secondary passage was performed to assess for any residual virus particles not detected in the primary passage. All media was transferred from the T25 flask to a T75 flask incubated for 1 h at 37 °C at 5% CO<sub>2</sub>, replenished with media and incubated for 7 dpi, and then photographed. Following the primary and secondary passaging, a final plaque assay was performed to determine viral titer and to assess viral clearance or reduction. For diagnostic comparison, qPCR samples were run on the Cepheid GeneXpert platform. Sample were inactivated in Xpert lysis buffer at room temperature for 10 min and then run with an Xpert Ebola assay cartridge<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>.</p></sec><sec id=\"Sec23\"><title>HandLens: the lateral flow reader app</title><p id=\"Par46\">First, the signal-containing section of the lateral flow strip is detected using OpenCV’s<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup> contour detection routines. This region is extracted and transformed into a smooth two-dimensional image using bilateral filtering. The result is enhanced using contrast limited adaptive histogram equalization<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, which has the property of increasing signal contrast in local regions. This signal is then linearized by integrating pixel intensity over each row. Finally, a signal is marked as positive for viral load if the signal intensity in the test band of the strip is above a certain user-defined threshold compared to the control strip, and negative if the signal intensity is too low. We also developed quantifiable signal graphs of each band and controlled for shadows and image contrast by applying a contrast-improvement algorithm (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3b</xref>).</p></sec><sec id=\"Sec24\"><title>Reporting summary</title><p id=\"Par47\">Further information on research design is available in the <xref rid=\"MOESM2\" ref-type=\"media\">Nature Research Reporting Summary</xref> linked to this article.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec25\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41467_2020_17994_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41467_2020_17994_MOESM2_ESM.pdf\"><caption><p>Reporting Summary</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM3\"><media xlink:href=\"41467_2020_17994_MOESM3_ESM.pdf\"><caption><p>Peer Review File</p></caption></media></supplementary-material></sec></sec></body><back><app-group><app id=\"App1\"><sec id=\"Sec26\"><title>Source data</title><p id=\"Par50\"><media position=\"anchor\" xlink:href=\"41467_2020_17994_MOESM4_ESM.xlsx\" id=\"MOESM4\"><caption><p>Source Data</p></caption></media></p></sec></app></app-group><fn-group><fn><p><bold>Peer review information</bold>\n<italic>Nature Communications</italic> thanks Jin Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Kayla G. Barnes, Anna E. Lachenauer.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary information</bold> is available for this paper at 10.1038/s41467-020-17994-9.</p></sec><ack><title>Acknowledgements</title><p>Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the United States Army or the National Institute of Health. We thank the staff of the Joint West Africa Research Group, the Walter Reed Army Institute of Research, the Henry M. Jackson Foundation for the Advancement of Military Medicine, and the U.S. Embassy in Abuja, Nigeria, for their collaborative support and our colleagues at the Irrua Specialist Teaching Hospital, Nigeria, and Kenema Government Hospital, Sierra Leone. We thank Dr. Christopher Moxon for his critical edits of the manuscript. This work is supported by grants from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) (U19AI110818 and R01AI114855 to the Broad Institute, P.C.S.), Henry M Jackson Foundation (W81XWH-18-2-0040 to P.C.S.), DARPA (D18AC00006 to the Broad Institute, P.C.S.), the Bill and Melinda Gates Foundation (OPP1192035 to Harvard University, P.C.S.), NIH-Fogarty (K01TW010853 to HTHCSPH, K.G.B.), and P.C.S. is supported by the Howard Hughes Medical Institute.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>K.G.B., A.E.L., and A.N. conceived of the study. P.C.S., C.T.H., C.M., and C.A.F. oversaw the development of the SHERLOCK assays. R.F.G., D.J.P., and S.F.S. provided input on study design and analysis. H.C.M., L.B., M.B., and B.S. provided validation data for the SHERLOCK assay. A.C. and S.S. developed the mobile application with help from A.E.L. R.G., B.D.K., and L.E.H. performed and oversaw the BSL4- safety validation. B.B. and G.P. generated and performed the diagnostics on the DRC samples. SHERLOCK laboratory experiments were performed by K.G.B., A.E.L., A.N., A.E.L., A.C., C.K.B., J.U., F.A., T.O., and M.K. Patient recruitment and care and data analysis of patient samples was carried out by K.G.B., A.E.L., K.J.S., S.B.M., J.U., F.A., T.O., I.O., J.D.S., M.M., M.F.A., A.B.T., Z.F.P., M.I., D.S.G., and K.M. All authors contributed to the drafting and editing of this manuscript.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>All data presented in this manuscript and generated for this work are included in the written text and figures including a list of all RPA primer sequences (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>), crRNA spacer sequences (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>), RT-qPCR primer sequences (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>), and RT-qPCR probe sequences (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>). All results generated using a fluorescence readout are the average of three replicates. Any other relevant data are available from the authors upon reasonable request. Source data are provided with this paper.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par48\">K.G.B., A.E.L., C.A.F., P.C.S., and C.M. are inventors on patent PCT/US2019/054561 held by the Broad Institute and related to this work. The patent covers all primers, crRNAs, and SHERLOCK technology. P.C.S. is a co-founder of, shareholder in, and advisor to Sherlock Biosciences, Inc., as well as a Board member of and shareholder in Danaher Corporation.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Goba</surname><given-names>A</given-names></name><etal/></person-group><article-title>An outbreak of Ebola virus disease in the Lassa fever zone</article-title><source>J. Infect. Dis.</source><year>2016</year><volume>214</volume><fpage>S110</fpage><lpage>S121</lpage><pub-id pub-id-type=\"doi\">10.1093/infdis/jiw239</pub-id><pub-id pub-id-type=\"pmid\">27402779</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Racsa</surname><given-names>LD</given-names></name><name><surname>Kraft</surname><given-names>CS</given-names></name><name><surname>Olinger</surname><given-names>GG</given-names></name><name><surname>Hensley</surname><given-names>LE</given-names></name></person-group><article-title>Viral hemorrhagic fever diagnostics</article-title><source>Clin. Infect. Dis.</source><year>2016</year><volume>62</volume><fpage>214</fpage><lpage>219</lpage><pub-id pub-id-type=\"doi\">10.1093/cid/civ792</pub-id><pub-id pub-id-type=\"pmid\">26354968</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shaffer</surname><given-names>JG</given-names></name><etal/></person-group><article-title>Lassa fever in post-conflict sierra leone</article-title><source>PLoS Negl. Trop. Dis.</source><year>2014</year><volume>8</volume><fpage>e2748</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pntd.0002748</pub-id><pub-id pub-id-type=\"pmid\">24651047</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Changula</surname><given-names>K</given-names></name><name><surname>Kajihara</surname><given-names>M</given-names></name><name><surname>Mweene</surname><given-names>AS</given-names></name><name><surname>Takada</surname><given-names>A</given-names></name></person-group><article-title>Ebola and Marburg virus diseases in Africa: increased risk of outbreaks in previously unaffected areas?</article-title><source>Microbiol Immunol.</source><year>2014</year><volume>58</volume><fpage>483</fpage><lpage>491</lpage><pub-id pub-id-type=\"doi\">10.1111/1348-0421.12181</pub-id><pub-id pub-id-type=\"pmid\">25040642</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bausch</surname><given-names>DG</given-names></name><etal/></person-group><article-title>Diagnosis and clinical virology of Lassa fever as evaluated by enzyme-linked immunosorbent assay, indirect fluorescent-antibody test, and virus isolation</article-title><source>J. Clin. Microbiol</source><year>2000</year><volume>38</volume><fpage>2670</fpage><lpage>2677</lpage><pub-id pub-id-type=\"doi\">10.1128/JCM.38.7.2670-2677.2000</pub-id><pub-id pub-id-type=\"pmid\">10878062</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bwaka</surname><given-names>MA</given-names></name><etal/></person-group><article-title>Ebola hemorrhagic fever in Kikwit, Democratic Republic of the Congo: clinical observations in 103 patients</article-title><source>J. Infect. Dis.</source><year>1999</year><volume>179</volume><fpage>S1</fpage><lpage>S7</lpage><pub-id pub-id-type=\"doi\">10.1086/514308</pub-id><pub-id pub-id-type=\"pmid\">9988155</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ksiazek</surname><given-names>TG</given-names></name><name><surname>West</surname><given-names>CP</given-names></name><name><surname>Rollin</surname><given-names>PE</given-names></name><name><surname>Jahrling</surname><given-names>PB</given-names></name><name><surname>Peters</surname><given-names>CJ</given-names></name></person-group><article-title>ELISA for the detection of antibodies to Ebola viruses</article-title><source>J. Infect. Dis.</source><year>1999</year><volume>179</volume><fpage>S192</fpage><lpage>S198</lpage><pub-id pub-id-type=\"doi\">10.1086/514313</pub-id><pub-id pub-id-type=\"pmid\">9988184</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cnops</surname><given-names>L</given-names></name><etal/></person-group><article-title>Risk of misinterpretation of Ebola virus PCR results after rVSV ZEBOV-GP vaccination</article-title><source>Clin. Infect. Dis.</source><year>2015</year><volume>60</volume><fpage>1725</fpage><lpage>1726</lpage><pub-id pub-id-type=\"doi\">10.1093/cid/civ131</pub-id><pub-id pub-id-type=\"pmid\">25694650</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jansen van Vuren</surname><given-names>P</given-names></name><etal/></person-group><article-title>Comparative evaluation of the diagnostic performance of the prototype Cepheid GeneXpert Ebola assay</article-title><source>J. Clin. Microbiol</source><year>2016</year><volume>54</volume><fpage>359</fpage><lpage>367</lpage><pub-id pub-id-type=\"doi\">10.1128/JCM.02724-15</pub-id><pub-id pub-id-type=\"pmid\">26637383</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pinsky</surname><given-names>BA</given-names></name><etal/></person-group><article-title>Analytical performance characteristics of the Cepheid GeneXpert Ebola assay for the detection of Ebola virus</article-title><source>PLoS ONE</source><year>2015</year><volume>10</volume><fpage>e0142216</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0142216</pub-id><pub-id pub-id-type=\"pmid\">26562786</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Asogun</surname><given-names>DA</given-names></name><etal/></person-group><article-title>Molecular diagnostics for lassa fever at Irrua specialist teaching hospital, Nigeria: lessons learnt from two years of laboratory operation</article-title><source>PLoS Negl. Trop. Dis.</source><year>2012</year><volume>6</volume><fpage>e1839</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pntd.0001839</pub-id><pub-id pub-id-type=\"pmid\">23029594</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Drosten</surname><given-names>C</given-names></name><etal/></person-group><article-title>Rapid detection and quantification of RNA of Ebola and Marburg viruses, Lassa virus, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, dengue virus, and yellow fever virus by real-time reverse transcription-PCR</article-title><source>J. Clin. Microbiol.</source><year>2002</year><volume>40</volume><fpage>2323</fpage><lpage>2330</lpage><pub-id pub-id-type=\"doi\">10.1128/JCM.40.7.2323-2330.2002</pub-id><pub-id pub-id-type=\"pmid\">12089242</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gootenberg</surname><given-names>JS</given-names></name><etal/></person-group><article-title>Nucleic acid detection with CRISPR-Cas13a/C2c2</article-title><source>Science</source><year>2017</year><volume>356</volume><fpage>438</fpage><lpage>442</lpage><pub-id pub-id-type=\"doi\">10.1126/science.aam9321</pub-id><pub-id pub-id-type=\"pmid\">28408723</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Myhrvold</surname><given-names>C</given-names></name><etal/></person-group><article-title>Field-deployable viral diagnostics using CRISPR-Cas13</article-title><source>Science</source><year>2018</year><volume>360</volume><fpage>444</fpage><lpage>448</lpage><pub-id pub-id-type=\"doi\">10.1126/science.aas8836</pub-id><pub-id pub-id-type=\"pmid\">29700266</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abudayyeh</surname><given-names>OO</given-names></name><etal/></person-group><article-title>C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector</article-title><source>Science</source><year>2016</year><volume>353</volume><fpage>aaf5573</fpage><pub-id pub-id-type=\"doi\">10.1126/science.aaf5573</pub-id><pub-id pub-id-type=\"pmid\">27256883</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>East-Seletsky</surname><given-names>A</given-names></name><etal/></person-group><article-title>Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection</article-title><source>Nature</source><year>2016</year><volume>538</volume><fpage>270</fpage><lpage>273</lpage><pub-id pub-id-type=\"doi\">10.1038/nature19802</pub-id><pub-id pub-id-type=\"pmid\">27669025</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gire</surname><given-names>SK</given-names></name><etal/></person-group><article-title>Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak</article-title><source>Science</source><year>2014</year><volume>345</volume><fpage>1369</fpage><lpage>1372</lpage><pub-id pub-id-type=\"doi\">10.1126/science.1259657</pub-id><pub-id pub-id-type=\"pmid\">25214632</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mbala-Kingebeni</surname><given-names>P</given-names></name><etal/></person-group><article-title>2018 Ebola virus disease outbreak in Equateur Province, Democratic Republic of the Congo: a retrospective genomic characterisation</article-title><source>Lancet Infect. Dis.</source><year>2019</year><volume>19</volume><fpage>641</fpage><lpage>647</lpage><pub-id pub-id-type=\"doi\">10.1016/S1473-3099(19)30124-0</pub-id><pub-id pub-id-type=\"pmid\">31000465</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Metsky</surname><given-names>HC</given-names></name><etal/></person-group><article-title>Capturing sequence diversity in metagenomes with comprehensive and scalable probe design</article-title><source>Nat. Biotechnol.</source><year>2019</year><volume>37</volume><fpage>160</fpage><lpage>168</lpage><pub-id pub-id-type=\"doi\">10.1038/s41587-018-0006-x</pub-id><pub-id pub-id-type=\"pmid\">30718881</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Park</surname><given-names>DJ</given-names></name><etal/></person-group><article-title>Ebola virus epidemiology, transmission, and evolution during seven months in Sierra Leone</article-title><source>Cell</source><year>2015</year><volume>161</volume><fpage>1516</fpage><lpage>1526</lpage><pub-id pub-id-type=\"doi\">10.1016/j.cell.2015.06.007</pub-id><pub-id pub-id-type=\"pmid\">26091036</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Andersen</surname><given-names>KG</given-names></name><etal/></person-group><article-title>Clinical sequencing uncovers origins and evolution of Lassa virus</article-title><source>Cell</source><year>2015</year><volume>162</volume><fpage>738</fpage><lpage>750</lpage><pub-id pub-id-type=\"doi\">10.1016/j.cell.2015.07.020</pub-id><pub-id pub-id-type=\"pmid\">26276630</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Siddle</surname><given-names>KJ</given-names></name><etal/></person-group><article-title>Genomic analysis of Lassa virus during an increase in cases in Nigeria in 2018</article-title><source>N. Engl. J. Med</source><year>2018</year><volume>379</volume><fpage>1745</fpage><lpage>1753</lpage><pub-id pub-id-type=\"doi\">10.1056/NEJMoa1804498</pub-id><pub-id pub-id-type=\"pmid\">30332564</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kugelman</surname><given-names>JR</given-names></name><etal/></person-group><article-title>Monitoring of Ebola virus Makona evolution through establishment of advanced genomic capability in Liberia</article-title><source>Emerg. Infect. Dis.</source><year>2015</year><volume>21</volume><fpage>1135</fpage><lpage>1143</lpage><pub-id pub-id-type=\"doi\">10.3201/eid2107.150522</pub-id><pub-id pub-id-type=\"pmid\">26079255</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mbala-Kingebeni</surname><given-names>P</given-names></name><etal/></person-group><article-title>Medical countermeasures during the 2018 Ebola virus disease outbreak in the North Kivu and Ituri Provinces of the Democratic Republic of the Congo: a rapid genomic assessment</article-title><source>Lancet Infect. Dis.</source><year>2019</year><volume>19</volume><fpage>648</fpage><lpage>657</lpage><pub-id pub-id-type=\"doi\">10.1016/S1473-3099(19)30118-5</pub-id><pub-id pub-id-type=\"pmid\">31000464</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nikisins</surname><given-names>S</given-names></name><etal/></person-group><article-title>International external quality assessment study for molecular detection of Lassa virus</article-title><source>PLoS Negl. Trop. Dis.</source><year>2015</year><volume>9</volume><fpage>e0003793</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pntd.0003793</pub-id><pub-id pub-id-type=\"pmid\">25996783</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bausch</surname><given-names>DG</given-names></name><etal/></person-group><article-title>Assessment of the risk of Ebola virus transmission from bodily fluids and fomites</article-title><source>J. Infect. Dis.</source><year>2007</year><volume>196</volume><fpage>S142</fpage><lpage>S147</lpage><pub-id pub-id-type=\"doi\">10.1086/520545</pub-id><pub-id pub-id-type=\"pmid\">17940942</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Emperador</surname><given-names>DM</given-names></name><name><surname>Yimer</surname><given-names>SA</given-names></name><name><surname>Mazzola</surname><given-names>LT</given-names></name><name><surname>Norheim</surname><given-names>G</given-names></name><name><surname>Kelly-Cirino</surname><given-names>C</given-names></name></person-group><article-title>Diagnostic applications for Lassa fever in limited-resource settings</article-title><source>BMJ Glob. Health</source><year>2019</year><volume>4</volume><fpage>e001119</fpage><pub-id pub-id-type=\"doi\">10.1136/bmjgh-2018-001119</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>M</given-names></name><etal/></person-group><article-title>Development and evaluation of a rapid and sensitive EBOV-RPA test for rapid diagnosis of Ebola virus disease</article-title><source>Sci. Rep.</source><year>2016</year><volume>6</volume><fpage>26943</fpage><pub-id pub-id-type=\"doi\">10.1038/srep26943</pub-id><pub-id pub-id-type=\"pmid\">27246147</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gootenberg</surname><given-names>JS</given-names></name><etal/></person-group><article-title>Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6</article-title><source>Science</source><year>2018</year><volume>360</volume><fpage>439</fpage><lpage>444</lpage><pub-id pub-id-type=\"doi\">10.1126/science.aaq0179</pub-id><pub-id pub-id-type=\"pmid\">29449508</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Matranga</surname><given-names>CB</given-names></name><etal/></person-group><article-title>Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples</article-title><source>Genome Biol.</source><year>2014</year><volume>15</volume><fpage>519</fpage><pub-id pub-id-type=\"doi\">10.1186/s13059-014-0519-7</pub-id><pub-id pub-id-type=\"pmid\">25403361</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boisen</surname><given-names>ML</given-names></name><etal/></person-group><article-title>Field validation of recombinant antigen immunoassays for diagnosis of Lassa fever</article-title><source>Sci. Rep.</source><year>2018</year><volume>8</volume><fpage>5939</fpage><pub-id pub-id-type=\"doi\">10.1038/s41598-018-24246-w</pub-id><pub-id pub-id-type=\"pmid\">29651117</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><mixed-citation publication-type=\"other\">Bradski, G. <italic>The OpenCV Library</italic> (Dr. Dobb’s Journal of Software Tools, 2000).</mixed-citation></ref><ref id=\"CR33\"><label>33.</label><mixed-citation publication-type=\"other\">Pizer, S. M., Pizer, A. E. & Austin, J.D. Adaptive histogram equalization and its variations. <italic>Comput. Vis. Graphics Image Process</italic>. <bold>39</bold>, 355–368 (1987).</mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"correction\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807847</article-id><article-id pub-id-type=\"pmc\">PMC7431546</article-id><article-id pub-id-type=\"publisher-id\">70340</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70340-3</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Author Correction</subject></subj-group></article-categories><title-group><article-title>Author Correction: Simultaneous Inhibition of Glycolysis and Oxidative Phosphorylation Triggers a Multi-Fold Increase in Secretion of Exosomes: Possible Role of 2′,3′-cAMP</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Ludwig</surname><given-names>Nils</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yerneni</surname><given-names>Saigopalakrishna S.</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Menshikova</surname><given-names>Elizabeth V.</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Gillespie</surname><given-names>Delbert G.</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Jackson</surname><given-names>Edwin K.</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Whiteside</surname><given-names>Theresa L.</given-names></name><address><email>whitesidetl@upmc.edu</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.21925.3d</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9000</institution-id><institution>Department of Pathology, </institution><institution>University of Pittsburgh School of Medicine, </institution></institution-wrap>Pittsburgh, PA 15213 USA </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.478063.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0456 9819</institution-id><institution>UPMC Hillman Cancer Center, </institution></institution-wrap>Pittsburgh, PA 15213 USA </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.147455.6</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2097 0344</institution-id><institution>Department of Biomedical Engineering, </institution><institution>Carnegie Mellon University, </institution></institution-wrap>Pittsburgh, PA USA </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.21925.3d</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9000</institution-id><institution>Department of Pharmacology and Chemical Biology, </institution><institution>University of Pittsburgh School of Medicine, </institution></institution-wrap>Pittsburgh, PA USA </aff><aff id=\"Aff5\"><label>5</label>Departments of Immunology and Otolaryngology, Pittsburgh, PA 15213 USA </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>14027</elocation-id><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><related-article related-article-type=\"corrected-article\" ext-link-type=\"doi\" xlink:href=\"10.1038/s41598-020-63658-5\" id=\"d30e71\"/><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><p id=\"Par1\">Correction to: <italic>Scientific Reports</italic>\n<ext-link ext-link-type=\"uri\" xlink:href=\"https://doi.org/10.1038/s41598-020-63658-5\">https://doi.org/10.1038/s41598-020-63658-5</ext-link>, published online 24 April 2020\n</p><p id=\"Par2\">The original version of this Article contained an error in the title of the paper, where the expression “2′,3′-cAMP” was incorrectly given as “2′3′-cAMP”.</p><p id=\"Par3\">The original version of this Article also contained errors in the Abstract.</p><p id=\"Par4\">“In cells lacking 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase; an enzyme that metabolizes 2′,3′-cAMP into 2′- and 3′-AMP), effects of IAA/DNP on exosome secretion were enhanced.”</p><p id=\"Par5\">now reads:</p><p id=\"Par6\">“In cells lacking 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase; an enzyme that metabolizes 2′,3′-cAMP into 2′-AMP), effects of IAA/DNP on exosome secretion were enhanced.”</p><p id=\"Par7\">These errors have now been corrected in the PDF and HTML versions of the Article and in the accompanying Supplementary Information file.</p></body></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807837</article-id><article-id pub-id-type=\"pmc\">PMC7431547</article-id><article-id pub-id-type=\"publisher-id\">70752</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70752-1</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Organically modified layered magnesium silicates to improve rheology of reservoir drilling fluids</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Patel</surname><given-names>Hasmukh A.</given-names></name><address><email>hasmukh.patel@aramcoamericas.com</email></address><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><contrib contrib-type=\"author\"><name><surname>Santra</surname><given-names>Ashok</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><aff id=\"Aff1\">Drilling Technology Team, Aramco Americas: Aramco Research Center – Houston, 16300 Park Row Dr, Houston, TX 77084 USA </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13851</elocation-id><history><date date-type=\"received\"><day>19</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Petroleum well drilling fluids are one of the most significant constituents in the subterranean drilling processes to meet an increasing global demand for oil and gas. Drilling fluids experience exceptional wellbore conditions, e.g. high temperature and high pressure that adversely affect the rheology of these fluids. Gas and oil well drilling operations have to adjourn due to changes in fluid rheology, since the drilling fluids may lose their effectiveness to suspend heavy particles and to carry drilled cuttings to the surface. The rheological properties of drilling fluids can be controlled by employing viscosifiers that should have exceptional stability in downhole environments. Here, we have developed next-generation viscosifiers—organically modified magnesium silicates (MSils)—for reservoir drilling fluids where organic functionalities are directly linked through the Si–C bond, unlike the industry’s traditional viscosifier, organoclay, that has electrostatic linkages. The successful formation of covalently-linked hexadecyl and phenyl functionalized magnesium silicates (MSil-C16 and MSil-Ph) were confirmed by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and thermogravimetric analysis (TGA). Identical drilling fluid formulations were designed for comparison using MSils and a commercial viscosifier. The rheological properties of fluids were measured at ambient conditions as well as at high temperatures (up to 150 °C) and high pressure (70 MPa). Owing to strong covalent linkages, drilling fluids that were formulated with MSils showed a 19.3% increase in yield point (YP) and a 31% decrease in apparent viscosity (AV) at 150 °C under 70 MPa pressure, as compared to drilling fluids that were formulated with traditional organoclay. The higher yield point and lower apparent viscosity are known to facilitate and increased drilling rate of penetration of the fluids and an enhanced equivalent circulation density (ECD), the dynamic density condition, for efficient oil and gas wells drilling procedures.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Inorganic chemistry</kwd><kwd>Materials chemistry</kwd><kwd>Physical chemistry</kwd><kwd>Surface chemistry</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">The properties of drilling fluids govern the successful completion of oil and gas well drilling operations. It has been well established that the non-productive time (NPT), owing to the deteriorating performance of drilling fluids, has largely amplified the cost of drilling operations and delayed the production of oil and gas from the reservoirs<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. The principal functions of drilling fluids are<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup> (i) suspending and transporting formation cuttings from the bottom of the wellbore to the surface, (ii) suspending formation cuttings during the shutdown of drilling operations, (iii) counter balance the formation pressures to prevent in-flow of gas, oil or water from rocks, (iv) forming a filter cake on the formation surface to improve wellbore stability, and (v) lubricating the drilling tools and drill pipes. There are mainly two types of drilling fluids employed in field operations, oil-based drilling fluids and water-based drilling fluids<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>–<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Water-based drilling fluids (WBMs) are often known for their environmentally benign characteristics, albeit unfavorably high viscosity and lack of stability under high temperature conditions have restricted their applications to certain hydrocarbon reservoirs<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. Oil-based drilling fluids, also known as oil-based muds (OBMs) or as invert emulsion fluids (IEF), have demonstrated wide acceptance in oil and gas drilling operations on account of their stability under extreme rock and reservoir conditions, e.g. high temperature and high pressure<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. The water-in-oil invert emulsions in OBMs have shown low to moderate viscosity that reduce the energy requirement to pump the fluids and significantly improve the rate of penetration. The key merits of OBMs over water-based drilling fluids are their abilities to perform in soluble salt, water sensitive formations, and offers low frictions<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>.</p><p id=\"Par3\">Drilling fluid formulations are composed of several additives, e.g. oil as a base fluid, an aqueous phase as an internally emulsified phase, viscosifiers, fluid loss additives, rheological modifiers, primary and secondary emulsifiers, wetting agents, pH controller, and weighting agents<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. These complex mixtures of additives in fluids address the stability of OBMs under the desired wellbore conditions and provide efficient drilling operations of oil and gas wells. One of the most vital among these additives is the viscosifier, because it preserves the viscosity of the fluids over wide range of temperatures. Numerous viscosifiers have been developed in last five decades and the majority of these viscosifiers are based on organically modified natural layered materials, also known as organoclays<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>.</p><p id=\"Par4\">The historical developments in the area of various viscosifiers that have been employed as additives in drilling fluids are summarized in Scheme <xref rid=\"Sch1\" ref-type=\"fig\">1</xref>a. Organoclays have been employed as a viscosifying additive in drilling fluid formulations since the 1970s. Organoclays are produced through an ion-exchange reaction between cationic clays and quaternary ammonium salts<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. The resulting organophilic clays can easily be dispersed in an oil- or diesel-based medium that imparts viscosity to the drilling fluids. Since organoclays have been synthesized from naturally abundant clay minerals, they are relatively low cost viscosifiers to manufacture. The refining of crude oil into value-added chemicals and polymers in the early 1980s has allowed for additional development in the area of modified polymers, which can also generate viscosity in the base fluid medium. However, the high cost and thermal degradation of polymers have restricted their wide scale deployment as a fiscally favorable additive in drilling fluid formulations. Researchers in the upstream petroleum sectors established techniques to control the particle size of organoclays to obtain nanoclays in the beginning of twenty-first century. The size reduction of organoclays allow for better dispersion of their nanometer-thick alumino-silicate platelets in the organic phase, however, the electrostatic interaction of organic moieties with layered materials remains as one of the unresolved characteristics. We have developed the next generation of viscosifiers to overcome the disadvantages associated with the current clay-based and polymeric viscosifying additives.<fig id=\"Sch1\"><label>Scheme 1</label><caption><p>Development of viscosifiers and their shortcomings. (<bold>a</bold>) Chronological development of viscosifiers for oil-based drilling fluids over the last 50 years. (<bold>b</bold>) Effect of the extreme downhole conditions, high temperature, on the traditional organoclays.</p></caption><graphic xlink:href=\"41598_2020_70752_Sch1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par5\">The organic functionalities in organoclays and nanoclays are attached through electrostatic bonding on the surface of layered materials (Scheme <xref rid=\"Sch1\" ref-type=\"fig\">1</xref>b). Drilling fluids often experience very high temperatures under downhole conditions, in addition to an alkaline/acidic environment. The organic functionalities are isolated from the layered materials, thereby losing their ability to contribute to the viscosifying properties in drilling fluids. Therefore, it is very important to have strong linkages between the layered materials and organic functionalities to preserve the rheological properties of the drilling fluids.</p><p id=\"Par6\">We have designed and synthesized layered materials that have covalently-linked organic functionalities. Two types of synthetic magnesium silicates (MSils) were prepared, bearing hexadecyl (MSil-C16) or phenyl groups (MSil-Ph) through a facile synthetic route. The synthesis of MSil without organic functionality (MSil-OH) was also demonstrated in order to compare the structural changes upon organic functionalization. The formation of layered structures was evaluated by X-ray diffraction and covalent bonding of organic moieties with nanometer-thick magnesium silicates was revealed by infrared spectroscopic analyses. The thermal stabilities of MSils were studied by thermogravimetric analysis. MSil-C16 and MSil-Ph were incorporated in the drilling fluids to demonstrate the effect of covalently-linked organic moieties. We have also compared the rheological properties of drilling fluids with commercial organoclay under identical conditions to establish the unique characteristics of MSils. The rheological properties were analyzed at high temperatures (up to 150 °C) and high pressures (70 MPa) to simulate the wellbore conditions.</p></sec><sec id=\"Sec2\"><title>Materials and methods</title><sec id=\"Sec3\"><title>Materials</title><p id=\"Par7\">Magnesium chloride hexahydrate (98%), phenyltrimethoxysilane (97%), hexadecyltrimethoxysilane (95%), tetraethyl orthosilicate (98%), methanol (99.8%), and sodium hydroxide (technical) were received from MilliporeSigma. Commercial organoclay (Claytone HT) was obtained from BYK-CHEMIE GMBH. Commercial drilling fluid additives were obtained from Schlumberger, USA. All chemicals were used as received.</p></sec><sec id=\"Sec4\"><title>Characterizations</title><p id=\"Par8\">Powder X-ray diffraction patterns of MSils were recorded by Rigaku benchtop Miniflex 600, equipped with monochromatic X-ray source (600 W) and a D/teX Ultra 1D silicon strip detector. Thermogravimetric analyses (TGA) of MSils were carried out on the SDT q600 TA instrument. The sample was heated up to 800 °C with a 10 °C/min heating rate under a 20 mL/min N<sub>2</sub> flow rate. Fourier transform infrared (FTIR) sprectra were recorded in attenuated total reflection (ATR) mode within the range 400–4,000 cm<sup>–1</sup> using a Bruker Tensor 37 FTIR (MiD IR/ATR) spectrometer. Viscoelastic properties of the OBMs were measured using a MCR 303 rheometer from Anton Parr. The storage modulus and loss modulus of the fluids were recorded at different temperatures under 3.45 MPa. The angular frequency was varied between 0.03 and 70 rad/s. The couette coaxial cylinder rotational viscometers (Model 35 Rheometer and iX77 Rheometer, Fann Instrument Company) were used to simulate wellbore conditions for studying the rheological properties of the OBMs. Model 35 Rheometer was employed to obtained rheological properties at ambient conditions while iX77 Rheometer was utilized to record the rheological properties at high temperatures (up to 150 °C) and high pressure (70 MPa). These rheometers offer a true simulation of the most significant flow process conditions encountered during drilling operations. Dial deflection torque readings (600, 300, 200, 100, 6, and 3 rpm) from the rheometers were recorded for the OBMs before and after ageing at 150 °C. Aged OBMs were tested at high temperature and high pressure to obtain data for each OBM. Plastic viscosity (PV), apparent viscosity (AV), and yield point (YP) were calculated from the dial reading recorded on the rheometers: PV = dial reading (600 rpm) – dial reading (300 rpm); YP = dial reading (300 rpm) – PV, AV = dial reading (600 rpm) ÷ 2. Gel strength of the OBMs were measured after holding the OBMs at 10 s and 10 min, followed by applying 3 rpm rotation in the rheometer. The dial readings were recorded and represented as gel strength of the drilling fluids.</p></sec><sec id=\"Sec5\"><title>Synthesis of MSil-OH, MSil-C16, and MSil-Ph</title><p id=\"Par9\">Organically modified synthetic magnesium silicates were prepared according to the reported technique with minor modifications<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>–<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. Detailed syntheses of MSils are given in Supplementary Information. Briefly, 0.08 mol of silane compound (tetraethyl orthosilicate, hexadecyltrimethoxysilane or phenyltrimethoxysilane) was added to a solution of magnesium chloride hexahydrate (0.06 mol) in 300 mL methanol with stirring at 25 °C. Subsequently, 0.5 M aqueous sodium hydroxide was metered through a peristaltic pump until the pH reached 11. The resulting precipitates were refluxed with stirring at 80 °C for 48 h. The reaction mixtures were cooled to room temperature, followed by filtrations and washing with de-ionized water. The products were dried under vacuum for 24 h and denoted as MSil-OH, MSil-C-16 and MSil-Ph.</p></sec><sec id=\"Sec6\"><title>Drilling fluid formulations</title><p id=\"Par10\">Drilling fluids (OBM1, OBM2, and OBM3) were prepared through high shear mixing of the additives (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>) in base fluid to form stable oil-in-water emulsions at 11,500 rpm. It is very important to follow the time and order of mixing for each additive to formulate the OBMs. The order of mixing and time of shearing after adding each component is as follows: Step 1 (1–2 min in each step): Diesel (178 g) → organoclay or MSil-C16 or MSil-Ph (2 g) → VersaMul (10 g) → VersaCoat ( 7 g) → Lime (10 g) → Priamine 1074 (3 g) → Shear for 20 min. Step 2 (1–2 min in each step): CaCl<sub>2</sub> brine (85 g) → VersaTrol HT (4 g) → Shear for 20 min. Step 3: Barite (280 g) → Shear for 20 min. Step 4: RevDust (50 g) → Shear for 5 min. Step 5 (ageing) hot rolled the OBMs at 150 °C under 3.45 MPa in a pressure cell for 16 h.<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Drilling fluid formulations—OBM1, OBM2, and OBM3—with various additives.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Fluid additives</th><th align=\"left\">Amount, g</th><th align=\"left\">OBM1</th><th align=\"left\">OBM2</th><th align=\"left\">OBM3</th></tr></thead><tbody><tr><td align=\"left\">Base fluid – oil phase</td><td char=\".\" align=\"char\">178</td><td align=\"left\">Diesel</td><td align=\"left\">Diesel</td><td align=\"left\">Diesel</td></tr><tr><td align=\"left\">Viscosifiers</td><td char=\".\" align=\"char\">2</td><td align=\"left\">Organoclay</td><td align=\"left\">MSil-C16</td><td align=\"left\">MSil-Ph</td></tr><tr><td align=\"left\">Lime</td><td char=\".\" align=\"char\">10</td><td align=\"left\">Ca(OH)<sub>2</sub></td><td align=\"left\">Ca(OH)<sub>2</sub></td><td align=\"left\">Ca(OH)<sub>2</sub></td></tr><tr><td align=\"left\">Primary emulsifier</td><td char=\".\" align=\"char\">10</td><td align=\"left\">VersaMul</td><td align=\"left\">VersaMul</td><td align=\"left\">VersaMul</td></tr><tr><td align=\"left\">Secondary emulsifier</td><td char=\".\" align=\"char\">7</td><td align=\"left\">VersaCoat</td><td align=\"left\">VersaCoat</td><td align=\"left\">VersaCoat</td></tr><tr><td align=\"left\">Rheological modifier</td><td char=\".\" align=\"char\">3</td><td align=\"left\">Priamine 1074</td><td align=\"left\">Priamine 1074</td><td align=\"left\">Priamine 1074</td></tr><tr><td align=\"left\">Internal aqueous phase</td><td char=\".\" align=\"char\">85</td><td align=\"left\">CalCl<sub>2</sub> brine</td><td align=\"left\">CalCl<sub>2</sub> brine</td><td align=\"left\">CalCl<sub>2</sub> brine</td></tr><tr><td align=\"left\">Fluid loss additive</td><td char=\".\" align=\"char\">4</td><td align=\"left\">VersaTrol HT</td><td align=\"left\">VersaTrol HT</td><td align=\"left\">VersaTrol HT</td></tr><tr><td align=\"left\">Weighting material</td><td char=\".\" align=\"char\">280</td><td align=\"left\">Barite</td><td align=\"left\">Barite</td><td align=\"left\">Barite</td></tr></tbody></table><table-wrap-foot><p>Oil to water phase ratio was 70:30 and the drilling fluid density is 1.61 g/cm<sup>3</sup>. Priamine 1074, Lime, VersaMul, VersaCoat, VersaTrol HT, and barite are commercial trade names of the drilling fluid additives. The density of CaCl<sub>2</sub> brine is 1.1 g/cm<sup>3</sup>. 50 g of Ca-montmorillonite (RevDust) was added into each OBM to replicate the drilled rock contamination effect that occurs during drilling operations<italic>.</italic></p></table-wrap-foot></table-wrap></p></sec></sec><sec id=\"Sec7\"><title>Results and discussion</title><p id=\"Par11\">A facile synthetic approach has applied for the preparation of organically modified magnesium silicates (MSils). The architectures of these layered materials were created from the combination of precipitation and sol–gel techniques (Scheme <xref rid=\"Sch2\" ref-type=\"fig\">2</xref>). Under alkaline conditions, magnesium salts are precipitated as brucite sheets (octahedral magnesium hydroxide/oxide) and tetrahedral silicates are attached on the brucite sheets during condensation reaction through the sol–gel process<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. The seed crystals of the brucite layers act as structure directing agents and resulting in nanometer-thick magnesium silicate platelets. Pendant groups of organosilanes also facilitate the formation of lamellar structures, due to the hydrophobic nature of the organic functionalities<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>.<fig id=\"Sch2\"><label>Scheme 2</label><caption><p>Synthesis and structural characteristics of MSils. (<bold>a</bold>) Reaction route to generate MSil-C16 and MSil-Ph under mild reaction conditions. (<bold>b</bold>) Covalently-linked layered silicate composed of ~ 1 nm thick tetrahedral-octahedral-tetrahedral platelets.</p></caption><graphic xlink:href=\"41598_2020_70752_Sch2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par12\">The formation of covalently-linked magnesium silicates—MSil-OH, MSil-C16, and MSil-Ph—was studied by recording and evaluating Fourier Transform Infrared Spectroscopic (FTIR) spectrum and Powder X-ray diffraction (XRD) patterns. Layered magnesium silicates and organic functional groups show characteristic vibration signals that confirmed the generation of the desired materials (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>a). The covalent bonding, Si–C, is clearly visible from the stretching at 1,183 cm<sup>–1</sup> in MSil-C16, and MSil-Ph. Stretching bands at 3,032–3,099 cm<sup>–1</sup>, 1506 cm<sup>–1</sup>, and 1,433 cm<sup>–1</sup> in MSil-Ph are attributed to C–H aromatic stretch, C–C stretch in the aromatic rings, and Si–H<sub>5</sub>C<sub>6</sub>, respectively. The organic moieties attached with layered materials can be identified from the vibrational band of the alkyl or aromatic functional groups. The vibrational bands<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup><sup>‒</sup><sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup> of aliphatic C–H and aliphatic C–C correspond to 2,931–2,859 cm<sup>–1</sup> and 1,470 cm<sup>–1</sup> for MSil-C16. Hydroxyl groups in the magnesium silicates show characteristic broad signals around 3,460–3,400 cm<sup>–1</sup>. Phyllosilicates<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup> (2:1) that are composed of MgO/OH sandwiched between silica tetrahedral provide distinctive stretching bands at 3,696 cm<sup>–1</sup> and 1,005 cm<sup>–1</sup> for MgO–H and Si–O–Si, respectively.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Formation of covalently-linked MSils. (<bold>a</bold>) FT-IR spectrum of MSil-OH, MSil-C16 and MSil-Ph show annotated characteristic stretching vibrations. (<bold>b</bold>) XRD patterns of MSil-OH, MSil-C16 and MSil-Ph (<italic>Inset: ICDD XRD pattern for Si</italic><sub><italic>4</italic></sub><italic>Mg</italic><sub><italic>3</italic></sub><italic>O</italic><sub><italic>12</italic></sub><italic>(OH)</italic><sub><italic>2</italic></sub><italic>, magnesium silicates hydrates, PDF card # 00–019-0,770</italic>) with crystallographic reflections assignment that show the formation of layered structures.</p></caption><graphic xlink:href=\"41598_2020_70752_Fig1_HTML\" id=\"MO3\"/></fig></p><p id=\"Par13\">Magnesium silicates are the most common class of 2:1 phyllosilicate mineral. In these minerals, Mg in octahedral coordination with O–H that are bound to two sheets of Si in tetrahedral coordination with O atoms. Since one of the O atoms is replaced by organic functionalities in organosilanes, it is expected to form Si tetrahedral with Si–C covalent linkages in layered magnesium silicates. Thus, organic functionalities are located in the interlayer space between layered materials. The formation of 2:1 phyllosilicates structure in these synthetic magnesium silicates was proven through crystallographic reflections in XRD patterns (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b). The [001] reflection represents basal spacing or interlayer spacing of magnesium silicates and it proves the information of organic functionalities situated within interlayer space<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. The d<sub>001</sub> for MSil-OH, MSil-C16, and MSil-Ph is 1.1, 1.6, and 1.3 nm, respectively and these various in interlayer spacing suggest that hexadecyl chains and phenyl groups are located between the inorganic platelets. The diffraction patterns of MSils have also compared with standard magnesium silicates—Si<sub>4</sub>Mg<sub>3</sub>O<sub>12</sub>(OH)<sub>2</sub>—as shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b (inset). The XRD patterns of standard magnesium silicates were obtained from ICDD (The international Center for Diffraction Data) data base. The diffraction peaks at [020, 110], [130, 200] and [060, 330] are fingerprint reflections of 2:1 phyllosilicate structure. The position of the [060] reflection demonstrates that Mg octahedral sheets are surrounded by three divalent cations and remained unaltered upon introduction of organic groups. It shows that the layered magnesium silicates can accommodate various functionalities without affecting tetrahedral − octahedral − tetrahedral structure<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>.</p><p id=\"Par14\">Drilling fluids have often encountered to extreme temperature and pressure under downhole conditions during oil and gas well drilling operations. Therefore, the additives that are utilized for drilling fluid formulation should show adequate thermal stability under these conditions. We have studied the thermal stability of MSil-OH, MSil-C16 and MSil-Ph up to 800 °C by thermogravimetric analysis (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). Initial mass loss in the TGA up to 100 °C corresponds to the removal of adsorbed waters from the magnesium silicates. The degradation of organic moieties in MSil-C16 and MSil-Ph started at 250 and 290 °C, respectively and therefore it is expected that these materials can withstand temperatures within these ranges. The temperature of oil and gas wells generally range from 75–260 °C, and gas wells drilling operations<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup> have encountered temperatures in the range of 150–260 °C. Owing to high thermal stability of MSil-Ph, it can be employed as an additive in drilling fluids in all temperature ranges. Moreover, MSil-Ph has covalently-linked phenyl groups, unlike traditional organoclays that have ionically-linked organic functionalities. MSil-OH shows mass loss (14.7%wt.) within 100–400 °C, which may correspond to tightly bound water molecules.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Thermogravimetric analyses of MSil-OH, MSil-C16 and MSil-Ph up to 800 °C.</p></caption><graphic xlink:href=\"41598_2020_70752_Fig2_HTML\" id=\"MO4\"/></fig></p><p id=\"Par15\">MSil-OH was synthesized to understand the structural changes upon incorporation of organic functionalities in MSils. MSil-OH is hydrophilic and therefore it cannot be dispersed in organic media. The drilling fluid formulations and their rheological properties studies only focuson the effect of two organophilic MSils. We have employed MSil-C16 and MSil-Ph as viscosifiers in OBMs and they were compared with a commercial viscosifier, organoclay. The OBMs are pumped through drilling strings (drilling pipes), carry cuttings, and return to the ground surface after passing between drill pipes and rock formation (Supplementary Information, Scheme S1). This type of fluid flow process requires measurement in a rheometer that is equipped with coaxial rotational cylinders (Couette type) and thus can provide true simulations of the flow characteristics of OBMs during drilling operations. Rheology of OBMs—OBM1, OBM2, and OBM3—were obtained at ambient conditions (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>).<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Dial readings, gel strength at 10 s. and 10 min., plastic viscosity, apparent viscosity, and yield point for OBMs at 25 °C and ambient pressure after ageing at 150 °C for 16 h.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Shear rate</th><th align=\"left\" colspan=\"3\">Dial readings</th></tr><tr><th align=\"left\">OBM1</th><th align=\"left\">OBM2</th><th align=\"left\">OBM3</th></tr></thead><tbody><tr><td align=\"left\">600 rpm</td><td align=\"left\">63.8</td><td align=\"left\">69.1</td><td align=\"left\">71.2</td></tr><tr><td align=\"left\">300 rpm</td><td align=\"left\">43.2</td><td align=\"left\">44.5</td><td align=\"left\">45.6</td></tr><tr><td align=\"left\">200 rpm</td><td align=\"left\">33.8</td><td align=\"left\">34.1</td><td align=\"left\">34.8</td></tr><tr><td align=\"left\">100 rpm</td><td align=\"left\">26.3</td><td align=\"left\">27.8</td><td align=\"left\">28.5</td></tr><tr><td align=\"left\">6 rpm</td><td align=\"left\">14</td><td align=\"left\">13.8</td><td align=\"left\">14.2</td></tr><tr><td align=\"left\">3 rpm</td><td align=\"left\">12.3</td><td align=\"left\">11.9</td><td align=\"left\">12.4</td></tr><tr><td align=\"left\" colspan=\"4\">Rheological properties</td></tr><tr><td align=\"left\">Gel strength 10 s., lb/100 ft<sup>2</sup></td><td align=\"left\">13</td><td align=\"left\">12.5</td><td align=\"left\">13.2</td></tr><tr><td align=\"left\">Gel strength 10 min., lb/100 ft<sup>2</sup></td><td align=\"left\">15.2</td><td align=\"left\">14.8</td><td align=\"left\">15.6</td></tr><tr><td align=\"left\">PV, cP</td><td align=\"left\">20.6</td><td align=\"left\">24.6</td><td align=\"left\">25.6</td></tr><tr><td align=\"left\">AV, cP</td><td align=\"left\">31.9</td><td align=\"left\">34.55</td><td align=\"left\">35.6</td></tr><tr><td align=\"left\">YP, lb/100 ft<sup>2</sup></td><td align=\"left\">22.6</td><td align=\"left\">19.9</td><td align=\"left\">20</td></tr></tbody></table></table-wrap></p><p id=\"Par16\">OBMs were aged at 150 °C in a pressure vessel under 3.45 MPa before measuring rheological properties. All the OBMs show shear thinning behavior as a function of shear rates. OBMs should have higher gel strength to suspend formation cuttings. OBM2 and OBM3 show equivalent or better gel strength compared to OBM1 at ambient conditions. This is attributed to excellent dispersion of organophilic layered materials that enhance the viscosity of the organic phase of the drilling fluids. Other rheological properties, such as PV, AV, and YP are also almost similar in all OBMs. This identical rheological property in all OBMs was expected because organoclay has been proven to be stable at low temperature. However, the changes in rheological properties in OBM1 that contains organoclay is clearly visible under higher temperature and high pressure conditions.</p><p id=\"Par17\">The fluid flow properties<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref>–<xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>—PV, AV, and YP—of the OBMs were derived from the slope of shear stress and shear rate based on the Bingham plastic model. The PV and AV of OBMs provide information on resistance of fluid flow. The resistance to initial fluid flow or stress needed to displace the fluid is known as YP. These properties were determined from the OBMs at different temperatures (65, 95, 125, and 150 °C) under 70 MPa pressure in the rheometer. OBM2 and OBM3 have demonstrated lower PV and AV as compared to OBM1 (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a, 3b). Owing to the lower PV and AV in OBM2 and OBM3, they can produce excellent rates of penetration during drilling operations.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Rheological properties of OBMs. (<bold>a</bold>) Plastic viscosity, (<bold>b</bold>) Apparent viscosity, and (<bold>c</bold>) Yield point, up to 150 °C at 70 MPa.</p></caption><graphic xlink:href=\"41598_2020_70752_Fig3_HTML\" id=\"MO5\"/></fig></p><p id=\"Par18\">The PV of OBM1, OBM2, and OBM3 was observed to be 7, 3, and 1.5 cP at 150 °C under 70 MPa. Likewise, the AV of OBM1, OBM2, and OBM3 was observed to be 13, 9.8, and 8.9 cP at 150 °C under 70 MPa. We have noticed about a 78% decrease in PV and a 31% decrease in AV for OBM3 when compared to OBM1. This phenomenon can be attributed to the smaller size of the silicate platelets of MSils with respect to organoclay. Furthermore, the density of organic functionality is higher in MSil since each silicon atom is attached with an organic moiety. There have been several techniques employed to measure the stability of invert emulsion fluids. In this study, electrical stability tests were conducted to check the invert emulsion stabilities in OBMs (Supplementary Information, Figure <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>). The electrical stability of OBM1, OBM2, and OBM3 is 569, 500, and 546 V, respectively. It is well established that electrical stability above 300 V for OBMs is considered stable for invert emulsions.</p><p id=\"Par19\">The interaction between particles and the forces between them are known to contribute largely toward the YP of OBMs. Thus, the thermal stability of YP over wide temperature ranges plays a key role in OBMs. OBMs should have a higher and stable YP under downhole conditions to suspend a large quantity of weighing materials (e.g. barite) and to transport cuttings of drilled rocks during drilling operations. The reduction in YP of OBMs at high temperature causes serious consequences, e.g. stuck pipes that put a halt on drilling operations and increases non-productive time. OBM2 and OBM3 have shown exceptional stability of YP at different temperatures. The YP of OBM1, OBM2, and OBM3 is 12, 13.6, and 14.3 lb/100 ft<sup>2</sup>, respectively at 150 °C (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>c), which is a 19.2% (OBM3) improvement compared to OBM1. It is clear from the YP of OBMs at varied temperatures that OBM3 has outperformed OBM2 and OBM3. Low shear yield point (LSYP) of OBM3 has exceeded OBM2 and OBM3 within the studied temperature ranges (Supplementary Information, Figure S2).</p><p id=\"Par20\">The low thermal stability of viscosifiers affects the viscosity of OBMs upon deviation in wellbore temperatures at different depths of the oil and gas wells. A drastic decrease in viscosity may result in poor hole cleaning, solid particles sagging, and disruption in fluid circulation. These difficulties are avoidable, if the OBMs show minimal changes in viscosity over a wide range of wellbore temperatures. A comparison between traditional rheology and flat rheology<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>–<xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup> has been illustrated in Scheme <xref rid=\"Sch3\" ref-type=\"fig\">3</xref>. In traditional rheology<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, the viscosities measured at different shear rates decreases with an increase in temperature. The viscosity readings at low rpm have also decreased at higher temperatures. There is an almost linear correlation between reductions in viscosity with increases in wellbore temperature. Nonetheless, if the viscosity readings at low rpm remained unchanged with increases in temperature and dial readings at higher rpm decreased at high temperature, the curves start to become flattened. This minimal sensitivity in flow properties over a range of temperatures has been known within the industry as flat rheology. Flat rheology of the OBMs improves the high temperature rate of penetration and provides better equivalent circulation density during oil and gas well drilling processes.<fig id=\"Sch3\"><label>Scheme 3</label><caption><p>Changes in the rheological properties of drilling fluids with respect to temperature – Traditional rheology <italic>vs</italic> Flat rheology.</p></caption><graphic xlink:href=\"41598_2020_70752_Sch3_HTML\" id=\"MO6\"/></fig></p><p id=\"Par21\">Interestingly, OBM3 shows flat rheological performance where it revealed minimal changes in low shear viscosity upon increases in temperature (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>a, Supplementary Information Figure S3). The dial readings at 3 rpm and 600 rpm are observed to be 11.8 cP and 17.3 cP for OBM3, whereas, dial readings of OBM1 at 3 rpm and 600 rpm are 10 cP and 26 cP, at 150 °C and 70 MPa. The order of flat rheology of the OBMs that have been studied in this research is OBM1 < OBM2 < OBM3. This unprecedented property of flat rheology in OBM1 and OBM2 could be ascribed to covalently-linked organic moieties on magnesium silicates, MSil-C16 and MSil-Ph. These two MSils have hexadecyl and phenyl functionalities that are linked through Si–C linkages. We believe that these strong chemical linkages in MSils have not allowed organic functionalities to be detached from the inorganic platelets. Ionic or electrostatic interactions between organic functionalities and the layered material in organoclay have dissociated under extreme conditions, which resulted in the changes in viscosity of drilling fluids at high temperatures.<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Observation of flat rheology and viscoelastic properties. (<bold>a</bold>) Rheological properties of OBM1, OBM2, and OBM3 under 70 MPa reveal flat rheological behavior for OBM3 with increase in temperature. (<bold>b</bold>) Storage modulus and loss modulus of OBM1, OBM2, and OBM3 under 3.45 MPa at 150 °C.</p></caption><graphic xlink:href=\"41598_2020_70752_Fig4_HTML\" id=\"MO7\"/></fig></p><p id=\"Par22\">The measurement of viscoelastic properties (storage modulus and loss modulus) were carried out to understand the thixotropic behaviors of OBMs at 150 °C under a confined pressure of 3.45 MPa (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>b). The strength of the gel formations improves suspension of high density materials and allow excellent suspension of drill cuttings, as we have already discussed in detail in this high temperature high pressure study of OBMs. Storage modulus (G’) and loss modulus (G’’) are represented as typical physical characteristics, e.g. gel-like and fluid-like behaviors. Higher G’ over G’’ at a given frequency reveals that the elastic property or gel strength of OBMs and fluid-like behavior is dominated if G’ is lower than G’’. The G’ has remained higher than G’’ for OBM1, OBM2, and OBM3 at all frequencies, suggesting that all OBMs have a gelation state. OBM1 demonstrated highest G’ when compared to OBM2 and OBM3 as a result of the excellent dispersion of MSil-Ph in invert emulsion fluids. High density phenyl functionalities on magnesium silicates allow formation of a colloidal dispersion in organic media. Traditional organoclay undergoes structural dissociation in OBM1 at high temperatures. The rigidity of phenyl functionality over hexadecyl functionality in MSils is responsible for the greater gel-like behavior in OBM1 compared to OBM2. It is significant to note that the synthetic nature of MSils, with minimum impurities and covalently-linked organic functionalities, contribute to the enriched rheological properties in OBMs compared to organoclay.</p></sec><sec id=\"Sec8\"><title>Conclusions</title><p id=\"Par23\">The application of MSils, comprised of covalently-linked organic functionalities on the nanometer-thick layered material, in reservoir drilling fluids have been successfully proven under extreme wellbore conditions. Organoclays have lost their functions as viscosifying fluids at high temperature because of their weak ionic linkages of organic moieties with alumino-silicates. As a result of facile synthetic routes, MSils have been prepared with minimum impurities and desired organic functionalities that were linked through Si–C bond. XRD patterns of MSil-OH, MSil-C16, and MSil-Ph have shown formation of 2:1 phyllosilicate structure and it has also revealed the site of organic functionalities within the interlayer spaces of layered materials, supported by an increase in the d<sub>001</sub> basal spacing. FT-IR spectrum showed characteristic vibration bands for structural features of MSils proving the formation of organically linked layered magnesium silicates. MSil-C16 and MSil-Ph displayed thermal stability of 250 °C and 290 °C, confirmed by TGA. Drilling fluid formulations—OBM1, OBM2, and OBM3—were prepared using various additives to obtain stable invert emulsion fluids. Rheological measurements of thermally aged OBMs were carried out to determined PV, AV, and YP of OBMs at different temperatures under 70 MPa. OBM3 has the lowest PV (1.5 cP) compared to OBM1 (7 cP) and OBM2 (3 cP) at 150 °C, which is expected to deliver excellent rate of penetration. Simultaneously, the YP of OBM3 was 14.3 lb/100 ft<sup>2</sup>, which is higher than OBM1 and OBM2 at high temperatures, a property that provides rock cutting carrying capacity. In addition to better PV, AV, and YP of OBMs that contained MSils, we have noticed minimal changes in viscosities of the fluids with an increase in temperature. OBM3 has exhibited flat rheological behaviors due to the presence of MSil-Ph in the formulations. The improved rheological properties in OBM2 and OBM3 correspond to the novel materials chemistries of MSils―synthetic nanometer-thick platelets and covalently-linked organic functionalities.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec9\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70752_MOESM1_ESM.pdf\"><caption><p>Supplementary information.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70752-1.</p></sec><ack><title>Acknowledgements</title><p>The authors would like to thank Carl Thaemlitz of Drilling Technology Team for fruitful discussion and suggestion throughout this research study.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>H.A.P. conceived and designed the research, carried out the synthesis of layered materials and performed rheology tests, analyzed the data, and composed the manuscript. A.S. contributed in predicting the high temperature rheology data and structure of the manuscript. Both authors discussed and commented on the manuscript.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par24\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Caenn</surname><given-names>R</given-names></name><name><surname>Darley</surname><given-names>HCH</given-names></name><name><surname>Gray</surname><given-names>RG</given-names></name></person-group><source>Composition and properties of drilling and completion fluids</source><year>2017</year><edition>7</edition><publisher-loc>Amsterdam</publisher-loc><publisher-name>Elsevier</publisher-name></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luo</surname><given-names>Z</given-names></name><name><surname>Pei</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>L</given-names></name><name><surname>Yu</surname><given-names>P</given-names></name><name><surname>Chen</surname><given-names>Z</given-names></name></person-group><article-title>Influence of an ionic liquid on rheological and filtration properties of water-based drilling fluids at high temperatures</article-title><source>Appl. Clay Sci.</source><year>2017</year><volume>136</volume><fpage>96</fpage><lpage>102</lpage><pub-id pub-id-type=\"doi\">10.1016/j.clay.2016.11.015</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chu</surname><given-names>Q</given-names></name><name><surname>Lin</surname><given-names>L</given-names></name></person-group><article-title>Effect of molecular flexibility on the rheological and filtration properties of synthetic polymers used as fluid loss additives in water-based drilling fluid</article-title><source>RSC Adv.</source><year>2019</year><volume>9</volume><fpage>8608</fpage><lpage>8619</lpage><pub-id pub-id-type=\"doi\">10.1039/C9RA00038K</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bayat</surname><given-names>AE</given-names></name><name><surname>Moghanloo</surname><given-names>PJ</given-names></name><name><surname>Piroozian</surname><given-names>A</given-names></name><name><surname>Rafati</surname><given-names>R</given-names></name></person-group><article-title>Experimental investigation of rheological and filtration properties of water based drilling fluids in presence of various nanoparticles</article-title><source>Colloids Surf. A Physicochem. Eng. Asp.</source><year>2018</year><volume>555</volume><fpage>256</fpage><lpage>263</lpage><pub-id pub-id-type=\"doi\">10.1016/j.colsurfa.2018.07.001</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luo</surname><given-names>Z</given-names></name><name><surname>Wang</surname><given-names>L</given-names></name><name><surname>Pei</surname><given-names>J</given-names></name><name><surname>Yu</surname><given-names>P</given-names></name><name><surname>Xi</surname><given-names>B</given-names></name></person-group><article-title>A novel star-shaped copolymer as a rheology modifier in water-based drilling fluids</article-title><source>J. Petrol. Sci. Eng.</source><year>2018</year><volume>168</volume><fpage>98</fpage><lpage>106</lpage><pub-id pub-id-type=\"doi\">10.1016/j.petrol.2018.05.003</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>K</given-names></name><etal/></person-group><article-title>Magnesium aluminum silicate nanoparticles as a high-performance rheological modifier in water-based drilling fluids</article-title><source>Appl. Clay Sci.</source><year>2018</year><volume>161</volume><fpage>427</fpage><lpage>435</lpage><pub-id pub-id-type=\"doi\">10.1016/j.clay.2018.05.012</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>X</given-names></name><etal/></person-group><article-title>An amphoteric polymer as a shale borehole stabilizer in water-based drilling Fluids</article-title><source>J. Petrol. Sci. Eng.</source><year>2018</year><volume>170</volume><fpage>112</fpage><lpage>120</lpage><pub-id pub-id-type=\"doi\">10.1016/j.petrol.2018.06.051</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hamed</surname><given-names>SB</given-names></name><name><surname>Belhadri</surname><given-names>M</given-names></name></person-group><article-title>Rheological properties of biopolymers drilling fluids</article-title><source>J. Pet. Sci. Eng.</source><year>2009</year><volume>67</volume><fpage>84</fpage><lpage>90</lpage><pub-id pub-id-type=\"doi\">10.1016/j.petrol.2009.04.001</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Saleh</surname><given-names>TA</given-names></name><name><surname>Ibrahim</surname><given-names>MA</given-names></name></person-group><article-title>Advances in functionalized nanoparticles based drilling inhibitors for oil production</article-title><source>Energy Rep.</source><year>2019</year><volume>5</volume><fpage>1293</fpage><lpage>1304</lpage><pub-id pub-id-type=\"doi\">10.1016/j.egyr.2019.06.002</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rana</surname><given-names>A</given-names></name><name><surname>Arfaj</surname><given-names>MK</given-names></name><name><surname>Yami</surname><given-names>AS</given-names></name><name><surname>Saleh</surname><given-names>TA</given-names></name></person-group><article-title>Cetyltrimethylammonium modified graphene as a clean swelling inhibitor in water-based oil-well drilling mud</article-title><source>J. Environ. Chem. Eng.</source><year>2020</year><volume>8</volume><fpage>103802</fpage><pub-id pub-id-type=\"doi\">10.1016/j.jece.2020.103802</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rana</surname><given-names>A</given-names></name><name><surname>Arfaj</surname><given-names>MK</given-names></name><name><surname>Saleh</surname><given-names>TA</given-names></name></person-group><article-title>Advanced developments in shale inhibitors for oil production with low environmental footprints – a review</article-title><source>Fuel</source><year>2019</year><volume>247</volume><fpage>237</fpage><lpage>249</lpage><pub-id pub-id-type=\"doi\">10.1016/j.fuel.2019.03.006</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><mixed-citation publication-type=\"other\">Rana, A., Saleh, T. A. & Arfaj, M. K. Improvement in rheological features, fluid loss and swelling inhibition of water-based drilling mud by using surfactant-modified graphene. <italic>Abu Dhabi International Petroleum Exhibition & Conference</italic>, Preprint at 10.2118/197774-MS (2019).</mixed-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ibrahim</surname><given-names>MA</given-names></name><name><surname>Saleh</surname><given-names>TA</given-names></name></person-group><article-title>Partially aminated acrylic acid grafted activated carbon as inexpensive shale hydration inhibitor</article-title><source>Carbohydr. Res.</source><year>2020</year><volume>491</volume><fpage>107960</fpage><pub-id pub-id-type=\"doi\">10.1016/j.carres.2020.107960</pub-id><pub-id pub-id-type=\"pmid\">32200318</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Patel</surname><given-names>HA</given-names></name><name><surname>Somani</surname><given-names>RS</given-names></name><name><surname>Bajaj</surname><given-names>HC</given-names></name><name><surname>Jasra</surname><given-names>RV</given-names></name></person-group><article-title>Preparation and characterization of phosphonium montmorillonite with enhanced thermal stability</article-title><source>Appl. Clay Sci.</source><year>2007</year><volume>35</volume><fpage>194</fpage><lpage>200</lpage><pub-id pub-id-type=\"doi\">10.1016/j.clay.2006.09.012</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Andrade</surname><given-names>MAS</given-names></name><name><surname>Pastore</surname><given-names>HO</given-names></name></person-group><article-title>Toward a delaminated organotalc: The use of polyamidoamine dendrons</article-title><source>ACS Appl. Mater. Interfaces.</source><year>2016</year><volume>8</volume><fpage>1884</fpage><lpage>1892</lpage><pub-id pub-id-type=\"doi\">10.1021/acsami.5b09963</pub-id><pub-id pub-id-type=\"pmid\">26713455</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Burkett</surname><given-names>SL</given-names></name><name><surname>Press</surname><given-names>A</given-names></name><name><surname>Mann</surname><given-names>S</given-names></name></person-group><article-title>Synthesis, characterization, and reactivity of layered inorganic-organic nanocomposites based on 2:1 trioctahedral phyllosilicates</article-title><source>Chem. Mater.</source><year>1997</year><volume>9</volume><fpage>1071</fpage><lpage>1073</lpage><pub-id pub-id-type=\"doi\">10.1021/cm9700615</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bocchini</surname><given-names>S</given-names></name><name><surname>Patel</surname><given-names>HA</given-names></name><name><surname>Frache</surname><given-names>A</given-names></name></person-group><article-title>One-pot synthesis of hexadecyl modified layered magnesium silicate and polyethylene based nanocomposite preparation</article-title><source>Appl. Clay Sci.</source><year>2013</year><volume>80–81</volume><fpage>320</fpage><lpage>325</lpage><pub-id pub-id-type=\"doi\">10.1016/j.clay.2013.05.010</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Claverie</surname><given-names>M</given-names></name><etal/></person-group><article-title>Synthetic talc and talc-like structures: Preparation, features and applications</article-title><source>Chem. Eur. J.</source><year>2018</year><volume>24</volume><fpage>519</fpage><lpage>542</lpage><pub-id pub-id-type=\"doi\">10.1002/chem.201702763</pub-id><pub-id pub-id-type=\"pmid\">28771845</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>da Fonseca</surname><given-names>MG</given-names></name><name><surname>Silva</surname><given-names>CR</given-names></name><name><surname>Airoldi</surname><given-names>C</given-names></name></person-group><article-title>Aminated phyllosilicates synthesized via a sol-gel process</article-title><source>Langmuir</source><year>1999</year><volume>15</volume><fpage>5048</fpage><lpage>5055</lpage><pub-id pub-id-type=\"doi\">10.1021/la9817866</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sales</surname><given-names>JAA</given-names></name><name><surname>Petrucelli</surname><given-names>GC</given-names></name><name><surname>Oliveira</surname><given-names>FJVE</given-names></name><name><surname>Airoldi</surname><given-names>C</given-names></name></person-group><article-title>Some features associated with organosilane groups grafted by the sol–gel process onto synthetic talc-like phyllosilicate</article-title><source>J. Colloid Interf. Sci.</source><year>2006</year><volume>297</volume><fpage>95</fpage><lpage>103</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jcis.2005.10.019</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Saleh</surname><given-names>TA</given-names></name></person-group><article-title>Mercury sorption by silica/carbon nanotubes and silica/activated carbon: a comparison study</article-title><source>J. Water Supply: Res. Technol. Aqua.</source><year>2015</year><volume>64</volume><issue>8</issue><fpage>892</fpage><lpage>903</lpage><pub-id pub-id-type=\"doi\">10.2166/aqua.2015.050</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Saleh</surname><given-names>TA</given-names></name></person-group><article-title>Simultaneous adsorptive desulfurization of diesel fuel over bimetallic nanoparticles loaded on activated carbon</article-title><source>J. Clean. Prod.</source><year>2018</year><volume>172</volume><fpage>2123</fpage><lpage>2132</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jclepro.2017.11.208</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Silva</surname><given-names>CR</given-names></name><name><surname>Fonseca</surname><given-names>MG</given-names></name><name><surname>Barone</surname><given-names>JS</given-names></name><name><surname>Airoldi</surname><given-names>C</given-names></name></person-group><article-title>Layered inorganic-organic talc-like nanocomposites</article-title><source>Chem. Mater.</source><year>2002</year><volume>14</volume><fpage>175</fpage><lpage>179</lpage><pub-id pub-id-type=\"doi\">10.1021/cm010474c</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Patel</surname><given-names>HA</given-names></name><name><surname>Sharma</surname><given-names>SK</given-names></name><name><surname>Jasra</surname><given-names>RV</given-names></name></person-group><article-title>Synthetic talc as a solid base catalyst for condensation of aldehydes and ketones</article-title><source>J. Mol. Catal. A-Chem.</source><year>2008</year><volume>286</volume><fpage>31</fpage><lpage>40</lpage><pub-id pub-id-type=\"doi\">10.1016/j.molcata.2008.01.042</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sharma</surname><given-names>SK</given-names></name><name><surname>Patel</surname><given-names>HA</given-names></name><name><surname>Jasra</surname><given-names>RV</given-names></name></person-group><article-title>Synthesis of jasminaldehyde using magnesium organo silicate as a solid base catalyst</article-title><source>J. Mol. Catal. A-Chem.</source><year>2008</year><volume>280</volume><fpage>61</fpage><lpage>67</lpage><pub-id pub-id-type=\"doi\">10.1016/j.molcata.2007.10.013</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Geng</surname><given-names>T</given-names></name><name><surname>Qiu</surname><given-names>Z</given-names></name><name><surname>Miao</surname><given-names>H</given-names></name><name><surname>Zhang</surname><given-names>W</given-names></name><name><surname>Lei</surname><given-names>K</given-names></name></person-group><article-title>Performance evaluation of synthetic-based drilling fluid with flat rheology</article-title><source>Int. J. Energy Power Eng.</source><year>2018</year><volume>7</volume><issue>5</issue><fpage>54</fpage><lpage>58</lpage><pub-id pub-id-type=\"doi\">10.11648/j.ijepe.20180705.11</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Elkatatny</surname><given-names>S</given-names></name><name><surname>Kamal</surname><given-names>MS</given-names></name><name><surname>Alakbari</surname><given-names>F</given-names></name><name><surname>Mahmoud</surname><given-names>M</given-names></name></person-group><article-title>Optimizing the rheological properties of water-based drilling fluid using clays and nanoparticles for drilling horizontal and multi-lateral wells</article-title><source>Appl. Rheol.</source><year>2018</year><volume>28</volume><fpage>43606</fpage></element-citation></ref><ref id=\"CR28\"><label>28.</label><mixed-citation publication-type=\"other\">Oort, E.V., Lee, J., Friedheim, J. & Toups, B. New flat-rheology synthetic-based mud for improved deepwater drilling. <italic>SPE Annual Technical Conference and Exhibition</italic>, Preprint at 10.2118/90987-MS (2004).</mixed-citation></ref><ref id=\"CR29\"><label>29.</label><mixed-citation publication-type=\"other\">Hilfiger, M., Thaemiltz, C. J. & Moellendick, E. Investigating the chemical nature of flat rheology. <italic>SPE Deepwater Drilling and Completions Conference</italic>, Preprint at 10.2118/180320-MS (2016).</mixed-citation></ref><ref id=\"CR30\"><label>30.</label><mixed-citation publication-type=\"other\">Rife, N., Young, S. & Lee, L. Flat rheology wellbore fluid. <italic>US Patent US2013/0331303 A1.</italic> (2013).</mixed-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xie</surname><given-names>B</given-names></name><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Liu</surname><given-names>W</given-names></name><name><surname>Luo</surname><given-names>M</given-names></name></person-group><article-title>Application a novel thermo-sensitive copolymer as a potential rheological modifier for deepwater water-based drilling fluids</article-title><source>Colloids Surf. A Physicochem. Eng. Asp.</source><year>2019</year><volume>581</volume><fpage>123848</fpage><pub-id pub-id-type=\"doi\">10.1016/j.colsurfa.2019.123848</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhao</surname><given-names>X</given-names></name><name><surname>Qiu</surname><given-names>Z</given-names></name><name><surname>Xu</surname><given-names>J</given-names></name><name><surname>Zhao</surname><given-names>C</given-names></name><name><surname>Gao</surname><given-names>J</given-names></name></person-group><article-title>Flat-rheology oil-based drilling fluid for deepwater drilling</article-title><source>Int. J. Heat Technol.</source><year>2017</year><volume>35</volume><issue>1</issue><fpage>19</fpage><lpage>24</lpage><pub-id pub-id-type=\"doi\">10.18280/ijht.350103</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>King</surname><given-names>HE</given-names></name><name><surname>Milner</surname><given-names>ST</given-names></name><name><surname>Lin</surname><given-names>MY</given-names></name><name><surname>Singh</surname><given-names>JP</given-names></name><name><surname>Mason</surname><given-names>TG</given-names></name></person-group><article-title>Structure and rheology of organoclay suspensions</article-title><source>Phys. Rev. E.</source><year>2007</year><volume>75</volume><fpage>021403</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevE.75.021403</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807827</article-id><article-id pub-id-type=\"pmc\">PMC7431548</article-id><article-id pub-id-type=\"publisher-id\">70880</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70880-8</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Smart textiles for multimodal wearable sensing using highly stretchable multiplexed optical fiber system</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Leal-Junior</surname><given-names>Arnaldo</given-names></name><address><email>leal-junior.arnaldo@ieee.org</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Avellar</surname><given-names>Leticia</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Frizera</surname><given-names>Anselmo</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Marques</surname><given-names>Carlos</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.412371.2</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2167 4168</institution-id><institution>Graduate Program in Electrical Engineering, </institution><institution>Federal University of Espírito Santo (UFES), </institution></institution-wrap>Fernando Ferrari Avenue, Vitória, 29075-910 Brazil </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.7311.4</institution-id><institution-id institution-id-type=\"ISNI\">0000000123236065</institution-id><institution>I3N and Physics Department, </institution><institution>Universidade de Aveiro, </institution></institution-wrap>3810-193 Aveiro, Portugal </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13867</elocation-id><history><date date-type=\"received\"><day>28</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>27</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">This paper presents the development and application of a multiparameter, quasi-distributed smart textile based on embedded highly stretchable polymer optical fiber (POF) sensors. The POF is fabricated using the light polymerization spinning process, resulting a highly stretchable optical fiber, so-called LPS-POF, with Young’s modulus and elastic limits of 15 MPa and 17%, respectively. The differential scanning calorimetry shows a thermal stability of the LPS-POF in temperature range of 13–40 °C. The developed sensors are based on the optical power variation, which results in a fully portable and low-cost technique. In order to obtain a multiplexed sensor system, a technique based on flexible light emitting diodes (LEDs) on–off keying modulation is applied, where each LED represents the response of one sensor. The smart textile comprises of LPS-POF and three flexible LEDs embedded in neoprene textile fabric. The performance of the system is evaluated for temperature, transverse force and angular displacement detection at different planes. The sensors presented high linearity (mean determination coefficient of 0.99) and high repeatability (inter-measurement deviations below 5%). The sensor is also applied in activity detection, where the principal component analysis (PCA) was applied in the sensors responses and, in conjunction with clustering techniques such as k-means, indicate the possibility of detecting basic activities such as walking, sitting on a chair and squatting.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Quality of life</kwd><kwd>Optical sensors</kwd><kwd>Optoelectronic devices and components</kwd><kwd>Polymers</kwd><kwd>Biomedical engineering</kwd></kwd-group><funding-group><award-group><funding-source><institution>Fundação Estadual de Amparo à Pesquisa do Estado do Espírito Santo</institution></funding-source><award-id>85426300</award-id><award-id>85426300</award-id><principal-award-recipient><name><surname>Frizera</surname><given-names>Anselmo</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>Petrobras</institution></funding-source><award-id>2017/00702-6</award-id></award-group></funding-group><funding-group><award-group><funding-source><institution>Conselho Nacional de Desenvolvimento Científico e Tecnológico</institution></funding-source><award-id>304049/2019-0</award-id><principal-award-recipient><name><surname>Frizera</surname><given-names>Anselmo</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>Fundação para a Ciência e a Tecnologia</institution></funding-source><award-id>CEECIND/00034/2018</award-id><principal-award-recipient><name><surname>Marques</surname><given-names>Carlos</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">The internet of things (IoT) concept mainly relies on the wireless connectivity of devices, which place demands towards a constant evolution in wireless systems and their miniaturization. Such evolution resulted in many developments in industry<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>, smart cities<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup> and remote healthcare<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup> applications. The latter plays an important role nowadays due to the demographic growth in conjunction with the population ageing<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>, where there is an increasing demand on smart systems for remote healthcare<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. In this scenario, it is desirable the continuous monitoring of human activities for remote assistance, which include diagnosis, transportation in case of emergencies and monitoring of patient’s health condition<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. The remote health monitoring leads to advantageous features such as reduction in the treatment cost and hospital (and clinical facilities) occupation<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. As another feature of the home monitoring, “staying-at-home” factor brings important emotional and psychological advantages for the patient due to the possibility of performing their daily activities and the sense of independent growth in the community<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>.</p><p id=\"Par3\">As a popular technology for the remote health monitoring, different wearable sensors have been proposed for the assessment of multiple parameters of the user<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. The parameters for wearable sensors in human monitoring include the movement assessment and analysis, body temperature, interaction forces/pressures (between human and objects), humidity and physiological parameters, including heartbeat and breath rates<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. It is also worth to mention the assessment of additional parameters in some cases such as arterial pulse, electromyography signals and pulse oximetry<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>.</p><p id=\"Par4\">As a growing approach for wearable sensors systems, smart textiles offer the advantages of higher transparency between the sensor and the user, i.e., the sensor system is lightweight, compact and does not inhibit the user’s movements<sup>10</sup>. The application of compact and embedded sensors in smart textiles and their advantages of easy installation and removal have a positive effect in the system’s usability<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. In light of these advantages, the developments on the flexible electronics have enable the development of flexible wearable sensors such as the ones summarized in previously reported reviews<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. The smart textile technologies continue to point towards an even higher miniaturization, low energy consumption and wireless connection, which are well-aligned with the requirements of the IoT devices<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. Such advances include resistive sensors embedded in fabrics patches<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup> and dual core microfibers for capacitive measurements<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>, including different embedment methods and circuitry<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>.</p><p id=\"Par5\">Optical fiber sensors have experienced a large growth in many fields of applications, including industrial<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>, structural health monitoring<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup> and healthcare<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. In such applications, the optical fiber sensors offer advantages such as compactness, lightweight, potential for multiplexing capabilities, intrinsically safe operation and electromagnetic interference immunity<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. Such advantages are especially important for wearable applications, where it is possible to obtain compact sensors with safe operation (since there is no electrical currents in the sensor’s head) and immune to electromagnetic interferences. Such interferences occur due to assistive devices (especially for users with health impairments) of the user as well as devices that emits electromagnetic waves, commonly used nowadays with the widespread of portable technologies.</p><p id=\"Par6\">These advantages motivate the development of photonic-integrated textiles, which, in their first reports, begun as clothing accessory or signaling devices<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. However, with the widespread of optical fiber sensors, the so-called photonics textiles are applied on the body temperature sensing<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>, breath and heart rates<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Many of the reported sensors are based on fiber Bragg gratings or other wavelength-encoded sensing approaches, which have a high precision and immunity to light source power deviations, but need an optical spectrum analyzer or an optical interrogator, where such devices are generally bulk and non-portable with high cost (when compared with other sensing techniques)<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. In addition, such sensors employ silica optical fibers, which, despite their lower optical loss, have a brittle nature with low impact resistance and strain limits<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. It is also noteworthy that in case of breakage, the glass fiber may puncture the user<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. In order to overcome these drawbacks, advances in the polymer processing, preparation and fabrication have enable the development of polymer optical fibers (POFs), which present higher strain limits, flexibility and impact toughness. Their rugged surface also make POFs easier to incorporate in textiles, where such features have been demonstrated in many works for wearable sensors for human health assessment<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. Furthermore, the smart textiles with POF sensors are mainly based on the intensity variation sensing principle, in which portable sensors with lower cost (when compared to wavelength-based sensors) are achieved.</p><p id=\"Par7\">As a drawback of the previously proposed systems, the intensity variation principle is sensitive to light source power deviations, leading to the necessity of techniques for compensation of light source power deviations<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Moreover, these sensors are not multiplexed, as they are mainly based on point detection of a single parameter, if more than one point or parameter needs to be simultaneously detected, there is the need of increasing the photodetectors, which leads to proportional reduction of the system’s portability and increase of wearable system cost. Although POFs have Young’s modulus one order of magnitude lower than silica fibers (leading to higher flexibility), the Young’s moduli of commercially available POFs are in the range of 1–4 GPa<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>, which are some order of magnitude higher than the conventionally applied materials in fabrics/textiles (in the range of tens of MPa)<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. Thus, the integration of commercial POFs (with diameters of 0.5–1.0 mm) in a textile may lead to a lower flexibility and freedom of movement of the clothing.</p><p id=\"Par8\">This paper presents a novel POF-integrated optoelectronic smart textile, which can overcome the limitations discussed above. The POF used in this work is fabricated through the light polymerization spinning (LPS) process, in which a mixture of monomers are polymerized with UV light, resulting in a higher degree of customization for the so-called LPS-POF<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. The proposed LPS-POF has Young’s modulus more than 100 times lower than commercial POFs, which is even lower than the elastic modulus of cotton and other textile/fabric materials. In addition, a multiplexing technique for intensity variation sensors based on side-coupling between the light source and the LPS-POF was applied to overcome the other limitation of the current proposed smart textile, i.e., the multipoint and multiparameter sensing<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. The proposed LPS-POF integrated multiparameter smart textile also takes advantage on the developments on flexible electronics, where light emitting diodes (LEDs) in flexible substrates are used as light source in order to further enhance the system’s flexibility, usability and transparence. The multiplexed system enables the measurements on multiple parameters in different parts of the user’s body, whereas the high flexibility of the materials enable not only higher flexibility of the sensors, but also does not inhibit the user’s natural movement. Thus, the proposed system was tested in the assessment of movement at different planes, temperature, interaction force between the user and the environment and activities monitoring.</p></sec><sec id=\"Sec2\"><title>Results</title><sec id=\"Sec3\"><title>Multi parameter LPS-POF smart textile development</title><p id=\"Par9\">The POF is fabricated by the LPS process in which there is a combination of monomers and additives instead of the extrusion of a polymer preform, conventionally used in the optical fiber fabrication. This process has intrinsic advantages when compared with the preform extrusion method such as higher customization and repeatability.</p><p id=\"Par10\">The LPS-POF has a diameter of 580 µm ± 30 µm with a core refractive index of 1.54, whereas the fiber cladding (with 20 µm thickness) has a refractive index of 1.45. It is worth noting that such a high diameter and refractive index difference leads to a large multimode behavior of the fiber. Although the large number of modes harms the application of many spectral-based sensors such as fiber Bragg gratings (FBGs), it does not inhibit the use as intensity variation-based sensors. In addition, as an acrylate-based fiber, the LPS-POF has lower optical losses at the visible wavelength, especially at 650 nm, where the optical loss is 4 dB/m. Thus, flexible LEDs at the visible wavelength range were used.</p><p id=\"Par11\">Regarding thermal and mechanical properties of the proposed fiber, the operation limits and the optical fiber behavior at different conditions were tested through the differential calorimetry scanning (DSC), tensile tests and dynamic mechanical analysis (DMA). The DSC test was performed at a temperature range of 13–200 °C using the DSC Q200 (TA Instruments, USA), where the heat flow variation of the LPS-POF is analyzed on the predefined temperature range as depicted in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>a. For the mechanical characterization, a universal tensile test machine (Biopdi, Brazil) was employed. In the stress–strain curve shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b, material parameters such as Young’s modulus and elongation at break are estimated. However, the polymer is a viscoelastic material, which presents time-dependent properties<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. For this reason, the DMA was employed in the LPS-POF to analyze the dynamic modulus, represented by the storage modulus and loss factor (as discussed in previous works<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>), at oscillatory movements with different frequencies. In DMA tests, an oscillatory load with controlled displacement and frequency is applied on the fiber sample, the variation of the dynamic properties, namely the storage modulus and loss factor, as a function of the frequency in the range of 0.01–100 Hz. This range was chosen based on the proposed application, i.e. human physiological parameters and movement/activities assessment, in which the frequency of movement is below 100 Hz<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. Figure <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>c shows the results obtained in the DMA tests for the frequency analysis. The insets in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> show a schematic representation of the thermal and mechanical loadings that the fiber is subjected in each characterization.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Characterization of the LPS-POF: (<bold>a</bold>) DSC results, (<bold>b</bold>) Stress–strain curves in static tensile tests and (<bold>c</bold>) Storage modulus and loss factor in DMA for different frequencies. Figure insets show a schematic representation of the thermal and mechanical loadings in the fiber.</p></caption><graphic xlink:href=\"41598_2020_70880_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par12\">Regarding the DSC results, there is a large endothermic baseline change, related to differences between the heat capacity of the sample and the reference in the DSC experiments (see Methods section). It is also worth noting a change in the curve slope at about 40 °C, which is related to the material’s glass transition temperature<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. Additionally, the melting point of the fiber is represented by an endothermic peak in the heat flow. However, such peak was not observed in the temperature range employed, which indicates that the LPS-POF melting point is higher than 200 °C. Thus, the thermal degradation, which generally occurs after the polymer melting, also does not appear in this temperature range. Nevertheless, there is a small exothermic peak at about 80 °C that can be related to the LPS-POF crystallization. Therefore, the thermal characterization of the LPS-POF indicate its suitability on small temperatures as the ones for on-body application (circa 36 °C) or room temperature assessment. For the mechanical characterization of the LPS-POF, the stress–strain curve in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b shows a large linear range for the fiber of about 17% with a Young’s modulus of 15.0 MPa. In addition, the dynamic analysis presented in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>c indicates the feasibility of the proposed LPS-POF on dynamic conditions in frequencies up to 21 Hz, such as in human movement. In the range of 0.01–21 Hz there is no significant variation in the material’s storage modulus (elastic component) and loss fact (ratio between the elastic and viscous component). However, in higher frequencies, there is a sharp increase of the LPS-POF storage modulus, reaching its maximum value of 450 MPa at 100 Hz. As the sensitivity of optical fiber sensors for mechanical parameters assessment is proportional to the fiber Young’s modulus, such increase in the fiber Young’s modulus leads to lower sensitivity for stress-related parameters sensing at oscillatory frequencies of 100 Hz. In addition, the variation of the sensitivity also results in a nonlinear behavior of the sensor.</p><p id=\"Par13\">The LPS-POF has promising mechanical features and properties for mechanical sensing with the possibility of embedding on textiles without changing the textile stiffness, which makes it suitable for multiparameter sensing in smart textiles. In order to achieve such multiparameter sensing using low cost intensity variation-based sensors, a multiplexing technique was employed in which there is a side coupling of the light source in the fiber and the end faces of the fiber are connected to photodetectors. In this case, the light source is a LED in flexible substrate to ensure a high flexibility to the system embedded in the textile fabric. Then, an on–off keying (OOK) modulation is applied to each flexible LED in a way that only one LED is activated at time. Thus, there is no simultaneous activation of each LED, where the position of each LED represents a sensor point in the fiber. For this reason, a quasi-distributed sensor system is achieved using only one fiber and photodetector, where the number of sensors in the system is equal to the number of LEDs side-coupled to the fiber. A microcontroller FRDM-KL2Z (NXP, Netherlands) is used for the activation signals to the LED and for the acquisition the data from the photodetector IF-D92 (Industrial Fiber Optics, USA) through its 16-bit analog-to-digital converter. As another key process in the microcontroller, there is a synchronization between the LED activation and the data acquisition from the photodetector, where the data acquired is positioned in a matrix (as shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). Each column represents the active LED and the lines are the data acquired through time. In this proof of concept, three flexible LEDs were used (as also shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>) and the matrix has three columns (for LEDs 1, 2 and 3). The data at each column is the photodetector signal when each LED is active, i.e., the data in column 1 is the one gathered when LED 1 is active, in column 2 when LED 2 is activated and so on. The proposed multiplexing technique not only allows the position assessment of mechanical disturbances in the LPS-POF, but also the multiparameter sensing by characterizing the sensors with respect to each of the desired parameter<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. In addition, the system has scalability and a higher number of sensors can be used with few centimeters distance between them<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>A picture and schematic representation of the proposed multifunctional smart textile. Figure also shows the acquisition matrix with the responses of the sensors.</p></caption><graphic xlink:href=\"41598_2020_70880_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par14\">Thereafter, the sensor system (comprised of the LPS-POF and the flexible LEDs) is sewed between two layers of a neoprene textile fabric. Figure <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> presents a picture of the proposed smart textile, where the optical fiber connector and the cables for the LEDs supply are also shown. Both of these cables connect in the microcontroller and its shield with circuit the board for the photodetectors and LEDs activation. Thus, the proposed system is fully portable solution in which the acquisition module comprised of the two photodetectors and the microcontroller can storage the data in a SD card or send it wirelessly using a Bluetooth connection to a host device (such as computer, tablet or smartphone) with acquisition frequency of 100 Hz. As an important parameter for wearable applications, the system can be considered a lightweight solution due to its total weight of 400 g, including microcontroller, photodetectors, LEDs and batteries. In addition, the proposed smart textile has low power consumption with an average consumption of 150 mA was obtained for the whole system (i.e., photodetectors, microcontroller and LEDs), which enable an autonomy as high as 10 h using commercially available 10,000 mAh power banks.</p><p id=\"Par15\">The proposed system is characterized and validated with respect to different movement and physiological parameters. First, different temperature profiles are applied on the sensors and their temperature responses are evaluated for the possibility of on-body temperature sensing. Then, controlled pressures are applied at each sensor in order to detect the interaction pressure between the user and the environment that can be used for activity monitoring. In addition, different movements of bending and torsion at different planes are applied on the textile in order to verify the system’s capacity for movement analysis applications. Finally, the smart textile is positioned on the lower back of a volunteer and the sensors responses are analyzed for commonly activities on daily routine, including walking, squatting and sitting, where the signals are analyzed using the Principal Component Analysis (PCA) in conjunction with clustering technique (such as k-means), which leads to the activities detection.</p></sec><sec id=\"Sec4\"><title>Smart textile responses to thermal and mechanical parameters</title><p id=\"Par16\">The LPS-POF embedded smart textile was tested in different conditions in order to verify its suitability on measuring multiple parameters, where the multiplexing technique based on the LEDs modulation enables the simultaneous measurement of multiple parameters in multiple points on the LPS-POF. In this case, the tests were performed in the textile with three measurement points. Figure <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> presents the temperature response of each sensor, where the tests were performed in a range of 20–40 °C due to both the intended application (room temperature monitoring and on-body applications) and the thermal restrictions in the fiber presented in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>a. The temperature characterization of each sensor (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a) shows a linear behavior of all sensors in the mean and standard deviations of five tests, where the sensors 1 presented the highest temperature sensitivity. The temperature increase in the LPS-POF leads to refractive index variation due to the thermo-optic effect, which results in variations in the transmitted optical power, the differences in the sensitivities can be related to anisotropy in the fiber material<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>. From the linear regressions obtained in the sensors characterizations in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a, it is possible to estimate a temperature distribution in the textile, as shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>b. In this case, a thermal blower is positioned in different locations along the fiber, leading to both distributed and concentrated temperature increase in different regions. The capability of sensing the temperature distribution in the textile was demonstrated, where the sensors also presented low cross-sensitivity between them, demonstrating the feasibility of the proposed multiplexing technique. Regarding the temperature responses, the solid lines are the responses of the sensors applying the linear regressions, whereas the shaded lines are the temperature uncertainty of the sensors, considering the standard deviations on the characterizations shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a. The positions and temperatures of the heating spots on the optical fiber are also presented in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>b, where it can be seen a temperature increase starting in the Sensor 3 and ending in Sensor 1. Thus, the proposed sensor system can be used for body temperature monitoring and can be applied in the interface between the user and an assistive device for microclimate assessment.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Temperature analysis of the LPS-POF embedded textile. (<bold>a</bold>) Temperature characterizations. (<bold>b</bold>) Temperature responses of each sensor for different heat spots.</p></caption><graphic xlink:href=\"41598_2020_70880_Fig3_HTML\" id=\"MO3\"/></fig></p><p id=\"Par17\">Thereafter, different forces are applied at each sensor to obtain their responses at transverse force conditions, which can be correlated to the applied pressure by considering the area of each sensor. In order to show the low crosstalk between sensors, Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>a shows the sensors responses as a function of time for a force of 100 N applied at one sensor at a time (starting from Sensor 1). It is possible to observe the low cross-sensitivity between sensors, i.e., when the force is applied directly on one sensor, there is no significant signal variation in the others, showing the feasibility of the proposed LED modulation multiplexing technique for intensity-based sensors. In Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>b, the force characterization is presented, where high linearity of all sensors and low standard deviation between tests is shown. By applying the linear regression in the sensors responses, it is possible to obtain a pressure/force map of the interaction between the sensor and the user (or environment), as shown in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>c. In this case, a rectangular object (resembling a chair support) is positioned on the textile and the force map is presented in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>c, which indicates the possibility of using the proposed smart textile with a mesh of sensors to evaluate the interaction pressure between the user and the environment, such as chairs and beds. Such evaluation is important to provide a remote monitoring of the user’s activities and prevent pressure ulcers.<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Force analysis of the LPS-POF embedded textile. (<bold>a</bold>) Transmitted optical power attenuation as function of time for forces applied at different sensors. (<bold>b</bold>) Force characterizations. (<bold>c</bold>) Force map from the sensors responses with a force applied on the textile.</p></caption><graphic xlink:href=\"41598_2020_70880_Fig4_HTML\" id=\"MO4\"/></fig></p><p id=\"Par18\">Comparing the force and temperature responses, the sensors presented higher optical signal variation in the force tests when compared with the temperature tests, see Figs. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a and <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>b. The signal variations are more than 3 times higher in the force tests than in the temperature experiments, which results in a higher signal-to-noise ratio of the force response (compared with the temperature response). Thus, if the sensors responses as function of time are compared, as shown in Figs. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>b and <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>a for temperature and force responses, respectively, there is a higher stability of the optical power variation in the force response.</p><p id=\"Par19\">The smart textile sensors responses under displacements applied at different planes are presented in Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>. Bending at different planes and torsions were applied with controlled angles. For the bending, the angles were 0° to 90° and 0° to − 90°, whereas, in the torsion assessment, angular displacements ranging from 0° to 180° were applied, as depicted in Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref> inset. Comparing the responses of each sensor, which are obtained at the angular displacements in different planes, it is possible to observe differences in the sensors behavior, which can be used for the classification of each movement in multiple planes. It is also noteworthy that Sensors 1 and 2 presented the highest bending sensitivity, related to the region where the bending was applied (see Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref> inset), resulting in a higher stress in Sensors 1 and 2. In the torsion case, the highest optical power variation was also obtained in Sensor 1, whereas, once again, the Sensor 3 presented the lowest signal variation. However, the differences in the sensors sensitivities obtained in each case can be used for the estimation of the multiplane displacements applied on the textile, using techniques such as transfer matrix for 3D plane displacements assessment<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>, where a system of equations obtained in the sensors characterizations are used for the angle assessment of each plane. It enables the remote human movement analysis using wearable sensors that do not inhibit the natural pattern of the user’s movements.<fig id=\"Fig5\"><label>Figure 5</label><caption><p>Sensors responses with angular displacement on different planes.</p></caption><graphic xlink:href=\"41598_2020_70880_Fig5_HTML\" id=\"MO5\"/></fig></p><p id=\"Par20\">As shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>, the sensors are also sensitive to temperature variations. Thus, temperature variations interfere on the bending and force assessment. Although the results presented in Figs. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref> and <xref rid=\"Fig5\" ref-type=\"fig\">5</xref> were obtained in constant temperature conditions, temperature variations can occur in practical applications. In order to mitigate the temperature influence on the sensors’ responses, two approaches are considered. The first approach is based on the difference between sensors responses for temperature and strain-related parameters (as previously validated in temperature-compensated systems<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>), considering their previously characterized sensitivities with respect to temperature, angle and force, in this case. Furthermore, the use of the smart textile in dynamic movement applications leads to an additional possibility of temperature compensation. In practical applications of the proposed textile, the temperature variation rate is lower than the one of the strain-related parameters (such as force and angle). This behavior leads to differences in the frequency components of the temperature and strain, where the lower frequencies are related to the temperature. Therefore, the temperature influence on the strain response is mitigated by filtering the low frequencies components on the sensor responses, as demonstrated in previous works<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>.</p><p id=\"Par21\">It is also worth noting that the smart textile characterization with respect to temperature and force show a high repeatability of the sensors with low standard deviation (0.004 a.u.) in the temperature assessment after 3 sequential tests (see the error bars in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a). Moreover, the error bars are not visible in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>b due to its low standard deviation (0.005 a.u.). As another indicator of the sensors’ consistency, the bending tests, whose results are shown in Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>, show the sensor reversibility, since the sensors responses returned to their initial condition without significant residual strains after the bending is performed. The maximum reversibility error (obtained by the comparison between the sensors responses before and after the bending) is 0.03 a.u., considering all three sensors.</p><p id=\"Par22\">In the last evaluation of the smart textile performance, the capability of detecting user’s activities is assessed by means of positioning the textile in a healthy volunteer. The textile is positioned on the user’s lower back. Its positioning in the lower back enables to acquire the signals from the activities characterized by strong correlation with user’s trunk displacement, as lower limb movements generally result in variations on the trunk, the smart textile can be used on the detection of gait-related activities as well. Thus, the user was asked to perform three common movements in daily routing: walking in self-selected speed, squatting and seat on a chair. The sensors responses for each case are presented in Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>a, where the sensors are analyzed in the time domain. The time domain response shows significant differences in the sensors responses, especially in the squatting activity, where Sensor 2 shows the highest signal attenuation, which is related to its positioning at the center of user’s lower back, leading to higher displacements in the fiber when compared with Sensors 1 and 3. Another offset in the sensors’ responses were found when the user sits on a chair, where the back support of the chair resulted in an attenuation on the transmitted optical power. The walking activity is the one that induced the lowest optical power variation in the sensors. In order to enhance the activity detection performance, the PCA was applied in the sensors responses. The PCA represents the data in a new coordinate system by using linear transformations and is a widely used technique for dimensionality reduction and as preprocessing for clustering techniques<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. In this case, the PCA was applied in the data to show the possibility of activity detection and identification using the proposed smart textile when used in conjunction with a clustering technique such as k-means<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. Figure <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>b shows the scatter plot of first two principal components for the sensors responses obtained on each activity, these two principal components represent 98% of all variability, mainly related to time-domain responses of Sensors 2 and 3, which indicate the possibility of dimensionality reduction by using just the first two principal components (instead of all components). Furthermore, the PCA results in three separate groups, which are identified as three clusters, representing each performed activity. It is important to mention that a higher number of observations was obtained in the walking and sitting activities due to their longer duration when compared with squatting activity, as shown Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>a. Thus, the PCA in conjunction with k-means (or other clustering technique) result in a feasible option for the activities detection using the proposed smart textile. It is also worth noting that the proposed textile has scalability, where a higher number of sensors can be used, which can lead to the detection of a higher number of activities, depending on the sensor system positioning on the user.<fig id=\"Fig6\"><label>Figure 6</label><caption><p>LPS-POF embedded smart textile for user’s activity monitoring. (<bold>a</bold>) Time-domain analysis. (<bold>b</bold>) Scatter plot for the activities monitoring.</p></caption><graphic xlink:href=\"41598_2020_70880_Fig6_HTML\" id=\"MO6\"/></fig></p><p id=\"Par23\">The experimental results obtained with the proposed smart textile shows the feasibility of this approach in a novel, multiparameter and transparent sensor system that can be used in multiple applications. It is possible to envisage that the next generation of the textile fabrics will be embedded with sensor system for the assessment of the user’s health condition and activities, where the proposed technology (i.e. LPS-POF embedded in conjunction with multiplexing techniques based on LEDs modulation) can be a key technology for transparent multipurpose sensor systems. The proposed smart textile can be also integrated with IoT modules for remote health monitoring. In addition, due to the scalability of the proposed technique, it is also possible to envisage the development of a complete clothing embedded with the proposed sensors for the assessment of multiple parameters, such as heart and breath rates, kinematic parameters of the human, interaction forces, body temperature, just to name a few. All these sensors transmitting to a home gateway and IoT modules for the remote health monitoring, which can also be integrated with novel classifiers and neural network in order to extract all the physical and physiological information of the user.</p></sec></sec><sec id=\"Sec5\"><title>Discussions</title><p id=\"Par24\">In this paper, a novel smart textile based on multiplexed intensity variation-based sensors was proposed. The multiplexing technique is based on an OOK modulation in the LEDs, which results in a quasi-distributed sensor system with low cross-sensitivity. In contrast with other popular quasi-distributed optical fiber sensors, such as FBGs, the proposed approach does not need neither specialized equipment for the sensor fabrication nor high cost (and generally bulk) interrogators for the signal acquisition. Therefore, the proposed technique is fully portable and low cost, which enable a plethora of applications in remote health monitoring by either transparent wearable sensors or smart homes. In addition, the LPS-POF used in the smart textile has remarkable flexibility (Young’s modulus of 15 MPa) in conjunction with high strain limits (about 17%), which can be embedded in clothing without restricting the user’s movement and results in sensors with high sensitivity and can be used in a large range of dynamic movements, as verified in the DMA experiments.</p><p id=\"Par25\">The LPS-POF and flexible LEDs were embedded in between two layers of neoprene textile fabric for the multiplexed sensor system. The temperature and interaction forces characterization showed the high sensitivity and linearity of the sensor system, which can also measure the temperature profile and force map with high repeatability and resolution. Then, the movement analysis using the proposed textile was performed by means of multiplane angle assessment using the textile sensors for bending at different planes as well as torsion, where the sensors presented different sensitivities with the possibility of simultaneous assessment of angular displacement at different planes. Finally, the smart textile was positioned on the user’s lower back and was able of identifying basic activities in daily life such as walking, siting and squatting by the analysis of the sensors responses using PCA in conjunction with clustering techniques. Therefore, the proposed device is a feasible option for novel transparent wearable sensors for remote health monitoring, which can be scalable for a fully instrumented clothing with smart sensors. Future works include the development and validation of the smart clothing for simultaneous and remote evaluation of multiple parameters.</p></sec><sec id=\"Sec6\"><title>Methods</title><sec id=\"Sec7\"><title>LPS-POF fabrication</title><p id=\"Par26\">The LPS-POF fabrication is performed in three steps: (1) addition of a liquid mixture of monomers and additive in a dosing system, where the main monomer is the Bisphenol-A acrylate due to its combination of high elasticity and optical transparency. (2) The liquid mixture passes through a spinneret in order to obtain the cylindrical shape. (3) The UV-curing is performed for the polymerization followed by an axial deformation stage to obtain the desired diameter for the optical fiber.</p></sec><sec id=\"Sec8\"><title>DSC tests</title><p id=\"Par27\">In order to characterize the fiber thermal transitions, DSC is performed, in which the LPS-POF enthalpy variation is compared with a thermally inert reference<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. The DSC equipment employed is the DSC Q200 (TA Instruments, USA), where the heat flow variation in the sample is analyzed with respect to the temperature. In this test, the offset in the heat flow variation curve is related to the polymer glass transition temperature, whereas an endothermic peak corresponds to its melting temperature.</p></sec><sec id=\"Sec9\"><title>Tensile and DMA tests</title><p id=\"Par28\">The tensile tests were performed using a universal testing machine (Biopdi, Brazil) in which the fiber was positioned in the machine’s clamps for the axial strain tests, where the displacement (strain) and force (stress) were continuously monitored. The test occurs until the fiber breakage, where it is possible to infer the strain limits of the LPS-POF. In addition, the Young’s modulus of the fiber is calculated as the ratio between the stress and strain in the elastic region (linear region) of the stress–strain curve. All the tensile tests were made with constant strain rate of 1 mm/min. Then, the DMA tests are performed by means of applying an oscillatory load in the sample with controlled displacement and frequency at constant temperature condition. The DMA 8000 (Perkin Helmer, USA) was used in the experiments in the axial strain configuration with a maximum displacement of 1 mm, where each measurement was performed 3 times in an isothermal period of 10 min. The temperature in the tests was 25 °C.</p></sec><sec id=\"Sec10\"><title>Temperature characterization setup</title><p id=\"Par29\">In the temperature characterization tests, Sensors 1, 2 and 3 were positioned in a thermoelectric Peltier plate TEC-12706 (Heibei IT, China) with closed loop temperature control TED 200C (Thorlabs, USA). The temperature range was 20–40 °C in 5 °C steps, where the isothermal period was 5 min in order to ensure a constant temperature at each sensor. For the temperature profile tests, a temperature-controlled hot air blower 858D (QWERTOUY, USA) was employed and positioned at different regions of the smart textile.</p></sec><sec id=\"Sec11\"><title>Force characterization setup</title><p id=\"Par30\">For the force characterization, calibrated weights with a known mass were positioned on the top of each sensor for about 10 s. All the sensors were tested in a range of 0–150 N. However, Sensors 1 and 3 have different forces due to the dimensions of the weights, which can apply a force on Sensor 2 due to the proximity of such sensors. In addition, the force map characterization was performed by position a weight with larger dimensions in the center of the smart textile, leading to a force distribution in the sensors.</p></sec><sec id=\"Sec12\"><title>Angular displacement characterization setup</title><p id=\"Par31\">The angular displacement tests were performed by manually bending the textile with the aid of a goniometer at the different planes, where the goniometer was previously aligned with the bending plane and the angular displacements in the range of 0° to 90° for bending and 0° to 180° for torsion were performed.</p></sec><sec id=\"Sec13\"><title>Human activity monitoring protocol</title><p id=\"Par32\">In the smart textile validation test, a healthy volunteer (male, 29 years) positioned the smart textile in his lower back with the aid of elastic bands. The test comprises of walking in a 5-m room at constant velocity and, at the end of the 5 m walk, the user performs a squat. Then, there is 180° turn and the user returns to the end of the room, where a chair is positioned. The user sits on the chair and the test ends at about 30 s later. We have the informed consent of all participants and the tests were made in accordance with the guidelines of the national health council with the protocols approved by Research Ethics Committee through the National Commission in Research Ethics—CONEP—(Certificate of Presentation for Ethical Appreciation—CAAE: 64797816.7.0000.5542). The responses of all three sensors were acquired for each activity, i.e. walking, squatting and sitting. The signal processing was performed using PCA, since it is a widely used statistical tool for multivariable data analysis (details on the PCA algorithm can be found in Jollife and Cadima<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>), where the data distributed in the principal components can be classified using techniques such as the k-means clustering.</p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><ack><title>Acknowledgements</title><p>Authors would like to thank LabPetro under the partnership agreement 0050.0022844.06.4 for the use of the DMA and DSC equipment. Authors also would like to thank Garry Berkovic, Ehud Shaffir and Oleg Palchik for providing the LPS-POF. This research is financed by FAPES (85426300, 84336650 and 2020-F057G), CNPq (304049/2019-0) and Petrobras (2017/00702-6). C. Marques acknowledges Fundação para a Ciência e a Tecnologia (FCT) through the CEECIND/00034/2018 (iFish project) and this work was developed within the scope of the project i3N, UIDB/50025/2020 & UIDP/50025/2020, financed by national funds through the FCT/MEC. This work is also funded by national funds (OE), through FCT, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>A.L.-J. and L. A. characterized the sensors, conceived and fabricated the smart textile. C.M., A.L.-J. and A.F. analyzed the data. All authors wrote the paper and revised the paper.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par33\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Karmakar</surname><given-names>A</given-names></name><name><surname>Dey</surname><given-names>N</given-names></name><name><surname>Baral</surname><given-names>T</given-names></name><name><surname>Chowdhury</surname><given-names>M</given-names></name><name><surname>Rehan</surname><given-names>M</given-names></name></person-group><article-title>Industrial internet of things: a review</article-title><source>Int Conf Opto-Electr. Appl. Opt. (Optronix)</source><year>2019</year><volume>3</volume><fpage>1</fpage><lpage>6</lpage></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zanella</surname><given-names>A</given-names></name><name><surname>Bui</surname><given-names>N</given-names></name><name><surname>Castellani</surname><given-names>A</given-names></name><name><surname>Vangelista</surname><given-names>L</given-names></name><name><surname>Zorzi</surname><given-names>M</given-names></name></person-group><article-title>Internet of things for smart cities</article-title><source>IEEE Internet Things J.</source><year>2014</year><volume>1</volume><fpage>22</fpage><lpage>32</lpage><pub-id pub-id-type=\"doi\">10.1109/JIOT.2014.2306328</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Domingues</surname><given-names>MF</given-names></name><etal/></person-group><article-title>Insole optical fiber sensor architecturefor remote gait analysis: an eHealth Solution</article-title><source>IEEE Internet Things J.</source><year>2017</year><volume>4662</volume><fpage>1</fpage><lpage>1</lpage></element-citation></ref><ref id=\"CR4\"><label>4.</label><mixed-citation publication-type=\"other\">United Nations, Department of Economic and Social Affairs, Population Division. World Population Ageing 2013. ST/ESA/SER.A/348 (2013).</mixed-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Majumder</surname><given-names>S</given-names></name><name><surname>Mondal</surname><given-names>T</given-names></name><name><surname>Deen</surname><given-names>M</given-names></name></person-group><article-title>Wearable sensors for remote health monitoring</article-title><source>Sensors</source><year>2017</year><volume>17</volume><fpage>130</fpage><pub-id pub-id-type=\"doi\">10.3390/s17010130</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>D</given-names></name></person-group><article-title>5G and intelligence medicine—how the next generation of wireless technology will reconstruct healthcare?</article-title><source>Precis. Clin. Med.</source><year>2019</year><volume>2</volume><fpage>205</fpage><lpage>208</lpage><pub-id pub-id-type=\"doi\">10.1093/pcmedi/pbz020</pub-id><pub-id pub-id-type=\"pmid\">31886033</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Na</surname><given-names>L</given-names></name><name><surname>Streim</surname><given-names>JE</given-names></name></person-group><article-title>Psychosocial well-being associated with activity of daily living stages among community-dwelling older adults</article-title><source>Gerontol. Geriatr. Med.</source><year>2017</year><volume>3</volume><fpage>233372141770001</fpage><pub-id pub-id-type=\"doi\">10.1177/2333721417700011</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mukhopadhyay</surname><given-names>SC</given-names></name></person-group><article-title>Wearable sensors for human activity monitoring: a review</article-title><source>IEEE Sens. J.</source><year>2015</year><volume>15</volume><fpage>1321</fpage><lpage>1330</lpage><pub-id pub-id-type=\"doi\">10.1109/JSEN.2014.2370945</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nag</surname><given-names>A</given-names></name><name><surname>Mukhopadhyay</surname><given-names>SC</given-names></name><name><surname>Kosel</surname><given-names>J</given-names></name></person-group><article-title>Wearable flexible sensors: a review</article-title><source>IEEE Sens. J.</source><year>2017</year><volume>17</volume><fpage>3949</fpage><lpage>3960</lpage><pub-id pub-id-type=\"doi\">10.1109/JSEN.2017.2705700</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quandt</surname><given-names>BM</given-names></name><etal/></person-group><article-title>Body-Monitoring and health supervision by means of optical fiber-based sensing systems in medical textiles</article-title><source>Adv. Healthc. Mater.</source><year>2015</year><volume>4</volume><fpage>330</fpage><lpage>355</lpage><pub-id pub-id-type=\"doi\">10.1002/adhm.201400463</pub-id><pub-id pub-id-type=\"pmid\">25358557</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Islam</surname><given-names>T</given-names></name><name><surname>Mukhopadhyay</surname><given-names>SC</given-names></name><name><surname>Suryadevara</surname><given-names>NK</given-names></name></person-group><article-title>Smart sensors and internet of things: a postgraduate paper</article-title><source>IEEE Sens. J.</source><year>2017</year><volume>17</volume><fpage>577</fpage><lpage>584</lpage><pub-id pub-id-type=\"doi\">10.1109/JSEN.2016.2630124</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Capineri</surname><given-names>L</given-names></name></person-group><article-title>Resistive sensors with smart textiles for wearable technology: From fabrication processes to integration with electronics</article-title><source>Procedia Eng.</source><year>2014</year><volume>87</volume><fpage>724</fpage><lpage>727</lpage><pub-id pub-id-type=\"doi\">10.1016/j.proeng.2014.11.748</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>L</given-names></name><etal/></person-group><article-title>Dual-core capacitive microfiber sensor for smart textile applications</article-title><source>ACS Appl. Mater. Interfaces</source><year>2019</year><volume>11</volume><fpage>33347</fpage><lpage>33355</lpage><pub-id pub-id-type=\"doi\">10.1021/acsami.9b10937</pub-id><pub-id pub-id-type=\"pmid\">31424908</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gonçalves</surname><given-names>C</given-names></name><name><surname>da Silva</surname><given-names>AF</given-names></name><name><surname>Gomes</surname><given-names>J</given-names></name><name><surname>Simoes</surname><given-names>R</given-names></name></person-group><article-title>Wearable e-textile technologies: a review on sensors, actuators and control elements</article-title><source>Inventions</source><year>2018</year><volume>3</volume><fpage>1</fpage><lpage>13</lpage><pub-id pub-id-type=\"doi\">10.3390/inventions3010014</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alwis</surname><given-names>L</given-names></name><name><surname>Sun</surname><given-names>T</given-names></name><name><surname>Grattan</surname><given-names>KTV</given-names></name></person-group><article-title>Developments in optical fibre sensors for industrial applications</article-title><source>Opt. Laser Technol.</source><year>2016</year><volume>78</volume><fpage>62</fpage><lpage>66</lpage><pub-id pub-id-type=\"doi\">10.1016/j.optlastec.2015.09.004</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Theodosiou</surname><given-names>A</given-names></name><name><surname>Komodromos</surname><given-names>M</given-names></name><name><surname>Kalli</surname><given-names>K</given-names></name></person-group><article-title>Carbon cantilever beam health inspection using a polymer fiber bragg grating array</article-title><source>J. Light. Technol.</source><year>2018</year><volume>36</volume><fpage>986</fpage><lpage>992</lpage><pub-id pub-id-type=\"doi\">10.1109/JLT.2017.2768414</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Leal-Junior</surname><given-names>AG</given-names></name><etal/></person-group><article-title>Polymer optical fiber sensors in healthcare applications: a comprehensive review</article-title><source>Sensors</source><year>2019</year><volume>19</volume><fpage>3156</fpage><pub-id pub-id-type=\"doi\">10.3390/s19143156</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Broadway</surname><given-names>C</given-names></name><name><surname>Min</surname><given-names>R</given-names></name><name><surname>Leal-Junior</surname><given-names>AG</given-names></name><name><surname>Marques</surname><given-names>C</given-names></name><name><surname>Caucheteur</surname><given-names>C</given-names></name></person-group><article-title>Toward commercial polymer fiber bragg grating sensors: review and applications</article-title><source>J. Light. Technol.</source><year>2019</year><volume>37</volume><fpage>2605</fpage><lpage>2615</lpage><pub-id pub-id-type=\"doi\">10.1109/JLT.2018.2885957</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Graham-Rowe</surname><given-names>D</given-names></name></person-group><article-title>Photonic fabrics take shape</article-title><source>Nat. Photonics</source><year>2007</year><volume>1</volume><fpage>6</fpage><lpage>7</lpage><pub-id pub-id-type=\"doi\">10.1038/nphoton.2006.35</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>H</given-names></name><name><surname>Yang</surname><given-names>H</given-names></name><name><surname>Li</surname><given-names>E</given-names></name><name><surname>Liu</surname><given-names>Z</given-names></name><name><surname>Wei</surname><given-names>K</given-names></name></person-group><article-title>Wearable sensors in intelligent clothing for measuring human body temperature based on optical fiber Bragg grating</article-title><source>Opt. Express</source><year>2012</year><volume>20</volume><fpage>11740</fpage><pub-id pub-id-type=\"doi\">10.1364/OE.20.011740</pub-id><pub-id pub-id-type=\"pmid\">22714162</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ciocchetti</surname><given-names>M</given-names></name><etal/></person-group><article-title>Smart textile based on fiber bragg grating sensors for respiratory monitoring: design and preliminary trials</article-title><source>Biosensors</source><year>2015</year><volume>5</volume><fpage>602</fpage><lpage>615</lpage><pub-id pub-id-type=\"doi\">10.3390/bios5030602</pub-id><pub-id pub-id-type=\"pmid\">26389961</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>Z</given-names></name><etal/></person-group><article-title>Simultaneous measurement of breathing rate and heart rate using a microbend multimode fiber optic sensor</article-title><source>J. Biomed. Opt.</source><year>2014</year><volume>19</volume><fpage>057001</fpage><pub-id pub-id-type=\"doi\">10.1117/1.JBO.19.5.057001</pub-id><pub-id pub-id-type=\"pmid\">24788372</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bilro</surname><given-names>L</given-names></name><name><surname>Alberto</surname><given-names>N</given-names></name><name><surname>Pinto</surname><given-names>JL</given-names></name><name><surname>Nogueira</surname><given-names>R</given-names></name></person-group><article-title>Optical sensors based on plastic fibers</article-title><source>Sensors</source><year>2012</year><volume>12</volume><fpage>12184</fpage><lpage>12207</lpage><pub-id pub-id-type=\"doi\">10.3390/s120912184</pub-id><pub-id pub-id-type=\"pmid\">23112707</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zupin</surname><given-names>Z</given-names></name><name><surname>Dimitrovski</surname><given-names>K</given-names></name></person-group><article-title>Mechanical Properties of Fabrics Made from Cotton and Biodegradable Yarns Bamboo, SPF, PLA in Weft</article-title><source>Woven Fabric Eng</source><year>2010</year><pub-id pub-id-type=\"doi\">10.5772/10479</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Leal-Junior</surname><given-names>A</given-names></name><etal/></person-group><article-title>Characterization of a new polymer optical fiber with enhanced sensing capabilities using a Bragg grating</article-title><source>Opt. Lett.</source><year>2018</year><volume>43</volume><fpage>4799</fpage><pub-id pub-id-type=\"doi\">10.1364/OL.43.004799</pub-id><pub-id pub-id-type=\"pmid\">30272743</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Leal-Junior</surname><given-names>AG</given-names></name><name><surname>Díaz</surname><given-names>CR</given-names></name><name><surname>Marques</surname><given-names>C</given-names></name><name><surname>Pontes</surname><given-names>MJ</given-names></name><name><surname>Frizera</surname><given-names>A</given-names></name></person-group><article-title>Multiplexing technique for quasi-distributed sensors arrays in polymer optical fiber intensity variation-based sensors</article-title><source>Opt. Laser Technol.</source><year>2019</year><volume>111</volume><fpage>81</fpage><lpage>88</lpage><pub-id pub-id-type=\"doi\">10.1016/j.optlastec.2018.09.044</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><mixed-citation publication-type=\"other\">Lakes, R. <italic>Viscoelastic Materials</italic> (Cambridge University Press, Cambridge, 2009). 10.1017/CBO9780511626722 .</mixed-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Leal-Junior</surname><given-names>AG</given-names></name><name><surname>Marques</surname><given-names>C</given-names></name><name><surname>Frizera</surname><given-names>A</given-names></name><name><surname>Pontes</surname><given-names>MJ</given-names></name></person-group><article-title>Dynamic mechanical analysis on a polymethyl methacrylate (PMMA) polymer optical fiber</article-title><source>IEEE Sens. J.</source><year>2018</year><volume>18</volume><fpage>2353</fpage><lpage>2361</lpage><pub-id pub-id-type=\"doi\">10.1109/JSEN.2018.2797086</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Kirtley</surname><given-names>C</given-names></name></person-group><source>Clinical Gait Analysis: Theory and Practice</source><year>2006</year><publisher-loc>Amsterdam</publisher-loc><publisher-name>Elsevier</publisher-name></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Leyva-Porras</surname><given-names>C</given-names></name></person-group><article-title>Application of differential scanning calorimetry (DSC) and modulated differential scanning calorimetry (MDSC) in food and drug industries</article-title><source>Polymers (Basel)</source><year>2019</year><volume>12</volume><fpage>5</fpage><pub-id pub-id-type=\"doi\">10.3390/polym12010005</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Avellar</surname><given-names>LM</given-names></name><name><surname>Leal-Junior</surname><given-names>AG</given-names></name><name><surname>Diaz</surname><given-names>CAR</given-names></name><name><surname>Marques</surname><given-names>C</given-names></name><name><surname>Frizera</surname><given-names>A</given-names></name></person-group><article-title>POF smart carpet: a multiplexed polymer optical fiber-embedded smart carpet for gait analysis</article-title><source>Sensors</source><year>2019</year><volume>19</volume><fpage>3356</fpage><pub-id pub-id-type=\"doi\">10.3390/s19153356</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Leal-Junior</surname><given-names>AG</given-names></name><name><surname>Díaz</surname><given-names>CR</given-names></name><name><surname>Marques</surname><given-names>C</given-names></name><name><surname>Pontes</surname><given-names>MJ</given-names></name><name><surname>Frizera</surname><given-names>A</given-names></name></person-group><article-title>3D-printed POF insole: development and applications of a low-cost, highly customizable device for plantar pressure and ground reaction forces monitoring</article-title><source>Opt. Laser Technol.</source><year>2019</year><volume>116</volume><fpage>256</fpage><lpage>264</lpage><pub-id pub-id-type=\"doi\">10.1016/j.optlastec.2019.03.035</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Roselló-Mechó</surname><given-names>X</given-names></name><name><surname>Delgado-Pinar</surname><given-names>M</given-names></name><name><surname>Díez</surname><given-names>A</given-names></name><name><surname>Andrés</surname><given-names>MV</given-names></name></person-group><article-title>Measurement of Pockels’ coefficients and demonstration of the anisotropy of the elasto-optic effect in optical fibers under axial strain</article-title><source>Opt. Lett.</source><year>2016</year><volume>41</volume><fpage>2934</fpage><pub-id pub-id-type=\"doi\">10.1364/OL.41.002934</pub-id><pub-id pub-id-type=\"pmid\">27367069</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Leal Junior</surname><given-names>AG</given-names></name><etal/></person-group><article-title>Simultaneous measurement of axial strain, bending and torsion with a single fiber bragg grating in CYTOP fiber</article-title><source>J. Light. Technol.</source><year>2018</year><volume>37</volume><fpage>971</fpage><lpage>980</lpage><pub-id pub-id-type=\"doi\">10.1109/JLT.2018.2884538</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Diáz</surname><given-names>CAR</given-names></name><etal/></person-group><article-title>Liquid level measurement based on FBG-embedded diaphragms with temperature compensation</article-title><source>IEEE Sens. J.</source><year>2018</year><volume>18</volume><fpage>193</fpage><lpage>200</lpage><pub-id pub-id-type=\"doi\">10.1109/JSEN.2017.2768510</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Leal-Junior</surname><given-names>AG</given-names></name><etal/></person-group><article-title>Simultaneous measurement of pressure and temperature with a single FBG embedded in a polymer diaphragm</article-title><source>Opt. Laser Technol.</source><year>2019</year><volume>112</volume><fpage>77</fpage><lpage>84</lpage><pub-id pub-id-type=\"doi\">10.1016/j.optlastec.2018.11.013</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jollife</surname><given-names>IT</given-names></name><name><surname>Cadima</surname><given-names>J</given-names></name></person-group><article-title>Principal component analysis: a review and recent developments</article-title><source>Philos. Trans. R. Soc. Math. Phys. Eng. Sci.</source><year>2016</year><volume>374</volume><fpage>20150202</fpage><pub-id pub-id-type=\"doi\">10.1098/rsta.2015.0202</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ding</surname><given-names>C</given-names></name><name><surname>He</surname><given-names>X</given-names></name></person-group><article-title>Cluster structure of K-means clustering via principal component analysis</article-title><source>Lect. Notes Comput. Sci.</source><year>2004</year><volume>3056</volume><fpage>414</fpage><lpage>418</lpage><pub-id pub-id-type=\"doi\">10.1007/978-3-540-24775-3_50</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Prado</surname><given-names>AR</given-names></name><etal/></person-group><article-title>Polymethyl methacrylate (PMMA) recycling for the production of optical fiber sensor systems</article-title><source>Opt. Express</source><year>2017</year><volume>25</volume><fpage>30051</fpage><pub-id pub-id-type=\"doi\">10.1364/OE.25.030051</pub-id><pub-id pub-id-type=\"pmid\">29221040</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807813</article-id><article-id pub-id-type=\"pmc\">PMC7431549</article-id><article-id pub-id-type=\"publisher-id\">70544</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70544-7</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Multi-AI competing and winning against humans in iterated Rock-Paper-Scissors game</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Lei</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Huang</surname><given-names>Wenbin</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Yuanpeng</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Evans</surname><given-names>Julian</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>He</surname><given-names>Sailing</given-names></name><address><email>Sailing@kth.se</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.13402.34</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1759 700X</institution-id><institution>National Engineering Research Center for Optical Instruments, Centre for Optical and Electromagnetic Research, </institution><institution>Zhejiang University, </institution></institution-wrap>Hangzhou, 310058 China </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.13402.34</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1759 700X</institution-id><institution>Ningbo Research Institute, Zhejiang University, </institution></institution-wrap>Ningbo, 315100 China </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5037.1</institution-id><institution-id institution-id-type=\"ISNI\">0000000121581746</institution-id><institution>Department of Electromagnetic Engineering, School of Electrical Engineering, </institution><institution>Royal Institute of Technology, </institution></institution-wrap>100 44 Stockholm, Sweden </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13873</elocation-id><history><date date-type=\"received\"><day>6</day><month>2</month><year>2020</year></date><date date-type=\"accepted\"><day>28</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Predicting and modeling human behavior and finding trends within human decision-making processes is a major problem of social science. Rock Paper Scissors (RPS) is the fundamental strategic question in many game theory problems and real-world competitions. Finding the right approach to beat a particular human opponent is challenging. Here we use an AI (artificial intelligence) algorithm based on Markov Models of one fixed memory length (abbreviated as “single AI”) to compete against humans in an iterated RPS game. We model and predict human competition behavior by combining many Markov Models with different fixed memory lengths (abbreviated as “multi-AI”), and develop an architecture of multi-AI with changeable parameters to adapt to different competition strategies. We introduce a parameter called “focus length” (a positive number such as 5 or 10) to control the speed and sensitivity for our multi-AI to adapt to the opponent’s strategy change. The focus length is the number of previous rounds that the multi-AI should look at when determining which Single-AI has the best performance and should choose to play for the next game. We experimented with 52 different people, each playing 300 rounds continuously against one specific multi-AI model, and demonstrated that our strategy could win against more than 95% of human opponents.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Human behaviour</kwd><kwd>Scientific data</kwd><kwd>Statistics</kwd><kwd>Computer science</kwd><kwd>Applied mathematics</kwd><kwd>Information technology</kwd><kwd>Information theory and computation</kwd><kwd>Statistical physics, thermodynamics and nonlinear dynamics</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">The Rock-Paper-Scissors (RPS) game has been widely used to study competitive phenomena in society and biology, such as ecological interactions, the maintenance of biodiversity in ecological systems<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup> and price dispersion of markets<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. There are two general approaches for RPS play, namely, Bayesian equilibrium and exploitation of player pattern<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. The payoff parameter <inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$a$$\\end{document}</tex-math><mml:math id=\"M2\"><mml:mi>a</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70544_Article_IEq1.gif\"/></alternatives></inline-formula>, defined as the incentive for winning divided by the incentive for drawing, is set to 2 to form a neutral RPS game<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>.</p><p id=\"Par3\">Previous research has found that there is a social circle in human competitive strategy when playing iterated RPS games<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. In this article we proposed a multi-AI algorithm that can exploit human strategy and win against human players in the same iterated RPS games and we conduct experiments with human players to confirm the results.</p><p id=\"Par4\">Our work may stimulate future more refined experimental and theoretical studies on the microscopic mechanisms of decision-making and learning in basic game systems<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>–<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>.</p><p id=\"Par5\">Markov chain models<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup> are the single models which our multi-AI is composed of. A Markov chain is a stochastic model describing a sequence of possible events in which the probability of each event depends only on the states attained in the previous events. Here, Markov chain is a special sort of belief network used to represent the sequences of states in a dynamic system. Previous research has used Markov process model to describe the stochastic evolution dynamic of the Rock–Scissors–Paper Game<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Here the iterated RPS game is considered as a Markov process and Markov chains are built throughout the process of 300 rounds of competition. The models are built to exploit attempted circular exploitation patterns.</p><p id=\"Par6\">The simplest discrete-time Markov chain is the first-order Markov chain, where the probability of moving to the next state depends only on the present state and not on the previous states:<disp-formula id=\"Equa\"><alternatives><tex-math id=\"M3\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Pr (X_{n + 1} = x|X_{1} = x_{1} ,X_{2} = x_{2} , \\ldots ,X_{n} = x_{n} ) = \\Pr (X_{n + 1} = x|X_{n} = x_{n} ),$$\\end{document}</tex-math><mml:math id=\"M4\" display=\"block\"><mml:mrow><mml:mo>Pr</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mrow><mml:mi>n</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mi>x</mml:mi><mml:mo stretchy=\"false\">|</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mo>,</mml:mo><mml:mo>…</mml:mo><mml:mo>,</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mi>n</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mi>n</mml:mi></mml:msub><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:mo>Pr</mml:mo><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mrow><mml:mi>n</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mi>x</mml:mi><mml:mo stretchy=\"false\">|</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mi>n</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mi>n</mml:mi></mml:msub><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mo>,</mml:mo></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70544_Article_Equa.gif\" position=\"anchor\"/></alternatives></disp-formula>where <inline-formula id=\"IEq2\"><alternatives><tex-math id=\"M5\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$X_{1}$$\\end{document}</tex-math><mml:math id=\"M6\"><mml:msub><mml:mi>X</mml:mi><mml:mn>1</mml:mn></mml:msub></mml:math><inline-graphic xlink:href=\"41598_2020_70544_Article_IEq2.gif\"/></alternatives></inline-formula>, <inline-formula id=\"IEq3\"><alternatives><tex-math id=\"M7\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$X_{2}$$\\end{document}</tex-math><mml:math id=\"M8\"><mml:msub><mml:mi>X</mml:mi><mml:mn>2</mml:mn></mml:msub></mml:math><inline-graphic xlink:href=\"41598_2020_70544_Article_IEq3.gif\"/></alternatives></inline-formula>, … <inline-formula id=\"IEq4\"><alternatives><tex-math id=\"M9\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$X_{n}$$\\end{document}</tex-math><mml:math id=\"M10\"><mml:msub><mml:mi>X</mml:mi><mml:mi>n</mml:mi></mml:msub></mml:math><inline-graphic xlink:href=\"41598_2020_70544_Article_IEq4.gif\"/></alternatives></inline-formula> are a sequence of random variables (“Rock”, “Paper”, and “Scissors” here). What you will play in the next round only depends on what you played this round, like a short memory pattern sequence.</p><p id=\"Par7\">Markov chains can be generalized to cases of short-term dependency, by taking into account recent past states in the chain. The m-th order Markov chain<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup> considers the current state to depend on m previous states, where m is finite, and is a process satisfying<disp-formula id=\"Equb\"><alternatives><tex-math id=\"M11\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\begin{aligned} & \\Pr (X_{n} = x_{n} |X_{n - 1} = x_{n - 1} ,X_{n - 2} = x_{n - 2} , \\ldots ,X_{1} = x_{1} ) \\\\ & \\quad = \\Pr (X_{n} = x_{n} |X_{n - 1} = x_{n - 1} ,X_{n - 2} = x_{n - 2} , \\ldots ,X_{n - m} = x_{n - m} )\\;{\\text{for}}\\;n > m \\\\ \\end{aligned}$$\\end{document}</tex-math><mml:math id=\"M12\" display=\"block\"><mml:mrow><mml:mtable><mml:mtr><mml:mtd/><mml:mtd columnalign=\"left\"><mml:mrow><mml:mo>Pr</mml:mo><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mi>n</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mi>n</mml:mi></mml:msub><mml:mo stretchy=\"false\">|</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mrow><mml:mi>n</mml:mi><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mrow><mml:mi>n</mml:mi><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mrow><mml:mi>n</mml:mi><mml:mo>-</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mrow><mml:mi>n</mml:mi><mml:mo>-</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:mo>…</mml:mo><mml:mo>,</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd columnalign=\"right\"><mml:mrow/></mml:mtd><mml:mtd columnalign=\"left\"><mml:mrow><mml:mspace width=\"1em\"/><mml:mo>=</mml:mo><mml:mo>Pr</mml:mo><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mi>n</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mi>n</mml:mi></mml:msub><mml:mo stretchy=\"false\">|</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mrow><mml:mi>n</mml:mi><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mrow><mml:mi>n</mml:mi><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mrow><mml:mi>n</mml:mi><mml:mo>-</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mrow><mml:mi>n</mml:mi><mml:mo>-</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:mo>…</mml:mo><mml:mo>,</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mrow><mml:mi>n</mml:mi><mml:mo>-</mml:mo><mml:mi>m</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mrow><mml:mi>n</mml:mi><mml:mo>-</mml:mo><mml:mi>m</mml:mi></mml:mrow></mml:msub><mml:mo stretchy=\"false\">)</mml:mo><mml:mspace width=\"0.277778em\"/><mml:mtext>for</mml:mtext><mml:mspace width=\"0.277778em\"/><mml:mi>n</mml:mi><mml:mo>></mml:mo><mml:mi>m</mml:mi></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd columnalign=\"right\"><mml:mrow/></mml:mtd></mml:mtr></mml:mtable></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70544_Article_Equb.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p>Here the m-th order Markov chain is like a model with memory length m, which ‘remembers’ the previous m states.</p><p id=\"Par8\">For <inline-formula id=\"IEq5\"><alternatives><tex-math id=\"M13\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$n < m$$\\end{document}</tex-math><mml:math id=\"M14\"><mml:mrow><mml:mi>n</mml:mi><mml:mo><</mml:mo><mml:mi>m</mml:mi></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70544_Article_IEq5.gif\"/></alternatives></inline-formula>, all m-th order single Markov chain models will select Rock, Paper, Scissors randomly, with 1/3 probability each.</p><p id=\"Par9\">As the competition goes, the combined model (multi-AI) will select one specific fixed memory length Markov Model that is better at predicting this particular human player’s decision strategy at this particular time.</p><p id=\"Par10\">Here for simplicity we use AI-m to denote our single Markov chain model of order m.</p><p id=\"Par11\">Through the experiments we found that different models work best against different human opponent’s competition strategies and the prediction results vary greatly so we built the 1st -5th order Markov chain models (i.e. AI-1 to AI-5) with different memory lengths for exploiting different human competition strategies. To make a multi-AI model that can differentiate and adapt to different human opponents, we combine the 1st-5th order single Markov models and introduce a “focus length” parameter to control the adaptation speed and sensitivity to form a multi-AI that can adapt to different human strategies and win against most of its opponents. The focus length, F, is the number of rounds that are used to evaluate the current performance of the single AIs. The multi-AI will produce the output associated with the single-AI that has the highest score in the last F rounds. Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> illustrates how this multi-AI model competes against a specific player as an example with focus length F = 5.<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>The internal calculation of our multi-AI algorithm with 5 models and focus length 5 playing against a human opponent.</p></caption><graphic position=\"anchor\" xlink:href=\"41598_2020_70544_Tab1_HTML\" id=\"MO5\"/></table-wrap></p><p id=\"Par12\">For example, for the fifth round, the multi-AI will look at all the previous 4 rounds and calculate each single models’ scores. Since AI 2 had the highest score, the dominant AI will be AI 2 and its output is used as the fifth round multi-AI output. For the 9th round, the multi-AI will look at the 4th–8th rounds (focus length F = 5), for which AI 3 has the highest total score, and the dominant AI will be switched to AI 3 and its output is used as the 9th round multi-AI output.</p><p id=\"Par13\">For the first round, the multi-AI will use the result from AI-1 and it selects Rock, Paper or Scissors randomly with 1/3 probability each.</p><p id=\"Par14\">Focus length parameter F is set to control the speed and sensitivity for our multi-AI model to adapt to the opponent’s strategy change. Our multi-AI model will look at the recent F rounds of history to decide which single model is currently performing the best and should produce the next output. For the first 4 rounds when our competition data is less than focus length F, which is set to 5 as before, multi-AI will simply consider all rounds to determine its next round’s dominant single AI model. In the specific case of Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>, the dominant AI for all of the first 4 rounds is AI-1. In round 5, AI-2 has the best cumulative score and thus is the dominant AI.</p><p id=\"Par15\">Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> shows how our multi-AI algorithm competes against a specific player when F = 5 as an example. The transition matrix for the last AI-2 seeing the player played “PS” in the past 2 rounds is:<table-wrap id=\"Taba\"><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">R</th><th align=\"left\">P</th><th align=\"left\">S</th></tr></thead><tbody><tr><td align=\"left\">PS</td><td align=\"left\">1/3</td><td align=\"left\">1/3</td><td align=\"left\">1/3</td></tr></tbody></table></table-wrap></p><p id=\"Par16\">Thus, for the next round, AI-2 has 1/3 probability to select Paper, Scissors and Rock.</p><p id=\"Par17\">Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref> shows an example for the selection between AI-1 and AI-4 when focus length F = 5. Although globally AI-1 has a higher score in total (from the first round AI-1 has a total score of 2, but its recent 5 rounds score is 0), AI-4 has a higher score (which is 3 in this case) locally during the recent 5 rounds. Thus, the next round multi-AI will pick AI 4’s result as our multi-AI output.<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Selection between AI-1 and AI-4 when focus length F = 5.</p></caption><graphic position=\"anchor\" xlink:href=\"41598_2020_70544_Tab2_HTML\" id=\"MO6\"/></table-wrap></p><p id=\"Par18\">The transition matrix for AI-2 after 20 rounds of competition is in Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref>.<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>The transition matrix for AI-2 after 20 rounds of competition.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">RR</th><th align=\"left\">RS</th><th align=\"left\">RP</th><th align=\"left\">PR</th><th align=\"left\">PP</th><th align=\"left\">PS</th><th align=\"left\">SR</th><th align=\"left\">SP</th><th align=\"left\">SS</th></tr></thead><tbody><tr><td align=\"left\">R</td><td align=\"left\">0</td><td align=\"left\">1/18</td><td align=\"left\">1/18</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">1/18</td><td align=\"left\">1/18</td><td align=\"left\">3/18</td><td align=\"left\">1/18</td></tr><tr><td align=\"left\">P</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">1/18</td><td align=\"left\">0</td><td align=\"left\">2/18</td><td align=\"left\">0</td><td align=\"left\">1/18</td><td align=\"left\">0</td></tr><tr><td align=\"left\">S</td><td align=\"left\">1/18</td><td align=\"left\">1/18</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">0</td><td align=\"left\">1/18</td><td align=\"left\">1/18</td><td align=\"left\">0</td><td align=\"left\">2/18</td></tr></tbody></table></table-wrap></p></sec><sec id=\"Sec2\"><title>Results</title><p id=\"Par19\">All experiments are conducted with money incentive. We did the experiment with 52 human subjects recruited at Zhejiang University and used a multi-AI model which has 5 or 10 single length Markov chain models. Figure <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> shows 4 typical results of our multi-AI strategy with a combination of the 1st–5th order Markov chain models (here focus length F is also set to 5, but it can be any other integer) competing against 4 typical players for 300 rounds.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>300 rounds AI competition results for 4 typical players.</p></caption><graphic xlink:href=\"41598_2020_70544_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par20\">The total score for the multi-AI against 52 people are shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>. Against one player, multi-AI with a combination of 5 models had a score of 151 (i.e., 198 wins, 55 draws and only 47 losses). The total times of wins for multi-AI is more than 4 times that for this human player. Figure <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>d is an example of a player who beat the AI by a margin of 4, and this is not a statistically meaningful margin as there are several lead changes.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Total scores for multi-AI competing against different players in 300 rounds game.</p></caption><graphic xlink:href=\"41598_2020_70544_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par23\">52 people’s preferences in choosing Rock Paper Scissors in their 300 rounds competition are shown in Table <xref rid=\"Tab4\" ref-type=\"table\">4</xref>. There is a slight preference in choosing Rock (Table <xref rid=\"Tab5\" ref-type=\"table\">5</xref>). <table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>52 people’s preferences in choosing Rock Paper Scissors in 300 rounds competitions.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">R</th><th align=\"left\">P</th><th align=\"left\">S</th></tr></thead><tbody><tr><td align=\"left\">MEAN (times)</td><td char=\".\" align=\"char\">106.6098</td><td char=\".\" align=\"char\">96.29268</td><td char=\".\" align=\"char\">97.09756</td></tr><tr><td align=\"left\">STDEV.S</td><td char=\".\" align=\"char\">13.08095</td><td char=\".\" align=\"char\">13.12239</td><td char=\".\" align=\"char\">12.11851</td></tr></tbody></table></table-wrap><table-wrap id=\"Tab5\"><label>Table 5</label><caption><p>Game results (total scores) of our multi-10AI competing with human in 300 rounds.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">AI1</th><th align=\"left\">AI2</th><th align=\"left\">AI3</th><th align=\"left\">AI4</th><th align=\"left\">AI5</th><th align=\"left\">AI6</th><th align=\"left\">AI7</th><th align=\"left\">AI8</th><th align=\"left\">AI9</th><th align=\"left\">AI10</th><th align=\"left\">AI1</th><th align=\"left\">10 single models average score</th><th align=\"left\">Multi-10AI score</th><th align=\"left\">Multi-5AI score</th></tr></thead><tbody><tr><td align=\"left\">Player1</td><td align=\"left\">− 13</td><td align=\"left\">37</td><td align=\"left\">24</td><td align=\"left\">20</td><td align=\"left\">22</td><td align=\"left\">14</td><td align=\"left\">− 6</td><td align=\"left\">− 2</td><td align=\"left\">− 10</td><td align=\"left\">3</td><td align=\"left\">8.9</td><td align=\"left\">10</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Player2</td><td align=\"left\">2</td><td align=\"left\">50</td><td align=\"left\">48</td><td align=\"left\">42</td><td align=\"left\">42</td><td align=\"left\">50</td><td align=\"left\">61</td><td align=\"left\">7</td><td align=\"left\">12</td><td align=\"left\">5</td><td align=\"left\">31.9</td><td align=\"left\">41</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Player3</td><td align=\"left\">46</td><td align=\"left\">58</td><td align=\"left\">52</td><td align=\"left\">53</td><td align=\"left\">24</td><td align=\"left\">29</td><td align=\"left\">11</td><td align=\"left\">− 15</td><td align=\"left\">− 14</td><td align=\"left\">12</td><td align=\"left\">25.6</td><td align=\"left\">8</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Player4</td><td align=\"left\">26</td><td align=\"left\">41</td><td align=\"left\">26</td><td align=\"left\">55</td><td align=\"left\">46</td><td align=\"left\">28</td><td align=\"left\">20</td><td align=\"left\">13</td><td align=\"left\">10</td><td align=\"left\">36</td><td align=\"left\">30.1</td><td align=\"left\">34</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Player5</td><td align=\"left\">35</td><td align=\"left\">39</td><td align=\"left\">92</td><td align=\"left\">107</td><td align=\"left\">85</td><td align=\"left\">67</td><td align=\"left\">88</td><td align=\"left\">61</td><td align=\"left\">78</td><td align=\"left\">70</td><td align=\"left\">72.2</td><td align=\"left\">73</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Player6</td><td align=\"left\">28</td><td align=\"left\">60</td><td align=\"left\">52</td><td align=\"left\">55</td><td align=\"left\">49</td><td align=\"left\">36</td><td align=\"left\">14</td><td align=\"left\">1</td><td align=\"left\">− 18</td><td align=\"left\">12</td><td align=\"left\">28.9</td><td align=\"left\">43</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Player7</td><td align=\"left\">56</td><td align=\"left\">63</td><td align=\"left\">68</td><td align=\"left\">65</td><td align=\"left\">55</td><td align=\"left\">46</td><td align=\"left\">54</td><td align=\"left\">− 1</td><td align=\"left\">− 14</td><td align=\"left\">17</td><td align=\"left\">40.9</td><td align=\"left\">48</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Player8</td><td align=\"left\">28</td><td align=\"left\">29</td><td align=\"left\">47</td><td align=\"left\">29</td><td align=\"left\">12</td><td align=\"left\">5</td><td align=\"left\">21</td><td align=\"left\">5</td><td align=\"left\">− 10</td><td align=\"left\">− 5</td><td align=\"left\">16.1</td><td align=\"left\">43</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Player9</td><td align=\"left\">32</td><td align=\"left\">63</td><td align=\"left\">58</td><td align=\"left\">72</td><td align=\"left\">62</td><td align=\"left\">72</td><td align=\"left\">32</td><td align=\"left\">26</td><td align=\"left\">20</td><td align=\"left\">15</td><td align=\"left\">45.2</td><td align=\"left\">59</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Player10</td><td align=\"left\">− 5</td><td align=\"left\">− 14</td><td align=\"left\">− 8</td><td align=\"left\">11</td><td align=\"left\">16</td><td align=\"left\">− 7</td><td align=\"left\">− 26</td><td align=\"left\">19</td><td align=\"left\">20</td><td align=\"left\">23</td><td align=\"left\">2.9</td><td align=\"left\">24</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Player11</td><td align=\"left\">40</td><td align=\"left\">15</td><td align=\"left\">33</td><td align=\"left\">16</td><td align=\"left\">9</td><td align=\"left\">24</td><td align=\"left\">25</td><td align=\"left\">− 9</td><td align=\"left\">6</td><td align=\"left\">37</td><td align=\"left\">19.6</td><td align=\"left\">9</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">MEAN</td><td align=\"left\">25.00</td><td align=\"left\">40.09</td><td align=\"left\">44.73</td><td align=\"left\">47.73</td><td align=\"left\">38.36</td><td align=\"left\">33.09</td><td align=\"left\">26.73</td><td align=\"left\">9.55</td><td align=\"left\">7.27</td><td align=\"left\">20.45</td><td align=\"left\">29.30</td><td align=\"left\">35.64</td><td char=\".\" align=\"char\">37.39</td></tr><tr><td align=\"left\">STDEVA</td><td align=\"left\">21.61</td><td align=\"left\">23.57</td><td align=\"left\">26.02</td><td align=\"left\">28.36</td><td align=\"left\">23.93</td><td align=\"left\">24.52</td><td align=\"left\">31.69</td><td align=\"left\">20.76</td><td align=\"left\">27.38</td><td align=\"left\">20.86</td><td align=\"left\">19.05</td><td align=\"left\">21.21</td><td char=\".\" align=\"char\">33.12</td></tr></tbody></table></table-wrap></p><p id=\"Par21\">41 players played against the multi-AI with focus length F = 5 and AI-1 to AI-5(see blue bars in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>) and 11 players played against the multi-AI with focus length F = 10 and AI-1 to AI-10 (see orange bars in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). From the overall results in Table <xref rid=\"Tab5\" ref-type=\"table\">5</xref>, we see that our multi-AI algorithm with F = 10 give similar scores, but has lower standard deviation than that with F = 5.</p><p id=\"Par22\">For simplicity, we let multi-5AI denote our multi-AI model with a combination of the 1st–5th order Markov chain models (here focus length F is also set to 5, but it can be any other integer), and multi-10AI denote our multi-AI model with a combination of the 1st–10th order Markov chain models (focus length F is also set to 10).</p><p id=\"Par24\">We looked at the performance of individual models within a multi-AI 300 game set. The AI which performs the best against a particular individual varies greatly, but overall AI 2–6 perform better than AI 1 or higher-order Markov chains of longer memory length. This general trend is consistent with human short memory holding around 7 items<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>.</p><p id=\"Par25\">Figures <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> and <xref rid=\"Fig4\" ref-type=\"fig\">4</xref> show AI with different memory lengths’ performance against specific human players.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Game results (total scores) of Multi-10AI competing with human in 300 rounds.</p></caption><graphic xlink:href=\"41598_2020_70544_Fig3_HTML\" id=\"MO3\"/></fig><fig id=\"Fig4\"><label>Figure 4</label><caption><p>Game results (total scores) of multi-AI with Markov chain lengths 1–5 competing with 8 typical players in 300 rounds.</p></caption><graphic xlink:href=\"41598_2020_70544_Fig4_HTML\" id=\"MO4\"/></fig></p><p id=\"Par26\">It is hard to build one single model that can exploit every different human’s behavior, and thus we decided to combine the single models to make it able to differentiate and adapt to more different human competition strategies and win against most of its opponents.</p></sec><sec id=\"Sec3\"><title>Discussion and conclusions</title><p id=\"Par27\">In this paper, we have introduced a multi-AI model that wins against human in iterated RPS games and experimentally confirmed our results. We found that using a single-length Markov model could beat most human players, but not all human players.</p><p id=\"Par28\">In single model experiments, we found the model with the best performance varies greatly for different people, which indicates that different people have different patterns. Although different humans have different patterns, and in total the patterns may be very hard to observe and exploit. Human competition behavior indeed has patterns and the patterns are exploitable by using proper simple models (single models that successfully predict this human’s behavior). We have obtained and exploited different human behaviors by building and combining single Markov chain models of different memory lengths and during the competition process it learns and switches to the best prediction model according to its focus length. We have introduced one possible architecture for human AI RPS games competition, and this model could be further improved by e.g., optimizing the voting weights of single Markov chain models, using the first part of the competition data to pre-train multi-AI model and switch to only two or three dominant single models after the pre-training process. Focus length is a hyper parameter and can be tested by more human experiments for further optimization. After rearranging single models and adjusting “focus length” our model can potentially be improved further. The competition behavior patterns and their successful exploitation may lead our future work to better modeling, predicting and adapting to different specific human’s competition behavior patterns.</p></sec><sec id=\"Sec4\"><title>Methods</title><sec id=\"Sec5\"><title>Experiment</title><p id=\"Par29\">Our methods and regulations for this experiment mostly follow the RPS social experiments conducted in the same university by Zhijian Wang et al.<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. The first author confirms that all methods and experiments were carried out in accordance with the guidelines and regulations of “5 Ethical Considerations in Sociological Research” in Code of Ethics made by The American Sociological Association (ASA)<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> and approved by the Ethics Committee of our center of Zhejiang University (China). The humans competing against our multi-AI in iterated RPS game experiment was approved by the relevant laboratory of our center of Zhejiang University and performed at Zhejiang University in the period of July 2019 to September 2019. A total number of 52 undergraduate and graduate students of Zhejiang University volunteered to serve as the human subjects of this experiment. These students were openly recruited at the university library and informed consent was collected from all the participating human subjects. The 52 human subjects (referred to as players in this paper) carried one experimental session by playing the RPS game for 300 rounds with fixed payoff parameter a = 2. To prepare for the experiments, the players were led and seated separately in a lab room, each facing a computer screen. They were not allowed to communicate with each other throughout the experiment. Written instructions were handed out to each player and the rules of the experiment were also orally explained by an experimental instructor. The rules of the experimental session are as follows:<list list-type=\"simple\"><list-item><label>i.</label><p id=\"Par30\">Each player plays the RPS game repeatedly with our computer program.</p></list-item><list-item><label>ii.</label><p id=\"Par31\">Each player earns virtual points during the experimental session where each winning round will earn the player 2 virtual points and each drawing round will earn the player 1 virtual point, and the number of virtual points won’t change if the player loses. These virtual points are then exchanged into money in RMB as a reward to the player, plus an additional 5 RMB as show-up fee.</p></list-item><list-item><label>iii.</label><p id=\"Par32\">After the decision has been made it cannot be changed.</p></list-item></list></p><p id=\"Par33\">Before the formal experimental session begins, the players were asked four questions to ensure that they completely understand the rules of the experimental session. The four questions are: (1) If your opponent chooses “Scissors” and you choose “Rock”, how much money will you earn for this round? (2) If your opponent chooses “Rock” and you choose “Rock”, how much money will you earn for this round? (3) If your opponent chooses “Rock” and you choose “Scissors”, how much money will you earn for this round? (4) Do you know that at each game round you will play with an AI opponent? During the experimental session, an information window and a decision window are displayed on the computer screen in front of each player. The window on the left of the computer screen is the information window. The current game round, the time limit (40 s) of making a choice, and the time left to make a choice are displayed on the upper panel of the information window. At the beginning of each game round, the color of this upper panel turns to green. If the player does not make a decision within 20 s, the color will change to yellow. The color will change to red if the decision time runs out (then the experimental instructor will urge the player to make a decision immediately). Fortunately, all the players made their decisions within 20 s. The color will change to blue if a decision has been made by the player. After the player has made his/her decision for this round, the lower panel of the information window will immediately show the opponent's choice, the player's choice, and the player's payoff for this game round. Each player's cumulated payoff is also shown on the screen. The players are asked to write down their own choices of each game round on the record sheet (Rock as <italic>R</italic>, Paper as <italic>P</italic>, and Scissors as <italic>S</italic>). The decision window is on the right side of the computer screen. The upper panel of the decision window lists the current game round, and the bottom panel lists the three candidate actions “Rock”, “Scissors”, “Paper” horizontally from left to right. The player can make a decision by clicking on the corresponding action word. After the decision has been made, the player will know the result for this game round and the decision window will be asking for the next game input. The reward money in RMB for each player is determined by the following formula. Suppose a player <italic>i</italic> earns <italic>x</italic><sub><italic>i</italic></sub> virtual points in the whole experimental session, the total reward <italic>y</italic><sub><italic>i</italic></sub> in RMB for this player is given by <italic>y</italic><sub><italic>i</italic></sub> = <italic>x</italic><sub><italic>i</italic></sub> × <italic>r</italic> + 5, where <italic>r</italic> is the exchange rate between virtual point and money in RMB. According to the mixed-strategy Nash equilibrium, each player’s expected payoff in one game round is (1 + <italic>a</italic>)/3. Therefore, we set the exchange rate to be <italic>r</italic> = 0.45/(1 + <italic>a</italic>) to ensure that, under the mixed-strategy NE assumption, the expected total earning for a player will be 50 RMB regardless of the particular experimental session. The payoff parameter <italic>a is set to 2</italic>. The numerical value of <italic>r is 0.15</italic>. The above-mentioned reward formula and how much virtual points you will earn if you win, draw or lose were listed in the printed instruction and also verbally mentioned at the instruction phase of the experiment by the experimental instructor.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec6\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70544_MOESM1_ESM.xlsx\"><caption><p>Supplementary Information 1.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41598_2020_70544_MOESM2_ESM.xlsx\"><caption><p>Supplementary Information 2.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p> is available for this paper at 10.1038/s41598-020-70544-7.</p></sec><ack><title>Acknowledgements</title><p>This work was partially supported by the National Key Research and Development Program of China (No. 2018YFC1407506), the National Natural Science Foundation of China (No. 11621101), and the Fundamental Research Funds for the Central Universities (Zhejiang University NGICS Platform). We thank Shuo Li for excellent assistance in arranging the experiments. Open access funding provided by Royal Institute of Technology.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>S.H. conceived and supervised the work. L.W. developed the algorithm and built the multi-AI. L.W., Y. L. and W.H. performed the experiments and collected data. L.W. processed and analyzed the data. The manuscript was discussed and written by L.W., J.E. and S.H. with comments and input from all authors.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>All 52 players experiment data with all process data of Markov chains we used are provided in the Supplementary Informations <xref rid=\"MOESM1\" ref-type=\"media\">1</xref> and <xref rid=\"MOESM2\" ref-type=\"media\">2</xref>.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par34\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>O'Dwyer</surname><given-names>JP</given-names></name></person-group><article-title>Contests between species aid biodiversity</article-title><source>Nature</source><year>2017</year><volume>548</volume><fpage>166</fpage><lpage>167</lpage><pub-id pub-id-type=\"doi\">10.1038/nature23103</pub-id><pub-id pub-id-type=\"pmid\">28746309</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grilli</surname><given-names>J</given-names></name><name><surname>Barabás</surname><given-names>G</given-names></name><name><surname>Michalska-Smith</surname><given-names>MJ</given-names></name><name><surname>Allesina</surname><given-names>S</given-names></name></person-group><article-title>Higher-order interactions stabilize dynamics in competitive network models</article-title><source>Nature</source><year>2017</year><volume>548</volume><fpage>210</fpage><lpage>213</lpage><pub-id pub-id-type=\"doi\">10.1038/nature23273</pub-id><pub-id pub-id-type=\"pmid\">28746307</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bergstrom</surname><given-names>CT</given-names></name><name><surname>Kerr</surname><given-names>B</given-names></name></person-group><article-title>Taking the bad with the good</article-title><source>Nature</source><year>2015</year><volume>521</volume><fpage>431</fpage><lpage>432</lpage><pub-id pub-id-type=\"doi\">10.1038/nature14525</pub-id><pub-id pub-id-type=\"pmid\">25992542</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><mixed-citation publication-type=\"other\">Allesina, S. & Levine, J. M. <italic>A Competitive Network Theory of Species Diversity</italic>. <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.pnas.org/content/108/14/5638.abstract\">http://www.pnas.org/content/108/14/5638.abstract</ext-link> (2011).</mixed-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reichenbach</surname><given-names>T</given-names></name><name><surname>Mobilia</surname><given-names>M</given-names></name><name><surname>Frey</surname><given-names>E</given-names></name></person-group><article-title>Mobility promotes and jeopardizes biodiversity in rock–paper–scissors games</article-title><source>Nature</source><year>2007</year><volume>448</volume><fpage>1046</fpage><lpage>1049</lpage><pub-id pub-id-type=\"doi\">10.1038/nature06095</pub-id><pub-id pub-id-type=\"pmid\">17728757</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>D</given-names></name><name><surname>McGreevy</surname><given-names>BP</given-names></name><name><surname>Barraclough</surname><given-names>DJ</given-names></name></person-group><article-title>Learning and decision making in monkeys during a rock–paper–scissors game</article-title><source>Cogn. Brain Res.</source><year>2005</year><volume>25</volume><fpage>416</fpage><lpage>430</lpage><pub-id pub-id-type=\"doi\">10.1016/j.cogbrainres.2005.07.003</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Semmann</surname><given-names>D</given-names></name><name><surname>Krambeck</surname><given-names>H-J</given-names></name><name><surname>Milinski</surname><given-names>M</given-names></name></person-group><article-title>Volunteering leads to rock–paper–scissors dynamics in a public goods game</article-title><source>Nature</source><year>2003</year><volume>425</volume><fpage>390</fpage><lpage>393</lpage><pub-id pub-id-type=\"doi\">10.1038/nature01986</pub-id><pub-id pub-id-type=\"pmid\">14508487</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kerr</surname><given-names>B</given-names></name><name><surname>Riley</surname><given-names>MA</given-names></name><name><surname>Feldman</surname><given-names>MW</given-names></name><name><surname>Bohannan</surname><given-names>BJM</given-names></name></person-group><article-title>Local dispersal promotes biodiversity in a real-life game of rock–paper–scissors</article-title><source>Nature</source><year>2002</year><volume>418</volume><fpage>171</fpage><lpage>174</lpage><pub-id pub-id-type=\"doi\">10.1038/nature00823</pub-id><pub-id pub-id-type=\"pmid\">12110887</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sinervo</surname><given-names>B</given-names></name><name><surname>Lively</surname><given-names>CM</given-names></name></person-group><article-title>The rock–paper–scissors game and the evolution of alternative male strategies</article-title><source>Nature</source><year>1996</year><volume>380</volume><fpage>240</fpage><lpage>243</lpage><pub-id pub-id-type=\"doi\">10.1038/380240a0</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cason</surname><given-names>TN</given-names></name><name><surname>Friedman</surname><given-names>D</given-names></name></person-group><article-title>Buyer search and price dispersion: a laboratory study</article-title><source>J. Econ. Theory</source><year>2003</year><volume>112</volume><fpage>232</fpage><lpage>260</lpage><pub-id pub-id-type=\"doi\">10.1016/S0022-0531(03)00135-0</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maskin</surname><given-names>E</given-names></name><name><surname>Tirole</surname><given-names>J</given-names></name></person-group><article-title>A theory of dynamic oligopoly, II: price competition, kinked demand curves, and Edgeworth cycles</article-title><source>Econometrica</source><year>1988</year><volume>56</volume><fpage>571</fpage><lpage>599</lpage><pub-id pub-id-type=\"doi\">10.2307/1911701</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>Z</given-names></name><name><surname>Xu</surname><given-names>B</given-names></name><name><surname>Zhou</surname><given-names>H-J</given-names></name></person-group><article-title>Social cycling and conditional responses in the Rock–Paper–Scissors game</article-title><source>Sci. Rep.</source><year>2014</year><volume>4</volume><fpage>5830</fpage><pub-id pub-id-type=\"doi\">10.1038/srep05830</pub-id><pub-id pub-id-type=\"pmid\">25060115</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Forder</surname><given-names>L</given-names></name><name><surname>Dyson</surname><given-names>BJ</given-names></name></person-group><article-title>Behavioural and neural modulation of win-stay but not lose-shift strategies as a function of outcome value in Rock, Paper, Scissors</article-title><source>Sci. Rep.</source><year>2016</year><volume>6</volume><fpage>33809</fpage><pub-id pub-id-type=\"doi\">10.1038/srep33809</pub-id><pub-id pub-id-type=\"pmid\">27658703</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><mixed-citation publication-type=\"other\">Hoffman, M., Suetens, S., Nowak, M. A. & Gneezy, U. In <italic>Proceedings of Fourth World Congress of the Game Theory Society (Istanbul, Turkey), session.</italic></mixed-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Claussen</surname><given-names>JC</given-names></name><name><surname>Traulsen</surname><given-names>A</given-names></name></person-group><article-title>Cyclic dominance and biodiversity in well-mixed populations</article-title><source>Phys. Rev. Lett.</source><year>2008</year><volume>100</volume><fpage>058104</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevLett.100.058104</pub-id><pub-id pub-id-type=\"pmid\">18352437</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Castellano</surname><given-names>C</given-names></name><name><surname>Fortunato</surname><given-names>S</given-names></name><name><surname>Loreto</surname><given-names>V</given-names></name></person-group><article-title>Statistical physics of social dynamics</article-title><source>Rev. Mod. Phys.</source><year>2009</year><volume>81</volume><fpage>591</fpage><lpage>646</lpage><pub-id pub-id-type=\"doi\">10.1103/RevModPhys.81.591</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ao</surname><given-names>P</given-names></name><name><surname>Chen</surname><given-names>T-Q</given-names></name><name><surname>Shi</surname><given-names>J-H</given-names></name></person-group><article-title>Dynamical decomposition of markov processes without detailed balance</article-title><source>Chin. Phys. Lett.</source><year>2013</year><volume>30</volume><fpage>070201</fpage><pub-id pub-id-type=\"doi\">10.1088/0256-307X/30/7/070201</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ao</surname><given-names>P</given-names></name></person-group><article-title>Emerging of stochastic dynamical equalities and steady state thermodynamics from darwinian dynamics</article-title><source>Commun. Theor. Phys.</source><year>2008</year><volume>49</volume><fpage>1073</fpage><lpage>1090</lpage><pub-id pub-id-type=\"doi\">10.1088/0253-6102/49/5/01</pub-id><pub-id pub-id-type=\"pmid\">21949462</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><mixed-citation publication-type=\"other\">Zhou, T., Han, X.-P. & Wang, B.-H. In <italic>Science Matters: Humanities as Complex Systems</italic>, 207–233 (World Scientific, 2008).</mixed-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grewal</surname><given-names>JK</given-names></name><name><surname>Krzywinski</surname><given-names>M</given-names></name><name><surname>Altman</surname><given-names>N</given-names></name></person-group><article-title>Markov models—Markov chains</article-title><source>Nat. Methods</source><year>2019</year><volume>16</volume><fpage>663</fpage><lpage>664</lpage><pub-id pub-id-type=\"doi\">10.1038/s41592-019-0476-x</pub-id><pub-id pub-id-type=\"pmid\">31363204</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>Q</given-names></name><name><surname>Fang</surname><given-names>D</given-names></name><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Jin</surname><given-names>C</given-names></name><name><surname>Ren</surname><given-names>Q</given-names></name></person-group><article-title>Stochastic evolution dynamic of the rock–scissors–paper game based on a quasi birth and death process</article-title><source>Sci. Rep.</source><year>2016</year><volume>6</volume><fpage>28585</fpage><pub-id pub-id-type=\"doi\">10.1038/srep28585</pub-id><pub-id pub-id-type=\"pmid\">27346701</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Skewes</surname><given-names>AD</given-names></name><name><surname>Welch</surname><given-names>RD</given-names></name></person-group><article-title>A Markovian analysis of bacterial genome sequence constraints</article-title><source>PeerJ</source><year>2013</year><volume>1</volume><fpage>e127</fpage><pub-id pub-id-type=\"doi\">10.7717/peerj.127</pub-id><pub-id pub-id-type=\"pmid\">24010012</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lisman</surname><given-names>JE</given-names></name><name><surname>Idiart</surname><given-names>MA</given-names></name></person-group><article-title>Storage of 7 +/- 2 short-term memories in oscillatory subcycles</article-title><source>Science</source><year>1995</year><volume>267</volume><fpage>1512</fpage><pub-id pub-id-type=\"doi\">10.1126/science.7878473</pub-id><pub-id pub-id-type=\"pmid\">7878473</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><mixed-citation publication-type=\"other\"><italic>American Sociological Association</italic>. <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.asanet.org/code-ethics\">http://www.asanet.org/code-ethics</ext-link> (2020).</mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807834</article-id><article-id pub-id-type=\"pmc\">PMC7431551</article-id><article-id pub-id-type=\"publisher-id\">70930</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70930-1</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Promotional effect of magnesium oxide for a stable nickel-based catalyst in dry reforming of methane</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Al-Fatesh</surname><given-names>Ahmed S.</given-names></name><address><email>aalfatesh@ksu.edu.sa</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kumar</surname><given-names>Rawesh</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Fakeeha</surname><given-names>Anis H.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kasim</surname><given-names>Samsudeen O.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Khatri</surname><given-names>Jyoti</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ibrahim</surname><given-names>Ahmed A.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Arasheed</surname><given-names>Rasheed</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Alabdulsalam</surname><given-names>Muhamad</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lanre</surname><given-names>Mahmud S.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Osman</surname><given-names>Ahmed I.</given-names></name><address><email>aosmanahmed01@qub.ac.uk</email></address><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Abasaeed</surname><given-names>Ahmed E.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Bagabas</surname><given-names>Abdulaziz</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.56302.32</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1773 5396</institution-id><institution>Chemical Engineering Department, College of Engineering, </institution><institution>King Saud University, </institution></institution-wrap>P.O. Box 800, Riyadh, 11421 Saudi Arabia </aff><aff id=\"Aff2\"><label>2</label>Sankalchand Patel University, Visnagar, Gujarat 384315 India </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.452562.2</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 8808 6435</institution-id><institution>National Petrochemical Technology Center (NPTC), Materials Science Research Institute (MSRI), </institution><institution>King Abdulaziz City for Science and Technology, </institution></institution-wrap>P.O. Box 6086, Riyadh, 11442 Saudi Arabia </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.4777.3</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0374 7521</institution-id><institution>School of Chemistry and Chemical Engineering, </institution><institution>Queen’s University Belfast, </institution></institution-wrap>Belfast, BT9 5AG Northern Ireland UK </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13861</elocation-id><history><date date-type=\"received\"><day>7</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>4</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">The generation of synthesis gas (hydrogen and carbon monoxide mixture) from two global warming gases of carbon dioxide and methane via dry reforming is environmentally crucial and for the chemical industry as well. Herein, magnesium-promoted NiO supported on mesoporous zirconia, 5Ni/xMg–ZrO<sub>2</sub> (x = 0, 3, 5, 7 wt%) were prepared by wet impregnation method and then were tested for syngas production via dry reforming of methane. The reaction temperature at 800 °C was found more catalytically active than that at 700 °C due to the endothermic feature of reaction which promotes efficient CH<sub>4</sub> catalytic decomposition over Ni and Ni–Zr interface as confirmed by CH<sub>4</sub>–TSPR experiment. NiO–MgO solid solution interacted with ZrO<sub>2</sub> support was found crucial and the reason for high CH<sub>4</sub> and CO<sub>2</sub> conversions. The highest catalyst stability of the 5Ni/3Mg–ZrO<sub>2</sub> catalyst was explained by the ability of CO<sub>2</sub> to partially oxidize the carbon deposit over the surface of the catalyst. A mole ratio of hydrogen to carbon monoxide near unity (H<sub>2</sub>/CO ~ 1) was obtained over 5Ni/ZrO<sub>2</sub> and 5Ni/5Mg–ZrO<sub>2</sub>, implying the important role of basic sites. Our approach opens doors for designing cheap and stable dry reforming catalysts from two potent greenhouse gases which could be of great interest for many industrial applications, including syngas production and other value-added chemicals.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Chemical engineering</kwd><kwd>Environmental sciences</kwd><kwd>Chemistry</kwd></kwd-group><funding-group><award-group><funding-source><institution>UK Research and Innovation</institution></funding-source><award-id>EP/S025545/1</award-id><principal-award-recipient><name><surname>Osman</surname><given-names>Ahmed I.</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">The production of syngas (a mixture of H<sub>2</sub> and CO) through dry reforming of methane is an excellent strategy to reduce the global warming effects of carbon dioxide (CO<sub>2</sub>) and methane (CH<sub>4</sub>). Noble metals such as palladium (Pd), platinum (Pt), and ruthenium (Ru) have been used for this purpose, but costly precursors and instability of catalyst, at high reaction temperature around 800 °C, have limited their application<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. On the other hand, cost-effective nickel (Ni) metal, supported on an appropriate supports such as alumina<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>, mesoporous silicates<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>–<xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>, and zirconia<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>–<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>, has been found to withstand at this reaction temperature (800 °C). In this context, many researchers have followed the surface modification methodology to optimise the catalyst performance because Ni-based catalyst is also prone to deactivation. The first series of modifications were carried out over alumina supports with K<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>, Mg, Ca, Ba, Sr <sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>–<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>, Y<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>, La<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>, Ce<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>, K-Ce<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>, Ti<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>, Zr<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>, Mo, W<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>, Mn<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>, Co & Cu<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>, Zn<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>, B<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>, Si<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>, and Sn<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Due to the extended pore network (from micro to meso) and easy pore tunable synthetic methodology of silicates, silica support is preferable over alumina support<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. Therefore, the second series of modifications were carried out over mesoporous silicates supports with Li<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>, K<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>, Mg<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>, Ca<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>, Ba<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, La<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>, Ce<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>, Zr<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>, Mn<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>, Co<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref>,<xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>, Cu<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>, Zn<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>, Al<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup> and Sn<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. However, neither alumina nor silica supports can utilize their lattice oxygen during carbon deposit oxidation at the surface, but zirconia support does and is thus are used as oxygen carrier materials. Zirconia support is characterized by its thermal stability, an expanded network, and the ability to utilize its mobile oxygen during the surface reaction<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. The third series of modifications were carried out over zirconia supports with K<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>, Mg<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref>–<xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>, K-MgO<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>, Ca<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref>,<xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>, La<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref>,<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>, and Ce<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref>–<xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup>. A brief literature survey of promoter/modifiers that were utilized over Ni-doped different supports is given in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>. <table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>A brief literature survey of promoter/modifier Ni-doped different supports.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Active metal</th><th align=\"left\" colspan=\"2\">Modified/promoter</th><th align=\"left\">Support: Al<sub>2</sub>O<sub>3</sub></th><th align=\"left\">Support: SiO<sub>2</sub></th><th align=\"left\">Support: ZrO<sub>2</sub></th></tr></thead><tbody><tr><td align=\"left\">Ni</td><td align=\"left\" rowspan=\"2\">Group I</td><td align=\"left\">Li</td><td align=\"left\"/><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup></td><td align=\"left\"/></tr><tr><td align=\"left\">Ni</td><td align=\"left\">K</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup></td></tr><tr><td align=\"left\">Ni</td><td align=\"left\" rowspan=\"4\">Group II</td><td align=\"left\">Mg</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>–<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref>–<xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup></td></tr><tr><td align=\"left\">Ni</td><td align=\"left\">Ca</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>–<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref>,<xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup></td></tr><tr><td align=\"left\">Ni</td><td align=\"left\">Sr</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>–<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup></td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">Ni</td><td align=\"left\">Ba</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>–<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup></td><td align=\"left\"/></tr><tr><td align=\"left\">Ni</td><td align=\"left\" rowspan=\"3\">Group III</td><td align=\"left\">Y</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup></td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">Ni</td><td align=\"left\">La</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref>,<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup></td></tr><tr><td align=\"left\">Ni</td><td align=\"left\">Ce</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref>–<xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup></td></tr><tr><td align=\"left\">Ni</td><td align=\"left\" rowspan=\"2\">Group IV</td><td align=\"left\">Ti</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup></td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">Ni</td><td align=\"left\">Zr</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup></td><td align=\"left\">–</td></tr><tr><td align=\"left\">Ni</td><td align=\"left\" rowspan=\"2\">Group VI</td><td align=\"left\">Mo</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup></td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">Ni</td><td align=\"left\">W</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup></td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">Ni</td><td align=\"left\">Group VII</td><td align=\"left\">Mn</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup></td><td align=\"left\"/></tr><tr><td align=\"left\">Ni</td><td align=\"left\">Group IX</td><td align=\"left\">Co</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref>,<xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup></td><td align=\"left\"/></tr><tr><td align=\"left\">Ni</td><td align=\"left\">Group XI</td><td align=\"left\">Cu</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup></td><td align=\"left\"/></tr><tr><td align=\"left\">Ni</td><td align=\"left\">Group XII</td><td align=\"left\">Zn</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup></td><td align=\"left\"/></tr><tr><td align=\"left\">Ni</td><td align=\"left\" rowspan=\"2\">Group XIII</td><td align=\"left\">B</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup></td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">Ni</td><td align=\"left\">Al</td><td align=\"left\">–</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup></td><td align=\"left\"/></tr><tr><td align=\"left\">Ni</td><td align=\"left\" rowspan=\"2\">Group XIV</td><td align=\"left\">Si</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup></td><td align=\"left\">–</td><td align=\"left\"/></tr><tr><td align=\"left\">Ni</td><td align=\"left\">Sn</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup></td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup></td><td align=\"left\"/></tr></tbody></table></table-wrap></p><p id=\"Par3\">Use of strong solid base as CaO and MgO showed significant improvement and facilitated the catalytic performance with prompt adsorption of slightly acidic CO<sub>2</sub> during dry reforming reaction over Ni-based catalysts. CaO coprecipitated Ni supported ZrO<sub>2</sub> was well studied for different types of carbon species deposited over the catalyst surface during dry reforming of methane<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>. MgO modified Ni system is known for outstanding coking tolerance<sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup>. Chunwen Sun et al. showed that MgO modification might help to stabilize the lattice oxygen sites of NiO which effectively decrease the carbon deposition or graphitic layer formation<sup><xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup>. Garcia et al. prepared the Ni/MgO–ZrO<sub>2</sub>–MgO (MgO loading in the range of 1–5 wt%) catalysts by co-precipitation method and found out that the CO<sub>2</sub> and CH<sub>4</sub> conversions were less than 35%<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>. Asencios and Assaf loaded Ni and Mg with different ratios on zirconia support by wet impregnation method and found out that catalyst with 20 wt% Ni and 20 mol% Mg has the best performance, where the activity was less than 80% in the oxidative reforming of methane<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup>. Most of the research outputs in the literature used high loading of Ni or MgO (as high as 35 mol%) for the dry reforming reaction as Montoya et al. via sol–gel method<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup> and Titus et al. via melt impregnation<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>.</p><p id=\"Par4\">Herein, we prepared four catalysts via incipient wetness impregnation method, where the support was mesoporous zirconia, nickel as the active catalyst, and magnesium oxide as a promoter. We varied the amount of magnesium oxide to find its optimum loading for the best catalytic performance. Furthermore, we optimised the performance by varying reaction temperature. Catalysts were characterized by TGA, N<sub>2</sub> physisorption, XRD, H<sub>2</sub>-TPR, and CO<sub>2</sub>-TPD. To understand the surface chemistry in optimizing the catalytic activity along with the stability of the modified catalyst, CO<sub>2</sub>-TPD, H<sub>2</sub>-TPD and O<sub>2</sub>-TPO of spent catalyst were also performed. A very fine-tuning, among catalytic activity and characterization results were performed; this will help to better understand the surface behaviour towards syngas production from dry reforming of methane.</p></sec><sec id=\"Sec25\"><title>Results and discussion</title><p id=\"Par5\">The catalytic activity of 5NixMgZr catalysts (x = 0, 3, 5, 7) in terms of CH<sub>4</sub> conversion, CO<sub>2</sub> conversion, and H<sub>2</sub>/CO mole ratio at 700 °C are shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>(A–C) and at 800 °C are shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>(D–F). The TGA results of spent catalysts are shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>(G,H), respectively. It is worth noting that without magnesium oxide modification, catalyst 5Ni/ZrO<sub>2</sub> shows lower catalytic activity than that of magnesium oxide modified catalyst in all cases. At the reaction temperature of 700 °C, 5Ni/xMg–ZrO<sub>2</sub> catalysts showed approximately 50–60% CH<sub>4</sub> conversion and 65–75% CO<sub>2</sub> conversion which were comparable to those in the recent publications<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref>,<xref ref-type=\"bibr\" rid=\"CR49\">49</xref>,<xref ref-type=\"bibr\" rid=\"CR54\">54</xref>,<xref ref-type=\"bibr\" rid=\"CR62\">62</xref>,<xref ref-type=\"bibr\" rid=\"CR63\">63</xref></sup>. The TGA results of these spent catalysts also showed carbon deposition. Interestingly, when the reaction temperature was set at 800 °C, it gave a stable performance with constant high conversion up to 500 min in the time-on-stream test (TOS) and no noticeable carbon deposition. Over 5Ni/3Mg–ZrO<sub>2</sub> catalyst, 85% CH<sub>4</sub> conversion, 92% CO<sub>2</sub> conversion and 0.94 H<sub>2</sub>/CO ratios were achieved constantly up to 500 min in the TOS. On the target of H<sub>2</sub>/CO = 1, the performance of the 5Ni/5Mg–ZrO<sub>2</sub> catalyst was found to be the best as it showed 82% CH<sub>4</sub> conversion, 87% CO<sub>2</sub> conversion. The 5Ni/7Mg–ZrO<sub>2</sub> catalyst performance was a little bit lower than that of 5Ni/5Mg–ZrO<sub>2</sub> (78% CH<sub>4</sub> conversion, 86% CO<sub>2</sub> conversion and H<sub>2</sub>/CO = 0.98). Whether to check the thermal decomposition of CH<sub>4</sub> at 800 °C, reaction without catalyst was carried out with substrate CH<sub>4</sub>. It gave 3% CH<sub>4</sub> conversion with 1.8% H<sub>2</sub> yield in 3 h time on stream. Further again, a blank reaction was carried out with substrates CH<sub>4</sub> and CO<sub>2</sub> together at 800 °C. It resulted in 1.6% CH<sub>4</sub> conversion, 3.6% CO<sub>2</sub> conversion, H<sub>2</sub> yield 0.63%, CO yield 4.25% and H<sub>2</sub>/CO = 0.14. As our catalytic systems are highly active towards DRM, so the thermal decomposition of CH<sub>4</sub> as an intermediate step in DRM could be neglected.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Catalytic activity profiles for methane dry reforming over various catalysts (<bold>A</bold>–<bold>F</bold>); (<bold>A</bold>) CH<sub>4</sub> conversion at 700 °C reaction temperature, (<bold>B</bold>) CO<sub>2</sub> conversion at 700 °C reaction temperature, (<bold>C</bold>) H<sub>2</sub>/CO mole ratio at 700 °C reaction temperature, (<bold>D</bold>) CH<sub>4</sub> conversion at 800 °C reaction temperature, (<bold>E</bold>) CO<sub>2</sub> conversion at 800 °C reaction temperature, (<bold>F</bold>) H<sub>2</sub>/CO mole ratio at 800 °C along with TGA curves of spent catalysts, (<bold>G</bold>) TGA results of spent catalyst carried out at 700 °C reaction temperature, (<bold>H</bold>) TGA results of spent catalysts carried out at 800 °C reaction temperature.</p></caption><graphic xlink:href=\"41598_2020_70930_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par6\">To understand the surface behaviour of the DRM reaction, we characterised the catalyst thoroughly and discussed the characterization results herein. The surface area analysis indicated that after the addition of MgO, type IV adsorption–desorption curve with H1 hysteresis loop (Figure <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>) was built up. It indicates the narrow distribution of mesopores.</p><p id=\"Par7\">XRD patterns of 5NixMgZr catalysts (x = 0, 3, 5, 7) are shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>(A–D). The diffraction lines at 2θ = 24.2°, 28.34°, 31.45°, 34.2°, and 55.4° were attributed to the monoclinic zirconia (m-ZrO<sub>2</sub>) whereas diffraction lines at 2θ = 30.48° and 50.24° were attributed to tetragonal zirconia (t-ZrO<sub>2</sub>). Cubic nickel oxide showed diffraction lines at 2θ = 37.2°, 43.28° and 62.9° for (111), (200) and (220) crystallographic planes, respectively. After the addition of basic promoter 3 wt% MgO, the crystalline peak intensity of ZrO<sub>2</sub> remarkably increased as well as the selected plane of NiO (200) about 43.28° bragg angle also intensified and shifted to the lower angle 43.12°. It indicated the rapid growth of NiO-MgO solid solution<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup> after addition of MgO. Further addition of MgO did not show such a rapid rise of NiO-MgO solid solution.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>XRD of catalyst samples: m = monoclinic zirconia (m-ZrO<sub>2</sub>), t = tetragonal zirconia (t-ZrO<sub>2</sub>), n = NiO.</p></caption><graphic xlink:href=\"41598_2020_70930_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par8\">The H<sub>2</sub>-TPR surface reduction profiles of fresh 5Ni/xMg–ZrO<sub>2</sub> catalysts are shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>A. 5Ni/ZrO<sub>2</sub> has one small reduction peak in the temperature range of 140–200 °C that attributed to the free NiO species, a shoulder reduction peak at the temperature range of 200–300 °C for “NiO weakly interacted with ZrO<sub>2</sub> support” and a strong peak at 300–450 °C for “NiO that interacted strongly with ZrO<sub>2</sub> support”. After the addition of 3.0 wt% MgO, these three peaks diminished and reduction peaks in the intermediate and high-temperature ranges appeared. The high reduction temperature for MgO modified samples could be correlated to the high inherent stability expected for NiO–MgO-solid solution with respect to pure NiO. From the XRD results, also after MgO modification, NiO–MgO-solid solution was found<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>. The intermediate temperature reduction peak in the range of 450–700 °C could be attributed to “NiO–MgO-solid solution weakly interacted with ZrO<sub>2</sub> support” whereas high-temperature reduction peak in the range of 700–900 °C could be claimed to “NiO–MgO-solid solution strongly interacted with ZrO<sub>2</sub> support”. As MgO loading was increased from 3.0 wt% to 5.0 wt%, the TCD signal intensity of the intermediate temperature reduction peak was decreased and high-temperature reduction peak was increased. These observations indicated that a higher amount of “NiO-MgO-solid solution strongly interacted on ZrO<sub>2</sub>” was present in 5Ni/5Mg–ZrO<sub>2</sub> than 5Ni/3Mg–ZrO<sub>2</sub>, thus 5 wt% MgO was the optimum loading. At 7 wt% MgO loading, both types of high-temperature peaks were suppressed in comparison to those for 5Ni/5Mg-ZrO<sub>2</sub>. The H<sub>2</sub>-TPR surface reduction profile of spent 5Ni/3Mg–ZrO<sub>2</sub> is shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>B. It showed that TPR peaks in the intermediate and high-temperature regions had got suppressed. Also, it was noticeable that a lower reduction temperature peak (0–400 °C) remained preserved as well as shifted to a lower temperature. The H<sub>2</sub>-TPR surface reduction profile of spent 5Ni/5Mg–ZrO<sub>2</sub> indicated the suppression and shifting of high-temperature region peaks to intermediate temperature regions (Fig. S2). These observations indicated that NiO supported on ZrO<sub>2</sub> was less involved whereas “NiO-MgO-solid solution interacted with ZrO<sub>2</sub> support” are significantly involved in DRM. Apart from that, the elimination of carbon deposit by hydrogen gas during methane gasification reaction (C + 2H<sub>2</sub> → CH<sub>4</sub>) over spent catalyst system was also possible<sup><xref ref-type=\"bibr\" rid=\"CR64\">64</xref></sup>.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>(<bold>A</bold>) The H<sub>2</sub>-TPR profile of 5Ni/xMg-ZrO<sub>2</sub>, (<bold>B</bold>) the H<sub>2</sub>-TPR profile of fresh and spent 5Ni/3Mg-ZrO<sub>2,</sub> (<bold>C</bold>) the CO<sub>2</sub>-TPD surface reduction profile of 5Ni/xMg-ZrO<sub>2</sub>, (<bold>D</bold>) CO<sub>2</sub>-TPD and O<sub>2</sub>-TPO TPD profile of fresh and spent 5Ni/3Mg-ZrO<sub>2</sub> catalyst.</p></caption><graphic xlink:href=\"41598_2020_70930_Fig3_HTML\" id=\"MO3\"/></fig></p><p id=\"Par9\">The CO<sub>2</sub>-TPD profiles of 5Ni/xMg–ZrO<sub>2</sub> are shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>C. Without magnesium oxide modification, the catalyst showed a sharp peak at lower temperature (weak basic sites) region and in intermediate temperature (medium basic sites) regions, but a broad peak in higher temperature regions (strong basic sites). This profile indicated a wide distribution of basic sites. However, after loading of 3.0 wt% MgO, only weak basic sites remained preserved; the rest disappeared. Surprisingly, basic modifier addition caused the disappearance of basicity. XRD of the same sample showed the appearance of NiO–MgO-solid solution as well as the rise of ZrO<sub>2</sub> crystallinity. This means that after the addition of basic 3.0 wt% MgO, basic MgO was engaged in the nurture of NiO–MgO solid solution and supported the crystallinity, thus it caused the disappearance of basicity. It caused the removal of intermediate strength as well as strong strength basic sites from the surface. Again, at 5 wt% MgO loading, peak reappeared in the intermediate temperature region whereas it broadened in high-temperature regions. As the TGA profile of the spent catalyst did not show markable carbon deposition, it is interesting to observe the basic profile of the spent catalyst.</p><p id=\"Par10\">The CO<sub>2</sub>-TPD profile of fresh as well as spent 5Ni/3Mg–ZrO<sub>2</sub> & 5Ni/5Mg–ZrO<sub>2</sub> catalyst are shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>D and Figure <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref> respectively. Figures <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>D and <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref> also include O<sub>2</sub>-TPO and “CO<sub>2</sub>-TPO followed by O<sub>2</sub>-TPO” of spent 5Ni/3Mg–ZrO<sub>2</sub> and 5Ni/5Mg–ZrO<sub>2</sub> catalysts, respectively. It is obvious from the fresh and spent CO<sub>2</sub>-TPD samples that there was a significant decrease in the intensity of basic sites after the reaction over the spent catalysts. However, unlike the fresh samples, the spent catalysts showed a small peak in CO<sub>2</sub>-TPD. Again, a consumption (negative) peak in O<sub>2</sub>-TPO of spent 5Ni/3Mg–ZrO<sub>2</sub> and spent 5Ni/5Mg–ZrO<sub>2</sub> catalyst samples were also seen at about the same temperature region. Interestingly, O<sub>2</sub>-TPO (carried out after CO<sub>2</sub>-TPD) of spent 5Ni/3Mg–ZrO<sub>2</sub> and spent 5Ni/5Mg–ZrO<sub>2</sub> catalysts had no such O<sub>2</sub> consumption peak. It can be explained that O<sub>2</sub> consumption peak in O<sub>2</sub>-TPO was due to oxidation of residual carbon by O<sub>2</sub> into CO<sub>2</sub>. So, the small evolution peak in CO<sub>2</sub>-TPD profile also indicated the oxidation of residual carbon deposit by CO<sub>2</sub>. As the carbon deposit on the surface of the catalyst was already oxidized by CO<sub>2</sub> during CO<sub>2</sub>-TPD profile so when O<sub>2</sub>-TPO was carried out after CO<sub>2</sub>-TPD, no evolution peak was found. This confirmed the oxidation of the carbon deposit by CO<sub>2</sub> over the surface of the catalyst<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref>,<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>. Oxidation of carbon deposit by lattice oxygen of ZrO<sub>2</sub> and thereafter simultaneous compensation of the oxygen vacant sites by CO<sub>2</sub> (through losing one of its oxygen to the vacant site) might be a possible route of oxidation of carbon deposit by CO<sub>2</sub>.</p><p id=\"Par11\">To study the conditions and sites of CH<sub>4</sub> decomposition, CH<sub>4</sub>-temperature programmed surface reaction (CH<sub>4</sub>-TPSR) experiment over ZrO<sub>2</sub>, 5Ni/ZrO<sub>2</sub> and 5Ni/3Mg–ZrO<sub>2</sub> were carried out (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>). It shows a decrease in the methane concentration with temperature over catalysts due to methane decomposition reaction on the surface. For ZrO<sub>2</sub>, a single prominent consumption peak at 870 °C temperature was noticed due to CH<sub>4</sub> interaction at ZrO<sub>2</sub> surface<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. After the addition of Ni, apart from the high-temperature peak, a lower temperature CH<sub>4</sub> consumption peak at about 350 °C and an intermediate temperature broad peak in the range of 400–800 °C were observed. Low temperature and intermediate temperature peaks could be claimed to the catalytic decomposition of CH<sub>4</sub> over Ni active sites as well as Ni–Zr interface<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. MgO containing catalysts (i.e. 5Ni/3Mg–ZrO<sub>2</sub>) also showed the intense peak at high temperature (about 800 °C), attributed to the effect of the temperature. At higher reaction temperature (about 800 °C), an endothermic feature of DRM reaction promotes more efficient catalytic decomposition of CH<sub>4</sub> over Ni and Ni–Zr interface over 5Ni/3Mg–ZrO<sub>2</sub> catalyst systems. This could explain the excellent CH<sub>4</sub> conversion over the magnesium modified catalyst system. It is worth noting that the high-temperature peak is near to the reaction temperature region according to the CH<sub>4</sub>-TPSR profiles. That means if dry reforming of methane was carried out in the temperature region of 700 °C, an advantage of high temperature favourable endothermic feature (about 800 °C) of DRM reaction would be missing as shown in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>. It might be an indication of lower catalytic conversion at the lower reaction temperature.<fig id=\"Fig4\"><label>Figure 4</label><caption><p>CH<sub>4</sub>-TPSR profile of catalysts.</p></caption><graphic xlink:href=\"41598_2020_70930_Fig4_HTML\" id=\"MO4\"/></fig></p><sec id=\"Sec3\"><title>Discussion</title><p id=\"Par12\">Thermal decomposition of CH<sub>4</sub> and thereby oxidation of carbon deposits by CO<sub>2</sub> towards dry reforming of methane is albeit possible with little activity i.e. 1.6% CH<sub>4</sub> conversion, 3.6% CO<sub>2</sub> conversion, H<sub>2</sub>/CO = 0.14. So, the catalytic role is utmost demanded in DRM. The summary of the catalytic activity of different catalysts towards dry reforming of methane is shown in Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>. At 700 °C reaction temperature, comparable CH<sub>4</sub> conversion, and CO<sub>2</sub> conversion, were observed. At high reaction temperature, about 800 °C, an endothermic feature of DRM reaction was ruled over. It efficiently promotes catalytic decomposition of CH<sub>4</sub> over Ni and Ni–Zr interface and thereafter oxidation of deposit by CO<sub>2</sub>. So, at 800 °C, all catalysts showed high CH<sub>4</sub> and CO<sub>2</sub> conversion as well as nearly no carbon deposit over the surface of the catalysts. Yang et al.<sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup> also claimed MgO modified Ni system as outstanding coking tolerance and Chunwen et al.<sup><xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup> explained the effective reduction of carbon deposit by MgO modified Ni system by stabilization of lattice oxygen sites of NiO by MgO.<fig id=\"Fig5\"><label>Figure 5</label><caption><p>Summary of catalytic activity for different catalyst systems.</p></caption><graphic xlink:href=\"41598_2020_70930_Fig5_HTML\" id=\"MO5\"/></fig></p><p id=\"Par13\">5Ni/ZrO<sub>2</sub> had free NiO species, “NiO species interacted with support” and a wide range of basicity. CO<sub>2</sub> uptake at basic sites, catalytic decomposition of CH<sub>4</sub> at Ni and Ni–Zr and oxidation of deposits by CO<sub>2</sub> pivoted the way of high-performance dry reforming reaction. It showed a constant 76% CH<sub>4</sub> conversion, constant 84% CO<sub>2</sub> conversion and 0.99 H<sub>2</sub>/CO ratios for 130 min, then a ratio of 0.98 for 300 min and finally a ratio of 0.96 for 500 min.</p><p id=\"Par14\">After modifying the catalyst with 3.0 wt% MgO, NiO–MgO-solid solution was built up. With a wide range of NiO–MgO-solid solution interaction (weakly as well as strongly with support ZrO<sub>2</sub>), 5Ni/3 Mg–ZrO<sub>2</sub> promoted the efficient catalytic decomposition of CH<sub>4</sub> over Ni, Ni–Zr interface and thereafter oxidation of deposit by CO<sub>2</sub><bold>.</bold> Thus, 5Ni/3Mg–ZrO<sub>2</sub> showed high 85% CH<sub>4</sub> conversion and 92% CO<sub>2</sub> conversion with H<sub>2</sub>/CO ratio ~ 0.96. The CO<sub>2</sub>-TPD, as well as O<sub>2</sub>-TPO profile of spent catalysts, showed an extra peak in TPD and a negative (consuming) peak in TPO, respectively which both related to the oxidation of residual carbon deposits on the surface of the catalyst. The CO<sub>2</sub>-TPD along with the O<sub>2</sub>-TPO results showed that CO<sub>2</sub> is capable of oxidizing carbon deposit over the surface of the catalyst. Removal of carbon deposits by hydrogen gas through methane gasification reaction (C + 2H<sub>2</sub> → CH<sub>4</sub>) is also possible<sup><xref ref-type=\"bibr\" rid=\"CR64\">64</xref></sup>. It resulted in stable catalytic activity up to 500 min in the TOS test. Furthermore, modifying the catalyst with 5 wt%MgO in 5Ni/5Mg–ZrO<sub>2</sub>, it showed more amount of “NiO–MgO solid solution strongly interacted with ZrO<sub>2</sub> support” as well as a wide variety of basic sites. That catalyst showed a constant conversion (82% CH<sub>4</sub> conversion and 89% CO<sub>2</sub> conversion) as well as H<sub>2</sub>/CO ratio = 1 for 250 min in the TOS then slightly decreased to 0.99 for another 250 min, with overall 500 min TOS. Thus, it could be concluded that 5 wt% MgO loading is optimum loading for an active and stable catalyst for methane dry reforming reaction. Further increase in magnesium oxide loading to 7 wt% MgO caused a decrease in NiO–MgO-solid solution that interacted weakly or strongly with the ZrO<sub>2</sub> support and consequently the loss of strong basic sites. Thus, decreasing the CH<sub>4</sub> conversion to 79% as well as CO<sub>2</sub> conversion to 86% and H<sub>2</sub>/CO ratio to 0.98 were noticed.</p></sec></sec><sec id=\"Sec26\"><title>Conclusion</title><p id=\"Par15\">Magnesium promoted NiO supported mesoporous zirconia, 5Ni/xMg–ZrO<sub>2</sub> (x = 0, 3, 5, 7) were prepared and tested for the methane dry reforming reaction. Higher activity was found at 800 °C than that at 700 °C due to favourable endothermic feature of DRM reaction which promotes efficient CH<sub>4</sub> decomposition over Ni and Ni–Zr interface and successive oxidation of carbon deposits by CO<sub>2</sub>. By modifying the catalyst (5Ni/ZrO<sub>2</sub>) with MgO as a promoter, NiO–MgO-solid solution was formed. It was found that for high constant CH<sub>4</sub> and CO<sub>2</sub> conversions, NiO–MgO-solid solution played a significant role during the DRM. The 5Ni/3Mg–ZrO<sub>2</sub> catalyst showed a constant 85% CH<sub>4</sub> conversion and 92% CO<sub>2</sub> conversion up to 500 min on stream at H<sub>2</sub>/CO mole ratio ~ 0.96. The highly constant performance of magnesium oxide modified catalysts was due to the ability of CO<sub>2</sub> to oxidize the carbon deposits during the DRM, thus maintaining the catalytic stability. However, with a further loading (> 5.0 wt% Mg) such as in 5Ni/5Mg–ZrO<sub>2</sub> which showed a higher amount of “NiO–MgO-solid solution strongly interacted with ZrO<sub>2</sub> support” along with a wide variety of basic sites as well. Thus, it showed a constant 82% CH<sub>4</sub> conversion and 89% CO<sub>2</sub> conversion and H<sub>2</sub>/CO mole ratio ~ 1. It is hoped that these findings could inspire finding more stable and less expensive synthesis gas production catalysts, including from two potent greenhouse gases emissions such as methane and carbon dioxide.</p></sec><sec id=\"Sec5\"><title>Experimental</title><sec id=\"Sec6\"><title>Materials</title><p id=\"Par16\">Nickel nitrate hexahydrate [Ni (NO<sub>3</sub>)<sub>2</sub>.6H<sub>2</sub>O, 98%, Alfa Aesar], magnesium acetate tetra-hydrate [Mg(O<sub>2</sub>CCH<sub>3</sub>)<sub>2</sub>.4H<sub>2</sub>O, 99.5–102.0%, Merck], mesoporous zirconia (<italic>meso</italic>-ZrO<sub>2</sub>, 1/8\" pellets, Alfa Aesar) were commercially available and were used without further purification. Ultrapure water was acquired from a Milli-Q water purification system (Millipore).</p></sec><sec id=\"Sec7\"><title>Catalyst preparation</title><p id=\"Par17\">A two-step procedure, based on incipient wetness impregnation as described elsewhere<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>, was followed for synthesizing the desired catalysts. The first step was to dope the support with a metal oxide promoter, while the second step was to load nickel oxide over the promoted support. The detailed description of each synthesis step is given below.</p></sec><sec id=\"Sec8\"><title>Synthesis of mesoporous zirconia promoted with magnesia (MgO-meso-ZrO<sub>2</sub>)</title><p id=\"Par18\">The required amount of Mg (CH<sub>3-</sub>CO<sub>2</sub>)<sub>2</sub>.4H<sub>2</sub>O for 3.0, 5.0, or 7.0 wt/wt% loading of MgO was mixed and pulverized with the required amount of <italic>meso</italic>-ZrO<sub>2</sub>. To this resultant solid mixture, drops of ultrapure water were added until the formation of a colourless paste, which was mechanically stirred until complete dryness at room temperature. The addition of water and drying processes were performed three times to ensure homogeneous distribution of Mg (CH<sub>3</sub>CO<sub>2</sub>)<sub>2</sub> within the matrix of <italic>meso</italic>-ZrO<sub>2</sub>. The solid mixture was then grounded and calcined in a muffle furnace, at 600 °C for 3 h in the static air atmosphere. The resultant materials were designated as xMg–ZrO<sub>2</sub> catalysts where x is wt% of MgO (x = 0, 3, 5, 7).</p></sec><sec id=\"Sec9\"><title>Synthesis of mesoporous zirconia supported nickel oxide promoted with magnesia (NiO/MgO-meso-ZrO<sub>2</sub>)</title><p id=\"Par19\">The required amount of Ni (NO<sub>3</sub>)<sub>2</sub>.6H<sub>2</sub>O to obtain 5.0 wt/wt% of NiO loading was mixed and was crushed with the required amount of MgO-<italic>meso</italic>-ZrO<sub>2</sub> of the desired MgO wt/wt% loading, forming a green solid mixture. Drops of ultrapure water were then added to get a paste. By continuous mechanical stirring, the paste was dried at room temperature. The wetting and drying processes were repeated three times. Afterwards, calcination was performed at 600 °C for 3 h in static air atmosphere. Overall, 5 wt% NiO loaded catalyst sample is designated as 5Ni/xMg–ZrO<sub>2</sub> catalysts where x is wt% of MgO (x = 0, 3, 5, 7).</p></sec><sec id=\"Sec10\"><title>Catalyst characterization</title><p id=\"Par20\">The details of instrument specifications and procedures are described in the supporting information and described elsewhere<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>.</p></sec><sec id=\"Sec11\"><title>Catalyst test</title><p id=\"Par21\">DRM was carried out in a fixed-bed stainless steel tubular micro-reactor (ID = 9 mm) at atmospheric pressure. A load of 0.10 g catalyst was activated under 20 SCCM H<sub>2</sub> flow at 800 °C for 60 min. Then 20 sccm of N<sub>2</sub> was fed to the reactor for 20 min at 800 °C to remove adsorbed H<sub>2</sub>. Afterwards, CH<sub>4</sub>, CO<sub>2</sub>, and N<sub>2</sub> were dosed at flow rates of 30, 30 and 5 sccm, respectively. A GC (GC-2014 Shimadzu) unit, equipped with a thermal conductivity detector and two columns, Porapak Q and Molecular Sieve 5A, was connected in series/bypass connections to have a complete analysis of the reaction products. The following equations were used to calculate the conversion of each reactant and the H<sub>2</sub>/CO mole ratio, respectively<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>.<disp-formula id=\"Equa\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\begin{aligned} & {\\text{CH}}_{4} \\;{\\text{conversion}} = { }\\frac{{{\\text{CH}}_{{4,{\\text{in}}}} - {\\text{CH}}_{{4,{\\text{out}}}} }}{{{\\text{CH}}_{{4,{\\text{in}}}} }}{ } \\times { }100{\\text{\\% }} \\\\ & {\\text{CO}}_{2} { }\\;{\\text{conversion}} = { }\\frac{{{\\text{CO}}_{{2,{\\text{in}}}} - {\\text{CO}}_{{2,{\\text{out}}}} }}{{{\\text{CO}}_{{2,{\\text{in}}}} }}{ } \\times { }100{\\text{\\% }} \\\\ & \\frac{{{\\text{H}}_{{2{ }}} }}{{{\\text{CO}}}}{ } = { }\\frac{{{\\text{mole}}\\;{\\text{ of }}\\;{\\text{H}}_{2} \\;{\\text{produced}}}}{{{\\text{mole }}\\;{\\text{of }}\\;{\\text{CO }}\\;{\\text{produced}}}} \\\\ \\end{aligned}$$\\end{document}</tex-math><mml:math id=\"M2\" display=\"block\"><mml:mrow><mml:mtable><mml:mtr><mml:mtd/><mml:mtd columnalign=\"left\"><mml:mrow><mml:msub><mml:mtext>CH</mml:mtext><mml:mn>4</mml:mn></mml:msub><mml:mspace width=\"0.277778em\"/><mml:mtext>conversion</mml:mtext><mml:mo>=</mml:mo><mml:mrow/><mml:mfrac><mml:mrow><mml:msub><mml:mtext>CH</mml:mtext><mml:mrow><mml:mn>4</mml:mn><mml:mo>,</mml:mo><mml:mtext>in</mml:mtext></mml:mrow></mml:msub><mml:mo>-</mml:mo><mml:msub><mml:mtext>CH</mml:mtext><mml:mrow><mml:mn>4</mml:mn><mml:mo>,</mml:mo><mml:mtext>out</mml:mtext></mml:mrow></mml:msub></mml:mrow><mml:msub><mml:mtext>CH</mml:mtext><mml:mrow><mml:mn>4</mml:mn><mml:mo>,</mml:mo><mml:mtext>in</mml:mtext></mml:mrow></mml:msub></mml:mfrac><mml:mrow/><mml:mo>×</mml:mo><mml:mrow/><mml:mn>100</mml:mn><mml:mrow><mml:mtext>\\%</mml:mtext><mml:mspace width=\"0.333333em\"/></mml:mrow></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd columnalign=\"right\"><mml:mrow/></mml:mtd><mml:mtd columnalign=\"left\"><mml:mrow><mml:msub><mml:mtext>CO</mml:mtext><mml:mn>2</mml:mn></mml:msub><mml:mrow/><mml:mspace width=\"0.277778em\"/><mml:mtext>conversion</mml:mtext><mml:mo>=</mml:mo><mml:mrow/><mml:mfrac><mml:mrow><mml:msub><mml:mtext>CO</mml:mtext><mml:mrow><mml:mn>2</mml:mn><mml:mo>,</mml:mo><mml:mtext>in</mml:mtext></mml:mrow></mml:msub><mml:mo>-</mml:mo><mml:msub><mml:mtext>CO</mml:mtext><mml:mrow><mml:mn>2</mml:mn><mml:mo>,</mml:mo><mml:mtext>out</mml:mtext></mml:mrow></mml:msub></mml:mrow><mml:msub><mml:mtext>CO</mml:mtext><mml:mrow><mml:mn>2</mml:mn><mml:mo>,</mml:mo><mml:mtext>in</mml:mtext></mml:mrow></mml:msub></mml:mfrac><mml:mrow/><mml:mo>×</mml:mo><mml:mrow/><mml:mn>100</mml:mn><mml:mrow><mml:mtext>\\%</mml:mtext><mml:mspace width=\"0.333333em\"/></mml:mrow></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd columnalign=\"right\"><mml:mrow/></mml:mtd><mml:mtd columnalign=\"left\"><mml:mrow><mml:mfrac><mml:msub><mml:mtext>H</mml:mtext><mml:mrow><mml:mn>2</mml:mn><mml:mrow/></mml:mrow></mml:msub><mml:mtext>CO</mml:mtext></mml:mfrac><mml:mrow/><mml:mo>=</mml:mo><mml:mrow/><mml:mfrac><mml:mrow><mml:mtext>mole</mml:mtext><mml:mspace width=\"0.277778em\"/><mml:mrow><mml:mspace width=\"0.333333em\"/><mml:mtext>of</mml:mtext><mml:mspace width=\"0.333333em\"/></mml:mrow><mml:mspace width=\"0.277778em\"/><mml:msub><mml:mtext>H</mml:mtext><mml:mn>2</mml:mn></mml:msub><mml:mspace width=\"0.277778em\"/><mml:mtext>produced</mml:mtext></mml:mrow><mml:mrow><mml:mrow><mml:mtext>mole</mml:mtext><mml:mspace width=\"0.333333em\"/></mml:mrow><mml:mspace width=\"0.277778em\"/><mml:mrow><mml:mtext>of</mml:mtext><mml:mspace width=\"0.333333em\"/></mml:mrow><mml:mspace width=\"0.277778em\"/><mml:mrow><mml:mtext>CO</mml:mtext><mml:mspace width=\"0.333333em\"/></mml:mrow><mml:mspace width=\"0.277778em\"/><mml:mtext>produced</mml:mtext></mml:mrow></mml:mfrac></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd columnalign=\"right\"><mml:mrow/></mml:mtd></mml:mtr></mml:mtable></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70930_Article_Equa.gif\" position=\"anchor\"/></alternatives></disp-formula></p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec12\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70930_MOESM1_ESM.docx\"><caption><p>Supplementary Information.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p> is available for this paper at 10.1038/s41598-020-70930-1.</p></sec><ack><title>Acknowledgements</title><p>The KSU authors would like to extend their sincere appreciation to the Deanship of Scientific Research at the King Saud University for funding this research group project # No. RGP-119. Dr Ahmed I. Osman would like to thank Prof. David Rooney for the given support and acknowledge the support given by the EPSRC project “Advancing Creative Circular Economies for Plastics via Technological-Social Transitions” (ACCEPT Transitions, EP/S025545/1). RK wants to acknowledge the administration of Sankalchand Patel University for providing research environment.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>Experiment test, A.S.A.-F. and S.O.K. writing—original draft, R.K. and A.S.A.-F., Preparation of Catalyst, A.A.B., M.A. and R.A. Characterization, A.S.A.-F., S.O.K., A.A.I., M.S.L., J.K., R.A. and A.H.F., writing—review and editing. R.K., M.A., A.A.B., A.E.A., A.I.O.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par22\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Osman</surname><given-names>AI</given-names></name></person-group><article-title>Catalytic hydrogen production from methane partial oxidation: mechanism and kinetic study</article-title><source>Chem. Eng. Technol.</source><year>2020</year><volume>43</volume><fpage>641</fpage><lpage>648</lpage><pub-id pub-id-type=\"doi\">10.1002/ceat.201900339</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhou</surname><given-names>L</given-names></name><name><surname>Li</surname><given-names>L</given-names></name><name><surname>Nini</surname><given-names>W</given-names></name><name><surname>Jun</surname><given-names>L</given-names></name><name><surname>Basset</surname><given-names>J-M</given-names></name></person-group><article-title>Effect of NiAl<sub>2</sub>O<sub>4</sub> formation on Ni/Al<sub>2</sub>O<sub>3</sub> stability during dry reforming of methane </article-title><source>ChemCatChem</source><year>2015</year><volume>7</volume><fpage>2508</fpage><lpage>2516</lpage><pub-id pub-id-type=\"doi\">10.1002/cctc.201500379</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>C</given-names></name><name><surname>Jie</surname><given-names>X</given-names></name><name><surname>Qiu</surname><given-names>Y</given-names></name><name><surname>Zhao</surname><given-names>Y</given-names></name><name><surname>Al-Megren</surname><given-names>HA</given-names></name><name><surname>Alshihri</surname><given-names>S</given-names></name><name><surname>Edwards</surname><given-names>PP</given-names></name><name><surname>Xiao</surname><given-names>T</given-names></name></person-group><article-title>The importance of inner cavity space within Ni@SiO<sub>2</sub> nanocapsule catalysts for excellent coking resistance in the high-space-velocity dry reforming of methane</article-title><source>Appl. Catal. B Environ.</source><year>2019</year><volume>259</volume><fpage>118019</fpage><lpage>118029</lpage><pub-id pub-id-type=\"doi\">10.1016/j.apcatb.2019.118019(2019)</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>D</given-names></name><name><surname>Quek</surname><given-names>XY</given-names></name><name><surname>Wah</surname><given-names>HA</given-names></name><name><surname>Zeng</surname><given-names>G</given-names></name><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Yang</surname><given-names>Y</given-names></name></person-group><article-title>Carbon dioxide reforming of methane over nickel-grafted SBA-15 and MCM-41 catalysts </article-title><source>Catal. Today</source><year>2009</year><volume>148</volume><fpage>243</fpage><lpage>265</lpage><pub-id pub-id-type=\"doi\">10.1016/j.cattod.2009.08.014</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quek</surname><given-names>XY</given-names></name><name><surname>Liu</surname><given-names>D</given-names></name><name><surname>Cheo</surname><given-names>WNE</given-names></name><name><surname>Wang</surname><given-names>H</given-names></name><name><surname>Chen</surname><given-names>Y</given-names></name><name><surname>Yang</surname><given-names>Y</given-names></name></person-group><article-title>Nickel-grafted TUD-1 mesoporous catalysts for carbon dioxide reforming of methane</article-title><source>Appl. Catal. B Environ.</source><year>2010</year><volume>95</volume><fpage>374</fpage><lpage>382</lpage><pub-id pub-id-type=\"doi\">10.1016/j.apcatb.2010.01.016</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Li</surname><given-names>F</given-names></name></person-group><article-title>Coke-resistant Ni@SiO<sub>2</sub> catalyst for dry reforming of methane</article-title><source>Appl. Catal. B Environ.</source><year>2015</year><volume>176–177</volume><fpage>513</fpage><lpage>521</lpage><pub-id pub-id-type=\"doi\">10.1016/j.apcatb.2015.04.039</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>Z</given-names></name><name><surname>Mo</surname><given-names>L</given-names></name><name><surname>Kathiraser</surname><given-names>Y</given-names></name><name><surname>Kawi</surname><given-names>S</given-names></name></person-group><article-title>Yolk–Satellite–Shell structured Ni–Yolk@Ni@SiO<sub>2</sub> nanocomposite: superb catalyst toward methane CO<sub>2</sub> reforming reaction</article-title><source>ACS Catal.</source><year>2014</year><volume>5</volume><fpage>1526</fpage><lpage>1536</lpage><pub-id pub-id-type=\"doi\">10.1021/cs401027p</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>M</given-names></name><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Wu</surname><given-names>Y</given-names></name><name><surname>Pan</surname><given-names>J</given-names></name><name><surname>Zhang</surname><given-names>Q</given-names></name><name><surname>Tan</surname><given-names>Y</given-names></name></person-group><article-title>Insight into the effects of the oxygen species over Ni/ZrO<sub>2</sub> catalyst surface on methane reforming with carbon dioxide</article-title><source>Appl. Catal. B Environ.</source><year>2019</year><volume>244</volume><fpage>427</fpage><lpage>437</lpage><pub-id pub-id-type=\"doi\">10.1016/j.apcatb.2018.11.068</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Delacruz</surname><given-names>VMG</given-names></name><name><surname>Pereñiguez</surname><given-names>R</given-names></name><name><surname>Ternero Juan</surname><given-names>F</given-names></name><name><surname>Holgado</surname><given-names>P</given-names></name><name><surname>Caballero</surname><given-names>A</given-names></name></person-group><article-title>Modifying the size of nickel metallic particles by H<sub>2</sub>/CO treatment in Ni/ZrO<sub>2</sub> Methane dry reforming catalysts</article-title><source>ACS Catal.</source><year>2011</year><volume>1</volume><issue>2</issue><fpage>82</fpage><lpage>88</lpage><pub-id pub-id-type=\"doi\">10.1021/cs100116m</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tathod</surname><given-names>AP</given-names></name><name><surname>Hayek</surname><given-names>N</given-names></name><name><surname>Shpasser</surname><given-names>D</given-names></name><name><surname>Simakov</surname><given-names>DSA</given-names></name><name><surname>Gazit</surname><given-names>OM</given-names></name></person-group><article-title>Mediating interaction strength between nickel and zirconia using a mixed oxide nanosheets interlayer for methane dry reforming</article-title><source>Appl. Catal. B Environ.</source><year>2019</year><volume>249</volume><fpage>106</fpage><lpage>115</lpage><pub-id pub-id-type=\"doi\">10.1016/j.apcatb.2019.02.040</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luna</surname><given-names>AEC</given-names></name><name><surname>Iriarte</surname><given-names>ME</given-names></name></person-group><article-title>Carbon dioxide reforming of methane over a metal modified Ni-Al<sub>2</sub>O<sub>3</sub> catalyst</article-title><source>Appl. Catal. A Gen.</source><year>2008</year><volume>343</volume><fpage>10</fpage><pub-id pub-id-type=\"doi\">10.1016/j.apcata.2007.11.041</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Juan</surname><given-names>JJ</given-names></name><name><surname>Martínez</surname><given-names>MCR</given-names></name><name><surname>Gómez</surname><given-names>MJI</given-names></name></person-group><article-title>Effect of potassium content in the activity of K-promoted Ni/Al<sub>2</sub>O<sub>3</sub> catalysts for the dry reforming of methane</article-title><source>Appl. Catal. A Gen.</source><year>2006</year><volume>301</volume><fpage>9</fpage><pub-id pub-id-type=\"doi\">10.1016/j.apcata.2005.11.006</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alipour</surname><given-names>Z</given-names></name><name><surname>Rezaei</surname><given-names>M</given-names></name><name><surname>Meshkani</surname><given-names>F</given-names></name></person-group><article-title>Effect of Ni loadings on the activity and coke formation of MgO-modified Ni/Al<sub>2</sub>O<sub>3</sub> nanocatalyst in dry reforming of methane</article-title><source>J. Energy Chem.</source><year>2014</year><volume>23</volume><fpage>633</fpage><pub-id pub-id-type=\"doi\">10.1016/S2095-4956(14)60194-7</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alipour</surname><given-names>Z</given-names></name><name><surname>Rezaeia</surname><given-names>M</given-names></name><name><surname>Meshkani</surname><given-names>F</given-names></name></person-group><article-title>Effects of support modifiers on the catalytic performance of Ni/Al<sub>2</sub>O<sub>3</sub> catalyst in CO<sub>2</sub> reforming of methane</article-title><source>Fuel</source><year>2014</year><volume>129</volume><fpage>197</fpage><pub-id pub-id-type=\"doi\">10.1016/j.fuel.2014.03.045</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alonso</surname><given-names>DSJ</given-names></name><name><surname>Gómez</surname><given-names>MJI</given-names></name><name><surname>Martínez</surname><given-names>MCR</given-names></name></person-group><article-title>K and Sr promoted Co alumina supported catalysts for the CO<sub>2</sub> reforming of methane</article-title><source>Catal. Today</source><year>2011</year><volume>176</volume><fpage>187</fpage><pub-id pub-id-type=\"doi\">10.1016/j.cattod.2010.11.093</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fatesh</surname><given-names>ASA</given-names></name><name><surname>Naeem</surname><given-names>MA</given-names></name><name><surname>Fakeeha</surname><given-names>AH</given-names></name><name><surname>Abasaeed</surname><given-names>AE</given-names></name></person-group><article-title>CO<sub>2</sub> reforming of methane to produce syngas over γ-Al<sub>2</sub>O<sub>3</sub>-supported Ni–Sr catalysts</article-title><source>Bull. Chem. Soc. Jpn.</source><year>2013</year><volume>86</volume><fpage>742</fpage><pub-id pub-id-type=\"doi\">10.1246/bcsj.20130002</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hassan</surname><given-names>M</given-names></name><name><surname>Kshudiram</surname><given-names>A</given-names></name><name><surname>Jarrod</surname><given-names>M</given-names></name><name><surname>James</surname><given-names>N</given-names></name><name><surname>Suresh</surname><given-names>T</given-names></name><name><surname>Bhargava</surname><given-names>K</given-names></name></person-group><article-title>Highly stable ytterbium promoted Ni/γ-Al<sub>2</sub>O<sub>3</sub> catalysts for carbon dioxide reforming of methane</article-title><source>Appl. Catal. B Environ.</source><year>2012</year><volume>119–120</volume><fpage>217</fpage><pub-id pub-id-type=\"doi\">10.1016/j.apcatb.2012.02.039</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xu</surname><given-names>J</given-names></name><name><surname>Zhou</surname><given-names>W</given-names></name><name><surname>Wang</surname><given-names>J</given-names></name><name><surname>Li</surname><given-names>Z</given-names></name><name><surname>Ma</surname><given-names>J</given-names></name></person-group><article-title>Characterization and analysis of carbon deposited during the dry reforming of methane over Ni/La<sub>2</sub>O<sub>3</sub>/Al<sub>2</sub>O<sub>3</sub> catalysts</article-title><source>Chin. J. Catal.</source><year>2009</year><volume>30</volume><fpage>1076</fpage><pub-id pub-id-type=\"doi\">10.1016/S1872-2067(08)60139-4</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Laosiripojana</surname><given-names>N</given-names></name><name><surname>Sutthisripok</surname><given-names>W</given-names></name><name><surname>Assabumrungrat</surname><given-names>S</given-names></name></person-group><article-title>Synthesis gas production from dry reforming of methane over CeO<sub>2</sub> doped Ni/Al<sub>2</sub>O<sub>3</sub>: influence of the doping ceria on the resistance toward carbon formation</article-title><source>Chem. Eng. J.</source><year>2005</year><volume>112</volume><fpage>13</fpage><pub-id pub-id-type=\"doi\">10.1016/j.cej.2005.06.003</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pechimuthu</surname><given-names>NA</given-names></name><name><surname>Pant</surname><given-names>KK</given-names></name><name><surname>Dhingra</surname><given-names>SC</given-names></name></person-group><article-title>Deactivation studies over Ni−K/CeO<sub>2</sub>−Al<sub>2</sub>O<sub>3</sub> catalyst for dry reforming of methane</article-title><source>Ind. Eng. Chem. Res.</source><year>2007</year><volume>46</volume><fpage>1731</fpage><pub-id pub-id-type=\"doi\">10.1021/ie061389n</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fatesh</surname><given-names>ASA</given-names></name><name><surname>Kumar</surname><given-names>R</given-names></name><name><surname>Kasim</surname><given-names>SO</given-names></name><name><surname>Ibrahim</surname><given-names>AA</given-names></name><name><surname>Fakeeha</surname><given-names>AH</given-names></name><name><surname>Abasaeed</surname><given-names>AE</given-names></name><name><surname>Alrasheed</surname><given-names>R</given-names></name><name><surname>Bagabas</surname><given-names>A</given-names></name><name><surname>Chaudhary</surname><given-names>ML</given-names></name><name><surname>Frusteri</surname><given-names>F</given-names></name><name><surname>Chowdhury</surname><given-names>B</given-names></name></person-group><article-title>The effect of modifier identity on the performance of Ni-based catalyst supported on γ-Al<sub>2</sub>O<sub>3</sub> in dry reforming of methane</article-title><source>Catal. Today</source><year>2020</year><volume>348</volume><fpage>236</fpage><lpage>242</lpage><pub-id pub-id-type=\"doi\">10.1016/j.cattod.2019.09.003</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Therdthianwong</surname><given-names>S</given-names></name><name><surname>Therdthianwong</surname><given-names>A</given-names></name><name><surname>Siangchin</surname><given-names>C</given-names></name><name><surname>Yongprapat</surname><given-names>S</given-names></name></person-group><article-title>Synthesis gas production from dry reforming of methane over Ni/Al<sub>2</sub>O<sub>3</sub> stabilized by ZrO<sub>2</sub></article-title><source>Int. J. Hydrog. Energy</source><year>2008</year><volume>33</volume><fpage>991</fpage><pub-id pub-id-type=\"doi\">10.1016/j.ijhydene.2007.11.029</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Therdthianwong</surname><given-names>S</given-names></name><name><surname>Siangchin</surname><given-names>C</given-names></name><name><surname>Therdthianwong</surname><given-names>A</given-names></name></person-group><article-title>Improvement of coke resistance of Ni/Al<sub>2</sub>O<sub>3</sub> catalyst in CH<sub>4</sub>/CO<sub>2</sub> reforming by ZrO<sub>2</sub> addition</article-title><source>Fuel Process. Technol.</source><year>2008</year><volume>89</volume><fpage>160</fpage><pub-id pub-id-type=\"doi\">10.1016/j.fuproc.2007.09.003</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Seoka</surname><given-names>S-H</given-names></name><name><surname>Choi</surname><given-names>SH</given-names></name><name><surname>Park</surname><given-names>ED</given-names></name><name><surname>Han</surname><given-names>SH</given-names></name><name><surname>Lee</surname><given-names>JS</given-names></name></person-group><article-title>Mn-promoted Ni/Al<sub>2</sub>O<sub>3</sub> catalysts for stable carbon dioxide reforming of methane</article-title><source>J. Catal.</source><year>2002</year><volume>209</volume><fpage>6</fpage><pub-id pub-id-type=\"doi\">10.1006/jcat.2002.3627</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rahemia</surname><given-names>N</given-names></name><name><surname>Rahemia</surname><given-names>N</given-names></name><name><surname>Haghighia</surname><given-names>M</given-names></name><name><surname>Babaluoa</surname><given-names>AA</given-names></name><name><surname>Jafarid</surname><given-names>MF</given-names></name><name><surname>Khorrame</surname><given-names>S</given-names></name></person-group><article-title>Non-thermal plasma assisted synthesis and physicochemical characterizations of Co and Cu doped Ni/Al<sub>2</sub>O<sub>3</sub> nanocatalysts used for dry reforming of methane</article-title><source>Int. J. Hydrogen Energy</source><year>2013</year><volume>38</volume><fpage>16048</fpage><pub-id pub-id-type=\"doi\">10.1016/j.ijhydene.2013.08.084</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Molood</surname><given-names>S</given-names></name><name><surname>Nataj</surname><given-names>M</given-names></name><name><surname>Aalvi</surname><given-names>SM</given-names></name><name><surname>Mazloom</surname><given-names>G</given-names></name></person-group><article-title>Catalytic performance of Ni supported on ZnO-Al<sub>2</sub>O<sub>3</sub> composites with different Zn content in methane dry reforming</article-title><source>J. Chem. Technol. Biotechnol.</source><year>2019</year><volume>94</volume><fpage>1305</fpage><pub-id pub-id-type=\"doi\">10.1002/jctb.5887</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fouskas</surname><given-names>A</given-names></name><name><surname>Kollia</surname><given-names>M</given-names></name><name><surname>Kambolis</surname><given-names>A</given-names></name><name><surname>Papadopoulou</surname><given-names>C</given-names></name><name><surname>Matralis</surname><given-names>H</given-names></name></person-group><article-title>Boron-modified Ni/Al<sub>2</sub>O<sub>3</sub> catalysts for reduced carbon deposition during dry reforming of methane</article-title><source>Appl. Catal. A</source><year>2014</year><volume>474</volume><fpage>125</fpage><pub-id pub-id-type=\"doi\">10.1016/j.apcata.2013.08.016</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><mixed-citation publication-type=\"other\">Amin, M. H., Tardio, J. & Bhargava, K. S. A comparison study on carbon dioxide reforming of methane over Ni catalysts supported on mesoporous SBA-15, MCM-41, KIT-6 and gamma-Al2. In <italic>Chemeca 2013 (41st : 2013 : Brisbane, Qld.). Chemeca 2013: Challenging Tomorrow. Barton</italic>, <italic>ACT: Engineers Australia</italic>, 543–548. <ext-link ext-link-type=\"uri\" xlink:href=\"https://search.informit.com.au/documentSummary;dn=883019063475312;res=IELENG\">https://search.informit.com.au/documentSummary;dn=883019063475312;res=IELENG</ext-link> (2013).</mixed-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pompeo</surname><given-names>F</given-names></name><name><surname>Nichio</surname><given-names>NN</given-names></name><name><surname>Gloria</surname><given-names>M</given-names></name><name><surname>Montesc</surname><given-names>GM</given-names></name></person-group><article-title>Characterization of Ni/SiO<sub>2</sub> and Ni/Li-SiO<sub>2</sub> catalysts for methane dry reforming</article-title><source>Catal. Today</source><year>2005</year><volume>107–108</volume><fpage>856</fpage><pub-id pub-id-type=\"doi\">10.1016/j.cattod.2005.07.024</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zapata</surname><given-names>B</given-names></name><name><surname>Valenzuela</surname><given-names>MA</given-names></name><name><surname>Palacios</surname><given-names>J</given-names></name><name><surname>Garcia</surname><given-names>ET</given-names></name></person-group><article-title>Effect of Ca, Ce or K oxide addition on the activity of Ni/SiO<sub>2</sub> catalysts for the methane decomposition reaction</article-title><source>Int. J. Hydrogen Energy</source><year>2010</year><volume>35</volume><fpage>12091</fpage><pub-id pub-id-type=\"doi\">10.1016/j.ijhydene.2009.09.072</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Assabumrungrat</surname><given-names>S</given-names></name><name><surname>Charoenseri</surname><given-names>S</given-names></name><name><surname>Laosiripojan</surname><given-names>N</given-names></name><name><surname>Kiatkittipong</surname><given-names>W</given-names></name><name><surname>Praserthdam</surname><given-names>P</given-names></name></person-group><article-title>Effect of oxygen addition on catalytic performance of Ni/SiO<sub>2</sub>·MgO toward carbon dioxide reforming of methane under periodic operation</article-title><source>Int. J. Hydrogen Energy</source><year>2009</year><volume>34</volume><fpage>6211</fpage><pub-id pub-id-type=\"doi\">10.1016/j.ijhydene.2009.05.128</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>Q</given-names></name><name><surname>Feng</surname><given-names>X</given-names></name><name><surname>Zhao</surname><given-names>L</given-names></name><name><surname>Song</surname><given-names>X</given-names></name><name><surname>Zhang</surname><given-names>P</given-names></name><name><surname>Gao</surname><given-names>L</given-names></name></person-group><article-title>Hollow hierarchical Ni/MgO-SiO<sub>2</sub> catalyst with high activity, thermal stability and coking resistance for catalytic dry reforming of methane</article-title><source>Int. J. Hydrogen Energy</source><year>2018</year><volume>43</volume><fpage>11056</fpage><pub-id pub-id-type=\"doi\">10.1016/j.ijhydene.2018.05.010</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jing</surname><given-names>Q</given-names></name><name><surname>Lou</surname><given-names>H</given-names></name><name><surname>Mo</surname><given-names>L</given-names></name><name><surname>Fei</surname><given-names>J</given-names></name><name><surname>Zheng</surname><given-names>X</given-names></name></person-group><article-title>Combination of CO<sub>2</sub> reforming and partial oxidation of methane over Ni/BaO-SiO<sub>2</sub> catalysts to produce low H<sub>2</sub>/CO ratio syngas using a fluidized bed reactor</article-title><source>J. Mol. Catal. A Chem.</source><year>2004</year><volume>212</volume><fpage>211</fpage><pub-id pub-id-type=\"doi\">10.1016/j.molcata.2003.10.041</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mo</surname><given-names>L</given-names></name><name><surname>Kai</surname><given-names>K</given-names></name><name><surname>Leong</surname><given-names>M</given-names></name><name><surname>Kawi</surname><given-names>S</given-names></name></person-group><article-title>A highly dispersed and anti-coking Ni–La<sub>2</sub>O<sub>3</sub>/SiO<sub>2</sub> catalyst for syngas production from dry carbon dioxide reforming of methane</article-title><source>Catal. Sci. Technol.</source><year>2014</year><volume>4</volume><fpage>2107</fpage><pub-id pub-id-type=\"doi\">10.1039/C3CY00869J</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>B</given-names></name><name><surname>Xu</surname><given-names>X</given-names></name><name><surname>Zhang</surname><given-names>S</given-names></name></person-group><article-title>Synthesis gas production in the combined CO<sub>2</sub> reforming with partial oxidation of methane over Ce-promoted Ni/SiO<sub>2</sub> catalysts</article-title><source>Int. J. Hydrogen Energy</source><year>2013</year><volume>38</volume><fpage>890</fpage><pub-id pub-id-type=\"doi\">10.1016/j.ijhydene.2012.10.103</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhu</surname><given-names>J</given-names></name><name><surname>Peng</surname><given-names>X</given-names></name><name><surname>Yao</surname><given-names>L</given-names></name><name><surname>Deng</surname><given-names>X</given-names></name><name><surname>Dong</surname><given-names>H</given-names></name><name><surname>Tong</surname><given-names>D</given-names></name><name><surname>Hu</surname><given-names>C</given-names></name></person-group><article-title>Synthesis gas production from CO<sub>2</sub> reforming of methane over Ni–Ce/SiO<sub>2</sub> catalyst: The effect of calcination ambience</article-title><source>Int. J. Hydrogen Energy</source><year>2013</year><volume>38</volume><fpage>117</fpage><pub-id pub-id-type=\"doi\">10.1016/j.ijhydene.2012.07.136</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yao</surname><given-names>L</given-names></name><name><surname>Shia</surname><given-names>J</given-names></name><name><surname>Xub</surname><given-names>H</given-names></name><name><surname>Shen</surname><given-names>W</given-names></name><name><surname>Hu</surname><given-names>C</given-names></name></person-group><article-title>Low-temperature CO<sub>2</sub> reforming of methane on Zr-promoted Ni/SiO<sub>2</sub> catalyst</article-title><source>Fuel Process. Technol.</source><year>2016</year><volume>144</volume><fpage>1</fpage><pub-id pub-id-type=\"doi\">10.1016/j.fuproc.2015.12.009</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yao</surname><given-names>L</given-names></name><name><surname>Zhu</surname><given-names>J</given-names></name><name><surname>Peng</surname><given-names>X</given-names></name><name><surname>Tong</surname><given-names>D</given-names></name><name><surname>Hu</surname><given-names>C</given-names></name></person-group><article-title>Comparative study on the promotion effect of Mn and Zr on the stability of Ni/SiO<sub>2</sub> catalyst for CO<sub>2</sub> reforming of methane</article-title><source>Int. J. Hydrogen Energy</source><year>2013</year><volume>38</volume><fpage>7268</fpage><pub-id pub-id-type=\"doi\">10.1016/j.ijhydene.2013.02.126</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bian</surname><given-names>Z</given-names></name><name><surname>Kawi</surname><given-names>S</given-names></name></person-group><article-title>Highly carbon-resistant Ni–Co/SiO<sub>2</sub> catalysts derived from phyllosilicates for dry reforming of methane</article-title><source>J. CO2 Util.</source><year>2017</year><volume>18</volume><fpage>345</fpage><pub-id pub-id-type=\"doi\">10.1016/j.jcou.2016.12.014</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhu</surname><given-names>J</given-names></name><name><surname>Peng</surname><given-names>X</given-names></name><name><surname>Yao</surname><given-names>L</given-names></name><name><surname>Shen</surname><given-names>J</given-names></name><name><surname>Tong</surname><given-names>D</given-names></name><name><surname>Hu</surname><given-names>C</given-names></name></person-group><article-title>The promoting effect of La, Mg, Co and Zn on the activity and stability of Ni/SiO<sub>2</sub> catalyst for CO<sub>2</sub> reforming of methane</article-title><source>Int. J. Hydrog. Energy</source><year>2011</year><volume>36</volume><fpage>7094</fpage><pub-id pub-id-type=\"doi\">10.1016/j.ijhydene.2011.02.133</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>T</given-names></name><name><surname>Zhang</surname><given-names>Q</given-names></name><name><surname>Cai</surname><given-names>W</given-names></name><name><surname>Zhang</surname><given-names>P</given-names></name><name><surname>Song</surname><given-names>X</given-names></name><name><surname>Sun</surname><given-names>Z</given-names></name><name><surname>Gao</surname><given-names>L</given-names></name></person-group><article-title>Phyllosilicate evolved hierarchical Ni- and Cu–Ni/SiO<sub>2</sub> nanocomposites for methane dry reforming catalysis</article-title><source>Appl. Catal. A</source><year>2015</year><volume>503</volume><fpage>94</fpage><pub-id pub-id-type=\"doi\">10.1016/j.apcata.2015.07.012</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>T</given-names></name><name><surname>Cai</surname><given-names>W</given-names></name><name><surname>Zhang</surname><given-names>P</given-names></name><name><surname>Song</surname><given-names>X</given-names></name><name><surname>Gao</surname><given-names>L</given-names></name></person-group><article-title>Cu–Ni@SiO<sub>2</sub> alloy nanocomposites for methane dry reforming catalysis</article-title><source>RSC Adv.</source><year>2013</year><volume>3</volume><fpage>23976</fpage><pub-id pub-id-type=\"doi\">10.1039/C3RA43203C</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>He</surname><given-names>S</given-names></name><name><surname>Wu</surname><given-names>H</given-names></name><name><surname>Yu</surname><given-names>W</given-names></name><name><surname>Mo</surname><given-names>L</given-names></name><name><surname>Lou</surname><given-names>H</given-names></name><name><surname>Zheng</surname><given-names>X</given-names></name></person-group><article-title>Combination of CO<sub>2</sub> reforming and partial oxidation of methane to produce syngas over Ni/SiO<sub>2</sub> and Ni–Al<sub>2</sub>O<sub>3</sub>/SiO<sub>2</sub> catalysts with different precursors</article-title><source>Int. J. Hydrogen Energy</source><year>2009</year><volume>34</volume><fpage>839</fpage><pub-id pub-id-type=\"doi\">10.1016/j.ijhydene.2008.10.072</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Baudouin</surname><given-names>D</given-names></name><name><surname>Candy</surname><given-names>JP</given-names></name><name><surname>Rodemerck</surname><given-names>U</given-names></name><name><surname>Krumeich</surname><given-names>F</given-names></name><name><surname>Veyre</surname><given-names>L</given-names></name><name><surname>Webb</surname><given-names>PB</given-names></name><name><surname>Thieuleux</surname><given-names>C</given-names></name><name><surname>Copéret</surname><given-names>C</given-names></name></person-group><article-title>Preparation of Sn-doped 2–3 nm Ni nanoparticles supported on SiO<sub>2</sub> via surface organometallic chemistry for low temperature dry reforming catalyst: The effect of tin doping on activity, selectivity and stability</article-title><source>Catal. Today</source><year>2014</year><volume>235</volume><fpage>237</fpage><pub-id pub-id-type=\"doi\">10.1016/j.cattod.2014.03.014</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Wang</surname><given-names>F</given-names></name><name><surname>Song</surname><given-names>Z</given-names></name><name><surname>Zhang</surname><given-names>S</given-names></name></person-group><article-title>Comparison of carbon deposition features between Ni/ZrO<sub>2</sub> and Ni/SBA-15 for the dry reforming of methane</article-title><source>React. Kinet. Mech. Catal.</source><year>2020</year><volume>129</volume><fpage>457</fpage><pub-id pub-id-type=\"doi\">10.1007/s11144-019-01707-5</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rezaei</surname><given-names>M</given-names></name><name><surname>Alavi</surname><given-names>SM</given-names></name><name><surname>Sahebdelfar</surname><given-names>S</given-names></name><name><surname>Bai</surname><given-names>P</given-names></name><name><surname>Liu</surname><given-names>X</given-names></name><name><surname>Yan</surname><given-names>Z</given-names></name></person-group><article-title>CO<sub>2</sub> reforming of CH<sub>4</sub> over nanocrystalline zirconia-supported nickel catalysts</article-title><source>Appl. Catal. B Environ.</source><year>2008</year><volume>77</volume><fpage>346</fpage><pub-id pub-id-type=\"doi\">10.1016/j.apcatb.2007.08.004</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>García</surname><given-names>V</given-names></name><name><surname>Fernández</surname><given-names>JJ</given-names></name><name><surname>Ruíz</surname><given-names>W</given-names></name><name><surname>Mondragón</surname><given-names>F</given-names></name><name><surname>Moreno</surname><given-names>A</given-names></name></person-group><article-title>Effect of MgO addition on the basicity of Ni/ZrO<sub>2</sub> and on its catalytic activity in carbon dioxide reforming of methane</article-title><source>Catal. Commun.</source><year>2009</year><volume>11</volume><fpage>240</fpage><pub-id pub-id-type=\"doi\">10.1016/j.catcom.2009.10.003</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Titus</surname><given-names>J</given-names></name><name><surname>Goepel</surname><given-names>M</given-names></name><name><surname>Schunk</surname><given-names>SA</given-names></name><name><surname>Wilde</surname><given-names>N</given-names></name><name><surname>Gläser</surname><given-names>R</given-names></name></person-group><article-title>The role of acid/base properties in Ni/MgO-ZrO<sub>2</sub>-based catalysts for dry reforming of methane</article-title><source>Catal. Commun.</source><year>2017</year><volume>100</volume><fpage>76</fpage><pub-id pub-id-type=\"doi\">10.1016/j.catcom.2017.06.027</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Asencios</surname><given-names>YJO</given-names></name><name><surname>Assaf</surname><given-names>EM</given-names></name></person-group><article-title>Combination of dry reforming and partial oxidation of methane on NiO–MgO–ZrO<sub>2</sub> catalyst: Effect of nickel content</article-title><source>Fuel Process. Technol.</source><year>2013</year><volume>106</volume><fpage>247</fpage><pub-id pub-id-type=\"doi\">10.1016/j.fuproc.2012.08.004</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Titus</surname><given-names>J</given-names></name><name><surname>Roussière</surname><given-names>T</given-names></name><name><surname>Wasserschaff</surname><given-names>G</given-names></name><name><surname>Schunk</surname><given-names>S</given-names></name><name><surname>Milanov</surname><given-names>A</given-names></name><name><surname>Schwab</surname><given-names>E</given-names></name><name><surname>Wagner</surname><given-names>G</given-names></name><name><surname>Oeckler</surname><given-names>O</given-names></name><name><surname>Gläser</surname><given-names>R</given-names></name></person-group><article-title>Dry reforming of methane with carbon dioxide over NiO–MgO–ZrO<sub>2</sub></article-title><source>Catal. Today</source><year>2016</year><volume>270</volume><fpage>68</fpage><pub-id pub-id-type=\"doi\">10.1016/j.cattod.2015.09.027</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nagaraja</surname><given-names>BM</given-names></name><name><surname>Bulushev</surname><given-names>DA</given-names></name><name><surname>Beloshapkin</surname><given-names>S</given-names></name><name><surname>Ross</surname><given-names>JR</given-names></name></person-group><article-title>H The effect of potassium on the activity and stability of Ni–MgO–ZrO<sub>2</sub> catalysts for the dry reforming of methane to give synthesis gas</article-title><source>Catal. Today</source><year>2011</year><volume>178</volume><fpage>132</fpage><pub-id pub-id-type=\"doi\">10.1016/j.cattod.2011.08.040</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>C</given-names></name><name><surname>Sun</surname><given-names>N</given-names></name><name><surname>Zhao</surname><given-names>N</given-names></name><name><surname>Wei</surname><given-names>W</given-names></name><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Zhao</surname><given-names>T</given-names></name><name><surname>Sun</surname><given-names>Y</given-names></name><name><surname>Sun</surname><given-names>C</given-names></name><name><surname>Liu</surname><given-names>H</given-names></name><name><surname>Snape</surname><given-names>CE</given-names></name></person-group><article-title>The properties of individual carbon residuals and their influence on the deactivation of Ni–CaO–ZrO<sub>2</sub> catalysts in CH<sub>4</sub> dry reforming</article-title><source>ChemCatChem</source><year>2014</year><volume>6</volume><fpage>640</fpage><pub-id pub-id-type=\"doi\">10.1002/cctc.201300754</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sun</surname><given-names>N</given-names></name><name><surname>Wen</surname><given-names>X</given-names></name><name><surname>Wang</surname><given-names>F</given-names></name><name><surname>Peng</surname><given-names>W</given-names></name><name><surname>Zhao</surname><given-names>N</given-names></name><name><surname>Xiao</surname><given-names>F</given-names></name><name><surname>Wei</surname><given-names>W</given-names></name><name><surname>Sun</surname><given-names>Y</given-names></name><name><surname>Kang</surname><given-names>J</given-names></name></person-group><article-title>Catalytic performance and characterization of Ni–CaO–ZrO<sub>2</sub> catalysts for dry reforming of methane</article-title><source>Appl. Surf. Sci.</source><year>2011</year><volume>257</volume><fpage>9169</fpage><pub-id pub-id-type=\"doi\">10.1016/j.apsusc.2011.05.127</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yabe</surname><given-names>T</given-names></name><name><surname>Mitarai</surname><given-names>K</given-names></name><name><surname>Oshima</surname><given-names>K</given-names></name><name><surname>Ogo</surname><given-names>S</given-names></name><name><surname>Sekine</surname><given-names>Y</given-names></name></person-group><article-title>Low-temperature dry reforming of methane to produce syngas in an electric field over La-doped Ni/ZrO<sub>2</sub> catalysts</article-title><source>Fuel Process. Technol.</source><year>2017</year><volume>158</volume><fpage>96</fpage><pub-id pub-id-type=\"doi\">10.1016/j.fuproc.2016.11.013</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sokolov</surname><given-names>S</given-names></name><name><surname>Kondratenko</surname><given-names>EV</given-names></name><name><surname>Pohl</surname><given-names>M-M</given-names></name><name><surname>Rodemerck</surname><given-names>U</given-names></name></person-group><article-title>Effect of calcination conditions on time on-stream performance of Ni/La<sub>2</sub>O<sub><bold>3</bold></sub>–ZrO<sub><bold>2</bold></sub> in low-temperature dry reforming of methane</article-title><source>Int. J. Hydrogen Energy</source><year>2013</year><volume>38</volume><fpage>16121</fpage><pub-id pub-id-type=\"doi\">10.1016/j.ijhydene.2013.10.013</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Montoya</surname><given-names>JA</given-names></name><name><surname>Pascual</surname><given-names>ER</given-names></name><name><surname>Gimon</surname><given-names>C</given-names></name><name><surname>Angel</surname><given-names>PD</given-names></name><name><surname>Monzón</surname><given-names>A</given-names></name></person-group><article-title>Methane reforming with CO<sub>2</sub> over Ni/ZrO<sub>2</sub>–CeO<sub>2</sub> catalysts prepared by sol–gel</article-title><source>Catal. Today</source><year>2000</year><volume>63</volume><fpage>71</fpage><pub-id pub-id-type=\"doi\">10.1016/S0920-5861(00)00447-8</pub-id></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arslan</surname><given-names>A</given-names></name><name><surname>Dogu</surname><given-names>T</given-names></name></person-group><article-title>Effect of calcination/reduction temperature of Ni impregnated CeO<sub>2</sub>-ZrO<sub>2</sub> catalysts on hydrogen yield and coke minimization in low temperature reforming of ethanol</article-title><source>Int. J. Hydrogen Energy</source><year>2016</year><volume>41</volume><fpage>16752</fpage><pub-id pub-id-type=\"doi\">10.1016/j.ijhydene.2016.07.082</pub-id></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Potdar</surname><given-names>RHS</given-names></name><name><surname>Jun</surname><given-names>KW</given-names></name></person-group><article-title>Carbon dioxide reforming of methane over co-precipitated Ni–CeO<sub>2</sub>, Ni–ZrO<sub>2</sub> and Ni–Ce–ZrO<sub>2</sub> catalysts</article-title><source>Catal. Today</source><year>2004</year><volume>93–95</volume><fpage>39</fpage><pub-id pub-id-type=\"doi\">10.1016/j.cattod.2004.05.012</pub-id></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>C</given-names></name><name><surname>Sun</surname><given-names>N</given-names></name><name><surname>Wei</surname><given-names>W</given-names></name><name><surname>Zhao</surname><given-names>Y</given-names></name></person-group><article-title>Carbon intermediates during CO<sub>2</sub> reforming of methane over Ni-CaO-ZrO<sub>2</sub> catalysts: a temperature-programmed surface reaction study</article-title><source>Int. J. Hydrogen Energy</source><year>2016</year><volume>41</volume><fpage>19014</fpage><pub-id pub-id-type=\"doi\">10.1016/j.ijhydene.2016.08.128</pub-id></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>Q</given-names></name><name><surname>Chai</surname><given-names>F</given-names></name><name><surname>Ma</surname><given-names>C</given-names></name><name><surname>Sun</surname><given-names>C</given-names></name><name><surname>Shi</surname><given-names>S</given-names></name><name><surname>Chen</surname><given-names>L</given-names></name></person-group><article-title>Enhanced coking tolerance of a MgO-modified Ni cermet anode for hydrocarbon fueled solid oxide fuel cells</article-title><source>J. Mater. Chem. A</source><year>2016</year><volume>4</volume><fpage>18031</fpage><pub-id pub-id-type=\"doi\">10.1039/C6TA08031F(2016)</pub-id></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sun</surname><given-names>CW</given-names></name><name><surname>Su</surname><given-names>R</given-names></name><name><surname>Chen</surname><given-names>J</given-names></name><name><surname>Lu</surname><given-names>L</given-names></name><name><surname>Guan</surname><given-names>PF</given-names></name></person-group><article-title>Carbon formation mechanism of C<sub>2</sub>H<sub>2</sub> in Ni-based catalysts revealed by in situ electron microscopy and molecular dynamics simulations</article-title><source>ACS Omega</source><year>2014</year><volume>3</volume><fpage>8413</fpage><pub-id pub-id-type=\"doi\">10.1021/acsomega.9b00958</pub-id></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mette</surname><given-names>K</given-names></name><name><surname>Kühl</surname><given-names>S</given-names></name><name><surname>Düdder</surname><given-names>H</given-names></name><name><surname>Kähler</surname><given-names>K</given-names></name><name><surname>Tarasov</surname><given-names>A</given-names></name><name><surname>Muhler</surname><given-names>M</given-names></name><name><surname>Behrens</surname><given-names>M</given-names></name></person-group><article-title>Stable performance of Ni catalysts in the dry reforming of methane at high temperatures for the efficient conversion of CO<sub>2</sub> into syngas</article-title><source>ChemCatChem</source><year>2014</year><volume>6</volume><fpage>100</fpage><pub-id pub-id-type=\"doi\">10.1002/cctc.201300699</pub-id></element-citation></ref><ref id=\"CR63\"><label>63.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kambolis</surname><given-names>A</given-names></name><name><surname>Matralis</surname><given-names>H</given-names></name><name><surname>Trovarelli</surname><given-names>A</given-names></name><name><surname>Papadopoulou</surname><given-names>C</given-names></name></person-group><article-title>Ni/CeO<sub>2</sub>-ZrO<sub>2</sub> catalysts for the dry reforming of methane</article-title><source>Appl. Catal. A</source><year>2010</year><volume>377</volume><fpage>16</fpage><pub-id pub-id-type=\"doi\">10.1016/j.apcata.2010.01.013</pub-id></element-citation></ref><ref id=\"CR64\"><label>64.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chung</surname><given-names>U-C</given-names></name></person-group><article-title>Effect of H<sub>2</sub> on formation behavior of carbon nanotubes</article-title><source>Bull. Korean Chem. Soc.</source><year>2004</year><volume>25</volume><issue>10</issue><fpage>1521</fpage><pub-id pub-id-type=\"doi\">10.4028/www.scientific.net/MSF.475-479.3559</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.3 20210610//EN\" \"JATS-archivearticle1-3-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\" article-type=\"review-article\" dtd-version=\"1.3\"><?properties open_access?><processing-meta base-tagset=\"archiving\" mathml-version=\"3.0\" table-model=\"xhtml\" tagset-family=\"jats\"><restricted-by>pmc</restricted-by></processing-meta><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Curr Clim Change Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Curr Clim Change Rep</journal-id><journal-title-group><journal-title>Current Climate Change Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2198-6061</issn><publisher><publisher-name>Springer International Publishing</publisher-name><publisher-loc>Cham</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32837849</article-id><article-id pub-id-type=\"pmc\">PMC7431553</article-id><article-id pub-id-type=\"publisher-id\">160</article-id><article-id pub-id-type=\"doi\">10.1007/s40641-020-00160-0</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Carbon Cycle and Climate (K Zickfeld, JR Melton and N Lovenduski, Section Editors)</subject></subj-group></article-categories><title-group><article-title>Tracking Improvement in Simulated Marine Biogeochemistry Between CMIP5 and CMIP6</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-2571-2114</contrib-id><name><surname>Séférian</surname><given-names>Roland</given-names></name><address><email>roland.seferian@meteo.fr</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0001-5782-2855</contrib-id><name><surname>Berthet</surname><given-names>Sarah</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-9879-2776</contrib-id><name><surname>Yool</surname><given-names>Andrew</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-0226-5243</contrib-id><name><surname>Palmiéri</surname><given-names>Julien</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0003-4732-4953</contrib-id><name><surname>Bopp</surname><given-names>Laurent</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-3572-3634</contrib-id><name><surname>Tagliabue</surname><given-names>Alessandro</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-6769-5957</contrib-id><name><surname>Kwiatkowski</surname><given-names>Lester</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0003-3954-506X</contrib-id><name><surname>Aumont</surname><given-names>Olivier</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0003-2448-6589</contrib-id><name><surname>Christian</surname><given-names>James</given-names></name><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-8794-0489</contrib-id><name><surname>Dunne</surname><given-names>John</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-9688-0692</contrib-id><name><surname>Gehlen</surname><given-names>Marion</given-names></name><xref ref-type=\"aff\" rid=\"Aff8\">8</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-3475-4842</contrib-id><name><surname>Ilyina</surname><given-names>Tatiana</given-names></name><xref ref-type=\"aff\" rid=\"Aff9\">9</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0003-2696-277X</contrib-id><name><surname>John</surname><given-names>Jasmin G.</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0003-2912-1837</contrib-id><name><surname>Li</surname><given-names>Hongmei</given-names></name><xref ref-type=\"aff\" rid=\"Aff9\">9</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0003-1273-2957</contrib-id><name><surname>Long</surname><given-names>Matthew C.</given-names></name><xref ref-type=\"aff\" rid=\"Aff10\">10</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-0032-9370</contrib-id><name><surname>Luo</surname><given-names>Jessica Y.</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-0769-3386</contrib-id><name><surname>Nakano</surname><given-names>Hideyuki</given-names></name><xref ref-type=\"aff\" rid=\"Aff11\">11</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0001-5241-4772</contrib-id><name><surname>Romanou</surname><given-names>Anastasia</given-names></name><xref ref-type=\"aff\" rid=\"Aff12\">12</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-7525-6882</contrib-id><name><surname>Schwinger</surname><given-names>Jörg</given-names></name><xref ref-type=\"aff\" rid=\"Aff13\">13</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0001-9549-8013</contrib-id><name><surname>Stock</surname><given-names>Charles</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-2627-1947</contrib-id><name><surname>Santana-Falcón</surname><given-names>Yeray</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0001-7984-8810</contrib-id><name><surname>Takano</surname><given-names>Yohei</given-names></name><xref ref-type=\"aff\" rid=\"Aff9\">9</xref><xref ref-type=\"aff\" rid=\"Aff14\">14</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0002-4600-2453</contrib-id><name><surname>Tjiputra</surname><given-names>Jerry</given-names></name><xref ref-type=\"aff\" rid=\"Aff13\">13</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0003-3336-0275</contrib-id><name><surname>Tsujino</surname><given-names>Hiroyuki</given-names></name><xref ref-type=\"aff\" rid=\"Aff11\">11</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0003-2563-7297</contrib-id><name><surname>Watanabe</surname><given-names>Michio</given-names></name><xref ref-type=\"aff\" rid=\"Aff15\">15</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0001-5187-9121</contrib-id><name><surname>Wu</surname><given-names>Tongwen</given-names></name><xref ref-type=\"aff\" rid=\"Aff16\">16</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0003-1066-6538</contrib-id><name><surname>Wu</surname><given-names>Fanghua</given-names></name><xref ref-type=\"aff\" rid=\"Aff16\">16</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">https://orcid.org/0000-0003-0314-4854</contrib-id><name><surname>Yamamoto</surname><given-names>Akitomo</given-names></name><xref ref-type=\"aff\" rid=\"Aff15\">15</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.30390.39</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2183 7107</institution-id><institution>CNRM, Université de Toulouse, </institution><institution>Météo-France, CNRS, </institution></institution-wrap>Toulouse, France </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.418022.d</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0603 464X</institution-id><institution>National Oceanography Centre, </institution></institution-wrap>European Way, Southampton, SO14 3ZH UK </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.440907.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1784 3645</institution-id><institution>LMD-IPSL, Ecole Normale Supérieure / Université PSL, CNRS, Ecole Polytechnique, Sorbonne Université, Paris, </institution><institution>PSL University, </institution></institution-wrap>Paris, France </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.10025.36</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 8470</institution-id><institution>School of Environmental Sciences, </institution><institution>University of Liverpool, </institution></institution-wrap>Liverpool, UK </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.462844.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2308 1657</institution-id><institution>LOCEAN Laboratory, </institution><institution>Sorbonne Université-CNRS-IRD-MNHN, </institution></institution-wrap>Paris, France </aff><aff id=\"Aff6\"><label>6</label>Canadian Centre for Climate Modelling and Analysis, Victoria, BC Canada </aff><aff id=\"Aff7\"><label>7</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.482795.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9269 5516</institution-id><institution>NOAA/Geophysical Fluid Dynamics Laboratory, </institution></institution-wrap>Princeton, NJ USA </aff><aff id=\"Aff8\"><label>8</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.460789.4</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 4910 6535</institution-id><institution>LSCE-IPSL, </institution><institution>Université Paris Saclay, </institution></institution-wrap>Gif-sur-Yvette, France </aff><aff id=\"Aff9\"><label>9</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.450268.d</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0721 4552</institution-id><institution>Max Planck Institute for Meteorology, </institution></institution-wrap>Hamburg, Germany </aff><aff id=\"Aff10\"><label>10</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.57828.30</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0637 9680</institution-id><institution>National Center for Atmospheric Research, </institution></institution-wrap>Boulder, CO USA </aff><aff id=\"Aff11\"><label>11</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.237586.d</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0597 9981</institution-id><institution>JMA Meteorological Research Institute, </institution></institution-wrap>Tsukuba, Japan </aff><aff id=\"Aff12\"><label>12</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.419078.3</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2284 9855</institution-id><institution>NASA Goddard Institute for Space Studies, </institution></institution-wrap>New York, USA </aff><aff id=\"Aff13\"><label>13</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.465508.a</institution-id><institution>NORCE Climate, </institution><institution>Bjerknes Centre for Climate Research, </institution></institution-wrap>Bergen, Norway </aff><aff id=\"Aff14\"><label>14</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.148313.c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0428 3079</institution-id><institution>Present Address: Los Alamos National Laboratory, </institution></institution-wrap>Los Alamos, NM USA </aff><aff id=\"Aff15\"><label>15</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.410588.0</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2191 0132</institution-id><institution>Research Center for Environmental Modeling and Application, </institution><institution>Japan Agency for Marine-Earth Science and Technology (JAMSTEC), </institution></institution-wrap>Yokohama, Japan </aff><aff id=\"Aff16\"><label>16</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.8658.3</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2234 550X</institution-id><institution>Beijing Climate Center, </institution><institution>China Meteorological Administration, </institution></institution-wrap>Beijing, China </aff></contrib-group><pub-date pub-type=\"epub\"><day>18</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>18</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"ppub\"><year>2020</year></pub-date><volume>6</volume><issue>3</issue><fpage>95</fpage><lpage>119</lpage><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license><ali:license_ref specific-use=\"textmining\" content-type=\"ccbylicense\">https://creativecommons.org/licenses/by/4.0/</ali:license_ref><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit <ext-link ext-link-type=\"uri\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><sec><title>Purpose of Review</title><p id=\"Par1\">The changes or updates in ocean biogeochemistry component have been mapped between CMIP5 and CMIP6 model versions, and an assessment made of how far these have led to improvements in the simulated mean state of marine biogeochemical models within the current generation of Earth system models (ESMs).</p></sec><sec><title>Recent Findings</title><p id=\"Par2\">The representation of marine biogeochemistry has progressed within the current generation of Earth system models. However, it remains difficult to identify which model updates are responsible for a given improvement. In addition, the full potential of marine biogeochemistry in terms of Earth system interactions and climate feedback remains poorly examined in the current generation of Earth system models.</p></sec><sec><title>Summary</title><p id=\"Par3\">Increasing availability of ocean biogeochemical data, as well as an improved understanding of the underlying processes, allows advances in the marine biogeochemical components of the current generation of ESMs. The present study scrutinizes the extent to which marine biogeochemistry components of ESMs have progressed between the 5th and the 6th phases of the Coupled Model Intercomparison Project (CMIP).</p></sec><sec><title>Electronic supplementary material</title><p>The online version of this article (10.1007/s40641-020-00160-0) contains supplementary material, which is available to authorized users.</p></sec></abstract><kwd-group xml:lang=\"en\"><title>Keywords</title><kwd>Marine Biogeochemistry</kwd><kwd>CMIP5</kwd><kwd>CMIP6</kwd><kwd>Biogeochemistry-Climate Feedbacks</kwd><kwd>Model Performance</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© Springer Nature Switzerland AG 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par4\">Marine biogeochemistry plays a key role in the Earth system. By regulating the exchange of CO2 and other climatically active gases with the atmosphere [<xref ref-type=\"bibr\" rid=\"CR1\">1</xref>], it is involved in a large range of climate feedbacks [<xref ref-type=\"bibr\" rid=\"CR2\">2</xref>]. As a result, changes in ocean biogeochemistry can have important consequences for climate [<xref ref-type=\"bibr\" rid=\"CR3\">3</xref>–<xref ref-type=\"bibr\" rid=\"CR5\">5</xref>]. Marine biogeochemistry is also deeply interwoven with the functioning of marine ecosystems and ultimately food webs [<xref ref-type=\"bibr\" rid=\"CR6\">6</xref>–<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>]. Marine ecosystems are affected by anthropogenic environmental change [<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>–<xref ref-type=\"bibr\" rid=\"CR11\">11</xref>], particularly through climate-induced changes in physical properties and CO<sub>2</sub>-induced ocean acidification [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>–<xref ref-type=\"bibr\" rid=\"CR16\">16</xref>]. Understanding and quantifying the response of ocean biogeochemistry to global changes, as well as its role in Earth system feedbacks [<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>, <xref ref-type=\"bibr\" rid=\"CR17\">17</xref>], are essential to improve our capacity to project ecosystem services and climate change in this century and beyond.</p><p id=\"Par5\">In this context, ocean biogeochemical models are acknowledged as powerful tools to study the ocean carbon cycle and its response to past and future climate and chemical changes [<xref ref-type=\"bibr\" rid=\"CR2\">2</xref>]. Since the pioneering assessment of anthropogenic carbon uptake by the ocean by Maier-Reimer and Hasselmann [<xref ref-type=\"bibr\" rid=\"CR18\">18</xref>] and Sarmiento et al. [<xref ref-type=\"bibr\" rid=\"CR19\">19</xref>], and the Ocean Carbon Model Intercomparison Project (OCMIP) of Orr et al. [<xref ref-type=\"bibr\" rid=\"CR20\">20</xref>], ocean biogeochemical models have been successfully integrated in many Earth system models (e.g. [<xref ref-type=\"bibr\" rid=\"CR21\">21</xref>–<xref ref-type=\"bibr\" rid=\"CR31\">31</xref>]).</p><p id=\"Par6\">Over the last few decades, the results from ocean biogeochemical models running within ESMs have increasingly been used to drive research on the carbon cycle. Their results have supported the assessment of carbon cycle feedbacks [<xref ref-type=\"bibr\" rid=\"CR32\">32</xref>–<xref ref-type=\"bibr\" rid=\"CR35\">35</xref>] and have improved the understanding of mechanisms behind the near-linear transient climate response to cumulative CO<sub>2</sub> emissions [<xref ref-type=\"bibr\" rid=\"CR36\">36</xref>]. Consequently, they have helped determine the change in carbon budgets that is compatible with a given level of warming since pre-industrial times. Ocean biogeochemical models have also been used to investigate potential geoengineering solutions to climate change such as solar radiation management [<xref ref-type=\"bibr\" rid=\"CR37\">37</xref>–<xref ref-type=\"bibr\" rid=\"CR39\">39</xref>], ocean fertilization [<xref ref-type=\"bibr\" rid=\"CR40\">40</xref>–<xref ref-type=\"bibr\" rid=\"CR47\">47</xref>], alkalinity addition [<xref ref-type=\"bibr\" rid=\"CR48\">48</xref>–<xref ref-type=\"bibr\" rid=\"CR52\">52</xref>] and reversibility experiments (e.g. [<xref ref-type=\"bibr\" rid=\"CR53\">53</xref>, <xref ref-type=\"bibr\" rid=\"CR54\">54</xref>]).</p><p id=\"Par7\">Recent advances in marine ecosystem modelling have also led to diversification in the use of ocean biogeochemistry models within ESMs to study a wide range of potential impacts [<xref ref-type=\"bibr\" rid=\"CR55\">55</xref>–<xref ref-type=\"bibr\" rid=\"CR58\">58</xref>]. These research activities are now grouped under the umbrella of the Inter-Sectoral Impact Model Intercomparison Project (ISIMIP), with the FishMIP initiative being a specific example for fisheries impacts [<xref ref-type=\"bibr\" rid=\"CR59\">59</xref>, <xref ref-type=\"bibr\" rid=\"CR60\">60</xref>].</p><p id=\"Par8\">Over recent years, models are increasingly being used in a semi-operational mode to aid with investigations of the predictability of key policy-relevant ocean biogeochemistry fields (e.g. net primary productivity, ocean acidity, ocean carbon uptake) [<xref ref-type=\"bibr\" rid=\"CR61\">61</xref>–<xref ref-type=\"bibr\" rid=\"CR67\">67</xref>]. Because of their close relationship with important living marine resources, skillful predictions of these properties have led to ocean biogeochemistry models being recognized as valuable tools when developing environmental policies (e.g. [<xref ref-type=\"bibr\" rid=\"CR68\">68</xref>]) or designing fisheries management [<xref ref-type=\"bibr\" rid=\"CR64\">64</xref>, <xref ref-type=\"bibr\" rid=\"CR65\">65</xref>, <xref ref-type=\"bibr\" rid=\"CR69\">69</xref>].</p><p id=\"Par9\">Because this large array of applications goes well beyond the conventional scientific investigation of the ocean carbon cycle, marine biogeochemical models have been developed in a number of directions over recent years. These developments are generally supported by progress in process understanding, which in turn is driven by an increasing number of observational databases [<xref ref-type=\"bibr\" rid=\"CR70\">70</xref>–<xref ref-type=\"bibr\" rid=\"CR72\">72</xref>]. However, from one generation to another, the development of marine biogeochemical models is driven not only by common scientific considerations but also by the internal priorities of individual modelling groups. As a consequence, it is difficult to anticipate how far the representation of marine biogeochemistry within the current generation of Earth system models differs from—and has improved upon—the previous one.</p><p id=\"Par10\">The present study maps the changes or updates in ocean biogeochemistry components that have arisen between CMIP5 and CMIP6 and assesses how far these have led to actual improvements in model skill against present-day observations. Overall, our assessment demonstrates that the simulated mean state of ocean biogeochemistry models in CMIP6 is more realistic than that produced by their CMIP5 analogues in many aspects, but that it remains difficult to clearly identify which changes in a given ocean biogeochemistry model are responsible for these improvements.</p></sec><sec id=\"Sec2\"><title>Mapping Changes or Updates in Ocean Biogeochemistry</title><p id=\"Par11\">In this section, we review the changes or updates implemented by participating modelling groups. The following method was employed to collect relevant model details as shown in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>. First, all of the modelling groups contributing both to CMIP5 and CMIP6 were approached. Next, a questionnaire in the form of a spreadsheet was proposed and developed. This sought details around (1) model resolution, (2) complexity in marine biology, (3) the representation of bacteria, (4) internal physiology, (5) organic matter cycling, (6) sediments, (7) nutrients and elemental cycling, (8) the level of interactions with the other components of the Earth system and (9) modelling approaches including spin-up protocols and tuning/calibration. The latter includes external inputs/outputs and biophysical interactions. The resulting master table of model properties is provided in Supplementary materials (Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>).<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Overview of the ocean and marine biogeochemical components of Earth system models as used in CMIP5 and CMIP6. The names of the ESM are given in the first line of the table where the CMIP6 ESMs are given in red cells and the CMIP5 predecessors are given in pink cells. The complexity of the marine biogeochemical models is described using (i) the trophic web, the number of living species or phytoplankton functional types; (ii) the internal physiology, the stoichiometry and the representation of internal photosynthetic pigment; (iii) the organic matter cycling, the number of organic carbon pools and their representation; (iv) the representation of marine sediments and (v) the nutrient cycling: the number of nutrients and the representation of oxygen and iron cycling</p></caption><graphic position=\"anchor\" xlink:href=\"40641_2020_160_Tab1a_HTML\" id=\"MO1\"/><table-wrap-foot><p>The reference paper of the reviewed ESMs is <sup>1</sup>Wu et al. [<xref ref-type=\"bibr\" rid=\"CR31\">31</xref>], <sup>2</sup>Wu et al. [<xref ref-type=\"bibr\" rid=\"CR73\">73</xref>], <sup>3</sup>Arora et al. [<xref ref-type=\"bibr\" rid=\"CR22\">22</xref>], <sup>4</sup>Swart et al. [<xref ref-type=\"bibr\" rid=\"CR74\">74</xref>], <sup>5</sup>Lindsay et al. [<xref ref-type=\"bibr\" rid=\"CR27\">27</xref>], <sup>6</sup>Danabasoglu et al. [<xref ref-type=\"bibr\" rid=\"CR75\">75</xref>], <sup>7</sup>Séférian et al. [<xref ref-type=\"bibr\" rid=\"CR29\">29</xref>, <xref ref-type=\"bibr\" rid=\"CR76\">76</xref>], <sup>8</sup>Séférian et al. [<xref ref-type=\"bibr\" rid=\"CR77\">77</xref>], <sup>9</sup>Dunne et al. [<xref ref-type=\"bibr\" rid=\"CR25\">25</xref>], <sup>10</sup>Held et al. [<xref ref-type=\"bibr\" rid=\"CR78\">78</xref>], <sup>11</sup>Krasting et al. [<xref ref-type=\"bibr\" rid=\"CR79\">79</xref>], <sup>12</sup>Romanou et al. [<xref ref-type=\"bibr\" rid=\"CR28\">28</xref>], <sup>13</sup>Ito et al. [<xref ref-type=\"bibr\" rid=\"CR80\">80</xref>], <sup>14</sup>Jones et al. [<xref ref-type=\"bibr\" rid=\"CR81\">81</xref>], <sup>15</sup>Sellar et al. [<xref ref-type=\"bibr\" rid=\"CR82\">82</xref>], <sup>16</sup>Dufresne et al. [<xref ref-type=\"bibr\" rid=\"CR24\">24</xref>], <sup>17</sup>Boucher et al. [<xref ref-type=\"bibr\" rid=\"CR83\">83</xref>], <sup>18</sup>Watanabe et al. [<xref ref-type=\"bibr\" rid=\"CR30\">30</xref>], <sup>19</sup>Hajima et al. [<xref ref-type=\"bibr\" rid=\"CR84\">84</xref>], <sup>20</sup>Giorgetta et al. [<xref ref-type=\"bibr\" rid=\"CR26\">26</xref>], <sup>21</sup>Mauritsen et al. [<xref ref-type=\"bibr\" rid=\"CR85\">85</xref>], <sup>22</sup>Adachi et al. [<xref ref-type=\"bibr\" rid=\"CR21\">21</xref>], <sup>23</sup>Yukimoto et al. [<xref ref-type=\"bibr\" rid=\"CR86\">86</xref>], <sup>24</sup>Bentsen et al. [<xref ref-type=\"bibr\" rid=\"CR23\">23</xref>], <sup>25</sup>Seland et al. [<xref ref-type=\"bibr\" rid=\"CR87\">87</xref>]. The reference paper of marine biogeochemical model is <sup>a</sup><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.ipsl.jussieu.fr/OCMIP/phase2\">http://www.ipsl.jussieu.fr/OCMIP/phase2</ext-link>, <sup>b</sup>Zahariev et al. [<xref ref-type=\"bibr\" rid=\"CR88\">88</xref>], <sup>c</sup>Hayashida et al. [<xref ref-type=\"bibr\" rid=\"CR89\">89</xref>], <sup>d</sup>Moore et al. [<xref ref-type=\"bibr\" rid=\"CR90\">90</xref>], <sup>e</sup>Aumont and Bopp [<xref ref-type=\"bibr\" rid=\"CR40\">40</xref>, <xref ref-type=\"bibr\" rid=\"CR41\">41</xref>], <sup>f</sup>Aumont et al. [<xref ref-type=\"bibr\" rid=\"CR91\">91</xref>], <sup>g</sup>Dunne et al. [<xref ref-type=\"bibr\" rid=\"CR25\">25</xref>], <sup>h</sup>Stock et al. [<xref ref-type=\"bibr\" rid=\"CR92\">92</xref>], <sup>i</sup>Dunne et al. [<xref ref-type=\"bibr\" rid=\"CR93\">93</xref>, <xref ref-type=\"bibr\" rid=\"CR94\">94</xref>], <sup>j</sup>Romanou et al. [<xref ref-type=\"bibr\" rid=\"CR28\">28</xref>], <sup>k</sup>Lerner et al. [<xref ref-type=\"bibr\" rid=\"CR95\">95</xref>], <sup>l</sup>Totterdell [<xref ref-type=\"bibr\" rid=\"CR96\">96</xref>], <sup>m</sup>Yool et al. [<xref ref-type=\"bibr\" rid=\"CR97\">97</xref>], <sup>p</sup>Watanabe et al. [<xref ref-type=\"bibr\" rid=\"CR30\">30</xref>], <sup>q</sup>Hajima et al. [<xref ref-type=\"bibr\" rid=\"CR98\">98</xref>], <sup>r</sup>Ilyina et al. [<xref ref-type=\"bibr\" rid=\"CR51\">51</xref>, <xref ref-type=\"bibr\" rid=\"CR52\">52</xref>], <sup>s</sup>Paulsen et al. [<xref ref-type=\"bibr\" rid=\"CR99\">99</xref>], <sup>t</sup>Nakano et al. [<xref ref-type=\"bibr\" rid=\"CR100\">100</xref>], <sup>u</sup>Tjiputra et al. [<xref ref-type=\"bibr\" rid=\"CR101\">101</xref>], <sup>v</sup>Tjiputra et al. [<xref ref-type=\"bibr\" rid=\"CR102\">102</xref>]. Bold text highlights when a model component has been completely updated</p></table-wrap-foot></table-wrap></p><p id=\"Par12\">Tables <xref rid=\"Tab1\" ref-type=\"table\">1</xref>, <xref rid=\"Tab2\" ref-type=\"table\">2</xref> and <xref rid=\"Tab3\" ref-type=\"table\">3</xref> map the key updates made between CMIP5 and CMIP6 (full details are available in Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>). Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> suggests that most of the changes have tried to address at least one missing process of major importance for marine biogeochemistry, as highlighted in IPCC AR5 ([<xref ref-type=\"bibr\" rid=\"CR2\">2</xref>], page 499), that is, representation of the lower trophic level including bacteria, organic matter cycling including sinking particles or variation in stoichiometric ratios.<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Overview of the ocean and marine biogeochemical components of Earth system models as used in CMIP5 and CMIP6. The names of the ESM are given in the first line of the table where the CMIP6 ESMs are given in red cells and the CMIP5 predecessors are given in pink cells. The Earth system interactions or couplings represented within the ESMs involving the marine biogeochemical models are described using three characteristics: the external inputs of nutrients or carbon-related fields conveyed by external boundary conditions are given with the chemical acronyms (C, P, N, Si, Fe), the representation of the gas exchange of greenhouse gases or reactive chemical species and the representation of Earth system feedbacks. In the last rows, ‘Fx’ indicates that the climate feedbacks or Earth system interactions ‘x’ as depicted in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> is represented in a given Earth system model</p></caption><graphic position=\"anchor\" xlink:href=\"40641_2020_160_Tab2_HTML\" id=\"MO3\"/></table-wrap><table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Overview of the ocean and marine biogeochemical components of Earth system models as used in CMIP5 and CMIP6. The names of the ESM are given in the first column of the table where the CMIP6 ESMs are given in red cells and the CMIP5 predecessors are given in pink cells. The modelling framework conducted by the various modelling groups for CMIP5 and CMIP6 is reviewed using two key characteristics: the duration of the spin-up simulation and the use of calibration/tuning procedure (further details about model calibration/tuning is given in Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>)</p></caption><graphic position=\"anchor\" xlink:href=\"40641_2020_160_Tab3_HTML\" id=\"MO4\"/></table-wrap></p><p id=\"Par13\">Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> includes a brief overview of the key updates in ocean physics between CMIP5 and CMIP6 because marine biogeochemistry is prominently driven by ocean circulation (large-scale circulation and mesoscale eddies) and vertical mixing.</p><p id=\"Par14\">Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> tracks not only updates in the horizontal and vertical resolution of physical ocean models but also changes in related ocean physical parameterization. As suggested by Griffies et al. [<xref ref-type=\"bibr\" rid=\"CR103\">103</xref>], an increase in horizontal or vertical resolution enables the representation of finer-scale ocean physical processes (e.g. mesoscale eddies) in relation with the activation of more realistic ocean physical parameterizations (such as vertical mixing, diurnal cycle or coupling with the atmosphere).</p><p id=\"Par15\">The first common difference between CMIP5 and CMIP6 ESMs comes from the ocean-sea ice components. Indeed, it is interesting to note that 8 ESM groups out of 12 use an upgraded version of the ocean models or employ a new ocean model (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>). These changes imply substantial updates or revisions in ocean physical parameterizations that may have an impact on large-scale circulation and vertical mixing.</p><p id=\"Par16\">In addition, another common difference between ocean models used in CMIP5 and CMIP6 is the grid resolution. It is interesting to note that all of the ocean models, with the exception of MPI-ESM1-2-LR, now resolve ocean dynamics at a minimum horizontal nominal resolution of 100 km. The highest horizontal nominal resolution in the available multi-model ensemble is 50 km (GFDL-ESM4). Despite this general increase in horizontal resolution, only GFDL-CM4 uses an eddy-permitting ocean model (~ 25 km). In addition, the current generation of ocean models also better represent vertical physical processes with a typically finer vertical resolution.</p><p id=\"Par17\">Another common difference between the two generations of models is the complexity of the marine ecosystem description and related parameterizations. Here, the complexity encompasses the diversity of model trophic web (i.e. the number of specific model phytoplankton and zooplankton types), the representation of bacteria, ecosystem functioning including macro- and micro-nutrient limitation (e.g. iron), and the variation in modelled stoichiometric ratios of carbon, nitrogen and other elements (e.g. photosynthetic pigment). Greater complexity does not necessarily imply a better representation of cycles and processes associated with each biogeochemical species, as it may introduce new degrees of freedom and/or non-linear (or at least not well controlled) interactions between parameterizations.</p><p id=\"Par18\">Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> shows that ocean biogeochemistry models span a wide range of complexity levels. The simplest models use ocean carbon cycle models based on the OCMIP protocol [<xref ref-type=\"bibr\" rid=\"CR20\">20</xref>] that do not include marine biota or nutrients. Meanwhile, the most complex models include a broad trophic structure that groups marine organisms into plankton functional types based on their biogeochemical role, with mechanistic representations of nutrient limitation and variable stoichiometric ratios.</p><p id=\"Par19\">Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> also highlights noticeable changes in biogeochemical parameterizations between CMIP5 and CMIP6. They concern 10 biogeochemical models out of 12 reviewed in this study. These changes may be related to the change in model complexity or to a revised set of parameterizations (e.g. nitrogen fixation, remineralization, grazing, flux feeding; see Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>).</p><p id=\"Par20\">We map updates and changes in ocean biogeochemical models along three major axes; axis 1. The trophic food web, the plankton internal physiology (e.g. variable stoichiometry, chlorophyll pigment) and nutrients cycling (iron cycle, nutrients cycles). This axis aims to track updates in biogeochemical dynamics and ecosystem functioning; axis 2. The external sources of nutrients; axis 3. The interactions of marine biogeochemistry with climate or ocean physics. The latter two axes track the level of integration of the marine biogeochemical model in the modelled Earth system.</p><p id=\"Par21\">It is important to stress that an increase or a decrease along one of those three axes does not necessarily imply an improvement in model performance or skill. In most cases, it reflects progress in process understanding (physical, biogeochemical or both), the inclusion of new Earth system interactions or the representation of climate feedbacks is required to investigate future scenarios.</p><p id=\"Par22\">Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> shows that the current generation of CMIP6 displays a greater diversity of marine biogeochemical models than CMIP5.</p><p id=\"Par23\">COBALTv2 (in GFDL-ESM 4), for instance, displays the highest trophic complexity level with 3 explicit phytoplankton classes, 1 implicit phytoplankton class, 3 explicit zooplankton classes and 1 explicit heterotrophic bacteria class; however, this model still employs a relatively simple parameterization of iron cycling. In comparison, PISCESv2-gas (in CNRM-ESM2-1) or PISCESv2 (in IPSL-CM6A-LR) includes 4 explicit plankton types (2 phytoplankton and 2 zooplankton), but two iron ligands and 5 iron forms [<xref ref-type=\"bibr\" rid=\"CR104\">104</xref>]. MARBL-BEC (in CESM2) also includes an iron ligand and has opted for increasing ecosystem complexity by introducing variable C:P stoichiometry, based on PO<sub>4</sub> concentrations [<xref ref-type=\"bibr\" rid=\"CR105\">105</xref>], while maintaining 4 plankton types. It is interesting to note that, while limiting the number of nutrients, CanESM5-CanOE have evolved toward a more comprehensive treatment of marine biogeochemistry with 4 explicit plankton types and using variable stoichiometry [<xref ref-type=\"bibr\" rid=\"CR89\">89</xref>]. In contrast with a general increase in complexity, NOAA-GFDL has started to use a reduced complexity marine biogeochemical model embedded in the high-resolution ocean model of GFDL-CM4. This approach implies a trade-off between computational costs and essential biogeochemical processes to represent the ocean carbon cycle as explained in Galbraith et al. [<xref ref-type=\"bibr\" rid=\"CR105\">105</xref>]. Such diversity tends to mirror progress in the understanding of the impact of variable stoichiometric ratios on ecosystem dynamics and carbon assimilation by phytoplankton cells [<xref ref-type=\"bibr\" rid=\"CR106\">106</xref>–<xref ref-type=\"bibr\" rid=\"CR110\">110</xref>].</p><p id=\"Par24\">Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> shows that all CMIP6 models except GFDL-CM4 have evolved toward a more comprehensive treatment of elemental cycling including nitrogen, oxygen and iron cycling. This moderate increase in model complexity is supported by recent observations in phytoplankton functioning, nutrient limitation or plankton physiology [<xref ref-type=\"bibr\" rid=\"CR111\">111</xref>–<xref ref-type=\"bibr\" rid=\"CR116\">116</xref>] and the availability of a larger array of observational data (bio-ARGO and GEOTRACES) supporting the model evaluation and development (e.g. Tagliabue et al. [<xref ref-type=\"bibr\" rid=\"CR117\">117</xref>]). On the other hand, this increase in complexity is also encouraged by the growing range of applications to which ESMs are being dedicated (e.g. marine resource applications as investigated in Lotze et al. [<xref ref-type=\"bibr\" rid=\"CR59\">59</xref>] or Park et al. [<xref ref-type=\"bibr\" rid=\"CR64\">64</xref>]).</p><p id=\"Par25\">Finally, Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> shows that all CMIP6 models have progressed toward a better representation of marine organic carbon cycling, sinking particles and marine sediments. In most cases, this component of marine biogeochemistry is parameterized using either a sediment box module or a meta-model based on downward fluxes of organic matter. Indeed, for several CMIP6 marine biogeochemical models, a more complex representation of sinking particles and organic matter pools (refractory classes or flux attenuation parameterization) replaces the generalized pools of organic matter used in the CMIP5 predecessors.</p><p id=\"Par26\">Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> also sheds light on noticeable changes in the representation of sediment interactions. Most of the reviewed CMIP6 ESMs now simulate this compartment with biogeochemical parameterization (e.g. balance, meta-model, sediment box) or with a comprehensive sediment module (12-layer sediments module).</p><p id=\"Par27\">Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref> also shows that the representation of the external sources of nutrients (i.e. the third axis of our model complexity breakdown) has grown in complexity between CMIP5 and CMIP6. It mirrors a more comprehensive treatment of boundary conditions between ESM components (atmosphere, rivers, glaciers, etc.). Most of the current generation of ocean biogeochemical models now consider inputs of biogeochemical elements via atmospheric deposition or from rivers. The iron delivery from sediment mobilization, hydrothermal sources or ice melting is additionally considered by a small set of models. This reflects recent advances in understanding the global iron cycle [<xref ref-type=\"bibr\" rid=\"CR111\">111</xref>–<xref ref-type=\"bibr\" rid=\"CR116\">116</xref>]. In contrast, despite a better understanding of the role of submarine water discharge in ocean nutrient supply [<xref ref-type=\"bibr\" rid=\"CR118\">118</xref>–<xref ref-type=\"bibr\" rid=\"CR121\">121</xref>], this particular boundary condition is not considered in the current generation of ocean biogeochemical models.</p><p id=\"Par28\">Besides, it is interesting to note that a couple of CMIP6 ESMs now includes a more comprehensive treatment of interactions between the marine biogeochemistry and the other Earth system components. For instance, GFDL-ESM 4 simulates interactively most of the primary source of iron for marine biogeochemistry (atmospheric dust deposition, iceberg melting and river supply), enabling the representation of biogeochemical couplings observed in the real world (e.g. [<xref ref-type=\"bibr\" rid=\"CR122\">122</xref>]).</p><p id=\"Par29\">Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref> highlights that the current generation of ESMs displays a wider range of Earth system feedbacks or interactions. In our review, we have decomposed Earth system interactions involving marine biogeochemistry along two axes: (1) the air-sea exchange of greenhouse gases or reactive chemical compounds interacting with Earth’s radiative budget (and hence climate); (2) the represented Earth system interactions involving marine biogeochemistry (including the air-sea exchange of greenhouse gases or reactive chemical compounds and biophysical interactions); that is, what is really contributing to the Earth system model climate. This latter has been mapped into 4 feedbacks: climate-carbon cycle feedbacks (F1), biogenic aerosol-cloud feedbacks (F2), non-CO<sub>2</sub> biogeochemical cycle feedbacks (F3) and phytoplankton-light feedbacks (F4).</p><p id=\"Par30\">The influence of ocean dimethylsulfide (DMS) emissions on cloud albedo is an example of the biogenic aerosol-cloud feedback (F2). DMS is a breakdown product of dimethylsulfoniopropionate (DMSP), a metabolite in many phytoplankton with a role as a cellular osmolyte/antioxidant [<xref ref-type=\"bibr\" rid=\"CR123\">123</xref>, <xref ref-type=\"bibr\" rid=\"CR124\">124</xref>]. It is exchanged with the atmosphere and is involved in the formation of sulfur aerosols once it is oxidized there. As the other sulfate aerosols, DMS may be involved in the formation of cloud condensation nuclei (CCN). The potential importance of ocean DMS emissions for the climate system is still largely debated [<xref ref-type=\"bibr\" rid=\"CR125\">125</xref>] because modern observations do not support its prominent role in the formation of CCN [<xref ref-type=\"bibr\" rid=\"CR126\">126</xref>–<xref ref-type=\"bibr\" rid=\"CR128\">128</xref>]. However, long-term measurement [<xref ref-type=\"bibr\" rid=\"CR129\">129</xref>] and mesocosm experiments (e.g. [<xref ref-type=\"bibr\" rid=\"CR17\">17</xref>]) suggest that global changes may impact the rate of ocean DMS emissions. Recent modelling studies argue for a potential role of ocean DMS in future climate change (e.g. [<xref ref-type=\"bibr\" rid=\"CR130\">130</xref>, <xref ref-type=\"bibr\" rid=\"CR131\">131</xref>]). Ocean NH<sub>x</sub> emissions are also involved in biogenic aerosol-cloud feedbacks (F2). Kirkby et al. [<xref ref-type=\"bibr\" rid=\"CR132\">132</xref>] suggest that NH<sub>x</sub> can also play an important role in the formation of secondary nitrate aerosols in the atmosphere. Similarly to DMS, these aerosols can serve as CCN and contribute to changes in cloud albedo. Non-CO<sub>2</sub> biogeochemical cycle feedbacks (F3) involve ocean emissions of non-CO<sub>2</sub> greenhouse gases (e.g. N<sub>2</sub>O or methane) or any chemical compounds contributing to the generation of greenhouses gases (e.g. methane, carbon monoxide). The phytoplankton-light feedbacks (F4) represent the suite of biophysical mechanisms that involve the influence of the marine biota on the upper ocean physics through the vertical redistribution of heat.</p><p id=\"Par31\">Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref> confirms that all ocean biogeochemical models account for the climate-carbon cycle feedback since CMIP5 (Earth system feedback F1 in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). In addition, Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> shows that the current generation of ocean biogeochemical models includes an air-sea gas exchange for a larger number of radiatively active biogeochemical compounds such as DMS, nitrous oxide (N<sub>2</sub>O) and ammonia (NH<sub>x</sub>). The inclusion of climate active gases or greenhouse gases other than CO<sub>2</sub> in the current generation of ocean biogeochemical models is a result of the increased recognition of the importance of these compounds in Earth system interactions with aerosols, atmospheric chemistry and, potentially, with clouds.<fig id=\"Fig1\"><label>Fig. 1</label><caption><p>Schematic representation of Earth system interactions and feedbacks between the ocean biogeochemistry and climate. F1 represents the well-established climate-carbon cycle feedbacks; F2 and F3 sketch the dominant pathways for the biogenic aerosol-cloud feedbacks and the non-CO<sub>2</sub> biogeochemical cycle feedbacks; F4 depicts the phytoplankton-light feedbacks (that is a biophysical interactions)</p></caption><graphic xlink:href=\"40641_2020_160_Fig1_HTML\" id=\"MO5\"/></fig></p><p id=\"Par32\">In particular, the inclusion of ocean NH<sub>x</sub> or N<sub>2</sub>O emissions in ocean biogeochemical models has been driven by a better understanding of the global nitrogen cycle and its role in climate change. In particular, the development of databases such as MEMENTO (https://memento.geomar.de/) has enabled better validation and calibration of N<sub>2</sub>O modules in global ocean biogeochemical models [<xref ref-type=\"bibr\" rid=\"CR133\">133</xref>–<xref ref-type=\"bibr\" rid=\"CR138\">138</xref>].</p><p id=\"Par33\">However, the inclusion of Earth system feedbacks as illustrated in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> has not in all cases progressed between CMIP5 and CMIP6. For example, biophysical interactions with the ocean radiative transfer (F4 in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>) are overlooked by more than half of the marine biogeochemical models examined, although this feedback is well documented and relatively well understood [<xref ref-type=\"bibr\" rid=\"CR139\">139</xref>, <xref ref-type=\"bibr\" rid=\"CR140\">140</xref>].</p><p id=\"Par34\">Our review of available ESMs suggests that the current generation of marine biogeochemical models has not much evolved toward comprehensive couplings between Earth system components and ocean biogeochemistry or toward improved treatment of biophysical and biogeochemical feedback with respect to their predecessors (F1 and F4 in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). The full impact of ocean biogeochemistry on climate and its role in Earth system feedback remains far from being entirely represented in the current generation of Earth system models, as it involves different spatial and temporal scales that models are not currently able to reach and also processes still poorly understood.</p><p id=\"Par35\">Finally, our review suggests that the modelling approaches have evolved between CMIP5 and CMIP6. These latter have been monitored with two key indicators: (1) the length of the spin-up simulation and (2) the use of calibration/tuning for marine biogeochemical parameters. These two key indicators were discussed in published literature (e.g. Séférian et al. [<xref ref-type=\"bibr\" rid=\"CR76\">76</xref>] or Hourdin et al. [<xref ref-type=\"bibr\" rid=\"CR141\">141</xref>]), reflecting, in general, an improved knowledge in model characteristics (strength and deficiency).</p><p id=\"Par36\">Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref> and Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref> highlight that most of the modelling groups have expanded the duration of the spin-up for CMIP6. This represents an important effort of the scientific community to converge toward recommended standards (e.g. [<xref ref-type=\"bibr\" rid=\"CR142\">142</xref>]). Only GFDL and IPSL have reduced the duration of their spin-up protocol for computing reasons: they manage to fulfil CMIP6 standard in a few hundreds of years. On the other hand, it is noticeable that several modelling groups have included a step of model calibration or tuning in CMIP6. Our review suggests that this step has been motivated by various reasons: bias reduction for key biogeochemical fields in CNRM, GFDL or NorESM or bias compensation to reduce the impact of known biases in simulated surface chlorophyll for ocean DMS and organic aerosols emissions in UKESM. There is no consensus between modelling groups on how model calibration or tuning takes place in the model preparation. Depending on modelling group, the calibration or tuning is either included in the model development or during the spin-up procedure (Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>).</p></sec><sec id=\"Sec3\"><title>Tracking Model Performance Across Two Generations of Models</title><p id=\"Par37\">Figures <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>, <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>, <xref rid=\"Fig4\" ref-type=\"fig\">4</xref> and <xref rid=\"Fig5\" ref-type=\"fig\">5</xref> illustrate the performance of the current generation of ESMs taking part in CMIP6, together with their predecessor CMIP5 models, for a range of climatological biogeochemical properties that are central to the carbon cycle and ecosystem applications: the sea-to-air flux of CO<sub>2</sub>, ocean chlorophyll, nitrate, silicate, oxygen and iron (see Methods in Supplementary materials). For Figs. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>, <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> and <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>, observation-based estimates of each property are shown at the top of the figure, followed by the biases found across the current and last generation models. We note that, in several cases, observation-based estimates are derived from significant processing of sparse observations or from algorithms relating the quantity of interest to directly observed quantities (e.g. sea-to-air CO<sub>2</sub> flux, satellite chlorophyll). As such, the observations themselves are also subject to uncertainty which will be discussed in the context of each comparison.<fig id=\"Fig2\"><label>Fig. 2</label><caption><p>Model-data intercomparison of <bold>a</bold> open ocean-sea-air carbon fluxes (fgco2, g C m<sup>−2</sup> year<sup>−1</sup>) and <bold>b</bold> open ocean surface chlorophyll (chl, mg Chl m<sup>−3</sup>) as simulated by ocean biogeochemical models embedded within CMIP6 Earth system models (the right column) and their former version as used for CMIP5 (the left column). <bold>a</bold> The first top panel shows observation-based estimates from Landschützer et al. [<xref ref-type=\"bibr\" rid=\"CR143\">143</xref>] averaged for the period 1995–2014 (see Methods in Supplementary materials). The other panels show model-data biases averaged for the same period. Coloured areas are indicative of the model-data absolute difference in magnitude of sea-air fluxes. Red regions indicate areas in models where the magnitude of the sea-air flux is greater than that observed, whereas blue regions indicate the reverse. <bold>b</bold> The first top panel shows satellite-based ocean chlorophyll estimates from ESA-CCI-OC [<xref ref-type=\"bibr\" rid=\"CR144\">144</xref>] averaged over 1998–2014. The other panels show model-data departure averaged over the period 1998–2014</p></caption><graphic xlink:href=\"40641_2020_160_Fig2_HTML\" id=\"MO6\"/></fig><fig id=\"Fig3\"><label>Fig. 3</label><caption><p>Model-data intercomparison of <bold>a</bold> surface nitrate concentrations (no3, μmol L<sup>−1</sup>) and <bold>b</bold> surface silicic acid concentrations (si, μmol L<sup>−1</sup>) as simulated by ocean biogeochemical models embedded within CMIP6 Earth system models (right columns) and their former version as used for CMIP5 (left columns). <bold>a</bold> and <bold>b</bold> The first top panel shows the optimal interpolation of nitrate (no3) and silicate (si) measurements as provided in the World Ocean Atlas Database 2013 (Garcia et al. [<xref ref-type=\"bibr\" rid=\"CR145\">145</xref>]). The other panels show model-data departure averaged over the period 1995–2014 (see Methods in Supplementary materials)</p></caption><graphic xlink:href=\"40641_2020_160_Fig3_HTML\" id=\"MO7\"/></fig><fig id=\"Fig4\"><label>Fig. 4</label><caption><p>Model-data intercomparison of oxygen concentrations at 150 m (o2, μmol L<sup>−1</sup>) as a proxy for oxygen minimum zones (OMZs) and as simulated by ocean biogeochemical models embedded within CMIP6 Earth system models (on the right column) and within their former version used for CMIP5 (on the left column). The first top panels in <bold>a</bold> and <bold>b</bold> show the observed oxygen concentrations at 150 m from the World Ocean Atlas 2013 (Garcia et al. [<xref ref-type=\"bibr\" rid=\"CR145\">145</xref>]). The other panels in <bold>a</bold> show oxygen concentrations at 150 m as simulated by CMIP5 and CMIP6 models averaged over the period 1995–2014, while panels in <bold>b</bold> show model-data departure averaged over the period 1995–2014 (see Methods in Supplementary materials)</p></caption><graphic xlink:href=\"40641_2020_160_Fig4_HTML\" id=\"MO8\"/></fig><fig id=\"Fig5\"><label>Fig. 5</label><caption><p>Model-data scatterplots for surface dissolved iron concentrations (log-log scale). Observational data are derived from the average of the 0–10 m of the measurement compilation used in Tagliabue et al. [<xref ref-type=\"bibr\" rid=\"CR117\">117</xref>]. Model concentrations are taken from the first ocean layer. Red dots and blue triangles indicate CMIP6 and CMIP5 models respectively. The red dashed line shows the 1:1 line; the red and blue solid lines highlight the model-data mismatch in terms of global mean concentrations for CMIP5 and CMIP6 models (see Methods in Supplementary materials). The global mean for observations and models are given in brackets. Model-data fit (squared correlation, <italic>R</italic><sup>2</sup>) is given in parenthesis with squared correlation coefficients for CMIP5 and CMIP6 models</p></caption><graphic xlink:href=\"40641_2020_160_Fig5_HTML\" id=\"MO9\"/></fig></p><p id=\"Par38\">In Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a</xref>, the sea-to-air flux of the critical greenhouse gas, CO<sub>2</sub>, is shown, with a data product based on the mapping of observational pCO<sub>2</sub> data drawn from the Landschützer et al. [<xref ref-type=\"bibr\" rid=\"CR143\">143</xref>] product (1995–2014). The key geographical features of this are strong outgassing (i.e. a net sea-to-air flux) in upwelling regions, most clearly in the tropics and along the equatorial region of the Pacific Ocean, and ingassing (i.e. a net air-to-sea flux) at temperate and subpolar latitudes. These features reflect processes that are governed by temperature, patterns of deep water formation, surface biological production and the thermohaline circulation.</p><p id=\"Par39\">In general, both CMIP5 and CMIP6 generations of models show a mixture of positive and negative biases across the globe with disagreement in the sign of the carbon fluxes over some regions. Common patterns are slightly negative biases both in the equatorial Pacific (i.e. weak outgassing) and in the North Atlantic (i.e. excessive ingassing). Both generations of models show a mix of relatively small positive and negative biases, except for the CMIP5 CanESM2 which shows the largest model-data error across the model ensemble. However, the comparison with observations has been substantially improved in CanESM5. More generally, Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a</xref> highlights that the improvement in simulated sea-to-air carbon flux is clearer when looking at the direction of the carbon flux. This improvement seems to be linked to an improved representation of ocean vertical mixing (see skill scores of the ocean mixed-layer depth below). Indeed, all CMIP6 models exhibit smaller domains where the direction of the sea-to-air carbon flux disagrees with observations, except for MPI-ESM1-2-LR, which used the same ocean model and displays the same pattern of model-data disagreement for CMIP5 and CMIP6.</p><p id=\"Par40\">Figure <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> b shows surface chlorophyll, compared with satellite-based estimates derived from ESA-CCI-OC ocean colour data [<xref ref-type=\"bibr\" rid=\"CR144\">144</xref>]. The key geographical features are relatively high concentrations in productive temperate, subpolar and upwelling regions, and extremely low concentrations in the unproductive subtropical gyres. The latter are dominated by perennially low-nutrient conditions, while the former experience frequent, or seasonal, introduction of nutrients by upwelling or deep mixing. While these general biome scale patterns are robust across satellite algorithms, we note that estimates diverge in the Southern Ocean [<xref ref-type=\"bibr\" rid=\"CR146\">146</xref>], where global satellite-based chlorophyll algorithms have been found to significantly underestimate observations [<xref ref-type=\"bibr\" rid=\"CR147\">147</xref>].</p><p id=\"Par41\">Several CMIP6 models compare more favourably with observations than their CMIP5 predecessors. All models displaying a pattern of generally negative bias in CMIP5 now exhibit large areas of both small positive and small negative biases. Models overestimating surface chlorophyll concentrations in CMIP5 now display reduced biases (< 0.4 mg Chl m<sup>−3</sup>). This improvement is small for MPI-ESM1-2-LR, which still overestimates surface concentrations of chlorophyll. Some CMIP6 models, such as CESM2, GISS-E2-1-G-CC and NorESM2-LM, display on the contrary larger model-data errors than their predecessors. Given the large diversity across the models, it is difficult to determine whether changes in physical ocean models or changes in ocean biogeochemical models are behind these changes.</p><p id=\"Par42\">However, it is interesting to note that three CMIP6 models (CNRM-ESM 2-1, IPSL-CM6A-LR and UKESM1-0-LL), which share a common ocean physics model, overlap in their patterns of positive and negative biases in spite of differences in marine biogeochemistry submodels (spatial correlation of model-data errors R<sup>2</sup> = ~0.5).</p><p id=\"Par43\">It is notable that most of the models reviewed here overestimate surface chlorophyll estimates in the Southern Ocean. This bias, however, is likely due in part to the underestimation of Southern Ocean chlorophyll by the global satellite chlorophyll algorithms [<xref ref-type=\"bibr\" rid=\"CR147\">147</xref>]. The substantial positive Southern Ocean bias in GFDL-ESM 4, for example, is significantly diminished when compared against Johnson’s Southern Ocean-specific satellite-based chlorophyll algorithms (e.g. [<xref ref-type=\"bibr\" rid=\"CR148\">148</xref>]).</p><p id=\"Par44\">Figure <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> a and b show the distribution of surface nitrate (NO<sub>3</sub>) and silicic acid (H<sub>4</sub>SiO<sub>4</sub>), which are represented in both CMIP5 and CMIP6 models. Figure <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> a shows that only GFDL, IPSL and MIROC models have consistently improved their mean states between CMIP5 and CMIP6 for nitrate concentrations. In some cases, model generations show the same spatial patterns of biases, while others, most noticeably UKESM1-0-LL (where entirely new marine biogeochemistry has been incorporated), show a large overestimation of surface nitrate concentration over the tropics.</p><p id=\"Par45\">A comparison of simulated surface concentrations of silicic acid with modern observations shows that all models except GISS and CESM models have improved their representation of the surface distribution of silicic acid (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b</xref>). The most striking improvement is seen between HadGEM2-ES and UKESM1-0-LL. Such an improvement is explained by the switch in the biogeochemical model component between CMIP5 and CMIP6, from Diat-HadOCC to MEDUSA-2.0 (see [<xref ref-type=\"bibr\" rid=\"CR96\">96</xref>], for further details). Figure <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> b sheds light upon another systematic bias in the Southern Ocean where all the models display large model-data errors independent of their generation. It suggests that processes other than ocean resolution or the complexity of the marine biogeochemical model may be at the origin of this systematic model deficiency. The pattern of error differs among models. UKESM1-0-LL, MPI-ESM 1-2-LR and GISS-E2-1-G-CC display a uniform bias in simulated silicic acid concentrations, whereas all the other models show a mixture of positive and negative biases in simulated concentrations.</p><p id=\"Par46\">Figure <xref rid=\"Fig4\" ref-type=\"fig\">4</xref> a presents the pattern of oxygen concentrations at a depth of 150 m where the signature of the oxygen minimum zone (OMZ) is expected to be visible. Note that 9 of 12 models simulated O<sub>2</sub> in CMIP5, and one further model added O<sub>2</sub> for CMIP6.</p><p id=\"Par47\">In general, CMIP6 models improve upon their CMIP5 predecessors in their representation of oxygen at 150 m (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4b</xref>). Model errors in the Southern Ocean have been reduced in CMIP6 with respect to CMIP5, highlighting a better representation of the deep ocean ventilation in the Southern Ocean or more accurate biogeochemical characteristics of outcropping water masses. Model-data errors have also been reduced in CMIP6 in large domains of the Indian Ocean where large OMZs occur although all models display a systematic overestimation of oxygen at 150 m in the Arabian Sea. The same feature is also observed in the tropical Pacific where a model-data error has been reduced in CMIP6 with respect to CMIP5. Contrasting with the other ocean domains, models’ performance has not improved in the Atlantic Ocean. For example, in the tropical Atlantic, some models have shifted in the sign of the model-data errors: from a negative bias in CMIP5 (stronger-than-observed OMZ) to a positive bias in CMIP6 (weaker-than-observed OMZ) or the opposite. In both cases, the absolute magnitude of the model-data errors in this region remains similar between model generations. This implies a systematic bias in ocean biogeochemical models which seems independent from ocean resolution or complexity of marine biogeochemistry models. Besides, our review of model performance highlights that open ocean hypoxia remains poorly represented in ocean biogeochemical models; the CMIP6 models still tend to overestimate this marine biogeochemical feature with respect to their CMIP5 predecessors. This is especially clear in the southern tropical Pacific, where all models except CESM2 and GFDL-ESM 4 overestimated the level of hypoxia of the OMZ (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>).</p><p id=\"Par48\">Improvement in GFDL-ESM 4 is explained by a suite of updates and changes in model physics (i.e. mixing and Southern Hemisphere climate) and biogeochemical parameterizations (i.e. the use of a revised remineralization scheme for organic matter depending on oxygen and temperature of Laufkötter et al. [<xref ref-type=\"bibr\" rid=\"CR148\">148</xref>]). In addition, COBALTv2 has lower net primary productivity than TOPAZv2 which allows the high-nutrient low-chlorophyll region to spread further meridionally in the tropical Pacific and reduce the eastern equatorial nutrient trapping and associated oxygen decline.</p><p id=\"Par49\">The surface distribution of dissolved iron is also an important feature of marine biogeochemistry. Its availability controls marine biological production in several ocean regions [<xref ref-type=\"bibr\" rid=\"CR149\">149</xref>]. As for oxygen, Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> highlights that marine iron cycling is not represented in all biogeochemical models. Nonetheless, this number has increased in CMIP6 (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>). It translates the current scientific consensus which recognizes the need to resolve the iron cycling in biogeochemical model in order to better simulate the marine biogeochemical dynamics, e.g. for glacial-interglacial climate change [<xref ref-type=\"bibr\" rid=\"CR150\">150</xref>] or for variability and response to climate change [<xref ref-type=\"bibr\" rid=\"CR151\">151</xref>].</p><p id=\"Par50\">Figure <xref rid=\"Fig5\" ref-type=\"fig\">5</xref> illustrates, however, that the performance of the current generation of models with respect to iron does not improve much with respect to that of the previous generation. Indeed, the model-data fit estimated with squared correlation coefficients remains < 0.25. This fit has not progressed much from CMIP5 to CMIP6, except possibly for IPSL and CNRM models which both employed PISCESv1 [<xref ref-type=\"bibr\" rid=\"CR40\">40</xref>, <xref ref-type=\"bibr\" rid=\"CR41\">41</xref>] for CMIP5 and PISCESv2 [<xref ref-type=\"bibr\" rid=\"CR91\">91</xref>] for CMIP6. As highlighted in Aumont et al. [<xref ref-type=\"bibr\" rid=\"CR91\">91</xref>], PISCESv2 includes a more detailed representation of the ocean iron cycle compared with PISCESv1.</p><p id=\"Par51\">The poor agreement between the observed and simulated distribution of dissolved iron relative to macronutrients (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>) partly reflects differences in the nature of the datasets. The relatively large number of nitrate measurements globally, for example, has allowed construction of robust climatological patterns [<xref ref-type=\"bibr\" rid=\"CR145\">145</xref>] that model climatologies can be compared against. The relative paucity of dissolved iron measurements, in contrast, requires a comparison of modelled climatologies against patchy individual measurements. Despite this, Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref> shows that some CMIP6 models better simulate the global average concentration of dissolved iron than their predecessors. This is particularly clear for UKESM1-0-LL, MPI-ESM 1-2-LR and GFDL-ESM 4. It is interesting to see the various modelling approaches for representing marine iron cycling. UKESM1-0-LL and MIROC-ES2L, for instance, use respectively Dutkiewicz et al. [<xref ref-type=\"bibr\" rid=\"CR152\">152</xref>] and Moore and Braucher [<xref ref-type=\"bibr\" rid=\"CR153\">153</xref>] parameterization for marine iron cycling that removes dissolved iron concentrations above an ad hoc threshold. Other ocean biogeochemical models use mechanistic iron cycling schemes that avoid the needs of ad hoc thresholds (e.g. PISCES-v2 and PISCES-v2-gas employs Völker and Tagliabue [<xref ref-type=\"bibr\" rid=\"CR154\">154</xref>] formulation and TOPAZv2 applies an empirical relationship to dissolved organic carbon (DOC) to derive ligand concentrations).</p><p id=\"Par52\">Table <xref rid=\"Tab4\" ref-type=\"table\">4</xref> provides a large-scale picture of the model’s ability to simulate key downward biogeochemical fluxes involved in global carbon and nutrients cycling. Most of the CMIP6 marine biogeochemical models better simulate the magnitude of the surface and 100 m biogeochemical fluxes than their CMIP5 predecessors. Indeed, CESM2, CNRM-ESM2-1, GISS-E2-1-G-CC, IPSL-CM6A-LR, MPI-ESM 1-2-LR and NorESM2-LR have improved the representation of at least one biogeochemical fluxes with respect to their CMIP5 predecessors; BCC-CSM2-MR, CanESM5, GFDL-ESM 4 and MIROC-ES2L display comparable performance; only CanESM5-CanOE, MRI-ESM 2-0 and NorESM2-LM have respectively degraded the representation of either the vertically integrated net primary productivity or the carbon export at 100 m compared with their CMIP5 predecessors.<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Comparison between observational and model estimates of biogeochemical fluxes over the modern period. For both CMIP5 and CMIP6 models, biogeochemical fluxes are calculated over the 1995–2014 period (see Methods in Supplementary materials)</p></caption><graphic position=\"anchor\" xlink:href=\"40641_2020_160_Tab4_HTML\" id=\"MO10\"/><table-wrap-foot><p>Observational estimates are derived from the following database: <sup>a</sup>Landschützer et al. [<xref ref-type=\"bibr\" rid=\"CR143\">143</xref>] product average over 1995–2014 and adjusted for the pre-industrial ocean source of CO<sub>2</sub> from river input to the ocean consistently with the methodology employed in [<xref ref-type=\"bibr\" rid=\"CR155\">155</xref>] that used a river flux adjustment of 0.78 Pg C year<sup>−1</sup> [<xref ref-type=\"bibr\" rid=\"CR156\">156</xref>]; <sup>b</sup>maximal range of remote-sensing estimates from Behrenfeld et al. [<xref ref-type=\"bibr\" rid=\"CR157\">157</xref>] and Kulk et al. [<xref ref-type=\"bibr\" rid=\"CR158\">158</xref>]; <sup>c</sup>Dunne et al. [<xref ref-type=\"bibr\" rid=\"CR159\">159</xref>] and <sup>d</sup>Tréguer and De La Rocha [<xref ref-type=\"bibr\" rid=\"CR160\">160</xref>]. When required, the modelled net ocean carbon uptake is corrected with the net riverine-induced outgassing diagnosed from the piControl simulation. Coloured cells indicate the relative deviation in model global estimates with respect to the observation median best estimates; hatched coloured cells indicate where model global estimates fall within the observational uncertainty range. Grey cells indicate missing or unrepresented biogeochemical fluxes</p></table-wrap-foot></table-wrap></p><p id=\"Par53\">Despite the general improvement, Table <xref rid=\"Tab4\" ref-type=\"table\">4</xref> highlights that several CMIP6 models fall outside the range of remote-sensing estimates of primary production ([<xref ref-type=\"bibr\" rid=\"CR157\">157</xref>, <xref ref-type=\"bibr\" rid=\"CR158\">158</xref>, <xref ref-type=\"bibr\" rid=\"CR161\">161</xref>]). It suggests that the current generation of marine biogeochemical models still has difficulties to model underlying processes involved in the carbon fixation by phytoplankton (such as nutrient colimitation, nitrogen fixation, remineralization), required to accurately simulate the magnitude of the vertically integrated net primary productivity. At the same time, it is also important to acknowledge that there are still large uncertainties in remote-sensing-based estimates of primary production, e.g. 38.8–42.1 Pg C year<sup>−1</sup> in the most recent estimates of Kulk et al. [<xref ref-type=\"bibr\" rid=\"CR158\">158</xref>] and 47.5–52.1 Pg C year<sup>−1</sup> according to Behrenfeld et al. [<xref ref-type=\"bibr\" rid=\"CR157\">157</xref>].</p><p id=\"Par54\">Figures <xref rid=\"Fig6\" ref-type=\"fig\">6</xref> and <xref rid=\"Fig7\" ref-type=\"fig\">7</xref> track changes in performance between CMIP5 and CMIP6 marine biogeochemical models. Figure <xref rid=\"Fig6\" ref-type=\"fig\">6</xref> highlights how far the CMIP6 models have improved their capability to simulate observed spatial patterns with respect to their CMIP5 predecessors; Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref> summarizes the overall model performance including information on model performance to reproduce observed distribution (pattern and magnitude).<fig id=\"Fig6\"><label>Fig. 6</label><caption><p>Scatter plot confronting the performance of CMIP6 models to replicate the geographical structure of observed fields with respect to that of their CMIP5 predecessors. The performance metrics are the model-data spatial correlation computed from yearly averaged data and model outputs. The variables of interest are mixed-layer depth (oml), air-sea CO<sub>2</sub> flux (fgco2), surface chlorophyll (chl), oxygen concentration at 150 m (o2) and surface concentrations of nitrate (no3) and silicic acid (si). The green (red) shading flags an improvement (degradation) of the model performance to replicate the observed geographical structure for a given field. The ocean mixed-layer depth is computed similarly in all models; it is based on a density criterion of 0.03 kg m<sup>−3</sup>. The ocean mixed-layer depth simulated by the various Earth system models is evaluated against the observational dataset of de Boyer Montégut et al. [<xref ref-type=\"bibr\" rid=\"CR162\">162</xref>]</p></caption><graphic xlink:href=\"40641_2020_160_Fig6_HTML\" id=\"MO12\"/></fig><fig id=\"Fig7\"><label>Fig. 7</label><caption><p>Portrait diagram highlighting the performance of CMIP6 models (one representative per modelling groups) with respect to their CMIP5 predecessors. The variables of interest are mixed-layer depth (oml), air-sea CO<sub>2</sub> flux (fgco2), surface chlorophyll (chl), oxygen concentration at 150 m (o2) and surface concentrations of nitrate (no3) and silicic acid (si). The skill score metric, Z-score, is computed for a given model and for a given field as follows: Z-score<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ =\\frac{{\\mathrm{RMSE}}_{\\mathrm{CMIP}6}(M)-{\\mathrm{RMSE}}_{\\mathrm{CMIP}5}(P)}{{\\mathrm{RMSE}}_{\\mathrm{CMIP}5}(P)}\\times 100 $$\\end{document}</tex-math><mml:math id=\"M2\" display=\"inline\"><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mtext>RMSE</mml:mtext><mml:mrow><mml:mtext>CMIP</mml:mtext><mml:mn>6</mml:mn></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>M</mml:mi></mml:mfenced><mml:mo>−</mml:mo><mml:msub><mml:mtext>RMSE</mml:mtext><mml:mrow><mml:mtext>CMIP</mml:mtext><mml:mn>5</mml:mn></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>P</mml:mi></mml:mfenced></mml:mrow><mml:mrow><mml:msub><mml:mtext>RMSE</mml:mtext><mml:mrow><mml:mtext>CMIP</mml:mtext><mml:mn>5</mml:mn></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>P</mml:mi></mml:mfenced></mml:mrow></mml:mfrac><mml:mo>×</mml:mo><mml:mn>100</mml:mn></mml:math><inline-graphic xlink:href=\"40641_2020_160_Article_IEq1.gif\"/></alternatives></inline-formula>, where RMSE<sub>CMIP6</sub>(<italic>M</italic>) is the global area-weighted average model-data root-mean-squared error (RMSE) of the model of the current generation contributing to CMIP6 and RMSE<sub>CMIP5</sub>(<italic>P</italic>) is the RMSE of its predecessor that has contributed to CMIP5. Greenish (reddish) colours and negative (positive) Z-scores indicate improved (degraded) field representations in CMIP6 model versions; darker colours indicate a greater change from CMIP5 to CMIP6. Grey indicates missing data for one or both generations of models. Air-sea CO<sub>2</sub> flux (fgco2) was adjusted for riverine-induced outgassing as in Table <xref rid=\"Tab4\" ref-type=\"table\">4</xref>. The ocean mixed-layer depth is computed similarly in all models; it is based on a density criterion of 0.03 kg m<sup>−3</sup>. The ocean mixed-layer depth simulated by the various Earth system models is evaluated against the observational dataset of de Boyer Montégut et al. [<xref ref-type=\"bibr\" rid=\"CR162\">162</xref>]</p></caption><graphic xlink:href=\"40641_2020_160_Fig7_HTML\" id=\"MO13\"/></fig></p><p id=\"Par55\">Both Figs. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref> and <xref rid=\"Fig7\" ref-type=\"fig\">7</xref> show that CMIP6 models have improved the representation of the ocean physics (here the ocean mixed-layer depth). The cross-generation picture of the model performance for marine biogeochemistry is more contrasted. Globally, Figs. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref> and <xref rid=\"Fig7\" ref-type=\"fig\">7</xref> show that most of the CMIP6 models outcompete their CMIP5 predecessors. However, this improvement remains modest. Except for some models displaying a noticeable improvement for one or two biogeochemical fields (surface nitrate for CESM2, surface chlorophyll for CNRM-ESM2-1, surface silicic acid for GFDL-ESM 4), most of the CMIP6 model display a slight increase in model-data spatial correlation (up to + 0.2, Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>) or an overall reduction in model-data RMSE of about 20% (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>). Besides, this improvement does not concern all models. For instance, GISS-E2-1-G-CC shows a noticeable degradation in performance for all of the biogeochemical fields analyzed here.</p></sec><sec id=\"Sec4\"><title>Conclusions</title><sec id=\"Sec5\"><title>Summary of 5 Years of Ocean Biogeochemical Model Development</title><p id=\"Par56\">Our review of available Earth system models highlights that the current generation of marine biogeochemical models used for CMIP6 displays a greater diversity than the previous one used for CMIP5. Several marine biogeochemical models have evolved toward a more comprehensive representation of marine biogeochemistry (i.e. CESM, CNRM, GFDL, IPSL, MIROC, UKESM), typically including an expanded array of biological taxa (e.g. diazotrophs) or elemental cycling (e.g. oxygen and iron cycles), variable stoichiometry, sediments (e.g. sediment box module) and the representation of (non-CO<sub>2</sub>) trace gases relevant to atmospheric chemistry. On the opposite, some groups have limited the increase in model complexity between CMIP5 and CMIP6 (i.e. BCC, GISS, MPI, MRI, NorESM). Finally, it is interesting to note that some groups have started to investigate the use of reduced complexity marine biogeochemical model (i.e. GFDL) or to intercompare in a traceable framework the impact of rising complexity on the simulated marine biogeochemistry (CanESM).</p><p id=\"Par57\">When assessed against observations, most of the CMIP6 models generally outperform their CMIP5 predecessors in many regions and for most of the marine biogeochemical fields reviewed here (Figs. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref> and <xref rid=\"Fig7\" ref-type=\"fig\">7</xref> and Table <xref rid=\"Tab4\" ref-type=\"table\">4</xref>). However, this model review has also highlighted several systematic model-data errors that are persistent even in CMIP6 models (e.g. oxygen concentrations at 150 m in tropical Atlantic, nutrient trapping in the Southern Ocean).</p><p id=\"Par58\">Our review also shows that the modelling approaches have evolved between CMIP5 and CMIP6. Indeed, most modelling groups have spun-up their model over a longer period for CMIP6 with respect to CMIP5 in order to fulfil the drift criterion as proposed by Jones et al. [<xref ref-type=\"bibr\" rid=\"CR142\">142</xref>]. In contrast, the use of tuning and calibration for marine biogeochemical models for CMIP remains a less common feature at the time of CMIP6.</p><p id=\"Par59\">Finally, our review of model mean state performance against their model properties (resolution, complexity) suggests that neither increasing resolution nor increasing complexity leads automatically to model improvement. Instead, improvement is a mixture of improved ocean physical processes and better representation of biogeochemical processes.</p><p id=\"Par60\">In the context of improving confidence in future climate projections, it is important to stress that the model mean state performance is not the only mean to understand multi-model uncertainty, comparisons against seasonal to multi-annual variations in observed quantities may ultimately prove most critical to building confidence in future climate projections (e.g. [<xref ref-type=\"bibr\" rid=\"CR13\">13</xref>, <xref ref-type=\"bibr\" rid=\"CR163\">163</xref>]).</p></sec><sec id=\"Sec6\"><title>What’s Next?</title><p id=\"Par61\">In this final section, we identify some directions where marine biogeochemical models could continue to improve or to progress.</p><p id=\"Par62\">The first step change to expect in the next generation of models is the emergence of high-resolution ocean biogeochemical models fit to investigate centennial-scale simulation. This step change may be supported in a number of ways: (1) the availability of greater computational resources; (2) the use of hybrid-resolution numerical schemes to decrease the cost of biogeochemical models (e.g. [<xref ref-type=\"bibr\" rid=\"CR164\">164</xref>]); (3) actually reduced complexity of marine biogeochemical models (e.g. such as miniBLING; [<xref ref-type=\"bibr\" rid=\"CR105\">105</xref>]); (4) the use of machine learning to either accelerate marine biogeochemical models or to reduce the numerical cost necessary to improve their performance (i.e. via tuning). These (and potentially other) step changes will help to understand the extent to which mesoscale or sub-mesoscale ocean physics might change the response of marine biogeochemistry to rising CO<sub>2</sub> and climate change—a missing factor in such models already highlighted from CMIP5 and IPCC AR5 [<xref ref-type=\"bibr\" rid=\"CR2\">2</xref>].</p><p id=\"Par63\">A second important step change is related to the phytoplankton physiology and evolution. This change may have two benefits. First, several recent studies show that the inclusion of a more comprehensive treatment of plankton physiology may improve model performance, in particular some systematic biases in the Southern Ocean (e.g. [<xref ref-type=\"bibr\" rid=\"CR108\">108</xref>, <xref ref-type=\"bibr\" rid=\"CR165\">165</xref>]). Then, this improvement is arguably a first step toward the representation of adaptation and fitness in ocean biogeochemical models [<xref ref-type=\"bibr\" rid=\"CR166\">166</xref>, <xref ref-type=\"bibr\" rid=\"CR167\">167</xref>]. This omission remains an important caveat for multi-stressors studies (e.g. [<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>]) or time-of-emergence studies [<xref ref-type=\"bibr\" rid=\"CR168\">168</xref>] as current models effectively assume no change in the underlying properties of modelled plankton.</p><p id=\"Par64\">Future developments should be pursued in the context of the internal cycling of micronutrients involved in phytoplankton physiology and metabolism such as iron, zinc or copper. Our review confirms that the current generation of marine biogeochemical models are still struggling to reproduce the major features of the oceanic iron distribution although the observations of dissolved iron in the ocean are growing rapidly [<xref ref-type=\"bibr\" rid=\"CR149\">149</xref>] and are made widely available by GEOTRACES [<xref ref-type=\"bibr\" rid=\"CR169\">169</xref>]. A key challenge for iron is that the dissolved iron commonly measured only appears to represent a trace residual of the underlying fluxes [<xref ref-type=\"bibr\" rid=\"CR170\">170</xref>], pointing to the need for more process studies and observations of fluxes. It is possible that iron isotopes may yield further insight into the role of external inputs and internal cycling in shaping iron distributions in the observations and models. Finally, the development of additional model components dealing with other trace metals, such as cobalt [<xref ref-type=\"bibr\" rid=\"CR171\">171</xref>], zinc [<xref ref-type=\"bibr\" rid=\"CR172\">172</xref>], manganese [<xref ref-type=\"bibr\" rid=\"CR173\">173</xref>] and copper [<xref ref-type=\"bibr\" rid=\"CR174\">174</xref>], may also prove beneficial in constraining the magnitude and dynamics of external inputs in particular.</p><p id=\"Par65\">An expanded array of biological taxa may also be expected in the next generation of ocean biogeochemical models. A potentially important change in the ocean ecosystem modelling paradigm is the inclusion and integration of mixotrophs which are an important grazer of bacterioplankton, and which also feed on phytoplankton, microzooplankton and (sometimes) mesozooplankton. Mixotrophic bacterivory among the phytoplankton may be important for alleviating nutrient stress and may increase primary production in oligotrophic waters. Some modelling studies indicate that mixotrophy has a profound impact on marine planktonic ecosystems and may enhance primary production, biomass transfer to higher trophic levels and the functioning of the biological carbon pump [<xref ref-type=\"bibr\" rid=\"CR175\">175</xref>].</p><p id=\"Par66\">This expanded array of biological taxa may take the concept of the marine biogeochemical model up to the marine ecosystem model, which will enable the representation of feedbacks of the marine trophic food web on marine biogeochemical cycles. The work of Lefort et al. [<xref ref-type=\"bibr\" rid=\"CR57\">57</xref>] provides an example of this type of marine ecosystem model realizing a comprehensive coupling between a marine biogeochemical model (PISCES) with a marine trophic food web model (APECOSM).</p><p id=\"Par67\">A third important step change is related to the couplings between Earth system components and ocean biogeochemistry. Our review highlights that models have evolved toward a more comprehensive treatment of biological boundary conditions (e.g. atmospheric deposition, riverine inputs, sediments, ice sheets, geothermal sources) but that these latter are currently largely represented using climatological data rather than dynamic connections. Progress toward more complete couplings between Earth system components such as rivers, ice sheet/iceberg calving and ice shelves or atmospheric aerosols can help to better simulate interactions between marine biogeochemistry, biogeochemical cycles and climate.</p><p id=\"Par68\">In the same manner, a more comprehensive treatment of biophysical and biogeochemical feedback could be realized in the next generation of marine biogeochemical models. The latter involves, for instance, ocean emissions of greenhouse gases or biogenic volatile organic compounds (BVOCs) that are already simulated by a small number of models (see Table <xref rid=\"Tab5\" ref-type=\"table\">5</xref>). However, our understanding of the global cycles of DMS, N<sub>2</sub>O and CH<sub>4</sub> (including, specifically, the processes that produce them) is much less developed compared with CO<sub>2</sub>. Therefore, better treatment of biophysical and biogeochemical feedback requires a larger array of observational data sets in order to improve our understanding of the processes underlying these ocean emissions.<table-wrap id=\"Tab5\"><label>Table 5</label><caption><p>Ocean natural emissions of non-CO<sub>2</sub> trace gases simulated by CMIP6 models</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th/><th>DMS (Tg S year<sup>−1</sup>)</th><th>N<sub>2</sub>O (Tg N year<sup>−1</sup>)</th><th>NHx [Tg N year<sup>−1</sup>)</th></tr></thead><tbody><tr><td>Observational estimates</td><td>17.6–34.4<sup>a</sup></td><td>1.9–9.4<sup>b</sup></td><td>2–5<sup>c</sup></td></tr><tr><td>CNRM-ESM 2-1</td><td>24.38</td><td>3.97</td><td>-</td></tr><tr><td>GFDL-ESM 4</td><td>-</td><td>-</td><td>3.10</td></tr><tr><td>UKESM1-0-LL</td><td>16.19</td><td>-</td><td>-</td></tr><tr><td>MIROC-ES2L</td><td>18.46</td><td>4.31</td><td>-</td></tr><tr><td>MPI-ESM 1-2-LR</td><td>-</td><td>8.89</td><td>-</td></tr><tr><td>NorESM2-LM</td><td>20.0</td><td>-</td><td>-</td></tr></tbody></table><table-wrap-foot><p><sup>a</sup>Lana et al. [<xref ref-type=\"bibr\" rid=\"CR176\">176</xref>]; <sup>b</sup>Buitenhuis et al. [<xref ref-type=\"bibr\" rid=\"CR177\">177</xref>]; <sup>c</sup>Paulot et al. [<xref ref-type=\"bibr\" rid=\"CR178\">178</xref>]</p></table-wrap-foot></table-wrap></p><p id=\"Par69\">From the perspective of tracking future model improvement, it is important to stress that our capacity to assess model performance resulting from any of the potential advances discussed above is contingent upon continued improvement in observational constraints. Existing constraints were adequate for detecting large skill differences between CMIP5 and CMIP6 models, but the overall improvement in models necessitates more precise comparisons to detect skill differences. Such comparisons are challenged by data sparsity and uncertainties in algorithms designed to derive global fields from sparse data or infer properties of interest from remotely sensed variables. Continued improvement in the quality and quantity of data-based constraints is critical.</p><p id=\"Par70\">That being said, our review of the available pairs of CMIP5-CMIP6 marine biogeochemical models strongly suggests that careful consideration is needed when selecting model complexity with regard to the fitness-for-purpose of models (i.e. carbon cycle feedbacks, multiple Earth system feedbacks, multi-stressors, adaptation and biodiversity). Indeed, when confronting model complexity against model mean state performance, our work suggests that complex models do not necessarily outperform simple models. This is consistent with the earlier study of Kwiatkowski et al. [<xref ref-type=\"bibr\" rid=\"CR179\">179</xref>], which directly led to the choice of marine biogeochemistry model in UKESM1-0-LL, where across many Earth system relevant metrics, the simplest model performed best. In this sense, our review shows that simple models (e.g. OCMIP nutrient restoring or NPZD type) remain viable when investigating carbon cycle feedbacks, although more complex models do still permit a better linkage with the marine biodiversity or a broader array of feedbacks and potentially more realistic Earth system behaviour.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Electronic supplementary material</title><sec id=\"Sec7\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"40641_2020_160_MOESM1_ESM.xlsx\"><label>ESM 1</label><caption><p>(XLSX 11927 kb)</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p>This article is part of the Topical Collection on <italic>Carbon Cycle and Climate</italic></p></fn><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><ack><title>Acknowledgements</title><p>R.S. on behalf of the author team thank the two anonymous referees for their useful comments that have improved the quality of this paper. R.S. thanks the author team for their contributions to this paper that occurred during the coronavirus SARS-CoV-2 pandemic. R.S. and S.B. thank the support of the team in charge of the CNRM-CM climate model. Supercomputing time was provided by the Météo-France/DSI supercomputing center.</p></ack><notes notes-type=\"funding-information\"><title>Funding Information</title><p>This work was supported by the European Union’s Horizon 2020 research and innovation program with the CRESCENDO project under the grant agreement No 641816, the TRIATLAS project under the grant agreement No 817578 and the COMFORT project under the grant agreement No 820989. JS and JT were supported by the Research Council of Norway through the projects INES (grant no. 270061) and COLUMBIA (grant no 275268). LK was supported by the Agence Nationale de la Recherche (grant no. ANR-18-ERC2-0001-01).</p></notes><notes><title>Compliance with Ethical Standards</title><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Conflict of Interest</title><p id=\"Par71\">On behalf of all authors, the corresponding author states that there is no conflict of interest.</p></notes><notes id=\"FPar2\"><title>Human and Animal Rights</title><p id=\"Par72\">This article does not contain any studies with human or animal subjects performed by any of the authors.</p></notes><notes id=\"FPar3\" notes-type=\"funding\"><title>Disclaimer</title><p id=\"Par73\">This article reflects only the authors’ view—the funding agencies as well as their executive agencies are not responsible for any use that may be made of the information that the article contains.</p></notes></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><mixed-citation publication-type=\"other\">Sarmiento JL, Gruber N. Ocean biogeochemical dynamics: Princeton University Press; 2006. p. 67. <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.mendeley.com/research/chapter-10-carbon-cycle-co2-climate/\">http://www.mendeley.com/research/chapter-10-carbon-cycle-co2-climate/</ext-link>.</mixed-citation></ref><ref id=\"CR2\"><label>2.</label><mixed-citation publication-type=\"other\">Ciais P, et al. Carbon and other biogeochemical cycles. Clim Chang 2013 - Phys Sci Basis. 2013:465–570. 10.1017/CBO9781107415324.015.</mixed-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lengaigne</surname><given-names>M</given-names></name><name><surname>Madec</surname><given-names>G</given-names></name><name><surname>Bopp</surname><given-names>L</given-names></name><name><surname>Menkes</surname><given-names>C</given-names></name><name><surname>Aumont</surname><given-names>O</given-names></name><name><surname>Cadule</surname><given-names>P</given-names></name></person-group><article-title>Bio-physical feedbacks in the Arctic Ocean using an Earth system model</article-title><source>Geophys Res Lett.</source><year>2009</year><volume>36</volume><fpage>L21602</fpage><pub-id pub-id-type=\"doi\">10.1029/2009GL040145</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Roy</surname><given-names>T</given-names></name><etal/></person-group><article-title>Regional impacts of climate change and atmospheric CO2 on future ocean carbon uptake: a multimodel linear feedback analysis</article-title><source>J Clim.</source><year>2011</year><volume>24</volume><fpage>2300</fpage><lpage>2318</lpage><pub-id pub-id-type=\"doi\">10.1175/2010JCLI3787.1</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schwinger</surname><given-names>J</given-names></name><etal/></person-group><article-title>Nonlinearity of ocean carbon cycle feedbacks in CMIP5 earth system models</article-title><source>J Clim.</source><year>2014</year><volume>27</volume><fpage>3869</fpage><lpage>3888</lpage><pub-id pub-id-type=\"doi\">10.1175/JCLI-D-13-00452.1</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chassot</surname><given-names>E</given-names></name><name><surname>Bonhommeau</surname><given-names>S</given-names></name><name><surname>Dulvy</surname><given-names>NK</given-names></name><name><surname>Mélin</surname><given-names>F</given-names></name><name><surname>Watson</surname><given-names>R</given-names></name><name><surname>Gascuel</surname><given-names>D</given-names></name><name><surname>Le Pape</surname><given-names>O</given-names></name></person-group><article-title>Global marine primary production constrains fisheries catches</article-title><source>Ecology Letters</source><year>2010</year><volume>13</volume><fpage>495</fpage><lpage>505</lpage><pub-id pub-id-type=\"doi\">10.1111/j.1461-0248.2010.01443.x</pub-id><pub-id pub-id-type=\"pmid\">20141525</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ryther</surname><given-names>JH</given-names></name></person-group><article-title>Photosynthesis and fish production in the sea</article-title><source>Science.</source><year>1969</year><volume>166</volume><fpage>72</fpage><lpage>76</lpage><pub-id pub-id-type=\"doi\">10.1126/science.166.3901.72</pub-id><pub-id pub-id-type=\"pmid\">5817762</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stock</surname><given-names>CA</given-names></name><name><surname>John</surname><given-names>JG</given-names></name><name><surname>Rykaczewski</surname><given-names>RR</given-names></name><name><surname>Asch</surname><given-names>RG</given-names></name><name><surname>Cheung</surname><given-names>WW</given-names></name><name><surname>Dunne</surname><given-names>JP</given-names></name><etal/></person-group><article-title>Reconciling fisheries catch and ocean productivity</article-title><source>Proc Natl Acad Sci.</source><year>2017</year><volume>114</volume><issue>8</issue><fpage>E1441</fpage><lpage>E1449</lpage><pub-id pub-id-type=\"doi\">10.1073/pnas.1610238114</pub-id><pub-id pub-id-type=\"pmid\">28115722</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bopp</surname><given-names>L</given-names></name><etal/></person-group><article-title>Multiple stressors of ocean ecosystems in the 21st century: projections with CMIP5 models</article-title><source>Biogeosciences.</source><year>2013</year><volume>10</volume><fpage>6225</fpage><lpage>6245</lpage><pub-id pub-id-type=\"doi\">10.5194/bg-10-6225-2013</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cocco</surname><given-names>V</given-names></name><etal/></person-group><article-title>Oxygen and indicators of stress for marine life in multi-model global warming projections</article-title><source>Biogeosciences.</source><year>2013</year><volume>10</volume><fpage>1849</fpage><lpage>1868</lpage><pub-id pub-id-type=\"doi\">10.5194/bg-10-1849-2013</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Doney</surname><given-names>SC</given-names></name><etal/></person-group><article-title>Climate change impacts on marine ecosystems</article-title><source>Annu Rev Mar Sci.</source><year>2012</year><volume>4</volume><fpage>11</fpage><lpage>37</lpage><pub-id pub-id-type=\"doi\">10.1146/annurev-marine-041911-111611</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gehlen</surname><given-names>M</given-names></name><name><surname>Séférian</surname><given-names>R</given-names></name><name><surname>Jones</surname><given-names>DOB</given-names></name><name><surname>Roy</surname><given-names>T</given-names></name><name><surname>Roth</surname><given-names>R</given-names></name><name><surname>Barry</surname><given-names>J</given-names></name><etal/></person-group><article-title>Projected pH reductions by 2100 might put deep North Atlantic biodiversity at risk</article-title><source>Biogeosciences.</source><year>2014</year><volume>11</volume><fpage>6955</fpage><lpage>6967</lpage><pub-id pub-id-type=\"doi\">10.5194/bg-11-6955-2014</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kwiatkowski</surname><given-names>L</given-names></name><name><surname>Orr</surname><given-names>JC</given-names></name></person-group><article-title>Diverging seasonal extremes for ocean acidification during the twenty-first century</article-title><source>Nat Clim Chang.</source><year>2018</year><volume>8</volume><fpage>141</fpage><lpage>145</lpage><pub-id pub-id-type=\"doi\">10.1038/s41558-017-0054-0</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Orr</surname><given-names>JC</given-names></name><etal/></person-group><article-title>Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms</article-title><source>Nature.</source><year>2005</year><volume>437</volume><fpage>681</fpage><lpage>686</lpage><pub-id pub-id-type=\"doi\">10.1038/nature04095</pub-id><pub-id pub-id-type=\"pmid\">16193043</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tagliabue</surname><given-names>BL</given-names></name><name><surname>Gehlen</surname><given-names>M</given-names></name></person-group><article-title>The response of marine carbon and nutrient cycles to ocean acidification: large uncertainties related to phytoplankton physiological assumptions</article-title><source>Glob Biogeochem Cycles</source><year>2011</year><volume>25</volume><fpage>GB3017</fpage><pub-id pub-id-type=\"doi\">10.1029/2010GB003929</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tynan</surname><given-names>E</given-names></name></person-group><article-title>Ocean acidification: emergence from pre-industrial conditions</article-title><source>Nat Geosci.</source><year>2016</year><volume>9</volume><fpage>804</fpage><pub-id pub-id-type=\"doi\">10.1038/ngeo2834</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hopkins</surname><given-names>FE</given-names></name><name><surname>Coauthors</surname></name></person-group><article-title>The impacts of ocean acidification on marine trace gases and the implications for atmospheric chemistry and climate</article-title><source>Proc. R. Soc. A Math. Phys. Eng. Sci</source><year>2020</year><volume>476</volume><fpage>20190769</fpage><pub-id pub-id-type=\"doi\">10.1098/rspa.2019.0769</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maier-Reimer</surname><given-names>E</given-names></name><name><surname>Hasselmann</surname><given-names>K</given-names></name></person-group><article-title>Transport and storage of C02 in the ocean - an inorganic ocean-circulation carbon cycle model</article-title><source>Clim Dyn.</source><year>1987</year><volume>2</volume><fpage>63</fpage><lpage>90</lpage><pub-id pub-id-type=\"doi\">10.1007/BF01054491</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sarmiento</surname><given-names>JL</given-names></name><name><surname>Orr</surname><given-names>JC</given-names></name><name><surname>Siegenthaler</surname><given-names>U</given-names></name></person-group><article-title>A perturbation simulation of CO2 uptake in an ocean general circulation model</article-title><source>J Geophys Res.</source><year>1992</year><volume>97</volume><fpage>3621</fpage><lpage>3645</lpage><pub-id pub-id-type=\"doi\">10.1029/91JC02849</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Orr</surname><given-names>JC</given-names></name><name><surname>Najjar</surname><given-names>R</given-names></name><name><surname>Sabine</surname><given-names>CL</given-names></name><name><surname>Joos</surname><given-names>F</given-names></name><name><surname>Internal OCMIP Report</surname><given-names>A-HOWTO</given-names></name><name><surname>Saclay</surname><given-names>LSCE/CEA</given-names></name></person-group><article-title>Gif-sur-Yvette, France</article-title><source>25 pp.</source><year>1999</year></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Adachi</surname><given-names>Y</given-names></name><name><surname>Yukimoto</surname><given-names>S</given-names></name><name><surname>Deushi</surname><given-names>M</given-names></name><name><surname>Obata</surname><given-names>A</given-names></name><name><surname>Nakano</surname><given-names>H</given-names></name><name><surname>Tanaka</surname><given-names>TY</given-names></name><etal/></person-group><article-title>Basic performance of a new earth system model of the Meteorological Research Institute (MRI-ESM 1)</article-title><source>Pap Meteorol Geophys.</source><year>2013</year><volume>64</volume><fpage>1</fpage><lpage>19</lpage><pub-id pub-id-type=\"doi\">10.2467/mripapers.64.1</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arora</surname><given-names>VK</given-names></name><name><surname>Scinocca</surname><given-names>JF</given-names></name><name><surname>Boer</surname><given-names>GJ</given-names></name><name><surname>Christian</surname><given-names>JR</given-names></name><name><surname>Denman</surname><given-names>KL</given-names></name><name><surname>Flato</surname><given-names>GM</given-names></name><etal/></person-group><article-title>Carbon emission limits required to satisfy future representative concentration pathways of greenhouse gases</article-title><source>Geophys Res Lett.</source><year>2011</year><volume>38</volume><fpage>L05805</fpage><pub-id pub-id-type=\"doi\">10.1029/2010GL046270</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bentsen</surname><given-names>M</given-names></name><name><surname>Bethke</surname><given-names>I</given-names></name><name><surname>Debernard</surname><given-names>JB</given-names></name><name><surname>Iversen</surname><given-names>T</given-names></name><name><surname>Kirkevåg</surname><given-names>A</given-names></name><name><surname>Seland</surname><given-names>Ø</given-names></name><etal/></person-group><article-title>The Norwegian Earth System Model, NorESM1-M – Part 1: description and basic evaluation of the physical climate</article-title><source>Geosci Model Dev.</source><year>2013</year><volume>6</volume><fpage>687</fpage><lpage>720</lpage><pub-id pub-id-type=\"doi\">10.5194/gmd-6-687-2013</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dufresne</surname><given-names>J</given-names></name><name><surname>Foujols</surname><given-names>M</given-names></name><name><surname>Denvil</surname><given-names>S</given-names></name><etal/></person-group><article-title>Climate change projections using the IPSL-CM5 Earth System Model: from CMIP3 to CMIP5</article-title><source>Clim Dyn.</source><year>2013</year><volume>40</volume><fpage>2123</fpage><lpage>2165</lpage><pub-id pub-id-type=\"doi\">10.1007/s00382-012-1636-1</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dunne</surname><given-names>JP</given-names></name><name><surname>John</surname><given-names>JG</given-names></name><name><surname>Shevliakova</surname><given-names>E</given-names></name><name><surname>Stouffer</surname><given-names>RJ</given-names></name><name><surname>Krasting</surname><given-names>JP</given-names></name><name><surname>Malyshev</surname><given-names>SL</given-names></name><etal/></person-group><article-title>GFDL’s ESM 2 Global Coupled Climate–Carbon Earth System Models. Part II: carbon system formulation and baseline simulation characteristics</article-title><source>J Clim.</source><year>2013</year><volume>26</volume><fpage>2247</fpage><lpage>2267</lpage><pub-id pub-id-type=\"doi\">10.1175/JCLI-D-12-00150.1</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Giorgetta</surname><given-names>MA</given-names></name><name><surname>Jungclaus</surname><given-names>J</given-names></name><name><surname>Reick</surname><given-names>CH</given-names></name><name><surname>Legutke</surname><given-names>S</given-names></name><name><surname>Bader</surname><given-names>J</given-names></name><name><surname>Böttinger</surname><given-names>M</given-names></name><etal/></person-group><article-title>Climate and carbon cycle changes from 1850 to 2100 in MPI-ESM simulations for the Coupled Model Intercomparison Project phase 5</article-title><source>J Adv Model Earth Syst.</source><year>2013</year><volume>5</volume><fpage>572</fpage><lpage>597</lpage><pub-id pub-id-type=\"doi\">10.1002/jame.20038</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lindsay</surname><given-names>K</given-names></name><name><surname>Bonan</surname><given-names>GB</given-names></name><name><surname>Doney</surname><given-names>SC</given-names></name><name><surname>Hoffman</surname><given-names>FM</given-names></name><name><surname>Lawrence</surname><given-names>DM</given-names></name><name><surname>Long</surname><given-names>MC</given-names></name><etal/></person-group><article-title>Preindustrial-control and twentieth-century carbon cycle experiments with the Earth System Model CESM1(BGC)</article-title><source>J Clim.</source><year>2014</year><volume>27</volume><fpage>8981</fpage><lpage>9005</lpage><pub-id pub-id-type=\"doi\">10.1175/JCLI-D-12-00565.1</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Romanou</surname><given-names>A</given-names></name><name><surname>Gregg</surname><given-names>WW</given-names></name><name><surname>Romanski</surname><given-names>J</given-names></name><name><surname>Kelley</surname><given-names>M</given-names></name><name><surname>Bleck</surname><given-names>R</given-names></name><name><surname>Healy</surname><given-names>R</given-names></name><etal/></person-group><article-title>Natural air-sea flux of CO<sub>2</sub> in simulations of the NASA-GISS climate model: sensitivity to the physical ocean model formulation</article-title><source>Ocean Model.</source><year>2013</year><volume>66</volume><fpage>26</fpage><lpage>44</lpage><pub-id pub-id-type=\"doi\">10.1016/j.ocemod.2013.01.008</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Séférian</surname><given-names>R</given-names></name><name><surname>Delire</surname><given-names>C</given-names></name><name><surname>Decharme</surname><given-names>B</given-names></name><name><surname>Voldoire</surname><given-names>A</given-names></name></person-group><article-title>Salas y Melia D, Chevallier M, et al. Development and evaluation of CNRM Earth system model – CNRM-ESM 1</article-title><source>Geosci Model Dev.</source><year>2016</year><volume>9</volume><fpage>1423</fpage><lpage>1453</lpage><pub-id pub-id-type=\"doi\">10.5194/gmd-9-1423-2016</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Watanabe</surname><given-names>S</given-names></name><name><surname>Hajima</surname><given-names>T</given-names></name><name><surname>Sudo</surname><given-names>K</given-names></name><name><surname>Nagashima</surname><given-names>T</given-names></name><name><surname>Takemura</surname><given-names>T</given-names></name><name><surname>Okajima</surname><given-names>H</given-names></name><etal/></person-group><article-title>MIROC-ESM 2010: model description and basic results of CMIP5-20c3m experiments</article-title><source>Geosci Model Dev.</source><year>2011</year><volume>4</volume><fpage>845</fpage><lpage>872</lpage><pub-id pub-id-type=\"doi\">10.5194/gmd-4-845-2011</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>T</given-names></name><name><surname>Li</surname><given-names>W</given-names></name><name><surname>Ji</surname><given-names>J</given-names></name><name><surname>Xin</surname><given-names>X</given-names></name><name><surname>Li</surname><given-names>L</given-names></name><name><surname>Wang</surname><given-names>Z</given-names></name><etal/></person-group><article-title>Global carbon budgets simulated by the Beijing climate center climate system model for the last century</article-title><source>J Geophys Res Atmos.</source><year>2013</year><volume>118</volume><fpage>4326</fpage><lpage>4347</lpage><pub-id pub-id-type=\"doi\">10.1002/jgrd.50320</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arora</surname><given-names>VK</given-names></name><etal/></person-group><article-title>Carbon-concentration and carbon-climate feedbacks in CMIP5 earth system models</article-title><source>J Clim.</source><year>2013</year><volume>26</volume><fpage>5289</fpage><lpage>5314</lpage><pub-id pub-id-type=\"doi\">10.1175/JCLI-D-12-00494.1</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><mixed-citation publication-type=\"other\">Arora VK, Katavouta A, Williams RG, Jones CD, Brovkin V, Friedlingstein P, et al. Carbon-concentration and carbon-climate feedbacks in CMIP6 models, and their comparison to CMIP5 models. Biogeosci Discuss. 2020. 10.5194/bg-2019-473 in review, 2019.</mixed-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Friedlingstein</surname><given-names>P</given-names></name><etal/></person-group><article-title>Climate–carbon cycle feedback analysis: results from the C <sup>4</sup> MIP Model Intercomparison</article-title><source>J Clim.</source><year>2006</year><volume>19</volume><fpage>3337</fpage><lpage>3353</lpage><pub-id pub-id-type=\"doi\">10.1175/JCLI3800.1</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Friedlingstein</surname><given-names>P</given-names></name><name><surname>Meinshausen</surname><given-names>M</given-names></name><name><surname>Arora</surname><given-names>VK</given-names></name><name><surname>Jones</surname><given-names>CD</given-names></name><name><surname>Anav</surname><given-names>A</given-names></name><name><surname>Liddicoat</surname><given-names>SK</given-names></name><etal/></person-group><article-title>Uncertainties in CMIP5 climate projections due to carbon cycle feedbacks</article-title><source>J Clim.</source><year>2014</year><volume>27</volume><fpage>511</fpage><lpage>526</lpage><pub-id pub-id-type=\"doi\">10.1175/JCLI-D-12-00579.1</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>MacDougall</surname><given-names>AH</given-names></name><name><surname>Gregory</surname><given-names>J</given-names></name><name><surname>Stouffer</surname><given-names>R</given-names></name><name><surname>Stordal</surname><given-names>F</given-names></name><name><surname>Eby</surname><given-names>M</given-names></name></person-group><article-title>The oceanic origin of path-independent carbon budgets</article-title><source>Sci Report.</source><year>2017</year><volume>15</volume><fpage>124</fpage><lpage>130</lpage><pub-id pub-id-type=\"doi\">10.1038/s41598-017-10557-x</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><mixed-citation publication-type=\"other\">Lauvset SK, Tjiputra J, Muri H. Climate engineering and the ocean: effects on biogeochemistry and primary production. Biogeosci Discuss. 2017:1–36. 10.5194/bg-2017-235.</mixed-citation></ref><ref id=\"CR38\"><label>38.</label><mixed-citation publication-type=\"other\">Plazzotta M, Séférian R, Douville H. Impact of solar radiation modification on allowable CO <sub>2</sub> emissions: what can we learn from multi-model simulations? Earth’s Futur. 2019. 10.1029/2019EF001165.</mixed-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tjiputra</surname><given-names>JF</given-names></name><name><surname>Grini</surname><given-names>A</given-names></name><name><surname>Lee</surname><given-names>H</given-names></name></person-group><article-title>Impact of idealized future stratospheric aerosol injection on the large-scale ocean and land carbon cycles</article-title><source>J Geophys Res G Biogeosci.</source><year>2016</year><volume>121</volume><fpage>2</fpage><lpage>27</lpage><pub-id pub-id-type=\"doi\">10.1002/2015JG003045</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><mixed-citation publication-type=\"other\">Aumont O, Bopp L. Globalizing results from ocean in situ iron fertilization studies. Glob Biogeochem Cycles. 2006a;20:GB2017. 10.1029/2005GB002591.</mixed-citation></ref><ref id=\"CR41\"><label>41.</label><mixed-citation publication-type=\"other\">Aumont O, Bopp L. Globalizing results from ocean in situ iron fertilization studies. Glob Biogeochem Cycles. 2006b;20:GB2017. 10.1029/2005GB002591.</mixed-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bertram</surname><given-names>C</given-names></name></person-group><article-title>Ocean iron fertilization in the context of the Kyoto protocol and the post-Kyoto process</article-title><source>Energy Policy.</source><year>2010</year><volume>38</volume><fpage>1130</fpage><lpage>1139</lpage><pub-id pub-id-type=\"doi\">10.1016/j.enpol.2009.10.065</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boyd</surname><given-names>P</given-names></name></person-group><article-title>Implications of large-scale iron fertilization of the oceans</article-title><source>Mar Ecol Prog Ser.</source><year>2008</year><volume>364</volume><fpage>213</fpage><lpage>218</lpage><pub-id pub-id-type=\"doi\">10.3354/meps07541</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>de Baar</surname><given-names>H</given-names></name><name><surname>Gerringa</surname><given-names>L</given-names></name><name><surname>Laan</surname><given-names>P</given-names></name><name><surname>Timmermans</surname><given-names>K</given-names></name></person-group><article-title>Efficiency of carbon removal per added iron in ocean iron fertilization</article-title><source>Mar Ecol Prog Ser.</source><year>2008</year><volume>364</volume><fpage>269</fpage><lpage>282</lpage><pub-id pub-id-type=\"doi\">10.3354/meps07548</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Harrison</surname><given-names>DP</given-names></name></person-group><article-title>Global negative emissions capacity of ocean macronutrient fertilization</article-title><source>Environ Res Lett.</source><year>2017</year><volume>12</volume><fpage>035001</fpage><pub-id pub-id-type=\"doi\">10.1088/1748-9326/aa5ef5</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Keller</surname><given-names>DP</given-names></name><name><surname>Feng</surname><given-names>EY</given-names></name><name><surname>Oschlies</surname><given-names>A</given-names></name></person-group><article-title>Potential climate engineering effectiveness and side effects during a high carbon dioxide-emission scenario</article-title><source>Nat Commun.</source><year>2014</year><volume>5</volume><fpage>3304</fpage><pub-id pub-id-type=\"doi\">10.1038/ncomms4304</pub-id><pub-id pub-id-type=\"pmid\">24569320</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yool</surname><given-names>A</given-names></name><name><surname>Shepherd</surname><given-names>JG</given-names></name><name><surname>Bryden</surname><given-names>HL</given-names></name><name><surname>Oschlies</surname><given-names>A</given-names></name></person-group><article-title>Low efficiency of nutrient translocation for enhancing oceanic uptake of carbon dioxide</article-title><source>J Geophys Res.</source><year>2009</year><volume>114</volume><fpage>C08009</fpage><pub-id pub-id-type=\"doi\">10.1029/2008JC004792</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ferrer-Gonzalez</surname><given-names>M</given-names></name><name><surname>Ilyina</surname><given-names>T</given-names></name><name><surname>Sonntag</surname><given-names>S</given-names></name><name><surname>Schmidt</surname><given-names>H</given-names></name></person-group><article-title>Enhanced rates of regional warming and ocean acidification after termination of large-scale ocean alkalinization</article-title><source>Geophys Res Lett.</source><year>2018</year><volume>45</volume><fpage>7120</fpage><lpage>7129</lpage><pub-id pub-id-type=\"doi\">10.1029/2018GL077847</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gattuso</surname><given-names>J-P</given-names></name><etal/></person-group><article-title>Ocean solutions to address climate change and its effects on marine ecosystems</article-title><source>Front Mar Sci.</source><year>2018</year><volume>5</volume><fpage>337</fpage><pub-id pub-id-type=\"doi\">10.3389/fmars.2018.00337</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>González</surname><given-names>MF</given-names></name><name><surname>Ilyina</surname><given-names>T</given-names></name></person-group><article-title>Impacts of artificial ocean alkalinization on the carbon cycle and climate in Earth system simulations</article-title><source>Geophys Res Lett.</source><year>2016</year><volume>43</volume><fpage>6493</fpage><lpage>6502</lpage><pub-id pub-id-type=\"doi\">10.1002/2016GL068576</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ilyina</surname><given-names>T</given-names></name><name><surname>Wolf-Gladrow</surname><given-names>D</given-names></name><name><surname>Munhoven</surname><given-names>G</given-names></name><name><surname>Heinze</surname><given-names>C</given-names></name></person-group><article-title>Assessing the potential of calcium-based artificial ocean alkalinization to mitigate rising atmospheric CO2 and ocean acidification</article-title><source>Geophys Res Lett.</source><year>2013</year><volume>40</volume><fpage>5909</fpage><lpage>5914</lpage><pub-id pub-id-type=\"doi\">10.1002/2013GL057981</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ilyina</surname><given-names>T</given-names></name><name><surname>Six</surname><given-names>K</given-names></name><name><surname>Segschneider</surname><given-names>J</given-names></name><name><surname>Maier-Reimer</surname><given-names>E</given-names></name><name><surname>Li</surname><given-names>H</given-names></name><name><surname>Núñez-Riboni</surname><given-names>I</given-names></name></person-group><article-title>Global ocean biogeochemistry model HAMOCC: model architecture and performance as component of the MPI-Earth System Model in different CMIP5 experimental realizations</article-title><source>J Adv Model Earth Syst.</source><year>2013</year><volume>5</volume><fpage>287</fpage><lpage>315</lpage><pub-id pub-id-type=\"doi\">10.1029/2012MS000178</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>John</surname><given-names>JG</given-names></name><name><surname>Stock</surname><given-names>CA</given-names></name><name><surname>Dunne</surname><given-names>JP</given-names></name></person-group><article-title>A more productive, but different, ocean after mitigation</article-title><source>Geophys Res Lett.</source><year>2015</year><volume>42</volume><fpage>9836</fpage><lpage>9845</lpage><pub-id pub-id-type=\"doi\">10.1002/2015GL066160</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schwinger</surname><given-names>J</given-names></name><name><surname>Tjiputra</surname><given-names>J</given-names></name></person-group><article-title>Ocean carbon cycle feedbacks under negative emissions</article-title><source>Geophys Res Lett.</source><year>2018</year><volume>45</volume><fpage>5062</fpage><lpage>5070</lpage><pub-id pub-id-type=\"doi\">10.1029/2018GL077790</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cheung</surname><given-names>WWL</given-names></name><name><surname>Sarmiento</surname><given-names>JL</given-names></name><name><surname>Dunne</surname><given-names>J</given-names></name><name><surname>Frölicher</surname><given-names>TL</given-names></name><name><surname>Lam</surname><given-names>VWY</given-names></name><name><surname>Deng Palomares</surname><given-names>ML</given-names></name><etal/></person-group><article-title>Shrinking of fishes exacerbates impacts of global ocean changes on marine ecosystems</article-title><source>Nat Clim Chang.</source><year>2012</year><volume>3</volume><fpage>254</fpage><lpage>258</lpage><pub-id pub-id-type=\"doi\">10.1038/nclimate1691</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Le Mézo</surname><given-names>P</given-names></name><name><surname>Lefort</surname><given-names>S</given-names></name><name><surname>Séférian</surname><given-names>R</given-names></name><name><surname>Aumont</surname><given-names>O</given-names></name><name><surname>Maury</surname><given-names>O</given-names></name><name><surname>Murtugudde</surname><given-names>R</given-names></name><etal/></person-group><article-title>Natural variability of marine ecosystems inferred from a coupled climate to ecosystem simulation</article-title><source>J Mar Syst.</source><year>2016</year><volume>153</volume><fpage>55</fpage><lpage>66</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jmarsys.2015.09.004</pub-id></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lefort</surname><given-names>S</given-names></name><name><surname>Aumont</surname><given-names>O</given-names></name><name><surname>Bopp</surname><given-names>L</given-names></name><name><surname>Arsouze</surname><given-names>T</given-names></name><name><surname>Gehlen</surname><given-names>M</given-names></name><name><surname>Maury</surname><given-names>O</given-names></name></person-group><article-title>Spatial and body-size dependent response of marine pelagic communities to projected global climate change</article-title><source>Glob Chang Biol.</source><year>2015</year><volume>21</volume><fpage>154</fpage><lpage>164</lpage><pub-id pub-id-type=\"doi\">10.1111/gcb.12679</pub-id><pub-id pub-id-type=\"pmid\">25044507</pub-id></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stock</surname><given-names>CA</given-names></name><etal/></person-group><article-title>On the use of IPCC-class models to assess the impact of climate on living marine resources</article-title><source>Prog Oceanogr.</source><year>2011</year><volume>88</volume><fpage>1</fpage><lpage>27</lpage><pub-id pub-id-type=\"doi\">10.1016/j.pocean.2010.09.001</pub-id></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lotze</surname><given-names>HK</given-names></name><name><surname>Tittensor</surname><given-names>DP</given-names></name><name><surname>Bryndum-Buchholz</surname><given-names>A</given-names></name><name><surname>Eddy</surname><given-names>TD</given-names></name><name><surname>Cheung</surname><given-names>WW</given-names></name><name><surname>Galbraith</surname><given-names>ED</given-names></name><etal/></person-group><article-title>Global ensemble projections reveal trophic amplification of ocean biomass declines with climate change</article-title><source>Proc Natl Acad Sci.</source><year>2019</year><volume>116</volume><issue>26</issue><fpage>12907</fpage><lpage>12912</lpage><pub-id pub-id-type=\"doi\">10.1073/pnas.1900194116</pub-id><pub-id pub-id-type=\"pmid\">31186360</pub-id></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tittensor</surname><given-names>DP</given-names></name><etal/></person-group><article-title>A protocol for the intercomparison of marine fishery and ecosystem models: Fish-MIP v1.0</article-title><source>Geosci Model Dev.</source><year>2018</year><volume>11</volume><fpage>1421</fpage><lpage>1442</lpage><pub-id pub-id-type=\"doi\">10.5194/gmd-11-1421-2018</pub-id></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>H</given-names></name><name><surname>Ilyina</surname><given-names>T</given-names></name><name><surname>Muller</surname><given-names>WA</given-names></name><name><surname>Sienz</surname><given-names>F</given-names></name></person-group><article-title>Decadal predictions of the North Atlantic CO2 uptake</article-title><source>Nat Commun.</source><year>2016</year><volume>7</volume><fpage>11076</fpage><pub-id pub-id-type=\"doi\">10.1038/ncomms11076</pub-id><pub-id pub-id-type=\"pmid\">27026490</pub-id></element-citation></ref><ref id=\"CR62\"><label>62.</label><mixed-citation publication-type=\"other\">Li H, Ilyina T, Müller W, Landschützer P. Predicting the variable ocean carbon sink, eaav6471. Sci Adv. 2019;5.</mixed-citation></ref><ref id=\"CR63\"><label>63.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lovenduski</surname><given-names>NS</given-names></name><name><surname>Yeager</surname><given-names>SG</given-names></name><name><surname>Lindsay</surname><given-names>K</given-names></name><name><surname>Long</surname><given-names>MC</given-names></name></person-group><article-title>Predicting near-term variability in ocean carbon uptake</article-title><source>Earth Syst Dynam.</source><year>2019</year><volume>10</volume><fpage>45</fpage><lpage>57</lpage><pub-id pub-id-type=\"doi\">10.5194/esd-10-45-2019</pub-id></element-citation></ref><ref id=\"CR64\"><label>64.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Park</surname><given-names>J-Y</given-names></name><name><surname>Stock</surname><given-names>CA</given-names></name><name><surname>Dunne</surname><given-names>J</given-names></name><name><surname>Yang</surname><given-names>X</given-names></name><name><surname>Rosati</surname><given-names>A</given-names></name></person-group><article-title>Seasonal to multiannual marine ecosystem prediction with a global Earth system model</article-title><source>Science.</source><year>2019</year><volume>365</volume><issue>6450</issue><fpage>284</fpage><lpage>288</lpage><pub-id pub-id-type=\"doi\">10.1126/science.aav6634</pub-id><pub-id pub-id-type=\"pmid\">31320541</pub-id></element-citation></ref><ref id=\"CR65\"><label>65.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Séférian</surname><given-names>R</given-names></name><name><surname>Bopp</surname><given-names>L</given-names></name><name><surname>Gehlen</surname><given-names>M</given-names></name><name><surname>Swingedouw</surname><given-names>D</given-names></name><name><surname>Mignot</surname><given-names>J</given-names></name><name><surname>Guilyardi</surname><given-names>E</given-names></name><etal/></person-group><article-title>Multiyear predictability of tropical marine productivity</article-title><source>Proc Natl Acad Sci.</source><year>2014</year><volume>111</volume><issue>32</issue><fpage>11646</fpage><lpage>11651</lpage><pub-id pub-id-type=\"doi\">10.1073/pnas.1315855111</pub-id><pub-id pub-id-type=\"pmid\">25071174</pub-id></element-citation></ref><ref id=\"CR66\"><label>66.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Séférian</surname><given-names>R</given-names></name><name><surname>Berthet</surname><given-names>CSM</given-names></name></person-group><article-title>Assessing the decadal predictability of land and ocean carbon uptake</article-title><source>Geophys Res Lett.</source><year>2018</year><volume>45</volume><fpage>2455</fpage><lpage>2466</lpage><pub-id pub-id-type=\"doi\">10.1002/2017GL076092</pub-id></element-citation></ref><ref id=\"CR67\"><label>67.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yeager</surname><given-names>SG</given-names></name><name><surname>Danabasoglu</surname><given-names>G</given-names></name><name><surname>Rosenbloom</surname><given-names>NA</given-names></name><name><surname>Strand</surname><given-names>W</given-names></name><name><surname>Bates</surname><given-names>SC</given-names></name><name><surname>Meehl</surname><given-names>GA</given-names></name><etal/></person-group><article-title>Predicting near-term changes in the Earth System: a large ensemble of initialized decadal prediction simulations using the Community Earth System Model</article-title><source>Bull Amer Meteor Soc.</source><year>2018</year><volume>99</volume><fpage>1867</fpage><lpage>1886</lpage><pub-id pub-id-type=\"doi\">10.1175/BAMS-D-17-0098.1</pub-id></element-citation></ref><ref id=\"CR68\"><label>68.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Peters</surname><given-names>GP</given-names></name><etal/></person-group><article-title>Towards real-time verification of CO2 emissions</article-title><source>Nat Clim Chang.</source><year>2017</year><volume>7</volume><fpage>848</fpage><lpage>850</lpage><pub-id pub-id-type=\"doi\">10.1038/s41558-017-0013-9</pub-id></element-citation></ref><ref id=\"CR69\"><label>69.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tommasi</surname><given-names>D</given-names></name><etal/></person-group><article-title>Managing living marine resources in a dynamic environment: the role of seasonal to decadal climate forecasts</article-title><source>Prog Oceanogr.</source><year>2017</year><volume>152</volume><fpage>15</fpage><lpage>49</lpage><pub-id pub-id-type=\"doi\">10.1016/j.pocean.2016.12.011</pub-id></element-citation></ref><ref id=\"CR70\"><label>70.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bakker</surname><given-names>DCE</given-names></name><etal/></person-group><article-title>A multi-decade record of high-quality fCO2 data in version 3 of the Surface Ocean CO2 atlas (SOCAT)</article-title><source>Earth Syst Sci Data.</source><year>2016</year><volume>8</volume><fpage>383</fpage><lpage>413</lpage><pub-id pub-id-type=\"doi\">10.5194/essd-8-383-2016</pub-id></element-citation></ref><ref id=\"CR71\"><label>71.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Buitenhuis</surname><given-names>ET</given-names></name><etal/></person-group><article-title>MAREDAT: towards a world atlas of MARine Ecosystem DATa</article-title><source>Earth Syst Sci Data.</source><year>2013</year><volume>5</volume><fpage>227</fpage><lpage>239</lpage><pub-id pub-id-type=\"doi\">10.5194/essd-5-227-2013</pub-id></element-citation></ref><ref id=\"CR72\"><label>72.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Key</surname><given-names>RM</given-names></name><etal/></person-group><article-title>Global Ocean Data Analysis Project, Version 2 (GLODAPv2)</article-title><source>Ornl/Cdiac-162, Ndp-093</source><year>2015</year><volume>8</volume><fpage>297</fpage><lpage>323</lpage><pub-id pub-id-type=\"doi\">10.3334/CDIAC/OTG.NDP093_GLODAPv2</pub-id></element-citation></ref><ref id=\"CR73\"><label>73.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>T</given-names></name><name><surname>Lu</surname><given-names>Y</given-names></name><name><surname>Fang</surname><given-names>Y</given-names></name><name><surname>Xin</surname><given-names>X</given-names></name><name><surname>Li</surname><given-names>L</given-names></name><name><surname>Li</surname><given-names>W</given-names></name><etal/></person-group><article-title>The Beijing Climate Center Climate System Model (BCC-CSM): main progress from CMIP5 to CMIP6</article-title><source>Geosci Model Dev.</source><year>2019</year><volume>12</volume><fpage>1573</fpage><lpage>1600</lpage><pub-id pub-id-type=\"doi\">10.5194/gmd-12-1573-2019</pub-id></element-citation></ref><ref id=\"CR74\"><label>74.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Swart</surname><given-names>NC</given-names></name><name><surname>Cole</surname><given-names>JNS</given-names></name><name><surname>Kharin</surname><given-names>VV</given-names></name><name><surname>Lazare</surname><given-names>M</given-names></name><name><surname>Scinocca</surname><given-names>JF</given-names></name><name><surname>Gillett</surname><given-names>NP</given-names></name><etal/></person-group><article-title>The Canadian Earth System Model version 5 (CanESM5.0.3)</article-title><source>Geosci Model Dev.</source><year>2019</year><volume>12</volume><fpage>4823</fpage><lpage>4873</lpage><pub-id pub-id-type=\"doi\">10.5194/gmd-12-4823-2019</pub-id></element-citation></ref><ref id=\"CR75\"><label>75.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Danabasoglu</surname><given-names>G</given-names></name><name><surname>Lamarque</surname><given-names>J-F</given-names></name><name><surname>Bacmeister</surname><given-names>J</given-names></name><name><surname>Bailey</surname><given-names>DA</given-names></name><name><surname>DuVivier</surname><given-names>AK</given-names></name><name><surname>Edwards</surname><given-names>J</given-names></name><etal/></person-group><article-title>The Community Earth System Model Version 2 (CESM2)</article-title><source>J Adv Model Earth Syst</source><year>2020</year><volume>12</volume><fpage>e2019MS001916</fpage><pub-id pub-id-type=\"doi\">10.1029/2019MS001916</pub-id></element-citation></ref><ref id=\"CR76\"><label>76.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Séférian</surname><given-names>R</given-names></name><name><surname>Gehlen</surname><given-names>M</given-names></name><name><surname>Bopp</surname><given-names>L</given-names></name><name><surname>Resplandy</surname><given-names>L</given-names></name><name><surname>Orr</surname><given-names>JC</given-names></name><name><surname>Marti</surname><given-names>O</given-names></name><etal/></person-group><article-title>Inconsistent strategies to spin up models in CMIP5: implications for ocean biogeochemical model performance assessment</article-title><source>Geosci Model Dev.</source><year>2016</year><volume>9</volume><fpage>1827</fpage><lpage>1851</lpage><pub-id pub-id-type=\"doi\">10.5194/gmd-9-1827-2016</pub-id></element-citation></ref><ref id=\"CR77\"><label>77.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Séférian</surname><given-names>R</given-names></name><name><surname>Nabat</surname><given-names>P</given-names></name><name><surname>Michou</surname><given-names>M</given-names></name><name><surname>Saint-Martin</surname><given-names>D</given-names></name><name><surname>Voldoire</surname><given-names>A</given-names></name><name><surname>Colin</surname><given-names>J</given-names></name><etal/></person-group><article-title>Evaluation of CNRM Earth-System model, CNRM-ESM2-1: role of Earth system processes in present-day and future climate</article-title><source>J Adv Model Earth Syst.</source><year>2019</year><volume>11</volume><fpage>4182</fpage><lpage>4227</lpage><pub-id pub-id-type=\"doi\">10.1029/2019MS001791</pub-id></element-citation></ref><ref id=\"CR78\"><label>78.</label><mixed-citation publication-type=\"other\">Held IM, Guo H, Adcroft A, Dunne JP, Horowitz LW, Krasting JP, et al. Structure and Performance of GFDL’s CM4.0 Climate Model. J Adv Model Earth Syst. 2019;11(11). 10.1029/2019MS001829.</mixed-citation></ref><ref id=\"CR79\"><label>79.</label><mixed-citation publication-type=\"other\">Krasting JP, John JG, Blanton C, McHugh C, Nikonov S, Radhakrishnan A, et al. NOAA-GFDL GFDL-ESM4 model output prepared for CMIP6 CMIP. Earth System Grid Federation. 2018. 10.22033/ESGF/CMIP6.1407.</mixed-citation></ref><ref id=\"CR80\"><label>80.</label><mixed-citation publication-type=\"other\">Ito G, Romanou A, Kiang NY, Faluvegi G, Aleinov I, Ruedy G, et al. Global carbon cycle and climate feedbacks in the NASA GISS ModelE2.1. Submitted to Journal of Advances in Modeling Earth Systems. in review.</mixed-citation></ref><ref id=\"CR81\"><label>81.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jones</surname><given-names>CD</given-names></name><name><surname>Hughes</surname><given-names>JK</given-names></name><name><surname>Bellouin</surname><given-names>N</given-names></name><name><surname>Hardiman</surname><given-names>SC</given-names></name><name><surname>Jones</surname><given-names>GS</given-names></name><name><surname>Knight</surname><given-names>J</given-names></name><etal/></person-group><article-title>The HadGEM2-ES implementation of CMIP5 centennial simulations</article-title><source>Geosci Model Dev.</source><year>2011</year><volume>4</volume><fpage>543</fpage><lpage>570</lpage><pub-id pub-id-type=\"doi\">10.5194/gmd-4-543-2011</pub-id></element-citation></ref><ref id=\"CR82\"><label>82.</label><mixed-citation publication-type=\"other\">Sellar AA, Jones CG, Mulcahy J, Tang Y, Yool A, Wiltshire A, et al. UKESM1: description and evaluation of the UK Earth System Model. J Adv Model Earth Syst. 2019. 10.1029/2019MS0017392019.</mixed-citation></ref><ref id=\"CR83\"><label>83.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Boucher</surname><given-names>O</given-names></name><name><surname>Servonnat</surname><given-names>J</given-names></name><name><surname>Albright</surname><given-names>AL</given-names></name><name><surname>Aumont</surname><given-names>O</given-names></name><name><surname>Balkanski</surname><given-names>Y</given-names></name><name><surname>Bastrikov</surname><given-names>V</given-names></name><etal/></person-group><source>Presentation and evaluation of the IPSL-CM6A-LR climate model</source><year>2020</year></element-citation></ref><ref id=\"CR84\"><label>84.</label><mixed-citation publication-type=\"other\">Hajima T, Watanabe M, Yamamoto A, Tatebe H, Noguchi MA, Abe M, et al. Description of the MIROC-ES2L Earth system model and evaluation of its climate–biogeochemical processes and feedback. Geosci Model Dev Discuss. 2019a. 10.5194/gmd-2019-275 in review.</mixed-citation></ref><ref id=\"CR85\"><label>85.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mauritsen</surname><given-names>T</given-names></name><name><surname>Bader</surname><given-names>J</given-names></name><name><surname>Becker</surname><given-names>T</given-names></name><name><surname>Behrens</surname><given-names>J</given-names></name><name><surname>Bittner</surname><given-names>M</given-names></name><name><surname>Brokopf</surname><given-names>R</given-names></name><etal/></person-group><article-title>Developments in the MPI-M Earth System Model version 1.2 (MPI-ESM 1.2) and its response to increasing CO2</article-title><source>J Adv Model Earth Syst.</source><year>2019</year><volume>11</volume><fpage>998</fpage><lpage>1038</lpage><pub-id pub-id-type=\"doi\">10.1029/2018MS001400</pub-id><pub-id pub-id-type=\"pmid\">32742553</pub-id></element-citation></ref><ref id=\"CR86\"><label>86.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yukimoto</surname><given-names>S</given-names></name><name><surname>Adachi</surname><given-names>Y</given-names></name><name><surname>Hosaka</surname><given-names>M</given-names></name><name><surname>Sakami</surname><given-names>T</given-names></name><name><surname>Yoshimura</surname><given-names>H</given-names></name><name><surname>Hirabara</surname><given-names>M</given-names></name><etal/></person-group><article-title>A new global climate model of the meteorological research institute: MRI-CGCM3 —model description and basic performance—</article-title><source>J Meteor Soc Japan.</source><year>2012</year><volume>90A</volume><fpage>23</fpage><lpage>64</lpage><pub-id pub-id-type=\"doi\">10.2151/jmsj.2012-A02</pub-id></element-citation></ref><ref id=\"CR87\"><label>87.</label><mixed-citation publication-type=\"other\">Seland Ø, Bentsen M, Seland Graff L, Olivié D, Toniazzo T, Gjermundsen A, et al. The Norwegian Earth System Model, NorESM2 – evaluation of theCMIP6 DECK and historical simulations. Geosci Model Dev Discuss. 2020. 10.5194/gmd-2019-378 in review.</mixed-citation></ref><ref id=\"CR88\"><label>88.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zahariev</surname><given-names>K</given-names></name><name><surname>Christian</surname><given-names>JR</given-names></name><name><surname>Denman</surname><given-names>KL</given-names></name></person-group><article-title>Preindustrial, historical, and fertilization simulations using a global ocean carbon model with new parameterizations of iron limitation, calcification, and N2 fixation</article-title><source>Prog Oceanogr.</source><year>2008</year><volume>77</volume><fpage>56</fpage><lpage>82</lpage><pub-id pub-id-type=\"doi\">10.1016/j.pocean.2008.01.007</pub-id></element-citation></ref><ref id=\"CR89\"><label>89.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hayashida</surname><given-names>H</given-names></name><name><surname>Christian</surname><given-names>JR</given-names></name><name><surname>Holdsworth</surname><given-names>AM</given-names></name><name><surname>Hu</surname><given-names>X</given-names></name><name><surname>Monahan</surname><given-names>AH</given-names></name><name><surname>Mortenson</surname><given-names>E</given-names></name><etal/></person-group><article-title>CSIB v1 (Canadian Sea-ice Biogeochemistry): a sea-ice biogeochemical model for the NEMO community ocean modelling framework</article-title><source>Geosci Model Dev.</source><year>2019</year><volume>12</volume><fpage>1965</fpage><lpage>1990</lpage><pub-id pub-id-type=\"doi\">10.5194/gmd-12-1965-2019</pub-id></element-citation></ref><ref id=\"CR90\"><label>90.</label><mixed-citation publication-type=\"other\">Moore JK, Doney SC, Lindsay K. Upper ocean ecosystem dynamics and iron cycling in a global three-dimensional model. Glob Biogeochem Cycles. 2004;18, GB4028. 10.1029/2004GB002220.</mixed-citation></ref><ref id=\"CR91\"><label>91.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aumont</surname><given-names>O</given-names></name><name><surname>Ethé</surname><given-names>C</given-names></name><name><surname>Tagliabue</surname><given-names>A</given-names></name><name><surname>Bopp</surname><given-names>L</given-names></name><name><surname>Gehlen</surname><given-names>M</given-names></name></person-group><article-title>PISCES-v2: an ocean biogeochemical model for carbon and ecosystem studies</article-title><source>Geosci Model Dev.</source><year>2015</year><volume>8</volume><fpage>2465</fpage><lpage>2513</lpage><pub-id pub-id-type=\"doi\">10.5194/gmd-8-2465-2015</pub-id></element-citation></ref><ref id=\"CR92\"><label>92.</label><mixed-citation publication-type=\"other\">Stock CA, Dunne JP, Fan S, Ginoux P, John J, Krasting JP, et al. Ocean biogeochemistry in GFDL’s earth system model 4.1 and its response to increasing atmospheric CO2. J Adv Model Earth Syst. submitted, in review.</mixed-citation></ref><ref id=\"CR93\"><label>93.</label><mixed-citation publication-type=\"other\">Dunne JP, Bociu I, Bronselaer B, Guo H, John JG, Krasting JP, et al. submitted-a Simple Global Ocean Biogeochemistry with Light, Iron, Nutrients and Gas version 2 (BLINGv2): model description and simulation characteristics in GFDL’s CM4.0. J Adv Model Earth Syst. in review.</mixed-citation></ref><ref id=\"CR94\"><label>94.</label><mixed-citation publication-type=\"other\">Dunne JP, Horowitz LW, Adcroft AJ, Ginoux P, Held IM, John JG, et al. The GFDL Earth System Model version 4.1 (GFDL-ESM4.1): model description and simulation characteristics. J Adv Model Earth Syst. submitted-b:2019MS002008.</mixed-citation></ref><ref id=\"CR95\"><label>95.</label><mixed-citation publication-type=\"other\">Lerner P, Romanou A, Kelley M, Romanski J, Ruedy R, Russell G. Drivers of air-sea CO2 flux seasonality and its long-term changes in the NASA-GISS model CMIP6 submission. J Adv Model Earth Syst. in review.</mixed-citation></ref><ref id=\"CR96\"><label>96.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Totterdell</surname><given-names>IJ</given-names></name></person-group><article-title>Description and evaluation of the Diat-HadOCC model v1.0: the ocean biogeochemical component of HadGEM2-ES</article-title><source>Geosci Model Dev.</source><year>2019</year><volume>12</volume><fpage>4497</fpage><lpage>4549</lpage><pub-id pub-id-type=\"doi\">10.5194/gmd-12-4497-2019</pub-id></element-citation></ref><ref id=\"CR97\"><label>97.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yool</surname><given-names>A</given-names></name><name><surname>Popova</surname><given-names>EE</given-names></name><name><surname>Anderson</surname><given-names>TR</given-names></name></person-group><article-title>MEDUSA-2.0: an intermediate complexity biogeochemical model of the marine carbon cycle for climate change and ocean acidification studies</article-title><source>Geosci Model Dev.</source><year>2013</year><volume>6</volume><fpage>1767</fpage><lpage>1811</lpage><pub-id pub-id-type=\"doi\">10.5194/gmd-6-1767-2013</pub-id></element-citation></ref><ref id=\"CR98\"><label>98.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hajima</surname><given-names>T</given-names></name><name><surname>Kawamiya</surname><given-names>M</given-names></name><name><surname>Watanabe</surname><given-names>M</given-names></name><etal/></person-group><article-title>Modeling in Earth system science up to and beyond IPCC AR5</article-title><source>Prog. in Earth and Planet. Sci.</source><year>2014</year><volume>1</volume><fpage>29</fpage><pub-id pub-id-type=\"doi\">10.1186/s40645-014-0029-y</pub-id></element-citation></ref><ref id=\"CR99\"><label>99.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Paulsen</surname><given-names>H</given-names></name><name><surname>Ilyina</surname><given-names>T</given-names></name><name><surname>Six</surname><given-names>KD</given-names></name><name><surname>Stemmler</surname><given-names>I</given-names></name></person-group><article-title>Incorporating a prognostic representation of marine nitrogen fixers into the global ocean biogeochemical model HAMOCC</article-title><source>J Adv Model Earth Syst.</source><year>2017</year><volume>9</volume><fpage>438</fpage><lpage>464</lpage><pub-id pub-id-type=\"doi\">10.1002/2016MS000737</pub-id></element-citation></ref><ref id=\"CR100\"><label>100.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nakano</surname><given-names>H</given-names></name><name><surname>Tsujino</surname><given-names>H</given-names></name><name><surname>Hirabara</surname><given-names>M</given-names></name><name><surname>Yasuda</surname><given-names>T</given-names></name><name><surname>Motoi</surname><given-names>T</given-names></name><name><surname>Ishii</surname><given-names>M</given-names></name><etal/></person-group><article-title>Uptake mechanism of anthropogenic CO2 in the Kuroshio Extension region in an ocean general circulation model</article-title><source>J Oceanogr.</source><year>2011</year><volume>67</volume><fpage>765</fpage><lpage>783</lpage><pub-id pub-id-type=\"doi\">10.1007/s10872-011-0075-7</pub-id></element-citation></ref><ref id=\"CR101\"><label>101.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tjiputra</surname><given-names>JF</given-names></name><name><surname>Roelandt</surname><given-names>C</given-names></name><name><surname>Bentsen</surname><given-names>M</given-names></name><name><surname>Lawrence</surname><given-names>DM</given-names></name><name><surname>Lorentzen</surname><given-names>T</given-names></name><name><surname>Schwinger</surname><given-names>J</given-names></name><etal/></person-group><article-title>Evaluation of the carbon cycle components in the Norwegian Earth System Model (NorESM)</article-title><source>Geosci Model Dev.</source><year>2013</year><volume>6</volume><fpage>301</fpage><lpage>325</lpage><pub-id pub-id-type=\"doi\">10.5194/gmd-6-301-2013</pub-id></element-citation></ref><ref id=\"CR102\"><label>102.</label><mixed-citation publication-type=\"other\">Tjiputra JF, Schwinger J, Bentsen M, Morée AL, Gao S, Bethke I, et al. Ocean biogeochemistry in the Norwegian Earth System Model version 2 (NorESM2). Geosci Model Dev Discuss. 2020. 10.5194/gmd-2019-347 in review.</mixed-citation></ref><ref id=\"CR103\"><label>103.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Griffies</surname><given-names>SM</given-names></name><name><surname>Adcroft</surname><given-names>AJ</given-names></name><name><surname>Banks</surname><given-names>H</given-names></name><name><surname>Boning</surname><given-names>CW</given-names></name><name><surname>Chassignet</surname><given-names>EP</given-names></name><name><surname>Danabasoglu</surname><given-names>G</given-names></name><etal/></person-group><person-group person-group-type=\"editor\"><name><surname>Hall</surname><given-names>J</given-names></name><name><surname>Harrison</surname><given-names>DE</given-names></name><name><surname>Stammer</surname><given-names>D</given-names></name><etal/></person-group><article-title>Problems and prospects in large-scale ocean circulation models</article-title><source>Proceedings of the OceanObs’09 Conference: Sustained Ocean Observations and Information for Society, 21–25 September 2009, Vol. 2</source><year>2010</year><publisher-loc>Venice</publisher-loc><publisher-name>ESA Publication WPP</publisher-name><fpage>306</fpage></element-citation></ref><ref id=\"CR104\"><label>104.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tagliabue</surname><given-names>A</given-names></name><name><surname>Völker</surname><given-names>C</given-names></name></person-group><article-title>Towards accounting for dissolved iron speciation in global ocean models</article-title><source>Biogeosciences.</source><year>2011</year><volume>8</volume><fpage>3025</fpage><lpage>3039</lpage><pub-id pub-id-type=\"doi\">10.5194/bg-8-3025-2011</pub-id></element-citation></ref><ref id=\"CR105\"><label>105.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Galbraith</surname><given-names>ED</given-names></name><name><surname>Dunne</surname><given-names>JP</given-names></name><name><surname>Gnanadesikan</surname><given-names>A</given-names></name><name><surname>Slater</surname><given-names>RD</given-names></name><name><surname>Sarmiento</surname><given-names>JL</given-names></name><name><surname>Dufour</surname><given-names>CO</given-names></name><etal/></person-group><article-title>Complex functionality with minimal computation: promise and pitfalls of reduced-tracer ocean biogeochemistry models</article-title><source>J Adv Model Earth Syst.</source><year>2015</year><volume>7</volume><fpage>2012</fpage><lpage>2028</lpage><pub-id pub-id-type=\"doi\">10.1002/2015MS000463</pub-id></element-citation></ref><ref id=\"CR106\"><label>106.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Karl</surname><given-names>DM</given-names></name><name><surname>Björkman</surname><given-names>KM</given-names></name><name><surname>Dore</surname><given-names>JE</given-names></name><name><surname>Fujieki</surname><given-names>L</given-names></name><name><surname>Hebel</surname><given-names>DV</given-names></name><name><surname>Houlihan</surname><given-names>T</given-names></name><etal/></person-group><article-title>Ecological nitrogen-to-phosphorus stoichiometry at station ALOHA</article-title><source>Deep Sea Res Part II Top Stud Oceanogr.</source><year>2001</year><volume>48</volume><fpage>1529</fpage><lpage>1566</lpage><pub-id pub-id-type=\"doi\">10.1016/S0967-0645(00)00152-1</pub-id></element-citation></ref><ref id=\"CR107\"><label>107.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Klausmeier</surname><given-names>CA</given-names></name><name><surname>Litchman</surname><given-names>E</given-names></name><name><surname>Daufresne</surname><given-names>T</given-names></name><name><surname>Levin</surname><given-names>SA</given-names></name></person-group><article-title>Optimal nitrogen-to-phosphorus stoichiometry of phytoplankton</article-title><source>Nature.</source><year>2004</year><volume>429</volume><fpage>171</fpage><lpage>174</lpage><pub-id pub-id-type=\"doi\">10.1038/nature02454</pub-id><pub-id pub-id-type=\"pmid\">15141209</pub-id></element-citation></ref><ref id=\"CR108\"><label>108.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kwiatkowski</surname><given-names>L</given-names></name><name><surname>Aumont</surname><given-names>O</given-names></name><name><surname>Bopp</surname><given-names>L</given-names></name><name><surname>Ciais</surname><given-names>P</given-names></name></person-group><article-title>The impact of variable phytoplankton stoichiometry on projections of primary production, food quality, and carbon uptake in the Global Ocean</article-title><source>Glob Biogeochem Cycles.</source><year>2018</year><volume>32</volume><fpage>516</fpage><lpage>528</lpage><pub-id pub-id-type=\"doi\">10.1002/2017GB005799</pub-id></element-citation></ref><ref id=\"CR109\"><label>109.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tanioka</surname><given-names>T</given-names></name><name><surname>Matsumoto</surname><given-names>K</given-names></name></person-group><article-title>Buffering of ocean export production by flexible elemental stoichiometry of particulate organic matter</article-title><source>Glob Biogeochem Cycles.</source><year>2017</year><volume>31</volume><fpage>1528</fpage><lpage>1542</lpage><pub-id pub-id-type=\"doi\">10.1002/2017GB005670</pub-id></element-citation></ref><ref id=\"CR110\"><label>110.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weber</surname><given-names>TS</given-names></name><name><surname>Deutsch</surname><given-names>C</given-names></name></person-group><article-title>Ocean nutrient ratios governed by plankton biogeography</article-title><source>Nature.</source><year>2010</year><volume>467</volume><fpage>550</fpage><lpage>554</lpage><pub-id pub-id-type=\"doi\">10.1038/nature09403</pub-id><pub-id pub-id-type=\"pmid\">20882009</pub-id></element-citation></ref><ref id=\"CR111\"><label>111.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ardyna</surname><given-names>M</given-names></name><etal/></person-group><article-title>Hydrothermal vents trigger massive phytoplankton blooms in the Southern Ocean</article-title><source>Nat Commun.</source><year>2019</year><volume>10</volume><fpage>2451</fpage><pub-id pub-id-type=\"doi\">10.1038/s41467-019-09973-6</pub-id><pub-id pub-id-type=\"pmid\">31165724</pub-id></element-citation></ref><ref id=\"CR112\"><label>112.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boyd</surname><given-names>PW</given-names></name><name><surname>Ellwood</surname><given-names>MJ</given-names></name></person-group><article-title>The biogeochemical cycle of iron in the ocean</article-title><source>Nat Geosci.</source><year>2010</year><volume>3</volume><fpage>675</fpage><lpage>682</lpage><pub-id pub-id-type=\"doi\">10.1038/ngeo964</pub-id></element-citation></ref><ref id=\"CR113\"><label>113.</label><mixed-citation publication-type=\"other\">Death R, Wadham JL, Monteiro F, Le Brocq AM, Tranter M, Ridgwell A, et al. Antarctic ice sheet fertilises the Southern Ocean. Biogeosciences. 2014;11:2635–2643. 10.5194/bg-11-2635-2014<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.biogeosciences.net/11/2635/2014/\">https://www.biogeosciences.net/11/2635/2014/</ext-link> (Accessed November 3, 2019).</mixed-citation></ref><ref id=\"CR114\"><label>114.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stibal</surname><given-names>M</given-names></name><name><surname>Šabacká</surname><given-names>M</given-names></name><name><surname>Žárský</surname><given-names>J</given-names></name></person-group><article-title>Biological processes on glacier and ice sheet surfaces</article-title><source>Nat Geosci.</source><year>2012</year><volume>5</volume><fpage>771</fpage><lpage>774</lpage><pub-id pub-id-type=\"doi\">10.1038/ngeo1611</pub-id></element-citation></ref><ref id=\"CR115\"><label>115.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tagliabue</surname><given-names>A</given-names></name><etal/></person-group><article-title>Hydrothermal contribution to the oceanic dissolved iron inventory</article-title><source>Nat Geosci.</source><year>2010</year><volume>3</volume><fpage>252</fpage><lpage>256</lpage><pub-id pub-id-type=\"doi\">10.1038/ngeo818</pub-id></element-citation></ref><ref id=\"CR116\"><label>116.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tagliabue</surname><given-names>AO</given-names></name><name><surname>Bopp</surname><given-names>L</given-names></name></person-group><article-title>The impact of different external sources of iron on the global carbon cycle</article-title><source>Geophys Res Lett.</source><year>2014</year><volume>41</volume><fpage>920</fpage><lpage>926</lpage><pub-id pub-id-type=\"doi\">10.1002/2013GL059059</pub-id></element-citation></ref><ref id=\"CR117\"><label>117.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tagliabue</surname><given-names>A</given-names></name><name><surname>Aumont</surname><given-names>O</given-names></name><name><surname>DeAth</surname><given-names>R</given-names></name><name><surname>Dunne</surname><given-names>JP</given-names></name><name><surname>Dutkiewicz</surname><given-names>S</given-names></name><name><surname>Galbraith</surname><given-names>E</given-names></name><etal/></person-group><article-title>How well do global ocean biogeochemistry models simulate dissolved iron distributions?</article-title><source>Glob Biogeochem Cycles.</source><year>2016</year><volume>30</volume><fpage>149</fpage><lpage>174</lpage><pub-id pub-id-type=\"doi\">10.1002/2015GB005289</pub-id></element-citation></ref><ref id=\"CR118\"><label>118.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>S</given-names></name><name><surname>Lu</surname><given-names>XX</given-names></name><name><surname>Xia</surname><given-names>X</given-names></name><name><surname>Zhang</surname><given-names>S</given-names></name><name><surname>Ran</surname><given-names>L</given-names></name><name><surname>Yang</surname><given-names>X</given-names></name><etal/></person-group><article-title>Dynamic biogeochemical controls on river pCO2 and recent changes under aggravating river impoundment: an example of the subtropical Yangtze River</article-title><source>Glob Biogeochem Cycles.</source><year>2016</year><volume>30</volume><fpage>880</fpage><lpage>897</lpage><pub-id pub-id-type=\"doi\">10.1002/2016GB005388</pub-id></element-citation></ref><ref id=\"CR119\"><label>119.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>J</given-names></name><name><surname>Su</surname><given-names>N</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name><name><surname>Du</surname><given-names>J</given-names></name></person-group><article-title>Submarine groundwater discharge and associated nutrient fluxes into the Southern Yellow Sea: a case study for semi-enclosed and oligotrophic seas-implication for green tide bloom</article-title><source>J Geophys Res Ocean.</source><year>2017</year><volume>122</volume><fpage>139</fpage><lpage>152</lpage><pub-id pub-id-type=\"doi\">10.1002/2016JC012282</pub-id></element-citation></ref><ref id=\"CR120\"><label>120.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moore</surname><given-names>WS</given-names></name><name><surname>Sarmiento</surname><given-names>JL</given-names></name><name><surname>Key</surname><given-names>RM</given-names></name></person-group><article-title>Submarine groundwater discharge revealed by 228Ra distribution in the upper Atlantic Ocean</article-title><source>Nat Geosci.</source><year>2008</year><volume>1</volume><fpage>309</fpage><lpage>311</lpage><pub-id pub-id-type=\"doi\">10.1038/ngeo183</pub-id></element-citation></ref><ref id=\"CR121\"><label>121.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rodellas</surname><given-names>V</given-names></name><name><surname>Garcia-Orellana</surname><given-names>J</given-names></name><name><surname>Masqué</surname><given-names>P</given-names></name><name><surname>Feldman</surname><given-names>M</given-names></name><name><surname>Weinstein</surname><given-names>Y</given-names></name></person-group><article-title>Submarine groundwater discharge as a major source of nutrients to the Mediterranean Sea</article-title><source>Proc Natl Acad Sci.</source><year>2015</year><volume>112</volume><fpage>3926</fpage><lpage>3930</lpage><pub-id pub-id-type=\"doi\">10.1073/pnas.1419049112</pub-id><pub-id pub-id-type=\"pmid\">25775554</pub-id></element-citation></ref><ref id=\"CR122\"><label>122.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schlosser</surname><given-names>C</given-names></name><etal/></person-group><article-title>Seasonal ITCZ migration dynamically controls the location of the (sub) tropical Atlantic biogeochemical divide</article-title><source>Proc Natl Acad Sci.</source><year>2014</year><volume>111</volume><fpage>1438</fpage><lpage>1442</lpage><pub-id pub-id-type=\"doi\">10.1073/pnas.1318670111</pub-id><pub-id pub-id-type=\"pmid\">24367112</pub-id></element-citation></ref><ref id=\"CR123\"><label>123.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stefels</surname><given-names>J</given-names></name></person-group><article-title>Physiological aspects of the production and conversion of DMSP in marine algae and higher plants</article-title><source>J Sea Res.</source><year>2000</year><volume>43</volume><fpage>183</fpage><lpage>197</lpage><pub-id pub-id-type=\"doi\">10.1016/S1385-1101(00)00030-7</pub-id></element-citation></ref><ref id=\"CR124\"><label>124.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sunda</surname><given-names>W</given-names></name><name><surname>Kieber</surname><given-names>D</given-names></name><name><surname>Kiene</surname><given-names>R</given-names></name><etal/></person-group><article-title>An antioxidant function for DMSP and DMS in marine algae</article-title><source>Nature.</source><year>2002</year><volume>418</volume><fpage>317</fpage><lpage>320</lpage><pub-id pub-id-type=\"doi\">10.1038/nature00851</pub-id><pub-id pub-id-type=\"pmid\">12124622</pub-id></element-citation></ref><ref id=\"CR125\"><label>125.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Green</surname><given-names>TK</given-names></name><name><surname>Hatton</surname><given-names>AD</given-names></name></person-group><article-title>The CLAW hypothesis: a new perspective on the role of biogenic sulphur in the regulation of global climate</article-title><source>Oceanogr Mar Biol.</source><year>2014</year><volume>52</volume><fpage>315</fpage><lpage>336</lpage><pub-id pub-id-type=\"doi\">10.1201/b17143-7</pub-id></element-citation></ref><ref id=\"CR126\"><label>126.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brévière</surname><given-names>EHG</given-names></name><etal/></person-group><article-title>Surface ocean-lower atmosphere study: scientific synthesis and contribution to Earth system science</article-title><source>Anthropocene.</source><year>2015</year><volume>12</volume><fpage>54</fpage><lpage>68</lpage><pub-id pub-id-type=\"doi\">10.1016/j.ancene.2015.11.001</pub-id></element-citation></ref><ref id=\"CR127\"><label>127.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quinn</surname><given-names>PK</given-names></name><name><surname>Bates</surname><given-names>TS</given-names></name></person-group><article-title>The case against climate regulation via oceanic phytoplankton sulphur emissions</article-title><source>Nature.</source><year>2011</year><volume>480</volume><fpage>51</fpage><lpage>56</lpage><pub-id pub-id-type=\"doi\">10.1038/nature10580</pub-id><pub-id pub-id-type=\"pmid\">22129724</pub-id></element-citation></ref><ref id=\"CR128\"><label>128.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quinn</surname><given-names>PK</given-names></name><name><surname>Coffman</surname><given-names>DJ</given-names></name><name><surname>Johnson</surname><given-names>JE</given-names></name><name><surname>Upchurch</surname><given-names>LM</given-names></name><name><surname>Bates</surname><given-names>TS</given-names></name></person-group><article-title>Small fraction of marine cloud condensation nuclei made up of sea spray aerosol</article-title><source>Nat Geosci.</source><year>2017</year><volume>10</volume><fpage>674</fpage><lpage>679</lpage><pub-id pub-id-type=\"doi\">10.1038/ngeo3003</pub-id></element-citation></ref><ref id=\"CR129\"><label>129.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Galí</surname><given-names>M</given-names></name><name><surname>Devred</surname><given-names>E</given-names></name><name><surname>Levasseur</surname><given-names>M</given-names></name><name><surname>Royer</surname><given-names>SJ</given-names></name><name><surname>Babin</surname><given-names>M</given-names></name></person-group><article-title>A remote sensing algorithm for planktonic dimethylsulfoniopropionate (DMSP) and an analysis of global patterns</article-title><source>Remote Sens Environ.</source><year>2015</year><volume>171</volume><fpage>171</fpage><lpage>184</lpage><pub-id pub-id-type=\"doi\">10.1016/j.rse.2015.10.012</pub-id></element-citation></ref><ref id=\"CR130\"><label>130.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schwinger</surname><given-names>J</given-names></name><name><surname>Tjiputra</surname><given-names>J</given-names></name><name><surname>Goris</surname><given-names>N</given-names></name><name><surname>Six</surname><given-names>KD</given-names></name><name><surname>Kirkevåg</surname><given-names>A</given-names></name><name><surname>Seland</surname><given-names>Ø</given-names></name><etal/></person-group><article-title>Amplification of global warming through pH dependence of DMS production simulated with a fully coupled Earth system model</article-title><source>Biogeosciences.</source><year>2017</year><volume>14</volume><fpage>3633</fpage><lpage>3648</lpage><pub-id pub-id-type=\"doi\">10.5194/bg-14-3633-2017</pub-id></element-citation></ref><ref id=\"CR131\"><label>131.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Six</surname><given-names>KD</given-names></name><name><surname>Kloster</surname><given-names>S</given-names></name><name><surname>Ilyina</surname><given-names>T</given-names></name><name><surname>Archer</surname><given-names>SD</given-names></name><name><surname>Zhang</surname><given-names>K</given-names></name><name><surname>Maier-Reimer</surname><given-names>E</given-names></name></person-group><article-title>Global warming amplified by reduced sulphur fluxes as a result of ocean acidification</article-title><source>Nat Clim Chang.</source><year>2013</year><volume>3</volume><fpage>975</fpage><lpage>978</lpage><pub-id pub-id-type=\"doi\">10.1038/nclimate1981</pub-id></element-citation></ref><ref id=\"CR132\"><label>132.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kirkby</surname><given-names>J</given-names></name><etal/></person-group><article-title>Role of sulphuric acid, ammonia and galactic cosmic rays in atmospheric aerosol nucleation</article-title><source>Nature.</source><year>2011</year><volume>476</volume><fpage>429</fpage><lpage>433</lpage><pub-id pub-id-type=\"doi\">10.1038/nature10343</pub-id><pub-id pub-id-type=\"pmid\">21866156</pub-id></element-citation></ref><ref id=\"CR133\"><label>133.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bianchi</surname><given-names>D</given-names></name><name><surname>Dunne</surname><given-names>JP</given-names></name><name><surname>Sarmiento</surname><given-names>JL</given-names></name><name><surname>Galbraith</surname><given-names>ED</given-names></name></person-group><article-title>Data-based estimates of suboxia, denitrification, and N2O production in the ocean and their sensitivities to dissolved O2</article-title><source>Glob Biogeochem Cycles.</source><year>2012</year><volume>26</volume><fpage>1</fpage><lpage>47</lpage><pub-id pub-id-type=\"doi\">10.1029/2011GB004209</pub-id></element-citation></ref><ref id=\"CR134\"><label>134.</label><mixed-citation publication-type=\"other\">Buitenhuis ET, Suntharalingam P, Le Quéré C. Constraints on global oceanic emissions of N2O from observations and models. Biogeosci Discuss. 2017:1–23. 10.5194/bg-2017-193.</mixed-citation></ref><ref id=\"CR135\"><label>135.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jin</surname><given-names>X</given-names></name><name><surname>Gruber</surname><given-names>N</given-names></name></person-group><article-title>Offsetting the radiative benefit of ocean iron fertilization by enhancing N <sub>2</sub> O emissions</article-title><source>Geophys Res Lett.</source><year>2003</year><volume>30</volume><fpage>1</fpage><lpage>4</lpage><pub-id pub-id-type=\"doi\">10.1029/2003GL018458</pub-id></element-citation></ref><ref id=\"CR136\"><label>136.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Landolfi</surname><given-names>A</given-names></name><name><surname>Somes</surname><given-names>CJ</given-names></name><name><surname>Koeve</surname><given-names>W</given-names></name><name><surname>Zamora</surname><given-names>LM</given-names></name><name><surname>Oschlies</surname><given-names>A</given-names></name></person-group><article-title>Oceanic nitrogen cycling andN2O flux perturbations in the Anthropocene</article-title><source>Glob Biogeochem Cycles.</source><year>2017</year><volume>31</volume><fpage>1236</fpage><lpage>1255</lpage><pub-id pub-id-type=\"doi\">10.1002/2017GB005633</pub-id></element-citation></ref><ref id=\"CR137\"><label>137.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martinez-Rey</surname><given-names>J</given-names></name><name><surname>Bopp</surname><given-names>L</given-names></name><name><surname>Gehlen</surname><given-names>M</given-names></name><name><surname>Tagliabue</surname><given-names>A</given-names></name><name><surname>Gruber</surname><given-names>N</given-names></name></person-group><article-title>Projections of oceanic N2O emissions in the 21st century using the IPSL Earth system model</article-title><source>Biogeosciences.</source><year>2015</year><volume>12</volume><fpage>4133</fpage><lpage>4148</lpage><pub-id pub-id-type=\"doi\">10.5194/bg-12-4133-2015</pub-id></element-citation></ref><ref id=\"CR138\"><label>138.</label><mixed-citation publication-type=\"other\">Nevison C, Butler JH, Elkins JW. Global distribution of N <sub>2</sub> O and the ΔN <sub>2</sub> O-AOU yield in the subsurface ocean. Glob Biogeochem Cycles. 2003. 10.1029/2003GB002068.</mixed-citation></ref><ref id=\"CR139\"><label>139.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hense</surname><given-names>I</given-names></name><name><surname>Stemmler</surname><given-names>I</given-names></name><name><surname>Sonntag</surname><given-names>S</given-names></name></person-group><article-title>Ideas and perspectives: climate-relevant marine biologically driven mechanisms in Earth system models</article-title><source>Biogeosciences.</source><year>2017</year><volume>14</volume><fpage>403</fpage><lpage>413</lpage><pub-id pub-id-type=\"doi\">10.5194/bg-14-403-2017</pub-id></element-citation></ref><ref id=\"CR140\"><label>140.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kahru</surname><given-names>M</given-names></name><name><surname>Leppanen</surname><given-names>J-M</given-names></name><name><surname>Rud</surname><given-names>O</given-names></name></person-group><article-title>Cyanobacterial blooms cause heating of the sea surface</article-title><source>Mar Ecol Prog Ser.</source><year>1993</year><volume>101</volume><fpage>1</fpage><lpage>7</lpage><pub-id pub-id-type=\"doi\">10.3354/meps101001</pub-id></element-citation></ref><ref id=\"CR141\"><label>141.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hourdin</surname><given-names>F</given-names></name><etal/></person-group><article-title>The art and science of climate model tuning</article-title><source>Bull Am Meteorol Soc.</source><year>2017</year><volume>98</volume><fpage>589</fpage><lpage>602</lpage><pub-id pub-id-type=\"doi\">10.1175/BAMS-D-15-00135.1</pub-id></element-citation></ref><ref id=\"CR142\"><label>142.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jones</surname><given-names>CD</given-names></name><name><surname>Arora</surname><given-names>V</given-names></name><name><surname>Friedlingstein</surname><given-names>P</given-names></name><name><surname>Bopp</surname><given-names>L</given-names></name><name><surname>Brovkin</surname><given-names>V</given-names></name><name><surname>Dunne</surname><given-names>J</given-names></name><etal/></person-group><article-title>C4MIP – the Coupled Climate–Carbon Cycle Model Intercomparison Project: experimental protocol for CMIP6</article-title><source>Geosci Model Dev.</source><year>2016</year><volume>9</volume><fpage>2853</fpage><lpage>2880</lpage><pub-id pub-id-type=\"doi\">10.5194/gmd-9-2853-2016</pub-id></element-citation></ref><ref id=\"CR143\"><label>143.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Landschützer</surname><given-names>P</given-names></name><name><surname>Gruber</surname><given-names>N</given-names></name><name><surname>Bakker</surname><given-names>DCE</given-names></name></person-group><article-title>Decadal variations and trends of the global ocean carbon sink</article-title><source>Glob Biogeochem Cycles.</source><year>2016</year><volume>30</volume><fpage>1396</fpage><lpage>1417</lpage><pub-id pub-id-type=\"doi\">10.1002/2015GB005359</pub-id></element-citation></ref><ref id=\"CR144\"><label>144.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Valente</surname><given-names>A</given-names></name><etal/></person-group><article-title>A compilation of global bio-optical in situ data for ocean-colour satellite applications</article-title><source>Earth Syst Sci Data.</source><year>2016</year><volume>8</volume><fpage>235</fpage><lpage>252</lpage><pub-id pub-id-type=\"doi\">10.5194/essd-8-235-2016</pub-id></element-citation></ref><ref id=\"CR145\"><label>145.</label><mixed-citation publication-type=\"other\">Garcia, H.E.R.A., Locarnini, T. P, Boyer, J. I, Antonov, O.K. Baranova, M.M. Zweng, J.R. Reagan, D.R. Johnson, 2014 World Ocean Atlas 2013, Volume 3: Dissolved oxygen, apparent oxygen utilization, and oxygen saturation. S. Levitus, Ed., A. Mishonov Technical Ed.; NOAA Atlas NESDIS 75, 27 pp.</mixed-citation></ref><ref id=\"CR146\"><label>146.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gregg</surname><given-names>WW</given-names></name><name><surname>Casey</surname><given-names>NW</given-names></name></person-group><article-title>Global and regional evaluation of the SeaWiFS chlorophyll data set</article-title><source>Remote Sens Environ.</source><year>2004</year><volume>93</volume><fpage>463</fpage><lpage>479</lpage><pub-id pub-id-type=\"doi\">10.1016/j.rse.2003.12.012</pub-id></element-citation></ref><ref id=\"CR147\"><label>147.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Johnson</surname><given-names>R</given-names></name><name><surname>Strutton</surname><given-names>PG</given-names></name><name><surname>Wright</surname><given-names>SW</given-names></name><name><surname>McMinn</surname><given-names>A</given-names></name><name><surname>Meiners</surname><given-names>KM</given-names></name></person-group><article-title>Three improved satellite chlorophyll algorithms for the Southern Ocean</article-title><source>J Geophys Res Oceans.</source><year>2013</year><volume>118</volume><issue>7</issue><fpage>3694</fpage><lpage>3703</lpage><pub-id pub-id-type=\"doi\">10.1002/jgrc.20270</pub-id></element-citation></ref><ref id=\"CR148\"><label>148.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Laufkötter</surname><given-names>C</given-names></name><name><surname>John</surname><given-names>JG</given-names></name><name><surname>Stock</surname><given-names>CA</given-names></name><name><surname>Dunne</surname><given-names>JP</given-names></name></person-group><article-title>Temperature and oxygen dependence of the remineralization of organic matter</article-title><source>Glob Biogeochem Cycles.</source><year>2017</year><volume>31</volume><issue>7</issue><fpage>1038</fpage><lpage>1050</lpage><pub-id pub-id-type=\"doi\">10.1002/2017GB005643</pub-id></element-citation></ref><ref id=\"CR149\"><label>149.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tagliabue</surname><given-names>A</given-names></name><name><surname>Bowie</surname><given-names>A</given-names></name><name><surname>Boyd</surname><given-names>P</given-names></name><etal/></person-group><article-title>The integral role of iron in ocean biogeochemistry</article-title><source>Nature.</source><year>2017</year><volume>543</volume><fpage>51</fpage><lpage>59</lpage><pub-id pub-id-type=\"doi\">10.1038/nature21058</pub-id><pub-id pub-id-type=\"pmid\">28252066</pub-id></element-citation></ref><ref id=\"CR150\"><label>150.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martin</surname><given-names>JH</given-names></name></person-group><article-title>Glacial-interglacial CO2 change: the iron hypothesis</article-title><source>Paleoceanography.</source><year>1990</year><volume>5</volume><issue>1</issue><fpage>1</fpage><lpage>13</lpage><pub-id pub-id-type=\"doi\">10.1029/PA005i001p00001</pub-id></element-citation></ref><ref id=\"CR151\"><label>151.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schneider</surname><given-names>B</given-names></name><name><surname>Bopp</surname><given-names>L</given-names></name><name><surname>Gehlen</surname><given-names>M</given-names></name><name><surname>Segschneider</surname><given-names>J</given-names></name><name><surname>Frölicher</surname><given-names>TL</given-names></name><name><surname>Cadule</surname><given-names>P</given-names></name><name><surname>Friedlingstein</surname><given-names>P</given-names></name><name><surname>Doney</surname><given-names>SC</given-names></name><name><surname>Behrenfeld</surname><given-names>MJ</given-names></name><name><surname>Joos</surname><given-names>F</given-names></name></person-group><article-title>Climate-induced interannual variability of marine primary and export production in three global coupled climate carbon cycle models</article-title><source>Biogeosciences</source><year>2008</year><volume>5</volume><fpage>597</fpage><lpage>614</lpage><pub-id pub-id-type=\"doi\">10.5194/bg-5-597-2008</pub-id></element-citation></ref><ref id=\"CR152\"><label>152.</label><mixed-citation publication-type=\"other\">Dutkiewicz S, Follows MJ, Parekh P. Interactions of the iron and phosphorus cycles: a three-dimensional model study. Glob Biogeochem Cycles. 2005;19, GB1021. 10.1029/2004GB002342.</mixed-citation></ref><ref id=\"CR153\"><label>153.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moore</surname><given-names>JK</given-names></name><name><surname>Braucher</surname><given-names>O</given-names></name></person-group><article-title>Sedimentary and mineral dust sources of dissolved iron to the world ocean</article-title><source>Biogeosciences.</source><year>2008</year><volume>5</volume><fpage>631</fpage><lpage>656</lpage><pub-id pub-id-type=\"doi\">10.5194/bg-5-631-2008</pub-id></element-citation></ref><ref id=\"CR154\"><label>154.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Völker</surname><given-names>C</given-names></name><name><surname>Tagliabue</surname><given-names>A</given-names></name></person-group><article-title>Modeling organic iron-binding ligands in a three-dimensional biogeochemical ocean model</article-title><source>Mar Chem.</source><year>2015</year><volume>173</volume><fpage>67</fpage><lpage>77</lpage><pub-id pub-id-type=\"doi\">10.1016/J.MARCHEM.2014.11.008</pub-id></element-citation></ref><ref id=\"CR155\"><label>155.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Le Quéré</surname><given-names>C</given-names></name><name><surname>Andrew</surname><given-names>RM</given-names></name><name><surname>Friedlingstein</surname><given-names>P</given-names></name><name><surname>Sitch</surname><given-names>S</given-names></name><name><surname>Hauck</surname><given-names>J</given-names></name><name><surname>Pongratz</surname><given-names>J</given-names></name><etal/></person-group><article-title>Global Carbon Budget 2018</article-title><source>Earth Syst. Sci. Data</source><year>2018</year><volume>10</volume><fpage>2141</fpage><lpage>2194</lpage><pub-id pub-id-type=\"doi\">10.5194/essd-10-2141-2018</pub-id></element-citation></ref><ref id=\"CR156\"><label>156.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Resplandy</surname><given-names>L</given-names></name><name><surname>Keeling</surname><given-names>RF</given-names></name><name><surname>Rödenbeck</surname><given-names>C</given-names></name><name><surname>Stephens</surname><given-names>BB</given-names></name><name><surname>Khatiwala</surname><given-names>S</given-names></name><name><surname>Rodgers</surname><given-names>KB</given-names></name><etal/></person-group><article-title>Revision of global carbon fluxes based on a reassessment of oceanic and riverine carbon transport</article-title><source>Nat Geosci.</source><year>2018</year><volume>11</volume><fpage>504</fpage><lpage>509</lpage><pub-id pub-id-type=\"doi\">10.1038/s41561-018-0151-3</pub-id></element-citation></ref><ref id=\"CR157\"><label>157.</label><mixed-citation publication-type=\"other\">Behrenfeld MJ, Boss E, Siegel DA, Shea DM. Carbon-based ocean productivity and phytoplankton physiology from space. Glob Biogeochem Cycles. 2005;19:GB1006. 10.1029/2004GB002299.</mixed-citation></ref><ref id=\"CR158\"><label>158.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kulk</surname><given-names>G</given-names></name><name><surname>Platt</surname><given-names>T</given-names></name><name><surname>Dingle</surname><given-names>J</given-names></name><name><surname>Jackson</surname><given-names>T</given-names></name><name><surname>Jönsson</surname><given-names>BF</given-names></name><name><surname>Bouman</surname><given-names>HA</given-names></name><etal/></person-group><article-title>Primary production, an index of climate change in the ocean: satellite-based estimates over two decades</article-title><source>Remote Sens.</source><year>2020</year><volume>12</volume><fpage>826</fpage><pub-id pub-id-type=\"doi\">10.3390/rs12050826</pub-id></element-citation></ref><ref id=\"CR159\"><label>159.</label><mixed-citation publication-type=\"other\">Dunne JP, Sarmiento JL, Gnanadesikan A. A synthesis of global particle export from the surface ocean and cycling through the ocean interior and on the seafloor. Glob Biogeochem Cycles. 2007;21(4). 10.1029/2006GB002907.</mixed-citation></ref><ref id=\"CR160\"><label>160.</label><mixed-citation publication-type=\"other\">Tréguer PJ, De La Rocha CL. The World Ocean silica cycle. Annu Rev Mar Sci. 2013;(1):477–501.</mixed-citation></ref><ref id=\"CR161\"><label>161.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carr</surname><given-names>ME</given-names></name><name><surname>Friedrichs</surname><given-names>MA</given-names></name><name><surname>Schmeltz</surname><given-names>M</given-names></name><name><surname>Noguchi Aita</surname><given-names>M</given-names></name><name><surname>Antoine</surname><given-names>D</given-names></name><name><surname>Arrigo</surname><given-names>KR</given-names></name><etal/></person-group><article-title>A comparison of global estimates of marine primary production from ocean color</article-title><source>Deep-Sea Res Part II Top Stud Oceanogr.</source><year>2006</year><volume>53</volume><fpage>741</fpage><lpage>770</lpage><pub-id pub-id-type=\"doi\">10.1016/j.dsr2.2006.01.028</pub-id></element-citation></ref><ref id=\"CR162\"><label>162.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>de Boyer Montégut</surname><given-names>C</given-names></name><name><surname>Madec</surname><given-names>G</given-names></name><name><surname>Fischer</surname><given-names>AS</given-names></name><name><surname>Lazar</surname><given-names>A</given-names></name><name><surname>Iudicone</surname><given-names>D</given-names></name></person-group><article-title>Mixed layer depth over the global ocean: an examination of profile data and a profile-based climatology</article-title><source>J Geophys Res C Ocean.</source><year>2004</year><volume>109</volume><fpage>1</fpage><lpage>20</lpage><pub-id pub-id-type=\"doi\">10.1029/2004JC002378</pub-id></element-citation></ref><ref id=\"CR163\"><label>163.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kessler</surname><given-names>A</given-names></name><name><surname>Tjiputra</surname><given-names>J</given-names></name></person-group><article-title>The Southern Ocean as a constraint to reduce uncertainty in future ocean carbon sinks</article-title><source>Earth Syst Dynam.</source><year>2016</year><volume>7</volume><fpage>295</fpage><lpage>312</lpage><pub-id pub-id-type=\"doi\">10.5194/esd-7-295-2016</pub-id></element-citation></ref><ref id=\"CR164\"><label>164.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Berthet</surname><given-names>S</given-names></name><name><surname>Séférian</surname><given-names>R</given-names></name><name><surname>Bricaud</surname><given-names>C</given-names></name><name><surname>Chevallier</surname><given-names>M</given-names></name><name><surname>Voldoire</surname><given-names>A</given-names></name><name><surname>Ethé</surname><given-names>C</given-names></name></person-group><article-title>Evaluation of an online grid-coarsening algorithm in a global eddy-admitting ocean biogeochemical model</article-title><source>J Adv Model Earth Syst.</source><year>2019</year><volume>11</volume><fpage>1759</fpage><lpage>1783</lpage><pub-id pub-id-type=\"doi\">10.1029/2019MS001644</pub-id></element-citation></ref><ref id=\"CR165\"><label>165.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Person</surname><given-names>R</given-names></name><name><surname>Aumont</surname><given-names>O</given-names></name><name><surname>Lévy</surname><given-names>M</given-names></name></person-group><article-title>The biological pump and seasonal variability of pCO 2 in the southern ocean: exploring the role of diatom adaptation to low Iron</article-title><source>J Geophys Res Ocean.</source><year>2018</year><volume>123</volume><fpage>3204</fpage><lpage>3226</lpage><pub-id pub-id-type=\"doi\">10.1029/2018JC013775</pub-id></element-citation></ref><ref id=\"CR166\"><label>166.</label><mixed-citation publication-type=\"other\">Tittensor DP, et al. Integrating climate adaptation and biodiversity conservation in the global protected ocean. Sci Adv. 2019;5:In revision:eaay9969. 10.1126/sciadv.aay9969.</mixed-citation></ref><ref id=\"CR167\"><label>167.</label><mixed-citation publication-type=\"other\">Ward BA, et al. Considering the role of adaptive evolution in models of the ocean and climate system. J Adv Model Earth Syst. 2019. 10.1029/2018MS001452.</mixed-citation></ref><ref id=\"CR168\"><label>168.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Henson</surname><given-names>S</given-names></name><name><surname>Beaulieu</surname><given-names>C</given-names></name><name><surname>Ilyina</surname><given-names>T</given-names></name><etal/></person-group><article-title>Rapid emergence of climate change in environmental drivers of marine ecosystems</article-title><source>Nat Commun.</source><year>2017</year><volume>8</volume><fpage>14682</fpage><pub-id pub-id-type=\"doi\">10.1038/ncomms14682</pub-id><pub-id pub-id-type=\"pmid\">28267144</pub-id></element-citation></ref><ref id=\"CR169\"><label>169.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schlitzer</surname><given-names>R</given-names></name><etal/></person-group><article-title>The GEOTRACES Intermediate Data Product 2017</article-title><source>Chem Geol.</source><year>2018</year><volume>493</volume><fpage>210</fpage><lpage>223</lpage><pub-id pub-id-type=\"doi\">10.1016/j.chemgeo.2018.05.040</pub-id></element-citation></ref><ref id=\"CR170\"><label>170.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tagliabue</surname><given-names>A</given-names></name><name><surname>Bowie</surname><given-names>AR</given-names></name><name><surname>DeVries</surname><given-names>T</given-names></name><name><surname>Ellwood</surname><given-names>MJ</given-names></name><name><surname>Landing</surname><given-names>WM</given-names></name><name><surname>Milne</surname><given-names>A</given-names></name><etal/></person-group><article-title>The interplay between regeneration and scavenging fluxes drives ocean iron cycling</article-title><source>Nat Commun.</source><year>2019</year><volume>10</volume><fpage>4960</fpage><pub-id pub-id-type=\"doi\">10.1038/s41467-019-12775-5</pub-id><pub-id pub-id-type=\"pmid\">31673108</pub-id></element-citation></ref><ref id=\"CR171\"><label>171.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tagliabue</surname><given-names>A</given-names></name><name><surname>Hawco</surname><given-names>NJ</given-names></name><name><surname>Bundy</surname><given-names>RM</given-names></name><name><surname>Landing</surname><given-names>WM</given-names></name><name><surname>Milne</surname><given-names>A</given-names></name><name><surname>Morton</surname><given-names>PL</given-names></name><etal/></person-group><article-title>The role of external inputs and internal cycling in shaping the global ocean cobalt distribution: insights from the first cobalt biogeochemical model</article-title><source>Glob Biogeochem Cycles.</source><year>2018</year><volume>32</volume><fpage>594</fpage><lpage>616</lpage><pub-id pub-id-type=\"doi\">10.1002/2017GB005830</pub-id></element-citation></ref><ref id=\"CR172\"><label>172.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weber</surname><given-names>T</given-names></name><name><surname>John</surname><given-names>S</given-names></name><name><surname>Tagliabue</surname><given-names>A</given-names></name><name><surname>DeVries</surname><given-names>T</given-names></name></person-group><article-title>Biological uptake and reversible scavenging of zinc in the global ocean</article-title><source>Science (80-)</source><year>2018</year><volume>361</volume><fpage>72 LP</fpage><lpage>72 76</lpage><pub-id pub-id-type=\"doi\">10.1126/science.aap8532</pub-id></element-citation></ref><ref id=\"CR173\"><label>173.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van Hulten</surname><given-names>M</given-names></name><name><surname>Middag</surname><given-names>R</given-names></name><name><surname>Dutay</surname><given-names>J-C</given-names></name><name><surname>de Baar</surname><given-names>H</given-names></name><name><surname>Roy-Barman</surname><given-names>M</given-names></name><name><surname>Gehlen</surname><given-names>M</given-names></name><etal/></person-group><article-title>Manganese in the west Atlantic Ocean in the context of the first global ocean circulation model of manganese</article-title><source>Biogeosciences.</source><year>2017</year><volume>14</volume><fpage>1123</fpage><lpage>1152</lpage><pub-id pub-id-type=\"doi\">10.5194/bg-14-1123-2017</pub-id></element-citation></ref><ref id=\"CR174\"><label>174.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Richon</surname><given-names>C</given-names></name><name><surname>Tagliabue</surname><given-names>A</given-names></name></person-group><article-title>Insights into the major processes driving the global distribution of copper in the ocean from a global model</article-title><source>Glob Biogeochem Cycles.</source><year>2019</year><volume>33</volume><fpage>1594</fpage><lpage>1610</lpage><pub-id pub-id-type=\"doi\">10.1029/2019GB006280</pub-id></element-citation></ref><ref id=\"CR175\"><label>175.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ward</surname><given-names>BA</given-names></name><name><surname>Follows</surname><given-names>MJ</given-names></name></person-group><article-title>Marine mixotrophy increases trophic transfer efficiency, mean organism size, and vertical carbon flux</article-title><source>PNAS.</source><year>2016</year><volume>113</volume><fpage>2958</fpage><lpage>2963</lpage><pub-id pub-id-type=\"doi\">10.1073/pnas.1517118113</pub-id><pub-id pub-id-type=\"pmid\">26831076</pub-id></element-citation></ref><ref id=\"CR176\"><label>176.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lana</surname><given-names>A</given-names></name><name><surname>Bell</surname><given-names>TG</given-names></name><name><surname>Simó</surname><given-names>R</given-names></name><name><surname>Vallina</surname><given-names>SM</given-names></name><name><surname>Ballabrera-Poy</surname><given-names>J</given-names></name><name><surname>Kettle</surname><given-names>AJ</given-names></name><etal/></person-group><article-title>An updated climatology of surface dimethlysulfide concentrations and emission fluxes in the global ocean</article-title><source>Glob Biogeochem Cycles.</source><year>2011</year><volume>25</volume><fpage>GB1004</fpage><pub-id pub-id-type=\"doi\">10.1029/2010GB003850</pub-id></element-citation></ref><ref id=\"CR177\"><label>177.</label><mixed-citation publication-type=\"other\">Buitenhuis ET, Suntharalingam P, Le Quéré C. Constraints on global oceanic emissions of N2O from observations and models. Biogeosciences. 2018;15:2161–2175. 10.5194/bg-15-2161-2018.</mixed-citation></ref><ref id=\"CR178\"><label>178.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Paulot</surname><given-names>F</given-names></name><name><surname>Jacob</surname><given-names>DJ</given-names></name><name><surname>Johnson</surname><given-names>MT</given-names></name><name><surname>Bell</surname><given-names>TG</given-names></name><name><surname>Baker</surname><given-names>AR</given-names></name><name><surname>Keene</surname><given-names>WC</given-names></name><etal/></person-group><article-title>Global oceanic emission of ammonia: constraints from seawater and atmospheric observations</article-title><source>Glob Biogeochem Cycles.</source><year>2015</year><volume>29</volume><fpage>1165</fpage><lpage>1178</lpage><pub-id pub-id-type=\"doi\">10.1002/2015GB005106</pub-id></element-citation></ref><ref id=\"CR179\"><label>179.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kwiatkowski</surname><given-names>L</given-names></name><etal/></person-group><article-title>iMarNet: an ocean biogeochemistry model intercomparison project within a common physical ocean modelling framework</article-title><source>Biogeosciences.</source><year>2014</year><volume>11</volume><fpage>7291</fpage><lpage>7304</lpage><pub-id pub-id-type=\"doi\">10.5194/bg-11-7291-2014</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807859</article-id><article-id pub-id-type=\"pmc\">PMC7431555</article-id><article-id pub-id-type=\"publisher-id\">70467</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70467-3</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Adoptive transfer of bone marrow-derived dendritic cells (BMDCs) alleviates OVA-induced allergic airway inflammation in asthmatic mice</article-title></title-group><contrib-group><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Xu</surname><given-names>Kan</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Wu</surname><given-names>Nan</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Min</surname><given-names>Zhihui</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Zheng</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhu</surname><given-names>Tao</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Liu</surname><given-names>Chunfang</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zeng</surname><given-names>Yuzhen</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Song</surname><given-names>Juan</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mao</surname><given-names>Ruolin</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-9558-0620</contrib-id><name><surname>Ji</surname><given-names>Hong</given-names></name><address><email>hgji@ucdavis.edu</email></address><xref ref-type=\"aff\" rid=\"Aff5\">5</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Jiang</surname><given-names>Zhilong</given-names></name><address><email>Jiang.zhilong@zs-hospital.sh.cn</email></address><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Chen</surname><given-names>Zhihong</given-names></name><address><email>czh60@hotmail.com</email></address><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.8547.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0125 2443</institution-id><institution>Geriatric Department of Zhongshan Hospital, </institution><institution>Shanghai Institute of Respiratory Disease, Fudan University, </institution></institution-wrap>Shanghai, China </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.8547.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0125 2443</institution-id><institution>Respiratory Division of Zhongshan Hospital, Shanghai Institute of Respiratory Disease, Fudan University, </institution></institution-wrap>No. 180 Fenglin Road, Shanghai, China </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.8547.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0125 2443</institution-id><institution>Research Center of Zhongshan Hospital, Fudan University, </institution></institution-wrap>Shanghai, China </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.412461.4</institution-id><institution>Department of Respiratory Medicine, </institution><institution>Second Affiliated Hospital of Chongqing Medical University, </institution></institution-wrap>Chongqing, China </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.27860.3b</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9684</institution-id><institution>Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, </institution><institution>University of California, </institution></institution-wrap>Davis, CA USA </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.27860.3b</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9684</institution-id><institution>California National Primate Research Center, </institution></institution-wrap>Davis, CA USA </aff><aff id=\"Aff7\"><label>7</label>Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13915</elocation-id><history><date date-type=\"received\"><day>12</day><month>9</month><year>2019</year></date><date date-type=\"accepted\"><day>30</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Airway dendritic cells (DCs) are recognized as important factors in the mechanisms of allergic inflammatory diseases. Suppressor of cytokine signaling 3 (SOCS3) is involved in regulating the functions of T cells and macrophages, but the roles of SOCS3-expressing DCs in the pathogeneses of allergic inflammatory diseases are still controversial. We compared the effects of adoptively transferred SOCS3<sup>−/−</sup> and SOCS3<sup>+/+</sup> bone marrow-derived DCs (BMDCs) on airway inflammation in ovalbumin (OVA)-sensitized asthmatic mice. Adoptive transfer of mature DCs (lipopolysaccharide [LPS]-induced DCs, DClps) with or without SOCS3 gene expression significantly ameliorated allergic airway inflammation. SOCS3<sup>−/−</sup> DCs slightly attenuated BMDC-induced immunogenic tolerance. DClps migrated to OVA-sensitized lungs with higher efficiency than immature DCs (DCim). DClps with or without SOCS3 greatly improved lung pathology scores and alleviated airway inflammatory cell infiltration after adoptive transfer into mice; they also increased interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) production and inhibited signal transducer and activator of transcription (STAT) 4 and STAT6 signaling in the lungs after OVA sensitization. In conclusion, the BMDC adoptive transfer-induced immunogenic tolerance in OVA-sensitized mice might not be due to SOCS3 gene depletion. BMDC adoptive transfer may be developed into a new approach that alleviates asthma by modulating the balance between immune tolerance and inflammation.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Asthma</kwd><kwd>Asthma</kwd><kwd>Therapeutics</kwd><kwd>Therapeutics</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">501100001809</institution-id><institution>National Natural Science Foundation of China (National Science Foundation of China)</institution></institution-wrap></funding-source><award-id>81470211</award-id><award-id>81970023</award-id></award-group></funding-group><funding-group><award-group><funding-source><institution>Shanghai Health committee(201840288)</institution></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Airway dendritic cells (DCs) play crucial roles in initiating effective adaptive immune responses against invading pathogens and inducing immune tolerance toward innocuous inhaled antigens. Exploiting the tolerogenic function of DCs might be a novel way to treat allergic airway diseases. However, deletion of DCs in the lungs is infeasible, as indicated by studies in which DC−/− mice have been found to exhibit severe viral respiratory infections and systematic illness<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. Fine-tuning the balance between tolerogenic and immunogenic lung DCs is a major goal in anti-inflammation research. Emerging literature has demonstrated that different DC subsets and discrete functional states of DCs might be responsible for promoting tolerance to inhaled antigenic substances. For example, Nakagome et al. reported that interleukin (IL)-10-treated DCs decrease airway allergic inflammation in mice<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. In addition, it has been shown that plasmacytoid DCs (pDCs) play an important role in inhalation tolerance. Mice in which pDCs are specifically depleted develop the features of severe asthma after exposure to nebulized harmless antigens<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. Steroids can modulate the functions of DCs in the lungs of patients with allergic asthma by activating indoleamine 2,3-dioxygenase (IDO) enzymes in DCs<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. Furthermore, vitamin D3-incubated bone marrow-derived DCs (BMDCs) express relatively low levels of major histocompatibility complex class II (MHCII) and costimulatory molecules, which ultimately attenuates DC-T cell interactions and T cell activation<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>.</p><p id=\"Par3\">Suppressor of cytokine signaling 3 (SOCS3) is central in negatively regulating signal transducer and activator of transcription (STAT) 3, STAT4, STAT1 and STAT5 signaling after stimulation with IL-6, IL-11, IL-27, etc. Kubo et al. found that SOCS3 mRNA expression is increased in eosinophils and CD4+ T cells in asthma and nonasthmatic eosinophilic bronchitis. T cell-specific deletion of SOCS3 impairs the T helper (Th) 2 response and increases Th1 responses<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. However, deletion of SOCS3 in hematopoietic cells results in severe inflammatory disease during adult life that is not rescued by IL-6 deletion<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. In addition, SOCS3 gene knockdown in macrophages results in activation of STAT1 and induction of type I interferon (IFN) responses upon IL-6 stimulation<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. Thus, the roles of the SOCS3 gene in DC functional states and the cognate interaction of SOCS3 with T cells have been controversial.</p><p id=\"Par4\">Herein, we critically assessed the effects of the SOCS3 gene in BMDCs on cell proliferation and activation by coculturing SOCS3<sup>−/−</sup> BMDCs with CD4 T cells. Then, DCs with SOCS3 gene deletion in different functional states were adoptively transferred into ovalbumin (OVA)-sensitized mice, and lung pathological injury and airway inflammatory cell infiltration were evaluated. The underlying cellular and molecular mechanisms were also studied.</p></sec><sec id=\"Sec2\"><title>Results</title><sec id=\"Sec3\"><title>SOCS3 deficiency increased the DC-induced proliferation and cytokine production of T lymphocytes</title><p id=\"Par5\">To investigate the role of SOCS3 in airway inflammation, we created conditional SOCS3-knockout (KO) mice according to the protocol in a previous study<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Briefly, SOCS3fl/fl mice were bred with mice transgenically expressing Cre under the control of the lysozyme 2 (Lyz2) promoter. The offspring SOCS3(Lyz2cre) mice lacked exon 2 of the SOCS3 locus in myeloid cells; this exon was deleted under the control of the Lyz2 promoter (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>A). To identify BMDCs with SOCS3 deficiency, we screened bone marrow cells expressing CD11c, CD80, and MHCII from each group and differentiated them into BMDCs in culture. Fluorescence-activated cell sorting (FACS) analysis showed that SOCS3 protein expression was significantly lower (62% lower) in SOCS3(Lyz2cre) mouse-derived BMDCs than in wild-type (WT) mouse-derived BMDCs (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>C). Western blot analysis confirmed that the expression of SOCS3 was decreased by 56% in SOCS3<sup>−/−</sup> BMDCs (Supplementary Data <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Generation of SOCS3(Lyz2cre) mice and identification of SOCS3<sup>−/−</sup> BMDCs. (<bold>A</bold>) Schematic diagram of the generation of SOCS3(Lyz2cre) mice. Floxp-flanked SOCS3 mice were back-crossed with Lyz2-Cre transgenic mice to create SOCS3 knockout mice with SOCS3 conditional knockout in myeloid cells, such as DCs or macrophages. (<bold>B</bold>) The genotypes of SOCS3(Lyz2cre) mice identified by analyzing mouse tails by PCR. The Cre + loci were identified as 700 bp. The FloxP-flanked exon 2 null SOCS3 loci were identified as 250 bp. (<bold>C</bold>) Expression of SOCS3 in BMDCs evaluated by flow cytometry. BMDCs were gated on CD11c + CD80 + MHCII + cells. A PE-conjugated anti-SOCS3 antibody was used to detect SOCS3 protein expression. In SOCS3<sup>−/−</sup> mice, SOCS3 protein expression was reduced by approximately 62.4%. One representative dot plot is shown (SOCS3 and MHCII expression was assessed by comparing with the corresponding fluorescence-minus-one (FMO) control).</p></caption><graphic xlink:href=\"41598_2020_70467_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par6\">We performed an allogeneic mixed lymphocyte reaction (MLR) to evaluate whether a lack of SOCS3 in BMDCs affects interactions with T lymphocytes by assessing the function and proliferative ability of T lymphocytes. We distinguished T cells by staining cells in MLR culture with an anti-CD3 antibody. Carboxyfluorescein diacetate succinimidyl ester (CFSE) analysis and differential cell counting both demonstrated that SOCS3<sup>−/−</sup> DCs induced more robust T cell proliferation than SOCS3<sup>+/+</sup>DCs (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A,B). Surprisingly, SOCS3 deficiency increased the expression of IFN-γ by T cells (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>C1), but it did not influence the expression of IL-4 (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>C2).<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Mixed lymphocyte reaction (MLR) with SOCS3<sup>−/−</sup> BMDCs. (<bold>A</bold>) Mitomycin-activated BMDCs cultured with CD4 + T cells (cell ratio of 1:4). T cell proliferation was assessed with CFSE staining. One representative plot is shown. (<bold>B</bold>) Number of CD4 + T cells after a 5-days MLR. (<bold>C</bold>) Cytokine release from CD4 + T cells after a 5-day MLR. (<bold>C-1</bold>) IFN-γ production; (<bold>C-2</bold>) IL-4 production. Data are representative of 3 independent experiments with similar results. The columns and error bars represent the mean and SEM (*P < 0.05, **P < 0.01, ns: no significant difference).</p></caption><graphic xlink:href=\"41598_2020_70467_Fig2_HTML\" id=\"MO2\"/></fig></p></sec><sec id=\"Sec4\"><title>Lack of SOCS3 did not alter the therapeutic effect of DCs on allergic airway inflammation</title><p id=\"Par7\">To further evaluate the effects of SOCS3 deletion, we generated an OVA-induced asthma mouse model and adoptively transferred DCs into the asthmatic mice (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>A). Transfer of DCs ameliorated lung tissue damage and decreased allergic airway inflammation; however, SOCS3 deficiency did not alter the therapeutic effect of DCs. In other words, DC therapy attenuated the inflammatory response in the lungs regardless of whether the mature DCs (lipopolysaccharide [LPS]-induced DCs, DClps) used for treatment expressed the SOCS3 gene (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>B,C). Notably, SOCS3<sup>−/−</sup> DC therapy seemed to have a smaller beneficial effect on lung tissue pathological scores than SOCS3<sup>+/+</sup>DC therapy. However, the difference was not statistically significant (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>C).<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Effect of adoptively transferred BMDCs with or without the SOCS3 gene on lung pathology in OVA-induced asthmatic mice. (<bold>A</bold>) Protocol for OVA-mediated induction of allergic asthma and SOCS3<sup>+/+</sup> DC or SOCS3<sup>−/−</sup> DC adoptive transfer into OVA-sensitized mice via intraperitoneal injection (DCs pulsed with LPS before transfer). (<bold>B</bold>) Representative photomicrographs of lung sections stained with H&E and examined at × 100 magnification. The same experiment was repeated 3 times with similar results (n = 6 in each group). C. The scores for lung tissue pathology. The columns and error bars represent the mean and SEM (**P < 0.01, ns: no significant difference).</p></caption><graphic xlink:href=\"41598_2020_70467_Fig3_HTML\" id=\"MO3\"/></fig></p><p id=\"Par8\">In addition, transfer of DCs reduced the total number of pulmonary inflammatory cells in the bronchoalveolar lavage (BAL) fluid (BALF) (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>A). An obvious reduction in neutrophil count and increase in lymphocyte count were observed. There was no change in eosinophil count after DC transfer (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>B). Correspondingly, the expression of IL-13 and immunoglobulin (Ig) E, which are related to the asthmatic Th2 response, was diminished after treatment with DCs (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>C). Unexpectedly, the inhibitory effect of BMDC adoptive transfer was not related to SOCS3 gene status (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>C).<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Adoptively transferred BMDCs with or without the SOCS3 gene alleviated allergic airway inflammation. (<bold>A</bold>) The total cell number in the bronchoalveolar lavage fluid (BALF). (<bold>B</bold>) The differential cell counts in the BALF. (<bold>C</bold>) The concentrations of Th2 cytokines in the BALF and serum IgE measured by ELISA. Data are representative of 3 independent experiments with similar results. The columns and error bars represent the mean and SEM (**P < 0.01, ns: no significant difference).</p></caption><graphic xlink:href=\"41598_2020_70467_Fig4_HTML\" id=\"MO4\"/></fig></p></sec><sec id=\"Sec5\"><title>Proinflammatory and chemotactic effects of DClps and immature DCs (DCim)</title><p id=\"Par9\">In the above experiment, we demonstrated that transfer of DCs greatly ameliorated lung tissue damage and decreased allergic airway inflammation. Next, we cultured DCs in vitro to further elucidate whether the therapeutic effect of DCs was dependent on DC maturation.</p><p id=\"Par10\">DCim were induced to differentiate into mature DCs in the presence of LPS (producing DClps) and were pulsed with OVA for 2 h. We then measured the levels of select related cytokines in DCim and DClps culture supernatants. ELISA revealed that the levels of IL-10 and transforming growth factor-β (TGF-β) were higher in DClps culture medium than in DCim culture medium and that the expression of MCP-3 and IFN-γ was unaffected by DC maturation (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>).<fig id=\"Fig5\"><label>Figure 5</label><caption><p>Activity of mature and immature DCs with or without the SOCS3 gene. BMDCs were generated from SOCS3<sup>+/+</sup> or SOCS3<sup>−/−</sup> mice and incubated with or without LPS (the cells incubated with LPS were considered mature DCs). The cells were then pulsed with OVA for 2 h, and the supernatant was collected for ELISA analysis. Data are representative of 3 independent experiments with similar results. The columns and error bars represent the mean and SEM (*P < 0.05, **P < 0.01, ns: no significant difference).</p></caption><graphic xlink:href=\"41598_2020_70467_Fig5_HTML\" id=\"MO5\"/></fig></p><p id=\"Par11\">Among the DClps groups, SOCS3 deficiency had no effect on the expression of MCP-3 or IFN-γ. Surprisingly, the expression of TGF-β was mildly elevated in SOCS3<sup>−/−</sup> DClps compared to SOCS3<sup>+/+</sup>DClps (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>).</p></sec><sec id=\"Sec6\"><title>The maturity of DCs affects the efficiency of DC pulmonary migration following intraperitoneal injection</title><p id=\"Par12\">We attempted to track DCs that had been labeled with the fluorescent dye PKH26 to test whether DCs could migrate to the lungs successfully and to assess whether the efficiency of pulmonary migration was related to the maturation state of DCs.</p><p id=\"Par13\">We injected mice with OVA-induced asthma intraperitoneally with PKH26-labeled DCim or DClps or with saline-diluted PKH26 as a control. We screened DCs by FACS with gating on CD11c + CD80 + MHCII + cells in the mononuclear cell populations of lung digests. PKH26-labeled BMDCs were also detected by FACS. On the first day after transplantation, 2.26% of DClps in the lungs were PKH26 positive. The proportion of PKH26-positive BMDCs in the DClps group peaked at 5.42% on day 3; this proportion was significantly higher than that in the DCim (1.52%) and control groups (0.86%). The number of PKH26-positive BMDCs in the DClps group was reduced on day 7 but was still higher than the numbers in the DCim and control groups (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>). These data showed that DCs delivered intraperitoneally accumulated in the lungs of OVA-sensitized asthmatic mice during the week after passive transfer. The efficiency of DC pulmonary migration was related to the maturation state of DCs, indicating that DClps could reach the lungs in more meaningful numbers than DCim.<fig id=\"Fig6\"><label>Figure 6</label><caption><p>Tracking adoptively transferred wild-type BMDCs in OVA-induced asthmatic mice. Wild-type BMDCs pulsed with or without LPS were labeled with PKH26 (WT-DClps and WT-DCim) or saline-diluted PKH26 and transperitoneally transferred into OVA-sensitized mice. On day 1, 3, or 7, mice were sacrificed. BMDCs were screened by gating on CD11c + CD80 + MHCII + cells in the mononuclear cell population of the lungs. WT-DClps labeled with PKH26 were tracked and reached 5.42% of the BMDC population in the lungs (the absolute number of WT-DClps migrating to the lungs was 1,220), and this proportion was higher than that of WT-DCim and saline control on day 3. The gating strategy and the fluorescence-minus-one (FMO) control (PKH26 negative) are presented. Histogram represents the percentage of PHK26 + BMDCs adoptively transferred into the lung within a week. Data are representative of 3 independent experiments, and three mice were used in each group. The columns and error bars represent the mean and SEM (**P < 0.01;*P < 0.05; ns: no significant difference).</p></caption><graphic xlink:href=\"41598_2020_70467_Fig6_HTML\" id=\"MO6\"/></fig></p></sec><sec id=\"Sec7\"><title>Administration of DClps in an OVA-induced mouse model significantly alleviated allergic airway inflammation</title><p id=\"Par14\">After we confirmed that the efficiency of pulmonary migration was determined by the maturation state of DCs, we attempted to further clarify whether the degree of DC maturation affected histopathological changes in the lungs.</p><p id=\"Par15\">In vivo experiments revealed that compared with DCim injection or no DC injection, DClps injection significantly reduced lung inflammatory cell infiltration and lung tissue pathological scores in mice with OVA-induced asthma (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>A). In addition, DClps injection significantly reduced the total number of pulmonary inflammatory cells in BALF, while there was no statistically significant difference between the DCim + OVA and OVA groups (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>B). Moreover, neutrophil infiltration was reduced and lymphocyte counts were increased in both the DClps + OVA and DCim + OVA groups (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>B).<fig id=\"Fig7\"><label>Figure 7</label><caption><p>Effect of adoptively transferring LPS-pulsed mature BMDCs on lung pathology in OVA-induced asthmatic mice. (<bold>A-1</bold>) Protocol for OVA-mediated induction of allergic asthma and transperitoneal adoptive transfer of LPS-pulsed matured BMDCs (wild type) into OVA-sensitized mice. (<bold>A-2</bold>) Representative photomicrographs of lung sections stained with H&E and examined at × 100 magnification. The same experiment was repeated 3 times with similar results (n = 6 in each group). (<bold>A-3</bold>) The scores for lung tissue pathology. (<bold>B</bold>) The total cell number in the bronchoalveolar lavage fluid (BALF) (top panel). The differential cell counts in the BALF (bottom panel). The columns and error bars represent the mean and SEM (**P < 0.01;*P < 0.05; ns: no significant difference).</p></caption><graphic xlink:href=\"41598_2020_70467_Fig7_HTML\" id=\"MO7\"/></fig></p></sec><sec id=\"Sec8\"><title>Administration of DClps reversed impairments in the STAT4 and STAT6 pathways</title><p id=\"Par16\">To define the roles of DCs in the Th1/Th2 immune signaling pathways, we evaluated the activation and expression of STAT-1, STAT-4, and STAT-6 in different groups of mice (the control [CON], OVA + WT DC [WT-DC], OVA + KO DC [KO-DC], and OVA groups). Western blot analysis revealed that administration of DCs significantly reduced the phosphorylation of STAT-4 and STAT-6 in the lungs of OVA-sensitized mice, and this effect was not related to the lack of SOCS3 (Fig. <xref rid=\"Fig8\" ref-type=\"fig\">8</xref>). There was also a trend toward reduced STAT1 phosphorylation after administration of DCs in mice with OVA-induced asthma. However, the reduction was not significant (Fig. <xref rid=\"Fig8\" ref-type=\"fig\">8</xref>). These findings indicate that DCs have the capacity to inhibit STAT6 and STAT4 signaling pathways to regulate allergen-induced Th2 immune responses.<fig id=\"Fig8\"><label>Figure 8</label><caption><p>STAT signaling pathways were inhibited by adoptive transfer of BMDCs with or without the SOCS3 gene. Lung tissue samples were homogenized after BMDC adoptive transfer into OVA-sensitized mice. Mononuclear cells were isolated from homogenates. STATs and phosphorylated STATs were detected by Western blotting (WB). The right columns represent the densitometry analysis of the WB results. (<bold>A</bold>) STAT1 and py-STAT1 detected by WB. (<bold>B</bold>) STAT4 and py-STAT4 detected by WB. C. STAT6 and py-STAT6 detected by WB. Data are representative of 3 independent experiments with similar results (*P < 0.05, ns: no significant difference).</p></caption><graphic xlink:href=\"41598_2020_70467_Fig8_HTML\" id=\"MO8\"/></fig></p></sec></sec><sec id=\"Sec9\"><title>Discussion</title><p id=\"Par17\">The role of the SOCS3 gene in inflammatory diseases, such as allergic asthma, has not been well defined. The SOCS3 protein is the most widely studied member of the SOCS family, which includes negative regulators of cytokine signaling. SOCS3 is central in negatively regulating Janus kinase (JAK) and STATs, such as STAT3, STAT4, STAT1 and STAT5. Previously published evidence has shown that SOCS3 knockdown leads to improvements in inflammation and airway hyperreactivity (AHR) in asthmatic mice and that T cell or CD4+ T cell activation is restricted after specific SOCS3 depletion<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. However, Duan et al. found that deletion of SOCS3 in macrophages enhanced the expression of STAT1-stimulated genes in response to IL-6. In addition, systemic administration of a SOCS3-specific antagonistic peptide (pJAK2) resulted in the induction of IFN responses<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Our previously published data revealed that SOCS3-deficient bone marrow-derived macrophages (BMDMs) expressed relatively high levels of TNF-α and that adoptive transfer of SOCS3-deficient BMDMs into WT mice enhanced the severity of acute lung injury (ALI)<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Such inconsistencies might be related to the different cell subsets in which the SOCS3 gene was specifically depleted in our experiments (myeloid cells) and other experiments (T cells). Based on previous data, BMDCs with or without SOCS3 gene expression were pulsed with LPS in the current study to promote maturation. We found that either SOCS3<sup>−/−</sup> DC transfer or SOCS3<sup>+/+</sup> DC transfer markedly alleviated OVA-induced lung injury and dramatically decreased the total number of inflammatory cells in BALF. When we examined the proportions of cells in BALF, we found that a lower proportion of neutrophils and a higher proportion of lymphocytes were present after BMDC adoptive transfer. Th2-type cytokines and IgE levels were also markedly decreased after BMDC transfer. Interestingly, compared with WT DCs, SOCS3<sup>−/−</sup> DCs slightly attenuated BMDC-induced immunogenic tolerance. In this regard, the effect of SOCS3 depletion on BMDC function was evaluated in an MLR. After mixing cultured BMDCs with CD4+ T cells, we found that the T cell proliferative capacity was enhanced in the SOCS3<sup>−/−</sup> BMDC group compared with the SOCS3<sup>+/+</sup>BMDC group and the control group. SOCS3<sup>−/−</sup> BMDCs induced CD4 + T cells to produce more IFN-γ but not IL-4. This finding partially explains why BMDC-induced immunogenic tolerance was slightly attenuated by SOCS3<sup>−/−</sup> BMDCs compared with SOCS3<sup>+/+</sup>BMDCs.</p><p id=\"Par18\">Different DC subsets and their discrete functional states might be responsible for promoting immunologic tolerance rather than inflammation. pDCs constitute a unique DC subset with the ability to induce regulatory T (Treg) cell responses and inhibit Th2 cell production, which has the potential to induce antigen-specific tolerance in asthma<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Kool et al.<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup> previously reported that selective removal of pDCs during allergen stimulation enhances airway inflammation, while adoptive transfer of pDCs before allergen stimulation inhibits airway inflammation. In contrast, adoptive transfer of conventional DCs (cDCs) or monocyte-derived DCs (moDCs) into OVA-sensitized mice augments airway inflammation. The various functional states of DCs affect allergic airway inflammation and AHR differently. Previous findings have shown that airway delivery of OVA-pulsed splenic CD8α + DCs reverses AHR and Th2 responses but not allergen-specific IgE and IgG1 responses<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. In addition, adoptive transfer of TGF-β and IL-10-treated DCs can significantly attenuated asthma presentation in sensitized mice<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. On the other hand, DCs differentiated with GM-CSF enhance AHR and eosinophil numbers and augment Th2 responses in recipient mice. Similar results have been observed in mice transferred with TNF-treated DCs<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. In this study, a subset of BMDCs were pulsed with LPS for 24 h to induce DClps development, while another subset of BMDCs that were not pulsed with LPS were called DCim. We found that adoptive transfer of DClps, but not DCim, via intraperitoneal injection greatly improved lung pathology scores and alleviated airway inflammatory cell infiltration. We subsequently evaluated the biological activities of DClps and DCim. DClps secreted more IL-10 and TGF-β than DCim, regardless of whether the SOCS3 gene was expressed. This finding is consistent with previous data showing that IL-10 plays a very important role in IL-10-differentiated DC (DC10)-mediated AHR improvement in allergic mice. The levels of MCP-3 and IFN-γ production were not significantly changed by BMDCs.</p><p id=\"Par19\">Gordon et al.<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup> assessed the effectiveness of DC10 delivery to asthmatic animals via various routes, specifically the transtracheal (t.t.), intraperitoneal (i.p.), intravenous (i.v.) and subcutaneous (s.c.) routes, and found that t.t. DC10 delivery and i.p. DC10 delivery were equally effective in reversing AHR and rapidly inhibiting eosinophil infiltration and the Th2 response. S.c. DC10 transfer inhibited airway Th2 responses to allergen attack, and i.v. DC10 transfer was completely ineffective in inducing tolerance in asthmatic mice<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. In the present study, PKH26-labeled BMDCs were transferred into OVA-sensitized mice via i.p. injection. We found that DClps could migrate to OVA-sensitized lungs more effectively than DCim. DClps began to migrate to the mouse lungs one day after delivery, and their numbers peaked on day 3 (5.42% vs 1.52% for DClps vs DCim; 5.42% vs 0.86% for DClps vs saline control). The labeled BMDCs in both groups disappeared on day 7. We found that migrated BMDCs prevented OVA-sensitized mice from reestablishing Th2 inflammation when BMDCs were administered intraperitoneally on days 14–16 during the OVA challenge period. This result indicates the rapid effects of BMDCs on immunological tolerance after adoptive cell transfer. However, the published data regarding the time frames of DC tolerance are inconsistent. For example, Nayyar et al.<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup> reported that the attenuation of AHR was first apparent 2 weeks after DC10 delivery and that the effect lasted for 3–10 weeks. We note that there are differences in experimental design, however; Nayyar et al. used mice with chronic OVA-induced asthma and performed DC10 transfer 2 weeks after OVA challenge, which was quite different from our approach.</p><p id=\"Par20\">The tolerogenicity of DCs is controlled by a complex network of environmental signals and intrinsic cellular mechanisms. DCs interact with T cells and determine the differentiation of distinct T cell subsets, such as the Th1, Th2, and Treg cell subsets. Th1 cells are primed mainly through the IFN-γ/STAT1 pathway and the IL-12/STAT4 pathway, while Th2 cells are primed mainly through the IL-4/STAT6 pathway. Medoff et al.<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup> demonstrated that STAT6 in BMDCs was sufficient for the production of C–C motif chemokine ligand (CCL) 17, CCL22, and CCL11, which are critical for Th2 lymphocyte recruitment to allergic airways, and found that STAT6 deficiency abrogated Th2 cell-selective chemokine production in an asthmatic mouse model. In the present study, we found that the phosphorylation of STAT4 and STAT6 in lung mononuclear cells was significantly decreased after BMDC adoptive transfer. There was also a decreasing trend in the phosphorylation of STAT1 after BMDC adoptive transfer. However, the difference was not significant. These results indicated that i.p. BMDC adoptive transfer reversed OVA-sensitized airway inflammation by inhibiting proinflammatory signal transcription and ultimately depressing both Th1- and Th2-mediated inflammation, especially Th2-mediated inflammation.</p><p id=\"Par21\">Overall, the findings suggested that BMDC adoptive transfer-induced immunogenic tolerance in OVA-sensitized mice might not be due to SOCS3 gene depletion. SOCS3<sup>−/−</sup> DCs slightly attenuated BMDC-induced immunogenic tolerance. In addition, DClps produced more IL-10 and TGF-β than DCim, leading to dramatic decreases in the phosphorylation of both STAT4 and STAT6, which are critical in initiating Th1- and Th2-mediated immunoinflammatory processes in asthma, respectively. Our study indicates that BMDC adoptive transfer may be developed into a new approach for the treatment of asthma, as it enables fine modulation of the balance between immune tolerance and inflammation. Further exploration is needed to elucidate the promising roles of BMDCs in alleviating allergic diseases. For example, the effects of distinct BMDC subsets on biological functional states, BMDC-T cell interactions, BMDC-epithelium interactions, and relevant molecular and biochemical mechanisms after BMDC transfer warrant additional investigation.</p></sec><sec id=\"Sec10\"><title>Methods</title><sec id=\"Sec11\"><title>Establishment and identification of myeloid cell-restricted SOCS3-KO mice</title><p id=\"Par22\">The animal protocol for this study was approved by the Ethics Committee of Zhongshan Hospital of Fudan University. All animals used in this study were maintained under specific pathogen-free conditions and treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978). The study was approved by the Ethics Boards of Zhongshan Hospital of Fudan University (Approval No. B2014-108).</p><p id=\"Par23\">The generation of SOCS3-KO mice has been described previously<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. Briefly, conditional SOCS3(Lyz2cre) mice were established by serial breeding of SOCS3fl/fl mice with Lyz2-Cre transgenic mice with Cre under the control of the myeloid cell-restricted Lyz2 promoter (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>A). Exon 2 was deleted by the Cre protein in SOCS3 Floxp+/+/Lyz2Cre+/− or SOCS3 Floxp+/+/Lyz2Cre+/+mice. Successful deletion of SOCS3 in SOCS3(Lyz2cre) mice was confirmed by PCR methods using 4 pairs of primers according to our previously published methods<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup> (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B). The Cre + loci were identified by 700-bp bands, and exon 2-deleted SOCS3-null loci were identified by 250-bp bands (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B). The mice identified as having the Cre+/+SOCS3<sup>−/−</sup> genotype were considered SOCS3(Lyz2cre) mice with myeloid cell-specific deletion of the SOCS3 gene.</p><p id=\"Par24\">To identify SOCS3 expression in BMDCs, bone marrow cells were collected from the femurs and tibiae of WT and SOCS3(Lyz2cre) mice. The bone marrow cells were seeded in RPMI-1640 medium supplemented with 1% antibiotics/antimycotics and 10% heat-inactivated fetal calf serum (FCS) containing 20 ng/ml GM-CSF. On days 3, 6, and 8, the nonadherent (dendritic) cell suspension was collected, and half of the supernatant was left. Complete medium supplemented with 20 ng/ml GM-CSF was added for further culture. On day 9, cells were collected for further study. The expression of cluster of differentiation (CD) 11c, CD80 and MHC-II was used to phenotypically identify BMDCs. A total of 10<sup>6</sup> cells were washed and subsequently stained with APC-Cy7-conjugated anti-CD11c (clone HL3, BD Pharmingen, USA), PE-Cy7-conjugated anti-CD80 (clone 16-10A1, BD Pharmingen, USA), and FITC-conjugated anti-MHC-II (clone M5/114, BD Pharmingen, USA) antibodies. The cells were stained with an anti-mouse SOCS3 antibody (ab16030, Abcam, UK) after being treated with a fixation/permeabilization agent (554722, BD Biosciences) to assess the SOCS3 expression deficiency in SOCS3-KO mice. The cells were analyzed on a FACSCanto II instrument (BD Biosciences, San Jose, CA, USA), and the data were analyzed with FlowJo software.</p></sec><sec id=\"Sec12\"><title>Generation of an OVA-induced mouse asthma model and adoptive transfer of DCs into asthmatic mice</title><p id=\"Par25\">To establish an asthmatic mouse model, mice were sensitized with two i.p. injections of 100 µg of OVA/alum (Sigma-Aldrich, Grade V; St. Louis, MO, USA) on day 0 and day 7 and were then exposed for 20 min to 100 μg of intranasal (i.n.) OVA on 5 consecutive days under light isoflurane anesthesia. The animals were treated with 1 × 10<sup>6</sup> DCs via i.p. injection on days 14–16 (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>A).</p><p id=\"Par26\">The mice were sacrificed within 24 h after the last OVA challenge, and BAL was immediately performed using 3 × 1 mL of 0.05 mM PBS-EDTA (Calbiochem, Darmstadt, Germany) as previously described<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>–<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. The cells in the BALF were collected with a Cytospin centrifuge (1,200 rpm for 10 min at 4 °C) and stained with Wright’s solution for differential cell counting. The supernatants were collected and frozen at -80 °C for IL-5 and IL-13 assessment by ELISA. Serum and tissue samples were obtained for further analyses.</p></sec><sec id=\"Sec13\"><title>Cell preparation and culture of BMDCs</title><p id=\"Par27\">BMDCs were generated from bone marrow cells collected from WT C57/B6 mice and SOCS3-KO mice. The bone marrow cells were collected from the femurs and tibiae of WT and SOCS3(Lyz2cre) mice. The bone marrow cells were then seeded in RPMI-1640 medium supplemented with 1% antibiotics/antimycotics and 10% heat-inactivated FCS containing 20 ng/ml GM-CSF. On days 3, 6, and 8, the nonadherent (dendritic) cell suspension was collected, and half of the supernatant was left. Complete medium supplemented with 20 ng/ml GM-CSF was added for further culture. On day 9, a subset of cells were collected for further study. Another subset of cells were incubated with LPS (100 ng/ml) for 24 h to generate DClps. The cells were then pulsed with OVA (1 µM) for 2 h at 37 °C.</p></sec><sec id=\"Sec14\"><title>T cell purification and DC stimulation of T cells</title><p id=\"Par28\">To establish an MLR, CD4 + T cells were first obtained from the spleen and lymph nodes of a native C57/B6 mouse. BMDCs were isolated and cultured as described above. SOCS3<sup>+/+</sup> and SOCS3<sup>−/−</sup> BMDCs were pretreated for 20 min at 37 °C in culture medium containing 200 μg/ml mitomycin C (Kyowa Hakko Kogyo, Tokyo, Japan). Murine CD4 + T cells were separated by negative isolation using a MagniSort™ Mouse CD4 T Cell Enrichment Kit (Thermo Fisher). Prior to culture, the purified CD4 + T cells were labeled with CFSE (Invitrogen, Ltd., UK) for subsequent assessment of T cell proliferation. Mitomycin C-treated SOCS3<sup>+/+</sup> or SOCS3<sup>−/−</sup> DCs were cocultured with CD4 + T cells at a ratio of 1:4 in a 37 °C, 5% CO2 atmosphere for 5 days. T cell proliferation was assessed by evaluating CFSE dilution on the last day of culture. After stimulation with phorbol 12-myristate 13-acetate (PMA; 100 ng/ml, Sigma) and ionomycin (5 μmol/L, Sigma) overnight, the supernatants of the cocultured cells were collected, and IL-4 and IFN-γ production was determined by ELISA.</p></sec><sec id=\"Sec15\"><title>Tracking of SOCS3<sup>−/−</sup> BMDCs in the lungs</title><p id=\"Par29\">SOCS3<sup>+/+</sup> and SOCS3<sup>−/−</sup> BMDCs were labeled with the fluorescent dye PKH26 (mini126, Sigma‐Aldrich) according to the manufacturer's protocol. Briefly, 2 × 10<sup>6</sup> BMDCs were suspended in 1 ml of diluted buffer from the manufacturer's labeling kit. The cell suspension was mixed with an equal volume of a labeling solution containing 4 × 10<sup>–6</sup> mol/L PKH26 dye in dilution buffer, and the mixture was incubated for 4 min at room temperature. The reaction was terminated by adding 2 ml of fetal bovine serum (FBS). After washing with a control medium, 5 × 10<sup>6</sup> DCs labeled with PKH26 were mixed with 1 mol/L PLA‐CMC solution and intraperitoneally injected into mice with OVA-induced asthma.</p><p id=\"Par30\">PKH26-labeled SOCS3<sup>+/+</sup> and SOCS3<sup>−/−</sup> DCs were injected intraperitoneally into asthmatic mice (1 × 10<sup>6</sup> cells/mouse) as described above. After 1, 3 or 7 days, we collected the lung tissue from each animal and generated single-cell suspensions by enzymatic digestion of the lungs (which included cutting the lungs into small pieces, incubating them with collagenase IV and DNase for one hour and filtering the resultant suspension through a 300-µm mesh filter). PKH26-labeled BMDCs in lung tissue samples were evaluated by assessing the expression of CD11c, CD80, MHC-II and PKH26 with a FACSCanto II instrument (BD Biosciences, San Jose, CA, USA).</p></sec><sec id=\"Sec16\"><title>Histochemistry and assessment of pathological lung injury</title><p id=\"Par31\">After the BALF was collected, the lungs were infused with 4% paraformaldehyde. Then, the trachea was clamped, and the lungs were excised. The left lung was embedded in paraffin. Five-micrometer paraffin sections were obtained and stained with hematoxylin and eosin (H&E). The right lung was collected and frozen at − 80 °C for protein assessment by Western blotting.</p><p id=\"Par32\">To quantify the degree of lung damage, H&E-stained slides were graded in a blinded fashion using a scoring system described previously<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. A score of 0 indicated that no detectable inflammation was present, a score of 1 indicated that occasional inflammatory cells were present, a score of 2 indicated that most bronchi or vessels were surrounded by a thin layer (one to five cells thick) of inflammatory cells, and a score of 3 indicated that most bronchi or vessels were surrounded by a thick layer (more than five cells thick) of inflammatory cells. Total lung inflammation was defined as the average of the peribronchial and perivascular inflammation scores. All slides were examined by two independent pathologists.</p><sec id=\"Sec17\"><title>ELISA</title><p id=\"Par33\">Cell culture supernatants and BALF samples were evaluated by using commercially available ELISA kits according to the manufacturers’ instructions. IL-4, IL-5, IL-10, IL-13, MCP-3, TGF-β, IFN-γ and IgE in supernatants were detected with ELISA kits (R&D Systems).</p></sec></sec><sec id=\"Sec18\"><title>Western blot analysis</title><p id=\"Par34\">The expression levels of STAT1, STAT4, STAT6 and the corresponding phosphorylated proteins in lung digests were analyzed by Western blot analysis. Cell lysates (40 μg) were separated by 10% sodium dodecyl sulfate-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Roche, USA). After incubation in a blocking buffer containing 5% skim milk in TBST (12.5 mM Tris–HCl pH 7.5, 68.5 mM NaCl, and 0.1% Tween 20) for 1 h, the blots were incubated overnight with primary antibodies including rabbit anti-STAT1 (D1K9Y, Cell Signaling Technology, USA), rabbit anti-phosphorylated STAT1 (Tyr701, Cell Signaling Technology, USA), rabbit anti-STAT4 (C46B10, Cell Signaling Technology, USA), rabbit anti-phosphorylated STAT4 (ab28815, Abcam, UK), rabbit anti-STAT6 (ab32520, Abcam, UK), and rabbit anti-phosphorylated STAT6 (ab28829, Abcam, UK). An anti-mouse GAPDH antibody was used as a loading control. The blots were washed with TBST, incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit Ig (Jackson ImmunoResearch) and then developed with an enhanced chemiluminescence (ECL) substrate solution (Millipore).</p></sec><sec id=\"Sec19\"><title>Statistical analysis</title><p id=\"Par35\">All data analysis and graph preparation were performed with GraphPad Prism 5.01 (GraphPad Software, San Diego, CA, USA). The data are presented as the mean ± standard error for each experimental group. Multigroup comparisons were performed by either one-way ANOVA with Tukey’s post hoc test or Wilcoxon signed rank tests (FACS analyses). P < 0.05 was regarded to indicate significance.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec20\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70467_MOESM1_ESM.pdf\"><caption><p>Supplementary file1</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Kan Xu, Nan Wu, and Zhihui Min.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70467-3.</p></sec><ack><title>Acknowledgements</title><p>This work was supported by the National Natural Science Foundation of China (81470211 and 81970023 to ZHC), the Shanghai Health Committee (201840288), the Shanghai Respiratory Research Institute and Yang Scientists Training Program of Zhongshan Hospital, the Shanghai Top-Priority Clinical Key Disciplines Construction Project (2017ZZ02013), and the Shanghai Municipal Key Clinical Specialty (shslczdzk02201).</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>Conceived and designed the study: C.Z; performed the biological experiments: K.X., N.W., Z.J., Z.M, and Z.L.; performed the statistical analysis: T.Z., C.L., Y.Z., J.S., and R.M.; and wrote and modified the paper: N.W., Z.J., H.J. and C.Z. All authors read and approved the final manuscript.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par36\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>GeurtsvanKessel</surname><given-names>CH</given-names></name><etal/></person-group><article-title>Clearance of influenza virus from the lung depends on migratory langerin+CD11b: but not plasmacytoid dendritic cells</article-title><source>J. Exp. Med.</source><year>2008</year><volume>205</volume><fpage>1621</fpage><lpage>1634</lpage><pub-id pub-id-type=\"doi\">10.1084/jem.20071365</pub-id><pub-id pub-id-type=\"pmid\">18591406</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nakagome</surname><given-names>K</given-names></name><etal/></person-group><article-title>In vivo IL-10 gene delivery suppresses airway eosinophilia and hyperreactivity by down-regulating APC functions and migration without impairing the antigen-specific systemic immune response in a mouse model of allergic airway inflammation</article-title><source>J. Immunol.</source><year>2005</year><volume>174</volume><fpage>6955</fpage><lpage>6966</lpage><pub-id pub-id-type=\"doi\">10.4049/jimmunol.174.11.6955</pub-id><pub-id pub-id-type=\"pmid\">15905538</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Smit</surname><given-names>JJ</given-names></name><name><surname>Rudd</surname><given-names>BD</given-names></name><name><surname>Lukacs</surname><given-names>NW</given-names></name></person-group><article-title>Plasmacytoid dendritic cells inhibit pulmonary immunopathology and promote clearance of respiratory syncytial virus</article-title><source>J. Exp. Med.</source><year>2006</year><volume>203</volume><fpage>1153</fpage><lpage>1159</lpage><pub-id pub-id-type=\"doi\">10.1084/jem.20052359</pub-id><pub-id pub-id-type=\"pmid\">16682497</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hammad</surname><given-names>H</given-names></name><etal/></person-group><article-title>Activation of the D Prostanoid 1 receptor suppresses asthma by modulation of lung dendritic cell function and induction of regulatory T cells</article-title><source>J. Exp. Med.</source><year>2007</year><volume>204</volume><fpage>357</fpage><lpage>367</lpage><pub-id pub-id-type=\"doi\">10.1084/jem.20061196</pub-id><pub-id pub-id-type=\"pmid\">17283205</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grohmann</surname><given-names>U</given-names></name><etal/></person-group><article-title>Reverse signaling through GITR Ligand enables dexamethasone to activate IDO in allergy</article-title><source>Nat. Med.</source><year>2007</year><volume>13</volume><fpage>579</fpage><lpage>586</lpage><pub-id pub-id-type=\"doi\">10.1038/nm1563</pub-id><pub-id pub-id-type=\"pmid\">17417651</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gordon</surname><given-names>JR</given-names></name><name><surname>Ma</surname><given-names>Y</given-names></name><name><surname>Churchman</surname><given-names>L</given-names></name><name><surname>Gordon</surname><given-names>SA</given-names></name><name><surname>Dawicki</surname><given-names>W</given-names></name></person-group><article-title>Regulatory dendritic cells for immunotherapy in immunologic diseases</article-title><source>Front. Immunol.</source><year>2014</year><volume>5</volume><fpage>7</fpage><pub-id pub-id-type=\"doi\">10.3389/fimmu.2014.00007</pub-id><pub-id pub-id-type=\"pmid\">24550907</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kubo</surname><given-names>M</given-names></name><name><surname>Inoue</surname><given-names>H</given-names></name></person-group><article-title>Suppressor of cytokine signaling 3 (SOCS3) in Th2 cells evokes Th2 Cytokines, IgE, and Eosinophilia</article-title><source>Curr. Allergy Asthma Rep.</source><year>2006</year><volume>6</volume><fpage>32</fpage><lpage>39</lpage><pub-id pub-id-type=\"doi\">10.1007/s11882-006-0007-6</pub-id><pub-id pub-id-type=\"pmid\">16476192</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>X</given-names></name><etal/></person-group><article-title>Key role of suppressor of cytokine signaling 3 in regulating Gp130 cytokine-induced signaling and limiting chondrocyte responses during murine inflammatory arthritis</article-title><source>Arthritis Rheumatol.</source><year>2014</year><volume>66</volume><fpage>2391</fpage><lpage>2402</lpage><pub-id pub-id-type=\"doi\">10.1002/art.38701</pub-id><pub-id pub-id-type=\"pmid\">24839265</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lang</surname><given-names>R</given-names></name><etal/></person-group><article-title>SOCS3 Regulates the plasticity of Gp130 signaling</article-title><source>Nat. Immunol.</source><year>2003</year><volume>4</volume><fpage>546</fpage><lpage>550</lpage><pub-id pub-id-type=\"doi\">10.1038/ni932</pub-id><pub-id pub-id-type=\"pmid\">12754506</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jiang</surname><given-names>Z</given-names></name><name><surname>Chen</surname><given-names>Z</given-names></name><name><surname>Li</surname><given-names>L</given-names></name><name><surname>Zhou</surname><given-names>W</given-names></name><name><surname>Zhu</surname><given-names>L</given-names></name></person-group><article-title>Lack of SOCS3 increases LPS-induced murine acute lung injury through modulation of Ly6C(+) macrophages</article-title><source>Respir. Res.</source><year>2017</year><volume>18</volume><fpage>217</fpage><pub-id pub-id-type=\"doi\">10.1186/s12931-017-0707-6</pub-id><pub-id pub-id-type=\"pmid\">29284516</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moriwaki</surname><given-names>A</given-names></name><etal/></person-group><article-title>T cell treatment with small interfering RNA for suppressor of cytokine signaling 3 modulates allergic airway responses in a murine model of asthma</article-title><source>Am. J. Respir. Cell. Mol. Biol.</source><year>2011</year><volume>44</volume><fpage>448</fpage><lpage>455</lpage><pub-id pub-id-type=\"doi\">10.1165/rcmb.2009-0051OC</pub-id><pub-id pub-id-type=\"pmid\">20508071</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Duan</surname><given-names>WN</given-names></name><etal/></person-group><article-title>Protective effects of SOCS3 overexpression in high glucoseinduced lung epithelial cell injury through the JAK2/STAT3 pathway</article-title><source>Mol. Med. Rep.</source><year>2017</year><volume>16</volume><fpage>2668</fpage><lpage>2674</lpage><pub-id pub-id-type=\"doi\">10.3892/mmr.2017.6941</pub-id><pub-id pub-id-type=\"pmid\">28713982</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>W</given-names></name><name><surname>Liang</surname><given-names>X</given-names></name><name><surname>Peterson</surname><given-names>AJ</given-names></name><name><surname>Munn</surname><given-names>DH</given-names></name><name><surname>Blazar</surname><given-names>BR</given-names></name></person-group><article-title>The indoleamine 2,3-dioxygenase pathway is essential for human plasmacytoid dendritic cell-induced adaptive t regulatory cell generation</article-title><source>J. Immunol.</source><year>2008</year><volume>181</volume><fpage>5396</fpage><lpage>5404</lpage><pub-id pub-id-type=\"doi\">10.4049/jimmunol.181.8.5396</pub-id><pub-id pub-id-type=\"pmid\">18832696</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>H</given-names></name><etal/></person-group><article-title>Regulatory dendritic cell expression of MHCII and IL-10 are jointly requisite for induction of tolerance in a murine model of OVA-asthma</article-title><source>Allergy</source><year>2013</year><volume>68</volume><fpage>1126</fpage><lpage>1135</lpage><pub-id pub-id-type=\"doi\">10.1111/all.12273</pub-id><pub-id pub-id-type=\"pmid\">23910017</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kool</surname><given-names>M</given-names></name><etal/></person-group><article-title>An anti-inflammatory role for plasmacytoid dendritic cells in allergic airway inflammation</article-title><source>J. Immunol.</source><year>2009</year><volume>183</volume><fpage>1074</fpage><lpage>1082</lpage><pub-id pub-id-type=\"doi\">10.4049/jimmunol.0900471</pub-id><pub-id pub-id-type=\"pmid\">19553531</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>H</given-names></name><name><surname>Dawicki</surname><given-names>W</given-names></name><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Town</surname><given-names>J</given-names></name><name><surname>Gordon</surname><given-names>JR</given-names></name></person-group><article-title>Tolerogenic dendritic cells induce CD4+CD25hiFoxp3+ regulatory T Cell differentiation from CD4+CD25-/loFoxp3- effector T Cells</article-title><source>J. Immunol.</source><year>2010</year><volume>185</volume><fpage>5003</fpage><lpage>5010</lpage><pub-id pub-id-type=\"doi\">10.4049/jimmunol.0903446</pub-id><pub-id pub-id-type=\"pmid\">20870943</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lu</surname><given-names>M</given-names></name><etal/></person-group><article-title>Therapeutic induction of tolerance by IL-10-differentiated dendritic cells in a mouse model of house dust mite-asthma</article-title><source>Allergy</source><year>2011</year><volume>66</volume><fpage>612</fpage><lpage>620</lpage><pub-id pub-id-type=\"doi\">10.1111/j.1398-9995.2010.02526.x</pub-id><pub-id pub-id-type=\"pmid\">21241316</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nayyar</surname><given-names>A</given-names></name><etal/></person-group><article-title>Induction of prolonged asthma tolerance by IL-10-differentiated dendritic cells: differential impact on airway hyperresponsiveness and the Th2 immunoinflammatory response</article-title><source>J. Immunol.</source><year>2012</year><volume>189</volume><fpage>72</fpage><lpage>79</lpage><pub-id pub-id-type=\"doi\">10.4049/jimmunol.1103286</pub-id><pub-id pub-id-type=\"pmid\">22634620</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Medoff</surname><given-names>BD</given-names></name><etal/></person-group><article-title>CD11b+ myeloid cells are the key mediators of Th2 cell homing into the airway in allergic inflammation</article-title><source>J. Immunol.</source><year>2009</year><volume>182</volume><fpage>623</fpage><lpage>635</lpage><pub-id pub-id-type=\"doi\">10.4049/jimmunol.182.1.623</pub-id><pub-id pub-id-type=\"pmid\">19109196</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cataldo</surname><given-names>DD</given-names></name><etal/></person-group><article-title>Matrix metalloproteinase-9 deficiency impairs cellular infiltration and bronchial hyperresponsiveness during allergen-induced airway inflammation</article-title><source>Am. J. Pathol.</source><year>2002</year><volume>161</volume><fpage>491</fpage><lpage>498</lpage><pub-id pub-id-type=\"doi\">10.1016/S0002-9440(10)64205-8</pub-id><pub-id pub-id-type=\"pmid\">12163374</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gueders</surname><given-names>MM</given-names></name><etal/></person-group><article-title>Matrix metalloproteinase-8 deficiency promotes granulocytic allergen-induced airway inflammation</article-title><source>J. Immunol.</source><year>2005</year><volume>175</volume><fpage>2589</fpage><lpage>2597</lpage><pub-id pub-id-type=\"doi\">10.4049/jimmunol.175.4.2589</pub-id><pub-id pub-id-type=\"pmid\">16081833</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schneider</surname><given-names>AM</given-names></name><name><surname>Li</surname><given-names>F</given-names></name><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Gordon</surname><given-names>JR</given-names></name></person-group><article-title>Induction of pulmonary allergen-specific IgA responses or airway hyperresponsiveness in the absence of allergic lung disease following sensitization with limiting doses of ovalbumin-alum</article-title><source>Cell. Immunol.</source><year>2001</year><volume>212</volume><fpage>101</fpage><lpage>109</lpage><pub-id pub-id-type=\"doi\">10.1006/cimm.2001.1854</pub-id><pub-id pub-id-type=\"pmid\">11748926</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kwak</surname><given-names>YG</given-names></name><etal/></person-group><article-title>Involvement of PTEN in airway hyperresponsiveness and inflammation in bronchial asthma</article-title><source>J. Clin. Invest.</source><year>2003</year><volume>111</volume><fpage>1083</fpage><lpage>1092</lpage><pub-id pub-id-type=\"doi\">10.1172/JCI16440</pub-id><pub-id pub-id-type=\"pmid\">12671058</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807858</article-id><article-id pub-id-type=\"pmc\">PMC7431556</article-id><article-id pub-id-type=\"publisher-id\">70830</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70830-4</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Coronary artery bypass grafting and perioperative stroke: imaging of atherosclerotic plaques in the ascending aorta with ungated high-pitch CT-angiography</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Asenbaum</surname><given-names>Ulrika</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Nolz</surname><given-names>Richard</given-names></name><address><email>richard.nolz@meduniwien.ac.at</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Puchner</surname><given-names>Stefan B.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Schoster</surname><given-names>Tobias</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Baumann</surname><given-names>Lukas</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Furtner</surname><given-names>Julia</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zimpfer</surname><given-names>Daniel</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Laufer</surname><given-names>Guenther</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Loewe</surname><given-names>Christian</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sandner</surname><given-names>Sigrid E.</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><aff id=\"Aff1\"><label>1</label>Department of Biomedical Imaging and Image-Guided Therapy, Medical University of Vienna, Vienna General Hospital, Waehringer Guertel 18-20, 1090 Vienna, Austria </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.22937.3d</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9259 8492</institution-id><institution>Center of Medical Statistics, Informatics, and Intelligent Systems (CEMSIIS), </institution><institution>Medical University of Vienna, </institution></institution-wrap>Vienna, Austria </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.22937.3d</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9259 8492</institution-id><institution>Division of Cardiac Surgery, </institution><institution>Medical University of Vienna, </institution></institution-wrap>Vienna, Austria </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13909</elocation-id><history><date date-type=\"received\"><day>29</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>3</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Perioperative stroke is a devastating complication after coronary artery bypass graft (CABG) surgery, with atherosclerosis of the ascending aorta as important risk factor. During surgical manipulation, detachment of plaques can lead to consecutive embolization into brain-supplying arteries. High-pitch computed tomography angiography (HP-CTA) represents a non-invasive imaging modality, which provides the opportunity for comprehensive imaging of the ascending aorta, including plaque detection and advanced characterization. In our present retrospective study on 719 individuals, who had undergone HP-CTA within 6 months prior to CABG, atherosclerotic disease of the ascending aorta was evaluated with respect to perioperative stroke rates. For image analysis, the ascending aorta was divided into a proximal and distal part, consisting of four segments, and evaluated for presence and distribution of calcified and mixed plaques. All patients with perioperative stroke presented with atherosclerotic disease of the ascending aorta. The stroke rate was significantly associated with the presence and extent of atherosclerotic disease. Patients burdened with mixed plaques presented with significantly higher perioperative stroke rates. This study demonstrates that HP-CTA allows accurate evaluation of plaque extent and composition in the ascending aorta, and therefore may improve risk stratification of stroke prior to CABG.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Cardiovascular diseases</kwd><kwd>Risk factors</kwd><kwd>Outcomes research</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par8\">Perioperative stroke is a devastating complication after coronary artery bypass graft (CABG) surgery<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>, with atherosclerosis of the ascending aorta as important risk factor<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. During surgical manipulation, detachment of plaques can lead to consecutive embolization into brain-supplying arteries<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. As non-calcified plaque components are more susceptible to rupture and erosion<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>, plaque characterization is particularly important. Thus, the extent, distribution, and composition of plaques have an influence on surgical planning to prevent atheroembolic stroke<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Consequently, preoperative plaque imaging is highly desirable to allow for planned modifications of surgical technique to reduce or avoid manipulation of the aorta.</p><p id=\"Par9\">Second-, and third-generation dual-source scanners enable fast acquisition of datasets of the entire aorta, whereby image quality becomes less dependent on heart rate and pulsation artefacts. By means of imaging of the ascending aorta prior CABG, the elimination of pulsation artifacts may be considered to be the key requirement of CTA. In this context, ungated high-pitch protocols demonstrated excellent image quality even without premedication for heart rate control (e.g. beta-blockers) and at similar or even lower radiation dose compared with both gated, and slow-pitch gated protocols<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>–<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Additionally, this technique provides the opportunity for comprehensive imaging of the ascending aorta, including plaque detection and advanced characterization based on Hounsfield unit (HU) measurements of their components<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>.</p><p id=\"Par10\">The aim of this study was to assess the extent and composition of atherosclerotic plaques in the ascending aorta with ungated high-pitch computed tomography angiography (CTA), with respect to perioperative stroke rates after CABG.</p></sec><sec id=\"Sec2\"><title>Materials and methods</title><sec id=\"Sec3\"><title>Study design</title><p id=\"Par11\">This was a single-center, retrospective, cross-sectional study of consecutive patients who underwent isolated CABG surgery between January 2009 and December 2016. This study was conducted in accordance with the declaration of Helsinki and was approved by the ethic committee of the Medical University Vienna (Ethikkommission der Medizinischen Universität Wien, 1090 Vienna, Austria) (No. 1239/2014). The IRB waived the need to obtain written, informed consent.</p></sec><sec id=\"Sec4\"><title>Study population</title><p id=\"Par12\">There were 2,320 consecutive patients who underwent isolated CABG during the specified time period. Patients to whom the following criteria applied were excluded from analysis: (1) pre- and/or postoperative, extracorporeal membrane oxygenation (n = 54); and (2) no preoperative CTA imaging (n = 1,025). Based on the heterogeneity of the study group due to the usage of different scanner types, and several modifications of imaging protocols during the defined time period, we also excluded patients who underwent preoperative non high-pitch CTA (retrospective, and prospective gated, n = 522). Thus, 719 consecutive patients who underwent ungated high-pitch CTA within a median time interval of 11 days (IQR 3–42 days) prior to CABG were analyzed. A part of the study population was included in a previously described cohort<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. Patient characteristics are given in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>.<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Patient characteristics.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">Overall (n = 719)</th><th align=\"left\">No perioperative stroke (n = 707)</th><th align=\"left\">Perioperative stroke (n = 12)</th><th align=\"left\"><italic>p</italic></th></tr></thead><tbody><tr><td align=\"left\">Sex, female</td><td char=\"(\" align=\"char\">132/719 (18.4%)</td><td char=\"(\" align=\"char\">126/707 (17.8%)</td><td align=\"left\">6/12 (50.0%)</td><td char=\".\" align=\"char\">0.012</td></tr><tr><td align=\"left\">Age, years</td><td char=\"(\" align=\"char\">68.0 (60.3–74.4)</td><td char=\"(\" align=\"char\">68.1 (60.9–74.3)</td><td align=\"left\">64.7 (61.9–72.8)</td><td char=\".\" align=\"char\">0.530</td></tr><tr><td align=\"left\">Height, cm</td><td char=\"(\" align=\"char\">173.0 (168.0–178.0)</td><td char=\"(\" align=\"char\">173.0 (168.0–178.0)</td><td align=\"left\">166.5 (160.5–174.5)</td><td char=\".\" align=\"char\">0.203</td></tr><tr><td align=\"left\">Weight, kg</td><td char=\"(\" align=\"char\">82.0 (74.0–94.0)</td><td char=\"(\" align=\"char\">82.0 (74.0–94.0)</td><td align=\"left\">79.0 (73.3–99.5)</td><td char=\".\" align=\"char\">0.698</td></tr><tr><td align=\"left\">BMI, kg/m<sup>2</sup></td><td char=\"(\" align=\"char\">28.0 (25.0–31.0)</td><td char=\"(\" align=\"char\">28.0 (25.0–31.0)</td><td align=\"left\">28.0 (23.8–31.8)</td><td char=\".\" align=\"char\">0.993</td></tr><tr><td align=\"left\">Smoking history</td><td char=\"(\" align=\"char\">339/719 (47.1%)</td><td char=\"(\" align=\"char\">330/707 (46.7%)</td><td align=\"left\">9/12 (75.0%)</td><td char=\".\" align=\"char\">0.051</td></tr><tr><td align=\"left\">Active smokers</td><td char=\"(\" align=\"char\">127/719 (17.7%)</td><td char=\"(\" align=\"char\">124/707 (17.5%)</td><td align=\"left\">3/12 (25.0%)</td><td char=\".\" align=\"char\">0.453</td></tr><tr><td align=\"left\">Family history of coronary heart disease</td><td char=\"(\" align=\"char\">117/719 (16.3%)</td><td char=\"(\" align=\"char\">115/707 (16.3%)</td><td align=\"left\">2/12 (16.7%)</td><td char=\".\" align=\"char\">1.00</td></tr><tr><td align=\"left\">Diabetes mellitus</td><td char=\"(\" align=\"char\">285/719 (39.7%)</td><td char=\"(\" align=\"char\">278/707 (39.3%)</td><td align=\"left\">7/12 (58.3%)</td><td char=\".\" align=\"char\" rowspan=\"2\">0.207</td></tr><tr><td align=\"left\">IDDM</td><td char=\"(\" align=\"char\">107/719 (14.9%)</td><td char=\"(\" align=\"char\">104/707 (14.7%)</td><td align=\"left\">3/12 (25.0%)</td></tr><tr><td align=\"left\">Dyslipidemia</td><td char=\"(\" align=\"char\">565/719 (78.6%)</td><td char=\"(\" align=\"char\">556/707 (78.6%)</td><td align=\"left\">9/12 (75.0%)</td><td char=\".\" align=\"char\">0.727</td></tr><tr><td align=\"left\">Renal insufficiency, mild to moderate</td><td char=\"(\" align=\"char\">96/719 (13.4%)</td><td char=\"(\" align=\"char\">91/707 (12.9%)</td><td align=\"left\">5/12 (41.7%)</td><td char=\".\" align=\"char\">0.014</td></tr><tr><td align=\"left\">Dialysis</td><td char=\"(\" align=\"char\">14/719 (1.9%)</td><td char=\"(\" align=\"char\">10/707 (1.4%)</td><td align=\"left\">4/12 (33.3%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">Serum creatinine, mg/dl</td><td char=\"(\" align=\"char\">1.0 (0.85–1.2)</td><td char=\"(\" align=\"char\">1.0 (0.85–1.2)</td><td align=\"left\">1.28 (0.83–3.05)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">Hypertension</td><td char=\"(\" align=\"char\">651/719 (90.5%)</td><td char=\"(\" align=\"char\">641/707 (90.7%)</td><td align=\"left\">10/12 (83.3%)</td><td char=\".\" align=\"char\">0.316</td></tr><tr><td align=\"left\">Carotid stenosis and history of stroke</td><td char=\"(\" align=\"char\">71/719 (9.9%)</td><td char=\"(\" align=\"char\">69/707 (9.8%)</td><td align=\"left\">2/12 (16.7%)</td><td char=\".\" align=\"char\">0.336</td></tr><tr><td align=\"left\">Asymptomatic carotid stenosis</td><td char=\"(\" align=\"char\">162/719 (22.5%)</td><td char=\"(\" align=\"char\">158/707 (22.3%)</td><td align=\"left\">4/12 (33.3%)</td><td char=\".\" align=\"char\">0.483</td></tr><tr><td align=\"left\">PAOD</td><td char=\"(\" align=\"char\">135/719 (18.8%)</td><td char=\"(\" align=\"char\">131/707 (18.5%)</td><td align=\"left\">4/12 (33.3%)</td><td char=\".\" align=\"char\">0.253</td></tr><tr><td align=\"left\">Chronic lung disease</td><td char=\"(\" align=\"char\">206/719 (28.7%)</td><td char=\"(\" align=\"char\">202/707 (28.6%)</td><td align=\"left\">4/12 (33.3%)</td><td char=\".\" align=\"char\">0.552</td></tr><tr><td align=\"left\">Previous CABG</td><td char=\"(\" align=\"char\">12/719 (1.7%)</td><td char=\"(\" align=\"char\">11/707(1.6%)</td><td align=\"left\">1/12 (8.3%)</td><td char=\".\" align=\"char\">0.184</td></tr><tr><td align=\"left\">Previous heart valve operation</td><td char=\"(\" align=\"char\">3/719 (0.4%)</td><td char=\"(\" align=\"char\">3/707 (0.4%)</td><td align=\"left\">0/12</td><td char=\".\" align=\"char\">1.000</td></tr><tr><td align=\"left\">Previous myocardial infarction</td><td char=\"(\" align=\"char\">378/719 (52.6%)</td><td char=\"(\" align=\"char\">369/707 (52.2%)</td><td align=\"left\">9/12 (75.0%)</td><td char=\".\" align=\"char\">0.117</td></tr><tr><td align=\"left\">Ejection fraction, %</td><td char=\"(\" align=\"char\">55.0 (45.0–60.0)</td><td char=\"(\" align=\"char\">55.0 (45.0–60.0)</td><td align=\"left\">60.0 (45.0–60.0)</td><td char=\".\" align=\"char\">0.341</td></tr><tr><td align=\"left\">Atrial arrhythmia</td><td char=\"(\" align=\"char\">95/719 (13.2%)</td><td char=\"(\" align=\"char\">94/707 (13.3%)</td><td align=\"left\">1/12 (8.3%)</td><td char=\".\" align=\"char\">1.000</td></tr><tr><td align=\"left\" colspan=\"5\"><bold>Coronary artery disease</bold></td></tr><tr><td align=\"left\">One-vessel</td><td char=\"(\" align=\"char\">21/719 (2.9%)</td><td char=\"(\" align=\"char\">21/707 (3.0%)</td><td align=\"left\">0/12</td><td char=\".\" align=\"char\" rowspan=\"3\">0.513</td></tr><tr><td align=\"left\">Two-vessel</td><td char=\"(\" align=\"char\">85/719 (11.8%)</td><td char=\"(\" align=\"char\">84/707 (11.9%)</td><td align=\"left\">1/12 (8.3%)</td></tr><tr><td align=\"left\">Three-vessel</td><td char=\"(\" align=\"char\">613/719 (85.3%)</td><td char=\"(\" align=\"char\">602/707 (85.1%)</td><td align=\"left\">11/12 (91.7%)</td></tr><tr><td align=\"left\" colspan=\"5\"><bold>Indication for surgery</bold></td></tr><tr><td align=\"left\">Elective</td><td char=\"(\" align=\"char\">457/719 (63.6%)</td><td char=\"(\" align=\"char\">450/707 (63.6%)</td><td align=\"left\">7/12 (58.3%)</td><td char=\".\" align=\"char\" rowspan=\"3\">0.796</td></tr><tr><td align=\"left\">Urgent</td><td char=\"(\" align=\"char\">234/719 (32.5%)</td><td char=\"(\" align=\"char\">229/707 (32.4%)</td><td align=\"left\">5/12 (41.7%)</td></tr><tr><td align=\"left\">Emergency</td><td char=\"(\" align=\"char\">28/719 (3.9%)</td><td char=\"(\" align=\"char\">28/707 (4.0%)</td><td align=\"left\">0/12</td></tr></tbody></table><table-wrap-foot><p>cm, centimeter; kg, kilogram; BMI, body mass index; kg/m<sup>2</sup>, kilogram per square meter; NIDDM, non-insulin-dependent diabetes mellitus; IDDM, insulin-dependent diabetes mellitus; mg/dl, milligrams per deciliter; PAOD, peripheral artery occlusive disease, CABG, coronary artery bypass grafting, NYHA, New York Heart Association classification; p, no perioperative stroke versus perioperative stroke.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec5\"><title>Ungated high-pitch computed tomography angiography</title><p id=\"Par13\">All examinations were performed on a second-, or third-generation dual-source computed tomography system. Following scanners were used: Somatom Definition Flash (Siemens Healthineers; n = 602, 83.7%), Somatom Drive (Siemens Healthineers; n = 79, 11.0%), and Somatom Force (Siemens Healthineers; n = 38, 5.3%). The institutional standard protocol (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>) included a single acquisition of the entire aorta with a high pitch to reduce motion artifacts, particularly at the level of the ascending aorta. No premedication for heart rate control was applied. A double-head power injector (Injektron CT2, Medtron AG, Saarbrücken, GER) was used for intravenous injection of a non-ionic contrast medium (Iomeron 400, Bracco-Austria, Vienna, Austria), via antecubital vein, followed by a 40 ml saline chaser bolus. Examinations on the Somatom Definition Flash scanner were performed after injection of 40 ml Iomeron 400 at an injection rate of 2.5 ml/s. For the Somatom Drive and the Somatom Force scanner, amount of contrast medium, injection rate, and tube voltage were adjusted according to body weight. The individual radiation dose was estimated using the dose-length product (DLP) given by the CT system.<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Technical details of dual-source computer tomography angiography.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" colspan=\"4\">Acquisition parameters</th></tr><tr><th align=\"left\">Scanner type</th><th align=\"left\">Somatom defintion flash<sup>a</sup></th><th align=\"left\">Somatom drive<sup>a</sup></th><th align=\"left\">Somatom force<sup>a</sup></th></tr></thead><tbody><tr><td align=\"left\">Tube voltage (refkV)</td><td align=\"left\">120</td><td align=\"left\">110</td><td align=\"left\">80</td></tr><tr><td align=\"left\">Tube current (refmAs, CD4D)</td><td align=\"left\">116</td><td align=\"left\">140</td><td align=\"left\">194</td></tr><tr><td align=\"left\">Collimation (mm)</td><td align=\"left\">128 × 0.6</td><td align=\"left\">128 × 0.6</td><td align=\"left\">192 × 0.6</td></tr><tr><td align=\"left\">Rotation time (s)</td><td align=\"left\">0.28</td><td align=\"left\">0.28</td><td align=\"left\">0.25</td></tr><tr><td align=\"left\">Pitch</td><td align=\"left\">2.4</td><td align=\"left\">2.4</td><td align=\"left\">1.9</td></tr><tr><td align=\"left\">Soft kernel</td><td align=\"left\">B31f.</td><td align=\"left\">I30f.</td><td align=\"left\">Bv40, Bv36</td></tr><tr><td align=\"left\">Iteration</td><td align=\"left\">None</td><td align=\"left\">Admire strength 3</td><td align=\"left\">Admire strength 3</td></tr></tbody></table><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" colspan=\"4\">Adjustment of contrast medium</th></tr><tr><th align=\"left\">Amount of contrast medium, ml</th><th align=\"left\">Body weight, kg</th><th align=\"left\">Tube voltage, kV</th><th align=\"left\">Injection rate, ml/s</th></tr></thead><tbody><tr><td align=\"left\">20</td><td align=\"left\"> < 60</td><td align=\"left\">70–80</td><td align=\"left\">1.3</td></tr><tr><td align=\"left\">30</td><td align=\"left\">60–80</td><td align=\"left\">90–100</td><td align=\"left\">2</td></tr><tr><td align=\"left\">40</td><td align=\"left\">80–100</td><td align=\"left\">110–120</td><td align=\"left\">2.5</td></tr></tbody></table><table-wrap-foot><p>refkV, reference kilovolt; refmAs, reference milliampere seconds; mm, millimeter; sec, seconds; ml, milliliter; kg, kilogram; kV, kilovolt; ml/sec, milliliter/second.</p><p><sup>a</sup>Siemens healthineers.</p></table-wrap-foot></table-wrap></p><p id=\"Par14\">Post processing included maximum intensity projections and was performed from thin transversal image slices on a Multimodality Workplace (MMWP, Siemens Healthineers, Erlangen, GER) in the coronal and sagittal views, with a slice thickness of 3 mm and 1 mm, and a reconstruction increment of 2 mm and 0.8 mm, respectively. All CTA images were transferred to a picture archiving and communication system (IMPAX, Agfa Healthcare, Mortsel, BEL).</p></sec><sec id=\"Sec6\"><title>Definitions and image interpretation</title><p id=\"Par15\">The ascending aorta was divided into a defined proximal and distal section that reflected the areas of crossclamping and arterial cannulation during a standard CABG procedure, as described by Johnson et al.<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. For this purpose, a “centerline” of the ascending aorta was delineated from the valvular level to the level of the left subclavian artery using semi-automatic image processing software (Syngo.Via, Siemens Healthineers, Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>a). The proximal section of the ascending aorta extended from the level of the origin of the most distal coronary artery to the level of the origin of the pulmonary artery bifurcation, determined orthogonally to the course of the “centerline”. Subsequently, the distal section of the ascending aorta extended from the pulmonary artery bifurcation to the origin of the brachiocephalic trunk (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b). In addition, both sections were divided into four segments (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>c).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Segmentation of the ascending aorta. “Centerline” of the ascending aorta from the valvular level to the level of the left subclavian artery with the corresponding curved planar reformations (<bold>a</bold>). Allocation of the ascending aorta into a proximal section, extending from the level of the origin of the most distal coronary artery to the level of the origin of the pulmonary artery bifurcation, and a distal section extending from the pulmonary artery bifurcation to the origin of the brachiocephalic trunk (<bold>b</bold>). Sections were determined orthogonally to the course of the “centerline”. For segmentation (<bold>c</bold>), a straight line was drawn through the center of the ascending and corresponding descending aorta at the level of the pulmonary artery bifurcation: (1) the anterior segment covered the sectors 45° on both sides of this straight line in the ventral part of the ascending aorta; (2) the posterior segment included the sectors 45° on both sides in the dorsal part of the ascending aorta. Subsequently, the remaining quarters to the right and to the left of the straight line were termed the (3) right, and the (4) left segment.</p></caption><graphic xlink:href=\"41598_2020_70830_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par16\">During a standard CABG procedure, crossclamping affects all segments of the proximal section of the ascending aorta, and cannulation is performed mainly in the distal anterior and left segment. Therefore, these six segments were defined as mechanically stressed segments.</p><p id=\"Par17\">The presence of atherosclerotic plaque was defined as intimal thickening ≥ 4 mm<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. Differentiation of atherosclerotic plaques was based on the HU of their components. Components were defined as calcified at a threshold value of ≥ 130 HU, and as non calcified < 130 HU, respectively<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Subsequently, plaques that consisted of exclusively calcified components were classified as calcified plaques. Due to the known increased risk of rupture of non-calcified plaque components<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>, plaques that consisting of any non-calcified components were classified as mixed plaques (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>a–c).<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Plaque characterization. Plaque that consisted of exclusively calcified components (HU ≥ 130) was classified as calcified plaque (<bold>a</bold>). Plaques that consisted of calcified (HU ≥ 130) and non-calcified (HU < 130) components (<bold>b</bold>), as well as exclusively non-calcified components (<bold>c</bold>), were graded as mixed plaques.</p></caption><graphic xlink:href=\"41598_2020_70830_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par18\">Based on these classifications, defined segments of the ascending aorta were screened for plaques, which were graded as a dichotomous variable. Image evaluation was performed by two board-certified radiologists (U.A., S.B.P.), with 10 and 15 years of experience in cardiovascular imaging, respectively, by consensus. Extent of disease was defined as the number of concomitantly involved segments.</p></sec><sec id=\"Sec7\"><title>Coronary artery bypass grafting and perioperative stroke</title><p id=\"Par19\">Patients referred for CABG underwent careful duplex evaluation of the carotid, vertebral, and subclavian arteries as part of their preoperative vascular assessment. Isolated CABG was performed as a standard procedure as described previously by Johnson et al.<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. Based on preoperative ungated high-pitch CTA imaging analysis for plaque distribution, modifications were made to the CABG procedure; most frequently this included the use of alternate cannulation sites, beating-heart, or off-pump procedures.</p><p id=\"Par20\">Patients with new-onset neurological disorders after CABG were routinely evaluated by a neurologist in service who ordered computed tomography of the brain at his/her discretion. Consequently, patient records were screened for neurological consultations and concomitant computed tomography scans of the brain to identify patients with stroke. Based on these examinations, stroke was defined according to the STS National Adult Cardiac Surgery Database as any confirmed new neurological deficit of abrupt onset caused by a disturbance in blood supply to the brain that did not resolve within 24 h, and which included additional detection of a new lesion by a computed tomography scan of the brain.</p><p id=\"Par21\">Stroke rates were compared with respect to plaque extent, distribution and morphology.</p></sec><sec id=\"Sec8\"><title>Statistics</title><p id=\"Par22\">Discrete variables were described with absolute and relative numbers and by using contingency tables; possible differences in discrete variables between groups were tested with the Chi-Square-test, the Fisher exact test, and the McNemar test as appropriate. Continuous variables were described as mean ± standard deviation, or median and interquartile range (IQRs), as appropriate. Normal distribution of data was tested with the Shapiro–Wilk-Test. Possible differences in continuous variables between groups were tested by the Wilcoxon test, the Kruskal–Wallis test, or the <italic>T </italic>test, as appropriate. All tests were two-sided. No formal Bonferroni correction was applied in this exploratory study. <italic>P</italic> values are given as calculated and should be interpreted with care, considering alpha error accumulation. Results were regarded as statistically significant, if <italic>p</italic> < 0.05. Statistical analyses were performed using SPSS for Windows (version 20.0; IBM Corporation).</p></sec></sec><sec id=\"Sec9\"><title>Results</title><sec id=\"Sec10\"><title>Plaque burden and distribution</title><p id=\"Par23\">The mean DLP was 515.6 ± 166.8 mGy × cm. Atherosclerotic plaques in the ascending aorta were detected in 159/719 patients (22.1%, Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref>). The proximal section of the ascending aorta was more frequently affected than the distal section (n = 112/719, 15.6% vs. n = 99/719, 13.8% of patients). Overall, the number of patients with calcified plaques (n = 142/719; 19.7%) was significantly higher than those with mixed plaques (n = 34/719; 4.7%, <italic>p</italic> < 0.001). In 133/719 patients (18.5%) at least one plaque was detected in one of the mechanically stressed segments. For these segments, the number of patients with calcified plaques was also significantly higher than those with mixed plaques (n = 114/719, 15.9% vs. n = 26/719, 3.6%, <italic>p</italic> < 0.001).<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Plaque burden per patient.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" colspan=\"5\">Patients (n = 719)</th></tr><tr><th align=\"left\"/><th align=\"left\">Any plaque number of patients (%)</th><th align=\"left\">Calcified plaque number of patients (%)</th><th align=\"left\">Mixed plaque number of patients (%)</th><th align=\"left\"><italic>p</italic></th></tr></thead><tbody><tr><td align=\"left\">Ascending aorta, overall</td><td char=\"(\" align=\"char\">159/719 (22.1%)</td><td char=\"(\" align=\"char\">142/719 (19.7%)</td><td char=\"(\" align=\"char\">34/719 (4.7%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">Ascending aorta, proximal</td><td char=\"(\" align=\"char\">112/719 (15.6%)</td><td char=\"(\" align=\"char\">102/719 (14.2%)</td><td char=\"(\" align=\"char\">15/719 (2.1%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">Ascending aorta, distal</td><td char=\"(\" align=\"char\">99/719 (13.8%)</td><td char=\"(\" align=\"char\">82/719 (11.4%)</td><td char=\"(\" align=\"char\">24/719 (3.3%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">Mechanically stressed segments</td><td char=\"(\" align=\"char\">133/719 (18.5%)</td><td char=\"(\" align=\"char\">114/719 (15.9%)</td><td char=\"(\" align=\"char\">26/719 (3.6%)</td><td char=\".\" align=\"char\"> < 0.001</td></tr></tbody></table><table-wrap-foot><p>Any plaque, patients with either calcified and/or mixed plaques; <italic>p</italic>, calcified plaque versus mixed plaque.</p></table-wrap-foot></table-wrap></p><p id=\"Par24\">Overall, atherosclerotic plaques were detected in 336 segments; 164 segments in the proximal section of the ascending aorta and 172 segments in the distal section of the ascending aorta. Atherosclerotic disease affected only one segment of the ascending aorta in 50.3% (n = 80/159) of the patients. Based on atherosclerotic plaque distribution, the most frequently affected segments were the proximal left and the distal posterior segment, in 40.9% (n = 65/159) and 39.0% (n = 62/159) of patients, respectively. Data about the number of involved segments and the distribution of atherosclerotic plaques stratified by plaque composition are presented in Table <xref rid=\"Tab4\" ref-type=\"table\">4</xref>.<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Plaque burden, composition, and distribution in patients with atherosclerotic disease of the ascending aorta.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" colspan=\"5\">Patients with atherosclerotic disease in the ascending aorta (n = 159)</th></tr><tr><th align=\"left\"/><th align=\"left\">Any plaque number of patients (%)</th><th align=\"left\">Calcified plaque number of patients (%)</th><th align=\"left\">Mixed plaque number of patients (%)</th><th align=\"left\"><italic>p</italic></th></tr></thead><tbody><tr><td align=\"left\" colspan=\"5\"><bold>Plaque distribution proximal section</bold></td></tr><tr><td align=\"left\">Anterior</td><td align=\"left\">53/159 (33.3)</td><td align=\"left\">43/159 (27.0)</td><td align=\"left\">10/159 (6.3)</td><td char=\".\" align=\"char\" rowspan=\"4\"> < 0.001</td></tr><tr><td align=\"left\">Right</td><td align=\"left\">18/159 (11.3)</td><td align=\"left\">14/159 (8.8)</td><td align=\"left\">4/159 (2.5)</td></tr><tr><td align=\"left\">Left</td><td align=\"left\">65/159 (40.9)</td><td align=\"left\">60/159 (37.7)</td><td align=\"left\">5/159 (3.2)</td></tr><tr><td align=\"left\">Posterior</td><td align=\"left\">28/159 (17.6)</td><td align=\"left\">25/159 (15.7)</td><td align=\"left\">3/159 (1.9)</td></tr><tr><td align=\"left\" colspan=\"5\"><bold>Plaque distribution distal section</bold></td></tr><tr><td align=\"left\">Anterior</td><td align=\"left\">36/159 (22.6)</td><td align=\"left\">24/159 (15.1)</td><td align=\"left\">12/159 (7.5)</td><td char=\".\" align=\"char\" rowspan=\"4\"> < 0.001</td></tr><tr><td align=\"left\">Right</td><td align=\"left\">27/159 (17.0)</td><td align=\"left\">18/159 (11.3)</td><td align=\"left\">9/159 (5.7)</td></tr><tr><td align=\"left\">Left</td><td align=\"left\">47/159 (29.5)</td><td align=\"left\">39/159 (24.5)</td><td align=\"left\">8/159 (5.0)</td></tr><tr><td align=\"left\">Posterior</td><td align=\"left\">62/159 (39.0)</td><td align=\"left\">46/159 (28.9)</td><td align=\"left\">16/159 (10.1)</td></tr><tr><td align=\"left\" colspan=\"5\"><bold>Number of affected segments</bold></td></tr><tr><td align=\"left\">1</td><td align=\"left\">80/159 (50.3)</td><td align=\"left\">82/159 (51.6)</td><td align=\"left\">20/159 (12.6)</td><td char=\".\" align=\"char\" rowspan=\"8\"> < 0.001</td></tr><tr><td align=\"left\">2</td><td align=\"left\">40/159 (25.2)</td><td align=\"left\">29/159 (18.2)</td><td align=\"left\">9/159 (5.7)</td></tr><tr><td align=\"left\">3</td><td align=\"left\">17/159 (10.7)</td><td align=\"left\">17/159 (10.7)</td><td align=\"left\">0/159</td></tr><tr><td align=\"left\">4</td><td align=\"left\">5/159 (3.2)</td><td align=\"left\">3/159 (1.9)</td><td align=\"left\">0/159</td></tr><tr><td align=\"left\">5</td><td align=\"left\">5/159 (3.2)</td><td align=\"left\">4/159 (2.5)</td><td align=\"left\">2/159 (1.3)</td></tr><tr><td align=\"left\">6</td><td align=\"left\">7/159 (4.4)</td><td align=\"left\">5/159 (3.2)</td><td align=\"left\">2/159 (1.3)</td></tr><tr><td align=\"left\">7</td><td align=\"left\">2/159 (1.3)</td><td align=\"left\">0/159</td><td align=\"left\">1/159 (0.6)</td></tr><tr><td align=\"left\">8</td><td align=\"left\">3/159 (1.9)</td><td align=\"left\">2/159 (1.3)</td><td align=\"left\">0/159</td></tr><tr><td align=\"left\">Total</td><td align=\"left\"> = 336 segments</td><td align=\"left\" colspan=\"2\"> = 336 segments</td><td align=\"left\"/></tr></tbody></table><table-wrap-foot><p>Any plaque, patients with either calcified and/or mixed plaques; <italic>p</italic>, calcified versus mixed plaque.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec11\"><title>Perioperative stroke</title><p id=\"Par25\">Of 719 patients studied, 12 patients (1.7%) experienced a perioperative stroke. The incidence of stroke was 7.6% (n = 12/159) in patients with atherosclerotic plaques compared to 0% (n = 0/560) in patients with no plaque (<italic>p</italic> < 0.001). In addition, stroke rates in patients with at least one plaque in mechanically stressed segments were 9.0% (n = 12/133) versus 0% (n = 0/586) in patients without (<italic>p</italic> < 0.001). In addition, we observed a significant association between stroke rate and the number of segments with atherosclerotic plaques (<italic>p</italic> < 0.001). When patients with and without calcified plaques were compared, stroke rates were 3.5% (n = 5/142) versus 1.2% (n = 7/577), which showed no significant difference (<italic>p</italic> = 0.068). Patients with mixed plaques demonstrated significantly higher stroke rates of 20.6% (n = 7/34), compared to 0.7% (n = 5/685) in patients without mixed plaques (<italic>p</italic> < 0.001).</p></sec></sec><sec id=\"Sec12\"><title>Discussion</title><p id=\"Par26\">In the present study we have shown that 22.1% (n = 159/719) of patients referred for CABG had atherosclerotic plaques in the ascending aorta, with calcified plaques significantly more frequently detected than mixed plaques. We observed a significantly higher perioperative stroke rate of 7.6% (n = 12/159) in patients with plaque compared to 0% (n = 0/560) in patients with no plaque in the ascending aorta. In addition, we found an association between plaque composition and stroke rate, which was significantly higher in patients with mixed plaques.</p><p id=\"Par27\">Atherosclerotic disease as an important risk factor for patients who are undergoing CABG mainly affects the distal section of the ascending aorta<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>, which is line with our findings, where the distal section was most frequently affected in 62.3% (n = 99/159) of patients with atherosclerotic disease.</p><p id=\"Par28\">Although intra-operative epi-aortic ultrasound is the most sensitive modality for the detection of atherosclerotic plaques, it is hampered by the lack of opportunity for preoperative planning<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. In this context, the reference method for imaging of the ascending aorta is conventional CTA, which proved to be superior compared to TEE in plaque detection<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. The major strength of CTA is the acquisition of three-dimensional, reproducible, surgeon-friendly images of the vascular anatomy<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. By means of detection and differentiation of composition of plaques, the elimination of pulsation artifacts may be considered to be the key requirement of CTA of the ascending aorta prior CABG. This feature can be achieved with different protocols, including gated non high-pitch, as well as gated or ungated high-pitch scans<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. In this context, ungated high-pitch protocols demonstrated excellent image quality at similar or even lower radiation dose compared with both, gated non high-pitch and high-pitch protocols<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Compared to the use of ECG gating, a further advantage of the ungated high-pitch protocol is time saving, in terms of patient preparation and setting optimal CT parameters<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>.</p><p id=\"Par29\">CABG candidates are frequently suffering from impaired renal function and considered to be at increased risk of contrast-induced nephropathy (CIN)<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. In this context, reduction of the amount of contrast media has been demonstrated for high-pitch compared to conventional CTA<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Although the topic related to the risk of CIN could appear controversial<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>, an optimized scan protocol to reduce any potential risk before CABG, as applied in our study, seems to be mandatory. The feasability and clinical usefullness of a low contrast scan-protocol was demonstrated in several studies focusing on high-pitch CTA prior transcatheter aortic valve implantation<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>.</p><p id=\"Par30\">To our knowledge, this is the first study with the use of ungated high-pitch CTA that addresses plaque composition in the ascending aorta prior to CABG. We could demonstrate that a significantly higher number of patients presented with calcified plaques compared to mixed plaques. This may be linked to the high number of patients (75%) treated with lipid-lowering drugs which can lead to an increase of calcifications in atherosclerotic plaques<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>.</p><p id=\"Par31\">Perioperative stroke is a life-threatening complication after CABG, with an incidence of 1.0–3.6%<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>, which is in line with our results. Stroke risk was found to be dependent on the presence and extent of atherosclerotic disease, with an incidence of 8.7% among patients with atherosclerotic plaques<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>, which is in line with the stroke rate of 7.6% (n = 12/159) in our study. Given the general assumption that perioperative strokes result mainly from cerebral embolization of atheromatous debris and not from calcified material<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>, we could demonstrate a significantly higher stroke rate in patients with mixed plaques.</p><p id=\"Par32\">During cannulation and aortic clamping mechanical manipulation in affected regions poses a risk for detachment of especially non-calcified plaque components and subsequent embolization<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. This mechanism is supported by our significantly higher stroke rate in patients with at least one atherosclerotic plaque in a mechanical stressed segment compared to patients without. In addition, our findings underline the importance of preoperative ungated high-pitch CTA imaging to optimize cannulation and aortic clamping strategies. In this context, several studies could demonstrate a reduction in stroke rates through adequate surgical planning and adaptation of the standard surgical CABG procedure to reduce or avoid mechanical manipulation<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>.</p><p id=\"Par33\">Beside the established vascular risk factors for stroke such as hypertension, dyslipidemia, smoking history, or diabetes mellitus, patients undergoing CABG are additionally burdened by secondary well-known risk factors like prosthetic cardiac valves, low ejection fraction, atrial fibrillation, and carotid stenosis<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. Comparing patients with and without perioperative stroke by means of these variables, our data did not demonstrate significant differences (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>).</p><p id=\"Par34\">The major limitation of our study was its retrospective design. Second, the outcome variable was a clinically evident neurologic deficit confirmed by computed tomography of the brain. No advanced neurocognitive testing was performed that would have allowed for detection of subtle differences in cognitive impairment. Given the fact, that diffusion weighted magnetic resonance imaging is the gold-standard to detect small, transient ischemic lesions<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>, computed tomography-negative, minor neurologic deficits could have been missed. Third, surgeons performed modifications of the surgical strategy at their discretion. The low event rates did not permit a meaningful comparison of stroke rates in patients who did or did not undergo modifications of the CABG procedure. Fourth, the low event rate significantly restricts the use of a multivariable model. Therefore, we are not able to demonstrate a relationship of further variables (e.g. sex, age, dyslipidemia, smoking history, diabetes mellitus, carotid stenosis or history of stroke) with perioperative stroke. Fifth, since eight patients who suffered from stroke presented with atherosclerotic plaques in more than one segment, our data do not allow a statement with respect to the causative diseased segment.</p><p id=\"Par36\">In summary, all patients with perioperative stroke presented with atherosclerotic disease of the ascending aorta. The stroke rate was significantly associated with the presence and extent of atherosclerotic disease. Patients burdened with mixed plaques presented with significantly higher perioperative stroke rates. To our knowledge, this is the first study to demonstrate that detection of plaque extent and composition in the ascending aorta may be helpful to improve risk stratification of stroke in patients prior to CABG. Further prospective studies, evaluating the relationship of atherosclerosis in the ascending aorta and minor neurological deficits related to CABG are warranted.</p></sec></body><back><glossary><title>Abbreviations</title><def-list><def-item><term>CTA</term><def><p id=\"Par2\">Computed tomography angiography</p></def></def-item><def-item><term>CABG</term><def><p id=\"Par3\">Coronary artery bypass grafting</p></def></def-item><def-item><term>TEE</term><def><p id=\"Par4\">Transesophageal echocardiography</p></def></def-item><def-item><term>TTE</term><def><p id=\"Par5\">Transthoracic echocardiography</p></def></def-item><def-item><term>HU</term><def><p id=\"Par6\">Hounsfield unit</p></def></def-item><def-item><term>ECMO</term><def><p id=\"Par7\">Extracorporeal membrane oxygenation</p></def></def-item></def-list></glossary><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>All authors contributed to this manuscript and approved the final manuscript. Study concepts: R.N. and S.E.S Study design: U.A., R.N. and S.E.S. Data acquisition: U.A., S.B.P., T.S., J.F. and S.E.S. Data analysis and interpretation: U.A., R.N., S.B.P., D.Z. and S.E.S. Statistical analysis: L.B. and R.N. Manuscript preparation: U.A. and R.N. Manuscript editing: R.N., J.F., D.Z., C.L., G.L. and S.E.S. Manuscript review: U.A., R.N., S.B.P., T.S., L.B., J.F., C.L., D.Z., G.L., and S.E.S. Manuscript approval: U.A., R.N., S.B.P., T.S., L.B., J.F., C.L., D.Z., G.L., and S.E.S.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>The dataset generated during the current study are available from the corresponding author on reasonable request.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par37\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stamou</surname><given-names>SC</given-names></name><etal/></person-group><article-title>Stroke after coronary artery bypass: incidence, predictors, and clinical outcome</article-title><source>Stroke</source><year>2001</year><volume>32</volume><fpage>1508</fpage><lpage>1513</lpage><pub-id pub-id-type=\"doi\">10.1161/01.STR.32.7.1508</pub-id><pub-id pub-id-type=\"pmid\">11441193</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tarakji</surname><given-names>KG</given-names></name><name><surname>Sabik</surname><given-names>JF</given-names></name><name><surname>Bhudia</surname><given-names>SK</given-names></name><name><surname>Batizy</surname><given-names>LH</given-names></name><name><surname>Blackstone</surname><given-names>EH</given-names></name></person-group><article-title>Temporal onset, risk factors, and outcomes associated with stroke after coronary artery bypass grafting</article-title><source>JAMA</source><year>2011</year><volume>305</volume><fpage>381</fpage><lpage>390</lpage><pub-id pub-id-type=\"doi\">10.1001/jama.2011.37</pub-id><pub-id pub-id-type=\"pmid\">21266685</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dávila-Román</surname><given-names>VG</given-names></name><etal/></person-group><article-title>Atherosclerosis of the ascending aorta. Prevalence and role as an independent predictor of cerebrovascular events in cardiac patients</article-title><source>Stroke</source><year>1994</year><volume>25</volume><fpage>2010</fpage><lpage>2016</lpage><pub-id pub-id-type=\"doi\">10.1161/01.STR.25.10.2010</pub-id><pub-id pub-id-type=\"pmid\">8091446</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hogue</surname><given-names>CW</given-names></name><name><surname>Murphy</surname><given-names>SF</given-names></name><name><surname>Schechtman</surname><given-names>KB</given-names></name><name><surname>Dávila-Román</surname><given-names>VG</given-names></name></person-group><article-title>Risk factors for early or delayed stroke after cardiac surgery</article-title><source>Circulation</source><year>1999</year><volume>100</volume><fpage>642</fpage><lpage>647</lpage><pub-id pub-id-type=\"doi\">10.1161/01.CIR.100.6.642</pub-id><pub-id pub-id-type=\"pmid\">10441102</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Amarenco</surname><given-names>P</given-names></name><etal/></person-group><article-title>The prevalence of ulcerated plaques in the aortic arch in patients with stroke</article-title><source>N. Engl. J. Med.</source><year>1992</year><volume>326</volume><fpage>221</fpage><lpage>225</lpage><pub-id pub-id-type=\"doi\">10.1056/NEJM199201233260402</pub-id><pub-id pub-id-type=\"pmid\">1727976</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kapetanakis</surname><given-names>EI</given-names></name><etal/></person-group><article-title>The impact of aortic manipulation on neurologic outcomes after coronary artery bypass surgery: a risk-adjusted study</article-title><source>Ann. Thorac. Surg.</source><year>2004</year><volume>78</volume><fpage>1564</fpage><lpage>1571</lpage><pub-id pub-id-type=\"doi\">10.1016/j.athoracsur.2004.05.019</pub-id><pub-id pub-id-type=\"pmid\">15511432</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Virmani</surname><given-names>R</given-names></name><name><surname>Burke</surname><given-names>AP</given-names></name><name><surname>Farb</surname><given-names>A</given-names></name><name><surname>Kolodgie</surname><given-names>FD</given-names></name></person-group><article-title>Pathology of the vulnerable plaque</article-title><source>J. Am. Coll. Cardiol.</source><year>2006</year><volume>47</volume><fpage>C13</fpage><lpage>C18</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jacc.2005.10.065</pub-id><pub-id pub-id-type=\"pmid\">16631505</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mann</surname><given-names>JM</given-names></name><name><surname>Davies</surname><given-names>MJ</given-names></name></person-group><article-title>Vulnerable plaque. Relation of characteristics to degree of stenosis in human coronary arteries</article-title><source>Circulation</source><year>1996</year><volume>94</volume><fpage>928</fpage><lpage>931</lpage><pub-id pub-id-type=\"doi\">10.1161/01.CIR.94.5.928</pub-id><pub-id pub-id-type=\"pmid\">8790027</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Park</surname><given-names>K-H</given-names></name><etal/></person-group><article-title>Clinical impact of computerised tomographic angiography performed for preoperative evaluation before coronary artery bypass grafting</article-title><source>Eur. J. Cardiothorac. Surg.</source><year>2010</year><volume>37</volume><fpage>1346</fpage><lpage>1352</lpage><pub-id pub-id-type=\"doi\">10.1016/j.ejcts.2009.12.040</pub-id><pub-id pub-id-type=\"pmid\">20138777</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nakamura</surname><given-names>M</given-names></name><etal/></person-group><article-title>Does intensive management of cerebral hemodynamics and atheromatous aorta reduce stroke after coronary artery surgery?</article-title><source>Ann. Thorac. Surg.</source><year>2008</year><volume>85</volume><fpage>513</fpage><lpage>519</lpage><pub-id pub-id-type=\"doi\">10.1016/j.athoracsur.2007.08.056</pub-id><pub-id pub-id-type=\"pmid\">18222254</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Karlo</surname><given-names>C</given-names></name><etal/></person-group><article-title>High-pitch dual-source CT angiography of the aortic valve-aortic root complex without ECG-synchronization</article-title><source>Eur. Radiol.</source><year>2011</year><volume>21</volume><fpage>205</fpage><lpage>212</lpage><pub-id pub-id-type=\"doi\">10.1007/s00330-010-1907-3</pub-id><pub-id pub-id-type=\"pmid\">20677006</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Beeres</surname><given-names>M</given-names></name><etal/></person-group><article-title>High-pitch dual-source CT angiography of the whole aorta without ECG synchronisation: initial experience</article-title><source>Eur. Radiol.</source><year>2012</year><volume>22</volume><fpage>129</fpage><lpage>137</lpage><pub-id pub-id-type=\"doi\">10.1007/s00330-011-2257-5</pub-id><pub-id pub-id-type=\"pmid\">21915607</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Apfaltrer</surname><given-names>P</given-names></name><etal/></person-group><article-title>Radiation dose and image quality at high-pitch CT angiography of the aorta: intraindividual and interindividual comparisons with conventional CT angiography</article-title><source>AJR Am. J. Roentgenol.</source><year>2012</year><volume>199</volume><fpage>1402</fpage><lpage>1409</lpage><pub-id pub-id-type=\"doi\">10.2214/AJR.12.8652</pub-id><pub-id pub-id-type=\"pmid\">23169737</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wielandner</surname><given-names>A</given-names></name><etal/></person-group><article-title>Is ECG triggering for motion artefact reduction in dual-source CT angiography of the ascending aorta still required with high-pitch scanning? The role of ECG-gating in high-pitch dual-source CT of the ascending aorta</article-title><source>Br. J. Radiol.</source><year>2016</year><volume>89</volume><fpage>20160174</fpage><pub-id pub-id-type=\"doi\">10.1259/bjr.20160174</pub-id><pub-id pub-id-type=\"pmid\">27245139</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nakajima</surname><given-names>S</given-names></name><etal/></person-group><article-title>Clinical application of effective atomic number for classifying non-calcified coronary plaques by dual-energy computed tomography</article-title><source>Atherosclerosis</source><year>2017</year><volume>261</volume><fpage>138</fpage><lpage>143</lpage><pub-id pub-id-type=\"doi\">10.1016/j.atherosclerosis.2017.03.025</pub-id><pub-id pub-id-type=\"pmid\">28372786</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schuhbäck</surname><given-names>A</given-names></name><etal/></person-group><article-title>Interobserver agreement for the detection of atherosclerotic plaque in coronary CT angiography: comparison of two low-dose image acquisition protocols with standard retrospectively ECG-gated reconstruction</article-title><source>Eur. Radiol.</source><year>2012</year><volume>22</volume><fpage>1529</fpage><lpage>1536</lpage><pub-id pub-id-type=\"doi\">10.1007/s00330-012-2389-2</pub-id><pub-id pub-id-type=\"pmid\">22661076</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sandner</surname><given-names>SE</given-names></name><etal/></person-group><article-title>Routine preoperative aortic computed tomography angiography is associated with reduced risk of stroke in coronary artery bypass grafting: a propensity-matched analysis</article-title><source>Eur. J. Cardiothorac. Surg.</source><year>2020</year><volume>57</volume><fpage>684</fpage><lpage>690</lpage><pub-id pub-id-type=\"pmid\">31504374</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Johnson</surname><given-names>WD</given-names></name><name><surname>Flemma</surname><given-names>RJ</given-names></name><name><surname>Lepley</surname><given-names>D</given-names></name><name><surname>Ellison</surname><given-names>EH</given-names></name></person-group><article-title>Extended treatment of severe coronary artery disease: a total surgical approach</article-title><source>Ann. Surg.</source><year>1969</year><volume>170</volume><fpage>460</fpage><lpage>470</lpage><pub-id pub-id-type=\"doi\">10.1097/00000658-196909010-00014</pub-id><pub-id pub-id-type=\"pmid\">5804381</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kronzon</surname><given-names>I</given-names></name><name><surname>Tunick</surname><given-names>PA</given-names></name></person-group><article-title>Aortic atherosclerotic disease and stroke</article-title><source>Circulation</source><year>2006</year><volume>114</volume><fpage>63</fpage><lpage>75</lpage><pub-id pub-id-type=\"doi\">10.1161/CIRCULATIONAHA.105.593418</pub-id><pub-id pub-id-type=\"pmid\">16818829</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Agatston</surname><given-names>AS</given-names></name><etal/></person-group><article-title>Quantification of coronary artery calcium using ultrafast computed tomography</article-title><source>J. Am. Coll. Cardiol.</source><year>1990</year><volume>15</volume><fpage>827</fpage><lpage>832</lpage><pub-id pub-id-type=\"doi\">10.1016/0735-1097(90)90282-T</pub-id><pub-id pub-id-type=\"pmid\">2407762</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Szilveszter</surname><given-names>B</given-names></name><name><surname>Celeng</surname><given-names>C</given-names></name><name><surname>Maurovich-Horvat</surname><given-names>P</given-names></name></person-group><article-title>Plaque assessment by coronary CT</article-title><source>Int. J. Cardiovasc. Imaging</source><year>2016</year><volume>32</volume><fpage>161</fpage><lpage>172</lpage><pub-id pub-id-type=\"doi\">10.1007/s10554-015-0741-8</pub-id><pub-id pub-id-type=\"pmid\">26280890</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van der Linden</surname><given-names>J</given-names></name><name><surname>Hadjinikolaou</surname><given-names>L</given-names></name><name><surname>Bergman</surname><given-names>P</given-names></name><name><surname>Lindblom</surname><given-names>D</given-names></name></person-group><article-title>Postoperative stroke in cardiac surgery is related to the location and extent of atherosclerotic disease in the ascending aorta</article-title><source>J. Am. Coll. Cardiol.</source><year>2001</year><volume>38</volume><fpage>131</fpage><lpage>135</lpage><pub-id pub-id-type=\"doi\">10.1016/S0735-1097(01)01328-6</pub-id><pub-id pub-id-type=\"pmid\">11451262</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rosenberger</surname><given-names>P</given-names></name><etal/></person-group><article-title>The influence of epiaortic ultrasonography on intraoperative surgical management in 6051 cardiac surgical patients</article-title><source>Ann. Thorac. Surg.</source><year>2008</year><volume>85</volume><fpage>548</fpage><lpage>553</lpage><pub-id pub-id-type=\"doi\">10.1016/j.athoracsur.2007.08.061</pub-id><pub-id pub-id-type=\"pmid\">18222262</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chatzikonstantinou</surname><given-names>A</given-names></name><etal/></person-group><article-title>CT angiography of the aorta is superior to transesophageal echocardiography for determining stroke subtypes in patients with cryptogenic ischemic stroke</article-title><source>Cerebrovasc. Dis.</source><year>2012</year><volume>33</volume><fpage>322</fpage><lpage>328</lpage><pub-id pub-id-type=\"doi\">10.1159/000335828</pub-id><pub-id pub-id-type=\"pmid\">22343088</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schernthaner</surname><given-names>RE</given-names></name><etal/></person-group><article-title>Dose modulated retrospective ECG-gated versus non-gated 64-row CT angiography of the aorta at the same radiation dose: comparison of motion artifacts, diagnostic confidence and signal-to-noise-ratios</article-title><source>Eur. J. Radiol.</source><year>2012</year><volume>81</volume><fpage>e585</fpage><lpage>e590</lpage><pub-id pub-id-type=\"doi\">10.1016/j.ejrad.2011.06.053</pub-id><pub-id pub-id-type=\"pmid\">21820829</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cooper</surname><given-names>WA</given-names></name><etal/></person-group><article-title>Impact of renal dysfunction on outcomes of coronary artery bypass surgery: results from the Society of Thoracic Surgeons National Adult Cardiac Database</article-title><source>Circulation</source><year>2006</year><volume>113</volume><fpage>1063</fpage><lpage>1070</lpage><pub-id pub-id-type=\"doi\">10.1161/CIRCULATIONAHA.105.580084</pub-id><pub-id pub-id-type=\"pmid\">16490821</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McDonald</surname><given-names>RJ</given-names></name><etal/></person-group><article-title>Intravenous contrast material-induced nephropathy: causal or coincident phenomenon?</article-title><source>Radiology</source><year>2013</year><volume>267</volume><fpage>106</fpage><lpage>118</lpage><pub-id pub-id-type=\"doi\">10.1148/radiol.12121823</pub-id><pub-id pub-id-type=\"pmid\">23360742</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bittner</surname><given-names>DO</given-names></name><etal/></person-group><article-title>Contrast volume reduction using third generation dual source computed tomography for the evaluation of patients prior to transcatheter aortic valve implantation</article-title><source>Eur. Radiol.</source><year>2016</year><volume>26</volume><fpage>4497</fpage><lpage>4504</lpage><pub-id pub-id-type=\"doi\">10.1007/s00330-016-4320-8</pub-id><pub-id pub-id-type=\"pmid\">26995206</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dankerl</surname><given-names>P</given-names></name><etal/></person-group><article-title>Computer-aided evaluation of low-dose and low-contrast agent third-generation dual-source CT angiography prior to transcatheter aortic valve implantation (TAVI)</article-title><source>Int. J. Comput. Assist. Radiol. Surg.</source><year>2017</year><volume>12</volume><fpage>795</fpage><lpage>802</lpage><pub-id pub-id-type=\"doi\">10.1007/s11548-016-1470-8</pub-id><pub-id pub-id-type=\"pmid\">27604759</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nakazato</surname><given-names>R</given-names></name><etal/></person-group><article-title>Statins use and coronary artery plaque composition: results from the International Multicenter CONFIRM Registry</article-title><source>Atherosclerosis</source><year>2012</year><volume>225</volume><fpage>148</fpage><lpage>153</lpage><pub-id pub-id-type=\"doi\">10.1016/j.atherosclerosis.2012.08.002</pub-id><pub-id pub-id-type=\"pmid\">22981406</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Roach</surname><given-names>GW</given-names></name><etal/></person-group><article-title>Adverse cerebral outcomes after coronary bypass surgery. Multicenter study of perioperative ischemia research group and the ischemia research and education foundation investigators</article-title><source>N. Engl. J. Med.</source><year>1996</year><volume>335</volume><fpage>1857</fpage><lpage>1863</lpage><pub-id pub-id-type=\"doi\">10.1056/NEJM199612193352501</pub-id><pub-id pub-id-type=\"pmid\">8948560</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Filsoufi</surname><given-names>F</given-names></name><name><surname>Rahmanian</surname><given-names>PB</given-names></name><name><surname>Castillo</surname><given-names>JG</given-names></name><name><surname>Bronster</surname><given-names>D</given-names></name><name><surname>Adams</surname><given-names>DH</given-names></name></person-group><article-title>Incidence, topography, predictors and long-term survival after stroke in patients undergoing coronary artery bypass grafting</article-title><source>Ann. Thorac. Surg.</source><year>2008</year><volume>85</volume><fpage>862</fpage><lpage>870</lpage><pub-id pub-id-type=\"doi\">10.1016/j.athoracsur.2007.10.060</pub-id><pub-id pub-id-type=\"pmid\">18291158</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Edelman</surname><given-names>JJ</given-names></name><name><surname>Yan</surname><given-names>TD</given-names></name><name><surname>Bannon</surname><given-names>PG</given-names></name><name><surname>Wilson</surname><given-names>MK</given-names></name><name><surname>Vallely</surname><given-names>MP</given-names></name></person-group><article-title>Coronary artery bypass grafting with and without manipulation of the ascending aorta: a meta-analysis</article-title><source>Heart Lung Circ.</source><year>2011</year><volume>20</volume><fpage>318</fpage><lpage>324</lpage><pub-id pub-id-type=\"doi\">10.1016/j.hlc.2011.02.003</pub-id><pub-id pub-id-type=\"pmid\">21511187</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vallely</surname><given-names>MP</given-names></name><etal/></person-group><article-title>Anaortic techniques reduce neurological morbidity after off-pump coronary artery bypass surgery</article-title><source>Heart Lung Circ.</source><year>2008</year><volume>17</volume><fpage>299</fpage><lpage>304</lpage><pub-id pub-id-type=\"doi\">10.1016/j.hlc.2007.11.138</pub-id><pub-id pub-id-type=\"pmid\">18294911</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Esenwa</surname><given-names>C</given-names></name><name><surname>Gutierrez</surname><given-names>J</given-names></name></person-group><article-title>Secondary stroke prevention: challenges and solutions</article-title><source>Vasc. Health Risk Manag.</source><year>2015</year><volume>11</volume><fpage>437</fpage><lpage>450</lpage><pub-id pub-id-type=\"pmid\">26300647</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gass</surname><given-names>A</given-names></name><name><surname>Ay</surname><given-names>H</given-names></name><name><surname>Szabo</surname><given-names>K</given-names></name><name><surname>Koroshetz</surname><given-names>WJ</given-names></name></person-group><article-title>Diffusion-weighted MRI for the ‘small stuff’: the details of acute cerebral ischaemia</article-title><source>Lancet Neurol.</source><year>2004</year><volume>3</volume><fpage>39</fpage><lpage>45</lpage><pub-id pub-id-type=\"doi\">10.1016/S1474-4422(03)00621-5</pub-id><pub-id pub-id-type=\"pmid\">14693110</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807868</article-id><article-id pub-id-type=\"pmc\">PMC7431557</article-id><article-id pub-id-type=\"publisher-id\">70852</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70852-y</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Effect of image quality fluctuations on the repeatability of thickness measurements in swept-source optical coherence tomography</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Yang</surname><given-names>Heon</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lee</surname><given-names>Hye Sun</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Bae</surname><given-names>Hyoung Won</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Seong</surname><given-names>Gong Je</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kim</surname><given-names>Chan Yun</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Lee</surname><given-names>Sang Yeop</given-names></name><address><email>yeopy@yuhs.ac</email></address><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><aff id=\"Aff1\"><label>1</label>Kong Eye Center, Seoul, Republic of Korea </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.15444.30</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0470 5454</institution-id><institution>Department of Ophthalmology, Institute of Vision Research, Severance Hospital, </institution><institution>Yonsei University College of Medicine, </institution></institution-wrap>Seoul, Republic of Korea </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.15444.30</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0470 5454</institution-id><institution>Department of Ophthalmology, Yongin Severance Hospital, </institution><institution>Yonsei University College of Medicine, </institution></institution-wrap>Yongin, Gyeonggi-do Republic of Korea </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.15444.30</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0470 5454</institution-id><institution>Biostatistics Collaboration Unit, Department of Research Affairs, </institution><institution>Yonsei University College of Medicine, </institution></institution-wrap>Seoul, Republic of Korea </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13897</elocation-id><history><date date-type=\"received\"><day>17</day><month>2</month><year>2020</year></date><date date-type=\"accepted\"><day>23</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">This study investigated the effect of image quality fluctuations on the repeatability of thickness measurements of the peripapillary retinal nerve fibre (PP-RNFL) and ganglion cell-inner plexiform (GC-IPL) layers using swept-source optical coherence tomography (SS-OCT). Three consecutive OCT scans each were performed on 56 healthy subject. Finally, 168 SS-OCT results were analysed. Based on the tertile values of the mean absolute difference of image quality score, all subjects were divided into the following three groups—low-(LIQD), moderate-(MIQD), and high-(HIQD) image quality score difference groups. A linear mixed model and intraclass correlation coefficients (ICCs) were used for analyses. Despite high ICC values (> 0.9), several sectors showed significant differences in the ICC values in intergroup comparisons. For LIQD-HIQD and MIQD-HIQD, most PP-RNFL sectors showed significant differences. For GC-IPL sectors, the LIQD-HIQD comparison showed significant differences in the temporosuperior (<italic>p</italic> = 0.012), inferior (<italic>p</italic> < .001), and temporoinferior (<italic>p</italic> = 0.042) sectors. Significant differences existed in the average GC-IPL (<italic>p</italic> = 0.009), nasoinferior (<italic>p</italic> = 0.035), and inferior GC-IPL sectors (<italic>p</italic> < .001) for MIQD-HIQD comparison. With higher image quality fluctuations, the repeatability of SS-OCT decreased in several sectors, which are considered clinically relevant in evaluating glaucoma status. Therefore, maintaining high-quality image status is essential to enhance the reliability of SS-OCT.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Diseases</kwd><kwd>Eye diseases</kwd><kwd>Optic nerve diseases</kwd><kwd>Medical research</kwd><kwd>Outcomes research</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Optical coherence tomography (OCT) is an indispensable ophthalmic imaging technology that effectively identifies retinal structural alterations. OCT technologies have undergone longitudinal development from time-domain OCT to spectral-domain OCT (SD-OCT) and swept-source OCT (SS-OCT). The recently developed SS-OCT uses a tunable light source with a central wavelength of 1,050 nm, and a photodiode detector with a semiconductor camera for light detection. These features permit a high scanning speed and a deep imaging range with uniform sensitivity. In glaucoma cases, these technological advances in OCT device have eased the measurement of changes in the peripapillary retinal nerve fibre layer (PP-RNFL) and ganglion cell-inner plexiform layer (GC-IPL) thickness. Both these layers are critical to evaluate the extent of damage of the glaucomatous optic nerve.</p><p id=\"Par3\">Diagnostic precision is of utmost importance when diagnosing a disease or monitoring its progression using OCT. Both image quality and repeatability/reproducibility of an OCT measurement affect its overall diagnostic precision. Segmentation error and misalignment of measurement area generate artefacts that affect the image quality and, ultimately, the OCT measurement values<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. Repeatability and reproducibility relate to the scatter of measured values and indicate whether a constant value is obtained when the same object is measured repeatedly. These parameters are helpful to monitor disease progression because repeated measurements are performed over time at the same anatomical region of an individual patient. Both SD-OCT and SS-OCT have demonstrated repeatability and reproducibility for clinical use<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>–<xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>, which is an important reason for the widespread use of OCT in the diagnosis and management of various ocular conditions, including glaucoma.</p><p id=\"Par4\">Although image quality and repeatability/reproducibility of OCT images are important factors when interpreting the results, only few studies have previously investigated the effect of image quality fluctuations on repeatability or reproducibility<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. Moreover, these studies were implemented using time-domain OCT or SD-OCT at the peripapillary area. Therefore, this study aimed to evaluate the effect of image quality fluctuations on the repeatability of SS-OCT measurement values in both the macular and peripapillary areas. The results of this study indicate the importance of maintaining image quality in SS-OCT while performing repeated measurements.</p></sec><sec id=\"Sec2\"><title>Results</title><p id=\"Par5\">Of the 58 healthy subjects who were selected for OCT imaging, two were excluded based on their image quality scores. SS-OCT data of 56 subjects (25 men and 31 women), comprising 168 results from the three consecutive OCT examinations, were analysed. Based on the tertile values of the mean absolute difference of image quality score, the subjects were stratified into three groups—low image quality score difference group (LIQD; with scores ranging between 0.06 and 0.86 in PP-RNFL, and between 0.067 and 0.747 in GC-IPL), moderate image quality score difference group (MIQD; with scores ranging between 0.873 and 1.927 in PP-RNFL, and between 0.753 and 1.227 in GC-IPL), and high image quality score difference group (HIQD; with scores ranging between 1.947 and 10.053 in PP-RNFL, and between 1.253 and 8.3 in GC-IPL). The three groups showed no significant differences in their demographic or clinical characteristics (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>).<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Comparison of demographics and clinical characteristics among groups.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">All subjects</th><th align=\"left\">LIQD</th><th align=\"left\">MIQD</th><th align=\"left\">HIQD</th><th align=\"left\"><italic>p</italic> value*</th></tr></thead><tbody><tr><td align=\"left\">Age, years</td><td align=\"left\">53.31 ± 15.92</td><td align=\"left\">51.52 ± 14.89</td><td align=\"left\">54.71 ± 11.36</td><td align=\"left\">53.83 ± 17.59</td><td char=\".\" align=\"char\">0.325</td></tr><tr><td align=\"left\">Sex (M:F)</td><td align=\"left\">26:30</td><td align=\"left\">10:8</td><td align=\"left\">7:12</td><td align=\"left\">9:10</td><td char=\".\" align=\"char\">0.223</td></tr><tr><td align=\"left\">Central corneal thickness, µm</td><td align=\"left\">542.22 ± 33.72</td><td align=\"left\">538.37 ± 28.64</td><td align=\"left\">542.79 ± 31.45</td><td align=\"left\">545.54 ± 23.61</td><td char=\".\" align=\"char\">0.641</td></tr><tr><td align=\"left\">Spherical equivalent, D</td><td align=\"left\">1.35 ± 2.17</td><td align=\"left\">0.87 ± 3.36</td><td align=\"left\">1.42 ± 2.26</td><td align=\"left\">1.76 ± 1.14</td><td char=\".\" align=\"char\">0.623</td></tr><tr><td align=\"left\">Axial length, mm</td><td align=\"left\">23.17 ± 1.2</td><td align=\"left\">23.05 ± 1.17</td><td align=\"left\">23.11 ± 1.52</td><td align=\"left\">23.35 ± 0.77</td><td char=\".\" align=\"char\">0.594</td></tr><tr><td align=\"left\">Intraocular pressure, mmHg</td><td align=\"left\">14.42 ± 2.84</td><td align=\"left\">13.82 ± 3.46</td><td align=\"left\">15.56 ± 2.63</td><td align=\"left\">13.88 ± 2.44</td><td char=\".\" align=\"char\">0.076</td></tr></tbody></table><table-wrap-foot><p><italic>LIQD</italic> low image quality difference group, <italic>MIQD</italic> moderate image quality difference group, <italic>HIQD</italic> high image quality difference group, <italic>SD</italic> standard deviation.</p><p>*Analysis of variance or chi-square test; all values are represented as mean ± SD or ratio.</p></table-wrap-foot></table-wrap></p><sec id=\"Sec3\"><title>Comparison of PP-RNFL and GC-IPL thicknesses among the three groups</title><p id=\"Par6\">Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref> shows results for the comparison of PP-RNFL and GC-IPL thicknesses among the three groups at each measurement sector. The linear mixed model showed no significant differences in PP-RNFL and GC-IPL thicknesses of different sectors among the three groups (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>). However, when the difference in image quality between OCT examinations was large, GC-IPL tended to be thick; this tendency was not seen in the peripapillary sectors.<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Comparison of thickness values measured using SS-OCT among the three groups.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">LIQD</th><th align=\"left\">MIQD</th><th align=\"left\">HIQD</th><th align=\"left\">Overall <italic>p</italic>*</th></tr></thead><tbody><tr><td align=\"left\">PPAver</td><td char=\"±\" align=\"char\">105.76 ± 2.01</td><td char=\"±\" align=\"char\">105.95 ± 1.96</td><td char=\"±\" align=\"char\">109.67 ± 1.96</td><td char=\".\" align=\"char\">0.294</td></tr><tr><td align=\"left\">4 T</td><td char=\"±\" align=\"char\">78.96 ± 2.84</td><td char=\"±\" align=\"char\">80.66 ± 2.77</td><td char=\"±\" align=\"char\">77.33 ± 2.77</td><td char=\".\" align=\"char\">0.697</td></tr><tr><td align=\"left\">4 S</td><td char=\"±\" align=\"char\">136.21 ± 3.258</td><td char=\"±\" align=\"char\">136.4 ± 3.17</td><td char=\"±\" align=\"char\">136.3 ± 3.17</td><td char=\".\" align=\"char\">0.999</td></tr><tr><td align=\"left\">4 N</td><td char=\"±\" align=\"char\">70.04 ± 3.788</td><td char=\"±\" align=\"char\">70.59 ± 3.687</td><td char=\"±\" align=\"char\">74.99 ± 3.69</td><td char=\".\" align=\"char\">0.589</td></tr><tr><td align=\"left\">4 I</td><td char=\"±\" align=\"char\">136 ± 3.284</td><td char=\"±\" align=\"char\">136.37 ± 3.197</td><td char=\"±\" align=\"char\">144.49 ± 3.19</td><td char=\".\" align=\"char\">0.117</td></tr><tr><td align=\"left\">12 T</td><td char=\"±\" align=\"char\">66.779 ± 2.26</td><td char=\"±\" align=\"char\">67.612 ± 2.2</td><td char=\"±\" align=\"char\">66.69 ± 2.2</td><td char=\".\" align=\"char\">0.948</td></tr><tr><td align=\"left\">12 TS</td><td char=\"±\" align=\"char\">92.523 ± 3.395</td><td char=\"±\" align=\"char\">92.407 ± 3.305</td><td char=\"±\" align=\"char\">95.93 ± 3.31</td><td char=\".\" align=\"char\">0.698</td></tr><tr><td align=\"left\">12 ST</td><td char=\"±\" align=\"char\">141.94 ± 5.299</td><td char=\"±\" align=\"char\">143.21 ± 5.158</td><td char=\"±\" align=\"char\">144.86 ± 5.16</td><td char=\".\" align=\"char\">0.925</td></tr><tr><td align=\"left\">12 S</td><td char=\"±\" align=\"char\">142.89 ± 5.881</td><td char=\"±\" align=\"char\">143.9 ± 5.724</td><td char=\"±\" align=\"char\">137.94 ± 5.72</td><td char=\".\" align=\"char\">0.736</td></tr><tr><td align=\"left\">12 SN</td><td char=\"±\" align=\"char\">125.81 ± 5.51</td><td char=\"±\" align=\"char\">124.17 ± 5.363</td><td char=\"±\" align=\"char\">126.54 ± 5.36</td><td char=\".\" align=\"char\">0.95</td></tr><tr><td align=\"left\">12 NS</td><td char=\"±\" align=\"char\">80.382 ± 4.685</td><td char=\"±\" align=\"char\">79.865 ± 4.56</td><td char=\"±\" align=\"char\">81.61 ± 4.56</td><td char=\".\" align=\"char\">0.962</td></tr><tr><td align=\"left\">12 N</td><td char=\"±\" align=\"char\">59.553 ± 2.879</td><td char=\"±\" align=\"char\">59.097 ± 2.802</td><td char=\"±\" align=\"char\">62.89 ± 2.8</td><td char=\".\" align=\"char\">0.584</td></tr><tr><td align=\"left\">12 NI</td><td char=\"±\" align=\"char\">69.555 ± 4.568</td><td char=\"±\" align=\"char\">71.495 ± 4.446</td><td char=\"±\" align=\"char\">78.49 ± 4.45</td><td char=\".\" align=\"char\">0.339</td></tr><tr><td align=\"left\">12 IN</td><td char=\"±\" align=\"char\">114.12 ± 4.993</td><td char=\"±\" align=\"char\">106.72 ± 4.86</td><td char=\"±\" align=\"char\">114.37 ± 4.86</td><td char=\".\" align=\"char\">0.458</td></tr><tr><td align=\"left\">12 I</td><td char=\"±\" align=\"char\">150.88 ± 5.699</td><td char=\"±\" align=\"char\">143.38 ± 5.547</td><td char=\"±\" align=\"char\">158.22 ± 5.55</td><td char=\".\" align=\"char\">0.177</td></tr><tr><td align=\"left\">12 IT</td><td char=\"±\" align=\"char\">144.88 ± 6.09</td><td char=\"±\" align=\"char\">155.52 ± 5.927</td><td char=\"±\" align=\"char\">151.35 ± 5.93</td><td char=\".\" align=\"char\">0.458</td></tr><tr><td align=\"left\">12 TI</td><td char=\"±\" align=\"char\">78.132 ± 3.722</td><td char=\"±\" align=\"char\">80.993 ± 3.623</td><td char=\"±\" align=\"char\">76.8 ± 3.62</td><td char=\".\" align=\"char\">0.707</td></tr><tr><td align=\"left\">GCLAver</td><td char=\"±\" align=\"char\">69.604 ± 1.206</td><td char=\"±\" align=\"char\">70.089 ± 1.174</td><td char=\"±\" align=\"char\">71.402 ± 1.174</td><td char=\".\" align=\"char\">0.544</td></tr><tr><td align=\"left\">TS</td><td char=\"±\" align=\"char\">71.632 ± 1.215</td><td char=\"±\" align=\"char\">72.165 ± 1.183</td><td char=\"±\" align=\"char\">72.685 ± 1.183</td><td char=\".\" align=\"char\">0.825</td></tr><tr><td align=\"left\">S</td><td char=\"±\" align=\"char\">68.888 ± 1.287</td><td char=\"±\" align=\"char\">68.93 ± 1.252</td><td char=\"±\" align=\"char\">70.747 ± 1.252</td><td char=\".\" align=\"char\">0.496</td></tr><tr><td align=\"left\">NS</td><td char=\"±\" align=\"char\">71.983 ± 1.44</td><td char=\"±\" align=\"char\">72.076 ± 1.402</td><td char=\"±\" align=\"char\">75.057 ± 1.402</td><td char=\".\" align=\"char\">0.224</td></tr><tr><td align=\"left\">NI</td><td char=\"±\" align=\"char\">69.546 ± 1.379</td><td char=\"±\" align=\"char\">69.868 ± 1.342</td><td char=\"±\" align=\"char\">71.046 ± 1.342</td><td char=\".\" align=\"char\">0.713</td></tr><tr><td align=\"left\">I</td><td char=\"±\" align=\"char\">64.464 ± 1.181</td><td char=\"±\" align=\"char\">65.254 ± 1.149</td><td char=\"±\" align=\"char\">65.903 ± 1.149</td><td char=\".\" align=\"char\">0.685</td></tr><tr><td align=\"left\">TI</td><td char=\"±\" align=\"char\">71.16 ± 1.358</td><td char=\"±\" align=\"char\">73.046 ± 1.322</td><td char=\"±\" align=\"char\">73.133 ± 1.322</td><td char=\".\" align=\"char\">0.508</td></tr></tbody></table><table-wrap-foot><p><italic>SS-OCT</italic> swept-source optical coherence tomography, <italic>LIQD</italic> low image quality difference group, <italic>MIQD</italic> moderate image quality difference group, <italic>HIQD</italic> high image quality difference group, <italic>SE</italic> standard error, <italic>PPAver</italic> average PP-RNFL thickness, <italic>T</italic> temporal, <italic>S</italic> superior, <italic>N</italic> nasal, <italic>I</italic> inferior, <italic>TS</italic> temporosuperior, <italic>ST</italic> superotemporal, <italic>SN</italic> superonasal, <italic>NS</italic> nasosuperior, <italic>NI</italic> nasoinferior, <italic>IN</italic> inferonasal, <italic>IT</italic> inferotemporal, <italic>TI</italic> temporoinferior, <italic>GCLAver</italic> average GC-IPL thickness.</p><p>*Linear mixed model; all values are represented as least-squares mean ± SE.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec4\"><title>Correlations between image quality and SS-OCT results at each measurement sector</title><p id=\"Par7\">Correlation analyses between image quality and OCT results at each measurement sector were performed for repeated measurements (Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref>). After adjusting for age and sex, five sectors showed significant negative correlations between image quality and PP-RNFL (average PP-RNFL, superotemporal, superior, inferior, and temporoinferior sectors) or GC-IPL (average GC-IPL, temporosuperior, nasoinferior, inferior, and temporoinferior sectors).<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Correlations and partial correlations between image quality and SS-OCT results.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\" colspan=\"2\">Correlation*</th><th align=\"left\" colspan=\"2\">Partial correlation<sup>†</sup></th></tr><tr><th align=\"left\">R</th><th align=\"left\"><italic>p</italic></th><th align=\"left\">r</th><th align=\"left\"><italic>p</italic></th></tr></thead><tbody><tr><td align=\"left\">PPAver</td><td char=\".\" align=\"char\"> − 0.258</td><td char=\".\" align=\"char\"><bold>0.001</bold></td><td char=\".\" align=\"char\"> − 0.225</td><td char=\".\" align=\"char\"><bold>0.004</bold></td></tr><tr><td align=\"left\">4 T</td><td char=\".\" align=\"char\"> − 0.054</td><td char=\".\" align=\"char\">0.489</td><td char=\".\" align=\"char\"> − 0.089</td><td char=\".\" align=\"char\">0.254</td></tr><tr><td align=\"left\">4 S</td><td char=\".\" align=\"char\"> − 0.151</td><td char=\".\" align=\"char\"><bold>0.049</bold></td><td char=\".\" align=\"char\"> − 0.147</td><td char=\".\" align=\"char\">0.058</td></tr><tr><td align=\"left\">4 N</td><td char=\".\" align=\"char\"> − 0.144</td><td char=\".\" align=\"char\">0.062</td><td char=\".\" align=\"char\"> − 0.061</td><td char=\".\" align=\"char\">0.436</td></tr><tr><td align=\"left\">4 I</td><td char=\".\" align=\"char\"> − 0.135</td><td char=\".\" align=\"char\">0.08</td><td char=\".\" align=\"char\"> − 0.113</td><td char=\".\" align=\"char\">0.147</td></tr><tr><td align=\"left\">12 T</td><td char=\".\" align=\"char\">0.02</td><td char=\".\" align=\"char\">0.795</td><td char=\".\" align=\"char\"> − 0.012</td><td char=\".\" align=\"char\">0.878</td></tr><tr><td align=\"left\">12 TS</td><td char=\".\" align=\"char\">0.031</td><td char=\".\" align=\"char\">0.691</td><td char=\".\" align=\"char\">0.025</td><td char=\".\" align=\"char\">0.753</td></tr><tr><td align=\"left\">12 ST</td><td char=\".\" align=\"char\"> − 0.137</td><td char=\".\" align=\"char\">0.077</td><td char=\".\" align=\"char\"> − 0.169</td><td char=\".\" align=\"char\"><bold>0.029</bold></td></tr><tr><td align=\"left\">12 S</td><td char=\".\" align=\"char\"> − 0.156</td><td char=\".\" align=\"char\"><bold>0.043</bold></td><td char=\".\" align=\"char\"> − 0.155</td><td char=\".\" align=\"char\"><bold>0.047</bold></td></tr><tr><td align=\"left\">12 SN</td><td char=\".\" align=\"char\">0.039</td><td char=\".\" align=\"char\">0.619</td><td char=\".\" align=\"char\">0.071</td><td char=\".\" align=\"char\">0.364</td></tr><tr><td align=\"left\">12 NS</td><td char=\".\" align=\"char\"> − 0.087</td><td char=\".\" align=\"char\">0.263</td><td char=\".\" align=\"char\"> − 0.026</td><td char=\".\" align=\"char\">0.738</td></tr><tr><td align=\"left\">12 N</td><td char=\".\" align=\"char\">0.016</td><td char=\".\" align=\"char\">0.834</td><td char=\".\" align=\"char\">0.129</td><td char=\".\" align=\"char\">0.099</td></tr><tr><td align=\"left\">12 NI</td><td char=\".\" align=\"char\"> − 0.178</td><td char=\".\" align=\"char\"><bold>0.021</bold></td><td char=\".\" align=\"char\"> − 0.094</td><td char=\".\" align=\"char\">0.227</td></tr><tr><td align=\"left\">12 IN</td><td char=\".\" align=\"char\"> − 0.191</td><td char=\".\" align=\"char\"><bold>0.013</bold></td><td char=\".\" align=\"char\"> − 0.148</td><td char=\".\" align=\"char\">0.058</td></tr><tr><td align=\"left\">12 I</td><td char=\".\" align=\"char\"> − 0.187</td><td char=\".\" align=\"char\"><bold>0.015</bold></td><td char=\".\" align=\"char\"> − 0.175</td><td char=\".\" align=\"char\"><bold>0.024</bold></td></tr><tr><td align=\"left\">12 IT</td><td char=\".\" align=\"char\"> − 0.018</td><td char=\".\" align=\"char\">0.821</td><td char=\".\" align=\"char\"> − 0.03</td><td char=\".\" align=\"char\">0.697</td></tr><tr><td align=\"left\">12 TI</td><td char=\".\" align=\"char\">0.254</td><td char=\".\" align=\"char\"><bold>0.001</bold></td><td char=\".\" align=\"char\">0.234</td><td char=\".\" align=\"char\"><bold>0.002</bold></td></tr><tr><td align=\"left\">GCLAver</td><td char=\".\" align=\"char\"> − 0.176</td><td char=\".\" align=\"char\"><bold>0.023</bold></td><td char=\".\" align=\"char\"> − 0.186</td><td char=\".\" align=\"char\"><bold>0.017</bold></td></tr><tr><td align=\"left\">TS</td><td char=\".\" align=\"char\"> − 0.181</td><td char=\".\" align=\"char\"><bold>0.019</bold></td><td char=\".\" align=\"char\"> − 0.175</td><td char=\".\" align=\"char\"><bold>0.025</bold></td></tr><tr><td align=\"left\">S</td><td char=\".\" align=\"char\"> − 0.113</td><td char=\".\" align=\"char\">0.146</td><td char=\".\" align=\"char\"> − 0.123</td><td char=\".\" align=\"char\">0.114</td></tr><tr><td align=\"left\">NS</td><td char=\".\" align=\"char\"> − 0.115</td><td char=\".\" align=\"char\">0.137</td><td char=\".\" align=\"char\"> − 0.134</td><td char=\".\" align=\"char\">0.086</td></tr><tr><td align=\"left\">NI</td><td char=\".\" align=\"char\"> − 0.169</td><td char=\".\" align=\"char\"><bold>0.029</bold></td><td char=\".\" align=\"char\"> − 0.189</td><td char=\".\" align=\"char\"><bold>0.015</bold></td></tr><tr><td align=\"left\">I</td><td char=\".\" align=\"char\"> − 0.192</td><td char=\".\" align=\"char\"><bold>0.013</bold></td><td char=\".\" align=\"char\"> − 0.207</td><td char=\".\" align=\"char\"><bold>0.008</bold></td></tr><tr><td align=\"left\">TI</td><td char=\".\" align=\"char\"> − 0.193</td><td char=\".\" align=\"char\"><bold>0.012</bold></td><td char=\".\" align=\"char\"> − 0.19</td><td char=\".\" align=\"char\"><bold>0.014</bold></td></tr></tbody></table><table-wrap-foot><p><italic>SS-OCT</italic> swept-source optical coherence tomography, <italic>PPAver</italic> average PP-RNFL thickness, <italic>T</italic> temporal, <italic>S</italic> superior, <italic>N</italic> nasal, <italic>I</italic> inferior, TS, temporosuperior, <italic>ST</italic> superotemporal, <italic>SN</italic> superonasal, <italic>NS</italic> nasosuperior, <italic>NI</italic> nasoinferior, <italic>IN</italic> inferonasal, <italic>IT</italic> inferotemporal, <italic>TI</italic> temporoinferior, <italic>GCLAver</italic> average GC-IPL thickness.</p><p>*Pearson’s correlation estimated using a linear mixed model; <sup>†</sup>Partial correlation after age- and sex-adjustments; significant <italic>p</italic> values are shown as bold-face text.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec5\"><title>Comparisons of repeatability among the three groups at each measurement sector</title><p id=\"Par8\">ICC of three consecutive measurement values was calculated and compared among the groups (Table <xref rid=\"Tab4\" ref-type=\"table\">4</xref>). The overall repeatability was high in all sectors for all groups (ICC > 0.8). The ICC values were the lowest for the HIQD group in every measurement sector. Figure <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> shows the representative results for difference in thickness at each measurement sectors of PP-RNFL by image quality difference. With increase in the image quality difference value, the difference between the measured values increased accordingly. Results of between-group comparisons showed significant differences in repeatability at only two sectors (temporoinferior for PP-RNFL; inferior for GC-IPL) in the LIQD and MIQD groups. In addition, results of comparisons between LIQD and HIQD groups, and between MIQD and HIQD groups, showed significant differences in repeatability at most sectors for PP-RNFL, except at the superior, nasal, superior nasal, and nasoinferior sectors. On comparison of repeatability in GC-IPL sectors, significant differences were seen at the temporosuperior, inferior, and temporoinferior sectors between LIQD and HIQD groups, and at the average GC-IPL, nasoinferior, and inferior sectors between MIQD and HIQD groups. No sector showed significant differences in repeatability when compared between LIQD and MIQD groups. The proportion of sectors affected by image quality fluctuations was higher in PP-RNFL than in GC-IPL.<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Comparison of repeatability among the three groups.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\" rowspan=\"2\">LIQD n = 18</th><th align=\"left\" rowspan=\"2\">MIQD n = 19</th><th align=\"left\" rowspan=\"2\">HIQD n = 19</th><th align=\"left\" colspan=\"3\"><italic>p</italic> value*</th></tr><tr><th align=\"left\">LIQD versus MIQD</th><th align=\"left\">LIQD versus HIQD</th><th align=\"left\">MIQD versus HIQD</th></tr></thead><tbody><tr><td align=\"left\">PPAver</td><td char=\"(\" align=\"char\">0.996 (0.992–0.999)</td><td char=\"(\" align=\"char\">0.996 (0.992–0.998)</td><td char=\"(\" align=\"char\">0.964 (0.925–0.985)</td><td char=\".\" align=\"char\">> .999</td><td char=\".\" align=\"char\"><bold>0.002</bold></td><td char=\".\" align=\"char\"><bold>0.001</bold></td></tr><tr><td align=\"left\">4 T</td><td char=\"(\" align=\"char\">0.996 (0.992–0.998)</td><td char=\"(\" align=\"char\">0.996 (0.992–0.998)</td><td char=\"(\" align=\"char\">0.982 (0.962–0.992)</td><td char=\".\" align=\"char\">> .999</td><td char=\".\" align=\"char\"><bold>0.03</bold></td><td char=\".\" align=\"char\"><bold>0.028</bold></td></tr><tr><td align=\"left\">4 S</td><td char=\"(\" align=\"char\">0.99 (0.978–0.996)</td><td char=\"(\" align=\"char\">0.988 (0.974–0.995)</td><td char=\"(\" align=\"char\">0.978 (0.954–0.991)</td><td char=\".\" align=\"char\">0.792</td><td char=\".\" align=\"char\">0.254</td><td char=\".\" align=\"char\">0.373</td></tr><tr><td align=\"left\">4 N</td><td char=\"(\" align=\"char\">0.996 (0.99–0.998)</td><td char=\"(\" align=\"char\">0.998 (0.996–0.999)</td><td char=\"(\" align=\"char\">0.993 (0.985–0.997)</td><td char=\".\" align=\"char\">0.319</td><td char=\".\" align=\"char\">0.421</td><td char=\".\" align=\"char\">0.067</td></tr><tr><td align=\"left\">4 I</td><td char=\"(\" align=\"char\">0.989 (0.976–0.995)</td><td char=\"(\" align=\"char\">0.99 (0.978–0.996)</td><td char=\"(\" align=\"char\">0.956 (0.906–0.982)</td><td char=\".\" align=\"char\">0.891</td><td char=\".\" align=\"char\"><bold>0.044</bold></td><td char=\".\" align=\"char\"><bold>0.029</bold></td></tr><tr><td align=\"left\">12 T</td><td char=\"(\" align=\"char\">0.995 (0.99–0.998)</td><td char=\"(\" align=\"char\">0.996 (0.992–0.998)</td><td char=\"(\" align=\"char\">0.956 (0.906–0.982)</td><td char=\".\" align=\"char\">0.748</td><td char=\".\" align=\"char\"><bold>0.002</bold></td><td char=\".\" align=\"char\"><bold>< .001</bold></td></tr><tr><td align=\"left\">12 TS</td><td char=\"(\" align=\"char\">0.99 (0.978–0.996)</td><td char=\"(\" align=\"char\">0.994 (0.987–0.997)</td><td char=\"(\" align=\"char\">0.956 (0.907–0.982)</td><td char=\".\" align=\"char\">0.462</td><td char=\".\" align=\"char\"><bold>0.031</bold></td><td char=\".\" align=\"char\"><bold>0.003</bold></td></tr><tr><td align=\"left\">12 ST</td><td char=\"(\" align=\"char\">0.977 (0.95–0.991)</td><td char=\"(\" align=\"char\">0.995 (0.989–0.998)</td><td char=\"(\" align=\"char\">0.878 (0.741–0.949)</td><td char=\".\" align=\"char\"><bold>0.028</bold></td><td char=\".\" align=\"char\"><bold>0.014</bold></td><td char=\".\" align=\"char\"><bold>< .001</bold></td></tr><tr><td align=\"left\">12 S</td><td char=\"(\" align=\"char\">0.993 (0.984–0.997)</td><td char=\"(\" align=\"char\">0.973 (0.944–0.989)</td><td char=\"(\" align=\"char\">0.965 (0.927–0.986)</td><td char=\".\" align=\"char\">0.051</td><td char=\".\" align=\"char\"><bold>0.02</bold></td><td char=\".\" align=\"char\">0.701</td></tr><tr><td align=\"left\">12 SN</td><td char=\"(\" align=\"char\">0.98 (0.957–0.992)</td><td char=\"(\" align=\"char\">0.987 (0.973–0.995)</td><td char=\"(\" align=\"char\">0.964 (0.923–0.985)</td><td char=\".\" align=\"char\">0.533</td><td char=\".\" align=\"char\">0.392</td><td char=\".\" align=\"char\">0.133</td></tr><tr><td align=\"left\">12 NS</td><td char=\"(\" align=\"char\">0.985 (0.967–0.994)</td><td char=\"(\" align=\"char\">0.996 (0.992–0.998)</td><td char=\"(\" align=\"char\">0.966 (0.928–0.986)</td><td char=\".\" align=\"char\">0.057</td><td char=\".\" align=\"char\">0.235</td><td char=\".\" align=\"char\"><bold>0.002</bold></td></tr><tr><td align=\"left\">12 N</td><td char=\"(\" align=\"char\">0.997 (0.993–0.999)</td><td char=\"(\" align=\"char\">0.997 (0.993–0.999)</td><td char=\"(\" align=\"char\">0.899 (0.784–0.958)</td><td char=\".\" align=\"char\">> .999</td><td char=\".\" align=\"char\"><bold>< .001</bold></td><td char=\".\" align=\"char\"><bold>< .001</bold></td></tr><tr><td align=\"left\">12 NI</td><td char=\"(\" align=\"char\">0.995 (0.989–0.998)</td><td char=\"(\" align=\"char\">0.99 (0.979–0.996)</td><td char=\"(\" align=\"char\">0.981 (0.959–0.992)</td><td char=\".\" align=\"char\">0.318</td><td char=\".\" align=\"char\">0.054</td><td char=\".\" align=\"char\">0.346</td></tr><tr><td align=\"left\">12 IN</td><td char=\"(\" align=\"char\">0.976 (0.948–0.99)</td><td char=\"(\" align=\"char\">0.988 (0.975–0.995)</td><td char=\"(\" align=\"char\">0.88 (0.745–0.95)</td><td char=\".\" align=\"char\">0.316</td><td char=\".\" align=\"char\"><bold>0.017</bold></td><td char=\".\" align=\"char\"><bold>< .001</bold></td></tr><tr><td align=\"left\">12 I</td><td char=\"(\" align=\"char\">0.987 (0.972–0.995)</td><td char=\"(\" align=\"char\">0.992 (0.983–0.997)</td><td char=\"(\" align=\"char\">0.965 (0.925–0.985)</td><td char=\".\" align=\"char\">0.484</td><td char=\".\" align=\"char\">0.151</td><td char=\".\" align=\"char\"><bold>0.03</bold></td></tr><tr><td align=\"left\">12 IT</td><td char=\"(\" align=\"char\">0.99 (0.979–0.996)</td><td char=\"(\" align=\"char\">0.972 (0.941–0.988)</td><td char=\"(\" align=\"char\">0.869 (0.721–0.945)</td><td char=\".\" align=\"char\">0.136</td><td char=\".\" align=\"char\"><bold>< .001</bold></td><td char=\".\" align=\"char\"><bold>0.02</bold></td></tr><tr><td align=\"left\">12 TI</td><td char=\"(\" align=\"char\">0.996 (0.992–0.998)</td><td char=\"(\" align=\"char\">0.984 (0.967–0.994)</td><td char=\"(\" align=\"char\">0.829 (0.637–0.929)</td><td char=\".\" align=\"char\"><bold>0.046</bold></td><td char=\".\" align=\"char\"><bold>< .001</bold></td><td char=\".\" align=\"char\"><bold>< .001</bold></td></tr><tr><td align=\"left\">GCLAver</td><td char=\"(\" align=\"char\">0.997 (0.994–0.999)</td><td char=\"(\" align=\"char\">0.999 (0.998–1)</td><td char=\"(\" align=\"char\">0.994 (0.986–0.997)</td><td char=\".\" align=\"char\">0.115</td><td char=\".\" align=\"char\">0.319</td><td char=\".\" align=\"char\"><bold>0.009</bold></td></tr><tr><td align=\"left\">TS</td><td char=\"(\" align=\"char\">0.997 (0.993–0.999)</td><td char=\"(\" align=\"char\">0.991 (0.98–0.996)</td><td char=\"(\" align=\"char\">0.983 (0.964–0.993)</td><td char=\".\" align=\"char\">0.114</td><td char=\".\" align=\"char\"><bold>0.012</bold></td><td char=\".\" align=\"char\">0.351</td></tr><tr><td align=\"left\">S</td><td char=\"(\" align=\"char\">0.998 (0.996–0.999)</td><td char=\"(\" align=\"char\">0.996 (0.992–0.998)</td><td char=\"(\" align=\"char\">0.993 (0.985–0.997)</td><td char=\".\" align=\"char\">0.329</td><td char=\".\" align=\"char\">0.072</td><td char=\".\" align=\"char\">0.413</td></tr><tr><td align=\"left\">NS</td><td char=\"(\" align=\"char\">0.999 (0.997–0.999)</td><td char=\"(\" align=\"char\">0.998 (0.996–0.999)</td><td char=\"(\" align=\"char\">0.997 (0.994–0.999)</td><td char=\".\" align=\"char\">0.319</td><td char=\".\" align=\"char\">0.115</td><td char=\".\" align=\"char\">0.554</td></tr><tr><td align=\"left\">NI</td><td char=\"(\" align=\"char\">0.995 (0.99–0.998)</td><td char=\"(\" align=\"char\">0.997 (0.993–0.999)</td><td char=\"(\" align=\"char\">0.987 (0.972–0.995)</td><td char=\".\" align=\"char\">0.456</td><td char=\".\" align=\"char\">0.168</td><td char=\".\" align=\"char\"><bold>0.035</bold></td></tr><tr><td align=\"left\">I</td><td char=\"(\" align=\"char\">0.997 (0.993–0.999)</td><td char=\"(\" align=\"char\">0.994 (0.986–0.997)</td><td char=\"(\" align=\"char\">0.93 (0.851–0.971)</td><td char=\".\" align=\"char\">0.319</td><td char=\".\" align=\"char\"><bold>< .001</bold></td><td char=\".\" align=\"char\"><bold>< .001</bold></td></tr><tr><td align=\"left\">TI</td><td char=\"(\" align=\"char\">0.994 (0.986–0.997)</td><td char=\"(\" align=\"char\">0.986 (0.969–0.994)</td><td char=\"(\" align=\"char\">0.976 (0.949–0.99)</td><td char=\".\" align=\"char\">0.222</td><td char=\".\" align=\"char\"><bold>0.042</bold></td><td char=\".\" align=\"char\">0.435</td></tr></tbody></table><table-wrap-foot><p><italic>ICC</italic> intraclass correlation coefficient, <italic>CI</italic> confidence interval, <italic>LIQ</italic> low image quality difference group, <italic>MIG</italic> moderate image quality difference group, <italic>HIQ</italic> high image quality difference group, <italic>LSM</italic> least-squares mean, <italic>SE</italic> standard error, <italic>PPAver</italic> average PP-RNFL thickness, <italic>T</italic> temporal, <italic>S</italic> superior, <italic>N</italic> nasal, <italic>I</italic> inferior, <italic>TS</italic> temporosuperior, <italic>ST</italic> superotemporal, <italic>SN</italic> superonasal, <italic>NS</italic> nasosuperior, <italic>NI</italic> nasoinferior, <italic>IN</italic> inferonasal, <italic>IT</italic> inferotemporal, <italic>TI</italic> temporoinferior, <italic>GCLAver</italic> average GC-IPL thickness.</p><p>*Z-test, data are represented as ICC (95% CI); significant <italic>p</italic> values are shown as bold-faced text.</p></table-wrap-foot></table-wrap><fig id=\"Fig1\"><label>Figure 1</label><caption><p>The representative results of differences between the measured values by image quality difference at each sector of PP-RNFL. The image quality difference for a, b, c, and d was 0.107, 0.88, 5.233, and 8.087, respectively. X-axis indicates the measurement sectors, and Y-axis indicates the difference between the masured values. The solid line indicates the difference between the first and second measurements. The thick dotted line indicates the difference between the second and third measurements. The thin dotted line indicates the difference between the first and third measurements. <italic>PP Aver</italic> average PP-RNFL thickness, <italic>T</italic> temporal, <italic>S</italic> superior, <italic>N</italic> nasal, <italic>I</italic> inferior, <italic>TS</italic> temporosuperior, <italic>ST</italic> superotemporal, <italic>SN</italic> superonasal, <italic>NS</italic> nasosuperior, <italic>NI</italic> nasoinferior, <italic>IN</italic> inferonasal, <italic>IT</italic> inferotemporal, <italic>TI</italic> temporoinferior, <italic>PP-RNFL</italic> peripapillary retinal nerve fibre layer.</p></caption><graphic xlink:href=\"41598_2020_70852_Fig1_HTML\" id=\"MO1\"/></fig></p></sec></sec><sec id=\"Sec6\"><title>Discussion</title><p id=\"Par9\">The results of this study, which investigated the association between image quality fluctuations and repeatability of SS-OCT measurements, showed that repeatability decreases with an increase in image quality fluctuation in several sectors of PP-RNFL and GC-IPL. These observations were made in healthy subjects with an OCT image quality > 60, which was calculated as per manufacturer’s recommendation for clinical use. Therefore, it can be said that our study was conducted under settings wherein the factors affecting OCT results, such as low image quality (image quality score < 60) and structural alteration by ocular disease, were controlled. In addition, when the study groups were compared based on the mean absolute difference among three consecutive OCT measurements, no significant differences were noted in the measured thickness at any of the measurement sectors (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>). This result also indicates that there was no large deviation in the measured values of our data set. Nevertheless, even with good image quality (recommended for clinical use) and high repeatability (based on ICC), the measurement repeatability was affected by image quality fluctuations in several sectors, especially in comparisons involving the HIQD group. Moreover, this phenomenon affected sectors that are considered important in glaucoma management. Thus, it is crucial to maintain not only a high level of image quality but also a constant value of image quality for the clinical application of SS-OCT.</p><p id=\"Par10\">Interestingly, although the HIQD group had the lowest ICC value of each measurement sector among the three groups, not all sectors showed significant differences on comparison with the LIQD or MIQD groups. In addition, only five sectors of the clock-hour map for PP-RNFL (superotemporal, nasal, inferonasal, inferotemporal, and temporoinferior sectors) showed ICC values under 0.9. If repeatability is exclusively determined by image quality, the repeatability of the OCT results obtained from subjects of HIQD group should be lower regardless of location of the measurement sectors. Segmentation is important for analysing the thickness of the retinal layer using OCT results. Although image quality is a critical factor for segmentation, ocular structural factors such as axial length, shape of optic disc, or tortuosity of retinal vessel also affect segmentation<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. The superotemporal, inferonasal, inferotemporal, and temporoinferior sectors contain retinal blood vessels, which contribute to the structural variation of the parapapillary area. Thus, the anatomic structure around the optic disc, which varies largely even in healthy eyes, could have influenced the repeatability.</p><p id=\"Par11\">Inter-individual diversity in the optic disc shape and peripapillary structures contribute to inaccuracies in the measurement of PP-RNFL thickness by OCT. In contrast, the macular area is well-known for its inter-individual similarities<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>–<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Such inaccuracies might influence clinical decision-making in glaucoma management. Therefore, several studies have emphasised on the usefulness of GC-IPL parameters for the diagnosis of glaucoma in myopic eyes<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>–<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. In the present study, the repeatability of GC-IPL sectors was relatively less affected by image quality fluctuations as compared to PP-RNFL sectors. This result further supports the usefulness of macular GC-IPL thickness evaluation for estimating glaucoma status, although further studies on patients with glaucoma are required to confirm this occurrence. Previous studies have shown a positive correlation between image quality and OCT-based measurement of macular or PP-RNFL thickness<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>–<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>, i.e., a reduction in image quality decreases the macular or PP-RNFL thickness, thereby leading to incorrect OCT interpretations of glaucoma progression. In this study, image quality correlated significantly in several sectors for both PP-RNFL and GC-IPL thickness, and this result did not change even after adjusting for age and sex. Therefore, image quality remains an essential factor in the interpretation of SS-OCT results. Unlike the correlation results reported previously, the negative correlation between the thickness values and image quality may be due to repeated measurements, small sample size, or unknown intrinsic characteristics of SS-OCT. It is possible that a study on patients with glaucoma may yield negative correlation between the thickness values and image quality.</p><p id=\"Par12\">Studies on the relationship between image quality fluctuations and repeatability of OCT measurements are limited. Lee et al. reported the effect of signal strength difference on the repeatability of PP-RNFL thickness in time-domain OCT<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>, and Kim et al. reported the effect of signal strength on PP-RNFL thickness and colour-coded classification in SD-OCT<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. Both studies inferred that substantial differences in the signal strength lower the repeatability. Our study presents similar results using SS-OCT. Compared to previous studies, the use of three consecutive measurements for statistical analysis provide more reliability to this study, and this strategy is more appropriate for identifying the impact of image quality fluctuation on OCT results.</p><p id=\"Par13\">This study has several limitations. First, although the data were collected prospectively, the number of subjects included was relatively small. Second, the effect of image quality fluctuation on repeatability was studied in healthy subjects. A similar study on patients with glaucoma will help to understand the clinical significance of image quality fluctuations on SS-OCT results. Third, the results of our study cannot be applied directly to other studies focused on other types of OCT. This is because the image quality score which was used for calculating image quality fluctuation in the present study was developed by the manufacturer of DRI OCT, although it is not difficult to predict that the accuracy of segmentation of the OCT will be lowered if the quality of the image deteriorates. Further studies involving other types of OCT seem necessary to clarify the effect of image quality fluctuation on repeatability in each type of OCT. Despite these limitations, our findings are meaningful because this is the first study to investigate the effect of image quality fluctuation on repeatability in SS-OCT using prospectively collected data.</p><p id=\"Par14\">In conclusion, this study reported that higher image quality fluctuation leads to lower repeatability of SS-OCT results in several sectors of PP-RNFL and GC-IPL. Interestingly, the identified sectors were clinically important for glaucoma management. In addition, the repeatability of GC-IPL sectors was relatively less affected than that of PP-RNFL sectors by image quality fluctuations. Thus, maintaining a high-quality image status is vital to enhance the reliability of SS-OCT for PP-RNFL and GC-IPL measurements, more so in the PP-RNFL region.</p></sec><sec id=\"Sec7\"><title>Methods</title><p id=\"Par15\">This study collected raw data retrospectively from the dataset used in a previous study to compare the repeatability and agreement between SD-OCT and SS-OCT in healthy eyes<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. The institutional review board of Yonsei University Severance Hospital, Seoul, Korea, approved this study (1-2019-0043), and the need for written informed consent was waived because of the retrospective study design. The study adhered to the tenets of the Declaration of Helsinki. The detailed characteristics of the subjects in dataset have been described previously<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. Normal subjects who had visited the glaucoma clinic at our hospital between August 2014 and December 2014 were enrolled Medical history, Snellen best-corrected visual acuity (BCVA), slit-lamp biomicroscopy findings, intraocular pressure (IOP; Goldmann applanation tonometry), and indirect ophthalmoscopy findings were obtained. In addition, the following data were acquired: axial length estimated using the IOL Master (Carl Zeiss Meditec AG, Jena, Germany); central corneal thickness calculated using ultrasound pachymetry (DGH-1000; DGH Technology Inc., Frazer, PA, USA); optic disc and RNFL thickness measurements performed using a + 90 diopter (D) lens, colour disc, and red-free photography (VISUCAM200, Carl Zeiss Meditec AG, Jena, Germany). Optic nerve function had been estimated using a Humphrey Visual Field analyser (24-2 Swedish Interactive Threshold Algorithm; Carl Zeiss Meditec, Inc., Dublin, CA, USA).</p><p id=\"Par16\">Healthy subjects of age > 19 years with a BCVA ≥ 20/25 and no evidence of glaucomatous optic disc changes, RNFL defects, or visual field changes with IOP < 21 mmHg were included retrospectively. The eye that was analysed in each patient was selected randomly. Exclusion criteria were the presence of cataract grade of Lens Opacities Classification System III > 3, axial length > 24.5 mm, refractive errors with spherical equivalent >  ±5D, or cylindrical error >  ±3D, and any medical or ophthalmic conditions that influenced the optic disc, RNFL, and visual field measurements.</p><sec id=\"Sec8\"><title>Thickness measurement using SS-OCT for repeatability</title><p id=\"Par17\">In this study, we used the DRI OCT-1 system (Topcon, Tokyo, Japan, analysis software version 9.1.2.28693), which had a high-speed wavelength tuning laser source with central wavelength of 1,050 nm. This SS-OCT system had an image acquisition speed of 100,000 A-scan/second, with an axial and transverse resolutions of 8 and 20 µm, respectively. Three consecutive SS-OCT scans were acquired on the same day with an interval of at least 5 min between the scans. A single technician performed all scans using an internal fixation target. Pupillary dilation was performed in all subjects. A three-dimensional (3D) optic disc and 3D wide scan protocols were used to measure PP-RNFL and GC-IPL thicknesses, respectively. The 3D optic disc scan covered a 6 × 6-mm area on the optic disc and comprised 512 A-scans × 256 B-scans. PP-RNFL thickness was measured in a 3.4-mm-diameter scan circle centred on the optic disc. The 3D wide scan protocol covered a 12 × 9-mm rectangular area centred between the optic disc and fovea and comprised 512 A-scans × 256 B-scans. PP-RNFL thicknesses was measured in each quadrant (evenly spaced 4 sectors), 12 clock-hour sectors (evenly spaced 12 sectors), and as an average. The quadrant PP-RNFL sector names started with the number 4, while the clock-hour sector names started with the number 12. The average GC-IPL thickness and measurement in each of six sectors (evenly configured sectors centred on the fovea) were collected. Built-in automated segmentation algorithms were used to distinguish each retinal layer. Two investigators (S.Y.L. and Y.H.) independently reconfirmed the image quality, segmentation, and alignment of the measurement window. SS-OCT images with image quality scores > 60 were selected for analysis according to the manufacturer’s recommendation.</p><p id=\"Par18\">The mean absolute difference among three consecutive OCT measurements were calculated as follows:<disp-formula id=\"Equa\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\begin{aligned} & Mean\\,absolute\\,difference\\,of\\,image\\,quality\\,score{\\text{:}} \\\\ & \\quad \\quad \\left( {\\left| {IQ1 - IQ2\\left| + \\right|IQ2 - IQ3\\left| + \\right|IQ1 - IQ3} \\right|} \\right)/3 \\\\ \\end{aligned}$$\\end{document}</tex-math><mml:math id=\"M2\" display=\"block\"><mml:mrow><mml:mtable><mml:mtr><mml:mtd/><mml:mtd columnalign=\"left\"><mml:mrow><mml:mi>M</mml:mi><mml:mi>e</mml:mi><mml:mi>a</mml:mi><mml:mi>n</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>a</mml:mi><mml:mi>b</mml:mi><mml:mi>s</mml:mi><mml:mi>o</mml:mi><mml:mi>l</mml:mi><mml:mi>u</mml:mi><mml:mi>t</mml:mi><mml:mi>e</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>d</mml:mi><mml:mi>i</mml:mi><mml:mi>f</mml:mi><mml:mi>f</mml:mi><mml:mi>e</mml:mi><mml:mi>r</mml:mi><mml:mi>e</mml:mi><mml:mi>n</mml:mi><mml:mi>c</mml:mi><mml:mi>e</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>o</mml:mi><mml:mi>f</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>i</mml:mi><mml:mi>m</mml:mi><mml:mi>a</mml:mi><mml:mi>g</mml:mi><mml:mi>e</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>q</mml:mi><mml:mi>u</mml:mi><mml:mi>a</mml:mi><mml:mi>l</mml:mi><mml:mi>i</mml:mi><mml:mi>t</mml:mi><mml:mi>y</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>s</mml:mi><mml:mi>c</mml:mi><mml:mi>o</mml:mi><mml:mi>r</mml:mi><mml:mi>e</mml:mi><mml:mtext>:</mml:mtext></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd columnalign=\"right\"><mml:mrow/></mml:mtd><mml:mtd columnalign=\"left\"><mml:mrow><mml:mspace width=\"1em\"/><mml:mspace width=\"1em\"/><mml:mfenced close=\")\" open=\"(\"><mml:mfenced close=\"|\" open=\"|\"><mml:mrow><mml:mi>I</mml:mi><mml:mi>Q</mml:mi><mml:mn>1</mml:mn><mml:mo>-</mml:mo><mml:mi>I</mml:mi><mml:mi>Q</mml:mi><mml:mn>2</mml:mn><mml:mfenced close=\"|\" open=\"|\"><mml:mo>+</mml:mo></mml:mfenced><mml:mi>I</mml:mi><mml:mi>Q</mml:mi><mml:mn>2</mml:mn><mml:mo>-</mml:mo><mml:mi>I</mml:mi><mml:mi>Q</mml:mi><mml:mn>3</mml:mn><mml:mfenced close=\"|\" open=\"|\"><mml:mo>+</mml:mo></mml:mfenced><mml:mi>I</mml:mi><mml:mi>Q</mml:mi><mml:mn>1</mml:mn><mml:mo>-</mml:mo><mml:mi>I</mml:mi><mml:mi>Q</mml:mi><mml:mn>3</mml:mn></mml:mrow></mml:mfenced></mml:mfenced><mml:mo stretchy=\"false\">/</mml:mo><mml:mn>3</mml:mn></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd columnalign=\"right\"><mml:mrow/></mml:mtd></mml:mtr></mml:mtable></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70852_Article_Equa.gif\" position=\"anchor\"/></alternatives></disp-formula>where IQ<sub>n</sub>—image quality score at the nth measurement.</p><p id=\"Par20\">The subjects were stratified into three groups based on the tertile values of the mean absolute difference of image quality score—LIQD (n = 18), MIQD (n = 19), and HIQD (n = 19). Because subjects in the LIQD group were included in the first third when the mean absolute difference of image quality score was listed in ascending order, they had similar image quality scores among the three consecutive OCT results. In contrast, subjects in the HIQD group showed substantial variation among the three image quality scores because these subjects were the last third subjects.</p></sec><sec id=\"Sec9\"><title>Statistical analyses</title><p id=\"Par21\">Analyses of variance and chi-square tests were performed for the comparison of continuous and categorical variables between the groups. A linear mixed model compared the thickness values among the three groups. To determine the repeatability of three consecutive measurements, intraclass correlation coefficients (ICCs) were used. The degree of repeatability was decided according to the ICC value—almost perfect (0.81–1), substantial (0.61–0.8), moderate (0.41–0.6), fair (0.21–0.4), and slight (0–0.2)<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. To compare the between-group ICC values, the z-score test was used<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>–<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. Pearson’s correlation coefficients with and without adjustment of age and sex were used to investigate correlation between the image quality and thickness value. Correlation coefficients were estimated using a linear mixed-effects model to consider three datasets in one individual. All statistical analyses were performed using SAS version 9.4 software (SAS Institute Inc., Cary, NC, USA) by a statistician (H.S.L). Statistical significance was defined as <italic>p</italic> value < 0.05.</p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>The authors are solely responsible for the content and writing of this manuscript; Drafting of the main manuscript: H.Y., S.Y.L.; Conception and design: H.Y., H.Y.B., G.J.S., C.Y.K., S.Y.L.; Analysis and interpretation: H.S.L., H.Y., S.Y.L.; Data collection: H.Y., S.Y.L.; Overall responsibility: S.Y.L.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par22\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Asrani</surname><given-names>S</given-names></name><name><surname>Essaid</surname><given-names>L</given-names></name><name><surname>Alder</surname><given-names>BD</given-names></name><name><surname>Santiago-Turla</surname><given-names>C</given-names></name></person-group><article-title>Artifacts in spectral-domain optical coherence tomography measurements in glaucoma</article-title><source>JAMA Ophthalmol.</source><year>2014</year><volume>132</volume><fpage>396</fpage><lpage>402</lpage><pub-id pub-id-type=\"doi\">10.1001/jamaophthalmol.2013.7974</pub-id><pub-id pub-id-type=\"pmid\">24525613</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>SY</given-names></name><etal/></person-group><article-title>Frequency, type and cause of artifacts in swept-source and cirrus HD optical coherence tomography in cases of glaucoma and suspected glaucoma</article-title><source>Curr. Eye Res.</source><year>2016</year><volume>41</volume><fpage>957</fpage><lpage>964</lpage><pub-id pub-id-type=\"doi\">10.3109/02713683.2015.1075219</pub-id><pub-id pub-id-type=\"pmid\">26431251</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mansberger</surname><given-names>SL</given-names></name><name><surname>Menda</surname><given-names>SA</given-names></name><name><surname>Fortune</surname><given-names>BA</given-names></name><name><surname>Gardiner</surname><given-names>SK</given-names></name><name><surname>Demirel</surname><given-names>S</given-names></name></person-group><article-title>Automated segmentation errors when using optical coherence tomography to measure retinal nerve fiber layer thickness in glaucoma</article-title><source>Am. J. Ophthalmol.</source><year>2017</year><volume>174</volume><fpage>1</fpage><lpage>8</lpage><pub-id pub-id-type=\"doi\">10.1016/j.ajo.2016.10.020</pub-id><pub-id pub-id-type=\"pmid\">27818206</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Matlach</surname><given-names>J</given-names></name><name><surname>Wagner</surname><given-names>M</given-names></name><name><surname>Malzahn</surname><given-names>U</given-names></name><name><surname>Gobel</surname><given-names>W</given-names></name></person-group><article-title>Repeatability of peripapillary retinal nerve fiber layer and inner retinal thickness among two spectral domain optical coherence tomography devices</article-title><source>Invest. Ophth. Vis. Sci.</source><year>2014</year><volume>55</volume><fpage>6536</fpage><lpage>6546</lpage><pub-id pub-id-type=\"doi\">10.1167/iovs.14-15072</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>SY</given-names></name><name><surname>Bae</surname><given-names>HW</given-names></name><name><surname>Kwon</surname><given-names>HJ</given-names></name><name><surname>Seong</surname><given-names>GJ</given-names></name><name><surname>Kim</surname><given-names>CY</given-names></name></person-group><article-title>Repeatability and agreement of swept source and spectral domain optical coherence tomography evaluations of thickness sectors in normal eyes</article-title><source>J. Glaucoma</source><year>2017</year><volume>26</volume><fpage>e46</fpage><lpage>e53</lpage><pub-id pub-id-type=\"doi\">10.1097/IJG.0000000000000536</pub-id><pub-id pub-id-type=\"pmid\">27599180</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>SY</given-names></name><name><surname>Bae</surname><given-names>HW</given-names></name><name><surname>Seong</surname><given-names>GJ</given-names></name><name><surname>Kim</surname><given-names>CY</given-names></name></person-group><article-title>Diagnostic ability of swept-source and spectral-domain optical coherence tomography for glaucoma</article-title><source>Yonsei Med. J.</source><year>2018</year><volume>59</volume><fpage>887</fpage><lpage>896</lpage><pub-id pub-id-type=\"doi\">10.3349/ymj.2018.59.7.887</pub-id><pub-id pub-id-type=\"pmid\">30091323</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dominguez-Vicent</surname><given-names>A</given-names></name><name><surname>Brautaset</surname><given-names>R</given-names></name><name><surname>Venkataraman</surname><given-names>AP</given-names></name></person-group><article-title>Repeatability of quantitative measurements of retinal layers with SD-OCT and agreement between vertical and horizontal scan protocols in healthy eyes</article-title><source>PLoS ONE</source><year>2019</year><volume>14</volume><fpage>e0221466</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0221466</pub-id><pub-id pub-id-type=\"pmid\">31437222</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>ES</given-names></name><name><surname>Kim</surname><given-names>H</given-names></name><name><surname>Kim</surname><given-names>JM</given-names></name></person-group><article-title>Effect of signal strength on reproducibility of peripapillary retinal nerve fiber layer thickness measurement and its classification by time-domain optical coherence tomography</article-title><source>Jpn. J. Ophthalmol.</source><year>2010</year><volume>54</volume><fpage>414</fpage><lpage>422</lpage><pub-id pub-id-type=\"doi\">10.1007/s10384-010-0850-9</pub-id><pub-id pub-id-type=\"pmid\">21052903</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>JH</given-names></name><etal/></person-group><article-title>Effect of signal strength on reproducibility of circumpapillary retinal nerve fiber layer thickness measurement and its classification by spectral-domain optical coherence tomography</article-title><source>Jpn. J. Ophthal.</source><year>2011</year><volume>55</volume><fpage>220</fpage><lpage>227</lpage><pub-id pub-id-type=\"doi\">10.1007/s10384-011-0023-5</pub-id><pub-id pub-id-type=\"pmid\">21559911</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Miki</surname><given-names>A</given-names></name><etal/></person-group><article-title>Prevalence and associated factors of segmentation errors in the peripapillary retinal nerve fiber layer and macular ganglion cell complex in spectral-domain optical coherence tomography images</article-title><source>J. Glaucoma</source><year>2017</year><volume>26</volume><fpage>995</fpage><lpage>1000</lpage><pub-id pub-id-type=\"doi\">10.1097/IJG.0000000000000771</pub-id><pub-id pub-id-type=\"pmid\">28858152</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ye</surname><given-names>C</given-names></name><name><surname>Yu</surname><given-names>M</given-names></name><name><surname>Leung</surname><given-names>CK</given-names></name></person-group><article-title>Impact of segmentation errors and retinal blood vessels on retinal nerve fibre layer measurements using spectral-domain optical coherence tomography</article-title><source>Acta Ophthalmol.</source><year>2016</year><volume>94</volume><fpage>e211</fpage><lpage>219</lpage><pub-id pub-id-type=\"doi\">10.1111/aos.12762</pub-id><pub-id pub-id-type=\"pmid\">26132774</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mwanza</surname><given-names>JC</given-names></name><etal/></person-group><article-title>Macular ganglion cell-inner plexiform layer: automated detection and thickness reproducibility with spectral domain-optical coherence tomography in glaucoma</article-title><source>Invest. Ophth. Vis. Sci.</source><year>2011</year><volume>52</volume><fpage>8323</fpage><lpage>8329</lpage><pub-id pub-id-type=\"doi\">10.1167/iovs.11-7962</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jeong</surname><given-names>JH</given-names></name><name><surname>Choi</surname><given-names>YJ</given-names></name><name><surname>Park</surname><given-names>KH</given-names></name><name><surname>Kim</surname><given-names>DM</given-names></name><name><surname>Jeoung</surname><given-names>JW</given-names></name></person-group><article-title>Macular ganglion cell imaging study: covariate effects on the spectral domain optical coherence tomography for glaucoma diagnosis</article-title><source>PLoS ONE</source><year>2016</year><volume>11</volume><fpage>e0160448</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0160448</pub-id><pub-id pub-id-type=\"pmid\">27490718</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>KE</given-names></name><name><surname>Park</surname><given-names>KH</given-names></name></person-group><article-title>Macular imaging by optical coherence tomography in the diagnosis and management of glaucoma</article-title><source>Brit. J. Ophthalmol.</source><year>2018</year><volume>102</volume><fpage>718</fpage><lpage>724</lpage><pub-id pub-id-type=\"doi\">10.1136/bjophthalmol-2017-310869</pub-id><pub-id pub-id-type=\"pmid\">29055905</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Akashi</surname><given-names>A</given-names></name><etal/></person-group><article-title>The ability of SD-OCT to differentiate early glaucoma with high myopia from highly myopic controls and nonhighly myopic controls</article-title><source>Invest. Ophth. Vis. Sci.</source><year>2015</year><volume>56</volume><fpage>6573</fpage><lpage>6580</lpage><pub-id pub-id-type=\"doi\">10.1167/iovs.15-17635</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Seol</surname><given-names>BR</given-names></name><name><surname>Jeoung</surname><given-names>JW</given-names></name><name><surname>Park</surname><given-names>KH</given-names></name></person-group><article-title>Glaucoma detection ability of macular ganglion cell-inner plexiform layer thickness in myopic preperimetric glaucoma</article-title><source>Invest. Ophth. Vis. Sci.</source><year>2015</year><volume>56</volume><fpage>8306</fpage><lpage>8313</lpage><pub-id pub-id-type=\"doi\">10.1167/iovs.15-18141</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>YK</given-names></name><etal/></person-group><article-title>Glaucoma-diagnostic ability of ganglion cell-inner plexiform layer thickness difference across temporal raphe in highly myopic eyes</article-title><source>Invest. Ophth. Vis. Sci.</source><year>2016</year><volume>57</volume><fpage>5856</fpage><lpage>5863</lpage><pub-id pub-id-type=\"doi\">10.1167/iovs.16-20116</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cheung</surname><given-names>CY</given-names></name><name><surname>Leung</surname><given-names>CK</given-names></name><name><surname>Lin</surname><given-names>D</given-names></name><name><surname>Pang</surname><given-names>CP</given-names></name><name><surname>Lam</surname><given-names>DS</given-names></name></person-group><article-title>Relationship between retinal nerve fiber layer measurement and signal strength in optical coherence tomography</article-title><source>Ophthalmology</source><year>2008</year><volume>115</volume><fpage>1347</fpage><lpage>1351</lpage><pub-id pub-id-type=\"doi\">10.1016/j.ophtha.2007.11.027</pub-id><pub-id pub-id-type=\"pmid\">18294689</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>Z</given-names></name><name><surname>Huang</surname><given-names>J</given-names></name><name><surname>Dustin</surname><given-names>L</given-names></name><name><surname>Sadda</surname><given-names>SR</given-names></name></person-group><article-title>Signal strength is an important determinant of accuracy of nerve fiber layer thickness measurement by optical coherence tomography</article-title><source>J. Glaucoma</source><year>2009</year><volume>18</volume><fpage>213</fpage><lpage>216</lpage><pub-id pub-id-type=\"doi\">10.1097/IJG.0b013e31817eee20</pub-id><pub-id pub-id-type=\"pmid\">19295375</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cheung</surname><given-names>CY</given-names></name><etal/></person-group><article-title>Determinants of quantitative optic nerve measurements using spectral domain optical coherence tomography in a population-based sample of non-glaucomatous subjects</article-title><source>Invest. Ophth. Vis. Sci.</source><year>2011</year><volume>52</volume><fpage>9629</fpage><lpage>9635</lpage><pub-id pub-id-type=\"doi\">10.1167/iovs.11-7481</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hardin</surname><given-names>JS</given-names></name><name><surname>Taibbi</surname><given-names>G</given-names></name><name><surname>Nelson</surname><given-names>SC</given-names></name><name><surname>Chao</surname><given-names>D</given-names></name><name><surname>Vizzeri</surname><given-names>G</given-names></name></person-group><article-title>Factors affecting cirrus-HD OCT optic disc scan quality: A review with case examples</article-title><source>J. Ophthalmol.</source><year>2015</year><volume>2015</volume><fpage>746150</fpage><pub-id pub-id-type=\"doi\">10.1155/2015/746150</pub-id><pub-id pub-id-type=\"pmid\">26351574</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Landis</surname><given-names>JR</given-names></name><name><surname>Koch</surname><given-names>GG</given-names></name></person-group><article-title>The measurement of observer agreement for categorical data</article-title><source>Biometrics</source><year>1977</year><volume>33</volume><fpage>159</fpage><lpage>174</lpage><pub-id pub-id-type=\"doi\">10.2307/2529310</pub-id><pub-id pub-id-type=\"pmid\">843571</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fanchin</surname><given-names>R</given-names></name><etal/></person-group><article-title>High reproducibility of serum anti-Mullerian hormone measurements suggests a multi-staged follicular secretion and strengthens its role in the assessment of ovarian follicular status</article-title><source>Hum. Reprod.</source><year>2005</year><volume>20</volume><fpage>923</fpage><lpage>927</lpage><pub-id pub-id-type=\"doi\">10.1093/humrep/deh688</pub-id><pub-id pub-id-type=\"pmid\">15640257</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hirai</surname><given-names>T</given-names></name><etal/></person-group><article-title>Quantitative blood flow measurements in gliomas using arterial spin-labeling at 3T: Intermodality agreement and inter- and intraobserver reproducibility study</article-title><source>AJNR. Am. J. Neuroradiol.</source><year>2011</year><volume>32</volume><fpage>2073</fpage><lpage>2079</lpage><pub-id pub-id-type=\"doi\">10.3174/ajnr.A2725</pub-id><pub-id pub-id-type=\"pmid\">21960503</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Neurosci</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Neurosci</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Neurosci.</journal-id><journal-title-group><journal-title>Frontiers in Neuroscience</journal-title></journal-title-group><issn pub-type=\"ppub\">1662-4548</issn><issn pub-type=\"epub\">1662-453X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32848585</article-id><article-id pub-id-type=\"pmc\">PMC7431558</article-id><article-id pub-id-type=\"doi\">10.3389/fnins.2020.00836</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Neuroscience</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Evaluating Sensory Acuity as a Marker of Balance Dysfunction After a Traumatic Brain Injury: A Psychophysical Approach</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Pilkar</surname><given-names>Rakesh</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/205480/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Karunakaran</surname><given-names>Kiran K.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/543913/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Veerubhotla</surname><given-names>Akhila</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1000579/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ehrenberg</surname><given-names>Naphtaly</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/652026/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ibironke</surname><given-names>Oluwaseun</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Nolan</surname><given-names>Karen J.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/375580/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Center for Mobility and Rehabilitation Engineering Research, Kessler Foundation</institution>, <addr-line>West Orange, NJ</addr-line>, <country>United States</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Physical Medicine and Rehabilitation, Rutgers New Jersey Medical School</institution>, <addr-line>Newark, NJ</addr-line>, <country>United States</country></aff><aff id=\"aff3\"><sup>3</sup><institution>New Jersey Institute of Technology</institution>, <addr-line>Newark, NJ</addr-line>, <country>United States</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Children’s Specialized Hospital</institution>, <addr-line>New Brunswick, NJ</addr-line>, <country>United States</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Massimo Sartori, University of Twente, Netherlands</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Giacomo Valle, ETH Zürich, Switzerland; George C. McConnell, Stevens Institute of Technology, United States</p></fn><corresp id=\"c001\">*Correspondence: Rakesh Pilkar, <email>rpilkar@kesslerfoundation.org</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>Present address: Rakesh Pilkar, Center for Mobility and Rehabilitation Engineering Research, Kessler Foundation, West Orange, NJ, United States</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Neuroprosthetics, a section of the journal Frontiers in Neuroscience</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>14</volume><elocation-id>836</elocation-id><history><date date-type=\"received\"><day>29</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>17</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Pilkar, Karunakaran, Veerubhotla, Ehrenberg, Ibironke and Nolan.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Pilkar, Karunakaran, Veerubhotla, Ehrenberg, Ibironke and Nolan</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>There is limited research on sensory acuity i.e., ability to perceive external perturbations via body-sway during standing in individuals with a traumatic brain injury (TBI). It is unclear whether sensory acuity diminishes after a TBI and if it is a contributing factor to balance dysfunction. The objective of this investigation is to first objectively quantify the sensory acuity in terms of perturbation perception threshold (PPT) and determine if it is related to functional outcomes of static and dynamic balance. Ten individuals with chronic TBI and 11 age-matched healthy controls (HC) performed PPT assessments at 0.33, 0.5, and 1 Hz horizontal perturbations to the base of support in the anterior-posterior direction, and a battery of functional assessments of static and dynamic balance and mobility [Berg balance scale (BBS), timed-up and go (TUG) and 5-m (5MWT) and 10-m walk test (10MWT)]. A psychophysical approach based on Single Interval Adjustment Matrix Protocol (SIAM), i.e., a <italic>yes-no</italic> task, was used to quantify the multi-sensory thresholds of perceived external perturbations to calculate PPT. A mixed-design analysis of variance (ANOVA) and <italic>post-hoc</italic> analyses were performed using independent and paired t-tests to evaluate within and between-group differences. Pearson correlation was computed to determine the relationship between the PPT and functional measures. The PPT values were significantly higher for the TBI group (0.33 Hz: 2.97 ± 1.0, 0.5 Hz: 2.39 ± 0.7, 1 Hz: 1.22 ± 0.4) compared to the HC group (0.33 Hz: 1.03 ± 0.6, 0.5 Hz: 0.89 ± 0.4, 1 Hz: 0.42 ± 0.2) for all three perturbation frequencies (<italic>p</italic> < 0.006 post Bonferroni correction). For the TBI group, the PPT for 1 Hz perturbations showed significant correlation with the functional measures of balance (BBS: <italic>r</italic> = −0.66, <italic>p</italic> = 0.037; TUG: <italic>r</italic> = 0.78, <italic>p</italic> = 0.008; 5MWT: <italic>r</italic> = 0.67, <italic>p</italic> = 0.034, 10MWT: <italic>r</italic> = 0.76, <italic>p</italic> = 0.012). These findings demonstrate that individuals with TBI have diminished sensory acuity during standing which may be linked to impaired balance function after TBI.</p></abstract><kwd-group><kwd>sensory threshold detection</kwd><kwd>posturography</kwd><kwd>traumatic brain injury</kwd><kwd>balance</kwd><kwd>rehabilitation</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">New Jersey Commission on Brain Injury Research<named-content content-type=\"fundref-id\">10.13039/100008474</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"4\"/><table-count count=\"4\"/><equation-count count=\"0\"/><ref-count count=\"32\"/><page-count count=\"9\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Balance control is regulated within the central nervous system by the complex integration of visual, vestibular, and somatosensory pathways and motor control (<xref rid=\"B8\" ref-type=\"bibr\">Hillier et al., 1997</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Greenwald et al., 2001</xref>). Traumatic brain injury (TBI) often damages the areas of the brain that regulate balance (<xref rid=\"B2\" ref-type=\"bibr\">Allison, 1999</xref>). Although the peripheral system may or may not be impaired as a secondary consequence of the same event, damage to the brain could result in impaired central motor processes such as intention to act, motor planning, and automatic postural response mechanisms (<xref rid=\"B2\" ref-type=\"bibr\">Allison, 1999</xref>). Further, impaired sensory integration, a central process, is postulated as one of the sources for imbalance after TBI (<xref rid=\"B28\" ref-type=\"bibr\">Sosnoff et al., 2011</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Fino et al., 2017</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Peterka et al., 2018</xref>). The body-position awareness, i.e., the detection of body-sway, is a fundamental necessity to maintain static and dynamic balance during activities of daily living (<xref rid=\"B5\" ref-type=\"bibr\">Fitzpatrick and McCloskey, 1994</xref>) and it is achieved by an accurate perception of the body’s interaction with the surrounding environment. TBI can impair the integration of the visual, vestibular, and somatosensory (proprioceptive) inputs (<xref rid=\"B2\" ref-type=\"bibr\">Allison, 1999</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Sarno et al., 2003</xref>) that permits body position awareness with respect to self and the environment. Therefore, impairments to sensory pathways (<xref rid=\"B4\" ref-type=\"bibr\">Fino et al., 2017</xref>) and their integration (<xref rid=\"B17\" ref-type=\"bibr\">Peterka et al., 2018</xref>) to facilitate perception of body-environment interaction can lead to poor understanding of the surroundings, impaired balance and a greater risk of falls after TBI. Falls occur when the center of mass (CoM) is displaced beyond the base of support and when the central nervous system fails to “detect and correct” this displacement in time (Institute of Medicine (US), and Division of Health Promotion and Disease Prevention, 1992). Therefore, accurate perception is even more critical in a dynamic setting which demands attention, adaptation to external stimuli and adequate reactive motor responses for achieving balance control and avoiding falls. Further, in the domain of perception and balance, sensory acuity, i.e., the ability to detect body-sway during external perturbations (<xref rid=\"B5\" ref-type=\"bibr\">Fitzpatrick and McCloskey, 1994</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Richerson et al., 2003</xref>), could stem from impaired sensory integration. Limited research specifically reports objective quantification of impairments to sensory integration after TBI (<xref rid=\"B17\" ref-type=\"bibr\">Peterka et al., 2018</xref>) and no research thus far has investigated sensory acuity in the individuals with TBI. An objective assessment of the sensory acuity, i.e., the ability to perceive external perturbations, is necessary to accurately detect, quantify, and treat sensory integration deficits that could lead to poor detection of body sway and imbalance in dynamic environment. Additionally, the outcome measure of sensory acuity can serve as a novel marker of balance function which goes beyond biomechanical and functional outcomes and may provide added information to develop rehabilitation programs aimed at improving balance and reducing falls in individuals with TBI.</p><p>Psychophysics provides a way to evaluate and quantify an individual’s sensory acuity to external stimuli (<xref rid=\"B7\" ref-type=\"bibr\">Han et al., 2016</xref>). In the realm of standing balance, psychophysical studies related to the perception of whole-body perturbations are commonly used to measure sensory acuity in terms of detection thresholds (<xref rid=\"B5\" ref-type=\"bibr\">Fitzpatrick and McCloskey, 1994</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Richerson et al., 2003</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Pilkar, 2011</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Puntkattalee et al., 2016</xref>). Most of the research on assessing balance deficits after a TBI is restricted to biomechanical [CoM, center of pressure (CoP)] and functional outcome measures (<xref rid=\"B14\" ref-type=\"bibr\">Lehmann et al., 1990</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Kaufman et al., 2006</xref>) and no research has reported psychophysical outcomes such as detection thresholds in individuals with TBI. The detection threshold quantifies the level of the external perturbation (magnitude, frequency, velocity, direction) below which the perception of the perturbation becomes unlikely (<xref rid=\"B19\" ref-type=\"bibr\">Pilkar, 2011</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Pilkar et al., 2016</xref>).</p><p>The purpose of this investigation is to objectively evaluate and quantify the multi-sensory acuity to external mechanical perturbations to the base of support during standing for individuals with TBI. This multi-sensory acuity will be quantified in terms of perturbation perception threshold (PPT) using a psychophysical approach when visual, vestibular and somatosensory systems are available. The secondary objective is to determine if our novel outcome measure, PPT, is related to the functional outcomes of static and dynamic balance. Our central hypothesis is that the balance dysfunction will be characterized by an impaired PPT in addition to deficits in functional outcomes after a TBI. More specifically, individuals with TBI will exhibit elevated PPTs compared to healthy controls when experiencing external perturbations. Our secondary hypothesis is that the PPT will be correlated to the functional outcome measures, as the diminished ability to perceive changes in the body position will affect the ability of individuals with TBI to perform static and dynamic balance tasks.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Participants</title><p>Eleven age-matched healthy controls (HC) with no neurological, orthopedic, or visual impairments and 10 individuals diagnosed with a TBI were recruited (see <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). The Kessler Foundation Institutional Review Board approved all procedures and informed consent was obtained prior to study participation. Inclusion criteria for the TBI group were: (1) age between 18 and 60; (2) diagnosed with a non-penetrating TBI (≥6 months); (3) ability to stand unsupported for at least 5 min; (4) willing and able to give informed consent. Exclusion criteria for the TBI group were: (1) history of injury to the lower limbs in the past 90 days; (2) cardiac disease; (3) a previous history of balance impairments prior to TBI.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Demographics for the study participants with data reported in terms of mean ± standard deviations.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Groups</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Age (years)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Sex</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Height (cm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Weight (kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">BMI</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">TSI (years)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">TBI severity</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HC (<italic>n</italic>\n<bold>=</bold> 11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">52.3 ± 5.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6 M, 5 F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">169.7 ± 7.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">87 ± 18.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">37.3 ± 27.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">–</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TBI (<italic>n</italic>\n<bold>=</bold> 10)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">55.6 ± 3.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7 M, 3 F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">172.2 ± 10.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">93.1 ± 25</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.1 ± 6.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.9 ± 17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5 mild, 2 moderate, 3 severe</td></tr></tbody></table><table-wrap-foot><attrib><italic>BMI, Body Mass Index; TSI, Time since injury at the time of testing. TBI severity was diagnosed at the time of injury.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S2.SS2\"><title>Procedures</title><sec id=\"S2.SS2.SSS1\"><title>Clinical Assessments of Static and Dynamic Balance Function</title><p>Participants from both the HC and TBI groups completed clinical assessments of static and dynamic balance function including: the Berg Balance Scale (BBS); the Timed-up and Go (TUG); 5-m walk test (5MWT); and 10-m walk test (10MWT). The BBS is a 14-item assessment scale that quantitatively assesses balance during static and dynamic functional movements in adults. Each item is scored from 0 to 4, with a score of 0 representing the inability to complete the task and a score of 4 representing independent completion of the task. The maximum possible score is 56 points. The 5MWT and 10MWT are assessments of how quickly and safely an individual traverses standard distances, and the TUG evaluates a participant’s ability to transition from sitting to brief locomotor tasks and then return to a seated position.</p></sec><sec id=\"S2.SS2.SSS2\"><title>PPT Assessments</title><p>PPT assessments for the HC and TBI groups were completed after completing clinical assessments of static and dynamic balance function. The NeuroCom Smart Equitest Clinical Research System (CRS) (Natus Medical Inc., Pleasanton, CA), was used to provide precise perturbations to the base of support in anterior-posterior (AP) direction (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Perturbations were applied to the base of support at three different frequencies- 0.3, 0.5, and 1 Hz, which were selected in order to keep the perturbations within the range of natural healthy sway (<2 Hz) (<xref rid=\"B27\" ref-type=\"bibr\">Soames and Atha, 1982</xref>). For each perturbation frequency, a total of 21 trials consisting of a randomized configuration of 14 perturbation trials and 7 non-perturbation trials (2:1) were performed. Each trial lasted 15-s which included 5 s of quiet standing (QS), followed by sinusoidal translations of the platform in the AP direction at the selected perturbation frequency and programmed amplitude for 5 s (or no movement for a non-perturbation trial), followed by 5 more seconds of QS (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). At the end of each trial, the participant was verbally asked if they felt the platform move. Depending on the correctness of their <italic>yes</italic> or <italic>no</italic> response (HIT: correctly detected perturbation, MISS: non-detected perturbation, Correct Rejection: correctly reported no perturbation and False Alarm: perturbation reported for a non-perturbation trial), the amplitude of the next trial was adjusted using the Single Interval Adjustment Matrix (SIAM) algorithm with parameter estimation by sequential testing (PEST) (<xref rid=\"B31\" ref-type=\"bibr\">Taylor, 1967</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Kaernbach, 1990</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Pilkar, 2011</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Pilkar et al., 2016</xref>). The process is shown in <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref> using the numbered sequences from 1 to 6. The PPT value for each frequency was computed using the psychometric curve (<xref rid=\"B1\" ref-type=\"bibr\">Algom, 1992</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Puntkattalee et al., 2016</xref>) by plotting the percentage of accuracy (HIT, correct rejections) as a function of perturbation amplitude (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). A sigmoid function was used to fit the data for each frequency, and the perturbation amplitude (x-axis) where the curve achieves a 75% probability of correct detection (y-axis) was chosen as the PPT value (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Algom, 1992</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Puntkattalee et al., 2016</xref>). This procedure was performed for all three frequencies for each participant. To familiarize the participants with perturbations and minimize the learning effect, five perturbation trials at suprathreshold amplitudes (≥4 mm peak-to-peak) were performed at each of the perturbation frequencies before the PPT assessments. No verbal response was recorded during these trials.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>The experimental set up and procedures are demonstrated for the PPT assessments of 0.5 Hz perturbations. (1) the assessment starts with the default perturbation amplitude of 4 mm, (2) perturbation amplitude is fed to the Neurocom computer which (3) sends out the command to the on-board controller for execution of the platform movement, (4) platform moves precisely at the desired amplitudes in the anterior-posterior direction, (5) the subject reports if he/she felt the platform movement, and (6) based on the correctness of subject’s response, SIAM algorithm computes the next perturbation amplitude and the steps 1–6 are repeated for the remaining 20 trials.</p></caption><graphic xlink:href=\"fnins-14-00836-g001\"/></fig><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p><bold>(A)</bold> Perturbation amplitude iterations based on a representative subject’s response for a set of PPT assessments and <bold>(B)</bold> corresponding psychometric curve with PPT shown by the green circle. Points d<sub>1</sub> and d<sub>2</sub> represent the median of perturbation amplitudes that were successfully detected (<italic>p</italic> = 1) and not detected (<italic>p</italic> = 0), respectively. HIT, perturbation presented and correctly reported; MISS, perturbation presented but not reported; Correct Rejection (CR), perturbation not presented and not reported; False Alarm (FA), perturbation not presented but reported.</p></caption><graphic xlink:href=\"fnins-14-00836-g002\"/></fig></sec></sec><sec id=\"S2.SS3\"><title>Statistical Analyses</title><p>The normality of the PPT outcome was evaluated using Shapiro-Wilk test of normality. It was found that the assumption of normality was valid for the PPT data for both groups for 0.33 (HC: <italic>p</italic> = 0.06; TBI: 0.21) and 1 Hz (HC: <italic>p</italic> = 0.56; TBI: 0.2) perturbations. For 0.5 Hz perturbations, PPT data was normally distributed for the HC group (<italic>p</italic> = 0.1). The TBI group showed approximately normal distribution (<italic>p</italic> = 0.01) which was also supported by the Q-Q plots showing approximately linear data fit. Hence, the PPT data were analyzed using a mixed-design Analysis of Variance (ANOVA) with a within-subjects factor of perturbation frequency (0.33, 0.5, and 1 Hz) and a between-subject factor of condition (healthy, TBI). Mauchly’s test for sphericity indicated that the assumption of sphericity was valid [χ<sup>2</sup>(2) = 1.36, <italic>p</italic> = 0.51] for the PPT measure, hence sphericity was assumed.</p><p>For the ANOVA tests, the significance level was set to 0.05. Based on the significance of the main effects, <italic>post-hoc</italic> tests were performed to compute a between-subjects comparison and a within-subject comparison. A Bonferroni correction was applied to avoid type-I errors and the new significance level was corrected to 0.006. The functional outcome measures of static and dynamic balance (BBS, TUG, 5MWT, 10MWT) were compared using independent sample t-tests. In addition, the functional outcome measures were correlated with PPT using a Pearson product-moment correlation (p ≤ 0.05). The results are reported in terms of mean ± standard deviations (sd) including the PPT outcome reported in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref> and the functional outcomes reported in <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>.</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Results of <italic>post-hoc</italic> analysis for between-group and within-group comparison of the PPT outcome (mean ± sd).</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Perturbation frequency (Hz)<hr/></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Within-group</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Groups</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td rowspan=\"1\" colspan=\"1\"/></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Healthy (<italic>n</italic>\n<bold>=</bold> 11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.03 ± 0.6<sup><italic>a,c</italic></sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.89 ± 0.4<sup><italic>a,b</italic></sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.42 ± 0.2<sup><italic>b,c</italic></sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><sup><italic>a</italic></sup><italic>t</italic>(10) = 1.1, <italic>p</italic> = 0.298</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><sup><italic>b</italic></sup><italic>t</italic>(10) = 4.14, <bold><italic>p</italic> = 0.002*</bold></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><sup><italic>c</italic></sup><italic>t</italic>(10) = 3.89, <bold><italic>p</italic> = 0.003*</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TBI (<italic>n</italic>\n<bold>=</bold> 10)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.97 ± 1.0<sup> a,c</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.39 ± 0.7<sup> a,b</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.22 ± 0.4<sup> b,c</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><sup><italic>a</italic></sup><italic>t</italic>(9) = 2.3, <italic>p</italic> = 0.047</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><sup><italic>c</italic></sup><italic>t</italic>(9) = 7.05, <bold><italic>p</italic> < 0.006*</bold></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><sup><italic>b</italic></sup><italic>t</italic>(9) = 5.79, <bold><italic>p</italic> < 0.006*</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Between-group</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>t</italic>(19) = −5.56, <italic>p</italic> < 0.006</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>t</italic>(19) = −6.01, <italic>p</italic> < 0.006</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>t</italic>(19) = −5.69, <italic>p</italic> < 0.006</td><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic>*p < 0.006 where 0.006 is the significance level adjusted after Bonferroni correction. <sup>a</sup>0.33 vs. 0.5 Hz. <sup>b</sup>0.5 vs. 1 Hz. <sup>c</sup>0.33 vs. 1 Hz. Bold values represent statistically significant differences.</italic></attrib></table-wrap-foot></table-wrap><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>Between-group comparison of the functional outcome measures (mean ± sd) of static and dynamic balance.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Groups</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">BBS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5MWT (s)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10MWT (s)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">TUG (s)</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HC (<italic>n</italic>\n<bold>=</bold> 11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">55.91 ± 0.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.96 ± 0.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.47 ± 0.91</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.41 ± 1.38</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TBI (<italic>n</italic>\n<bold>=</bold> 10)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">48.8 ± 6.43</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.61 ± 1.18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.34 ± 2.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.7 ± 3.6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Between-group</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>t</italic>(10) = 3.68, <bold><italic>p</italic> = 0.007</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>t</italic>(10) = −4.15, <bold><italic>p</italic> = 0.001</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>t</italic>(10) = −4.64, <bold><italic>p</italic> < 0.005</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>t</italic>(10) = −4.52, <bold><italic>p</italic> < 0.005</bold></td></tr></tbody></table><table-wrap-foot><attrib><italic>Bold values represent statistically significant differences.</italic></attrib></table-wrap-foot></table-wrap></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Perception of Perturbation Threshold (PPT)</title><p>The SIAM algorithm successfully converged to the threshold amplitudes for each participant in both groups. The PPTs computed using a classical psychometric approach showed a decreasing trend with increasing perturbation frequency for both groups (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). For both groups, PPTs computed for 0.33 Hz showed the highest variability while 1 Hz perturbations showed the lowest variability based on the standard deviations. A mixed-design ANOVA showed significant main effects of perturbation frequency [<italic>F</italic>(2, 38) = 42.14. <italic>p</italic> < 0.005], and condition [<italic>F</italic>(1, 19) = 44.35, <italic>p</italic> < 0.005] on PPT, and interactions between perturbation frequency and condition [<italic>F</italic>(2, 38) = 9.65, <italic>p</italic> < 0.005].</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Box plot representation of PPT values computed using SIAM for three sets of perturbation frequencies (x-axis) for HC (<italic>n</italic> = 11) and TBI (<italic>n</italic> = 10). Horizontal lines in each box represent the median values. Data points shown with red circles are the outliers. <sup>∗</sup><italic>p</italic> < 0.006 (significance level post Bonferroni correction).</p></caption><graphic xlink:href=\"fnins-14-00836-g003\"/></fig><p><italic>Post-hoc</italic> analysis showed that there was a significant difference in PPT values between the HC group and the TBI group for all three frequency sets (<italic>p</italic> < 0.006) (see <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). Furthermore, <italic>post-hoc</italic> analysis showed no significant difference between frequencies of 0.33 Hz and 0.5 Hz for the within-group comparison for the HC group (<italic>p</italic> = 0.298) and the TBI group (<italic>p</italic> = 0.047). The PPT values obtained for 0.5 Hz were significantly different than those obtained for 1 Hz for both the HC group (<italic>p</italic> = 0.002) and the TBI group (<italic>p</italic> < 0.006) (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). Similarly, the PPT values for 0.33 Hz were significantly higher than 1 Hz for both groups (HC: <italic>p</italic> = 0.003; TBI: <italic>p</italic> < 0.006) (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>).</p></sec><sec id=\"S3.SS2\"><title>Correlation Between the Functional Outcomes and the PPT</title><p>The TBI group showed significantly lower scores on functional assessments compared to the HC group (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). The BBS was significantly lower for the TBI group (48.8 ± 6.43) than the HC group (55.91 ± 0.3) (<italic>p</italic> = 0.007). The time required to complete the 5MWT was significantly higher for the TBI group (4.61 ± 1.18 s) compared to the HC (2.96 ± 0.58 s) group (<italic>p</italic> = 0.001) and similar group differences were observed for the 10MWT (<italic>p</italic> < 0.005) and TUG test (<italic>p</italic> < 0.005) (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>).</p><p>A Pearson product-moment correlation was run to determine the relationship between the PPT and functional measures of static and dynamic balance (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref> and <xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). For individuals with TBI, there was a significant positive correlation between the PPT (1 Hz) and the time required to complete 5MWT (<italic>p</italic> = 0.034), 10MWT (<italic>p</italic> = 0.012), and TUG (<italic>p</italic> = 0.008). For the TBI group, no significant correlation was found between 0.33 Hz PPT and time to complete 5MWT (<italic>p</italic> = 0.34), 10MWT (<italic>p</italic> = 0.13), and TUG (<italic>p</italic> = 0.29). Similarly, no significant correlation was found between 0.5 Hz PPT and 5MWT (<italic>p</italic> = 0.28), 10MWT (<italic>p</italic> = 0.18), and TUG (<italic>p</italic> = 0.14). For the HC group, no significant correlation was found between the PPT (all frequencies) and the time required to complete 5MWT, 10MWT, and TUG (see <xref rid=\"T4\" ref-type=\"table\">Table 4</xref> and <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). Furthermore, a significant negative correlation was found between the 1 Hz PPT and the BBS for the TBI group (<italic>p</italic> = 0.037), while no correlation was found for 0.33 Hz (<italic>p</italic> = 0.09) and 0.5 Hz PPT data (<italic>p</italic> = 0.17) (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref> and <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). For the HC group, PPT data (all frequencies) showed no correlation with the BBS (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref> and <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>).</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Demonstration of a linear relationship between the PPT (1 Hz) and time required to complete <bold>(A)</bold> 5MWT, <bold>(B)</bold> 10MWT, <bold>(C)</bold> TUG, and <bold>(D)</bold> scores for the BBS for the TBI group. No significant correlations were found for the HC group PPT data for all three frequencies. Also, no correlations were found for the 0.33 Hz and 0.5 Hz PPT data for the TBI group as reported in <xref rid=\"T4\" ref-type=\"table\">Table 4</xref>.</p></caption><graphic xlink:href=\"fnins-14-00836-g004\"/></fig><table-wrap id=\"T4\" position=\"float\"><label>TABLE 4</label><caption><p>Results of Pearson’s r correlation analysis between PPT and functional outcomes for both groups.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Groups</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PPT (0.33 Hz)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PPT (0.5 Hz)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">PPT (1 Hz)</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5MWT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HC (<italic>n</italic> = 11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.19, <italic>p</italic> = 0.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.07, <italic>p</italic> = 0.84</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = −0.16, <italic>p</italic> = 0.64</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">TBI (<italic>n</italic> = 10)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.34, <italic>p</italic> = 0.34</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.38, <italic>p</italic> = 0.28</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.67, <bold><italic>p</italic> = 0.034</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">10MWT</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HC (<italic>n</italic> = 11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.26, <italic>p</italic> = 0.44</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.1, <italic>p</italic> = 0.77</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = −0.11, <italic>p</italic> = 0.75</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">TBI (<italic>n</italic> = 10)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.51, <italic>p</italic> = 0.13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.46, <italic>p</italic> = 0.18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.76, <bold><italic>p</italic> = 0.012</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TUG</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HC (<italic>n</italic> = 11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.11, <italic>p</italic> = 0.75</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.34, <italic>p</italic> = 0.31</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.1, <italic>p</italic> = 0.76</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">TBI (<italic>n</italic> = 10)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.38, <italic>p</italic> = 0.29</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.5, <italic>p</italic> = 0.14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = 0.78, <bold><italic>p</italic> = 0.008</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BBS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">HC (<italic>n</italic> = 11)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = −0.36, <italic>p</italic> = 0.28</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = −0.38, <italic>p</italic> = 0.25</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = −0.25, <italic>p</italic> = 0.46</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">TBI (<italic>n</italic> = 10)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = −0.57, <italic>p</italic> = 0.09</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = −0.48, <italic>p</italic> = 0.17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>r</italic> = −0.66, <bold><italic>p</italic> = 0.037</bold></td></tr></tbody></table><table-wrap-foot><attrib><italic>Bold values represent statistically significant differences.</italic></attrib></table-wrap-foot></table-wrap></sec></sec><sec id=\"S4\"><title>Discussion</title><p>The primary objective of this investigation was to quantify the sensory acuity to perturbations to the base of support during standing in individuals with TBI. Accurate perception of body-sway is critical in a dynamic setting to adapt to external stimuli and generate adequate motor responses for achieving balance control. Sensory acuity directly relates to perceptual mechanisms and impairment to sensory afferents as well as their integration after TBI could significantly contribute to impaired sensory acuity and balance dysfunction. Limited research specifically reports objective quantification of impairments to sensory integration after TBI (<xref rid=\"B17\" ref-type=\"bibr\">Peterka et al., 2018</xref>) and no research thus far has investigated sensory acuity in the individuals with TBI. This investigation presents an objective measure of sensory acuity in terms of PPT which goes beyond the biomechanical and functional markers of balance dysfunction and it is related to the process of sensory integration. The sensory organization test (SOT) has been widely used to assess contributions of visual and somatosensory inputs in maintaining balance during standing (<xref rid=\"B16\" ref-type=\"bibr\">Nashner and Peters, 1990</xref>). More recently, Peterka et al. proposed a novel central sensorimotor integration (CSMI) tests to quantify sensory integration by measuring the relative contributions of different sensory systems to balance control (<xref rid=\"B17\" ref-type=\"bibr\">Peterka et al., 2018</xref>). Though these tests provide an objective way to quantify sensory integration, the sensory acuity to external perturbations and its relation to balance function still remains to be studied in individuals with TBI. Further, sensory acuity in terms of detection threshold assessments to the whole-body stimuli have been reported (<xref rid=\"B5\" ref-type=\"bibr\">Fitzpatrick and McCloskey, 1994</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Richerson et al., 2003</xref>, <xref rid=\"B24\" ref-type=\"bibr\">2006</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Puntkattalee et al., 2016</xref>), however limited data exists for individuals with TBI (<xref rid=\"B20\" ref-type=\"bibr\">Pilkar et al., 2016</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Tanis et al., 2018</xref>). For the first time, a classical psychophysical approach was used to determine sensory acuity in terms of PPT in a sample of individuals with impaired balance. A lower PPT for a set of perturbations at a given frequency suggests a better perceptual ability to detect base of support perturbations during standing. The TBI group showed significantly elevated PPT values compared to the HC group for all three perturbation frequencies suggesting their diminished ability to perceive and report changes in their support surface during standing. Multi-sensory deficits are common due to brain lesions after TBI (<xref rid=\"B2\" ref-type=\"bibr\">Allison, 1999</xref>), and these deficits can lead to impaired sensory integration, reduced ability to use the optimal sensory system in different environmental contexts or over-reliance on a single sensory system, which is usually the visual system (<xref rid=\"B2\" ref-type=\"bibr\">Allison, 1999</xref>). However, <xref rid=\"B5\" ref-type=\"bibr\">Fitzpatrick and McCloskey (1994)</xref> showed that the visual thresholds for perceiving movement are higher than the proprioceptive thresholds at slower velocities of base of support movements in healthy individuals. In the context of PPT assessments, the perturbation frequencies are within the natural sway and amplitudes are kept small (<4 mm) by the algorithm as perturbations are confined by the sensory-threshold boundaries. Therefore, such perturbations may be difficult to perceive if only the visual system is used. As a result, sole reliance on the visual system while vestibular and somatosensory systems are impaired could significantly impact one’s ability to perceive the altered posture in relation to itself and the environment. In situations where multiple sensory modalities are available (e.g., PPT assessments), participants will yield thresholds that are equivalent to the sensory modality with the greatest acuity (<xref rid=\"B5\" ref-type=\"bibr\">Fitzpatrick and McCloskey, 1994</xref>). The vestibular system is only known to be engaged at much greater postural disturbances (<xref rid=\"B5\" ref-type=\"bibr\">Fitzpatrick and McCloskey, 1994</xref>) and visual system requires larger threshold amplitude (<xref rid=\"B5\" ref-type=\"bibr\">Fitzpatrick and McCloskey, 1994</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Richerson et al., 2003</xref>). Hence, the majority of the contributions toward PPT where the perturbation frequencies and amplitudes are kept within the natural sway, could stem from the proprioceptive afferents. It is postulated that contributions from the tactile afferents to be minimal as all participants wore shoes on the platform and presence of footwear has shown to attenuate the tactile information compared to the barefoot condition (<xref rid=\"B25\" ref-type=\"bibr\">Robbins et al., 1995</xref>).</p><p>The PPT assessments were performed for three sets of perturbation frequencies. Similar to previously reported studies (<xref rid=\"B24\" ref-type=\"bibr\">Richerson et al., 2006</xref>), our selection of frequencies is based on the rationale that the our primary objective was to quantify the sensory acuity and not study the reactive postural strategies to the external stimuli. Therefore, frequencies ≤1 Hz kept the perturbations within the natural sway (<xref rid=\"B27\" ref-type=\"bibr\">Soames and Atha, 1982</xref>) which were appropriate for our assessments. Of the three perturbation frequencies, 1 Hz perturbations are curious based on two results −- (1) PPT for 1 Hz were significantly lower than 0.33 and 0.5 Hz with no significant difference between 0.33 and 0.5 Hz perturbations; and (2) a significant negative correlation was found between 1 Hz PPT and functional measures of balance (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>) while no such relationship was observed for 0.33 and 0.5 Hz data for the TBI. These results may suggest that 0.33 and 0.5 Hz perturbations might not be differentiable by the sensory systems resulting in no significant perceptual differences for both groups. Further, these slow perturbations may not be sufficient enough to engage the sensory mechanisms that are relevant to influence the functional tasks hence showed no correlation with functional outcomes. On the other hand, 1 Hz perturbations could be sufficient enough to tap into impaired sensory mechanisms of TBI group (but still not large enough to tap into intact sensory system of the HC group) that are also relevant to functional balance tasks. This may have led to the linear relationships between the PPT at 1 Hz and functional measures suggesting that a lower PPT (i.e., the enhanced sensory acuity) could be critical for achieving adequate postural and functional control after TBI. For future investigations, 1 Hz may serve as guidance for selecting perturbation frequencies for similar experiments. Perturbations between 0.5 and 1 Hz could be explored to further confirm the dependency of sensory acuity on perturbation frequency as seen in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>. The PPT assessments for perturbations faster than 1 Hz may induce additional postural strategies (hip) and may require extremely small and precise amplitudes to reach to threshold detection, however such perturbations may not be practically deliverable using the existing Neurocom CRS system or in fact, may not yield the PPT.</p><p>Enhanced perception (i.e., lower PPT) requires integration and interpretation of the multi-sensory afferents as well as the capability to handle attentional demands. Therefore, in addition to impaired sensory integration, an elevated PPT could also stem from the deficits in attention that occur in 39–62% of TBI survivors (<xref rid=\"B15\" ref-type=\"bibr\">Marsh et al., 2016</xref>). Selective attention is essential for dynamic aspects of activities of daily living (<xref rid=\"B29\" ref-type=\"bibr\">Straudi et al., 2017</xref>) and individuals with balance impairments due to deficits in their automatic postural responses (APRs) (<xref rid=\"B32\" ref-type=\"bibr\">Woollacott and Shumway-Cook, 2002</xref>) rely more heavily on attentional mechanisms during standing. Attentional deficits post TBI and potentially impaired APRs due to impaired sensory integration could interfere with a TBI survivor’s ability to safely complete motor tasks (<xref rid=\"B21\" ref-type=\"bibr\">Ponsford and Kinsella, 1992</xref>). Our novel PPT outcome therefore not only reflects the perceptual and attentional indicators of balance deficit but also presents a potentially quantifiable link between the sensory acuity and functional tasks. The absence of significant correlations between the PPT and functional measures for the HC group could potentially imply less reliance on attentional mechanisms and more on their unaffected APRs (<xref rid=\"B22\" ref-type=\"bibr\">Puntkattalee et al., 2016</xref>) as well as intact attentional mechanisms.</p><p>The literature on TBI balance suggests that the level and characteristics of balance impairments are related to the severity and location of the brain damage (<xref rid=\"B2\" ref-type=\"bibr\">Allison, 1999</xref>). The PPT outcome reported in this investigation as a measure of sensory acuity is a manifestation of cognitive (attention) and sensory components. Therefore, the results reported could be influenced by injury characteristics such as time since injury (TSI), severity, location of lesions, etc. Injury characteristics that directly affect cognition (attention), sensory and motor components are expected to show impaired sensory acuity (elevated PPT values). It is expected that the individuals with TBI in the acute stage with the severe symptoms will most likely show elevated PPT values and with the recovery of sensorimotor function over time due to neuroplasticity or rehabilitation, PPT would decrease. Moreover, the individuals with damages to the spinocerebellar tract and the anterior lobe of the cerebellum could show elevated values of PPT as legions to these areas are known to affect the transmission and perception of somatosensation needed to detect the location of body segments in relation to each other and the location of the body in relation to the base of support (<xref rid=\"B2\" ref-type=\"bibr\">Allison, 1999</xref>).</p><sec id=\"S4.SS1\"><title>Limitations and Future Considerations</title><p>The limitations of the current work are its small sample size and heterogeneity within the TBI group in terms of the severity of the injury as well as sex. Heterogeneity within the population is a common challenge in characterizing the balance after TBI due to the complexity of injury and deficits (<xref rid=\"B2\" ref-type=\"bibr\">Allison, 1999</xref>). A larger homogeneous sample of TBI (based on the severity of injury, TSI, legions, and functional capability) with equal distribution of male and female participants needs to be assessed at multiple time-points to comprehensively understand the PPT as an outcome measure. Furthermore, the current unidirectional (applying perturbations only in AP direction) approach of the posturography assessment limits the understanding of the role sensory acuity plays in maintaining balance. It has been suggested that the keys to improving balance after a TBI include training methods that are specific and require multiple adaptive responses (<xref rid=\"B9\" ref-type=\"bibr\">Horak et al., 1997</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Huang et al., 2006</xref>), and as a result, a multidirectional approach for perturbation-based assessment and training is recommended. Finally, the presented method to evaluate sensory acuity employs multi-sensory approach which may not be able to isolate the impairments specific to individual sensory system. However, this investigation focuses on objective evaluation of sensory acuity and its potential connection to the balance dysfunction after TBI. Therefore, use of multi-sensory approach is applicable as most of the functional balance tasks employ a multi-sensory approach. The PPT outcome presented in this investigation can serve as an additional marker of balance dyfunction in addition to the functional and biomechanical (CoP, CoM) outcomes after TBI. The interventions that specifically target the sensory mechanisms have shown to be effective in improving standing balance. E.g., <xref rid=\"B3\" ref-type=\"bibr\">Charkhkar et al. (2020)</xref> showed that the enhanced perception of the plantar pressures under the prosthetic feet achieved using artificial sensory feedback can significantly improve the postural stability of lower limb amputees. Similarly, <xref rid=\"B18\" ref-type=\"bibr\">Petrini et al. (2019)</xref> showed that real-time tactile and proprioceptive feedback provided by sensory neuroprosthetic promoted improved mobility, fall prevention, and agility during active tasks in transfemoral (above-knee) amutees. These investigations show that the manipulation and augmentation of sensory feedback is critical to enhance balance and mobility. Our novel outcome, PPT, can be integrated into balance training paradigms to provide perturbations that engage and enhance proprioception and somatosensation and improve balance after TBI.</p></sec></sec><sec id=\"S5\"><title>Conclusion</title><p>The current work presented the PPT as a new metric for the objective assessment of the sensory acuity to perceive external horizontal perturbations to the base of support during standing in individuals with a TBI. The TBI group showed significantly elevated PPTs compared to the HC group, suggesting their diminished ability to perceive changes to perturbation-induced sway. A significant correlation between the PPTs and functional outcomes was found for the TBI group, demonstrating the critical role perceptual ability may play in achieving improved balance function after injury. Therefore, sub-threshold perturbations that engage perceptual mechanisms could be important to include along with the supra-threshold perturbations that engage the compensatory mechanisms during balance rehabilitation after TBI.</p></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>The datasets generated for this study are subject to the following licenses/restrictions: Data availability will be subjected to the funding agency guidelines. Requests to access these datasets should be directed to <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.state.nj.us/health/njcbir/\">https://www.state.nj.us/health/njcbir/</ext-link>.</p></sec><sec id=\"S7\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by the Kessler Foundation Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.</p></sec><sec id=\"S8\"><title>Author Contributions</title><p>RP and KN designed the study. NE and OI performed the data collection. RP, KK, and AV performed the data analysis and prepared the manuscript. KK and RP performed the statistical analysis. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This study was supported by the New Jersey Commission on Brain Injury Research (NJCBIR) Multi-Investigator Grant CBIR15MIG004.</p></fn></fn-group><ack><p>We would like to thank Dan Tanis and Will Weber for their contributions to participant recruitment and data collection.</p></ack><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Algom</surname><given-names>D.</given-names></name></person-group> (<year>1992</year>). <source><italic>Psychophysical Approaches to Cognition.</italic></source>\n<publisher-loc>Amsterdam</publisher-loc>: <publisher-name>North-Holland</publisher-name>.</mixed-citation></ref><ref id=\"B2\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Allison</surname><given-names>L.</given-names></name></person-group> (<year>1999</year>). <article-title>Imbalance following traumatic brain injury in adults: causes and characteristics.</article-title>\n<source><italic>Neurol. Rep.</italic></source>\n<volume>23</volume>\n<fpage>13</fpage>–<lpage>18</lpage>. <pub-id pub-id-type=\"doi\">10.1097/01253086-199923010-00010</pub-id></mixed-citation></ref><ref id=\"B3\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Charkhkar</surname><given-names>H.</given-names></name><name><surname>Christie</surname><given-names>B. P.</given-names></name><name><surname>Triolo</surname><given-names>R. J.</given-names></name></person-group> (<year>2020</year>). <article-title>Sensory neuroprosthesis improves postural stability during sensory organization test in lower-limb amputees.</article-title>\n<source><italic>Sci. Rep.</italic></source>\n<volume>10</volume>:<issue>6984</issue>.</mixed-citation></ref><ref id=\"B4\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fino</surname><given-names>P. C.</given-names></name><name><surname>Peterka</surname><given-names>R. J.</given-names></name><name><surname>Hullar</surname><given-names>T. E.</given-names></name><name><surname>Murchison</surname><given-names>C.</given-names></name><name><surname>Horak</surname><given-names>F. B.</given-names></name><name><surname>Chesnutt</surname><given-names>J. C.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Assessment and rehabilitation of central sensory impairments for balance in mTBI using auditory biofeedback: a randomized clinical trial.</article-title>\n<source><italic>BMC Neurol.</italic></source>\n<volume>17</volume>:<issue>41</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12883-017-0812-7</pub-id>\n<pub-id pub-id-type=\"pmid\">28231824</pub-id></mixed-citation></ref><ref id=\"B5\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fitzpatrick</surname><given-names>R.</given-names></name><name><surname>McCloskey</surname><given-names>D. I.</given-names></name></person-group> (<year>1994</year>). <article-title>Proprioceptive, visual and vestibular thresholds for the perception of sway during standing in humans.</article-title>\n<source><italic>J. Physiol.</italic></source>\n<volume>478</volume>\n<fpage>173</fpage>–<lpage>186</lpage>. <pub-id pub-id-type=\"doi\">10.1113/jphysiol.1994.sp020240</pub-id>\n<pub-id pub-id-type=\"pmid\">7965833</pub-id></mixed-citation></ref><ref id=\"B6\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Greenwald</surname><given-names>B. D.</given-names></name><name><surname>Cifu</surname><given-names>D. X.</given-names></name><name><surname>Marwitz</surname><given-names>J. H.</given-names></name><name><surname>Enders</surname><given-names>L. J.</given-names></name><name><surname>Brown</surname><given-names>A. W.</given-names></name><name><surname>Englander</surname><given-names>J. S.</given-names></name><etal/></person-group> (<year>2001</year>). <article-title>Factors associated with balance deficits on admission to rehabilitation after traumatic brain injury: a multicenter analysis.</article-title>\n<source><italic>J. Head Trauma Rehabil.</italic></source>\n<volume>16</volume>\n<fpage>238</fpage>–<lpage>252</lpage>. <pub-id pub-id-type=\"doi\">10.1097/00001199-200106000-00003</pub-id>\n<pub-id pub-id-type=\"pmid\">11346446</pub-id></mixed-citation></ref><ref id=\"B7\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Han</surname><given-names>J.</given-names></name><name><surname>Waddington</surname><given-names>G.</given-names></name><name><surname>Adams</surname><given-names>R.</given-names></name><name><surname>Anson</surname><given-names>J.</given-names></name><name><surname>Liu</surname><given-names>Y.</given-names></name></person-group> (<year>2016</year>). <article-title>Assessing proprioception: a critical review of methods.</article-title>\n<source><italic>J. Sport Health Sci.</italic></source>\n<volume>5</volume>\n<fpage>80</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jshs.2014.10.004</pub-id>\n<pub-id pub-id-type=\"pmid\">30356896</pub-id></mixed-citation></ref><ref id=\"B8\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hillier</surname><given-names>S. L.</given-names></name><name><surname>Sharpe</surname><given-names>M. H.</given-names></name><name><surname>Metzer</surname><given-names>J.</given-names></name></person-group> (<year>1997</year>). <article-title>Outcomes 5 years post-traumatic brain injury (with further reference to neurophysical impairment and disability).</article-title>\n<source><italic>Brain Inj.</italic></source>\n<volume>11</volume>\n<fpage>661</fpage>–<lpage>675</lpage>. <pub-id pub-id-type=\"doi\">10.1080/026990597123214</pub-id>\n<pub-id pub-id-type=\"pmid\">9376834</pub-id></mixed-citation></ref><ref id=\"B9\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Horak</surname><given-names>F. B.</given-names></name><name><surname>Henry</surname><given-names>S. M.</given-names></name><name><surname>Shumway-Cook</surname><given-names>A. S.</given-names></name></person-group> (<year>1997</year>). <article-title>Postural perturbations: new insights for treatment of balance disorders.</article-title>\n<source><italic>Physical Therapy.</italic></source>\n<volume>77</volume>\n<fpage>517</fpage>–<lpage>533</lpage>. <pub-id pub-id-type=\"doi\">10.1093/ptj/77.5.517</pub-id>\n<pub-id pub-id-type=\"pmid\">9149762</pub-id></mixed-citation></ref><ref id=\"B10\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>H.</given-names></name><name><surname>Wolf</surname><given-names>S.</given-names></name><name><surname>He</surname><given-names>J.</given-names></name></person-group> (<year>2006</year>). <article-title>Recent developments in biofeedback for neuromotor rehabilitation.</article-title>\n<source><italic>J. Neuroeng. Rehabil.</italic></source>\n<volume>3</volume>:<issue>11</issue>.</mixed-citation></ref><ref id=\"B11\"><mixed-citation publication-type=\"book\"><collab>Institute of Medicine (US), and Division of Health Promotion and Disease Prevention</collab>, (<year>1992</year>). “<article-title>. Falls in older persons: risk factors and prevention</article-title>,” in <source><italic>The Second Fifty Years: Promoting Health and Preventing Disability</italic></source>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>Berg</surname><given-names>R. L.</given-names></name><name><surname>Cassells</surname><given-names>J. S.</given-names></name></person-group>, (<publisher-loc>Washington, DC</publisher-loc>: <publisher-name>Institute of Medicine (US)</publisher-name>).</mixed-citation></ref><ref id=\"B12\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kaernbach</surname><given-names>C.</given-names></name></person-group> (<year>1990</year>). <article-title>A single-interval adjustment-matrix (SIAM) procedure for unbiased adaptive testing.</article-title>\n<source><italic>J. Acoust. Soc. Am.</italic></source>\n<volume>88</volume>\n<fpage>2645</fpage>–<lpage>2655</lpage>.<pub-id pub-id-type=\"pmid\">2283438</pub-id></mixed-citation></ref><ref id=\"B13\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kaufman</surname><given-names>K. R.</given-names></name><name><surname>Brey</surname><given-names>R. H.</given-names></name><name><surname>Chou</surname><given-names>L. S.</given-names></name><name><surname>Rabatin</surname><given-names>A.</given-names></name><name><surname>Brown</surname><given-names>A. W.</given-names></name><name><surname>Basford</surname><given-names>J. R.</given-names></name></person-group> (<year>2006</year>). <article-title>Comparison of subjective and objective measurements of balance disorders following traumatic brain injury.</article-title>\n<source><italic>Med. Eng. Phys.</italic></source>\n<volume>28</volume>\n<fpage>234</fpage>–<lpage>239</lpage>.<pub-id pub-id-type=\"pmid\">16043377</pub-id></mixed-citation></ref><ref id=\"B14\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lehmann</surname><given-names>J. F.</given-names></name><name><surname>Boswell</surname><given-names>S.</given-names></name><name><surname>Price</surname><given-names>R.</given-names></name><name><surname>Burleigh</surname><given-names>A.</given-names></name><name><surname>deLateur</surname><given-names>B. J.</given-names></name><name><surname>Jaffe</surname><given-names>K. M.</given-names></name><etal/></person-group> (<year>1990</year>). <article-title>Quantitative evaluation of sway as an indicator of functional balance in post-traumatic brain injury.</article-title>\n<source><italic>Arch. Phys. Med. Rehabil.</italic></source>\n<volume>71</volume>\n<fpage>955</fpage>–<lpage>962</lpage>.<pub-id pub-id-type=\"pmid\">2241541</pub-id></mixed-citation></ref><ref id=\"B15\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Marsh</surname><given-names>N. V.</given-names></name><name><surname>Ludbrook</surname><given-names>M. R.</given-names></name><name><surname>Gaffaney</surname><given-names>L. C.</given-names></name></person-group> (<year>2016</year>). <article-title>Cognitive functioning following traumatic brain injury: a five-year follow-up.</article-title>\n<source><italic>NeuroRehabilitation.</italic></source>\n<volume>38</volume>\n<fpage>71</fpage>–<lpage>78</lpage>. <pub-id pub-id-type=\"doi\">10.3233/nre-151297</pub-id>\n<pub-id pub-id-type=\"pmid\">26889800</pub-id></mixed-citation></ref><ref id=\"B16\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nashner</surname><given-names>L. M.</given-names></name><name><surname>Peters</surname><given-names>J. F.</given-names></name></person-group> (<year>1990</year>). <article-title>Dynamic posturography in the diagnosis and management of dizziness and balance disorders.</article-title>\n<source><italic>Neurol. Clin.</italic></source>\n<volume>8</volume>\n<fpage>331</fpage>–<lpage>349</lpage>. <pub-id pub-id-type=\"doi\">10.1016/s0733-8619(18)30359-1</pub-id><pub-id pub-id-type=\"pmid\">2193215</pub-id></mixed-citation></ref><ref id=\"B17\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Peterka</surname><given-names>R. J.</given-names></name><name><surname>Murchison</surname><given-names>C. F.</given-names></name><name><surname>Parrington</surname><given-names>L.</given-names></name><name><surname>Fino</surname><given-names>P. C.</given-names></name><name><surname>King</surname><given-names>L. A.</given-names></name></person-group> (<year>2018</year>). <article-title>Implementation of a central sensorimotor integration test for characterization of human balance control during stance.</article-title>\n<source><italic>Front. Neurol.</italic></source>\n<volume>9</volume>:<issue>1045</issue>. <pub-id pub-id-type=\"doi\">10.3389/fneur.2018.01045</pub-id>\n<pub-id pub-id-type=\"pmid\">30619027</pub-id></mixed-citation></ref><ref id=\"B18\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Petrini</surname><given-names>F. M.</given-names></name><name><surname>Valle</surname><given-names>G.</given-names></name><name><surname>Bumbasirevic</surname><given-names>M.</given-names></name><name><surname>Barberi</surname><given-names>F.</given-names></name><name><surname>Bortolotti</surname><given-names>D.</given-names></name><name><surname>Cvancara</surname><given-names>P.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Enhancing functional abilities and cognitive integration of the lower limb prosthesis.</article-title>\n<source><italic>Sci. Transl. Med.</italic></source>\n<volume>11</volume>:<issue>eaav8939</issue>. <pub-id pub-id-type=\"doi\">10.1126/scitranslmed.aav8939</pub-id>\n<pub-id pub-id-type=\"pmid\">31578244</pub-id></mixed-citation></ref><ref id=\"B19\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Pilkar</surname><given-names>R.</given-names></name></person-group> (<year>2011</year>). <source><italic>Analyzing Human Postural Control Responses to the Short Near-Threshold Sinusoidal Translations of the Base of Support.</italic></source>\n<publisher-loc>Ann Arbor, MI</publisher-loc>: <publisher-name>Clarkson University</publisher-name>.</mixed-citation></ref><ref id=\"B20\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pilkar</surname><given-names>R.</given-names></name><name><surname>Arzouni</surname><given-names>N.</given-names></name><name><surname>Ramanujam</surname><given-names>A.</given-names></name><name><surname>Chervin</surname><given-names>K.</given-names></name><name><surname>Nolan</surname><given-names>K. J.</given-names></name></person-group> (<year>2016</year>). <article-title>Postural responses after utilization of a computerized biofeedback based intervention aimed at improving static and dynamic balance in traumatic brain injury: a case study.</article-title>\n<source><italic>Conf. Proc. IEEE Eng. Med. Biol. Soc.</italic></source>\n<volume>2016</volume>\n<fpage>25</fpage>–<lpage>28</lpage>.<pub-id pub-id-type=\"pmid\">28268272</pub-id></mixed-citation></ref><ref id=\"B21\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ponsford</surname><given-names>J.</given-names></name><name><surname>Kinsella</surname><given-names>G.</given-names></name></person-group> (<year>1992</year>). <article-title>Attentional deficits following closed-head injury.</article-title>\n<source><italic>J. Clin. Exp. Neuropsychol.</italic></source>\n<volume>14</volume>\n<fpage>822</fpage>–<lpage>838</lpage>. <pub-id pub-id-type=\"doi\">10.1080/01688639208402865</pub-id>\n<pub-id pub-id-type=\"pmid\">1474148</pub-id></mixed-citation></ref><ref id=\"B22\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Puntkattalee</surname><given-names>M. J.</given-names></name><name><surname>Whitmire</surname><given-names>C. J.</given-names></name><name><surname>Macklin</surname><given-names>A. S.</given-names></name><name><surname>Stanley</surname><given-names>G. B.</given-names></name><name><surname>Ting</surname><given-names>L. H.</given-names></name></person-group> (<year>2016</year>). <article-title>Directional acuity of whole-body perturbations during standing balance.</article-title>\n<source><italic>Gait Posture</italic></source>\n<volume>48</volume>\n<fpage>77</fpage>–<lpage>82</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.gaitpost.2016.04.008</pub-id>\n<pub-id pub-id-type=\"pmid\">27477713</pub-id></mixed-citation></ref><ref id=\"B23\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Richerson</surname><given-names>S. J.</given-names></name><name><surname>Faulkner</surname><given-names>L. W.</given-names></name><name><surname>Robinson</surname><given-names>C. J.</given-names></name><name><surname>Redfern</surname><given-names>M. S.</given-names></name><name><surname>Purucker</surname><given-names>M. C.</given-names></name></person-group> (<year>2003</year>). <article-title>Acceleration threshold detection during short anterior and posterior perturbations on a translating platform.</article-title>\n<source><italic>Gait Posture</italic></source>\n<volume>18</volume>\n<fpage>11</fpage>–<lpage>19</lpage>. <pub-id pub-id-type=\"doi\">10.1016/s0966-6362(02)00189-3</pub-id><pub-id pub-id-type=\"pmid\">14654203</pub-id></mixed-citation></ref><ref id=\"B24\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Richerson</surname><given-names>S. J.</given-names></name><name><surname>Morstatt</surname><given-names>S. M.</given-names></name><name><surname>O’Neal</surname><given-names>K. K.</given-names></name><name><surname>Patrick</surname><given-names>G.</given-names></name><name><surname>Robinson</surname><given-names>C. J.</given-names></name></person-group> (<year>2006</year>). <article-title>Effect of lateral perturbations on psychophysical acceleration detection thresholds.</article-title>\n<source><italic>J. Neuroeng. Rehabil.</italic></source>\n<volume>3</volume>:<issue>2</issue>.</mixed-citation></ref><ref id=\"B25\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Robbins</surname><given-names>S.</given-names></name><name><surname>Waked</surname><given-names>E.</given-names></name><name><surname>McClaran</surname><given-names>J.</given-names></name></person-group> (<year>1995</year>). <article-title>Proprioception and stability: foot position awareness as a function of age and footwear.</article-title>\n<source><italic>Age Ageing</italic></source>\n<volume>24</volume>\n<fpage>67</fpage>–<lpage>72</lpage>.<pub-id pub-id-type=\"pmid\">7762465</pub-id></mixed-citation></ref><ref id=\"B26\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sarno</surname><given-names>S.</given-names></name><name><surname>Erasmus</surname><given-names>L. P.</given-names></name><name><surname>Lipp</surname><given-names>B.</given-names></name><name><surname>Schlaegel</surname><given-names>W.</given-names></name></person-group> (<year>2003</year>). <article-title>Multisensory integration after traumatic brain injury: a reaction time study between pairings of vision, touch and audition.</article-title>\n<source><italic>Brain Inj.</italic></source>\n<volume>17</volume>\n<fpage>413</fpage>–<lpage>426</lpage>.<pub-id pub-id-type=\"pmid\">12745713</pub-id></mixed-citation></ref><ref id=\"B27\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Soames</surname><given-names>R. W.</given-names></name><name><surname>Atha</surname><given-names>J.</given-names></name></person-group> (<year>1982</year>). <article-title>The spectral characteristics of postural sway behaviour.</article-title>\n<source><italic>Eur. J. Appl. Physiol. Occup. Physiol.</italic></source>\n<volume>49</volume>\n<fpage>169</fpage>–<lpage>177</lpage>.<pub-id pub-id-type=\"pmid\">6981506</pub-id></mixed-citation></ref><ref id=\"B28\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sosnoff</surname><given-names>J. J.</given-names></name><name><surname>Broglio</surname><given-names>S. P.</given-names></name><name><surname>Shin</surname><given-names>S.</given-names></name><name><surname>Ferrara</surname><given-names>M. S.</given-names></name></person-group> (<year>2011</year>). <article-title>Previous mild traumatic brain injury and postural-control dynamics.</article-title>\n<source><italic>J. Athl. Train.</italic></source>\n<volume>46</volume>\n<fpage>85</fpage>–<lpage>91</lpage>.<pub-id pub-id-type=\"pmid\">21214355</pub-id></mixed-citation></ref><ref id=\"B29\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Straudi</surname><given-names>S.</given-names></name><name><surname>Severini</surname><given-names>G.</given-names></name><name><surname>Sabbagh Charabati</surname><given-names>A.</given-names></name><name><surname>Pavarelli</surname><given-names>C.</given-names></name><name><surname>Gamberini</surname><given-names>G.</given-names></name><name><surname>Scotti</surname><given-names>A.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>The effects of video game therapy on balance and attention in chronic ambulatory traumatic brain injury: an exploratory study.</article-title>\n<source><italic>BMC Neurol.</italic></source>\n<volume>17</volume>:<issue>86</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12883-017-0871-9</pub-id>\n<pub-id pub-id-type=\"pmid\">28490322</pub-id></mixed-citation></ref><ref id=\"B30\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tanis</surname><given-names>D.</given-names></name><name><surname>Pilkar</surname><given-names>R.</given-names></name><name><surname>Ibironke</surname><given-names>O.</given-names></name><name><surname>Nolan</surname><given-names>K. J.</given-names></name></person-group> (<year>2018</year>). <article-title>Improving perception of perturbations and balance using a novel dynamic computerized biofeedback based intervention after traumatic brain injury.</article-title>\n<source><italic>Conf. Proc. IEEE Eng. Med. Biol. Soc.</italic></source>\n<volume>2018</volume>\n<fpage>5594</fpage>–<lpage>5597</lpage>.<pub-id pub-id-type=\"pmid\">30441604</pub-id></mixed-citation></ref><ref id=\"B31\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Taylor</surname><given-names>M. M.</given-names></name></person-group> (<year>1967</year>). <article-title>Creelman CD. PEST: efficient estimates on probability functions.</article-title>\n<source><italic>J. Acoust. Soc. Am.</italic></source>\n<volume>41</volume>\n<fpage>782</fpage>–<lpage>787</lpage>.</mixed-citation></ref><ref id=\"B32\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Woollacott</surname><given-names>M.</given-names></name><name><surname>Shumway-Cook</surname><given-names>A.</given-names></name></person-group> (<year>2002</year>). <article-title>Attention and the control of posture and gait: a review of an emerging area of research.</article-title>\n<source><italic>Gait Posture</italic></source>\n<volume>16</volume>\n<fpage>1</fpage>–<lpage>14</lpage>.<pub-id pub-id-type=\"pmid\">12127181</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Cell Infect Microbiol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Cell Infect Microbiol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Cell. Infect. Microbiol.</journal-id><journal-title-group><journal-title>Frontiers in Cellular and Infection Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">2235-2988</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850505</article-id><article-id pub-id-type=\"pmc\">PMC7431559</article-id><article-id pub-id-type=\"doi\">10.3389/fcimb.2020.00421</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Cellular and Infection Microbiology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Reciprocal Cooperation of Type A Procyanidin and Nitrofurantoin Against Multi-Drug Resistant (MDR) UPEC: A pH-Dependent Study</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Vasudevan</surname><given-names>Sahana</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/418963/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Thamil Selvan</surname><given-names>Gopalakrishnan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/473030/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Bhaskaran</surname><given-names>Sunil</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Hari</surname><given-names>Natarajan</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1026489/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Solomon</surname><given-names>Adline Princy</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/203188/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Quorum Sensing Laboratory, Centre for Research in Infectious Diseases (CRID), School of Chemical and Biotechnology, SASTRA Deemed to be University</institution>, <addr-line>Thanjavur</addr-line>, <country>India</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Scientific Affairs, Indus Biotech Private Limited</institution>, <addr-line>Pune</addr-line>, <country>India</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Nuclear Magnetic Resonance Laboratory, School of Chemical and Biotechnology, SASTRA Deemed to be University</institution>, <addr-line>Thanjavur</addr-line>, <country>India</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Paola Scavone, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Uruguay</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Sheryl S. Justice, The Ohio State University, United States; Rafael Vignoli, University of the Republic, Uruguay</p></fn><corresp id=\"c001\">*Correspondence: Adline Princy Solomon <email>adlineprinzy@biotech.sastra.edu</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Clinical Microbiology, a section of the journal Frontiers in Cellular and Infection Microbiology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>421</elocation-id><history><date date-type=\"received\"><day>24</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>08</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Vasudevan, Thamil Selvan, Bhaskaran, Hari and Solomon.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Vasudevan, Thamil Selvan, Bhaskaran, Hari and Solomon</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Uropathogenic <italic>Escherichia coli</italic> (UPEC) accounts for the majority of complicated and uncomplicated urinary tract infections. The use of phytomolecules in the treatment of UTI is fast gaining attention. The current report identifies a multidrug-resistant strain (QSLUPEC7), which is a strong biofilm producer, among the considered clinical isolates. The antimicrobial and antibiofilm activity was evaluated for the phytomolecule, Type A procyanidin (TAP) from <italic>Cinnamomum zeylanicum</italic> against QSLUPEC7. TAP treatment did not affect the growth of the MDR strain but affected the biofilm formation (~70% inhibition). The confocal microscopic examination reveals the biofilm inhibition and the live cells in the biofilm corroborates the antimicrobial results. Further, the synergy studies of TAP and nitrofurantoin (NIT) were carried out at different pH. TAP acts synergistically with nitrofurantoin at different pH considered. A closer look in the results reveals that at pH 5.8, maximum growth inhibition is recorded. The gene expression analysis shows that TAP alone and in combination with NIT downregulates the major fimbriae adhesins of UPEC. The results conclude that the TAP has an antibiofilm activity against the multidrug-resistant strain of UPEC, without affecting the growth. Also, TAP reciprocally cooperates with nitrofurantoin at different pH by downregulating the adhesins of UPEC.</p></abstract><kwd-group><kwd>UPEC</kwd><kwd>type A procyanidin</kwd><kwd>nitrofurantoin</kwd><kwd>anti-biofilm</kwd><kwd>adhesins</kwd></kwd-group><counts><fig-count count=\"3\"/><table-count count=\"1\"/><equation-count count=\"2\"/><ref-count count=\"45\"/><page-count count=\"10\"/><word-count count=\"6630\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Urinary tract infections (UTI) are the collective term for pathogenic infections of the urinary tract, which are estimated to cost $5 billion annually (Tan and Chlebicki, <xref rid=\"B39\" ref-type=\"bibr\">2016</xref>). The opportunistic intracellular pathogen, Uropathogenic <italic>Escherichia coli</italic> (UPEC) accounts for the 80–85% of cases of UTIs. The armament of virulence factors, both structural and secreted, of UPEC directs the adhesion and invasion of UPEC to epithelial cells. These virulence factors, along with the biofilm formation, facilitates UPEC growth, persistence in extreme pH variation, and toxin secretion (Flores-Mireles et al., <xref rid=\"B12\" ref-type=\"bibr\">2015</xref>). In addition to virulence factors, host factors such as urine pH and iron availability in the bladder also influence UPEC behavior (Nielubowicz and Mobley, <xref rid=\"B31\" ref-type=\"bibr\">2010</xref>). The therapeutic response of the current treatments is affected by both high urinary concentrations and urinary pH (Cunha, <xref rid=\"B9\" ref-type=\"bibr\">2016</xref>). pH affects not only the growth of the uropathogens but also the efficacy of antibiotics (Burian et al., <xref rid=\"B5\" ref-type=\"bibr\">2012</xref>). Therefore, it is crucial to take into account the pH of the urine before any treatments. The effect of urinary pH in the antimicrobial action of nitrofurantoin is well-documented (Fransen et al., <xref rid=\"B14\" ref-type=\"bibr\">2017</xref>). Nitrofurantoin is a broad-spectrum antibiotic, exclusively used as a therapy for uncomplicated UTI–cystitis (Gardiner et al., <xref rid=\"B15\" ref-type=\"bibr\">2019</xref>) and a prophylactic agent for recurrent UTI (Muller et al., <xref rid=\"B26\" ref-type=\"bibr\">2017</xref>). It is from the Nitrofurans family of flavonoids and works best at an optimum pH of 5.5–6.5 against UPEC (Fransen et al., <xref rid=\"B14\" ref-type=\"bibr\">2017</xref>). A strategy to improve the efficacy of antibiotics in the face of antibiotic resistance and changing pH is to use combinations of plant-derived compounds with antibiotics, which enhances and restores the antibacterial activity of the traditional antibiotics. This improves antibiotic efficiency as well as reduces the concentration drastically without any gain of resistance (Stapleton et al., <xref rid=\"B38\" ref-type=\"bibr\">2004</xref>; Coutinho et al., <xref rid=\"B8\" ref-type=\"bibr\">2009</xref>; Li, <xref rid=\"B22\" ref-type=\"bibr\">2016</xref>). Plant extracts such as polyphenols are known to cause cell wall lysis and inhibit efflux pumps (Chusri et al., <xref rid=\"B6\" ref-type=\"bibr\">2009</xref>). Cinnamon bark (<italic>Cinnamomum zeylanicum</italic>) is traditionally known for possessing potent biological activities such as antibacterial, antitermitic, larvicidal, antifungal, insecticidal, and nematicidal activities (Nabavi et al., <xref rid=\"B29\" ref-type=\"bibr\">2015</xref>). Cinnamons are known to possess oligomeric procyanidins which confer different biological properties (Rauf et al., <xref rid=\"B34\" ref-type=\"bibr\">2019</xref>). Previous reports show the procyanidins to affect dental caries and suppression of various virulence factors from sorghum episperm (Xu et al., <xref rid=\"B45\" ref-type=\"bibr\">2011</xref>). Type A procyanidin (TAP) extracted from cinnamon was previously shown to improve immune responses and antiviral activity (Bhaskaran and Vishwaraman, <xref rid=\"B3\" ref-type=\"bibr\">2014</xref>). The current study explores the kinetics of synergistic action of the TAP and nitrofurantoin at different pH against MDR UPEC.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Chemicals and Reagents Used</title><p>The cinnamon derived type A procyanidin (TAP) was obtained from Indus Biotech Pvt. Ltd., Pune, India. Nitrofurantoin (NIT) antibiotic was purchased from Sigma Aldrich, USA (98.0–102.0% purity). The antibiotic discs were purchased from HiMedia. The stock concentration (1 mg/mL) of TAP was prepared in sterile distilled water and the stock concentration (50 mg/mL) of nitrofurantoin was prepared in DMSO according to CLSI guidelines. The concentration of DMSO was maintained at >0.5% for all assays.</p></sec><sec><title>Microbial Strains and Conditions</title><p>A total of 13 UPEC clinical isolates (QSLUPEC1–QSLUPEC13) were obtained from the microbial repository of JSS medical college, Mysore. These strains were collected at different time points from the patients reported to have urinary tract infections. The isolates were confirmed to be <italic>E. coli</italic> by standard microbiological screening methods. They were also checked for their expression of the <italic>fimX</italic> gene, which is type 1 pili regulator of <italic>E. coli</italic> (Bateman et al., <xref rid=\"B1\" ref-type=\"bibr\">2013</xref>). The strains were maintained as glycerol stock at −80°C. Cation adjusted Muller Hilton Broth (CAMHB) was used for the determination of the antibacterial activity and synergy studies. The biofilm formation and inhibitory effect was done in the Luria Broth media (LB).</p></sec><sec><title>Screening of Multidrug Resistant Strains</title><p>To screen the MDR strains, the clinical isolates were screened for resistance to the antibiotics. The following nine antibiotics which belong to different classes were tested for resistance: Co-Trimoxazole (COT), Trimethoprim (TMP), Ampicillin (AMP), Nalidixic acid (NAL), Streptomycin (STS), Cefuroxime (CXM), Cefotaxime (CTX), Norfloxacin (NOR), and Ciprofloxacin (CIP).</p><p>A standard disc diffusion assay was carried out for screening the resistant strains. Briefly, the bacterial suspensions were prepared from 16 h old plate. The turbidity was set equivalent to 0.5 McFarland standard (OD<sub>595</sub> = 0.08–0.1). The OD adjusted bacterial suspensions were swabbed onto Mueller-Hinton agar plates, incubated for 24 h at 37°C. The zone of inhibition was measured and interpreted with CLSI guidelines [CLSI M100–ED30: (Clinical and Laboratory Standards Institute (CLSI), <xref rid=\"B7\" ref-type=\"bibr\">2020</xref>)]. According to the standard definition by Magiorakos et al. (<xref rid=\"B23\" ref-type=\"bibr\">2012</xref>), the strains resistant to three and more than three antimicrobial classes were defined as MDR.</p></sec><sec><title>Biofilm Formation Assay</title><p>The biofilm-forming capability of the 13 clinical isolates was assessed semi-quantitatively using the standard Tissue Culture Plate (TCP) method and qualitatively using Congo Red Agar (CRA) method (Hassan et al., <xref rid=\"B18\" ref-type=\"bibr\">2011</xref>). For the tissue culture plate method, the overnight plate cultures of each of the clinical isolates were adjusted to 0.5 McFarland units (~1.5 × 10<sup>8</sup> CFU/mL) with saline media. The prepared suspensions were added to 96 well microtiter plates containing LB media (1:10 dilution). After 24 h incubation at 37°C, absorbance was measured at OD<sub>655</sub> using ELISA plate reader (BioRad i-Mark, Japan). Then, the planktonic cells were removed by gently tapping the plate and subsequent washing with water twice. The wells were stained with 100 μL of 0.2% crystal violet stain and incubated for 20 min. After air-drying the plate for 30 min, the bound crystal violet was suspended in 33% acetic acid. The optical density was measured at the wavelength of 595 nm. The specific biofilm formation index was calculated as follows:</p><disp-formula id=\"E1\"><mml:math id=\"M1\"><mml:mtable columnalign=\"left\"><mml:mtr><mml:mtd><mml:mi>S</mml:mi><mml:mi>B</mml:mi><mml:mi>F</mml:mi><mml:mo>=</mml:mo><mml:mtext> </mml:mtext><mml:mfrac><mml:mrow><mml:mi>A</mml:mi><mml:mi>B</mml:mi><mml:mo>-</mml:mo><mml:mi>C</mml:mi><mml:mi>W</mml:mi></mml:mrow><mml:mrow><mml:mi>G</mml:mi></mml:mrow></mml:mfrac></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula><p>where SBF denotes Specific Biofilm formation index, AB denotes OD<sub>595</sub> of the stained attached bacteria, CW denotes OD<sub>595</sub> of the well containing LB media (blank), and G denotes OD<sub>655</sub> of the cell growth in the suspended culture (Naves et al., <xref rid=\"B30\" ref-type=\"bibr\">2008</xref>).</p><p>Qualitatively, biofilm formation was assessed using Congo Red Agar (CRA) method. The congo red agar medium was prepared as described previously (Hassan et al., <xref rid=\"B18\" ref-type=\"bibr\">2011</xref>). The bacterial suspensions were streaked in the CRA media and incubated for 24 h at 37°C. The colonies were visualized, and the strains with black colonies and dry consistency were interpreted as biofilm producers.</p></sec><sec><title>Biofilm Inhibitory Activity of TAP</title><p>From the above assays, the strain that has the dual strength of being a strong biofilm producer and multidrug-resistant was chosen to understand the biological activity of TAP.</p><p>The biofilm inhibitory efficacy of the TAP was tested at different concentrations (128–2 μg/ml). The bacterial cultures inoculated in the above-mentioned conditions were incubated at 37°C for 24 h without agitation. The bacterial inoculated broth was taken as control and the uninoculated broth was taken as blank. The biofilm was processed using the crystal violet assay. Briefly, the planktonic cells were removed, and the adhered cells were fixed with methanol. The adhered biofilm cells were incubated with 0.2% crystal violet for 30 min at room temperature. The excess stain was washed, and biofilm stained crystal violet was eluted using 33% acetic acid. The optical density was measured at the wavelength of 595 nm. The treated biofilm at different concentrations formed is compared to the untreated culture. The minimum biofilm inhibitory concentration –MBIC<sub>50</sub> and MBIC<sub>90</sub> are the lowest concentrations at which the compound inhibits 50 and 90% biofilm as compared to the untreated control, respectively.</p></sec><sec><title>Microscopic Analysis of Biofilm Inhibition</title><p>Confocal microscopy imaging was used to examine the biofilm inhibition of TAP. The bacterial suspensions were prepared as described above. The biofilm was allowed to form in clean, sterile coverslips in the presence and absence of the above-mentioned treatment. After 24 h incubation, the planktonic cells from coverslips were removed by rinsing with sterile water and stained with <italic>Bac</italic>Light Bacterial Viability Kit (L7012) as per the kit protocol. A 40X objective lens was used to capture two and three-dimensional images using a confocal laser scanning microscope (Olympus FLUOVIEW, FV1000).</p></sec><sec><title>Checker Board Analysis</title><p>The double-dose response of TAP and nitrofurantoin was determined using checkerboard analysis. The overnight plate culture of the MDR strain was adjusted to 0.5 McFarland units (~1.5 × 10<sup>8</sup> CFU/mL) with saline media. The prepared suspensions were added to 96 well microtiter plates containing CAMHB media (1:10 dilution). The different dilutions were prepared by dissolving TAP in the bacterial growth media (CAMHB). The concentration of TAP and nitrofurantoin for synergy studies were from 64 to 1 μg/ml: 2-fold dilution. The plates were incubated at 37°C for 24 h. After the incubation, the growth inhibition was measured by comparing the OD<sub>595</sub> of control and the plant polyphenols treated cells.</p><disp-formula id=\"E2\"><mml:math id=\"M2\"><mml:mtable columnalign=\"left\"><mml:mtr><mml:mtd><mml:mi>%</mml:mi><mml:mtext> </mml:mtext><mml:mi>G</mml:mi><mml:mi>r</mml:mi><mml:mi>o</mml:mi><mml:mi>w</mml:mi><mml:mi>t</mml:mi><mml:mi>h</mml:mi><mml:mtext> </mml:mtext><mml:mi>I</mml:mi><mml:mi>n</mml:mi><mml:mi>h</mml:mi><mml:mi>i</mml:mi><mml:mi>b</mml:mi><mml:mi>i</mml:mi><mml:mi>t</mml:mi><mml:mi>i</mml:mi><mml:mi>o</mml:mi><mml:mi>n</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>C</mml:mi><mml:mi>o</mml:mi><mml:mi>n</mml:mi><mml:mi>t</mml:mi><mml:mi>r</mml:mi><mml:mi>o</mml:mi><mml:mi>l</mml:mi><mml:mtext> </mml:mtext><mml:mi>O</mml:mi><mml:msub><mml:mrow><mml:mi>D</mml:mi></mml:mrow><mml:mrow><mml:mn>595</mml:mn></mml:mrow></mml:msub><mml:mo>-</mml:mo><mml:mi>T</mml:mi><mml:mi>e</mml:mi><mml:mi>s</mml:mi><mml:mi>t</mml:mi><mml:mtext> </mml:mtext><mml:mi>O</mml:mi><mml:msub><mml:mrow><mml:mi>D</mml:mi></mml:mrow><mml:mrow><mml:mn>595</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:mi>C</mml:mi><mml:mi>o</mml:mi><mml:mi>n</mml:mi><mml:mi>t</mml:mi><mml:mi>r</mml:mi><mml:mi>o</mml:mi><mml:mi>l</mml:mi><mml:mtext> </mml:mtext><mml:mi>O</mml:mi><mml:msub><mml:mrow><mml:mi>D</mml:mi></mml:mrow><mml:mrow><mml:mn>595</mml:mn></mml:mrow></mml:msub><mml:mtext> </mml:mtext></mml:mrow></mml:mfrac><mml:mtext> </mml:mtext><mml:mo>×</mml:mo><mml:mn>100</mml:mn></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula><p>The synergism was evaluated using the BI (Bliss independence model) and FIC (Fractional Inhibitory Concentration) index. The calculations are similar to the previous reports (Kaur et al., <xref rid=\"B21\" ref-type=\"bibr\">2016</xref>).</p></sec><sec><title>Time-Course Action of the Combination</title><p>The synergism kinetics at different pH (5.2, 5.8, 6.4, 7.0, and 7.6) was performed in broth microdilution method for nitrofurantoin and TAP using 96 well microtiter plate. The bacterial suspension was prepared as mentioned above and was added (1:10 dilution) to the CAMHB media containing different concentrations of the TAP and nitrofurantoin (64–1 μg/ml: 2-fold dilution) at varying pH. The plates were incubated at 37°C and absorbance was measured at 595 nm at a time interval of 1–8 h and also after 24 h.</p></sec><sec><title>Gene Expression Analysis</title><p>The biofilm inhibitory effect of the synergistic combination was understood using the gene expression analysis of the biofilm regulatory genes of UPEC. The planktonic cells (log phase) of UPEC were treated with Nitrofurantoin (8 μg/mL), TAP (32 μg/mL), a combination of TAP (32 μg/mL) and Nitrofurantoin (8 μg/mL) and incubated for 24 h at 37°C. The pH of 5.8 was maintained in all the treatments. Total RNA was extracted by following the manufacturer's guidelines of RNeasy® Protect Bacteria Mini Kit (Qiagen). Standard agarose gel electrophoresis procedure was developed to verify the integrity and NanoDrop (Thermo Scientific, USA) was done to evaluate the purity of isolated RNA. From the isolated RNA, cDNA was synthesized using iScript™ cDNA Synthesis Kit (Manufacturer's protocol was followed).</p><p>The expression level of genes responsible for adhesins of UPEC was analyzed using qRT-PCR. The respective primers and the melting temperature of each of the gene used were listed in <xref ref-type=\"supplementary-material\" rid=\"SM1\">Table S1</xref>. Reference gene and negative control are 16srRNA and without cDNA were maintained, respectively. Calculations of relative gene expression were done with 2<sup>−ΔΔCT</sup> method (Hema et al., <xref rid=\"B19\" ref-type=\"bibr\">2017</xref>).</p></sec><sec><title>Statistical Analysis</title><p>GraphPad Prism software version 8.0.2 (GraphPad Software Inc., San Diego, CA, United States) was used for carrying out the statistical analysis. The significance was checked with Student <italic>t</italic>-test with <italic>p</italic> set at <italic>p</italic> ≤ 0.05. All the assays were carried out in biological and technical triplicates, and the results were expressed as mean ± SD.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>Antibiogram of the Clinical Isolates</title><p>For the clinical isolates tested, the most common resistance was observed in nalidixic acid, ciprofloxacin and ampicillin, followed by cefuroxime, cefotaxime and norfloxacin, tetracycline, co-trimoxazole, trimethoprim, and streptomycin.</p><p>It is interesting to note that no isolate was resistant to all nine antibiotics considered. But all were resistant to at least three antibiotics. Thus, all the strains were multidrug-resistant according to the definition by Magiorakos et al. (<xref rid=\"B23\" ref-type=\"bibr\">2012</xref>), QSLUPEC1 and QSLUPEC6 were resistant to at least one agent in three antimicrobial classes whereas the majority of the strains, QSLUPEC2, QSLUPEC9, QSLUPEC10, QSLUPEC11, QSLUPEC12, and QSLUPEC13 were resistant to in four antimicrobial classes. The clinical isolates, QSLUPEC3, QSLUPEC5, QSLUPEC7, and QSLUPEC8 were resistant to at least one agent in five antibiotic categories (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Table S2</xref>).</p></sec><sec><title>Biofilm Forming Capacity</title><p>The biofilm-forming capacity of the clinical isolates was analyzed using TCP method and CRA method. The data are summarized in <xref ref-type=\"supplementary-material\" rid=\"SM1\">Table S3</xref>. According to Naves et al. (<xref rid=\"B30\" ref-type=\"bibr\">2008</xref>), the strains having SBF > 1.10 is considered to be strong biofilm producers, 0.7–1.09 are moderate biofilm producers, 0.35–0.69 are weak biofilm producers. The SBF <0.35 are not capable of biofilm formation.</p><p>Of the 13 clinical isolates, seven isolates were strong biofilm producers, five were moderate biofilm producers, and only one was a weak biofilm producer. The CRA method correlated with the TCP method except for one clinical isolate. For QSLUPEC2, which showed a moderate biofilm production in the TCP method and observed pink colonies in the case of CRA method.</p><p>The correlation between biofilm formation and antibiotic resistance were next analyzed. Out of the three classified MDR isolates, three were strong biofilm formers (QSLUPEC3, QSLUPEC5, and QSLUPEC7). QSLUPEC8 was a moderate biofilm producer. In order to validate the biofilm inhibition and synergy activity of TAP, QSLUPEC7, which is multidrug-resistant and also a strong biofilm producer, was chosen.</p></sec><sec><title>Biofilm Impairment by TAP</title><p>The antimicrobial and antibiofilm activity of TAP against the QSLUPEC7 was evaluated. TAP did not have any antimicrobial activity across the concentrations considered (Data not shown). The maximum biofilm inhibitory activity was recorded at 128 μg/mL, and a monotonic dose curve was observed (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). The confocal micrographs also substantiated the quantitative results. There was a significant reduction in the biofilm formation with TAP treatment (~71%). The absence of the red fluorescence supported the fact that there was no cell death with the TAP treatment even at the highest concentration considered (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Biofilm Inhibition of TAP. <bold>(A)</bold> The monotonic dose-response curve of biofilm inhibitory action of TAP. The maximum biofilm inhibition was observed at 128 μg/mL. <bold>(B)</bold> The confocal micrographs of the biofilm inhibition at 128 μg/mL. Z-axis length is 10 μm.</p></caption><graphic xlink:href=\"fcimb-10-00421-g0001\"/></fig></sec><sec><title>Potentiation of Nitrofurantoin by TAP—pH-Dependent Study</title><p>The antibiofilm activity at a very low concentration of TAP directed toward the evaluation of potentiating activity of TAP with the known frontline antibiotic, NIT. Previous reports state that the effective pH of nitrofurantoin activity is 5.5–6.5 (Fransen et al., <xref rid=\"B14\" ref-type=\"bibr\">2017</xref>). Thus, the synergy activity was tested in different pH (5.2, 5.8, 6.4, 7.0, and 7.6) in a time-dependent manner. <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref> shows the time-dependent antibacterial activity at a different pH and synergy concentration. It is observed that the antimicrobial action increases in a time-dependent manner. Thus, TAP potentiates the activity of nitrofurantoin significantly at pH 5.8 and pH 7.6, as compared to the other pH values considered. <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> shows the synergy scores calculated using the BLISS independence model in a pH and time-dependent manner. The Bliss score and FIC index values indicate that synergistic action of the TAP and nitrofurantoin. Taking both the synergy models into the account, a strong synergism was observed for pH 5.8, at a lower concentration of 8 μg/mL of NIT and 32 μg/mL. Even though the FIC index indicated synergy in the case of pH 5.2, the BLISS calculation indicated weak synergy. A moderate synergy was observed for pH 6.4 and pH 7.0. A stronger synergism was observed at pH 7.6 at a concentration of 16 μg/mL of NIT and 64 μg/mL.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>pH and time-dependent synergy activity of TAP and NIT. <bold>(A–E)</bold> shows the individual and synergy antibacterial action at the recorded concentrations of synergy activity. <bold>(A,B)</bold> the concentrations of TAP and NIT are 32 and 8 μg/mL, respectively. <bold>(C–E)</bold> the concentrations of TAP and NIT are 64 and 16 μg/mL, respectively. Student unpaired <italic>t</italic>-test was used for the significance analysis. <italic>p</italic> < 0.05 was considered significant. ***<italic>p</italic> = 0.002 and ****<italic>p</italic> ≤ 0.0001. <bold>(F)</bold> Shows the violin plot to depict the distribution of the synergy activity at different time points. At pH 5.8, the activity is maintained across all time points.</p></caption><graphic xlink:href=\"fcimb-10-00421-g0002\"/></fig><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>BLISS and FIC calculations at different pH.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Sl. No</bold>.</th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>pH</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Conc. of NIT (μg/mL)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Conc. of TAP (μg/mL)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Time Point (h)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>BLISS Score</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Interpretation</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>FIC Index</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Interpretation</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">62.7</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weak synergy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.03125</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Synergy</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">41.25</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weak synergy</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">377.4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Strong synergy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.03125</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Synergy</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">418.1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Strong synergy</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">64</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">120.5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Moderate synergy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.25</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Synergy</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">154</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Moderate synergy</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">64</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">245</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Strong synergy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.125</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Synergy</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">186.6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Moderate synergy</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">64</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">153</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Moderate synergy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.125</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Synergy</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">208</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Strong synergy</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table></table-wrap></sec><sec><title>Synergy Activity—Time Course Action</title><p><xref ref-type=\"fig\" rid=\"F2\">Figure 2E</xref> shows a violin plot of the synergy activity at different pH considered in a time-dependent fashion. The maximum inhibition of 43% was recorded for pH 5.2 at 8 h and maintained till 24 h. A similar observation was made for pH 6.4 and 7.0, where the inhibition of 50–65% was maintained from 4 to 24 h. A notable observation is that the potentiation activity of TAP was enhanced at acidic of pH 5.8 as well as slightly alkaline (pH 7.6). The time scale mapping reveals that at pH 5.8 and 7.6, the antimicrobial activity is enhanced from the initial time point till 24 h. A time-dependent gradual increase in the growth inhibition was observed with >75% inhibition at pH 5.8 and 7.6.</p></sec><sec><title>Gene Expression Analysis</title><p>The above studies established the anti-biofilm and potentiating activity of TAP. In order to understand the biofilm inhibitory role of TAP and the synergy combination, gene expression analysis was carried out at pH 5.8 (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). The genes that control bacterial adherence was considered as they are mainly responsible for the bacteria to adhere to the urinary tract. The combination treatment showed a ~4 log<sub>10</sub>-fold reduction in the expression of <italic>focA</italic>, which encodes for the significant fimbrin subunit and belongs to the F1C fimbriae family. This was followed by P-fimbriae component, <italic>papG</italic> with a ~3.5 log<sub>10</sub>-fold reduction. The type I fimbrial adhesion system, <italic>fimA</italic> and <italic>fimH</italic> were also downregulated with ~2 log<sub>10</sub>-fold reduction. When compared to the other fimbriae systems considered, the S fimbriae (<italic>sfaA</italic> and <italic>sfaS</italic>) did not have a significant downregulation. There was only ~1 log<sub>10</sub>-fold reduction observed. A noteworthy observation is that the adhesion systems considered were slightly upregulated with only nitrofurantoin treatment. There was a proportionate downregulation of the fimbriae genes found with the TAP treatment. The downregulation of the adhesins was enhanced in the synergy treatment as compared to the individual treatments.</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Gene expression Studies. <bold>(A)</bold> Heatmap of the adhesion genes downregulated by the combination. <bold>(B)</bold> Tableau Graph that represents the fimbriae systems considered. The graph shows that the synergy treatment significantly downregulated the adhesin genes as compared to the NIT treatment. The log<sub>10</sub>fold difference of the different genes upon different treatments is depicted. All the assays were done in triplicates on different occasions. The combination treatment and TAP downregulated adhesins considered. Student unpaired <italic>t</italic>-test was used for the significance analysis. <italic>p</italic> < 0.05 was considered significant. **<italic>p</italic> ≤ 0.001 and ***<italic>p</italic> ≤ 0.0001.</p></caption><graphic xlink:href=\"fcimb-10-00421-g0003\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Urinary Tract Infections are placed at fourth rank among the infections associated with healthcare (Tenney et al., <xref rid=\"B40\" ref-type=\"bibr\">2018</xref>). It is one of the most common diseases in the community as well as hospital settings. The primary uropathogen responsible for both complicated and uncomplicated UTI is Uropathogenic <italic>E. coli</italic> (UPEC) (Flores-Mireles et al., <xref rid=\"B12\" ref-type=\"bibr\">2015</xref>). With the arsenal of virulence factors, UPEC invades the host system and causes infection (Terlizzi et al., <xref rid=\"B41\" ref-type=\"bibr\">2017</xref>). Different classes of antibiotics are administered to contain the infection (Terlizzi et al., <xref rid=\"B41\" ref-type=\"bibr\">2017</xref>). But the antibiotic treatments are failing due to a sharp increase in antibiotic resistance, and there is a need for alternative therapies. Biofilm is considered to be the major virulence factor of UPEC which promotes adherence to the host and thereby colonizing the host leading to a severe case of infection (Flores-Mireles et al., <xref rid=\"B12\" ref-type=\"bibr\">2015</xref>).</p><p>The current study reports the antibiotic susceptibility pattern of the clinical isolates of UPEC and screening of Multidrug-resistant (MDR) strains among the clinical isolates. Multidrug resistance is defined when the bacterial strain is resistant to at least one agent in three and more than three antimicrobial categories (Magiorakos et al., <xref rid=\"B23\" ref-type=\"bibr\">2012</xref>). According to this definition, the current study classifies all the 13 clinical isolates of UPEC are multidrug-resistant. The strains are resistant to the fluoroquinolones, second and third-generation cephalosporins, folate pathway antagonist, and quinolones. All the strains are nalidixic-resistant.</p><p>The clinical isolates are then analyzed for their biofilm-forming capacity. Biofilm forming capacity is evaluated both quantitatively (crystal violet method) as well as qualitatively (congo red agar method). The results of both methods correlated well with each other. The obtained strains are classified into strong, moderate and weak biofilm producers by their specific biofilm formation index value. Among the strong biofilm producers, three isolates were MDR (QSLUPEC3, QSLUPEC5, and QSLUPEC7). The strong biofilm production correlates significantly with the resistance to multiple class of antibiotics such as cephalosporins (second and third generation), quinolones, aminopenicillin, and fluoroquinolone. A strong biofilm producer being multidrug-resistant strain is well-documented previously (Murugan et al., <xref rid=\"B28\" ref-type=\"bibr\">2011</xref>; Ponnusamy et al., <xref rid=\"B32\" ref-type=\"bibr\">2012</xref>; Mittal et al., <xref rid=\"B25\" ref-type=\"bibr\">2015</xref>). Biofilm gives multiple advantages to the pathogen for its survival, such as protection against host defense mechanism and antibiotic tolerance and resistance. Also, it is documented that the crucial event of the adhesion of the bacterial cells to uroepithelial cells is regulated through biofilm-forming factors, adhesins (Wu et al., <xref rid=\"B44\" ref-type=\"bibr\">1996</xref>). Previous reports have proven that the biofilm formation is correlated to the increased hemolysin production and type 1 fimbriae expression, which are essential virulence factors (Soto et al., <xref rid=\"B36\" ref-type=\"bibr\">2007</xref>). Thus, screening of the multidrug-resistant, strong biofilm producer as an ideal candidate to check the drug action becomes an eventuality. Hence for the further assays, the clinical isolate QSLUPEC7 was chosen, since it was a strong biofilm producing MDR strain.</p><p>Plant polyphenols are secondary metabolites that are mainly produced as plant defensive mechanisms (Daglia, <xref rid=\"B10\" ref-type=\"bibr\">2012</xref>). They have various activities such as antioxidants, anti-allergic, anti-inflammatory, anti-cancer, anti-hypertensive, and antimicrobial agents (Daglia, <xref rid=\"B10\" ref-type=\"bibr\">2012</xref>). It has also been established as an anti-biofilm agent, and researchers showed that biofilm mechanisms such as quorum sensing and other regulatory systems had been downregulated by the plant polyphenols without any effect on their growth (Slobodníková et al., <xref rid=\"B35\" ref-type=\"bibr\">2016</xref>). A-type procyanidins from cranberry have been extensively studied for their anti-adhesive properties, especially against UPEC (Foo et al., <xref rid=\"B13\" ref-type=\"bibr\">2000</xref>; Rane et al., <xref rid=\"B33\" ref-type=\"bibr\">2014</xref>). They possess double interflavanyl linkages which confer the antiadhesive properties to these phytomolecules. It was shown that type-A procyanidin trimers attach to the P-fimbriae of UPEC preventing the adhesion to the uroepithelial cells. The anti-adherence activity for the type A proanthocyanidin trimer from cranberry was seen at a concentration of 2.4 mg/ mL (Foo et al., <xref rid=\"B13\" ref-type=\"bibr\">2000</xref>). In the current study, the anti-biofilm activity of the type-A procyanidin pentamer from cinnamon is shown at a 15 μg/mL (MBIC<sub>50</sub>). The presence of the four interflavanyl linkages present in the pentamer may be attributed to the anti-biofilm activity of the TAP at a lower concentration.</p><p>Proanthocyanidins from cranberries were proven to have synergistic activity with antibiotics against both Gram-positive and Gram-negative organisms. Against <italic>Staphylococcus aureus</italic>, proanthocyanidins were shown to have synergy with the β-lactam antibiotics, as it acts on peptidoglycan synthesis (Diarra et al., <xref rid=\"B11\" ref-type=\"bibr\">2013</xref>). A recent reported that the synergistic action of cranberry proanthocyanidins against Gram-negative organisms was through the repression of the intrinsic resistance mechanisms (Maisuria et al., <xref rid=\"B24\" ref-type=\"bibr\">2019</xref>). In a study against <italic>Pseudomonas aeruginosa</italic>, it was proved that the potentiating activity of the proanthocyanidins due to its iron-chelating property and anti-biofilm property (Ulrey et al., <xref rid=\"B42\" ref-type=\"bibr\">2014</xref>). Thus, biofilm inhibition can potentiate the action of the existing antibiotic (Vasudevan et al., <xref rid=\"B43\" ref-type=\"bibr\">2018</xref>). The present study extends the antibiotic potentiating activity of the cinnamon derived proanthocyanidin to nitrofurantoin. Since it was reported that pH plays a vital role in the antibacterial activity of nitrofurantoin (Fransen et al., <xref rid=\"B14\" ref-type=\"bibr\">2017</xref>), the synergy studies were conducted in a range of pH from 5.2 to 7.6. In order to establish synergy mathematically, two models were considered—Fractional Inhibitory Concentration Index and Bliss independence model (Kaur et al., <xref rid=\"B21\" ref-type=\"bibr\">2016</xref>). There was at least 4-fold reduction in the MIC of NIT at all the pH considered. The maximum inhibitory activity of 80% was observed at pH 5.8, with a reduced concentration of NIT (8 μg/mL) in a time-dependent fashion. At pH 7.6, a similar result was obtained but at a higher concentration (16 μg/mL) but less than the CLSI guidelines of 32 μg/mL. There were conflicting results with respect to pH 5.2 where FIC index showed synergy, but Bliss score showed weak synergy. Thus, it is necessary to take into consideration more than one synergy model for the precise interpretation of the synergy activity.</p><p>The establishment of antibiofilm activity of TAP and the synergy with nitrofurantoin led us to unravel the mechanism of action of TAP and the combination. Adhesins are the portal of entry for UPEC to invade and persist in the dynamic host environment (Mulvey, <xref rid=\"B27\" ref-type=\"bibr\">2002</xref>). They play an important role in the establishment of various virulence pathways, including biofilm formation (Behzadi, <xref rid=\"B2\" ref-type=\"bibr\">2018</xref>). The antibiofilm activity of TAP can be attributed to the downregulation of the majority of the adhesins. Each of the adhesins has an essential and unique role in the pathogenesis process. The most downregulated adhesin is <italic>focA</italic> which is the primary fimbrin unit of F1C fimbriae unit. This is responsible for the ascending UTIs and has an affinity toward the different host cells, which include bladder and kidney epithelial cells (Mulvey, <xref rid=\"B27\" ref-type=\"bibr\">2002</xref>). It should be noted that TAP did not have much effect on S-fimbriae genes which are homologous to F1C fimbriae. <italic>papG</italic> is closely associated with pyelonephritis, which was downregulated by TAP. This encodes for PapG protein which can adhere to erythrocytes and it was shown previously that the cranberry proanthocyanidin binds to P-fimbriae to exert anti-adherence property (Foo et al., <xref rid=\"B13\" ref-type=\"bibr\">2000</xref>; Howell et al., <xref rid=\"B20\" ref-type=\"bibr\">2005</xref>). The type I fimbriae system, <italic>fimA</italic> and <italic>fimH</italic>, is also downregulated. The type 1 fimbriae play a significant role in the bacterial adhesion (Bouckaert et al., <xref rid=\"B4\" ref-type=\"bibr\">2006</xref>), and several studies were conducted to develop FimH inhibitors to evade UPEC pathogenesis (Han et al., <xref rid=\"B17\" ref-type=\"bibr\">2010</xref>, <xref rid=\"B16\" ref-type=\"bibr\">2012</xref>; Spaulding et al., <xref rid=\"B37\" ref-type=\"bibr\">2017</xref>). It was shown that there is a cross-talk in the expression of adhesins regulation. The appearance of one adhesin suppresses the other. Thus, it is required to identify the compounds which can affect multiple adhesins. TAP alone and in combination with NIT was able to downregulate the major adhesins significantly that mediate UPEC attachment to host cells.</p></sec><sec sec-type=\"conclusions\" id=\"s5\"><title>Conclusions</title><p>A wide range of research is being done on the antimicrobial activity of the phytomolecules for the past few years as we are forced to identify alternative strategies to combat the antimicrobial resistance crisis. The ideal characteristics of these polyphenols selected for this study are their bioavailability, diverse structures, and non-toxic nature. This can help in developing them as excellent antimicrobial agents. Various studies also reveal the synergistic effects of Polyphenols and antibiotics. Through this study, the anti-biofilm effect and synergistic effect with the first-line antibiotic used for the uncomplicated UTIs, Nitrofurantoin was established. In the face of dynamic host condition, it is necessary to identify a cocktail of drugs which can evade the bacterial pathogenesis. The synergy studies revealed that the antibacterial activity was enhanced at a lower concentration of the nitrofurantoin at varying pH. Gene expression studies demonstrated the downregulatory effect on UPEC adhesins. Thus, the potentiating effect may be attributed to the biofilm inhibitory and downregulation of adhesins. Further validation studies are required to understand the target(s), the change in the gene expression of the adhesins at different pH and <italic>in vivo</italic> studies which are in process. This study reveals a promising lead to explore the combinatorial effect of Phyto-molecules and the currently used antibiotics.</p></sec><sec sec-type=\"data-availability\" id=\"s6\"><title>Data Availability Statement</title><p>All datasets presented in this study are included in the article/<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Material</xref>.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>SB was employed by Indus Biotech Private Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>The authors would like to thank the management of SASTRA Deemed to be University for providing the required infrastructure to complete the research work. The authors like to thank Dr. Sumana MN, Prof. and HOD, Department of Microbiology, JSS Medical College, Mysore, India for providing the clinical isolates. Authors would also like to thank Ms. Sadhana. S., Postgraduate from SASTRA Deemed to be University for her technical assistance.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> SV wish to expresses her sincere thanks to DST-INSPIRE (IF170369) for the financial support. The Confocal Laser Scanning Microscope (CLSM) Imaging facility used in this study was sponsored by DST-FIST program (SR/FST/LSI-058/2010).</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s9\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fcimb.2020.00421/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fcimb.2020.00421/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Data_Sheet_1.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bateman</surname><given-names>S. L.</given-names></name><name><surname>Stapleton</surname><given-names>A. E.</given-names></name><name><surname>Stamm</surname><given-names>W. E.</given-names></name><name><surname>Hooton</surname><given-names>T. M.</given-names></name><name><surname>Seed</surname><given-names>P. C.</given-names></name></person-group> (<year>2013</year>). <article-title>The type 1 pili regulator gene fimX and pathogenicity island PAI-X as molecular markers of uropathogenic <italic>Escherichia coli</italic></article-title>. <source>Microbiol.</source>\n<volume>159</volume>, <fpage>1606</fpage>–<lpage>1617</lpage>. <pub-id pub-id-type=\"doi\">10.1099/mic.0.066472-0</pub-id><pub-id pub-id-type=\"pmid\">23744903</pub-id></mixed-citation></ref><ref id=\"B2\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Behzadi</surname><given-names>P.</given-names></name></person-group> (<year>2018</year>). <article-title>“Uropathogenic <italic>Escherichia coli</italic> and fimbrial adhesins virulome,”</article-title> in <source>Urinary Tract Infection - The Result of the Strength of the Pathogen, or the Weakness of the Host</source>, eds <person-group person-group-type=\"editor\"><name><surname>Jarzembowski</surname><given-names>T.</given-names></name><name><surname>Daca</surname><given-names>A.</given-names></name><name><surname>Debska-Slizien</surname><given-names>M. A.</given-names></name></person-group> (<publisher-loc>London</publisher-loc>: <publisher-name>InTech</publisher-name>), <fpage>65</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.5772/intechopen.71374</pub-id></mixed-citation></ref><ref id=\"B3\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Bhaskaran</surname><given-names>S.</given-names></name><name><surname>Vishwaraman</surname><given-names>M.</given-names></name></person-group> (<year>2014</year>). <source>Standardized Composition, Method of Manufacture and Use in the Resolution of RNA Virus Infection.</source> U.S. Patent No 8,835,415 B2. <publisher-loc>Washington, DC</publisher-loc>: <publisher-name>U.S. Patent and Trademark Office</publisher-name>.</mixed-citation></ref><ref id=\"B4\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bouckaert</surname><given-names>J.</given-names></name><name><surname>Mackenzie</surname><given-names>J.</given-names></name><name><surname>De Paz</surname><given-names>J. L.</given-names></name><name><surname>Chipwaza</surname><given-names>B.</given-names></name><name><surname>Choudhury</surname><given-names>D.</given-names></name><name><surname>Zavialov</surname><given-names>A.</given-names></name><etal/></person-group>. (<year>2006</year>). <article-title>The affinity of the FimH fimbrial adhesin is receptor-driven and quasi-independent of <italic>Escherichia coli</italic> pathotypes</article-title>. <source>Mol. Microbiol.</source>\n<volume>61</volume>, <fpage>1556</fpage>–<lpage>1568</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-2958.2006.05352.x</pub-id><pub-id pub-id-type=\"pmid\">16930149</pub-id></mixed-citation></ref><ref id=\"B5\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Burian</surname><given-names>A.</given-names></name><name><surname>Erdogan</surname><given-names>Z.</given-names></name><name><surname>Jandrisits</surname><given-names>C.</given-names></name><name><surname>Zeitlinger</surname><given-names>M.</given-names></name></person-group> (<year>2012</year>). <article-title>Impact of pH on activity of trimethoprim, fosfomycin, amikacin, colistin and ertapenem in human urine</article-title>. <source>Pharmacology</source>\n<volume>90</volume>, <fpage>281</fpage>–<lpage>287</lpage>. <pub-id pub-id-type=\"doi\">10.1159/000342423</pub-id><pub-id pub-id-type=\"pmid\">23037005</pub-id></mixed-citation></ref><ref id=\"B6\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chusri</surname><given-names>S.</given-names></name><name><surname>Villanueva</surname><given-names>I.</given-names></name><name><surname>Voravuthikunchai</surname><given-names>S. P.</given-names></name><name><surname>Davies</surname><given-names>J.</given-names></name></person-group> (<year>2009</year>). <article-title>Enhancing antibiotic activity: a strategy to control <italic>Acinetobacter</italic> infections</article-title>. <source>J. Antimicrob. Chemother.</source>\n<volume>64</volume>, <fpage>1203</fpage>–<lpage>1211</lpage>. <pub-id pub-id-type=\"doi\">10.1093/jac/dkp381</pub-id><pub-id pub-id-type=\"pmid\">19861335</pub-id></mixed-citation></ref><ref id=\"B7\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><collab>Clinical and Laboratory Standards Institute (CLSI)</collab></person-group> (<year>2020</year>). <article-title><italic>Performance Standards for Antimicrobial Susceptibility Testing, 30th</italic> E<italic>d</italic>n</article-title>. <source>CLSI supplement M100</source>. <publisher-loc>Wayne, PA</publisher-loc>: <publisher-name>Clinical and Laboratory Standards Institute</publisher-name>.</mixed-citation></ref><ref id=\"B8\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coutinho</surname><given-names>H. D. M.</given-names></name><name><surname>Costa</surname><given-names>J. G. M.</given-names></name><name><surname>Lima</surname><given-names>E. O.</given-names></name><name><surname>Falcão-Silva</surname><given-names>V. S.</given-names></name><name><surname>Siqueira</surname><given-names>J. P.</given-names></name></person-group> (<year>2009</year>). <article-title>Herbal therapy associated with antibiotic therapy: potentiation of the antibiotic activity against methicillin - resistant <italic>Staphylococcus aureus</italic> by <italic>Turnera ulmifolia</italic> L</article-title>. <source>BMC Complement. Altern. Med.</source>\n<volume>9</volume>:<fpage>13</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1472-6882-9-13</pub-id><pub-id pub-id-type=\"pmid\">19426487</pub-id></mixed-citation></ref><ref id=\"B9\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cunha</surname><given-names>B. A.</given-names></name></person-group> (<year>2016</year>). <article-title>An infectious disease and pharmacokinetic perspective on oral antibiotic treatment of uncomplicated urinary tract infections due to multidrug-resistant Gram-negative uropathogens: the importance of urinary antibiotic concentrations and urinary pH</article-title>. <source>Eur. J. Clin. Microbiol. Infect. Dis.</source>\n<volume>35</volume>, <fpage>521</fpage>–<lpage>526</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s10096-016-2577-0</pub-id><pub-id pub-id-type=\"pmid\">26861814</pub-id></mixed-citation></ref><ref id=\"B10\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Daglia</surname><given-names>M.</given-names></name></person-group> (<year>2012</year>). <article-title>Polyphenols as antimicrobial agents</article-title>. <source>Curr. Opin. Biotechnol.</source>\n<volume>23</volume>, <fpage>174</fpage>–<lpage>181</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.copbio.2011.08.007</pub-id><pub-id pub-id-type=\"pmid\">21925860</pub-id></mixed-citation></ref><ref id=\"B11\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Diarra</surname><given-names>M. S.</given-names></name><name><surname>Block</surname><given-names>G.</given-names></name><name><surname>Rempel</surname><given-names>H.</given-names></name><name><surname>Oomah</surname><given-names>B. D.</given-names></name><name><surname>Harrison</surname><given-names>J.</given-names></name><name><surname>Mc Callum</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title><italic>In vitro</italic> and <italic>in vivo</italic> antibacterial activities of cranberry press cake extracts alone or in combination with β-lactams against <italic>Staphylococcus aureus</italic></article-title>. <source>BMC Complement. Altern. Med.</source>\n<volume>13</volume>:<fpage>90</fpage>\n<pub-id pub-id-type=\"doi\">10.1186/1472-6882-13-90</pub-id><pub-id pub-id-type=\"pmid\">23622254</pub-id></mixed-citation></ref><ref id=\"B12\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Flores-Mireles</surname><given-names>A. L.</given-names></name><name><surname>Walker</surname><given-names>J. N.</given-names></name><name><surname>Caparon</surname><given-names>M.</given-names></name><name><surname>Hultgren</surname><given-names>S. J.</given-names></name></person-group> (<year>2015</year>). <article-title>Urinary tract infections: epidemiology, mechanisms of infection and treatment options</article-title>. <source>Nat. Rev. Microbiol.</source>\n<volume>13</volume>, <fpage>269</fpage>–<lpage>284</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrmicro3432</pub-id><pub-id pub-id-type=\"pmid\">25853778</pub-id></mixed-citation></ref><ref id=\"B13\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Foo</surname><given-names>L. Y.</given-names></name><name><surname>Lu</surname><given-names>Y.</given-names></name><name><surname>Howell</surname><given-names>A. B.</given-names></name><name><surname>Vorsa</surname><given-names>N.</given-names></name></person-group> (<year>2000</year>). <article-title>A-type proanthocyanidin trimers from cranberry that inhibit adherence of uropathogenic P-fimbriated <italic>Escherichia coli</italic></article-title>. <source>J. Nat. Prod.</source>\n<volume>63</volume>, <fpage>1225</fpage>–<lpage>1228</lpage>. <pub-id pub-id-type=\"doi\">10.1021/np000128u</pub-id><pub-id pub-id-type=\"pmid\">11000024</pub-id></mixed-citation></ref><ref id=\"B14\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fransen</surname><given-names>F.</given-names></name><name><surname>Melchers</surname><given-names>M. J. B.</given-names></name><name><surname>Lagarde</surname><given-names>C. M. C.</given-names></name><name><surname>Meletiadis</surname><given-names>J.</given-names></name><name><surname>Mouton</surname><given-names>J. W.</given-names></name></person-group> (<year>2017</year>). <article-title>Pharmacodynamics of nitrofurantoin at different pH levels against pathogens involved in urinary tract infections</article-title>. <source>J. Antimicrob. Chemother.</source>\n<volume>72</volume>, <fpage>3366</fpage>–<lpage>3373</lpage>. <pub-id pub-id-type=\"doi\">10.1093/jac/dkx313</pub-id><pub-id pub-id-type=\"pmid\">28961900</pub-id></mixed-citation></ref><ref id=\"B15\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gardiner</surname><given-names>B. J.</given-names></name><name><surname>Stewardson</surname><given-names>A. J.</given-names></name><name><surname>Abbott</surname><given-names>I. J.</given-names></name><name><surname>Peleg</surname><given-names>A. Y.</given-names></name></person-group> (<year>2019</year>). <article-title>Nitrofurantoin and fosfomycin for resistant urinary tract infections: old drugs for emerging problems</article-title>. <source>Aust. Prescr.</source>\n<volume>42</volume>, <fpage>14</fpage>–<lpage>19</lpage>. <pub-id pub-id-type=\"doi\">10.18773/austprescr.2019.002</pub-id><pub-id pub-id-type=\"pmid\">30765904</pub-id></mixed-citation></ref><ref id=\"B16\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Han</surname><given-names>Z.</given-names></name><name><surname>Pinkner</surname><given-names>J. S.</given-names></name><name><surname>Ford</surname><given-names>B.</given-names></name><name><surname>Chorell</surname><given-names>E.</given-names></name><name><surname>Crowley</surname><given-names>J. M.</given-names></name><name><surname>Cusumano</surname><given-names>C. K.</given-names></name><etal/></person-group>. (<year>2012</year>). <article-title>Lead optimization studies on FimH antagonists: discovery of potent and orally bioavailable ortho-substituted biphenyl mannosides</article-title>. <source>J. Med. Chem.</source>\n<volume>55</volume>, <fpage>3945</fpage>–<lpage>3959</lpage>. <pub-id pub-id-type=\"doi\">10.1021/jm300165m</pub-id><pub-id pub-id-type=\"pmid\">22449031</pub-id></mixed-citation></ref><ref id=\"B17\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Han</surname><given-names>Z.</given-names></name><name><surname>Pinkner</surname><given-names>J. S.</given-names></name><name><surname>Ford</surname><given-names>B.</given-names></name><name><surname>Obermann</surname><given-names>R.</given-names></name><name><surname>Nolan</surname><given-names>W.</given-names></name><name><surname>Wildman</surname><given-names>S. A.</given-names></name><etal/></person-group>. (<year>2010</year>). <article-title>Structure-based drug design and optimization of mannoside bacterial FimH antagonists</article-title>. <source>J. Med. Chem.</source>\n<volume>53</volume>, <fpage>4779</fpage>–<lpage>4792</lpage>. <pub-id pub-id-type=\"doi\">10.1021/jm100438s</pub-id><pub-id pub-id-type=\"pmid\">20507142</pub-id></mixed-citation></ref><ref id=\"B18\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hassan</surname><given-names>A.</given-names></name><name><surname>Usman</surname><given-names>J.</given-names></name><name><surname>Kaleem</surname><given-names>F.</given-names></name><name><surname>Omair</surname><given-names>M.</given-names></name><name><surname>Khalid</surname><given-names>A.</given-names></name><name><surname>Iqbal</surname><given-names>M.</given-names></name></person-group> (<year>2011</year>). <article-title>Evaluation of different detection methods of biofilm formation in the clinical isolates</article-title>. <source>Brazilian J. Infect. Dis.</source>\n<volume>15</volume>, <fpage>305</fpage>–<lpage>311</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S1413-8670(11)70197-0</pub-id><pub-id pub-id-type=\"pmid\">21860999</pub-id></mixed-citation></ref><ref id=\"B19\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hema</surname><given-names>M.</given-names></name><name><surname>Vasudevan</surname><given-names>S.</given-names></name><name><surname>Balamurugan</surname><given-names>P.</given-names></name><name><surname>Adline Princy</surname><given-names>S.</given-names></name></person-group> (<year>2017</year>). <article-title>Modulating the global response regulator, LuxO of <italic>V. cholerae</italic> quorum sensing system using a pyrazine dicarboxylic acid derivative (PDCApy): an antivirulence approach</article-title>. <source>Front. Cell. Infect. Microbiol.</source>\n<volume>7</volume>:<fpage>441</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fcimb.2017.00441</pub-id><pub-id pub-id-type=\"pmid\">29075619</pub-id></mixed-citation></ref><ref id=\"B20\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Howell</surname><given-names>A. B.</given-names></name><name><surname>Reed</surname><given-names>J. D.</given-names></name><name><surname>Krueger</surname><given-names>C. G.</given-names></name><name><surname>Winterbottom</surname><given-names>R.</given-names></name><name><surname>Cunningham</surname><given-names>D. G.</given-names></name><name><surname>Leahy</surname><given-names>M.</given-names></name></person-group> (<year>2005</year>). <article-title>A-type cranberry proanthocyanidins and uropathogenic bacterial anti-adhesion activity</article-title>. <source>Phytochemistry</source>\n<volume>66</volume>, <fpage>2281</fpage>–<lpage>2291</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.phytochem.2005.05.022</pub-id><pub-id pub-id-type=\"pmid\">16055161</pub-id></mixed-citation></ref><ref id=\"B21\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kaur</surname><given-names>G.</given-names></name><name><surname>Balamurugan</surname><given-names>P.</given-names></name><name><surname>Maheswari</surname><given-names>C. U.</given-names></name><name><surname>Anitha</surname><given-names>A.</given-names></name><name><surname>Princy</surname><given-names>S. A.</given-names></name></person-group> (<year>2016</year>). <article-title>Combinatorial effects of aromatic 1, 3-disubstituted ureas and fluoride on <italic>in vitro</italic> inhibition of <italic>Streptococcus</italic> mutans biofilm formation</article-title>. <source>Front. Microbiol.</source>\n<volume>7</volume>:<fpage>861</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2016.00861</pub-id><pub-id pub-id-type=\"pmid\">27375583</pub-id></mixed-citation></ref><ref id=\"B22\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>Y.</given-names></name></person-group> (<year>2016</year>). <article-title>“Overall safety of peroxides,”</article-title> in Tooth <italic>W</italic>hitening <italic>a</italic>n <italic>E</italic>vidence-<italic>B</italic>ased <italic>P</italic>erspect, ed <person-group person-group-type=\"editor\"><name><surname>Perdigão</surname><given-names>J.</given-names></name></person-group> (<publisher-loc>Cham</publisher-loc>: <publisher-name>Springer</publisher-name>), <fpage>35</fpage>–<lpage>44</lpage>. <pub-id pub-id-type=\"doi\">10.1007/978-3-319-38849-6_3</pub-id></mixed-citation></ref><ref id=\"B23\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Magiorakos</surname><given-names>A. P.</given-names></name><name><surname>Srinivasan</surname><given-names>A.</given-names></name><name><surname>Carey</surname><given-names>R. B.</given-names></name><name><surname>Carmeli</surname><given-names>Y.</given-names></name><name><surname>Falagas</surname><given-names>M. E.</given-names></name><name><surname>Giske</surname><given-names>C. G.</given-names></name><etal/></person-group>. (<year>2012</year>). <article-title>Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance</article-title>. <source>Clin. Microbiol. Infect.</source>\n<volume>18</volume>, <fpage>268</fpage>–<lpage>281</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1469-0691.2011.03570.x</pub-id><pub-id pub-id-type=\"pmid\">21793988</pub-id></mixed-citation></ref><ref id=\"B24\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maisuria</surname><given-names>V. B.</given-names></name><name><surname>Okshevsky</surname><given-names>M.</given-names></name><name><surname>Déziel</surname><given-names>E.</given-names></name><name><surname>Tufenkji</surname><given-names>N.</given-names></name></person-group> (<year>2019</year>). <article-title>Proanthocyanidin interferes with intrinsic antibiotic resistance mechanisms of gram-negative bacteria</article-title>. <source>Adv. Sci.</source>\n<volume>6</volume>:<fpage>1802333</fpage>. <pub-id pub-id-type=\"doi\">10.1002/advs.201802333</pub-id><pub-id pub-id-type=\"pmid\">31406662</pub-id></mixed-citation></ref><ref id=\"B25\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mittal</surname><given-names>S.</given-names></name><name><surname>Sharma</surname><given-names>M.</given-names></name><name><surname>Chaudhary</surname><given-names>U.</given-names></name></person-group> (<year>2015</year>). <article-title>Biofilm and multidrug resistance in uropathogenic <italic>Escherichia coli</italic></article-title>. <source>Pathog. Glob. Health</source>\n<volume>109</volume>, <fpage>26</fpage>–<lpage>29</lpage>. <pub-id pub-id-type=\"doi\">10.1179/2047773215Y.0000000001</pub-id><pub-id pub-id-type=\"pmid\">25605466</pub-id></mixed-citation></ref><ref id=\"B26\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Muller</surname><given-names>A. E.</given-names></name><name><surname>Verhaegh</surname><given-names>E. M.</given-names></name><name><surname>Harbarth</surname><given-names>S.</given-names></name><name><surname>Mouton</surname><given-names>J. W.</given-names></name><name><surname>Huttner</surname><given-names>A.</given-names></name></person-group> (<year>2017</year>). <article-title>Nitrofurantoin's efficacy and safety as prophylaxis for urinary tract infections: a systematic review of the literature and meta-analysis of controlled trials</article-title>. <source>Clin. Microbiol. Infect.</source>\n<volume>23</volume>, <fpage>355</fpage>–<lpage>362</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cmi.2016.08.003</pub-id><pub-id pub-id-type=\"pmid\">27542332</pub-id></mixed-citation></ref><ref id=\"B27\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mulvey</surname><given-names>M. A.</given-names></name></person-group> (<year>2002</year>). <article-title>Adhesion and entry of uropathogenic <italic>Escherichia coli</italic></article-title>. <source>Cell. Microbiol.</source>\n<volume>4</volume>, <fpage>257</fpage>–<lpage>271</lpage>. <pub-id pub-id-type=\"doi\">10.1046/j.1462-5822.2002.00193.x</pub-id><pub-id pub-id-type=\"pmid\">12027955</pub-id></mixed-citation></ref><ref id=\"B28\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Murugan</surname><given-names>S.</given-names></name><name><surname>Devi</surname><given-names>P. U.</given-names></name><name><surname>John</surname><given-names>P. N.</given-names></name></person-group> (<year>2011</year>). <article-title>Antimicrobial susceptibility pattern of biofilm producing <italic>Escherichia coli</italic> of urinary tract infections</article-title>. <source>Curr. Res. Bacteriol.</source>\n<volume>4</volume>, <fpage>73</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.3923/crb.2011.73.80</pub-id></mixed-citation></ref><ref id=\"B29\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nabavi</surname><given-names>S. F.</given-names></name><name><surname>Di Lorenzo</surname><given-names>A.</given-names></name><name><surname>Izadi</surname><given-names>M.</given-names></name><name><surname>Sobarzo-Sánchez</surname><given-names>E.</given-names></name><name><surname>Daglia</surname><given-names>M.</given-names></name><name><surname>Nabavi</surname><given-names>S. M.</given-names></name></person-group> (<year>2015</year>). <article-title>Antibacterial effects of cinnamon: from farm to food, cosmetic and pharmaceutical industries</article-title>. <source>Nutrients</source>\n<volume>7</volume>, <fpage>7729</fpage>–<lpage>7748</lpage>. <pub-id pub-id-type=\"doi\">10.3390/nu7095359</pub-id><pub-id pub-id-type=\"pmid\">26378575</pub-id></mixed-citation></ref><ref id=\"B30\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Naves</surname><given-names>P.</given-names></name><name><surname>Del Prado</surname><given-names>G.</given-names></name><name><surname>Huelves</surname><given-names>L.</given-names></name><name><surname>Gracia</surname><given-names>M.</given-names></name><name><surname>Ruiz</surname><given-names>V.</given-names></name><name><surname>Blanco</surname><given-names>J.</given-names></name><etal/></person-group>. (<year>2008</year>). <article-title>Measurement of biofilm formation by clinical isolates of <italic>Escherichia coli</italic> is method-dependent</article-title>. <source>J. Appl. Microbiol.</source>\n<volume>105</volume>, <fpage>585</fpage>–<lpage>590</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-2672.2008.03791.x</pub-id><pub-id pub-id-type=\"pmid\">18363684</pub-id></mixed-citation></ref><ref id=\"B31\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nielubowicz</surname><given-names>G. R.</given-names></name><name><surname>Mobley</surname><given-names>H. L. T.</given-names></name></person-group> (<year>2010</year>). <article-title>Host-pathogen interactions in urinary tract infection</article-title>. <source>Nat. Rev. Urol.</source>\n<volume>7</volume>, <fpage>430</fpage>–<lpage>441</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrurol.2010.101</pub-id><pub-id pub-id-type=\"pmid\">20647992</pub-id></mixed-citation></ref><ref id=\"B32\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ponnusamy</surname><given-names>P.</given-names></name><name><surname>Natarajan</surname><given-names>V.</given-names></name><name><surname>Sevanan</surname><given-names>M.</given-names></name></person-group> (<year>2012</year>). <article-title><italic>In vitro</italic> biofilm formation by uropathogenic <italic>Escherichia coli</italic> and their antimicrobial susceptibility pattern</article-title>. <source>Asian Pac. J. Trop. Med.</source>\n<volume>5</volume>, <fpage>210</fpage>–<lpage>213</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S1995-7645(12)60026-1</pub-id><pub-id pub-id-type=\"pmid\">22305786</pub-id></mixed-citation></ref><ref id=\"B33\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rane</surname><given-names>H. S.</given-names></name><name><surname>Bernardo</surname><given-names>S. M.</given-names></name><name><surname>Howell</surname><given-names>A. B.</given-names></name><name><surname>Lee</surname><given-names>S. A.</given-names></name></person-group> (<year>2014</year>). <article-title>Cranberry-derived proanthocyanidins prevent formation of <italic>Candida albicans</italic> biofilms in artificial urine through biofilm- and adherence-specific mechanisms</article-title>. <source>J. Antimicrob. Chemother.</source>\n<volume>69</volume>, <fpage>428</fpage>–<lpage>436</lpage>. <pub-id pub-id-type=\"doi\">10.1093/jac/dkt398</pub-id><pub-id pub-id-type=\"pmid\">24114570</pub-id></mixed-citation></ref><ref id=\"B34\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rauf</surname><given-names>A.</given-names></name><name><surname>Imran</surname><given-names>M.</given-names></name><name><surname>Abu-Izneid</surname><given-names>T.</given-names></name><name><surname>Iahtisham-Ul-Haq</surname></name><name><surname>Patel</surname><given-names>S.</given-names></name><name><surname>Pan</surname><given-names>X.</given-names></name><etal/></person-group>. (<year>2019</year>). <article-title>Proanthocyanidins: a comprehensive review</article-title>. <source>Biomed. Pharmacother.</source>\n<volume>116</volume>:<fpage>108999</fpage>. <pub-id pub-id-type=\"doi\">10.1016/j.biopha.2019.108999</pub-id><pub-id pub-id-type=\"pmid\">31146109</pub-id></mixed-citation></ref><ref id=\"B35\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Slobodníková</surname><given-names>L.</given-names></name><name><surname>Fialová</surname><given-names>S.</given-names></name><name><surname>Rendeková</surname><given-names>K.</given-names></name><name><surname>Kováč</surname><given-names>J.</given-names></name><name><surname>Mučaji</surname><given-names>P.</given-names></name></person-group> (<year>2016</year>). <article-title>Antibiofilm activity of plant polyphenols</article-title>. <source>Molecules</source>\n<volume>21</volume>, <fpage>1</fpage>–<lpage>15</lpage>. <pub-id pub-id-type=\"doi\">10.3390/molecules21121717</pub-id><pub-id pub-id-type=\"pmid\">27983597</pub-id></mixed-citation></ref><ref id=\"B36\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Soto</surname><given-names>S. M.</given-names></name><name><surname>Smithson</surname><given-names>A.</given-names></name><name><surname>Martinez</surname><given-names>J. A.</given-names></name><name><surname>Horcajada</surname><given-names>J. P.</given-names></name><name><surname>Mensa</surname><given-names>J.</given-names></name><name><surname>Vila</surname><given-names>J.</given-names></name></person-group> (<year>2007</year>). <article-title>Biofilm formation in uropathogenic <italic>Escherichia coli</italic> strains: relationship with prostatitis, urovirulence factors and antimicrobial resistance</article-title>. <source>J. Urol.</source>\n<volume>177</volume>, <fpage>365</fpage>–<lpage>368</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.juro.2006.08.081</pub-id><pub-id pub-id-type=\"pmid\">17162092</pub-id></mixed-citation></ref><ref id=\"B37\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Spaulding</surname><given-names>C. N.</given-names></name><name><surname>Klein</surname><given-names>R. D.</given-names></name><name><surname>Ruer</surname><given-names>S.</given-names></name><name><surname>Kau</surname><given-names>A. L.</given-names></name><name><surname>Schreiber</surname><given-names>H. L.</given-names></name><name><surname>Cusumano</surname><given-names>Z. T.</given-names></name><etal/></person-group>. (<year>2017</year>). <article-title>Selective depletion of uropathogenic <italic>E. coli</italic> from the gut by a FimH antagonist</article-title>. <source>Nature</source>\n<volume>546</volume>, <fpage>528</fpage>–<lpage>532</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature22972</pub-id><pub-id pub-id-type=\"pmid\">28614296</pub-id></mixed-citation></ref><ref id=\"B38\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stapleton</surname><given-names>P. D.</given-names></name><name><surname>Shah</surname><given-names>S.</given-names></name><name><surname>Anderson</surname><given-names>J. C.</given-names></name><name><surname>Hara</surname><given-names>Y.</given-names></name><name><surname>Hamilton-Miller</surname><given-names>J. M. T.</given-names></name><name><surname>Taylor</surname><given-names>P. W.</given-names></name></person-group> (<year>2004</year>). <article-title>Modulation of β-lactam resistance in <italic>Staphylococcus aureus</italic> by catechins and gallates</article-title>. <source>Int. J. Antimicrob. Agents</source>\n<volume>23</volume>, <fpage>462</fpage>–<lpage>467</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijantimicag.2003.09.027</pub-id><pub-id pub-id-type=\"pmid\">15120724</pub-id></mixed-citation></ref><ref id=\"B39\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tan</surname><given-names>C. W.</given-names></name><name><surname>Chlebicki</surname><given-names>M. P.</given-names></name></person-group> (<year>2016</year>). <article-title>Urinary tract infections in adults</article-title>. <source>Singapore Med. J.</source>\n<volume>57</volume>, <fpage>485</fpage>–<lpage>490</lpage>. <pub-id pub-id-type=\"doi\">10.11622/smedj.2016153</pub-id><pub-id pub-id-type=\"pmid\">27662890</pub-id></mixed-citation></ref><ref id=\"B40\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tenney</surname><given-names>J.</given-names></name><name><surname>Hudson</surname><given-names>N.</given-names></name><name><surname>Alnifaidy</surname><given-names>H.</given-names></name><name><surname>Ting</surname><given-names>J.</given-names></name><name><surname>Li</surname><given-names>C.</given-names></name><name><surname>Harriet</surname><given-names>K.</given-names></name></person-group> (<year>2018</year>). <article-title>Risk factors for aquiring multidrug-resistant organisms in urinary tract infections : a systematic literature review</article-title>. <source>Saudi Pharm. J.</source>\n<volume>26</volume>, <fpage>678</fpage>–<lpage>684</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jsps.2018.02.023</pub-id><pub-id pub-id-type=\"pmid\">29991911</pub-id></mixed-citation></ref><ref id=\"B41\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Terlizzi</surname><given-names>M. E.</given-names></name><name><surname>Gribaudo</surname><given-names>G.</given-names></name><name><surname>Maffei</surname><given-names>M. E.</given-names></name></person-group> (<year>2017</year>). <article-title>UroPathogenic <italic>Escherichia coli</italic> (UPEC) infections: virulence factors, bladder responses, antibiotic, and non-antibiotic antimicrobial strategies</article-title>. <source>Front. Microbiol.</source>\n<volume>8</volume>:<fpage>566</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2017.01566</pub-id><pub-id pub-id-type=\"pmid\">28421059</pub-id></mixed-citation></ref><ref id=\"B42\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ulrey</surname><given-names>R. K.</given-names></name><name><surname>Barksdale</surname><given-names>S. M.</given-names></name><name><surname>Zhou</surname><given-names>W.</given-names></name><name><surname>van Hoek</surname><given-names>M. L.</given-names></name></person-group> (<year>2014</year>). <article-title>Cranberry proanthocyanidins have anti-biofilm properties against <italic>Pseudomonas aeruginosa</italic></article-title>. <source>BMC Complement. Altern. Med.</source>\n<volume>14</volume>:<fpage>499</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1472-6882-14-499</pub-id><pub-id pub-id-type=\"pmid\">25511463</pub-id></mixed-citation></ref><ref id=\"B43\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Vasudevan</surname><given-names>S.</given-names></name><name><surname>Swamy</surname><given-names>S. S.</given-names></name><name><surname>Kaur</surname><given-names>G.</given-names></name><name><surname>Princy</surname><given-names>S. A.</given-names></name><name><surname>Balamurugan</surname><given-names>P.</given-names></name></person-group> (<year>2018</year>) <article-title>“Synergism between quorum sensing inhibitors antibiotics: combating the antibiotic resistance crisis,”</article-title> in <source>Biotechnological Applications of Quorum Sensing Inhibitors</source>, ed <person-group person-group-type=\"editor\"><name><surname>Kalia</surname><given-names>V. C.</given-names></name></person-group> (<publisher-loc>Singapore</publisher-loc>: <publisher-name>Springer</publisher-name>), <fpage>205</fpage>–<lpage>225</lpage>. <pub-id pub-id-type=\"doi\">10.1007/978-981-10-9026-4_10</pub-id></mixed-citation></ref><ref id=\"B44\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>X.-R.</given-names></name><name><surname>Sun Ii</surname><given-names>T.-T.</given-names></name><name><surname>Medina</surname><given-names>J. J.</given-names></name></person-group> (<year>1996</year>). <article-title><italic>In vitro</italic> binding of type 1-fimbriated <italic>Escherichia coli</italic> to uroplakins Ia and Tb: relation to urinary tract infections</article-title>. <source>Cell Biol.</source>\n<volume>93</volume>, <fpage>9630</fpage>–<lpage>9635</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.93.18.9630</pub-id></mixed-citation></ref><ref id=\"B45\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xu</surname><given-names>L.</given-names></name><name><surname>Liu</surname><given-names>R.</given-names></name><name><surname>Li</surname><given-names>D.</given-names></name><name><surname>Tu</surname><given-names>S.</given-names></name><name><surname>Chen</surname><given-names>J.</given-names></name></person-group> (<year>2011</year>). <article-title>An <italic>in vitro</italic> study on the dental caries preventing effect of oligomeric procyanidins in sorghum episperm</article-title>. <source>Food Chem.</source>\n<volume>126</volume>, <fpage>911</fpage>–<lpage>916</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.foodchem.2010.11.075</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Oncol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Oncol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Oncol.</journal-id><journal-title-group><journal-title>Frontiers in Oncology</journal-title></journal-title-group><issn pub-type=\"epub\">2234-943X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850461</article-id><article-id pub-id-type=\"pmc\">PMC7431560</article-id><article-id pub-id-type=\"doi\">10.3389/fonc.2020.01571</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Oncology</subject><subj-group><subject>Perspective</subject></subj-group></subj-group></article-categories><title-group><article-title>COVID-19 Emergency and Post-Emergency in Italian Cancer Patients: How Can Patients Be Assisted?</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Crispo</surname><given-names>Anna</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/334287/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Montagnese</surname><given-names>Concetta</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/973248/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Perri</surname><given-names>Francesco</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1045837/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Grimaldi</surname><given-names>Maria</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1045644/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Bimonte</surname><given-names>Sabrina</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/469928/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Augustin</surname><given-names>Livia Silvia</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Amore</surname><given-names>Alfonso</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1045661/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Celentano</surname><given-names>Egidio</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Di Napoli</surname><given-names>Marilena</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Cascella</surname><given-names>Marco</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/141814/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Pignata</surname><given-names>Sandro</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1045750/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Epidemiology and Biostatistics Unit, Istituto Nazionale Tumori, IRCCS Fondazione G. Pascale</institution>, <addr-line>Naples</addr-line>, <country>Italy</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Head and Neck Medical and Experimental Oncology Unit, Istituto Nazionale Tumori, IRCCS Fondazione G. Pascale</institution>, <addr-line>Naples</addr-line>, <country>Italy</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Anesthesia and Pain Medicine, Istituto Nazionale Tumori, IRCCS Fondazione G. Pascale</institution>, <addr-line>Naples</addr-line>, <country>Italy</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Medical Oncology Unit, Istituto Nazionale Tumori, IRCCS Fondazione G. Pascale</institution>, <addr-line>Naples</addr-line>, <country>Italy</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Antonio Russo, Paolo Giaccone University Hospital in Palermo, Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Cristian FIori, University of Turin, Italy; Riccardo Campi, Careggi University Hospital, Italy; Luigi Cavanna, Ospedaliera di Piacenza, Italy</p></fn><corresp id=\"c001\">*Correspondence: Anna Crispo, <email>a.crispo@istitutotumori.na.it</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Cancer Epidemiology and Prevention, a section of the journal Frontiers in Oncology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>11</day><month>8</month><year>2020</year></pub-date><!-- PMC Release delay is 0 months and 0 days and was based on the <pub-date pub-type=\"epub\"/>. --><volume>10</volume><elocation-id>1571</elocation-id><history><date date-type=\"received\"><day>14</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>21</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Crispo, Montagnese, Perri, Grimaldi, Bimonte, Augustin, Amore, Celentano, Di Napoli, Cascella and Pignata.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Crispo, Montagnese, Perri, Grimaldi, Bimonte, Augustin, Amore, Celentano, Di Napoli, Cascella and Pignata</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Italy and worldwide are experiencing an outbreak of a new coronavirus-related disease, named COVID-19, declared by the WHO COVID-19 a pandemic. The fragility of cancer patients is well-known, with many cases affecting aged patients or those with several comorbidities that frequently result in a loss of independency and functionality. Therefore, cancer patients have been greatly affected by this health emergency and, due to their vulnerability to COVID-19, oncologic patient visits have been often delayed or canceled leading to possible under-treatment. Different solutions can be adopted for reducing travels to cancer screening centers and the overall impact of cancer screening visits. As a consequence, it has been recommended that, when possible, the follow-up visits for cancer patients treated with oral anticancer drugs could be performed telematically. Furthermore, many patients refuse hospital visits, even if necessary, because of fear of contagion. Moreover, in some regions in Italy even the very first non-urgent visits have been postponed with the consequent delay in diagnosis, which may negatively affect disease prognosis. For these reasons, new approaches are needed such as the telemedicine tool. Throughout organized and appropriate tools, it would be possible to manage patients’ visits and treatments, to avoid the dangerous extension of waiting lists when the standard activities will resume. In this context, a number of hospital visits can be substituted with visits at small local health centers, and general practitioners’office, taking in turn, advantage of well-defined telemedicine path which will be developed in the post-emergency phase.</p></abstract><kwd-group><kwd>COVID-19</kwd><kwd>coronavirus</kwd><kwd>cancer patients</kwd><kwd>telemedicine</kwd><kwd>infections</kwd></kwd-group><counts><fig-count count=\"0\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"30\"/><page-count count=\"5\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Background</title><sec id=\"S1.SS1\"><title>Epidemiology</title><p>Italy and worldwide are experiencing an outbreak of a new coronavirus-related disease named COVID-19 due to SARS-CoV2 virus. Cases that develop severe pneumonitis are characterized by dyspnea, cyanosis and fever, which may configure the clinical framework of Acute Distress Respiratory Syndrome (ARDS) (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B2\" ref-type=\"bibr\">2</xref>). Because COVID-19 spread to more than 100 countries and accounted for several tens of thousands of cases within a few months, WHO declared COVID-19 a pandemic (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>).</p><p>Italy currently is the second European country after Spain for confirmed COVID-19 cases (197,675 and 207,634, respectively), and the second country in the world for COVID-19 death after United States (26,644 and 47,980, respectively) (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Of note, although in Italy the outbreak of SARS-CoV-2 infection grew progressively from end of Feb 2020 to end of Mar 2020 over the entire national territory, large regional differences were recorded.</p><p>On March 9, 2020, the lockdown was extended for the entire national territory and progressively severe limitations were adopted. The dynamics of the epidemics followed a geographical differentiation, with a North to South gradient that is likely to depend on the different timing of onset of the local outbreaks before the implementation of national containment measures.</p><p>A report from the Italian Superior Institute of Health based on 3,200 patients who died of SARS-CoV-2 related mortality to old age and comorbidities including cancer (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Patients suffering from oncologic or onco-hematologic pathologies, as well as other diseases associated with immunosuppression (e.g., congenital immunodeficiency, solid organ transplants or hematopoietic stem cells, autoimmune diseases following immunosuppressive treatment), are particularly at risk for morbidity and mortality from respiratory viral infections such as influenza for which the risk of hospitalization of cancer patients is approximately four times higher than in healthy subjects of comparable age (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). Although the data are currently extremely limited, it seems that patients with oncologic or onco-hematologic diseases are exposed either to greater risk of contracting SARS-CoV-2 infection or to have a worse prognosis. In fact, these patients are characterized by a greater risk of events such as hospitalization in intensive care unit and/or exitus.</p><p>As of now no validated vaccines or antiviral drugs against SARS-CoV-2 infection exist (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>), the hospital in our country have provided and establish dedicated areas (e.g., waiting rooms) for cancer patients. Preventive measures have been doubled by implementing active-surveillance that allows cases to be identified earlier, isolating them according to appropriate management and containment procedures. Another measure to reduce contagion of vulnerable patients was to postpone visits for non-urgent cancer patients. This however, poses the question on how to best follow patients in their homes (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>, <xref rid=\"B9\" ref-type=\"bibr\">9</xref>). Finally in order to change the clinical practice for oncologic patients in terms of assistance and controls, a multidisciplinary team is needed to improve the diagnostic and therapeutic management for cancer patients (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>).</p></sec><sec id=\"S1.SS2\"><title>Telemedicine</title><p>The term “telemedicine” was created with the aim of identifying a particular sector of application of Information and Communication Systems, ICT, with the aim of providing a better performance for healthcare activities. The term is used both to describe computer and telecommunication systems that allow people to work together over time and space, and to refer generally to the use of information technology in medicine. Worldwide, there has been an important increase in the use of telemedicine in the health sector in recent years. Telecommunication systems provide remote healthcare by improving access to care and changing its organization (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B12\" ref-type=\"bibr\">12</xref>).</p><p>The literature has analyzed the efficacy and efficiency of telemedicine applied to the oncology field with assessments through systematic reviews, meta-analyses and clinical trials. Specifically, the aim of telemedicine in oncology is to reduce the access disparity and patients care (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). O’Gorman et al. within his own study shows that the use of telemedicine was higher in rural areas of northern Ontario in Canada compared to other parts of the province suggesting that telemedicine is utilized to improve access to medical care services, especially in sparsely populated and least served regions (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>).</p><p>Patrick D. Hoek et al. (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>) through a clinical trial, highlighted the most problematic aspects in the application of teleconsultation, showing how it is necessary to seek ways of optimizing multidisciplinary care done through teleconsultation, making the times and frequency of teleconsultations more suitable for patients with advanced cancer, underlining the need of doing research in this area for improving of the most critical aspects of telemedicine (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>).</p><p>In Italy, the application of telemedicine in the oncology field has not yet found widespread use despite its great potential. For example the Ministry of Health released in 2014 the national guidelines on telemedicine define the roles and tasks of telemedicine (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>).</p><p>A recent published document (AIOM, CIPOMO) for COVID-19 emergency indicates the need to postpone the follow-up outpatient activities for disease-free patients (e.g., follow-up to 6- 12 months), providing a telephone triage and/or telematic patient rescheduling based on clinical severity (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). This indication opens the opportunity to apply telemedicine methodologies in emergency situations such as the current one but also in the post-emergency phase.</p><p>Advanced computer technology widely available at medical institutions allows a reduction of required human resources and makes the routine collection of data feasible in busy clinical practices (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>–<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). In particular, computer technology has been extensively applied for immediate record and presentation of PRO results to clinicians, utilizing useful tools for data collection and presentation, already validated and easily transferable to other fields of clinical activities (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>–<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). This is applicable for research purposes, for symptom screening and patient monitoring thereby contributing to hospital quality assurance.</p></sec><sec id=\"S1.SS3\"><title>COVID Infection and Cancer: Individualized Management</title><p>According to the latest data in the literature concerning the characteristics of COVID-19 infection, we can assume that age and comorbidities strongly influence prognosis, and mainly, the possibility to develop a severe disease with respiratory failure and ARDS (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B25\" ref-type=\"bibr\">25</xref>). In particular, some comorbidities, such as chronic obstructive pulmonary disease and cardiovascular diseases place the patients at higher risk of developing a severe form of COVID-19 and ultimately death.</p><p>On the other hand, due to the low number of data available, little is acknowledged about the impact of COVID-19 infection in patients affected by cancer. Only few retrospective studies have faced this issue and their results were in favor of a heavy impact of cancer on prognosis. So, it is presumable that COVID-19 tends to have a poorer outcome in cancer patients (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>, <xref rid=\"B27\" ref-type=\"bibr\">27</xref>).</p><p>Solid tumors are genetically characterized by a strong immune-suppression which at first involves tumor microenvironment, then in a more advanced phase the whole organism. This immune-suppressive status is strongly due to the over production, mainly by the tumor cells, of cytokines such as IL-6, TNF-alpha and TGF-beta (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). Moreover, patients affected by solid tumors often suffer from blood coagulation disorders because of chemotherapy, surgery, hormone therapy, biological agents (e.g., anti-VEGFR drugs), and bedding. In addition, cancer cells are capable of producing the “cancer pro-coagulant” which is a cysteine proteinase able to induce a thrombotic diathesis, mainly in very advanced tumors (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>).</p><p>Finally, most patients affected by cancer, in particular those suffering from lung cancer, head and neck carcinomas, urologic cancers, and gastrointestinal cancers are elderly patients (>70 years old) and often present a number of comorbidities at diagnosis. Moreover, a fair number of such patients, especially those having lung cancer and head/neck carcinomas, are heavy smokers. Both age and smoker status seem to poorly impact prognosis of patients affected by COVID-19 (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>).</p></sec><sec id=\"S1.SS4\"><title>Cancer Patient Management</title><p>COVIDd-19 (from now on indicated interchangeably as COVID or COVID-19) is determining a complete reorganization of hospitals with two new different paths for COVID and non-COVID. Many hospital units have been converted to COVID units with the risk that non-COVID patients may not receive adequate therapy for their disease.</p><p>The fragility of cancer patients is well known, with many cases affecting aged patients or those with several comorbidities that frequently results in a loss of independency and functionality. Based on these patients characteristics and the data coming from Protezione Civile indicating that around 20% of cancer deaths from COVID infection occurs in patients with previous cancer or active of cancer (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>), a number of actions in the hospitals have been set up to maintain cancer patients out of hospitals when there is no urgent need. It has been recommended that, when possible, the follow up visits for cancer patients treated with oral anticancer drugs are performed telematically. Furthermore, many patients refuse hospital visits, even if necessary, because of fear of contagion: in some Italian regions the first non-urgent visits have been postponed with the consequent delay in diagnosis which will affect disease prognosis.</p><p>The practical experience of oncologic units could offer assistance in the way of telemedicine that should be organized with appropriate tools in order to plan for personnel, including patient count and a clear definition of patients who may benefit from this approach. Telemedicine may also be useful to increase or preserve cancer screening programs.</p><p>It is desirable that this type of experience could produce telematic clinical strategies that will allow, in the Covid-19 post-emergency, to continue caring for cancer patients in an appropriate setting where a number of hospital visits are substituted with visits at small local health centers and general practitioners’ office and taking advantage of well-defined telemedicine path which will be developed in this phase.</p></sec></sec><sec id=\"S2\"><title>Conclusion</title><p>The main conclusion of the above considerations is that patients suffering from solid tumors tend to develop a severe form of COVID-19 infection, which can often lead to death, due to the cancer “<italic>per se</italic>,” and the comorbidities linked to cancer. Thus, the management of patients affected by cancer during COVID-19 pandemic is particularly challenging.</p><p>The first precaution to take is to reduce the risk of contagion in these patients. The general policy of Cancer Centres (CC) in Italy has been to remain COVID-19 free to ensure sufficient clinical and intensive-care capacity for critical cancer surgeries or management of side effects from systemic anticancer treatment. This goal could be achieved by immediately transferring the new infected cases from CC to specialized hospitals for the treatment of SARS-CoV-2 infections (Covid-Centers In this scenario, we can hypothesize to act in a similar way.</p><p>Importantly, the follow-up of outpatients with telemedicine should be strongly individualized and based on disease severity and initial treatment priorities. The possibility of obtaining diagnostic test results via e-mail, as well as images related to radiological tests cannot be excluded. The contribution of the general practitioner in visiting patients with suspicious symptoms at home should also be considered.</p><p>In this pandemic situation a multidisciplinary approach for the management of cancer patients should be improved where possible in order to give the most priority to patients, better equilibrating oncologic and COVID-19 needs.</p><p>The management of patients considered infected depends on their symptoms. In asymptomatic patients, it is reasonable to propose quarantine at home and careful telephone monitoring of symptoms. Patients with mild symptoms, such as slight-fever, cough and mild asthenia, should be early hospitalized in CC facilities, since they can deteriorate rapidly. Telemedicine would also allow for prompt treatment of COVID-19 patients with cancer right in their homes with prophylactic heparin treatment and other drugs employed in hospital settings during the early phase of the disease.</p></sec><sec sec-type=\"data-availability\" id=\"S3\"><title>Data Availability Statement</title><p>The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.</p></sec><sec id=\"S4\"><title>Author Contributions</title><p>AC and SP: conceptualization and methodology. CM, FP, MG SB, and LA: formal analysis. AC, CM, FP, AA, LA, EC, MC, MD, and SP: investigation. CM, MG, FP, SB, and LA: resources. AC, MC, and SP: supervision. AC, CM, FP, MG, LA, MC, and SP: writing – original draft. AC, MC, LA, and SP: writing – review and editing. All authors have read and agreed this manuscript.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Du</surname><given-names>Y</given-names></name><name><surname>Tu</surname><given-names>L</given-names></name><name><surname>Zhu</surname><given-names>P</given-names></name><name><surname>Mu</surname><given-names>M</given-names></name><name><surname>Wang</surname><given-names>R</given-names></name><name><surname>Yang</surname><given-names>P</given-names></name><etal/></person-group>\n<article-title>Clinical features of 85 fatal cases of COVID-19 from Wuhan: a retrospective observational study.</article-title>\n<source><italic>Am J Respir Crit Care Med.</italic></source> (<year>2020</year>) <volume>201</volume>:<fpage>1372</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1164/rccm.202003-0543OC</pub-id>\n<pub-id pub-id-type=\"pmid\">32242738</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>Z</given-names></name><name><surname>Yang</surname><given-names>B</given-names></name><name><surname>Li</surname><given-names>Q</given-names></name><name><surname>Wen</surname><given-names>L</given-names></name><name><surname>Zhang</surname><given-names>R.</given-names></name></person-group>\n<article-title>Clinical features of 69 cases with Coronavirus disease 2019 in Wuhan, China.</article-title>\n<source><italic>Clin Infect Dis.</italic></source> (2020) 16:ciaa272. <pub-id pub-id-type=\"doi\">10.1093/cid/ciaa272</pub-id>\n<pub-id pub-id-type=\"pmid\">32176772</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moorthy</surname><given-names>V</given-names></name><name><surname>Henao Restrepo</surname><given-names>AM</given-names></name><name><surname>Preziosi</surname><given-names>MP</given-names></name><name><surname>Swaminathan</surname><given-names>S.</given-names></name></person-group>\n<article-title>Data sharing for novel coronavirus (COVID-19).</article-title>\n<source><italic>Bull World Health Organ.</italic></source> (<year>2020</year>) <volume>98</volume>:<issue>150</issue>. <pub-id pub-id-type=\"doi\">10.2471/BLT.20.251561</pub-id>\n<pub-id pub-id-type=\"pmid\">32132744</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><collab>WHO</collab>\n<source><italic>Coronavirus Disease 2019 (COVID-19): Situation Report 98.</italic></source> (2020). Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200427-sitrep-98-covid-19.pdf?sfvrsn=90323472_4\">https://www.who.int/docs/default-source/coronaviruse/situation-reports/20200427-sitrep-98-covid-19.pdf?sfvrsn=90323472_4</ext-link> (accessed <month>4</month>\n<day>27</day>, <year>2020</year>).</mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><collab>Istituto Superiore di Sanità</collab>\n<source><italic>Report on the Characteristics of COVID-19 Positive Deceased Patients in Italy.</italic></source> (2020). Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.epicentro.iss.it/coronavirus/bollettino/Report-COVID-2019_20_marzo.pdf\">https://www.epicentro.iss.it/coronavirus/bollettino/Report-COVID-2019_20_marzo.pdf</ext-link>\n<comment>(accessed April 27, 2020)</comment>.</mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Phua</surname><given-names>J</given-names></name><name><surname>Weng</surname><given-names>L</given-names></name><name><surname>Ling</surname><given-names>L</given-names></name><name><surname>Egi</surname><given-names>M</given-names></name><name><surname>Lim</surname><given-names>CM</given-names></name><name><surname>Divatia</surname><given-names>JV</given-names></name><etal/></person-group>\n<article-title>Intensive care management of coronavirus disease 2019 (COVID-19): challenges and recommendations.</article-title>\n<source><italic>Lancet Respir Med.</italic></source> (<year>2020</year>) <volume>8</volume>: <fpage>506</fpage>–<lpage>17</lpage>.<pub-id pub-id-type=\"pmid\">32272080</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Checcucci</surname><given-names>E</given-names></name><name><surname>Piramide</surname><given-names>F</given-names></name><name><surname>Pecoraro</surname><given-names>A</given-names></name><name><surname>Amparore</surname><given-names>D</given-names></name><name><surname>Campi</surname><given-names>R</given-names></name><name><surname>Fiori</surname><given-names>C</given-names></name><etal/></person-group>\n<article-title>The vaccine journey for COVID-19: a comprehensive systematic review of current clinical trials in humans.</article-title>\n<source><italic>Panminerva Med.</italic></source> (2020). 26. <pub-id pub-id-type=\"doi\">10.23736/S0031-0808.20.03958-0</pub-id>\n<comment>[Epub ahead of print]</comment>. <pub-id pub-id-type=\"pmid\">32456404</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Campi</surname><given-names>R</given-names></name><name><surname>Amparore</surname><given-names>D</given-names></name><name><surname>Capitanio</surname><given-names>U</given-names></name><name><surname>Checcucci</surname><given-names>E</given-names></name><name><surname>Salonia</surname><given-names>A</given-names></name><name><surname>Fiori</surname><given-names>C</given-names></name><etal/></person-group>\n<article-title>Assessing the burden of nondeferrable major Uro-oncologic surgery to guide prioritisation strategies during the COVID-19 pandemic: insights from three Italian high-volume referral centres.</article-title>\n<source><italic>Eur Urol.</italic></source> (2020) <volume>78</volume>:<fpage>11</fpage>–<lpage>5</lpage>.<pub-id pub-id-type=\"pmid\">32307215</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moletta</surname><given-names>L</given-names></name><name><surname>Pierobon</surname><given-names>ES</given-names></name><name><surname>Capovilla</surname><given-names>G</given-names></name><name><surname>Costantini</surname><given-names>M</given-names></name><name><surname>Salvador</surname><given-names>R</given-names></name><name><surname>Merigliano</surname><given-names>S</given-names></name><etal/></person-group>\n<article-title>International guidelines and recommendations for surgery during Covid-19 pandemic: a systematic review.</article-title>\n<source><italic>Int J Surg.</italic></source> (<year>2020</year>) <volume>79</volume>: <fpage>180</fpage>–<lpage>8</lpage>.<pub-id pub-id-type=\"pmid\">32454253</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sciarra</surname><given-names>A</given-names></name><name><surname>Salciccia</surname><given-names>S</given-names></name><name><surname>Maggi</surname><given-names>M</given-names></name><name><surname>Del Giudice</surname><given-names>F</given-names></name><name><surname>Busetto</surname><given-names>GM</given-names></name><name><surname>Musio</surname><given-names>D</given-names></name><etal/></person-group>\n<article-title>Elective procedures for prostate cancer in the time of Covid-19: a multidisciplinary team experience.</article-title>\n<source><italic>Prostate Cancer Prostatic Dis.</italic></source> (<year>2020</year>) <volume>8</volume>:<fpage>1</fpage>–<lpage>3</lpage>.</mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Craig</surname><given-names>J</given-names></name><name><surname>Patterson</surname><given-names>V.</given-names></name></person-group>\n<article-title>Introduction to telemedicine.</article-title>\n<source><italic>J Telemed Telecare</italic></source> (<year>2005</year>) <volume>11</volume>:<fpage>3</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1258/1357633053430494</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rajani</surname><given-names>R</given-names></name><name><surname>Perry</surname><given-names>M.</given-names></name></person-group>\n<article-title>The reality of medical work: the case for a new perspective on telemedicine.</article-title>\n<source><italic>Virtual Real.</italic></source> (<year>1999</year>) <volume>4</volume>:<fpage>243</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1007/BF01421807</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Flodgren</surname><given-names>G</given-names></name><name><surname>Rachas</surname><given-names>A</given-names></name><name><surname>Farmer</surname><given-names>AJ</given-names></name><name><surname>Inzitari</surname><given-names>M</given-names></name><name><surname>Shepperd</surname><given-names>S.</given-names></name></person-group>\n<article-title>Interactive telemedicine: effects on professional practice and health care outcomes.</article-title>\n<source><italic>Cochrane Database Syst Rev.</italic></source> (<year>2015</year>) <volume>2015</volume>:<issue>CD002098</issue>. <pub-id pub-id-type=\"doi\">10.1002/14651858.CD002098</pub-id>\n<pub-id pub-id-type=\"pmid\">10796678</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>O’Gorman</surname><given-names>LD</given-names></name><name><surname>Hogenbirk</surname><given-names>JC.</given-names></name></person-group>\n<article-title>Driving distance to telemedicine units in Northern Ontario as a measure of potential access to healthcare.</article-title>\n<source><italic>Telemed J E Health.</italic></source> (<year>2016</year>) <volume>22</volume>(4):<fpage>269</fpage>–<lpage>75</lpage>. <pub-id pub-id-type=\"doi\">10.1089/tmj.2015.0133</pub-id>\n<pub-id pub-id-type=\"pmid\">26451902</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hoek</surname><given-names>PD</given-names></name><name><surname>Schers</surname><given-names>HJ</given-names></name><name><surname>Bronkhorst</surname><given-names>EM</given-names></name><name><surname>Vissers</surname><given-names>KCP</given-names></name><name><surname>Hasselaar</surname><given-names>JGJ.</given-names></name></person-group>\n<article-title>The effect of weekly specialist palliative care teleconsultations in patients with advanced cancer -a randomized clinical trial.</article-title>\n<source><italic>BMC Med.</italic></source> (<year>2017</year>) <volume>15</volume>:<issue>119</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12916-017-0866-9</pub-id>\n<pub-id pub-id-type=\"pmid\">28625164</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><collab>Ministero della Salute</collab>\n<source><italic>TELEMEDICINA Linee di Indirizzo Nazionali.</italic></source> (<year>2012</year>). Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.salute.gov.it/portale/documentazione/p6_2_2_1.jsp?lingua=italiano&id=2129\">http://www.salute.gov.it/portale/documentazione/p6_2_2_1.jsp?lingua=italiano&id=2129</ext-link> (accessed <month>4</month>\n<day>27</day>, <year>2020</year>).</mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><collab>AIOM</collab>\n<source><italic>Indicazioni AIOM CIPOMO su COVID-19 per L’oncologia.</italic></source> (<year>2020</year>). Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.aiom.it/wp-content/uploads/2020/03/20200313_COVID-19_indicazioni_AIOM-CIPOMO-COMU.pdf\">https://www.aiom.it/wp-content/uploads/2020/03/20200313_COVID-19_indicazioni_AIOM-CIPOMO-COMU.pdf</ext-link>\n<comment>(accessed April 27, 2020)</comment>.</mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cox</surname><given-names>A</given-names></name><name><surname>Lucas</surname><given-names>G</given-names></name><name><surname>Marcu</surname><given-names>A</given-names></name><name><surname>Piano</surname><given-names>M</given-names></name><name><surname>Grosvenor</surname><given-names>W</given-names></name><name><surname>Mold</surname><given-names>F</given-names></name><etal/></person-group>\n<article-title>Cancer survivors’ experience with telehealth: a systematic review and thematic synthesis.</article-title>\n<source><italic>J Med Internet Res.</italic></source> (<year>2017</year>) <volume>19</volume>:<issue>e11</issue>. <pub-id pub-id-type=\"doi\">10.2196/jmir.6575</pub-id>\n<pub-id pub-id-type=\"pmid\">28069561</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rogante</surname><given-names>M</given-names></name><name><surname>Giacomozzi</surname><given-names>C</given-names></name><name><surname>Grigioni</surname><given-names>M</given-names></name><name><surname>Kairy</surname><given-names>D.</given-names></name></person-group>\n<article-title>Telemedicine in palliative care: a review of systematic reviews.</article-title>\n<source><italic>Ann Ist Super Sanita.</italic></source> (<year>2016</year>) <volume>52</volume>:<fpage>434</fpage>–<lpage>42</lpage>. <pub-id pub-id-type=\"doi\">10.4415/ANN_16_03_16</pub-id>\n<pub-id pub-id-type=\"pmid\">27698303</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kruse</surname><given-names>CS</given-names></name><name><surname>Krowski</surname><given-names>N</given-names></name><name><surname>Rodriguez</surname><given-names>B</given-names></name><name><surname>Tran</surname><given-names>L</given-names></name><name><surname>Vela</surname><given-names>J</given-names></name><name><surname>Brooks</surname><given-names>M.</given-names></name></person-group>\n<article-title>Telehealth and patient satisfaction: a systematic review and narrative analysis.</article-title>\n<source><italic>BMJ Open.</italic></source> (<year>2017</year>) <volume>7</volume>:<issue>e016242</issue>. <pub-id pub-id-type=\"doi\">10.1136/bmjopen-2017-016242</pub-id>\n<pub-id pub-id-type=\"pmid\">28775188</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Holzner</surname><given-names>B</given-names></name><name><surname>Giesinger</surname><given-names>JM</given-names></name><name><surname>Pinggera</surname><given-names>J</given-names></name><name><surname>Zugal</surname><given-names>S</given-names></name><name><surname>Schöpf</surname><given-names>F</given-names></name><name><surname>Oberguggenberger</surname><given-names>AS</given-names></name><etal/></person-group>\n<article-title>The computer-based health evaluation software (CHES): a software for electronic patient-reported outcome monitoring.</article-title>\n<source><italic>BMC Med Inform Decis Mak.</italic></source> (<year>2012</year>) <volume>12</volume>:<issue>126</issue>. <pub-id pub-id-type=\"doi\">10.1186/1472-6947-12-126</pub-id>\n<pub-id pub-id-type=\"pmid\">23140270</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gutteling</surname><given-names>JJ</given-names></name><name><surname>Busschbach</surname><given-names>JJ</given-names></name><name><surname>de Man</surname><given-names>RA</given-names></name><name><surname>Darlington</surname><given-names>AS.</given-names></name></person-group>\n<article-title>Logistic feasibility of health related quality of life measurement in clinical practice: results of a prospective study in a large population of chronic liver patients.</article-title>\n<source><italic>Health Qual Life Outcomes.</italic></source> (<year>2008</year>) <volume>6</volume>:<issue>97</issue>. <pub-id pub-id-type=\"doi\">10.1186/1477-7525-6-97</pub-id>\n<pub-id pub-id-type=\"pmid\">19000316</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Taenzer</surname><given-names>P</given-names></name><name><surname>Bultz</surname><given-names>BD</given-names></name><name><surname>Carlson</surname><given-names>LE</given-names></name><name><surname>Speca</surname><given-names>M</given-names></name><name><surname>DeGagne</surname><given-names>T</given-names></name><name><surname>Olson</surname><given-names>K</given-names></name><etal/></person-group>\n<article-title>Impact of computerized quality of life screening on physician behaviour and patient satisfaction in lung cancer outpatients.</article-title>\n<source><italic>Psychooncology.</italic></source> (<year>2000</year>) <volume>9</volume>: <fpage>203</fpage>–<lpage>13</lpage>.<pub-id pub-id-type=\"pmid\">10871716</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wright</surname><given-names>EP</given-names></name><name><surname>Selby</surname><given-names>PJ</given-names></name><name><surname>Crawford</surname><given-names>M</given-names></name><name><surname>Gillibrand</surname><given-names>A</given-names></name><name><surname>Johnston</surname><given-names>C</given-names></name><name><surname>Perren</surname><given-names>TJ</given-names></name><etal/></person-group>\n<article-title>Feasibility and compliance of automated measurement of quality of life in oncology practice.</article-title>\n<source><italic>J Clin Oncol.</italic></source> (<year>2003</year>) <volume>21</volume>:<fpage>374</fpage>–<lpage>82</lpage>. <pub-id pub-id-type=\"doi\">10.1200/JCO.2003.11.044</pub-id>\n<pub-id pub-id-type=\"pmid\">12525532</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jazieh</surname><given-names>AR</given-names></name><name><surname>Alenazi</surname><given-names>TH</given-names></name><name><surname>Alhejazi</surname><given-names>A</given-names></name><name><surname>Al Safi</surname><given-names>F</given-names></name><name><surname>Al Olayan</surname><given-names>A.</given-names></name></person-group>\n<article-title>Outcome of oncology patients infected with Coronavirus.</article-title>\n<source><italic>JCO Glob Oncol.</italic></source> (<year>2020</year>) <volume>6</volume>:<fpage>471</fpage>–<lpage>75</lpage>. <pub-id pub-id-type=\"doi\">10.1200/GO.20.00064</pub-id>\n<pub-id pub-id-type=\"pmid\">32196389</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ye</surname><given-names>Z</given-names></name><name><surname>Hong</surname><given-names>Y</given-names></name><name><surname>Wu</surname><given-names>X</given-names></name><name><surname>Hong</surname><given-names>D</given-names></name><name><surname>Zhang</surname><given-names>Y</given-names></name><name><surname>Dong</surname><given-names>X</given-names></name><etal/></person-group>\n<article-title>[Management of a colon cancer patient infected with corona virus disease 2019].</article-title>\n<source><italic>Zhejiang Da Xue Xue Bao Yi Xue Ban.</italic></source> (2020) <volume>49</volume>:0.</mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Perri</surname><given-names>F</given-names></name><name><surname>Ionna</surname><given-names>F</given-names></name><name><surname>Longo</surname><given-names>F</given-names></name><name><surname>Della Vittoria Scarpati</surname><given-names>G</given-names></name><name><surname>De Angelis</surname><given-names>C</given-names></name><name><surname>Ottaiano</surname><given-names>A</given-names></name><etal/></person-group>\n<article-title>Immune response against head and neck cancer: biological mechanisms and implication on therapy.</article-title>\n<source><italic>Transl Oncol.</italic></source> (<year>2020</year>) <volume>13</volume>:<fpage>262</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.tranon.2019.11.008</pub-id>\n<pub-id pub-id-type=\"pmid\">31869751</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hisada</surname><given-names>Y</given-names></name><name><surname>Mackman</surname><given-names>N.</given-names></name></person-group>\n<article-title>Cancer-associated pathways and biomarkers of venous thrombosis.</article-title>\n<source><italic>Blood.</italic></source> (<year>2017</year>) <volume>130</volume>:<fpage>1499</fpage>–<lpage>506</lpage>. <pub-id pub-id-type=\"doi\">10.1182/blood-2017-03-743211</pub-id>\n<pub-id pub-id-type=\"pmid\">28807983</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xu</surname><given-names>X</given-names></name><name><surname>Lai</surname><given-names>Y</given-names></name><name><surname>Hua</surname><given-names>ZC.</given-names></name></person-group>\n<article-title>Apoptosis and apoptotic body: disease message and therapeutic target potentials.</article-title>\n<source><italic>Biosci Rep.</italic></source> (<year>2019</year>) <volume>39</volume>:<issue>BSR20180992</issue>. <pub-id pub-id-type=\"doi\">10.1042/BSR20180992</pub-id>\n<pub-id pub-id-type=\"pmid\">30530866</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhao</surname><given-names>Z</given-names></name><name><surname>Bai</surname><given-names>H</given-names></name><name><surname>Duan</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>J.</given-names></name></person-group>\n<article-title>Recommendations of individualized medical treatment and common adverse events management for lung cancer patients during the outbreak of COVID-19 epidemic.</article-title>\n<source><italic>Thorac Cancer.</italic></source> (<year>2020</year>) <volume>11</volume>:<fpage>1752</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1111/1759-7714.13424</pub-id>\n<pub-id pub-id-type=\"pmid\">32291968</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Oncol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Oncol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Oncol.</journal-id><journal-title-group><journal-title>Frontiers in Oncology</journal-title></journal-title-group><issn pub-type=\"epub\">2234-943X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850460</article-id><article-id pub-id-type=\"pmc\">PMC7431561</article-id><article-id pub-id-type=\"doi\">10.3389/fonc.2020.01565</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Oncology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>NAP1L1: A Novel Human Colorectal Cancer Biomarker Derived From Animal Models of <italic>Apc</italic> Inactivation</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Queiroz</surname><given-names>Cleberson J. S.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/863932/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Song</surname><given-names>Fei</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Reed</surname><given-names>Karen R.</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Al-Khafaji</surname><given-names>Nadeem</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Clarke</surname><given-names>Alan R.</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Vimalachandran</surname><given-names>Dale</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Miyajima</surname><given-names>Fabio</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff7\"><sup>7</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/389878/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Pritchard</surname><given-names>D. Mark</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/416682/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Jenkins</surname><given-names>John R.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/867680/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Institute of Systems, Molecular and Integrative Biology, Henry Wellcome Laboratory, University of Liverpool</institution>, <addr-line>Liverpool</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Faculty of Medicine, Federal University of Mato Grosso (UFMT)</institution>, <addr-line>Cuiaba</addr-line>, <country>Brazil</country></aff><aff id=\"aff3\"><sup>3</sup><institution>INFRAFRONTIER GmbH</institution>, <addr-line>Neuherberg</addr-line>, <country>Germany</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Wales Gene Park, Division of Cancer and Genetics, Cardiff University School of Medicine</institution>, <addr-line>Cardiff</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff5\"><sup>5</sup><institution>European Cancer Stem Cell Research Institute, Cardiff University School of Biosciences</institution>, <addr-line>Cardiff</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff6\"><sup>6</sup><institution>Department of Colorectal Surgery, Countess of Chester Hospital NHS Foundation Trust</institution>, <addr-line>Chester</addr-line>, <country>United Kingdom</country></aff><aff id=\"aff7\"><sup>7</sup><institution>Molecular Epidemiology Laboratory, Oswaldo Cruz Foundation</institution>, <addr-line>Eusebio</addr-line>, <country>Brazil</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Boris Zhivotovsky, Karolinska Institutet (KI), Sweden</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Giovanna Caderni, University of Florence, Italy; Gabriele Multhoff, Technical University of Munich, Germany</p></fn><corresp id=\"c001\">*Correspondence: D. Mark Pritchard, <email>mark.pritchard@liverpool.ac.uk</email>; <email>mark.pritchard@liv.ac.uk</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>Deceased</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>1565</elocation-id><history><date date-type=\"received\"><day>11</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>20</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Queiroz, Song, Reed, Al-Khafaji, Clarke, Vimalachandran, Miyajima, Pritchard and Jenkins.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Queiroz, Song, Reed, Al-Khafaji, Clarke, Vimalachandran, Miyajima, Pritchard and Jenkins</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><sec><title>Introduction</title><p>Colorectal cancer (CRC) is the second leading cause of cancer death worldwide and most deaths result from metastases. We have analyzed animal models in which <italic>Apc</italic>, a gene that is frequently mutated during the early stages of colorectal carcinogenesis, was inactivated and human samples to try to identify novel potential biomarkers for CRC.</p></sec><sec><title>Materials and Methods</title><p>We initially compared the proteomic and transcriptomic profiles of the small intestinal epithelium of transgenic mice in which <italic>Apc</italic> and/or <italic>Myc</italic> had been inactivated. We then studied the mRNA and immunohistochemical expression of one protein that we identified to show altered expression following <italic>Apc</italic> inactivation, nucleosome assembly protein 1–like 1 (NAP1L1) in human CRC samples and performed a prognostic correlation between biomarker expression and survival in CRC patients.</p></sec><sec><title>Results</title><p><italic>Nap1l1</italic> mRNA expression was increased in mouse small intestine following <italic>Apc</italic> deletion in a <italic>Myc</italic> dependant manner and was also increased in human CRC samples. Immunohistochemical NAP1L1 expression was decreased in human CRC samples relative to matched adjacent normal colonic tissue. In a separate cohort of 75 CRC patients, we found a strong correlation between NAP1L1 nuclear expression and overall survival in those patients who had stage III and IV cancers.</p></sec><sec><title>Conclusion</title><p><italic>NAP1L1</italic> expression is increased in the mouse small intestine following <italic>Apc</italic> inactivation and its expression is also altered in human CRC. Immunohistochemical NAP1L1 nuclear expression correlated with overall survival in a cohort of CRC patients. Further studies are now required to clarify the role of this protein in CRC.</p></sec></abstract><kwd-group><kwd>colorectal cancer</kwd><kwd>biomarkers</kwd><kwd><italic>Apc</italic></kwd><kwd>NAP1L1</kwd><kwd>prognosis</kwd><kwd>survival</kwd></kwd-group><counts><fig-count count=\"5\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"41\"/><page-count count=\"11\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Colorectal cancer (CRC) is the third most common cancer type and the second leading cause of cancer death worldwide (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Deaths usually result from recurrent and metastatic disease. Most international guidelines recommend chemotherapy to reduce the risk of recurrence in stage III tumors and to prolong survival in stage IV cancers (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). Conversely, chemotherapy is generally not used in stage I and most stage II tumors. However, some patients with low-risk stage III disease may respond well following courses of chemotherapy that are shorter than the 6-month standard schedule, although the definition of “low-risk” has not been well established in this scenario (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Additionally, almost 20% of patients who have stage II tumors and who are therefore considered to have low-risk disease, relapse and eventually die from cancer progression (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). There is currently no accurate biomarker to better define prognosis within stage groups. Therefore, biomarkers for prognostic stratification in CRC have the potential of improving the treatment decision-making process (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B6\" ref-type=\"bibr\">6</xref>). We hypothesized that studying molecular mechanisms that are known to be involved in CRC development might yield promising novel biomarkers for this disease.</p><p><italic>Adenomatous polyposis coli</italic> (<italic>APC</italic>) inactivating mutations are the earliest and most common genetic alterations in the adenoma-carcinoma sequence that leads to CRC (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Such mutations result in the accumulation of β-catenin within cells and activation of the Wnt signaling pathway (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). Animal models of <italic>Apc</italic> inactivation demonstrate epithelial transformation and tumor formation mirroring cancer development (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>–<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). One of these models is the <italic>AhCre<sup>+</sup>Apc<sup>fl/fl</sup></italic> mouse, an animal bearing loxp-flanked <italic>Apc</italic> alleles and a <italic>Cre</italic>-recombinase transgene. When this animal is exposed to β-naphthoflavone, Cre-recombinase transcription is activated resulting in the deletion of loxp-flanked <italic>Apc</italic> alleles specifically in the small intestinal epithelium, thus causing an acute activation of the Wnt pathway (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). The <italic>Apc</italic><sup>Min/+</sup> mouse has a germline mutation in one <italic>Apc</italic> allele simulating a familial adenomatous polyposis (FAP) patient, and spontaneously develops multiple intestinal neoplasms during its life-span (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>). The <italic>Myc</italic> gene is a Wnt-target that plays an essential role in the development of malignant phenotypes upon <italic>Apc</italic> inactivation (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>, <xref rid=\"B15\" ref-type=\"bibr\">15</xref>). We hypothesized that the analysis of mouse models of <italic>Apc</italic> and <italic>Apc/Myc</italic> deletion could lead to the discovery of genes or proteins with potential clinical use as human CRC biomarkers.</p><p>Our group has previously published a proteomic evaluation of an animal model of CRC based on acute <italic>Apc</italic> deletion (<italic>AhCre<sup>+</sup>Apc<sup>fl/fl</sup></italic> mouse) (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). A 4-plex iTRAQ labeling analysis identified 126 proteins that were significantly altered upon <italic>Apc</italic> deletion (76 up- and 50 down-regulated) and which were proposed as Wnt targets. We have now performed an additional analysis of this dataset by comparing the protein list with the transcriptomic data generated using Affymetrix arrays and intestinal tissues from the same mice (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>). This study used <italic>Apc</italic>–deficient (<italic>AhCre<sup>+</sup>Apc<sup>fl/fl</sup>Myc<sup>+/+</sup>)</italic> and double-mutant <italic>Apc</italic>-<italic>Myc</italic> deficient (<italic>AhCre<sup>+</sup>Apc<sup>fl/fl</sup>Myc<sup>fl/fl</sup></italic>) mice to identify <italic>Myc</italic> dependant Wnt pathway genes following <italic>Apc</italic> inactivation. We investigated whether there were any genes/proteins that showed congruent findings in both analyses according to strict criteria. One protein, nucleosome assembly protein 1 like 1 (NAP1L1) was identified that met our criteria.</p><p>We therefore analyzed the expression of <italic>Nap1l1</italic> in <italic>Apc</italic>–deficient (<italic>AhCre<sup>+</sup>Apc<sup>fl/fl</sup>Myc<sup>+/+</sup></italic>) and double-mutant <italic>Apc</italic>-<italic>Myc</italic> deficient (<italic>AhCre<sup>+</sup>Apc<sup>fl/fl</sup>Myc<sup>fl/fl</sup></italic>) mice to assess whether its expression was <italic>Myc</italic>-dependant and therefore a potential biomarker of Wnt activation. Following confirmation of our findings, we investigated the mRNA and protein expression of NAP1L1 in tumor and adjacent normal mucosa samples from patients with CRC as well as colonic tissues from unaffected individuals.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Animal Samples</title><p>Mouse experiments were performed with UK Home Office approval with personal and project licenses (30/2737 and 30/3279) and according to ARRIVE guidelines and following local ethical review by the Cardiff University Animal Welfare Ethical Review Panel. The transgenic mice that were used in this study were generated and maintained at the University of Cardiff as previously described in (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>). Small intestinal epithelial cell extracts were generated from these mice following injection of beta-naphthlaflavone as described by Hammoudi et al. (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>).</p></sec><sec id=\"S2.SS2\"><title>qPCR RT-PCR</title><p>For the mouse small intestinal tissue samples and human CRC samples obtained in the United Kingdom (Wales cohorts 1 and 2), total RNA was used to synthesize first strand cDNA using a VersoTM cDNA Kit (Thermo Scientific) and anchored oligo-dT primers (Thermo Scientific) according to the manufacturer’s instructions. Single-stranded cDNA samples were amplified in a Polymerase chain reaction (PCR) using sequence-specific primers (Eurogentec) and probes from the Universal Probe Library (Roche) that were designed using the Universal ProbeLibrary Assay Design Centre, using PCR Master mix (Roche) and a light cycler 480 (Roche) (see <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Table 2</xref> for primers and probes used).</p><p>For the human CRC samples recruited in Brazil, RNA was extracted using the RNeasy<sup>®</sup> Mini Kit (Qiagen, Hilden, Germany). cDNA was produced using TaqMan<sup>®</sup> Reverse Transcription Reagents kit (Applied Biosystems, Carlsbad, CA, United States) according to the manufacturer’s protocol. Quantitative PCR reactions were performed using the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, United States) (see <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Table 3</xref> for expression assay specifications).</p></sec><sec id=\"S2.SS3\"><title>Proteomic and Microarray Comparison Analysis</title><p>We set out to determine the subset of genes/proteins, which demonstrated upregulation of both protein and mRNA following <italic>Apc</italic> deletion, but no increase in expression in <italic>AhCre<sup>+</sup>Apc<sup>fl/fl</sup>Myc<sup>fl/fl</sup></italic> mice. The MAXD/View-Program data files from the microarray analyses were used to calculate gene expression fold changes in the intestinal tissues from the various transgenic mice. These data were then combined with our previously published proteomic profile data (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>) in which we identified proteins which showed a greater than 1.2 fold increase in expression following <italic>Apc</italic> deletion, using iTRAQ analysis.</p><p>The following features from the DNA microarray analysis were used to identify candidate <italic>Myc</italic> dependant Wnt pathway proteins: (i) a statistically significant (<italic>p</italic> < 0.05) greater than 2 fold increase in expression in <italic>Apc</italic>–deficient (<italic>AhCre<sup>+</sup>Apc<sup>fl/fl</sup></italic>) mice compared to wild-type mice (APC:WT); (ii) no significant increase in expression in the double-mutant <italic>Apc-Myc</italic> deficient (<italic>AhCre</italic> + <italic>Apc<sup>fl/fl</sup> Myc<sup>fl/fl</sup></italic>) mice when compared to the wild-type mice (a ratio value of 1:1, with boundaries of 0.75 and 1.25) (APCMYC:WT) and (iii) a <italic>AhCre<sup>+</sup> Apc<sup>fl/fl</sup> Myc<sup>fl/fl</sup></italic> to <italic>AhCre<sup>+</sup>Apc<sup>fl/fl</sup></italic>, ratio < 0.5 with <italic>p</italic> < 0.05 (APCMYC:APC) and findings being confirmed by at least three Affymetrix probes corresponding to the protein.</p></sec><sec id=\"S2.SS4\"><title>Human Samples and Ethics</title><sec id=\"S2.SS4.SSS1\"><title>United Kingdom Cohorts</title><p>Total RNA samples from CRC tumor tissues and adjacent uninvolved colonic mucosa (18 patients) were obtained from the Wales Cancer Bank (Wales cohort 1) with the ethical approval and informed consent that is associated with this tissue bank, and these samples were used in the initial gene expression studies. There were 9 male and 9 female patients, with 6 samples from stage I, 6 from stage II and 6 from stage III CRC and mean patient age was 69.3 years. Another set of 30 matched sample pairs was subsequently obtained from the same Tissue Bank (Wales cohort 2) and these were analyzed separately for validation of the findings. Wales cohort 2 had 9 samples with stage I, 8 samples with stage II and 13 samples with stage III CRC and the mean patient age was 69.4 years.</p><p>A tissue microarray (TMA) was constructed using samples from 19 CRC patients recruited at the Countess of Chester Hospital NHS Foundation Trust (Chester, United Kingdom) again with informed consent and local ethics committee approval (12/NW/0011). Cancer tissues were available for all cases (5 cases with stage I, 3 cases with stage II, 5 cases with stage III and 6 cases with stage IV CRC), whilst normal adjacent mucosa was only obtained for 8 of them. The analysis of this cohort was part of the immunohistochemistry (IHC) validation study and the findings are presented in <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 2</xref>.</p></sec><sec id=\"S2.SS4.SSS2\"><title>Brazil Cohort</title><p>Fresh frozen tissues from tumors removed from 25 CRC patients and normal colonic tissues from 10 normal individuals (who had a normal colonoscopy on a bowel cancer screening program) were collected, after informed consent was obtained, and were analyzed in the gene expression studies. The CRC samples were from 17 males and 8 females with stage I: 7; II: 3; III: 8 and IV: 7 CRC and the mean patient age was 55.9 years. The normal samples were from 3 males and 7 females and the patients had a mean age of 54.7 years.</p><p>For the initial Brazilian IHC study, samples from 32 patients were prospectively collected in Cuiaba – Brazil between January 2013 and August 2015. Informed consent was obtained. Fragments from the tumor and from the normal adjacent mucosa (at least 10 cm from the tumor) were fixed in 10% buffered formalin for 24 h, and then processed into paraffin blocks. Patient characteristics are described in the results section below.</p><p>For the IHC prognostic study, samples were obtained for 75 CRC patients from the archives of two pathology laboratories in Cuiaba, Brazil (Laboratorio São Nicolau and the Julio Muller University Hospital Pathology Lab). Inclusion criteria were: (i) 4 or more years since diagnosis, (ii) presence of tumor tissue in the paraffin block, (iii) traceable patient survival information, and (iv) survival for at least 30 days after surgery. We tracked the patient’s current health service to obtain mortality information. Alternatively, if no clinical information was available, we checked the Brazilian electronic death database “<italic>Sistema de Informacao de Mortalidade</italic>” which records all deaths and their causes. Overall survival was recorded as the interval between diagnosis and death from any cause (when death had occurred) or the date when the database was last checked (when death had not occurred).</p><p>This research was approved by the Committee for Research Ethics of the Hospital Universitario Julio Muller – Federal University of Mato Grosso, Cuiaba – Brazil, and by the Brazilian National Commission for Research Ethics (CONEP), decision number: 1.628.901.</p></sec></sec><sec id=\"S2.SS5\"><title>Immunohistochemistry in the Validation Study</title><p>Four μm sections from paraffin blocks were subjected to standard protocols for IHC. Tris-buffered saline-tween 0.1% (TBS-T) was used for all washes. In summary the main steps were: dewaxing in xylene twice; endogenous peroxidases block using 3% hydrogen peroxide in methanol; rehydration in decreasing concentrations of ethanol solutions and, finally, distilled water; heat-induced epitope retrieval using 10 mM citrate buffer pH 6.0 in a microwave oven at 800 W for 20 min; block using 10% normal goat serum (Dako, Ely, United Kingdom) in TBS-T for 45 min at room temperature; primary rabbit anti-NAP1L1 antibody (cat. number ab33076, Abcam, Cambridge, United Kingdom) 1:4,000 in 10% normal goat serum in TBS-T overnight at 4°C in a humid chamber; biotinylated secondary goat anti-rabbit antibody (Dako, Ely, United Kingdom) solution 1:200 in 10% normal goat serum for 30 min at room temperature; avidin-biotin-peroxidase complex (Vectastain Elite ABC kit – Peterborough, United Kingdom) for 30 min at room temperature; visualization using 3,3′-diaminobenzidine (Sigmafast DAB tablets – Sigma, Gillingham, United Kingdom) substrate solution; application of hematoxylin counterstain; dehydration in increasing concentrations of ethanol; clearing in xylene and mounting using DPX mounting medium (Sigma) and glass coverslips. Stained sections were viewed and photographed using a microscope and camera set (Leica Biosystems, Milton Keynes, United Kingdom). The same staining protocol was used for the additional United Kingdom patient cohort described in <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 2</xref>.</p></sec><sec id=\"S2.SS6\"><title>Immunohistochemistry in the Prognostic Study</title><p>The protocol adopted in this part of the research was the routine technique used in the São Nicolau Laboratory (Cuiaba/Brazil), a pathology lab with extensive expertise in IHC. All branded solutions and buffers were purchased from Cell-Marque<sup>TM</sup>/Sigma-Aldrich (Rocklin, CA, United States). Four μm tissue sections were dewaxed in xylene and rehydrated as previously described. After a wash step in distilled water, slides were immersed in Trilogy<sup>TM</sup> pre-treatment solution and incubated at 96°C for 30 min for epitope retrieval. After this, the slides were washed in phosphate buffered saline (PBS), Peroxide block<sup>TM</sup> solution was added and samples were incubated for 20 min. After another PBS wash, the primary antibody solution (same concentration as those described above) was placed onto the samples and incubated for 20 min at room temperature. After washes in PBS, HiDef Detection<sup>TM</sup> amplifier (secondary antibody solution) was applied to the slides for 15 min. After a further PBS wash, the former step was repeated using HiDef Detection<sup>TM</sup> detector (a horseradish peroxidase polymer solution). Finally, color development was performed by incubating the slides with DAB substrate<sup>TM</sup> chromogen. Stained slides were counterstained, dehydrated and mounted as described above. Slides were photographed using an Axio Scope.A1 microscope coupled with an AxioCamHRc camera (Zeiss, Oberkochen, Germany). Some samples were stained using both the protocol described here and that in the previous section and these confirmed that the staining patterns were similar (see <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 1</xref>).</p></sec><sec id=\"S2.SS7\"><title>Immunohistochemistry Scoring System</title><p>Scoring was performed electronically using the software ImageJ (publicly available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://imagej.nih.gov/ij/\">rsbweb.nih.gov/ij/</ext-link>) (IMAGEJ). The images were initially edited in Image J to exclude non-epithelial/non-cancerous/stromal tissues. For cytoplasmic assessment, the plugin <italic>IHC Profiler</italic> was used (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). Based on the readings produced by this tool, we calculated a modified IHC Profiler score = [(% of negative) × 0] + [(% of low-positive) × 100] + [(% of positive) × 200] + [(% of high-positive) × 300], with final scores ranging from 0 to 300. For nuclear scoring, the plugin <italic>ImmunoRatio</italic> was used (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). It assesses the percentage of positive nuclei based on a threshold setting. Two microscopy fields (×400) containing at least 100 stained epithelial (in control cases) or cancer cells each were analyzed per sample.</p></sec><sec id=\"S2.SS8\"><title>Statistical Analysis</title><p>Comparisons of continuous variables were carried out using Mann–Whitney <italic>U</italic> test or Kruskal–Wallis test followed by the Dunn-Bonferroni test for <italic>post hoc</italic> comparison. Categorical data were compared using the Chi-square test (or Fisher’s exact test in case of less than five expected counts per cell in the contingency table). For the survival analysis, groups were assessed using the Kaplan-Meier method, and survival curves were compared by log-rank tests. When significant differences were observed, Cox proportional hazards model was used for multivariate analysis. Two-sided <italic>p</italic>-values <0.05 were accepted as significant in the entire study. All statistical analyses were performed using the software IBM<sup>®</sup> SPSS<sup>®</sup> Statistics version 22 and R packages.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Combination of Proteomic and Transcriptomic Datasets</title><p>Our previously published proteomic data (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>) was integrated with the DNA microarray data from the double-mutant <italic>Apc</italic>- and <italic>Myc</italic>-deficient (<italic>AhCre<sup>+</sup>Apc<sup>fl/fl</sup>Myc<sup>fl/fl</sup></italic>) mice as shown in <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Table 1</xref>. Of the 93 up-regulated proteins from the iTRAQ analysis, only one also showed up-regulation of mRNA in the intestine of <italic>AhCre<sup>+</sup>Apc<sup>fl/fl</sup>Myc<sup>+/+</sup></italic> mice and no change in the intestine of <italic>AhCre<sup>+</sup>Apc<sup>fl/fl</sup>Myc<sup>fl/fl</sup></italic> mice using the criteria described in the <italic>Materials and Methods</italic> section. This protein was NAP1L1.</p></sec><sec id=\"S3.SS2\"><title>Evaluation of <italic>Nap1l1</italic> mRNA Expression in Mouse Small Intestine</title><p>In order to validate whether <italic>Nap1l1</italic> mRNA was upregulated following conditional <italic>Apc</italic> deletion in the mouse intestinal epithelium, qRT-PCR was carried out using mRNA extracted from small intestinal epithelial cell extracts from <italic>AhCre<sup>+</sup>Apc<sup><italic>fl/f</italic></sup>Myc<sup>fl/fl</sup></italic>, <italic>AhCre<sup>+</sup>Apc<sup>fl/fl</sup></italic>, and <italic>AhCre<sup>+</sup>Myc<sup>fl/fl</sup></italic> mice, 4 days post induction. We compared relative mRNA expression in these transgenic mouse cohorts with the mRNA expression levels in <italic>AhCre<sup>+</sup>Apc<sup>+/+</sup>Myc<sup>+/+</sup></italic> (wild-type) mice. Results are shown as fold change relative to the wild-type mice (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). This analysis confirmed that <italic>Nap1l1</italic> mRNA expression was significantly increased following <italic>Apc</italic> deletion in a <italic>Myc</italic>-dependent manner.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Relative expression of <italic>Nap1l1</italic> mRNA extracted from small intestinal epithelial samples from induced <italic>AhCre<sup>+</sup>Apc<sup>+/+</sup>Myc<sup>+/+</sup></italic>, <italic>AhCre<sup>+</sup>Apc<sup>fl/fl</sup></italic>, <italic>AhCre<sup>+</sup> Apc<sup>fl/fl</sup> Myc<sup>fl/fl</sup></italic>, and <italic>AhCre<sup>+</sup>Myc<sup>fl/fl</sup></italic> mouse models (four mice in each group) showing the fold change in mRNA expression for each gene relative to <italic>AhCre<sup>+</sup>Apc<sup>+/+</sup>Myc<sup>+/+</sup></italic>. Error bars: standard error (SE). <italic>p</italic> < 0.05 for <italic>AhCre<sup>+</sup>Apc<sup>fl/fl</sup></italic> compared to other groups.</p></caption><graphic xlink:href=\"fonc-10-01565-g001\"/></fig></sec><sec id=\"S3.SS3\"><title>Evaluation of <italic>NAP1L1</italic> mRNA Expression in Human Colorectal Cancer Samples</title><p><italic>NAP1L1</italic> was then analyzed in three cohorts of human CRC samples. We assessed the expression of <italic>NAP1L1</italic> firstly in mRNA from CRC tissues and matched normal mucosa from 18 patients supplied by the Wales Cancer Bank. <italic>NAP1L1</italic> demonstrated statistically significant elevated mRNA levels in CRC samples (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>, Wales cohort 1). We next performed a confirmatory study using a different set of 30 samples from the same Tissue Bank, and we observed consistent results (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>, Wales cohort 2). In order to further validate the findings, we repeated the experiment using a different platform (in terms of equipment and reagents) and compared a cohort of 10 normal colonic samples (individuals without any colonoscopic evidence of colorectal neoplasia) and 25 CRC samples from Brazil (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>, Brazil cohort). Once more, significantly increased levels of <italic>NAP1L1</italic> mRNA were observed in CRC specimens and this time the comparison was with normal colonic tissue from patients who had no evidence of colorectal neoplasia.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>qPCR analysis of <italic>NAP1L1</italic> expression in CRC tumors presented as fold-change relative to normal tissues in different cohorts. Each column represents the relative quantification (fold-change) of <italic>NAP1L1</italic> mRNA expression in CRC compared to the respective normal control (normal control expression mean = 1). Wales cohort 1, mean fold-change = 2.7; <italic>p</italic> < 0.05 (18 paired colorectal cancer and adjacent normal colonic tissue samples). Wales cohort 2, mean fold-change = 5.8; <italic>p</italic> < 0.001 (30 paired colorectal cancer and adjacent normal colonic tissue samples). Brazil cohort, mean fold-change = 7.9; <italic>p</italic> < 0.001 (10 normal samples from healthy individuals without colorectal neoplasia and 25 colorectal cancer samples). Mann–Whitney <italic>U</italic> test. Error bars: ±1 SE.</p></caption><graphic xlink:href=\"fonc-10-01565-g002\"/></fig></sec><sec id=\"S3.SS4\"><title>Confirmation of Differential Immuno-Expression of NAP1L1 in Human Colorectal Tissues</title><p>Immunohistochemistry for NAP1L1 was then performed in colorectal tissue samples from a different cohort of 32 patients, as described in section “Materials and Methods.” Cancer tissues and the matched unaffected mucosa collected 10 cm from the primary lesion were analyzed. Scoring was performed electronically using the software ImageJ (publicly available at <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.rsbweb.nih.gov/ij/\">rsbweb.nih.gov/ij/</ext-link>) and the plugins IHC Profiler for cytoplasmic scoring (resulting in a modified H-score ranging from 0 to 300)(<xref rid=\"B17\" ref-type=\"bibr\">17</xref>) and ImmunoRatio for nuclear scoring (ranging from 0 to 100%)(<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). The samples were subdivided into two groups who had early stage (11 samples encompassing stages I and II) and late stage (21 samples including stages III and IV) CRC.</p><p>We initially assessed the expression of β-catenin to confirm Wnt pathway activation and to validate the electronic scoring methods (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). A clear and statistically significant increase in both nuclear and cytoplasmic localization of β-catenin was observed in cancer tissues compared to the adjacent mucosa, as expected based on previous literature (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>–<xref rid=\"B21\" ref-type=\"bibr\">21</xref>), thus validating our scoring system. Using the same scoring methods, we observed an opposite staining pattern for NAP1L1. A clear and statistically significant decrease in both the nuclear and cytoplasmic expression of NAP1L1 was seen in CRC tissues relative to the adjacent mucosa (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). No difference was detected between early and late stage tumor groups for both markers.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Staining patterns and scoring results for β-catenin. Nuclear and cytoplasmic staining of β-catenin increased in cancer tissues when compared to the adjacent mucosa. No difference was observed between different stages of cancer. ***<italic>p</italic> < 0.001 (Kruskal–Wallis test followed by <italic>post hoc</italic> Dunn-Bonferroni test for pair-wise comparisons). Error bars: ±2 SE. Sample numbers: normal = 32, stages I–II = 11, stages III–IV = 21. Magnification: 630×.</p></caption><graphic xlink:href=\"fonc-10-01565-g003\"/></fig><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Staining patterns and scoring results for NAP1L1. NAP1L1 nuclear and cytoplasmic scores were decreased in the cancer groups when compared to normal adjacent tissues. No difference was observed between different stages of cancer. ***<italic>p</italic> < 0.001 (Kruskal–Wallis test followed by <italic>post hoc</italic> Dunn-Bonferroni test for pair-wise comparisons). Error bars: ±2 SE. Sample numbers: normal = 32, stages I–II = 11, stages III–IV = 21. Magnification: 630×.</p></caption><graphic xlink:href=\"fonc-10-01565-g004\"/></fig><p>We also performed a confirmatory analysis using a different cohort of 19 patients from the Countess of Chester Hospital NHS Foundation Trust (Chester, United Kingdom). This analysis used a slightly different manual scoring method as described in <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 2</xref>. The findings were very similar to those demonstrated in the Brazilian patients and again demonstrated decreased NAP1L1 expression in the CRC samples (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 2</xref>).</p></sec><sec id=\"S3.SS5\"><title>NAP1L1 Nuclear Expression Is a Strong Predictor of Survival in Late Stage CRC</title><p>Having demonstrated decreased NAP1L1 immunohistochemical expression in CRC samples, we investigated whether the expression pattern had any effect on patient outcome. We analyzed a further cohort of 75 CRC cases diagnosed between 2004 and 2012. Median follow-up was 84.7 months (range 48–153 months). Given the relatively small number of cases, cancer stages were again combined into two groups: early stage (stages I and II) and late-stage (stages III and IV). Immunohistochemistry was conducted as described in section “Materials and Methods.” <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> describes the characteristics of the patients included in this analysis.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Characteristics of the patients included in the prognostic analysis.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristics</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Patients (<italic>n</italic> = 75)</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Mean age</bold> (range)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">59.7(33−84)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Gender</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">43(57.3%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32(42.7%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Stage</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I–II</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28(37.3%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">III–IV</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">47(62.7%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Grade</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Well differentiated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23(30.6%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Moderately differentiated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50(66.6%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Poorly differentiated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2(2.6%)</td></tr></tbody></table></table-wrap><p>Initially, using mortality status as the binary event of interest, ROC curves were generated. The area under the curve (AUC) was 0.58 for the nuclear score and 0.60 for the cytoplasmic score. Cut-offs were determined either by manually assessing the best balance between sensitivity and specificity or electronically by the use of the software Cutoff Finder (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>) and X-Tile (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). All methods suggested the same cut-off for nuclear staining: 32% (of positive nuclei). Use of this threshold resulted in a sensitivity of 61% and a specificity of 67.5% for discriminating mortality status. For cytoplasmic staining, sensitivity/specificity optimization suggested a cut-off of 135 (in a range from 0 to 300), yielding a sensitivity of 57% and a specificity of 54%. The electronic tools suggested higher cut-offs (167 and 168). Although these resulted in a higher specificity (92%), the sensitivity was low (29%). Despite these differences, the prognostic results were similar, so the cytoplasmic cut-off of 135 was selected to describe the results.</p><p>The prognostic cohort was therefore divided into two groups with low-expression and high-expression of NAP1L1. Groups were similar in terms of age, gender, stage and grade. <xref rid=\"T2\" ref-type=\"table\">Table 2</xref> shows the clinicopathological characteristics of the groups according to the nuclear expression of NAP1L1. Similar balanced distribution was also observed for cytoplasmic expression.</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Clinicopathological characteristics according to NAP1L1 nuclear expression.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Characteristics</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Low nuclear expression (<italic>n</italic> = 34)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">High nuclear expression (<italic>n</italic> = 41)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Two-sided <italic>p</italic>-values</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Mean age</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">61.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">57.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.157</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Gender</bold></td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.648</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Male</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Stage</bold></td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.338</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">I–II</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">III–IV</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Grade</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.351</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Well differentiated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mod differentiated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Poorly differentiated</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic>No significant difference between groups was observed. Mean age was compared by <italic>t</italic>-test. Categorical variables were compared by Chi-square test or Fisher’s exact test.</italic></attrib></table-wrap-foot></table-wrap><p>Using the Kaplan–Meier method, cumulative survivals for the two groups (high and low nuclear expression) were compared. Initially, groups were assessed as a whole, regardless of disease stage (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>). A clear difference in cumulative survival was observed according to nuclear NAP1L1 staining (<italic>p</italic> = 0.012, log-rank test). In the multivariate analysis including age, gender, stage and grade (Cox proportional hazards model), the nuclear score was independently associated with cumulative survival. The high nuclear expression group exhibited a hazard ratio (HR): 0.39 ([95%CI: 0.17–0.87]; <italic>p</italic> = 0.02), denoting a 61% reduction in cumulative mortality in this group. As a result, the estimated 5-year survival was 44.4% in the low expression group and 75% in the high expression group. Median duration of survival was 32 months in the low expression group, whilst it was not reached for the high expression cohort. The only additional variable also associated with survival was tumor stage (HR: 2.55 [95%CI: 1.01–6.43]; <italic>p</italic> = 0.047), an expected finding since stage is a known prognostic factor in CRC. These results strongly suggest an association between NAP1L1 nuclear staining and survival in CRC patients. Conversely, cytoplasmic NAP1L1 staining was not associated with survival (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>) or with any other clinicopathological variable.</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Cumulative survival according to NAP1L1 expression. In panel <bold>(A)</bold>, nuclear staining is assessed. A highly significant (<italic>p</italic> = 0.012) and clinically relevant (HR = 0.39 [95%CI: 0.17–0.87]) difference in survival between groups was observed favoring the high expression group. In panel <bold>(B)</bold>, cytoplasmic staining, no significant difference was observed. In panel <bold>(C)</bold>, nuclear expression for early stage disease (stages I and II) is assessed. No difference in survival was observed. Panel <bold>(D)</bold> shows the results for late stage disease (stages III and IV). A statistically significant difference was observed favoring the high nuclear expression group (HR: 0.28 [95%CI: 0.11–0.71]). Squares and circles = censored cases. HR: hazard ratio. CI: confidence interval.</p></caption><graphic xlink:href=\"fonc-10-01565-g005\"/></fig><p>We then analyzed survival according to NAP1L1 nuclear expression in different stage groups (<xref ref-type=\"fig\" rid=\"F5\">Figures 5C,D</xref>). For early stage disease, no significant difference in survival was found. By contrast, a highly significant difference in survival was observed for the cohort containing stages III and IV tumors. Multivariate analysis once again demonstrated that NAP1L1 nuclear score was an independent prognostic factor in CRC patients. The calculated HR (0.28 [95%CI: 0.11–0.71]; <italic>p</italic> = 0.008) was even more notable than that observed for the entire cohort, now suggesting a 72% reduction in cumulative mortality. The 5-year survival advantage for high expression tumors was also greater: 70%, versus 34% for low expression cancers. Median survival was only 23 months in the low expression group and, again, was not reached in the high expression cohort.</p></sec></sec><sec id=\"S4\"><title>Discussion</title><p>The discovery of novel CRC biomarkers to assist in early diagnosis, prognostic stratification and prediction of response to treatment remains an unmet medical need. We hypothesized that the study of animal models of CRC based on transgenic <italic>Apc</italic> gene inactivation could lead to the discovery of novel useful CRC biomarkers in humans.</p><p>By combining transcriptomic and proteomic analyses of small intestinal tissue from transgenic mice in which <italic>Apc</italic> and/or <italic>Myc</italic> had been specifically deleted, we identified NAP1L1 as the only gene/protein that showed significantly altered expression in <italic>Apc</italic> and <italic>Myc</italic>-dependent manners in all analyses. We confirmed these findings using qPCR in mouse small intestine and additionally demonstrated that <italic>NAP1L1</italic> mRNA expression was increased in human CRC. It was unfortunately not possible to study whether there was any altered NAP1L1 expression in the colon of the <italic>AhCre</italic> mouse model as there is no Cre mediated recombination in the colon of these mice following injection of β-naphthoflavone and they have no colonic phenotype (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>).</p><p>NAP1L1 is a highly conserved histone chaperone protein which is one of five NAP1-like proteins in mammals (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>, <xref rid=\"B22\" ref-type=\"bibr\">22</xref>). It has been suggested to play a role in mediating nucleosome formation and regulation of the H2A-H2B complex as well as nucleosome assembly (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>), cell cycle progression, and cell proliferation (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). It has also been linked to embryogenesis and tissue differentiation (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>–<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Few researchers have previously studied <italic>NAP1L1</italic> expression in cancer cell lines or tissues. Drozdov et al. compared small intestinal neuroendocrine tumors (NETs) and normal enterochromaffin cell preparations, and showed a 13.7-fold increase in <italic>NAP1L1</italic> expression in tumor tissues (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). However, no analysis of the adjacent mucosa was performed. Kidd et al. also suggested that <italic>NAP1L1</italic> was increased in NETs but not in CRCs (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). Line et al. evaluated <italic>NAP1L1</italic> mRNA expression in CRC and adjacent tissues as a secondary endpoint in a study primarily aimed at finding sero-reactive biomarkers (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). They showed that, among 15 cases of CRC, seven exhibited moderate increases in <italic>NAP1L1</italic> expression (ranging from 2.9 to 9.3-fold) and eight cases showed expression levels similar to those in the corresponding adjacent mucosa. A recent paper has also demonstrated that NAP1L1 is a prognostic biomarker and contributes to doxorubicin chemotherapy resistance in hepatocellular carcinoma (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>).</p><p>Immunohistochemistry is used in routine clinical practice to assess the expression of proteins with prognostic or predictive value in other types of cancer such as breast (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>) and lung carcinomas (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>), soft tissue sarcomas (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>), and lymphomas (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). Given the absence of a standard scoring method for NAP1L1, we initially decided to assess both the nuclear and the cytoplasmic expression of the protein in our samples using electronic tools. Our results showed that NAP1L1 expression was decreased both in the nucleus and the cytoplasm of CRC tissues when compared to the normal adjacent mucosa.</p><p>This was a somewhat unexpected finding, given the increased expression of <italic>NAP1L1</italic> mRNA in animal models and in human tissues. Such discrepancy between mRNA and protein expression has however previously been demonstrated for other cancer markers (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>–<xref rid=\"B37\" ref-type=\"bibr\">37</xref>). Several processes could be responsible, such as posttranscriptional modifications, protein degradation, secretion via exocytosis or alterations in subcellular protein localization. For example a recent paper has reported that NAP1L1 undergoes alternative cleavage and polyadenylation in the more advanced stages of CRC (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>). The full length isoform of NAP1L1 was overrepresented in the cytoplasmic fraction of a CRC cell line which had a more metastatic phenotype. This may therefore represent one mechanism to explain the altered NAP1L1 subcellular localization that is reported in CRC specimens in our current manuscript. Counterintuitively, our finding of increased gene expression in the initial screen may have been a response to reduced protein content and not the primary event. Further research is required in order to clarify this issue. Qiao et al. (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>) demonstrated that knock down of <italic>NAP1L1</italic> increased cellular proliferation, disrupted normal cell development and distribution, and caused global deregulation of gene expression. These are classical hallmarks of cancer and of activated Wnt signaling. Qiao et al. also demonstrated that <italic>Nap1l1</italic> knockdown resulted in reduced RassF10 expression, low expression of which has been associated with poor survival in CRC patients in another study (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>). Thus reduced NAP1L1 protein expression could be mechanistically associated with tumor progression.</p><p>We also assessed whether NAP1L1 expression was associated with prognosis. We retrospectively retrieved blocks from a cohort of CRC patients with more than 4 years follow up. Cut-offs for nuclear and cytoplasmic expression of NAP1L1 were established and a survival analysis was performed. Nuclear expression of NAP1L1 correlated with overall survival in CRC. High nuclear expression was independently associated with an increase in median survival and 5-year survival estimates. Subgroup analysis however showed that the survival correlation was limited to late stage tumors (stages III and IV). No association between NAP1L1 nuclear expression and clinicopathologic variables (age, gender, stage and grade) was observed. These findings suggest that the expression of NAP1L1 could potentially help in discriminating low and high-risk disease in stage III CRC cases and, also, to determine the aggressiveness of the disease in stage IV cancers. In both cases, this information could help to better define the best treatment approach.</p><p>We acknowledge some limitations in this study. Although positive and relevant findings were observed, the use of small clinical sample sizes may have limited our observations. This also meant that it was necessary to evaluate cancer stages as combined groups rather than individually. Better prognostic stratification in stage II disease is urgently needed to improve the treatment decision-making process and our data did not permit this. Moreover, analyzing larger cohorts of stage III and stage IV diseases separately would also be desirable, as these stages are associated with markedly different clinical outcomes.</p><p>This study provides proof of concept that the analysis of animal models of Wnt pathway activation may yield potentially useful CRC biomarkers in humans. We undertook a comprehensive assessment of NAP1L1 expression in animal models and clinical samples and our findings suggest that it could be a prognostic biomarker for CRC. Confirmatory research studying larger sample cohorts and a better assessment of the role of this protein in CRC carcinogenesis is now recommended before this marker can be introduced routinely into clinical practice.</p></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>The datasets generated for this study can be found in the Broad Institute Gene Set Enrichment Analysis (GSEA) M1755, M1756, M1757, and M1578.</p></sec><sec id=\"S6\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by the respective institutions, Brazilian National Commission for Research Ethics (CONEP); Wales Cancer Bank and Countess of Chester Hospital NHS Foundation Trust. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Cardiff University Animal Welfare Ethical Review Panel and UK Home Office.</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>JJ, DP, KR, and AC designed the research project. JJ, FM, and DP supervised CQ’s Ph.D. studies. JJ and DV supervised NA-K’s MRes studies. KR managed the mouse inter-crosses and collection of murine samples. FS performed the qPCR of human samples. CQ and NA-K performed the human sample collection, qPCR, immunohistochemistry staining, and scoring and data analysis. CQ, DP, and JJ drafted the manuscript and all other authors critically reviewed the manuscript and approved the final version submitted. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> CQ was supported by a scholarship from the Science Without Borders Programme (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior, CAPES, Ministry of Education, Brazil). KR was supported by Cancer Research UK (ARC Programme grant C1295/A15937).</p></fn></fn-group><ack><p>We acknowledge the contribution made by Dr. Abeer Hammoudi to the initial proteomic experiments. We would like to acknowledge the Laboratorio São Nicolau (Dr. Ivana Menezes, Cuiaba – Brazil) for helping in the collection of clinical samples and in the performance of immunohistochemistry for the prognostic study. We thank Elaine Taylor for assistance with mouse husbandry, Mark Bishop and Matthew Zverev for technical support and genotyping of murine samples. Some of the content of this manuscript formed part of CQ’s doctoral thesis to obtain a Ph.D. degree at the University of Liverpool (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>).</p></ack><sec id=\"S10\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fonc.2020.01565/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fonc.2020.01565/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Table_1.XLSX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM2\"><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bray</surname><given-names>F</given-names></name><name><surname>Ferlay</surname><given-names>J</given-names></name><name><surname>Soerjomataram</surname><given-names>I</given-names></name><name><surname>Siegel</surname><given-names>RL</given-names></name><name><surname>Torre</surname><given-names>LA</given-names></name><name><surname>Jemal</surname><given-names>A.</given-names></name></person-group>\n<article-title>Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</article-title>\n<source><italic>CA Cancer J Clin.</italic></source> (<year>2018</year>) <volume>68</volume>:<fpage>394</fpage>–<lpage>424</lpage>. <pub-id pub-id-type=\"doi\">10.3322/caac.21492</pub-id>\n<pub-id pub-id-type=\"pmid\">30207593</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luzietti</surname><given-names>E</given-names></name><name><surname>Pellino</surname><given-names>G</given-names></name><name><surname>Nikolaou</surname><given-names>S</given-names></name><name><surname>Qiu</surname><given-names>S</given-names></name><name><surname>Mills</surname><given-names>S</given-names></name><name><surname>Warren</surname><given-names>O</given-names></name><etal/></person-group>\n<article-title>Comparison of guidelines for the management of rectal cancer.</article-title>\n<source><italic>BJS Open.</italic></source> (<year>2018</year>) <volume>2</volume>: <fpage>433</fpage>–<lpage>51</lpage>.<pub-id pub-id-type=\"pmid\">30511044</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grothey</surname><given-names>A</given-names></name><name><surname>Sobrero</surname><given-names>AF</given-names></name><name><surname>Shields</surname><given-names>AF</given-names></name><name><surname>Yoshino</surname><given-names>T</given-names></name><name><surname>Paul</surname><given-names>J</given-names></name><name><surname>Taieb</surname><given-names>J</given-names></name><etal/></person-group>\n<article-title>Duration of adjuvant chemotherapy for stage III colon cancer.</article-title>\n<source><italic>N Engl J Med.</italic></source> (<year>2018</year>) <volume>378</volume>:<fpage>1177</fpage>–<lpage>88</lpage>.<pub-id pub-id-type=\"pmid\">29590544</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schilsky</surname><given-names>RL.</given-names></name></person-group>\n<article-title>A new IDEA in adjuvant chemotherapy for colon cancer.</article-title>\n<source><italic>N Engl J Med.</italic></source> (<year>2018</year>) <volume>378</volume>:<fpage>1242</fpage>–<lpage>4</lpage>.<pub-id pub-id-type=\"pmid\">29590539</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bockelman</surname><given-names>C</given-names></name><name><surname>Engelmann</surname><given-names>BE</given-names></name><name><surname>Kaprio</surname><given-names>T</given-names></name><name><surname>Hansen</surname><given-names>TF</given-names></name><name><surname>Glimelius</surname><given-names>B.</given-names></name></person-group>\n<article-title>Risk of recurrence in patients with colon cancer stage II and III: a systematic review and meta-analysis of recent literature.</article-title>\n<source><italic>Acta Oncol.</italic></source> (<year>2015</year>) <volume>54</volume>:<fpage>5</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.3109/0284186x.2014.975839</pub-id>\n<pub-id pub-id-type=\"pmid\">25430983</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coghlin</surname><given-names>C</given-names></name><name><surname>Murray</surname><given-names>GI.</given-names></name></person-group>\n<article-title>Biomarkers of colorectal cancer: recent advances and future challenges.</article-title>\n<source><italic>Proteomics Clin Appl.</italic></source> (<year>2015</year>) <volume>9</volume>:<fpage>64</fpage>–<lpage>71</lpage>. <pub-id pub-id-type=\"doi\">10.1002/prca.201400082</pub-id>\n<pub-id pub-id-type=\"pmid\">25311658</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><collab>Cancer Genome Atlas Network.</collab><article-title>Comprehensive molecular characterization of human colon and rectal cancer.</article-title>\n<source><italic>Nature.</italic></source> (<year>2012</year>) <volume>487</volume>:<fpage>330</fpage>–<lpage>7</lpage>.<pub-id pub-id-type=\"pmid\">22810696</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Baarsma</surname><given-names>HA</given-names></name><name><surname>Konigshoff</surname><given-names>M</given-names></name><name><surname>Gosens</surname><given-names>R.</given-names></name></person-group>\n<article-title>The WNT signaling pathway from ligand secretion to gene transcription: molecular mechanisms and pharmacological targets.</article-title>\n<source><italic>Pharmacol Ther.</italic></source> (<year>2013</year>) <volume>138</volume>:<fpage>66</fpage>–<lpage>83</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.pharmthera.2013.01.002</pub-id>\n<pub-id pub-id-type=\"pmid\">23328704</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sansom</surname><given-names>OJ</given-names></name><name><surname>Reed</surname><given-names>KR</given-names></name><name><surname>Hayes</surname><given-names>AJ</given-names></name><name><surname>Ireland</surname><given-names>H</given-names></name><name><surname>Brinkmann</surname><given-names>H</given-names></name><name><surname>Newton</surname><given-names>IP</given-names></name><etal/></person-group>\n<article-title>Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration.</article-title>\n<source><italic>Genes Dev.</italic></source> (<year>2004</year>) <volume>18</volume>:<fpage>1385</fpage>–<lpage>90</lpage>.<pub-id pub-id-type=\"pmid\">15198980</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cordero</surname><given-names>J</given-names></name><name><surname>Vidal</surname><given-names>M</given-names></name><name><surname>Sansom</surname><given-names>O.</given-names></name></person-group>\n<article-title>APC as a master regulator of intestinal homeostasis and transformation: from flies to vertebrates.</article-title>\n<source><italic>Cell Cycle.</italic></source> (<year>2009</year>) <volume>8</volume>:<fpage>2926</fpage>–<lpage>31</lpage>.<pub-id pub-id-type=\"pmid\">19657225</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dow</surname><given-names>LE</given-names></name><name><surname>O’Rourke</surname><given-names>KP</given-names></name><name><surname>Simon</surname><given-names>J</given-names></name><name><surname>Tschaharganeh</surname><given-names>DF</given-names></name><name><surname>van Es</surname><given-names>JH</given-names></name><name><surname>Clevers</surname><given-names>H</given-names></name><etal/></person-group>\n<article-title>Apc restoration promotes cellular differentiation and reestablishes Crypt homeostasis in colorectal cancer.</article-title>\n<source><italic>Cell.</italic></source> (<year>2015</year>) <volume>161</volume>:<fpage>1539</fpage>–<lpage>52</lpage>.<pub-id pub-id-type=\"pmid\">26091037</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Su</surname><given-names>LK</given-names></name><name><surname>Kinzler</surname><given-names>KW</given-names></name><name><surname>Vogelstein</surname><given-names>B</given-names></name><name><surname>Preisinger</surname><given-names>AC</given-names></name><name><surname>Moser</surname><given-names>AR</given-names></name><name><surname>Luongo</surname><given-names>C</given-names></name><etal/></person-group>\n<article-title>Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene.</article-title>\n<source><italic>Science.</italic></source> (<year>1992</year>) <volume>256</volume>:<fpage>668</fpage>–<lpage>70</lpage>.<pub-id pub-id-type=\"pmid\">1350108</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moser</surname><given-names>AR</given-names></name><name><surname>Pitot</surname><given-names>HC</given-names></name><name><surname>Dove</surname><given-names>WF.</given-names></name></person-group>\n<article-title>A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse.</article-title>\n<source><italic>Science.</italic></source> (<year>1990</year>) <volume>247</volume>:<fpage>322</fpage>–<lpage>4</lpage>.<pub-id pub-id-type=\"pmid\">2296722</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sansom</surname><given-names>OJ</given-names></name><name><surname>Meniel</surname><given-names>VS</given-names></name><name><surname>Muncan</surname><given-names>V</given-names></name><name><surname>Phesse</surname><given-names>TJ</given-names></name><name><surname>Wilkins</surname><given-names>JA</given-names></name><name><surname>Reed</surname><given-names>KR</given-names></name><etal/></person-group>\n<article-title>Myc deletion rescues Apc deficiency in the small intestine.</article-title>\n<source><italic>Nature.</italic></source> (<year>2007</year>) <volume>446</volume>:<fpage>676</fpage>–<lpage>9</lpage>.<pub-id pub-id-type=\"pmid\">17377531</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Myant</surname><given-names>K</given-names></name><name><surname>Sansom</surname><given-names>OJ.</given-names></name></person-group>\n<article-title>Wnt/Myc interactions in intestinal cancer: partners in crime.</article-title>\n<source><italic>Exp Cell Res.</italic></source> (<year>2011</year>) <volume>317</volume>:<fpage>2725</fpage>–<lpage>31</lpage>.<pub-id pub-id-type=\"pmid\">21851818</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hammoudi</surname><given-names>A</given-names></name><name><surname>Song</surname><given-names>F</given-names></name><name><surname>Reed</surname><given-names>KR</given-names></name><name><surname>Jenkins</surname><given-names>RE</given-names></name><name><surname>Meniel</surname><given-names>VS</given-names></name><name><surname>Watson</surname><given-names>AJ</given-names></name><etal/></person-group>\n<article-title>Proteomic profiling of a mouse model of acute intestinal Apc deletion leads to identification of potential novel biomarkers of human colorectal cancer (CRC).</article-title>\n<source><italic>Biochem Biophys Res Commun.</italic></source> (<year>2013</year>) <volume>440</volume>:<fpage>364</fpage>–<lpage>70</lpage>.<pub-id pub-id-type=\"pmid\">23998936</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Varghese</surname><given-names>F</given-names></name><name><surname>Bukhari</surname><given-names>AB</given-names></name><name><surname>Malhotra</surname><given-names>R</given-names></name><name><surname>De</surname><given-names>A.</given-names></name></person-group>\n<article-title>IHC Profiler: an open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples.</article-title>\n<source><italic>PLoS One.</italic></source> (<year>2014</year>) <volume>9</volume>:<issue>e96801</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0096801</pub-id>\n<pub-id pub-id-type=\"pmid\">24802416</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tuominen</surname><given-names>VJ</given-names></name><name><surname>Ruotoistenmäki</surname><given-names>S</given-names></name><name><surname>Viitanen</surname><given-names>A</given-names></name><name><surname>Jumppanen</surname><given-names>M</given-names></name><name><surname>Isola</surname><given-names>J.</given-names></name></person-group>\n<article-title>ImmunoRatio: a publicly available web application for quantitative image analysis of estrogen receptor (ER), progesterone receptor (PR), and Ki-67.</article-title>\n<source><italic>Breast Cancer Res.</italic></source> (<year>2010</year>) <volume>12</volume>:<issue>R56</issue>.</mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wong</surname><given-names>SC</given-names></name><name><surname>Lo</surname><given-names>ES</given-names></name><name><surname>Chan</surname><given-names>AK</given-names></name><name><surname>Lee</surname><given-names>KC</given-names></name><name><surname>Hsiao</surname><given-names>WL.</given-names></name></person-group>\n<article-title>Nuclear beta catenin as a potential prognostic and diagnostic marker in patients with colorectal cancer from Hong Kong.</article-title>\n<source><italic>Mol Pathol.</italic></source> (<year>2003</year>) <volume>56</volume>:<fpage>347</fpage>–<lpage>52</lpage>.<pub-id pub-id-type=\"pmid\">14645698</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wong</surname><given-names>SC</given-names></name><name><surname>Lo</surname><given-names>ES</given-names></name><name><surname>Lee</surname><given-names>KC</given-names></name><name><surname>Chan</surname><given-names>JK</given-names></name><name><surname>Hsiao</surname><given-names>WL.</given-names></name></person-group>\n<article-title>Prognostic and diagnostic significance of beta-catenin nuclear immunostaining in colorectal cancer.</article-title>\n<source><italic>Clin Cancer Res.</italic></source> (<year>2004</year>) <volume>10</volume>:<fpage>1401</fpage>–<lpage>8</lpage>.<pub-id pub-id-type=\"pmid\">14977843</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>Z</given-names></name><name><surname>He</surname><given-names>X</given-names></name><name><surname>Jia</surname><given-names>M</given-names></name><name><surname>Liu</surname><given-names>Y</given-names></name><name><surname>Qu</surname><given-names>D</given-names></name><name><surname>Wu</surname><given-names>D</given-names></name><etal/></person-group>\n<article-title>β-catenin overexpression in the nucleus predicts progress disease and unfavourable survival in colorectal cancer: a meta-analysis.</article-title>\n<source><italic>PLoS One.</italic></source> (<year>2013</year>) <volume>8</volume>:<issue>e63854</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0063854</pub-id>\n<pub-id pub-id-type=\"pmid\">23717499</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Budczies</surname><given-names>J</given-names></name><name><surname>Klauschen</surname><given-names>F</given-names></name><name><surname>Sinn</surname><given-names>BV</given-names></name><name><surname>Gyorffy</surname><given-names>B</given-names></name><name><surname>Schmitt</surname><given-names>WD</given-names></name><name><surname>Darb-Esfahani</surname><given-names>S</given-names></name><etal/></person-group>\n<article-title>Cutoff Finder: a comprehensive and straightforward Web application enabling rapid biomarker cutoff optimization.</article-title>\n<source><italic>PLoS One.</italic></source> (<year>2012</year>) <volume>7</volume>:<issue>e51862</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0051862</pub-id>\n<pub-id pub-id-type=\"pmid\">23251644</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Camp</surname><given-names>RL</given-names></name><name><surname>Dolled-Filhart</surname><given-names>M</given-names></name><name><surname>Rimm</surname><given-names>DL.</given-names></name></person-group>\n<article-title>X-tile: a new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization.</article-title>\n<source><italic>Clin Cancer Res.</italic></source> (<year>2004</year>) <volume>10</volume>:<fpage>7252</fpage>–<lpage>9</lpage>.<pub-id pub-id-type=\"pmid\">15534099</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ireland</surname><given-names>H</given-names></name><name><surname>Kemp</surname><given-names>R</given-names></name><name><surname>Houghton</surname><given-names>C</given-names></name><name><surname>Howard</surname><given-names>L</given-names></name><name><surname>Clarke</surname><given-names>AR</given-names></name><name><surname>Sansom</surname><given-names>OJ</given-names></name><etal/></person-group>\n<article-title>Inducible Cre-mediated control of gene expression in the murine gastrointestinal tract: effect of loss of beta-catenin.</article-title>\n<source><italic>Gastroenterology.</italic></source> (<year>2004</year>) <volume>126</volume>:<fpage>1236</fpage>–<lpage>46</lpage>.<pub-id pub-id-type=\"pmid\">15131783</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Okuwaki</surname><given-names>M</given-names></name><name><surname>Kato</surname><given-names>K</given-names></name><name><surname>Nagata</surname><given-names>K.</given-names></name></person-group>\n<article-title>Functional characterization of human nucleosome assembly protein 1-like proteins as histone chaperones.</article-title>\n<source><italic>Genes Cells.</italic></source> (<year>2010</year>) <volume>15</volume>:<fpage>13</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-2443.2009.01361.x</pub-id>\n<pub-id pub-id-type=\"pmid\">20002496</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Clark</surname><given-names>J</given-names></name><name><surname>Alvarez</surname><given-names>DF</given-names></name><name><surname>Alexeyev</surname><given-names>M</given-names></name><name><surname>King</surname><given-names>JA</given-names></name><name><surname>Huang</surname><given-names>L</given-names></name><name><surname>Yoder</surname><given-names>MC</given-names></name><etal/></person-group>\n<article-title>Regulatory role for nucleosome assembly protein-1 in the proliferative and vasculogenic phenotype of pulmonary endothelium.</article-title>\n<source><italic>Am J Physiol Lung Cell Mol Physiol.</italic></source> (<year>2008</year>) <volume>294</volume>:<fpage>L431</fpage>–<lpage>9</lpage>.<pub-id pub-id-type=\"pmid\">17981956</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Drozdov</surname><given-names>I</given-names></name><name><surname>Kidd</surname><given-names>M</given-names></name><name><surname>Nadler</surname><given-names>B</given-names></name><name><surname>Camp</surname><given-names>RL</given-names></name><name><surname>Mane</surname><given-names>SM</given-names></name><name><surname>Hauso</surname><given-names>O</given-names></name><etal/></person-group>\n<article-title>Predicting neuroendocrine tumor (carcinoid) neoplasia using gene expression profiling and supervised machine learning.</article-title>\n<source><italic>Cancer.</italic></source> (<year>2009</year>) <volume>115</volume>:<fpage>1638</fpage>–<lpage>50</lpage>.<pub-id pub-id-type=\"pmid\">19197975</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kidd</surname><given-names>M</given-names></name><name><surname>Modlin</surname><given-names>IM</given-names></name><name><surname>Mane</surname><given-names>SM</given-names></name><name><surname>Camp</surname><given-names>RL</given-names></name><name><surname>Eick</surname><given-names>G</given-names></name><name><surname>Latich</surname><given-names>I.</given-names></name></person-group>\n<article-title>The role of genetic markers–NAP1L1, MAGE-D2, and MTA1–in defining small-intestinal carcinoid neoplasia.</article-title>\n<source><italic>Ann Surg Oncol.</italic></source> (<year>2006</year>) <volume>13</volume>:<fpage>253</fpage>–<lpage>62</lpage>.<pub-id pub-id-type=\"pmid\">16424981</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Line</surname><given-names>A</given-names></name><name><surname>Slucka</surname><given-names>Z</given-names></name><name><surname>Stengrevics</surname><given-names>A</given-names></name><name><surname>Silina</surname><given-names>K</given-names></name><name><surname>Li</surname><given-names>G</given-names></name><name><surname>Rees</surname><given-names>RC.</given-names></name></person-group>\n<article-title>Characterisation of tumour-associated antigens in colon cancer.</article-title>\n<source><italic>Cancer Immunol Immunother.</italic></source> (<year>2002</year>) <volume>51</volume>:<fpage>574</fpage>–<lpage>82</lpage>.<pub-id pub-id-type=\"pmid\">12384809</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Le</surname><given-names>Y</given-names></name><name><surname>Kan</surname><given-names>A</given-names></name><name><surname>Li</surname><given-names>QJ</given-names></name><name><surname>He</surname><given-names>MK</given-names></name><name><surname>Chen</surname><given-names>HL</given-names></name><name><surname>Shi</surname><given-names>M.</given-names></name></person-group>\n<article-title>NAP1L1 is a prognostic biomarker and contribute to doxorubicin chemotherapy resistance in human hepatocellular carcinoma.</article-title>\n<source><italic>Cancer Cell Int.</italic></source> (<year>2019</year>) <volume>19</volume>:<issue>228</issue>.</mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zaha</surname><given-names>DC.</given-names></name></person-group>\n<article-title>Significance of immunohistochemistry in breast cancer.</article-title>\n<source><italic>World J Clin Oncol.</italic></source> (<year>2014</year>) <volume>5</volume>:<fpage>382</fpage>–<lpage>92</lpage>.<pub-id pub-id-type=\"pmid\">25114853</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pekar-Zlotin</surname><given-names>M</given-names></name><name><surname>Hirsch</surname><given-names>FR</given-names></name><name><surname>Soussan-Gutman</surname><given-names>L</given-names></name><name><surname>Ilouze</surname><given-names>M</given-names></name><name><surname>Dvir</surname><given-names>A</given-names></name><name><surname>Boyle</surname><given-names>T</given-names></name><etal/></person-group>\n<article-title>Fluorescence in situ hybridization, immunohistochemistry, and next-generation sequencing for detection of EML4-ALK rearrangement in lung cancer.</article-title>\n<source><italic>Oncologist</italic></source> (<year>2015</year>) <volume>20</volume>:<fpage>316</fpage>–<lpage>22</lpage>.<pub-id pub-id-type=\"pmid\">25721120</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hornick</surname><given-names>JL.</given-names></name></person-group>\n<article-title>Novel uses of immunohistochemistry in the diagnosis and classification of soft tissue tumors.</article-title>\n<source><italic>Mod Pathol.</italic></source> (<year>2014</year>) <volume>27</volume>:<fpage>S47</fpage>–<lpage>63</lpage>.<pub-id pub-id-type=\"pmid\">24384853</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Higgins</surname><given-names>RA</given-names></name><name><surname>Blankenship</surname><given-names>JE</given-names></name><name><surname>Kinney</surname><given-names>MC.</given-names></name></person-group>\n<article-title>Application of immunohistochemistry in the diagnosis of non-Hodgkin and Hodgkin lymphoma.</article-title>\n<source><italic>Arch Pathol Lab Med.</italic></source> (<year>2008</year>) <volume>132</volume>:<fpage>441</fpage>–<lpage>61</lpage>.<pub-id pub-id-type=\"pmid\">18318586</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Toga</surname><given-names>T</given-names></name><name><surname>Nio</surname><given-names>Y</given-names></name><name><surname>Hashimoto</surname><given-names>K</given-names></name><name><surname>Higami</surname><given-names>T</given-names></name><name><surname>Maruyama</surname><given-names>R.</given-names></name></person-group>\n<article-title>The dissociated expression of protein and messenger RNA of DPC4 in human invasive ductal carcinoma of the pancreas and their implication for patient outcome.</article-title>\n<source><italic>Anticancer Res.</italic></source> (<year>2004</year>) <volume>24</volume>:<fpage>1173</fpage>–<lpage>8</lpage>.<pub-id pub-id-type=\"pmid\">15154643</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>G</given-names></name><name><surname>Gharib</surname><given-names>TG</given-names></name><name><surname>Huang</surname><given-names>CC</given-names></name><name><surname>Taylor</surname><given-names>JM</given-names></name><name><surname>Misek</surname><given-names>DE</given-names></name><name><surname>Kardia</surname><given-names>SL</given-names></name><etal/></person-group>\n<article-title>Discordant protein and mRNA expression in lung adenocarcinomas.</article-title>\n<source><italic>Mol Cell Proteomics.</italic></source> (<year>2002</year>) <volume>1</volume>:<fpage>304</fpage>–<lpage>13</lpage>.<pub-id pub-id-type=\"pmid\">12096112</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pietkiewicz</surname><given-names>P</given-names></name><name><surname>Gornowicz-Porowska</surname><given-names>J</given-names></name><name><surname>Bowszyc-Dmochowska</surname><given-names>M</given-names></name><name><surname>Jagielska</surname><given-names>J</given-names></name><name><surname>Helak-Lapaj</surname><given-names>C</given-names></name><name><surname>Kaczmarek</surname><given-names>E</given-names></name><etal/></person-group>\n<article-title>Discordant expression of desmoglein 2 and 3 at the mRNA and protein levels in nodular and superficial basal cell carcinoma revealed by immunohistochemistry and fluorescent in situ hybridization.</article-title>\n<source><italic>Clin Exp Dermatol.</italic></source> (<year>2014</year>) <volume>39</volume>: <fpage>628</fpage>–<lpage>35</lpage>.<pub-id pub-id-type=\"pmid\">24934917</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fischl</surname><given-names>H</given-names></name><name><surname>Neve</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>Z</given-names></name><name><surname>Patel</surname><given-names>R</given-names></name><name><surname>Louey</surname><given-names>A</given-names></name><name><surname>Tian</surname><given-names>B</given-names></name><etal/></person-group>\n<article-title>hnRNPC regulates cancer-specific alternative cleavage and polyadenylation profiles.</article-title>\n<source><italic>Nucleic Acids Res.</italic></source> (<year>2019</year>) <volume>47</volume>:<fpage>7580</fpage>–<lpage>91</lpage>.<pub-id pub-id-type=\"pmid\">31147722</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Qiao</surname><given-names>H</given-names></name><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Feng</surname><given-names>C</given-names></name><name><surname>Duo</surname><given-names>S</given-names></name><name><surname>Ji</surname><given-names>F</given-names></name><name><surname>Jiao</surname><given-names>J.</given-names></name></person-group>\n<article-title>Nap1l1 controls embryonic neural progenitor cell proliferation and differentiation in the developing brain.</article-title>\n<source><italic>Cell Rep.</italic></source> (<year>2018</year>) <volume>22</volume>:<fpage>2279</fpage>–<lpage>93</lpage>.<pub-id pub-id-type=\"pmid\">29490266</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>F</given-names></name><name><surname>Li</surname><given-names>P</given-names></name><name><surname>Feng</surname><given-names>Y</given-names></name><name><surname>Hu</surname><given-names>YL</given-names></name><name><surname>Liu</surname><given-names>YF</given-names></name><name><surname>Guo</surname><given-names>YB</given-names></name><etal/></person-group>\n<article-title>Low expression of RASSF10 is associated with poor survival in patients with colorectal cancer.</article-title>\n<source><italic>Hum Pathol.</italic></source> (<year>2017</year>) <volume>62</volume>:<fpage>108</fpage>–<lpage>14</lpage>.<pub-id pub-id-type=\"pmid\">28041974</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Queiroz</surname><given-names>CJS.</given-names></name></person-group>\n<source><italic>Validation of Novel Colorectal Cancer Biomarkers Derived From Animal Models of Apc Inactivation: Analysis of Cohorts From the UK and Brazil.</italic></source> (2017). PhD thesis, University of Liverpool, Liverpool.</mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Nat Commun</journal-id><journal-id journal-id-type=\"iso-abbrev\">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type=\"epub\">2041-1723</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807771</article-id><article-id pub-id-type=\"pmc\">PMC7431562</article-id><article-id pub-id-type=\"publisher-id\">17635</article-id><article-id pub-id-type=\"doi\">10.1038/s41467-020-17635-1</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Resonant thermal energy transfer to magnons in a ferromagnetic nanolayer</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-4528-3983</contrib-id><name><surname>Kobecki</surname><given-names>Michal</given-names></name><address><email>michal.kobecki@tu-dortmund.de</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Scherbakov</surname><given-names>Alexey V.</given-names></name><address><email>alexey.shcherbakov@tu-dortmund.de</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-5498-9702</contrib-id><name><surname>Linnik</surname><given-names>Tetiana L.</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-6901-2657</contrib-id><name><surname>Kukhtaruk</surname><given-names>Serhii M.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-2394-7892</contrib-id><name><surname>Gusev</surname><given-names>Vitalyi E.</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Pattnaik</surname><given-names>Debi P.</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-2035-2324</contrib-id><name><surname>Akimov</surname><given-names>Ilya A.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-8774-6662</contrib-id><name><surname>Rushforth</surname><given-names>Andrew W.</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-8173-8212</contrib-id><name><surname>Akimov</surname><given-names>Andrey V.</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-0893-5949</contrib-id><name><surname>Bayer</surname><given-names>Manfred</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5675.1</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0416 9637</institution-id><institution>Experimentelle Physik 2, </institution><institution>Technische Universität Dortmund, </institution></institution-wrap>Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.423485.c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0548 8017</institution-id><institution>Ioffe Institute, </institution></institution-wrap>Politechnycheskaya 26, St. Petersburg, Russian Federation 194021 </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.466789.2</institution-id><institution>Department of Theoretical Physics, </institution><institution>V.E. Lashkaryov Institute of Semiconductor Physics, </institution></institution-wrap>Pr. Nauky 41, Kyiv, 03028 Ukraine </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.34566.32</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2172 3046</institution-id><institution>LAUM, CNRS UMR 6613, </institution><institution>Le Mans Université, </institution></institution-wrap>72085 Le Mans, France </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.4563.4</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 8868</institution-id><institution>School of Physics and Astronomy, </institution><institution>University of Nottingham, </institution></institution-wrap>Nottingham, NG7 2RD UK </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>4130</elocation-id><history><date date-type=\"received\"><day>29</day><month>1</month><year>2020</year></date><date date-type=\"accepted\"><day>2</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Energy harvesting is a concept which makes dissipated heat useful by transferring thermal energy to other excitations. Most of the existing principles are realized in systems which are heated continuously. We present the concept of high-frequency energy harvesting where the dissipated heat in a sample excites resonant magnons in a thin ferromagnetic metal layer. The sample is excited by femtosecond laser pulses with a repetition rate of 10 GHz, which results in temperature modulation at the same frequency with amplitude ~0.1 K. The alternating temperature excites magnons in the ferromagnetic nanolayer which are detected by measuring the net magnetization precession. When the magnon frequency is brought onto resonance with the optical excitation, a 12-fold increase of the amplitude of precession indicates efficient resonant heat transfer from the lattice to coherent magnons. The demonstrated principle may be used for energy harvesting in various nanodevices operating at GHz and sub-THz frequency ranges.</p></abstract><abstract id=\"Abs2\" abstract-type=\"web-summary\"><p id=\"Par2\">Most of the energy harvesting principles are realized in heated-continuously systems. Here, the authors present a concept of high-frequency energy harvesting where the dissipated heat in a sample excites resonant magnons in a ferromagnetic metal layer.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Energy harvesting</kwd><kwd>Ferromagnetism</kwd><kwd>Spintronics</kwd><kwd>Magneto-optics</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100002347</institution-id><institution>Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)</institution></institution-wrap></funding-source><award-id>Nanomagnetron</award-id><principal-award-recipient><name><surname>Kobecki</surname><given-names>Michal</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100001659</institution-id><institution>Deutsche Forschungsgemeinschaft (German Research Foundation)</institution></institution-wrap></funding-source><award-id>TRR160 19-52-12065</award-id><principal-award-recipient><name><surname>Kobecki</surname><given-names>Michal</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100002261</institution-id><institution>Russian Foundation for Basic Research (RFBR)</institution></institution-wrap></funding-source><award-id>TRR160 19-52-12065</award-id><principal-award-recipient><name><surname>Kobecki</surname><given-names>Michal</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100001663</institution-id><institution>Volkswagen Foundation (VolkswagenStiftung)</institution></institution-wrap></funding-source><award-id>97758</award-id><principal-award-recipient><name><surname>Kobecki</surname><given-names>Michal</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100000266</institution-id><institution>RCUK | Engineering and Physical Sciences Research Council (EPSRC)</institution></institution-wrap></funding-source><award-id>EP/H003487/1</award-id><principal-award-recipient><name><surname>Kobecki</surname><given-names>Michal</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par3\">The transfer of thermal energy to mechanical, electrical, or magnetic excitations is of great interest and is widely considered for energy harvesting when waste heat is transferred to more usable types of energy<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. The rational utilization of heat is a critical task for nanoscale electronics, where operations are accompanied by extensive production of parasitic heat. Nanoscale devices for communication and computing operate with digital signals, which are generated by ultrafast current or optical pulses with high repetition rate. The heat generated in this case is also modulated at the same frequency and could be transferred to nonthermal types of excitation, which become the elements of the energy-harvesting process. This process would be most efficient in the case of resonance, when the modulated heat is transferred to a subsystem with the same intrinsic frequency. Resonant heat transfer is widely used in photoacoustics and was proposed by Bell in 1881<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. There the modulated optical signal is absorbed in a system with acoustic resonance at the frequency of modulation and as a result the modulated heat resonantly excites the acoustic wave. Modulation frequencies in photoacoustics do not exceed the MHz frequency range<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>, while thermomodulation with frequencies up to 400 GHz<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup> is used in picosecond ultrasonics for studying sub-THz coherent phonon dynamics.</p><p id=\"Par4\">Here we propose to convert high-frequency (GHz) modulated heat to magnons, which are collective spin excitations in magnetically ordered materials. The manipulation of coherent high-frequency magnons on the nanoscale is one of the most prospective concepts for information technologies<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>, also in the quantum regime<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. While the most common way to excite coherent magnons is nonthermal and based on microwave techniques<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>, thermal methods have proven successful in ferromagnetic metals. These methods are based on ultrafast modulation of the magnetic anisotropy induced by rapid lattice heating, for example, due to the absorption of optical pulses<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. In ferromagnetic metals, the intrinsic magnon frequency depends on the external magnetic field and may be varied between ~1 and ~100 GHz<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. This range covers the clocking frequency for most electronic devices and, thus, magnons are suitable to receive dissipated heat modulated at the resonant frequency.</p><p id=\"Par5\">In the present paper, we introduce the concept of transferring heat dissipated in a semiconductor from the relaxation of hot electrons excited by a 10 GHz laser to coherent magnons. In our experiments, the energy harvester is a metallic ferromagnetic film of 5 nm thickness grown on a semiconductor substrate. We demonstrate an efficient heat–magnon transfer by measuring a 12-fold increase of the fundamental magnon mode amplitude at the resonance conditions. The experiments and related theoretical analysis show that GHz modulation of the temperature on the scale of ~0.1 K is sufficient for the excitation of magnons with amplitude reliable for the operation of spintronic devices.</p></sec><sec id=\"Sec2\" sec-type=\"results\"><title>Results</title><sec id=\"Sec3\"><title>Samples and experimental technique</title><p id=\"Par6\">The ferromagnetic energy harvester is a 5 nm layer of Galfenol (Fe<sub>0.81</sub>Ga<sub>0.19</sub>) grown by magnetron sputtering on a (001) semi-insulating GaAs substrate and covered by a 2 nm Cr cap layer to prevent oxidation. The chosen composition of Fe and Ga is characterized by a high Curie temperature (<italic>T</italic><sub>c</sub> ≈ 900 K) and large saturation magnetization <italic>M</italic><sub>0</sub><sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. Experiments were carried out at ambient conditions at room temperature with an external magnetic field, <bold>B</bold>, applied in the layer plane at an angle −<italic>π</italic>/8 from the [100] crystallographic direction, which corresponds to the maximal sensitivity of the magnetization, <bold>M</bold>, to the temperature-induced changes of the magnetic anisotropy<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. In the studied layer, the lowest, fundamental mode of the quantized magnon spectrum is well separated from the higher-order modes due to the large exchange mode splitting<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. As a result, the magnon spectrum consists of a narrow single spectral line of Lorentzian shape with a width of ≈500 MHz. Examples of the magnon spectrum for several values of <italic>B</italic> measured by monitoring the magnetization precession excited by a single optical pulse are shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref> (for details see “Methods” section). The experimentally measured dependence of magnon frequency <italic>f</italic> on <italic>B</italic> is shown by the symbols in the right panel of Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>.<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Magnon resonances and temperature modulation.</title><p><bold>a</bold> Fast Fourier transform spectra showing the fundamental magnon mode for several values of magnetic field, <italic>B</italic> (left panel) and the field dependence of its frequency, <italic>f</italic> (right panel) for the 5-nm-thick Fe<sub>0.81</sub>Ga<sub>0.19</sub> energy-harvesting layer. Symbols show <italic>f</italic>(<italic>B</italic>) obtained by fast Fourier transformation of Kerr rotation signals measured in a single pulse pump–probe experiment. Solid lines are calculated dependences for Δ<italic>T</italic> = 0 K (upper curve) and Δ<italic>T</italic> = 200 K (lower curve). The dashed horizontal lines show the frequencies of the harmonics in the temperature modulation spectrum induced by the 10 GHz optical excitation. The vertical arrows indicate the expected resonances for the fundamental magnon mode at these harmonics. <bold>b</bold> Scheme of the experiment. <bold>c</bold> Calculated temporal evolution of the Fe<sub>0.81</sub>Ga<sub>0.19</sub> lattice temperature induced by the 10 GHz optical excitation of the Fe<sub>0.81</sub>Ga<sub>0.19</sub>/GaAs heterostructure at the pump excitation power <italic>W</italic> = 95 mW (upper panel) and its Fourier spectrum (lower panel).</p></caption><graphic xlink:href=\"41467_2020_17635_Fig1_HTML\" id=\"d30e525\"/></fig></p><p id=\"Par7\">Figure <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref> illustrates a schematic of the pump–probe experiment for magnon energy harvesting from high-frequency modulated heat. A femtosecond pump laser with repetition rate <italic>f</italic><sub>0</sub> = 10 GHz and average power up to <italic>W</italic> = 150 mW is used for modulation of the lattice temperature. A second laser with repetition rate of 1 GHz and average power of 18 mW is used to probe the response of the magnons to the high-frequency heat modulation by means of the transient polar Kerr rotation (KR) effect. The pump and probe beams are focused on the Fe<sub>81</sub>Ga<sub>19</sub> layer into overlapping spots of 17 and 14 μm diameter, respectively, using different sectors of the same reflective microscope objective (for details see the “Methods” section).</p></sec><sec id=\"Sec4\"><title>Temperature evolution</title><p id=\"Par8\">Figure <xref rid=\"Fig1\" ref-type=\"fig\">1c</xref> (upper panel) shows the calculated temporal evolution of the lattice temperature <italic>T</italic>(<italic>t</italic>) in the Fe<sub>0.81</sub>Ga<sub>0.19</sub> layer under 10 GHz pump optical excitation. The calculations were performed by solving the heat equations taking into account the thermal resistance at the Fe<sub>0.81</sub>Ga<sub>0.19</sub>/GaAs interface (see “Methods” section and Supplementary Notes <xref rid=\"MOESM1\" ref-type=\"media\">1</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>). The temperature modulation amplitude <inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\delta T\\sim W/(C_{\\mathrm{f}}r^2)$$\\end{document}</tex-math><mml:math id=\"M2\"><mml:mi>δ</mml:mi><mml:mi>T</mml:mi><mml:mo>~</mml:mo><mml:mi>W</mml:mi><mml:mo>/</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>C</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">f</mml:mi></mml:mrow></mml:msub><mml:msup><mml:mrow><mml:mi>r</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq1.gif\"/></alternatives></inline-formula>, where <italic>C</italic><sub>f</sub> and <italic>r</italic> are the heat capacity and radius of the excitation spot. It is seen that the lattice temperature oscillates with amplitude <italic>δT</italic> on a stationary background that exceeds the room temperature <italic>T</italic><sub>0</sub> by Δ<italic>T</italic>. Both Δ<italic>T</italic> and <italic>δT</italic> increase linearly with <italic>W</italic>: <italic>δT</italic> = <italic>pW</italic> and Δ<italic>T</italic> = <italic>PW</italic>, where <italic>p</italic> and <italic>P</italic> are constants, which for our structure are equal to 4.9 and 510 K W<sup>−1</sup>, respectively. The temperature modulation is not harmonic and its spectrum, which is shown in the lower panel of Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1c</xref>, consists of discreet harmonics at the frequencies <italic>f</italic><sub><italic>n</italic></sub> = <italic>nf</italic><sub>0</sub>, where <italic>n</italic> is an integer. The harmonic amplitude decreases with the increase of <italic>n</italic>.</p><p id=\"Par9\">The idea of the experiments illustrated in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref> is to exploit the periodic thermal modulation for exciting coherent magnons and to monitor the magnetization precession by transient KR measurements of the out-of-plane magnetization projection, <italic>δM</italic><sub><italic>z</italic></sub>, for various values of <italic>B</italic>. We expect a resonant increase of the precession amplitude at the resonances when <italic>B</italic> = <italic>B</italic><sub><italic>n</italic></sub>, which correspond to <italic>f</italic> = <italic>f</italic><sub><italic>n</italic></sub>. The expected values of <italic>B</italic><sub><italic>n</italic></sub> for the first three harmonics are shown in the right panel of Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref> by the vertical arrows. The idea is analogous to the excitation of a harmonic oscillator with tunable eigenfrequency <italic>f</italic> by a periodic force, which acts on the oscillator with repetition rate <italic>f</italic><sub>0</sub>. For magnetization precession, this force is generated by the modulated temperature <italic>T</italic>(<italic>t</italic>), and the magnon eigenfrequency <italic>f</italic> is controlled by the external magnetic field.</p></sec><sec id=\"Sec5\"><title>Resonant magnon signals and their spectra</title><p id=\"Par10\">Figure <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> shows the transient KR signals measured for <italic>W</italic> = 95 mW and various values of <italic>B</italic>. The signals measured in the vicinity of the first resonance (<italic>n</italic> = 1) have a harmonic shape. At these conditions, we fit the signals by a sine function <inline-formula id=\"IEq2\"><alternatives><tex-math id=\"M3\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\delta M_z\\left( t \\right) = A_{B}\\,{\\mathrm{sin}}\\left( {\\omega _1t + \\varphi _B} \\right)$$\\end{document}</tex-math><mml:math id=\"M4\"><mml:mi>δ</mml:mi><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi>z</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:msub><mml:mrow><mml:mi>A</mml:mi></mml:mrow><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:msub><mml:mspace width=\"0.25em\"/><mml:mi mathvariant=\"normal\">sin</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:msub><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mi>t</mml:mi><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mi>φ</mml:mi></mml:mrow><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfenced></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq2.gif\"/></alternatives></inline-formula>, where <italic>ω</italic><sub>1</sub> = 2<italic>πf</italic><sub>1</sub>, and <italic>A</italic><sub><italic>B</italic></sub> and <italic>φ</italic><sub><italic>B</italic></sub> are the magnetic field-dependent amplitude and phase of the harmonic oscillations. Times <italic>t</italic> = 0 and 100 ps correspond to excitation of the sample by the pump pulses. It is seen that the amplitude of the signal is maximal when <italic>B</italic> = <italic>B</italic><sub>1</sub> = 44 mT, which corresponds to the first resonance <italic>f</italic> = <italic>f</italic><sub>1</sub> (see Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>). In the vicinity of the resonance, the phase changes from <italic>φ</italic><sub><italic>B</italic></sub> = −<italic>π</italic>/2 for <italic>B</italic> < <italic>B</italic><sub>1</sub> to <italic>φ</italic><sub><italic>B</italic></sub> = +<italic>π</italic>/2 for <italic>B</italic> > <italic>B</italic><sub>1</sub>. At the resonance <italic>φ</italic><sub><italic>B</italic></sub> ≈ 0. Similar results are observed for the signals measured in the vicinity of the second resonance (<italic>n</italic> = 2). The amplitude of the oscillations at <italic>B</italic> = <italic>B</italic><sub>2</sub> = 250 mT are 1.4 times smaller than for the first resonance and the same conclusions as for the first resonance can be made concerning the field dependences of the amplitude and phase of the signal around <italic>B</italic><sub>2</sub>. Away from the resonances the signals are periodic but not harmonic. An example of a nonresonant signal measured at <italic>B</italic> = 190 mT is presented in the right panel of Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>. The amplitude of the signal is much smaller than in the case of resonance and the temporal shape is impossible to fit with a single sine function.<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Magnon signals in time domain.</title><p>Transient Kerr rotation signals measured in the vicinity of the first (left panel) and second (central panel) resonances and at the intermediate magnetic field of 190 mT, corresponding to off-resonance (right panel). The amplification of the precession amplitude observed at <italic>B</italic> = <italic>B</italic><sub>1</sub> = 44 mT and <italic>B</italic> = <italic>B</italic><sub>2</sub> = 250 mT is due to thermally induced resonant driving of the precession <bold>M</bold> by the oscillating effective field <bold>B</bold><sub>eff</sub>. The torque <bold>Q</bold> acting on the magnetization (see sketch at the top right) is maximal at the resonance conditions: <italic>f</italic> = <italic>nf</italic><sub>0</sub>.</p></caption><graphic xlink:href=\"41467_2020_17635_Fig2_HTML\" id=\"d30e977\"/></fig></p><p id=\"Par11\">To plot the dependence of the measured signal amplitude as a function of <italic>B</italic>, we present the root-mean-square (RMS) amplitude <inline-formula id=\"IEq3\"><alternatives><tex-math id=\"M5\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\tilde A_B$$\\end{document}</tex-math><mml:math id=\"M6\"><mml:msub><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi>A</mml:mi></mml:mrow><mml:mrow><mml:mo>~</mml:mo></mml:mrow></mml:mover></mml:mrow><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:msub></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq3.gif\"/></alternatives></inline-formula> of the measured KR signal. The symbols in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref> show the field dependences <inline-formula id=\"IEq4\"><alternatives><tex-math id=\"M7\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\tilde A_B(B)$$\\end{document}</tex-math><mml:math id=\"M8\"><mml:msub><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi>A</mml:mi></mml:mrow><mml:mrow><mml:mo>~</mml:mo></mml:mrow></mml:mover></mml:mrow><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>B</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq4.gif\"/></alternatives></inline-formula> for three pump excitation powers <italic>W</italic>. It is clearly seen that the dependences have peaks at the resonant values of <italic>B</italic><sub><italic>n</italic></sub> corresponding to <italic>n</italic> = 1, 2, and 3. Figure <xref rid=\"Fig3\" ref-type=\"fig\">3b</xref> shows zoomed fragments of the field dependences of the amplitude and phase, respectively, obtained for the first resonance at <italic>W</italic> = 95 mW. The 12-fold increase of <inline-formula id=\"IEq5\"><alternatives><tex-math id=\"M9\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\tilde A_B$$\\end{document}</tex-math><mml:math id=\"M10\"><mml:msub><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi>A</mml:mi></mml:mrow><mml:mrow><mml:mo>~</mml:mo></mml:mrow></mml:mover></mml:mrow><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:msub></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq5.gif\"/></alternatives></inline-formula> at the resonance condition is clearly seen by comparison with the out-of-resonance RMS amplitude shown by the horizontal dashed line in the upper panel of Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b</xref>. No peaks in the dependence of the precession amplitude on <italic>B</italic> are detected in the case of single pulse excitation, where <inline-formula id=\"IEq6\"><alternatives><tex-math id=\"M11\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\tilde A_B$$\\end{document}</tex-math><mml:math id=\"M12\"><mml:msub><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi>A</mml:mi></mml:mrow><mml:mrow><mml:mo>~</mml:mo></mml:mrow></mml:mover></mml:mrow><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:msub></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq6.gif\"/></alternatives></inline-formula> gradually decreases with the increase of <italic>B</italic>.<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Magnon spectra.</title><p><bold>a</bold> Measured magnetic field dependences of the root-mean-square (RMS) amplitude of the Kerr rotation signal, <inline-formula id=\"IEq7\"><alternatives><tex-math id=\"M13\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\tilde A}_{B}$$\\end{document}</tex-math><mml:math id=\"M14\"><mml:msub><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi>A</mml:mi></mml:mrow><mml:mrow><mml:mo>~</mml:mo></mml:mrow></mml:mover></mml:mrow><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:msub></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq7.gif\"/></alternatives></inline-formula>, for three values of pump excitation power, <italic>W</italic>. The vertical arrows indicate the magnetic fields <italic>B</italic><sub><italic>n</italic></sub> at which the resonance condition <italic>f</italic> = <italic>f</italic><sub><italic>n</italic></sub> is fulfilled; the horizontal bars indicate zero signal levels. <bold>b</bold> Measured (symbols) and calculated (solid lines) zoomed fragments of the field dependences of <inline-formula id=\"IEq8\"><alternatives><tex-math id=\"M15\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\tilde A}_{B}$$\\end{document}</tex-math><mml:math id=\"M16\"><mml:msub><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi>A</mml:mi></mml:mrow><mml:mrow><mml:mo>~</mml:mo></mml:mrow></mml:mover></mml:mrow><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:msub></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq8.gif\"/></alternatives></inline-formula> (upper panel) and the phase (lower panel) in the vicinity of the first resonance (<italic>n</italic> = 1) for <italic>W</italic> = 95 mW; the dashed horizontal line shows <inline-formula id=\"IEq9\"><alternatives><tex-math id=\"M17\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\tilde A}_{B}$$\\end{document}</tex-math><mml:math id=\"M18\"><mml:msub><mml:mrow><mml:mover accent=\"true\"><mml:mrow><mml:mi>A</mml:mi></mml:mrow><mml:mrow><mml:mo>~</mml:mo></mml:mrow></mml:mover></mml:mrow><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:msub></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq9.gif\"/></alternatives></inline-formula> at the intermediate off-resonance field. <bold>c</bold> Calculated field dependences of the RMS precession amplitude for the background temperatures Δ<italic>T</italic> obtained from the experimental dependences in <bold>a</bold>. <bold>d</bold> Power dependence of the relative precession amplitude (left axis) and the corresponding precession angle (right axis) at the first resonance (<italic>n</italic> = 1) when <italic>f</italic> = <italic>f</italic><sub>0</sub>.</p></caption><graphic xlink:href=\"41467_2020_17635_Fig3_HTML\" id=\"d30e1228\"/></fig></p><p id=\"Par12\">The values of the measured resonance fields <italic>B</italic><sub><italic>n</italic></sub> shift to slightly higher fields when <italic>W</italic> increases. We explain this shift by the heat-induced decrease of the magnon frequencies<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup> and respective increase of the magnetic field value required for achieving the resonance conditions. We use the values of this shift to obtain the background temperature Δ<italic>T</italic> of the Galfenol film by comparison with the known dependence of <italic>f</italic>(<italic>B</italic>) on temperature. Two dependences of <italic>f</italic>(<italic>B</italic>) calculated using the known dependence of the Galfenol magnetic parameters on temperature<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup> are demonstrated in the right panel of Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref> by the solid lines (for details see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>). The corresponding values of the background temperature obtained from the experimentally measured shifts of the resonances are Δ<italic>T</italic> = 28, 44, and 77 K for <italic>W</italic> = 35, 55, and 95 mW, respectively. They are 40% higher than the values calculated theoretically from the heat equations. We attribute this difference to the additional background heating by the probe beam that is not considered in the theoretical modeling.</p></sec></sec><sec id=\"Sec6\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par13\">In the analysis, we consider thermal modulation of the magnetic anisotropy as the main mechanism for magnon excitation in our experiment and do not take into account other mechanisms (e.g., thermal strain). This approach is based on previous experiments and theoretical analysis where various mechanisms of laser-pulse-induced excitation of magnons in Galfenol were considered<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. We also exclude the effect of thermal gradients inside a ferromagnetic film (we estimate 4% temperature difference across the film). This gradient in special cases can induce alternating current (a.c.) spin transfer<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup> modulated at the laser repetition rate, <italic>f</italic><sub>0</sub>. In our experiment, due to the uniform in-plane magnetization in a single thin ferromagnetic layer, this transfer does not produce a torque on the magnetization<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. The dominating role of the thermal modulation of the magnetocrystalline anisotropy is also confirmed by the dependence of the precession amplitude excited by a single laser pulse on the direction of the external magnetic field. This dependence demonstrates a four-fold in-plane symmetry with a slight uniaxial distortion, which corresponds to the magnetocrystalline anisotropy of the studied layer<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>.</p><p id=\"Par14\">We describe the effect of thermal modulation on the magnetization, <bold>M</bold>, by considering the magnetization precession motion in a time-dependent effective field <bold>B</bold><sub>eff</sub> as schematically shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref><sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. The magnetization precession is described by the Landau–Lifshitz–Gilbert (LLG) equation<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>:<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M19\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\frac{{{\\mathrm{d}}{\\mathbf{m}}}}{{{\\mathrm{d}}t}} = - \\gamma _0{\\mathbf{m}} \\, \\times \\, {\\mathbf{B}}_{{\\mathrm{eff}}}(t) + \\alpha _0{\\mathbf{m}} \\, \\times \\frac{{{\\mathrm{d}}{\\mathbf{m}}}}{{{\\mathrm{d}}t}},$$\\end{document}</tex-math><mml:math id=\"M20\"><mml:mfrac><mml:mrow><mml:mi mathvariant=\"normal\">d</mml:mi><mml:mi mathvariant=\"bold\">m</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">d</mml:mi><mml:mi>t</mml:mi></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>γ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mi mathvariant=\"bold\">m</mml:mi><mml:mspace width=\"0.25em\"/><mml:mo>×</mml:mo><mml:mspace width=\"0.25em\"/><mml:msub><mml:mrow><mml:mi mathvariant=\"bold\">B</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">eff</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>t</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mi>α</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mi mathvariant=\"bold\">m</mml:mi><mml:mspace width=\"0.25em\"/><mml:mo>×</mml:mo><mml:mfrac><mml:mrow><mml:mi mathvariant=\"normal\">d</mml:mi><mml:mi mathvariant=\"bold\">m</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">d</mml:mi><mml:mi>t</mml:mi></mml:mrow></mml:mfrac><mml:mo>,</mml:mo></mml:math><graphic xlink:href=\"41467_2020_17635_Article_Equ1.gif\" position=\"anchor\"/></alternatives></disp-formula>where <bold>m</bold> = <bold>M</bold>/<italic>M</italic><sub>0</sub> is the normalized magnetization and <italic>α</italic><sub>0</sub> is the Gilbert damping parameter. The effective magnetic field is determined as <bold>B</bold><sub>eff</sub> = −∇<sub><bold><italic>m</italic></bold></sub><italic>F</italic><sub><bold>M</bold></sub>(<bold><italic>m</italic></bold>, <italic>t</italic>), where <italic>F</italic><sub><bold>M</bold></sub> is the normalized free energy density<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>:<disp-formula id=\"Equ2\"><label>2</label><alternatives><tex-math id=\"M21\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$F_{\\mathbf{M}}\\left( m \\right) = - \\left( {{\\mathbf{m}} \\cdot {\\mathbf{B}}} \\right) + B_{\\mathrm{d}}m_z^2 + K_1\\left( {m_x^2m_y^2 + m_z^2m_y^2 + m_x^2m_z^2} \\right) - K_{\\mathrm{u}}\\left( {{\\mathbf{m}} \\cdot {\\mathbf{s}}} \\right)^2,$$\\end{document}</tex-math><mml:math id=\"M22\"><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"bold\">M</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>m</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mo>−</mml:mo><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi mathvariant=\"bold\">m</mml:mi><mml:mo>⋅</mml:mo><mml:mi mathvariant=\"bold\">B</mml:mi></mml:mrow></mml:mfenced><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mi>B</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">d</mml:mi></mml:mrow></mml:msub><mml:msubsup><mml:mrow><mml:mi>m</mml:mi></mml:mrow><mml:mrow><mml:mi>z</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:msubsup><mml:mrow><mml:mi>m</mml:mi></mml:mrow><mml:mrow><mml:mi>x</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:msubsup><mml:mrow><mml:mi>m</mml:mi></mml:mrow><mml:mrow><mml:mi>y</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:mo>+</mml:mo><mml:msubsup><mml:mrow><mml:mi>m</mml:mi></mml:mrow><mml:mrow><mml:mi>z</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:msubsup><mml:mrow><mml:mi>m</mml:mi></mml:mrow><mml:mrow><mml:mi>y</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:mo>+</mml:mo><mml:msubsup><mml:mrow><mml:mi>m</mml:mi></mml:mrow><mml:mrow><mml:mi>x</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:msubsup><mml:mrow><mml:mi>m</mml:mi></mml:mrow><mml:mrow><mml:mi>z</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup></mml:mrow></mml:mfenced><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">u</mml:mi></mml:mrow></mml:msub><mml:msup><mml:mrow><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi mathvariant=\"bold\">m</mml:mi><mml:mo>⋅</mml:mo><mml:mi mathvariant=\"bold\">s</mml:mi></mml:mrow></mml:mfenced></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msup><mml:mo>,</mml:mo></mml:math><graphic xlink:href=\"41467_2020_17635_Article_Equ2.gif\" position=\"anchor\"/></alternatives></disp-formula>where the first term is the Zeeman energy, the second term is the demagnetization energy <inline-formula id=\"IEq10\"><alternatives><tex-math id=\"M23\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\left( {B_{\\mathrm{d}} = \\frac{{\\mu _0M_0}}{2}} \\right)$$\\end{document}</tex-math><mml:math id=\"M24\"><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:msub><mml:mrow><mml:mi>B</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">d</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mi>μ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:mfrac></mml:mrow></mml:mfenced></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq10.gif\"/></alternatives></inline-formula>, and the following two terms describe the cubic and uniaxial magnetocrystalline anisotropy with respective coefficients <italic>K</italic><sub>1</sub> and <italic>K</italic><sub>u</sub>, with the unit vector <bold>s</bold> ∥ [110] along the uniaxial anisotropy axis. In the chosen coordinate system, the <italic>x</italic>, <italic>y</italic>, and <italic>z</italic> axes correspond to the main crystallographic directions [100], [010], and [001] (normal to the layer plane), respectively. At equilibrium, that is, without temperature modulation, the value of <bold>B</bold><sub>eff</sub> determines the fundamental magnon frequency, while its direction sets the equilibrium orientation of the magnetization <bold>m</bold>. The change of temperature alters <bold>B</bold><sub>eff</sub> through the temperature-dependent parameters <italic>M</italic><sub>0</sub>, <italic>K</italic><sub>1</sub>, and <italic>K</italic><sub>u</sub>. Within the studied temperature range their dependence on <italic>T</italic> is linear<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>: <inline-formula id=\"IEq11\"><alternatives><tex-math id=\"M25\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$X = X^{{\\mathrm{RT}}} + \\beta _X\\left( {T - T_0} \\right)$$\\end{document}</tex-math><mml:math id=\"M26\"><mml:mi>X</mml:mi><mml:mo>=</mml:mo><mml:msup><mml:mrow><mml:mi>X</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">RT</mml:mi></mml:mrow></mml:msup><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mi>β</mml:mi></mml:mrow><mml:mrow><mml:mi>X</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>T</mml:mi><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>T</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:mfenced></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq11.gif\"/></alternatives></inline-formula>, where <italic>X</italic> = <italic>M</italic><sub>0</sub>, <italic>K</italic><sub>1</sub>, or <italic>K</italic><sub>u</sub>, the index RT indicates the room-temperature values and <italic>β</italic><sub><italic>X</italic></sub> is the corresponding thermal coefficient. The room-temperature values <inline-formula id=\"IEq12\"><alternatives><tex-math id=\"M27\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$M_0^{{\\mathrm{RT}}}$$\\end{document}</tex-math><mml:math id=\"M28\"><mml:msubsup><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">RT</mml:mi></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq12.gif\"/></alternatives></inline-formula> = 1.96 T, <inline-formula id=\"IEq13\"><alternatives><tex-math id=\"M29\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$K_1^{{\\mathrm{RT}}}$$\\end{document}</tex-math><mml:math id=\"M30\"><mml:msubsup><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">RT</mml:mi></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq13.gif\"/></alternatives></inline-formula> = 20 mT, and <inline-formula id=\"IEq14\"><alternatives><tex-math id=\"M31\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$K_{\\mathrm{u}}^{{\\mathrm{RT}}}$$\\end{document}</tex-math><mml:math id=\"M32\"><mml:msubsup><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">u</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">RT</mml:mi></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq14.gif\"/></alternatives></inline-formula> = 9 mT for our sample are found from fitting the experimental dependence <italic>f</italic>(<italic>B</italic>) (shown in the right panel of Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. The thermal coefficients, <inline-formula id=\"IEq15\"><alternatives><tex-math id=\"M33\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\beta _{M_0} = - 0.97$$\\end{document}</tex-math><mml:math id=\"M34\"><mml:msub><mml:mrow><mml:mi>β</mml:mi></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mo>−</mml:mo><mml:mn>0.97</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq15.gif\"/></alternatives></inline-formula> mT K<sup>−1</sup>, <inline-formula id=\"IEq16\"><alternatives><tex-math id=\"M35\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\beta _{K_1} = - 0.046$$\\end{document}</tex-math><mml:math id=\"M36\"><mml:msub><mml:mrow><mml:mi>β</mml:mi></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mo>−</mml:mo><mml:mn>0.046</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq16.gif\"/></alternatives></inline-formula> mT K<sup>−1</sup>, and <inline-formula id=\"IEq17\"><alternatives><tex-math id=\"M37\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\beta _{K_{\\mathrm{u}}} = - 0.025$$\\end{document}</tex-math><mml:math id=\"M38\"><mml:msub><mml:mrow><mml:mi>β</mml:mi></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">u</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mo>−</mml:mo><mml:mn>0.025</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq17.gif\"/></alternatives></inline-formula> mT K<sup>−1</sup> for Galfenol are taken from previous studies<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. The Gilbert damping coefficient <italic>α</italic><sub>0</sub> = 0.006 is obtained from the precession kinetics excited by a single laser pulse.</p><p id=\"Par15\">The modulation amplitude <italic>δT</italic> << Δ<italic>T</italic> and for solving Eq. (<xref rid=\"Equ1\" ref-type=\"\">1</xref>), we assume that the magnitude of <bold>B</bold><sub>eff</sub> is constant and the temperature modulation affects only its direction. We also consider only the ground magnon mode with zero wave vector, that is, we assume a uniform precession, due to the large diameter (17 μm) of the excitation spot, which limits the in-plane wave vector of magnons to <italic>q</italic><sub>||</sub> ≤ 4700 cm<sup>−1</sup>. In the 5-nm-thick Galfenol layer, the frequency range for such <italic>q</italic><sub>||</sub> does not exceed 270 MHz<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>, which is clearly below the 500 MHz spectral width of the fundamental magnon mode. For details of the magnon dispersion and contribution of magnons with finite wave vectors in a 100-nm-thick layer, see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>.</p><p id=\"Par16\">With these assumptions, the LLG equation can be reduced to a system of equations (see the “Methods” section for details) for the magnetization components, where the thermal modulation of the anisotropy coefficients alters the direction of <bold>B</bold><sub>eff</sub> and generates a tangential driving torque, <bold>Q</bold>, acting on the magnetization<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup> as schematically shown in the inset of Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>. The vector <bold>B</bold><sub>eff</sub> follows the temporal temperature evolution <italic>T</italic>(<italic>t</italic>) shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1c</xref>, which can be written as sum of background heating and a Fourier series expansion for the small periodic temperature modulation:<disp-formula id=\"Equ3\"><label>3</label><alternatives><tex-math id=\"M39\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$T\\left( t \\right) = T_0 + {\\Delta}T + \\delta T \\sum\\limits_{n = 1}^\\infty u_n\\,{{\\sin}} \\left( {\\omega _nt + \\psi _n} \\right).$$\\end{document}</tex-math><mml:math id=\"M40\"><mml:mi>T</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:msub><mml:mrow><mml:mi>T</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:mi>T</mml:mi><mml:mo>+</mml:mo><mml:mi>δ</mml:mi><mml:mi>T</mml:mi><mml:munderover accent=\"false\" accentunder=\"false\"><mml:mrow><mml:mo mathsize=\"big\"> ∑</mml:mo></mml:mrow><mml:mrow><mml:mi>n</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>∞</mml:mi></mml:mrow></mml:munderover><mml:msub><mml:mrow><mml:mi>u</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mspace width=\"0.25em\"/><mml:mi>sin</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:msub><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mi>t</mml:mi><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mi>ψ</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfenced><mml:mo>.</mml:mo></mml:math><graphic xlink:href=\"41467_2020_17635_Article_Equ3.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p id=\"Par17\">Here the amplitude and phase of the <italic>n</italic>th harmonic are <inline-formula id=\"IEq18\"><alternatives><tex-math id=\"M41\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$u_n = \\frac{2}{\\pi }\\frac{{\\omega _1\\tau _0}}{{\\sqrt {1 + \\omega _n^2\\tau _0^2} }}$$\\end{document}</tex-math><mml:math id=\"M42\"><mml:msub><mml:mrow><mml:mi>u</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mi>π</mml:mi></mml:mrow></mml:mfrac><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>τ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:msqrt><mml:mrow><mml:mn>1</mml:mn><mml:mo>+</mml:mo><mml:msubsup><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:msubsup><mml:mrow><mml:mi>τ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup></mml:mrow></mml:msqrt></mml:mrow></mml:mfrac></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq18.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq19\"><alternatives><tex-math id=\"M43\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\psi _n = {\\mathrm{arctan}}( {\\frac{1}{{\\omega _n\\tau _0}}} )$$\\end{document}</tex-math><mml:math id=\"M44\"><mml:msub><mml:mrow><mml:mi>ψ</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mi mathvariant=\"normal\">arctan</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>τ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:mfrac></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq19.gif\"/></alternatives></inline-formula>, respectively, <inline-formula id=\"IEq20\"><alternatives><tex-math id=\"M45\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\omega _n = 2\\pi f_n$$\\end{document}</tex-math><mml:math id=\"M46\"><mml:msub><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>2</mml:mn><mml:mi>π</mml:mi><mml:msub><mml:mrow><mml:mi>f</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq20.gif\"/></alternatives></inline-formula>, <italic>τ</italic><sub>0</sub> is the characteristic cooling time of the Galfenol layer, which is determined by the thermal boundary resistance, <italic>R</italic>, at the interface with GaAs<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. The solution of the linearized Eq. (<xref rid=\"Equ1\" ref-type=\"\">1</xref>) for the <italic>δm</italic><sub><italic>z</italic></sub> component expanded as a Fourier series has the form:<disp-formula id=\"Equ4\"><label>4</label><alternatives><tex-math id=\"M47\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\delta m_z\\left( t \\right) = \\sum\\limits_{n = 1}^\\infty A_n\\left( B \\right){\\mathrm{sin}}\\left( {\\omega _nt + \\varphi _n\\left( B \\right)} \\right),$$\\end{document}</tex-math><mml:math id=\"M48\"><mml:mi>δ</mml:mi><mml:msub><mml:mrow><mml:mi>m</mml:mi></mml:mrow><mml:mrow><mml:mi>z</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:munderover accent=\"false\" accentunder=\"false\"><mml:mrow><mml:mo mathsize=\"big\"> ∑</mml:mo></mml:mrow><mml:mrow><mml:mi>n</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>∞</mml:mi></mml:mrow></mml:munderover><mml:msub><mml:mrow><mml:mi>A</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:mfenced><mml:mi mathvariant=\"normal\">sin</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:msub><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mi>t</mml:mi><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mi>φ</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:mfenced></mml:mrow></mml:mfenced><mml:mo>,</mml:mo></mml:math><graphic xlink:href=\"41467_2020_17635_Article_Equ4.gif\" position=\"anchor\"/></alternatives></disp-formula>where <italic>A</italic><sub><italic>n</italic></sub>(<italic>B</italic>) and <italic>φ</italic><sub><italic>n</italic></sub>(<italic>B</italic>) are the time-independent amplitude and phase for the <italic>n</italic>th harmonic. Like in the case of a simple harmonic oscillator, at the resonant conditions <italic>f</italic>(<italic>B</italic>) = <italic>f</italic><sub><italic>n</italic></sub> the precession amplitude reaches maximum with zero phase-shift relative to the driving force <inline-formula id=\"IEq21\"><alternatives><tex-math id=\"M49\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\varphi _n\\left( B \\right) \\approx 0$$\\end{document}</tex-math><mml:math id=\"M50\"><mml:msub><mml:mrow><mml:mi>φ</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:mfenced><mml:mo>≈</mml:mo><mml:mn>0</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq21.gif\"/></alternatives></inline-formula>. The analytical expressions for <italic>A</italic><sub><italic>n</italic></sub>(<italic>B</italic>) and <italic>φ</italic><sub><italic>n</italic></sub>(<italic>B</italic>) are presented in the “Methods” section.</p><p id=\"Par18\">Figure <xref rid=\"Fig3\" ref-type=\"fig\">3c</xref> shows the field dependences of the RMS amplitude of the calculated <italic>δm</italic><sub><italic>z</italic></sub>(<italic>t</italic>) for three values of the background temperature Δ<italic>T</italic>, which correspond to the <italic>W</italic> indicated near the experimentally measured data in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref>. The agreement of the measured and calculated spectral shapes is explicitly demonstrated in the zoomed dependences of the RMS amplitude and phase in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b</xref>, where the calculated dependences (solid lines) for <italic>n</italic> = 1 are presented together with the experimental values. Good agreement is observed also for higher <italic>n</italic> and <italic>W</italic>.</p><p id=\"Par19\">The measured power dependence of the resonant magnon amplitude for <italic>n</italic> = 1 in the studied sample is shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3d</xref>. The precession amplitudes are obtained from the absolute values of the KR angle in the transient KR signals<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. The field-independent small amplitude background, which is due to the response of the electron system and not related to the magnetization<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>, has been subtracted from the experimental values. At <italic>W</italic> = 100 mW the precession amplitude reaches 0.1% of the saturation magnetization <italic>M</italic><sub>0</sub>. To get this value, the parameters of the magnetic anisotropy have to be modulated by the oscillating temperature with the amplitude <italic>δT</italic> = 0.54 K. This is in perfect agreement with the values obtained by solving the thermal equations, which for the same power give <italic>δT</italic> = 0.49 K. The achieved precession amplitude, which corresponds to the precession angle Θ ≈ 0.25° (with ellipticity of 0.2) and the generated a.c. induction Δ<italic>M</italic><sub><italic>z</italic></sub> ~ 1 mT, is large enough for prospective spintronics applications: detection of spin currents by spin pumping<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>; manipulating single spin states of NV centers in nanodiamonds by microwave magnetic fields<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>; excitation of propagating spin waves in magnonic devices<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>.</p><p id=\"Par20\">For the practical use of resonant energy harvesting in a device excited optically or electrically, it is important to achieve high efficiency of the transformation of the modulated part of the thermal energy into the oscillations of magnetization. This efficiency is defined by the ratio of the magnetization angle Θ of precession and the power <italic>W</italic> injected into a device. In our experiment, we demonstrate Θ/<italic>W</italic> = 2.5 °W<sup>−1</sup>, which is only one order of magnitude less than the efficiency of conventional mesoscopic microwave devices<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup> and of the same order as devices utilizing surface acoustic waves for spin pumping<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. At resonance, the magnetization precession remains harmonic up to the maximum used power, and, thus, nonlinear (anharmonic) effects do not affect the transformation efficiency. However, the shift of the resonance frequency with the increasing Δ<italic>T</italic> at higher <italic>W</italic> affects the linear power dependence of the precession amplitude at fixed magnetic field. This temperature-induced “nonlinearity” is known from the conventional microwave experiments on ferromagnetic resonance<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. The effect of this nonlinearity is very weak in the studied structure. For instance, for the second resonance (<italic>n</italic> = 2) the 20% change of the background temperature without changing thermal modulation amplitude results in a ~3% decrease of the precession amplitude. Thus, stability of the background temperature is not critical for the suggested concept.</p><p id=\"Par21\">The dependences of <italic>δT</italic> on the parameters of the used structure and materials define the efficiency of the heat transfer to the magnons. For optical excitation, shorter penetration depths and smaller heat capacities of the ferromagnetic layer increase <italic>δT</italic> and correspondingly Θ<italic>/W</italic>. It is convenient to define the dimensionless resonant harvesting efficiency, <italic>ζ</italic> = <italic>δT</italic>/Δ<italic>T</italic>, which governs the ratio of the useful temperature modulation <italic>δT</italic> relative to the parasitic heating, Δ<italic>T</italic>. Obviously, it is favorable to have <italic>ζ</italic> as large as possible. For instance, faster cooling to the bulk of the substrate leads to an increase of <italic>δT</italic> with a simultaneous decrease of Δ<italic>T</italic> and correspondingly to an increase of <italic>ζ</italic>. In our particular experiment, where both the Fe<sub>0.81</sub>Ga<sub>0.19</sub> nanolayer and the GaAs substrate are excited optically, we get <italic>ζ</italic> ~ 10<sup>−2</sup>. The value of <italic>ζ</italic> increases with the decrease of the thickness of the ferromagnetic layer and the increase of the substrate thermoconductivity (see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). For instance, for the same ferromagnetic layer on a silicon substrate <italic>ζ</italic> increases by a factor of 1.5. An efficient way to increase <italic>ζ</italic> and the efficiency of the heat–magnon transfer would be to use a multilayer structure where Δ<italic>T</italic> is governed by the rapid escape of heat to the substrate while the high-frequency thermal waves, which define <italic>δT</italic>, are localized inside or in the vicinity of the harvester layer. To demonstrate that the concept is applicable for electrical nanodevices, we have considered the case of a thin conducting layer (e.g., graphene) where Joule heat can be generated as a result of passing GHz current pulses and transferred to the ferromagnetic harvester through a thin spacer layer. For high efficiency, the spacer layer should have high thermal conductivity, while the ferromagnetic layer should have low specific heat capacity. In the example presented in the Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>, we get <italic>ζ</italic> = 4 × 10<sup>−3</sup>.</p><p id=\"Par22\">In our experiments, we excite resonances up to 30 GHz. To reach higher, sub-THz frequencies, the corresponding bandwidth should be available for both temperature and magnon modulation. For temperature modulation, we refer to the picosecond acoustic experiment<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup> where the temperature in a 12-nm Al film was modulated at a frequency up to 400 GHz. The thermal properties of ferromagnetic metals do not differ much from normal metals and thus similar modulation in thin films is achievable. For optical excitation of metals, it is essential that optically excited hot electrons pass their energy to the lattice in a time <1 ps. Theoretically, for the parameters fixed as above, the amplitude of the temperature oscillations is proportional to the laser power and inversely proportional to the frequency up to sub-THz frequencies. For the magnons in thin (Fe,Ga) films, narrow-band precession with frequencies up to 100 GHz was demonstrated at strong magnetic fields<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Moreover, metallic ferrimagnetic materials (e.g., Mn-Te compounds) possess narrow magnon resonances at frequencies up to hundreds of GHz at low magnetic fields<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. At the lower frequency end, there is no limit for temperature modulation, while for magnons the lower frequency varies from hundreds of MHz (e.g., for garnets) up to several GHz in metallic ferromagnets. The available choice of a proper magnetic material is not limited to the Galfenol used in our study. This ferromagnet is actively studied nowadays<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup> and presents a good example for realization of the demonstrated concept due to its pronounced magnetocrystalline anisotropy and narrow magnon resonances (long lifetime of precession). However, other ferro- and ferrimagnetic materials possessing similar properties will also work as resonant harvesters for transferring parasitic heat modulated at high frequencies to magnons.</p><p id=\"Par23\">To conclude, we have demonstrated how the heat generated during 10 GHz pulsed optical excitation is used for the excitation of resonant magnons. The amplitude of the magnetization precession at the fundamental magnon frequency increases enormously when the repetition rate of the optical pulses is equal to the magnon frequency. The resonances also occur at the higher harmonic frequencies, that is, 20 and 30 GHz. An amplitude of the temperature modulation of order ~0.1 K is sufficient to generate a.c. magnetization with an amplitude of 1 mT<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>, which is sufficient to be exploited in various applications including information and quantum technologies. The concept of resonant heat transfer can be considered as a prospective method to generate magnons in ferromagnetic layers deposited on processors and other microchips operating at GHz and sub-THz clock frequencies.</p></sec><sec id=\"Sec7\"><title>Methods</title><sec id=\"Sec8\"><title>Pump–probe measurements with 10 GHz repetition rate</title><p id=\"Par24\">We used two synchronized Titanium sapphire lasers (Taccor x10 and Gigajet 20c from Laser Quantum) generating 50 fs pulses with repetition rates of 10 GHz (pump) and 1 GHz (probe) at center wavelengths of 810 and 780 nm, respectively. The beams were focused onto the sample using a reflective microscope objective with a magnification factor of 15 comprising 4 sectors through which light could enter and exit the objective<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. The incidence angles of the laser beams are 17°. The spot diameters for the pump and probe beams were set to 17 and 14 μm, respectively. The diameters are defined at the 1/<italic>e</italic><sup>2</sup> level of the Gaussian spatial energy distributions. The coherent response of the magnetization was measured by monitoring the polar KR of the reflected probe pulses using a differential scheme based on a Wollaston prism and a balanced optical receiver with 10 MHz bandwidth. Temporal resolution was achieved by means of an asynchronous optical sampling (ASOPS) technique<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>, where we used an offset frequency of 20 kHz between the 10 GHz laser and the tenth harmonics of the 1 GHz laser in order to resolve the transient signals in 100 ps time window between pump pulses with the time resolution of 50 fs. The sample was mounted between the poles of a dipole electromagnet.</p></sec><sec id=\"Sec9\"><title>Measurements of the magnon spectrum with single pulse excitation</title><p id=\"Par25\">The magnon spectrum of the studied sample was obtained by a conventional magneto-optical pump–probe scheme based on two mode-locked Erbium-doped ring fiber lasers (FemtoFiber Ultra 780 and FemtoFiber Ultra 1050 from Toptica). The lasers generate pulses of 150 fs duration with a repetition rate of 80 MHz at wavelengths of 1046 nm (pump pulses) and 780 nm (probe pulses). The magnetization precession was excited by the pump pulses with an energy of 3 nJ per pulse focused on the Galfenol film surface to a spot of 17 μm diameter. The linearly polarized probe pulses with energy of 30 pJ per pulse hitting at normal incidence to the sample surface were focused to a spot of 1 μm diameter in the center of the pump spot. The coherent response was measured by transient polar KR in the same way as for the 10 GHz measurements. Temporal resolution was achieved also by the ASOPS technique with a frequency offset of 800 Hz, which in combination with the 80 MHz repetition rate allowed measurement of the time-resolved signals in the time window of 12.5 ns with 150 fs time resolution. The magnon spectra and the magnetic field dependence of the central frequency were obtained by fast Fourier transforming the corresponding transient KR signals.</p></sec><sec id=\"Sec10\"><title>Modeling of temperature evolution</title><p id=\"Par26\">In the case of periodic laser-pulse excitation, the temperature oscillates around the background temperature <italic>T</italic><sub>0</sub> + Δ<italic>T</italic> with amplitude <italic>δT</italic>. The amplitude <italic>δT</italic> is estimated by solving the standard heat equations for the lattice temperatures of Fe<sub>0.81</sub>Ga<sub>0.19</sub> and GaAs for periodic excitation by laser pulses using Comsol Multiphysics®<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup> (see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>). We estimate the background heating Δ<italic>T</italic> from the solution of the 3D stationary heat equation for continuous wave laser excitation (see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>). We use the following parameters for GaAs<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>: refractive index 3.7 + i0.09, which results in an absorption length of 740 nm; heat capacity 1.76 × 10<sup>6</sup> J m<sup>−3</sup> K<sup>−1</sup> and thermal conductivity 55 W m<sup>−1</sup> K<sup>−1 </sup><sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. We assume that the optical and thermal parameters of Fe<sub>0.81</sub>Ga<sub>0.19</sub> are close to the parameters of Fe: refractive index 2.9 + i3.4<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>; heat capacity 3.8 × 10<sup>6</sup> J m<sup>−3</sup> K<sup>−1</sup> and thermal conductivity 80 W m<sup>−1</sup> K<sup>−1 </sup><sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. As a result the reflection coefficient of the (Fe,Ga)/GaAs heterostructure is 0.4 and the absorption coefficients for the Fe<sub>0.81</sub>Ga<sub>0.19</sub> layer and GaAs substrate are 0.1 and 0.49, respectively. The calculated values of Δ<italic>T</italic> and <italic>δT</italic> at the excitation power <italic>W</italic> = 95 mW are 48 and 0.47 K, respectively.</p><p id=\"Par27\">In our calculations, we consider the thermal boundary resistance, <italic>R</italic>, which determines the flux through the Fe<sub>0.81</sub>Ga<sub>0.19</sub>/GaAs interface<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. In order to estimate <italic>R</italic>, we measured the reflectivity signal from the studied structure in the case of low-frequency (80 MHz) laser excitation. Using the experimentally observed decay time of the transient reflectivity <italic>τ</italic><sub>0</sub> = 200 ps, we found the thermal boundary resistance <italic>R</italic> = 10<sup>−8</sup> m<sup>2</sup> K W<sup>−1</sup>.</p></sec><sec id=\"Sec11\"><title>Modeling the periodic laser-pulse-induced magnetization precession</title><p id=\"Par28\">To calculate the magnetization precession amplitudes <italic>A</italic><sub><italic>n</italic></sub>(<italic>B</italic>) and phases <italic>φ</italic><sub><italic>n</italic></sub>(<italic>B</italic>), we apply the approach developed in ref. <sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup> where the detailed theory of magnetization precession due to ultrafast heating of a Galfenol film was considered. We analyze the precession by rewriting the LLG equation in a spherical coordinate system with in-plane azimuthal angle, <italic>ϕ</italic>, and polar angle, <italic>θ</italic> (<italic>θ</italic> = 0 corresponds to the layer normal, <italic>z</italic>), which describes the direction of the normalized magnetization, <bold>m</bold>. Assuming that the changes of <italic>δϕ</italic> and <italic>δθ</italic> from the equilibrium angles <italic>ϕ</italic><sub>0</sub> and <italic>θ</italic><sub>0</sub> are small and induced by the modulation of the cubic and uniaxial anisotropy constants, the LLG equation in linear approximation has the form:<disp-formula id=\"Equ5\"><label>5</label><alternatives><tex-math id=\"M51\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\begin{array}{l}\\frac{{\\partial \\delta \\phi }}{{\\partial t}} = \\gamma _0F_{\\theta \\theta }\\delta \\theta + \\alpha _0\\frac{{\\partial \\delta \\theta }}{{\\partial t}},\\\\ \\frac{{\\partial \\delta \\theta }}{{\\partial t}} = \\gamma _0F_{\\phi \\phi }\\delta \\phi - \\gamma _0F_{\\phi K_1}\\delta K_1(t) - \\gamma _0F_{\\phi K_{\\mathrm{u}}}\\delta K_{\\mathrm{u}}\\left( t \\right) - \\alpha _0\\frac{{\\partial \\delta \\phi }}{{\\partial t}},\\end{array}$$\\end{document}</tex-math><mml:math id=\"M52\"><mml:mtable><mml:mtr><mml:mtd columnalign=\"left\"><mml:mfrac><mml:mrow><mml:mi>∂</mml:mi><mml:mi>δ</mml:mi><mml:mi>ϕ</mml:mi></mml:mrow><mml:mrow><mml:mi>∂</mml:mi><mml:mi>t</mml:mi></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:msub><mml:mrow><mml:mi>γ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi>θ</mml:mi><mml:mi>θ</mml:mi></mml:mrow></mml:msub><mml:mi>δ</mml:mi><mml:mi>θ</mml:mi><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mi>α</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mfrac><mml:mrow><mml:mi>∂</mml:mi><mml:mi>δ</mml:mi><mml:mi>θ</mml:mi></mml:mrow><mml:mrow><mml:mi>∂</mml:mi><mml:mi>t</mml:mi></mml:mrow></mml:mfrac><mml:mo>,</mml:mo></mml:mtd></mml:mtr><mml:mtr><mml:mtd columnalign=\"left\"><mml:mfrac><mml:mrow><mml:mi>∂</mml:mi><mml:mi>δ</mml:mi><mml:mi>θ</mml:mi></mml:mrow><mml:mrow><mml:mi>∂</mml:mi><mml:mi>t</mml:mi></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:msub><mml:mrow><mml:mi>γ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi>ϕ</mml:mi><mml:mi>ϕ</mml:mi></mml:mrow></mml:msub><mml:mi>δ</mml:mi><mml:mi>ϕ</mml:mi><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>γ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi>ϕ</mml:mi><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mi>δ</mml:mi><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>t</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>γ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi>ϕ</mml:mi><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">u</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mi>δ</mml:mi><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">u</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>t</mml:mi></mml:mrow></mml:mfenced><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>α</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mfrac><mml:mrow><mml:mi>∂</mml:mi><mml:mi>δ</mml:mi><mml:mi>ϕ</mml:mi></mml:mrow><mml:mrow><mml:mi>∂</mml:mi><mml:mi>t</mml:mi></mml:mrow></mml:mfrac><mml:mo>,</mml:mo></mml:mtd></mml:mtr></mml:mtable></mml:math><graphic xlink:href=\"41467_2020_17635_Article_Equ5.gif\" position=\"anchor\"/></alternatives></disp-formula>where the <inline-formula id=\"IEq22\"><alternatives><tex-math id=\"M53\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$F_{i,j} = \\frac{{\\partial ^2}}{{\\partial i\\partial j}}F_{\\mathbf{M}}(i,j = \\theta ,\\phi ,K_1,K_{\\mathrm{u}})$$\\end{document}</tex-math><mml:math id=\"M54\"><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi><mml:mo>,</mml:mo><mml:mi>j</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msup><mml:mrow><mml:mi>∂</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:mrow><mml:mrow><mml:mi>∂</mml:mi><mml:mi>i</mml:mi><mml:mi>∂</mml:mi><mml:mi>j</mml:mi></mml:mrow></mml:mfrac><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"bold\">M</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>i</mml:mi><mml:mo>,</mml:mo><mml:mi>j</mml:mi><mml:mo>=</mml:mo><mml:mi>θ</mml:mi><mml:mo>,</mml:mo><mml:mi>ϕ</mml:mi><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">u</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq22.gif\"/></alternatives></inline-formula> are calculated for the equilibrium (in-plane) orientation of <bold>m</bold>, <italic>ϕ</italic><sub>0</sub>(<bold>B</bold>, <italic>T</italic>), <italic>θ</italic><sub>0</sub> = <italic>π</italic>/2, and <italic>α</italic><sub>0</sub> is the Gilbert damping parameter. At the in-plane equilibrium orientation of <bold>m</bold>, <inline-formula id=\"IEq23\"><alternatives><tex-math id=\"M55\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$F_{\\theta \\phi } = F_{\\theta K_1} = F_{\\theta K_{\\mathrm{u}}} = 0$$\\end{document}</tex-math><mml:math id=\"M56\"><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi>θ</mml:mi><mml:mi>ϕ</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi>θ</mml:mi><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi>θ</mml:mi><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">u</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>0</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq23.gif\"/></alternatives></inline-formula> and there is no torque due to the thermal change of <italic>M</italic><sub>0</sub>.</p><p id=\"Par29\">In Eq. (<xref rid=\"Equ4\" ref-type=\"\">4</xref>) the precession amplitude and phase are determined as <inline-formula id=\"IEq24\"><alternatives><tex-math id=\"M57\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$A_n(B) = \\sqrt {a_n^2\\left( B \\right) + b_n^2(B)}$$\\end{document}</tex-math><mml:math id=\"M58\"><mml:msub><mml:mrow><mml:mi>A</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>B</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:msqrt><mml:mrow><mml:msubsup><mml:mrow><mml:mi>a</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:mfenced><mml:mo>+</mml:mo><mml:msubsup><mml:mrow><mml:mi>b</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>B</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:msqrt></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq24.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq25\"><alternatives><tex-math id=\"M59\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\varphi _n\\left( B \\right) = {\\mathrm{arctan}}\\left( {a_n(B)/b_n(B)} \\right)$$\\end{document}</tex-math><mml:math id=\"M60\"><mml:msub><mml:mrow><mml:mi>φ</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mi mathvariant=\"normal\">arctan</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:msub><mml:mrow><mml:mi>a</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>B</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>/</mml:mo><mml:msub><mml:mrow><mml:mi>b</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>B</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:mfenced></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq25.gif\"/></alternatives></inline-formula>, where <inline-formula id=\"IEq26\"><alternatives><tex-math id=\"M61\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$a_n\\left( B \\right) = \\left( { - \\omega _n\\xi Q_n^b + 2\\tau _M^{ - 1}\\omega _n^2Q_n^a} \\right)/G$$\\end{document}</tex-math><mml:math id=\"M62\"><mml:msub><mml:mrow><mml:mi>a</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mi>ξ</mml:mi><mml:msubsup><mml:mrow><mml:mi>Q</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mi>b</mml:mi></mml:mrow></mml:msubsup><mml:mo>+</mml:mo><mml:mn>2</mml:mn><mml:msubsup><mml:mrow><mml:mi>τ</mml:mi></mml:mrow><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:msubsup><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:msubsup><mml:mrow><mml:mi>Q</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mi>a</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:mfenced><mml:mo>/</mml:mo><mml:mi>G</mml:mi></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq26.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq27\"><alternatives><tex-math id=\"M63\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$b_n\\left( B \\right) = \\left( {\\omega _n\\xi Q_n^a + 2\\tau _M^{ - 1}\\omega _n^2Q_n^b} \\right)/G$$\\end{document}</tex-math><mml:math id=\"M64\"><mml:msub><mml:mrow><mml:mi>b</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>B</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:msub><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:mi>ξ</mml:mi><mml:msubsup><mml:mrow><mml:mi>Q</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mi>a</mml:mi></mml:mrow></mml:msubsup><mml:mo>+</mml:mo><mml:mn>2</mml:mn><mml:msubsup><mml:mrow><mml:mi>τ</mml:mi></mml:mrow><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:msubsup><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:msubsup><mml:mrow><mml:mi>Q</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mi>b</mml:mi></mml:mrow></mml:msubsup></mml:mrow></mml:mfenced><mml:mo>/</mml:mo><mml:mi>G</mml:mi></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq27.gif\"/></alternatives></inline-formula>, with <inline-formula id=\"IEq28\"><alternatives><tex-math id=\"M65\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$G = \\xi ^2 + 4\\omega _n^2\\tau _M^{ - 2}$$\\end{document}</tex-math><mml:math id=\"M66\"><mml:mi>G</mml:mi><mml:mo>=</mml:mo><mml:msup><mml:mrow><mml:mi>ξ</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msup><mml:mo>+</mml:mo><mml:mn>4</mml:mn><mml:msubsup><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:msubsup><mml:mrow><mml:mi>τ</mml:mi></mml:mrow><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq28.gif\"/></alternatives></inline-formula>, and <inline-formula id=\"IEq29\"><alternatives><tex-math id=\"M67\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\xi = \\omega _n^2 - \\omega ^2$$\\end{document}</tex-math><mml:math id=\"M68\"><mml:mi>ξ</mml:mi><mml:mo>=</mml:mo><mml:msubsup><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:mo>−</mml:mo><mml:msup><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq29.gif\"/></alternatives></inline-formula>. Here <inline-formula id=\"IEq30\"><alternatives><tex-math id=\"M69\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\omega=2{\\pi}f(B)$$\\end{document}</tex-math><mml:math id=\"M70\"><mml:mi>ω</mml:mi><mml:mo>=</mml:mo><mml:mn>2</mml:mn><mml:mi>π</mml:mi><mml:mi>f</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>B</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq30.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq31\"><alternatives><tex-math id=\"M71\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\tau _M^{ - 1} = \\alpha _0\\gamma _0(F_{\\theta \\theta } + F_{\\phi \\phi })/2$$\\end{document}</tex-math><mml:math id=\"M72\"><mml:msubsup><mml:mrow><mml:mi>τ</mml:mi></mml:mrow><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:mo>=</mml:mo><mml:msub><mml:mrow><mml:mi>α</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>γ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi>θ</mml:mi><mml:mi>θ</mml:mi></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi>ϕ</mml:mi><mml:mi>ϕ</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>/</mml:mo><mml:mn>2</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq31.gif\"/></alternatives></inline-formula> are the field-dependent frequency and decay time of the fundamental magnon mode<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. The Fourier series expansion coefficients of the effective driving force <inline-formula id=\"IEq32\"><alternatives><tex-math id=\"M73\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$Q_n^a$$\\end{document}</tex-math><mml:math id=\"M74\"><mml:msubsup><mml:mrow><mml:mi>Q</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mi>a</mml:mi></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq32.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq33\"><alternatives><tex-math id=\"M75\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$Q_n^b$$\\end{document}</tex-math><mml:math id=\"M76\"><mml:msubsup><mml:mrow><mml:mi>Q</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mi>b</mml:mi></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq33.gif\"/></alternatives></inline-formula> are determined by the temporal modulation of the temperature given by Eq. (<xref rid=\"Equ2\" ref-type=\"\">2</xref>) and are equal to <inline-formula id=\"IEq34\"><alternatives><tex-math id=\"M77\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$Q_n^a = \\frac{2}{\\pi } \\frac{{\\omega _1\\tau _0}}{{1 + \\omega _n^2\\tau _0^2}}Q$$\\end{document}</tex-math><mml:math id=\"M78\"><mml:msubsup><mml:mrow><mml:mi>Q</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mi>a</mml:mi></mml:mrow></mml:msubsup><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mi>π</mml:mi></mml:mrow></mml:mfrac><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>τ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:mn>1</mml:mn><mml:mo>+</mml:mo><mml:msubsup><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:msubsup><mml:mrow><mml:mi>τ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup></mml:mrow></mml:mfrac><mml:mi>Q</mml:mi></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq34.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq35\"><alternatives><tex-math id=\"M79\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$Q_n^b = \\frac{2}{\\pi } \\frac{{\\omega _1\\omega _n\\tau _0^2}}{{1 + \\omega _n^2\\tau _0^2}}Q$$\\end{document}</tex-math><mml:math id=\"M80\"><mml:msubsup><mml:mrow><mml:mi>Q</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mi>b</mml:mi></mml:mrow></mml:msubsup><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mi>π</mml:mi></mml:mrow></mml:mfrac><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow></mml:msub><mml:msubsup><mml:mrow><mml:mi>τ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup></mml:mrow><mml:mrow><mml:mn>1</mml:mn><mml:mo>+</mml:mo><mml:msubsup><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi>n</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:msubsup><mml:mrow><mml:mi>τ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup></mml:mrow></mml:mfrac><mml:mi>Q</mml:mi></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq35.gif\"/></alternatives></inline-formula>, where <inline-formula id=\"IEq36\"><alternatives><tex-math id=\"M81\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$Q = \\gamma _0(\\sin \\left( {4\\phi _0} \\right)\\beta _{K_1}/2 - {\\mathrm{cos}}(2\\phi _0)\\beta _{K_{\\mathrm{u}}})\\delta T$$\\end{document}</tex-math><mml:math id=\"M82\"><mml:mi>Q</mml:mi><mml:mo>=</mml:mo><mml:msub><mml:mrow><mml:mi>γ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>sin</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mn>4</mml:mn><mml:msub><mml:mrow><mml:mi>ϕ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:mfenced><mml:msub><mml:mrow><mml:mi>β</mml:mi></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mo>/</mml:mo><mml:mn>2</mml:mn><mml:mo>−</mml:mo><mml:mi mathvariant=\"normal\">cos</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mn>2</mml:mn><mml:msub><mml:mrow><mml:mi>ϕ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:msub><mml:mrow><mml:mi>β</mml:mi></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mi>K</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">u</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mi>δ</mml:mi><mml:mi>T</mml:mi></mml:math><inline-graphic xlink:href=\"41467_2020_17635_Article_IEq36.gif\"/></alternatives></inline-formula>.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec12\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41467_2020_17635_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41467_2020_17635_MOESM2_ESM.pdf\"><caption><p>Peer Review File</p></caption></media></supplementary-material></sec></sec></body><back><fn-group><fn><p><bold>Peer review information</bold>\n<italic>Nature Communications</italic> thanks Tengfei Luo, Andreas Mandelis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary information</bold> is available for this paper at 10.1038/s41467-020-17635-1.</p></sec><ack><title>Acknowledgements</title><p>We are grateful to Ilya Razdolski for fruitful discussions. The work was supported by the Bundesministerium fur Bildung und Forschung through the project VIP+ “Nanomagnetron,” the Deutsche Forschungsgemeinschaft and the Russian Foundation for Basic Research (Grant No. 19-52-12065) in the frame of the International Collaborative Research Center TRR 160 and by the Engineering and Physical Sciences Research Council (Grant No. EP/H003487/1). The cooperation between TU Dortmund, the Lashkaryov Institute, and the Ioffe Institute was supported by the Volkswagen Foundation (Grant No. 97758).</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>M.K. constructed the experimental setup, carried out the experiment, and performed the data analysis. A.V.S., I.A.A., and A.V.A. designed the experiment and supervised the experiment and theoretical modeling, T.L.L., S.M.K., and V.E.G. performed theoretical analysis and numerical modeling, A.W.R. and D.P.P. designed and deposited the Galfenol samples, D.P.P. characterized the Galfenol samples, M.K., A.V.S., T.L.L., S.M.K., V.E.G., I.A.A., A.W.R., A.V.A., and M.B. discussed the results and wrote the manuscript.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>Raw pump–probe data (presented in Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> and  <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>) and processed data (presented in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>) that support the findings of this study are available in Mendeley Depository with the identifier 10.17632/h2yw7v8kb2.1</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par30\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Benenti</surname><given-names>G</given-names></name><name><surname>Casati</surname><given-names>G</given-names></name><name><surname>Saito</surname><given-names>K</given-names></name><name><surname>Whitney</surname><given-names>RS</given-names></name></person-group><article-title>Fundamental aspects of steady-state conversion of heat to work at the nanoscale</article-title><source>Phys. Rep.</source><year>2017</year><volume>694</volume><fpage>1</fpage><lpage>124</lpage><pub-id pub-id-type=\"doi\">10.1016/j.physrep.2017.05.008</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bell</surname><given-names>AG</given-names></name></person-group><article-title>Upon the production of sound by radiant energy</article-title><source>Philos. Mag. J. Sci.</source><year>1881</year><volume>11</volume><fpage>510</fpage><lpage>528</lpage><pub-id pub-id-type=\"doi\">10.1080/14786448108627053</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manomar</surname><given-names>S</given-names></name><name><surname>Razansky</surname><given-names>D</given-names></name></person-group><article-title>Photoacoustics: a historical review</article-title><source>Adv. Opt. Photonics</source><year>2016</year><volume>8</volume><fpage>586</fpage><lpage>617</lpage><pub-id pub-id-type=\"doi\">10.1364/AOP.8.000586</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Klieber</surname><given-names>C</given-names></name><etal/></person-group><article-title>Narrow-band acoustic attenuation measurements in vitreous silica at frequencies between 20 and 400 GHz</article-title><source>Appl. Phys. Lett.</source><year>2011</year><volume>98</volume><fpage>211908</fpage><pub-id pub-id-type=\"doi\">10.1063/1.3595275</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chumak</surname><given-names>AV</given-names></name><name><surname>Vasyuchka</surname><given-names>VI</given-names></name><name><surname>Serga</surname><given-names>AA</given-names></name><name><surname>Hillebrands</surname><given-names>B</given-names></name></person-group><article-title>Magnon spintronics</article-title><source>Nat. Phys.</source><year>2015</year><volume>11</volume><fpage>453</fpage><lpage>461</lpage><pub-id pub-id-type=\"doi\">10.1038/nphys3347</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yi Li</surname></name><etal/></person-group><article-title>Strong coupling between magnons and microwave photons in on-chip ferromagnet-superconductor thin-film devices</article-title><source>Phys. Rev. Lett.</source><year>2019</year><volume>123</volume><fpage>107701</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevLett.123.107701</pub-id><pub-id pub-id-type=\"pmid\">31573284</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><mixed-citation publication-type=\"other\">Gurevich, A. G. & Melkov, G. A. <italic>Magnetization Oscillations and Waves</italic> (CRC Press, Boca Raton, 1996).</mixed-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van Kampen</surname><given-names>M</given-names></name><etal/></person-group><article-title>All-optical probe of coherent spin waves</article-title><source>Phys. Rev. Lett.</source><year>2002</year><volume>88</volume><fpage>227201</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevLett.88.227201</pub-id><pub-id pub-id-type=\"pmid\">12059451</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vomir</surname><given-names>M</given-names></name><name><surname>Andrade</surname><given-names>LHF</given-names></name><name><surname>Guidoni</surname><given-names>L</given-names></name><name><surname>Beaurepaire</surname><given-names>E</given-names></name><name><surname>Bigot</surname><given-names>J-Y</given-names></name></person-group><article-title>Real space trajectory of the ultrafast magnetization dynamics in ferromagnetic metals</article-title><source>Phys. Rev. Lett.</source><year>2005</year><volume>94</volume><fpage>237601</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevLett.94.237601</pub-id><pub-id pub-id-type=\"pmid\">16090502</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Frait.</surname><given-names>Z</given-names></name><name><surname>MacFaden</surname><given-names>H</given-names></name></person-group><article-title>Ferromagnetic resonance in metals. Frequency dependence</article-title><source>Phys. Rev.</source><year>1965</year><volume>139</volume><fpage>A1173</fpage><lpage>A1181</lpage><pub-id pub-id-type=\"doi\">10.1103/PhysRev.139.A1173</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Restorff</surname><given-names>JB</given-names></name><etal/></person-group><article-title>Tetragonal magnetostriction and magnetoelastic coupling in Fe-Al, Fe-Ga, Fe-Ge, Fe-Si, Fe-Ga-Al, and Fe-Ga-Ge alloys</article-title><source>J. Appl. Phys.</source><year>2012</year><volume>111</volume><fpage>023905</fpage><pub-id pub-id-type=\"doi\">10.1063/1.3674318</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scherbakov</surname><given-names>AV</given-names></name><etal/></person-group><article-title>Optical excitation of single- and multimode magnetization precession in Fe-Ga nanolayers</article-title><source>Phys. Rev. Appl.</source><year>2019</year><volume>11</volume><fpage>031003</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevApplied.11.031003</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Clark</surname><given-names>AE</given-names></name><etal/></person-group><article-title>Temperature dependence of the magnetic anisotropy and magnetostriction of Fe<sub>100−<italic>x</italic></sub>Ga<sub><italic>x</italic></sub> (<italic>x</italic> = 8.6, 16.6, 28.5)</article-title><source>J. Appl. Phys.</source><year>2005</year><volume>97</volume><fpage>10M316</fpage><pub-id pub-id-type=\"doi\">10.1063/1.1856731</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kats</surname><given-names>VN</given-names></name><etal/></person-group><article-title>Ultrafast changes of magnetic anisotropy driven by laser-generated coherent and noncoherent phonons in metallic films</article-title><source>Phys. Rev. B</source><year>2016</year><volume>93</volume><fpage>214422</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevB.93.214422</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Melnikov</surname><given-names>A</given-names></name><etal/></person-group><article-title>Ultrafast transport of laser-excited spin-polarized carriers in Au/Fe/MgO(001)</article-title><source>Phys. Rev. Lett.</source><year>2011</year><volume>107</volume><fpage>076601</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevLett.107.076601</pub-id><pub-id pub-id-type=\"pmid\">21902412</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kimling</surname><given-names>J</given-names></name><etal/></person-group><article-title>Picosecond spin Seebeck effect</article-title><source>Phys. Rev. Lett.</source><year>2017</year><volume>118</volume><fpage>057207</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevLett.118.057201</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Razdolskii</surname><given-names>I</given-names></name><etal/></person-group><article-title>Nanoscale interface confinement of ultrafast spin transfer torque driving non-uniform spin dynamics</article-title><source>Nat. Commun.</source><year>2017</year><volume>8</volume><fpage>15007</fpage><pub-id pub-id-type=\"doi\">10.1038/ncomms15007</pub-id><pub-id pub-id-type=\"pmid\">28406144</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kamimaki</surname><given-names>A</given-names></name><name><surname>Iihama</surname><given-names>S</given-names></name><name><surname>Sasaki</surname><given-names>Y</given-names></name><name><surname>Ando</surname><given-names>Y</given-names></name><name><surname>Mizukami</surname><given-names>S</given-names></name></person-group><article-title>Reciprocal excitation of propagating spin waves by a laser pulse and their reciprocal mapping in magnetic metal films</article-title><source>Phys. Rev. B</source><year>2017</year><volume>96</volume><fpage>014438</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevB.96.014438</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Khokhlov</surname><given-names>NE</given-names></name><etal/></person-group><article-title>Optical excitation of propagating magnetostatic waves in an epitaxial galfenol film by ultrafast magnetic anisotropy change</article-title><source>Phys. Rev. Appl.</source><year>2019</year><volume>12</volume><fpage>044044</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevApplied.12.044044</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krenzer</surname><given-names>B</given-names></name><name><surname>Hanisch</surname><given-names>A</given-names></name><name><surname>Duvenbeck</surname><given-names>A</given-names></name><name><surname>Rethfeld</surname><given-names>B</given-names></name><name><surname>Horn-von Hoegen</surname><given-names>M</given-names></name></person-group><article-title>Heat transport in nanoscale heterosystems: a numerical and analytical study</article-title><source>J. Nanomater.</source><year>2008</year><volume>2008</volume><fpage>590609</fpage><pub-id pub-id-type=\"doi\">10.1155/2008/590609</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Razdolski</surname><given-names>I</given-names></name><etal/></person-group><article-title>Analysis of the time-resolved magneto-optical Kerr effect for ultrafast magnetization dynamics in ferromagnetic thin films</article-title><source>J. Phys. C</source><year>2017</year><volume>29</volume><fpage>174002</fpage></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ando</surname><given-names>K</given-names></name><etal/></person-group><article-title>Inverse spin-Hall effect induced by spin pumping in metallic system</article-title><source>J. Appl. Phys.</source><year>2011</year><volume>109</volume><fpage>103913</fpage><pub-id pub-id-type=\"doi\">10.1063/1.3587173</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weiler</surname><given-names>M</given-names></name><etal/></person-group><article-title>Spin pumping with coherent elastic waves</article-title><source>Phys. Rev. Lett.</source><year>2012</year><volume>108</volume><fpage>176601</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevLett.108.176601</pub-id><pub-id pub-id-type=\"pmid\">22680888</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fuchs</surname><given-names>GD</given-names></name><etal/></person-group><article-title>Excited-state spectroscopy using single spin manipulation in diamond</article-title><source>Phys. Rev. Lett.</source><year>2008</year><volume>101</volume><fpage>117601</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevLett.101.117601</pub-id><pub-id pub-id-type=\"pmid\">18851332</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Demidov</surname><given-names>VE</given-names></name><etal/></person-group><article-title>Excitation of coherent propagating spin waves by pure spin currents</article-title><source>Nat. Commun.</source><year>2016</year><volume>7</volume><fpage>10446</fpage><pub-id pub-id-type=\"doi\">10.1038/ncomms10446</pub-id><pub-id pub-id-type=\"pmid\">26818232</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gupta</surname><given-names>S</given-names></name><etal/></person-group><article-title>Important role of magnetization precession angle measurement in inverse spin Hall effect induced by spin pumping</article-title><source>Appl. Phys. Lett.</source><year>2017</year><volume>110</volume><fpage>022404</fpage><pub-id pub-id-type=\"doi\">10.1063/1.4973704</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Damon</surname><given-names>RW</given-names></name></person-group><article-title>Thermally‐induced nonlinear effects in the ferromagnetic resonance</article-title><source>Phys. Stat. Sol.</source><year>1965</year><volume>8</volume><fpage>K89</fpage><lpage>K93</lpage><pub-id pub-id-type=\"doi\">10.1002/pssb.19650080228</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mizukami</surname><given-names>S</given-names></name></person-group><article-title>Long-lived ultrafast spin precession in manganese alloys films with a large perpendicular magnetic anisotropy</article-title><source>Phys. Rev. Lett.</source><year>2011</year><volume>106</volume><fpage>117201</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevLett.106.117201</pub-id><pub-id pub-id-type=\"pmid\">21469891</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parkes</surname><given-names>DE</given-names></name><etal/></person-group><article-title>Magnetostrictive thin films for microwave spintronics</article-title><source>Sci. Rep.</source><year>2013</year><volume>3</volume><fpage>2220</fpage><pub-id pub-id-type=\"doi\">10.1038/srep02220</pub-id><pub-id pub-id-type=\"pmid\">23860685</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Salasyuk</surname><given-names>AS</given-names></name><etal/></person-group><article-title>Generation of a localized microwave magnetic field by coherent phonons in a ferromagnetic nanograting</article-title><source>Phys. Rev. B</source><year>2018</year><volume>97</volume><fpage>060404(R)</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevB.97.060404</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kreilkamp</surname><given-names>LE</given-names></name><etal/></person-group><article-title>Terahertz dynamics of lattice vibrations in Au/CdTe plasmonic crystals: photoinduced segregation of Te and enhancement of optical response</article-title><source>Phys. Rev. B</source><year>2016</year><volume>93</volume><fpage>125404</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevB.93.125404</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bartels</surname><given-names>A</given-names></name><name><surname>Hudert</surname><given-names>F</given-names></name><name><surname>Janke</surname><given-names>C</given-names></name><name><surname>Dekorsy</surname><given-names>T</given-names></name><name><surname>Köhler</surname><given-names>K</given-names></name></person-group><article-title>Femtosecond time-resolved optical pump-probe spectroscopy at kilohertz-scan-rates over nanosecond-time-delays without mechanical delay line</article-title><source>Appl. Phys. Lett.</source><year>2006</year><volume>88</volume><fpage>041117</fpage><pub-id pub-id-type=\"doi\">10.1063/1.2167812</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><mixed-citation publication-type=\"other\">COMSOL. COMSOL Multiphysics® v. 5.4. <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.comsol.com\">http://www.comsol.com</ext-link> (COMSOL AB, Stockholm).</mixed-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aspnes</surname><given-names>DE</given-names></name><name><surname>Kelso</surname><given-names>SM</given-names></name><name><surname>Logan</surname><given-names>RA</given-names></name><name><surname>Bhat</surname><given-names>R</given-names></name></person-group><article-title>Optical properties of Al<sub><italic>x</italic></sub>Ga<sub>1−<italic>x</italic></sub>As</article-title><source>J. Appl. Phys.</source><year>1986</year><volume>60</volume><fpage>754</fpage><lpage>767</lpage><pub-id pub-id-type=\"doi\">10.1063/1.337426</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>B</given-names></name><etal/></person-group><article-title>Dynamics and control of gold-encapped gallium arsenide nanowires imaged by 4D electron microscopy</article-title><source>Proc. Natl Acad. Sci. USA</source><year>2017</year><volume>114</volume><fpage>12876</fpage><lpage>12881</lpage><pub-id pub-id-type=\"doi\">10.1073/pnas.1708761114</pub-id><pub-id pub-id-type=\"pmid\">29158393</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carlson</surname><given-names>RO</given-names></name><name><surname>Slack</surname><given-names>GA</given-names></name><name><surname>Silverman</surname><given-names>SJ</given-names></name></person-group><article-title>Thermal conductivity of GaAs and GaAs<sub>1−<italic>x</italic></sub>P<sub><italic>x</italic></sub> laser semiconductors</article-title><source>J. Appl. Phys.</source><year>1965</year><volume>36</volume><fpage>505</fpage><lpage>507</lpage><pub-id pub-id-type=\"doi\">10.1063/1.1714018</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Johnson</surname><given-names>PB</given-names></name><name><surname>Christy</surname><given-names>RW</given-names></name></person-group><article-title>Optical constants of transition metals: Ti, V, Cr, Mn, Fe, Co, Ni, and Pd</article-title><source>Phys. Rev. B</source><year>1974</year><volume>9</volume><fpage>5056</fpage><lpage>5070</lpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevB.9.5056</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Lide</surname><given-names>DR</given-names></name></person-group><source>CRC Handbook of Chemistry and Physics</source><year>2003</year><publisher-loc>Boca Raton</publisher-loc><publisher-name>CRC Press</publisher-name></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807871</article-id><article-id pub-id-type=\"pmc\">PMC7431563</article-id><article-id pub-id-type=\"publisher-id\">70846</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70846-w</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Zinc tolerant plant growth promoting bacteria alleviates phytotoxic effects of zinc on maize through zinc immobilization</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-4345-1536</contrib-id><name><surname>Jain</surname><given-names>Devendra</given-names></name><address><email>devroshan@gmail.com</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kour</surname><given-names>Ramandeep</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-2869-4342</contrib-id><name><surname>Bhojiya</surname><given-names>Ali Asger</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Meena</surname><given-names>Ram Hari</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-5274-6080</contrib-id><name><surname>Singh</surname><given-names>Abhijeet</given-names></name><address><email>abhijeetdhaliwal@gmail.com</email></address><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-6065-6445</contrib-id><name><surname>Mohanty</surname><given-names>Santosh Ranjan</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rajpurohit</surname><given-names>Deepak</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-1799-0189</contrib-id><name><surname>Ameta</surname><given-names>Kapil Dev</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.444738.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0369 7278</institution-id><institution>Department of Molecular Biology and Biotechnology, Rajasthan College of Agriculture, </institution><institution>Maharana Pratap University of Agriculture and Technology, </institution></institution-wrap>Udaipur, Rajasthan 313001 India </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.444738.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0369 7278</institution-id><institution>Department of Soil Science and Agricultural Chemistry, Rajasthan College of Agriculture, </institution><institution>Maharana Pratap University of Agriculture and Technology, </institution></institution-wrap>Udaipur, Rajasthan 313001 India </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.411639.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0571 5193</institution-id><institution>Department of Biosciences, </institution><institution>Manipal University Jaipur, </institution></institution-wrap>Jaipur, Rajasthan 303007 India </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.464869.1</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9288 3664</institution-id><institution>AINP on Soil Biodiversity-Bio-Fertilizers, </institution><institution>Indian Institute of Soil Science, </institution></institution-wrap>Nabibagh, Berasia Road, Bhopal, Madhya Pradesh 462038 India </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.444738.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0369 7278</institution-id><institution>Department of Horticulture, Rajasthan College of Agriculture, </institution><institution>Maharana Pratap University of Agriculture and Technology, </institution></institution-wrap>Udaipur, Rajasthan 313001 India </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.444372.2</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1788 5984</institution-id><institution>Department of Agriculture and Veterinary Sciences, </institution><institution>Mewar University, </institution></institution-wrap>Chittaurgarh, Rajasthan India </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13865</elocation-id><history><date date-type=\"received\"><day>22</day><month>7</month><year>2019</year></date><date date-type=\"accepted\"><day>7</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">The increasing heavy metal contamination in agricultural soils has become a serious concern across the globe. The present study envisages developing microbial inoculant approach for agriculture in Zn contaminated soils. Potential zinc tolerant bacteria (ZTB) were isolated from zinc (Zn) contaminated soils of southern Rajasthan, India. Isolates were further screened based on their efficiency towards Zn tolerance and plant growth promoting activities. Four strains viz<italic>.</italic> ZTB15, ZTB24, ZTB28 and ZTB29 exhibited high degree of tolerance to Zn up to 62.5 mM. The Zn accumulation by these bacterial strains was also evidenced by AAS and SEM–EDS studies. Assessment of various plant growth promotion traits viz<italic>.,</italic> IAA, GA<sub>3</sub>, NH<sub>3</sub>, HCN, siderophores, ACC deaminase, phytase production and P, K, Si solubilization studies revealed that these ZTB strains may serve as an efficient plant growth promoter under in vitro conditions. Gluconic acid secreted by ZTB strains owing to mineral solubilization was therefore confirmed using high performance liquid chromatography. A pot experiment under Zn stress conditions was performed using maize (<italic>Zea mays</italic>) variety (FEM-2) as a test crop. Zn toxicity reduced various plant growth parameters; however, inoculation of ZTB strains alleviated the Zn toxicity and enhanced the plant growth parameters. The effects of Zn stress on antioxidant enzyme activities in maize under in vitro conditions were also investigated. An increase in superoxide dismutase, peroxidase, phenylalanine ammonia lyase, catalase and polyphenol oxidase activity was observed on inoculation of ZTB strains. Further, ZIP gene expression studies revealed high expression in the ZIP metal transporter genes which were declined in the ZTB treated maize plantlets. The findings from the present study revealed that ZTB could play an important role in bioremediation in Zn contaminated soils.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Biotechnology</kwd><kwd>Microbiology</kwd></kwd-group><funding-group><award-group><funding-source><institution>All India Network Project on Soil Biodiversity and Bio-fertilizers</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>Rashtriya Krishi Vikas Yojana</institution></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par7\">Heavy metals are the natural elements having high atomic mass and density (approximately 5 times greater than water)<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. Heavy metal contamination and exposure is a serious concern for environment and health mainly due to the activities like mining, smelting, use of metals and metal-containing compounds in various applications including agriculture. Many heavy metal ions are essential as trace elements in ppm quantities, but at high concentrations, they turn into toxic elements<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. These heavy metals are neither remove nor degrade from the environment, unlike the other pollutants that can be degraded by either chemically or biologically means. Excessive levels of heavy metals like zinc, cadmium, copper, lead, nickel and mercury are considered as toxic pollutants<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>.</p><p id=\"Par8\">Elevated concentrations of Zn at toxic levels in the agricultural land from different anthropogenic practices such as application of metal contaminated sewage sludge or from mining activities might represent a potential risk for sustainable and quality food production<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. In such contaminated soils Zn ions are found at higher concentrations causing toxicity. The applications of plant growth promoting metal tolerant rhizobacteria can be used to decrease such metal toxicity<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. Considering the significant diversity and capacities of Zn resistance and removal from natural environment, it is essential to identify the candidate microorganisms and also to understand the molecular mechanism of metal removal processes<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>.</p><p id=\"Par9\">Bioremediation is a natural process that uses living organisms or enzymes to detoxify heavy metals from the environment have received great deals of attention<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. Though the bioremediation approach is relatively slow and time taking, but it is superior over conventional chemical process and most importantly it maintains soil fertility<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. High concentrations of heavy metals into the environment create selective pressure for the emergence of bacterial strains with tolerance to the metals. These bacteria can affect the reactivity and mobility of such heavy metals and can be used to detoxify some metals preventing further metal contamination<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>.</p><p id=\"Par10\">Zinc (Zn<sup>+2</sup> cations) cannot diffuse across cell membrane hence specific Zn transporters (Zn-regulated transporter (ZRT), iron-regulated transporter (IRT)-like proteins broadly classified as ZIP protein family) are required for Zn ion homeostasis by regulating Zn uptake and transport<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Furthermore, ZIP genes are accountable for the translocation, detoxification and storage of Zn or Fe in the plant cells<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>.</p><p id=\"Par11\">The concentration of Zn over its threshold limit is toxic and reduces plant growth due to reduced photosynthesis, enzyme activity, plant mineral nutrition etc. Hence, heavy metal-tolerant microbes had attended a great deal of interest by researches for Zn bioremediation<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Important Zn tolerant PGPR strains include the genus <italic>Cupriavidus</italic>,<italic> Pseudomonas</italic>,<italic> Streptomyces</italic>,<italic> Micrococcus</italic>,<italic> Sphingomonas</italic>,<italic> Klebsiella</italic>,<italic> Serratia</italic>,<italic> Proteus </italic>etc<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>–<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. However, there is a need to isolate novel PGPR strains which can perform well in all types of Zn contaminated conditions and importantly their PGP traits should remain active even under Zn stress condition.</p><p id=\"Par12\">The objectives of the present study were to explore the role of ZTB in maize seedlings grown under Zn stress conditions. The effects of these strains were observed upon growth, photosynthetic activities efficiencies and on Zn uptake. Moreover, the role of different Zn transporters genes (ZIPs) involved in Zn uptake and translocation were also analyzed through real time PCR.</p></sec><sec id=\"Sec2\"><title>Results</title><sec id=\"Sec3\"><title>Analysis of soil samples</title><p id=\"Par13\">In the present study, the rhizospheric soil samples were collected from the Zawar, Udaipur (zinc-lead ore mine tailings areas). The physico-chemical characteristics of the soil samples are described in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>. The soil collected in the present study was neutral to slightly alkaline in nature. The rhizospheric soils of Zn contaminated soils contains moderate to high range of EC, OC, total N, total P and total K which might be due to associated PGPRs. The higher Zn contents are attributed to the Zn smelting and mining activities in this area. The diethylene triamine pentacetate acid (DTPA) extractable concentrations of Zn were found to be 35.99 mg/kg and 39.99 mg/kg in Mochia and Balaria mining regions respectively.<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Soil sample sites and chemical properties of experimented soil.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Place</th><th align=\"left\">Satellite location</th><th align=\"left\">EC<sup>a</sup> (dS/m)</th><th align=\"left\">pH<sup>a</sup></th><th align=\"left\">OC (g/kg)</th><th align=\"left\">Av. N (kg/ha)</th><th align=\"left\">Av. P (kg/ha)</th><th align=\"left\">Av. K (kg/ha)</th><th align=\"left\">DTPA-Zn (mg/kg)</th></tr></thead><tbody><tr><td align=\"left\">Mochia, Zawar</td><td align=\"left\"><p>24° 21′ 37.6\" N</p><p>73° 41′ 45.3\" E</p></td><td char=\".\" align=\"char\">0.57</td><td char=\".\" align=\"char\">7.19</td><td char=\".\" align=\"char\">0.55</td><td char=\".\" align=\"char\">94.82</td><td char=\".\" align=\"char\">20.20</td><td char=\".\" align=\"char\">199.36</td><td char=\".\" align=\"char\">35.99</td></tr><tr><td align=\"left\">Balaria, Zawar</td><td align=\"left\"><p>24° 35′ 38.8\" N</p><p>73° 75′ 21.1\" E</p></td><td char=\".\" align=\"char\">0.62</td><td char=\".\" align=\"char\">7.25</td><td char=\".\" align=\"char\">0.60</td><td char=\".\" align=\"char\">81.09</td><td char=\".\" align=\"char\">18.22</td><td char=\".\" align=\"char\">169.44</td><td char=\".\" align=\"char\">39.99</td></tr></tbody></table><table-wrap-foot><p><sup>a</sup>1:2 soil to water ratio, OC,  organic carbon; Av. N, available nitrogen (Kjeldahl digestion); Av. P, available phosphorus (Olsen’s P<sub>2</sub>O<sub>5</sub>); Av. K, available potassium (ammonium acetate extractable K<sub>2</sub>O).</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec4\"><title>Zinc tolerance (MIC) for ZTB strains</title><p id=\"Par14\">The ZTB strains isolated from zinc-lead ore mine tailings areas were subjected to determination of their MIC against Zn and four isolates viz<italic>.,</italic> ZTB15, ZTB24, ZTB28, ZTB29 showed the high MIC value of 63.0 mM Zn in the medium were selected for the characterization.</p></sec><sec id=\"Sec5\"><title>ZTB strains characterization</title><p id=\"Par15\">The biochemical characterization of ZTB strains viz<italic>.,</italic> ZTB15, ZTB24, ZTB28 and ZTB29 were summarized in Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>. Among these 4 strains, all 4 strains were positive for citrate utilization, 3 strains were positive for starch hydrolysis, 1 strain (ZTB24) was positive for nitrate reduction, one strain (ZTB15) was positive for gelatin hydrolysis, all 4 strains were positive for catalase activity and 1 strain (ZTB28) was positive for oxidase activity.<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Biochemical characterization of zinc tolerant bacteria.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Strain name</th><th align=\"left\">Starch hydrolysis</th><th align=\"left\">Citrate utilization</th><th align=\"left\">Nitrate reduction</th><th align=\"left\">Gelatin liquefaction</th><th align=\"left\">Catalase activity</th><th align=\"left\">Oxidase</th></tr></thead><tbody><tr><td align=\"left\">ZTB 15</td><td align=\"left\"><bold>−</bold></td><td align=\"left\"><bold> + </bold></td><td align=\"left\"><bold>−</bold></td><td align=\"left\"><bold> + </bold></td><td align=\"left\"><bold> + </bold></td><td align=\"left\"><bold>−</bold></td></tr><tr><td align=\"left\">ZTB 24</td><td align=\"left\"><bold> + </bold></td><td align=\"left\"><bold> + </bold></td><td align=\"left\"><bold> + </bold></td><td align=\"left\"><bold>−</bold></td><td align=\"left\"><bold> + </bold></td><td align=\"left\"><bold>−</bold></td></tr><tr><td align=\"left\">ZTB 28</td><td align=\"left\"><bold> + </bold></td><td align=\"left\"><bold> + </bold></td><td align=\"left\"><bold>−</bold></td><td align=\"left\"><bold>−</bold></td><td align=\"left\"><bold> + </bold></td><td align=\"left\"><bold> + </bold></td></tr><tr><td align=\"left\">ZTB 29</td><td align=\"left\"><bold> + </bold></td><td align=\"left\"><bold> + </bold></td><td align=\"left\"><bold>−</bold></td><td align=\"left\"><bold>−</bold></td><td align=\"left\"><bold> + </bold></td><td align=\"left\"><bold>−</bold></td></tr></tbody></table><table-wrap-foot><p><bold> +, Positive; −, negative.</bold></p></table-wrap-foot></table-wrap></p><p id=\"Par16\">The partial 16S rDNA sequence of ZTB15, ZTB24, ZTB28 and ZTB29 strains were sequenced and analyzed using the BLAST tool. The BLAST results revealed greatest sequence identity of ZTB strains with the previously reported type strains of genus <italic>Serratia</italic>. The phylogenetic position of these ZTB strains is shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>. The NCBI GenBank accession number assigned to the ZTB strains are following:<list list-type=\"order\"><list-item><p id=\"Par17\">ZTB15: <italic>Serratia</italic> sp. (Accession Number: MK773869)</p></list-item><list-item><p id=\"Par18\">ZTB24: <italic>Serratia</italic> sp. (Accession Number: MK773870)</p></list-item><list-item><p id=\"Par19\">ZTB28: <italic>Serratia</italic> sp. (Accession Number: MK773872)</p></list-item><list-item><p id=\"Par20\">ZTB29: <italic>Serratia</italic> sp. (Accession Number: MK773873)</p></list-item></list><fig id=\"Fig1\"><label>Figure 1</label><caption><p>Phylogenetic tree of ZTB strains based on 16S rDNA.</p></caption><graphic xlink:href=\"41598_2020_70846_Fig1_HTML\" id=\"MO1\"/></fig></p></sec><sec id=\"Sec6\"><title>Zinc biosorption potential of ZTB strains</title><p id=\"Par21\">The results of Zn biosorption were summarized in Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref>. The results obtained revealed that all the selected ZTB strains were able to remove Zn from the nutrient broth medium efficiently and the highest biosorption of Zn was recorded in the bacterial strain ZTB15 followed by ZTB29, ZTB28 and ZTB24. The results of Zn biosorption were calculated as percentage biosorption. In nutrient broth medium supplemented with concentration of Zn (20 mg/L), ZTB15 was able to remove the highest amount of Zn from the medium i.e. 92.46%. At higher concentration of Zn (40 mg/L), ZTB15 was able to remove 93.51% of Zn from the medium which was the highest among all the ZTB strains. It was closely followed by ZTB28 and ZTB29 with 91.87% and 91.01% respectively of Zn biosorption efficiency. The ZTB strain ZTB24 was able to remove 76.04% of Zn.<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Effect of Zn concentration on biosorption of Zn by ZTB.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Strain name</th><th align=\"left\" colspan=\"2\">Concentration of Zn (mg/L) in the supernatant after biosorptionby ZTB after 72 h</th><th align=\"left\" colspan=\"2\">% Biosorption of Zn by ZTB after 72 h</th></tr><tr><th align=\"left\">Media with 20 mg/L Zn</th><th align=\"left\">Media with 40 mg/L Zn</th><th align=\"left\">Media with 20 mg/L Zn</th><th align=\"left\">Media with 40 mg/L Zn</th></tr></thead><tbody><tr><td align=\"left\">ZTB 15</td><td char=\".\" align=\"char\">1.508 ± 0.196<sup>a</sup></td><td char=\".\" align=\"char\">2.598 ± 0.252<sup>a</sup></td><td char=\".\" align=\"char\">92.46</td><td char=\".\" align=\"char\">93.51</td></tr><tr><td align=\"left\">ZTB 24</td><td char=\".\" align=\"char\">3.285 ± 0.020<sup>c</sup></td><td char=\".\" align=\"char\">9.586 ± 0.121<sup>c</sup></td><td char=\".\" align=\"char\">83.58</td><td char=\".\" align=\"char\">76.04</td></tr><tr><td align=\"left\">ZTB 28</td><td char=\".\" align=\"char\">1.831 ± 0.050<sup>b</sup></td><td char=\".\" align=\"char\">3.851 ± 0.059<sup>b</sup></td><td char=\".\" align=\"char\">90.85</td><td char=\".\" align=\"char\">91.87</td></tr><tr><td align=\"left\">ZTB 29</td><td char=\".\" align=\"char\">1.825 ± 0.309<sup>ab</sup></td><td char=\".\" align=\"char\">3.597 ± 0.252<sup>b</sup></td><td char=\".\" align=\"char\">90.88</td><td char=\".\" align=\"char\">91.01</td></tr></tbody></table><table-wrap-foot><p>Data is presented as means of 3 replicates ± SD (standard deviation). The Mean value followed by same letter in column of each treatment is not significant difference at <italic>p</italic> = 0.05 by Tukey–Kramer HSD test.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec7\"><title>Scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM–EDS) studies of ZTB strains</title><p id=\"Par22\">Scanning electron microscopy (SEM) was used to examine the morphology of bacterial cells after 72 h exposure to 100 mg/L of Zn whereas the EDS analysis of the bacterium established the presence of elemental content on the microbial biomass (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). It was illustrated from the SEM micrographs that the ZTB produces very high amount of exopolysaccharide (EPS) in response to very high concentration of Zn whereas these strains without Zn did not produced EPS (supplementary data sheet). The EDS micrograph of control (heavy metal free) biomass showed only prominent peaks of alkali and alkaline earth metal indicating the presence of these elements in the bacterial biomass (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). Further, in the Zn treated bacterial cell samples the peak of Zn metal along with the earlier peaks of the alkali and alkaline earth metal were appeared in the ZTB strains. The EDS analysis showed that the amount of Zn accumulated in the ZTB cells was a maximum of 3.97% and a minimum of 2.72%.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Scanning electron microscopy (SEM) images (<bold>a</bold>) ZTB15, (<bold>c</bold>) ZTB24, (<bold>e</bold>) ZTB28, (<bold>g</bold>) ZTB29 and energy dispersive X-ray (EDX) spectra of (<bold>b</bold>) ZTB15, (<bold>d</bold>) ZTB24, (<bold>f</bold>) ZTB28, (<bold>h</bold>) ZTB29 of Zn treated cells.</p></caption><graphic xlink:href=\"41598_2020_70846_Fig2_HTML\" id=\"MO2\"/></fig></p></sec><sec id=\"Sec8\"><title>Plant growth promoting traits of the ZTB strains</title><p id=\"Par23\">All the 4 ZTB isolates were subjected to various plant growth promoting activities viz<italic>.,</italic> IAA production, ACC deaminase activity, siderophore production, phosphate solubilization, potash solubilization, silica solubilization, ammonia production, phytase production and volatile compounds production i.e. HCN production were summarized in Table <xref rid=\"Tab4\" ref-type=\"table\">4</xref>. All the 4 ZTB strains were positive for IAA production and the isolate ZTB29 showed significantly higher IAA production (12.45 µg/mL). All the 4ZTB isolates showed positive results in ACC deaminase activity and ammonia production and showed negative results in production of HCN. All the 4 ZTB strains were positive for GA<sub>3</sub> production and the isolate ZTB24 produced the highest amount of GA<sub>3</sub> (60.60 µg/mL). All the 4 ZTB strains were able to solubilize tricalcium phosphate forming holo zones from which phosphate solubilization index (PSI) was calculated. The phosphate solubilization ability of the ZTB15 strain was significantly higher (4.6) in comparison to other three strains. All the 4 ZTB strains were able to solubilize potash and form the clear zones on the medium, the potash solubilization index (KSI) was calculated (supplementary data sheet). The highest KSI was showed by isolate ZTB29 (8.0). The silica solubilization ability of the isolate ZTB28 was significantly higher with silica solubilization index (SSI) of 3.52 cm in comparison to other three strains. All the 4 ZTB strains were found positive for phytase production. The phytase production index (PPI) was highest for isolate ZTB15 (12.12). All the 4 ZTB strains were found positive for siderophore and the siderophore production index was highest for isolate ZTB15 (2.08). Thus presence of these important PGP traits in ZTB strains could provide plant growth promotion and Zn tolerance under Zn stress.<table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Plant growth promoting activities of ZTB.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">PGPR activity</th><th align=\"left\" colspan=\"4\">ZTB strains</th></tr><tr><th align=\"left\">ZTB15</th><th align=\"left\">ZTB24</th><th align=\"left\">ZTB28</th><th align=\"left\">ZTB29</th></tr></thead><tbody><tr><td align=\"left\">IAA production (µg/mL)</td><td align=\"left\">4.83 ± 0.02</td><td align=\"left\">4.32 ± 0.040</td><td align=\"left\">8.03 ± 0.02</td><td align=\"left\">12.54 ± 0.07</td></tr><tr><td align=\"left\">ACC deaminase activity</td><td align=\"left\"> +  + </td><td align=\"left\"> + </td><td align=\"left\"> +  + </td><td align=\"left\"> + </td></tr><tr><td align=\"left\">Ammonia production (µg/mL)</td><td align=\"left\">1.42 ± 0.23</td><td align=\"left\">1.49 ± 0.56</td><td align=\"left\">1.48 ± 0.18</td><td align=\"left\">1.45 ± 0.86</td></tr><tr><td align=\"left\">HCN production</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td></tr><tr><td align=\"left\">GA<sub>3</sub> (µg/mL)</td><td align=\"left\">28.20 ± 1.31</td><td align=\"left\">60.60 ± 1.50</td><td align=\"left\">40.86 ± 1.23</td><td align=\"left\">28.10 ± 1.01</td></tr><tr><td align=\"left\">Phosphate solublization index</td><td align=\"left\">4.60 ± 0.10</td><td align=\"left\">3.45 ± 0.10</td><td align=\"left\">4.10 ± 0.20</td><td align=\"left\">3.85 ± 0.04</td></tr><tr><td align=\"left\">Potassium solublization index</td><td align=\"left\">4.20 ± 0.05</td><td align=\"left\">6.30 ± 0.05</td><td align=\"left\">6.33 ± 0.03</td><td align=\"left\">8.00 ± 0.10</td></tr><tr><td align=\"left\">Silica solublization index</td><td align=\"left\">2.23 ± 0.02</td><td align=\"left\">2.90 ± 0.01</td><td align=\"left\">3.52 ± 0.01</td><td align=\"left\">2.30 ± 0.01</td></tr><tr><td align=\"left\">Phytase production index</td><td align=\"left\">12.12 ± 0.01</td><td align=\"left\">11.42 ± 0.01</td><td align=\"left\">7.50 ± 0.02</td><td align=\"left\">11.42 ± 0.01</td></tr><tr><td align=\"left\">Siderophore index (Z/C)</td><td align=\"left\">2.08 ± 0.01</td><td align=\"left\">1.66 ± 0.01</td><td align=\"left\">1.11 ± 0.01</td><td align=\"left\">2.00 ± 0.60</td></tr></tbody></table><table-wrap-foot><p> +, Positive; ++, medium positive; +++, high positive; −, negative; Data is presented as means of 3 replicates ± SD (standard deviation).</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec9\"><title>Gluconic acid production</title><p id=\"Par24\">The analysis of ZTB culture supernatant grown under Zn stress conditions allows the detection of gluconic acid produced by ZTB strains. The standard 50% Gluconic acid showed single peak in HPLC chromatogram and was detected at the retention time of 2.22 min (supplementary data sheet). All the four bacterial isolates were able to produce gluconic acid ranging from 293.66 to 382.37 mg/mL. Based on comparison with the standard 50% gluconic acid used in this study, it was found that strain ZTB15, ZTB24, ZTB28 and ZTB29 generated about 293.66, 297.82, 301.13 and 382.37 mg/mL gluconic acid respectively.</p></sec><sec id=\"Sec10\"><title>In vitro plant growth promotion by ZTB on maize under Zn stress</title><p id=\"Par25\">The pot culture experiments were conducted under net house conditions in plastic pots filled with sterile planting mixture. The plant growth promoting activities of ZTB isolates under Zn stress conditions were studied on maize plantlets treated with ZTB inoculants (seed bacterization method) under Zn stress conditions (1,000 mg Zn/kg planting mixture). Four ZTB strains viz; ZTB15, ZTB24, ZTB28 and ZTB29 were selected and pot experiment data were recorded under Zn stress condition after 30 days of germination were summarized in Table <xref rid=\"Tab5\" ref-type=\"table\">5</xref>. In uninoculated control (pot containing 1,000 mg Zn/kg planting mixture) the overall all plant growth and chlorophyll content was significantly decreased due to the Zn stress compared to control plantlets (without any Zn stress) (Supplementary Data Sheet). Whereas, higher plant growth and chlorophyll content were observed in maize plantlets treated with ZTB strains compared to uninoculated control. The maize plantlets inoculated with ZTB28 showed best response compare to the other strains and uninoculated control. All the treatments significantly influenced the observed parameters. All the ZTB strains significantly influenced the observed parameters and contributed to plant growth under Zn stress conditions.<table-wrap id=\"Tab5\"><label>Table 5</label><caption><p>In vitro studies on the effect of zinc tolerant bacteria on growth and biomass of maize seedling under Zn stress conditions (1,000 mg Zn/kg planting mixture).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Treatment details</th><th align=\"left\">Average shoot length (cm)</th><th align=\"left\">Average root length (cm )</th><th align=\"left\">Average root number</th><th align=\"left\">Average leaf number</th><th align=\"left\">Total chlorophyll (µg/mL)</th></tr></thead><tbody><tr><td align=\"left\">T1: control without Zn and ZTB inoculation</td><td align=\"left\">11.50 ± 0.93<sup>c</sup></td><td align=\"left\">38.50 ± 4.03<sup>b</sup></td><td align=\"left\">10.52 ± 0.98<sup>cd</sup></td><td align=\"left\">6.00 ± 1.0<sup>a</sup></td><td align=\"left\">34.14 ± 4.14<sup>b</sup></td></tr><tr><td align=\"left\">T2: control with Zn and without ZTB inoculation</td><td align=\"left\">8.90 ± 1.03<sup>bc</sup></td><td align=\"left\">36.50 ± 3.20<sup>b</sup></td><td align=\"left\">10.13 ± 0.86<sup>d</sup></td><td align=\"left\">5.00 ± 0.58<sup>a</sup></td><td align=\"left\">32.83 ± 4.91<sup>b</sup></td></tr><tr><td align=\"left\">T3: with Zn and ZTB15 inoculation</td><td align=\"left\">13.2 ± 1.47<sup>b</sup></td><td align=\"left\">47.23 ± 2.07<sup>a</sup></td><td align=\"left\">13.33 ± 1.32<sup>bc</sup></td><td align=\"left\">5.30 ± 1.15<sup>a</sup></td><td align=\"left\">47.10 ± 4.0<sup>a</sup></td></tr><tr><td align=\"left\">T4: with Zn and ZTB24 inoculation</td><td align=\"left\">13.26 ± 1.25<sup>b</sup></td><td align=\"left\">48.56 ± 2.22<sup>a</sup></td><td align=\"left\">14.33 ± 1.25<sup>b</sup></td><td align=\"left\">6.30 ± 0.58<sup>a</sup></td><td align=\"left\">47.10 ± 3.77<sup>a</sup></td></tr><tr><td align=\"left\">T5: with Zn and ZTB28 inoculation</td><td align=\"left\">16.59 ± 0.90<sup>a</sup></td><td align=\"left\">52.96 ± 3.04<sup>a</sup></td><td align=\"left\">17.67 ± 1.23<sup>a</sup></td><td align=\"left\">6.67 ± 1.15<sup>a</sup></td><td align=\"left\">57.87 ± 3.99<sup>a</sup></td></tr><tr><td align=\"left\">T6: with Zn and ZTB29 inoculation</td><td align=\"left\">13.85 ± 1.10<sup>ab</sup></td><td align=\"left\">50.23 ± 1.94<sup>a</sup></td><td align=\"left\">14.33 ± 1.08<sup>b</sup></td><td align=\"left\">5.30 ± 1.15<sup>a</sup></td><td align=\"left\">48.67 ± 4.26<sup>a</sup></td></tr><tr><td align=\"left\">CD at 5%</td><td align=\"left\">2.21</td><td align=\"left\">4.42</td><td align=\"left\">2.21</td><td align=\"left\">2.01</td><td align=\"left\">6.73</td></tr><tr><td align=\"left\">CV%</td><td align=\"left\">14.08</td><td align=\"left\">7.97</td><td align=\"left\">13.61</td><td align=\"left\">28.93</td><td align=\"left\">12.44</td></tr></tbody></table><table-wrap-foot><p>Data are recorded after 30 days of germination; data is presented as means of 4 replicates ± SD (standard deviation). The Mean value followed by same letter in column of each treatment is not significant difference at <italic>p</italic> = 0.05 by Tukey–Kramer HSD test.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec11\"><title>Antioxidant enzymes activities</title><p id=\"Par26\">The stress related enzymes viz<italic>.,</italic> catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) were also studied in 14 days old seedlings (Table <xref rid=\"Tab6\" ref-type=\"table\">6</xref>). After 14 days of growth under in vitro conditions, the expression of stress related enzymes viz<italic>.,</italic> catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) were significantly lower in control plantlets (without Zn stress) and the plantlet treated with selected ZTB strains compared to the uninoculated control under Zn stress conditions. The maize plantlet treated with selected ZTB isolates showed lower activity of SOD ranged from 0.33 to 0.39 unit/mg fresh weight compare to 0.27 unit/mg fresh weight of uninoculated control under Zn stress conditions and highest activity of SOD were observed in the plantlets treated with ZTB24 whereas lowest activity was observed in plantlets treated with ZTB28.<table-wrap id=\"Tab6\"><label>Table 6</label><caption><p>In vitro studies on the effect of ZTB on stress related enzymes of maize seedling under Zn stress conditions (1,000 mg Zn /kg planting mixture).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Treatment details</th><th align=\"left\">SOD (unit/mg) fresh weight</th><th align=\"left\">POD (µmole/min/g)</th><th align=\"left\">PAL (µmole/min/g)</th><th align=\"left\">Catalase (µmole/min/g)</th><th align=\"left\">PPO (µmole/min/g)</th></tr></thead><tbody><tr><td align=\"left\">T1: control without Zn and ZTB inoculation</td><td align=\"left\">0.21 ± 0.02f</td><td align=\"left\">1.80 ± 0.18<sup>g</sup></td><td align=\"left\">0.0203 ± 0.002<sup>fg</sup></td><td align=\"left\">18.50 ± 0.41<sup>d</sup></td><td align=\"left\">0.0127 ± 0.001<sup>d</sup></td></tr><tr><td align=\"left\">T2: control with Zn and without ZTB inoculation</td><td align=\"left\">0.27 ± 0.02<sup>ab</sup></td><td align=\"left\">1.95 ± 0.30<sup>ab</sup></td><td align=\"left\">0.0213 ± 0.001<sup>ab</sup></td><td align=\"left\">19.23 ± 0.25<sup>a</sup></td><td align=\"left\">0.0141 ± 0.001<sup>c</sup></td></tr><tr><td align=\"left\">T3: with Zn and ZTB15 inoculation</td><td align=\"left\">0.36 ± 0.03<sup>cde</sup></td><td align=\"left\">2.82 ± 0.20<sup>bc</sup></td><td align=\"left\">0.0233 ± 0.006<sup>g</sup></td><td align=\"left\">20.92 ± 1.95<sup>cd</sup></td><td align=\"left\">0.0170 ± 0.002<sup>a</sup></td></tr><tr><td align=\"left\">T4: with Zn and ZTB24 inoculation</td><td align=\"left\">0.39 ± 0.03<sup>bc</sup></td><td align=\"left\">2.27 ± 0.25<sup>ef</sup></td><td align=\"left\">0.0283 ± 0.002<sup>cdef</sup></td><td align=\"left\">22.58 ± 1.26<sup>c</sup></td><td align=\"left\">0.0163 ± 0.002<sup>b</sup></td></tr><tr><td align=\"left\">T5: with Zn and ZTB28 inoculation</td><td align=\"left\">0.33 ± 0.03<sup>def</sup></td><td align=\"left\">2.20 ± 0.25<sup>ef</sup></td><td align=\"left\">0.0314 ± 0.001<sup>bc</sup></td><td align=\"left\">21.17 ± 2.05<sup>cd</sup></td><td align=\"left\">0.0174 ± 0.001<sup>a</sup></td></tr><tr><td align=\"left\">T6: with Zn and ZTB29 inoculation</td><td align=\"left\">0.37 ± 0.03<sup>cd</sup></td><td align=\"left\">2.71 ± 0.25<sup>cd</sup></td><td align=\"left\">0.0301 ± 0.005<sup>cd</sup></td><td align=\"left\">26.53 ± 1.51<sup>ab</sup></td><td align=\"left\">0.0176 ± 0.002<sup>a</sup></td></tr><tr><td align=\"left\">CD at 5%</td><td align=\"left\">0.050</td><td align=\"left\">0.460</td><td align=\"left\">0.010</td><td align=\"left\">1.430</td><td align=\"left\">0.001</td></tr><tr><td align=\"left\">CV%</td><td align=\"left\">8.61</td><td align=\"left\">11.15</td><td align=\"left\">14.10</td><td align=\"left\">3.65</td><td align=\"left\">4.42</td></tr></tbody></table><table-wrap-foot><p>*Value is mean of 4 replicates. The Mean value followed by same letter in column of each treatment is not significant difference at <italic>p</italic> = 0.05 by Tukey–Kramer HSD test.</p></table-wrap-foot></table-wrap></p><p id=\"Par27\">The maize plantlet treated with selected ZTB isolates showed lower activity of POD ranged from 2.20 to 2.82 µmole/min/g compare to 1.95 µmole/min/g of uninoculated control under Zn stress conditions and highest activity of POD were observed in the plantlets treated with ZTB15 whereas lowest activity was observed in plantlets treated with ZTB28. Similarly, the PAL activity were ranged from 0.0233 to 0.0314 µmole/min/g compare to 0.0213 µmole/min/g of uninoculated control under Zn stress conditions and highest amount of PAL produced by ZTB28 whereas lowest amount of PAL produced by ZTB15. The expression of CAT in maize plantlet were ranged from 20.92 to 26.53 µmole/min/g compare to 19.23 µmole/min/g of uninoculated control under Zn stress conditions and highest amount of CAT produced by ZTB29 whereas lowest amount of CAT produced by ZTB15. Whereas, in case of PPO, the ZTB treated plantlets expressed the PPO enzyme ranged from 0.0163 to 0.0176 µmole/min/g compare to 0.0141 µmole/min/g of uninoculated control under Zn stress conditions and highest amount of PPO produced by ZTB29 whereas lowest amount of PPO produced by ZTB24.</p></sec><sec id=\"Sec12\"><title>Analysis of Zn uptake in maize seedling using atomic absorption spectroscopy</title><p id=\"Par28\">Accumulation of Zn in the maize plantlet after 30 days of germination under Zn stress conditions (1,000 mg Zn/kg planting mixture) were summarized in Table <xref rid=\"Tab7\" ref-type=\"table\">7</xref>. The results indicated that the treatment with ZTB strains reduced the Zn uptakes in maize seedling significantly compare to the untreated plants. Moreover, inoculated and un-inoculated shoot system exhibited greater Zn accumulation than the roots.<table-wrap id=\"Tab7\"><label>Table 7</label><caption><p>In vitro studies on the effect of ZTB on Zn accumulation in maize seedling under Zn stress conditions (1,000 mg Zn /kg planting mixture).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Treatment details</th><th align=\"left\">Zn concentration in shoot (µg/g fresh weight)</th><th align=\"left\">Zn concentration in root (µg/g fresh weight)</th></tr></thead><tbody><tr><td align=\"left\">T1: control without Zn and ZTB inoculation</td><td align=\"left\">65.01 ± 5.0<sup>d</sup></td><td align=\"left\">46.03 ± 6.5<sup>e</sup></td></tr><tr><td align=\"left\">T2: control with Zn and without ZTB inoculation</td><td align=\"left\">632.64 ± 6.0<sup>a</sup></td><td align=\"left\">487.90 ± 11.5<sup>a</sup></td></tr><tr><td align=\"left\">T3: with Zn and ZTB15 inoculation</td><td align=\"left\">356.28 ± 5.1<sup>b</sup></td><td align=\"left\">299.70 ± 10.1<sup>b</sup></td></tr><tr><td align=\"left\">T4: with Zn and ZTB24 inoculation</td><td align=\"left\">335.31 ± 7.6<sup>bc</sup></td><td align=\"left\">280.20 ± 5.5<sup>bc</sup></td></tr><tr><td align=\"left\">T5: with Zn and ZTB28 inoculation</td><td align=\"left\">333.12 ± 7.5<sup>c</sup></td><td align=\"left\">262.20 ± 7.0<sup>c</sup></td></tr><tr><td align=\"left\">T6: with Zn and ZTB29 inoculation</td><td align=\"left\">339.57 ± 7.1<sup>bc</sup></td><td align=\"left\">218.70 ± 4.45<sup>d</sup></td></tr><tr><td align=\"left\">CD at 5%</td><td align=\"left\">2.44</td><td align=\"left\">6.22</td></tr><tr><td align=\"left\">CV%</td><td align=\"left\">0.39</td><td align=\"left\">1.29</td></tr></tbody></table><table-wrap-foot><p>Each value is mean of 4 replicates. The Mean value followed by same letter in column of each treatment is not significant difference at <italic>p</italic> = 0.05 by Tukey–Kramer HSD test.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec13\"><title>Gene expression analysis</title><p id=\"Par29\">Zinc treated maize plantlet showed a significant increase in the ZIP metal transporter gene expression under Zn stress as compared to control without Zn stress (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>). Gene expression studies in 14 days old maize seedlings revealed up regulation of ZIP1, ZIP4, ZIP5 and ZIP8 metal transporter genes in Zn-treated seedlings in response to control without Zn stress. The ZTB inoculation substantially reduced the metal transporter ZIP expression in maize in the presence of Zn.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Expression pattern of ZmZIP genes in 14 days maize plantlet under Zn stress conditions (1,000 mg Zn/kg planting mixture) (<bold>A</bold>) ZIP1, (<bold>B</bold>) ZIP4, (<bold>C</bold>) ZIP5 and (<bold>D</bold>) ZIP8 (treatments: control: without Zn and ZTB inoculation; control + Zn: with Zn and without ZTB inoculation; ZTB-15: ZTB-24: ZTB-28: ZTB-29: with Zn and ZTB inoculation).</p></caption><graphic xlink:href=\"41598_2020_70846_Fig3_HTML\" id=\"MO3\"/></fig></p><p id=\"Par30\">The seedlings treated with ZTB15 showed decline in the expression of metal transporter ZIP1, ZIP4, ZIP5 and ZIP8 genes by 54.12, 7.80, 63.41 and 43.56% respectively in response to Zn treated seedlings. The seedlings treated with ZTB24 showed decline in the expression of metal transporter ZIP1, ZIP4, ZIP5 and ZIP8 genes by 62.07, 74.09, 36.80 and 31.40% respectively in response to Zn treated seedlings. The seedlings treated with ZTB28 showed decline in the expression of metal transporter ZIP1, ZIP4, ZIP5 and ZIP8 genes by 31.67, 24.44, 39.12 and 15.97% respectively in response to Zn treated seedlings. Similar decline in the expression of metal transporter ZIP1, ZIP4, ZIP5 and ZIP8 genes was recorded by 64.63, 17.92, 31.76 and 39.02% respectively on application of ZTB29 in response to Zn treated seedlings.</p></sec></sec><sec id=\"Sec14\"><title>Discussion</title><p id=\"Par31\">Zinc contaminated soil has negative impacts on plants as well as soil microbiome however, such soils are enriched with Zn tolerant PGPR hence, the soil sampling is one of the critical criteria for the isolation of such Zn tolerant PGPRs<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. Many areas in Rajasthan including the Zawar region are contaminated with the toxic amounts of Zn and other heavy metals due to ore mining and other human activities<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. The rhizospheric soil samples of the present study site were contaminated with a high degree of Zn metals/metalloids and could serve a better source for isolation of plant growth promoting ZTB strains. The DTPA extractable Zn is considered to be the bio-available Zn and having a concern with respect to the toxicity to the environment and plant uptake and utilization<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. Yang et al.<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup> studied the soil properties of the different Zn contaminated sites of China and reported that the soil organic matter, available nitrogen and phosphorus were significantly high in rhizosphere soil compare to their bulk soils at Zn contaminated sites reveled the role of associated heavy metal tolerant plant growth promoting rhizospheric bacteria.</p><p id=\"Par32\">Microbial remediation of Zn is due to the several mechanisms viz<italic>.,</italic> biosorption of Zn on the cell surface of microbe through the exopolysaccharide (EPS) secretion, Zn bioaccumulation in the microbe due to the cobalt, zinc and cadmium (CZC) transporter genes and Zn bioprecipitation through the production of sulfide precipitates. The ZTB strains tolerated high Zn concentrations also produced exopolysaccharide (EPS) and the presence of CZC genes were also confirmed through PCR (supplementary data sheet). The resistance by the bacteria to the toxic concentration of Zn is achieved through the two efflux mechanisms mediated by P-type ATPase efflux system and resistance-nodulation-division (RND)-driven transporters system<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Haroun et al.<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup> reported the tolerance of same bacterial strains against heavy metals and the highest degree of tolerance was observed with Zn.</p><p id=\"Par33\">The four ZTB trains showed maximum MIC, belongs to genus <italic>Serratia</italic> based on the 16SrDNA analysis. The <italic>Serratia</italic> sp. was previously reported for the heavy metal tolerance and bioremediation. Cristani et al. reported the role of <italic>Serratia marcescens</italic> in toxic metal bioremediation viz<italic>.,</italic> Pb, Cd and Cr from polluted environments<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. However, the isolation and characterization of <italic>Serratia</italic> sp. in Zn tolerance and bioremediation were not reported earlier and to our best knowledge this is the first detailed report on the application of <italic>Serratia</italic> sp. in Zn bioremediation.</p><p id=\"Par34\">The active mode of Zn accumulation by bacteria is designated as bioaccumulations which varies from organism to organism and mainly depend on to the intrinsic biochemical and structural properties of the bacteria. Results of Zn metal removal studies showed that the 4 ZTB strains remove Zn efficiently from the medium. The capacity of microbes to remove toxic heavy metals from growth medium is significantly influenced by growth conditions. Benmalek and Fardeau reported the Zn biosorption efficiency of <italic>Micrococcus</italic> spp. is 59.55 to 78.90% when 25–100 mg/L of Zn was added in the medium<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. In the present study, similar findings were reported and the Zn accumulation capacities of ZTB were significantly high.</p><p id=\"Par35\">The most possible reasons behind such high heavy metal resistance are due to the phenomena of either bioaccumulation or biosorption. Bioaccumulation of Zn by ZTB strains was evident in AAS studies. The ZTB strains were found to produce significant amount of EPS under Zn stress. EPS mediated Zn biosorption mainly occurs due to the interaction between positively charged Zn ions and negatively charged EPS on the cell surfaces<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. The present finding was supported by the earlier workers<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup> and also confirmed about the ability of alleviating heavy metal stress by microbes.</p><p id=\"Par36\">All ZTB isolates were subjected to various plant growth promoting activities and exhibited multiple PGP traits in vitro which are similar to the earlier findings<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. Plant growth promoting rhizobacteria positively alters plant growth and its productivity by the production of growth regulators viz<italic>.</italic>, IAA, GA<sub>3</sub>, siderophore etc. which increase the nutrient availability to plants<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. Compared to other published reports, ZTB strain of the present study possess maximum PGPR activities so far reported and could be helpful for bioremediation in Zn contaminated agricultural land near Zn mining areas.</p><p id=\"Par37\">Gluconic acid is produced in the periplasm and secreted outside the bacterial cells hence can be studied in the supernatant<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. In the present study all the ZTB strains were able to produce high amount of gluconic acid which non-specifically solubilize Zn, phosphorus, potassium, calcium, manganese etc. from their respective minerals<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup> and moreover may chelate toxic metals resulting in the formation of metallo-organic molecules<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>.</p><p id=\"Par38\">The effects of ZTB strains on the growth of maize plants under Zn<sup>2+</sup> stress were studied. Under Zn<sup>2+</sup> stress, a substantial reduction in shoot length, root length, fresh weight, and total chlorophyll was noticed, however inoculation with ZTB strains resulted in significant enhancement in all the plant growth parameters. Zn stresses (1,000 mg Zn/kg planting mixture) resulted in reduced maize plant growth and also induce oxidative damage<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>. The inoculation of plant growth promoting heavy metal tolerant bacteria reduces the metal toxicity and also improves the nutritional status in plants by complex unknown mechanism<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. Islam et al. reported the inoculation of maize with PGP <italic>Proteus mirabilis</italic> could reduce the negative consequences of oxidative stress caused by heavy metal toxicity<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>.</p><p id=\"Par39\">Crop plants employ detoxifying antioxidative system to maintain ROS at an optimum level. The exposure to the toxic concentrations of heavy metal causes ROS production resulting in high oxidative damage to the crop plant. The antioxidant enzyme i.e. SOD, POD, PAL, PPO and CAT activities in heavy metal stressed plants are depending on the concentration and type of heavy metal, plant species, exposure etc. and most of the cases relatively high compare to control conditions<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. The higher antioxidant enzyme activity with ZTB strains inoculation in the present study might be due to the increased expression of plant antioxidant enzymes compare to un-inoculated plants. These finding were very well supported by the previously published research where the bacterial inoculation activates the gene expression profile of metal detoxifying enzymes to cope up the metal stress<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref>,<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>.</p><p id=\"Par40\">The improved growth of maize plantlet under Zn stress conditions was due to the reduced accumulation and uptake of Zn in the maize plantlet by ZTB inoculation led to the reduced Zn toxicity. This could happen due to the reduced bioavailability and bioaccumulation of Zn by ZTB strains. Similar findings of metal tolerant PGPR inoculation were found very effective upon inoculation in different crops and also conferred metal tolerance<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>.</p><p id=\"Par41\">The ZIP family transporter genes are responsible for Zn uptake from soil by roots, translocation within root system and from root to shoot and also storage of Zn in various plant parts such as fruits, grains etc. Importantly the relative ZIP gene expression varied between shoot and root<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. The finding of the quantitative real-time reverse transcription PCR of the ZIP genes from the present study was very well supported by the findings of Khanna et al.<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. They reported that the enhanced expression of the different metal transporter genes which were further declined in metal tolerant PGPR supplemented plantlets. Hence, the metal tolerant PGPR reduces the heavy metal toxicity and improve the growth of plants on metal contaminated sites.</p></sec><sec id=\"Sec15\"><title>Conclusion</title><p id=\"Par42\">The current study was framed to explore zinc tolerant bacteria (ZTB) for improving plant growth under Zn toxicity and define the mechanistic processes regulating the Zn tolerance. Four potential bacterial strains were isolated from a Zn contaminated agricultural field and were identified as <italic>Serratia</italic> sp. In addition to their high tolerance to Zn, these strains also exhibited various plant growth promoting activities. Moreover, experimental evidences also suggested that ZTB strains produced gluconic acid as natural chelating agents of heavy metals and forms metal complexes. To evaluate the extent of PGPR attributes under Zn toxicity rendered by the ZTB, maize plants were inoculated with the strains. Decreased growth of Zn stressed maize plantlet was possibly attributable to the activation of plant defense mechanism and also to the reduced synthesis of plant growth promoting substances. However, the application of ZTB strains significantly improved the growth, antioxidant enzymes activities and decreased the accumulation of Zn in maize plantlet under Zn stress conditions. The results indicated that the ZTB can be used as microbial inoculants for improving agriculture in Zn contaminated soil and bioremediation of heavy metals in polluted industrial sites. Further to confirm the efficacy of these ZTB, dedicated field studies are required on different crops under Zn stress conditions for the determination of Zn bioremediation potential of these isolates.</p></sec><sec id=\"Sec16\"><title>Methods</title><sec id=\"Sec17\"><title>Soil samples and physico-chemical properties</title><p id=\"Par43\">Rhizospheric soil samples were collected from the Zawar mines areas of Udaipur regions of Southern Rajasthan, India. The collected soil samples were air dried, sieved, and kept for the analysis. Physical and chemical properties of collected soil samples like electrical conductivity (EC), pH, organic carbon (OC) and soil nutrients viz<italic>.,</italic> available nitrogen (Av. N), available phosphorus (Av. P), and available potassium (Av. K) and DTPA extracted Zn were analyzed<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref>–<xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>.</p></sec><sec id=\"Sec18\"><title>Isolation of zinc tolerant bacteria and determination of minimum inhibitory concentration (MIC) of zinc</title><p id=\"Par44\">The Zn tolerant bacteria were isolated by serial dilution and pour plate methods using nutrient agar amended with 1 mM concentration of zinc sulphate heptahydrate. The plates were incubated at 30 °C for 24 h. The MIC of heavy metals at which no colony growth occurred was determined by the agar dilution method<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. All the ZTB isolates were grown on nutrient agar plates with gradually increasing the concentration of the Zn ions. The lowest concentration of Zn ions that inhibited the growth of ZTB was taken as the MIC of that metal.</p></sec><sec id=\"Sec19\"><title>Characterization of the isolates</title><p id=\"Par45\">Morphological and biochemical identification tests of the ZTB were carried out by using the standard protocol outlined in Bergey’s Manual of Systemic Bacteriology. Molecular identification of the ZTB was done using 16S rDNA amplification and sequencing. The universal primers for 16S r DNA viz<italic>.</italic> 27 F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1,492 R (3′-TACGGYTACCTTGTTACGACTT-5′) were used for amplification. The amplified PCR product was further purified gel extraction kit (Sigma) and sequenced directly in an automated DNA Sequencer (ABI Prism 310 Genetic Analyzer, Applied Biosystems, Inc., Foster City, CA). This was followed by assembling the sequences by BioEdit software package<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. Phylogenetic analysis was performed using the obtained aligned sequence followed by the BLAST with the 16S ribosomal RNA sequence (Bacteria and Archaea) nucleotide database. The closest species related to the sequence were retrieved and analyzed by MEGA software 6.0<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. The neighbor joining method was employed with bootstrap values generated from 1,000 replicates for construction of phylogenetic tree.</p></sec><sec id=\"Sec20\"><title>Determination of biosorption potential of zinc tolerant isolates</title><p id=\"Par46\">Biosorption potential of ZTB strains showing higher MIC was determined by atomic absorption spectroscopy (AAS) as the amount of metal present in the supernatant after the treatment with ZTB strains<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. Nutrient broth supplemented with Zn was inoculated with the 1% of overnight grown ZTB isolate and incubated for 72 h in a shaking condition. The cell free supernatants were used to determine the concentration of Zn biosorption using AAS. The biomass of ZTB strains were also recorded after 72 h biosorption and summarized in (supplementary data sheet).</p></sec><sec id=\"Sec21\"><title>Scanning electron microscopy-energy dispersive spectroscopy (SEM–EDS)</title><p id=\"Par47\">The pelleted ZTB cells after biosorption were fixed with 3% glutaraldehyde and further dried under freeze drier (Labtech, India) and placed on the stud surface, there after sputtered with gold particles and imaged with SEM (Carl Zeiss, EVO 181, Germany) operating at 30.0 kV. Further, EDS (Inca Penta FETx3 energy dispersive X-ray system, UK) were also performed to analyses the elemental composition of the surface of ZTB strains after Zn biosorption.</p></sec><sec id=\"Sec22\"><title>Screening for multiple plant growth promoting activities</title><p id=\"Par48\">All the selected ZTB inoculates was screened for multiple plant growth promoting activities viz<italic>.,</italic> IAA, ammonia, HCN, Siderophore production and solubilization of phosphorus, potassium and silica standard procedures<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>. The ACC deaminase activity was quantitatively analyzed based on their ability to use ACC as a sole nitrogen source<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>. Production of GA<sub>3</sub> was carried out using the standard procedure of Berryos et al.<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>. The phytase producing ability of the ZTB were tested on phytase screening medium (PSM) described by Kerovuo et al.<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup>.</p></sec><sec id=\"Sec23\"><title>Gluconic acid production</title><p id=\"Par49\">The ZTB were tested for the production of gluconic acid by using the injecting the culture filtrate of the isolates in to a HPLC system (Waters, Austria) on a reverse-phase C18 column (Nucleosil 100-5 C18, 250 × 4.6 mm, 5 μm). Elution was performed with an isocratic flow consisting of acetonitrile: water (30:70 v/v) with a flow rate of 1.0 mL/min at 210 nm using UV/Vis detector<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>.</p></sec><sec id=\"Sec24\"><title>In vitro studies on the effect of zinc tolerant bacteria on growth and biomass of maize seedling under Zn stress conditions</title><p id=\"Par50\">The pot experiment for selected ZTB isolates showing high MIC values was conducted in plastic pots filled with sterile coco peat/vermiculite/perlite mixture (0.5 kg pot<sup>−1</sup>). Maize cultivable variety (FEM-2) recommended for this agro climatic zone seeds will be used for the in vitro studies. The seeds treated with bacterial inoculant was sown under Zn stress conditions, whereas the uninoculated control under Zn stress condition will also be maintained. The Zn<sup>2+</sup> concentration in the pots was maintained to 1,000 mg Zn/kg planting mixture. The experimental setup was designed as per previous studies by Gupta et al.<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. The pots containing Zn contaminated soil were left for two weeks for stabilization of Zn. Six treatments were set up in quadruplicate in a complete randomized design comprising one plant per pot. The details of the treatments are: T1 (control, uncontaminated soil), T2 (soil containing 1,000 mg Zn/kg planting mixture), T3 (soil containing 1,000 mg Zn/kg planting mixture + ZTB15), T4 (soil containing 1,000 mg Zn/kg planting mixture + ZTB24), T5 (soil containing 1,000 mg Zn/kg planting mixture + ZTB28), T6 soil containing 1,000 mg Zn/kg planting mixture + ZTB29). The pots were watered once in 2 days with sterile distilled water until the completion of the study. The maize seed was surface sterilized using 70% ethanol followed by 3% hypochlorite solution for 3 min and used for germination in pot experiment. The concentration of Zn in the pots will be maintained as 1,000 mg Zn/kg planting mixture and the pots will be left for 2 weeks for metal stabilization. The pots will be set in triplicate in a complete randomized design. Different plant growth parameters like average shoot length, root length, root number, leaf number, chlorophyll content of leaf number will be analyzed. The stress related enzymes viz<italic>.,</italic> catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) will be studies in 14 days old seedlings as per the standard protocols previously published<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>. Analysis of Zn content in the maize plantlet was analyzed using atomic absorption spectroscopy (AAS). Briefly 1.0 g of plant material was digested in di-acid mixture containing concentrated nitric acid and concentrated sulphuric acid at 70 °C followed by diluting the samples in distilled water. The extract was filtered and analyzed for total Zn content using AAS.</p></sec><sec id=\"Sec25\"><title>Gene expression studies using real-time RT PCR</title><p id=\"Par51\">The 14 day old maize plantlets grown under different treatments were utilized for gene expression studies of different ZIP transporter genes using quantitative real time RT-PCR (Bio-Rad “CFX96 Real-Time PCR System”). The primer details are given in Table <xref rid=\"Tab8\" ref-type=\"table\">8</xref>. Total RNA was isolated from 14 days old maize plantlet with TRIzol (Invitrogen). The cDNA synthesis was done from 5 μg of total RNA as a template using iScript cDNA Synthesis Kit (Bio Red, USA). Real-time RT-PCR was performed in a 20 μL reaction containing a 5 μL cDNA,0.4 μM of gene-specific primers and 10 μL of 2X SYBR Green JumpStart Taq ReadyMix (Sigma). The data was assessed in triplicates and <italic>actin</italic> gene was used as house-keeping control gene for normalization purposes. The data was calculated using threshold cycle (Ct) of the amplification curve. The relative gene expression level was assessed using the 2−ΔΔct method<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. The sizes of the amplified fragments were confirmed by gel electrophoresis.<table-wrap id=\"Tab8\"><label>Table 8</label><caption><p>Primers for real-time RT-PCR<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Primer names</th><th align=\"left\">Primer sequences</th></tr></thead><tbody><tr><td align=\"left\">RTZmZIP1F</td><td align=\"left\">5′-CCTCTCTGCGTTGGTTGCTCT-3′</td></tr><tr><td align=\"left\">RTZmZIP1R</td><td align=\"left\">5′-TTGATGGTTGTTTTCTGGTCGT-3′</td></tr><tr><td align=\"left\">RTZmZIP4F</td><td align=\"left\">5′-CCTTCTTCTCGCTCACCGCT-3′</td></tr><tr><td align=\"left\">RTZmZIP4R</td><td align=\"left\">5′-AGCCTCGGGTTGCTGAAGT-3′</td></tr><tr><td align=\"left\">RTZmZIP5F</td><td align=\"left\">5′-GCACATAGGCATAGCCACGC-3′</td></tr><tr><td align=\"left\">RTZmZIP5R</td><td align=\"left\">5′-ACGCCCAAAGATAGCCCGAT-3′</td></tr><tr><td align=\"left\">RTZmZIP8F</td><td align=\"left\">5′-CGTGTCATCGCTCAGGTTCTTG-3′</td></tr><tr><td align=\"left\">RTZmZIP8R</td><td align=\"left\">5′-CCCTCGAACATTTGGTGGAAG-3′</td></tr><tr><td align=\"left\">ZmActin1F</td><td align=\"left\">5′-ATGTTTCCTGGGATTGCCGAT-3′</td></tr><tr><td align=\"left\">ZmActin1R</td><td align=\"left\">5′-CCAGTTTCGTCATACTCTCCCTTG-3′</td></tr></tbody></table></table-wrap></p></sec><sec id=\"Sec26\"><title>Statistical analyses</title><p id=\"Par52\">All the observations recorded were subjected to the statistical analysis viz<italic>.</italic> standard deviation (SD), critical difference (CD), coefficient of variation (CV), etc. using Microsoft Excel 2003. The significant difference among variable treatment were determined by the analysis performed in JMP software<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup> version 11 using Turkey–Kramer HSD test at <italic>p</italic> = 0.05.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec27\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70846_MOESM1_ESM.docx\"><caption><p>Supplementary Information.</p></caption></media></supplementary-material></p></sec></sec></body><back><glossary><title>Abbreviations</title><def-list><def-item><term>Zinc</term><def><p id=\"Par2\">Zn</p></def></def-item><def-item><term>ZTB</term><def><p id=\"Par3\">Zinc tolerant bacteria</p></def></def-item><def-item><term>PGP</term><def><p id=\"Par4\">Plant growth promoting</p></def></def-item><def-item><term>AAS</term><def><p id=\"Par5\">Atomic absorption spectroscopy</p></def></def-item><def-item><term>SEM</term><def><p id=\"Par6\">Scanning electron microscopy</p></def></def-item></def-list></glossary><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70846-w.</p></sec><ack><title>Acknowledgements</title><p>The financial assistance from All India Network Project on soil biodiversity and bio-fertilizers and Rashtriya Krishi Vikas Yojana (RKVY) research project are highly acknowledged. The support of Dean, RCA and Director, Directorate of Research, MPUAT is highly acknowledged for providing necessary facilities and services during the conduct of this research.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>D.J. designed the research. D.J., R.K., K.D.-A., A.A.-B. performed the experiments and interpreted the data. R.H.M. and D.R. performed soil and AAS analysis. A.S. performed HPLC and SEM studies. D.J., A.A.-B., S.R.M. wrote the manuscript. All authors reviewed the manuscript.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par53\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ychounwou</surname><given-names>PB</given-names></name><name><surname>Yedjou</surname><given-names>CG</given-names></name><name><surname>Patlolla</surname><given-names>AK</given-names></name><name><surname>Sutton</surname><given-names>DJ</given-names></name></person-group><article-title>Heavy metal toxicity and the environment</article-title><source>Exp. Suppl.</source><year>2012</year><volume>101</volume><fpage>133</fpage><lpage>164</lpage><pub-id pub-id-type=\"pmid\">22945569</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mihdhir</surname><given-names>AA</given-names></name><name><surname>Assaeed</surname><given-names>SA</given-names></name><name><surname>Abulreesh</surname><given-names>HH</given-names></name><name><surname>Osman</surname><given-names>EH</given-names></name></person-group><article-title>Detection, identification and characterization of some heavy metals tolerant bacteria</article-title><source>J. Microb. Biochem. Technol.</source><year>2016</year><volume>8</volume><issue>3</issue><fpage>226</fpage><lpage>230</lpage></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xie</surname><given-names>X</given-names></name><name><surname>Fu</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>H</given-names></name><name><surname>Liu</surname><given-names>J</given-names></name></person-group><article-title>Heavy metal resistance by two bacteria strains isolated from a copper mine tailing in China</article-title><source>Afr. J. Biotechnol.</source><year>2010</year><volume>9</volume><issue>26</issue><fpage>4055</fpage><lpage>4066</lpage></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>XL</given-names></name><name><surname>Christie</surname><given-names>P</given-names></name></person-group><article-title>Changes in soil solution Zn and pH and uptake of Zn by arbuscular mycorrhizal red clover in Zn-contaminated soil</article-title><source>Chemosphere</source><year>2001</year><volume>42</volume><fpage>201</fpage><lpage>207</lpage><pub-id pub-id-type=\"pmid\">11237299</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wani</surname><given-names>PA</given-names></name><name><surname>Khan</surname><given-names>MS</given-names></name><name><surname>Zaidi</surname><given-names>A</given-names></name></person-group><article-title>Impact of zinc-tolerant plant growth-promoting rhizobacteria on lentil grown in zinc-amended soil</article-title><source>Agron. Sustain. Dev.</source><year>2007</year><volume>28</volume><fpage>449</fpage><lpage>455</lpage></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Khanna</surname><given-names>K</given-names></name><name><surname>Jamwal</surname><given-names>VL</given-names></name><name><surname>Gandhi</surname><given-names>SG</given-names></name><name><surname>Ohri</surname><given-names>P</given-names></name><name><surname>Bhardwaj</surname><given-names>R</given-names></name></person-group><article-title>Metal resistant PGPR lowered Cd uptake and expression of metal transporter genes with improved growth and photosynthetic pigments in <italic>Lycopersicon esculentum</italic> under metal toxicity</article-title><source>Sci. Rep.</source><year>2019</year><volume>9</volume><fpage>5855</fpage><pub-id pub-id-type=\"pmid\">30971817</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Singh</surname><given-names>KK</given-names></name><name><surname>Hasan</surname><given-names>HS</given-names></name><name><surname>Talat</surname><given-names>M</given-names></name><name><surname>Singh</surname><given-names>VK</given-names></name><name><surname>Gangwar</surname><given-names>SK</given-names></name></person-group><article-title>Removal of Cr(VI) from aqueous solutions using wheat bran</article-title><source>J. Chem. Eng.</source><year>2009</year><volume>151</volume><fpage>113</fpage><lpage>121</lpage></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Saranraj</surname><given-names>P</given-names></name><name><surname>Sttela</surname><given-names>D</given-names></name></person-group><article-title>Bioremediation of sugar mill effluent by immobilized bacterial consortium</article-title><source>Int. J. Res. Pure Appl. Microbiol.</source><year>2012</year><volume>2</volume><fpage>43</fpage><lpage>48</lpage></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tsezos</surname><given-names>M</given-names></name><name><surname>Volesky</surname><given-names>B</given-names></name></person-group><article-title>Biosorption of uranium and thorium</article-title><source>Biotechnol. Bioeng.</source><year>1981</year><volume>23</volume><fpage>583</fpage><lpage>604</lpage></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Colangelo</surname><given-names>EP</given-names></name><name><surname>Guerinot</surname><given-names>ML</given-names></name></person-group><article-title>Put the metal to the petal: metal uptake and transport throughout plants</article-title><source>Curr. Opin. Plant Biol.</source><year>2006</year><volume>9</volume><issue>3</issue><fpage>322</fpage><lpage>330</lpage><pub-id pub-id-type=\"pmid\">16616607</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>S</given-names></name><name><surname>Zhou</surname><given-names>X</given-names></name><name><surname>Huang</surname><given-names>Y</given-names></name><name><surname>Zhu</surname><given-names>L</given-names></name><name><surname>Zhang</surname><given-names>S</given-names></name><name><surname>Zhao</surname><given-names>Y</given-names></name><name><surname>Guo</surname><given-names>J</given-names></name><name><surname>Chen</surname><given-names>J</given-names></name><name><surname>Chen</surname><given-names>R</given-names></name></person-group><article-title>Identification and characterization of the zinc-regulated transporters, iron-regulated transporter-like protein (ZIP) gene family in maize</article-title><source>BMC Plant Biol.</source><year>2013</year><volume>13</volume><fpage>114</fpage><pub-id pub-id-type=\"pmid\">23924433</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kour</surname><given-names>R</given-names></name><name><surname>Jain</surname><given-names>D</given-names></name><name><surname>Bhojiya</surname><given-names>AA</given-names></name><etal/></person-group><article-title>Zinc biosorption, biochemical and molecular characterization of plant growth-promoting zinc-tolerant bacteria</article-title><source>3Biotech.</source><year>2019</year><volume>9</volume><fpage>421</fpage><pub-id pub-id-type=\"doi\">10.1007/s13205-019-1959-2</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ortiz-Ojeda</surname><given-names>P</given-names></name><name><surname>Ogata-Gutiérrez</surname><given-names>K</given-names></name><name><surname>Zúñiga-Dávila</surname><given-names>D</given-names></name></person-group><article-title>Evaluation of plant growth promoting activity and heavy metal tolerance of psychrotrophic bacteria associated with maca (<italic>Lepidium meyenii </italic>Walp.) rhizosphere</article-title><source>AIMS Microbiol.</source><year>2017</year><volume>3</volume><issue>2</issue><fpage>279</fpage><lpage>292</lpage><pub-id pub-id-type=\"doi\">10.3934/microbiol.2017.2.279</pub-id><pub-id pub-id-type=\"pmid\">31294161</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bhojiya</surname><given-names>AA</given-names></name><name><surname>Joshi</surname><given-names>H</given-names></name></person-group><article-title>Study of potential plant growth-promoting activities and heavy metal tolerance of <italic>Pseudomonas aeruginosa</italic> HMR16 isolated from Zawar, Udaipur India.</article-title><source>Curr. Trends Biotechnol. Pharm.</source><year>2016</year><volume>10</volume><issue>2</issue><fpage>161</fpage><lpage>168</lpage></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>B</given-names></name><name><surname>Shen</surname><given-names>J</given-names></name><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Pan</surname><given-names>F</given-names></name><name><surname>Yang</surname><given-names>X</given-names></name><name><surname>Feng</surname><given-names>Y</given-names></name></person-group><article-title>The endophytic bacterium, <italic>Sphingomonas</italic> SaMR12, improves the potential for zinc phytoremediation by its host <italic>Sedum alfredii</italic></article-title><source>PLoS ONE</source><year>2014</year><volume>9</volume><issue>9</issue><fpage>e106826</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0106826</pub-id><pub-id pub-id-type=\"pmid\">25198772</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Afzal</surname><given-names>AM</given-names></name><name><surname>Rasool</surname><given-names>MH</given-names></name><name><surname>Waseem</surname><given-names>M</given-names></name><name><surname>Aslam</surname><given-names>B</given-names></name></person-group><article-title>Assessment of heavy metal tolerance and biosorptive potential of <italic>Klebsiella variicola</italic> isolated from industrial effluents</article-title><source>AMB Express</source><year>2017</year><volume>7</volume><fpage>184</fpage><pub-id pub-id-type=\"doi\">10.1186/s13568-017-0482-2</pub-id><pub-id pub-id-type=\"pmid\">28963704</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Redmile-Gordon</surname><given-names>M</given-names></name><name><surname>Chen</surname><given-names>L</given-names></name></person-group><article-title>Zinc toxicity stimulates microbial production of extracellular polymers in a copiotrophic acid soil</article-title><source>Int. Biodeter. Biodegr.</source><year>2017</year><volume>119</volume><fpage>413</fpage><lpage>418</lpage></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mishra</surname><given-names>J</given-names></name><name><surname>Singh</surname><given-names>R</given-names></name><name><surname>Arora</surname><given-names>NK</given-names></name></person-group><article-title>Alleviation of heavy metal stress in plants and remediation of soil by rhizosphere microorganisms</article-title><source>Front. Microbiol.</source><year>2017</year><volume>8</volume><fpage>1706</fpage><pub-id pub-id-type=\"pmid\">28932218</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ali</surname><given-names>A</given-names></name><name><surname>Guo</surname><given-names>D</given-names></name><name><surname>Mahar</surname><given-names>A</given-names></name><name><surname>Ma</surname><given-names>F</given-names></name><name><surname>Li</surname><given-names>R</given-names></name><name><surname>Shen</surname><given-names>F</given-names></name><name><surname>Wang</surname><given-names>P</given-names></name><name><surname>Zengqiang</surname><given-names>Z</given-names></name></person-group><article-title><italic>Streptomyces pactum</italic> assisted phytoremediation in Zn/Pb smelter contaminated soil of Feng County and its impact on enzymatic activities</article-title><source>Sci. Rep.</source><year>2017</year><volume>7</volume><fpage>46087</fpage><pub-id pub-id-type=\"pmid\">28387235</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>Y</given-names></name><name><surname>Dong</surname><given-names>M</given-names></name><name><surname>Cao</surname><given-names>Y</given-names></name><name><surname>Wang</surname><given-names>J</given-names></name><name><surname>Tang</surname><given-names>M</given-names></name><name><surname>Ban</surname><given-names>Y</given-names></name></person-group><article-title>Comparisons of soil properties, enzyme activities and microbial communities in heavy metal contaminated bulk and rhizosphere soils of <italic>Robinia pseudoacacia</italic> L. in the northern foot of Qinling mountain</article-title><source>Forests.</source><year>2017</year><volume>8</volume><fpage>430</fpage></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Spain</surname><given-names>A</given-names></name><name><surname>Alm</surname><given-names>E</given-names></name></person-group><article-title>Implications of microbial heavy metal tolerance in the environment</article-title><source>Rev. Undergrad. Res.</source><year>2003</year><volume>2</volume><fpage>1</fpage><lpage>6</lpage></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haroun</surname><given-names>AA</given-names></name><name><surname>Kamaluddeen</surname><given-names>KK</given-names></name><name><surname>Alhaji</surname><given-names>I</given-names></name><name><surname>Magaji</surname><given-names>Y</given-names></name><name><surname>Oaikhena</surname><given-names>EE</given-names></name></person-group><article-title>Evaluation of heavy metal tolerance level (MIC) and bioremediation potentials of <italic>Pseudomonas aeruginosa</italic> isolated from Makera-Kakuri industrial drain in Kaduna Nigeria.</article-title><source>Eur. Exp. Biol.</source><year>2017</year><volume>7</volume><issue>5</issue><fpage>28</fpage></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cristani</surname><given-names>M</given-names></name><name><surname>Naccari</surname><given-names>C</given-names></name><name><surname>Nostro</surname><given-names>A</given-names></name><name><surname>Pizzimenti</surname><given-names>A</given-names></name><name><surname>Trombetta</surname><given-names>D</given-names></name><name><surname>Pizzimenti</surname><given-names>F</given-names></name></person-group><article-title>Possible use of <italic>Serratia marcescens</italic> in toxic metal biosorption (removal)</article-title><source>Environ. Sci. Pollut. Res.</source><year>2012</year><volume>19</volume><fpage>161</fpage><lpage>168</lpage></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Benmalek</surname><given-names>Y</given-names></name><name><surname>Fardeau</surname><given-names>M-L</given-names></name></person-group><article-title>Isolation and characterization of metal-resistant bacterial strain from wastewater and evaluation of its capacity in metal-ions removal using living and dry bacterial cells</article-title><source>Int. J. Environ. Sci. Technol.</source><year>2016</year><volume>13</volume><fpage>2153</fpage><lpage>2162</lpage></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gupta</surname><given-names>P</given-names></name><name><surname>Diwan</surname><given-names>B</given-names></name></person-group><article-title>Bacterial exopolysaccharide mediated heavy metal removal: a review on biosynthesis, mechanism and remediation strategies</article-title><source>Biotechnol. Rep.</source><year>2017</year><volume>13</volume><fpage>58</fpage><lpage>71</lpage></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pramanik</surname><given-names>K</given-names></name><name><surname>Mitra</surname><given-names>S</given-names></name><name><surname>Sarkar</surname><given-names>A</given-names></name><name><surname>Soren</surname><given-names>T</given-names></name><name><surname>Maiti</surname><given-names>TK</given-names></name></person-group><article-title>Characterization of cadmium-resistant <italic>Klebsiella pneumonia</italic> MCC 3091 promoted rice seedling growth by alleviating phytotoxicity of cadmium</article-title><source>Environ. Sci. Pollut. Res.</source><year>2017</year><volume>24</volume><issue>31</issue><fpage>24419</fpage><lpage>24437</lpage></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pramanik</surname><given-names>K</given-names></name><name><surname>Mitra</surname><given-names>S</given-names></name><name><surname>Sarkar</surname><given-names>A</given-names></name><name><surname>Maiti</surname><given-names>TK</given-names></name></person-group><article-title>Alleviation of phytotoxic effects of cadmium on rice seedlings by cadmium resistant PGPR strain <italic>Enterobacter aerogenes</italic> MCC 3092</article-title><source>J. Hazard. Mater.</source><year>2018</year><volume>5</volume><issue>351</issue><fpage>317</fpage><lpage>329</lpage></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nakbanpote</surname><given-names>W</given-names></name><name><surname>Panitlurtumpai</surname><given-names>N</given-names></name><name><surname>Sangdee</surname><given-names>A</given-names></name><name><surname>Sakulpone</surname><given-names>N</given-names></name><name><surname>Sirisom</surname><given-names>P</given-names></name><name><surname>Pimthong</surname><given-names>A</given-names></name></person-group><article-title>Salt-tolerant and plant growth-promoting bacteria isolated from Zn/Cd contaminated soil: identification and effect on rice under saline conditions</article-title><source>J. Plant Interact.</source><year>2014</year><volume>9</volume><issue>1</issue><fpage>379</fpage><lpage>387</lpage></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tiwari</surname><given-names>S</given-names></name><name><surname>Lata</surname><given-names>C</given-names></name></person-group><article-title>Heavy metal stress, signaling, and tolerance due to plant-associated microbes: an overview</article-title><source>Front. Plant Sci.</source><year>2018</year><volume>9</volume><fpage>452</fpage><pub-id pub-id-type=\"pmid\">29681916</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Perez</surname><given-names>E</given-names></name><name><surname>Sulbaran</surname><given-names>M</given-names></name><name><surname>Ball</surname><given-names>M</given-names></name><name><surname>Yarzabal</surname><given-names>LA</given-names></name></person-group><article-title>Isolation and characterization of mineral phosphate-solubilizing bacteria naturally colonizing a limonitic crust in the south-eastern Venezuelan region</article-title><source>Soil Biol. Biochem.</source><year>2007</year><volume>39</volume><fpage>2905</fpage><lpage>2914</lpage></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Uroz</surname><given-names>S</given-names></name><name><surname>Calvaruso</surname><given-names>C</given-names></name><name><surname>Turpault</surname><given-names>MP</given-names></name><name><surname>FreyKlett</surname><given-names>P</given-names></name></person-group><article-title>Mineral weathering by bacteria: ecology, actors and mechanisms</article-title><source>Trends Microbiol.</source><year>2009</year><volume>17</volume><issue>8</issue><fpage>378</fpage><lpage>387</lpage><pub-id pub-id-type=\"pmid\">19660952</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Seneviratne</surname><given-names>M</given-names></name><name><surname>Seneviratne</surname><given-names>G</given-names></name><name><surname>Madawala</surname><given-names>H</given-names></name><name><surname>Vithanage</surname><given-names>M</given-names></name></person-group><person-group person-group-type=\"editor\"><name><surname>Singh</surname><given-names>JS</given-names></name><name><surname>Seneviratne</surname><given-names>G</given-names></name></person-group><article-title>Role of rhizospheric microbes in heavy metal uptake by plants</article-title><source>Agro-Environmental Sustainability: Managing Environmental Pollution</source><year>2017</year><publisher-loc>Cham</publisher-loc><publisher-name>Springer</publisher-name><fpage>147</fpage><lpage>163</lpage></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cui</surname><given-names>Y</given-names></name><name><surname>Zhao</surname><given-names>N</given-names></name></person-group><article-title>Oxidative stress and change in plant metabolism of maize (<italic>Zea mays</italic> L.) growing in contaminated soil with elemental sulphur and toxic effect of zinc</article-title><source>Plant Soil Environ.</source><year>2011</year><volume>57</volume><issue>1</issue><fpage>34</fpage><lpage>39</lpage></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Islam</surname><given-names>F</given-names></name><name><surname>Yasmeen</surname><given-names>T</given-names></name><name><surname>Riaz</surname><given-names>M</given-names></name><name><surname>Arif</surname><given-names>MS</given-names></name><name><surname>Ali</surname><given-names>S</given-names></name><name><surname>Raza</surname><given-names>SH</given-names></name></person-group><article-title><italic>Proteus mirabilis</italic> alleviates zinc toxicity by preventing oxidative stress in maize (<italic>Zea mays</italic>) plants</article-title><source>Ecotoxicol. Environ. Saf.</source><year>2014</year><volume>110</volume><fpage>143</fpage><lpage>152</lpage><pub-id pub-id-type=\"pmid\">25240234</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sharma</surname><given-names>SS</given-names></name><name><surname>Dietz</surname><given-names>KJ</given-names></name></person-group><article-title>The relationship between metal toxicity and cellular redox imbalance</article-title><source>Trends Plant Sci.</source><year>2008</year><volume>14</volume><fpage>43</fpage><lpage>50</lpage><pub-id pub-id-type=\"pmid\">19070530</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Islam</surname><given-names>F</given-names></name><name><surname>Yasmeen</surname><given-names>T</given-names></name><name><surname>Ali</surname><given-names>Q</given-names></name><name><surname>Mubin</surname><given-names>M</given-names></name><name><surname>Ali</surname><given-names>S</given-names></name><name><surname>Arif</surname><given-names>MS</given-names></name><name><surname>Hussain</surname><given-names>S</given-names></name><name><surname>Riaz</surname><given-names>M</given-names></name><name><surname>Abbas</surname><given-names>F</given-names></name></person-group><article-title>Copper-resistant bacteria reduces oxidative stress and uptake of copper in lentil plants: potential for bacterial bioremediation</article-title><source>Environ. Sci. Pollut. Res. Int.</source><year>2016</year><volume>23</volume><issue>1</issue><fpage>220</fpage><lpage>233</lpage><pub-id pub-id-type=\"pmid\">26387695</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gupta</surname><given-names>P</given-names></name><name><surname>Rani</surname><given-names>R</given-names></name><name><surname>Chandra</surname><given-names>A</given-names></name><name><surname>Kumar</surname><given-names>V</given-names></name></person-group><article-title>Potential applications of <italic>Pseudomonas</italic> sp. (strain CPSB21) to ameliorate Cr<sup>6+</sup> stress and phytoremediation of tannery effluent contaminated agricultural soils</article-title><source>Sci. Rep.</source><year>2018</year><volume>8</volume><fpage>4860</fpage><pub-id pub-id-type=\"pmid\">29559691</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Han</surname><given-names>H</given-names></name><name><surname>Wang</surname><given-names>Q</given-names></name><name><surname>He</surname><given-names>LY</given-names></name><name><surname>Sheng</surname><given-names>XF</given-names></name></person-group><article-title>Increased biomass and reduced rapeseed Cd accumulation of oilseed rape in the presence of Cd-immobilizing and polyamine-producing bacteria</article-title><source>J. Hazard. Mater.</source><year>2018</year><volume>353</volume><fpage>280</fpage><lpage>289</lpage><pub-id pub-id-type=\"pmid\">29677530</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ozlu</surname><given-names>E</given-names></name><name><surname>Kumar</surname><given-names>S</given-names></name></person-group><article-title>Response of soil organic carbon, pH, electrical conductivity, and water stable aggregates to long-term annual manure and inorganic fertilizer</article-title><source>Soil Sci. Soc. Am. J.</source><year>2018</year><volume>82</volume><fpage>1243</fpage><lpage>1251</lpage><pub-id pub-id-type=\"doi\">10.2136/sssaj2018.02.0082</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Qaswar</surname><given-names>M</given-names></name><name><surname>Ahmed</surname><given-names>W</given-names></name><name><surname>Jing</surname><given-names>H</given-names></name><name><surname>Hongzhu</surname><given-names>F</given-names></name><etal/></person-group><article-title>Soil carbon (C), nitrogen (N) and phosphorus (P) stoichiometry drives phosphorus lability in paddy soil under long term fertilization: a fractionation and path analysis study</article-title><source>PLoS ONE</source><year>2019</year><volume>14</volume><issue>6</issue><fpage>0218195</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0218195</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lindsay</surname><given-names>WL</given-names></name><name><surname>Norvell</surname><given-names>WA</given-names></name></person-group><article-title>Development of a DTPA soil test for zinc, iron, manganese, and copper</article-title><source>Soil Sci. Soc. Am. J.</source><year>1978</year><volume>42</volume><issue>3</issue><fpage>421</fpage><pub-id pub-id-type=\"doi\">10.2136/sssaj1978.03615995004200030009</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luli</surname><given-names>GW</given-names></name><name><surname>Talnagi</surname><given-names>JW</given-names></name><name><surname>Strohl</surname><given-names>WR</given-names></name><name><surname>Pfister</surname><given-names>RM</given-names></name></person-group><article-title>Hexavalent chromium resistant bacteria isolated from river sediments</article-title><source>Appl. Environ. Microbiol.</source><year>1983</year><volume>46</volume><issue>4</issue><fpage>846</fpage><lpage>854</lpage><pub-id pub-id-type=\"pmid\">6639032</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tippmann</surname><given-names>H-F</given-names></name></person-group><article-title>Analysis for free: comparing programs for sequence analysis</article-title><source>Brief Bioinform.</source><year>2004</year><volume>5</volume><issue>1</issue><fpage>82</fpage><lpage>87</lpage><pub-id pub-id-type=\"pmid\">15153308</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tamura</surname><given-names>K</given-names></name><name><surname>Stecher</surname><given-names>G</given-names></name><name><surname>Peterson</surname><given-names>D</given-names></name><name><surname>Filipski</surname><given-names>A</given-names></name><name><surname>Kumar</surname><given-names>S</given-names></name></person-group><article-title>Molecular evolutionary genetics analysis version 6.0</article-title><source>Mol. Biol. Evol.</source><year>2013</year><volume>30</volume><fpage>2725</fpage><lpage>2729</lpage><pub-id pub-id-type=\"pmid\">24132122</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krishna</surname><given-names>MP</given-names></name><name><surname>Varghese</surname><given-names>R</given-names></name><name><surname>Babu</surname><given-names>VA</given-names></name><name><surname>Jyothy</surname><given-names>S</given-names></name><name><surname>Hatha</surname><given-names>AA</given-names></name></person-group><article-title>Bioremediation of zinc using <italic>Bacillus</italic> sp. isolated from metal-contaminated industrial zone</article-title><source>Prospect. Biosci.</source><year>2013</year><volume>2</volume><fpage>11</fpage><lpage>18</lpage></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dinesh</surname><given-names>R</given-names></name><name><surname>Srinivasan</surname><given-names>V</given-names></name><name><surname>Hamza</surname><given-names>S</given-names></name><name><surname>Sarathambal</surname><given-names>C</given-names></name><name><surname>Gowda</surname><given-names>SJ</given-names></name><name><surname>Ganeshamurthy</surname><given-names>AN</given-names></name><name><surname>Gupta</surname><given-names>SB</given-names></name><name><surname>Nair</surname><given-names>AV</given-names></name><name><surname>Subila</surname><given-names>KP</given-names></name><name><surname>Lijina</surname><given-names>A</given-names></name><name><surname>Divya</surname><given-names>VC</given-names></name></person-group><article-title>Isolation and characterization of potential Zn solubilizing bacteria from soil and its effects on soil Zn release rates, soil available Zn and plant Zn content</article-title><source>Geoderma</source><year>2018</year><volume>321</volume><fpage>173</fpage><lpage>186</lpage></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Penrose</surname><given-names>DM</given-names></name><name><surname>Glick</surname><given-names>BR</given-names></name></person-group><article-title>Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria</article-title><source>Physiol. Plant</source><year>2003</year><volume>118</volume><fpage>10</fpage><lpage>15</lpage><pub-id pub-id-type=\"pmid\">12702008</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Berryos</surname><given-names>J</given-names></name><name><surname>Illanes</surname><given-names>A</given-names></name><name><surname>Aroca</surname><given-names>G</given-names></name></person-group><article-title>Spectrophotometric method for determining gibberellic acid in fermentation broths</article-title><source>Biotechnol. Lett.</source><year>2004</year><volume>26</volume><fpage>67</fpage><lpage>70</lpage><pub-id pub-id-type=\"pmid\">15005155</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kerovuo</surname><given-names>J</given-names></name><name><surname>Lauraeus</surname><given-names>M</given-names></name><name><surname>Nurminen</surname><given-names>P</given-names></name><name><surname>Kalkkinen</surname><given-names>N</given-names></name><name><surname>Apajalahti</surname><given-names>J</given-names></name></person-group><article-title>Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from <italic>Bacillus subtilis</italic></article-title><source>Appl. Environ. Microbiol.</source><year>1998</year><volume>64</volume><issue>6</issue><fpage>2079</fpage><lpage>2085</lpage><pub-id pub-id-type=\"pmid\">9603817</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Singh</surname><given-names>OV</given-names></name><name><surname>Pereira</surname><given-names>BMJ</given-names></name><name><surname>Singh</surname><given-names>RP</given-names></name></person-group><article-title>Isolation and characterization of a potent fungal strain <italic>Aspergillus niger</italic> ORS-4 for gluconic acid production</article-title><source>J. Sci. Ind. Res.</source><year>1999</year><volume>58</volume><fpage>594</fpage><lpage>600</lpage></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>D</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Chen</surname><given-names>M</given-names></name><name><surname>He</surname><given-names>X</given-names></name><name><surname>Li</surname><given-names>J</given-names></name><name><surname>An</surname><given-names>R</given-names></name></person-group><article-title>Defense mechanisms of two pioneer submerged plants during their optimal performance period in the bioaccumulation of lead: a comparative study</article-title><source>Int. J. Environ. Res. Public Health.</source><year>2018</year><volume>15</volume><issue>12</issue><fpage>2844</fpage><pub-id pub-id-type=\"doi\">10.3390/ijerph15122844</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><collab>SAS</collab></person-group><source>JMP: User’s Guide, Version 8</source><year>2009</year><publisher-loc>Cary</publisher-loc><publisher-name>SAS Institute, INC.</publisher-name></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807818</article-id><article-id pub-id-type=\"pmc\">PMC7431564</article-id><article-id pub-id-type=\"publisher-id\">70825</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70825-1</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Allergic diseases do not impair the cognitive development of children but do damage the mental health of their caregivers</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Kuo</surname><given-names>Ho-Chang</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Chang</surname><given-names>Ling-Sai</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Tsai</surname><given-names>Zi-Yu</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Wang</surname><given-names>Liang-Jen</given-names></name><address><email>wangliangjen@gmail.com</email></address><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.413804.a</institution-id><institution>Department of Pediatrics and Kawasaki Disease Center, </institution><institution>Kaohsiung Chang Gung Memorial Hospital, </institution></institution-wrap>Kaohsiung, Taiwan </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.145695.a</institution-id><institution>Chang Gung University College of Medicine, </institution></institution-wrap>Kaohsiung, 83301 Taiwan </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.145695.a</institution-id><institution>Department of Child and Adolescent Psychiatry, </institution><institution>Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, </institution></institution-wrap>No.123, Ta-Pei Road, Kaohsiung, Taiwan </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13854</elocation-id><history><date date-type=\"received\"><day>1</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>3</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">This study aimed to investigate whether children with atopic diseases exhibited different neurodevelopment function from healthy controls and whether their caregivers had differential parental stress. In total, we recruited 109 patients with atopic diseases (mean age 6.8 years, 54.1% male) and 82 healthy children (mean age 6.3 years, 54.9% male). Based on the children’s age, they underwent developmental, cognitive evaluations and attention deficit/hyperactivity disorder (ADHD) symptoms. The parenting stress of children’s caregivers was evaluated using the Chinese Health Questionnaire (CHQ-12) and Family APGAR. Of the children with atopic diseases, 87.2%, 74.3%, 29.4%, and 8.3% of them had allergic rhinitis, asthma, atopic dermatitis, and urticaria, respectively. None of these conditions were associated with children’s cognitive profiles or ADHD symptoms. However, the caregivers of patients who had asthma suffered from higher CHQ-12 scores than those of patients without asthma. Furthermore, the number of atopic diseases had a dose–response effect on caregivers’ CHQ-12 scores. In conclusion, allergic diseases did not impair the cognitive development of children. However, caregivers of patients with asthma or multiple atopic diseases may suffer a greater mental health burden with regard to caring for their children. Such caregivers may require support to effectively fulfill their parenting roles.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Allergy</kwd><kwd>Asthma</kwd></kwd-group><funding-group><award-group><funding-source><institution>Ministry of Science and Technology, Taiwan</institution></funding-source><award-id>MOST 108-2314-B-182 -037 -MY3</award-id><principal-award-recipient><name><surname>Kuo</surname><given-names>Ho-Chang</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>Kaohsiung Chang Gung Memorial Hospital</institution></funding-source><award-id>CMRPG8C1082</award-id><award-id>CMRPG8E1613</award-id><award-id>CMRPG8D1561-2</award-id><award-id>CMRPG8D0521</award-id></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Atopy refers to the genetic susceptibility to developing allergy-related diseases<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. Atopic diseases, including allergic rhinitis<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>, asthma<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>, atopic eczema/dermatitis<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>, and urticaria<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> are common in children and present many management challenges for their caregivers<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>–<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. Allergies are immune responses and usually involve chronic inflammation<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. However, whether inflammation and irritation increase the risk of neuropsychological consequences in children with atopic diseases is still unclear<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>–<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. For example, allergic rhinitis is associated with significantly impaired mental health and impaired cognitive function<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Asthma is a chronic respiratory disease, but has no negative impact on patients’ intellectual quotient (IQ)<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>.\n</p><p id=\"Par3\">In addition, some studies have shown that the relationship between immune response and the central nervous system (CNS) may make children more susceptible to neuropsychological disorders, such as attention deficit hyperactivity disorder (ADHD)<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. Various allergic diseases, such as allergic rhinitis, atopic dermatitis, or asthma, have been associated with ADHD<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>–<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. Several epidemiological studies using large databases have suggested that ADHD is associated with atopic dermatitis in children<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. However, another population study revealed that children with atopic dermatitis did not have a significantly increased prevalence of ADHD<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>.</p><p id=\"Par4\">In addition, caring for children with atopic diseases can be a time-consuming task that can lead to mental health burdens on the caregiver and cause a decline in his or her psychosocial function<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. Deficient sleep with poor quality in the caregivers of children with chronic illnesses may have a significant impact on their health and well-being, as well as on their caregiving responsibilities<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. However, it is unclear whether specific atopic diseases affect patients' cognitive development or impose pressure on caregivers. Moreover, atopic diseases are usually comorbid, and whether the number of atopic diseases showed a dose-related effect on patients’ neurocognitive outcomes and caregivers’ parental stress remains unclear.</p><p id=\"Par5\">We hypothesized that chronic atopic diseases may have detrimental effects on neurodevelopment, and caregivers of patients with atopic diseases may experience great mental health burden with regard to caring for their children. Therefore, we conducted a clinical survey to investigate whether children with atopic diseases and healthy controls exhibit different neurodevelopmental functions, and whether their caregivers have different parental stressors. We also investigated whether the number of atopic diseases showed a dose-related effect on the outcomes of the above mentioned children and parents.</p></sec><sec id=\"Sec2\"><title>Results</title><p id=\"Par6\">The clinical cohort consisted of 109 patients with atopic diseases (mean age 6.8 years, 54.1% male) and 82 healthy children (mean age 6.3 years, 54.9% male) (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>), with no significant differences in age or gender between the atopic disease children and the controls. We observed no significant differences in the cognitive scores or SNAP-IV scores between the children with atopic diseases and the healthy controls. With regard to caregiver characteristics, no significant difference in age, sex, or relation to the patients was observed between the caregivers of patients with atopic diseases and those of the controls. Furthermore, the caregivers of the atopic disease children had higher CHQ scores than the control group, but we found no difference in family APGAR scores.<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Characteristics and development of children with allergic diseases and control subjects.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Variables</th><th align=\"left\" colspan=\"2\">Allergic diseases (N = 109)</th><th align=\"left\" colspan=\"2\">Controls (N = 82)</th><th align=\"left\" rowspan=\"2\">Statistics</th><th align=\"left\" rowspan=\"2\">P-value</th></tr><tr><th align=\"left\">Mean or N</th><th align=\"left\">SD or %</th><th align=\"left\">Mean or N</th><th align=\"left\">SD or %</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"7\"><bold>Characteristics of children</bold></td></tr><tr><td align=\"left\">Age (years)</td><td align=\"left\">6.8</td><td char=\".\" align=\"char\">2.3</td><td align=\"left\">6.3</td><td char=\".\" align=\"char\">3.0</td><td char=\".\" align=\"char\">1.113</td><td char=\".\" align=\"char\">0.267</td></tr><tr><td align=\"left\"><bold>Sex</bold></td><td align=\"left\"/><td char=\".\" align=\"char\"/><td align=\"left\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\">0.011</td><td char=\".\" align=\"char\">0.918</td></tr><tr><td align=\"left\">Female</td><td align=\"left\">50</td><td char=\".\" align=\"char\">45.9</td><td align=\"left\">37</td><td char=\".\" align=\"char\">45.1</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Male</td><td align=\"left\">59</td><td char=\".\" align=\"char\">54.1</td><td align=\"left\">45</td><td char=\".\" align=\"char\">54.9</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\" colspan=\"7\"><bold>Allergic diseases</bold></td></tr><tr><td align=\"left\">Allergic rhinitis</td><td align=\"left\">95</td><td char=\".\" align=\"char\">87.2</td><td align=\"left\">–</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Asthma</td><td align=\"left\">81</td><td char=\".\" align=\"char\">74.3</td><td align=\"left\">–</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Atopic dermatitis</td><td align=\"left\">32</td><td char=\".\" align=\"char\">29.4</td><td align=\"left\">–</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Urticaria</td><td align=\"left\">9</td><td char=\".\" align=\"char\">8.3</td><td align=\"left\">–</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\" colspan=\"7\"><bold>Number of allergic diseases</bold></td></tr><tr><td align=\"left\">1</td><td align=\"left\">23</td><td char=\".\" align=\"char\">21.1</td><td align=\"left\">–</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">2</td><td align=\"left\">65</td><td char=\".\" align=\"char\">59.6</td><td align=\"left\">–</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">≥ 3</td><td align=\"left\">21</td><td char=\".\" align=\"char\">19.3</td><td align=\"left\">–</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\" colspan=\"7\"><bold>Outcomes</bold></td></tr><tr><td align=\"left\">Intelligence Quotient</td><td align=\"left\">108.9</td><td char=\".\" align=\"char\">13.7</td><td align=\"left\">108.0</td><td char=\".\" align=\"char\">16.5</td><td char=\".\" align=\"char\">0.423</td><td char=\".\" align=\"char\">0.673</td></tr><tr><td align=\"left\">SNAP-IV</td><td align=\"left\">24.6</td><td char=\".\" align=\"char\">10.7</td><td align=\"left\">22.7</td><td char=\".\" align=\"char\">14.2</td><td char=\".\" align=\"char\">0.928</td><td char=\".\" align=\"char\">0.355</td></tr><tr><td align=\"left\" colspan=\"7\"><bold>Characteristics of caregivers</bold></td></tr><tr><td align=\"left\">Age (years)</td><td align=\"left\">38.8</td><td char=\".\" align=\"char\">4.9</td><td align=\"left\">38.0</td><td char=\".\" align=\"char\">4.9</td><td char=\".\" align=\"char\">1.146</td><td char=\".\" align=\"char\">0.253</td></tr><tr><td align=\"left\"><bold>Sex</bold></td><td align=\"left\"/><td char=\".\" align=\"char\"/><td align=\"left\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\">0.063</td><td char=\".\" align=\"char\">0.802</td></tr><tr><td align=\"left\">Female</td><td align=\"left\">88</td><td char=\".\" align=\"char\">80.7</td><td align=\"left\">65</td><td char=\".\" align=\"char\">79.3</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Male</td><td align=\"left\">21</td><td char=\".\" align=\"char\">19.3</td><td align=\"left\">17</td><td char=\".\" align=\"char\">20.7</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"><bold>Education levels</bold></td><td align=\"left\"/><td char=\".\" align=\"char\"/><td align=\"left\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\">0.894</td><td char=\".\" align=\"char\">0.640</td></tr><tr><td align=\"left\">High school or lower</td><td align=\"left\">37</td><td char=\".\" align=\"char\">33.9</td><td align=\"left\">28</td><td char=\".\" align=\"char\">34.1</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">College</td><td align=\"left\">56</td><td char=\".\" align=\"char\">51.4</td><td align=\"left\">38</td><td char=\".\" align=\"char\">46.3</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Master or above</td><td align=\"left\">16</td><td char=\".\" align=\"char\">14.7</td><td align=\"left\">16</td><td char=\".\" align=\"char\">19.5</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"><bold>Relation to the patients</bold></td><td align=\"left\"/><td char=\".\" align=\"char\"/><td align=\"left\"/><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\">0.106</td><td char=\".\" align=\"char\">0.948</td></tr><tr><td align=\"left\">Mother</td><td align=\"left\">84</td><td char=\".\" align=\"char\">77.1</td><td align=\"left\">62</td><td char=\".\" align=\"char\">75.6</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Father</td><td align=\"left\">18</td><td char=\".\" align=\"char\">16.5</td><td align=\"left\">15</td><td char=\".\" align=\"char\">18.3</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Others</td><td align=\"left\">7</td><td char=\".\" align=\"char\">6.4</td><td align=\"left\">5</td><td char=\".\" align=\"char\">6.1</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\" colspan=\"7\"><bold>Mental Health</bold></td></tr><tr><td align=\"left\">CHQ</td><td align=\"left\">3.1</td><td char=\".\" align=\"char\">2.0</td><td align=\"left\">2.1</td><td char=\".\" align=\"char\">2.4</td><td char=\".\" align=\"char\">3.069</td><td char=\".\" align=\"char\">0.003</td></tr><tr><td align=\"left\">Family APGAR</td><td align=\"left\">7.5</td><td char=\".\" align=\"char\">2.8</td><td align=\"left\">7.4</td><td char=\".\" align=\"char\">2.7</td><td char=\".\" align=\"char\">0.158</td><td char=\".\" align=\"char\">0.874</td></tr></tbody></table><table-wrap-foot><p>Data are expressed as mean ± SD or n (%).</p></table-wrap-foot></table-wrap></p><p id=\"Par7\">Of the children with atopic diseases, 87.2%, 74.3%, 29.4%, and 8.3% of them suffered from allergic rhinitis, asthma, atopic dermatitis, and urticaria, respectively. Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref> shows the effects of allergic diseases on children’s cognitive function and caregivers’ mental health. We found that asthma was positively correlated to caregivers’ CHQ scores (t = 3.069, p = 0.003). However, allergic rhinitis, asthma, atopic dermatitis, and urticaria did not exhibit an individual effect on children’s cognitive scores, SNAP-IV scores, or caregivers’ family APGAR scores. Furthermore, the caregivers’ education levels of a college’s degree or above were associated with higher cognitive scores, lower CHQ scores, and higher family APGAR scores.<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>The effects of allergic diseases on children’s cognitive function and caregivers’ mental health.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\" colspan=\"2\">Intelligence quotient</th><th align=\"left\" colspan=\"2\">SNAP-IV</th><th align=\"left\" colspan=\"2\">CHQ</th><th align=\"left\" colspan=\"2\">Family APGAR</th></tr><tr><th align=\"left\">B (95% CI)</th><th align=\"left\">p-value</th><th align=\"left\">B (95% CI)</th><th align=\"left\">p-value</th><th align=\"left\">B (95% CI)</th><th align=\"left\">p-value</th><th align=\"left\">B (95% CI)</th><th align=\"left\">p-value</th></tr></thead><tbody><tr><td align=\"left\">Age (years)</td><td align=\"left\">0.79 (− 0.13, 1.71)</td><td align=\"left\">0.091</td><td align=\"left\">0.24 (− 0.72, 1.20)</td><td align=\"left\">0.621</td><td align=\"left\">0.03 (− 0.17, 0.11)</td><td align=\"left\">0.712</td><td align=\"left\">0.01(− 0.16, 0.18)</td><td align=\"left\">0.928</td></tr><tr><td align=\"left\">Sex (female vs. male)</td><td align=\"left\">0.16 (− 4.12, 4.44)</td><td align=\"left\">0.941</td><td align=\"left\">− 3.29 (− 7.26, 0.69)</td><td align=\"left\">0.104</td><td align=\"left\">0.00 (− 0.65, 0.65)</td><td align=\"left\">0.997</td><td align=\"left\">− 0.56 (− 1.35, 0.23)</td><td align=\"left\">0.161</td></tr><tr><td align=\"left\">Age of caregivers</td><td align=\"left\">0.24 (− 0.25, 0.73)</td><td align=\"left\">0.340</td><td align=\"left\">− 0.18 (− 0.64, 0.29)</td><td align=\"left\">0.462</td><td align=\"left\">− 0.04 (− 0.11, 0.04)</td><td align=\"left\">0.336</td><td align=\"left\">− 0.11 (− 0.20, − 0.02)</td><td align=\"left\">0.018</td></tr><tr><td align=\"left\" colspan=\"9\"><bold>Education of caregivers</bold></td></tr><tr><td align=\"left\">High school or lower</td><td align=\"left\">Reference</td><td align=\"left\"/><td align=\"left\">Reference</td><td align=\"left\"/><td align=\"left\">Reference</td><td align=\"left\"/><td align=\"left\">Reference</td><td align=\"left\"/></tr><tr><td align=\"left\">College</td><td align=\"left\">6.79 (2.04, 11.53)</td><td align=\"left\">0.005</td><td align=\"left\">− 0.44(− 4.81, 3.92)</td><td align=\"left\">0.842</td><td align=\"left\">− 0.79 (− 1.51, − 0.07)</td><td align=\"left\">0.033</td><td align=\"left\">1.36 (0.49, 2.24)</td><td align=\"left\">0.003</td></tr><tr><td align=\"left\">Master or above</td><td align=\"left\">10.90 (4.19, 17.60)</td><td align=\"left\">0.002</td><td align=\"left\">− 4.36 (− 10.55,1.83)</td><td align=\"left\">0.166</td><td align=\"left\">− 1.45 (− 2.46, − 0.44)</td><td align=\"left\">0.005</td><td align=\"left\">2.40 (1.17, 3.63)</td><td align=\"left\">0.000</td></tr><tr><td align=\"left\" colspan=\"9\"><bold>Allergic diseases</bold></td></tr><tr><td align=\"left\">Allergic rhinitis</td><td align=\"left\">− 0.70 (− 7.24, 5.83)</td><td align=\"left\">0.832</td><td align=\"left\">− 1.22 (− 6.84, 4.40)</td><td align=\"left\">0.668</td><td align=\"left\">0.10(− 0.90, 1.10)</td><td align=\"left\">0.841</td><td align=\"left\">− 0.52 (− 1.74, 0.70)</td><td align=\"left\">0.399</td></tr><tr><td align=\"left\">Asthma</td><td align=\"left\">1.55 (− 4.86, 8.00)</td><td align=\"left\">0.634</td><td align=\"left\">1.66(− 3.89 7.21)</td><td align=\"left\">0.555</td><td align=\"left\">− 1.00 (− 1.99, − 0.00)</td><td align=\"left\">0.049</td><td align=\"left\">0.15 (− 1.06, 1.36)</td><td align=\"left\">0.807</td></tr><tr><td align=\"left\">Atopic dermatitis</td><td align=\"left\">− 0.12 (− 6.05, 5.81)</td><td align=\"left\">0.968</td><td align=\"left\">− 1.49 (− 6.64, 3.66)</td><td align=\"left\">0.569</td><td align=\"left\">− 0.18(− 1.09, − 0.74)</td><td align=\"left\">0.705</td><td align=\"left\">− 0.07 (− 1.18, 1.03)</td><td align=\"left\">0.897</td></tr><tr><td align=\"left\">Urticaria</td><td align=\"left\">− 7.48 (− 18.02, 3.06)</td><td align=\"left\">0.163</td><td align=\"left\">− 6.61(− 16.97, 3.76)</td><td align=\"left\">0.210</td><td align=\"left\">− 0.08 (− 1.67, 1.51)</td><td align=\"left\">0.923</td><td align=\"left\">− 0.58 (− 2.51, 1.35)</td><td align=\"left\">0.557</td></tr></tbody></table></table-wrap></p><p id=\"Par8\">Of the children with AD, 21.1%, 59.6%, and 19.3% of them had one atopic disease (A1), two atopic diseases (A2), and three or more atopic diseases (A3), respectively. The relationships between the number of atopic diseases and children’s development and caregivers’ mental health are shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>. Compared to caregivers of children without atopic disease (A0), the caregivers of the A2 (mean difference = 1.05, p = 0.005) and A3 groups (mean difference = 1.08, p = 0.045) had significantly higher CHQ scores. Except for CHQ, we observed no significant difference in cognitive scores, SNAP-IV, or family APGAR scores between the four groups.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>The relationship of numbers of atopic diseases and children’s development and caregivers’ mental health. A0: children without atopic disease, A1: child with one atopic disease, A2: children with two atopic diseases, A3: children with three or more atopic diseases.</p></caption><graphic xlink:href=\"41598_2020_70825_Fig1_HTML\" id=\"MO1\"/></fig></p></sec><sec id=\"Sec3\"><title>Discussion</title><p id=\"Par9\">This study is the first to investigate the potential effect of atopic diseases on cognitive development and parental stress. Inconsistent with our hypothesis, our data revealed that allergic rhinitis, asthma, atopic dermatitis, and urticaria were not associated with children’s cognitive profiles or ADHD symptoms. However, the caregivers of patients who had asthma experienced a greater mental health burden than those of patients without asthma. Furthermore, the numbers of atopic diseases had a dose–response effect on caregivers’ mental health.</p><p id=\"Par10\">Our data revealed that allergic rhinitis, asthma, atopic dermatitis, and urticaria were not associated with children’s cognitive profiles. While allergic rhinitis is a chronic disease that effects quality of life, we identified no negative effects on IQ<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. Although asthma is a chronic disease and causes many respiratory problems, it also has no negative impact on IQ<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Nevertheless, certain neurocognitive symptoms are increased in children with moderate-to-severe atopic dermatitis, compared to healthy controls<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. The relationships between urticaria and intelligence have not been well-established. Atopy is defined as a personal and/or familial tendency, usually in childhood or adolescence, to become sensitized and produce IgE antibodies in response to ordinary exposure to allergens, usually proteins. We consider that chronic atopic diseases may have detrimental effects on neurodevelopment. However, in line with previous studies, we have found that none of allergic rhinitis, asthma, atopic dermatitis, or urticaria have detrimental effects on children’s cognitive function.</p><p id=\"Par11\">In addition to cognitive function, we found that allergic rhinitis, asthma, atopic dermatitis, and urticaria were not associated with children’s ADHD symptoms. This finding contradicts current opinions raised by many researchers. A number of previous evidence has supported the association between ADHD and allergic/autoimmune diseases<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. Various allergic diseases, like allergic rhinitis, atopic dermatitis, and asthma, have been associated with ADHD<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>–<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. We have proposed some possible explanations for this discrepancy. Previous epidemiological studies primarily investigated the relationship between ADHD diagnosis and atopic diseases. This kind of reimbursement data may be influenced by a detection bias. For example, patients with atopic diseases who regularly followed up in the pediatrics outpatient-department (OPD) may be more likely referred to a child psychiatrist and be diagnosed as ADHD. In contrast, the ADHD symptoms in our study were presented as SNAP-IV score (continuous variable). Furthermore, the participants were consecutively recruited in OPD, and no significant difference in ADHD symptom severity was found between patients and controls.</p><p id=\"Par12\">This study is the first to provide evidence related to the profiles of parenting stress in the caregivers of children with atopic diseases. Our data revealed that caregivers of children who had asthma or multiple atopic diseases suffered from greater parenting stress than the caregivers of children who did not have any atopic diseases. Negative experiences with asthma care and the unpredictability of the disease outcomes impair the ability of caregivers to adapt successfully to their caregiving role and encourage perceptions that they cannot cope with this illness<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. Moreover, individuals with multiple atopic diseases cope with a significant psychosocial burden, in addition to dealing with the medical aspects of the disease<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. Caring for children with multiple atopic diseases can be a time-consuming task that can impair caregivers’ personal relationships, as well as decrease psychosocial functioning<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Deficient sleep with poor quality in the caregivers of children with chronic illnesses may have a significant impact on their health and well-being, as well as on their caregiving responsibilities<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. Pursuant to our findings, healthcare providers should better target support to these caregivers of children with asthma or multiple atopic diseases so that they can better care for their children.</p><p id=\"Par13\">This study has certain limitations. First, this is a cross-sectional study, so the duration of illness and possible medications were not recorded. Whether the children’s outcomes and caregivers’ mental health were influenced by the treatment outcome is unknown. Second, atopic diseases were only recorded as a categorical variable (with or without), but the symptom severity on cognition or parenting stress was not assessed herein. Third, we did not record physical or neurodevelopmental comorbidities. Whether other comorbidities (i.e., developmental delay or epilepsy) may actually influence or moderate children’s cognitive development warrants further investigation. Fourth, the sample size was small (especially the control group), which reduced the statistical power of this study.</p><p id=\"Par14\">Allergic rhinitis, asthma, atopic dermatitis, and urticaria were not associated with children’s cognitive profiles or ADHD symptoms. This result is good news for caregivers and patients with atopic diseases, reassuring them that their atopic diseases will have no effect on their cognitive development or ADHD symptoms. However, the caregivers of patients who had asthma or multiple atopic diseases may feel stress about the physical or psychological burden of caring for their children. These caregivers may require support or help to overcome that stress.</p></sec><sec id=\"Sec4\"><title>Methods</title><sec id=\"Sec5\"><title>Participants in a clinical setting</title><p id=\"Par15\">We recruited a total of 109 patients with atopic diseases from the Department of Pediatrics, Kaohsiung Chang Gung Memorial Hospital, Taiwan or communities near the hospital. Patients who had one or more atopic diseases (allergic rhinitis, asthma, atopic dermatitis, or urticaria) were recruited by a properly trained allergist clinician.</p><p id=\"Par16\">The 82 control subjects consisted of healthy children from the communities surrounding Kaohsiung Chang Gung Memorial Hospital or of children suffering from upper respiratory tract infection (URI) whose symptoms were currently in remission. We excluded any patients with atopic diseases (allergic rhinitis, asthma, atopic dermatitis, or urticaria) or other major physical illnesses (such as genetic, metabolic, or infectious conditions).</p></sec><sec id=\"Sec6\"><title>Neurocognitive assessments</title><p id=\"Par17\">A developmental or cognitive assessment was administered to each patient and control subject by an experienced child psychologist in a room designed to reduce testing condition variables. Subjects under the age of 4 years old were assessed using the Mullen Scales of Early Learning (MSEL)<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>; subjects between the ages of 4 and 7 years old were examined using the Wechsler Preschool and Primary Scale of Intelligence-Fourth Edition (WPPSI-IV)<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>; and subjects older than 7 years old were tested using the Wechsler Intelligence Scale for Children-Fourth Edition (WISC-IV)<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. The Early Learning Composite score of MSLE and the Full-Scale Intelligence Quotient (FSIQ) of WPPSI-IV and WISC-IV were considered the intelligence quotient score. The patients’ caregivers were requested to complete the following questionnaires which assess the ADHD symptoms severity and mental health burden.</p><p id=\"Par18\"><italic>The Chinese Version of the Swanson, Nolan, and Pelham IV Scale (SNAP-IV)</italic>, a 26-item questionnaire, is commonly used for evaluating ADHD symptoms and severity. The questionnaire can be completed by either parents or teachers<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>. The 26 items consist of nine items for inattention, nine items for hyperactivity and impulsivity and eight items that concern oppositional defiant disorder symptoms, as defined in the DSM-IV-TR. Each item is scored on a four-point Likert scale (from 0 to 3). The Chinese version of the SNAP-IV has been reported to have satisfactory reliability and concurrent validity<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>.</p><p id=\"Par19\"><italic>The Chinese Health Questionnaire (CHQ-12)</italic>, a 12-item self-report questionnaire, was modified from the General Health Questionnaire<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. This instrument has been widely used to identify those who have minor psychiatric disorders in both primary care and community settings. This measure has been proven to have good reliability and validity<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>.</p><p id=\"Par20\"><italic>Family APGAR</italic>, a five-item measure often used to measure family well-being<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>, was completed by the primary caregiver in each of the five areas by using a three-point Likert scale, ranging from 0 (low satisfaction) to 2 (high satisfaction). The Mandarin version has adequate internal reliability and validity.</p></sec><sec id=\"Sec7\"><title>Statistical analysis</title><p id=\"Par21\">All data processing and statistical analyses were performed using the Statistical Package for Social Science (SPSS) software, Version 21.0 (SPSS, Chicago, IL, USA). Two-tailed <italic>p</italic> values < 0.05 were considered statistically significant.</p><p id=\"Par22\">We adopted the Chi-square test to compare differences in categorical variables between patients with atopic diseases and healthy controls. We compared continuous variables between the two groups through an independent <italic>t</italic>-test. Multiple linear regression was carried out to observe the effect of atopic diseases on children’s cognitive function and caregivers’ mental health. The dependent factors were set as a cognitive score, total scores of SNAP-IV, CHQ, and family APGAR. The cognitive scores were set as the ELC scores of the MSEL or the FSIQ scores of the WPPSI or WISC-IV. The four atopic diseases (allergic rhinitis, asthma, atopic dermatitis, or urticaria) were considered the independent variables, and we controlled the confounding effects of children’s age and sex and caregivers’ age and education levels.</p><p id=\"Par23\">To investigate whether the number of atopic diseases had dose–response or dose-related effects on children’s and caregivers’ outcomes, we categorized the participants into children who had no atopic disease (A0), children who had one atopic disease (A1), children who had two atopic diseases (A2), and children who had three or more atopic diseases (A3). Furthermore, we used one-way ANOVA with an LSD post-hoc test to examine the difference in cognitive, SNAP-IV, CHQ, and family APGAR scores between the aforementioned groups.</p></sec><sec id=\"Sec8\"><title>Ethical approval</title><p id=\"Par24\">This study was approved by the Chang Gung Memorial Hospital’s Internal Review Board (IRB No.201700509B0), and we obtained written informed consent from the parents or guardians of all participating children. All methods were performed in accordance with the relevant guidelines and regulations by the Declaration of Helsinki.</p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><ack><title>Acknowledgements</title><p>This study was supported by the following grants: MOST 108-2314-B-182 -037 -MY3 from the Ministry of Science and Technology of Taiwan and Chang Gung Memorial Hospital (CMRPG8C1082, CMRPG8E1613, CMRPG8D1561-2, CMRPG8J0611 and CMRPG8D0521). Said institutions had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>H.-C.K. and L.-J.W. conceived the manuscript and designed the study, performed data analysis and writing the manuscript. L.-S.C. and Z.-Y.T. assisted in recruiting patients and collecting the data. All authors contributed to writing of the manuscript and agree with its final version.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par25\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thomsen</surname><given-names>SF</given-names></name></person-group><article-title>Epidemiology and natural history of atopic diseases</article-title><source>Eur. Clin. Respir. J.</source><year>2015</year><volume>2</volume><fpage>24642</fpage><pub-id pub-id-type=\"doi\">10.3402/ecrj.v2.24642</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kakli</surname><given-names>HA</given-names></name><name><surname>Riley</surname><given-names>TD</given-names></name></person-group><article-title>Allergic rhinitis</article-title><source>Prim. Care</source><year>2016</year><volume>43</volume><fpage>465</fpage><lpage>475</lpage><pub-id pub-id-type=\"doi\">10.1016/j.pop.2016.04.009</pub-id><pub-id pub-id-type=\"pmid\">27545735</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mims</surname><given-names>JW</given-names></name></person-group><article-title>Asthma: Definitions and pathophysiology</article-title><source>Int. Forum Allergy Rhinol.</source><year>2015</year><volume>5</volume><issue>Suppl 1</issue><fpage>S2</fpage><lpage>6</lpage><pub-id pub-id-type=\"doi\">10.1002/alr.21609</pub-id><pub-id pub-id-type=\"pmid\">26335832</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bieber</surname><given-names>T</given-names></name></person-group><article-title>Atopic dermatitis</article-title><source>N. Engl. J. Med.</source><year>2008</year><volume>358</volume><fpage>1483</fpage><lpage>1494</lpage><pub-id pub-id-type=\"doi\">10.1056/NEJMra074081</pub-id><pub-id pub-id-type=\"pmid\">18385500</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ghazanfar</surname><given-names>MN</given-names></name><name><surname>Kibsgaard</surname><given-names>L</given-names></name><name><surname>Thomsen</surname><given-names>SF</given-names></name><name><surname>Vestergaard</surname><given-names>C</given-names></name></person-group><article-title>Risk of comorbidities in patients diagnosed with chronic urticaria: A nationwide registry-study</article-title><source>World Allergy Organ. J.</source><year>2020</year><volume>13</volume><fpage>100097</fpage><pub-id pub-id-type=\"doi\">10.1016/j.waojou.2019.100097</pub-id><pub-id pub-id-type=\"pmid\">32021661</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>O'Connell</surname><given-names>EJ</given-names></name></person-group><article-title>The burden of atopy and asthma in children</article-title><source>Allergy</source><year>2004</year><volume>59</volume><issue>Suppl 78</issue><fpage>7</fpage><lpage>11</lpage><pub-id pub-id-type=\"doi\">10.1111/j.1398-9995.2004.00563.x</pub-id><pub-id pub-id-type=\"pmid\">15245350</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Verschoor</surname><given-names>D</given-names></name><name><surname>von Gunten</surname><given-names>S</given-names></name></person-group><article-title>Allergy and atopic diseases: An update on experimental evidence</article-title><source>Int. Arch. Allergy Immunol.</source><year>2019</year><volume>180</volume><fpage>235</fpage><lpage>243</lpage><pub-id pub-id-type=\"doi\">10.1159/000504439</pub-id><pub-id pub-id-type=\"pmid\">31694044</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cohen</surname><given-names>S</given-names></name><etal/></person-group><article-title>Co-morbidities and cognitive status in a cohort of teenagers with asthma</article-title><source>Pediatr. Pulmonol.</source><year>2016</year><volume>51</volume><fpage>901</fpage><lpage>907</lpage><pub-id pub-id-type=\"doi\">10.1002/ppul.23443</pub-id><pub-id pub-id-type=\"pmid\">27177276</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Goodwin</surname><given-names>RD</given-names></name><name><surname>Robinson</surname><given-names>M</given-names></name><name><surname>Sly</surname><given-names>PD</given-names></name><name><surname>Holt</surname><given-names>PG</given-names></name></person-group><article-title>Childhood atopy and mental health: A prospective, longitudinal investigation</article-title><source>Psychol. Med.</source><year>2017</year><volume>47</volume><fpage>317</fpage><lpage>325</lpage><pub-id pub-id-type=\"doi\">10.1017/S0033291716001896</pub-id><pub-id pub-id-type=\"pmid\">27762174</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abd El-Hamid</surname><given-names>ZB</given-names></name><etal/></person-group><article-title>Impact of allergy on children with attention deficit hyperactivity disorder</article-title><source>Eur. Ann. Allergy Clin. Immunol.</source><year>2018</year><volume>50</volume><fpage>262</fpage><lpage>267</lpage><pub-id pub-id-type=\"doi\">10.23822/EurAnnACI.1764-1489.72</pub-id><pub-id pub-id-type=\"pmid\">30067000</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>LJ</given-names></name><etal/></person-group><article-title>Attention deficit-hyperactivity disorder is associated with allergic symptoms and low levels of hemoglobin and serotonin</article-title><source>Sci. Rep.</source><year>2018</year><volume>8</volume><fpage>10229</fpage><pub-id pub-id-type=\"doi\">10.1038/s41598-018-28702-5</pub-id><pub-id pub-id-type=\"pmid\">29980754</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Daramola</surname><given-names>OO</given-names></name><name><surname>Ayoola</surname><given-names>OO</given-names></name><name><surname>Ogunbiyi</surname><given-names>AO</given-names></name></person-group><article-title>The comparison of intelligence quotients of atopic and nonatopic children in ibadan, Nigeria</article-title><source>Indian J. Dermatol.</source><year>2010</year><volume>55</volume><fpage>221</fpage><lpage>224</lpage><pub-id pub-id-type=\"doi\">10.4103/0019-5154.70665</pub-id><pub-id pub-id-type=\"pmid\">21063510</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kremer</surname><given-names>B</given-names></name><name><surname>den Hartog</surname><given-names>HM</given-names></name><name><surname>Jolles</surname><given-names>J</given-names></name></person-group><article-title>Relationship between allergic rhinitis, disturbed cognitive functions and psychological well-being</article-title><source>Clin. Exp. Allergy</source><year>2002</year><volume>32</volume><fpage>1310</fpage><lpage>1315</lpage><pub-id pub-id-type=\"doi\">10.1046/j.1365-2745.2002.01483.x</pub-id><pub-id pub-id-type=\"pmid\">12220469</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ghaffari</surname><given-names>J</given-names></name><name><surname>Abbaskhanian</surname><given-names>A</given-names></name><name><surname>Jalili</surname><given-names>M</given-names></name></person-group><article-title>IQ scores of children with moderate asthma: A comparison with healthy children</article-title><source>Oman Med. J.</source><year>2014</year><volume>29</volume><fpage>71</fpage><lpage>74</lpage><pub-id pub-id-type=\"doi\">10.5001/omj.2014.16</pub-id><pub-id pub-id-type=\"pmid\">24498486</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chang</surname><given-names>HY</given-names></name><etal/></person-group><article-title>Allergic diseases in preschoolers are associated with psychological and behavioural problems</article-title><source>Allergy Asthma Immunol. Res.</source><year>2013</year><volume>5</volume><fpage>315</fpage><lpage>321</lpage><pub-id pub-id-type=\"doi\">10.4168/aair.2013.5.5.315</pub-id><pub-id pub-id-type=\"pmid\">24003389</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Strom</surname><given-names>MA</given-names></name><name><surname>Fishbein</surname><given-names>AB</given-names></name><name><surname>Paller</surname><given-names>AS</given-names></name><name><surname>Silverberg</surname><given-names>JI</given-names></name></person-group><article-title>Association between atopic dermatitis and attention deficit hyperactivity disorder in U.S. children and adults</article-title><source>Br. J. Dermatol.</source><year>2016</year><volume>175</volume><fpage>920</fpage><lpage>929</lpage><pub-id pub-id-type=\"doi\">10.1111/bjd.14697</pub-id><pub-id pub-id-type=\"pmid\">27105659</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Miyazaki</surname><given-names>C</given-names></name><etal/></person-group><article-title>Allergic diseases in children with attention deficit hyperactivity disorder: A systematic review and meta-analysis</article-title><source>BMC Psychiatry</source><year>2017</year><volume>17</volume><fpage>120</fpage><pub-id pub-id-type=\"doi\">10.1186/s12888-017-1281-7</pub-id><pub-id pub-id-type=\"pmid\">28359274</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Feng</surname><given-names>B</given-names></name><etal/></person-group><article-title>Association of pediatric allergic rhinitis with the ratings of attention-deficit/hyperactivity disorder</article-title><source>Am. J. Rhinol. Allergy</source><year>2017</year><volume>31</volume><fpage>161</fpage><lpage>167</lpage><pub-id pub-id-type=\"doi\">10.2500/ajra.2017.31.4439</pub-id><pub-id pub-id-type=\"pmid\">28490400</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schans</surname><given-names>JV</given-names></name><name><surname>Cicek</surname><given-names>R</given-names></name><name><surname>de Vries</surname><given-names>TW</given-names></name><name><surname>Hak</surname><given-names>E</given-names></name><name><surname>Hoekstra</surname><given-names>PJ</given-names></name></person-group><article-title>Association of atopic diseases and attention-deficit/hyperactivity disorder: A systematic review and meta-analyses</article-title><source>Neurosci. Biobehav. Rev.</source><year>2017</year><volume>74</volume><fpage>139</fpage><lpage>148</lpage><pub-id pub-id-type=\"doi\">10.1016/j.neubiorev.2017.01.011</pub-id><pub-id pub-id-type=\"pmid\">28111269</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Riis</surname><given-names>JL</given-names></name><name><surname>Vestergaard</surname><given-names>C</given-names></name><name><surname>Deleuran</surname><given-names>MS</given-names></name><name><surname>Olsen</surname><given-names>M</given-names></name></person-group><article-title>Childhood atopic dermatitis and risk of attention deficit/hyperactivity disorder: A cohort study</article-title><source>J. Allergy Clin. Immunol.</source><year>2016</year><volume>138</volume><fpage>608</fpage><lpage>610</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jaci.2016.01.027</pub-id><pub-id pub-id-type=\"pmid\">27025346</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Horev</surname><given-names>A</given-names></name><name><surname>Freud</surname><given-names>T</given-names></name><name><surname>Manor</surname><given-names>I</given-names></name><name><surname>Cohen</surname><given-names>AD</given-names></name><name><surname>Zvulunov</surname><given-names>A</given-names></name></person-group><article-title>Risk of attention-deficit/hyperactivity disorder in children with atopic dermatitis</article-title><source>Acta Dermatovenerol. Croat.</source><year>2017</year><volume>25</volume><fpage>210</fpage><lpage>214</lpage><pub-id pub-id-type=\"pmid\">29252173</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cices</surname><given-names>LK</given-names></name><name><surname>Fishbein</surname><given-names>A</given-names></name><name><surname>Paller</surname><given-names>AS</given-names></name></person-group><article-title>Neurocognitive function in moderate-severe pediatric atopic dermatitis: A case-control study</article-title><source>Pediatr. Dermatol.</source><year>2019</year><volume>36</volume><fpage>110</fpage><lpage>114</lpage><pub-id pub-id-type=\"doi\">10.1111/pde.13710</pub-id><pub-id pub-id-type=\"pmid\">30506922</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>EJ</given-names></name><name><surname>Beck</surname><given-names>KM</given-names></name><name><surname>Sekhon</surname><given-names>S</given-names></name><name><surname>Bhutani</surname><given-names>T</given-names></name><name><surname>Koo</surname><given-names>J</given-names></name></person-group><article-title>The impact of pediatric atopic dermatitis on families: A review</article-title><source>Pediatr. Dermatol.</source><year>2019</year><volume>36</volume><fpage>66</fpage><lpage>71</lpage><pub-id pub-id-type=\"doi\">10.1111/pde.13727</pub-id><pub-id pub-id-type=\"pmid\">30556595</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carroll</surname><given-names>CL</given-names></name><name><surname>Balkrishnan</surname><given-names>R</given-names></name><name><surname>Feldman</surname><given-names>SR</given-names></name><name><surname>Fleischer</surname><given-names>AB</given-names><suffix>Jr</suffix></name><name><surname>Manuel</surname><given-names>JC</given-names></name></person-group><article-title>The burden of atopic dermatitis: Impact on the patient, family, and society</article-title><source>Pediatr. Dermatol.</source><year>2005</year><volume>22</volume><fpage>192</fpage><lpage>199</lpage><pub-id pub-id-type=\"doi\">10.1111/j.1525-1470.2005.22303.x</pub-id><pub-id pub-id-type=\"pmid\">15916563</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Meltzer</surname><given-names>LJ</given-names></name><name><surname>Booster</surname><given-names>GD</given-names></name></person-group><article-title>Sleep disturbance in caregivers of children with respiratory and atopic disease</article-title><source>J. Pediatr. Psychol.</source><year>2016</year><volume>41</volume><fpage>643</fpage><lpage>650</lpage><pub-id pub-id-type=\"doi\">10.1093/jpepsy/jsw016</pub-id><pub-id pub-id-type=\"pmid\">27017428</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ghaffari</surname><given-names>J</given-names></name><name><surname>Abbaskhanian</surname><given-names>A</given-names></name><name><surname>Jalili</surname><given-names>M</given-names></name><name><surname>Yazdani Charati</surname><given-names>J</given-names></name></person-group><article-title>IQ Score of children with persistent or perennial allergic rhinitis: A comparison with healthy children</article-title><source>Iran J. Child Neurol.</source><year>2014</year><volume>8</volume><fpage>44</fpage><lpage>48</lpage><pub-id pub-id-type=\"pmid\">25143773</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>MH</given-names></name><etal/></person-group><article-title>Comorbidity of allergic and autoimmune diseases among patients with ADHD</article-title><source>J. Atten. Disord.</source><year>2017</year><volume>21</volume><fpage>219</fpage><lpage>227</lpage><pub-id pub-id-type=\"doi\">10.1177/1087054712474686</pub-id><pub-id pub-id-type=\"pmid\">23400216</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bellin</surname><given-names>MH</given-names></name><etal/></person-group><article-title>Stress and quality of life in urban caregivers of children with poorly controlled asthma: A longitudinal analysis</article-title><source>J. Pediatr. Health Care</source><year>2015</year><volume>29</volume><fpage>536</fpage><lpage>546</lpage><pub-id pub-id-type=\"doi\">10.1016/j.pedhc.2015.04.018</pub-id><pub-id pub-id-type=\"pmid\">26036621</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>SH</given-names></name><name><surname>Huang</surname><given-names>JL</given-names></name><name><surname>Yeh</surname><given-names>KW</given-names></name><name><surname>Tsai</surname><given-names>YF</given-names></name></person-group><article-title>The stress of caring for children with asthma: A qualitative study of primary caregivers</article-title><source>J. Nurs. Res.</source><year>2015</year><volume>23</volume><fpage>298</fpage><lpage>307</lpage><pub-id pub-id-type=\"pmid\">26562461</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Mullen</surname><given-names>EM</given-names></name></person-group><source>Mullen Scales of Early Learning Item Administration Book</source><year>1995</year><publisher-loc>Circle Pines</publisher-loc><publisher-name>American Guidance Service</publisher-name></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Wechsler</surname><given-names>D</given-names></name></person-group><source>Wechsler Preschool and Primary Scales of Intelligence (WPPSI-IV)</source><year>2012</year><edition>4</edition><publisher-loc>San Antonio</publisher-loc><publisher-name>Psychological Corporation</publisher-name></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Baron</surname><given-names>IS</given-names></name></person-group><article-title>Test review: Wechsler Intelligence Scale for Children-Fourth Edition (WISC-IV)</article-title><source>Child Neuropsychol.</source><year>2005</year><volume>11</volume><fpage>471</fpage><lpage>475</lpage><pub-id pub-id-type=\"doi\">10.1080/09297040590951587</pub-id><pub-id pub-id-type=\"pmid\">16306021</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bussing</surname><given-names>R</given-names></name><etal/></person-group><article-title>Parent and teacher SNAP-IV ratings of attention deficit hyperactivity disorder symptoms: Psychometric properties and normative ratings from a school district sample</article-title><source>Assessment</source><year>2008</year><volume>15</volume><fpage>317</fpage><lpage>328</lpage><pub-id pub-id-type=\"doi\">10.1177/1073191107313888</pub-id><pub-id pub-id-type=\"pmid\">18310593</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gau</surname><given-names>SS</given-names></name><etal/></person-group><article-title>Psychometric properties of the Chinese version of the Swanson, Nolan, and Pelham, version IV scale—parent form</article-title><source>Int. J. Methods Psychiatr. Res.</source><year>2008</year><volume>17</volume><fpage>35</fpage><lpage>44</lpage><pub-id pub-id-type=\"doi\">10.1002/mpr.237</pub-id><pub-id pub-id-type=\"pmid\">18286459</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cheng</surname><given-names>TA</given-names></name><name><surname>Williams</surname><given-names>P</given-names></name></person-group><article-title>The design and development of a screening questionnaire (CHQ) for use in community studies of mental disorders in Taiwan</article-title><source>Psychol. Med.</source><year>1986</year><volume>16</volume><fpage>415</fpage><lpage>422</lpage><pub-id pub-id-type=\"doi\">10.1017/S0033291700009247</pub-id><pub-id pub-id-type=\"pmid\">3726013</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Smilkstein</surname><given-names>G</given-names></name><name><surname>Ashworth</surname><given-names>C</given-names></name><name><surname>Montano</surname><given-names>D</given-names></name></person-group><article-title>Validity and reliability of the family APGAR as a test of family function</article-title><source>J. Fam. Pract.</source><year>1982</year><volume>15</volume><fpage>303</fpage><lpage>311</lpage><pub-id pub-id-type=\"pmid\">7097168</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807824</article-id><article-id pub-id-type=\"pmc\">PMC7431565</article-id><article-id pub-id-type=\"publisher-id\">70877</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70877-3</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Electrochemical immunosensor with Cu(I)/Cu(II)-chitosan-graphene nanocomposite-based signal amplification for the detection of newcastle disease virus</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Huang</surname><given-names>Jiaoling</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Xie</surname><given-names>Zhixun</given-names></name><address><email>xiezhixun@126.com</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Huang</surname><given-names>Yihong</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Xie</surname><given-names>Liji</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Luo</surname><given-names>Sisi</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Fan</surname><given-names>Qing</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zeng</surname><given-names>Tingting</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Yanfang</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Sheng</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Minxiu</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Xie</surname><given-names>Zhiqin</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Deng</surname><given-names>Xianwen</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.418337.a</institution-id><institution>Guangxi Key Laboratory of Veterinary Biotechnology, </institution><institution>Guangxi Veterinary Research Institute, </institution></institution-wrap>51 You Ai North Road, Nanning, 530001 Guangxi China </aff><aff id=\"Aff2\"><label>2</label>Liuzhou Centre for Animal Disease Control and Prevention, Beijing, China </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13869</elocation-id><history><date date-type=\"received\"><day>22</day><month>1</month><year>2020</year></date><date date-type=\"accepted\"><day>12</day><month>5</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">An electrochemical immunoassay for the ultrasensitive detection of Newcastle disease virus (NDV) was developed using graphene and chitosan-conjugated Cu(I)/Cu(II) (Cu(I)/Cu(II)-Chi-Gra) for signal amplification. Graphene (Gra) was used for both the conjugation of an anti-Newcastle disease virus monoclonal antibody (MAb/NDV) and the immobilization of anti-Newcastle disease virus polyclonal antibodies (PAb/NDV). Cu(I)/Cu(II) was selected as an electroactive probe, immobilized on a chitosan-graphene (Chi-Gra) hybrid material, and detected by differential pulse voltammetry (DPV) after a sandwich-type immune response. Because Gra had a large surface area, many antibodies were loaded onto the electrochemical immunosensor to effectively increase the electrical signal. Additionally, the introduction of Gra significantly increased the loading amount of electroactive probes (Cu(I)/Cu(II)), and the electrical signal was further amplified. Cu(I)/Cu(II) and Cu(I)/Cu(II)-Chi-Gra were compared in detail to characterize the signal amplification ability of this platform. The results showed that this immunosensor exhibited excellent analytical performance in the detection of NDV in the concentration range of 10<sup>0.13</sup> to 10<sup>5.13</sup> EID<sub>50</sub>/0.1 mL, and it had a detection limit of 10<sup>0.68</sup> EID<sub>50</sub>/0.1 mL, which was calculated based on a signal-to-noise (S/N) ratio of 3. The resulting immunosensor also exhibited high sensitivity, good reproducibility and acceptable stability.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Analytical chemistry</kwd><kwd>Immunochemistry</kwd><kwd>Graphene</kwd></kwd-group><funding-group><award-group><funding-source><institution>Guangxi Science Base and Talents Special Program</institution></funding-source><award-id>AD17195083</award-id><principal-award-recipient><name><surname>Xie</surname><given-names>Zhixun</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>Guangxi Science Great Special Program</institution></funding-source><award-id>AA17204057</award-id><principal-award-recipient><name><surname>Xie</surname><given-names>Zhixun</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>Guangxi BaGui Scholars Program Foundation</institution></funding-source><award-id>2019-79</award-id><principal-award-recipient><name><surname>Xie</surname><given-names>Zhixun</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>Guangxi Science and Technology Projects</institution></funding-source><award-id>AB16380054</award-id><principal-award-recipient><name><surname>Xie</surname><given-names>Liji</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>Guangxi Shi Bai Qian Talents Engineering Foundation</institution></funding-source><award-id>[2020]24</award-id><principal-award-recipient><name><surname>Xie</surname><given-names>Liji</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Newcastle disease virus (NDV) is a viral disease of poultry that belongs to avian paramyxovirus 1. It is a single-strand, non-segmented, and negative-sense RNA virus<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>, and it is a great threat to the poultry industry<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. The first important step in NDV prevention and control is to develop a rapid and sensitive method for diagnosis. Currently, several methods for detecting NDV, included virus isolation<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>, reverse transcription polymerase chain reaction (RT-PCR)<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>, real-time RT-PCR<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>, immunochromatographic strip (ICS) tests<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>, and reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>, have been reported. However, these diagnostic methods had some disadvantages; for example, virus isolation is the gold standard for the detection of NDV, but the procedure is time-consuming. For RT-PCR, appropriate laboratory facilities and a trained technician are needed. Real-time RT-PCR requires complicated operations as well as expensive reagents and equipment. Therefore, these diagnostic methods are limited in practical applications.</p><p id=\"Par3\">Electrochemical immunosensors are powerful tools that have good specificity, high sensitivity, good precision, and simple instrumentation; give rapid and reliable responses; and are relatively low cost. Their use in clinical diagnosis, food analysis, environmental monitoring and archaeological studies should be highly valuable<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. Furthermore, electrochemical immunosensors are based on antibody-antigen reactions. Therefore, immobilizing antibodies or antigens on a transducer as a biorecognition element plays a very important role in the construction of electrochemical immunosensors. Different methods for immobilizing antibodies/antigens on a transducer, including chemical and physical adsorption, have been discussed<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. It has been reported that chitosan (Chi) is a suitable matrix for immobilizing biorecognition elements due to its biocompatibility, hydrophilicity, mouldability, chemical reactivity, and biodegradability<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. However, Chi is non-conductive and has low solubility in different solutions; thus, many kinds of nanomaterials have been combined with Chi to increase its conductivity for the fabrication of electrochemical immunosensors<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. Modifying transducers with conductive materials enhances the electron transfer between the electrode surface and electrolyte<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Furthermore, modifying them with nanomaterials provides a rougher surface that enables the biorecognition element to attach closely to the electrode surface. Many kinds of nanomaterials, including Gra<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>, multi-walled carbon nanotubes<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>, gold nanoparticles<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>, magnetic nanoparticles<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>, quantum dots<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup> and hybrid nanostructures<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>, have been used in immunosensors.</p><p id=\"Par4\">Gra has a one-atom-thick planar structure composed of sp<sup>2−</sup> hybridized carbon atoms packed in a honeycomb-like lattice<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. Due to this unique structure, Gra has an exceptionally high surface-to-volume ratio, electrical conductivity, and thermal conductivity and good mechanical properties<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. Gra has been used to improve the sensitivity and stability of immunosensors many times<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. However, the direct immobilization of protein molecules on Gra is difficult. As previously mentioned, Chi can easily immobilize protein molecules and form a film on transducers. Due to these properties, nanocomposites consisting of Chi and Gra are an ideal immunosensor material, and our group successfully synthesized a silver nanoparticle-chitosan-graphene composite to construct an electrochemical immunosensor<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>.</p><p id=\"Par5\">However, copper is much less expensive than silver nanoparticles, and Cu(II) ions can be adsorbed by Chi from aqueous solutions via chelation because of its unique three-dimensional structure<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. Additionally, the synthesis of CuO (Cu(II)) and Cu<sub>2</sub>O (Cu(I)) using Chi as a stabilizing and reducing agent has been reported<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref>–<xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. Furthermore, Cu(II) ions provide a good stripping voltammetric signal<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. In addition, Cu(I) has a direct band gap of 2.0 eV and is a p-type semiconductor that is very important in superconductors and electrode materials<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. As previously mentioned, Cu(I) and Cu(II) can be used as electroactive materials. The more electroactive a material carried by an immunosensor is, the more sensitive the immunoassay is. Therefore, in this study, Gra, which has a high loading capacity, was used to load a large amount of electroactive probes on an immunosensor. Hybrid Cu(I)/ Cu(II)-modified Gra effectively amplifies signals. In this work, a sandwich-type electrochemical immunosensor was designed using a gold nanoparticle-chitosan-graphene (AuNP-Chi-Gra) nanocomposite as the platform and a Cu(I)/Cu(II)-chitosan-graphene (Cu(I)/Cu(II)-Chi-Gra) nanocomposite as the label for detecting NDV with a low detection limit (10<sup>0.68</sup> EID<sub>50</sub>/0.1 mL) and high sensitivity in a relatively wide linear range (from 10<sup>0.13</sup> to 10<sup>5.13</sup> EID<sub>50</sub>/0.1 mL). The developed immunosensor shows potential for applications in the clinical screening of other pathogenic microorganisms and point-of-care diagnostics.</p></sec><sec id=\"Sec2\"><title>Results and discussion</title><sec id=\"Sec3\"><title>Morphological characterization of the nanocomposites</title><p id=\"Par6\">Figure <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> shows scanning electron microscopy (SEM) images and energy dispersive spectrometry (EDS) analyses of Gra, Chi-Gra and Cu(I)/Cu(II)-Chi-Gra. The image of Gra confirms that its structure had many folds (a). After Gra was modified with Chi, the folded structure was filled with Chi, and the surface of the Chi-Gra composite became smooth (b). The presence of Chi on Gra was confirmed by EDS analysis (e). N was observed in the sample because Chi is a natural, biocompatible polymer with many amino groups. Interestingly, the Cu(I)/Cu(II)-Chi-Gra nanocomposite exhibited many upturned folded edges and had a porous matrix (c). Due to this characteristic structure, the exposed surface of the Cu(I)/Cu(II)-Chi-Gra nanocomposite was larger than those of the Chi-Gra composite and Gra. The active surface area increased, resulting in a high surface/volume ratio for antibody immobilization. Furthermore, this porous structure facilitated electrochemical signal amplification. The successful incorporation of Cu(I)/Cu(II) into the Chi-Gra surface was also confirmed by EDS analysis (f).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>SEM images of Gra (<bold>a</bold>), Chi-Gra (<bold>b</bold>) and Cu(I)/Cu(II)-Chi-Gra (<bold>c</bold>). The zones examined by EDS (marked by red boxes) and the results of the analysis are also shown for the Gra (<bold>d</bold>), Chi-Gra (<bold>e</bold>) and Cu(I)/Cu(II)-Chi-Gra (<bold>f</bold>) samples.</p></caption><graphic xlink:href=\"41598_2020_70877_Fig1_HTML\" id=\"MO1\"/></fig></p></sec><sec id=\"Sec4\"><title>Chemical characterization of the nanocomposites</title><p id=\"Par7\">Fourier transform infrared (FT-IR) spectra of Chi, Gra, Chi-Gra, CuSO<sub>4</sub> and Cu(I)/Cu(II)-Chi-Gra are presented in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>. As shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>a (black line, Chi), the stretching vibrations of the –OH bonds in Chi were observed at 3,425 cm<sup>−1</sup>, and this band overlapped with the –NH<sub>2</sub> stretching peaks<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. The signals originating from the C-H stretching vibrations were observed at approximately 2,920 cm<sup>−1</sup> and 2,878 cm<sup>−1</sup><sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. The NH<sub>2</sub> group and γ-NH<sub>2</sub> bending vibrations appeared at 1653 cm<sup>−1</sup> and 1596 cm<sup>−1</sup>, respectively<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. Furthermore, the peak at 1,424 cm<sup>−1</sup> was attributed to the OH bending vibration. The stretching vibrations of the C–C–O bonds in the Chi backbone were observed at approximately 1,154 cm<sup>−1</sup>, 1,081 cm<sup>−1</sup> and 1,034 cm<sup>−1</sup>. As shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>a (red line), the characteristic absorption bands of pure Gra appeared at 1555 cm<sup>−1</sup>, 1,459 cm<sup>−1</sup>, and 1,420 cm<sup>−1</sup> (benzene ring backbone stretching vibrations); 1659 cm<sup>−1</sup> (C=O stretching vibration); 2,916 cm<sup>−1</sup> (C–H stretching vibration); and 3,406 cm<sup>−1</sup> (O–H stretching vibration). Chi adsorption on Gra resulted in the appearance of the characteristic absorption bands of pure Gra in the FT-IR spectrum of Chi-Gra (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>a; blue line), but compared with pure Gra, the characteristic absorption bands of Chi-Gra had lower intensities, which helped confirm that Chi was successfully adsorbed on Gra. Comparing the spectra of Chi-Gra and Cu(I)/Cu(II)-Chi-Gra (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>b; red and blue lines) revealed some changes in the intensities and shifts in the peaks. Furthermore, the main absorption peaks of pure CuSO<sub>4</sub> (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>b; black line) were also observed in the FT-IR spectrum of Cu(I)/Cu(II)-Chi-Gra (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>b; blue line), providing evidence of the interaction between CuSO<sub>4</sub> and Chi-Gra. Chi-Gra binds Cu<sup>2+</sup> well because Chi-Gra contains many negatively charged groups (carboxylic (O=C–OH), hydroxyl (–C–OH) and carbonyl (–C=O)) that can strongly interact with the positively charged Cu<sup>2+</sup> ion in CuSO<sub>4</sub>.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>FT-IR spectra of (<bold>a</bold>) Chi, Gra, and Chi-Gra and (<bold>b</bold>) Chi-Gra, CuSO<sub>4</sub>, and Cu(I)/Cu(II)-Chi-Gra.</p></caption><graphic xlink:href=\"41598_2020_70877_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par8\">In addition, X-ray photoelectron spectroscopy (XPS) was used to identify the valence state of Cu. The XPS spectrum of Cu(I)/Cu(II)-Chi-Gra is shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a. The formation of Cu<sub>2</sub>O was confirmed by the presence of the Cu 2p<sub>3/2</sub> peak at 931.73 eV and the Cu 2p<sub>1/2</sub> peak at 951.39 eV<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. Furthermore, the presence of Cu 2p<sub>3/2</sub> and Cu 2p<sub>1/2</sub> peaks with binding energies of 933.26 eV and 953.14 eV, respectively, proved the formation of CuO<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. The presence of CuSO<sub>4</sub> was confirmed by the Cu 2p<sub>3/2</sub> peak at 934.91 eV and Cu 2p<sub>1/2</sub> peak at 954.62 eV<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>. In addition, to obtain a clearer XPS survey, 10 times the amount of CuSO<sub>4</sub> was added to Chi-Gra to prepare rich[Cu(I)/Cu(II)]-Chi-Gra, and the XPS spectrum of rich[Cu(I)/Cu(II)]-Chi-Gra shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>b confirmed that the valence states of the Cu element were Cu<sup>+</sup> (Cu(I)) and Cu<sup>2+</sup> (Cu(II)). The concentration of Cu(II) in rich[Cu(I)/Cu(II)]-Chi-Gra was higher than that in Cu(I)/Cu(II)-Chi-Gra because the ability of Chi to chelate Cu<sup>2+</sup> is stronger than the ability of Chi to reduce Cu<sup>2+</sup> to Cu<sup>+</sup>. Additionally, the presence of Cu4, Cu4′, Cu5 and Cu5′ in rich[Cu(I)/Cu(II)]-Chi-Gra might be due to the different Cu<sup>2+</sup>-chelating abilities of the various functional groups in Chi-Gra. Under competitive conditions, functional groups with a stronger Cu<sup>2+</sup>-chelating ability chelate Cu<sup>2+</sup> first, and functional groups with a weaker Cu<sup>2+</sup>-chelating ability chelate Cu<sup>2+</sup> last. When the amount of Cu<sup>2+</sup> is too low, the functional groups with a weaker Cu<sup>2+</sup>-chelating ability lose Cu<sup>2+</sup>, but these functional groups can chelate Cu<sup>2+</sup> when a sufficient amount of Cu<sup>2+</sup> is present. Therefore, Cu4, Cu4′, Cu5 and Cu5′ were present in rich[Cu(I)/Cu(II)]-Chi-Gra, but absent in Cu(I)/Cu(II)-Chi-Gra.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>XPS spectra of (<bold>a</bold>) Cu(I)/Cu(II)-Chi-Gra and (<bold>b</bold>) rich[Cu(I)/Cu(II)]-Chi-Gra.</p></caption><graphic xlink:href=\"41598_2020_70877_Fig3_HTML\" id=\"MO3\"/></fig></p></sec><sec id=\"Sec5\"><title>Electrochemical characterization of the immunosensor</title><p id=\"Par9\">Cyclic voltammetry (CV) was used to investigate the surface of the glassy carbon electrode (GCE) during the process. The electrochemical behaviour was monitored in 5 mM Fe(CN)<sub>6</sub><sup>3−/4−</sup> (1:1) and 0.01 M phosphate-buffered saline (PBS) (pH = 7.4, containing 0.1 M KCl) in the potential range of − 0.2 to 0.6 V at a scan rate of 50 mV/s<sup>−1</sup>, and the results are shown in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>a. A pair of well-defined voltammetric peaks was obtained for the bare GCE (curve a-1). Coating the bare GCE with AuNP-Chi (curve a-2) and AuNP-Chi-Gra (curve a-3) caused an increase in the redox peak current. A comparison of the curves indicated that the AuNPs and Gra had good conductivity and electrocatalytic effects. After attaching MAb/NDV to the modified GCE (curve a-4), the current decreased. This decrease can be explained by the following two factors: (i) AuNP-Chi-Gra could conjugate MAb/NDV via Au–S covalent bonds, and (ii) electron transfer was hindered by MAb/NDV. Subsequently, BSA was used to block the immunosensor, and the redox peaks decreased even further (curve a-5), because BSA is hydrophobic and electron transfer was further inhibited.<fig id=\"Fig4\"><label>Figure 4</label><caption><p>(<bold>a</bold>) CV curves of the electrode at different stages obtained at a scan rate of 50 mV/s: (a-1) GCE, (a-2) AuNP-Chi-GCE, (a-3) AuNP-Chi-Gra-GCE, (a-4) MAb/NDV-AuNP-Chi-Gra-GCE, and (a-5) BSA-MAb/NDV-AuNP-Chi-Gra-GCE. The supporting electrolyte was 5 mM Fe(CN)<sub>6</sub><sup>3−/4−</sup> + 0.1 M KCl + 0.01 M PBS (pH = 7.4). (<bold>b</bold>) CV curves of the immunosensor measurement process: (b-1) BSA-MAb/NDV-AuNP-Chi-Gra-GCE and (b-2) PAb/NDV-Cu(I)/Cu(II)-Chi-Gra-NDV-BSA-MAb/NDV-AuNP-Chi-Gra-GCE. The supporting electrolyte was 0.1 M KCl + 0.01 M PBS (pH = 7.4). The sample included 15 µL of 10<sup>5.13</sup> EID<sub>50</sub>/0.1 mL NDV (F48E9). (<bold>c</bold>) DPV of the immunosensor measurement process: (c-1) BSA-MAb/NDV-AuNP-Chi-Gra-GCE, (c-2) PAb/NDV-Cu(I)/Cu(II)-Chi-NDV-BSA-MAb/NDV-AuNP-Chi-Gra-GCE, and (c-3) PAb/NDV-Cu(I)/Cu(II)-Chi-Gra-NDV-BSA-MAb/NDV-AuNP-Chi-Gra-GCE. The supporting electrolyte was 0.1 M KCl + 0.01 M PBS (pH = 7.4). The sample included 15 µL of 10<sup>2.13</sup> EID<sub>50</sub>/0.1 mL NDV (F48E9). (<bold>d</bold>) Influence of the incubation time on the current response of the immunosensor to (d-1) NDV and (d-2) PAb/NDV-Cu(I)/Cu(II)-Chi-Gra. The sample included 15 µL of 10<sup>5.13</sup> EID<sub>50</sub>/0.1 mL NDV (F48E9).</p></caption><graphic xlink:href=\"41598_2020_70877_Fig4_HTML\" id=\"MO4\"/></fig></p><p id=\"Par10\">To investigate the immunosensor detection programme, CV was performed in 0.01 mmol/L PBS (pH = 7.4) containing 0.1 mmol/L KCl, and the results are shown in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>b. For CV curve b-1 in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>b, which was obtained with the BSA-MAb/NDV-AuNP-Chi-Gra film-modified GCE, the background current was low, and no CV redox waves were observed because of the absence of electrochemically active substances in the working solution. After the immunosensor was incubated with 10<sup>5.13</sup> EID<sub>50</sub>/0.1 mL NDV and sandwiched for the immunoreaction with PAb/NDV-Cu(I)/Cu(II)-Chi-Gra, stable redox peaks were observed at 0.13 and − 0.08 V vs. saturated calomel electrode (SCE) (curve b-2 in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>b), due to the redox reaction of Cu(I)/Cu(II). The peak at 0.13 V was caused by the oxidation of Cu(I) to Cu(II), and the reduction of Cu(II) to Cu(I) produced the peak at − 0.08 V. These results indicated the efficient redox activity of Cu(I)/Cu(II)-functionalized Gra.</p></sec><sec id=\"Sec6\"><title>Comparison of different signal amplification strategies</title><p id=\"Par11\">Signal amplification strategies are very important for immunosensors. Two signal label materials (PAb/NDV-Cu(I)/Cu(II)-Chi-Gra and PAb/NDV-Cu(I)/Cu(II)-Chi) were prepared, and differential pulse voltammetry (DPV) was performed from − 0.3 to 0.4 V at a 50 mV/s scan rate using a 10<sup>2.13</sup> EID<sub>50</sub>/0.1 mL sample to evaluate the effects of the signal amplification materials. The results are shown in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>c. As indicated by curve c-1, in the absence of a signal labelling material, a low background current was obtained, and no anodic peak was observed for the immunosensor. In contrast, the immunosensor conjugated with PAb/NDV-Cu(I)/Cu(II)-Chi-Gra (curve c-3) exhibited a greater current shift than the immunosensor conjugated with PAb/NDV-Cu(I)/Cu(II)-Chi (curve c-2). The increase in the current shift was due to the use of Gra, which has with a high surface/volume ratio, as the carrier, leading to the immobilization of Cu(I)/Cu(II) on the GCE and facilitating electrochemical signal amplification. These results confirmed that the immunosensor with Gra could load more of the electroactive signal labelling material and PAb/NDV than the immunosensor without Gra. Accordingly, the signal of the immunosensor was greatly amplified by using Gra.</p></sec><sec id=\"Sec7\"><title>Optimization of the experimental conditions</title><p id=\"Par12\">During NDV capture and the specific reaction with the signal labelling material (PAb/NDV-Cu(I)/Cu(II)-Chi-Gra), the incubation time is an important factor. Thus, the incubation times of NDV and PAb/NDV-Cu(I)/Cu(II)-Chi-Gra were optimized separately. To optimize the NDV incubation time, different incubation times (5, 10, 15, 20, 30, 40, 50, and 60 min) were used, and after incubation with NDV, the immunosensors were incubated with PAb/NDV-Cu(I)/Cu(II)-Chi-Gra for 60 min. Finally, the immunosensors were used for DPV detection. Each test was repeated five times. The results are shown in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>d, curve d-1. As the NDV incubation time was increased up to 30 min, the electrochemical response increased; after 30 min, a constant value was reached, indicating that the immunoreaction was complete, and all the NDV in the sample was captured by the immunosensor. Thus, the optimal incubation time for NDV was 30 min.</p><p id=\"Par13\">To optimize the PAb/NDV-Cu(I)/Cu(II)-Chi-Gra incubation time, the immunosensors were first incubated with NDV (10<sup>5.13</sup> EID<sub>50</sub>/0.1 mL) for 30 min and then incubated with PAb/NDV-Cu(I)/Cu(II)-Chi-Gra for 5, 10, 15, 20, 30, 40, 50, and 60 min, respectively. Finally, the immunosensors were used for DPV detection. Each test was repeated five times. The results are shown in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>d, curve d-2. In the second immunoreaction step, as the PAb/NDV-Cu(I)/Cu(II)-Chi-Gra incubation time was increased, the electrochemical response current increased, reaching a steady-state value at 40 min, which indicates that the reaction between NDV and PAb/NDV-Cu(I)/Cu(II)-Chi-Gra was complete. Thus, the optimal incubation time for PAb/NDV-Cu(I)/Cu(II)-Chi-Gra was 40 min. Compared with NDV, PAb/NDV-Cu(I)/Cu(II)-Chi-Gra required more time to complete the reaction, which might be due to the greater steric hindrance of PAb/NDV-Cu(I)/Cu(II)-Chi-Gra.</p></sec><sec id=\"Sec8\"><title>Analytical performance of the immunosensor</title><p id=\"Par14\">The response of the prepared immunosensor was measured at different concentrations of NDV (F48E9) under the optimal experimental conditions. The results are shown in Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>a. The electrochemical response current increased as the concentration of NDV increased, and the peak of the electrochemical response current was proportional to the concentration in the range of 10<sup>0.13</sup> to 10<sup>5.13</sup> EID<sub>50</sub>/0.1 mL. The linear regression equation, which is shown in Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>b, was I (μA) = 0.75 log EID<sub>50</sub>/0.1 mL + 1.05, with a correlation coefficient of 0.97075, and the limit of determination for NDV was 10<sup>0.68</sup> EID<sub>50</sub>/0.1 mL, which was calculated based on a signal-to-noise ratio of 3 (S/N = 3). These results demonstrated that the immunosensor was sensitive enough to quantitatively monitor NDV.<fig id=\"Fig5\"><label>Figure 5</label><caption><p>(<bold>a</bold>) Typical DPV signals acquired in the presence of different concentrations of NDV with PAb/NDV-Cu(I)/Cu(II)-Chi-Gra as the label: (a-1) 0, (a-2) 10<sup>0.13</sup> EID<sub>50</sub>/0.1 mL, (a-3) 10<sup>1.13</sup> EID<sub>50</sub>/0.1 mL, (a-4) 10<sup>2.13</sup> EID<sub>50</sub>/0.1 mL, (a-5) 10<sup>3.13</sup> EID<sub>50</sub>/0.1 mL, (a-6) 10<sup>4.13</sup> EID<sub>50</sub>/0.1 mL, and (a-7) 10<sup>5.13</sup> EID<sub>50</sub>/0.1 mL. (<bold>b</bold>) Relationship between the antigen concentration and sensor current response corresponding to (<bold>a</bold>). (<bold>c</bold>) Typical DPV signals before incubation with NDV (c-1) and in the presence of different concentrations of NDV ((c-2) 0, (c-3) 10<sup>0.13</sup> EID<sub>50</sub>/0.1 mL, (c-4) 10<sup>1.13</sup> EID<sub>50</sub>/0.1 mL, (c-5) 10<sup>2.13</sup> EID<sub>50</sub>/0.1 mL, (c-6) 10<sup>3.13</sup> EID<sub>50</sub>/0.1 mL, (c-7) 10<sup>4.13</sup> EID<sub>50</sub>/0.1 mL, and (c-8) 10<sup>5.13</sup> EID<sub>50</sub>/0.1 mL) with PAb/NDV-Cu(I)/Cu(II)-Chi as the label. (<bold>d</bold>) Relationship between the antigen concentration and sensor current response corresponding to (<bold>c</bold>). Error bar =  ± standard deviation.</p></caption><graphic xlink:href=\"41598_2020_70877_Fig5_HTML\" id=\"MO5\"/></fig></p><p id=\"Par15\">The results for the immunosensor with PAb/NDV-Cu(I)/Cu(II)-Chi-Gra as the signal label were compared with those for the immunosensor with PAb/NDV-Cu(I)/Cu(II)-Chi as the signal label, and the results obtained with PAb/NDV-Cu(I)/Cu(II)-Chi are shown in Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>c. The electrochemical response current increased linearly with increasing NDV concentration, and the calibration curve in the range of 10<sup>0.13</sup> to 10<sup>5.13</sup> EID<sub>50</sub>/0.1 mL (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>d) was: I (μA) = 0.15 log EID<sub>50</sub>/0.1 mL + 1.10. The limit of determination for NDV was 10<sup>2.09</sup> EID<sub>50</sub>/0.1 mL (S/N = 3). This result indicated that Gra can improve the immunosensor sensitivity. In addition, as shown in Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>c (curve c-2), the background signal was high when PAb/NDV-Cu(I)/Cu(II)-Chi was used as the signal label because without Gra, the excess Chi could not be removed from PAb/NDV-Cu(I)/Cu(II)-Chi by centrifugation, and the excess Chi chelated with Cu(I)/Cu(II) was attached to the GCE by non-specific binding.</p></sec><sec id=\"Sec9\"><title>Comparison of methods</title><p id=\"Par16\">The results of a comparative study between the designed method and other methods for NDV detection are summarized in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>a. The table shows that the developed electrochemical immunosensor has acceptable sensitivity and advantages over the other methods in terms of rapid detection, intuitiveness, user-friendliness and cost.<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Comparison of the proposed immunosensor with other sensors for NDV detection (a); results of clinical samples (b); analysis data sheet of positive samples (c); recovery results of clinical samples with different concentrations of NDV (d).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">(a) Method</th><th align=\"left\">Detection time</th><th align=\"left\">Detection limit</th><th align=\"left\">References</th><th align=\"left\"/><th align=\"left\"/></tr></thead><tbody><tr><td align=\"left\">Virus isolation</td><td align=\"left\">4–7 days</td><td align=\"left\">1 EID<sub>50</sub>/mL</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup></td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">RT-PCR</td><td align=\"left\">5 h</td><td align=\"left\">10<sup>4.0</sup> EID<sub>50</sub>/0.1 mL</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup></td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">Real-time RT-PCR</td><td align=\"left\">3 h</td><td align=\"left\">10<sup>1</sup> EID<sub>50</sub>/mL</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup></td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">ICS</td><td align=\"left\">15 min</td><td align=\"left\">10<sup>4.9</sup> EID<sub>50</sub>/0.1 mL</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup></td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">RT-LAMP</td><td align=\"left\">3 h</td><td align=\"left\">1.3 Haemagglutination units</td><td align=\"left\"><sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup></td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">Proposed immunosensor</td><td align=\"left\">70 min</td><td align=\"left\">10<sup>0.68</sup> EID<sub>50</sub>/0.1 mL</td><td align=\"left\">This study</td><td align=\"left\"/><td align=\"left\"/></tr></tbody></table><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">(b) Method</th><th align=\"left\">Total number of samples</th><th align=\"left\">Number of positive samples</th><th align=\"left\">Positive rate/%</th><th align=\"left\"/><th align=\"left\"/></tr></thead><tbody><tr><td align=\"left\">Proposed immunosensor</td><td align=\"left\">120</td><td align=\"left\">7</td><td char=\".\" align=\"char\">5.8</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Virus isolation</td><td align=\"left\">120</td><td align=\"left\">7</td><td char=\".\" align=\"char\">5.8</td><td char=\".\" align=\"char\"/><td char=\".\" align=\"char\"/></tr></tbody></table><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">(c) NO</th><th align=\"left\" colspan=\"3\">Results of the proposed immunosensor</th><th align=\"left\" rowspan=\"2\">Results of virus isolation</th><th align=\"left\" rowspan=\"2\"/></tr><tr><th align=\"left\">Measured concentration/EID<sub>50</sub>/0.1 mL</th><th align=\"left\">Average/EID<sub>50</sub>/0.1 mL</th><th align=\"left\">RSD/% (n = 5)</th></tr></thead><tbody><tr><td align=\"left\">1</td><td align=\"left\">40.74, 39.90, 41.27, 39.15, 42.65</td><td char=\".\" align=\"char\">40.74</td><td char=\".\" align=\"char\">3.28</td><td align=\"left\">Positive</td><td align=\"left\"/></tr><tr><td align=\"left\">2</td><td align=\"left\">92.47, 90.73, 93.04, 91.38, 94.31</td><td char=\".\" align=\"char\">92.39</td><td char=\".\" align=\"char\">1.52</td><td align=\"left\">Positive</td><td align=\"left\"/></tr><tr><td align=\"left\">3</td><td align=\"left\">107.46, 105.92, 108.17, 110.29, 109.67</td><td char=\".\" align=\"char\">108.30</td><td char=\".\" align=\"char\">1.61</td><td align=\"left\">Positive</td><td align=\"left\"/></tr><tr><td align=\"left\">4</td><td align=\"left\">367.41, 370.35, 361.91, 374.34, 354.73</td><td char=\".\" align=\"char\">365.75</td><td char=\".\" align=\"char\">2.09</td><td align=\"left\">Positive</td><td align=\"left\"/></tr><tr><td align=\"left\">5</td><td align=\"left\">409.32, 417.93, 406.78, 423.32, 428.46</td><td char=\".\" align=\"char\">417.16</td><td char=\".\" align=\"char\">2.19</td><td align=\"left\">Positive</td><td align=\"left\"/></tr><tr><td align=\"left\">6</td><td align=\"left\">742.16, 737.59, 731.81, 749.19, 728.94</td><td char=\".\" align=\"char\">737.94</td><td char=\".\" align=\"char\">1.10</td><td align=\"left\">Positive</td><td align=\"left\"/></tr><tr><td align=\"left\">7</td><td align=\"left\">1,490.28, 1,481.38, 1,463.57, 1,447.34, 1,452.85</td><td char=\".\" align=\"char\">1,467.08</td><td char=\".\" align=\"char\">1.25</td><td align=\"left\">Positive</td><td align=\"left\"/></tr></tbody></table><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">(d) NO</th><th align=\"left\" rowspan=\"2\">Initial NDV concentration in sample/EID<sub>50</sub>/0.1 mL</th><th align=\"left\" rowspan=\"2\">Added NDV amount/EID<sub>50</sub>/0.1 mL</th><th align=\"left\" colspan=\"2\">Total found</th><th align=\"left\" rowspan=\"2\">Recovery rate/% (n = 5)</th></tr><tr><th align=\"left\">Average/EID<sub>50</sub>/0.1 mL</th><th align=\"left\">RSD/% (n = 5)</th></tr></thead><tbody><tr><td align=\"left\">1</td><td char=\".\" align=\"char\">40.74</td><td align=\"left\">50</td><td char=\".\" align=\"char\">87.36</td><td char=\".\" align=\"char\">2.74</td><td char=\".\" align=\"char\">96.28</td></tr><tr><td align=\"left\">2</td><td char=\".\" align=\"char\">92.51</td><td align=\"left\">100</td><td char=\".\" align=\"char\">190.83</td><td char=\".\" align=\"char\">2.38</td><td char=\".\" align=\"char\">99.13</td></tr><tr><td align=\"left\">3</td><td char=\".\" align=\"char\">108.30</td><td align=\"left\">500</td><td char=\".\" align=\"char\">610.17</td><td char=\".\" align=\"char\">1.76</td><td char=\".\" align=\"char\">100.31</td></tr><tr><td align=\"left\">4</td><td char=\".\" align=\"char\">365.75</td><td align=\"left\">1,000</td><td char=\".\" align=\"char\">1,363.72</td><td char=\".\" align=\"char\">1.47</td><td char=\".\" align=\"char\">99.85</td></tr><tr><td align=\"left\">5</td><td char=\".\" align=\"char\">417.16</td><td align=\"left\">5,000</td><td char=\".\" align=\"char\">5,421.03</td><td char=\".\" align=\"char\">2.39</td><td char=\".\" align=\"char\">100.07</td></tr><tr><td align=\"left\">6</td><td char=\".\" align=\"char\">737.94</td><td align=\"left\">10,000</td><td char=\".\" align=\"char\">11,219.82</td><td char=\".\" align=\"char\">3.56</td><td char=\".\" align=\"char\">104.49</td></tr><tr><td align=\"left\">7</td><td char=\".\" align=\"char\">1,467.08</td><td align=\"left\">50,000</td><td char=\".\" align=\"char\">50,734.94</td><td char=\".\" align=\"char\">2.71</td><td char=\".\" align=\"char\">98.58</td></tr></tbody></table></table-wrap></p></sec><sec id=\"Sec10\"><title>Selectivity, repeatability, reproducibility and stability of the immunosensor</title><p id=\"Par17\">Selectivity is a significant parameter for an immunosensor. Therefore, to determine the selectivity of the fabricated immunosensor, some possible interferents, including aviadenovirus group I (AAV, 10<sup>6.37</sup> EID<sub>50</sub>/0.1 mL), infectious bronchitis virus (IBV, 10<sup>7.02</sup> EID<sub>50</sub>/0.1 mL), infectious laryngotracheitis virus (ILTV, 10<sup>5.84</sup> EID<sub>50</sub>/0.1 mL), avian influenza virus subtype H7 (AIV H7, 10<sup>6.45</sup> EID<sub>50</sub>/0.1 mL), avian reovirus (ARV, 10<sup>6.51</sup> EID<sub>50</sub>/0.1 mL), infectious bursal disease (IBD, 10<sup>7.34</sup> EID<sub>50</sub>/0.1 mL), glucose (1.0 µg/mL), vitamin C (1.0 µg/mL) and BSA (1.0 µg/mL), were investigated. The results are depicted in Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>a. When the fabricated immunosensor was exposed to possible interferents (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>a (samples a-2 ~ a-10): AAV, IBV, ILTV, AIV H7, ARV, IBD, glucose, vitamin C, and BSA), the detection currents were as low as that for the negative control (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>a (sample a-1): ddH<sub>2</sub>0). The immunosensor exhibited a higher signal when incubated with a sample including NDV (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>a (samples a-11, a-16)) than when incubated with samples containing the possible interferents (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>a (samples a-2 ~ a-10)). Additionally, the responses of the fabricated immunosensor to 10<sup>5.13</sup> and 10<sup>3.13</sup> EID<sub>50</sub>/0.1 mL NDV solutions containing other interfering substances were measured (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>a (samples a-12 ~ a-15, a-17 ~ a-20)), and the current variation due to the interfering substances was less than 5% of that obtained without interferences. The results show that the developed immunosensor had good selectivity for NDV.<fig id=\"Fig6\"><label>Figure 6</label><caption><p>(<bold>a</bold>) Selectivity of the immunosensor: (a-1) ddH<sub>2</sub>0, (a-2) AAV (10<sup>6.37</sup> EID<sub>50</sub>/0.1 mL), (a-3) IBV (10<sup>7.02</sup> EID<sub>50</sub>/0.1 mL), (a-4) ILTV (10<sup>5.84</sup> EID<sub>50</sub>/0.1 mL), (a-5) AIV H7 (10<sup>6.45</sup> EID<sub>50</sub>/0.1 mL), (a-6) ARV (10<sup>6.51</sup> EID<sub>50</sub>/0.1 mL), (a-7) IBD (10<sup>7.34</sup> EID<sub>50</sub>/0.1 mL), (a-8) glucose (1.0 µg/mL), (a-9) vitamin C (1.0 µg/mL), (a-10) BSA (1.0 µg/mL), (a-11) NDV (10<sup>5.13</sup> EID<sub>50</sub>/0.1 mL), (a-12) NDV (10<sup>5.13</sup> EID<sub>50</sub>/0.1 mL) + AAV (10<sup>6.37</sup> EID<sub>50</sub>/0.1 mL), (a-13) NDV (10<sup>5.13</sup> EID<sub>50</sub>/0.1 mL) + IBV (10<sup>7.02</sup> EID<sub>50</sub>/0.1 mL), (a-14) NDV (10<sup>5.13</sup> EID<sub>50</sub>/0.1 mL) + AIV H7 (10<sup>6.45</sup> EID<sub>50</sub>/0.1 mL), (a-15) NDV (10<sup>5.13</sup> EID<sub>50</sub>/0.1 mL) + ARV (10<sup>6.51</sup> EID<sub>50</sub>/0.1 mL), (a-16) NDV (10<sup>3.13</sup> EID<sub>50</sub>/0.1 mL), (a-17) NDV (10<sup>3.13</sup> EID<sub>50</sub>/0.1 mL) + ILTV (10<sup>5.84</sup> EID<sub>50</sub>/0.1 mL), (a-18) NDV (10<sup>3.13</sup> EID<sub>50</sub>/0.1 mL) + IBD (10<sup>7.34</sup> EID<sub>50</sub>/0.1 mL), (a-19) NDV (10<sup>3.13</sup> EID<sub>50</sub>/0.1 mL) + vitamin C (1.0 µg/mL), and (a-20) NDV (10<sup>3.13</sup> EID<sub>50</sub>/0.1 mL) + BSA (1.0 µg/mL); (<bold>b</bold>) repeatability, (<bold>c</bold>) reproducibility, and (<bold>d</bold>) storage stability of the immunosensor.</p></caption><graphic xlink:href=\"41598_2020_70877_Fig6_HTML\" id=\"MO6\"/></fig></p><p id=\"Par18\">Under the optimal experimental conditions, equivalently prepared immunosensors were used to detect 10<sup>3.13</sup> EID<sub>50</sub> NDV 20 times to evaluate the repeatability of the developed immunosensor, and the results are shown in Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>b. The relative standard deviation was 2.58%, demonstrating the good repeatability of the immunosensor. The reproducibility of the immunosensor was evaluated by preparing six different batches of the immunosensor independently. A series of six different batches of the immunosensor were prepared for the detection of 10<sup>3.13</sup> EID<sub>50</sub> NDV, and the results are shown in Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>c. The relative standard deviation was found to be 2.84%, showing the excellent reproducibility.</p><p id=\"Par19\">Long-term storage stability tests show the robustness of an immunosensor. The current responses of the developed immunosensor were periodically checked to evaluate its stability. The immunosensor was stored in PBS (pH = 7.4) at 4 °C when it was not in use. Every week, electrochemical measurements were performed with the developed immunosensor, and the average value was calculated based on five assays. The results shown in Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>d indicated that the immunosensor response current decreased by only 4.1% after 2 weeks. After four weeks, the immunosensor current response decreased by 9.5% relative to its initial current, which indicated that the immunosensor had acceptable storage stability.</p></sec><sec id=\"Sec11\"><title>Application of the proposed immunosensor for the detection of NDV</title><p id=\"Par20\">Oral and cloacal swab samples, which were gently collected from fowls at different live bird markets in Guangxi Province, were used as clinical samples. A viral transport medium composed of 0.05 mmol/L PBS containing 10 mg/mL gentamycin, 10 mg/mL kanamycin, 10 mg/mL streptomycin, 5% (v/v) foetal bovine serum and 10,000 units/mL penicillin was used to prepare the clinical samples, and the clinical samples were placed in an ice box.</p><p id=\"Par21\">With the permission of the owners of the live bird markets, a total of 120 clinical samples were collected from chickens, the samples were assayed using the proposed immunosensor, and seven NDV-positive samples were detected. Virus isolation<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup> was employed to confirm the test results. The positive results detected by the developed immunosensor were in agreement with the results of virus isolation, and the results are summarized in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>b,c. To test the recovery by the proposed immunosensor, NDV standards were added to the clinical samples that had been confirmed as positive. The results (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>d) showed that the fabricated immunosensor had acceptable recovery (96.28 ~ 104.49). Considering the acceptable recovery in real samples, the immunosensor was found to be practical for sample detection.</p></sec></sec><sec id=\"Sec12\"><title>Materials and methods</title><sec id=\"Sec13\"><title>Reagents and materials</title><p id=\"Par22\">MAb/NDV and PAb/NDV were purchased from Abcam (Cambridge, UK). Copper sulfate (CuSO<sub>4</sub>), hydrochloroauric acid (HAuCl<sub>4</sub>), graphite powder (< 45 mm), KMnO<sub>4</sub>, NaNO<sub>3</sub> and H<sub>2</sub>SO<sub>4</sub> were supplied by the Guoyao Group Chemical Reagents Co., Ltd., Shanghai. Bovine serum albumin (BSA) was purchased from Sigma (USA). All chemicals used were of analytical reagent grade. Double-distilled deionized water was used in all experiments. In addition, 10 mmol/L PBS (pH = 7.4) was prepared by mixing stock solutions of 10 mmol/L NaH<sub>2</sub>PO<sub>4</sub> and 10 mmol/L Na<sub>2</sub>HPO<sub>4</sub>.</p></sec><sec id=\"Sec14\"><title>Instruments</title><p id=\"Par23\">SEM was performed on a HITACHI UHR FE-SEM SU8000 Series (SU8020) instrument. FT-IR spectra were collected on a Nicolet IS10 instrument. XPS analysis was performed on an X-ray photoelectron spectrometer (ESCALAB 250Xi, Thermo Scientific). A CHI660D electrochemical workstation (Beijing CH Instruments, Beijing, China) with a standard three-electrode cell (a working electrode, an SCE as the reference electrode and a platinum wire as the auxiliary electrode) was employed to study the electrochemical characteristics. Electrochemical detection was performed at room temperature (25 ± 0.5 °C).</p></sec><sec id=\"Sec15\"><title>Gra synthesis</title><p id=\"Par24\">A modified Hummers method was used to prepare Gra oxide<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. In short, NaNO<sub>3</sub> (2.5 g) and graphite powder (1.0 g) were added to concentrated H<sub>2</sub>SO<sub>4</sub> (100 mL) and stirred for 2 h. KMnO<sub>4</sub> (5 g) was slowly added to the mixture under continuous stirring, and the mixture was then cooled with ice. Next, the mixture was stirred at 35 °C for 24 h. Double-distilled deionized water (100 mL) was slowly added to the reacted slurry, which was then stirred at 80 °C for another 3 h. Next, more double-distilled deionized water (300 mL) was added to the reacted slurry. Then, 6 mL of H<sub>2</sub>O<sub>2</sub> (30%) was added (bubbles appeared, and the slurry immediately turned bright yellow). The resulting solution was continuously stirred for 3 h and then precipitated for 24 h at room temperature. The supernatant was subsequently decanted. The resulting yellow slurry was washed with 0.5 mol/L HCl (500 mL) and centrifuged. The solution was washed with double-distilled deionized water and centrifuged until the pH of the solution was neutral (pH = 7.0). Gra oxide was obtained after the solution was ultrasonicated for 2 h. To obtain Gra, Gra oxide was reduced at 95 °C for 3 h using NaBH<sub>4</sub> as a reducing agent.</p></sec><sec id=\"Sec16\"><title>Preparation of the Chi-Gra nanocomposite</title><p id=\"Par25\">Chi<italic>-</italic>Gra was prepared according to a previously reported method<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Briefly, Chi powder was dissolved in a 1.0% (v/v) acetic acid solution under stirring for 0.5 h at room temperature until it was completely dispersed. The Chi solution (0.5 wt.%) was thus prepared. Then, Gra (10 mg) was added to the Chi solution (10 mL), ultrasonicated for 1 h, and stirred for 24 h at 25 °C. Finally, the Chi-Gra nanocomposite was obtained.</p></sec><sec id=\"Sec17\"><title>Preparation of the AuNP-Chi-Gra nanocomposite</title><p id=\"Par26\">The AuNP-Chi-Gra nanocomposite was prepared as previously described<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. Furthermore, 0.5 mL of HAuCl<sub>4</sub> (1 mM) was added to Chi-Gra (5 mL) under stirring at 25 °C for 4 h. Then, the solution was incubated at 80 °C for 1 h with vigorous stirring. Au<sup>3+</sup> was subsequently reduced to AuNPs by Chi at 80 °C. Finally, the AuNP-Chi-Gra nanocomposite was obtained.</p></sec><sec id=\"Sec18\"><title>Preparation of the Cu(I)/Cu(II)-Chi-Gra nanocomposite</title><p id=\"Par27\">The Cu(I)/Cu(II)-Chi-Gra nanocomposite was prepared according to the method used to prepare the AuNP-Chi-Gra nanocomposite with certain modifications. CuSO<sub>4</sub>·5H<sub>2</sub>O was used as the source of copper. First, 10 mg of CuSO<sub>4</sub>·5H<sub>2</sub>O was added to 5 mL of the Chi-Gra nanocomposite under continuous stirring at 25 °C for 8 h. Then, the mixture was incubated at 95 °C for 4 h under continuous stirring. Finally, the Cu(I)/Cu(II)-Chi-Gra nanocomposite was obtained.</p></sec><sec id=\"Sec19\"><title>Preparation of PAb/NDV-Cu(I)/Cu(II)-Chi-Gra nanocomposite bioconjugates</title><p id=\"Par28\">First, 5 mL of the Cu(I)/Cu(II)-Chi-Gra nanocomposite obtained from the above preparation method was centrifuged (12,000 rpm, 10 min), the supernatant was discarded, and the residue was washed with double-distilled deionized water three times to remove the excess Chi, Cu<sup>2+</sup> and SO<sub>4</sub><sup>2−</sup> that did not combine with Gra. Then, 5.0 mL of a PBS buffer (pH = 7.4) was added to the residue to disperse the Cu(I)/Cu(II)-Chi-Gra nanocomposite, and the mixture was sonicated for 10 min to obtain a homogeneous suspension. Next, 1 mL of PAb/NDV (10 µg/mL) was added to the homogeneous suspension, and the mixture was vigorously stirred for 5 min at 4 °C. Then, 1 mL of 1% glutaraldehyde was slowly added to the solution under continuous stirring. The solution was subsequently incubated at 4 °C for 8 h. The reaction mixture was washed with PBS (pH = 7.4) and centrifuged (12,000 rpm, 10 min) three times. The supernatant was discarded, the resulting mixture was dispersed in PBS (5.0 mL, pH = 7.4), and 1 mL of a 2.0% (w/v) BSA solution was added to the suspension, which was then incubated at 4 °C for 8 h. The obtained PAb/NDV-Cu(I)/Cu(II)-Chi-Gra nanocomposite was stored at 4 °C for further use.</p></sec><sec id=\"Sec20\"><title>Fabrication of the electrochemical immunosensor</title><p id=\"Par29\">First, 0.05 mm alumina was used to polish a GCE (Ø = 3 mm) until it had a mirror-like surface. Then, the GCE was rinsed with double-distilled deionized water and ultrasonicated in baths of double-distilled deionized water, ethyl alcohol, and double-distilled deionized water to remove any physically adsorbed substances. Next, the GCE was placed in H<sub>2</sub>SO<sub>4</sub> (0.05 M) and chemically cleaned until the background signal stabilized. Finally, the GCE was thoroughly rinsed with double-distilled deionized water and dried with nitrogen gas to obtain a clean GCE.</p><p id=\"Par30\">Figure <xref rid=\"Fig7\" ref-type=\"fig\">7</xref> shows the procedures used to construct the immunosensor. The process was as follows: the AuNP-Chi-Gra (8 μL) nanocomposite was dropped onto the clean GCE surface, dried at 4 °C overnight to obtain the modified electrode (AuNP-Chi-Gra-GCE), washed with double-distilled deionized water, immersed in a 1 µg/mL (200 µL) MAb/NDV PBS solution (pH = 7.4) and incubated at 4 °C for 8 h. The resulting electrode (MAb/NDV-AuNP-Chi-Gra-GCE) was immersed in a 1.0% (w/w) BSA solution for 1 h at 37 °C to block the remaining active sites. The final modified electrode was stored at 4 °C when not in use.<fig id=\"Fig7\"><label>Figure 7</label><caption><p>Preparation procedures of AuNP-Chi-Gra, Cu(I)/Cu(II)-Chi-Gra (<bold>a</bold>) and the immunosensor (<bold>b</bold>).</p></caption><graphic xlink:href=\"41598_2020_70877_Fig7_HTML\" id=\"MO7\"/></fig></p></sec><sec id=\"Sec21\"><title>Electrochemical immunosensor detection</title><p id=\"Par31\">A well-known sandwich immunoassay was used to detect NDV. First, the MAb/NDV-AuNP-Chi-Gra-GCE immunosensor was incubated with 15 μL of the sample for 30 min and then washed with a PBS buffer (pH = 7.4) to remove non-specifically adsorbed conjugates. Next, the modified electrode was incubated with 200 μL of the PAb/NDV-Cu(I)/Cu(II)-Chi-Gra nanocomposite for 40 min and washed with a PBS buffer (pH = 7.4). Finally, the resulting electrode was placed in a 0.01 mol/L PBS (pH = 7.4) KCl solution, and DPV experiments were performed (− 0.3 to 0.4 V, 50 mV/s) to detect NDV.</p></sec><sec id=\"Sec22\"><title>Ethics statement</title><p id=\"Par32\">The authors confirm that relevant guidelines were followed for the care and use of animals. This work was approved and conducted by the Animal Ethics Committee of the Guangxi Veterinary Research Institute, which supervises all live bird markets in Guangxi Province. Oral and cloacal swab samples, which were gently collected from fowls at different live bird markets in Guangxi Province, were used as clinical samples. Before sampling, the fowls were not anaesthetized, and after sampling, they were returned to their cages and observed for 30 min.</p></sec></sec><sec id=\"Sec23\"><title>Conclusions</title><p id=\"Par33\">In summary, AuNP-Chi-Gra was used as a platform, and PAb/NDV-Cu(I)/Cu(II)-Chi-Gra was used as a label for signal amplification in this work. Based on the well-known sandwich immunoreaction, a novel electrochemical immunosensor was developed for the quantitative detection of NDV. It exhibited a linear response over a wide range (10<sup>0.13</sup> to 10<sup>5.13</sup> EID<sub>50</sub>/mL), had a low detection limit (10<sup>0.68</sup> EID<sub>50</sub>/0.1 mL), and was more sensitive than an immunosensor with PAb/NDV-Cu(I)/Cu(II)-Chi as the signal label (the limit of detection for NDV was 10<sup>2.09</sup> EID<sub>50</sub>/0.1 mL). This newly designed immunosensor might have widespread application potential because it had acceptable reproducibility, selectivity and stability; could be obtained by a facile fabrication procedure; and was ultrasensitive for the detection of NDV.</p></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><ack><title>Acknowledgements</title><p>This research project was funded by the Guangxi Science and Technology Projects (AB16380054), Guangxi Science Base and Talents Special Program (AD17195083), Guangxi Science Great Special Program (AA17204057), Guangxi BaGui Scholars Program Foundation (2019-79) and Guangxi Shi Bai Qian Talents Engineering Foundation ([2020]24).</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>J.L.H. and Z.X.X. designed and conceived the experiments; J.L.H., Z.X.X., Y.H.H. and L.J.X. performed the experiments; and J.L.H., S.S.L., Q.F., T.T.Z., Y.F.Z., S.W., M.X.Z., Z.Q.X., and X.W.D. analysed the data and contributed reagents/materials/analysis tools. All authors reviewed the manuscript.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par34\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brown</surname><given-names>VR</given-names></name><name><surname>Bevins</surname><given-names>SN</given-names></name></person-group><article-title>A review of virulent Newcastle disease viruses in the United States and the role of wild birds in viral persistence and spread</article-title><source>Vet. Res.</source><year>2017</year><volume>48</volume><fpage>68</fpage><pub-id pub-id-type=\"doi\">10.1186/s13567-017-0475-9</pub-id><pub-id pub-id-type=\"pmid\">29073919</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ganar</surname><given-names>K</given-names></name><name><surname>Das</surname><given-names>M</given-names></name><name><surname>Sinha</surname><given-names>S</given-names></name><name><surname>Kumar</surname><given-names>S</given-names></name></person-group><article-title>Newcastle disease virus: Current status and our understanding</article-title><source>Virus Res.</source><year>2014</year><volume>184</volume><fpage>71</fpage><lpage>81</lpage><pub-id pub-id-type=\"doi\">10.1016/j.virusres.2014.02.016</pub-id><pub-id pub-id-type=\"pmid\">24589707</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>XF</given-names></name><name><surname>Wan</surname><given-names>HQ</given-names></name><name><surname>Ni</surname><given-names>XX</given-names></name><name><surname>Wu</surname><given-names>YT</given-names></name><name><surname>Liu</surname><given-names>WB</given-names></name></person-group><article-title>Pathotypical and genotypical characterization of strains of Newcastle disease virus isolated from outbreaks in chicken and goose flocks in some regions of China during 1985–2001</article-title><source>Adv. Virol.</source><year>2003</year><volume>148</volume><fpage>1387</fpage><lpage>1403</lpage><pub-id pub-id-type=\"doi\">10.1007/s00705-003-0014-z</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ali</surname><given-names>A</given-names></name><name><surname>Reynolds</surname><given-names>DL</given-names></name></person-group><article-title>A multiplex reverse transcription-polymerase chain reaction assay for Newcastle disease virus and avian pneumovirus (Colorado strain)</article-title><source>Avian Dis.</source><year>2000</year><volume>44</volume><fpage>938</fpage><lpage>943</lpage><pub-id pub-id-type=\"doi\">10.2307/1593068</pub-id><pub-id pub-id-type=\"pmid\">11195650</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fratnik Steyer</surname><given-names>A</given-names></name><name><surname>Rojs</surname><given-names>OZ</given-names></name><name><surname>Krapez</surname><given-names>U</given-names></name><name><surname>Slavec</surname><given-names>B</given-names></name><name><surname>Barlic-Maganja</surname><given-names>D</given-names></name></person-group><article-title>A diagnostic method based on MGB probes for rapid detection and simultaneous differentiation between virulent and vaccine strains of avian paramyxovirus type 1</article-title><source>J. Virol. Methods</source><year>2010</year><volume>166</volume><fpage>28</fpage><lpage>36</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jviromet.2010.02.012</pub-id><pub-id pub-id-type=\"pmid\">20170680</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>Q</given-names></name><etal/></person-group><article-title>Evaluation of an immunochromatographic strip for detection of avian avulavirus 1 (Newcastle disease virus)</article-title><source>J. Vet. Diagn. Invest.</source><year>2019</year><volume>31</volume><fpage>475</fpage><lpage>480</lpage><pub-id pub-id-type=\"doi\">10.1177/1040638719837320</pub-id><pub-id pub-id-type=\"pmid\">30973087</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>Q</given-names></name><etal/></person-group><article-title>An improved reverse transcription loop-mediated isothermal amplification assay for sensitive and specific detection of Newcastle disease virus</article-title><source>Adv. Virol.</source><year>2009</year><volume>154</volume><fpage>1433</fpage><lpage>1440</lpage><pub-id pub-id-type=\"doi\">10.1007/s00705-009-0464-z</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hosu</surname><given-names>O</given-names></name><name><surname>Selvolini</surname><given-names>G</given-names></name><name><surname>Cristea</surname><given-names>C</given-names></name><name><surname>Marrazza</surname><given-names>G</given-names></name></person-group><article-title>Electrochemical immunosensors for disease detection and diagnosis</article-title><source>Curr. Med. Chem.</source><year>2018</year><volume>25</volume><fpage>4119</fpage><lpage>4137</lpage><pub-id pub-id-type=\"doi\">10.2174/0929867324666170727104429</pub-id><pub-id pub-id-type=\"pmid\">28748767</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Felix</surname><given-names>FS</given-names></name><name><surname>Angnes</surname><given-names>L</given-names></name></person-group><article-title>Electrochemical immunosensors—a powerful tool for analytical applications</article-title><source>Biosens. Bioelectron.</source><year>2018</year><volume>102</volume><fpage>470</fpage><lpage>478</lpage><pub-id pub-id-type=\"doi\">10.1016/j.bios.2017.11.029</pub-id><pub-id pub-id-type=\"pmid\">29182930</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aydin</surname><given-names>EB</given-names></name><name><surname>Aydin</surname><given-names>M</given-names></name><name><surname>Sezginturk</surname><given-names>MK</given-names></name></person-group><article-title>Electrochemical immunosensor based on chitosan/conductive carbon black composite modified disposable ITO electrode: An analytical platform for p53 detection</article-title><source>Biosens. Bioelectron.</source><year>2018</year><volume>121</volume><fpage>80</fpage><lpage>89</lpage><pub-id pub-id-type=\"doi\">10.1016/j.bios.2018.09.008</pub-id><pub-id pub-id-type=\"pmid\">30199712</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aydin</surname><given-names>M</given-names></name><name><surname>Aydin</surname><given-names>EB</given-names></name><name><surname>Sezginturk</surname><given-names>MK</given-names></name></person-group><article-title>A highly selective electrochemical immunosensor based on conductive carbon black and star PGMA polymer composite material for IL-8 biomarker detection in human serum and saliva</article-title><source>Biosens. Bioelectron.</source><year>2018</year><volume>117</volume><fpage>720</fpage><lpage>728</lpage><pub-id pub-id-type=\"doi\">10.1016/j.bios.2018.07.010</pub-id><pub-id pub-id-type=\"pmid\">30014946</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>B</given-names></name><etal/></person-group><article-title>An amperometric beta-glucan biosensor based on the immobilization of bi-enzyme on Prussian blue-chitosan and gold nanoparticles-chitosan nanocomposite films</article-title><source>Biosens. Bioelectron.</source><year>2014</year><volume>55</volume><fpage>113</fpage><lpage>119</lpage><pub-id pub-id-type=\"doi\">10.1016/j.bios.2013.12.004</pub-id><pub-id pub-id-type=\"pmid\">24368228</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bhattarai</surname><given-names>JK</given-names></name><etal/></person-group><article-title>Preparation, modification, characterization, and biosensing application of nanoporous gold using electrochemical techniques</article-title><source>Nanomaterials</source><year>2018</year><pub-id pub-id-type=\"doi\">10.3390/nano8030171</pub-id><pub-id pub-id-type=\"pmid\">29547580</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sun</surname><given-names>D</given-names></name><etal/></person-group><article-title>Electrochemical immunosensors with AuPt-vertical graphene/glassy carbon electrode for alpha-fetoprotein detection based on label-free and sandwich-type strategies</article-title><source>Biosens. Bioelectron.</source><year>2019</year><volume>132</volume><fpage>68</fpage><lpage>75</lpage><pub-id pub-id-type=\"doi\">10.1016/j.bios.2019.02.045</pub-id><pub-id pub-id-type=\"pmid\">30856429</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rezaei</surname><given-names>B</given-names></name><etal/></person-group><article-title>An electrochemical immunosensor for cardiac Troponin I using electrospun carboxylated multi-walled carbon nanotube-whiskered nanofibres</article-title><source>Talanta</source><year>2018</year><volume>182</volume><fpage>178</fpage><lpage>186</lpage><pub-id pub-id-type=\"doi\">10.1016/j.talanta.2018.01.046</pub-id><pub-id pub-id-type=\"pmid\">29501138</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sun</surname><given-names>B</given-names></name><etal/></person-group><article-title>Investigate electrochemical immunosensor of cortisol based on gold nanoparticles/magnetic functionalized reduced graphene oxide</article-title><source>Biosens. Bioelectron.</source><year>2017</year><volume>88</volume><fpage>55</fpage><lpage>62</lpage><pub-id pub-id-type=\"doi\">10.1016/j.bios.2016.07.047</pub-id><pub-id pub-id-type=\"pmid\">27499382</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tuteja</surname><given-names>SK</given-names></name><etal/></person-group><article-title>A label-free electrochemical immunosensor for the detection of cardiac marker using graphene quantum dots (GQDs)</article-title><source>Biosens. Bioelectron.</source><year>2016</year><volume>86</volume><fpage>548</fpage><lpage>556</lpage><pub-id pub-id-type=\"doi\">10.1016/j.bios.2016.07.052</pub-id><pub-id pub-id-type=\"pmid\">27448545</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bhardwaj</surname><given-names>H</given-names></name><name><surname>Pandey</surname><given-names>MK</given-names></name><name><surname>Rajesh</surname><given-names>SG</given-names></name></person-group><article-title>Electrochemical Aflatoxin B1 immunosensor based on the use of graphene quantum dots and gold nanoparticles</article-title><source>Mikrochim. Acta</source><year>2019</year><volume>186</volume><fpage>592</fpage><pub-id pub-id-type=\"doi\">10.1007/s00604-019-3701-5</pub-id><pub-id pub-id-type=\"pmid\">31372749</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stankovich</surname><given-names>S</given-names></name><etal/></person-group><article-title>Graphene-based composite materials</article-title><source>Nature</source><year>2006</year><volume>442</volume><fpage>282</fpage><lpage>286</lpage><pub-id pub-id-type=\"doi\">10.1038/nature04969</pub-id><pub-id pub-id-type=\"pmid\">16855586</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Geim</surname><given-names>AK</given-names></name><name><surname>Novoselov</surname><given-names>KS</given-names></name></person-group><article-title>The rise of graphene</article-title><source>Nat. Mater.</source><year>2007</year><volume>6</volume><fpage>183</fpage><lpage>191</lpage><pub-id pub-id-type=\"doi\">10.1038/nmat1849</pub-id><pub-id pub-id-type=\"pmid\">17330084</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>J</given-names></name><etal/></person-group><article-title>Three-dimensional electrochemical immunosensor for sensitive detection of carcinoembryonic antigen based on monolithic and macroporous graphene foam</article-title><source>Biosens. Bioelectron.</source><year>2015</year><volume>65</volume><fpage>281</fpage><lpage>286</lpage><pub-id pub-id-type=\"doi\">10.1016/j.bios.2014.10.016</pub-id><pub-id pub-id-type=\"pmid\">25461170</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>L</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Yu</surname><given-names>J</given-names></name><name><surname>Ge</surname><given-names>S</given-names></name><name><surname>Song</surname><given-names>X</given-names></name></person-group><article-title>All-graphene composite materials for signal amplification toward ultrasensitive electrochemical immunosensing of tumor marker</article-title><source>Biosens. Bioelectron.</source><year>2015</year><volume>71</volume><fpage>108</fpage><lpage>114</lpage><pub-id pub-id-type=\"doi\">10.1016/j.bios.2015.04.032</pub-id><pub-id pub-id-type=\"pmid\">25897879</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>J</given-names></name><etal/></person-group><article-title>Silver nanoparticles coated graphene electrochemical sensor for the ultrasensitive analysis of avian influenza virus H7</article-title><source>Anal. Chim. Acta</source><year>2016</year><volume>913</volume><fpage>121</fpage><lpage>127</lpage><pub-id pub-id-type=\"doi\">10.1016/j.aca.2016.01.050</pub-id><pub-id pub-id-type=\"pmid\">26944996</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Guzman</surname><given-names>J</given-names></name><name><surname>Saucedo</surname><given-names>I</given-names></name><name><surname>Revilla</surname><given-names>J</given-names></name><name><surname>Navarro</surname><given-names>R</given-names></name><name><surname>Guibal</surname><given-names>E</given-names></name></person-group><article-title>Copper sorption by chitosan in the presence of citrate ions: Influence of metal speciation on sorption mechanism and uptake capacities</article-title><source>Int. J. Biol. Macromol.</source><year>2003</year><volume>33</volume><fpage>57</fpage><lpage>65</lpage><pub-id pub-id-type=\"doi\">10.1016/s0141-8130(03)00067-9</pub-id><pub-id pub-id-type=\"pmid\">14599585</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Di Tocco</surname><given-names>A</given-names></name><etal/></person-group><article-title>Development of an electrochemical biosensor for the determination of triglycerides in serum samples based on a lipase/magnetite-chitosan/copper oxide nanoparticles/multiwalled carbon nanotubes/pectin composite</article-title><source>Talanta</source><year>2018</year><volume>190</volume><fpage>30</fpage><lpage>37</lpage><pub-id pub-id-type=\"doi\">10.1016/j.talanta.2018.07.028</pub-id><pub-id pub-id-type=\"pmid\">30172514</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Singh</surname><given-names>J</given-names></name><etal/></person-group><article-title>Bienzyme-functionalized monodispersed biocompatible cuprous oxide/chitosan nanocomposite platform for biomedical application</article-title><source>J. Phys. Chem. B</source><year>2013</year><volume>117</volume><fpage>141</fpage><lpage>152</lpage><pub-id pub-id-type=\"doi\">10.1021/jp309639w</pub-id><pub-id pub-id-type=\"pmid\">23270337</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>H</given-names></name><etal/></person-group><article-title>Facile synthesis of cuprous oxide nanowires decorated graphene oxide nanosheets nanocomposites and its application in label-free electrochemical immunosensor</article-title><source>Biosens. Bioelectron.</source><year>2017</year><volume>87</volume><fpage>745</fpage><lpage>751</lpage><pub-id pub-id-type=\"doi\">10.1016/j.bios.2016.09.014</pub-id><pub-id pub-id-type=\"pmid\">27649330</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oliveira</surname><given-names>PR</given-names></name><etal/></person-group><article-title>Electrochemical determination of copper ions in spirit drinks using carbon paste electrode modified with biochar</article-title><source>Food Chem.</source><year>2015</year><volume>171</volume><fpage>426</fpage><lpage>431</lpage><pub-id pub-id-type=\"doi\">10.1016/j.foodchem.2014.09.023</pub-id><pub-id pub-id-type=\"pmid\">25308690</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kumar</surname><given-names>S</given-names></name><name><surname>Koh</surname><given-names>J</given-names></name><name><surname>Kim</surname><given-names>H</given-names></name><name><surname>Gupta</surname><given-names>MK</given-names></name><name><surname>Dutta</surname><given-names>PK</given-names></name></person-group><article-title>A new chitosan-thymine conjugate: Synthesis, characterization and biological activity</article-title><source>Int. J. Biol. Macromol.</source><year>2012</year><volume>50</volume><fpage>493</fpage><lpage>502</lpage><pub-id pub-id-type=\"doi\">10.1016/j.ijbiomac.2012.01.015</pub-id><pub-id pub-id-type=\"pmid\">22285484</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shanmugasundaram</surname><given-names>N</given-names></name><etal/></person-group><article-title>Collagen-chitosan polymeric scaffolds for the in vitro culture of human epidermoid carcinoma cells</article-title><source>Biomaterials</source><year>2001</year><volume>22</volume><fpage>1943</fpage><lpage>1951</lpage><pub-id pub-id-type=\"doi\">10.1016/s0142-9612(00)00220-9</pub-id><pub-id pub-id-type=\"pmid\">11426872</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Qi</surname><given-names>L</given-names></name><name><surname>Xu</surname><given-names>Z</given-names></name><name><surname>Jiang</surname><given-names>X</given-names></name><name><surname>Hu</surname><given-names>C</given-names></name><name><surname>Zou</surname><given-names>X</given-names></name></person-group><article-title>Preparation and antibacterial activity of chitosan nanoparticles</article-title><source>Carbohyd. Res.</source><year>2004</year><volume>339</volume><fpage>2693</fpage><lpage>2700</lpage><pub-id pub-id-type=\"doi\">10.1016/j.carres.2004.09.007</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morales</surname><given-names>J</given-names></name><name><surname>Espinos</surname><given-names>JP</given-names></name><name><surname>Caballero</surname><given-names>A</given-names></name><name><surname>Gonzalez-Elipe</surname><given-names>AR</given-names></name><name><surname>Mejias</surname><given-names>JA</given-names></name></person-group><article-title>XPS study of interface and ligand effects in supported Cu2O and CuO nanometric particles</article-title><source>J. Phys. Chem. B</source><year>2005</year><volume>109</volume><fpage>7758</fpage><lpage>7765</lpage><pub-id pub-id-type=\"doi\">10.1021/jp0453055</pub-id><pub-id pub-id-type=\"pmid\">16851901</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Qiu</surname><given-names>H</given-names></name><name><surname>Zhang</surname><given-names>S</given-names></name><name><surname>Pan</surname><given-names>B</given-names></name><name><surname>Zhang</surname><given-names>W</given-names></name><name><surname>Lv</surname><given-names>L</given-names></name></person-group><article-title>Effect of sulfate on Cu(II) sorption to polymer-supported nano-iron oxides: Behavior and XPS study</article-title><source>J. Colloid Interface Sci.</source><year>2012</year><volume>366</volume><fpage>37</fpage><lpage>43</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jcis.2011.09.070</pub-id><pub-id pub-id-type=\"pmid\">22014398</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>H</given-names></name><name><surname>Zhang</surname><given-names>B</given-names></name><name><surname>Bulin</surname><given-names>C</given-names></name><name><surname>Li</surname><given-names>R</given-names></name><name><surname>Xing</surname><given-names>R</given-names></name></person-group><article-title>High-efficient synthesis of graphene oxide based on improved hummers method</article-title><source>Sci. Rep.</source><year>2016</year><volume>6</volume><fpage>36143</fpage><pub-id pub-id-type=\"doi\">10.1038/srep36143</pub-id><pub-id pub-id-type=\"pmid\">27808164</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>KJ</given-names></name><name><surname>Niu</surname><given-names>DJ</given-names></name><name><surname>Xie</surname><given-names>WZ</given-names></name><name><surname>Wang</surname><given-names>W</given-names></name></person-group><article-title>A disposable electrochemical immunosensor for carcinoembryonic antigen based on nano-Au/multi-walled carbon nanotubes-chitosans nanocomposite film modified glassy carbon electrode</article-title><source>Anal. Chim. Acta</source><year>2010</year><volume>659</volume><fpage>102</fpage><lpage>108</lpage><pub-id pub-id-type=\"doi\">10.1016/j.aca.2009.11.023</pub-id><pub-id pub-id-type=\"pmid\">20103110</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Nat Commun</journal-id><journal-id journal-id-type=\"iso-abbrev\">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type=\"epub\">2041-1723</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807804</article-id><article-id pub-id-type=\"pmc\">PMC7431566</article-id><article-id pub-id-type=\"publisher-id\">17980</article-id><article-id pub-id-type=\"doi\">10.1038/s41467-020-17980-1</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Adaptation to the cervical environment is associated with increased antibiotic susceptibility in <italic>Neisseria gonorrhoeae</italic></article-title></title-group><contrib-group><contrib contrib-type=\"author\" equal-contrib=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-4326-2911</contrib-id><name><surname>Ma</surname><given-names>Kevin C.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-6255-690X</contrib-id><name><surname>Mortimer</surname><given-names>Tatum D.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-1372-1301</contrib-id><name><surname>Hicks</surname><given-names>Allison L.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-4599-9164</contrib-id><name><surname>Wheeler</surname><given-names>Nicole E.</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-4162-0228</contrib-id><name><surname>Sánchez-Busó</surname><given-names>Leonor</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-0688-2521</contrib-id><name><surname>Golparian</surname><given-names>Daniel</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Taiaroa</surname><given-names>George</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rubin</surname><given-names>Daniel H. F.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Yi</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-7363-6665</contrib-id><name><surname>Williamson</surname><given-names>Deborah A.</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Unemo</surname><given-names>Magnus</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Harris</surname><given-names>Simon R.</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-5646-1314</contrib-id><name><surname>Grad</surname><given-names>Yonatan H.</given-names></name><address><email>ygrad@hsph.harvard.edu</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.38142.3c</institution-id><institution-id institution-id-type=\"ISNI\">000000041936754X</institution-id><institution>Department of Immunology and Infectious Diseases, </institution><institution>Harvard T.H. Chan School of Public Health, </institution></institution-wrap>Boston, MA USA </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.10306.34</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0606 5382</institution-id><institution>Centre for Genomic Pathogen Surveillance, Wellcome Sanger Institute, </institution><institution>Wellcome Genome Campus, </institution></institution-wrap>Hinxton, Cambridgeshire UK </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.15895.30</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0738 8966</institution-id><institution>WHO Collaborating Centre for Gonorrhoea and other STIs, Swedish Reference Laboratory for STIs, Faculty of Medicine and Health, </institution><institution>Örebro University, </institution></institution-wrap>Örebro, Sweden </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.1008.9</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2179 088X</institution-id><institution>Microbiological Diagnostic Unit Public Health Laboratory, Department of Microbiology and Immunology, </institution><institution>The University of Melbourne at The Peter Doherty Institute for Infection and Immunity, </institution></institution-wrap>Melbourne, VIC Australia </aff><aff id=\"Aff5\"><label>5</label>Microbiotica Ltd, Biodata Innovation Centre, Wellcome Genome Campus, Hinxton, Cambridgeshire UK </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.62560.37</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0378 8294</institution-id><institution>Division of Infectious Diseases, </institution><institution>Brigham and Women’s Hospital and Harvard Medical School, </institution></institution-wrap>Boston, MA USA </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>4126</elocation-id><history><date date-type=\"received\"><day>26</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>24</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\"><italic>Neisseria gonorrhoeae</italic> is an urgent public health threat due to rapidly increasing incidence and antibiotic resistance. In contrast with the trend of increasing resistance, clinical isolates that have reverted to susceptibility regularly appear, prompting questions about which pressures compete with antibiotics to shape gonococcal evolution. Here, we used genome-wide association to identify loss-of-function (LOF) mutations in the efflux pump <italic>mtrCDE</italic> operon as a mechanism of increased antibiotic susceptibility and demonstrate that these mutations are overrepresented in cervical relative to urethral isolates. This enrichment holds true for LOF mutations in another efflux pump, <italic>farAB</italic>, and in urogenitally-adapted versus typical <italic>N. meningitidis</italic>, providing evidence for a model in which expression of these pumps in the female urogenital tract incurs a fitness cost for pathogenic <italic>Neisseria</italic>. Overall, our findings highlight the impact of integrating microbial population genomics with host metadata and demonstrate how host environmental pressures can lead to increased antibiotic susceptibility.</p></abstract><abstract id=\"Abs2\" abstract-type=\"web-summary\"><p id=\"Par2\">Antibiotic resistance in <italic>Neisseria gonorrhoeae</italic> is rising, yet sometimes strains emerge that have reverted to susceptibility. Here, the authors find that selective pressures from the host may influence susceptibility through loss-of-function mutations in genes that encode for efflux pumps.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Microbial genetics</kwd><kwd>Pathogens</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/100000060</institution-id><institution>U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)</institution></institution-wrap></funding-source><award-id>1 F32 AI145157-01</award-id><award-id>1R01AI132606-01</award-id><principal-award-recipient><name><surname>Mortimer</surname><given-names>Tatum D.</given-names></name><name><surname>Grad</surname><given-names>Yonatan H.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100000925</institution-id><institution>Department of Health | National Health and Medical Research Council (NHMRC)</institution></institution-wrap></funding-source><award-id>GNT1123854</award-id><principal-award-recipient><name><surname>Williamson</surname><given-names>Deborah A.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/100001341</institution-id><institution>Richard and Susan Smith Family Foundation (Smith Family Foundation)</institution></institution-wrap></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par3\"><italic>Neisseria gonorrhoeae</italic> is the causative agent of the sexually transmitted disease gonorrhea. Antibiotics have played a key role in shaping gonococcal evolution<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>, with <italic>N. gonorrhoeae</italic> gaining resistance to each of the first-line antibiotics used to treat it<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>–<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. As <italic>N. gonorrhoeae</italic> is an obligate human pathogen, the mucosal niches it infects—most commonly including the urethra, cervix, pharynx, and rectum—must also influence its evolution<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. The gonococcal phylogeny suggests the interaction of these factors, with an ancestral split between a drug-susceptible lineage circulating primarily in heterosexuals and a drug-resistant lineage circulating primarily in men who have sex with men<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>.</p><p id=\"Par4\">Despite the deeply concerning increase in antibiotic resistance reported in gonococcal populations globally<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>, some clinical isolates of <italic>N. gonorrhoeae</italic> have become more susceptible to antibiotics<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. This unexpected phenomenon prompts questions about which environmental pressures could be drivers of increased susceptibility and the mechanisms by which suppression or reversion of resistance may occur. To address these questions, we analyzed the genomes of a global collection of clinical isolates together with patient demographic and clinical data to identify mutations associated with increased susceptibility and define the environments in which they appear. We find that loss-of-function mutations in the efflux pump component <italic>mtrC</italic> are significantly associated with both antibiotic susceptibility and cervical infections, demonstrating how antibiotic and mucosal niche selective pressures intersect. In support of a model in which efflux pump expression incurs a cost in this niche, we also observe enrichment of loss-of-function mutations in cervical isolates in another efflux pump in <italic>N. gonorrhoeae</italic> and in urogenitally-adapted <italic>N. meningitidis</italic>. Our findings demonstrate how shifts in environmental pressures experienced by pathogenic <italic>Neisseria</italic> can lead to loss of efflux pump function and suppression of antibiotic resistance.</p></sec><sec id=\"Sec2\" sec-type=\"results\"><title>Results</title><sec id=\"Sec3\"><title>Unknown genetic loci influence antibiotic susceptibility</title><p id=\"Par5\">We first assessed how well variation in antibiotic resistance phenotype was captured by the presence and absence of known resistance markers. To do so, we assembled and examined a global dataset comprising the genomes and minimum inhibitory concentrations (MICs) of 4852 isolates collected across 65 countries and 38 years (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>, Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>). We modeled log-transformed MICs using multiple regression on a panel of experimentally characterized resistance markers for the three most clinically relevant antibiotics<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup> (Supplementary Data <xref rid=\"MOESM4\" ref-type=\"media\">1</xref>). This enabled us to make quantitative predictions of MIC based on known genotypic markers and to assess how well these markers predicted true MIC values. For the macrolide azithromycin, we observed that 434/4505 (9.63%) isolates had predicted MICs that deviated by two dilutions or more from their reported phenotypic values. The majority (59.4%) of these isolates had MICs that were lower than expected, indicative of increased susceptibility unexplained by the genetic determinants in our model. Overall MIC variance explained by known resistance mutations was relatively low (adjusted <italic>R</italic><sup>2</sup> = 0.667), in agreement with prior studies that employed whole-genome supervised learning algorithms to predict azithromycin resistance<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. MIC variance explained by known resistance mutations was also low for ceftriaxone (adjusted <italic>R</italic><sup>2</sup> = 0.674) but higher for ciprofloxacin (adjusted <italic>R</italic><sup>2</sup> = 0.937), with 2.02% and 2.90% of strains, respectively, exhibiting two dilutions or lower reported MICs compared to predictions, similarly indicating unexplained susceptibility. The predictive modeling results, therefore, suggested unknown modifiers that promote susceptibility for multiple classes of antibiotics in <italic>N. gonorrhoeae</italic>.<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Population structure and susceptibility profile of <italic>N. gonorrhoeae</italic> global meta-analysis collection.</title><p>A midpoint rooted recombination-corrected maximum likelihood phylogeny of 4852 genomes based on 68697 SNPs (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>) was annotated with binarized resistance (ciprofloxacin) or decreased susceptibility (azithromycin, ceftriaxone) values. Annotation rings are listed in order of ciprofloxacin, ceftriaxone, and azithromycin from innermost to outermost. For ciprofloxacin, MIC < 1 μg/ml is light purple, and MIC ≥ 1 μg/ml is dark purple. For ceftriaxone, MIC < 0.125 μg/ml is light blue, and MIC ≥ 0.125 μg/ml is dark blue. For azithromycin, MIC ≤ 1 μg/ml is light pink, and MIC > 1 μg/ml is dark pink. Branch length represents total number of substitutions after removal of predicted recombination.</p></caption><graphic xlink:href=\"41467_2020_17980_Fig1_HTML\" id=\"d30e551\"/></fig></p></sec><sec id=\"Sec4\"><title>GWAS identifies a susceptibility-associated variant in <italic>mtrC</italic></title><p id=\"Par6\">To identify novel antibiotic susceptibility loci in an unbiased manner, we conducted a bacterial genome-wide association study (GWAS). We used a linear mixed model framework to control for population structure, and we used unitigs constructed from genome assemblies to capture SNPs, indels, and accessory genome elements (see “Methods”)<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>–<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. Unitigs are a flexible representation of the genetic variation across a dataset that are constructed using compacted de Bruijn graphs and have been previously applied as markers for microbial GWAS<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. We performed a GWAS on the sequences of 4505 isolates with associated azithromycin MICs using a Bonferroni-corrected significance threshold of 3.38 × 10<sup>−7</sup>. The linear mixed model adequately controlled for population structure (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>), and the proportion of phenotypic MIC variance attributable to genotype (i.e., narrow-sense heritability) estimated by the linear mixed model was high (<italic>h</italic><sup>2</sup> = 0.97). In line with this, we observed highly significant unitigs with positive effect sizes corresponding to the known resistance substitutions C2611T and A2059G (<italic>E. coli</italic> numbering) in the 23S ribosomal RNA gene (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. The next most significant variant was a unitig associated with increased susceptibility that mapped to <italic>mtrC</italic> (β, or effect size on the log<sub>2</sub>-transformed MIC scale = −2.82, 95% CI [−3.06, −2.57]; <italic>p</italic>-value = 2.81 × 10<sup>−108</sup>). Overexpression of the <italic>mtrCDE</italic> efflux pump operon has been shown to decrease gonococcal susceptibility to a range of hydrophobic acids and antimicrobial agents<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>, and conversely, knockout of the pump results in multi-drug hypersusceptibility<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. To assess whether this <italic>mtrC</italic> variant was associated with increased susceptibility to other antibiotics, we performed GWAS for ceftriaxone (for which MICs were available from 4497 isolates) and for ciprofloxacin (4135 isolates). We recovered known ceftriaxone resistance mutations including recombination in the <italic>penA</italic> gene and ciprofloxacin resistance substitutions in DNA gyrase (<italic>gyrA</italic>). In agreement with the known pleiotropic effect of the MtrCDE efflux pump<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>, we observed the same <italic>mtrC</italic> unitig at genome-wide significance associated with increased susceptibility to both ceftriaxone (<italic>β</italic> = −1.18, 95% CI [−1.34, −1.02]; <italic>p</italic>-value = 2.00 × 10<sup>−44</sup>) and ciprofloxacin (<italic>β</italic> = −1.29, 95% CI [−1.54, −1.04]; <italic>p</italic>-value = 1.87 × 10<sup>−23</sup>) (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). Across all three drugs, heritability estimates for this <italic>mtrC</italic> variant were comparable to that of prevalent major resistance determinants (azithromycin <italic>h</italic><sup>2</sup>: 0.323; ceftriaxone <italic>h</italic><sup>2</sup>: 0.208; ciprofloxacin <italic>h</italic><sup>2</sup>: 0.155), indicating that unexplained susceptibility in our model could be partially addressed by the inclusion of this mutation.<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>GWAS identifies a variant mapping to <italic>mtrC</italic> associated with increased susceptibility.</title><p>The Manhattan plot shows negative log<sub>10</sub>-transformed <italic>p</italic>-values (calculated using likelihood-ratio tests in the GWAS) for the association of unitigs with MICs to azithromycin (pink, <italic>n</italic> = 4505), ceftriaxone (blue, <italic>n</italic> = 4497), and ciprofloxacin (purple, <italic>n</italic> = 4135). The sign of the GWAS regression coefficient <italic>β</italic> (with positive indicating an association with increased resistance and negative indicating an association with increased susceptibility) is indicated by an X for <italic>β</italic> < 0 and a dot for <italic>β</italic> > 0. Labels indicate known influential resistance determinants, and the <italic>mtrC</italic> variant associated with increased susceptibility was highlighted in gray. A full list of the annotated significant unitigs for each antibiotic can be found in Supplementary Data <xref rid=\"MOESM5\" ref-type=\"media\">2</xref>. Inset: schematic of the <italic>mtr</italic> genetic regulon including structural genes <italic>mtrCDE</italic>, the activator <italic>mtrA</italic>, and the repressor <italic>mtrR</italic>. The approximate genomic location within <italic>mtrC</italic> and specific nucleotide change of the <italic>mtrC</italic> GWAS variant relative to the gonococcal NCCP11945 reference genome (i.e., a two base pair deletion in a ‘GC’ dinucleotide repeat) is shown.</p></caption><graphic xlink:href=\"41467_2020_17980_Fig2_HTML\" id=\"d30e740\"/></fig></p><p id=\"Par7\">Annotation of the <italic>mtrC</italic> unitig revealed that it represented a two base pair deletion in a ‘GC’ dinucleotide hexarepeat, leading to early termination of <italic>mtrC</italic> translation and probable loss of MtrCDE activity<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup> (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> inset). We also checked whether the two base pair deletion would affect recognition by any of the gonococcal methylases<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>, but no methylase target motif sites mapped to the hexarepeat or its direct surrounding sequences. A laboratory-generated gonococcal mutant with a four base pair deletion in this same <italic>mtrC</italic> dinucleotide hexarepeat exhibited multi-drug susceptibility<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>, and clinical gonococcal isolates hypersensitive to erythromycin were shown to have mutations mapping to this locus<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. To directly test the hypothesis that the two base pair deletion also contributed to increased susceptibility for the panel of antibiotics we examined, we complemented the mutation in a clinical isolate belonging to the multidrug-resistant lineage ST-1901<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup> and observed significant increases in MICs for all three antibiotics, as predicted by the GWAS (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>).</p><p id=\"Par8\">We searched for additional <italic>mtrC</italic> loss-of-function (LOF) mutations and found six clinical isolates with genomes encoding indels outside of the dinucleotide hexarepeat that also were associated with increased susceptibility (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2A</xref>). Ten isolates that had acquired the two base pair deletion also have a two base pair insertion elsewhere in <italic>mtrC</italic> that restores the original coding frame, suggesting that loss of MtrC function may be reverted by further mutation or recombination (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2A</xref>). In line with this, <italic>mtrC</italic> LOF mutations have emerged numerous times throughout the phylogeny (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>), indicative of possible repeated losses of a dinucleotide in the hexarepeat region due to DNA polymerase slippage, which may occur at a higher rate than single nucleotide nonsense mutations<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. In total, including all strains with <italic>mtrC</italic> frameshift mutations and excluding revertants, we identified 185 isolates (3.82%) that encoded a LOF allele of <italic>mtrC</italic> (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). Presence of the <italic>mtrC</italic> LOF mutation in isolates with known resistance markers was correlated with significantly reduced MICs (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>), and inclusion of <italic>mtrC</italic> LOF mutations in our linear model increased adjusted <italic>R</italic><sup>2</sup> values (azithromycin: 0.667–0.704; ceftriaxone: 0.674–0.690; ciprofloxacin: 0.937–0.939), decreased the proportion of strains with unexplained susceptibility (azithromycin: 5.73%–3.88%; ceftriaxone: 2.02%–1.73%; ciprofloxacin: 2.90%–2.42%), and significantly improved model fit (<italic>p</italic>-value < 2.2 × 10<sup>−16</sup> for all three antibiotics; Likelihood-ratio <italic>χ</italic><sup>2</sup> test for nested models). <italic>mtrC</italic> LOF strains were identified in 28 of the 66 countries surveyed and ranged in isolation date from 2002 to 2017. Because most strains in this dataset were collected within the last two decades, we also examined a dataset of strains collected in Denmark from 1928 to 2013 to understand the historical prevalence of <italic>mtrC</italic> LOF mutations<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. We observed an additional 10 strains with the ‘GC’ two base pair deletion ranging in isolation date from 1951 to 2000, indicating that <italic>mtrC</italic> LOF strains have either repeatedly arisen or persistently circulated for decades. Our results demonstrate that a relatively common mechanism of gonococcal acquired antibiotic susceptibility is a two base pair deletion in <italic>mtrC</italic> and that such mutations are globally and temporally widespread.</p></sec><sec id=\"Sec5\"><title>Loss of MtrCDE pump is associated with cervical infection</title><p id=\"Par9\">The MtrCDE pump has been demonstrated to play a critical role in gonococcal survival in the presence of human neutrophils and in the female murine genital tract model of gonococcal infection, and overexpression of <italic>mtrCDE</italic> results in substantial fitness benefits for dealing with both antimicrobial and environmental pressures<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref>–<xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. The relative frequency of the <italic>mtrC</italic> LOF mutations we observe (occurring in ~1 in every 25 isolates) thus seems unusual for a mutation predicted to be deleterious for human infection. <italic>mtrC</italic> LOF strains do not grow more or less quickly in vitro than <italic>mtrC</italic> wild-type strains, indicating that this mutation does not confer a simple fitness benefit due to reduced energetic cost<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref>,<xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. Instead, we hypothesized that there are unique environments that select for non-functional efflux pump.</p><p id=\"Par10\">We aggregated patient-level metadata across included studies on sex partner preferences and anatomical site of infection. Sexual behavior and <italic>mtrC</italic> genotypic information was available for 1975 isolates from individual patients. There was a significant association between <italic>mtrC</italic> LOF and sexual behavior (<italic>p</italic>-value = 0.04021; two-sided Fisher’s exact test) (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref>, Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>), and <italic>mtrC</italic> LOF occurred more often in isolates from men who have sex with women (MSW) (28/626, 4.47%) compared to isolates from men who have sex with men (MSM) (31/1189, 2.61%) (OR = 1.75, 95% CI [1.00–3.04], <italic>p</italic>-value = 0.037; two-sided Fisher’s exact test). To understand whether anatomical selective pressures contributed to this enrichment, we analyzed the site of infection and <italic>mtrC</italic> genotypic information available for 2730 isolates. <italic>mtrC</italic> LOF mutations were significantly associated with site of infection (<italic>p</italic>-value = 6.49 × 10<sup>−5</sup>; two-sided Fisher’s exact test) and were overrepresented particularly in cervical isolates: 16 out of 129 (12.4%) cervical isolates contained an <italic>mtrC</italic> LOF mutation compared to 82 out of 2249 urethral isolates (3.65%; OR = 3.74, 95% CI [1.98–6.70], <italic>p</italic>-value = 4.71 × 10<sup>−5</sup>; two-sided Fisher’s exact test), 3 out of 106 pharyngeal isolates (2.83%; OR = 4.83, 95% CI [1.33–26.63], <italic>p</italic>-value = 0.00769; two-sided Fisher’s exact test), and 4 out of 246 rectal isolates (1.63%; OR = 8.52, 95% CI [2.67–35.787], <italic>p</italic>-value = 2.39 × 10<sup>−5</sup>; two-sided Fisher’s exact test) (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b</xref>, Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>). Because our meta-analysis collection comprised datasets potentially biased by preferential sampling for drug-resistant strains, we validated our epidemiological associations on a set of 2186 sequenced isolates, corresponding to all cultured isolates of <italic>N. gonorrhoeae</italic> in the state of Victoria, Australia in 2017<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. We again observed significant associations between <italic>mtrC</italic> LOF and sexual behavior (<italic>p</italic>-value = 0.0180; two-sided Fisher’s exact test) as well as the anatomical site of infection (<italic>p</italic>-value = 0.0256; two-sided Fisher’s exact test) (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>, Supplementary Tables <xref rid=\"MOESM1\" ref-type=\"media\">6</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">7</xref>). <italic>mtrC</italic> LOF mutations were again overrepresented in cervical isolates: 9 out of 227 (3.96%) cervical isolates contained an <italic>mtrC</italic> LOF mutation compared to 15 out of 882 urethral isolates (1.70%; OR = 2.38, 95% CI [0.91–5.91], <italic>p</italic>-value = 0.0679; two-sided Fisher’s exact test), 3 out of 386 pharyngeal isolates (0.78%; OR = 5.26, 95% CI [1.29–30.51], <italic>p</italic>-value = 0.0117; two-sided Fisher’s exact test), and 7 out of 632 rectal isolates (1.11%; OR = 3.68, 95% CI [1.20–11.78], <italic>p</italic>-value = 0.0173; two-sided Fisher’s exact test). These results indicate that environmental pressures unique to female urogenital infection may select for loss of the primary gonococcal efflux pump resulting in broadly increased susceptibility to antibiotics and host-derived antimicrobial peptides.<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Gonococcal <italic>mtrC</italic>, <italic>mtrA</italic>, and <italic>farA</italic> LOF mutations are associated with cervical infection.</title><p><bold>a</bold> Sexual behavior of patients infected with isolates with either intact or LOF alleles of <italic>mtrC</italic> (left), <italic>mtrA</italic> (middle), or <italic>farA</italic> (right). <bold>b</bold> Site of infection in patients infected with isolates with either intact or LOF alleles of <italic>mtrC</italic> (left), <italic>mtrA</italic> (middle), or <italic>farA</italic> (right). <italic>mtrA</italic> alleles were predicted as LOF only in the absence of other epistatic Mtr overexpression mutations. Statistical significance between genotype and patient metadata was assessed by two-sided Fisher’s exact test: *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, and ***<italic>p</italic> < 0.001. Exact <italic>p-</italic>values from left to right for analyses in <bold>a</bold> were 0.04021, 1.81 × 10<sup>−11</sup>, 5.06 × 10<sup>−10</sup> and for <bold>b</bold> were 6.49 × 10<sup>−5</sup>, 1.64 × 10<sup>−12</sup>, 1.78 × 10<sup>−12</sup>. WSM = women who have sex with men, MSW = men who have sex with women, MSMW = men who have sex with men and women, MSM = men who have sex with men.</p></caption><graphic xlink:href=\"41467_2020_17980_Fig3_HTML\" id=\"d30e1077\"/></fig></p></sec><sec id=\"Sec6\"><title><italic>mtrA</italic> LOF offers an additional level of adaptive regulation</title><p id=\"Par11\">The association of <italic>mtrC</italic> LOF mutations with cervical specimens suggests that other mutations that downregulate expression of the <italic>mtrCDE</italic> operon should also promote adaptation to the cervical niche. The MtrCDE efflux pump regulon comprises the MtrR repressor and the MtrA activator (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> inset), the latter of which exists in two allelic forms: a wild-type functional gene capable of inducing <italic>mtrCDE</italic> expression and a variant rendered non-functional by an 11-bp deletion near the 5’ end of the gene<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup> (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2B</xref>). Knocking out <italic>mtrA</italic> has a detrimental effect on fitness in the gonococcal mouse model, and epistatic <italic>mtrR</italic> mutations resulting in overexpression of <italic>mtrCDE</italic> compensate for this fitness defect by masking the effect of the <italic>mtrA</italic> knockout<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. Prior work assessing the genomic diversity of <italic>mtrA</italic> in a set of 922 primarily male urethral specimens found only four isolates with the 11-bp deletion (0.43%)<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, seemingly in agreement with the in vivo importance of <italic>mtrA</italic>. However, in our global meta-analysis dataset, 362/4842 isolates (7.48%) were predicted to be <italic>mtrA</italic> LOF, of which the majority (357/362, 98.6%) were due to the 11-bp deletion. Of the 4842 isolates, 268 (5.53%) had <italic>mtrA</italic> LOF mutations in non-<italic>mtrCDE</italic> overexpression backgrounds (as defined by the absence of known <italic>mtrR</italic> promoter or coding sequence mutations or <italic>mtrCDE</italic> mosaic alleles) and therefore not epistatically masked (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">8</xref>). We repeated our epidemiological associations on these <italic>mtrA</italic> LOF strains without concurrent overexpression mutations and observed highly significant associations with reported patient sexual behavior (<italic>p</italic>-value = 1.81 × 10<sup>−11</sup>; two-sided Fisher’s exact test) and site of infection (<italic>p</italic>-value = 1.64 × 10<sup>−12</sup>; two-sided Fisher’s exact test) (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>, Supplementary Tables <xref rid=\"MOESM1\" ref-type=\"media\">9</xref>–<xref rid=\"MOESM1\" ref-type=\"media\">10</xref>). As with <italic>mtrC</italic> LOF mutations, <italic>mtrA</italic> LOF mutations were significantly overrepresented in cervical isolates: 25 out of 129 (19.4%) cervical isolates contained an <italic>mtrA</italic> LOF mutation compared to 61 out of 2248 urethral isolates (2.71%; OR = 8.60, 95% CI [4.96–14.57], <italic>p</italic>-value = 4.60 × 10<sup>−13</sup>; two-sided Fisher’s exact test), 4 out of 106 pharyngeal isolates (3.78%; OR = 6.09, 95% CI [2.00–24.93], <italic>p</italic>-value = 0.000240; two-sided Fisher’s exact test), and 4 out of 246 rectal isolates (1.63%; OR = 14.43, 95% CI [4.81–58.52], <italic>p</italic>-value = 3.00 × 10<sup>−9</sup>; two-sided Fisher’s exact test). In the Australian validation cohort<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>, the majority (81/85, 95.3%) of <italic>mtrA</italic> LOF strains had concurrent <italic>mtrCDE</italic> overexpression mutations, so it was not possible to test for these associations (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">11</xref>). In such genetic backgrounds where overexpression mutations mask the effect of <italic>mtrA</italic> LOF, <italic>mtrC</italic> LOF is the preferred method of efflux pump downregulation: the majority of <italic>mtrC</italic> LOF mutations in both the global dataset (174/180, 96.7%) and the Australian cohort (33/35, 94.3%) occurred in backgrounds with known <italic>mtr</italic> overexpression mutations (Supplementary Tables <xref rid=\"MOESM1\" ref-type=\"media\">12</xref>–<xref rid=\"MOESM1\" ref-type=\"media\">13</xref>). Phylogenetic analysis showed that the distribution of <italic>mtrA</italic> LOF differed from that of <italic>mtrC</italic> LOF with fewer introductions but more sustained transmission and that the two mutations were largely non-overlapping (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>); only four strains had both <italic>mtrA</italic> and <italic>mtrC</italic> LOF mutations. Our results indicate that multiple adaptive paths for MtrCDE efflux pump downregulation exist depending on genetic interactions with other concurrent mutations in the <italic>mtrCDE</italic> regulon.</p></sec><sec id=\"Sec7\"><title>FarAB efflux pump LOF is associated with cervical infection</title><p id=\"Par12\">The associations we observed in the <italic>mtrCDE</italic> regulon raised the question of the mechanism by which the cervical environment could select for pump downregulation. Recent work on <italic>Pseudomonas</italic> suggested one possible model: overexpression of homologous <italic>P. aeruginosa</italic> efflux pumps belonging to the same resistance/nodulation/cell division (RND) proton/substrate antiporter family as MtrCDE results in a fitness cost due to increased cytoplasmic acidification<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. This fitness cost was only observed in anaerobic conditions, where aerobic respiration cannot be used to dissipate excess protons efficiently<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. Analogous conditions in the female urogenital tract, potentially augmented by environmental acidity, could create a similar selective pressure during human infection that leads to pump downregulation or loss.</p><p id=\"Par13\">This model predicts that adaptation to these conditions would similarly result in the downregulation of FarAB, the other proton-substrate antiporter efflux pump in <italic>N. gonorrhoeae</italic>. FarAB is a member of the major facilitator superfamily (MFS) of efflux pumps and effluxes long-chain fatty acids<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. In our global dataset, 332/4838 (6.86%) of isolates were predicted to have <italic>farA</italic> LOF mutations, of which the majority (316/332; 95.2%) were due to indels in a homopolymeric stretch of eight ‘T’ nucleotides near the 5’ end of the gene (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2C</xref>). <italic>farA</italic> LOF mutations were associated with patient sexual behavior (<italic>p</italic>-value = 5.06 × 10<sup>−10</sup>; two-sided Fisher’s exact test) and site of infection (<italic>p</italic>-value = 1.78 × 10<sup>−12</sup>; two-sided Fisher’s exact test) and overrepresented in cervical isolates: 33 out of 129 (25.6%) cervical isolates contained a <italic>farA</italic> LOF mutation compared to 117 out of 2246 urethral isolates (5.21%; OR = 6.25, 95% CI [3.90–9.83], <italic>p</italic>-value = 3.24 × 10<sup>−13</sup>; two-sided Fisher’s exact test), 3 out of 106 pharyngeal isolates (2.83%; OR = 11.70, 95% CI [3.50–61.61], <italic>p</italic>-value = 3.80 × 10<sup>−7</sup>; two-sided Fisher’s exact test), and 12 out of 246 rectal isolates (4.88%; OR = 6.66, 95% CI [3.19-14.80], <italic>p</italic>-value = 1.57 × 10<sup>−8</sup>; two-sided Fisher’s exact test) (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>, Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">14</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">15</xref>). <italic>farA</italic> LOF mutations were prevalent also in our Australian validation dataset<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup> (225/2180; 10.32%) and again associated with sexual behavior (<italic>p</italic>-value < 2.20 × 10<sup>−16</sup>; two-sided Fisher’s exact test) and site of infection (<italic>p</italic>-value < 2.20 × 10<sup>−16</sup>; two-sided Fisher’s exact test) (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>, Supplementary Tables <xref rid=\"MOESM1\" ref-type=\"media\">16</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">17</xref>). The phylogenetic distribution of <italic>farA</italic> LOF indicated sustained transmission (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>) and overlapped with that of <italic>mtrA</italic> LOF mutations (48.9% of isolates with <italic>mtrA</italic> LOF mutations also had <italic>farA</italic> LOF mutations), potentially indicating additive contributions to cervical adaptation. Furthermore, MtrR activates <italic>farAB</italic> expression by repressing the <italic>farR</italic> repressor<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. This cross-talk between the two efflux pump operons indicates that in <italic>mtrCDE</italic> overexpression strains where MtrR activity is impaired, the effect of <italic>farA</italic> LOF—like <italic>mtrA</italic> LOF—may be masked. We did not observe frequent LOF mutations in the sodium gradient-dependent MATE family efflux pump NorM<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup> or in the ATP-dependent ABC family pump MacAB<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup> (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). The prevalence and cervical enrichment of <italic>farA</italic> LOF mutations and the relative rarity of LOF mutations in other non-proton motive force-driven pumps suggest that cytoplasmic acidification may be a mechanism by which the female urogenital tract selects for efflux pump loss.</p></sec><sec id=\"Sec8\"><title>Meningococcal <italic>mtrC</italic> LOF is driven by urogenital adaptation</title><p id=\"Par14\"><italic>N. meningitidis</italic>, a species closely related to <italic>N. gonorrhoeae</italic>, colonizes the oropharyngeal tract and can cause invasive disease, including meningitis and septicemia<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. We characterized <italic>mtrC</italic> diversity in a collection of 14,798 <italic>N. meningitidis</italic> genomes, reasoning that the cervical environmental pressures that select for efflux pump LOF in the gonococcus will be rarely encountered by the meningococcus. In agreement with this, the ‘GC’ hexarepeat associated with most gonococcal <italic>mtrC</italic> LOF mutations was less conserved in <italic>N. meningitidis</italic>; only 9684/14798 (65.4%) isolates contained an intact hexarepeat compared to 4644/4847 (95.8%) of <italic>N. gonorrhoeae</italic> isolates (<italic>p</italic>-value < 2.2 × 10<sup>−16</sup>; two-sided Fisher’s exact test). In this same collection, we observed <italic>mtrC</italic> LOF due to deletions in the hexarepeat region in only 82 meningococcal isolates (0.55%), with a similar frequency of 25/4059 (0.62%) in a curated dataset comprising all invasive meningococcal disease isolates collected in the UK from 2009 to 2013<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. The observed interruption of ‘GC’ dinucleotide repeats, predicted to result in a lower mutation rate<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>, and the relative rarity of <italic>mtrC</italic> LOF mutations suggests that efflux pump loss is not generally adaptive in <italic>N. meningitidis</italic>. However, a urogenitally-adapted meningococcal lineage has recently emerged in the US associated with outbreaks of non-gonococcal urethritis in heterosexual patients<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref>–<xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. In isolates from this lineage, the prevalence of <italic>mtrC</italic> LOF mutations was 18/207 (8.70%), substantially higher than typical <italic>N. meningitidis</italic> and comparable to the prevalence of gonococcal <italic>mtrC</italic> LOF mutations in MSW in our global dataset (4.47%). We compared the frequency of <italic>mtrC</italic> LOF mutations in the urogenital lineage to geographically and genetically matched isolates (i.e., all publicly available <italic>n</italic> = 456 PubMLST ST-11 North American isolates) and observed a significant difference in prevalence (18 out of 207 or 8.70% versus 2 out of 249 or 0.80%; OR = 11.71, 95% CI [2.75–105.37], <italic>p</italic>-value = 3.31 × 10<sup>−5</sup>; two-sided Fisher’s exact test). Most <italic>mtrC</italic> LOF mutations occurred due to the same hexarepeat two base pair deletion that we previously observed for <italic>N. gonorrhoeae</italic>, and in line with this, <italic>mtrC</italic> LOF in this urogenital lineage arose multiple times independently similarly to gonococcal <italic>mtrC</italic> LOF mutations (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). <italic>farA</italic> LOF mutations were not observed in this meningococcal lineage. We conclude that MtrCDE efflux pump LOF is rare in typical meningococcal strains that inhabit the oropharynx but elevated in frequency in a unique urogenitally-adapted lineage circulating in heterosexuals, indicative of potential ongoing adaptation to the cervical niche. Our results suggest that efflux pump loss is broadly adaptive for cervical colonization across pathogenic <italic>Neisseria</italic>.<fig id=\"Fig4\"><label>Fig. 4</label><caption><title><italic>mtrC</italic> LOF mutations are enriched in a lineage of ST-11 urogenitally-adapted <italic>N. meningitidis</italic>.</title><p>A core-genome maximum likelihood phylogeny based on 25045 SNPs was estimated of all North American ST-11 <italic>N. meningitidis</italic> strains from PubMLST (<italic>n</italic> = 456; accessed 2019–09–03) rooted with meningococcal reference genome MC58. Membership in the ST-11 urogenital clade (dark blue) is defined as in Retchless et al.<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. Genomes with <italic>mtrC</italic> LOF mutations are indicated in pink. Branch length represents substitutions per site.</p></caption><graphic xlink:href=\"41467_2020_17980_Fig4_HTML\" id=\"d30e1561\"/></fig></p></sec></sec><sec id=\"Sec9\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par15\">In an era in which widespread antimicrobial pressure has led to the emergence of extensively drug-resistant <italic>N. gonorrhoeae</italic><sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>, isolates that appear to have reverted to susceptibility still arise<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>, demonstrating that antibiotic and host environmental pressures interact to shape the evolution of <italic>N. gonorrhoeae</italic>. Here, we showed that frameshift-mediated truncations in the <italic>mtrC</italic> component of the MtrCDE efflux pump are the primary mechanism for epistatic increases in antibiotic susceptibility across a global collection of clinical gonococcal isolates, as suggested by prior work<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. <italic>mtrC</italic> LOF mutations are enriched in cervical isolates and a frameshifted form of the pump activator MtrA exhibits similar trends, supporting a model in which reduced or eliminated <italic>mtrCDE</italic> efflux pump expression contributes to adaptation to the female genital tract. We hypothesized that the mechanism by which this occurs is through increased cytoplasmic acidification in anaerobic conditions<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup> and demonstrated that LOF mutations in <italic>farA</italic>, encoding a subunit of the other proton motive force-driven pump FarAB, were likewise enriched in cervical isolates. The LOF mutations we observed in <italic>mtrC</italic> and <italic>farA</italic> primarily occurred in short homopolymeric sequences (though with low numbers of repeated units) and thus may occur at higher rates than insertions or deletions in non-repetitive regions or nonsense mutations, similar to other resistance suppressor mutations<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>, though this will need to be confirmed in future experiments. In total, 42.6% of cervical isolates in the global dataset and 32.6% in the validation dataset contained a LOF mutation in either <italic>mtrC, farA</italic>, or <italic>mtrA</italic>, indicating that efflux pump downregulation via multiple genetic mechanisms is prevalent in cervical infection. These results complement prior studies suggesting that <italic>mtrR</italic> LOF resulting in increased resistance to fecal lipids plays a critical role in gonococcal adaptation to the rectal environment<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref>,<xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup> and taken together suggest a model in which the fitness benefit of efflux pump expression is highly context dependent.</p><p id=\"Par16\">Other selective forces could also have contributed to the observed enrichment of LOF mutations in cervical isolates. For instance, iron levels modulate <italic>mtrCDE</italic> expression through Fur (the ferric uptake regulator) and MpeR<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>. Iron limitation results in increased expression of <italic>mtrCDE</italic>, and conversely, iron enrichment result in decreased expression, suggesting a fitness cost for <italic>mtrCDE</italic> expression during high iron conditions. Variation in environmental iron levels, such as in the menstrual cycle, may provide another selective pressure for LOF mutations to arise particularly when MtrR function is impaired through active site or promoter mutations. Differing rates of antibiotic use for gonorrhea in men and women due to increased asymptomatic infection in women might also select for <italic>mtrC</italic> LOF mutations, but this would not explain the associations we observed for the non-antibiotic substrate efflux pump <italic>farAB</italic> or the increased frequency of <italic>mtrC</italic> LOF mutations in urogenitally-adapted meningococci. RNA sequencing from men and women infected with gonorrhea demonstrated a 4-fold lower expression of <italic>mtrCDE</italic> in women, re-affirming the idea that efflux pump expression in the female genital tract incurs a fitness cost<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>.</p><p id=\"Par17\">Despite significant associations, only a proportion of cervical isolates exhibited these LOF genotypes, suggesting variation in cervix-associated pressures or indicating that cervical culture specimens were obtained before niche pressures could select for pump downregulation. This variation could also lead to mixed populations of efflux pump wild-type and LOF strains; however, because only one clonal isolate per site per patient is typically sequenced in clinical surveillance studies, we would be unable to detect this intra-host patient diversity. Targeted amplicon sequencing of LOF loci directly from patients in future studies would help to assess whether this intra-host diversity plays a role in infection and transmission. In particular, this intra-host pathogen diversity could facilitate transmission from the female genital tract to other sites of infection, where efflux pump activity incurs less of a fitness cost. In those new sites, isolates with wild-type efflux pump loci in the mixed population could selectively expand relative to LOF efflux pump strains and also serve as possible recombination donors of wild-type alleles. This standing genetic variation would, therefore, facilitate gonococcal adaptation across different mucosal niches. Additionally, while the cervix is the primary site of infection and source for culture in women, the selective pressures at play may include other sites more broadly in the female genital tract and may be influenced by the presence of other microbial species both pathogenic and commensal.</p><p id=\"Par18\">Our model extended to the other pathogenic <italic>Neisseria</italic> species, <italic>N. meningitidis</italic>, in that a urogenital clade transmitting in primarily heterosexual populations appeared to be undergoing further urogenital adaptation via the same <italic>mtrC</italic> frameshift mutation that was most commonly observed for <italic>N. gonorrhoeae</italic>. In the absence of data on cases of cervicitis, we hypothesized that for this meningococcal lineage, efflux pump LOF emerged in the female urogenital tract and was transmitted to heterosexual men resulting in the enrichment we observed. Efflux pumps are common across Gram-negative bacteria<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>, and their loss may be a general adaptive strategy for species that face similar pressures as <italic>N. gonorrhoeae</italic> and urogenitally-adapted <italic>N. meningitidis</italic>. In support of this, clinical isolates of <italic>Pseudomonas aeruginosa</italic> with truncations in genes homologous to <italic>mtrC</italic><sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref>,<xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup> and exhibiting antibiotic hypersensitivity have been obtained from cystic fibrosis patients, in whom the thick mucus in airway environments can likewise exhibit increased acidity and decreased oxygen availability<sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref>,<xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>.</p><p id=\"Par19\">Our results also suggest potential therapeutic avenues for addressing the emergence of multidrug-resistant gonococcal strains. Selective knockdown of MtrCDE homologs in other bacteria via antisense RNA<sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup> and bacteriophages<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup> has successfully re-sensitized resistant strains and enhanced antibiotic efficacy, and ectopic expression in <italic>N. gonorrhoeae</italic> of the <italic>mtrR</italic> repressor in a cephalosporin-resistant strain enhances gonococcal killing by β-lactam antibiotics in the mouse model<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>. Our population-wide estimated effect sizes for <italic>mtrC</italic> LOF mutations provide a prediction for the re-sensitization effect of MtrCDE knockdown across multiple genetic backgrounds and suggest particularly strong effects for the macrolide azithromycin (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>). Because the correlation between MIC differences and clinical efficacy is still not well understood<sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref>,<xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup>, follow up studies to assess treatment efficacy differences in patients with and without <italic>mtrC</italic> LOF strains can help to quantify the expected effect of MtrCDE knockdown in the clinical context.</p><p id=\"Par20\">In summary, by analysis of population genomics and patient clinical data, we have shown that pathogenic <italic>Neisseria</italic> can use multiple avenues of efflux pump perturbation as an adaptive strategy to respond to host environmental pressures and illustrate how these host pressures may result in increased antibiotic susceptibility in <italic>N. gonorrhoeae</italic>.</p></sec><sec id=\"Sec10\"><title>Methods</title><sec id=\"Sec11\"><title>Genomics pipeline</title><p id=\"Par21\">Reads for isolates with either associated azithromycin, ciprofloxacin, or ceftriaxone MIC metadata were downloaded from datasets listed in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>. Reads were inspected using FastQC version 0.11.7 (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.bioinformatics.babraham.ac.uk/projects/fastqc/\">https://www.bioinformatics.babraham.ac.uk/projects/fastqc/</ext-link>) and removed if GC content diverged notably from expected values (~52–54%) or if base quality was systematically poor. We mapped read data to the NCCP11945 reference genome (RefSeq accession: NC_011035.1) using BWA-MEM (version 0.7.17-r1188)<sup><xref ref-type=\"bibr\" rid=\"CR62\">62</xref>,<xref ref-type=\"bibr\" rid=\"CR63\">63</xref></sup> and deduplicated reads using Picard (version 2.8.0) (<ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/broadinstitute/picard\">https://github.com/broadinstitute/picard</ext-link>). BamQC in Qualimap (version 2.2.1)<sup><xref ref-type=\"bibr\" rid=\"CR64\">64</xref></sup> was run to identify samples with less than 70% of reads aligned or samples with less than 40X coverage, which were discarded. We used Pilon (version 1.16)<sup><xref ref-type=\"bibr\" rid=\"CR65\">65</xref></sup> to call variants with mindepth set to 10 and minmq set to 20 and generated pseudogenomes from Pilon VCFs by including all PASS sites and alternate alleles with AF > 0.9; all other sites were assigned as ‘N’. Samples with greater than 15% of sites across the genome missing were also excluded. We created de novo assemblies using SPAdes (version 3.12.0 run using 8 threads, paired end reads where available, and the --careful flag set)<sup><xref ref-type=\"bibr\" rid=\"CR66\">66</xref></sup> and quality filtered contigs to ensure coverage greater than 10X, length greater than 500 base pairs, and total genome size approximately equal to the FA1090 genome size (2.0–2.3 Mbp). We annotated assemblies with Prokka (version 1.13)<sup><xref ref-type=\"bibr\" rid=\"CR67\">67</xref></sup>, and clustered core genes using Roary (version 3.12)<sup><xref ref-type=\"bibr\" rid=\"CR68\">68</xref></sup> (flags -z -e -n -v -s -i 92) and core intergenic regions using piggy (version 1.2)<sup><xref ref-type=\"bibr\" rid=\"CR69\">69</xref></sup>. A recombination-corrected phylogeny of all isolates was constructed by running Gubbins (version 2.3.4) on the aligned pseudogenomes and visualized in iTOL (version 4.4.2)<sup><xref ref-type=\"bibr\" rid=\"CR70\">70</xref>–<xref ref-type=\"bibr\" rid=\"CR72\">72</xref></sup>. All isolates with associated metadata and accession numbers are listed in Supplementary Data <xref rid=\"MOESM6\" ref-type=\"media\">3</xref> and <xref rid=\"MOESM7\" ref-type=\"media\">4</xref>.</p></sec><sec id=\"Sec12\"><title>Resistance allele calling</title><p id=\"Par22\">Known resistance determinants in single-copy genes were called by identifying expected SNPs in the pseudogenomes. For categorizing mosaic alleles of <italic>mtr</italic>, we ran BLASTn (version 2.6.0)<sup><xref ref-type=\"bibr\" rid=\"CR73\">73</xref></sup> on the de novo assemblies using a query sequence from FA1090 (Genbank accession: NC_002946.2) comprising the <italic>mtr</italic> intergenic promoter region and <italic>mtrCDE</italic>. BLAST results were aligned using MAFFT (version 7.450)<sup><xref ref-type=\"bibr\" rid=\"CR74\">74</xref></sup> and clustered into distinct allelic families using FastBAPS (version 1.0.0)<sup><xref ref-type=\"bibr\" rid=\"CR75\">75</xref></sup>. We confirmed that horizontally-transferred <italic>mtr</italic> alleles associated with resistance from prior studies<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> corresponded to distinct clusters in FastBAPS. A similar approach was used to cluster <italic>penA</italic> alleles after running BLASTn with a <italic>penA</italic> reference sequence from FA1090. Variant calling in the multi-copy 23S rRNA locus was done by mapping to a modified NCCP11945 reference genome containing only one copy of the 23S rRNA and analyzing variant allele frequencies<sup><xref ref-type=\"bibr\" rid=\"CR76\">76</xref></sup>. We identified truncated MtrR proteins using Prokka annotations, and mutations in the <italic>mtr</italic> promoter region associated with upregulation of <italic>mtrCDE</italic> (A deletion and TT insertion in inverted repeat, <italic>mtr</italic> 120) using an alignment of the <italic>mtr</italic> promoter from piggy output.</p></sec><sec id=\"Sec13\"><title>Phenotype processing and linear models</title><p id=\"Par23\">We doubled GISP azithromycin MICs before 2005 to account for the GISP MIC protocol testing change<sup><xref ref-type=\"bibr\" rid=\"CR77\">77</xref></sup>. Samples with binary resistance phenotypes (i.e., “SUS” and “RES”) were discarded. For samples with MICs listed as above or below a threshold (indicated by greater than or less than symbols), the MIC was set to equal the provided threshold. MICs were log<sub>2</sub>-transformed for use as continuous outcome variables in linear modeling and GWAS. We modeled transformed MICs using a panel of known resistance markers<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup> and included the recently characterized mosaic <italic>mtrCDE</italic> alleles<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> and <italic>rplD</italic> G70D substitution<sup><xref ref-type=\"bibr\" rid=\"CR78\">78</xref></sup> conferring azithromycin resistance, as well as isolate country of origin. Formulas called by the lm function in R (version 3.5.1) for each drug were (with codon or nucleotide site indicated after each gene or rRNA, respectively):</p><p id=\"Par24\">Azithromycin: Log_AZI ~ Country + MtrR 39 + MtrR 45 + MtrR LOF + <italic>mtrR</italic> promoter + <italic>mtrRCDE</italic> BAPS + RplD G70D + 23S rRNA 2059 + 23S rRNA 2611.</p><p id=\"Par25\">Ceftriaxone: Log_CRO ~ Country + MtrR 39 + MtrR 45 + MtrR LOF + <italic>mtrR</italic> promoter + <italic>penA</italic> BAPS + PonA 421 + PenA 501 + PenA 542 + PenA 551 + PorB 120 + PorB 121.</p><p id=\"Par26\">Ciprofloxacin: Log_CIP ~ Country + MtrR 39 + MtrR 45 + MtrR LOF + <italic>mtrR</italic> promoter + GyrA 91 + GyrA 95 + ParC 86 + ParC 87 + ParC 91 + PorB 120 + PorB 121.</p><p id=\"Par27\">To visualize the continuous MICs using thresholds as on Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>, we binarized MICs using the CLSI resistance breakpoint for ciprofloxacin, the CLSI non-susceptibility breakpoint for azithromycin, and the CDC GISP surveillance breakpoint for ceftriaxone.</p></sec><sec id=\"Sec14\"><title>GWAS and unitig annotation</title><p id=\"Par28\">We used a regression-based GWAS approach to identify novel susceptibility mutations. In particular, we employed a linear mixed model with a random effect to control for the confounding influence of population structure and a fixed effect to control for isolate country of origin. Though the outcome variable (log<sub>2</sub>-transformed MICs) is the same, in contrast to the linear modeling approach described above, which models the linear, additive effect of multiple, known resistance mutations, regression in a GWAS is usually run independently and univariately on each variant for all identified variants in the genome, providing a systematic way to identify novel contributors to the outcome variable. Linear mixed model GWAS was run using Pyseer (version 1.2.0 with default allele frequency filters) on the 480,902 unitigs generated from GATB (version 1.3.0); the recombination-corrected phylogeny from Gubbins was used to parameterize the Pyseer population structure random effects term and isolate country of origin was included as a fixed effect covariate. To create the Manhattan plot, we mapped all unitigs from the GWAS using BWA-MEM (modified parameters: -B 2 and -O 3) to the pan-susceptible WHO F strain reference genome (Genbank accession: GCA_900087635.2) edited to contain only one locus of the 23S rRNA. Significant unitigs were annotated using Pyseer’s annotation pipeline. Unitigs mapping to multiple sites in the genome and in or near the highly variable <italic>pilE</italic> (encoding pilus subunit) or <italic>piiC</italic> (encoding opacity protein family) genes were excluded, as were unitigs less than twenty base pairs in length. Due to redundancy and linkage, variants will be spanned by multiple overlapping unitigs with similar frequencies and <italic>p</italic>-values. For ease of interpretation, we grouped unitigs within 50 base pairs of each other and represented each cluster by the most significant unitig. Unitigs with allele frequency greater than 50% were also excluded as they represented the majority allele. Unitig clusters were then annotated by gene or adjacent genes for unitigs mapping to intergenic regions and further analyzed for predicted functional effect relative to the WHO F reference genome in Geneious Prime (version 2019.2.1, <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.geneious.com\">https://www.geneious.com</ext-link>).</p></sec><sec id=\"Sec15\"><title>Identifying LOF and upregulation alleles</title><p id=\"Par29\">To identify predicted LOF alleles of efflux pump proteins, we ran BLASTn on the de novo assemblies using a query sequence from FA1090 (reference genome FA19 was used for <italic>mtrA</italic>). Sequences that were full-length or approximately full-length (+ /−5 nucleotides) beginning with expected start codons were translated using Python (version 3.6.5) and Biopython (version 1.69)<sup><xref ref-type=\"bibr\" rid=\"CR79\">79</xref></sup>. Peptides shorter than 90% of the expected full-length size of the protein were further analyzed using Geneious Prime (version 2019.2.1, <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.geneious.com\">https://www.geneious.com</ext-link>) to identify the nucleotide mutations resulting in predicted LOF by alignment of the nucleotide sequences. We called <italic>mtrCDE</italic> overexpression status by identifying the presence of any of the known <italic>mtrR</italic> promoter mutations, MtrR coding sequence mutations, and mosaic <italic>mtrCDE</italic> alleles.</p></sec><sec id=\"Sec16\"><title>Experimental validation</title><p id=\"Par30\"><italic>N. gonorrhoeae</italic> culture was conducted on GCB agar (Difco) plates supplemented with 1% Kellogg’s supplements<sup><xref ref-type=\"bibr\" rid=\"CR80\">80</xref></sup> at 37 °C in a 5% CO<sub>2</sub> atmosphere. Antimicrobial susceptibility testing was conducted on GCB agar supplemented with 1% IsoVitaleX (Becton Dickinson) using Etests (bioMérieux) at 37 °C in a 5% CO<sub>2</sub> atmosphere. We selected a clinical isolate (NY0195<sup><xref ref-type=\"bibr\" rid=\"CR81\">81</xref></sup>) from the multidrug-resistant lineage ST-1901<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup> that contained an <italic>mtrC</italic> LOF mutation mediated by a two base pair hexarepeat deletion and confirmed via Etests that its MIC matched, within one dilution, its reported MIC. Isolate NY0195 contained mosaic <italic>penA</italic> allele XXXIV conferring cephalosporin reduced susceptibility and the <italic>gyrA</italic> S91F substitution conferring ciprofloxacin resistance<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. We complemented the <italic>mtrC</italic> LOF mutation in this strain by transforming it via electroporation<sup><xref ref-type=\"bibr\" rid=\"CR80\">80</xref></sup> with a 2 kb PCR product containing a <italic>Neisserial</italic> DNA uptake sequence and an in-frame <italic>mtrC</italic> allele, obtained by colony PCR from a neighboring isolate (GCGS0759). After obtaining transformants by selecting on an azithromycin 0.05 μg/mL GCB plate supplemented with Kellogg’s supplement, we confirmed successful transformation by Sanger sequencing of the <italic>mtrC</italic> gene. No spontaneous mutants on azithromycin 0.05 μg/mL plates were observed after conducting control transformations in the absence of GCGS0759 <italic>mtrC</italic> PCR product. We conducted antimicrobial susceptibility testing in triplicate using Etests, assessing statistical significance between parental and transformant MICs by a two-sample <italic>t</italic>-test.</p></sec><sec id=\"Sec17\"><title>Metadata analysis</title><p id=\"Par31\">Patient metadata were collected from the following publications from Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref> that had information on site of infection: Demczuk et al.<sup><xref ref-type=\"bibr\" rid=\"CR82\">82</xref></sup>, Demczuk et al.<sup><xref ref-type=\"bibr\" rid=\"CR83\">83</xref></sup>, Ezewudo et al.<sup><xref ref-type=\"bibr\" rid=\"CR84\">84</xref></sup>, Grad et al.<sup><xref ref-type=\"bibr\" rid=\"CR85\">85</xref></sup>, Grad et al.<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>, Kwong et al.<sup><xref ref-type=\"bibr\" rid=\"CR86\">86</xref></sup>, Lee et al.<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>, and Mortimer et al.<sup><xref ref-type=\"bibr\" rid=\"CR81\">81</xref></sup>. Sites of infection were standardized across datasets using a common ontology (i.e., specified as urethra, rectum, pharynx, cervix, or other). Two-sided Fisher’s exact test in R (version 3.5.1) was used to infer whether there was nonrandom association between <italic>mtrC</italic> LOF presence and either anatomical site of infection or sexual behavior. For sexual behavior analysis, isolates cultured from multiple sites on the same patient were counted as only one data point.</p></sec><sec id=\"Sec18\"><title>Meningococcal <italic>mtrC</italic> analysis</title><p id=\"Par32\"><italic>mtrC</italic> alleles from <italic>N. meningitidis</italic> assembled genomes were downloaded from PubMLST (<italic>n</italic> = 14798; accessed 2019–09–03) by setting (species = “Neisseria meningitidis”), filtering by (Sequence bin size > = 2 Mbp), and exporting sequences for Locus “NEIS1634”<sup><xref ref-type=\"bibr\" rid=\"CR87\">87</xref></sup>. <italic>mtrC</italic> LOF alleles were identified as described above. We generated a core-genome maximum likelihood phylogeny of all North American ST-11 <italic>N. meningitidis</italic> strains from PubMLST (<italic>n</italic> = 456; accessed 2019–09–03) rooted with meningococcal reference genome MC58 (Genbank accession: AE002098.2) using Roary (version 3.12) (flags -z -e -n -v -s -i 92) and annotated it using metadata from Retchless et al.<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup> (see Supplementary Data <xref rid=\"MOESM8\" ref-type=\"media\">5</xref> for PubMLST IDs). Overrepresentation of <italic>mtrC</italic> LOF alleles in the US urogenital lineage compared to selected control datasets was assessed using two-sided Fisher’s exact test in R (version 3.5.1).</p></sec><sec id=\"Sec19\"><title>Reporting summary</title><p id=\"Par33\">Further information on research design is available in the <xref rid=\"MOESM9\" ref-type=\"media\">Nature Research Reporting Summary</xref> linked to this article.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec20\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41467_2020_17980_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41467_2020_17980_MOESM2_ESM.pdf\"><caption><p>Peer Review File</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM3\"><media xlink:href=\"41467_2020_17980_MOESM3_ESM.pdf\"><caption><p>Descriptions of Additional Supplementary Files</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM4\"><media xlink:href=\"41467_2020_17980_MOESM4_ESM.xlsx\"><caption><p>Supplementary Data 1</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM5\"><media xlink:href=\"41467_2020_17980_MOESM5_ESM.xlsx\"><caption><p>Supplementary Data 2</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM6\"><media xlink:href=\"41467_2020_17980_MOESM6_ESM.xlsx\"><caption><p>Supplementary Data 3</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM7\"><media xlink:href=\"41467_2020_17980_MOESM7_ESM.xlsx\"><caption><p>Supplementary Data 4</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM8\"><media xlink:href=\"41467_2020_17980_MOESM8_ESM.xlsx\"><caption><p>Supplementary Data 5</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM9\"><media xlink:href=\"41467_2020_17980_MOESM9_ESM.pdf\"><caption><p>Reporting Summary</p></caption></media></supplementary-material></sec></sec></body><back><fn-group><fn><p><bold>Peer review information</bold>\n<italic>Nature Communications</italic> thanks Hank Seifert and the other, anonymous reviewer(s) for their contribution to the peer review of this work. Peer review reports are available.</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Kevin C. Ma, Tatum D. Mortimer.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary information</bold> is available for this paper at 10.1038/s41467-020-17980-1.</p></sec><ack><title>Acknowledgements</title><p>This work was supported by the NIH/NIAID grant 1R01AI132606-01 and the Smith Family Foundation. T.D.M. is additionally supported by the NIH/NIAID F32AI145157, and K.C.M. is additionally supported by the NSF GRFP. D.H.F.R. was supported by award Number T32GM007753 from the National Institute of General Medical Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. D.A.W. is supported by an Early Career Fellowship from the National Health and Medical Research Council of Australia (GNT1123854). Portions of this research were conducted on the O2 high-performance computing cluster, supported by the Research Computing Group at Harvard Medical School. This publication made use of the Meningitis Research Foundation Meningococcus Genome Library (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.meningitis.org/research/genome\">http://www.meningitis.org/research/genome</ext-link>) developed by Public Health England, the Wellcome Trust Sanger Institute, and the University of Oxford as a collaboration and funded by the Meningitis Research Foundation. The authors additionally thank Crista Wadsworth, Samantha Palace, and other members of the Grad Lab for helpful comments during development of the project.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>K.C.M., T.D.M., A.L.H., N.E.W., and L.S.B. performed and interpreted genomic analyses. K.C.M., D.H.F.R., and Y.W. performed experimental analyses. D.G. and M.U. provided data and conducted genomic analyses on historical isolates. G.T. and D.A.W. provided data and interpreted results for the validation dataset. S.R.H., M.U., D.A.W., and Y.H.G. supervised the project. K.C.M., T.D.M., and Y.H.G. wrote the paper with contributions from all authors.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>In Supplementary Data <xref rid=\"MOESM6\" ref-type=\"media\">3</xref>–<xref rid=\"MOESM7\" ref-type=\"media\">4</xref>, we have included accession numbers (via publicly hosted database NCBI SRA) for accessing all raw sequence data used for <italic>N. gonorrhoeae</italic> analyses. Intermediate outputs from the genomics pipeline (e.g., de novo assemblies) may also be available from the authors upon request. In Supplementary Data <xref rid=\"MOESM8\" ref-type=\"media\">5</xref>, we have included accession numbers (via publicly hosted database PubMLST: <ext-link ext-link-type=\"uri\" xlink:href=\"https://pubmlst.org/neisseria/\">https://pubmlst.org/neisseria/</ext-link>) for accessing all sequence data used for <italic>N. meningitidis</italic> analyses. Source data underlying all figures are available in Supplementary Data <xref rid=\"MOESM4\" ref-type=\"media\">1</xref>–<xref rid=\"MOESM5\" ref-type=\"media\">2</xref> or at <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/gradlab/mtrC-GWAS\">https://github.com/gradlab/mtrC-GWAS</ext-link>.</p></notes><notes notes-type=\"data-availability\"><title>Code availability</title><p>Code to reproduce the analyses and figures is available at <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/gradlab/mtrC-GWAS\">https://github.com/gradlab/mtrC-GWAS</ext-link> or from the authors upon request.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par34\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Olesen</surname><given-names>SW</given-names></name><etal/></person-group><article-title>Azithromycin susceptibility among <italic>Neisseria gonorrhoeae</italic> Isolates and seasonal macrolide use</article-title><source>J. Infect. Dis.</source><year>2019</year><volume>219</volume><fpage>619</fpage><lpage>623</lpage><pub-id pub-id-type=\"pmid\">30239814</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Olesen</surname><given-names>SW</given-names></name><name><surname>Grad</surname><given-names>YH</given-names></name></person-group><article-title>Deciphering the impact of bystander selection for antibiotic resistance in <italic>Neisseria gonorrhoeae</italic></article-title><source>J. Infect. Dis.</source><year>2020</year><volume>221</volume><fpage>1033</fpage><lpage>1035</lpage><pub-id pub-id-type=\"pmid\">30957162</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><mixed-citation publication-type=\"other\">Sanchez-Buso, L. et al. The impact of antimicrobials on gonococcal evolution. <italic>Nat. Microbiol</italic>. 10.1038/s41564-019-0501-y (2019).</mixed-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Unemo</surname><given-names>M</given-names></name><name><surname>Shafer</surname><given-names>WM</given-names></name></person-group><article-title>Antimicrobial resistance in <italic>Neisseria gonorrhoeae</italic> in the 21st century: past, evolution, and future</article-title><source>Clin. Microbiol Rev.</source><year>2014</year><volume>27</volume><fpage>587</fpage><lpage>613</lpage><pub-id pub-id-type=\"pmid\">24982323</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><mixed-citation publication-type=\"other\">Wadsworth, C. B., Arnold, B. J., Sater, M. R. A. & Grad, Y. H. Azithromycin resistance through interspecific acquisition of an epistasis-dependent efflux pump component and transcriptional regulator in <italic>Neisseria gonorrhoeae</italic>. <italic>MBio</italic>. 10.1128/mBio.01419-18 (2018).</mixed-citation></ref><ref id=\"CR6\"><label>6.</label><mixed-citation publication-type=\"other\">Rouquette-Loughlin, C. E. et al. Mechanistic basis for decreased antimicrobial susceptibility in a clinical isolate of <italic>Neisseria gonorrhoeae</italic> possessing a mosaic-like mtr efflux pump locus. <italic>MBio</italic>. 10.1128/mBio.02281-18 (2018).</mixed-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quillin</surname><given-names>SJ</given-names></name><name><surname>Seifert</surname><given-names>HS</given-names></name></person-group><article-title><italic>Neisseria gonorrhoeae</italic> host adaptation and pathogenesis</article-title><source>Nat. Rev. Microbiol</source><year>2018</year><volume>16</volume><fpage>226</fpage><lpage>240</lpage><pub-id pub-id-type=\"pmid\">29430011</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wi</surname><given-names>T</given-names></name><etal/></person-group><article-title>Antimicrobial resistance in <italic>Neisseria gonorrhoeae</italic>: global surveillance and a call for international collaborative action</article-title><source>PLoS Med.</source><year>2017</year><volume>14</volume><fpage>e1002344</fpage><pub-id pub-id-type=\"pmid\">28686231</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grad</surname><given-names>YH</given-names></name><etal/></person-group><article-title>Genomic epidemiology of gonococcal resistance to extended-spectrum cephalosporins, macrolides, and fluoroquinolones in the United States, 2000-2013</article-title><source>J. Infect. Dis.</source><year>2016</year><volume>214</volume><fpage>1579</fpage><lpage>1587</lpage><pub-id pub-id-type=\"pmid\">27638945</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><mixed-citation publication-type=\"other\">Yahara, K. et al. Genomic surveillance of <italic>Neisseria gonorrhoeae</italic> to investigate the distribution and evolution of antimicrobial-resistance determinants and lineages. <italic>Microb. Genom.</italic>10.1099/mgen.0.000205 (2018).</mixed-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eyre</surname><given-names>DW</given-names></name><etal/></person-group><article-title>WGS to predict antibiotic MICs for <italic>Neisseria gonorrhoeae</italic></article-title><source>J. Antimicrob. Chemother.</source><year>2017</year><volume>72</volume><fpage>1937</fpage><lpage>1947</lpage><pub-id pub-id-type=\"pmid\">28333355</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><mixed-citation publication-type=\"other\">Demczuk, W. et al. Equations to predict antimicrobial MICs in <italic>Neisseria gonorrhoeae</italic> using molecular antimicrobial resistance determinants. <italic>Antimicrob. Agents Chemother</italic>. 10.1128/AAC.02005-19 (2020).</mixed-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hicks</surname><given-names>AL</given-names></name><etal/></person-group><article-title>Evaluation of parameters affecting performance and reliability of machine learning-based antibiotic susceptibility testing from whole genome sequencing data</article-title><source>PLoS Comput. Biol.</source><year>2019</year><volume>15</volume><fpage>e1007349</fpage><pub-id pub-id-type=\"pmid\">31479500</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Earle</surname><given-names>SG</given-names></name><etal/></person-group><article-title>Identifying lineage effects when controlling for population structure improves power in bacterial association studies</article-title><source>Nat. Microbiol</source><year>2016</year><volume>1</volume><fpage>16041</fpage><pub-id pub-id-type=\"pmid\">27572646</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><mixed-citation publication-type=\"other\">Lees, J. A., Galardini, M., Bentley, S. D., Weiser, J. N. & Corander, J. pyseer: a comprehensive tool for microbial pangenome-wide association studies. <italic>Bioinformatics</italic>10.1093/bioinformatics/bty539 (2018).</mixed-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jaillard</surname><given-names>M</given-names></name><etal/></person-group><article-title>A fast and agnostic method for bacterial genome-wide association studies: Bridging the gap between k-mers and genetic events</article-title><source>PLoS Genet.</source><year>2018</year><volume>14</volume><fpage>e1007758</fpage><pub-id pub-id-type=\"pmid\">30419019</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><mixed-citation publication-type=\"other\">Mortimer, T. D. & Grad, Y. H. Applications of genomics to slow the spread of multidrug-resistant <italic>Neisseria gonorrhoeae</italic>. <italic>Ann. N. Y. Acad. Sci</italic>. 10.1111/nyas.13871 (2018).</mixed-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Veal</surname><given-names>WL</given-names></name><name><surname>Nicholas</surname><given-names>RA</given-names></name><name><surname>Shafer</surname><given-names>WM</given-names></name></person-group><article-title>Overexpression of the MtrC-MtrD-MtrE efflux pump due to an mtrR mutation is required for chromosomally mediated penicillin resistance in <italic>Neisseria gonorrhoeae</italic></article-title><source>J. Bacteriol.</source><year>2002</year><volume>184</volume><fpage>5619</fpage><lpage>5624</lpage><pub-id pub-id-type=\"pmid\">12270819</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Golparian</surname><given-names>D</given-names></name><name><surname>Shafer</surname><given-names>WM</given-names></name><name><surname>Ohnishi</surname><given-names>M</given-names></name><name><surname>Unemo</surname><given-names>M</given-names></name></person-group><article-title>Importance of multidrug efflux pumps in the antimicrobial resistance property of clinical multidrug-resistant isolates of <italic>Neisseria gonorrhoeae</italic></article-title><source>Antimicrob. Agents Chemother.</source><year>2014</year><volume>58</volume><fpage>3556</fpage><lpage>3559</lpage><pub-id pub-id-type=\"pmid\">24733458</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Veal</surname><given-names>WL</given-names></name><etal/></person-group><article-title>Loss-of-function mutations in the mtr efflux system of <italic>Neisseria gonorrhoeae</italic></article-title><source>Microbiology</source><year>1998</year><volume>144</volume><fpage>621</fpage><lpage>627</lpage><pub-id pub-id-type=\"pmid\">9534233</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sanchez-Buso</surname><given-names>L</given-names></name><name><surname>Golparian</surname><given-names>D</given-names></name><name><surname>Parkhill</surname><given-names>J</given-names></name><name><surname>Unemo</surname><given-names>M</given-names></name><name><surname>Harris</surname><given-names>SR</given-names></name></person-group><article-title>Genetic variation regulates the activation and specificity of Restriction-Modification systems in <italic>Neisseria gonorrhoeae</italic></article-title><source>Sci. Rep.</source><year>2019</year><volume>9</volume><fpage>14685</fpage><pub-id pub-id-type=\"pmid\">31605008</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eisenstein</surname><given-names>BI</given-names></name><name><surname>Sparling</surname><given-names>PF</given-names></name></person-group><article-title>Mutations to increased antibiotic sensitivity in naturally-occurring gonococci</article-title><source>Nature</source><year>1978</year><volume>271</volume><fpage>242</fpage><lpage>244</lpage><pub-id pub-id-type=\"pmid\">414141</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shimuta</surname><given-names>K</given-names></name><etal/></person-group><article-title>Emergence and evolution of internationally disseminated cephalosporin-resistant <italic>Neisseria gonorrhoeae</italic> clones from 1995 to 2005 in Japan</article-title><source>BMC Infect. Dis.</source><year>2015</year><volume>15</volume><fpage>378</fpage><pub-id pub-id-type=\"pmid\">26381611</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bichara</surname><given-names>M</given-names></name><name><surname>Pinet</surname><given-names>I</given-names></name><name><surname>Schumacher</surname><given-names>S</given-names></name><name><surname>Fuchs</surname><given-names>RP</given-names></name></person-group><article-title>Mechanisms of dinucleotide repeat instability in <italic>Escherichia coli</italic></article-title><source>Genetics</source><year>2000</year><volume>154</volume><fpage>533</fpage><lpage>542</lpage><pub-id pub-id-type=\"pmid\">10655209</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Golparian</surname><given-names>D</given-names></name><etal/></person-group><article-title>Genomic evolution of <italic>Neisseria gonorrhoeae</italic> since the preantibiotic era (1928–2013): antimicrobial use/misuse selects for resistance and drives evolution</article-title><source>BMC Genomics</source><year>2020</year><volume>21</volume><fpage>116</fpage><pub-id pub-id-type=\"pmid\">32013864</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Handing</surname><given-names>JW</given-names></name><name><surname>Ragland</surname><given-names>SA</given-names></name><name><surname>Bharathan</surname><given-names>UV</given-names></name><name><surname>Criss</surname><given-names>AK</given-names></name></person-group><article-title>The MtrCDE efflux pump contributes to survival of <italic>Neisseria gonorrhoeae</italic> from human neutrophils and their antimicrobial components</article-title><source>Front. Microbiol.</source><year>2018</year><volume>9</volume><fpage>2688</fpage><pub-id pub-id-type=\"pmid\">30515136</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Warner</surname><given-names>DM</given-names></name><name><surname>Folster</surname><given-names>JP</given-names></name><name><surname>Shafer</surname><given-names>WM</given-names></name><name><surname>Jerse</surname><given-names>AE</given-names></name></person-group><article-title>Regulation of the MtrC-MtrD-MtrE efflux-pump system modulates the in vivo fitness of <italic>Neisseria gonorrhoeae</italic></article-title><source>J. Infect. Dis.</source><year>2007</year><volume>196</volume><fpage>1804</fpage><lpage>1812</lpage><pub-id pub-id-type=\"pmid\">18190261</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Warner</surname><given-names>DM</given-names></name><name><surname>Shafer</surname><given-names>WM</given-names></name><name><surname>Jerse</surname><given-names>AE</given-names></name></person-group><article-title>Clinically relevant mutations that cause derepression of the <italic>Neisseria gonorrhoeae</italic> MtrC-MtrD-MtrE Efflux pump system confer different levels of antimicrobial resistance and in vivo fitness</article-title><source>Mol. Microbiol</source><year>2008</year><volume>70</volume><fpage>462</fpage><lpage>478</lpage><pub-id pub-id-type=\"pmid\">18761689</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Folster</surname><given-names>JP</given-names></name><etal/></person-group><article-title>MtrR modulates rpoH expression and levels of antimicrobial resistance in <italic>Neisseria gonorrhoeae</italic></article-title><source>J. Bacteriol.</source><year>2009</year><volume>191</volume><fpage>287</fpage><lpage>297</lpage><pub-id pub-id-type=\"pmid\">18978065</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>S</given-names></name><etal/></person-group><article-title>Gonococcal MtrE and its surface-expressed Loop 2 are immunogenic and elicit bactericidal antibodies</article-title><source>J. Infect.</source><year>2018</year><volume>77</volume><fpage>191</fpage><lpage>204</lpage><pub-id pub-id-type=\"pmid\">29902495</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Williamson</surname><given-names>DA</given-names></name><etal/></person-group><article-title>Bridging of <italic>Neisseria gonorrhoeae</italic> lineages across sexual networks in the HIV pre-exposure prophylaxis era</article-title><source>Nat. Commun.</source><year>2019</year><volume>10</volume><fpage>3988</fpage><pub-id pub-id-type=\"pmid\">31488838</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rouquette</surname><given-names>C</given-names></name><name><surname>Harmon</surname><given-names>JB</given-names></name><name><surname>Shafer</surname><given-names>WM</given-names></name></person-group><article-title>Induction of the mtrCDE-encoded efflux pump system of <italic>Neisseria gonorrhoeae</italic> requires MtrA, an AraC-like protein</article-title><source>Mol. Microbiol</source><year>1999</year><volume>33</volume><fpage>651</fpage><lpage>658</lpage><pub-id pub-id-type=\"pmid\">10417654</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><mixed-citation publication-type=\"other\">Vidyaprakash, E., Abrams, A. J., Shafer, W. M. & Trees, D. L. Whole-genome sequencing of a large panel of contemporary <italic>Neisseria gonorrhoeae</italic> clinical isolates indicates that a wild-type mtrA gene is common: implications for inducible antimicrobial resistance. <italic>Antimicrob. Agents Chemother.</italic>10.1128/AAC.00262-17 (2017).</mixed-citation></ref><ref id=\"CR34\"><label>34.</label><mixed-citation publication-type=\"other\">Olivares Pacheco, J., Alvarez-Ortega, C., Alcalde Rico, M. & Martinez, J. L. Metabolic compensation of fitness costs is a general outcome for antibiotic-resistant <italic>Pseudomonas aeruginosa</italic> mutants overexpressing efflux pumps. <italic>MBio</italic>. 10.1128/mBio.00500-17 (2017).</mixed-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zgurskaya</surname><given-names>HI</given-names></name></person-group><article-title>Multicomponent drug efflux complexes: architecture and mechanism of assembly</article-title><source>Future Microbiol</source><year>2009</year><volume>4</volume><fpage>919</fpage><lpage>932</lpage><pub-id pub-id-type=\"pmid\">19722844</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>EH</given-names></name><name><surname>Shafer</surname><given-names>WM</given-names></name></person-group><article-title>The farAB-encoded efflux pump mediates resistance of gonococci to long-chained antibacterial fatty acids</article-title><source>Mol. Microbiol</source><year>1999</year><volume>33</volume><fpage>839</fpage><lpage>845</lpage><pub-id pub-id-type=\"pmid\">10447892</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>EH</given-names></name><name><surname>Rouquette-Loughlin</surname><given-names>C</given-names></name><name><surname>Folster</surname><given-names>JP</given-names></name><name><surname>Shafer</surname><given-names>WM</given-names></name></person-group><article-title>FarR regulates the farAB-encoded efflux pump of <italic>Neisseria gonorrhoeae</italic> via an MtrR regulatory mechanism</article-title><source>J. Bacteriol.</source><year>2003</year><volume>185</volume><fpage>7145</fpage><lpage>7152</lpage><pub-id pub-id-type=\"pmid\">14645274</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rouquette-Loughlin</surname><given-names>C</given-names></name><name><surname>Dunham</surname><given-names>SA</given-names></name><name><surname>Kuhn</surname><given-names>M</given-names></name><name><surname>Balthazar</surname><given-names>JT</given-names></name><name><surname>Shafer</surname><given-names>WM</given-names></name></person-group><article-title>The NorM efflux pump of <italic>Neisseria gonorrhoeae</italic> and <italic>Neisseria meningitidis</italic> recognizes antimicrobial cationic compounds</article-title><source>J. Bacteriol.</source><year>2003</year><volume>185</volume><fpage>1101</fpage><lpage>1106</lpage><pub-id pub-id-type=\"pmid\">12533487</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rouquette-Loughlin</surname><given-names>CE</given-names></name><name><surname>Balthazar</surname><given-names>JT</given-names></name><name><surname>Shafer</surname><given-names>WM</given-names></name></person-group><article-title>Characterization of the MacA-MacB efflux system in <italic>Neisseria gonorrhoeae</italic></article-title><source>J. Antimicrob. Chemother.</source><year>2005</year><volume>56</volume><fpage>856</fpage><lpage>860</lpage><pub-id pub-id-type=\"pmid\">16162665</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stephens</surname><given-names>DS</given-names></name><name><surname>Greenwood</surname><given-names>B</given-names></name><name><surname>Brandtzaeg</surname><given-names>P</given-names></name></person-group><article-title>Epidemic meningitis, meningococcaemia, and <italic>Neisseria meningitidi</italic>s</article-title><source>Lancet</source><year>2007</year><volume>369</volume><fpage>2196</fpage><lpage>2210</lpage><pub-id pub-id-type=\"pmid\">17604802</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hill</surname><given-names>DM</given-names></name><etal/></person-group><article-title>Genomic epidemiology of age-associated meningococcal lineages in national surveillance: an observational cohort study</article-title><source>Lancet Infect. Dis.</source><year>2015</year><volume>15</volume><fpage>1420</fpage><lpage>1428</lpage><pub-id pub-id-type=\"pmid\">26515523</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bichara</surname><given-names>M</given-names></name><name><surname>Schumacher</surname><given-names>S</given-names></name><name><surname>Fuchs</surname><given-names>RP</given-names></name></person-group><article-title>Genetic instability within monotonous runs of CpG sequences in <italic>Escherichia coli</italic></article-title><source>Genetics</source><year>1995</year><volume>140</volume><fpage>897</fpage><lpage>907</lpage><pub-id pub-id-type=\"pmid\">7672590</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bazan</surname><given-names>JA</given-names></name><etal/></person-group><article-title>Notes from the field: Increase in <italic>Neisseria meningitidis</italic>-associated urethritis among men at two sentinel clinics - Columbus, Ohio, and Oakland County, Michigan, 2015</article-title><source>MMWR Morb. Mortal. Wkly Rep.</source><year>2016</year><volume>65</volume><fpage>550</fpage><lpage>552</lpage><pub-id pub-id-type=\"pmid\">27254649</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Retchless</surname><given-names>AC</given-names></name><etal/></person-group><article-title>Expansion of a urethritis-associated <italic>Neisseria meningitidis</italic> clade in the United States with concurrent acquisition of <italic>N. gonorrhoeae</italic> alleles</article-title><source>BMC Genomics</source><year>2018</year><volume>19</volume><fpage>176</fpage><pub-id pub-id-type=\"pmid\">29499642</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tzeng</surname><given-names>YL</given-names></name><etal/></person-group><article-title>Emergence of a new <italic>Neisseria meningitidis</italic> clonal complex 11 lineage 11.2 clade as an effective urogenital pathogen</article-title><source>Proc. Natl Acad. Sci. USA</source><year>2017</year><volume>114</volume><fpage>4237</fpage><lpage>4242</lpage><pub-id pub-id-type=\"pmid\">28373547</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Whiley</surname><given-names>DM</given-names></name><name><surname>Jennison</surname><given-names>A</given-names></name><name><surname>Pearson</surname><given-names>J</given-names></name><name><surname>Lahra</surname><given-names>MM</given-names></name></person-group><article-title>Genetic characterisation of <italic>Neisseria gonorrhoeae</italic> resistant to both ceftriaxone and azithromycin</article-title><source>Lancet Infect. Dis.</source><year>2018</year><volume>18</volume><fpage>717</fpage><lpage>718</lpage><pub-id pub-id-type=\"pmid\">29976521</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><mixed-citation publication-type=\"other\">Kime, L. et al. Transient silencing of antibiotic resistance by mutation represents a significant potential source of unanticipated therapeutic failure. <italic>MBio</italic>. 10.1128/mBio.01755-19 (2019).</mixed-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morse</surname><given-names>SA</given-names></name><etal/></person-group><article-title>Gonococcal strains from homosexual men have outer membranes with reduced permeability to hydrophobic molecules</article-title><source>Infect. Immun.</source><year>1982</year><volume>37</volume><fpage>432</fpage><lpage>438</lpage><pub-id pub-id-type=\"pmid\">6811431</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shafer</surname><given-names>WM</given-names></name><name><surname>Balthazar</surname><given-names>JT</given-names></name><name><surname>Hagman</surname><given-names>KE</given-names></name><name><surname>Morse</surname><given-names>SA</given-names></name></person-group><article-title>Missense mutations that alter the DNA-binding domain of the MtrR protein occur frequently in rectal isolates of <italic>Neisseria gonorrhoeae</italic> that are resistant to faecal lipids</article-title><source>Microbiology</source><year>1995</year><volume>141</volume><issue>Pt 4</issue><fpage>907</fpage><lpage>911</lpage><pub-id pub-id-type=\"pmid\">7773394</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mercante</surname><given-names>AD</given-names></name><etal/></person-group><article-title>MpeR regulates the mtr efflux locus in <italic>Neisseria gonorrhoeae</italic> and modulates antimicrobial resistance by an iron-responsive mechanism</article-title><source>Antimicrob. Agents Chemother.</source><year>2012</year><volume>56</volume><fpage>1491</fpage><lpage>1501</lpage><pub-id pub-id-type=\"pmid\">22214775</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><mixed-citation publication-type=\"other\">Nudel, K. et al. Transcriptome Analysis of <italic>Neisseria gonorrhoeae</italic> during natural infection reveals differential expression of antibiotic resistance determinants between men and women. <italic>mSphere</italic>10.1128/mSphereDirect.00312-18 (2018).</mixed-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Du</surname><given-names>D</given-names></name><etal/></person-group><article-title>Multidrug efflux pumps: structure, function and regulation</article-title><source>Nat. Rev. Microbiol.</source><year>2018</year><volume>16</volume><fpage>523</fpage><lpage>539</lpage><pub-id pub-id-type=\"pmid\">30002505</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vettoretti</surname><given-names>L</given-names></name><etal/></person-group><article-title>Efflux unbalance in <italic>Pseudomonas aeruginosa</italic> isolates from cystic fibrosis patients</article-title><source>Antimicrob. Agents Chemother.</source><year>2009</year><volume>53</volume><fpage>1987</fpage><lpage>1997</lpage><pub-id pub-id-type=\"pmid\">19258280</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chalhoub</surname><given-names>H</given-names></name><etal/></person-group><article-title>Mechanisms of intrinsic resistance and acquired susceptibility of <italic>Pseudomonas aeruginosa</italic> isolated from cystic fibrosis patients to temocillin, a revived antibiotic</article-title><source>Sci. Rep.</source><year>2017</year><volume>7</volume><fpage>40208</fpage><pub-id pub-id-type=\"pmid\">28091521</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Garnett</surname><given-names>JP</given-names></name><etal/></person-group><article-title>Hyperglycaemia and Pseudomonas aeruginosa acidify cystic fibrosis airway surface liquid by elevating epithelial monocarboxylate transporter 2 dependent lactate-H(+) secretion</article-title><source>Sci. Rep.</source><year>2016</year><volume>6</volume><fpage>37955</fpage><pub-id pub-id-type=\"pmid\">27897253</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Worlitzsch</surname><given-names>D</given-names></name><etal/></person-group><article-title>Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients</article-title><source>J. Clin. Invest</source><year>2002</year><volume>109</volume><fpage>317</fpage><lpage>325</lpage><pub-id pub-id-type=\"pmid\">11827991</pub-id></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ayhan</surname><given-names>DH</given-names></name><etal/></person-group><article-title>Sequence-specific targeting of bacterial resistance genes increases antibiotic efficacy</article-title><source>PLoS Biol.</source><year>2016</year><volume>14</volume><fpage>e1002552</fpage><pub-id pub-id-type=\"pmid\">27631336</pub-id></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chan</surname><given-names>BK</given-names></name><etal/></person-group><article-title>Phage selection restores antibiotic sensitivity in MDR Pseudomonas aeruginosa</article-title><source>Sci. Rep.</source><year>2016</year><volume>6</volume><fpage>26717</fpage><pub-id pub-id-type=\"pmid\">27225966</pub-id></element-citation></ref><ref id=\"CR59\"><label>59.</label><mixed-citation publication-type=\"other\">Chen, S. et al. Could dampening expression of the <italic>Neisseria gonorrhoeae</italic> mtrCDE-encoded efflux pump be a strategy to preserve currently or resurrect formerly used antibiotics to treat gonorrhea? <italic>MBio</italic>. 10.1128/mBio.01576-19 (2019).</mixed-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cole</surname><given-names>MJ</given-names></name><etal/></person-group><article-title>Gentamicin, azithromycin and ceftriaxone in the treatment of gonorrhoea: the relationship between antibiotic MIC and clinical outcome</article-title><source>J. Antimicrob. Chemother.</source><year>2020</year><volume>75</volume><fpage>449</fpage><lpage>457</lpage><pub-id pub-id-type=\"pmid\">31670808</pub-id></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tapsall</surname><given-names>JW</given-names></name><etal/></person-group><article-title>Failure of azithromycin therapy in gonorrhea and discorrelation with laboratory test parameters</article-title><source>Sex. Transm. Dis.</source><year>1998</year><volume>25</volume><fpage>505</fpage><lpage>508</lpage><pub-id pub-id-type=\"pmid\">9858344</pub-id></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>H</given-names></name><name><surname>Durbin</surname><given-names>R</given-names></name></person-group><article-title>Fast and accurate short read alignment with Burrows-Wheeler transform</article-title><source>Bioinformatics</source><year>2009</year><volume>25</volume><fpage>1754</fpage><lpage>1760</lpage><pub-id pub-id-type=\"pmid\">19451168</pub-id></element-citation></ref><ref id=\"CR63\"><label>63.</label><mixed-citation publication-type=\"other\">Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at <ext-link ext-link-type=\"uri\" xlink:href=\"https://arxiv.org/abs/1303.3997\">https://arxiv.org/abs/1303.3997</ext-link> (2013).</mixed-citation></ref><ref id=\"CR64\"><label>64.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Garcia-Alcalde</surname><given-names>F</given-names></name><etal/></person-group><article-title>Qualimap: evaluating next-generation sequencing alignment data</article-title><source>Bioinformatics</source><year>2012</year><volume>28</volume><fpage>2678</fpage><lpage>2679</lpage><pub-id pub-id-type=\"pmid\">22914218</pub-id></element-citation></ref><ref id=\"CR65\"><label>65.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Walker</surname><given-names>BJ</given-names></name><etal/></person-group><article-title>Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement</article-title><source>PLoS ONE</source><year>2014</year><volume>9</volume><fpage>e112963</fpage><pub-id pub-id-type=\"pmid\">25409509</pub-id></element-citation></ref><ref id=\"CR66\"><label>66.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bankevich</surname><given-names>A</given-names></name><etal/></person-group><article-title>SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing</article-title><source>J. Comput. Biol.</source><year>2012</year><volume>19</volume><fpage>455</fpage><lpage>477</lpage><pub-id pub-id-type=\"pmid\">22506599</pub-id></element-citation></ref><ref id=\"CR67\"><label>67.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Seemann</surname><given-names>T</given-names></name></person-group><article-title>Prokka: rapid prokaryotic genome annotation</article-title><source>Bioinformatics</source><year>2014</year><volume>30</volume><fpage>2068</fpage><lpage>2069</lpage><pub-id pub-id-type=\"pmid\">24642063</pub-id></element-citation></ref><ref id=\"CR68\"><label>68.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Page</surname><given-names>AJ</given-names></name><etal/></person-group><article-title>Roary: rapid large-scale prokaryote pan genome analysis</article-title><source>Bioinformatics</source><year>2015</year><volume>31</volume><fpage>3691</fpage><lpage>3693</lpage><pub-id pub-id-type=\"pmid\">26198102</pub-id></element-citation></ref><ref id=\"CR69\"><label>69.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thorpe</surname><given-names>HA</given-names></name><name><surname>Bayliss</surname><given-names>SC</given-names></name><name><surname>Sheppard</surname><given-names>SK</given-names></name><name><surname>Feil</surname><given-names>EJ</given-names></name></person-group><article-title>Piggy: a rapid, large-scale pan-genome analysis tool for intergenic regions in bacteria</article-title><source>Gigascience</source><year>2018</year><volume>7</volume><fpage>1</fpage><lpage>11</lpage><pub-id pub-id-type=\"pmid\">29635296</pub-id></element-citation></ref><ref id=\"CR70\"><label>70.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Letunic</surname><given-names>I</given-names></name><name><surname>Bork</surname><given-names>P</given-names></name></person-group><article-title>Interactive Tree Of Life (iTOL) v4: recent updates and new developments</article-title><source>Nucleic Acids Res.</source><year>2019</year><volume>47</volume><fpage>W256</fpage><lpage>W259</lpage><pub-id pub-id-type=\"pmid\">30931475</pub-id></element-citation></ref><ref id=\"CR71\"><label>71.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Croucher</surname><given-names>NJ</given-names></name><etal/></person-group><article-title>Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins</article-title><source>Nucleic Acids Res</source><year>2015</year><volume>43</volume><fpage>e15</fpage><pub-id pub-id-type=\"pmid\">25414349</pub-id></element-citation></ref><ref id=\"CR72\"><label>72.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stamatakis</surname><given-names>A</given-names></name></person-group><article-title>RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies</article-title><source>Bioinformatics</source><year>2014</year><volume>30</volume><fpage>1312</fpage><lpage>1313</lpage><pub-id pub-id-type=\"pmid\">24451623</pub-id></element-citation></ref><ref id=\"CR73\"><label>73.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Altschul</surname><given-names>SF</given-names></name><name><surname>Gish</surname><given-names>W</given-names></name><name><surname>Miller</surname><given-names>W</given-names></name><name><surname>Myers</surname><given-names>EW</given-names></name><name><surname>Lipman</surname><given-names>DJ</given-names></name></person-group><article-title>Basic local alignment search tool</article-title><source>J. Mol. Biol.</source><year>1990</year><volume>215</volume><fpage>403</fpage><lpage>410</lpage><pub-id pub-id-type=\"pmid\">2231712</pub-id></element-citation></ref><ref id=\"CR74\"><label>74.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Katoh</surname><given-names>K</given-names></name><name><surname>Misawa</surname><given-names>K</given-names></name><name><surname>Kuma</surname><given-names>K</given-names></name><name><surname>Miyata</surname><given-names>T</given-names></name></person-group><article-title>MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform</article-title><source>Nucleic Acids Res.</source><year>2002</year><volume>30</volume><fpage>3059</fpage><lpage>3066</lpage><pub-id pub-id-type=\"pmid\">12136088</pub-id></element-citation></ref><ref id=\"CR75\"><label>75.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tonkin-Hill</surname><given-names>G</given-names></name><name><surname>Lees</surname><given-names>JA</given-names></name><name><surname>Bentley</surname><given-names>SD</given-names></name><name><surname>Frost</surname><given-names>SDW</given-names></name><name><surname>Corander</surname><given-names>J</given-names></name></person-group><article-title>Fast hierarchical Bayesian analysis of population structure</article-title><source>Nucleic Acids Res.</source><year>2019</year><volume>47</volume><fpage>5539</fpage><lpage>5549</lpage><pub-id pub-id-type=\"pmid\">31076776</pub-id></element-citation></ref><ref id=\"CR76\"><label>76.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Johnson</surname><given-names>SR</given-names></name><name><surname>Grad</surname><given-names>Y</given-names></name><name><surname>Abrams</surname><given-names>AJ</given-names></name><name><surname>Pettus</surname><given-names>K</given-names></name><name><surname>Trees</surname><given-names>DL</given-names></name></person-group><article-title>Use of whole-genome sequencing data to analyze 23S rRNA-mediated azithromycin resistance</article-title><source>Int J. Antimicrob. Agents</source><year>2017</year><volume>49</volume><fpage>252</fpage><lpage>254</lpage><pub-id pub-id-type=\"pmid\">28038960</pub-id></element-citation></ref><ref id=\"CR77\"><label>77.</label><mixed-citation publication-type=\"other\">Kersh, E. N. et al. Rationale for a <italic>Neisseria gonorrhoeae</italic> susceptible only interpretive breakpoint for azithromycin. <italic>Clin. Infect Dis.</italic>10.1093/cid/ciz292 (2019).</mixed-citation></ref><ref id=\"CR78\"><label>78.</label><mixed-citation publication-type=\"other\">Ma, K. C. et al. Increased power from bacterial genome-wide association conditional on known effects identifies <italic>Neisseria gonorrhoeae</italic> macrolide resistance mutations in the 50S ribosomal protein L4. Preprint at <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.biorxiv.org/content/10.1101/2020.03.24.006650v1\">https://www.biorxiv.org/content/10.1101/2020.03.24.006650v1</ext-link> (2020).</mixed-citation></ref><ref id=\"CR79\"><label>79.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cock</surname><given-names>PJ</given-names></name><etal/></person-group><article-title>Biopython: freely available Python tools for computational molecular biology and bioinformatics</article-title><source>Bioinformatics</source><year>2009</year><volume>25</volume><fpage>1422</fpage><lpage>1423</lpage><pub-id pub-id-type=\"pmid\">19304878</pub-id></element-citation></ref><ref id=\"CR80\"><label>80.</label><mixed-citation publication-type=\"other\">Dillard, J. P. Genetic Manipulation of <italic>Neisseria gonorrhoeae</italic>. <italic>Curr Protoc. Microbiol.</italic> Chapter 4, Unit4A 2, 10.1002/9780471729259.mc04a02s23 (2011).</mixed-citation></ref><ref id=\"CR81\"><label>81.</label><mixed-citation publication-type=\"other\">Mortimer, T. D. et al. The distribution and spread of susceptible and resistant <italic>Neisseria gonorrhoeae</italic> across demographic groups in a major metropolitan center. Preprint at <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.medrxiv.org/content/10.1101/2020.04.30.20086413v1\">https://www.medrxiv.org/content/10.1101/2020.04.30.20086413v1</ext-link> (2020).</mixed-citation></ref><ref id=\"CR82\"><label>82.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Demczuk</surname><given-names>W</given-names></name><etal/></person-group><article-title>Whole-Genome Phylogenomic Heterogeneity of Neisseria gonorrhoeae Isolates with Decreased Cephalosporin Susceptibility Collected in Canada between 1989 and 2013.</article-title><source>J. Clin. Microbiol.</source><year>2015</year><volume>53</volume><fpage>191</fpage><lpage>200</lpage><pub-id pub-id-type=\"pmid\">25378573</pub-id></element-citation></ref><ref id=\"CR83\"><label>83.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Demczuk</surname><given-names>W</given-names></name><etal/></person-group><article-title>Genomic Epidemiology and Molecular Resistance Mechanisms of Azithromycin-Resistant Neisseria gonorrhoeae in Canada from 1997 to 2014</article-title><source>J. Clin. Microbiol.</source><year>2016</year><volume>54</volume><fpage>1304</fpage><lpage>1313</lpage><pub-id pub-id-type=\"pmid\">26935729</pub-id></element-citation></ref><ref id=\"CR84\"><label>84.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ezewudo</surname><given-names>MN</given-names></name><etal/></person-group><article-title>Population structure of Neisseria gonorrhoeae based on whole genome data and its relationship with antibiotic resistance</article-title><source>PeerJ</source><year>2015</year><volume>3</volume><fpage>e806</fpage><pub-id pub-id-type=\"pmid\">25780762</pub-id></element-citation></ref><ref id=\"CR85\"><label>85.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grad</surname><given-names>YH</given-names></name><etal/></person-group><article-title>Genomic epidemiology of Neisseria gonorrhoeae with reduced susceptibility to cefixime in the USA: a retrospective observational study.</article-title><source>The Lancet Infectious Diseases</source><year>2014</year><volume>14</volume><fpage>220</fpage><lpage>226</lpage><pub-id pub-id-type=\"pmid\">24462211</pub-id></element-citation></ref><ref id=\"CR86\"><label>86.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kwong</surname><given-names>JC</given-names></name><etal/></person-group><article-title>Whole-genome sequencing reveals transmission of gonococcal antibiotic resistance among men who have sex with men: an observational study</article-title><source>Sex Transm Infect</source><year>2018</year><volume>94</volume><fpage>151</fpage><lpage>157</lpage><pub-id pub-id-type=\"pmid\">29247013</pub-id></element-citation></ref><ref id=\"CR87\"><label>87.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jolley</surname><given-names>KA</given-names></name><name><surname>Bray</surname><given-names>JE</given-names></name></person-group><article-title>& Maiden, M. C. J. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications</article-title><source>Wellcome Open Res</source><year>2018</year><volume>3</volume><fpage>124</fpage><pub-id pub-id-type=\"pmid\">30345391</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.3 20210610//EN\" \"JATS-archivearticle1-3-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:ali=\"http://www.niso.org/schemas/ali/1.0/\" article-type=\"research-article\" dtd-version=\"1.3\"><?properties open_access?><processing-meta base-tagset=\"archiving\" mathml-version=\"3.0\" table-model=\"xhtml\" tagset-family=\"jats\"><restricted-by>pmc</restricted-by></processing-meta><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Cell Discov</journal-id><journal-id journal-id-type=\"iso-abbrev\">Cell Discov</journal-id><journal-title-group><journal-title>Cell Discovery</journal-title></journal-title-group><issn pub-type=\"epub\">2056-5968</issn><publisher><publisher-name>Springer Singapore</publisher-name><publisher-loc>Singapore</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32864161</article-id><article-id pub-id-type=\"pmc\">PMC7431567</article-id><article-id pub-id-type=\"publisher-id\">183</article-id><article-id pub-id-type=\"doi\">10.1038/s41421-020-0183-x</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>A bioenergetic shift is required for spermatogonial differentiation</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Wei</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Zhaoran</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Chang</surname><given-names>Chingwen</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yang</surname><given-names>Zhichang</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Pengxiang</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Fu</surname><given-names>Haihui</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wei</surname><given-names>Xiao</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Eric</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Tan</surname><given-names>Suxu</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Huang</surname><given-names>Wen</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sun</surname><given-names>Liangliang</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ni</surname><given-names>Ting</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-7896-1184</contrib-id><name><surname>Yang</surname><given-names>Yi</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-0939-1269</contrib-id><name><surname>Wang</surname><given-names>Yuan</given-names></name><address><email>wangyu81@msu.edu</email></address><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.22069.3f</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0369 6365</institution-id><institution>Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, </institution><institution>East China Normal University, </institution></institution-wrap>Shanghai, 200241 China </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.17088.36</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2150 1785</institution-id><institution>Department of Animal Sciences, College of Agriculture and Natural Resources, </institution><institution>Michigan State University, </institution></institution-wrap>East Lansing, MI 48824 USA </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.17088.36</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2150 1785</institution-id><institution>Department of Chemistry, College of Natural Science, </institution><institution>Michigan State University, </institution></institution-wrap>East Lansing, MI 48824 USA </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.8547.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0125 2443</institution-id><institution>State Key Laboratory of Genetic Engineering & MOE Key Laboratory of Contemporary Anthropology, Collaborative Innovation Center of Genetics and Development, School of Life Sciences, </institution><institution>Fudan University, </institution></institution-wrap>Shanghai, 200438 China </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.28056.39</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2163 4895</institution-id><institution>Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, </institution><institution>East China University of Science and Technology, </institution></institution-wrap>Shanghai, 200237 China </aff></contrib-group><pub-date pub-type=\"epub\"><day>18</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>18</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>6</volume><elocation-id>56</elocation-id><history><date date-type=\"received\"><day>4</day><month>2</month><year>2020</year></date><date date-type=\"accepted\"><day>22</day><month>5</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license><ali:license_ref specific-use=\"textmining\" content-type=\"ccbylicense\">https://creativecommons.org/licenses/by/4.0/</ali:license_ref><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"https://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">A bioenergetic balance between glycolysis and mitochondrial respiration is particularly important for stem cell fate specification. It however remains to be determined whether undifferentiated spermatogonia switch their preference for bioenergy production during differentiation. In this study, we found that ATP generation in spermatogonia was gradually increased upon retinoic acid (RA)-induced differentiation. To accommodate this elevated energy demand, RA signaling concomitantly switched ATP production in spermatogonia from glycolysis to mitochondrial respiration, accompanied by increased levels of reactive oxygen species. Disrupting mitochondrial respiration significantly blocked spermatogonial differentiation. Inhibition of glucose conversion to glucose-6-phosphate or pentose phosphate pathway also repressed the formation of c-Kit<sup>+</sup> differentiating germ cells, suggesting that metabolites produced from glycolysis are required for spermatogonial differentiation. We further demonstrated that the expression levels of several metabolic regulators and enzymes were significantly altered upon RA-induced differentiation, with both RNA-seq and quantitative proteomic analyses. Taken together, our data unveil a critically regulated bioenergetic balance between glycolysis and mitochondrial respiration that is required for spermatogonial proliferation and differentiation.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Stem cells</kwd><kwd>Cell biology</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100002855</institution-id><institution>Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)</institution></institution-wrap></funding-source><award-id>2016YFA0100300</award-id><principal-award-recipient><name><surname>Wang</surname><given-names>Yuan</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100001809</institution-id><institution>National Natural Science Foundation of China (National Science Foundation of China)</institution></institution-wrap></funding-source><award-id>91854123</award-id><award-id>31771655</award-id><principal-award-recipient><name><surname>Wang</surname><given-names>Yuan</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par2\">Spermatogonial stem cells (SSCs) maintain a pool of undifferentiated progenitor spermatogonia during spermatogenesis, the postnatal germ cell development<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. During this process, undifferentiated spermatogonia proliferate transiently and respond to developmental cues, such as retinoic acid (RA) to become differentiating spermatogonia, which further develop into spermatocytes and haploid spermatids through meiosis<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. It is therefore critical to understand how spermatogonial proliferation and differentiation are balanced to sustain proper spermatogenesis. SSCs are rare and only occupy ~0.03% of germ cell populations in adult mice<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. In contrast, in vitro cultured spermatogonia can proliferate long-term, and thus represents a feasible platform to investigate both genetic and environmental influence on postnatal germ cell development<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. CD90 and CD9 surface antigens are often used to enrich undifferentiated spermatogonia and SSCs (refs. <sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>) that express marker genes, such as ID4, GFRα1, and PLZF at high levels<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>–<xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>–<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. The c-Kit expression marks the appearance of differentiating spermatogonia and early-stage spermatocytes<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. FGF2 and GDNF are required to maintain spermatogonial proliferation<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref>–<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>, whereas upon treatment with RA (refs. <sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>), CD90<sup>+</sup>/CD9<sup>+</sup>/c-Kit<sup>−</sup> spermatogonia start to differentiate into c-Kit<sup>+</sup> cells, in which STRA8 and SYCP3 are upregulated<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref>–<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Undifferentiated spermatogonia with an SSC capacity can be further evaluated for their potential to undergo meiosis and regenerate complete spermatogenesis in vivo by testicular transplantation into <italic>Kit</italic><sup><italic>w/w-v</italic></sup> mice, a commonly used recipient mouse model with defective endogenous germ cell development<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>–<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>.</p><p id=\"Par3\">All mammalian cells produce ATP with various proportional contributions from glycolysis and mitochondrial oxidative phosphorylation (OXPHOS). These cellular processes not only generate energy, but also provide essential metabolic intermediates that are needed for cell division, lineage development, and epigenetic modifications<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. A tightly regulated balance between glycolysis and OXPHOS is particularly important for stem cell self-renewal and differentiation. It has been shown that the activation of glycolysis and inhibition of OXPHOS increase the reprograming efficiency of somatic cells toward induced pluripotent stem cells<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>–<xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. Specifically for germ cells, accumulating evidence suggests that an intricate network of transcriptional factors, metabolic drivers, and signaling molecules acts together to maintain a biogenetic balance between glycolysis and OXPHOS (refs. <sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>). For example, single-cell RNA-seq analyses have revealed differential expressions of metabolic drivers during different stages of spermatogenesis<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. In addition, forkhead box (FOX)O1 regulates the expression of MYC/MYCN transcription factors that in turn modulate cell cycle and glycolysis to impact SSC self-renewal<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>.</p><p id=\"Par4\">Most studies suggest that adult stem cells reside at a niche with low oxygen tension, and thus rely on glycolysis for energy production to avoid DNA damage caused by reactive oxygen species (ROS) from OXPHOS (refs. <sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>). However, SSCs and undifferentiated spermatogonia are located at the base of seminiferous tubules with layers of spermatocytes and spermatids sequentially migrating from basal toward luminal regions<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. As blood vessels run between seminiferous tubules, oxygen reaches to the lumen only by diffusion<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref>,<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. Compared to meiotic and post-meiotic germ cells, spermatogonia appear to have relatively easier access to oxygen<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref>,<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. Indeed, it was reported that ROS was required for mouse SSC self-renewal by activating P38/MAPK and JNK pathways<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. However, recent studies also showed that hypoxia culture conditions, and increased glycolysis favored SSC establishment and long-term maintenance<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. It remains undetermined whether the balance between glycolysis and OXPHOS changes during spermatogonial differentiation, and whether a high level of OXPHOS is required for this process.</p><p id=\"Par5\">We hereby conducted experiments to unveil metabolic changes and regulatory mechanisms in spermatogonial differentiation. Using an in vitro differentiation platform, we found that undifferentiated spermatogonia adapted a distinct bioenergetic preference from their differentiating populations. In addition, we identified several novel metabolic regulators that were differentially expressed in undifferentiated and differentiating spermatogonia. Disturbed bioenergetic balance led to the disrupted spermatogonial proliferation and differentiation, thereby supporting an essential role of metabolic regulation in spermatogonial fate specification.</p></sec><sec id=\"Sec2\" sec-type=\"results\"><title>Results</title><sec id=\"Sec3\"><title>Establishing an in vitro spermatogonial differentiation platform</title><p id=\"Par6\">To understand metabolic changes during postnatal germ cell development, we first established an in vitro spermatogonial culture and differentiation platform. Undifferentiated spermatogonia exhibited typical clustered grape-like clones<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup> (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>) and ~90% of them expressed the surface antigens CD90 and CD9, assessed by flow cytometry analyses (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref>). Upon RA-induced differentiation, the cells in spermatogonial colonies became loosely connected (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>) and started to differentiate into c-Kit<sup>+</sup> cells (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref>). In addition, undifferentiated spermatogonia highly expressed SSC markers, such as ID4, GFRα1, and PLZF that were downregulated in response to RA treatment (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1c, d</xref>). By contrast, c-Kit, STRA8, and SYCP3 were significantly increased at both RNA and protein levels in RA-induced c-Kit<sup>+</sup> differentiating cells (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b–d</xref>). No statistically significant differences were found in the expression of marker genes (<italic>Gfrα1, Plzf, Id4</italic>, and <italic>Ngn3</italic>) between in vitro maintained spermatogonia and in vivo developed CD9<sup>+</sup>/c-Kit<sup>−</sup> cells (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1e</xref>). Similarly, <italic>c-Kit</italic> and <italic>Sycp3</italic> displayed comparable levels between in vitro RA-treated population and c-Kit<sup>+</sup> cells collected from testes (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1e</xref>). However, we observed increased <italic>Gfrα1</italic> (albeit not statistically significant) and <italic>Stra8</italic> expression levels from in vitro cultured cells compared to their in vivo counterparts (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1e</xref>). One potential explanation may relate to the high heterogeneity of in vivo isolated populations. For example, isolated c-Kit<sup>+</sup> cells include differentiating spermatogonia at different developmental stages that express <italic>Stra8</italic> at various levels. CD9<sup>+</sup>/c-Kit<sup>−</sup> cells contain undifferentiated spermatogonia at different developmental stages and may also be contaminated with a few somatic cells (e.g., mesenchymal cells) that express CD9 (ref. <sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>). In vitro culture favors the growth of undifferentiated spermatogonia and synchronizes them to respond to RA treatment in a more homogenous manner. Another possibility of elevated <italic>Stra8</italic> expression may be due to the high RA induction dose in vitro (Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">1a</xref>). Nevertheless, undifferentiated spermatogonia that were maintained for more than four months in vitro robustly reconstituted spermatogenesis after seminiferous tubule transplantation, and formed ACR (Acrosin)+ and PNA+ haploid spermatids in testes of <italic>Kit</italic><sup><italic>w/w-v</italic></sup> mice (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1f–h</xref>), a model with defective endogenous germ cell development<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>–<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. By contrast, only residue derivatives from differentiating spermatogonia in the RA-treated group were observed at four weeks, but no spermatids were detected at two-month post transplantation (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1f–h</xref>), suggesting efficient differentiation induced by RA. Taken together, we successfully established an in vitro differentiation platform of spermatogonial culture.<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Establishing spermatogonial culture and in vitro differentiation platform.</title><p><bold>a</bold> A typical view of undifferentiated (−RA) and differentiating (+RA) spermatogonial clones. Scale bar, 100 μm. Insets are blow-up images with higher magnificent powers. <bold>b</bold> Spermatogonia in culture and upon RA-induced differentiation were analyzed by flow cytometry. <bold>c</bold>, <bold>d</bold> Marker gene expressions in undifferentiated and differentiating spermatogonia were examined by real-time RT-PCR (<bold>c</bold>) and IF (<bold>d</bold>) assays. <bold>e</bold> Marker gene expressions of in vitro cultured spermatogonia (SG) and differentiating population (dSG) were compared with in vivo developed CD9<sup>+</sup>/c-Kit<sup>−</sup> and c-Kit<sup>+</sup> cells, respectively, by real-time PCR assays. <bold>c</bold>, <bold>e</bold> Data are presented as mean ± SEM. *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01; N.S., no statistical significance; <italic>n</italic> = 3. <bold>f</bold> Seminiferous tubule transplantation of in vitro cultured GFP<sup>+</sup> spermatogonia with or without RA treatment into <italic>Kit</italic><sup><italic>w/w-v</italic></sup> testes. Green fluorescence in testes was detected at two-month post transplantation. A bar graph on the right shows the number of colonies/10−e5 transplanted cells averaged from three recipients. Scale bar, 1 mm. <bold>g</bold><italic>Kit</italic><sup><italic>w/w-v</italic></sup> testes transplanted with undifferentiated spermatogonia and those with RA treatment. Histology was performed at four-week or two-month post transplantation. <bold>h</bold><italic>Kit</italic><sup><italic>w/w-v</italic></sup> testes transplanted with undifferentiated spermatogonia and those treated with RA. IHF assays with an ACR antibody, PNA, and DAPI staining were performed at two-month post transplantation. <bold>g</bold>, <bold>h</bold> Scale bar, 100 µm.</p></caption><graphic xlink:href=\"41421_2020_183_Fig1_HTML\" id=\"d32e777\"/></fig></p></sec><sec id=\"Sec4\"><title>Metabolic changes upon spermatogonial differentiation</title><p id=\"Par7\">To determine metabolic dynamics during spermatogonial differentiation, we first assessed ATP levels and ROS production along RA induction. We found that ATP levels were first decreased but upregulated significantly at day 3 post RA treatment (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a</xref>), indicating a dynamical change in the balance of energy production and consumption during spermatogonial differentiation. Intriguingly, ROS levels started to increase at 24 h and were significantly elevated at 48 h post RA induction (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2b</xref>, Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">1b</xref>), suggesting increased mitochondrial OXPHOS upon spermatogonial differentiation.<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Distinct bioenergetic preference in undifferentiated and differentiating spermatogonia.</title><p><bold>a</bold> ATP levels were measured in undifferentiated spermatogonia (−RA) and in those treated with RA (+RA) for various time periods. <bold>b</bold> ROS levels were examined by flow cytometry in undifferentiated spermatogonia and c-Kit<sup>+</sup> cells induced by RA treatment for one or two days. <bold>c</bold> Cytosolic NADH and NAD+ levels were examined by flow cytometry, and their ratios were calculated for in vitro cultured SoNar spermatogonia with or without RA for 24 h and/or oxamate pretreatment for 5 mins. <bold>d</bold>, <bold>e</bold> LDH activities were determined on undifferentiated spermatogonia and differentiating spermatogonia induced by RA treatment (<bold>d</bold>) or isolated by flow cytometry from mice at postnatal day 11 (<bold>e</bold>). <bold>f</bold>–<bold>h</bold> ECAR (<bold>f</bold>), GlycoPER (<bold>g</bold>), and OCR (<bold>h</bold>) were measured in undifferentiated spermatogonia and in spermatogonia after RA treatment for 24 or 48 h. <bold>a</bold>, <bold>d</bold>–<bold>f</bold>, <bold>h</bold> Fold changes relative to undifferentiated spermatogonia were calculated. <bold>a</bold>, <bold>b</bold>, <bold>d</bold>–<bold>h</bold> Data are presented as mean ± SEM. *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01; ***<italic>P</italic> < 0.001; <italic>n</italic> = 3 for <bold>a</bold>, <bold>b</bold>; <italic>n</italic> = 4 for <bold>d</bold>, <bold>e</bold>; <italic>n</italic> ≥ 5 for <bold>f</bold>, <bold>h</bold>.</p></caption><graphic xlink:href=\"41421_2020_183_Fig2_HTML\" id=\"d32e905\"/></fig></p><p id=\"Par8\">To further determine the changing metabolic preference during spermatogonial differentiation, we established a primary spermatogonial line containing a fluorescent cytosolic SoNar sensor, the intensity of which reflects the cytosolic NAD+ and NADH redox state<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. In general, glycolysis produces NADH, which may be shuttled to mitochondria and oxidized to NAD+, or be recycled to NAD+ by lactate dehydrogenase (LDH) when generating lactate from pyruvate in the cytosol<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. Therefore, the cytosolic NAD+/NADH ratio is determined by the net balance of these three pathways<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. We found that undifferentiated spermatogonia displayed a much higher NAD+/NADH ratio than that of differentiating spermatogonia induced by RA for 24 h (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2c</xref>). Upon treatment of oxamate that blocks LDH activities, the NAD+/NADH ratio was dramatically reduced in undifferentiated spermatogonia (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2c</xref>), suggesting active conversions from pyruvate to lactate in those cells. By contrast, differentiating spermatogonia did not respond well to the oxamate inhibition (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2c</xref>). Because germ cells at distinct developmental stages may express different LDH isoforms<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref>,<xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>, we analyzed LDH activities in response to the oxamate inhibition and confirmed that in both groups LDH activities were efficiently inhibited by the oxamate treatment (Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">1c</xref>). Consistent with these findings, when measured directly, we found significantly higher LDH activities in undifferentiated spermatogonia than in differentiating population treated with RA (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2d</xref>). We further confirmed these observations by assessing LDH activities on in vivo developed CD9<sup>+</sup>/c-Kit<sup>−</sup> undifferentiated spermatogonia and c-Kit<sup>+</sup> differentiating spermatogonia from wild-type mice (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2e</xref>, Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">1d</xref>). In addition, we recently reported that ROS levels were significantly higher in c-Kit<sup>+</sup> differentiating spermatogonia than those from CD90<sup>+</sup>/c-Kit<sup>−</sup> undifferentiated spermatogonia developed in vivo<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. These data thus support our findings that glycolysis decreases, but OXPHOS elevates upon spermatogonial differentiation under physiological conditions. Although interstitial somatic cells, such as Leydig cells from testes also express c-Kit<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>, these cells are largely stripped from seminiferous tubules with sequential trypsin and collagenase treatment during multistep spermatogonial isolation. Sorted c-Kit<sup>+</sup> cells highly expressed germ cell markers, as displayed by real-time RT-PCR analyses (Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">1e</xref>). Finally, to exclude the possibility that these altered metabolic changes were due to direct effects of RA treatment, we collected c-Kit<sup>−</sup> and c-Kit<sup>+</sup> cells from the same RA-treated population, and measured their LDH activities. Significantly higher LDH activities were detected in c-Kit<sup>−</sup> spermatogonia than those from c-Kit<sup>+</sup> cells (Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">1f</xref>), thereby confirming that the observed metabolic shift is not an artifact from RA treatment, but rather determined by the developmental stages of spermatogonia. Taken together, these data reveal that the redox state of spermatogonia changes upon differentiation, and aerobic glycolysis is highly active in undifferentiated spermatogonia to enable the regeneration of NAD+ from pyruvate to lactate.</p><p id=\"Par9\">Using a Seahorse extracellular flux analyzer, we next examined the extracellular acidification rate (ECAR) in undifferentiated spermatogonia and differentiating populations after RA induction for 24 and 48 h. In principle, ECAR reflects both glycolysis and proton contribution from Krebs cycle<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. We found that the glycolytic capacity was reduced in differentiating spermatogonia at 24 h and further decreased at 48 h post RA-induced differentiation, as calculated by the maximum change in ECAR after sequential addition of glucose, oligomycin (to inhibit OXPHOS), and 2-deoxyglucose (2-DG, to inhibit glycolysis; Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2f</xref>, Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">2a</xref>), suggesting a more active glycolysis in undifferentiated spermatogonia. To further distinguish the acidification effects of glycolysis from those generated through Krebs cycle, we measured proton efflux rate (PER) from undifferentiated spermatogonia and cells after RA induction in the presence of glucose, L-glutamine, and pyruvate with the sequential addition of rotenone/antimycin A (to block OXPHOS) and 2-DG. Both basal glycolysis (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2g</xref>, left panel) excluding the mitochondrion-derived acidification and compensatory glycolysis (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2g</xref>, right panel) were dropped gradually along differentiation, thereby supporting a higher glycolytic level in undifferentiated spermatogonia. In addition, we analyzed oxygen consumption rate (OCR) that represents the level of mitochondrial respiration. We found that basal OCR elevated dramatically in differentiating spermatogonia with RA treatment for 48 h (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2h</xref>, middle panel; Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">2b</xref>). The maximum mitochondrial respiration, which was measured as the changes of OCR after sequential addition of oligomycin and FCCP followed by antimycin A supplementation, was significantly increased in cells at 24 and 48 h post RA treatment (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2h</xref>, right panel; Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">2b</xref>), suggesting upregulated mitochondrial respiration upon spermatogonial differentiation. In summary, our data unveil a metabolic shift from glycolysis to mitochondrial respiration upon spermatogonial differentiation.</p></sec><sec id=\"Sec5\"><title>Inhibition of glucose-6-phosphate production and pentose pathway in glycolysis represses spermatogonial differentiation</title><p id=\"Par10\">To identify critical metabolic pathways for spermatogonial differentiation, we treated cells with inhibitors at various steps of glycolysis (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref>). Consistent with the observation above that differentiating spermatogonia prefer OXPHOS for energy production, enoblock, an inhibitor that blocks the formation from 2-phosphoglycerate to phosphoenol pyruvate had little effects on spermatogonial differentiation, as demonstrated by the similar percentage of c-Kit<sup>+</sup> cells upon RA induction compared to mock controls (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a, b</xref>). Similarly, oxamate that inhibited pyruvate to lactate conversion did not have any obvious influence on spermatogonial differentiation at a concentration of 60 mM or lower (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a, c</xref>). At a high concentration of 120 mM, oxamate caused significant cell death likely due to chemical toxicity (data not shown).<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>G6P formation from glucose in glycolysis is required for spermatogonial differentiation.</title><p><bold>a</bold> Overview of inhibitors used to block various steps of glycolysis. <bold>b</bold>–<bold>f</bold> The percentages of CD90<sup>+</sup> and c-Kit<sup>+</sup> cells were determined using flow cytometry on spermatogonia in the absence (−RA) or presence (+RA) of RA for 24 h. Inhibitors include enoblock (Eno; <bold>b</bold>), oxamate (<bold>c</bold>), 2-DG (<bold>d</bold>), loniamine (Lon; <bold>e</bold>), and 6-aminonicotinamide (6-AN; <bold>f</bold>). <bold>g</bold> Gene expression levels were measured by real-time RT-PCR on spermatogonia treated with inhibitors for 24 h. <bold>h</bold> The numbers of spermatogonia were counted in the presence of 10 mM 2-DG for three days (D3), and fold changes of cell numbers after 2-DG treatment was calculated in comparison to mock controls. <bold>g</bold>, <bold>h</bold> Data are presented as mean ± SEM. *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01; N.S., no statistical significance; <italic>n</italic> = 3.</p></caption><graphic xlink:href=\"41421_2020_183_Fig3_HTML\" id=\"d32e1109\"/></fig></p><p id=\"Par11\">Surprisingly, 2-DG, a glucose analog that inhibits the initial step of glycolysis from glucose to glucose-6-phosphate (G6P), significantly blocked spermatogonial differentiation within 24 h of treatment (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a, d</xref>). The percentage of c-Kit<sup>+</sup> cells upon RA induction dropped to 23.8% in 2-DG-treated cells, compared to 93.6% in the control group (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a, d</xref>). Treatment of lonidamine, another inhibitor of hexokinase that catalyzes glucose to G6P, led to similar results (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a, e</xref>), demonstrating that the formation of G6P is critical for spermatogonial differentiation. Because G6P provides building blocks for nucleotide synthesis through pentose phosphate pathway (PPP), we hypothesized that the reduced spermatogonial differentiation was likely due to a decreased supply of metabolites, such as ribose 5-phosphate for nucleotide synthesis. We tested this hypothesis with 6-aminonicotinamide, an inhibitor of G6P dehydrogenase that catalyzes G6P into 6-phosphogluconolactone at the first step of PPP (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref>). Indeed, 6-aminonicotinamide treatment significantly blocked spermatogonial differentiation, reducing c-Kit<sup>+</sup> cells from 93.6% to 27.9% (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3f</xref>). Taken together, our data suggest that active G6P formation in glycolysis and PPP are required for spermatogonial differentiation.</p><p id=\"Par12\">Using these inhibitors at the same concentrations, we further examined their effects on spermatogonial maintenance. No obvious alteration in the percentage of CD90<sup>+</sup>/c-Kit<sup>−</sup> undifferentiated spermatogonia was detected (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b, d, f</xref>). We only observed a slightly reduced colony size after 24 h treatment of lonidamine (Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">3a, b</xref>). As SSC population doubles about every five to six days<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>, short-term glycolysis inhibition may not lead to measurable defects in spermatogonial maintenance. Nevertheless, lonidamine treatment significantly decreased gene expression levels of <italic>Gfrα1</italic> and <italic>Id4</italic> (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3g</xref>), two SSC markers. Expression of <italic>Ngn3</italic>, a gene expressed mainly in early differentiating spermatogonia<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>, was elevated upon treatment of 2-DG, 6-aminonicotinamide, or enoblock (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3g</xref>). We further found that prolonged treatments of 2-DG for two and three days led to dramatic reductions in both spermatogonial colony size and cell number (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3h</xref>, Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">4a</xref>), suggesting that inhibition of glycolysis also impairs spermatogonial self-renewal.</p></sec><sec id=\"Sec6\"><title>Inhibition of OXPHOS blocks spermatogonial differentiation</title><p id=\"Par13\">Using a similar strategy, we determined the requirement of specific enzyme complexes in OXPHOS during spermatogonial differentiation by targeting the mitochondrial respiration chain with various inhibitors. These inhibitors included rotenone against complex I, antimycin A inhibiting complex III, oligomycin repressing the function of ATP synthase, and FCCP, a chemical that disrupts ATP synthesis by abolishing the mitochondrial membrane potential (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4a</xref>). All these inhibitors significantly blocked the formation of c-Kit<sup>+</sup> cells within 24 h post RA induction (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4b–d</xref>), supporting a critical requirement of OXPHOS in spermatogonial differentiation.<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>Spermatogonial differentiation needs OXPHOS.</title><p><bold>a</bold> Overview of inhibitors used to block various steps of mitochondrial respiration. <bold>b</bold>–<bold>d</bold> The percentages of CD90<sup>+</sup> and c-Kit<sup>+</sup> cells were analyzed by flow cytometry on in vitro cultured spermatogonia in the absence (−RA) or presence (+RA) of RA, with or without the following inhibitors: rotenone (Rot; <bold>b</bold>), antimycin A (Ant) and oligomycin (Olig; <bold>c</bold>), and FCCP (<bold>d</bold>). <bold>e</bold> Gene expression levels were measured by real-time RT-PCR assays on spermatogonia treated with inhibitors for 24 h. <bold>f</bold> The numbers of spermatogonia were counted in the presence of 6 μM rotenone or 2 μg/mL oligomycin for two and three days, and fold changes of cell numbers after the treatment of inhibitors were calculated in comparison to mock controls. <bold>e</bold>, <bold>f</bold> Data are presented as mean ± SEM. *<italic>P</italic> < 0.05; ***<italic>P</italic> < 0.001; N.S., no statistical significance; <italic>n</italic> = 3.</p></caption><graphic xlink:href=\"41421_2020_183_Fig4_HTML\" id=\"d32e1252\"/></fig></p><p id=\"Par14\">The same inhibitor treatments did not change the percentage of CD90<sup>+</sup>/c-Kit<sup>−</sup> undifferentiated spermatogonia (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4b–d</xref>), and the spermatogonial morphology was normal after 24 h treatment (Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">3b, c</xref>). We further examined the expression of genes that mark SSC identity by real-time RT-PCR. We found that rotenone treatment showed no effects, but antimycin A, FCCP, and oligomycin all decreased the expression levels of <italic>Gfrα1</italic> (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4e</xref>). Furthermore, prolonged treatment of rotenone or oligomycin for two and three days significantly reduced spermatogonial cell number and colony size (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4f</xref>, Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">4b</xref>), suggesting mitochondrial respiration is needed for long-term maintenance of spermatogonial proliferation.</p></sec><sec id=\"Sec7\"><title>Differential expression of metabolic regulators during spermatogonial differentiation</title><p id=\"Par15\">To systematically investigate the molecular mechanism underlying the metabolic changes during spermatogonial differentiation, we performed RNA-seq analyses to assess the genome-wide expression profiles of undifferentiated spermatogonia and the ones after 36-h RA induction. We confirmed that SSC markers, such as <italic>Gfrα1, Id4, Plzf, Etv5</italic>, and <italic>Sall4</italic>, were highly expressed in undifferentiated spermatogonia (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5a</xref>, Supplementary Table S<xref rid=\"MOESM2\" ref-type=\"media\">1</xref>). By contrast, genes that indicate spermatogonial differentiation, such as <italic>Sycp3, Stra8</italic>, and <italic>c-Kit</italic>, were significantly increased upon RA induction (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5a</xref>, Supplementary Table S<xref rid=\"MOESM2\" ref-type=\"media\">1</xref>). Interestingly, these two cell populations also showed dramatic differences in the levels of many metabolic enzymes, including those for glycolysis (e.g., HKs, Aldoa, <italic>Eno1/2</italic>, and <italic>Ldha/b</italic>; Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5a</xref>, Supplementary Table S<xref rid=\"MOESM2\" ref-type=\"media\">1</xref>). More importantly, several known metabolic regulators, such as <italic>Pdk1/2</italic>, <italic>Srf</italic>, <italic>c-Myc</italic>, and <italic>Mycn</italic>, were expressed at distinct levels in undifferentiated spermatogonia from RA-induced population (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5a</xref>, Supplementary Table S<xref rid=\"MOESM2\" ref-type=\"media\">1</xref>). Consistent with these findings, gene ontology (GO) analyses using KEGG database revealed that glycolysis was among the top differentially regulated pathways between undifferentiated and differentiating spermatogonia (Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">5a</xref>).<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>Differential expressions of metabolic regulators during spermatogonial differentiation.</title><p><bold>a</bold> A heat map graph extracted from RNA-seq analyses shows transcript levels of genes involved in spermatogonial stemness and differentiation, as well as those of metabolic enzymes, known metabolic regulators, and FOX family members. SG, undifferentiated spermatogonia without RA treatment; dSG, differentiating spermatogonia after RA treatment for 36 h. <bold>b</bold> Expression levels of enzymes that catalyze glycolysis were measured by real-time RT-PCR on spermatogonia in the absence or presence of RA for 24 h or 48 h. <bold>c</bold> Expression levels of hexokinase isoform 2 (<italic>Hk2</italic>) and 3 (<italic>Hk3</italic>) were measured by real-time RT-PCR on spermatogonia in the absence or presence of RA for 24 and 48 h. <bold>d</bold> Transcript levels of known metabolic regulators and selected FOX family members were examined by real-time RT-PCR. Their expression levels in differentiating spermatogonia induced by RA for 24 h were calculated relative to those in undifferentiated spermatogonia without RA treatment (SG). <bold>e</bold> Transcript levels of enzymes in glycolysis and mitochondrial OXPHOS, as well as metabolic regulators were assessed by real-time RT-PCR assays. Their expression levels in c-Kit<sup>+</sup> cells from testes at postnatal day 12 were calculated relative to those in CD9<sup>+</sup>/c-Kit<sup>−</sup> undifferentiated spermatogonia (SG) from the same mice. <bold>f</bold> Volcano plots generated from differentially expressed proteins between undifferentiated spermatogonia (SG) and spermatogonia with RA treatment for 36 h (dSG). Solid dots represent metabolic enzymes and markers genes for undifferentiated and differentiating spermatogonia with annotation. <bold>g</bold> Differentially expressed proteins are classified by GO analyses with DAVID online software. <bold>b</bold>–<bold>e</bold> Gene expression levels were normalized to β-actin expression of each sample and calculated relative to control groups. Data are presented as mean ± SEM from three or more independent experiments. *<italic>P</italic> < 0.05; **<italic>P</italic> < 0.01; ***<italic>P</italic> < 0.001; N.S., no statistical significance.</p></caption><graphic xlink:href=\"41421_2020_183_Fig5_HTML\" id=\"d32e1407\"/></fig></p><p id=\"Par16\">We further confirmed these RNA-seq results by real-time RT-PCR using total RNAs collected from independent experiments. The levels of several enzymes in glycolysis were indeed downregulated at different stages of spermatogonial differentiation (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5b</xref>). For example, expression levels of <italic>Ldha</italic> and <italic>Ldhb</italic>, enzymes that catalyze pyruvate to lactate conversion, were indeed decreased after 24-h RA induction (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5b</xref>), consistent with our observation that aerobic glycolysis for energy production is highly active in undifferentiated spermatogonia. Although <italic>Ldha</italic> and <italic>Ldhb</italic> transcript levels in differentiating spermatogonia at 48 h with RA treatment became comparable to undifferentiated spermatogonia (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5b</xref>), their protein levels or activities seemed to remain at lower levels as indicated by LDH activity measurement assays and ECAR from Seahorse analyses (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>), suggesting posttranscriptional regulation of LDH enzymatic activities. Notably, differential expression of hexokinase isoforms was also observed. The isoform <italic>Hk2</italic> was downregulated during the entire differentiation process (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5c</xref>); <italic>Hk3</italic> expression on the other hand remained unchanged at 24 h but was significantly upregulated after 48 h RA induction (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5c</xref>), suggesting differential regulatory mechanisms of stage-specific isozymes during spermatogonial differentiation. Interestingly, many members of the FOX family exhibited distinct expression profiles in undifferentiated spermatogonia from those under RA-induced differentiation (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5a, d</xref>). To confirm whether these enzymes and regulators display similar expression patterns during in vivo spermatogonial differentiation, we performed real-time RT-PCR on sorted CD9<sup>+</sup>/c-Kit<sup>−</sup> undifferentiated and c-Kit<sup>+</sup> differentiating spermatogonia from mouse testes. Indeed, we found that glycolytic enzymes expressed at higher levels in CD9<sup>+</sup>/c-Kit<sup>−</sup> undifferentiated spermatogonia, while mitochondrial regulators were upregulated in c-Kit<sup>+</sup> cells (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5e</xref>). In addition, several metabolic regulators (such as <italic>Fox</italic> genes and <italic>Mycn</italic>) displayed similar expression patterns during in vivo spermatogonial development to those found in vitro (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5e</xref>).</p><p id=\"Par17\">Because metabolic regulators and enzymes can also be regulated through protein translation, posttranslational modification, or differential expression of enzyme isoforms, transcript levels may reflect some but not all changes of these genes between undifferentiated and differentiating spermatogonia. To understand the changes in gene expression at the protein level during spermatogonial differentiation, we further performed quantitative proteomics on undifferentiated spermatogonia and RA-induced differentiating population (Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">5b, c</xref>). A total of 4879 proteins were quantified, and 502 proteins displayed differential expression between these two groups (Supplementary Fig. S<xref rid=\"MOESM1\" ref-type=\"media\">5d</xref> and Table S<xref rid=\"MOESM3\" ref-type=\"media\">2</xref>). Consistent with our observation from RNA-seq analyses, several key enzymes in glycolysis, including HK2, ALDOA, PGK1, PKM, and LDHA, were significantly decreased in differentiating spermatogonia (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5f</xref>). The expression of PDK1, a kinase that inhibits citrate cycle via phosphorylating pyruvate dehydrogenase<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>, was also downregulated (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5f</xref>). Notably, GO enrichment analyses demonstrated that proteins involved in the metabolic process ranked at the top among all differentially regulated cellular processes between undifferentiated and differentiating spermatogonia (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5g</xref>). Taken together, our data reveal differential expression patterns of metabolic regulators between undifferentiated and differentiating spermatogonia at both transcript and protein levels, and these likely drive the metabolic shift from glycolysis to mitochondrial respiration during spermatogonial differentiation.</p></sec></sec><sec id=\"Sec8\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par18\">Previous studies report that increased glycolysis and hypoxia favor the establishment and long-term maintenance of SSCs (refs. <sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>), but ROS is also required for SSC self-renewal<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. To date, no study has directly compared bioenergetic preference between undifferentiated spermatogonia and their differentiating derivatives, nor is clear whether undifferentiated spermatogonia mainly rely on aerobic glycolysis for energy production. Here, we demonstrate that spermatogonial maintenance needs both glycolysis and mitochondrial respiration. Inhibition of glycolysis and OXPHOS in undifferentiated spermatogonia leads to reduced spermatogonial colony size and decreased expressions of SSC marker genes. Glycolysis apparently provides ATP as well as building blocks for other cellular products, whereas OXPHOS likely generates ROS as a signaling molecule for spermatogonial self-renewal<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup>. Nevertheless, consistent with what we know about other types of somatic stem cells, undifferentiated spermatogonia display a higher glycolytic capacity than their differentiating derivatives that prefer OXPHOS for energy production. We observed that spermatogonial differentiation was dramatically blocked by various inhibitors of mitochondrial respiration. Our data thus suggest that the levels of mitochondrial respiration and ROS production need to be critically regulated for proper spermatogonial proliferation and differentiation. It is plausible that spermatogonia need increased energy supply from OXPHOS to go through a transient proliferation to become differentiating progenitors that will further prepare for meiosis to form spermatocytes.</p><p id=\"Par19\">Although spermatogonia switch their preference for bioenergy production upon RA induction, differentiating spermatogonia still need glycolysis to produce various metabolic intermediates to get ready for meiosis. This is unique to germ cells, as somatic cells generally reduce or even stop proliferation upon differentiation and do not go through meiosis before maturation. We observed that 2-DG and lonidamine, two inhibitors that repress glucose to G6P conversion, significantly blocked spermatogonial differentiation. An increased <italic>Hk3</italic> expression likely compensates <italic>Hk2</italic> reduction during spermatogonial differentiation to meet the need of G6P formation from glycolysis for meiotic preparation. On the other hand, undifferentiated spermatogonia with prolonged treatment of 2-DG showed a significantly reduced number of spermatogonial cells. Lonidamine inhibition further decreased <italic>Id4</italic> and <italic>Gfrα1</italic> expression levels. These data are consistent with a previous report that 2-DG repressed long-term SSC growth and maintenance<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. Interestingly, 2-DG treatment in spermatogonia upregulated <italic>Ngn3</italic> expression. Undifferentiated spermatogonia become c-Kit<sup>+</sup> cells by going through a stage of committed progenitors, for which NGN3 is defined as a marker<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>. It is plausible that 2-DG blocks spermatogonial differentiation at the NGN3<sup>+</sup> cell stage. Taken together, our data reveal that the initial step of glycolysis is crucial for both spermatogonial proliferation and differentiation from NGN3<sup>+</sup> committed spermatogonial progenitors to c-Kit<sup>+</sup> cells.</p><p id=\"Par20\">Mitochondrial respiration increases upon differentiation induced by RA treatment for 24 or 48 h, as supported by upregulated ROS levels and OCR, indicating that OXPHOS is particularly important for spermatogonial differentiation. Indeed, inhibitors of OXPHOS significantly reduced the formation of c-Kit<sup>+</sup> differentiated cells. By contrast, obvious alteration in undifferentiated spermatogonia was observed mainly after prolonged treatments for two or three days. Mitochondrial respiration generally generates higher amounts of both ATP molecules and ROS than glycolysis. However, the ATP level dropped first and then elevated on day 3 during differentiation. The discrepancy between a declined ATP level and elevated ROS production suggests a higher energy consumption than production at the early stage of RA-induced differentiation, during which elevated energy demand is required for undifferentiated spermatogonial progenitors to go through transient and rapid proliferation before meiosis.</p><p id=\"Par21\">How do spermatogonia switch their bioenergetic preference from glycolysis to OXPHOS upon RA-induced differentiation? Our RNA-seq and proteomic data provided further clues to answer this question. We found that the metabolic process ranked among the top differentially altered pathways based upon GO enrichment analyses. Expression levels of key enzymes (HK2, ALDOA, PKM, and LDHA) in glycolysis were downregulated during differentiation at both RNA and protein levels. According to our transcriptome profiling, several known metabolic regulators, including <italic>c-Myc</italic> and <italic>Mycn</italic>, two genes that are critical for maintaining glycolysis in undifferentiated spermatogonia<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>, were decreased upon differentiation. Many FOX family members were also differentially expressed between undifferentiated spermatogonia and RA-induced cells. The FOX family of transcription factors is an evolutionarily ancient gene family. More than 40 FOX family members in mammals have been identified and are classified into subfamilies from FOXA to FOXP (ref. <sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>). Of these, FOXO subfamily members have been implicated as metabolic sensors in stem cell regulation<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>. Recently, FOXK1 and K2 were also reported to regulate aerobic glycolysis in somatic cells<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>. We did not observe any significant change in the expressions of <italic>Foxk1</italic> and <italic>Foxk2</italic> upon spermatogonial differentiation. However, we found six novel <italic>Fox</italic> genes that exhibited more than threefold differences in their expressions between undifferentiated and differentiating spermatogonia (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5d</xref>, Supplementary Table S<xref rid=\"MOESM2\" ref-type=\"media\">1</xref>). RNA-seq analyses from published studies also showed a similar trend of differential <italic>Fox</italic> expression between undifferentiated and differentiating spermatogonia<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref>,<xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>. Our data thus suggest a potential requirement of these novel FOX members, as potential metabolic regulators of the bioenergetic switch during spermatogonial differentiation.</p><p id=\"Par22\">Admittedly, because culture conditions lack of physiological hypoxia environment, oxygen gradient across the testicular lumen, and supporting cells from the niche, studies of in vitro spermatogonial proliferation and differentiation cannot completely recapitulate what may happen in vivo. Nevertheless, our conclusion about a metabolic shift from glycolysis to OXPHOS during spermatogonial differentiation is supported by several lines of in vivo evidence. First, multiple RNA-seq analyses unveil that regulators in glycolysis are decreased in differentiating spermatogonia isolated from testes, while mitochondrial regulators appear to be upregulated, compared to SSCs (refs. <sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref>,<xref ref-type=\"bibr\" rid=\"CR53\">53</xref>,<xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>). We observed the same phenomena by real-time RT-PCR on sorted undifferentiated spermatogonia and their progenitors developed in vivo. Second, we found that LDH activities decreased (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2e</xref>), but ROS levels<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup> elevated in c-Kit<sup>+</sup> differentiating spermatogonia developed in vivo compared to CD9<sup>+</sup>/c-Kit<sup>−</sup> undifferentiated population. These data strongly support that a metabolic shift indeed occurs in vivo during spermatogonial differentiation. Further research is warranted to investigate how environmental and developmental stimuli are hardwired with metabolic regulators to fine-tune the differentiation and self-renewal programs of SSCs during spermatogenesis.</p></sec><sec id=\"Sec9\" sec-type=\"materials|methods\"><title>Materials and methods</title><sec id=\"Sec10\"><title>Experimental animals and establishment of spermatogonial cell culture</title><p id=\"Par23\">Testes were collected around postnatal day 5 from DBA mice or F1 of DBA crossed with GFP mice (Stock #: 003516, Jackson Lab, Sacramento, CA, USA). Spermatogonial culture was established and cultured according to published protocols<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. Briefly, seminiferous tubules were incubated at 37 °C in PBS containing 1 mg/mL type IV collagenase (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min and then in PBS/0.05% trypsin (Thermo Fisher Scientific) for 5 min, with occasional agitation. After enzyme digestion, CD9<sup>+</sup>/c-Kit<sup>−</sup> undifferentiated spermatogonia were sorted by an Aria II flow cytometer (BD Bioscience, San Jose, CA, USA) and then maintained in Stempro34 (Thermo Fisher Scientific) supplemented with 20 ng/ml β-FGF (233-FB, R&D Systems, Minneapolis, MN,USA), 10<sup>3</sup> U/mL ESGRO (Thermo Fisher Scientific), and 20 ng/mL GDNF/EGF (10561-HNCH/10605-HNAE, both from Sino Biological, Beijing, China). The use of animals and experimental protocols in this study were approved by the Institutional Animal Care and Use Committee of East China Normal University (project no. M20190317) and Michigan State University (08/17-137-00).</p></sec><sec id=\"Sec11\"><title>Seminiferous tubule transplantation</title><p id=\"Par24\">Approximately 1 × 10<sup>4</sup> (~3 µL as total volume) in vitro cultured GFP<sup>+</sup> spermatogonia with trypan blue were transplanted into the seminiferous tubules of <italic>KIT</italic><sup><italic>W/W-v</italic></sup> testes (Jackson Lab) at three to five weeks of age. Mice were sacrificed two months later. GFP fluorescence in transplanted testes was examined under an Olympus microscope IX71, followed by histological analysis and immunohistofluorescence of testicular sections.</p></sec><sec id=\"Sec12\"><title>Immunofluorescence, immunohistofluorescence, and histology studies</title><p id=\"Par25\">Cells on Matrigel (BD Bioscience) coated cover slides were fixed in 4% paraformaldehyde (Sangon Biotech, Shanghai, China) at 4 °C overnight, and immunofluorescence (IF) assays were performed according to published protocols<sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>. The following antibodies were used: PLZF (SC-28319, Santa Cruz Biotech, Dallas, TX, USA), STRA8 (ab49602, Abcam, Cambridge, MA, USA), SYCP3 (ab15093, Abcam), Alexa Fluor 488- or TRITC-conjugated anti-mouse and anti-rabbit secondary antibodies (115-545-146, 115-025-146; 111-545-144, and 111-025-144, Jackson ImmunoResearch, West Grove, PA, USA). Nuclei were stained with 0.5 µg/mL DAPI before being visualized using an Olympus microscope BX53. Images were processed with the Image-J software. For histology studies, testes were fixed in Bouin’s fixative at 4 °C overnight for staining with hematoxylin and eosin, as previously described<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>. For immunohistofluorescence (IHF), testes were fixed with 4% paraformaldehyde in PBS at 4 °C overnight and embedded in paraffin. Testis sections were stained with an ACR antibody (HPA048687, Atlas Antibodies, Sweden), followed by staining with an Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch) and Rhodamine-labeled Peanut Agglutinin/PNA (RL-1072, Vector Laboratories, USA). Images were collected using a fluorescent microscope (Leica, DM400BLED368424).</p></sec><sec id=\"Sec13\"><title>Spermatogonial differentiation and usages of inhibitors</title><p id=\"Par26\">Spermatogonial cells were maintained in Stempro34 medium with cytokines. Undifferentiated spermatogonia were transferred into Matrigel-coated dishes and spermatogonial differentiation was induced with RA (R2625, Sigma, St. Louis, MO, USA) in the spermatogonial culture medium. All RA treatments were performed with 100 nM RA for 24 h unless specified differently. To test the effects of inhibitors on spermatogonial proliferation, inhibitors were added at 12 h after cell passage. To examine the effects of inhibitors during spermatogonial differentiation, inhibitors were added simultaneously with RA treatment unless otherwise specified. The final concentration of inhibitors used in this study was listed as following unless otherwise specified: lonidamine (400 μM, HY-B0486) and AP-III-α4/ENOblock (20 µM, HY-15858) from MedChemExpress, Monmouth Junction, NJ, USA; 6-aminonicotinamide (1 mM C4497, APExBIO, Houston, TX, USA); 2-DG (10 mM, D8375); oxamate (30 μM, O2751), rotenone (12 μM, R8875), antimycin A (10 μM, A8674), and FCCP (20 μM, C2920) are from Sigma-Aldrich (St. Louis, MO, USA); and oligomycin A (2 µg/mLS1478, Selleck, Houston, TX, USA).</p></sec><sec id=\"Sec14\"><title>Flow cytometry</title><p id=\"Par27\">Cells were digested into single cells using 0.05% trypsin and incubated in DMEM containing 10% fetal bovine serum (Thermo Fisher Scientific), stained with fluorochrome-conjugated antibodies, and washed with PBS before being analyzed, or sorted by a BD Fortessa or an Aria II flow cytometer (BD Biosciences). To determine the NAD+/NADH ratio, spermatogonia containing a SoNar probe were excited by 488 and 405 nm lasers, and fluorescence signals were detected through 530/30 FITC and 530/30 Alexa Fluor 430 channels, using BD Fortessa or Aria II flow cytometers. To determine ROS levels, cells were stained with 5 µM H<sub>2</sub>DCFDA (D399, Thermo Fisher Scientific) in PBS containing 1% BSA at 37 °C for 40 min in the dark followed by washing with PBS twice before measurement, using flow cytometers. Antibodies were used with 2 µg/mL per 1–2 million cells as a final working concentration: APC-CD9 (17-0091-82, eBioscience, USA); APC-CD90.2 (105311), and FITC-c-Kit (105806) from Biolegend, USA; and PE-cy7-CD90.2 (561642), PE-c-Kit (553355), and APC-c-Kit (553356) from BD Bioscience, USA.</p></sec><sec id=\"Sec15\"><title>ATP measurement</title><p id=\"Par28\">Cellular ATP levels were measured using an ATP Assay Kit (Beyotime Biotech, Shanghai, China) according to the manufacturer’s instructions. Briefly, supernatants (60 µL) from cells were mixed with 60 µL of ATP detection buffer, and luminance signals were measured using an Infinite™ M200 Microplate Reader (TECAN, Männedorf, Switzerland). Protein concentration for each sample was determined by Bradford protein assay, and ATP concentration (µM) per mg of protein was calculated. Three or more technical replicates were examined for each independent experiment.</p></sec><sec id=\"Sec16\"><title>LDH activity assay</title><p id=\"Par29\">To measure LDH activity, a Lactate Dehydrogenase Assay Kit (C0017, Beyotime Biotech, Shanghai, China) was used according to the manufacturer’s instructions. Briefly, cells were treated with 150 μL LDH release reagent and 120 μL supernatants were collected into a 96-well plate. After incubation with 60 μL LDH reaction reagent, absorbance was measured at the wavelength of 490 nm with a SPECTROstar Nano Microplate Reader (BMG Labtech, Germany). LDH activities (mU/ml) were calculated using standard curves generated from an LDH standard (L8080, Solarbio, Shanghai, China). Protein concentration for each sample was determined by Bradford protein assay, and LDH activities per mg of protein were calculated. To measure the inhibitory effects of oxamate, LDH activities were determined after incubation of cell lysate with 60 mM oxamate for 15 min on ice. Three or more technical replicates were used for each independent experiment.</p></sec><sec id=\"Sec17\"><title>Seahorse analyses</title><p id=\"Par30\">For OCR and ECAR analyses, 2–3 × 10<sup>4</sup> cells were plated on Matrigel-coated Seahorse XF96 microplates (Agilent Technologies, California, CA, USA, 101085-004). OCR, ECAR, and PER were determined using a XF96 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA, USA). For OCR detection, injection port A on the sensor cartridge was loaded with 150 μM oligomycin A, port B with 200 μM FCCP, and port C with 100 μM antimycin A. During sensor calibration, cells were incubated at 37 °C in a CO<sub>2</sub>-free incubator with 180 μL assay medium (XF Base Medium with 10 mM glucose, 3.75 mM sodium pyruvate, and 2 mM L-glutamine). Basal respiration rates and maximum respiration were calculated following the manufacture’s protocols. For ECAR test, injection port A was loaded with 100 mM glucose, port B with 150 μM oligomycin, and port C with 100 mM 2-DG. Cells were incubated at 37 °C in 180 μL assay medium (XF Base Medium plus 2 mM L-glutamine) before measurement. Glycolytic capacity was calculated according to the manufacturer’s instructions. For PER detection, injection port A on the sensor cartridge was loaded with 100 μM rotenone and 100 μM antimycin A, and port B with 100 mM 2-DG. Cells were incubated at 37 °C in 180 μL of assay medium (XF DMEM with 10 mM glucose, 3.75 mM sodium pyruvate, and 2 mM L-glutamine) for 1 h and fresh medium was changed before measurement. Basal glycolysis, percentage of PER from glycolysis, and compensatory glycolysis were generated automatically through Seahorse Analyzer software. OCR, ECAR, and PER data were normalized to protein concentrations as determined by Bradford protein assay for each sample.</p></sec><sec id=\"Sec18\"><title>RNA sequencing and bioinformatics analyses</title><p id=\"Par31\">For RNA-seq analyses, undifferentiated and differentiating spermatogonia were collected in Trizol (Thermo Fisher Scientific), and total RNAs were extracted. The cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s instructions. RNA-seq libraries were generated with a NEB Next Directional RNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA, USA). Resulting libraries were size-selected by agarose gel electrophoresis and subsequently sequenced using an Illumina HiSeq-X platform with a 2 × 150 bp modality. Paired-end RNA-seq reads with 150 bp in each end were aligned to the <italic>Mus musculus</italic> genome (GRCm38) using Subread Aligner<sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup> with its default parameter settings, and reads were counted using Feature Counts v1.5.3 (ref. <sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup>). DESeq2 (ref. <sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>) was used to identify differentially expressed genes with false discovery rate (FDR) < 0.05 and fold change ≥ 2. KEGG enrichment pathway analyses were performed using KOBAS (ref. <sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup>).</p></sec><sec id=\"Sec19\"><title>Quantitative proteomics and bioinformatics analyses</title><p id=\"Par32\">For proteomic analyses, one million undifferentiated and differentiating spermatogonia (induced by RA for 36 h) were lifted from feeder cells with gentle pipetting and suspended in 100 µL cell lysis buffer (2% SDS, 100 mM NH<sub>4</sub>HCO<sub>3</sub>, protease inhibitors, and phosphatase inhibitors in PBS). Biological triplicates from each treatment group were collected. Cell lysis was processed through ultrasonication (Branson Sonifier 250, VWR Scientific, Batavia, IL), denaturing, and centrifugation to collect the supernatant. A total of 10 µg of proteins from each sample were utilized for reduction (by DL-Dithiothreitol) and alkylation (by Iodoacetamide). Single-spot solid-phase sample preparation with magnetic beads (SP3) was used to remove salts and SDS following a published protocol<sup><xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup>. Labeled peptides were combined and processed by zip-tip desalting and high-pH reverse-phase liquid chromatography (RPLC) fractionation on an Easy nano-LC 1200 (Thermo Fisher Scientific) with a capillary column (75 μm i.d. × 50 cm, C18, 2 μm, 100 Å). Fractions were collected every 2 min with 400 nL of eluates into the tube containing the acidic aqueous phase. A total of 30 fractions were collected and analyzed by low-pH nanoRPLC-MS/MS with the same LC system as fractionation. A total of 80% of peptides from each fraction were loaded onto the analytical column (75 μm i.d. × 50 cm, C18, 2 μm, 100 Å) and then separated through a 3-h linear gradient with a flow rate at 200 nL/min. A Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) was used for the MS/MS analysis with ESI voltage at 2 kV and MS parameters as follows: the resolution for full MS was 60,000, AGC at 3E6, the maximum injection time at 50 ms, and scan range at 300–1800 <italic>m</italic>/<italic>z</italic>. A Top10 data-dependent acquisition method was applied with following parameters: quadrupole isolation window was set at 2 <italic>m</italic>/<italic>z</italic>; normalized collision energy at 28 and 30%; MS/MS scan resolution at 30,000, AGC at 1e5, the maximum injection time at 50 ms, dynamic exclusion window at 30 s, fixed first mass at 100, and MS/MS intensity threshold for MS/MS was set 5e4. The database search was processed by Maxquant (v 1.5.5.1)<sup><xref ref-type=\"bibr\" rid=\"CR62\">62</xref></sup> with Uniport database for <italic>M. musculus</italic> (UP000000589). All parameters were set as default. Reporter ion MS2 was selected with TMT6plex for quantification. Filter by PIF was checked with 0.75 as minimum reporter PIF. The FDR was evaluated through the target–decoy database search. Reporter ion intensity of the first TMT channel (channel 126) was used to normalize reporter ion intensities of the other channels for fold change calculation: each individual reporter ion intensity was divided by the corresponding reporter ion intensity of the channel 126, converting the reporter ion intensity to protein ratio. Protein ratios of each TMT channel were divided by the corresponding median to make sure the ratios of each channel center at 1. The Perseus software was employed to generate volcano plots and perform <italic>t</italic>-test analyses<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. The differentially expressed proteins between the differentiated and undifferentiated cells were determined with FDR at 0.1 and s0 at 0.1 using the Perseus software. GO terms of the differentially expressed proteins were analyzed by DAVID software<sup><xref ref-type=\"bibr\" rid=\"CR63\">63</xref></sup>.</p></sec><sec id=\"Sec20\"><title>RT-PCR and real-time PCR</title><p id=\"Par33\">Total RNAs were extracted using Trizol and reverse-transcribed using a cDNA Synthesis Kit (TaKaRa Biotechnology Co., Ltd., Shiga, Japan). Real-time PCR assays were performed and normalized to β-actin expression as previously described<sup><xref ref-type=\"bibr\" rid=\"CR64\">64</xref></sup> on a Biorad thermal cycler (Biorad, Hercules, CA, USA) or a Stratagene Mx3000P (Stratagene, San Diego, CA, USA). Sequences of primers used in this study are provided in Supplementary Table S<xref rid=\"MOESM4\" ref-type=\"media\">3</xref>.</p></sec><sec id=\"Sec21\"><title>Statistical analysis</title><p id=\"Par34\">Data were presented as mean ± SEM. All experiments were performed independently at least three times unless specified otherwise. Unpaired Student’s <italic>t</italic>-test (comparison between two groups) or one-way ANOVA (multiple groups) were conducted using the Prism Graphic software with the exception of RNA-seq analyses and proteomics.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec22\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41421_2020_183_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media></supplementary-material>\n<supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41421_2020_183_MOESM2_ESM.xlsx\"><caption><p>Supplementary Table S1</p></caption></media></supplementary-material>\n<supplementary-material content-type=\"local-data\" id=\"MOESM3\"><media xlink:href=\"41421_2020_183_MOESM3_ESM.xlsx\"><caption><p>Supplementary Table S2</p></caption></media></supplementary-material>\n<supplementary-material content-type=\"local-data\" id=\"MOESM4\"><media xlink:href=\"41421_2020_183_MOESM4_ESM.xlsx\"><caption><p>Supplementary Table S3</p></caption></media></supplementary-material>\n</p></sec></sec></body><back><fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary Information</bold> accompanies the paper at (10.1038/s41421-020-0183-x).</p></sec><ack><title>Acknowledgements</title><p>This work was supported by grants from the Ministry of Science and Technology of China (2016YFA0100300), the National Natural Science Foundation of China (91854123 and 31771655), and NIFA through AgbioResearch Hatch Fund at Michigan State University.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>W.C., Z.Z., C.C., Z.Y., P.W., X.W., and Y.Y. performed research; H.F., E.C., S.T., L.S., W.H., and T.N. analyzed data; and Y.W. designed experiments, analyzed results, and wrote the manuscript.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>RNA-seq and proteomic raw data have been deposited in the publicly accessible database GEO (GSE150583) and ProteomeXchange (PXD019136).</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Conflict of interest</title><p id=\"Par35\">The authors declare that they have no conflict of interest.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wylie</surname><given-names>C</given-names></name></person-group><article-title>Germ cells</article-title><source>Cell</source><year>1999</year><volume>96</volume><fpage>165</fpage><lpage>174</lpage><pub-id pub-id-type=\"pmid\">9988212</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ewen</surname><given-names>KA</given-names></name><name><surname>Koopman</surname><given-names>P</given-names></name></person-group><article-title>Mouse germ cell development: from specification to sex determination</article-title><source>Mol. Cell Endocrinol.</source><year>2010</year><volume>323</volume><fpage>76</fpage><lpage>93</lpage><pub-id pub-id-type=\"pmid\">20036311</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Griswold</surname><given-names>MD</given-names></name><name><surname>Oatley</surname><given-names>JM</given-names></name></person-group><article-title>Concise review: defining characteristics of mammalian spermatogenic stem cells</article-title><source>Stem Cells</source><year>2013</year><volume>31</volume><fpage>8</fpage><lpage>11</lpage><pub-id pub-id-type=\"pmid\">23074087</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mecklenburg</surname><given-names>JM</given-names></name><name><surname>Hermann</surname><given-names>BP</given-names></name></person-group><article-title>Mechanisms regulating spermatogonial differentiation</article-title><source>Results Probl. Cell Differ.</source><year>2016</year><volume>58</volume><fpage>253</fpage><lpage>287</lpage><pub-id pub-id-type=\"pmid\">27300182</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>QE</given-names></name><name><surname>Oatley</surname><given-names>JM</given-names></name></person-group><article-title>Spermatogonial stem cell functions in physiological and pathological conditions</article-title><source>Curr. Top. Dev. Biol.</source><year>2014</year><volume>107</volume><fpage>235</fpage><lpage>267</lpage><pub-id pub-id-type=\"pmid\">24439809</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kanatsu-Shinohara</surname><given-names>M</given-names></name><etal/></person-group><article-title>Long-term proliferation in culture and germline transmission of mouse male germline stem cells</article-title><source>Biol. Reprod.</source><year>2003</year><volume>69</volume><fpage>612</fpage><lpage>616</lpage><pub-id pub-id-type=\"pmid\">12700182</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kubota</surname><given-names>H</given-names></name><name><surname>Avarbock</surname><given-names>MR</given-names></name><name><surname>Brinster</surname><given-names>RL</given-names></name></person-group><article-title>Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells</article-title><source>Biol. Reprod.</source><year>2004</year><volume>71</volume><fpage>722</fpage><lpage>731</lpage><pub-id pub-id-type=\"pmid\">15115718</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kanatsu-Shinohara</surname><given-names>M</given-names></name><name><surname>Toyokuni</surname><given-names>S</given-names></name><name><surname>Shinohara</surname><given-names>T</given-names></name></person-group><article-title>CD9 is a surface marker on mouse and rat male germline stem cells</article-title><source>Biol. Reprod.</source><year>2004</year><volume>70</volume><fpage>70</fpage><lpage>75</lpage><pub-id pub-id-type=\"pmid\">12954725</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kubota</surname><given-names>H</given-names></name><name><surname>Avarbock</surname><given-names>MR</given-names></name><name><surname>Brinster</surname><given-names>RL</given-names></name></person-group><article-title>Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells</article-title><source>Proc. Natl Acad. Sci. USA</source><year>2003</year><volume>100</volume><fpage>6487</fpage><lpage>6492</lpage><pub-id pub-id-type=\"pmid\">12738887</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Buageaw</surname><given-names>A</given-names></name><etal/></person-group><article-title>GDNF family receptor alpha1 phenotype of spermatogonial stem cells in immature mouse testes</article-title><source>Biol. Reprod.</source><year>2005</year><volume>73</volume><fpage>1011</fpage><lpage>1016</lpage><pub-id pub-id-type=\"pmid\">16014811</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chan</surname><given-names>F</given-names></name><etal/></person-group><article-title>Functional and molecular features of the Id4+ germline stem cell population in mouse testes</article-title><source>Genes Dev.</source><year>2014</year><volume>28</volume><fpage>1351</fpage><lpage>1362</lpage><pub-id pub-id-type=\"pmid\">24939937</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Costoya</surname><given-names>JA</given-names></name><etal/></person-group><article-title>Essential role of Plzf in maintenance of spermatogonial stem cells</article-title><source>Nat. Genet.</source><year>2004</year><volume>36</volume><fpage>653</fpage><lpage>659</lpage><pub-id pub-id-type=\"pmid\">15156143</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kaucher</surname><given-names>AV</given-names></name><name><surname>Oatley</surname><given-names>MJ</given-names></name><name><surname>Oatley</surname><given-names>JM</given-names></name></person-group><article-title>NEUROG3 is a critical downstream effector for STAT3-regulated differentiation of mammalian stem and progenitor spermatogonia</article-title><source>Biol. Reprod.</source><year>2012</year><volume>86</volume><fpage>1</fpage><lpage>11</lpage></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oatley</surname><given-names>MJ</given-names></name><name><surname>Kaucher</surname><given-names>AV</given-names></name><name><surname>Racicot</surname><given-names>KE</given-names></name><name><surname>Oatley</surname><given-names>JM</given-names></name></person-group><article-title>Inhibitor of DNA binding 4 is expressed selectively by single spermatogonia in the male germline and regulates the self-renewal of spermatogonial stem cells in mice</article-title><source>Biol. Reprod.</source><year>2011</year><volume>85</volume><fpage>347</fpage><lpage>356</lpage><pub-id pub-id-type=\"pmid\">21543770</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ikami</surname><given-names>K</given-names></name><etal/></person-group><article-title>Hierarchical differentiation competence in response to retinoic acid ensures stem cell maintenance during mouse spermatogenesis</article-title><source>Development</source><year>2015</year><volume>142</volume><fpage>1582</fpage><lpage>1592</lpage><pub-id pub-id-type=\"pmid\">25858458</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Masaki</surname><given-names>K</given-names></name><etal/></person-group><article-title>FGF2 has distinct molecular functions from GDNF in the mouse germline niche</article-title><source>Stem Cell Rep.</source><year>2018</year><volume>10</volume><fpage>1782</fpage><lpage>1792</lpage></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kubota</surname><given-names>H</given-names></name><name><surname>Avarbock</surname><given-names>MR</given-names></name><name><surname>Brinster</surname><given-names>RL</given-names></name></person-group><article-title>Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells</article-title><source>Proc. Natl Acad. Sci. USA</source><year>2004</year><volume>101</volume><fpage>16489</fpage><lpage>16494</lpage><pub-id pub-id-type=\"pmid\">15520394</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Meng</surname><given-names>X</given-names></name><etal/></person-group><article-title>Regulation of cell fate decision of undifferentiated spermatogonia by GDNF</article-title><source>Science</source><year>2000</year><volume>287</volume><fpage>1489</fpage><lpage>1493</lpage><pub-id pub-id-type=\"pmid\">10688798</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Anderson</surname><given-names>EL</given-names></name><etal/></person-group><article-title>Stra8 and its inducer, retinoic acid, regulate meiotic initiation in both spermatogenesis and oogenesis in mice</article-title><source>Proc. Natl Acad. Sci. USA</source><year>2008</year><volume>105</volume><fpage>14976</fpage><lpage>14980</lpage><pub-id pub-id-type=\"pmid\">18799751</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bowles</surname><given-names>J</given-names></name><etal/></person-group><article-title>Retinoid signaling determines germ cell fate in mice</article-title><source>Science</source><year>2006</year><volume>312</volume><fpage>596</fpage><lpage>600</lpage><pub-id pub-id-type=\"pmid\">16574820</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oatley</surname><given-names>JM</given-names></name><name><surname>Avarbock</surname><given-names>MR</given-names></name><name><surname>Telaranta</surname><given-names>AI</given-names></name><name><surname>Fearon</surname><given-names>DT</given-names></name><name><surname>Brinster</surname><given-names>RL</given-names></name></person-group><article-title>Identifying genes important for spermatogonial stem cell self-renewal and survival</article-title><source>Proc. Natl Acad. Sci. USA</source><year>2006</year><volume>103</volume><fpage>9524</fpage><lpage>9529</lpage><pub-id pub-id-type=\"pmid\">16740658</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>F</given-names></name><etal/></person-group><article-title>Mouse SYCP2 is required for synaptonemal complex assembly and chromosomal synapsis during male meiosis</article-title><source>J. Cell Biol.</source><year>2006</year><volume>173</volume><fpage>497</fpage><lpage>507</lpage><pub-id pub-id-type=\"pmid\">16717126</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yuan</surname><given-names>L</given-names></name><etal/></person-group><article-title>The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility</article-title><source>Mol. Cell.</source><year>2000</year><volume>5</volume><fpage>73</fpage><lpage>83</lpage><pub-id pub-id-type=\"pmid\">10678170</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brinster</surname><given-names>RL</given-names></name><name><surname>Avarbock</surname><given-names>MR</given-names></name></person-group><article-title>Germline transmission of donor haplotype following spermatogonial transplantation</article-title><source>Proc. Natl Acad. Sci. USA</source><year>1994</year><volume>91</volume><fpage>11303</fpage><lpage>11307</lpage><pub-id pub-id-type=\"pmid\">7972054</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ogawa</surname><given-names>T</given-names></name><name><surname>Dobrinski</surname><given-names>I</given-names></name><name><surname>Avarbock</surname><given-names>MR</given-names></name><name><surname>Brinster</surname><given-names>RL</given-names></name></person-group><article-title>Transplantation of male germ line stem cells restores fertility in infertile mice</article-title><source>Nat. Med.</source><year>2000</year><volume>6</volume><fpage>29</fpage><lpage>34</lpage><pub-id pub-id-type=\"pmid\">10613820</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ohta</surname><given-names>H</given-names></name><name><surname>Tohda</surname><given-names>A</given-names></name><name><surname>Nishimune</surname><given-names>Y</given-names></name></person-group><article-title>Proliferation and differentiation of spermatogonial stem cells in the w/wv mutant mouse testis</article-title><source>Biol. Reprod.</source><year>2003</year><volume>69</volume><fpage>1815</fpage><lpage>1821</lpage><pub-id pub-id-type=\"pmid\">12890724</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ryall</surname><given-names>JG</given-names></name><name><surname>Cliff</surname><given-names>T</given-names></name><name><surname>Dalton</surname><given-names>S</given-names></name><name><surname>Sartorelli</surname><given-names>V</given-names></name></person-group><article-title>Metabolic reprogramming of stem cell epigenetics</article-title><source>Cell. Stem Cell.</source><year>2015</year><volume>17</volume><fpage>651</fpage><lpage>662</lpage><pub-id pub-id-type=\"pmid\">26637942</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>J</given-names></name><name><surname>Ocampo</surname><given-names>A</given-names></name><name><surname>Belmonte</surname><given-names>JCI</given-names></name></person-group><article-title>Cellular metabolism and induced pluripotency</article-title><source>Cell</source><year>2016</year><volume>166</volume><fpage>1371</fpage><lpage>1385</lpage><pub-id pub-id-type=\"pmid\">27610564</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Folmes</surname><given-names>CD</given-names></name><etal/></person-group><article-title>Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming</article-title><source>Cell Metab.</source><year>2011</year><volume>14</volume><fpage>264</fpage><lpage>271</lpage><pub-id pub-id-type=\"pmid\">21803296</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Nuebel</surname><given-names>E</given-names></name><name><surname>Daley</surname><given-names>GQ</given-names></name><name><surname>Koehler</surname><given-names>CM</given-names></name><name><surname>Teitell</surname><given-names>MA</given-names></name></person-group><article-title>Metabolic regulation in pluripotent stem cells during reprogramming and self-renewal</article-title><source>Cell. Stem Cell.</source><year>2012</year><volume>11</volume><fpage>589</fpage><lpage>595</lpage><pub-id pub-id-type=\"pmid\">23122286</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shyh-Chang</surname><given-names>N</given-names></name><name><surname>Daley</surname><given-names>GQ</given-names></name><name><surname>Cantley</surname><given-names>LC</given-names></name></person-group><article-title>Stem cell metabolism in tissue development and aging</article-title><source>Development</source><year>2013</year><volume>140</volume><fpage>2535</fpage><lpage>2547</lpage><pub-id pub-id-type=\"pmid\">23715547</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>K</given-names></name><etal/></person-group><article-title>Redox homeostasis: the linchpin in stem cell self-renewal and differentiation</article-title><source>Cell Death Dis.</source><year>2013</year><volume>4</volume><fpage>e537</fpage><pub-id pub-id-type=\"pmid\">23492768</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Green</surname><given-names>CD</given-names></name><etal/></person-group><article-title>A comprehensive roadmap of murine spermatogenesis defined by single-cell RNA-seq</article-title><source>Dev. Cell.</source><year>2018</year><volume>46</volume><fpage>651</fpage><lpage>667</lpage><pub-id pub-id-type=\"pmid\">30146481</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hermann</surname><given-names>BP</given-names></name><etal/></person-group><article-title>The mammalian spermatogenesis single-cell transcriptome, from spermatogonial stem cells to spermatids</article-title><source>Cell Rep.</source><year>2018</year><volume>25</volume><fpage>1650</fpage><lpage>1667</lpage><pub-id pub-id-type=\"pmid\">30404016</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kanatsu-Shinohara</surname><given-names>M</given-names></name><etal/></person-group><article-title>Myc/Mycn-mediated glycolysis enhances mouse spermatogonial stem cell self-renewal</article-title><source>Genes Dev.</source><year>2016</year><volume>30</volume><fpage>2637</fpage><lpage>2648</lpage><pub-id pub-id-type=\"pmid\">28007786</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cross</surname><given-names>BA</given-names></name><name><surname>Silver</surname><given-names>IA</given-names></name></person-group><article-title>Neurovascular control of oxygen tension in the testis and epididymis</article-title><source>J. Reprod. Fertil.</source><year>1962</year><volume>3</volume><fpage>377</fpage><lpage>395</lpage><pub-id pub-id-type=\"pmid\">13882374</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wenger</surname><given-names>RH</given-names></name><name><surname>Katschinski</surname><given-names>DM</given-names></name></person-group><article-title>The hypoxic testis and post-meiotic expression of PAS domain proteins</article-title><source>Semin Cell Dev. Biol.</source><year>2005</year><volume>16</volume><fpage>547</fpage><lpage>553</lpage><pub-id pub-id-type=\"pmid\">15936961</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morimoto</surname><given-names>H</given-names></name><etal/></person-group><article-title>ROS are required for mouse spermatogonial stem cell self-renewal</article-title><source>Cell. Stem Cell.</source><year>2013</year><volume>12</volume><fpage>774</fpage><lpage>786</lpage><pub-id pub-id-type=\"pmid\">23746981</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Helsel</surname><given-names>AR</given-names></name><name><surname>Oatley</surname><given-names>MJ</given-names></name><name><surname>Oatley</surname><given-names>JM</given-names></name></person-group><article-title>Glycolysis-optimized conditions enhance maintenance of regenerative integrity in mouse spermatogonial stem cells during long-term culture</article-title><source>Stem Cell Rep.</source><year>2017</year><volume>8</volume><fpage>1430</fpage><lpage>1441</lpage></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Holley</surname><given-names>RJ</given-names></name><etal/></person-group><article-title>Comparative quantification of the surfaceome of human multipotent mesenchymal progenitor cells</article-title><source>Stem Cell Rep.</source><year>2015</year><volume>4</volume><fpage>473</fpage><lpage>488</lpage></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhao</surname><given-names>Y</given-names></name><etal/></person-group><article-title>SoNar, a highly responsive NAD+/NADH Sensor, allows high-throughput metabolic screening of anti-tumor agents</article-title><source>Cell Metab.</source><year>2015</year><volume>21</volume><fpage>777</fpage><lpage>789</lpage><pub-id pub-id-type=\"pmid\">25955212</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hao</surname><given-names>X</given-names></name><etal/></person-group><article-title>Metabolic imaging reveals a unique preference of symmetric cell division and homing of leukemia-initiating cells in an endosteal niche</article-title><source>Cell Metab.</source><year>2019</year><volume>29</volume><fpage>950</fpage><lpage>965</lpage><pub-id pub-id-type=\"pmid\">30581117</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boussouar</surname><given-names>F</given-names></name><name><surname>Benahmed</surname><given-names>M</given-names></name></person-group><article-title>Lactate and energy metabolism in male germ cells</article-title><source>Trends Endocrinol. Metab.</source><year>2004</year><volume>15</volume><fpage>345</fpage><lpage>350</lpage><pub-id pub-id-type=\"pmid\">15350607</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Meistrich</surname><given-names>ML</given-names></name><name><surname>Trostle</surname><given-names>PK</given-names></name><name><surname>Frapart</surname><given-names>M</given-names></name><name><surname>Erickson</surname><given-names>RP</given-names></name></person-group><article-title>Biosynthesis and localization of lactate dehydrogenase X in pachytene spermatocytes and spermatids of mouse testes</article-title><source>Dev. Biol.</source><year>1977</year><volume>60</volume><fpage>428</fpage><lpage>441</lpage><pub-id pub-id-type=\"pmid\">924020</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>W</given-names></name><etal/></person-group><article-title>MFN2 plays a distinct role from MFN1 in regulating spermatogonial differentiation</article-title><source>Stem Cell Rep.</source><year>2020</year><volume>14</volume><fpage>803</fpage><lpage>817</lpage></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rothschild</surname><given-names>G</given-names></name><etal/></person-group><article-title>A role for kit receptor signaling in Leydig cell steroidogenesis</article-title><source>Biol. Reprod.</source><year>2003</year><volume>69</volume><fpage>925</fpage><lpage>932</lpage><pub-id pub-id-type=\"pmid\">12773427</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Song</surname><given-names>HW</given-names></name><name><surname>Wilkinson</surname><given-names>MF</given-names></name></person-group><article-title>Transcriptional control of spermatogonial maintenance and differentiation</article-title><source>Semin Cell Dev. Biol.</source><year>2014</year><volume>30</volume><fpage>14</fpage><lpage>26</lpage><pub-id pub-id-type=\"pmid\">24560784</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kolobova</surname><given-names>E</given-names></name><name><surname>Tuganova</surname><given-names>A</given-names></name><name><surname>Boulatnikov</surname><given-names>I</given-names></name><name><surname>Popov</surname><given-names>KM</given-names></name></person-group><article-title>Regulation of pyruvate dehydrogenase activity through phosphorylation at multiple sites</article-title><source>Biochem J.</source><year>2001</year><volume>358</volume><fpage>69</fpage><lpage>77</lpage><pub-id pub-id-type=\"pmid\">11485553</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bigarella</surname><given-names>CL</given-names></name><name><surname>Liang</surname><given-names>R</given-names></name><name><surname>Ghaffari</surname><given-names>S</given-names></name></person-group><article-title>Stem cells and the impact of ROS signaling</article-title><source>Development</source><year>2014</year><volume>141</volume><fpage>4206</fpage><lpage>4218</lpage><pub-id pub-id-type=\"pmid\">25371358</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hannenhalli</surname><given-names>S</given-names></name><name><surname>Kaestner</surname><given-names>KH</given-names></name></person-group><article-title>The evolution of Fox genes and their role in development and disease</article-title><source>Nat. Rev. Genet.</source><year>2009</year><volume>10</volume><fpage>233</fpage><lpage>240</lpage><pub-id pub-id-type=\"pmid\">19274050</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liang</surname><given-names>R</given-names></name><name><surname>Ghaffari</surname><given-names>S</given-names></name></person-group><article-title>Stem cells seen through the FOXO lens: an evolving paradigm</article-title><source>Curr. Top. Dev. Biol.</source><year>2018</year><volume>127</volume><fpage>23</fpage><lpage>47</lpage><pub-id pub-id-type=\"pmid\">29433739</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sukonina</surname><given-names>V</given-names></name><etal/></person-group><article-title>FOXK1 and FOXK2 regulate aerobic glycolysis</article-title><source>Nature</source><year>2019</year><volume>566</volume><fpage>279</fpage><lpage>283</lpage><pub-id pub-id-type=\"pmid\">30700909</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Garbuzov</surname><given-names>A</given-names></name><etal/></person-group><article-title>Purification of GFRalpha1+ and GFRalpha1- spermatogonial stem cells reveals a niche-dependent mechanism for fate determination</article-title><source>Stem Cell Rep.</source><year>2018</year><volume>10</volume><fpage>553</fpage><lpage>567</lpage></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hammoud</surname><given-names>SS</given-names></name><etal/></person-group><article-title>Transcription and imprinting dynamics in developing postnatal male germline stem cells</article-title><source>Genes Dev.</source><year>2015</year><volume>29</volume><fpage>2312</fpage><lpage>2324</lpage><pub-id pub-id-type=\"pmid\">26545815</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>J</given-names></name><etal/></person-group><article-title>GASZ and mitofusin-mediated mitochondrial functions are crucial for spermatogenesis</article-title><source>EMBO Rep.</source><year>2016</year><volume>17</volume><fpage>220</fpage><lpage>234</lpage><pub-id pub-id-type=\"pmid\">26711429</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>J</given-names></name><etal/></person-group><article-title>NRF1 coordinates with DNA methylation to regulate spermatogenesis</article-title><source>FASEB J.</source><year>2017</year><volume>31</volume><fpage>4959</fpage><lpage>4970</lpage><pub-id pub-id-type=\"pmid\">28754714</pub-id></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liao</surname><given-names>Y</given-names></name><name><surname>Smyth</surname><given-names>GK</given-names></name><name><surname>Shi</surname><given-names>W</given-names></name></person-group><article-title>The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote</article-title><source>Nucleic Acids Res.</source><year>2013</year><volume>41</volume><fpage>e108</fpage><pub-id pub-id-type=\"pmid\">23558742</pub-id></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liao</surname><given-names>Y</given-names></name><name><surname>Smyth</surname><given-names>GK</given-names></name><name><surname>Shi</surname><given-names>W</given-names></name></person-group><article-title>featureCounts: an efficient general purpose program for assigning sequence reads to genomic features</article-title><source>Bioinformatics</source><year>2014</year><volume>30</volume><fpage>923</fpage><lpage>930</lpage><pub-id pub-id-type=\"pmid\">24227677</pub-id></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Love</surname><given-names>MI</given-names></name><name><surname>Huber</surname><given-names>W</given-names></name><name><surname>Anders</surname><given-names>S</given-names></name></person-group><article-title>Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2</article-title><source>Genome Biol.</source><year>2014</year><volume>15</volume><fpage>550</fpage><pub-id pub-id-type=\"pmid\">25516281</pub-id></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xie</surname><given-names>C</given-names></name><etal/></person-group><article-title>KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases</article-title><source>Nucleic Acids Res.</source><year>2011</year><volume>39</volume><fpage>W316</fpage><lpage>W322</lpage><pub-id pub-id-type=\"pmid\">21715386</pub-id></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hughes</surname><given-names>CS</given-names></name><etal/></person-group><article-title>Ultrasensitive proteome analysis using paramagnetic bead technology</article-title><source>Mol. Syst. Biol.</source><year>2014</year><volume>10</volume><fpage>757</fpage><pub-id pub-id-type=\"pmid\">25358341</pub-id></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cox</surname><given-names>J</given-names></name><name><surname>Mann</surname><given-names>M</given-names></name></person-group><article-title>MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification</article-title><source>Nat. Biotechnol.</source><year>2008</year><volume>26</volume><fpage>1367</fpage><lpage>1372</lpage><pub-id pub-id-type=\"pmid\">19029910</pub-id></element-citation></ref><ref id=\"CR63\"><label>63.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang da</surname><given-names>W</given-names></name><name><surname>Sherman</surname><given-names>BT</given-names></name><name><surname>Lempicki</surname><given-names>RA</given-names></name></person-group><article-title>Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources</article-title><source>Nat. Protoc.</source><year>2009</year><volume>4</volume><fpage>44</fpage><lpage>57</lpage><pub-id pub-id-type=\"pmid\">19131956</pub-id></element-citation></ref><ref id=\"CR64\"><label>64.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>X</given-names></name><etal/></person-group><article-title>Transcriptional repression by theS BRG1-SWI/SNF complex affects the pluripotency of human embryonic stem cells</article-title><source>Stem Cell Rep.</source><year>2014</year><volume>3</volume><fpage>460</fpage><lpage>474</lpage></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807904</article-id><article-id pub-id-type=\"pmc\">PMC7431568</article-id><article-id pub-id-type=\"publisher-id\">70898</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70898-y</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Early overnutrition sensitizes the growth hormone axis to the impact of diet-induced obesity via sex-divergent mechanisms</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Sanchez-Garrido</surname><given-names>M. A.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ruiz-Pino</surname><given-names>F.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Pozo-Salas</surname><given-names>A. I.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Castellano</surname><given-names>J. M.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Vazquez</surname><given-names>M. J.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Luque</surname><given-names>R. M.</given-names></name><address><email>bc2luhur@uco.es</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Tena-Sempere</surname><given-names>M.</given-names></name><address><email>fi1tesem@uco.es</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.428865.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0445 6160</institution-id><institution>Maimónides Institute of Biomedical Research of Córdoba (IMIBIC), </institution></institution-wrap>Córdoba, Spain </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.411901.c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2183 9102</institution-id><institution>Department of Cell Biology, Physiology and Immunology, Faculty of Medicine, </institution><institution>University of Córdoba, </institution></institution-wrap>Avda. Menéndez Pidal s/n., 14004 Córdoba, Spain </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.411349.a</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1771 4667</institution-id><institution>Reina Sofia University Hospital, </institution></institution-wrap>Córdoba, Spain </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.413448.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9314 1427</institution-id><institution>CIBER Fisiopatología de La Obesidad y Nutrición (CIBEROBN), </institution><institution>Instituto de Salud Carlos III, </institution></institution-wrap>14004 Córdoba, Spain </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.1374.1</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2097 1371</institution-id><institution>Institute of Biomedicine, </institution><institution>University of Turku, </institution></institution-wrap>20520 Turku, Finland </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13898</elocation-id><history><date date-type=\"received\"><day>2</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>13</day><month>5</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">In addition to its essential role in the physiological control of longitudinal growth, growth-hormone (GH) is endowed with relevant metabolic functions, including anabolic actions in muscle, lipolysis in adipose-tissue and glycemic modulation. Adult obesity is known to negatively impact GH-axis, thereby promoting a vicious circle that may contribute to the exacerbation of the metabolic complications of overweight. Yet, to what extent early-overnutrition sensitizes the somatotropic-axis to the deleterious effects of obesity remains largely unexplored. Using a rat-model of sequential exposure to obesogenic insults, namely postnatal-overfeeding during lactation and high-fat diet (HFD) after weaning, we evaluated in both sexes the individual and combined impact of these nutritional challenges upon key elements of the somatotropic-axis. While feeding HFD per se had a modest impact on the adult GH-axis, early overnutrition had durable effects on key elements of the somatotropic-system, which were sexually different, with a significant inhibition of pituitary gene expression of GH-releasing hormone-receptor (GHRH-R) and somatostatin receptor-5 (SST5) in males, but an increase in pituitary GHRH-R, SST2, SST5, GH secretagogue-receptor (GHS-R) and ghrelin expression in females. Notably, early-overnutrition sensitized the GH-axis to the deleterious impact of HFD, with a significant suppression of pituitary GH expression in both sexes and lowering of circulating GH levels in females. Yet, despite their similar metabolic perturbations, males and females displayed rather distinct alterations of key somatotropic-regulators/ mediators. Our data document a synergistic effect of postnatal-overnutrition on the detrimental impact of HFD-induced obesity on key elements of the adult GH-axis, which is conducted via mechanisms that are sexually-divergent.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Physiology</kwd><kwd>Endocrinology</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/501100000780</institution-id><institution>European Commission</institution></institution-wrap></funding-source><award-id>REP-655232 (ReprObesity)</award-id></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/501100004587</institution-id><institution>Instituto de Salud Carlos III</institution></institution-wrap></funding-source><award-id>PIE-00005</award-id></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/501100003329</institution-id><institution>Ministerio de Economía y Competitividad</institution></institution-wrap></funding-source><award-id>BFU2014-57581-P</award-id><principal-award-recipient><name><surname>Tena-Sempere</surname><given-names>M.</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Growth hormone (GH) plays a pivotal role in longitudinal growth during development<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. In addition, GH also exerts important actions on metabolic processes by promoting anabolic effects in the muscle and catabolic actions in the adipose tissue<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. It is well established that adult GH deficiency causes alterations in body composition and lipid metabolism<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>–<xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. Adult GH deficiency is usually accompanied by increased abdominal fat accumulation, which is attenuated after GH replacement<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>–<xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. It is also known that abdominal obesity negatively affects GH secretion. This effect has been documented in obese humans and rodents, where spontaneous, as well as stimulated, GH secretion is markedly reduced<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>–<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. Due to the lipolytic effect of GH on the adipose tissue, it has been suggested that such obesity-related disruption of GH secretion may promote a vicious cycle that might enhance adiposity and exacerbate the metabolic comorbidities linked to obesity<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. Interestingly, weight loss in obese subjects reverses the decline in GH levels and improves stimulated GH output<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>, suggesting that the metabolic disturbances associated to weight gain may be responsible for the blunted basal and stimulated GH secretion in obesity.</p><p id=\"Par3\">The underlying mechanisms for the reduction in circulating GH levels in obesity are complex and not fully understood. An array of metabolic hormones and signals has been shown to alter GH output by acting at hypothalamic and/or pituitary levels. Several studies have provided evidence of the detrimental impact of increased circulating levels of insulin-like growth factor-1 (IGF-1), glucose, insulin, free fatty acids (FFA) and other metabolic factors on the GH axis<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Of note, plasma levels of all these metabolic factors are commonly elevated in obesity and, therefore, may ultimately contribute to obesity-associated GH deficiency.</p><p id=\"Par4\">Over the last decades, compelling evidence has suggested that early perturbations of the nutritional environment (e.g., during the perinatal period) might permanently influence metabolic health and other physiological parameters in adulthood<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Epidemiological and experimental studies have revealed a strong association between early nutritional alterations and the incidence of obesity and its related comorbidities later in life<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref>–<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. These findings suggest that the adaptive changes that occur during early stages of development may have a durable impact on key metabolic pathways that may predispose to metabolic abnormalities in subsequent stages of development. In addition, it has been suggested that early overfeeding may sensitize for the deleterious effects of a high-fat diet (HFD) in adulthood. In line with this hypothesis, our group and others have recently reported that early postnatal overnutrition exacerbates HFD-induced metabolic disturbances in adult life<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>–<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. Interestingly, recent studies have also documented that early overnutrition may compromise the correct functioning of various neurohormonal axes in adulthood<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref>,<xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, including the GH axis<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. In this regard, it has been shown that early overnutrition increases somatic growth during development by altering hypothalamic GH-releasing hormone (GHRH) and pituitary GH expression in mice, changes that persisted in adult life<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. However, whether early overfeeding may sensitize the GH axis for the deleterious effects of HFD in adulthood remains to date unexplored. Yet, such possibility is of considerable translational interest, given the escalating trends of child obesity<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>.</p><p id=\"Par5\">In the above context, the present study aimed at characterizing the potential impact of early postnatal overnutrition on the susceptibility of the GH axis to the impact of HFD-induced obesity in adult life, by means of the analysis of key hypothalamic, pituitary and liver components of the somatotropic axis, as well as relevant circulating hormones, in male and female rats subjected to these two obesogenic insults, with special attention being paid to the identification of potential sex-dependent differences in the pathophysiological mechanisms underlying the eventual alterations of the GH axis in such conditions.</p></sec><sec id=\"Sec2\"><title>Results</title><p id=\"Par6\">Analyses of the elements of the somatotropic axis, including circulating levels of GH and IGF-1, as well as the gene expression levels of key factors at the hypothalamus [GHRH, somatostatin (SRIF), Neuropeptide-Y (NPY) and ghrelin], pituitary [GH, somatostatin receptors (SSTs), Leptin-receptor (Lep-R), Insulin-receptor (Ins-R), ghrelin, ghrelin-receptor (GHS-R), and Ghrelin-O-Acyl Transferase (GOAT)] and liver [GH-receptor (GH-R) and IGF-1], were implemented in serum and tissue samples from cohorts of male and female rats, generated in the context of large longitudinal studies addressing the impact of sequential obesogenic insults, namely postnatal overfeeding (SL) and exposure to HFD after weaning, on various reproductive and metabolic parameters. While metabolic characterization of these models was partially reported in previous publications of our group<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, key metabolic parameters are summarized also here and presented in Supplemental Figure <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>, for reference purposes.</p><sec id=\"Sec3\"><title>Metabolic profiles of adult male and female rats after early overnutrition or/and HFD</title><p id=\"Par7\">Postnatal overfeeding (SL) resulted in an increase in delta body weight (i.e., absolute BW gain) between PND 23 and 120 in both males and females (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). However, representation of delta body weight curves during this period revealed that males were more sensitive to the body weight gain effect of SL than females, with an increase being detectable in SL/CD males versus NL/CD from PND 50 onwards (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). Despite this obesogenic effect, SL per se did not significantly affect circulating leptin or insulin levels in either sex (NL/CD vs. SL/CD; Suppl. Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>). However, SL males, but not females, exhibited elevated basal glucose levels as well as increased body length (NL/CD vs. SL/CD; Suppl. Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">S4</xref>). After 12 weeks of exposure to HFD, NL males and females (NL/HFD) were heavier than their respective NL/CD controls, with enhanced delta body weight and increased daily energy intake (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> and Suppl. Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>). In addition, NL/HFD rats displayed elevated serum leptin levels, although such an increase reached statistical significance only in males (Suppl. Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>). In contrast, basal insulin levels were oppositely affected in NL/HFD males (significant decrease) versus females (moderate, but not statistically significant increase; Suppl. Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>). NL/HFD males also showed increased circulating glucose levels and body size as compared to control (NL/CD) males, while no significant differences in basal glucose levels or body length were detected between NL/CD and NL/HFD females (Suppl. Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">S4</xref>). Finally, the combination of both obesogenic factors (SL/HFD) caused the highest increase in body weight as well as basal leptin and glucose levels in both sexes (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> and Suppl. Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>), while further enhancement of body length by SL/HFD was only observed in males (Suppl. Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S4</xref>). Moreover, no significant differences were detected in circulating insulin levels in SL/HFD male and female rats versus their corresponding controls (Suppl. Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Cumulative delta body weight (BW) curves between PND 23 and 120 (<bold>A</bold>; the insets represent the absolute BW gain between PND 23 and 120), circulating GH levels (<bold>B</bold>) and pituitary GH mRNA levels (<bold>C</bold>) from male (<italic>left panels</italic>) and female rats (<italic>right panels</italic>) subjected or not to overfeeding during lactation (SL vs. NL) and/or HFD after weaning (HFD vs. CD). mRNA copy numbers were determined by qPCR and adjusted by a Normalization Factor (NF) in each sample obtained from the expression levels of three housekeeping genes (<italic>β-actin</italic>, <italic>Hprt</italic> and C<italic>yclophilin A</italic>). Data are presented as mean ± standard error of the mean (SEM), n = 8 per experimental group. Statistically significant differences were assessed by two-way ANOVA to analyze the effects of litter size and diet and their interactions. When significant differences were found, the data were further analyzed using Newman-Keuls tests to identify simple effects. *<italic>P</italic> < .05; **<italic>P</italic> < .01 effect of HFD; a, <italic>P</italic> < .05, effect of litter size; b, <italic>P</italic> < .05 interaction of litter size/HFD.</p></caption><graphic xlink:href=\"41598_2020_70898_Fig1_HTML\" id=\"MO1\"/></fig></p></sec><sec id=\"Sec4\"><title>Impact of early overnutrition on the GH axis in adult male and female rats</title><p id=\"Par8\">Early overnutrition (SL) per se did not cause significant alterations in circulating GH levels compared with NL/CD controls in both sexes, with GH levels being consistently higher in male than in female groups, in line with previous references<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref>,<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. Accordingly, no changes were detected in pituitary GH mRNA levels between SL/CD and NL/CD male and female rats (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). In good agreement, circulating IGF-1 levels (also higher in males than in females), as well as hepatic expression of IGF-1 and GH-R levels in SL/CD rats did not differ from those of the control NL/CD group in both sexes (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). Yet, SL/CD males displayed a significant reduction in pituitary GHRH-R and SST5 mRNA levels compared to NL/CD controls (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>). In contrast, pituitary ghrelin, GHS-R and SST2 expression in SL/CD males were not significantly altered (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>). In contrast, SL/CD females displayed a significant increase in pituitary GHRH-R, GHS-R, SST2, SST5 and ghrelin mRNA levels versus control NL/CD rats (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>). In addition, early overfeeding did not alter pituitary leptin and insulin receptor expression in either sex, neither it affected the expression levels of Pit-1, SST1, SST3 or GOAT mRNAs (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> & Suppl. Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">S4</xref>), while SST4 expression was undetectable at the pituitary (<italic>data not shown</italic>). Interestingly, at the hypothalamic level, SL/CD males showed a significant reduction in SRIF and NPY expression versus NL/CD controls, which was not detected in females (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>). Hypothalamic GHRH mRNA levels were not altered by SL in both sexes, whereas hypothalamic ghrelin expression was significantly reduced by SL only in females (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>).<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Circulating IGF-1 levels (<bold>A</bold>) and liver IGF-1 and GH-R mRNA levels (<bold>B</bold>) from male (<italic>left panels</italic>) and female rats (<italic>right panels</italic>) subjected or not to overfeeding during lactation (SL vs. NL) and/or HFD after weaning (HFD vs. CD). mRNA copy numbers were determined by qPCR and adjusted by a Normalization Factor (NF) in each sample obtained from the expression levels of three housekeeping genes (<italic>β-actin</italic>, <italic>Hprt</italic> and C<italic>yclophilin A</italic>). Data are presented as mean ± standard error of the mean (SEM), n = 8 per experimental group. Statistically significant differences were assessed by two-way ANOVA to analyze the effects of litter size and diet and their interactions. When significant differences were found, data were further analyzed using Newman-Keuls tests to identify simple effects. *<italic>P</italic> < .05 effect of HFD.</p></caption><graphic xlink:href=\"41598_2020_70898_Fig2_HTML\" id=\"MO2\"/></fig><fig id=\"Fig3\"><label>Figure 3</label><caption><p>Pituitary GHRH-R, GHS-R, SST2, SST5, ghrelin and Pit-1 mRNA levels in male (<italic>left panels</italic>) and female rats (<italic>right panels</italic>) subjected or not to overfeeding during lactation (SL vs. NL) and/or HFD after weaning (HFD vs. CD). mRNA copy numbers were determined by qPCR and adjusted by a Normalization Factor (NF) in each sample obtained from the expression levels of three housekeeping genes (<italic>β-actin</italic>, <italic>Hprt</italic> and C<italic>yclophilin A</italic>). Data are presented as mean ± standard error of the mean (SEM), n = 8 per experimental group. Statistically significant differences were assessed by two-way ANOVA to analyze the effects of litter size and diet and their interactions. When significant differences were found, the data were further analyzed using Newman-Keuls tests to identify simple effects. *<italic>P</italic> < .05; **<italic>P</italic> < .01 effect of HFD; a, <italic>P</italic> < .05, effect of litter size; b, <italic>P</italic> < .05 interaction of litter size/HFD.</p></caption><graphic xlink:href=\"41598_2020_70898_Fig3_HTML\" id=\"MO3\"/></fig><fig id=\"Fig4\"><label>Figure 4</label><caption><p>Hypothalamic GHRH, SRIF, NPY and ghrelin mRNA levels in male (<italic>left panels</italic>) and female rats (<italic>right panels</italic>) subjected or not to overfeeding during lactation (SL vs. NL) and/or HFD after weaning (HFD vs. CD). mRNA copy numbers were determined by qPCR and adjusted by a Normalization Factor (NF) in each sample obtained from the expression levels of three housekeeping genes (<italic>β-actin</italic>, <italic>Hprt</italic> and C<italic>yclophilin A</italic>). Data are presented as mean ± standard error of the mean, n = 5–6 per experimental group. Statistically significant differences were assessed by two-way ANOVA to analyze the effects of litter size and diet and their interactions. When significant differences were found, the data were further analyzed using Newman–Keuls tests to identify simple effects. *<italic>P</italic> < .05 effect of HFD; a, <italic>P</italic> < .05 effect of litter size.</p></caption><graphic xlink:href=\"41598_2020_70898_Fig4_HTML\" id=\"MO4\"/></fig></p></sec><sec id=\"Sec5\"><title>Effect of HFD on the GH axis in adult male and female rats</title><p id=\"Par9\">At PND-120, male rats fed on HFD showed a trend for decreased circulating GH levels (<italic>P</italic> = 0.09; NL/CD controls vs. NL/HFD), without significant changes being detected on pituitary expression of GH (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). In females, HFD alone did not alter serum GH levels or pituitary GH expression (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). Similarly, no significant changes were detected in serum IGF-1 levels and hepatic IGF-1 and GH-R expression in NL/HFD males and females vs NL/CD controls (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). In males, HFD did not significantly affect pituitary expression of GHRH-R, SST2, SST5 and ghrelin, but caused a non-significant increase in GHS-R mRNA levels (<italic>P</italic> = 0.07, Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>). Likewise, NL/HFD females did not display significant alterations in pituitary GHRH-R, GHS-R, SST5 and ghrelin mRNA levels, but HFD caused a significant increase in pituitary SST2 expression (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>). Similar to SL, HFD did not alter the pituitary expression of the genes encoding leptin or insulin receptors in males or females, nor did it modify pituitary Pit-1, SST1, SST3 or GOAT mRNA levels (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> & Suppl. Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">S4</xref>), whereas SST4 mRNA expression was below the limit of detection at the pituitary in any of the experimental groups (<italic>data not shown</italic>). At the hypothalamic level, HFD failed to alter GHRH expression in either males or females (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>). In the same line, hypothalamic SRIF, NPY and ghrelin mRNA levels did not significantly differ between NL/CD and NL/HFD groups of both sexes (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>).</p></sec><sec id=\"Sec6\"><title>Combined effect of early overnutrition and HFD on the GH axis in adult rats</title><p id=\"Par10\">The combination of both obesogenic factors, SL and HFD, caused a significant reduction in pituitary GH mRNA levels compared to their respective SL/CD controls in both males and females (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). Such a reduction was accompanied by a non-significant decrease (> 50%) in circulating GH levels in females, whereas basal GH levels were not overtly affected by the combination of both factors in males (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). Despite these alterations in pituitary GH expression and serum GH levels, no changes in liver IGF-1 expression or circulating IGF-1 levels were detected between SL/CD and SL/HFD rats in both sexes (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). Yet, SL/HFD males showed a significant suppression of liver GH-R mRNA levels versus the control (SL/CD) group; an effect that was not detected in females (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). Analyses of pituitary components of the GH axis in males revealed that the combination of both obesogenic insults significantly increased pituitary mRNA levels of GHRH-R and GHS-R as compared to SL/CD controls (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>). In addition, SL/HFD males showed a pronounced and significant reduction in pituitary SST2 mRNA levels, the major receptor that drives the inhibitory effects of SRIF on pituitary GH expression and release (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>). SL/HFD males also displayed increased pituitary expression of leptin and insulin receptors (Suppl. Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>). In contrast, SL/HFD females did not display alterations in pituitary GHRH-R, GHS-R, SST2, leptin or insulin receptor mRNA levels versus the corresponding SL controls (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> & Suppl. Figure <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>). However, the combination of postnatal overnutrition and HFD in females caused a significant decrease in pituitary ghrelin expression, which was not detected in males (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>). In contrast, in SL/HFD male and female rats, no significant changes were detected in pituitary Pit-1, SST1, SST3 and GOAT mRNA levels versus the control groups (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> & Suppl. Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">S4</xref>); SST4 expression was not detectable (<italic>data not shown</italic>). Of note, the combination of both nutritional insults significantly reduced hypothalamic GHRH expression only in males (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>). SL/HFD males and females did not show alterations in hypothalamic SRIF and ghrelin expression versus SL/CD controls (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>). However, SL/HFD females exhibited a significant increase in hypothalamic NPY mRNA levels (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>).</p></sec></sec><sec id=\"Sec7\"><title>Discussion</title><p id=\"Par11\">The prevalence of obesity and its related metabolic disorders, such as type 2 diabetes and cardiovascular disease, has increased dramatically during the last decades worldwide, representing an enormous health and economic burden. Evidence from numerous studies has documented that nutritional alterations during early stages of development may contribute to enhance the obesity rates and the occurrence of obesity-related comorbidities<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. Adverse metabolic conditions during the perinatal stage likely disrupt proper development of key neurohormonal systems due to the relative plasticity of some of their components during these early periods of development; phenomena that may have long-term consequences in energy homeostasis and modify the response to later metabolic challenges. In previous reports, we documented the impact of early postnatal overfeeding and HFD, alone or in combination, on metabolic and reproductive health in adult male and female rats<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, with increased sensitivity to the deleterious impact of HFD being observed in animals overfed during the perinatal stage; a phenomenon also shown by others<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref>–<xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. In the present study, we expand those studies by showing for the first time that perinatal overnutrition sensitizes also the GH axis for the detrimental effects of HFD-induced obesity; perturbations that may contribute to exacerbate the metabolic abnormalities linked to HFD consumption. Our study also documents striking sex-dependent differences in the regulatory mechanisms of the GH axis in obesity at the pituitary and hypothalamic level, which were associated with a differential impact of the combination of obesogenic insults on body length, which was overtly altered in SL/HFD males, but not in obese females.</p><p id=\"Par12\">It is widely accepted that there is a negative correlation between obesity and GH secretion. The deleterious impact of obesity on the somatotropic axis has been demonstrated in various species, from rodent to humans<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR41\">41</xref>–<xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. Of note, most of the studies addressing the detrimental impact of obesity on the GH axis have been conducted in males<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>–<xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>, while females remained scarcely studied. In our current study, HFD alone caused rather modest alterations in the somatotropic axis in both sexes. This is possibly due to the relatively moderate nature of our HFD insult (45% fat content, causing a 10% elevation of BW) and the pulsatile nature of GH secretion<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>, which makes difficult to detect significant changes in mean values obtained from single blood samples. In any event, pituitary GH mRNA levels were not altered in HFD-induced obese rats of either sex, in keeping with previous reports showing similar pituitary GH content in lean and obese rats<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref>,<xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>. Furthermore, no other element of the somatotropic axis analyzed in HFD rats at adulthood was overtly altered, except for an increase in pituitary SST2 levels in females, suggesting that the impact, if any, of the HFD challenge alone on the GH axis is rather modest in our experimental model. In contrast, despite the fact that their metabolic profiles in adulthood were not as severely affected as in the HFD group, SL rats subjected to early postnatal overnutrition displayed durable alterations of several elements of the somatotropic axis, which were remarkably sexually different. These findings illustrate (1) the sensitivity of the GH axis to early nutritional challenges, which may have long-lasting consequences in somatotropic and related functions; and (2) the clear sex differences in the impact of such early nutritional challenges on the GH axis.</p><p id=\"Par13\">In addition, early onset overnutrition markedly sensitized the elements of the GH axis to the deleterious impact of obesity, in spite of the mild-to-null impact of HFD alone on various somatotropic parameters. Thus, SL/HFD males displayed suppressed GH mRNA expression in the pituitary, together with reduced hypothalamic GHRH and liver GH-R expression, while in SL/HFD females, a significant lowering of circulating GH and pituitary GH mRNA expression was observed, which was associated to suppressed expression of ghrelin at the pituitary and increased expression of NPY at the hypothalamus; the later might also contribute to the increased daily energy intake of SL/HFD versus control female rats. As none of these changes were detected in either SL or HFD rats, our data document the synergistic interaction of both obesogenic insults in perturbing the GH axis. Admittedly, the greater perturbation of the somatotropic axis in SL/HFD rats may derive, at least partially, from the higher severity of their metabolic perturbations, as compared to rats exposed to a single obesogenic insult (SL or HFD). However, considering that both male and female SL/HFD rats showed roughly similar alterations in key metabolic parameters, such as body weight gain, increased basal glucose levels and hyper-leptinemia, the observed sex differences in deregulated somatotropic factors are unlikely to be merely caused by this higher metabolic deterioration in SL/HFD animals, but rather represent a genuine phenomenon that documents the sexually-divergent mechanisms underlying the impact of early onset obesity on the GH axis.</p><p id=\"Par14\">The differential changes in the patterns of expression detected between male and female SL/HFD rats are likely indicative of distinct pathophysiological mechanisms for the perturbation of the hypothalamic and pituitary signals that govern GH output. Thus, while in SL/HFD males the suppression of pituitary GH mRNA expression may be driven by the inhibition of the hypothalamic expression of GHRH, as major physiological stimulus of GH synthesis and secretion<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>, in females, defective GH mRNA expression and circulating levels were not linked to suppressed GHRH expression at the hypothalamus, but rather to decreased pituitary expression of ghrelin and increased hypothalamic NPY mRNA levels. Lower ghrelin expression might be mechanistically relevant, as the pituitary ghrelin system has been reported to be an important local modulator of GH synthesis and release<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref>–<xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>. In addition, NPY has been suggested to negatively influence GH secretion in different experimental models and conditions<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref>,<xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>. Supporting this potential inhibitory effect of NPY on GH release, exogenous administration of NPY into the third ventricle has been shown to reduce plasma GH levels in ovariectomized rats<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. Furthermore, in vitro studies have reported the negative effect of NPY on pituitary GH secretion<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>. Collectively, these findings suggest that the lowering of pituitary ghrelin and the rise in hypothalamic NPY expression detected in SL/HFD females might negatively affect pituitary GH expression.</p><p id=\"Par15\">In the same vein, other components of the somatotropic axis were affected by the combination of both obesogenic insults in a sexually divergent manner. Thus, SL/HFD males displayed increased expression of pituitary receptors for GH-stimulating signals, including GHRH-R and GHS-R, and decreased expression of SST2, the main inhibitory receptor subtype involved in the GH-inhibiting actions of SRIF. These pituitary changes may suggest a compensatory mechanism of the somatotropes in response to reduced GH levels in this experimental group. In contrast, the gene expression profiles of the receptors for GH-stimulating and -inhibiting signals were unaltered in SL/HFD females. In addition, while circulating IGF-1 and hepatic expression of IGF-1 gene, whose levels have been correlated with changes in circulating GH in various experimental models<sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>, were not altered in SL/HFD rats of either sex, liver expression of GH-R was significantly reduced in males, but not females exposed to both obesogenic insults. This would suggest an impaired hepatic sensitivity to GH particularly in males. Given the well-known actions of GH in the liver for proper glucose homeostasis<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>, the contribution of such alteration upon the systemic metabolic perturbations seen in obese male rats merits further investigation. Interestingly, hepatic insulin resistance has been previously proposed as an early metabolic defect in states of severe GH deficiency<sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup>.</p><p id=\"Par16\">Compelling evidence suggests that somatotropes may play a role as metabolic sensors of the organism<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref>–<xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup>. Various metabolic hormones, such as leptin and insulin, have been reported to regulate somatotrope function and, consequently, GH secretion, which in turn would influence body composition<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref>,<xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup>. Selective deletion of pituitary leptin receptors in mice has been associated with GH deficiency and development of obesity<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref>,<xref ref-type=\"bibr\" rid=\"CR62\">62</xref></sup>; such an effect was more pronounced in males<sup><xref ref-type=\"bibr\" rid=\"CR62\">62</xref></sup>. In our study, a clear sexual difference was found regarding the pituitary expression of leptin receptor mRNA, whose levels were significantly up-regulated only in SL/HFD males. Given the positive effects of leptin on pituitary GH, this might suggest the existence of a compensatory mechanism in response to decreased GH mRNA levels. On the other hand, pituitary insulin receptor expression was also enhanced in SL/HFD males, but not females. Insulin carries out inhibitory actions on pituitary GH synthesis and secretion<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup>. Although no significant differences in circulating insulin levels were detected between SL and SL/HFD males, such an increase in pituitary insulin receptor expression, and therefore in pituitary insulin sensitivity, might also contribute to the reduction in pituitary GH mRNA levels observed in SL/HFD males.</p><p id=\"Par17\">The question arising from our data is what are the potential mechanisms underlying the sexually different impact of early obesity on the somatotropic axis. While these were not directly addressed by our current study, it is tenable to speculate that epigenetic processes, taking place during early stages of development, might differentially affect gene expression of key elements of the GH axis in males and females, which may predispose to sex-specific responses to HFD at later developmental stages<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref>,<xref ref-type=\"bibr\" rid=\"CR29\">29</xref>–<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. In addition, sex-specific changes in gut microbiota composition induced by early onset obesity may also contribute, via epigenetic mechanisms, to disrupt proper adaptation to nutritional insults later in life<sup><xref ref-type=\"bibr\" rid=\"CR63\">63</xref></sup>, which may compromise the correct functioning of the somatotropic axis in the long-term. Finally, changes in sex steroids might also contribute to the observed sexually-divergent patterns. It is known that sex hormones can modulate GH output and actions, and alter GH signaling pathways in peripheral tissues<sup><xref ref-type=\"bibr\" rid=\"CR64\">64</xref></sup>; alterations in sex steroid levels and gonadal function are bound to obesity, with a differential impact between males and females<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. Hence, sex-specific obesity-related changes in gonadal steroid levels might alter also the gene expression of certain elements of the somatotropic axis, thus promoting sex differences.</p><p id=\"Par18\">In sum, our findings demonstrate that exposure to an obesogenic nutritional environment during early periods of development not only enhanced the metabolic perturbations caused by HFD, but also exacerbated the detrimental effects of HFD-induced obesity on the somatotropic axis in both sexes. Yet, our data also disclose clear discrepancies between males and females concerning the neurohormonal mechanisms that deregulate the GH axis in obesity conditions due to the combination of early overnutrition and HFD. Whereas in males the decline in pituitary GH expression is mostly attributed to decreased hypothalamic GHRH expression, in females, pituitary down-regulation of ghrelin expression along with increased hypothalamic expression of NPY may be key mechanisms responsible for the suppression of the GH axis (see Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>). Pending additional, confirmatory measurements of the actual changes of tissue protein levels and time-course analyses of putative alterations of the elements of the GH axis after exposure to obesogenic insults at various developmental windows, our present study, addressing mainly changes in hormonal and gene expression levels, highlights the importance of early nutritional programming as a contributing factor for the vulnerability of the GH axis to HFD, thus suggesting that the early nutritional environment may be an important modifier of the GH axis in adulthood in conditions of diet-induced obesity.<fig id=\"Fig5\"><label>Figure 5</label><caption><p>Graphical summary of the most salient alterations in key elements of the GH axis caused by the obesogenic insults, SL (rearing in small litters) and HFD (exposure to high fat content diet from weaning onwards), in male and female rats, with particular focus on the impact of combined exposure to these obesogenic insults and sexually-divergent perturbations. The figure was created by the authors, using free medical images obtained from Servier Medical Art (<ext-link ext-link-type=\"uri\" xlink:href=\"https://smart.servier.com\">https://smart.servier.com</ext-link>).</p></caption><graphic xlink:href=\"41598_2020_70898_Fig5_HTML\" id=\"MO5\"/></fig></p></sec><sec id=\"Sec8\"><title>Materials and methods</title><sec id=\"Sec9\"><title>Animals and diets</title><p id=\"Par19\">Wistar male and female rats bred in the vivarium of the University of Córdoba were used. Animals were maintained under constant conditions of light (14 h of light, from 07:00 h) and temperature (22 °C), with free access to pelleted food and tap water. Animals were fed a control diet [CD; D12450B: 10% of calories from fat, 20% from protein, and 70% from carbohydrate], or a high-fat diet [HFD; D12451: 45% of calories from fat, 20% from protein, and 35% from carbohydrate; Research Diets Inc.]. Experimental procedures were approved by Córdoba University Ethical Committee and conducted in accordance with European Union guidelines.</p></sec><sec id=\"Sec10\"><title>Experimental design</title><p id=\"Par20\">In order to explore whether early postnatal overnutrition may affect the somatotropic axis and alter its sensitivity to the deleterious effects of HFD in adult male and female rats, expression and hormonal analyses were implemented in serum and tissue samples from cohorts of male and female rats, generated in the context of large longitudinal studies addressing the impact of these sequential obesogenic insults on various reproductive and metabolic parameters<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. As model of early postnatal overnutrition, we used a well-established experimental protocol of litter size manipulation during the lactation period, which has been widely used by our numerous groups, including ours, to induce conditions of postnatal overfeeding<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>–<xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR65\">65</xref>,<xref ref-type=\"bibr\" rid=\"CR66\">66</xref></sup>. Pregnant dams were obtained by mating with adult males of proven fertility. On postnatal day (PND) 1, male and female pups were selected, cross-fostered, and grouped into two different litter sizes: normal litters (NL; 12 pups per litter) and small litters (SL; four pups per litter) in order to induce early postnatal normo- and over-nutrition, respectively. On PND 23, the animals were weaned and pups from each litter size were housed in groups of four rats per cage. From weaning onward, the animals were fed either a CD or a HFD ad libitum. This experimental design allowed us to generate four experimental groups in both males and females: NL/CD, NL/HFD, SL/CD and SL/HFD. One week before the animals were euthanized, daily energy intake was calculated from mean food ingestion per day (during 1-wk) using the kcal/g index provided by the manufacturer (3.85 kcal/g for CD; 4.73 kcal/g for HFD). In the morning of PND 120, all animals (males and females) from the different experimental groups were weighed and humanely killed by decapitation and trunk blood, pituitary, hypothalamus, and liver were collected for further analysis. An overview of the experimental design is represented in Suppl. Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>.</p></sec><sec id=\"Sec11\"><title>Hormonal measurements</title><p id=\"Par21\">After euthanasia, trunk blood was collected from all animals and centrifuged at 3.000 rpm for 20 min at 4 °C. Serum was stored at − 80 °C until evaluation of GH, IGF-1, leptin and insulin levels. GH was measured using a rat ELISA kit (Millipore Corporation; Billerica, MA). The sensitivity of the assay was 0.07 ng/mL, and the intra- and inter-assay coefficients of variation (CV) were 1.7 and 4.9, respectively. Serum IGF-1 levels were also determined using a commercial ELISA kit (IDS; Boldon, UK). In this case, the sensitivity was 63 ng/mL and the intra- and inter-assay CV were 4.3 and 6.3, respectively. Serum leptin and insulin levels were measured using RIA kits from Linco Research (St. Charles, MO); the sensitivity of the assays was 0.5 (leptin) and 0.1 (insulin) ng/mL.</p></sec><sec id=\"Sec12\"><title>RNA isolation and retro-transcription (RT)</title><p id=\"Par22\">After decapitation of the animals, whole hypothalami, pituitaries, and liver extracts were immediately collected, frozen in liquid nitrogen and stored at − 80 °C until used for RNA isolation. Tissues were processed for recovery of total RNA using the Absolutely RNA RT-PCR Miniprep Kit (Stratagene; La Jolla, CA), with deoxyribonuclease treatment. Total RNA concentration and purification was assessed by using a NanoDrop-2000 spectrophotometer (Thermo Scientific; Waltham, MA). For the retro-transcription, 1 μg of RNA was reversed transcribed using random hexamer primers, with enzyme and buffers supplied in the First Strand cDNA Synthesis kit (MRI Fermentas; Hanover, MD). Duplicate aliquots were amplified by quantitative real time PCR, in which samples were run against specific synthetic standards of each transcript analyzed to estimate mRNA copy number (<italic>see below</italic>).</p></sec><sec id=\"Sec13\"><title>Quantitative real-time RT-PCR</title><p id=\"Par23\">Analyses were conducted following previously published protocols<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Primers sets (see Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>) were designed using Primer 3 software (Rosen, S., and H. J. Skaletsky, 2000; Whitehead Institute for Biomedical Research), using rat genomic sequences as templates. PCR assays were conducted with the 2X Master Mix PCR reagent (MRI Fermentas; Hanover, MD), using specific primer pairs and cDNA from RT reactions as template. Profiles for PCR amplification were as follows: one cycle of 95 °C for 10 min, followed by 35 cycles of 95 °C for 1 min, 61 °C for 1 min, and 72 °C for 1 min; a final cycle of 72 °C for 10 min was included. PCR products were run on agarose gels for visual verification under ethidium bromide staining, and subjected to column purification (QIAGEN, Valencia, CA) and sequencing to confirm amplicon specificity.<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Specific sequence of primers used in the study for quantitative real-time PCR and product sizes.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Primer</th><th align=\"left\">Sequence</th><th align=\"left\"/><th align=\"left\">Product size (bp)</th></tr></thead><tbody><tr><td align=\"left\"><italic>GH</italic></td><td align=\"left\"><p>5′-TTA CCT GCC ATG CCC TTG T-3’</p><p>5′-TGT AGG CAC GCT CGA ACT CT-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">106</td></tr><tr><td align=\"left\"><italic>GHRH-R</italic></td><td align=\"left\"><p>5′-AGT CCT CTC TGT TGG GGT GAA-3’</p><p>5′-ACA GCG GGA TAA GGA GAA GTG-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">146</td></tr><tr><td align=\"left\"><italic>SST1</italic></td><td align=\"left\"><p>5′-TGC CCT TTC TGG TCA CTT CC-3’</p><p>5′-AGC GGT CCA CAC TAA GCA CA-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">134</td></tr><tr><td align=\"left\"><italic>SST2</italic></td><td align=\"left\"><p>5′-CCC ATC CTG TAC GCC TTC TT-3’</p><p>5′-GTC TCA TTC AGC CGG GAT TT-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">134</td></tr><tr><td align=\"left\"><italic>SST3</italic></td><td align=\"left\"><p>5′-TTG GCC TCT ACT TCC TGG TG-3’</p><p>5′-ATC CTC CTC CTC CTC CGT CT-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">199</td></tr><tr><td align=\"left\"><italic>SST4</italic></td><td align=\"left\"><p>5′-ACA ACT TCC GAC GCT CTT TC-3’</p><p>5′-CTC TTC CTC AGC ACC TCC A-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">199</td></tr><tr><td align=\"left\"><italic>SST5</italic></td><td align=\"left\"><p>5′-TCA TTG TGG TCA AGG TGA AGG-3’</p><p>5′-AAG AAA TAG AGG CCG GCA GA-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">199</td></tr><tr><td align=\"left\"><italic>Ghrelin</italic></td><td align=\"left\"><p>5′-TCC AAG AAG CCA CCA GCT AA-3’</p><p>5′-AAC ATC GAA GGG AGC ATT GA-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">126</td></tr><tr><td align=\"left\"><italic>Pit-1</italic></td><td align=\"left\"><p>5′-GGA AGA GGA AAC GGA GGA CA-3’</p><p>5′-TCG GTT GCA GAA CCA CAC TC-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">158</td></tr><tr><td align=\"left\"><italic>Insulin-R</italic></td><td align=\"left\"><p>5′-TCA TGG ATG GAG GCT ATC TGG-3’</p><p>5′-CCT TGA GCA GGT TGA CGA TTT-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">129</td></tr><tr><td align=\"left\"><italic>Leptin-R</italic></td><td align=\"left\"><p>5′-GGA AGG AGT TGG AAA ACC AAA G-3’</p><p>5′-TCC GAG CAG TAG GAC ACA AGA-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">127</td></tr><tr><td align=\"left\"><italic>GHRH</italic></td><td align=\"left\"><p>5′-TGG GTG TTC TTT GTG CTC CT-3’</p><p>5′-CTT TGT TCC TGG TTC CTC TCC-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">197</td></tr><tr><td align=\"left\"><italic>SRIF</italic></td><td align=\"left\"><p>5′-TCT GCA TCG TCC TGG CTT T-3’</p><p>5′-CTT GGC CAG TTC CTG TTT CC-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">113</td></tr><tr><td align=\"left\"><italic>NPY</italic></td><td align=\"left\"><p>5′-TCG CTC TAT CCC TGC TCG T-3’</p><p>5′-GGG GCA TTT TCT GTG CTT TC-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">220</td></tr><tr><td align=\"left\"><italic>GH-R</italic></td><td align=\"left\"><p>5′-ACT GGC AGC ATG TGA AGA AGA-3’</p><p>5′-GGA ACT GGT ACT GGG GGT AAA-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">147</td></tr><tr><td align=\"left\"><italic>IGF-1</italic></td><td align=\"left\"><p>5′-TTG TGG ATG AGT GTT GCT TCC-3’</p><p>5′-GGT CTT GTT TCC TGC ACT TCC-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">179</td></tr><tr><td align=\"left\"><italic>GOAT</italic></td><td align=\"left\"><p>5′-ATT TGT GAA GGG AAG GTG GAG-3’</p><p>5′-CAG GAG AGC AGG GAA AAA GAG-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">120</td></tr><tr><td align=\"left\"><italic>Cyclophilin-A</italic></td><td align=\"left\"><p>5′-TGG TCT TTG GGA AGG TGA AAG-3’</p><p>5′-TGT CCA CAG TCG GAG ATG GT-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">97</td></tr><tr><td align=\"left\"><italic>β-actin</italic></td><td align=\"left\"><p>5′-CCT AAG GCC AAC CGT GAA A-3’</p><p>5′-CCA GAG GCA TAC AGG GAC AA-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">104</td></tr><tr><td align=\"left\"><italic>Hprt</italic></td><td align=\"left\"><p>5′-AGC TTG CTG GTG AAA AGG AC-3’</p><p>5′-TCC ACT TTC GCT GAT GAC AC-3’</p></td><td align=\"left\"><p>Sense</p><p>Antisense</p></td><td align=\"left\">153</td></tr></tbody></table><table-wrap-foot><p><italic>GH</italic> growth hormone; <italic>GHRH-R</italic> GH-releasing hormone-receptor; <italic>SST1, SST2 SST3, SST4</italic> and <italic>SST5</italic> somatostatin receptor 1, 2, 3, 4 and 5; <italic>GHRH</italic> GH-releasing hormone; <italic>SRIF</italic> somatostatin; <italic>NPY</italic> neuropeptide Y; <italic>GH-R</italic> growth hormone receptor; <italic>IGF-1</italic> insulin-like growth factor-1; <italic>GOAT</italic> ghrelin-O-acyltransferase; <italic>Hprt</italic> hypoxanthine phosphoribosyl-transferase</p></table-wrap-foot></table-wrap></p><p id=\"Par24\">Quantitative real-time PCRs (qPCR) were performed using the qPCR Stratagene Mx3000p instrument (Agilent, Santa Clara, CA, USA) with Brilliant III SYBR Green Master Mix (Agilent). The development, validation and application of the qPCR to measure the expression levels of different transcripts have been previously reported<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Briefly, absolute gene expression levels were calculated using a specific standard curve for each transcript analyzed (1, 10<sup>1</sup>, 10<sup>2</sup>, 10<sup>3</sup>, 10<sup>4</sup>, 10<sup>5</sup>, and 10<sup>6</sup> copies of synthetic template). A No-RT sample was used as a negative control. For each qPCR reaction, 10 μl of master mix, 0,3 μl of each primer (10 μM stock), 8,4 μl of distilled H<sub>2</sub>O and 1 μl of cDNA (50 ng) were mixed with a program consisting of the following steps: (1) 95 °C for 3 min, (2) 40 cycles of denaturing (95 °C for 20 s) and annealing/extension (61 °C for 20 s) and, (3) a graded temperature-dependent dissociation step (55 °C to 95 °C, increasing 0.5 °C/30 s) to verify that only one product was amplified. To control for variations in the amount of RNA used and the efficiency of the RT-reaction, the expression level of each transcript was adjusted by a Normalization Factor (NF) in each sample obtained from the expression levels of three housekeeping genes (<italic>β-actin</italic>, <italic>Hprt</italic> and C<italic>yclophilin A</italic>) using the Genorm program. Expression level of the housekeeping genes analyzed did not differ between experimental groups (data not shown).</p></sec><sec id=\"Sec14\"><title>Data presentation and statistical analysis</title><p id=\"Par25\">As mentioned above, experiments addressing the effects of early postnatal overnutrition on absolute mRNA copy number of the transcript of interest within the whole pituitary, hypothalamic and hepatic extracts were adjusted by a NF in each sample using the absolute mRNA copy number of three housekeeping genes. RNA analyses were performed in duplicate from at least 5 independent samples per group, in line with previous studies of the group addressing pituitary changes in mRNAs expression levels of GH-related factors<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. The total number of animals included in each experimental group and the minimal numbers of independent samples (n = 8) for hormonal determinations in the study are shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>. All data are expressed as the mean ± SEM. For the analysis of the effects of the two major variables under study (litter size and diet) and their interactions, two-way analysis of variance (ANOVA) was performed. Significance level was set at <italic>P</italic> ≤ 0.05 (GraphPad Software Inc., La Jolla, CA). When significant differences were found, the data were further analyzed through post hoc comparison using Newman–Keuls tests to identify simple effects, in keeping with previous studies<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. Considering the inherent variability of the factors under analysis, when <italic>P</italic> values ranged between < 0.1 and > 0.05, a trend for significance was indicated, where appropriate.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec15\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70898_MOESM1_ESM.pdf\"><caption><p>Supplementary information.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors jointly supervised this work: R.M. Luque and M. Tena-Sempere.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70898-y.</p></sec><ack><title>Acknowledgements</title><p>The authors acknowledge the superb technical assistance of Ms. Ana Belen Rodriguez in conduction of animal experiments. This work was supported by Grants BFU2014-57581-P and BFU2017-83934-P (Ministerio de Economía y Competitividad, Spain; co-funded with EU funds from FEDER Program); Project PIE-00005 (Instituto de Salud Carlos III, Ministerio de Sanidad, Spain); Project P12-FQM-01943 (Junta de Andalucía, Spain); and Project REP-655232 (ReprObesity; European Union). CIBER Fisiopatología de la Obesidad y Nutrición is an initiative of Instituto de Salud Carlos III, Spain.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>M.A.S.G., had a leading role in the design and conduction of animal studies, the implementation of expression analyses, as well as in the evaluation and presentation of data; F.R.P., and A.I.P.S., assisted M.A.S.G., in the conduction of animal experiments and processing of biological samples; J.M.C., and M.J.V., actively participated in hormonal analyses, as well as in the discussion and interpretation of the whole dataset; R.M.L., actively contributed to the experimental design, the integral analysis of the data, and manuscript preparation; M.T.S., was responsible for the design and coordination of the complete study, with a leading role in the analysis and interpretation of the whole dataset. M.T.-S., was responsible in manuscript preparation, with the active participation of M.A.S.G., and R.M.L., All authors revised and approved the paper.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>The data supporting the findings and conclusions of this study are included in this article and its supplementary files. All relevant original data are available from the corresponding authors, upon reasonable request.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par26\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Isaksson</surname><given-names>OG</given-names></name><name><surname>Lindahl</surname><given-names>A</given-names></name><name><surname>Nilsson</surname><given-names>A</given-names></name><name><surname>Isgaard</surname><given-names>J</given-names></name></person-group><article-title>Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth</article-title><source>Endocr. Rev.</source><year>1987</year><volume>8</volume><fpage>426</fpage><lpage>438</lpage><pub-id pub-id-type=\"pmid\">3319530</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moller</surname><given-names>N</given-names></name><name><surname>Jorgensen</surname><given-names>JO</given-names></name></person-group><article-title>Effects of growth hormone on glucose, lipid, and protein metabolism in human subjects</article-title><source>Endocr. Rev.</source><year>2009</year><volume>30</volume><fpage>152</fpage><lpage>177</lpage><pub-id pub-id-type=\"pmid\">19240267</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Richelsen</surname><given-names>B</given-names></name><etal/></person-group><article-title>Growth hormone treatment of obese women for 5 wk: effect on body composition and adipose tissue LPL activity</article-title><source>Am. J. Physiol.</source><year>1994</year><volume>266</volume><fpage>E211</fpage><lpage>E216</lpage><pub-id pub-id-type=\"pmid\">8141279</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luque</surname><given-names>RM</given-names></name><etal/></person-group><article-title>Metabolic impact of adult-onset, isolated, growth hormone deficiency (AOiGHD) due to destruction of pituitary somatotropes</article-title><source>PLoS ONE</source><year>2011</year><volume>6</volume><fpage>e15767</fpage><pub-id pub-id-type=\"pmid\">21283519</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Johannsson</surname><given-names>G</given-names></name></person-group><article-title>Management of adult growth hormone deficiency</article-title><source>Endocrinol. Metab. Clin. N. Am.</source><year>2007</year><volume>36</volume><fpage>203</fpage><lpage>220</lpage></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Beshyah</surname><given-names>SA</given-names></name><etal/></person-group><article-title>Abnormal body composition and reduced bone mass in growth hormone deficient hypopituitary adults</article-title><source>Clin. Endocrinol.</source><year>1995</year><volume>42</volume><fpage>179</fpage><lpage>189</lpage></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vance</surname><given-names>ML</given-names></name><name><surname>Mauras</surname><given-names>N</given-names></name></person-group><article-title>Growth hormone therapy in adults and children</article-title><source>N. Engl. J. Med.</source><year>1999</year><volume>341</volume><fpage>1206</fpage><lpage>1216</lpage><pub-id pub-id-type=\"pmid\">10519899</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bengtsson</surname><given-names>BA</given-names></name><etal/></person-group><article-title>Treatment of adults with growth hormone (GH) deficiency with recombinant human GH</article-title><source>J. Clin. Endocrinol. Metab.</source><year>1993</year><volume>76</volume><fpage>309</fpage><lpage>317</lpage><pub-id pub-id-type=\"pmid\">8432773</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chrisoulidou</surname><given-names>A</given-names></name><etal/></person-group><article-title>Effects of 7 years of growth hormone replacement therapy in hypopituitary adults</article-title><source>J. Clin. Endocrinol. Metab.</source><year>2000</year><volume>85</volume><fpage>3762</fpage><lpage>3769</lpage><pub-id pub-id-type=\"pmid\">11061536</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cummings</surname><given-names>DE</given-names></name><name><surname>Merriam</surname><given-names>GR</given-names></name></person-group><article-title>Growth hormone therapy in adults</article-title><source>Annu. Rev. Med.</source><year>2003</year><volume>54</volume><fpage>513</fpage><lpage>533</lpage><pub-id pub-id-type=\"pmid\">12471175</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Molitch</surname><given-names>ME</given-names></name><etal/></person-group><article-title>Evaluation and treatment of adult growth hormone deficiency: an endocrine society clinical practice guideline</article-title><source>J. Clin. Endocrinol. Metab.</source><year>2006</year><volume>91</volume><fpage>1621</fpage><lpage>1634</lpage><pub-id pub-id-type=\"pmid\">16636129</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luque</surname><given-names>RM</given-names></name><etal/></person-group><article-title>Does the pituitary somatotrope play a primary role in regulating GH output in metabolic extremes?</article-title><source>Ann. N. Y. Acad. Sci.</source><year>2011</year><volume>1220</volume><fpage>82</fpage><lpage>92</lpage><pub-id pub-id-type=\"pmid\">21388406</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Williams</surname><given-names>T</given-names></name><etal/></person-group><article-title>Impaired growth hormone responses to growth hormone-releasing factor in obesity. A pituitary defect reversed with weight reduction</article-title><source>N. Engl. J. Med.</source><year>1984</year><volume>311</volume><fpage>1403</fpage><lpage>1407</lpage><pub-id pub-id-type=\"pmid\">6436706</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bonert</surname><given-names>VS</given-names></name><name><surname>Elashoff</surname><given-names>JD</given-names></name><name><surname>Barnett</surname><given-names>P</given-names></name><name><surname>Melmed</surname><given-names>S</given-names></name></person-group><article-title>Body mass index determines evoked growth hormone (GH) responsiveness in normal healthy male subjects: diagnostic caveat for adult GH deficiency</article-title><source>J. Clin. Endocrinol. Metab.</source><year>2004</year><volume>89</volume><fpage>3397</fpage><lpage>3401</lpage><pub-id pub-id-type=\"pmid\">15240621</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cattaneo</surname><given-names>L</given-names></name><etal/></person-group><article-title>Characterization of the hypothalamo-pituitary-IGF-I axis in rats made obese by overfeeding</article-title><source>J. Endocrinol.</source><year>1996</year><volume>148</volume><fpage>347</fpage><lpage>353</lpage><pub-id pub-id-type=\"pmid\">8699149</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Schepper</surname><given-names>JA</given-names></name><etal/></person-group><article-title>Cafeteria diet-induced obesity is associated with a low spontaneous growth hormone secretion and normal plasma insulin-like growth factor-I concentrations</article-title><source>Growth Horm. IGF Res.</source><year>1998</year><volume>8</volume><fpage>397</fpage><lpage>401</lpage><pub-id pub-id-type=\"pmid\">10984301</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alvarez</surname><given-names>P</given-names></name><etal/></person-group><article-title>Effect of withdrawal of somatostatin plus GH-releasing hormone as a stimulus of GH secretion in obesity</article-title><source>Clin. Endocrinol.</source><year>2002</year><volume>56</volume><fpage>487</fpage><lpage>492</lpage></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Girod</surname><given-names>JP</given-names></name><name><surname>Brotman</surname><given-names>DJ</given-names></name></person-group><article-title>The metabolic syndrome as a vicious cycle: Does obesity beget obesity?</article-title><source>Med. Hypotheses</source><year>2003</year><volume>60</volume><fpage>584</fpage><lpage>589</lpage><pub-id pub-id-type=\"pmid\">12615528</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rasmussen</surname><given-names>MH</given-names></name><etal/></person-group><article-title>Massive weight loss restores 24-hour growth hormone release profiles and serum insulin-like growth factor-I levels in obese subjects</article-title><source>J. Clin. Endocrinol. Metab.</source><year>1995</year><volume>80</volume><fpage>1407</fpage><lpage>1415</lpage><pub-id pub-id-type=\"pmid\">7536210</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cordoba-Chacon</surname><given-names>J</given-names></name><etal/></person-group><article-title>Peripubertal-onset but not adult-onset obesity increases IGF-I and drives development of lean mass, which may lessen the metabolic impairment in adult obesity</article-title><source>Am. J. Physiol. Endocrinol. Metab.</source><year>2012</year><volume>303</volume><fpage>E1151</fpage><lpage>E1157</lpage><pub-id pub-id-type=\"pmid\">22932784</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luque</surname><given-names>RM</given-names></name><name><surname>Kineman</surname><given-names>RD</given-names></name></person-group><article-title>Impact of obesity on the growth hormone axis: evidence for a direct inhibitory effect of hyperinsulinemia on pituitary function</article-title><source>Endocrinology</source><year>2006</year><volume>147</volume><fpage>2754</fpage><lpage>2763</lpage><pub-id pub-id-type=\"pmid\">16513828</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Guilloteau</surname><given-names>P</given-names></name><name><surname>Zabielski</surname><given-names>R</given-names></name><name><surname>Hammon</surname><given-names>HM</given-names></name><name><surname>Metges</surname><given-names>CC</given-names></name></person-group><article-title>Adverse effects of nutritional programming during prenatal and early postnatal life, some aspects of regulation and potential prevention and treatments</article-title><source>J. Physiol. Pharmacol.</source><year>2009</year><volume>60</volume><issue>Suppl 3</issue><fpage>17</fpage><lpage>35</lpage><pub-id pub-id-type=\"pmid\">19996479</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Barker</surname><given-names>DJ</given-names></name><etal/></person-group><article-title>Type 2 (non-insulin-dependent) diabetes mellitus, hypertension and hyperlipidaemia (syndrome X): relation to reduced fetal growth</article-title><source>Diabetologia</source><year>1993</year><volume>36</volume><fpage>62</fpage><lpage>67</lpage><pub-id pub-id-type=\"pmid\">8436255</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Frankel</surname><given-names>S</given-names></name><etal/></person-group><article-title>Birthweight, body-mass index in middle age, and incident coronary heart disease</article-title><source>Lancet</source><year>1996</year><volume>348</volume><fpage>1478</fpage><lpage>1480</lpage><pub-id pub-id-type=\"pmid\">8942776</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Inadera</surname><given-names>H</given-names></name></person-group><article-title>Developmental origins of obesity and type 2 diabetes: molecular aspects and role of chemicals</article-title><source>Environ. Health Prev. Med.</source><year>2013</year><volume>18</volume><fpage>185</fpage><lpage>197</lpage><pub-id pub-id-type=\"pmid\">23382021</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dearden</surname><given-names>L</given-names></name><name><surname>Ozanne</surname><given-names>SE</given-names></name></person-group><article-title>Early life origins of metabolic disease: developmental programming of hypothalamic pathways controlling energy homeostasis</article-title><source>Front. Neuroendocrinol.</source><year>2015</year><volume>39</volume><fpage>3</fpage><lpage>16</lpage><pub-id pub-id-type=\"pmid\">26296796</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sanchez-Garrido</surname><given-names>MA</given-names></name><etal/></person-group><article-title>Obesity-induced hypogonadism in the male: premature reproductive neuroendocrine senescence and contribution of Kiss1-mediated mechanisms</article-title><source>Endocrinology</source><year>2014</year><volume>155</volume><fpage>1067</fpage><lpage>1079</lpage><pub-id pub-id-type=\"pmid\">24424048</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sanchez-Garrido</surname><given-names>MA</given-names></name><etal/></person-group><article-title>Metabolic and gonadotropic impact of sequential obesogenic insults in the female: influence of the loss of ovarian secretion</article-title><source>Endocrinology</source><year>2015</year><volume>156</volume><fpage>2984</fpage><lpage>2998</lpage><pub-id pub-id-type=\"pmid\">25984764</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>Z</given-names></name><etal/></person-group><article-title>Neonatal overnutrition in mice exacerbates high-fat diet-induced metabolic perturbations</article-title><source>J. Endocrinol.</source><year>2013</year><volume>219</volume><fpage>131</fpage><lpage>143</lpage><pub-id pub-id-type=\"pmid\">23959078</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kayser</surname><given-names>BD</given-names></name><name><surname>Goran</surname><given-names>MI</given-names></name><name><surname>Bouret</surname><given-names>SG</given-names></name></person-group><article-title>Perinatal overnutrition exacerbates adipose tissue inflammation caused by high-fat feeding in C57BL/6J mice</article-title><source>PLoS ONE</source><year>2015</year><volume>10</volume><fpage>e0121954</fpage><pub-id pub-id-type=\"pmid\">25835281</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Glavas</surname><given-names>MM</given-names></name><etal/></person-group><article-title>Early overnutrition results in early-onset arcuate leptin resistance and increased sensitivity to high-fat diet</article-title><source>Endocrinology</source><year>2010</year><volume>151</volume><fpage>1598</fpage><lpage>1610</lpage><pub-id pub-id-type=\"pmid\">20194730</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cai</surname><given-names>G</given-names></name><etal/></person-group><article-title>Overfeeding during a critical postnatal period exacerbates hypothalamic-pituitary-adrenal axis responses to immune challenge: a role for adrenal melanocortin 2 receptors</article-title><source>Sci. Rep.</source><year>2016</year><volume>6</volume><fpage>21097</fpage><pub-id pub-id-type=\"pmid\">26868281</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lisboa</surname><given-names>PC</given-names></name><name><surname>Conceicao</surname><given-names>EP</given-names></name><name><surname>de Oliveira</surname><given-names>E</given-names></name><name><surname>Moura</surname><given-names>EG</given-names></name></person-group><article-title>Postnatal overnutrition programs the thyroid hormone metabolism and function in adulthood</article-title><source>J. Endocrinol.</source><year>2015</year><volume>226</volume><fpage>219</fpage><lpage>226</lpage><pub-id pub-id-type=\"pmid\">26203167</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kappeler</surname><given-names>L</given-names></name><etal/></person-group><article-title>Early postnatal nutrition determines somatotropic function in mice</article-title><source>Endocrinology</source><year>2009</year><volume>150</volume><fpage>314</fpage><lpage>323</lpage><pub-id pub-id-type=\"pmid\">18801897</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nehus</surname><given-names>E</given-names></name><name><surname>Mitsnefes</surname><given-names>M</given-names></name></person-group><article-title>Childhood obesity and the metabolic syndrome</article-title><source>Pediatr. Clin. N. Am.</source><year>2019</year><volume>66</volume><fpage>31</fpage><lpage>43</lpage></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cordoba-Chacon</surname><given-names>J</given-names></name><etal/></person-group><article-title>Somatostatin and its receptors contribute in a tissue-specific manner to the sex-dependent metabolic (fed/fasting) control of growth hormone axis in mice</article-title><source>Am. J. Physiol. Endocrinol. Metab.</source><year>2011</year><volume>300</volume><fpage>E46</fpage><lpage>54</lpage><pub-id pub-id-type=\"pmid\">20943754</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cordoba-Chacon</surname><given-names>J</given-names></name><etal/></person-group><article-title>Cortistatin is not a somatostatin analogue but stimulates prolactin release and inhibits GH and ACTH in a gender-dependent fashion: potential role of ghrelin</article-title><source>Endocrinology</source><year>2011</year><volume>152</volume><fpage>4800</fpage><lpage>4812</lpage><pub-id pub-id-type=\"pmid\">21971153</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McMillen</surname><given-names>IC</given-names></name><etal/></person-group><article-title>The early origins of later obesity: pathways and mechanisms</article-title><source>Adv. Exp. Med. Biol.</source><year>2009</year><volume>646</volume><fpage>71</fpage><lpage>81</lpage><pub-id pub-id-type=\"pmid\">19536665</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Agosti</surname><given-names>M</given-names></name><name><surname>Tandoi</surname><given-names>F</given-names></name><name><surname>Morlacchi</surname><given-names>L</given-names></name><name><surname>Bossi</surname><given-names>A</given-names></name></person-group><article-title>Nutritional and metabolic programming during the first thousand days of life</article-title><source>Pediatr. Med. Chir.</source><year>2017</year><volume>39</volume><fpage>157</fpage><pub-id pub-id-type=\"pmid\">28673078</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ji</surname><given-names>C</given-names></name><etal/></person-group><article-title>Postnatal overfeeding promotes early onset and exaggeration of high-fat diet-induced nonalcoholic fatty liver disease through disordered hepatic lipid metabolism in rats</article-title><source>J. Nutr. Biochem.</source><year>2014</year><volume>25</volume><fpage>1108</fpage><lpage>1116</lpage><pub-id pub-id-type=\"pmid\">25154569</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pijl</surname><given-names>H</given-names></name><etal/></person-group><article-title>Altered neuroregulation of GH secretion in viscerally obese premenopausal women</article-title><source>J. Clin. Endocrinol. Metab.</source><year>2001</year><volume>86</volume><fpage>5509</fpage><lpage>5515</lpage><pub-id pub-id-type=\"pmid\">11701729</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Berryman</surname><given-names>DE</given-names></name><name><surname>Glad</surname><given-names>CA</given-names></name><name><surname>List</surname><given-names>EO</given-names></name><name><surname>Johannsson</surname><given-names>G</given-names></name></person-group><article-title>The GH/IGF-1 axis in obesity: pathophysiology and therapeutic considerations</article-title><source>Nat. Rev. Endocrinol.</source><year>2013</year><volume>9</volume><fpage>346</fpage><lpage>356</lpage><pub-id pub-id-type=\"pmid\">23568441</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Briard</surname><given-names>N</given-names></name><etal/></person-group><article-title>Hypothalamic mediated action of free fatty acid on growth hormone secretion in sheep</article-title><source>Endocrinology</source><year>1998</year><volume>139</volume><fpage>4811</fpage><lpage>4819</lpage><pub-id pub-id-type=\"pmid\">9832417</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Goldenberg</surname><given-names>N</given-names></name><name><surname>Barkan</surname><given-names>A</given-names></name></person-group><article-title>Factors regulating growth hormone secretion in humans</article-title><source>Endocrinol. Metab. Clin. N. Am.</source><year>2007</year><volume>36</volume><fpage>37</fpage><lpage>55</lpage></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cattaneo</surname><given-names>L</given-names></name><etal/></person-group><article-title>Hypothalamo-pituitary-IGF-1 axis in female rats made obese by overfeeding</article-title><source>Life Sci.</source><year>1997</year><volume>61</volume><fpage>881</fpage><lpage>889</lpage><pub-id pub-id-type=\"pmid\">9284081</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bielohuby</surname><given-names>M</given-names></name><etal/></person-group><article-title>Lack of dietary carbohydrates induces hepatic growth hormone (GH) resistance in rats</article-title><source>Endocrinology</source><year>2011</year><volume>152</volume><fpage>1948</fpage><lpage>1960</lpage><pub-id pub-id-type=\"pmid\">21427215</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vance</surname><given-names>ML</given-names></name></person-group><article-title>Growth-hormone-releasing hormone</article-title><source>Clin. Chem.</source><year>1990</year><volume>36</volume><fpage>415</fpage><lpage>420</lpage><pub-id pub-id-type=\"pmid\">2107038</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gahete</surname><given-names>MD</given-names></name><etal/></person-group><article-title>Metabolic regulation of ghrelin O-acyl transferase (GOAT) expression in the mouse hypothalamus, pituitary, and stomach</article-title><source>Mol. Cell. Endocrinol.</source><year>2010</year><volume>317</volume><fpage>154</fpage><lpage>160</lpage><pub-id pub-id-type=\"pmid\">20035826</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gahete</surname><given-names>MD</given-names></name><etal/></person-group><article-title>Ghrelin gene products, receptors, and GOAT enzyme: biological and pathophysiological insight</article-title><source>J. Endocrinol.</source><year>2014</year><volume>220</volume><fpage>R1</fpage><lpage>24</lpage><pub-id pub-id-type=\"pmid\">24194510</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kamegai</surname><given-names>J</given-names></name><etal/></person-group><article-title>The role of pituitary ghrelin in growth hormone (GH) secretion: GH-releasing hormone-dependent regulation of pituitary ghrelin gene expression and peptide content</article-title><source>Endocrinology</source><year>2004</year><volume>145</volume><fpage>3731</fpage><lpage>3738</lpage><pub-id pub-id-type=\"pmid\">15087428</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Suzuki</surname><given-names>N</given-names></name><name><surname>Okada</surname><given-names>K</given-names></name><name><surname>Minami</surname><given-names>S</given-names></name><name><surname>Wakabayashi</surname><given-names>I</given-names></name></person-group><article-title>Inhibitory effect of neuropeptide Y on growth hormone secretion in rats is mediated by both Y1- and Y2-receptor subtypes and abolished after anterolateral deafferentation of the medial basal hypothalamus</article-title><source>Regul. Pept.</source><year>1996</year><volume>65</volume><fpage>145</fpage><lpage>151</lpage><pub-id pub-id-type=\"pmid\">8884982</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>L</given-names></name><etal/></person-group><article-title>Actions of NPY, and its Y1 and Y2 receptors on pulsatile growth hormone secretion during the fed and fasted state</article-title><source>J. Neurosci.</source><year>2014</year><volume>34</volume><fpage>16309</fpage><lpage>16319</lpage><pub-id pub-id-type=\"pmid\">25471570</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McDonald</surname><given-names>JK</given-names></name><name><surname>Lumpkin</surname><given-names>MD</given-names></name><name><surname>Samson</surname><given-names>WK</given-names></name><name><surname>McCann</surname><given-names>SM</given-names></name></person-group><article-title>Neuropeptide Y affects secretion of luteinizing hormone and growth hormone in ovariectomized rats</article-title><source>Proc. Natl. Acad. Sci. USA</source><year>1985</year><volume>82</volume><fpage>561</fpage><lpage>564</lpage><pub-id pub-id-type=\"pmid\">3855566</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Adams</surname><given-names>EF</given-names></name><etal/></person-group><article-title>Neuropeptide Y directly inhibits growth hormone secretion by human pituitary somatotropic tumours</article-title><source>Acta Endocrinol. (Copenh.)</source><year>1987</year><volume>115</volume><fpage>149</fpage><lpage>154</lpage><pub-id pub-id-type=\"pmid\">2884793</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Iida</surname><given-names>K</given-names></name><etal/></person-group><article-title>Tissue-specific regulation of growth hormone (GH) receptor and insulin-like growth factor-I gene expression in the pituitary and liver of GH-deficient (lit/lit) mice and transgenic mice that overexpress bovine GH (bGH) or a bGH antagonist</article-title><source>Endocrinology</source><year>2004</year><volume>145</volume><fpage>1564</fpage><lpage>1570</lpage><pub-id pub-id-type=\"pmid\">14726438</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>SH</given-names></name><name><surname>Park</surname><given-names>MJ</given-names></name></person-group><article-title>Effects of growth hormone on glucose metabolism and insulin resistance in human</article-title><source>Ann. Pediatr. Endocrinol. Metab.</source><year>2017</year><volume>22</volume><fpage>145</fpage><lpage>152</lpage><pub-id pub-id-type=\"pmid\">29025199</pub-id></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cordoba-Chacon</surname><given-names>J</given-names></name><name><surname>Gahete</surname><given-names>MD</given-names></name><name><surname>McGuinness</surname><given-names>OP</given-names></name><name><surname>Kineman</surname><given-names>RD</given-names></name></person-group><article-title>Differential impact of selective GH deficiency and endogenous GH excess on insulin-mediated actions in muscle and liver of male mice</article-title><source>Am. J. Physiol. Endocrinol. Metab.</source><year>2014</year><volume>307</volume><fpage>E928</fpage><lpage>934</lpage><pub-id pub-id-type=\"pmid\">25269484</pub-id></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vazquez-Borrego</surname><given-names>MC</given-names></name><etal/></person-group><article-title>The pituitary gland is a novel major site of action of metformin in non-human primates: a potential path to expand and integrate its metabolic actions</article-title><source>Cell Physiol. Biochem.</source><year>2018</year><volume>49</volume><fpage>1444</fpage><lpage>1459</lpage><pub-id pub-id-type=\"pmid\">30205369</pub-id></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Childs</surname><given-names>GV</given-names></name><etal/></person-group><article-title>The somatotrope as a metabolic sensor: deletion of leptin receptors causes obesity</article-title><source>Endocrinology</source><year>2011</year><volume>152</volume><fpage>69</fpage><lpage>81</lpage><pub-id pub-id-type=\"pmid\">21084451</pub-id></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vazquez-Borrego</surname><given-names>MC</given-names></name><etal/></person-group><article-title>Multiple signaling pathways convey central and peripheral signals to regulate pituitary function: Lessons from human and non-human primate models</article-title><source>Mol. Cell Endocrinol.</source><year>2018</year><volume>463</volume><fpage>4</fpage><lpage>22</lpage><pub-id pub-id-type=\"pmid\">29253530</pub-id></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gahete</surname><given-names>MD</given-names></name><etal/></person-group><article-title>Insulin and IGF-I inhibit GH synthesis and release in vitro and in vivo by separate mechanisms</article-title><source>Endocrinology</source><year>2013</year><volume>154</volume><fpage>2410</fpage><lpage>2420</lpage><pub-id pub-id-type=\"pmid\">23671263</pub-id></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Allensworth-James</surname><given-names>ML</given-names></name><name><surname>Odle</surname><given-names>A</given-names></name><name><surname>Haney</surname><given-names>A</given-names></name><name><surname>Childs</surname><given-names>G</given-names></name></person-group><article-title>Sex differences in somatotrope dependency on leptin receptors in young mice: ablation of LEPR causes severe growth hormone deficiency and abdominal obesity in males</article-title><source>Endocrinology</source><year>2015</year><volume>156</volume><fpage>3253</fpage><lpage>3264</lpage><pub-id pub-id-type=\"pmid\">26168341</pub-id></element-citation></ref><ref id=\"CR63\"><label>63.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Indrio</surname><given-names>F</given-names></name><etal/></person-group><article-title>Epigenetic matters: the link between early nutrition, microbiome, and long-term health development</article-title><source>Front. Pediatr.</source><year>2017</year><volume>5</volume><fpage>178</fpage><pub-id pub-id-type=\"pmid\">28879172</pub-id></element-citation></ref><ref id=\"CR64\"><label>64.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fernandez-Perez</surname><given-names>L</given-names></name><etal/></person-group><article-title>Sex steroids and growth hormone interactions</article-title><source>Endocrinol. Nutr.</source><year>2016</year><volume>63</volume><fpage>171</fpage><lpage>180</lpage><pub-id pub-id-type=\"pmid\">26775014</pub-id></element-citation></ref><ref id=\"CR65\"><label>65.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Castellano</surname><given-names>JM</given-names></name><etal/></person-group><article-title>Early metabolic programming of puberty onset: impact of changes in postnatal feeding and rearing conditions on the timing of puberty and development of the hypothalamic kisspeptin system</article-title><source>Endocrinology</source><year>2011</year><volume>152</volume><fpage>3396</fpage><lpage>3408</lpage><pub-id pub-id-type=\"pmid\">21712362</pub-id></element-citation></ref><ref id=\"CR66\"><label>66.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Habbout</surname><given-names>A</given-names></name><name><surname>Li</surname><given-names>N</given-names></name><name><surname>Rochette</surname><given-names>L</given-names></name><name><surname>Vergely</surname><given-names>C</given-names></name></person-group><article-title>Postnatal overfeeding in rodents by litter size reduction induces major short- and long-term pathophysiological consequences</article-title><source>J. Nutr.</source><year>2013</year><volume>143</volume><fpage>553</fpage><lpage>562</lpage><pub-id pub-id-type=\"pmid\">23446961</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807810</article-id><article-id pub-id-type=\"pmc\">PMC7431569</article-id><article-id pub-id-type=\"publisher-id\">70593</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70593-y</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Campylobacter infections expected to increase due to climate change in Northern Europe</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Kuhn</surname><given-names>Katrin Gaardbo</given-names></name><address><email>kuh@ssi.dk</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Nygård</surname><given-names>Karin Maria</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Guzman-Herrador</surname><given-names>Bernardo</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sunde</surname><given-names>Linda Selje</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rimhanen-Finne</surname><given-names>Ruska</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Trönnberg</surname><given-names>Linda</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Jepsen</surname><given-names>Martin Rudbeck</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ruuhela</surname><given-names>Reija</given-names></name><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wong</surname><given-names>Wai Kwok</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ethelberg</surname><given-names>Steen</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff8\">8</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.6203.7</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0417 4147</institution-id><institution>Infectious Disease Epidemiology and Prevention, </institution><institution>Statens Serum Institut, </institution></institution-wrap>Artillerivej 5, Copenhagen, Denmark </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.418193.6</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 1541 4204</institution-id><institution>Department of Infectious Disease Epidemiology, </institution><institution>Norwegian Institute of Public Health, </institution></institution-wrap>Oslo, Norway </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.14758.3f</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 1013 0499</institution-id><institution>Department of Health Security, </institution><institution>National Institute for Health and Welfare, </institution></institution-wrap>Helsinki, Finland </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.419734.c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9580 3113</institution-id><institution>Department of Monitoring and Evaluation, </institution><institution>Public Health Agency of Sweden, </institution></institution-wrap>Solna, Sweden </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5254.6</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0674 042X</institution-id><institution>Section for Geography, IGN, </institution><institution>University of Copenhagen, </institution></institution-wrap>Copenhagen, Denmark </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.8657.c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2253 8678</institution-id><institution>Weather and Climate Change Impact Research, </institution><institution>Finnish Meteorological Institute, </institution></institution-wrap>Helsinki, Finland </aff><aff id=\"Aff7\"><label>7</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.436622.7</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2236 7549</institution-id><institution>Department of Hydrology, </institution><institution>Norwegian Water Resources and Energy Directorate, </institution></institution-wrap>Oslo, Norway </aff><aff id=\"Aff8\"><label>8</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5254.6</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0674 042X</institution-id><institution>Global Health Section, Department of Public Health, </institution><institution>University of Copenhagen, </institution></institution-wrap>Copenhagen, Denmark </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13874</elocation-id><history><date date-type=\"received\"><day>21</day><month>1</month><year>2020</year></date><date date-type=\"accepted\"><day>27</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Global climate change is predicted to alter precipitation and temperature patterns across the world, affecting a range of infectious diseases and particularly foodborne infections such as <italic>Campylobacter</italic>. In this study, we used national surveillance data to analyse the relationship between climate and campylobacteriosis in Denmark, Finland, Norway and Sweden and estimate the impact of climate changes on future disease patterns. We show that <italic>Campylobacter</italic> incidences are linked to increases in temperature and especially precipitation in the week before illness, suggesting a non-food transmission route. These four countries may experience a doubling of <italic>Campylobacter</italic> cases by the end of the 2080s, corresponding to around 6,000 excess cases per year caused only by climate changes. Considering the strong worldwide burden of campylobacteriosis, it is important to assess local and regional impacts of climate change in order to initiate timely public health management and adaptation strategies.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Epidemiology</kwd><kwd>Climate-change impacts</kwd><kwd>Projection and prediction</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/501100005416</institution-id><institution>Norges Forskningsråd</institution></institution-wrap></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">The zoonotic pathogen <italic>Campylobacter</italic> is the most commonly reported cause of human bacterial gastroenteritis throughout Europe, including the Nordic countries (Denmark, Finland, Iceland, Norway and Sweden). Since 2008, the reported incidence of <italic>Campylobacter</italic> infections in Europe has increased and is now three times greater than that of salmonellosis<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. Domestic cases of campylobacteriosis are commonly linked to consumption of contaminated food or drink such as poultry, unpasteurized milk and cross-contaminated vegetables<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>–<xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. Lately environmental and behavioural factors such as recreational water contact, occupational exposure (e.g. poultry farms and abattoirs) and contact to household pets<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>–<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup> have also emerged as important <italic>Campylobacter</italic> transmission routes. Infection rates are highest in children under five and young adults, and in all temperate regions, particularly the Nordic countries, the incidence of disease varies seasonally and tends to peak during the late spring and summer months<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. This peak may represent a combination of fluctuating infection rates in poultry<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup> (caused either by a direct impact of temperature on <italic>Campylobacter</italic> growth rates or indirect effects such as changes in contamination sources) and increased human exposure to environmental reservoirs as well as different eating and hygiene patterns during the summer months<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>.\n</p><p id=\"Par3\">Global climate change is predicted to alter temperatures and precipitation across the world with strong direct and indirect impacts on human health<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Because weather and climate are important determinants of infectious diseases, including food-borne, it is relevant and important to estimate future changes in disease patterns related to climate or environmental changes<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. Many published reports highlight a strong effect of climate changes on important infectious diseases such as malaria, West Nile virus, cholera, and tuberculosis<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>–<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>, but—considering their overall contribution to the global disease burden—the impact of climate changes on zoonotic diseases has been less well studied. For instance, it is predicted that climate changes are likely to increase the future incidence of salmonellosis in selected areas<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>–<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>, but evidence of a link between climate and <italic>Campylobacter</italic> is conflicting and of varying strength<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>–<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. Generally, it appears that human campylobacteriosis—not considering the seasonal variation—is driven by a complex mixture of climatic drivers where precipitation in particular may be important<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. This is especially highlighted for extreme rainfall<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup> which has been linked to large <italic>Campylobacter</italic> outbreaks<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR29\">29</xref>–<xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. Although campylobacteriosis is rarely fatal, the global disease burden—particularly from an economic perspective—is currently high<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>–<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup> and, irrespective of climate, predicted to increase in the future<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. This natural increase, reflecting changing populations, exposure patterns and microbiological features such as antimicrobial resistance, combined with climatic changes, can result in a future heavier disease burden and higher costs to societies. Establishing how extreme weather events and climate changes affect campylobacteriosis can form the basis of well-guided early warning systems in vulnerable areas and better targeting of prevention and control measures, potentially reducing the public health and economic impact of <italic>Campylobacter</italic> in these areas.</p><p id=\"Par4\">In this study, we analyse the temporal and spatial relationship between climatic factors and the incidence of <italic>Campylobacter</italic> in the Nordic countries by fitting observed reported disease cases and weather events in a Poisson regression model and applying these relationships to estimate the future incidence of disease associated with projected climate changes.</p></sec><sec id=\"Sec2\"><title>Results</title><sec id=\"Sec3\"><title>Campylobacter and climate at baseline 2000–2015</title><p id=\"Par5\">A total of 64,034 reported cases of <italic>Campylobacter</italic> were included in the final database. The average number of cases per municipality during the study period was 154 (median: 0 and maximum: 274). During the baseline period from 2000 to 2015, the average annual number of <italic>Campylobacter</italic> cases per 100,000 persons in the four countries was 42, ranging from 25 in Norway to 60 in Denmark.</p><p id=\"Par6\">For all locations, the average weekly temperature observed during the study period was 4.4 °C (ranging from − 35.5 to 32.8 °C) and the average daily precipitation was 2.2 mm (ranging from 0 to 105 mm). Overall, each municipality experienced a total of 2.4 heat waves and 3.3 heavy precipitation events (excluding snow) during the study period. Fitting the six regression models on the 10% of omitted data showed that the number of <italic>Campylobacter</italic> cases was best predicted using the standard Poisson regression model for both winter and summer periods with a time-lag of 1 week (i.e. using the previous week’s climate data to predict the following week’s cases of <italic>Campylobacter</italic>). The models predicted 79.7 and 84.5% data records correctly in the winter and summer months, respectively (data not shown). The final models for <italic>Campylobacter</italic> and climate showed that the number of <italic>Campylobacter</italic> cases in any week during the summer increased significantly with increasing temperature, precipitation and number of heavy precipitation events in the previous week (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>). Conversely, an increase in the number of heat waves in any week during the summer as well as increases in precipitation during the winter months decreased the number of <italic>Campylobacter</italic> cases reported 1 week later (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>).<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Models for the relationship between weather and variations in <italic>Campylobacter</italic> cases.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Variable</th><th align=\"left\">Coefficient</th><th align=\"left\">95% CI</th><th align=\"left\">p</th><th align=\"left\">Overall statistics</th></tr></thead><tbody><tr><td align=\"left\"><bold>Summer period model (April–September)</bold></td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\">N. obs = 78,842; X<sup>2</sup> = 24,036; p < 0.001; R<sup>2</sup> = 0.51</td></tr><tr><td align=\"left\">Temperature</td><td align=\"left\">0.09</td><td align=\"left\">0.09–0.10</td><td align=\"left\">< 0.001</td><td align=\"left\" rowspan=\"4\"/></tr><tr><td align=\"left\">Precipitation</td><td align=\"left\">0.30</td><td align=\"left\">0.29–0.32</td><td align=\"left\">< 0.001</td></tr><tr><td align=\"left\">Heat wave</td><td align=\"left\">− 0.10</td><td align=\"left\">− 0.15 to − 0.05</td><td align=\"left\">< 0.001</td></tr><tr><td align=\"left\">Heavy precipitation</td><td align=\"left\">0.79</td><td align=\"left\">0.76–0.82</td><td align=\"left\">< 0.001</td></tr><tr><td align=\"left\"><bold>Winter period model (October–May)</bold></td><td align=\"left\"/><td align=\"left\"/><td align=\"left\"/><td align=\"left\">N. obs = 66,648; x<sup>2</sup> = 18,235; p < 0.001; R<sup>2</sup> = 0.33</td></tr><tr><td align=\"left\">Temperature</td><td align=\"left\">0.18</td><td align=\"left\">0.17–0.18</td><td align=\"left\">< 0.001</td><td align=\"left\" rowspan=\"3\"/></tr><tr><td align=\"left\">Precipitation</td><td align=\"left\">− 0.18</td><td align=\"left\">− 0.19 to − 0.18</td><td align=\"left\">< 0.001</td></tr><tr><td align=\"left\">Heavy precipitation</td><td align=\"left\">− 0.05</td><td align=\"left\">− 0.07 to − 0.03</td><td align=\"left\">< 0.001</td></tr></tbody></table><table-wrap-foot><p>Explanatory variables are related to the outcome with a 1 week time-lag (the weather in 1 week determines the following week’s number of cases).</p></table-wrap-foot></table-wrap></p><p id=\"Par7\">To predict the number of <italic>Campylobacter</italic> cases in 2000–2015, the Poisson models were applied to observed climate data from 2000–2015 (separately for winter and summer months). We used the resulting predictions as the disease baseline. Compared to observed weekly number of cases at municipality level during 2000–2015, these predictions were overall 93.3% accurate (data not shown). We estimated the effects of arbitrary climate changes in our models by varying the different explanatory variables (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>). For instance, a 1 mm increase in precipitation (all other variables remaining unchanged) in any municipality in any week during the summer will result in a 38% increase in the number of <italic>Campylobacter</italic> cases in that municipality the following week.<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Quantitative translation of weather impact on <italic>Campylobacter</italic> cases in any municipality in the Nordic countries.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Scenario</th><th align=\"left\">Number of cases</th><th align=\"left\">Additional cases</th><th align=\"left\">% change</th></tr></thead><tbody><tr><td align=\"left\">Normal</td><td align=\"left\">16</td><td align=\"left\">n.a.</td><td align=\"left\">n.a.</td></tr><tr><td align=\"left\">1 °C increase in mean temperature</td><td align=\"left\">18</td><td align=\"left\">2</td><td align=\"left\">13</td></tr><tr><td align=\"left\">1 mm increase in precipitation during the summer</td><td align=\"left\">22</td><td align=\"left\">6</td><td align=\"left\">38</td></tr><tr><td align=\"left\">One additional heat wave<sup>a</sup> in the summer</td><td align=\"left\">14</td><td align=\"left\">-2</td><td align=\"left\">-13</td></tr><tr><td align=\"left\">One additional heavy precipitation<sup>b</sup> event in the summer</td><td align=\"left\">24</td><td align=\"left\">8</td><td align=\"left\">50</td></tr></tbody></table><table-wrap-foot><p>The hypothetical situation where one scenario event takes place in any municipality in a given week, and the resulting number of (additional) Campylobacter cases the following week because of this event.</p><p><sup>a</sup>Heat wave: three consecutive days with temperatures exceeding 99th percentile of the daily maximum temperature from 2000 to 2015.</p><p><sup>b</sup>Heavy precipitation: a day with precipitation exceeding 95th percentile of daily precipitation from 2000 to 2015.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec4\"><title>Predicting <italic>Campylobacter</italic> in the future using climate change projections</title><p id=\"Par8\">Our predictions indicate that <italic>Campylobacter</italic> cases in the four Nordic countries combined can increase by 25% by the end of the 2040s and 196% by the end of the 2080s compared to the predicted baseline of 2000–2015 (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). These impacts vary with country and time period with the highest increases predicted in Denmark and Norway during the late part of the time period (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Number of <italic>Campylobacter</italic> cases estimated at baseline and predicted for future scenarios.</p></caption><graphic xlink:href=\"41598_2020_70593_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par9\">Our models also predict a change in the future seasonal distribution of cases. At present, <italic>Campylobacter</italic> cases increase during the spring and summer and almost half of the annual total cases are reported between July and September alone (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). During 2040–2059, this pattern will remain similar although the high seasons extends until November. For the later scenarios, the seasonal variation has become less pronounced with the number of cases increasing from April and remaining higher until November. Here, only 32% of cases will be reported in July–September.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Seasonal distribution of <italic>Campylobacter</italic> cases observed at baseline and predicted for the future.</p></caption><graphic xlink:href=\"41598_2020_70593_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par10\">Using estimations of future population sizes, we translated the predicted number of cases to incidences (Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref>), showing that the average annual incidence of <italic>Campylobacter</italic> per 100,000 persons is expected to increase from 42 in 2000–2015 to 117 in 2080–2089 (Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref>). These results were displayed at regional or municipality level, showing that, in Denmark, incidences will increase more in western municipalities (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a). In Finland, the central regions are predicted to experience the highest increases in <italic>Campylobacter</italic> (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>b), while in Norway, the strongest changes are in central and northern regions (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>c). For Sweden, the overall changes are less pronounced but some central and southern regions are still predicted to experience increases in the incidence of <italic>Campylobacter</italic> (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>d).<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Average annual <italic>Campylobacter</italic> incidence and total number of excess cases observed at baseline and predicted for future decades.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Country</th><th align=\"left\">2000–2015</th><th align=\"left\" colspan=\"2\">2040–2049</th><th align=\"left\" colspan=\"2\">2050–2059</th><th align=\"left\" colspan=\"2\">2060–2069</th><th align=\"left\" colspan=\"2\">2070–2079</th><th align=\"left\" colspan=\"2\">2080–2089</th></tr><tr><th align=\"left\">Incidence</th><th align=\"left\">Incidence</th><th align=\"left\">Excess cases</th><th align=\"left\">Incidence</th><th align=\"left\">Excess cases</th><th align=\"left\">Incidence</th><th align=\"left\">Excess cases</th><th align=\"left\">Incidence</th><th align=\"left\">Excess cases</th><th align=\"left\">Incidence</th><th align=\"left\">Excess cases</th></tr></thead><tbody><tr><td align=\"left\">Denmark</td><td align=\"left\">60</td><td align=\"left\">72</td><td align=\"left\">94</td><td align=\"left\">85</td><td align=\"left\">294</td><td align=\"left\">103</td><td align=\"left\">590</td><td align=\"left\">164</td><td align=\"left\">1,092</td><td align=\"left\">170</td><td align=\"left\">1884</td></tr><tr><td align=\"left\">Finland</td><td align=\"left\">45</td><td align=\"left\">45</td><td align=\"left\">195</td><td align=\"left\">58</td><td align=\"left\">795</td><td align=\"left\">71</td><td align=\"left\">1,150</td><td align=\"left\">94</td><td align=\"left\">1547</td><td align=\"left\">107</td><td align=\"left\">876</td></tr><tr><td align=\"left\">Norway</td><td align=\"left\">25</td><td align=\"left\">45</td><td align=\"left\">446</td><td align=\"left\">58</td><td align=\"left\">956</td><td align=\"left\">70</td><td align=\"left\">1,318</td><td align=\"left\">82</td><td align=\"left\">1635</td><td align=\"left\">97</td><td align=\"left\">2,216</td></tr><tr><td align=\"left\">Sweden</td><td align=\"left\">31</td><td align=\"left\">41</td><td align=\"left\">− 157</td><td align=\"left\">54</td><td align=\"left\">-9</td><td align=\"left\">64</td><td align=\"left\">141</td><td align=\"left\">89</td><td align=\"left\">315</td><td align=\"left\">93</td><td align=\"left\">960</td></tr><tr><td align=\"left\">Average</td><td align=\"left\">42</td><td align=\"left\">51</td><td align=\"left\">145</td><td align=\"left\">64</td><td align=\"left\">509</td><td align=\"left\">77</td><td align=\"left\">800</td><td align=\"left\">107</td><td align=\"left\">1,147</td><td align=\"left\">117</td><td align=\"left\">1,484</td></tr></tbody></table><table-wrap-foot><p>Number of cases/100,000 population.</p><p>Compared to the expected number of cases during the time period.</p></table-wrap-foot></table-wrap><fig id=\"Fig3\"><label>Figure 3</label><caption><p>Predicted <italic>Campylobacter</italic> incidences in (<bold>a</bold>) Denmark, (<bold>b</bold>) Finland, (<bold>c</bold>) Norway and (<bold>d</bold>) Sweden, 2000–2090.</p></caption><graphic xlink:href=\"41598_2020_70593_Fig3_HTML\" id=\"MO3\"/></fig></p><p id=\"Par11\">By subtracting the expected number of cases due to ‘natural variation’ in 2040–2089 from the climate-modelled number of cases, we calculated the excess number of cases caused by climate changes alone (i.e. how many cases in the future are caused by climate only and not by natural variation in the form of demographic changes, microbiology, diagnostic practices etc.). These results show that climate changes alone can result in an average 145 excess annual cases of <italic>Campylobacter</italic> by 2040–2049 and almost 1,500 by the end of the 2080s in each country per year (Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref>). The effect varies with country, with less pronounced effects particularly in Sweden (Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref>).</p></sec></sec><sec id=\"Sec5\"><title>Discussion</title><p id=\"Par12\">Using national surveillance data collected from four Nordic countries, we investigated the relationship between the temporal and geographical distribution of <italic>Campylobacter</italic> and climate factors and, based on these relationships, estimated a future scenario for the likely effect of predicted climate changes on campylobacteriosis.</p><p id=\"Par13\">Our models show that the temporal and geographical distribution of <italic>Campylobacter</italic> in Denmark, Finland, Norway and Sweden can be associated with temperature and precipitation. Specifically, increases in temperature and heavy rainfall may increase the number of <italic>Campylobacter</italic> cases reported the following week. On the other hand, according to our models, heat waves and winter precipitation (i.e. both rain and snowfall) may decrease the number of cases reported. Generally, these results support the theory that transmission via the environment could be important for cases which are not explained by consumption or handling of poultry<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. People in the Nordic countries are weather dependent and spend an increasing amount of time outdoors when weather conditions are suitable, resulting in a higher likelihood of contact to environmental infection sources. At the same time, heavy rainfall can mobilise and distribute bacteria, facilitating transmission from contact to water and mud rather than dry surfaces<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref>,<xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>.</p><p id=\"Par14\">Our predicted climate changes were based on projections from the EUR-11 HIRHAM-5 RCM for the RCP8.5 scenario<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref>,<xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. According to these, by the year 2,100, average annual temperatures in the Nordic countries may increase by an average of 4.2 °C, ranging from 3.5 °C in the southernmost parts to 5 °C in northern and central areas. Precipitation is predicted to increase by 25% with only small variations between the countries, and heavy precipitation events can increase by up to 45% across the region. The frequency of heat waves in this region is also affected by climate changes, with an overall 20% increase predicted for most areas (but up to 45% in some parts of Norway).</p><p id=\"Par15\">By incorporating these climate changes into our models, we estimate an overall increase in campylobacteriosis of almost 200% by the end of this century. This translates to nearly 6,000 excess <italic>Campylobacter</italic> cases per year in these four countries which could potentially be linked only to climate changes. Campylobacteriosis is a highly seasonal disease in the Nordic countries, and our models also indicate a future change in the seasonality of transmission. Rather than the current large increase limited to the summer months, the future pattern can show a less pronounced peak during the summer but greater increases earlier in the year, i.e. a prolonged <italic>Campylobacter</italic> season with higher incidences over longer time.</p><p id=\"Par16\">Only few published studies have analysed the association between climate and <italic>Campylobacter</italic> cases—and some with conflicting results. Generally, it appears that <italic>Campylobacter</italic> in chicken flocks fluctuates with temperature and humidity<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup> while incidences in humans may be linked to fluctuations in both temperature and rainfall and especially heavy precipitation events<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR45\">45</xref>–<xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>. Heat waves have been linked to reduced incidences of <italic>Campylobacter</italic><sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Projections of future patterns in campylobacteriosis related to climate changes are also few in the published literature, but available results suggest increases of between 3 and 20% by the end of the century in different geographical locations<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR46\">46</xref>,<xref ref-type=\"bibr\" rid=\"CR49\">49</xref>,<xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>. The results from this study of <italic>Campylobacter</italic> in Scandinavia to some extent confirm these findings while also providing further evidence of a climate dependence. The increases predicted as a result of climate changes are higher than from other studies, however this is expected as the methods, data resolution, geographical areas, demographic and climate change projections are all different.</p><p id=\"Par17\">Given the data available, the results presented here should be considered with respect to several limitations. Our models predicted present levels of campylobacteriosis with an overall accuracy of more than 90%. That they are not more accurate most likely reflects underlying variations in data quality, both with respect to disease and climate data, as well as the fact that other unaccounted factors play different roles in different locations. An important limitation is that disease data have been collected using different methods and different definitions in the four countries. Firstly, our database of observed <italic>Campylobacter</italic> cases from 2000 to 2015 included only domestic cases for Norway and Sweden but both domestic and cases of unknown origin from Denmark and Finland. Assuming that some of these unknown cases were in fact acquired abroad, this may have clouded the association between climate and disease patterns. The ‘time’ variable is also particularly important. The date used for disease data refers to the week in which the sample was taken (Norway and Finland) or in which the sample was received in the diagnostic laboratory (Denmark and Sweden)—and the time-lag between actual symptom onset and these dates is unknown (presumably several days). Therefore, it is difficult to establish a true infection date estimate. Further, the climate data were aggregated from daily to weekly data, which reduces data size but also the likelihood of capturing variations. Spatially, there was a mismatch between the disease data collected at relatively high resolution (down to point-level) and then aggregated to municipality level, and climate data at low resolution (25 × 25 km), which reduced the geographical correlation between disease and climate data and the ability of the models to capture high-resolution variations. This is especially important for extreme weather events, which often occur as well-confined local events. Also, it is likely that for some cases, the exposure leading to infection did not occur at the reported geographical location of each cases. Again, this limitation reduces the chance of catching the association between local weather events which could have affected the risk of exposure. Worth noting is also that we simulated climate changes using outputs from one model and one emission scenario only, limiting the predictions to a single situation rather than a range of different ones. However, this is unlikely to have impacted the general accuracy of the predictions as the both the EUR-11 HIRHAM-5 model and the RCP8.5 scenario are considered robust and often used for similar scientific purposes. Overall, our predictions were made using a relatively simple Poisson model (see Supplementary Methods), and the results should also be considered with reference to these specific modelling constraints. The underlying assumptions for linear and Poisson regressions, particularly the assumption of linearity and equal mean and variance, may not be universally valid in the dataset used (i.e. for different geographical locations). Standard Poisson regression may in this case not be optimal for investigating climate/disease relationships, and more sophisticated dynamic models should also be assessed.</p><p id=\"Par18\">In the context of exploring linkages between climate and disease, it is important to note that many such associations are likely to be indirect. For <italic>Campylobacter</italic> in particular, disease transmission reflects chicken flock infection rates and human behaviour (barbecues, outdoor activities) both of which are also strongly dependent on weather and therefore likely to be altered in a changing climate. In addition, disease incidences are also determined by the structure and function of the socio-economic and public health systems which, given different constraints, may also appear different in the future. In relation to this, our results likely over-estimate the future number of cases as public health systems will adapt to higher incidences by taking stronger measures to reduce the incidence. Finally, because <italic>Campylobacter</italic> is a zoonotic infection, in order to understand disease patterns in the present and future, it is necessary to adopt a One Health approach where evidence and knowledge from the public health, food safety, veterinary and environmental sectors are considered together.</p><p id=\"Par19\">To our knowledge, this is one of the first attempts to describe an association between campylobacteriosis and climate factors using high-quality routinely collected surveillance data and modelling the effect of climate changes on this disease at local and national levels. Overall, the results from our models correlate with published evidence for a <italic>Campylobacter</italic>-climate association. Considering their limitations, the models show that climate changes—particularly increases in rainfall and rainfall intensity—could potentially lead to an increase in the incidence of <italic>Campylobacter</italic> in the Nordic countries. Considering the strong burden of campylobacteriosis across the world, the effects of climate changes on this disease are important to further assess for policy makers to identify potentially vulnerable areas as well as future strategies for public health management and adaptation measures.</p></sec><sec id=\"Sec6\"><title>Methods</title><p id=\"Par20\">Denmark, Finland, Norway and Sweden constitute part of the Nordics, a 7-country region in Northern Europe. In 2015, approximately 26 million people inhabited these four countries in a total of 1,121 municipalities. Human <italic>Campylobacter</italic> infection is under statutory surveillance in all four countries where clinicians or laboratories must notify a confirmed case to national public health authorities: Statens Serum Institut in Denmark (through the national microbiology database, MiBa), the National Institute for Health and Welfare in Finland, The Norwegian Institute of Public Health (through the Norwegian Surveillance System for Communicable Diseases, MSIS) and the Public Health Agency of Sweden (in the national surveillance system SmiNet). The laboratory criteria for a <italic>Campylobacter</italic> diagnosis is similar across the countries: isolation of <italic>Campylobacter</italic> spp. by culture from faeces or blood. Since 2013, the Swedish criteria have also included samples positive for <italic>Campylobacter</italic> using direct PCR on stool samples. During the study period, PCR diagnosis was not routinely established in the other countries. Each case notification contains as a minimum the patient’s name, age and sex, geographical location (address) and a date corresponding to sample taken/sample analysed/diagnosis/registration. For this study, we used <italic>Campylobacter</italic> infections notified in Denmark, Finland and Sweden from 1st January 2000 until 31st December 2015 and Norway from 1st January 2000 until 31st December 2014<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>. For Sweden and Norway, we only included “domestically acquired” infections (i.e. excluded travel acquired infections or infections with unknown origin). For Denmark and Finland, we included both domestic infections and those of unknown origin as the proportion of unknown infections was more than 80%<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup> and the exclusion of these would have resulted in a significant loss of data. The disease data were aggregated to total number of cases per week per municipality for the study period.</p><p id=\"Par21\">Baseline climate data from 2000 to 2015 at 0.25° × 0.25° resolution (approximately 25 × 25 km) for all four countries were extracted from the E-OBS daily gridded dataset developed by the European Climate Assessment & Dataset project<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>. The climate data were aggregated to municipality level using zonal statistics in a Geographical Information System (GIS). From the daily data, for each municipality, we calculated weekly average temperature, precipitation, number of heat waves and number of heavy precipitation events. Heavy precipitation was defined as a day with precipitation exceeding the 95th percentile of daily precipitation using as baseline the municipal average from 2000–2015 (only days with precipitation > 1 mm were considered). In order to explore how precipitation patterns and associations varied throughout the year, we calculated precipitation for both ‘winter’ (October–March) and ‘summer’ months (April–September). A heat wave was defined as three consecutive days in which the temperature exceeded the 99th percentile of the daily maximum temperature using as baseline the municipal average from 2000 to 2015. The definition of heat waves was applied only for the summer months (April–September).</p><p id=\"Par22\">The final database of disease and climate included per municipality per week and year from 2000 to 2015: number of reported <italic>Campylobacter</italic> cases, precipitation and temperature, the number of heat waves and the number of days with heavy precipitation. A total of 163,997 data records were included in the database, representing 416 municipalities. Of these, a random sample of 10% (16,400 records) were omitted from the initial data analysis and used to validate the predictive fit of the final model.</p><p id=\"Par23\">Future climate change scenarios for the period 2040–2089 for all four countries were projected using bias-corrected<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup> outputs from the EUR-11 HIRHAM-5 regional atmospheric climate model (RCM) with a resolution of 12.5 km, generated by the EURO-CORDEX ensemble and based on the latest scenarios defined by the Intergovernmental Panel on Climate Change (IPCC)<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. We used the RCP8.5 emission ‘worst case business as usual’ scenario corresponding to a high increasing greenhouse gas emissions pathway, not including specific climate mitigation targets<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>. We chose this scenario because observed climatic changes correspond to those predicted by the RCP8.5 scenario<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>. Absolute daily values for temperature and precipitation in 2040–8989 for all municipalities were calculated by comparing the EUR-11 projections to the municipal average from 2000 to 2015. From these, the weekly average temperature, precipitation, number of heat waves and number of heavy precipitation events at municipality level were calculated and finally aggregated to ten-year averages at a weekly basis.</p><p id=\"Par24\">Data were explored using descriptive statistics. We modelled the relationship between <italic>Campylobacter</italic> cases and climate using three approaches: standard Poisson regression, zero-inflated Poisson regression and standard Poisson regression omitting all observations with zero <italic>Campylobacter</italic> cases. These approaches were chosen to identify which model best fitted the observed data, considering the large amount of zero counts in the data. Time-lags were investigated to identify the optimal interval between when a <italic>Campylobacter</italic> case was reported and when the climate variables were measured. The models were constructed using a backwards stepwise multivariate approach where the full model containing all explanatory variables was run and the least significant variables removed one by one until only significant variables remained. The final model was adjusted for the effect of week (season), year (changing notification rates) and municipality (geographical variation) and for interaction between the climate variables. Separate models were constructed for the winter (October–March) and summer (April–September) periods to account for the effect of precipitation and temperatures varying throughout the year. Model predictive fits were evaluated using the 10% of data records omitted from the analysis. The models and underlying assumptions are described in detail in the Supplementary Methods.</p><p id=\"Par25\">The best fit final models were applied to the baseline climate data and the EUR-11 climate change scenario data, resulting in the estimated number of average cases per municipality per week for 2000–2015 and in ten-year intervals from 2040 to 2089. We compared the weekly predicted number of cases per municipality for the baseline of 2000–2015 to the observed number in order to estimate the predictive accuracy (in percent) of the models. We converted the future predicted number of <italic>Campylobacter</italic> cases per municipality or region to future incidences per region/municipality using projected population sizes obtained from the National Statistical Offices in each country.</p><p id=\"Par26\">The excess number of <italic>Campylobacter</italic> cases, caused by climate change alone (i.e. adjusted for ‘natural variation’), was calculated firstly by estimating the expected monthly and annual average number of cases per municipality for 2040–2089 (natural variation) using the average proportional change in the number of cases for each municipality from year to year during the baseline 2000–2015, assuming similar variability for the years 2040–2089. In order to calculate excess cases, we then subtracted these from the number of cases predicted from the initial climate change scenario modelling.</p><p id=\"Par27\">Data were analysed using the STATA software 14.0 (Statacorp., Lakeway, TX, USA) and GIS analyses undertaken using QGIS 2.14.11. (QuantumGIS, Open Source Geospatial Federation Project, <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.qgis.org\">https://www.qgis.org</ext-link>).</p><sec id=\"Sec7\"><title>Ethical considerations</title><p id=\"Par28\">This study was approved under the general agreement for non-interventional database studies between the Danish Data Protection Agency and Statens Serum Institut, reference number 2008-54-0474. According to Danish regulations, ethical committee approval is not required for studies which do not involve analysis of biological material from human subjects. The use of national surveillance data does not require informed consent from subjects.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec8\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70593_MOESM1_ESM.docx\"><caption><p>Supplementary file1</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70593-y.</p></sec><ack><title>Acknowledgements</title><p>We are very grateful to all members of the KLIMAFORSK Group for providing data, advice on data sources, analyses and interpretation, and comments on all outputs. This study was included under the KLIMAFORSK project which received funding from the Research Council of Norway.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>K.G.K. and S.E. designed the study, K.G.K. collected data, performed the analyses, constructed predictive models and maps; K.N., B.G.-H., L.S.S., R.R.-F. and L.T. extracted national disease data and interpreted epidemiological outputs; M.R.J., R.R. and W.K.W. extracted, processed and interpreted climate data; all of the authors jointly wrote the paper.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>The data generated and analysed during the current study are not publicly available because they are classified as sensitive surveillance data, but are available from the corresponding author on reasonable request. All methods were carried out in accordance with relevant guidelines and regulations.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par29\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><collab>European Food Safety Authority & European Centre for Disease Prevention and Control</collab><etal/></person-group><article-title>The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2015</article-title><source>EFSA J.</source><year>2016</year><volume>14</volume><fpage>1</fpage><lpage>231</lpage></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boysen</surname><given-names>L</given-names></name><etal/></person-group><article-title>Source attribution of human campylobacteriosis in Denmark</article-title><source>Epidemiol. Infect.</source><year>2014</year><volume>142</volume><fpage>1599</fpage><lpage>1608</lpage><pub-id pub-id-type=\"pmid\">24168860</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>MacDonald</surname><given-names>E</given-names></name><etal/></person-group><article-title>Risk factors for sporadic domestically acquired campylobacter infections in Norway 2010–2011: A national prospective case-control study</article-title><source>PLoS ONE</source><year>2015</year><volume>10</volume><fpage>e0139636</fpage><pub-id pub-id-type=\"pmid\">26431341</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuhn</surname><given-names>KG</given-names></name><name><surname>Nielsen</surname><given-names>EM</given-names></name><name><surname>Mølbak</surname><given-names>K</given-names></name><name><surname>Ethelberg</surname><given-names>S</given-names></name></person-group><article-title>Determinants of sporadic Campylobacter infections in Denmark: A nationwide case–control study among children and young adults</article-title><source>Clin. Epidemiol.</source><year>2018</year><volume>10</volume><fpage>1695</fpage><lpage>1707</lpage><pub-id pub-id-type=\"pmid\">30538574</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gras</surname><given-names>LM</given-names></name><etal/></person-group><article-title>Increased risk for <italic>Campylobacter jejuni</italic> and <italic>C. coli</italic> infection of pet origin in dog owners and evidence for genetic association between strains causing infection in humans and their pets</article-title><source>Epidemiol. Infect.</source><year>2013</year><volume>141</volume><fpage>2526</fpage><lpage>2535</lpage><pub-id pub-id-type=\"pmid\">23445833</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ravel</surname><given-names>A</given-names></name><name><surname>Pintar</surname><given-names>K</given-names></name><name><surname>Nesbitt</surname><given-names>A</given-names></name><name><surname>Pollari</surname><given-names>F</given-names></name></person-group><article-title>Non food-related risk factors of campylobacteriosis in Canada: A matched case–control study</article-title><source>BMC Public Health</source><year>2016</year><volume>16</volume><fpage>1016</fpage><pub-id pub-id-type=\"pmid\">27677338</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuhn</surname><given-names>KG</given-names></name><name><surname>Nielsen</surname><given-names>EM</given-names></name><name><surname>Mølbak</surname><given-names>K</given-names></name><name><surname>Ethelberg</surname><given-names>S</given-names></name></person-group><article-title>Epidemiology of campylobacteriosis in Denmark 2000–2015</article-title><source>Zoonoses Public Health</source><year>2018</year><volume>65</volume><fpage>59</fpage><lpage>66</lpage><pub-id pub-id-type=\"pmid\">28597535</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Strachan</surname><given-names>NJC</given-names></name><etal/></person-group><article-title>Identifying the seasonal origins of human campylobacteriosis</article-title><source>Epidemiol. Infect.</source><year>2013</year><volume>141</volume><fpage>1267</fpage><lpage>1275</lpage><pub-id pub-id-type=\"pmid\">22989449</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Patrick</surname><given-names>ME</given-names></name><etal/></person-group><article-title>Effects of climate on incidence of <italic>Campylobacter</italic> spp. in humans and prevalence in broiler flocks in Denmark</article-title><source>Appl. Environ. Microbiol.</source><year>2004</year><volume>70</volume><fpage>7474</fpage><lpage>7480</lpage><pub-id pub-id-type=\"pmid\">15574950</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>David</surname><given-names>JM</given-names></name><etal/></person-group><article-title>Do contamination of and exposure to chicken meat and water drive the temporal dynamics of Campylobacter cases?</article-title><source>Epidemiol. Infect.</source><year>2017</year><volume>145</volume><fpage>3191</fpage><lpage>3203</lpage><pub-id pub-id-type=\"pmid\">29022517</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McMichael</surname><given-names>AJ</given-names></name><name><surname>Woodruff</surname><given-names>RE</given-names></name><name><surname>Hales</surname><given-names>S</given-names></name></person-group><article-title>Climate change and human health: present and future risks</article-title><source>Lancet Lond. Engl.</source><year>2006</year><volume>367</volume><fpage>859</fpage><lpage>869</lpage></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Watts</surname><given-names>N</given-names></name><etal/></person-group><article-title>The Lancet Countdown on health and climate change: From 25 years of inaction to a global transformation for public health</article-title><source>Lancet</source><year>2018</year><volume>391</volume><fpage>581</fpage><lpage>630</lpage><pub-id pub-id-type=\"pmid\">29096948</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Baylis</surname><given-names>M</given-names></name></person-group><article-title>Potential impact of climate change on emerging vector-borne and other infections in the UK</article-title><source>Environ. Health Glob. Access Sci. Source</source><year>2017</year><volume>16</volume><fpage>112</fpage></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Murdock</surname><given-names>CC</given-names></name><name><surname>Sternberg</surname><given-names>ED</given-names></name><name><surname>Thomas</surname><given-names>MB</given-names></name></person-group><article-title>Malaria transmission potential could be reduced with current and future climate change</article-title><source>Sci. Rep.</source><year>2016</year><volume>6</volume><fpage>27771</fpage><pub-id pub-id-type=\"pmid\">27324146</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Semenza</surname><given-names>JC</given-names></name><etal/></person-group><article-title>Climate change projections of West Nile virus infections in Europe: Implications for blood safety practices</article-title><source>Environ. Health Glob. Access. Sci. Source</source><year>2016</year><volume>15</volume><issue>Suppl 1</issue><fpage>28</fpage></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Walker</surname><given-names>JT</given-names></name></person-group><article-title>The influence of climate change on waterborne disease and Legionella: A review</article-title><source>Perspect. Public Health</source><year>2018</year><volume>138</volume><fpage>282</fpage><lpage>286</lpage><pub-id pub-id-type=\"pmid\">30156484</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>CY</given-names></name><name><surname>Zhang</surname><given-names>A</given-names></name></person-group><article-title>Climate and air pollution alter incidence of tuberculosis in Beijing, China</article-title><source>Ann. Epidemiol.</source><year>2019</year><volume>37</volume><fpage>71</fpage><lpage>76</lpage><pub-id pub-id-type=\"pmid\">31473123</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Akil</surname><given-names>L</given-names></name><name><surname>Ahmad</surname><given-names>HA</given-names></name><name><surname>Reddy</surname><given-names>RS</given-names></name></person-group><article-title>Effects of climate change on Salmonella infections</article-title><source>Foodborne Pathog. Dis.</source><year>2014</year><volume>11</volume><fpage>974</fpage><lpage>980</lpage><pub-id pub-id-type=\"pmid\">25496072</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jiang</surname><given-names>C</given-names></name><etal/></person-group><article-title>Climate change, extreme events and increased risk of salmonellosis in Maryland, USA: Evidence for coastal vulnerability</article-title><source>Environ. Int.</source><year>2015</year><volume>83</volume><fpage>58</fpage><lpage>62</lpage><pub-id pub-id-type=\"pmid\">26093493</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Semenza</surname><given-names>JC</given-names></name><etal/></person-group><article-title>Climate change impact assessment of food- and waterborne diseases</article-title><source>Crit. Rev. Environ. Sci. Technol.</source><year>2012</year><volume>42</volume><fpage>857</fpage><lpage>890</lpage><pub-id pub-id-type=\"pmid\">24808720</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kovats</surname><given-names>RS</given-names></name><etal/></person-group><article-title>Climate variability and campylobacter infection: An international study</article-title><source>Int. J. Biometeorol.</source><year>2005</year><volume>49</volume><fpage>207</fpage><lpage>214</lpage><pub-id pub-id-type=\"pmid\">15565278</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Milazzo</surname><given-names>A</given-names></name><etal/></person-group><article-title>The effects of ambient temperature and heatwaves on daily Campylobacter cases in Adelaide, Australia, 1990–2012</article-title><source>Epidemiol. Infect.</source><year>2017</year><volume>145</volume><fpage>2603</fpage><lpage>2610</lpage><pub-id pub-id-type=\"pmid\">28693637</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tam</surname><given-names>CC</given-names></name><name><surname>Rodrigues</surname><given-names>LC</given-names></name><name><surname>O’Brien</surname><given-names>SJ</given-names></name><name><surname>Hajat</surname><given-names>S</given-names></name></person-group><article-title>Temperature dependence of reported Campylobacter infection in England, 1989–1999</article-title><source>Epidemiol. Infect.</source><year>2006</year><volume>134</volume><fpage>119</fpage><lpage>125</lpage><pub-id pub-id-type=\"pmid\">16409658</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weisent</surname><given-names>J</given-names></name><name><surname>Seaver</surname><given-names>W</given-names></name><name><surname>Odoi</surname><given-names>A</given-names></name><name><surname>Rohrbach</surname><given-names>B</given-names></name></person-group><article-title>The importance of climatic factors and outliers in predicting regional monthly campylobacteriosis risk in Georgia, USA</article-title><source>Int. J. Biometeorol.</source><year>2014</year><volume>58</volume><fpage>1865</fpage><lpage>1878</lpage><pub-id pub-id-type=\"pmid\">24458769</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yun</surname><given-names>J</given-names></name><etal/></person-group><article-title>Association between the ambient temperature and the occurrence of human Salmonella and Campylobacter infections</article-title><source>Sci. Rep.</source><year>2016</year><volume>6</volume><fpage>28442</fpage><pub-id pub-id-type=\"pmid\">27324200</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Louis</surname><given-names>VR</given-names></name><etal/></person-group><article-title>Temperature-driven campylobacter seasonality in England and Wales</article-title><source>Appl. Environ. Microbiol.</source><year>2005</year><volume>71</volume><fpage>85</fpage><lpage>92</lpage><pub-id pub-id-type=\"pmid\">15640174</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Semenza</surname><given-names>JC</given-names></name><etal/></person-group><article-title>Determinants and drivers of infectious disease threat events in Europe</article-title><source>Emerg. Infect. Dis.</source><year>2016</year><volume>22</volume><fpage>581</fpage><lpage>589</lpage><pub-id pub-id-type=\"pmid\">26982104</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Soneja</surname><given-names>S</given-names></name><etal/></person-group><article-title>Extreme precipitation events and increased risk of campylobacteriosis in Maryland, USA</article-title><source>Environ. Res.</source><year>2016</year><volume>149</volume><fpage>216</fpage><lpage>221</lpage><pub-id pub-id-type=\"pmid\">27214137</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Auld</surname><given-names>H</given-names></name><name><surname>MacIver</surname><given-names>D</given-names></name><name><surname>Klaassen</surname><given-names>J</given-names></name></person-group><article-title>Heavy rainfall and waterborne disease outbreaks: the Walkerton example</article-title><source>J. Toxicol. Environ. Health A</source><year>2004</year><volume>67</volume><fpage>1879</fpage><lpage>1887</lpage><pub-id pub-id-type=\"pmid\">15371222</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thomas</surname><given-names>KM</given-names></name><etal/></person-group><article-title>A role of high impact weather events in waterborne disease outbreaks in Canada, 1975–2001</article-title><source>Int. J. Environ. Health Res.</source><year>2006</year><volume>16</volume><fpage>167</fpage><lpage>180</lpage><pub-id pub-id-type=\"pmid\">16611562</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nichols</surname><given-names>G</given-names></name><name><surname>Lane</surname><given-names>C</given-names></name><name><surname>Asgari</surname><given-names>N</given-names></name><name><surname>Verlander</surname><given-names>NQ</given-names></name><name><surname>Charlett</surname><given-names>A</given-names></name></person-group><article-title>Rainfall and outbreaks of drinking water related disease and in England and Wales</article-title><source>J. Water Health</source><year>2009</year><volume>7</volume><fpage>1</fpage><lpage>8</lpage><pub-id pub-id-type=\"pmid\">18957770</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><mixed-citation publication-type=\"other\">Guzman-Herrador, B. <italic>et al.</italic> Waterborne outbreaks in the Nordic countries, 1998–2012. <italic>Euro Surveill. Bull. Eur. Sur Mal. Transm. Eur. Commun. Dis. Bull.</italic><bold>20</bold> (2015).</mixed-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Harder-Lauridsen</surname><given-names>NM</given-names></name><name><surname>Kuhn</surname><given-names>KG</given-names></name><name><surname>Erichsen</surname><given-names>AC</given-names></name><name><surname>Mølbak</surname><given-names>K</given-names></name><name><surname>Ethelberg</surname><given-names>S</given-names></name></person-group><article-title>Gastrointestinal illness among triathletes swimming in non-polluted versus polluted seawater affected by heavy rainfall, Denmark, 2010–2011</article-title><source>PLoS ONE</source><year>2013</year><volume>8</volume><fpage>e78371</fpage><pub-id pub-id-type=\"pmid\">24244306</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuhn</surname><given-names>KG</given-names></name><etal/></person-group><article-title>Epidemiological and serological investigation of a waterborne <italic>Campylobacter jejuni</italic> outbreak in a Danish town</article-title><source>Epidemiol. Infect.</source><year>2017</year><volume>145</volume><fpage>701</fpage><lpage>709</lpage><pub-id pub-id-type=\"pmid\">27903324</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gibney</surname><given-names>KB</given-names></name><name><surname>O’Toole</surname><given-names>J</given-names></name><name><surname>Sinclair</surname><given-names>M</given-names></name><name><surname>Leder</surname><given-names>K</given-names></name></person-group><article-title>Disease burden of selected gastrointestinal pathogens in Australia, 2010</article-title><source>Int. J. Infect. Dis.</source><year>2014</year><volume>28</volume><fpage>176</fpage><lpage>185</lpage><pub-id pub-id-type=\"pmid\">25281904</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Havelaar</surname><given-names>AH</given-names></name><etal/></person-group><article-title>Disease burden of foodborne pathogens in the Netherlands, 2009</article-title><source>Int. J. Food Microbiol.</source><year>2012</year><volume>156</volume><fpage>231</fpage><lpage>238</lpage><pub-id pub-id-type=\"pmid\">22541392</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Havelaar</surname><given-names>AH</given-names></name><etal/></person-group><article-title>World Health Organization Global Estimates and Regional Comparisons of the Burden of Foodborne Disease in 2010</article-title><source>PLOS Med.</source><year>2015</year><volume>12</volume><fpage>1</fpage><lpage>13</lpage></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>de Noordhout</surname><given-names>CM</given-names></name><etal/></person-group><article-title>Current and future Disability-Adjusted Life Years (DALYs) of Salmonella and Campylobacter in Belgium</article-title><source>Arch. Public Health</source><year>2015</year><volume>73</volume><fpage>K3</fpage></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>de Noordhout</surname><given-names>CM</given-names></name><etal/></person-group><article-title>Burden of salmonellosis, campylobacteriosis and listeriosis: A time series analysis, Belgium, 2012 to 2020</article-title><source>Eurosurveillance</source><year>2017</year><volume>22</volume><fpage>30615</fpage></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zeigler</surname><given-names>M</given-names></name><etal/></person-group><article-title>Outbreak of campylobacteriosis associated with a long-distance obstacle adventure race-Nevada, October 2012</article-title><source>MMWR Morb. Mortal. Wkly. Rep.</source><year>2014</year><volume>63</volume><fpage>375</fpage><lpage>378</lpage><pub-id pub-id-type=\"pmid\">24785983</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stuart</surname><given-names>TL</given-names></name><etal/></person-group><article-title>Campylobacteriosis outbreak associated with ingestion of mud during a mountain bike race</article-title><source>Epidemiol. Infect.</source><year>2010</year><volume>138</volume><fpage>1695</fpage><lpage>1703</lpage><pub-id pub-id-type=\"pmid\">20334726</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jacob</surname><given-names>D</given-names></name><etal/></person-group><article-title>EURO-CORDEX: New high-resolution climate change projections for European impact research</article-title><source>Reg. Environ. Change</source><year>2014</year><volume>14</volume><fpage>563</fpage><lpage>578</lpage></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kotlarski</surname><given-names>S</given-names></name><etal/></person-group><article-title>Regional climate modeling on European scales: A joint standard evaluation of the EURO-CORDEX RCM ensemble</article-title><source>Geosci. Model Dev.</source><year>2014</year><volume>7</volume><fpage>1297</fpage><lpage>1333</lpage></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sandberg</surname><given-names>M</given-names></name><etal/></person-group><article-title>Risk factors for Campylobacter colonization in Danish broiler flocks, 2010 to 2011</article-title><source>Poult. Sci.</source><year>2015</year><volume>94</volume><fpage>447</fpage><lpage>453</lpage><pub-id pub-id-type=\"pmid\">25638473</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sanderson</surname><given-names>RA</given-names></name><etal/></person-group><article-title>Spatio-temporal models to determine association between Campylobacter cases and environment</article-title><source>Int. J. Epidemiol.</source><year>2018</year><volume>47</volume><fpage>202</fpage><lpage>216</lpage><pub-id pub-id-type=\"pmid\">29069406</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sterk</surname><given-names>A</given-names></name><name><surname>Schijven</surname><given-names>J</given-names></name><name><surname>de Nijs</surname><given-names>T</given-names></name><name><surname>Husman</surname><given-names>AMR</given-names></name></person-group><article-title>Direct and indirect effects of climate change on the risk of infection by water-transmitted pathogens</article-title><source>Environ. Sci. Technol.</source><year>2013</year><volume>47</volume><fpage>12648</fpage><lpage>12660</lpage><pub-id pub-id-type=\"pmid\">24125400</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Djennad</surname><given-names>A</given-names></name><etal/></person-group><article-title>Seasonality and the effects of weather on Campylobacter infections</article-title><source>BMC Infect. Dis.</source><year>2019</year><volume>19</volume><fpage>255</fpage><pub-id pub-id-type=\"pmid\">30866826</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lake</surname><given-names>IR</given-names></name><etal/></person-group><article-title>Exploring Campylobacter seasonality across Europe using The European Surveillance System (TESSy), 2008 to 2016</article-title><source>Eurosurveillance</source><year>2019</year><volume>24</volume><fpage>1800028</fpage></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cullen</surname><given-names>E</given-names></name></person-group><article-title>The impact of climate change on the future incidence of specified foodborne diseases in Ireland</article-title><source>Epidemiology</source><year>2009</year><volume>20</volume><fpage>S227</fpage></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McBride</surname><given-names>G</given-names></name><name><surname>Tait</surname><given-names>A</given-names></name><name><surname>Slaney</surname><given-names>D</given-names></name></person-group><article-title>Projected changes in reported campylobacteriosis and cryptosporidiosis rates as a function of climate change: A New Zealand study</article-title><source>Stoch. Environ. Res. Risk Assess.</source><year>2014</year><volume>28</volume><fpage>2133</fpage><lpage>2147</lpage></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuhn</surname><given-names>KG</given-names></name><etal/></person-group><article-title>Campylobacteriosis in the Nordic countries from 2000 to 2015: Trends in time and space</article-title><source>Scand. J. Public Health.</source><year>2019</year><pub-id pub-id-type=\"doi\">10.1177/1403494819875020</pub-id><pub-id pub-id-type=\"pmid\">31763953</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van den Besselaar</surname><given-names>EJM</given-names></name><name><surname>Haylock</surname><given-names>MR</given-names></name><name><surname>van der Schrier</surname><given-names>G</given-names></name><name><surname>Klein Tank</surname><given-names>AMG</given-names></name></person-group><article-title>A European daily high-resolution observational gridded data set of sea level pressure</article-title><source>J. Geophys. Res.</source><year>2011</year><volume>116</volume><fpage>1</fpage><lpage>11</lpage></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dosio</surname><given-names>A</given-names></name><name><surname>Paruolo</surname><given-names>P</given-names></name></person-group><article-title>Bias correction of the ENSEMBLES high-resolution climate change projections for use by impact models: Evaluation on the present climate</article-title><source>J. Geophys. Res. Atmos.</source><year>2011</year><volume>116</volume><fpage>1</fpage><lpage>22</lpage></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Riahi</surname><given-names>K</given-names></name><etal/></person-group><article-title>RCP 8.5—A scenario of comparatively high greenhouse gas emissions</article-title><source>Clim. Change</source><year>2011</year><volume>109</volume><fpage>33</fpage></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brown</surname><given-names>PT</given-names></name><name><surname>Caldeira</surname><given-names>K</given-names></name></person-group><article-title>Greater future global warming inferred from Earth’s recent energy budget</article-title><source>Nature</source><year>2017</year><volume>552</volume><fpage>45</fpage><lpage>50</lpage><pub-id pub-id-type=\"pmid\">29219964</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807840</article-id><article-id pub-id-type=\"pmc\">PMC7431570</article-id><article-id pub-id-type=\"publisher-id\">70837</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70837-x</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Liberalizing the killing of endangered wolves was associated with more disappearances of collared individuals in Wisconsin, USA</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Santiago-Ávila</surname><given-names>Francisco J.</given-names></name><address><email>santiagoavil@wisc.edu</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Chappell</surname><given-names>Richard J.</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Treves</surname><given-names>Adrian</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.14003.36</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2167 3675</institution-id><institution>Nelson Institute for Environmental Studies, </institution><institution>University of Wisconsin – Madison, </institution></institution-wrap>Madison, USA </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.14003.36</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2167 3675</institution-id><institution>Department of Biostatistics & Medical Informatics, </institution><institution>University of Wisconsin – Madison, </institution></institution-wrap>Madison, USA </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13881</elocation-id><history><date date-type=\"received\"><day>5</day><month>2</month><year>2020</year></date><date date-type=\"accepted\"><day>4</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Although poaching (illegal killing) is an important cause of death for large carnivores globally, the effect of lethal management policies on poaching is unknown for many populations. Two opposing hypotheses have been proposed: liberalizing killing may decrease poaching incidence (‘tolerance hunting’) or increase it (‘facilitated poaching’). For gray wolves in Wisconsin, USA, we evaluated how five causes of death and disappearances of monitored, adult wolves were influenced by policy changes. We found slight decreases in reported wolf poaching hazard and incidence during six liberalized killing periods, but that was outweighed by larger increases in hazard and incidence of disappearance. Although the observed increase in the hazard of disappearance cannot be definitively shown to have been caused by an increase in cryptic poaching, we discuss two additional independent lines of evidence making this the most likely explanation for changing incidence among n = 513 wolves’ deaths or disappearances during 12 replicated changes in policy. Support for the facilitated poaching hypothesis suggests the increase (11–34%) in disappearances reflects that poachers killed more wolves and concealed more evidence when the government relaxed protections for endangered wolves. We propose a refinement of the hypothesis of ‘facilitated poaching’ that narrows the cognitive and behavioral mechanisms underlying wolf-killing.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Conservation biology</kwd><kwd>Environmental impact</kwd></kwd-group><funding-group><award-group><funding-source><institution>Nelson Institute for Environmental Studies</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>Therese Foundation, Inc.</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>UCLA Law School Animal Law</institution></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Globally, loss of large predators has contributed to simplification of trophic structures, lower biodiversity and degradation of ecosystem functions<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. It is widely acknowledged that humans are responsible for more large carnivore deaths than any other cause<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>, although the scientific debate about the sustainability of this killing remains far from settled for many large carnivore populations<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. Many policies for large carnivores focus on limiting or regulating human-caused mortality, and many management decisions rely on estimates of human-caused mortality and on understanding the policy effects on such mortality. Therefore, biased estimates of human-caused mortality patterns can undermine policy goals and evaluations (e.g., recolonization by endangered species, restoring ecosystem processes), and harm carnivore population recovery or stability<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>–<xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. More broadly, accurate estimates of policy effects on illegal killing (‘poaching’ hereafter) of wild animals can improve enforcement of laws for nature protection and adherence to national and international treaties relating to the protection or the restoration of endangered species, ecosystems, biodiversity, and interdiction of unregulated wildlife trade.</p><p id=\"Par3\">Of all direct killing by humans, poaching is the primary cause of death in many carnivore populations<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>–<xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>, slowing population growth<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>–<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup> or hindering recolonization of historic range<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Poaching is extremely difficult to detect, measure and prevent. Given its illegal nature, poachers often conceal evidence from management authorities tasked with monitoring marked animals. When authorities find the body of a poached animal they might detect that the individual was poached, but many wild animals die undetected. Measurement uncertainty rises from low but non-zero in the latter case to very high when poachers conceal evidence or when marked animals elude monitoring by those authorities. ‘Cryptic poaching’<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> refers to this type of unreported, concealed illegal killing. Several studies have estimated it as rivaling or exceeding the subset of reported poaching detected and measured by authorities<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. Therefore, in places where poaching is often cryptic, official estimates of mortality process and pattern are systematically biased to under-estimate the risk of poaching, unless analysts adequately account for uncertainty. Such accounting has been facilitated by a variety of statistical techniques developed since 2011<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>.</p><p id=\"Par4\">Traditional methods for estimating mortality hazard (i.e., the instantaneous probability of an event such as poaching occurring), incidence (i.e., probability of an event such as poaching occurring in the presence of other mortality sources), and for partitioning those rate parameters among various human and non-human causes typically require data from marked individuals (e.g., collared animals recaptured dead or alive). Yet these traditional methods assume that the marked individuals that are never recaptured or recovered (disappeared hereafter) had suffered from similar causes of death as those recaptured. That assumption proved highly inaccurate for wolves, because systematic measurement biases caused by disappearances and other forms of uncertainty disproportionately affect poaching rather than other causes of death. Failure to account for these biases has led to under-estimating poaching, misidentifying the major cause of death and failing to intervene effectively against wolf-poaching<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. Liberg et al.<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>, to our knowledge, was the first to correct for this underestimation and quantify cryptic poaching. Their analysis of Scandinavian wolf deaths estimated poaching at half of total mortality (51%), with two-thirds attributed to cryptic poaching. Similarly, a later estimate for Wisconsin wolves put the proportion of cryptic poaching at 50%<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. Efforts to correct poaching estimates for four endangered wolf populations in the contiguous USA found traditional methods overestimated the relative risk of legal killing by 5–16% and underestimated it for poaching by 17–44%<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. That study also concluded that poaching (observed and cryptic) was the major cause of death for all studied wolf populations.</p><p id=\"Par5\">Quantifying poaching hazard, incidence, and patterns (including its cryptic variant), and how these might be affected by policy, can improve the design of management interventions and thereby hasten restoration and conservation. However, the scientific literature has just begun evaluating the influence of policies on poaching hazard and incidence. A long-held assumption has been that some predator control (e.g.: special permits for killing or hunting seasons) might increase tolerance for controversial species and thus reduce poaching; a claim first argued in a legal brief by the U.S. government in 2006 (Humane Society of US v. Kempthorne, docket DC 06-1279) and articulated as a scientific hypothesis in<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>, and later developed and renamed ‘tolerance hunting’<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>–<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. In Wisconsin, USA a series of studies have taken up the question using mortality data. One early study examined reported poaching variation<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup> to hypothesize that frustration with inconsistent management may lead to increased poaching, and colleagues modeled wolf demographic parameters in relation to policy changes (for others, see<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>). However, these studies provide weak inference due to several shortcomings: reliance on correlative analyses, failure to consider cryptic poaching, plus unresolved concerns about modelling of density-dependence and its potential confounding effects of various changes in monitoring methods entangled with so-called ‘recovery periods’<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>.</p><p id=\"Par6\">A parallel and independent analysis of the Wisconsin and Michigan wolf populations found that periods with policy that liberalized wolf-killing were followed by significant decreases in potential population growth rates independent of the number of wolves killed legally<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. The authors inferred increases in poaching during six periods of policy that liberalized wolf-killing had caused several decreases in growth rates and that the resumption of more protective policies caused several increases in growth rates. These authors suggested what we now call the ‘facilitated poaching’ hypothesis, which proposes that would-be poachers respond to the policy changes as a signal to increase their activities, possibly associated with cognitive processes relating to values (e.g., lower value of wolves in the eyes of would-be poachers), social norms (e.g., greater acceptability of poaching, or less enforcement against poaching), or perceived control (e.g., would-be poachers perceive themselves helping authorities to kill wolves). This hypothesis is supported by four quantitative surveys of residents of Wisconsin from 2001 to 2013 and two qualitative focus groups from 2011 to 2012, which revealed increased inclinations to poach after Wisconsin wolf policies liberalized killing<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. Three critiques of the ‘facilitated poaching’ hypothesis were published, and one critique of the ‘frustration’ hypothesis, so the scientific debate is lively but it remains based on indirect evidence and weak to moderate strengths of inference<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>.</p><p id=\"Par7\">Subsequent research linking wolf mortality to population growth rates in Finland found poaching rates increased as a response to increases in wolf population size<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. Follow-up research by the same team found the total number of legally hunted wolves at the local scale and the country scale decreased the probability of poaching, while increases in the number of permits issued to kill wolves (the ‘bag limit’) increased the probability of poaching<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. The authors hypothesized that the declines in probability of poaching, given more wolves killed through legal hunting, might reflect a decrease in the number of individual wolves exposed to poaching because they were instead legally killed prior to potential poaching, essentially “just cleaning up the numbers”<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. However, their analyses did not statistically account for the uncertainties in causes of death and disappearance.</p><p id=\"Par8\">No study explicitly modeled the durations and periods of policy that individual wolves were exposed to legal killing<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. That would allow for the estimation of mortality hazard and incidence (from various causes) for individual wolves that <italic>experienced</italic> the policy over time. With individual-level estimates of hazard and incidence for marked individual wolves, we can more confidently draw inference about population-level effects on the growth rate and patterns of poaching. Nor did these studies model the effect of legal killing on wolf disappearances (those animals ‘lost-to-follow-up’; LTF). LTF animals could not have been killed by legal means or by conspicuous causes, otherwise their carcasses would have been recovered<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. Thus, LTF could conceal a component of cryptic poaching, in addition to those collared individuals that moved out of radio-telemetry range or those who died from natural causes but whose radio-transmitters suffered mechanical failure beforehand.</p><p id=\"Par9\">Here we test the hypothesis that poaching (both observed and cryptic) of adult wolves in Wisconsin, USA was influenced by changes in government policies via effects on individual wolf deaths and disappearances (from 1979 to 2012, 513 collared adults), which we modeled by mortality hazard and incidence in a competing risks framework. Widely used in the biomedical literature for the estimation of risk and prognosis for health interventions, competing risk analyses allowed us to estimate both hazards and incidences of various causes of death or disappearance in relation to wolves’ exposure time to policy. Therefore, competing risk analyses illuminate one fate (‘endpoint’ hereafter) among many, to understand the effects of policy for individual wolves, for all endpoints, especially LTF and its cryptic poaching component. Following recommendations for the most rigorous approach to competing risk analysis<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref>–<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>, we report results on all endpoint-specific hazards and CIFs and synthesize findings from both. In interpreting and discussing the results of our analyses, specifically point estimates and compatibility intervals, we follow the recommendations of researchers who argued for expanding discussion beyond traditional, arbitrary thresholds of ‘statistical significance’<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>. Instead, we provide point estimates and compatibility intervals (i.e., ‘confidence intervals’) for our MAIN imputation scenario. We present and discuss the distributions of parameters of interest as well as simulation scenarios for 26 wolves with incomplete data (see “<xref rid=\"Sec14\" ref-type=\"sec\">Materials and methods</xref>” section). In our discussion, we focus on the resulting point estimates as the most compatible values given our data and assumptions. We then discuss the implications of our model assumptions and uncertainty in our data, in particular for those results relevant to policy effects on mortality hazards and incidences.</p><p id=\"Par10\">Our results suggest that reduced protections under the Endangered Species Act (ESA) for wolves in the form of policies allowing selective liberalized killing may increase wolf mortality risk and incidence beyond the wolves legally killed. Given the ubiquity of large carnivore poaching, our research and methods can improve the effectiveness of many jurisdictions’ policies on environmental crimes, endangered species, and protections for wild animals.</p></sec><sec id=\"Sec2\"><title>Results</title><p id=\"Par11\">The six periods (Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S2</xref>) during which policy that liberalized wolf-killing were associated with various significant changes in endpoints for collared adult wolves<bold>,</bold> whether one examined hazards from Cox models, subhazards from Fine-Gray (FG) competing risk models, or their cumulative incidence functions (CIFs).</p><sec id=\"Sec3\"><title>Policy and covariate effects on endpoint hazards</title><p id=\"Par12\">The 6 policy periods with liberalized killing (Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S2</xref>) were 10–33% more hazardous for wolves to be lost-to-follow up (LTF) than policy periods with full protection. Liberalized killing periods were also more hazardous for legal killing, not surprisingly. Liberalized killing periods were less hazardous for monitored wolves reported poached than periods of full protection. We compare those three effects directly below, in light of existing theory.</p><p id=\"Par13\">Liberalized killing periods were more hazardous for two causes of death, the nonhuman and uncertain endpoints, and less hazardous for collisions, than periods of full protection. Winters were at least twice as hazardous as summers for the three most common endpoints (LTF, poached and nonhuman).</p><sec id=\"Sec4\"><title>Lost to follow-up (LTF)</title><p id=\"Par14\">Liberalized killing periods were 18% (HR 1.18) more hazardous than periods of full protection, yet compatible with a relatively narrow 13% decrease to a 60% increase in hazard (HR 95% CI 0.87–1.60). The resulting LTF HR distribution suggests an 85% probability of an increase in LTF hazard from the liberalized killing policy period (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>, Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>). These estimates depend on imputing the LTF (or ‘censored’) endpoint for 26 collared wolves whose records were incomplete in 2012 (Supplementary Tables <xref rid=\"MOESM4\" ref-type=\"media\">S3</xref>, <xref rid=\"MOESM4\" ref-type=\"media\">S4</xref>). We report the conservative MAIN imputation scenario in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> (see Supplementary Tables <xref rid=\"MOESM4\" ref-type=\"media\">S5</xref>, <xref rid=\"MOESM4\" ref-type=\"media\">S6</xref> for model diagnostics), which is consistent with the distribution of LTF in the overall sample, but also offer two alternative scenarios (LOW and HIGH) that generate HR estimates resulting in narrower bounds, spanning 10–33% increases in hazard for LTF endpoints (Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S7</xref>).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Hazard ratio (HR) of wolves lost to follow-up (LTF, n = 243 in MAIN* scenario) during liberalized killing policy periods (blue) relative to periods of full protection and during winter (orange) relative to summer. Bell curves illustrate the HR distributions with the same color of dashed lines and text as the bell curves to which they correspond for HR point estimates (n = 513). The vertical black solid line at HR = 1 (no effect) is provided for comparison to dashed lines indicating HR point estimates for covariates. Probabilities (%) of a HR of < 1 (left side) or > 1 (right side) are shown with color-coded text for each HR distribution. *We built LTF (or censored) endpoint imputation models (IMs) in three scenarios for 26 collared wolves with missing endpoints (5.1% of collared wolves, see “<xref rid=\"Sec14\" ref-type=\"sec\">Materials and methods</xref>” section and Supplemental Text). Our MAIN imputation scenario resulted in 12 of the 26 wolves going LTF (average <italic>T</italic> = 947 days), which is consistent with the expected proportion from the aggregate data in which 46% had an LTF endpoint. Results for the LOW and HIGH scenarios are presented in Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S7</xref> and narrowed the bounds of the LTF CI.</p></caption><graphic xlink:href=\"41598_2020_70837_Fig1_HTML\" id=\"MO1\"/></fig><table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Hazard ratio (HR) point estimates from the stratified joint Cox Model 5 (M5) for n = 513 monitored wolves (for MAIN* LTF imputation scenario), by endpoint.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"3\">Variable</th><th align=\"left\" colspan=\"12\">Endpoint</th></tr><tr><th align=\"left\" colspan=\"2\">Lost to follow-up (LTF)</th><th align=\"left\" colspan=\"2\">Reported poached</th><th align=\"left\" colspan=\"2\">Legal</th><th align=\"left\" colspan=\"2\">Nonhuman</th><th align=\"left\" colspan=\"2\">Collision</th><th align=\"left\" colspan=\"2\">Uncertain</th></tr><tr><th align=\"left\">HR</th><th align=\"left\">95% CI</th><th align=\"left\">HR</th><th align=\"left\">95% CI</th><th align=\"left\">HR</th><th align=\"left\">95% CI</th><th align=\"left\">HR</th><th align=\"left\">95% CI</th><th align=\"left\">HR</th><th align=\"left\">95% CI</th><th align=\"left\">HR</th><th align=\"left\">95% CI</th></tr></thead><tbody><tr><td align=\"left\">Liberalized killing periods (lib_kill)</td><td align=\"left\">1.18</td><td align=\"left\">0.87–1.60</td><td align=\"left\">0.81</td><td align=\"left\">0.48–1.35</td><td align=\"left\">1.57</td><td align=\"left\">0.60–4.07</td><td align=\"left\">1.09</td><td align=\"left\">0.64–1.87</td><td align=\"left\">0.43</td><td align=\"left\">0.14–1.36</td><td align=\"left\">1.36</td><td align=\"left\">0.55–3.34</td></tr><tr><td align=\"left\">Winter periods (winter)</td><td align=\"left\">3.13</td><td align=\"left\">1.99–4.93</td><td align=\"left\">4.7</td><td align=\"left\">2.47–8.91</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">2.03</td><td align=\"left\">1.08–3.81</td><td align=\"left\">0.48</td><td align=\"left\">0.20–1.13</td><td align=\"left\">1</td><td align=\"left\">–</td></tr><tr><td align=\"left\" colspan=\"13\"><bold>Census periods (method_change)</bold></td></tr><tr><td align=\"left\">Census method 1</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Census method 2</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">0.35</td><td align=\"left\">0.16–0.76</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Census method 3</td><td align=\"left\"/><td align=\"left\"/><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td></tr><tr><td align=\"left\" colspan=\"13\"><bold>Time-varying coefficient (tvc)</bold></td></tr><tr><td align=\"left\">Liberalized killing periods (lib_kill tvc) (change per year)</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">2.07</td><td align=\"left\">1–4.29</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Winter periods (winter tvc) (change per year)</td><td align=\"left\">0.69</td><td align=\"left\">0.48–0.69</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td></tr></tbody></table><table-wrap-foot><p>We present HRs and compatibility intervals (95% CI) for all covariate-endpoint interactions. Model selection criteria revealed that M5 was the best model (Supplementary Figs. <xref rid=\"MOESM4\" ref-type=\"media\">S1</xref>, <xref rid=\"MOESM4\" ref-type=\"media\">S2</xref>, Tables <xref rid=\"MOESM4\" ref-type=\"media\">S5</xref>, <xref rid=\"MOESM4\" ref-type=\"media\">S6</xref> for model diagnostics).</p></table-wrap-foot></table-wrap></p><p id=\"Par15\">For LTF endpoints, winters were 213% more hazardous than summers, with a broad range 99–393% (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>, Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>). The estimated LTF HR of 3.13 at baseline with a time-varying coefficient of 0.69/year means that winter was very hazardous after initial collaring, but then decreased substantially (3.13 × 0.69 = 2.16 and 3.13 × 0.69 × 0.69 = 1.49), to a 116% and 49% increase in hazard (relative to summer) at 1 and 2 years after collaring, respectively.</p></sec><sec id=\"Sec5\"><title>Reported poached</title><p id=\"Par16\">Liberalized killing periods were 19% less hazardous for the endpoint of reported poaching (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>), yet a 52% decrease to a 35% increase in hazard were also compatible with the data (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>, Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). The resulting poached HR distribution suggests a 79% probability of a decrease in the hazard from the liberalized killing policy signal (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). Winters were 370% more hazardous for the endpoint reported poached, with broad compatible estimates spanning increases between 147 and 791% (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>, Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>).<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Hazard ratio (HR) of wolves reported poached (n = 88) during liberalized killing policy periods (blue) relative to periods of full protection; winter (orange) relative to summer; census period 2 (1995–2000) relative to census period 1 (1979–1994). Bell curves, vertical lines, text and color coding as in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>.</p></caption><graphic xlink:href=\"41598_2020_70837_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par17\">We found an effect of census period on reported poaching; a 65% decrease in hazard during census period 2 1995–2000 (relative to census period 1, 1979–1994), with narrow compatible estimates spanning 84–24% decrease in hazard (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>, Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>).</p></sec><sec id=\"Sec6\"><title>Legal killing</title><p id=\"Par18\">As the policy intended, liberalized killing periods were 57% more hazardous for the endpoint <italic>legal</italic> than periods of full protection, with compatible estimates spanning a broad range of 40% decrease to a 307% increase. The estimated <italic>legal</italic> HR of 1.57 at baseline with a time-varying coefficient of 2.07/year means that liberalized killing policies were associated with substantial increase over monitoring time (1.57 × 2.07 = 3.25 and 1.57 × 2.07 × 2.07 = 6.73), to a 225% and 573% increase in hazard (relative to full protection periods) at 1 and 2 years after collaring, respectively. The broad range of compatibility estimates indicates variability in the time it took for marked wolves to die from this cause, in policy periods ranging from x to y days of liberalized killing.</p></sec><sec id=\"Sec7\"><title>Nonhuman, collisions and uncertain</title><p id=\"Par19\">Liberalized killing periods were 9% more hazardous for the endpoint of dying by nonhuman cause (57% decrease for collisions; 36% increase for uncertain), with a narrow range of 36% decrease to an 87% increase also compatible with our data (a broad range of − 86% to + 36% for collisions; and a broad range of − 45% to + 234% for uncertain). Winters were associated with an increase in the nonhuman endpoint HR of 103%, with compatible estimates spanning increases of 8–281% (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>).</p><p id=\"Par20\">Next, we evaluated how the same covariates as above affected the incidence of each endpoint, using FG models to consider the effect of competing risks on a single endpoint. In FG models of incidence, common endpoints gain importance and rarer endpoints lose importance proportional to their prevalence in the population. By comparison with hazard ratios that do not consider monitored wolves experiencing other endpoints, subhazard ratios consider all endpoints in the dataset up to the time point in question.</p></sec></sec><sec id=\"Sec8\"><title>Policy and covariate effects on endpoint incidences in a competing risk framework</title><p id=\"Par21\">FG models revealed liberalized killing periods were associated with an increase in incidence of LTF of 11–34% relative to periods with full protections (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>, Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S7</xref>). As intended by the policy, the former periods were associated with an increase in the incidence of legal killing. By contrast, liberalized killing periods were associated with a 24% decrease in incidence of reported poaching. FG models also suggest an increase in incidence of nonhuman and uncertain endpoints, along with a decrease in the incidence of death by collision.<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Subhazard ratio (SHR) point estimates from FG models for 513 monitored wolves for MAIN imputation scenario, by endpoint.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"3\">Variable</th><th align=\"left\" colspan=\"12\">Endpoint</th></tr><tr><th align=\"left\" colspan=\"2\">Lost to follow-up (LTF)</th><th align=\"left\" colspan=\"2\">Reported Poached</th><th align=\"left\" colspan=\"2\">Legal</th><th align=\"left\" colspan=\"2\">Nonhuman</th><th align=\"left\" colspan=\"2\">Collision</th><th align=\"left\" colspan=\"2\">Uncertain</th></tr><tr><th align=\"left\">SHR</th><th align=\"left\">95% CI</th><th align=\"left\">SHR</th><th align=\"left\">95% CI</th><th align=\"left\">SHR</th><th align=\"left\">95% CI</th><th align=\"left\">SHR</th><th align=\"left\">95% CI</th><th align=\"left\">SHR</th><th align=\"left\">95% CI</th><th align=\"left\">SHR</th><th align=\"left\">95% CI</th></tr></thead><tbody><tr><td align=\"left\">Liberalized killing periods (lib_kill)</td><td align=\"left\">1.19</td><td align=\"left\">0.86–1.65</td><td align=\"left\">0.76</td><td align=\"left\">0.44–1.31</td><td align=\"left\">1.57</td><td align=\"left\">0.59–4.12</td><td align=\"left\">1.17</td><td align=\"left\">0.67–2.03</td><td align=\"left\">0.42</td><td align=\"left\">0.13–1.37</td><td align=\"left\">1.32</td><td align=\"left\">0.55–3.16</td></tr><tr><td align=\"left\">Winter periods (winter)</td><td align=\"left\">2.89</td><td align=\"left\">1.89–4.42</td><td align=\"left\">3.36</td><td align=\"left\">1.88–6.00</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">2.04</td><td align=\"left\">1.17–3.54</td><td align=\"left\">0.53</td><td align=\"left\">0.25–1.14</td><td align=\"left\">0.39</td><td align=\"left\">0.16–0.96</td></tr><tr><td align=\"left\" colspan=\"13\"><bold>Census periods (method_change)</bold></td></tr><tr><td align=\"left\">Census method 1</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Census method 2</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">0.37</td><td align=\"left\">0.17–0.81</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Census method 3</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td></tr><tr><td align=\"left\" colspan=\"13\"><bold>Time-varying coefficient (tvc)</bold></td></tr><tr><td align=\"left\">Liberalized killing periods (lib_kill tvc) (change per year)</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">2.07</td><td align=\"left\">1–6.17</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td></tr><tr><td align=\"left\">Winter periods (winter tvc) (change per year)</td><td align=\"left\">0.69</td><td align=\"left\">0.48–0.69</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td><td align=\"left\">1</td><td align=\"left\">–</td></tr></tbody></table><table-wrap-foot><p>We present SHRs and compatibility intervals (95% CI) for all covariate-endpoint interactions.</p></table-wrap-foot></table-wrap></p><sec id=\"Sec9\"><title>LTF</title><p id=\"Par22\">Along with a suggested increase in LTF hazard (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>), disappearances of wolves were 19% more likely during policy periods with liberalized killing, relative to periods of full protections (i.e., the proportion of wolves over time going LTF increases), with compatible estimates from the MAIN imputation scenario spanning a 14% decrease to a 65% increase in incidence (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>). That range of compatible SHR values was narrowed by our imputation scenarios for the 26 wolves with missing data to a narrower 11%-34% increase in LTF incidence (Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S7</xref>).</p><p id=\"Par23\">LTF incidence also increased by 189% during winter (relative to summer), with a nroad compatibility interval suggesting increases of 89–342%. The model also detects a non-proportional change (winter tvc) amounting to a 31% decrease in LTF incidence during winter periods for every year of monitoring a given marked wolf (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>).</p></sec><sec id=\"Sec10\"><title>Reported poached</title><p id=\"Par24\">Liberalized killing periods were associated with a decrease of 24% in the incidence of reported poaching, with broad compatible estimates spanning a 56% decrease to a 31% increase in incidence (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>). Winters were associated with an increase in incidence of 236%, with compatible estimates spanning a broad range of 88–500% (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>). During census period 2 (1995–2000), the incidence of reported poaching decreased by 63%, with compatible estimates spanning a narrow range of decreases of 83–29% (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>).</p></sec><sec id=\"Sec11\"><title>Legal killing</title><p id=\"Par25\">Consistent with the increase in hazard and the objective of the policy change, liberalized killing periods were associated with a 57% increase in the incidence of legal killing, with compatible estimates spanning a broad range of 41% decrease to a 312% increase. Along with this main policy effect, we obtain a similar (to the HR) non-proportional change amounting to a 107% increase in incidence during those policy periods for every year of monitoring a given wolf (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>).</p></sec><sec id=\"Sec12\"><title>Nonhuman, collision and uncertain</title><p id=\"Par26\">Liberalized killing periods were associated with a 17% increase in the incidence of death by nonhuman cause (58% decrease for collisions; 32% increase for uncertain), with broad compatible estimates spanning a 33% decrease to an 103% increase (− 87% to + 37% for collisions; − 45% to + 216% for uncertain). Winters seemed to increase the incidence of a nonhuman endpoint by 104%, with compatible estimates spanning increases of 17–254% (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>). The FG model for uncertain suggests an additional (to that of the Cox model) effect of census period associated with a 61% decrease in incidence of this endpoint, with compatible estimates spanning a narrow range of 84–4% decrease in incidence (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>).</p><p id=\"Par27\">We focus on the FG-derived CIFs (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>) because these consider the prevalence of endpoints in the population at which scale the hypotheses make predictions. The most relevant endpoints (LTF and poached) CIFs suggest liberalized killing periods were associated with an increase in the cumulative incidence of LTF of approximately 0.10 relative to full protection periods (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>; range 0.05–0.15 for LOW–HIGH imputation scenarios [Supplementary Tables <xref rid=\"MOESM4\" ref-type=\"media\">S8</xref>, <xref rid=\"MOESM4\" ref-type=\"media\">S9</xref> Supplementary Figs. <xref rid=\"MOESM4\" ref-type=\"media\">S21</xref>, <xref rid=\"MOESM4\" ref-type=\"media\">S22</xref>]). This increase in LTF incidence is comparable to that of legal killing because of the high prevalence of LTF (i.e., the total number of LTF endpoints in the wolf population). Moreover, the increase in incidence of LTF associated with liberalized killing periods was 5 times larger than the associated decrease in the cumulative incidence of wolves reported poached during the same periods (0.02–0.03 decrease; Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>).<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Cumulative incidence functions (CIFs) for 513 monitored wolves. Lines show separate endpoints for lost-to-follow-up, LTF (n = 243, orange), reported poached (n = 88, maroon), and legal kills (n = 32, black) in two periods, derived from Fine-Gray models for MAIN imputation scenario. For each endpoint, we illustrate the cumulative incidence for liberalized killing periods (dashed lines) and periods of full protection (solid lines). We derived CIF curves according to the M5 stratified joint Cox model (Supplementary Figs. <xref rid=\"MOESM4\" ref-type=\"media\">S3</xref>–<xref rid=\"MOESM4\" ref-type=\"media\">S8</xref>) and from each endpoint-specific semi-parametric FG models (Supplementary Tables <xref rid=\"MOESM4\" ref-type=\"media\">S8</xref>, <xref rid=\"MOESM4\" ref-type=\"media\">S9</xref> for LTF and legal SHRs used for estimating FG CIFs, and Supplementary Figs. <xref rid=\"MOESM4\" ref-type=\"media\">S9</xref>–<xref rid=\"MOESM4\" ref-type=\"media\">S14</xref>) and non-parametric FG models (Supplementary Figs. <xref rid=\"MOESM4\" ref-type=\"media\">S15</xref>–<xref rid=\"MOESM4\" ref-type=\"media\">S20</xref>). Visual comparison of the three sets of CIF curves for each policy period-endpoint combination suggests consistent results between Cox and FG CIFs for most endpoints as well as compliance with FG model assumptions (i.e., proportionality of endpoint subhazards) for all endpoints except nonhuman (Supplementary Fig. <xref rid=\"MOESM4\" ref-type=\"media\">S5</xref>).</p></caption><graphic xlink:href=\"41598_2020_70837_Fig3_HTML\" id=\"MO3\"/></fig></p></sec></sec></sec><sec id=\"Sec13\"><title>Discussion</title><p id=\"Par28\">Traditional time-to-event models in wildlife science discard or censor data from marked animals that disappeared (no collar or carcass recovered) as uninformative. This approach fails to account for the high certainty attached to rates of legal causes of death compared to the low certainty about rates of other causes that are not well reported, including the least documented form termed cryptic poaching. Instead, the assumption that individuals that are lost-to-monitoring suffer from similar hazards and endpoints as monitored individuals, or survive through migration or dispersal, produce systematic biases. These biases may underestimate mortality and its anthropogenic component, but more perniciously, these biases may obscure or mislead during the evaluation of any policy effects on mortality hazard and incidence. Our competing risk analyses illuminate how to evaluate policy effects on mortality without introducing the aforementioned assumptions leading to overwhelming biases.</p><p id=\"Par29\">The distribution of the LTF hazard ratio HR suggests a liberalized killing policy signal was associated with an 85% likelihood of increasing the risk of disappearance (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>) for monitored adults in Wisconsin. The situation is partially reversed for reported poaching, with the liberalized killing policy signal being associated with a 79% probability of a smaller decrease in hazard of being reported poached (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). Neither of these changes reached statistical significance (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>), yet apparent similarities in these HR distributions are misleading because the incidence of disappearances (LTF) is so much greater than the incidence of observed poaching. We discuss the limitations of our study but when considering all the evidence, we infer that the policy of liberalizing wolf killing in Wisconsin from 2003 onward resulted in more cryptic poaching.</p><p id=\"Par30\">We found that periods with policies that liberalized wolf-killing were most compatible with increases in the hazard (10–33%) and more importantly, in the incidence (11–34%) of disappearances (LTF) among monitored wolves. The same periods were associated with decreases in the hazard (19%) and incidence (24%) of reported poaching of monitored wolves, along with an association with census method never before reported (discussed further below). Given the low number of observations as well as illustrated CIFs (Supplementary Figs. <xref rid=\"MOESM4\" ref-type=\"media\">S17</xref>–<xref rid=\"MOESM4\" ref-type=\"media\">S19</xref>), we are unable to discern any policy effects for the collision, uncertain, and nonhuman endpoints.</p><p id=\"Par31\">The suggested decline in reported poaching does not compensate for the LTF increase because LTF had a much higher prevalence in the dataset (n = 243; 47% compared to n = 88; 17.2% for reported poached). The importance of the relative prevalence of LTF and reported poaching is shown in cumulative incidence curves in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>. Indeed, the increase in cumulative incidence of LTF associated with liberalized killing policies seems comparable only to that of legal killing. Indeed, legal killing crossed HR = 1 (95% CI 0.60–4.07; Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>), indicating that variance and uncertainty in something we know increased in those periods does not weaken the inference that hazard increased for legal killing. Rather, the compatibility interval indicates the variability in time it took for a monitored wolf to be killed and how many survived such periods. Moreover, we would argue the shape of the HR distributions (Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>, <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>) are important; both are sharply peaked and with considerably less uncertainty than our seasonal covariate.</p><p id=\"Par32\">The LTF endpoint is certainly an aggregation of three components: (1) individuals that had moved out of range of aerial radio-telemetry (i.e., long-distance migration), (2) collars that stopped transmitting (i.e., mechanical failures), and (3) unreported poached individuals (‘cryptic poaching’)<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. Thus, the increase in LTF incidence associated with liberalized killing policies could result from increases in any of these components. We discuss at length each component below but we foreshadow our inference that the major component is cryptic poaching simply because there is no known mechanism through which a policy would cause wolves to migrate out of state or cause mechanical failure of collars. Based on our point estimates and resulting CIFs, our findings are consistent with the hypothesis that ‘culling increases poaching’<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>, compatible with growing evidence of cryptic poaching of predators around the world, and inconsistent with the U.S. federal government’s claim in federal court that liberalizing wolf-killing would reduce poaching and protect the endangered wolves we studied here.</p><p id=\"Par33\">A plausible hypothesis for how liberalized killing periods may increase the incidence of emigration of monitored wolves would be through legal killing possibly causing disruption of wolf behavioral and social dynamics<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref>–<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>, leading to breeding pair dissolution or pack disbanding and perhaps increasing the number of dispersers leaving the state before the next monitoring period. However, the scientific evidence on the effects of anthropogenic mortality on wolf dispersal to date suggests the opposite effect; that low-to-moderate levels of anthropogenic mortality may instead be compensated by increased immigration from adjacent populations, and increased pup survival<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. In their analysis of the effects of anthropogenic mortality on the wolf population in northern Alaska, Adams et al.<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup> conclude that immigration was the main mechanism allowing otherwise unsustainable killing to continue for several years. Consistent with this, 7 times more radio-collared wolves entered Wisconsin from neighboring Michigan than went in the reverse direction, especially during the years with liberalized wolf-killing policies<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. If liberalizing wolf-killing prompted more emigration by Wisconsin wolves, one might expect more vehicle collisions also. We found the opposite (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>). Therefore, emigration out of state seems an unlikely mechanism for the increase in LTF incidence.</p><p id=\"Par34\">Regarding collar failure, we are unable to suggest a possible mechanism associating the incidence of collar failure with liberalized killing periods for several reasons. First, given the technological advances related to collaring and monitoring between 1979 and 2003 (when liberalized killing periods begin; Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S2</xref>) it seems unlikely that the incidence (or risk) of collar failure would be higher during these later time periods relative to periods of full protections. Moreover, if increased collar failure was a possible mechanism we would expect the incidence (or risk) of collar failures to increase as a function of monitoring time instead of the observed proportional increase in incidence during liberalized killing periods relative to full protection periods. To this we add that policy periods were implemented multiple times during those later time periods (Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S2</xref>), which makes a confounding effect of collar failure implausible. Regarding the potential for lower temperatures to reduce battery life and affect LTF in winter, looking at both seasonal effects would seem to suggest cold temperatures having two contradictory effects on battery/mechanical failure: (1) a decrease in battery life relative to summer (main winter HR/SHR), but (2) also a decrease in this difference over time (illustrated through the season time-varying effect). Further, the magnitude of the seasonal effect on LTF, an increased risk of 213% in winter relative to summer, seems large enough to implicate a mechanism other than battery life.</p><p id=\"Par35\">The ‘facilitated poaching’ hypothesis proposes a human cognitive mechanism through which liberalized killing policies affect human behavior. The policy signal might decrease the value of wolves for potential poachers or increase the acceptability of poaching by their associates. Social survey research reported rising inclinations to poach wolves in Wisconsin three times after the implementation of liberalized killing policies<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>, consistent with the ‘facilitated poaching’ hypothesis. In the three studies cited, certainty about the policy effect compared to confounding effects increased as the intervals between resampling respondents decreased with each successive study.</p><p id=\"Par36\">The increases in incidence of monitored wolf disappearance (LTF) during liberalized killing periods suggest how the LTF component of cryptic poaching may obscure (at least part of) the additional mortality necessary to explain the slow-down in the population’s annual growth rate from 2003 to 2011<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>, and is consistent with increases in mortality over and above legal killing during said policy periods that were inferred to be responsible for population growth slow-downs<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Thus, the ‘facilitated poaching’ hypothesis seems the most plausible explanation for the rise in incidence of disappearances among Wisconsin’s monitored wolves from 2003 to 2012. If this is indeed the case, LTF hazard rose because wolves faced an increased rate of cryptic poaching and incidence rose because the proportion of cryptically poached wolves increased.</p><p id=\"Par37\">The decrease we found in the incidence of reported poaching during liberalized killing policy periods might be interpreted as consistent with the ‘tolerance hunting’ hypothesis, which suggests that some lethal predator control may increase tolerance for the species and thus reduce poaching<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. However, we cannot distinguish changes in reporting from changes in poaching with these data. In any case, looking only at the policy effect on reported poaching dismisses cryptic poaching, especially in light of evidence that most poaching goes unreported and thus underestimated in the Wisconsin wolf population and others<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Given the slight decreases in cumulative incidence of reported poaching that we found (approximately 0.02–0.03) and the more robust increases in LTF incidence (range 0.05–0.15 across our scenarios), it would suffice for just a portion of the suggested increase in LTF incidence to be attributable to cryptic poaching to (over)compensate for any decreases in reported poaching. For example, limiting the observed LTF incidence increase to the proportion of LTF wolves later found by means other than telemetry and found to have been poached (33%; a conservative minimum estimate) would still amount to a 0.02–0.05 incidence increase in cryptic poaching. Thus, our results undermine any claims of reductions in <italic>total</italic> (i.e., observed and cryptic) poaching from liberalized killing policies. (i.e., ‘tolerance hunting’), contra Olson et al.<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>.</p><p id=\"Par38\">Additionally, our results for reported poaching also seem consistent with another hypothesized relationship between legal killing and reported poaching from research in Finland. The notion of “cleaning up the numbers”<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>, predicted a decline in reported poaching after increases in liberalized killing simply because fewer wolves are ;eft alive to be exposed to poaching. There seems to be no need to attribute this decline to human cognitive mechanisms (i.e., tolerance); wolves are simply killed legally at a higher rate (higher hazard and incidence) than they are reported poached during these periods (Tables <xref rid=\"Tab1\" ref-type=\"table\">1</xref>, <xref rid=\"Tab2\" ref-type=\"table\">2</xref>, Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>). Indeed, we found that monitoring time (wolf collar transmitting) was associated with an increase in both the hazard and incidence of wolves being killed by government agents during liberalized killing periods. This result should not be ignored by decision-makers because it implies an (unplanned) accelerating incidence of legal killing during prolonged periods of liberalized killing. That is, once government agents are allowed to kill wolves, the likelihood of complaints or wolf deaths increased over time. Our present results seem to implicate human behavior in such a pattern, but further research is needed.</p><p id=\"Par39\">The ‘facilitated poaching’ explanation implies several non-mutually exclusive hypothetical mechanisms by which would-be poachers might respond to a policy signal to increase cryptic poaching. For instance, (a) would-be poachers feel emboldened by a perceived relaxation of anti-poaching laws during liberalized killing periods. This mechanism seems unlikely given cryptic poaching rose 2.5–7.5 times more than reported poaching decreased (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>). Alternatively (b), would-be poachers perceive a shift to social norms favoring their activities. This too seems unlikely given cryptic poaching remains covert while tolerance for poaching would seem to favor more overt poaching. Finally, (c) would-be poachers interpret wolves as having lower value because the government killing wolves suggests the population is too large. The latter hypothetical mechanism seems viable still and can be measured by classic valuation surveys, even perhaps conducted on the broad public rather than having to find would-be poachers to survey.</p><p id=\"Par40\">Our results also inform the literature on the effect of relaxing protections on environmental crimes. If we are correct in arguing the ‘facilitated poaching’ hypothesis is behind the suggested increases in cryptic poaching, then it seems that legalizing the killing of large nonhuman animals may drive their killing underground and perhaps motivate it, a hypothesis that has found support in research on the elephant ivory trade<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. The increase in incidence of cryptic poaching we infer without an increase in reported poaching favors the idea that poachers remained averse to the risk posed by the state’s authority to curb poaching (such as with increased enforcement or reestablishment of full federal protections). Here the observed decline in reported poaching incidence in census years 1995–2000 bears mention. Those years were associated with a change in methods to triple or more the number of wolf census-takers each winter<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. Increasing human presence could have reduced either poaching activity or reporting (although there was no quantification of telemetry effort). The decline in unknown causes of death during the latter census period tends to support a view that additional volunteer census-takers each winter found more wolf carcasses—without any associated change in LTF during that same period. The role of census method requires further study therefore.</p><p id=\"Par41\">Given the scientific evidence suggesting continued declines in tolerance for wolves after the legalization of wolf-hunting in 2012<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>, we hypothesize that cryptic poaching hazard and incidence may have increased after our study period. However, our study, results and scientific inferences are subject to various limitations. Our results are conditioned by any bias inherent in the WDNR data used, such as missing data for 26 wolves that we had to simulate and, in particular, measurement error for date of endpoint. The LTF endpoint is particularly susceptible to measurement error because wolves go LTF between monitoring intervals of weeks or months of unsuccessful monitoring, in most cases without reliable evidence with which to provide estimates of an actual LTF date. Time to event of LTF is the critical parameter in our analyses, not the number of wolves that went LTF, but regardless, the high proportion (119/231 = 52%) of wolves experiencing LTF during the first period of full protection (1979–31 March 2003, Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S2</xref>) dismisses the concern that the absolute number of collared wolves in later periods potentially confounded our results. Our study is also limited by the lack of individual-level variables (e.g., sex, breeding or dispersal status) that may affect wolf mortality. Moreover, the lack of randomization in the observational study is a weakness. Although we have adjusted for all reasonable and available predictors, the observed effects could still be at least partially attributed to residual confounding variables. However, these results are still interesting and useful for hypothesis generation in the absence of a randomized trial on liberalized wolf killing policy. Until randomized experiments are conducted, ours and other investigators’ inferences are limited by being based on correlative associations, although our analyses enjoyed the benefits to inference that come from longitudinal analyses of long time series covering multiple policy changes.</p></sec><sec id=\"Sec14\"><title>Materials and methods</title><sec id=\"Sec15\"><title>Data sources</title><p id=\"Par42\">Our dataset includes all collared wolves monitored by telemetry (virtually all VHF radio-transmitters) in Wisconsin (WI) between 1979 and April 2012, published previously in full detail<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. The dataset includes 486 wolves fitted with collars by the Wisconsin Department of Natural Resources (WDNR) or its agents, plus 27 collared wolves initially captured in the neighboring state of Michigan, which later migrated to Wisconsin (for a total n = 513 individuals).</p><p id=\"Par43\">Our dataset includes 257 wolves that were reported by the WDNR as ‘lost-to-follow-up’ (LTF) because they were not detected via repeated aerial telemetry. LTF may occur for various reasons: (a) individuals that have moved permanently out of telemetry range (i.e., migrants), (b) collars that stopped transmitting because of battery depletion or mechanical failure, and (c) unreported poaching followed by destruction of the transmitter (cryptic poaching). The WDNR suspended telemetry monitoring and assigned an LTF to a wolf if their personnel were unable to detect the collar signal after several months of statewide aerial or ground telemetry. However, the WDNR did not quantify telemetry effort. Dead wolves (n = 242) were recovered by the mortality signals emitted from their collars, after legal killing by management agents, or after private citizens reported a dead wolf between monitoring flights<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. Some LTF wolves were subsequently recovered by means other than telemetry, such as reporting by private citizens. For these cases we used the estimated date of LTF for the endpoint (i.e., death from various causes or disappearance). For fuller treatment of disappearances, detection, and causes of individual wolf death see<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>.</p></sec><sec id=\"Sec16\"><title>Estimating conditional hazards</title><p id=\"Par44\">Our analyses exploit the survival history of monitored wolves, measured in days from date of collaring until date of endpoint (i.e., date of death, last monitoring date, or end of our analysis period on April 15, 2012) for each monitored individual.</p><p id=\"Par45\">We modeled endpoint-specific hazard and subhazard in a competing risk framework, which are extensions of survival (or ‘time-to-event’) analyses. Survival analyses estimate ‘time-to-event’ functions, which describe the probability of observing a time interval (T) to an endpoint (‘event’) within a specified analysis time (<italic>t</italic>) that a subject was observed, such that <inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$S\\left(t\\right)=P(T>t)$$\\end{document}</tex-math><mml:math id=\"M2\"><mml:mrow><mml:mi>S</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mi>t</mml:mi></mml:mfenced><mml:mo>=</mml:mo><mml:mi>P</mml:mi><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>T</mml:mi><mml:mo>></mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq1.gif\"/></alternatives></inline-formula> . Alternatively, these techniques allow for calculating the hazard function, <inline-formula id=\"IEq2\"><alternatives><tex-math id=\"M3\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${h}_{k}(t)$$\\end{document}</tex-math><mml:math id=\"M4\"><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mi>k</mml:mi></mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq2.gif\"/></alternatives></inline-formula>, or the instantaneous rate of occurrence of a particular endpoint <italic>k</italic> conditional on not experiencing any endpoint until time <italic>t</italic><sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR43\">43</xref>,<xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. We also used the (conditional) hazard functions for all endpoints to estimate the probability of any endpoint up to a particular time <italic>T</italic>, i.e., the incidence over time for particular endpoints, such as LTF or death by vehicle collision, nonhuman cause, etc.</p><p id=\"Par46\">Semi-parametric, Cox proportional hazard models estimate how the endpoint-specific <inline-formula id=\"IEq3\"><alternatives><tex-math id=\"M5\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${h}_{k}(t)$$\\end{document}</tex-math><mml:math id=\"M6\"><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mi>k</mml:mi></mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq3.gif\"/></alternatives></inline-formula> changes as a function of survival time and a set of hypothetical covariates; <inline-formula id=\"IEq4\"><alternatives><tex-math id=\"M7\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$S\\left(t\\right)={e}^{-{h}_{k}(t,x,\\beta )}$$\\end{document}</tex-math><mml:math id=\"M8\"><mml:mrow><mml:mi>S</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mi>t</mml:mi></mml:mfenced><mml:mo>=</mml:mo><mml:msup><mml:mrow><mml:mi>e</mml:mi></mml:mrow><mml:mrow><mml:mo>-</mml:mo><mml:msub><mml:mi>h</mml:mi><mml:mi>k</mml:mi></mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo>,</mml:mo><mml:mi>x</mml:mi><mml:mo>,</mml:mo><mml:mi>β</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:msup></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq4.gif\"/></alternatives></inline-formula>, where <italic>x</italic> is a vector of covariates acting on the hazard, and <italic>β</italic> is a vector of their respective parameter estimates. The estimation of covariate effects on the endpoint-specific hazard is modeled as <inline-formula id=\"IEq5\"><alternatives><tex-math id=\"M9\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${h}_{k}\\left(t\\right)= {h}_{0k}(t){e}^{({\\beta }_{1}{x}_{1}+\\dots +{\\beta }_{j}{x}_{j})}$$\\end{document}</tex-math><mml:math id=\"M10\"><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mi>k</mml:mi></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>t</mml:mi></mml:mfenced><mml:mo>=</mml:mo><mml:msub><mml:mi>h</mml:mi><mml:mrow><mml:mn>0</mml:mn><mml:mi>k</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:msup><mml:mrow><mml:mi>e</mml:mi></mml:mrow><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:msub><mml:mi>β</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:msub><mml:mi>x</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mo>+</mml:mo><mml:mo>⋯</mml:mo><mml:mo>+</mml:mo><mml:msub><mml:mi>β</mml:mi><mml:mi>j</mml:mi></mml:msub><mml:msub><mml:mi>x</mml:mi><mml:mi>j</mml:mi></mml:msub><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:msup></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq5.gif\"/></alternatives></inline-formula>, where <inline-formula id=\"IEq6\"><alternatives><tex-math id=\"M11\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${h}_{0k}(t)$$\\end{document}</tex-math><mml:math id=\"M12\"><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mrow><mml:mn>0</mml:mn><mml:mi>k</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq6.gif\"/></alternatives></inline-formula> is an unestimated baseline hazard function (i.e., semi-parametric) and <inline-formula id=\"IEq7\"><alternatives><tex-math id=\"M13\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\beta }_{j}$$\\end{document}</tex-math><mml:math id=\"M14\"><mml:msub><mml:mi>β</mml:mi><mml:mi>j</mml:mi></mml:msub></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq7.gif\"/></alternatives></inline-formula> represent estimates of hazard ratios (HRs) for each covariate <inline-formula id=\"IEq8\"><alternatives><tex-math id=\"M15\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${x}_{j}$$\\end{document}</tex-math><mml:math id=\"M16\"><mml:msub><mml:mi>x</mml:mi><mml:mi>j</mml:mi></mml:msub></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq8.gif\"/></alternatives></inline-formula> (HR < 1 represents a reduction in hazard and HR > 1 an increase in hazard).</p><p id=\"Par47\">The estimated HRs, <inline-formula id=\"IEq9\"><alternatives><tex-math id=\"M17\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\beta }_{j}$$\\end{document}</tex-math><mml:math id=\"M18\"><mml:msub><mml:mi>β</mml:mi><mml:mi>j</mml:mi></mml:msub></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq9.gif\"/></alternatives></inline-formula>, are assumed proportional throughout the analysis time, <italic>t</italic>, (only differ multiplicatively between categorical covariate levels). Furthermore, we include time-varying effects on hazards and incidences by including interactions between covariates and monitoring time (in days) (see “<xref rid=\"Sec19\" ref-type=\"sec\">Model covariates</xref>” section)<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref>–<xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. These models allow us to estimate covariate effects on the rate of occurrence of an endpoint looking only at those wolves reaching that endpoint (so that the presence of other endpoints would not affect these estimates). Inference from hazards is limited in the presence of other endpoints competing to bring about the end of monitoring because interaction between endpoint hazards is unaccounted for. Interactions between endpoints are crucial for our tests of hypotheses that relate legal killing to poaching (i.e., illegal killing, both reported poaching and cryptic poaching through the LTF endpoint) at an individual level.</p></sec><sec id=\"Sec17\"><title>Estimating unconditional incidences</title><p id=\"Par48\">Competing risk analyses extend standard survival analysis by considering multiple endpoints simultaneously (e.g.: multiple causes of death or disappearance). These models are useful for estimating the incidence of a particular endpoint while accounting for the potential occurrence of all other competing endpoints (e.g., the incidence of wolf-poaching in the presence of other causes of death or LTF). In a competing risk framework, individuals can potentially experience one of multiple mutually exclusive endpoints at each interval <italic>T</italic>. Because only one endpoint can occur first, we refer to the endpoints as ‘competing’ over time, and to the respective probabilities over time as ‘competing risks’.</p><p id=\"Par49\">Rather than estimating the endpoint-specific HRs, as in the Cox model explained above, competing risk analyses estimate the cumulative incidence function (CIF) for each endpoint, defined by the failure probability <inline-formula id=\"IEq10\"><alternatives><tex-math id=\"M19\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$Prob(T\\le t,D=k)$$\\end{document}</tex-math><mml:math id=\"M20\"><mml:mrow><mml:mi>P</mml:mi><mml:mi>r</mml:mi><mml:mi>o</mml:mi><mml:mi>b</mml:mi><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>T</mml:mi><mml:mo>≤</mml:mo><mml:mi>t</mml:mi><mml:mo>,</mml:mo><mml:mi>D</mml:mi><mml:mo>=</mml:mo><mml:mi>k</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq10.gif\"/></alternatives></inline-formula>; the cumulative probability of endpoint <italic>k</italic> occurring over time in the presence of other competing endpoints<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>. Competing risk analysis accounts for the CIF of any endpoint being a function of all endpoint-specific hazards, <inline-formula id=\"IEq11\"><alternatives><tex-math id=\"M21\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${h}_{k}(t)$$\\end{document}</tex-math><mml:math id=\"M22\"><mml:mrow><mml:msub><mml:mi>h</mml:mi><mml:mi>k</mml:mi></mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>t</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq11.gif\"/></alternatives></inline-formula>, reflecting the rate of occurrence of that endpoint as well as how it is influenced by others<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>.</p><p id=\"Par50\">Although CIFs can be derived by using all endpoint-specific HRs derived from Cox models, such a procedure cannot estimate the magnitude of the relative difference between covariate CIFs for each endpoint. Using Fine-Gray (FG) models instead of Cox models allows us to estimate differences in CIFs for a given endpoint conditional on covariates<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>. FG models are also semi-parametric (i.e., the baseline subhazard function is not estimated) and assume proportionality of subhazard functions, defined as the risk of failure at time <italic>t</italic> from endpoint <italic>k</italic> in subjects that have yet to reach an endpoint or have experienced any other endpoint<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>. Therefore, FG models estimate the subhazard functions of endpoint-specific CIFs using similar regression techniques as the Cox model (but on the subhazard rather than the hazard thus yielding SHR rather than HR for ratios that compare to a standard), but parameter interpretation changes. Subhazards are interpreted as relative incidence in the presence of other endpoints<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref>–<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>.</p><p id=\"Par51\">In sum, endpoint-specific Cox models and their HRs allow us to test the hypothesis that liberalized wolf-killing affected the rate of occurrence of any endpoint; for example, if liberalized killing increased or decreased the rate of occurrence of reported poaching or LTF. By contrast, the FG models and their SHRs allow us to account for the simultaneous presence of all competing endpoints to test if and how much liberalized killing affected the probability and incidence of reported poaching or LTF, in addition to the potential simultaneous effects of other covariates described after data preparation. CIFs allow us to visualize those effects on incidence while considering the prevalence of each endpoint in the population.</p></sec><sec id=\"Sec18\"><title>Data preparation</title><p id=\"Par52\">For wolves monitored until death, our endpoints classify the cause of death by 5 mutually exclusive causes of death similar to<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>: “collision” (trauma caused by vehicles; n = 24, 4.7%), “legal” (lethal control by management agencies; n = 32, 6.2%), “poached” (illegal human-caused killing; n = 88, 17.2%), “nonhuman” (causes unrelated to people, e.g.: other wolves or diseases; n = 77, 15.0%) and “uncertain” (uncertain cause but the wolf carcass was recovered, i.e.: difficult to discern in necropsy; n = 21, 4.1%). We added a sixth distinct category of LTF endpoint (n = 231, 45.0%, and see Supplementary Data <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>) and we address 40 collared wolves missing endpoint dates (7.7%) below.</p><p id=\"Par53\">We defined the date of endpoint either as the recorded date of death for wolves monitored by telemetry until death (n = 242, 47.2% of sample) or as the date of last telemetry contact for LTF wolves (n = 231, 45.0%). Some of the LTF wolves were found dead later (n = 51), through means other than telemetry (e.g., visual detection), which might bias to a later date of ‘death’, if carcasses were found long after the actual date of death which was not uncommon<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. Given the sensitivity of time-to-event models to the accuracy of endpoint dates and because most (n = 206, 78% of the LTF subsample) were never detected again, our step to restrict the record histories of LTF wolves to the last date of monitoring is an important yet imperfect improvement in measurement precision.</p><p id=\"Par54\">Accounting for all individuals at risk of experiencing an endpoint at any particular time <italic>T</italic> (the ‘risk set’) is essential for obtaining unbiased estimates of HR, SHR, and CIF<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref>,<xref ref-type=\"bibr\" rid=\"CR44\">44</xref>,<xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>. Omitting a class of individuals (e.g., LTF) strongly biased risk estimates for four populations of wolves, and in the Wisconsin wolf population specifically, as summarized above<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>.</p></sec><sec id=\"Sec19\"><title>Model covariates</title><p id=\"Par55\">We included three time-dependent categorical covariates in our models. Time-dependent covariates are variables that change value due to external events at a known date, either for individual wolves or all wolves. For example, we modeled policy period as time-dependent by changing the covariate value at the dates of policy change for a particular individual’s history of monitoring. To assign categorical values of the time-dependent covariates to each monitored wolf, we split each history at each specified date of change in covariate value. We refer to the splits for a monitored wolf as ‘spells’, because they refer to briefer time periods within an individual’s total monitoring time <italic>T</italic>. So, the time-dependent categorical covariates have a duration that overlaps the monitoring period for collared wolves during that period, but the wolves have individual spells that might be less than or equal to the duration (see example in Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S1</xref>).</p><p id=\"Par56\">Our main covariate of interest is policy that liberalized wolf-killing (<italic>lib_kill</italic> where 1 = liberalized killing, 0 = full protection). Gray wolves experienced full protection under the ESA from 1979 to March 31, 2003. From April 1, 2003, wolves in WI and MI were subject to 11 alternating sequential, non-overlapping periods in which wolf-killing policies were first liberalized and then restricted for varied durations (Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S2</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. Although WDNR or its agents occasionally killed a wolf during full protection periods, in capture-related accidents or after verified threats to human safety, these were rare and few. By contrast, liberalized killing periods were characterized by an announcement of policy change that allowed managers or private landowners to kill wolves for perceived or verified losses of domestic animals. Liberalized killing periods included:<list list-type=\"bullet\"><list-item><p id=\"Par57\">‘Downlisting’ to threatened status (one period starting April 1, 2003; 670 days, Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S2</xref>)—allows for lethal control in defense of human property or safety as well as for population management or conservation purposes under ESA section Rule 4(d).</p></list-item><list-item><p id=\"Par58\">Issuing of sub-permits for “take” (“to harass, harm, pursue, hunt, shoot, wound, kill, trap, capture, or collect, or to attempt to engage in any such conduct” [ESA]) of wolves by managers and sometimes private landowners (periods within 2005 and 2006; 263 days, Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S2</xref>) under ESA sections 9 and 10.</p></list-item><list-item><p id=\"Par59\">‘Delisting’, or removing ESA protections entirely (periods of 2007, 2009 and 2012; 701 days, Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S2</xref>).</p></list-item></list></p><p id=\"Par60\">Choosing to end our study on April 14, 2012 presented several advantages. First, the WDNR summarized wolf census data and population reports for the preceding year on April 15th. Second, we could compare our results to prior work<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup>. Third, the April 2012 passage of Act 169 enacting the first wolf-hunting seasons since wolf bounties were terminated in the 1950s<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup> was a qualitatively different policy signal than those of the liberalized killing periods (Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S2</xref>).</p><p id=\"Par61\">Our second binary covariate, <italic>winter,</italic> produced spells for October–March (‘1’, winter) and April-September (‘0’, summer). Our inclusion of this variable is warranted by robust independent evidence of seasonal differences in both overall and endpoint-specific mortality<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR51\">51</xref>,<xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>. Most LTF endpoints occurred during winter months (143/231 = 62% of LTF wolves, with n = 40 wolves censored).</p><p id=\"Par62\">Our third covariate had three levels for periods with different methods of censusing wolves (<italic>method_change</italic>). In the winter of 1994–1995 the wolf census methods changed, and did so again sometime between summer 2000 and winter 2003–2004, with changes in monitoring techniques and protocols for data handling<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Those changes affected effort and training of wolf census-takers, so might have affected the detection and monitoring effort for collared wolves also. Although there is some ambiguity in the literature over the exact dates of these changes, we opted for the following splits based on year of endpoint: 1979–1994 (‘1’), 1995–2000 (‘2’) and 2001–2012 (‘3’).</p></sec><sec id=\"Sec20\"><title>Imputation for 2012 records without endpoint data</title><p id=\"Par63\">We right-censored the interval for individuals that did not experience an endpoint during the analysis period (start of monitoring until April 14, 2012), meaning they are considered as part of the risk set from collaring until the end of the analysis period. Our dataset includes 40 wolves without attributed mortality of disappearance data, because we could not find their endpoint (i.e., cause of death or disappearance) in public records after December 31st, 2011 (see supplementary data files for WDNR monitoring records for 2012 and 2013). Although 14 of those 40 wolves were later found dead in mortality reports between May 2012 and October 2013 (Supplementary Data <xref rid=\"MOESM2\" ref-type=\"media\">S2</xref>), those reports did not reveal the last date of monitoring but rather a lengthy interval without a record of monitoring followed by discovery of the dead animal. Therefore, we conservatively censored those 14 wolves at April 14, 2012 to consider them as within the risk set (monitored) for the corresponding time intervals, yet without experiencing an endpoint during that time. For the other 26 censored wolves that vanished from public records after December 31st, 2011, our repeated efforts to obtain data were not fulfilled by the WDNR. We submitted four separate requests to the WDNR (1 open records request, 1 state Natural Heritage Inventory request, a personal request to research staff who have published analyses with those data, and we enlisted the aid of the lieutenant governor and governor’s offices to request those data) for all collared wolves monitored in the state in 2012. Therefore, we simulated their endpoints in three scenarios described below.</p><p id=\"Par64\">We imputed either an LTF or censored status to the <italic>n</italic> = 26 wolves with missing endpoints based on the rationale that if any of these monitored wolves had suffered a death rather than a disappearance, their deaths should have appeared in mortality records spanning January 1, 2012 (when missing records for these wolves begin) to October 31, 2013, as happened with the 14 wolves with missing endpoint but found in subsequent mortality reports and therefore censored. Thus, the two remaining possibilities are that these wolves were either LTF or survived our analysis period and beyond October 31, 2013 which means they must be included in the risk set but be censored for endpoint analyses because they do not fit our 6 categories of endpoint.</p><p id=\"Par65\">For our simulation scenarios, we developed a series of FG imputation models (IMs) with LTF as the endpoint of interest using the above covariates for the full, original dataset (i.e., with all 40 wolves with missing data classified as ‘censored’ on April 14, 2012). We then used the most appropriate FG model (accounting for Akaike’s Information Criterion (AIC), Bayesian Information Criterion (BIC), log-likelihood (LL), parsimony and proportionality assumptions) to predict the probability of LTF incidence by April 14, 2012 for each of the 26 wolves. Because we assumed all 26 wolves were alive on April 14, 2012 (i.e., each is imputed their maximum <italic>survival</italic> time) for all models, whereas they might actually have disappeared earlier in 2012, our approach is conservative because it likely underestimates the relative incidence of LTF.</p><p id=\"Par66\">To calculate each of the 26 wolves’ probability of LTF, we first calculated the baseline CIF for the best IM and multiplied it by the exponentiated <italic>lib_kill</italic> and <italic>winter</italic> coefficients in Model 2 to obtain a probability of LTF for each wolf during winter periods with liberalized killing, as wolves experienced during the period beginning January 28, 2012 until April 14, 2012 (Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S2</xref>). Then we ran 1,000 simulations for each wolf going LTF, using a Bernoulli distribution with the LTF probability for each wolf as the probability of success (‘LTF’). For our MAIN imputation scenario, each wolf was imputed an LTF endpoint (on April 14, 2012) if the simulated occurrence of the LTF endpoint was higher than the probability of LTF predicted from the FG model (used as an imputation threshold), <inline-formula id=\"IEq12\"><alternatives><tex-math id=\"M23\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${p}_{i,SIM}\\left(ltf\\right)>{p}_{i,FG}(ltf)$$\\end{document}</tex-math><mml:math id=\"M24\"><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mo>,</mml:mo><mml:mi>S</mml:mi><mml:mi>I</mml:mi><mml:mi>M</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>l</mml:mi><mml:mi>t</mml:mi><mml:mi>f</mml:mi></mml:mfenced><mml:mo>></mml:mo><mml:msub><mml:mi>p</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mo>,</mml:mo><mml:mi>F</mml:mi><mml:mi>G</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>l</mml:mi><mml:mi>t</mml:mi><mml:mi>f</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq12.gif\"/></alternatives></inline-formula>, otherwise we censored that wolf. To analyze sensitivity to the MAIN scenario, we also developed HIGH and LOW scenarios following a similar imputation process (Supplementary Data <xref rid=\"MOESM3\" ref-type=\"media\">S3</xref>). For the HIGH imputation scenario, we increased the threshold probability for going LTF by half the difference between <inline-formula id=\"IEq13\"><alternatives><tex-math id=\"M25\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${p}_{i,FG}(ltf)$$\\end{document}</tex-math><mml:math id=\"M26\"><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mo>,</mml:mo><mml:mi>F</mml:mi><mml:mi>G</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>l</mml:mi><mml:mi>t</mml:mi><mml:mi>f</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq13.gif\"/></alternatives></inline-formula> and 1; <inline-formula id=\"IEq14\"><alternatives><tex-math id=\"M27\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${p}_{i,HI}\\left(ltf\\right)={p}_{i,FG}\\left(ltf\\right)+(1-{p}_{i,FG}\\left(ltf\\right)/2$$\\end{document}</tex-math><mml:math id=\"M28\"><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mo>,</mml:mo><mml:mi>H</mml:mi><mml:mi>I</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>l</mml:mi><mml:mi>t</mml:mi><mml:mi>f</mml:mi></mml:mfenced><mml:mo>=</mml:mo><mml:msub><mml:mi>p</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mo>,</mml:mo><mml:mi>F</mml:mi><mml:mi>G</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>l</mml:mi><mml:mi>t</mml:mi><mml:mi>f</mml:mi></mml:mfenced><mml:mrow><mml:mo>+</mml:mo><mml:mo stretchy=\"false\">(</mml:mo><mml:mn>1</mml:mn><mml:mo>-</mml:mo></mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mo>,</mml:mo><mml:mi>F</mml:mi><mml:mi>G</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>l</mml:mi><mml:mi>t</mml:mi><mml:mi>f</mml:mi></mml:mfenced><mml:mo stretchy=\"false\">/</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq14.gif\"/></alternatives></inline-formula>. For the LOW imputation scenario, we decreased the threshold probability for going LTF by half of <inline-formula id=\"IEq15\"><alternatives><tex-math id=\"M29\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${p}_{i,FG}(ltf)$$\\end{document}</tex-math><mml:math id=\"M30\"><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mo>,</mml:mo><mml:mi>F</mml:mi><mml:mi>G</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>l</mml:mi><mml:mi>t</mml:mi><mml:mi>f</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq15.gif\"/></alternatives></inline-formula>; <inline-formula id=\"IEq16\"><alternatives><tex-math id=\"M31\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${p}_{i,LO}\\left(ltf\\right)={p}_{i,FG}\\left(ltf\\right)-{p}_{i,FG}(ltf)/2$$\\end{document}</tex-math><mml:math id=\"M32\"><mml:mrow><mml:msub><mml:mi>p</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mo>,</mml:mo><mml:mi>L</mml:mi><mml:mi>O</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>l</mml:mi><mml:mi>t</mml:mi><mml:mi>f</mml:mi></mml:mfenced><mml:mo>=</mml:mo><mml:msub><mml:mi>p</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mo>,</mml:mo><mml:mi>F</mml:mi><mml:mi>G</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>l</mml:mi><mml:mi>t</mml:mi><mml:mi>f</mml:mi></mml:mfenced><mml:mo>-</mml:mo><mml:msub><mml:mi>p</mml:mi><mml:mrow><mml:mi>i</mml:mi><mml:mo>,</mml:mo><mml:mi>F</mml:mi><mml:mi>G</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mi>l</mml:mi><mml:mi>t</mml:mi><mml:mi>f</mml:mi><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow><mml:mo stretchy=\"false\">/</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70837_Article_IEq16.gif\"/></alternatives></inline-formula>. The LOW and HIGH scenarios provided bounds on the point estimates of relative hazard and incidence for the simulated LTF process in the MAIN scenario.</p></sec><sec id=\"Sec21\"><title>Statistical tests</title><p id=\"Par67\">To model all endpoint-specific HRs, we employed Lunn & McNeil’s (1995 Method B) data augmentation method. Namely, we augmented the data by our 6 endpoint categories and employed stratified joint Cox multiple regression (on endpoint) with interactions between covariates and each endpoint. Our initial model included all interactions. We then discarded the weakest first to follow model selection procedures while retaining the policy variable in all models (7 models total, Supplementary Table <xref rid=\"MOESM4\" ref-type=\"media\">S5</xref>). The approach provides us with covariate HRs for all endpoints and we use those HRs for estimating the CIFs by policy period for each endpoint. We model HR distributions of covariates for our poaching and LTF by exponentiating a normal distribution parameterized with the covariate coefficients and standard deviations obtained from their respective Cox models.</p><p id=\"Par68\">We also ran separate FG univariate and multivariate models, which mirrored the best stratified joint Cox model, to estimate FG CIFs for each endpoint. We compared CIFs visually to identify the most appropriate CIF model estimate (Cox or FG), following<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>.</p><p id=\"Par69\">Given the complete survival history of each individual wolf was split into multiple spells, we clustered all our regression analyses using a unique identifier for each wolf, following methods in<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>. Clustering on wolf identity accounts for auto-correlation (e.g., all spells are analyzed within-subjects) and avoids pseudo-replication of observations. We evaluated compliance with the proportionality assumptions for each model through the inclusion of time-varying coefficients (tvc). A tvc is an interaction of each parameter with analysis time which models changes in that parameter’s effect over time; i.e., non-proportionality. Endpoint-specific models with significant non-proportionality in a covariate (tvc) cannot provide predictions of risk or incidence due to computational limitations. We further verified proportionality using Schoenfeld residuals<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref>,<xref ref-type=\"bibr\" rid=\"CR44\">44</xref>,<xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>, which should show a random pattern against time as evidence of compliance with the PH assumption. We selected the best regression models considering AIC, BIC, LL, parsimony, and compliance with model assumptions. When we set aside a best model because of non-proportionality, we present and discuss the best model but our CIF calculations use parameters from the same Cox or FG model without the tvc. We visually assessed goodness-of-fit for each selected endpoint-specific Cox model by Cox-Snell residual plots, which should show the Nelson-Aalen cumulative hazard closely following the line of Cox-Snell residuals if the model is a good fit. We conducted all statistical analyses in Stata 15 (StatCorp, College Station, TX, 2015; see supplementary materials for statistical code).</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec22\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70837_MOESM1_ESM.xlsx\"><caption><p>Supplementary Dataset 1.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41598_2020_70837_MOESM2_ESM.xlsx\"><caption><p>Supplementary Dataset 2.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM3\"><media xlink:href=\"41598_2020_70837_MOESM3_ESM.xlsx\"><caption><p>Supplementary Dataset 3.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM4\"><media xlink:href=\"41598_2020_70837_MOESM4_ESM.docx\"><caption><p>Supplementary Information</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70837-x.</p></sec><ack><title>Acknowledgements</title><p>We thank the Wisconsin Department of Natural Resources and US Fish & Wildlife Service for data collection. We thank the Nelson Institute for Environmental Studies, the UCLA Law School Animal Law & Policy Grants Program and Therese Foundation, Inc. for funding. This article does not necessarily reflect the views of the institutions or agencies involved.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>Conceptualization: FSA, AT. Data curation: FSA, AT. Formal analysis: FSA. Funding acquisition: FSA, AT. Investigation: FSA, AT. Methodology: FSA. Project administration: FSA. Resources: FSA, RJC, AT. Software: FSA. Supervision: RJC, AT. Validation: FSA. Writing—original draft: FSA. Writing—review & editing: FSA, RJC, AT.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>All data and statistical code is available in the main text, supplementary materials or from [INSTITUTIONAL DATA REPOSITORY].</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par70\">FSA and RJC declare no competing interests. AT declares no competing interests, and provides his CV (<ext-link ext-link-type=\"uri\" xlink:href=\"https://faculty.nelson.wisc.edu/treves/archive_BAS/Treves_vita_Jan2020.pdf\">https://faculty.nelson.wisc.edu/treves/archive_BAS/Treves_vita_Jan2020.pdf</ext-link>) and all funding awarded as of 6 Jan 2020 (<ext-link ext-link-type=\"uri\" xlink:href=\"https://faculty.nelson.wisc.edu/treves/archive_BAS/funding.pdf\">https://faculty.nelson.wisc.edu/treves/archive_BAS/funding.pdf</ext-link>) for transparency, so readers can decide if they perceive a competing interest.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Terborgh</surname><given-names>J</given-names></name><etal/></person-group><article-title>The role of top carnivores in regulating terrestrial ecosystems</article-title><source>Endanger. Species Update</source><year>1999</year><volume>18</volume><fpage>2001</fpage></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Estes</surname><given-names>JA</given-names></name><etal/></person-group><article-title>Trophic downgrading of planet Earth</article-title><source>Science</source><year>2011</year><volume>333</volume><fpage>301</fpage><lpage>306</lpage><pub-id pub-id-type=\"pmid\">21764740</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ripple</surname><given-names>WJ</given-names></name><etal/></person-group><article-title>Status and ecological effects of the world’s largest carnivores</article-title><source>Science</source><year>2014</year><volume>343</volume><fpage>1241484</fpage><pub-id pub-id-type=\"pmid\">24408439</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Woodroffe</surname><given-names>R</given-names></name><name><surname>Ginsberg</surname><given-names>JR</given-names></name></person-group><article-title>Edge effects and the extinction of populations inside protected areas</article-title><source>Science</source><year>1998</year><volume>280</volume><fpage>2126</fpage><lpage>2128</lpage><pub-id pub-id-type=\"pmid\">9641920</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Treves</surname><given-names>A</given-names></name><name><surname>Langenberg</surname><given-names>JA</given-names></name><name><surname>López-Bao</surname><given-names>JV</given-names></name><name><surname>Rabenhorst</surname><given-names>MF</given-names></name></person-group><article-title>Gray wolf mortality patterns in Wisconsin from 1979 to 2012</article-title><source>J. Mamm.</source><year>2017</year><volume>98</volume><fpage>17</fpage><lpage>32</lpage></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Creel</surname><given-names>S</given-names></name><etal/></person-group><article-title>Questionable policy for large carnivore hunting</article-title><source>Science</source><year>2015</year><volume>350</volume><fpage>1473</fpage><lpage>1475</lpage><pub-id pub-id-type=\"pmid\">26680181</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chapron</surname><given-names>G</given-names></name><etal/></person-group><article-title>Recovery of large carnivores in Europe’s modern human-dominated landscapes</article-title><source>Science</source><year>2014</year><volume>346</volume><fpage>1517</fpage><lpage>1519</lpage><pub-id pub-id-type=\"pmid\">25525247</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Suutarinen</surname><given-names>J</given-names></name><name><surname>Kojola</surname><given-names>I</given-names></name></person-group><article-title>Poaching regulates the legally hunted wolf population in Finland</article-title><source>Biol. Conserv.</source><year>2017</year><volume>215</volume><fpage>11</fpage><lpage>18</lpage></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Treves</surname><given-names>A</given-names></name><name><surname>Artelle</surname><given-names>KA</given-names></name><name><surname>Darimont</surname><given-names>CT</given-names></name><name><surname>Parsons</surname><given-names>DR</given-names></name></person-group><article-title>Mismeasured mortality: Correcting estimates of wolf poaching in the United States</article-title><source>J. Mamm.</source><year>2017</year><pub-id pub-id-type=\"doi\">10.1093/jmammal/gyx052</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liberg</surname><given-names>O</given-names></name><etal/></person-group><article-title>Shoot, shovel and shut up: Cryptic poaching slows restoration of a large carnivore in Europe</article-title><source>Proc. R. Soc. B Biol. Sci.</source><year>2012</year><volume>279</volume><fpage>910</fpage><lpage>915</lpage></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Robinson</surname><given-names>HS</given-names></name><name><surname>Goodrich</surname><given-names>JM</given-names></name><name><surname>Miquelle</surname><given-names>DG</given-names></name><name><surname>Miller</surname><given-names>CS</given-names></name><name><surname>Seryodkin</surname><given-names>IV</given-names></name></person-group><article-title>Mortality of Amur tigers: The more things change, the more they stay the same</article-title><source>Integr. Zool.</source><year>2015</year><volume>10</volume><fpage>344</fpage><lpage>353</lpage><pub-id pub-id-type=\"pmid\">26096683</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chapron</surname><given-names>G</given-names></name><name><surname>Treves</surname><given-names>A</given-names></name></person-group><article-title>Blood does not buy goodwill: Allowing culling increases poaching of a large carnivore</article-title><source>Proc. R. Soc. Lond. B Biol. Sci.</source><year>2016</year><volume>283</volume><fpage>20152939</fpage></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chapron</surname><given-names>G</given-names></name><etal/></person-group><article-title>The impact on tigers of poaching versus prey depletion</article-title><source>J. Appl. Ecol.</source><year>2008</year><volume>45</volume><fpage>1667</fpage><lpage>1674</lpage></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chapron</surname><given-names>G</given-names></name><name><surname>Treves</surname><given-names>A</given-names></name></person-group><article-title>Correction to ‘blood does not buy goodwill: Allowing culling increases poaching of a large carnivore’</article-title><source>Proc. R. Soc. B Biol. Sci.</source><year>2016</year><volume>283</volume><fpage>20162577</fpage></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Adams</surname><given-names>LG</given-names></name><name><surname>Stephenson</surname><given-names>RO</given-names></name><name><surname>Dale</surname><given-names>BW</given-names></name><name><surname>Ahgook</surname><given-names>RT</given-names></name><name><surname>Demma</surname><given-names>DJ</given-names></name></person-group><article-title>Population dynamics and harvest characteristics of wolves in the central brooks range, Alaska</article-title><source>Wildl. Monogr.</source><year>2008</year><volume>170</volume><fpage>1</fpage><lpage>25</lpage></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Treves</surname><given-names>A</given-names></name><name><surname>Krofel</surname><given-names>M</given-names></name><name><surname>Lopez-Bao</surname><given-names>JV</given-names></name></person-group><article-title>Missing wolves, misguided policy</article-title><source>Science</source><year>2016</year><volume>350</volume><fpage>1473</fpage><lpage>1475</lpage></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Treves</surname><given-names>A</given-names></name></person-group><article-title>Hunting for large carnivore conservation</article-title><source>J. Appl. Ecol.</source><year>2009</year><volume>46</volume><fpage>1350</fpage><lpage>1356</lpage></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chapron</surname><given-names>G</given-names></name><name><surname>Treves</surname><given-names>A</given-names></name></person-group><article-title>Reply to comments by Olson et al. 2017 and Stien 2017</article-title><source>Proc. R. Soc. B Biol. Sci.</source><year>2017</year><pub-id pub-id-type=\"doi\">10.1098/rspb.2017.1743</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Epstein</surname><given-names>Y</given-names></name></person-group><article-title>Killing wolves to save them? Legal responses to ‘tolerance hunting’in the European Union and United States</article-title><source>Rev. Eur. Comp. Int. Environ. Law</source><year>2017</year><volume>26</volume><fpage>19</fpage><lpage>29</lpage></element-citation></ref><ref id=\"CR20\"><label>20.</label><mixed-citation publication-type=\"other\">Epstein, Y. & Chapron, G. <italic>The Hunting of Strictly Protected Species: The Tapiola Case and the Limits of Derogation under Article 16 of the Habitats Directive The Hunting of Strictly Protected Species</italic>, Vol. 26, 78–87 (2018).</mixed-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Olson</surname><given-names>ER</given-names></name><etal/></person-group><article-title>Pendulum swings in wolf management led to conflict, illegal kills, and a legislated wolf hunt</article-title><source>Conserv. Lett.</source><year>2015</year><volume>8</volume><fpage>351</fpage><lpage>360</lpage></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stenglein</surname><given-names>JL</given-names></name><name><surname>Zhu</surname><given-names>J</given-names></name><name><surname>Clayton</surname><given-names>MK</given-names></name><name><surname>Van Deelen</surname><given-names>TR</given-names></name></person-group><article-title>Are the numbers adding up? Exploiting discrepancies among complementary population models</article-title><source>Ecol. Evol.</source><year>2015</year><volume>5</volume><fpage>368</fpage><lpage>376</lpage><pub-id pub-id-type=\"pmid\">25691964</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><mixed-citation publication-type=\"other\"><italic>Summary Report of Independent Peer Reviews of the Proposed Rule and Draft Biological Report for Nationwide Wolf Delisting for the US Fish and Wildlife Service</italic>. (2019).</mixed-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Treves</surname><given-names>A</given-names></name><name><surname>Naughton-Treves</surname><given-names>L</given-names></name><name><surname>Shelley</surname><given-names>V</given-names></name></person-group><article-title>Longitudinal analysis of attitudes toward wolves</article-title><source>Conserv. Biol.</source><year>2013</year><volume>27</volume><fpage>315</fpage><lpage>323</lpage><pub-id pub-id-type=\"pmid\">23293913</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Browne-Nuñez</surname><given-names>C</given-names></name><name><surname>Treves</surname><given-names>A</given-names></name><name><surname>MacFarland</surname><given-names>D</given-names></name><name><surname>Voyles</surname><given-names>Z</given-names></name><name><surname>Turng</surname><given-names>C</given-names></name></person-group><article-title>Tolerance of wolves in Wisconsin: A mixed-methods examination of policy effects on attitudes and behavioral inclinations</article-title><source>Biol. Conserv.</source><year>2015</year><volume>189</volume><fpage>59</fpage><lpage>71</lpage></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Treves</surname><given-names>A</given-names></name><name><surname>Krofel</surname><given-names>M</given-names></name><name><surname>Ohrens</surname><given-names>O</given-names></name><name><surname>van Eeden</surname><given-names>LM</given-names></name></person-group><article-title>Predator control needs a standard of unbiased randomized experiments with cross-over design</article-title><source>Front. Ecol. Evol.</source><year>2019</year><volume>7</volume><fpage>1</fpage><lpage>14</lpage></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Suutarinen</surname><given-names>J</given-names></name><name><surname>Kojola</surname><given-names>I</given-names></name></person-group><article-title>One way or another: Predictors of wolf poaching in a legally harvested wolf population</article-title><source>Anim. Conserv.</source><year>2018</year><pub-id pub-id-type=\"doi\">10.1111/acv.12409</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Refsnider</surname><given-names>RL</given-names></name></person-group><person-group person-group-type=\"editor\"><name><surname>Wydeven</surname><given-names>AP</given-names></name><name><surname>Van Deelan</surname><given-names>TR</given-names></name><name><surname>Heske</surname><given-names>E</given-names></name></person-group><article-title>The role of the Endangered Species Act in midwest wolf recovery</article-title><source>Recovery of Gray Wolves in the Great Lakes Region of the United States</source><year>2009</year><publisher-loc>New York</publisher-loc><publisher-name>Springer</publisher-name><fpage>311</fpage><lpage>329</lpage></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Latouche</surname><given-names>A</given-names></name><name><surname>Allignol</surname><given-names>A</given-names></name><name><surname>Beyersmann</surname><given-names>J</given-names></name><name><surname>Labopin</surname><given-names>M</given-names></name><name><surname>Fine</surname><given-names>JP</given-names></name></person-group><article-title>A competing risks analysis should report results on all cause-specific hazards and cumulative incidence functions</article-title><source>J. Clin. Epidemiol.</source><year>2013</year><volume>66</volume><fpage>648</fpage><lpage>653</lpage><pub-id pub-id-type=\"pmid\">23415868</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Austin</surname><given-names>PC</given-names></name><name><surname>Lee</surname><given-names>DS</given-names></name><name><surname>Fine</surname><given-names>JP</given-names></name></person-group><article-title>Introduction to the analysis of survival data in the presence of competing risks</article-title><source>Circulation</source><year>2016</year><volume>133</volume><fpage>601</fpage><lpage>609</lpage><pub-id pub-id-type=\"pmid\">26858290</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dignam</surname><given-names>JJ</given-names></name><name><surname>Kocherginsky</surname><given-names>MN</given-names></name></person-group><article-title>Choice and interpretation of statistical tests used when competing risks are present</article-title><source>J. Clin. Oncol.</source><year>2008</year><volume>26</volume><fpage>4027</fpage><lpage>4034</lpage><pub-id pub-id-type=\"pmid\">18711194</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dignam</surname><given-names>JJ</given-names></name><name><surname>Zhang</surname><given-names>Q</given-names></name><name><surname>Kocherginsky</surname><given-names>MNM</given-names></name></person-group><article-title>The use and interpretation of competing risks regression models</article-title><source>Clin. Cancer Res.</source><year>2012</year><volume>18</volume><fpage>2301</fpage><lpage>2308</lpage><pub-id pub-id-type=\"pmid\">22282466</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Amrhein</surname><given-names>V</given-names></name><name><surname>Greenland</surname><given-names>S</given-names></name><name><surname>McShane</surname><given-names>B</given-names></name></person-group><article-title>Retire statistical significance</article-title><source>Nature</source><year>2019</year><volume>567</volume><fpage>305</fpage><lpage>307</lpage><pub-id pub-id-type=\"pmid\">30894741</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Borg</surname><given-names>BL</given-names></name><name><surname>Brainerd</surname><given-names>SM</given-names></name><name><surname>Meier</surname><given-names>TJ</given-names></name><name><surname>Prugh</surname><given-names>LR</given-names></name></person-group><article-title>Impacts of breeder loss on social structure, reproduction and population growth in a social canid</article-title><source>J. Anim. Ecol.</source><year>2015</year><volume>84</volume><fpage>177</fpage><lpage>187</lpage><pub-id pub-id-type=\"pmid\">25041127</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brainerd</surname><given-names>SM</given-names></name><etal/></person-group><article-title>The effects of breeder loss on wolves</article-title><source>J. Wildl. Manage.</source><year>2008</year><volume>72</volume><fpage>89</fpage><lpage>98</lpage></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Milleret</surname><given-names>C</given-names></name><etal/></person-group><article-title>Let’s stay together? Intrinsic and extrinsic factors involved in pair bond dissolution in a recolonizing wolf population</article-title><source>J. Anim. Ecol.</source><year>2017</year><volume>86</volume><fpage>43</fpage><lpage>54</lpage><pub-id pub-id-type=\"pmid\">27559712</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Fuller</surname><given-names>TK</given-names></name><name><surname>Mech</surname><given-names>LD</given-names></name><name><surname>Cochrane</surname><given-names>JF</given-names></name></person-group><person-group person-group-type=\"editor\"><name><surname>Mech</surname><given-names>D</given-names></name><name><surname>Boitani</surname><given-names>L</given-names></name></person-group><article-title>Wolf population dynamics</article-title><source>Wolves: Behavior, Ecology and Conservation</source><year>2003</year><publisher-loc>Chicago</publisher-loc><publisher-name>The University of Chicago Press</publisher-name></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hogberg</surname><given-names>J</given-names></name><name><surname>Treves</surname><given-names>A</given-names></name><name><surname>Shaw</surname><given-names>B</given-names></name><name><surname>Naughton-Treves</surname><given-names>L</given-names></name></person-group><article-title>Changes in attitudes toward wolves before and after an inaugural public hunting and trapping season: Early evidence from Wisconsin’s wolf range</article-title><source>Environ. Conserv.</source><year>2015</year><volume>43</volume><fpage>45</fpage><lpage>55</lpage></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Olson</surname><given-names>ER</given-names></name><etal/></person-group><article-title>(2017) Flawed analysis and unconvincing interpretation: A comment on Chapron and Treves 2016</article-title><source>Proc. R. Soc. B Biol. Sci.</source><year>2017</year><pub-id pub-id-type=\"doi\">10.1098/rspb.2017.0273</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Hsiang</surname><given-names>S</given-names></name><name><surname>Sekar</surname><given-names>N</given-names></name></person-group><source>Does Legalization Reduce Black Market Activity? Evidence from a Global Ivory Experiment and Elephant Poaching Data</source><year>2016</year><publisher-loc>Cambridge</publisher-loc><publisher-name>National Bureau of Economic Research</publisher-name></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Wydeven</surname><given-names>AP</given-names></name><etal/></person-group><person-group person-group-type=\"editor\"><name><surname>Wydeven</surname><given-names>AP</given-names></name><name><surname>Van Deelan</surname><given-names>TR</given-names></name><name><surname>Heske</surname><given-names>E</given-names></name><etal/></person-group><article-title>History, population growth, and management of wolves in Wisconsin</article-title><source>Recovery of Gray Wolves in the Great Lakes Region of the United States</source><year>2009</year><publisher-loc>New York</publisher-loc><publisher-name>Springer</publisher-name><fpage>87</fpage><lpage>105</lpage></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Wydeven</surname><given-names>AP</given-names></name><name><surname>Treves</surname><given-names>A</given-names></name><name><surname>Brost</surname><given-names>B</given-names></name><name><surname>Wiedenhoeft</surname><given-names>JE</given-names></name></person-group><source>Characteristics of Wolf Packs in Wisconsin: Identification of Traits Influencing Depredation</source><year>2004</year><publisher-loc>Washington DC</publisher-loc><publisher-name>People Predators From Conflict to Coexistence Island Press</publisher-name><fpage>28</fpage><lpage>50</lpage></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Heisey</surname><given-names>DM</given-names></name><name><surname>Patterson</surname><given-names>BR</given-names></name></person-group><article-title>A review of methods to estimate cause-specific mortality in presence of competing risks</article-title><source>J. Wildl. Manage.</source><year>2006</year><volume>70</volume><fpage>1544</fpage><lpage>1555</lpage></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Hosmer</surname><given-names>DW</given-names></name><name><surname>Lemeshow</surname><given-names>S</given-names></name><name><surname>May</surname><given-names>S</given-names></name></person-group><source>Applied Survival Analysis: Regression Modelling of Time to Event Data</source><year>2008</year><edition>2</edition><publisher-loc>Hoboken</publisher-loc><publisher-name>Wiley</publisher-name></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lunn</surname><given-names>M</given-names></name><name><surname>McNeil</surname><given-names>D</given-names></name></person-group><article-title>Applying Cox regression to competing risks</article-title><source>Biometrics</source><year>1995</year><volume>51</volume><fpage>524</fpage><lpage>532</lpage><pub-id pub-id-type=\"pmid\">7662841</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Putter</surname><given-names>H</given-names></name><name><surname>Fiocco</surname><given-names>M</given-names></name><name><surname>Geskus</surname><given-names>RB</given-names></name></person-group><article-title>Tutorial in biostatistics: Competing risks and multi-state models</article-title><source>Stat. Med.</source><year>2007</year><volume>26</volume><fpage>2389</fpage><lpage>2430</lpage><pub-id pub-id-type=\"pmid\">17031868</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fine</surname><given-names>JP</given-names></name><name><surname>Gray</surname><given-names>RJ</given-names></name></person-group><article-title>A proportional hazards model for the subdistribution of a competing risk</article-title><source>J. Am. Stat. Assoc.</source><year>1999</year><volume>94</volume><fpage>496</fpage><lpage>509</lpage></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Kalbfleisch</surname><given-names>JD</given-names></name><name><surname>Prentice</surname><given-names>RL</given-names></name></person-group><source>The Statistical Analysis of Failure Time Data</source><year>2002</year><publisher-loc>Hoboken</publisher-loc><publisher-name>Wiley</publisher-name></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stenglein</surname><given-names>JL</given-names></name><etal/></person-group><article-title>Mortality patterns and detection bias from carcass data: An example from wolf recovery in Wisconsin</article-title><source>J. Wildl. Manage.</source><year>2015</year><volume>79</volume><fpage>1173</fpage><lpage>1184</lpage></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Santiago-Ávila</surname><given-names>FJ</given-names></name><name><surname>Lynn</surname><given-names>WS</given-names></name><name><surname>Treves</surname><given-names>A</given-names></name></person-group><person-group person-group-type=\"editor\"><name><surname>Hovardas</surname><given-names>T</given-names></name></person-group><article-title>Inappropriate consideration of animal interests in predator management: Towards a comprehensive moral code</article-title><source>Large Carnivore Conservation and Management: Human Dimensions and Governance</source><year>2018</year><publisher-loc>Abingdon</publisher-loc><publisher-name>Routledge</publisher-name><fpage>227</fpage><lpage>251</lpage></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stenglein</surname><given-names>JL</given-names></name><name><surname>Wydeven</surname><given-names>AP</given-names></name><name><surname>Van Deelen</surname><given-names>TR</given-names></name></person-group><article-title>Compensatory mortality in a recovering top carnivore: Wolves in Wisconsin, USA (1979–2013)</article-title><source>Oecologia</source><year>2018</year><volume>187</volume><fpage>99</fpage><lpage>111</lpage><pub-id pub-id-type=\"pmid\">29627957</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schmidt</surname><given-names>JH</given-names></name><name><surname>Johnson</surname><given-names>DS</given-names></name><name><surname>Lindberg</surname><given-names>MS</given-names></name><name><surname>Adams</surname><given-names>LG</given-names></name></person-group><article-title>Estimating demographic parameters using a combination of known-fate and open N-mixture models</article-title><source>Ecology</source><year>2015</year><volume>96</volume><fpage>2583</fpage><lpage>2589</lpage><pub-id pub-id-type=\"pmid\">26649379</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coviello</surname><given-names>V</given-names></name><name><surname>Boggess</surname><given-names>M</given-names></name></person-group><article-title>Cumulative incidence estimation in the presence of competing risks</article-title><source>Stata J.</source><year>2004</year><volume>4</volume><fpage>103</fpage><lpage>112</lpage></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lin</surname><given-names>DY</given-names></name><name><surname>Wei</surname><given-names>L-J</given-names></name></person-group><article-title>The robust inference for the Cox proportional hazards model</article-title><source>J. Am. Stat. Assoc.</source><year>1989</year><volume>84</volume><fpage>1074</fpage><lpage>1078</lpage></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807854</article-id><article-id pub-id-type=\"pmc\">PMC7431571</article-id><article-id pub-id-type=\"publisher-id\">70397</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70397-0</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Post-stimulatory activity in primate auditory cortex evoked by sensory stimulation during passive listening</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Cooke</surname><given-names>James E.</given-names></name><address><email>james.cooke@ucl.ac.uk</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lee</surname><given-names>Julie J.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Bartlett</surname><given-names>Edward L.</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Xiaoqin</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Bendor</surname><given-names>Daniel</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.83440.3b</institution-id><institution-id institution-id-type=\"ISNI\">0000000121901201</institution-id><institution>Institute of Behavioural Neuroscience (IBN), </institution><institution>University College London (UCL), </institution></institution-wrap>London, WC1H 0AP UK </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.83440.3b</institution-id><institution-id institution-id-type=\"ISNI\">0000000121901201</institution-id><institution>Institute of Ophthalmology, </institution><institution>University College London (UCL), </institution></institution-wrap>London, WC1H 0AP UK </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.169077.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1937 2197</institution-id><institution>Departments of Biological Sciences and Biomedical Engineering, </institution><institution>Purdue University, </institution></institution-wrap>West Lafayette, 47907 USA </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.21107.35</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2171 9311</institution-id><institution>Departments of Biomedical Engineering, </institution><institution>Johns Hopkins University, </institution></institution-wrap>Baltimore, 21205 USA </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13885</elocation-id><history><date date-type=\"received\"><day>16</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>17</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Under certain circumstances, cortical neurons are capable of elevating their firing for long durations in the absence of a stimulus. Such activity has typically been observed and interpreted in the context of performance of a behavioural task. Here we investigated whether post-stimulatory activity is observed in auditory cortex and the medial geniculate body of the thalamus in the absence of any explicit behavioural task. We recorded spiking activity from single units in the auditory cortex (fields A1, R and RT) and auditory thalamus of awake, passively-listening marmosets. We observed post-stimulatory activity that lasted for hundreds of milliseconds following the termination of the acoustic stimulus. Post-stimulatory activity was observed following both adapting, sustained and suppressed response profiles during the stimulus. These response types were observed across all cortical fields tested, but were largely absent from the auditory thalamus. As well as being of shorter duration, thalamic post-stimulatory activity emerged following a longer latency than in cortex, indicating that post-stimulatory activity may be generated within auditory cortex during passive listening. Given that these responses were observed in the absence of an explicit behavioural task, post-stimulatory activity in sensory cortex may play a functional role in processes such as echoic memory and temporal integration that occur during passive listening.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Neuroscience</kwd><kwd>Auditory system</kwd><kwd>Sensory processing</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/501100000781</institution-id><institution>European Research Council</institution></institution-wrap></funding-source><award-id>CHIME</award-id></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/100010269</institution-id><institution>Wellcome Trust</institution></institution-wrap></funding-source><award-id>109004/Z/15/Z</award-id><principal-award-recipient><name><surname>Lee</surname><given-names>Julie J.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>DC06357</award-id><award-id>DC003180</award-id><principal-award-recipient><name><surname>Bartlett</surname><given-names>Edward L.</given-names></name><name><surname>Wang</surname><given-names>Xiaoqin</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/100004412</institution-id><institution>Human Frontier Science Program</institution></institution-wrap></funding-source><award-id>534669</award-id><principal-award-recipient><name><surname>Bendor</surname><given-names>Daniel</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Neurons in a number of cortical areas have been found to fire for durations on the order of seconds in the absence of sensory stimulation<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. This phenomenon was first described in the context of working memory tasks<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. In such tasks, subjects are typically presented with a cue and are required to respond after a delay period, during which the cue is no longer available. Neurons in the dorsolateral prefrontal cortex of macaque have been found to fire continuously during such delay periods, providing a possible substrate for the maintenance of information in working memory<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>–<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. In keeping with this proposed function, suppressing this activity has been shown to impair performance on working memory tasks<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>–<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>.</p><p id=\"Par3\">Such activity has been found to carry spatial information such as the location of visual stimulus<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR3\">3</xref>–<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> as well as the direction of a forthcoming saccade<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. Persistent activity has been reported in multiple cortical areas with differing functional properties. The intrinsic timescales of neural activity have been found to vary between cortical regions<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup> in a manner that predicts the functional properties of persistent activity across cortical regions<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. “In addition to prefrontal activity, sensory areas have been found to encode stimulus information across a delay period in the form of post-stimulatory activity. In a tone discrimination task where the tones were separated by a one-second delay period, neurons in the auditory cortex of the macaque were found to elevate their firing during the delay period<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. The presence of persistent delay period activity in auditory cortex during working memory tasks has been widely observed in the macaque<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>–<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. Such activity has also been observed in rodent auditory cortex during working memory tasks and reference memory tasks<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup> as well as during auditory-signaled response preparation<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup> and following delay conditioning<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. In addition to carrying stimulus information, persistent activity in auditory cortex has been found to relate to reinforcers and behavioral responses<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>–<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. Post-stimulatory activity has also been reported in human auditory cortex, as measured by magnetoencephalography (MEG)<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>. These findings demonstrate the ability of auditory cortical neurons to maintain their firing for hundreds of milliseconds in the absence of a coincident stimulus.”</p><p id=\"Par4\">During passive listening, the termination of an auditory stimulus routinely evokes transient offset responses in auditory cortical neurons, typically lasting up to tens of milliseconds<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref>–<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. In the cat, the tonal receptive fields of offset responses are often similar to the tuning of responses evoked at the onset of the stimulus although onset and offset receptive fields can also vary their tuning properties<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. In the mouse however, the tuning of onset and offset responses is largely distinct<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. Despite this lack of consistency in receptive field dynamics, the ability of auditory cortical networks to routinely generate activity following the termination of an effective auditory stimulus is a consistent feature across species. The maximum duration of activity that can be evoked following stimulus offset, however, is currently unknown.</p><p id=\"Par5\">Here, we investigated whether it is possible to evoke post-stimulatory activity in auditory cortical neurons in the absence of a behavioural task and aimed to characterise the properties of such activity. We analysed activity from single units in the auditory cortex (fields A1, R and RT) and thalamus of awake, passively-listening marmosets that were presented an array of auditory stimuli during experiments not concerned with investigating post-stimulatory activity<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>–<xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. We observed post-stimulatory activity lasting for hundreds of milliseconds following the termination of the acoustic stimulus in a sub-population of auditory cortical neurons. This activity followed a variety of response profiles during sensory stimulation, including adapting, sustained and suppressed responses. Post-stimulus activity had a shorter latency and was of longer duration in cortex than in thalamus, indicating that the mechanisms underlying this activity may be primarily cortical.</p></sec><sec id=\"Sec2\"><title>Results</title><sec id=\"Sec3\"><title>Post-stimulatory activity is observed in auditory cortex units during passive listening</title><p id=\"Par6\">The data analysed in this report were based on a database of 1557 single units recorded from the auditory cortex of 4 passively listening marmosets during the presentation of a variety of auditory stimuli in previous experiments. The stimuli presented were unmodulated pure tones, amplitude-modulated tones, white noise, band-pass noise and click trains. Of these units, 1,188 met our criteria to be included in further analysis (see “<xref rid=\"Sec7\" ref-type=\"sec\">Methods</xref>”). Units were required to show an elevation in firing > 2 standard deviations over mean baseline firing on at least half of the trials that was longer in duration than for baseline activity observed during the pre-stimulus period. For the initial analysis of post-stimulatory activity units were not separated by the cortical field they were recorded from. Post-stimulatory activity was observed in a subset of these single units in response to particular stimuli (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>A). Short-duration offset responses were commonly observed, with 77.78% (N = 924/1,188) of units showing significant post-stimulus activity beginning within 50 ms of stimulus offset (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B). By searching the stimulus space, it was possible to evoke post-stimulatory activity in 39.31% (N = 467/1,188) of the units recorded (see “<xref rid=\"Sec7\" ref-type=\"sec\">Methods</xref>”) (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>C).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Post-stimulatory activity during passive listening. (<bold>A</bold>) Example unit presented with bandpass noise stimuli of varying bandwidth. This units displays short-duration offset responses to the majority of stimuli presented but also post-stimulatory activity in response to particular bandwidths. Spike rasters showing a representative offset response are indicated by the +. Spike rasters showing post-stimulatory activity are indicated by the *. The within-stimulus period is indicated by the black bar while the duration of significant post-stimulus activity on these trials in indicated by grey shading. (<bold>B</bold>) The black trace corresponds to the PSTH of spiking activity indicated by the + symbol in A while the grey traces are the PSTHs associated with all other stimuli. The black bar indicates the stimulus duration. The coloured area of the PSTH indicates the duration of the post-stimulus response that was elevated above baseline. (<bold>C</bold>) PSTH of spiking activity indicated by the * symbol in (<bold>A</bold>), showing post-stimulatory activity in the same unit.</p></caption><graphic xlink:href=\"41598_2020_70397_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par7\">Stimulus parameters such as stimulus type and post-stimulus interval were varied across the neurons recorded. In order to quantify the duration of the observed post-stimulus activity, the analysis was initially restricted to stimuli with a post-stimulus interval (PSI) of 300 ms. This subset consisted of responses to tone and noise stimuli lasting 200 ms. For the population of 805 units recorded with these stimulus parameters, the longest duration post-stimulus response observed for each unit exceeded this 300 ms interval in 21.86% of units (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A). When the PSI was extended to 500 ms for tone and noise stimuli 5.23% of the 172 units tested exceeded this 500 ms interval (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>B). For units presented with tone, noise and click stimuli followed by a PSI of > 500 ms (N = 65), the range of post-stimulus response durations spanned from 12 to 1681 ms (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>C).<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Duration of post-stimulus activity. (<bold>A</bold>) Left panel: Distribution of longest duration post-stimulus activity for 805 single units presented with 200 ms tone and noise stimuli followed by a 300 ms post-stimulus interval (PSI). The black bar indicates units that showed post-stimulatory activity lasting for the entire PSI. Proportion of units here and throughout the paper refers to the fraction of the total number of units included in the particular analysis. Right panel: Normalised population PSTH for these responses. (<bold>B</bold>) Left panel: Distribution of longest duration post-stimulus activity for 172 single units presented with 500 ms tone and noise stimuli followed by a 500 ms PSI. Right panel: Normalised population PSTH for these responses. (<bold>C</bold>) Distribution of longest duration post-stimulus activity for 65 single units presented with tone, noise and click stimuli that were followed by a PSI > 500 ms.</p></caption><graphic xlink:href=\"41598_2020_70397_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par8\">Post-stimulatory activity was observed for all stimulus types tested. For stimuli with a PSI of 300 ms, tone stimuli produced significantly longer duration post-stimulatory activity (N units = 694, mean = 186.93 ms) than noise stimuli (N units = 308, mean = 167.96 ms; Two-Sample t-test, p < 0.001) (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>A). For stimuli with a PSI of 500 ms, no significant difference was found between the duration of post-stimulus activity in response to tone (N units = 80, mean = 196.3 ms) and noise stimuli (N units = 110, mean = 201.49 ms) (Two-Sample t-test, p = 0.8) (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>B). For stimuli with a PSI of > 500 ms, neurons were tested with tone (N units = 59, mean = 280.93 ms), noise (N units = 44, mean = 246.05 ms) and click train (N units = 29, mean = 256.21 ms) stimuli. No significant difference was observed in the duration of post-stimulus activity produced by these different stimulus types (ANOVA(2); p = 0.84, Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>C).<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Distribution of post-stimulatory activity durations produced by different stimulus types. (<bold>A</bold>) Comparison of the distributions of post-stimulatory activity duration produced by tone and noise stimuli followed by a 300 ms PSI. Post-stimulus activity in response to tone stimuli was found to be of longer duration than that produced by noise stimuli when tested with this PSI. (<bold>B</bold>) Comparison of post-stimulatory activity duration produced by tone and noise stimuli followed by a 500 ms PSI. No significant difference in the means of these distributions were observed. (<bold>C</bold>) Comparison of post-stimulatory activity duration produced by tone, noise and click stimuli followed by a 500 ms PSI. No significant difference in the means of these distributions were observed.</p></caption><graphic xlink:href=\"41598_2020_70397_Fig3_HTML\" id=\"MO3\"/></fig></p></sec><sec id=\"Sec4\"><title>Post-stimulatory activity is observed following adapting, sustained and suppressed responses</title><p id=\"Par9\">The response profiles observed within auditory stimulation were examined next in order to gain insight into the dynamics that might produce this post-stimulatory activity. For the units that showed post-stimulatory activity (N = 467), within-stimulus evoked activity was quantified by taking the ratio of the within-stimulus firing rate and the baseline firing rate preceding sensory stimulation to produce an evoked ratio (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>A). An evoked ratio > 1 would indicate that the within-stimulus firing rate was increased with respect to baseline firing while values < 1 would indicate suppression of firing in the within-stimulus period. The dynamics of responses with an evoked ratio of > 1 were first analysed. For each response in this group the median within-stimulus spike time was calculated as a measure of the extent of adaptation that occurred during sensory stimulation. This median spike time was normalised by the duration of the stimulus, producing a normalised median spike time (NMST) for each unit between 1 and 0. This measure of adaptation results in fast adapting responses being associated with values near to zero and ramping responses being associated with values near one. For units with an evoked ratio of > 1 (N units = 332 of 467) the majority of units had NMSTs in the middle of this range, indicating a prevalence of sustained response profiles (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>B). In order to separate the overlapping adapting and sustained populations, adapting responses were defined as those with an NMST of < 0.35 (N units = 78 of 332) while sustained responses were defined as those with an NMST of > 0.35 (N units = 254 of 332, Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>B). Ramping responses with NMSTs near 1 were rare and were classed as sustained responses here. Post-stimulatory activity was observed following both adapting and sustained responses (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>C,D). NMSTs were also calculated for the mean response to all stimuli for a single unit and showed a qualitatively similar distribution to the response NMSTs that were calculated for the stimulus that produced the longest duration post-stimulatory activity (N units = 332, adapting N = 76, sustained N = 256, Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>E). These two measures also showed a significant positive correlation (r = 0.7, p < 0.001) indicating that within-stimulus response profiles that precede post-stimulatory activity for a single effective stimulus are representative of the average response dynamics of the units (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>F).<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Post-stimulus activity following adapting and sustained responses. (<bold>A</bold>) Distribution of evoked ratios (N = 467). The vertical line at 1 indicates the threshold separating evoked (N units = 332) from suppressed responses (N units = 135). (<bold>B</bold>) Distribution of normalised median spike times of responses to the stimulus that evoked post-stimulatory activity (N units = 332). The vertical line at 0.35 indicates the threshold separating adapting (N units = 78) from sustained responses (N units = 254). (<bold>C</bold>) Upper panels: Raster plots for two units, both presented with 500 ms pure tone stimuli at their best frequency with varying sound levels. The left panel shows an adapting response while the right panel shows a sustained response. Lower panels: Corresponding PSTHs for the two units shown in upper panels. The PSTH in black is the response to the stimulus that produced the longest post-stimulatory activity for that unit. Titles above PSTHs show the normalised median spike time for this response. (<bold>D</bold>) Normalised population PSTH for adapting (left panel) and sustained (right panel) responses. (<bold>E</bold>) Distribution of normalised median spike times calculated from the mean responses across all stimuli for each unit (N units = 332, adapting N = 76, sustained N = 256). (<bold>F</bold>) Scatter plot between unit and response normalised median spike times showing a positive correlation.</p></caption><graphic xlink:href=\"41598_2020_70397_Fig4_HTML\" id=\"MO4\"/></fig></p><p id=\"Par10\">The relationship between within-stimulus response magnitude and the duration of post-stimulus activity was next examined. Within-stimulus tuning curves were calculated by measuring the peak firing rate during the stimulus time period in the mean PSTH for each stimulus (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>A). Post-stimulus tuning curves were calculated by measuring the duration of significant post-stimulus activity produced by each stimulus. A correlation coefficient (cc) was then calculated for these tuning curve pairs for each unit by correlating the peak firing rates across stimuli during the within period with the duration of post-stimulus activity. Tuning curves showed a significant positive correlation across the population of units classified as having adapting responses to their best stimulus (N units = 78, mean = 0.28, t-test; p < 0.001) (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>B). This was also the case for units that showed sustained responses to their best stimulus (N units = 254, mean = 0.37, t-test; p < 0.001) (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>C). Tuning curves for units that showed a sustained response (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>C) showed a significantly greater correlation across the population than units that showed an adapting response to their best stimulus (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>B) (Adapting mean cc = 0.28, Sustained mean cc = 0.37, two sample t-test; p = 0.001).<fig id=\"Fig5\"><label>Figure 5</label><caption><p>Within and post-stimulus tuning curves. (<bold>A</bold>). Rasterplots of responses (upper panels) and tuning curves (lower panels) for two units. The left panel shows the responses of a unit presented with 200 ms pure tone stimuli of varying sound level presented at the neuron’s best frequency. The right panel shows the responses of a unit presented with click train stimuli lasting 500 ms with varying click rates. Black curves show the within-stimulus tuning curves, calculated by taking the normalised peak firing rate in response to each stimulus. The purple curves show the post-stimulus tuning curve for the unit, calculated by measuring the duration of significantly elevated firing in the post-stimulus interval. (<bold>B</bold>) The distribution of correlation coefficients (ccs) between the within and post-stimulus tuning curves for units showing adapting responses to their best stimulus. The broken line indicates the median cc for the distribution. (<bold>C</bold>) Distribution of ccs between the within and post-stimulus tuning curves for units showing sustained responses to their best stimulus.</p></caption><graphic xlink:href=\"41598_2020_70397_Fig5_HTML\" id=\"MO5\"/></fig></p><p id=\"Par11\">Not all units that showed significant post-stimulus activity showed evoked within-stimulus auditory activity in the within-stimulus interval. These units, associated with an evoked ratio of < 1, were examined next (N units 135 of 467). Units in this population typically demonstrated within-stimulus suppression (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>A,B). Elevated post-stimulus activity can therefore be observed following adapting, sustained and suppressed within-stimulus response profiles. For stimuli with a PSI of 300 ms, significant variation in post-stimulus response duration was observed between adapting (N units = 66, mean = 181.24 ms), sustained (N units = 167, mean = 190.98 ms) and suppressed (N units = 96, mean = 161.3 ms) response subtypes (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>C) (ANOVA(2); p = 0.003). Pairwise significance tests between group means (Tukey–Kramer test, corrected for multiple comparisons) indicated that sustained responses were followed by longer duration post-stimulus activity (mean = 190.98) than suppressed responses (mean = 161.3) but was not significantly different from adapting responses (mean = 181.24) (adapting vs sustained: p = 0.58, adapting vs suppressed: p = 0.16, sustained vs suppressed: p = 0.002). For stimuli with a PSI of 500 ms, no significant differences were observed between the durations of post-stimulus activity for adapting (N units = 12, mean = 136.67 ms), sustained (N units = 76, mean = 194.99 ms) and suppressed (N units = 31, mean = 153.97 ms) response types (ANOVA(2); p = 0.08) (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>D).<fig id=\"Fig6\"><label>Figure 6</label><caption><p>Post-stimulus activity following stimulus induced suppression. (<bold>A</bold>) Raster plots and corresponding PSTHs for two suppressed units. The responses in the upper two panels are from a neuron presented with 2 s amplitude modulated (AM) pure tones presented at the neuron’s best frequency, with the rate of amplitude modulation varied. The responses in the lower two panels are from a neuron presented with 200 ms pure tone stimuli of varying frequency. PSTHs in bold indicate the responses to the stimulus that produced the longest duration post-stimulus activity for that unit. (<bold>B</bold>) Normalised population PSTH for units showing within-stimulus period suppression when presented with noise, tone or click stimuli lasting 200 ms. The stimulus that evoked the longest duration post-stimulus activity was used for each unit, resulting in a variety of stimuli contributing to the population PSTH. (<bold>C</bold>) Cumulative distribution of longest duration post-stimulus activity for adapting, sustained and suppressed responses for stimuli with a post-stimulus interval of 300 ms. (<bold>D</bold>) Cumulative distribution of post-stimulus response durations for stimuli with a post-stimulus interval of 500 ms.</p></caption><graphic xlink:href=\"41598_2020_70397_Fig6_HTML\" id=\"MO6\"/></fig></p><p id=\"Par12\">A significant positive correlation was observed between baseline firing rates and the duration of post-stimulus activity across adapting (N units = 78, r = 0.32, p < 0.001), sustained (N units = 254, r = 0.23, p < 0.001) and suppressed units (N units = 135, r = 0.1, p < 0.01). We quantified the burstiness of baseline firing using the coefficient of variation, <italic>CV</italic>:<disp-formula id=\"Equa\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${CV = \\frac{\\sigma }{\\mu }}$$\\end{document}</tex-math><mml:math id=\"M2\" display=\"block\"><mml:mrow><mml:mi>C</mml:mi><mml:mi>V</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mi>σ</mml:mi><mml:mi>μ</mml:mi></mml:mfrac></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70397_Article_Equa.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p id=\"Par13\">where the standard deviation of the inter-spike-intervals is <italic>σ</italic> and the mean inter-spike-interval is <italic>μ</italic>. A significant positive correlation was also observed between the coefficient of variation and the duration of post-stimulus activity across adapting (N units = 78, r = 0.23, p < 0.001), sustained (N units = 254, r = 0.22, p < 0.001) and suppressed units (N units = 135, r = 0.22, p < 0.001).</p></sec><sec id=\"Sec5\"><title>Post-stimulus activity emerges in cortex</title><p id=\"Par14\">Single units (N = 379) were also recorded from the auditory thalamus of 3 passively listening marmosets (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>A). The criteria for inclusion in further analysis was met by N = 361 units (see “<xref rid=\"Sec7\" ref-type=\"sec\">Methods</xref>”). When presented with stimuli lasting 200 ms followed by a 300 ms PSI (N units = 303), thalamic post-stimulus activity was significantly shorter (median = 57 ms) than the post-stimulus activity reported above in cortex (N units = 805, median = 191 ms) (rank-sum test, p < 0.001) (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>A). Thalamic post-stimulus activity was also significantly shorter (N units = 48, median = 38.5 ms) than cortical activity (N units = 237, median = 196 ms) for units presented with stimuli followed by a PSI of > 300 ms (rank-sum test, p < 0.001) (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>B). The latency of post-stimulus activity, the time following stimulus termination before firing became significantly elevated, was significantly shorter in cortex (N units = 467, latency = 4 ms) than in thalamus (N units = 351, latency = 23 ms) (rank-sum test, p < 0.001) (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>C).<fig id=\"Fig7\"><label>Figure 7</label><caption><p>Post-stimulus activity in auditory thalamus. (<bold>A</bold>) Cumulative distribution of longest duration post-stimulus activity for thalamic units (broken line) presented with 200 ms stimuli followed by a 300 ms post-stimulus interval. Ns refer to thalamic units. The cumulative distribution of cortical response durations is shown for comparison (solid line). (<bold>B</bold>) Cumulative distribution of longest duration post-stimulus activity for thalamic units presented with stimuli that were followed by a PSI > 300 ms. Again, the cumulative distribution of cortical response durations is shown for comparison (solid line). (<bold>C</bold>) Cumulative distribution of post-stimulus activity latency in thalamus (broken line) and cortex (solid line).</p></caption><graphic xlink:href=\"41598_2020_70397_Fig7_HTML\" id=\"MO7\"/></fig></p><p id=\"Par15\">We next investigated whether variation in the duration of post-stimulus activity exists between cortical fields. Single units were recorded in areas A1, R and RT in core auditory cortex of two subjects, with units in each field being exposed to all stimulus types (Fig. <xref rid=\"Fig8\" ref-type=\"fig\">8</xref>A). In order to compare durations across fields it was necessary to only include units for which the maximum duration of post-stimulus activity could be accurately estimated, this is, where the duration of activity did not exceed the post-stimulus interval. The duration of post-stimulus activity showed significant variation across cortical fields (Kruskall–Wallis test, H(2) = 15.11, p < 0.001) (Fig. <xref rid=\"Fig8\" ref-type=\"fig\">8</xref>B). Post-hoc multiple comparisons of mean ranks indicated that post-stimulus activity of significantly longer duration in RT compared to A1 and R (median durations, A1 = 193.5 ms, R = 192 ms, RT = 233 ms; A1 vs R, p = 0.91; A1 vs RT, p < 0.001; R vs RT, p = 0.004). The pattern of longest duration responses being found in RT also held across individual subjects (Subject 1: Kruskall–Wallis test, H(2) = 12, p = 0.003; median durations, A1 = 159 ms, R = 175 ms, RT = 230.5 ms; A1 vs R, p = 0.82; A1 vs RT, p = 0.002; R vs RT, p = 0.017; Subject 2: Kruskall–Wallis test, H(2) = 4.45, p = 0.11; median durations, A1 = 172.5 ms, R = 177 ms, RT = 208 ms; A1 vs R, p = 0.996; A1 vs RT, p = 0.11; R vs RT, p = 0.16). Finally, we investigated whether the proportion of units showing each of the three within-stimulus response profiles (adapting, sustained, suppressed) varied across cortical fields. In A1 the majority of units that showed post-stimulus activity showed sustained responses in the within-stimulus period (54.95%; Fig. <xref rid=\"Fig8\" ref-type=\"fig\">8</xref>C). The proportion of units showing sustained responses was even greater in R (69.26%) and greater still in RT (73.14%). This indicates that response profiles preceding post-stimulus activity become more sustained in the more anterior auditory cortical fields.<fig id=\"Fig8\"><label>Figure 8</label><caption><p>Variation in post-stimulus activity duration across auditory cortical fields. (<bold>A</bold>) Median (solid line) and inter-quartile ranges (coloured areas) of post-stimulus activity across the anterior–posterior extent of auditory cortex. Boundaries of cortical fields A1, R and RT are indicated with dashed lines. Data shown is combined from two subjects where recordings were made across the entire anterior–posterior extent of auditory cortex. (<bold>B</bold>) Cumulative distributions of post-stimulus activity across cortical fields showing a significant increase in duration in more anterior fields, R and RT. (<bold>C</bold>) Proportions of response types observed in different cortical fields, showing a trend towards a greater proportion of sustained responses in more anterior fields.</p></caption><graphic xlink:href=\"41598_2020_70397_Fig8_HTML\" id=\"MO8\"/></fig></p></sec></sec><sec id=\"Sec6\"><title>Discussion</title><p id=\"Par16\">Here we report the presence of post-stimulatory activity in a population of auditory cortical neurons in the absence of a behavioural task. We observed that, in the awake marmoset auditory cortex, these responses could last hundreds of milliseconds and in some cases over a second. Post-stimulatory activity has previously been observed in subcortical structures in non-primate species. In the dorsal cochlear nucleus of the cat pause-build units have been found to show modulation of spontaneous activity on the order of hundreds of milliseconds following the termination of auditory stimuli<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref>–<xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. In the inferior colliculus of the anaesthetised mouse long-duration stimuli lasting over 30 s have been found to produce sound-evoked after discharges lasting several minutes<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. The inheritance of post-stimulus activity from subcortical structures does not appear to account for the activity that we observed in cortex as we found post-stimulus activity to be of shorter duration in auditory thalamus than in cortex. Post-stimulus activity was also found to have a longer latency following stimulus offset in thalamus compared to cortex. This indicates that units displaying such activity in thalamus may simply inherit this activity via cortical feedback projections. We also observed that the duration of this activity increased in RT, a secondary cortical area. These findings indicate that the circuit architecture required to generate these responses may be intrinsic to cortical networks.</p><p id=\"Par17\">Post-stimulus activity was observed following several different response profiles during sensory stimulation. As previously reported, units in auditory cortex are capable of showing adapting and sustained responses<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>. The evoked responses of units observed here fell along a continuum from adapting, through sustained to ramping. Post-stimulatory activity was observed following both adapting and sustained response profiles, as well as following suppression. Given the heterogeneity of the stimulus-related activity that preceded the post-stimulus firing, the possibility that a single mechanism is responsible for this activity appears unlikely. In the prefrontal cortex, the mechanistic basis of post-stimulatory activity has not been fully elucidated but three broad classes of mechanism have been proposed to account for this pattern of activity; intrinsic neuronal properties at the cellular level, synaptic dynamics at the network level and the effect of activity from subcortical afferents at the systems level<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>.</p><p id=\"Par18\">The post-stimulus activity observed in sustained units could be accounted for by slow NMDA receptor-mediated excitatory synaptic currents which can last for hundreds of milliseconds<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>. These currents have been found to have a twofold longer decay time in rat prefrontal cortex compared to primary visual cortex, in keeping with these slow currents playing a role in the generation of prefrontal post-stimulatory activit<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup>. It is not known whether NMDA currents in the marmoset auditory cortex are slower than in other cortical areas however. Cortical networks have a highly recurrent architecture<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup> and reverberant activity in such a network has long been considered a candidate mechanism for the generation of post-stimulatory activity<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref>–<xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup>. A recurrent mechanism of this kind would account for the temporal fluctuations in post-stimulus activity that were widely observed in these units. It has been suggested that hybrid mechanisms are most likely responsible for post-stimulatory activity throughout the brain<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup> and, in keeping with this, combining slow excitatory currents with recurrent activity has been found to be important for the generation and stabilisation of post-stimulatory activity in computational models of such activity<sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref>,<xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>. Similarly, asynchronous excitatory activity mediated by AMPA receptors has been found to be important for the maintenance of post-stimulatory reverberant activity in hippocampal cultures<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>. Sustained responses in auditory cortex that persist following stimulus offset and post-stimulatory activity in prefrontal cortex may therefore both be implemented by a hybrid of excitatory mechanisms. In keeping with an excitatory mechanism, the duration of post-stimulus activity following sustained responses was longer than for the other responses types. Furthermore, for sustained responses the peak firing rate in the within stimulus period predicted the duration of post-stimulus activity, indicating a positive relationship between excitatory drive during sensory stimulation and the duration of post-stimulatory activity observed. Future experiments could systematically vary sound duration in order to separate the mechanistic contribution of onset vs offset response dynamics to the generation of these responses.</p><p id=\"Par19\">Purely excitatory mechanisms do not seem capable of accounting for the full response profiles of adapting and suppressed response types however. Inhibition is known to play a role in the generation of adapting onset responses<sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup> and thus may be responsible for the observed alteration in post-stimulus tuning observed for this class of responses. Greater adaptation of inhibitory synaptic input has been observed during sensory stimulation in neurons of the rat somatosensory cortex<sup><xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup>. Such a pattern of adaptation could lead to excitation dominating during the postsynaptic period, providing a mechanism for post-stimulus firing in these units. Rebound calcium burst activity lasting hundred of milliseconds has been observed following sensory stimulation in the auditory thalamus<sup><xref ref-type=\"bibr\" rid=\"CR62\">62</xref></sup> and may contribute to the emergence of the post-stimulus activity observed in this area. This mechanism may also contribute to the generation of post-stimulus activity in adapting and suppressed cortical units as rebound activity is produced following inhibitory activity<sup><xref ref-type=\"bibr\" rid=\"CR63\">63</xref></sup>. We also observed units that showed dramatic suppression in response to auditory stimuli, followed by the longest duration post-stimulus activity that we observed. This dramatic suppression is in keeping with a role for inhibition in generating this post-stimulus activity and this class of responses may share a common mechanism with the adapting response type. An imbalance in balance of excitation and inhibition during the post-stimulus period in favour of excitation may therefore represent a common mechanism for all three response types observed here.</p><p id=\"Par20\">The function of post-stimulus activity has been studied in a variety of behavioural contexts but the data presented here indicate that this activity may also play a role during passive listening. Echoic memory is a form of short-term sensory memory that differs from working memory in that it is posited to be active under all behavioral conditions, including passive listening<sup><xref ref-type=\"bibr\" rid=\"CR64\">64</xref></sup>. The content of echoic memory is generally stored on the order of seconds<sup><xref ref-type=\"bibr\" rid=\"CR65\">65</xref>–<xref ref-type=\"bibr\" rid=\"CR67\">67</xref></sup>, a timescale that fits with the duration of post-stimulus activity observed here. An alternate possibility is that the activity reported here reflects the recruitment of circuits that exist in order to retain information in working memory and provide no function during passive listening. A functional role for this activity appears plausible, however, given the requirement of temporal integration in auditory perception in all species, from insects<sup><xref ref-type=\"bibr\" rid=\"CR68\">68</xref></sup> to humans<sup><xref ref-type=\"bibr\" rid=\"CR69\">69</xref></sup>. Beyond the auditory domain, multi-sensory integration requires combining sensory information from multiple cortical areas with different latencies and temporal dynamics<sup><xref ref-type=\"bibr\" rid=\"CR70\">70</xref></sup>. Such post-stimulatory activity may provide a substrate for integrating disparate sensory signals into a coherent percept of an object. Investigating the function of activity observed in the absence of a behavioural task is particularly challenging however. Experiments addressing the function of this activity could exploit behavioural measures that provide an implicit measure of the processing of sensory information in the absence of a task, such as fear-conditioned freezing<sup><xref ref-type=\"bibr\" rid=\"CR71\">71</xref></sup>. Combining this approach with temporally precise suppression of this activity through optogenetic methods, it may be possible to investigate the potential role of this activity in echoic memory and temporal integration. Developing an approach that will enable the investigation of the potential roles of post-stimulatory activity during passive listening will be crucial in elucidating the functional properties of such activity.</p></sec><sec id=\"Sec7\"><title>Methods</title><p id=\"Par21\">The data analysed in this study were obtained from several previous experiments conducted in the Laboratory of Auditory Neurophysiology at Johns Hopkins University School of Medicine in the laboratory of Prof. Xiaoqin Wang<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref>–<xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. All experimental procedures were approved by the Johns Hopkins University Animal Use and Care Committee and were carried out in accordance with relevant guidelines and regulations. The methods to record single-unit activity in awake marmosets were previously described<sup><xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup> and are briefly summarised below.</p><p id=\"Par22\">Single-unit recordings from auditory cortex and thalamus were conducted in 7 awake, passively listening marmosets sitting on a semi-restraint device with their head immobilized, within a double-walled soundproof chamber (Industrial Acoustics). The inside wall of the chamber was covered by 3-inch acoustic absorption foam (Sonex). For auditory cortical recordings high-impedance tungsten microelectrodes (3–5 M, A-M Systems) were inserted perpendicular to the cortical surface. Electrodes were mounted on a micromanipulator (Narishige) and advanced by a manual hydraulic microdrive (Trent Wells). Action potentials were detected on-line using a template-based spike sorter (Multi-Spike Detector; Alpha Omega Engineering) and continuously monitored by the experimenter while data recording progressed. Typically 5–15 electrode penetrations were made within a miniature recording hole (diameter 1 mm) over the course of several days, after which the hole was sealed with dental cement and another hole opened for new electrode penetrations. Neurons were recorded from all cortical layers, but most commonly from supragranular layers.</p><sec id=\"Sec8\"><title>Generation of acoustic stimuli</title><p id=\"Par23\">Acoustic stimuli were generated digitally and delivered by a free-field loudspeaker located one meter directly in front of the animal. All sound stimuli were generated at a 100 kHz sampling rate and low-pass filtered at 50 kHz. The sound level of individual frequency components used in this study was no higher than 80 dB SPL. Frequency tuning curves and rate-level functions were generated using pure-tone stimuli of 200 ms in duration with post-stimulus intervals of 500 ms, and had a minimum of five repetitions each. Stimulus presentation order was fully randomised. Pure-tone stimuli intensity levels were generally 10–20 dB above threshold for neurons with monotonic rate-level functions, or at preferred levels for non-monotonic neurons. Broadband rectangular clicks or narrowband clicks made of brief pulses of white noise or a tone (at an integer multiple of the frequency) were used to generate click trains. Rectangular click trains had a width of 0.1 ms while narrowband clicks had each pulse convolved with a Gaussian envelope with a standard deviation of 0.1–0.4.</p></sec><sec id=\"Sec9\"><title>Identification of cortical fields</title><p id=\"Par24\">Single units with significant neuronal discharges to narrowband stimuli, such as tones and band-pass noise, were used to generate cortical characteristic frequency maps. The characteristic frequency of each location on the map was determined by the median characteristic frequency of all electrode tracks within 0.25 mm. Electrode track characteristic frequencies were calculated by computing the median characteristic frequency of units within the track. The anterior–posterior position is reported relative to the boundary between A1 and R. Both the boundary between A1 and R as well as the boundary between R and RT were identified from the frequency reversal between the cochleotopic gradients of the two fields, as their maps are mirror-reversed<sup><xref ref-type=\"bibr\" rid=\"CR72\">72</xref>–<xref ref-type=\"bibr\" rid=\"CR75\">75</xref></sup>. Please see<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup> for more details.</p></sec><sec id=\"Sec10\"><title>Quantification of post-stimulus activity duration</title><p id=\"Par25\">Quantification of post-stimulus activity was based on significant difference from baseline firing. Baseline firing rate was calculated from a pre-stimulus interval that was separate from the post-stimulus interval of the preceding stimulus (Fig. <xref rid=\"Fig9\" ref-type=\"fig\">9</xref>A). A wide range of pre-stimulus interval, post-stimulus interval, stimulus duration and stimulus type combinations were used resulting in 82 different stimulation conditions. Several subsets with consistent durations were used in our analysis. Stimuli that had a post-stimulus interval of 300 ms had a pre-stimulus interval of 200 ms, creating an inter-tone interval of 500 ms. Stimuli that had a post-stimulus interval of 500 ms had a pre-stimulus interval of 500 ms, creating an inter-tone interval of 1,000 ms. For stimuli with a post-stimulus interval of > 500 ms, a range of pre-stimulus intervals from 200 to 8,000 ms were used that varied with the duration of the particular stimulus. Units were required to have a baseline firing rate, calculated across all pre-stimulus periods, of > 1 spike per second and to have been presented with 5 or more repetitions of each stimulus to be included in all further analysis. This criterion was met by 1,188 units of 1,557. The duration of post-stimulus activity was quantified by convolving spike trains for a given stimulus with a bi-directional Gaussian filter (σ = 5 ms, total bandwidth = 20 ms) in order to estimate the mean instantaneous firing rate before, during and after the presentation of the stimulus (Fig. <xref rid=\"Fig9\" ref-type=\"fig\">9</xref>B). The same method was used in order to generate a mean firing rate trace for the pre-stimulus period, combining the data from all pre-stimulus periods for the unit (Fig. <xref rid=\"Fig9\" ref-type=\"fig\">9</xref>C). The mean and standard deviations of baseline firing were calculated from this trace (Fig. <xref rid=\"Fig9\" ref-type=\"fig\">9</xref>D). Significant post-stimulus activity was required to be > 2 standard deviations over mean baseline firing.<fig id=\"Fig9\"><label>Figure 9</label><caption><p>Quantification of post-stimulus activity duration. (<bold>A</bold>) Pre-stimulus firing for one neuron for all stimuli presented. (<bold>B</bold>) Bi-directional Gaussian filter (σ = 5 ms, total bandwidth = 20 ms), used to calculate instantaneous firing rate during rate. (<bold>C</bold>) Pre-stimulus instantaneous firing rate, calculated by convolving the spike trains in A with the filter in B and normalizing by the number of stimulus repetitions. The dashed line is the threshold for significantly elevated firing, positioned at 2 standard deviations (SD) above mean firing rate. (<bold>D</bold>) The distribution of firing rate values in the trace in (<bold>C</bold>), showing the mean firing rate (red line), 1 SD above the mean (blue line) and 2 SD above the mean (broken black line). The latter is equivalent to the threshold for significantly elevated firing, as shown in (<bold>C</bold>). (<bold>E</bold>) Left panel: Firing for the same neuron on a subset of trials, corresponding to five repetitions of a single stimulus. The black bar indicates the duration of the 500 ms bandpass noise stimulus. Right panel: Instantaneous firing rate calculated by convolving the spike train in (<bold>E</bold>) with the filter in (<bold>B</bold>). The broken black line indicates the threshold shown in (<bold>C</bold>) and (<bold>D</bold>), as calculated from pre-stimulus firing. The area shaded in blue above the threshold indicates the duration of significantly elevated post-stimulatory activity.</p></caption><graphic xlink:href=\"41598_2020_70397_Fig9_HTML\" id=\"MO9\"/></fig></p><p id=\"Par26\">Significant post-stimulus activity was defined as periods of firing that crossed the threshold (Fig. <xref rid=\"Fig9\" ref-type=\"fig\">9</xref>E). Firing during this period was required to be over the threshold on at least half of the trials, in order to avoid large single trial events being mistaken for reliable post-stimulus activity. The observed post-stimulus activity could be highly variable over time. Therefore, momentary drops below this threshold (< 20 ms) were not considered in our estimate of post-stimulus activity duration. In order to control for chance fluctuations in firing rates, the duration of elevated firing during spontaneous activity during the pre-stimulus period was also calculated. The duration of post-stimulus activity was required to be above the range of values that were observed by chance in the pre-stimulus period for each neuron.</p></sec><sec id=\"Sec11\"><title>Ethical approval</title><p id=\"Par27\">All experimental procedures were carried out at Johns Hopkins University in AAALAC approved facilities and were approved by the Johns Hopkins University Animal Use and Care Committee (IACUC). All methods were carried out in accordance with relevant guidelines and regulations.</p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><ack><p>We would like to acknowledge the members of the Bendor lab for their comments and suggestions, and Dr. Elias Issa for his contribution of electrophysiological data. This work was supported by an ERC starting Grant (CHIME) and a Medical Research Council (MR/M022889/1) grant to DB, NIH Grants (DC003180, DC005808, DC014503) to XW, a NIH RO3 Grant (DC06357) to EB and a Wellcome Trust doctoral fellowship (109004/Z/15/Z) to JJL.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>D.B. and E.L.B. collected the data. J.E.C. and J.J.L. processed the data and performed the statistical analyses. J.E.C. prepared the figures and drafted the manuscript. J.E.C., J.J.L., E.L.B., X.W. and D.B. were involved in study conception, study design and interpretation of the data.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par28\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fuster</surname><given-names>JM</given-names></name><name><surname>Alexander</surname><given-names>GE</given-names></name></person-group><article-title>Neuron activity related to short-term memory</article-title><source>Science</source><year>1971</year><volume>173</volume><fpage>652</fpage><lpage>654</lpage><pub-id pub-id-type=\"pmid\">4998337</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kubota</surname><given-names>K</given-names></name><name><surname>Niki</surname><given-names>H</given-names></name></person-group><article-title>Prefrontal cortical unit activity and delayed alternation performance in monkeys</article-title><source>J. Neurophysiol.</source><year>1971</year><volume>34</volume><fpage>337</fpage><lpage>347</lpage><pub-id pub-id-type=\"pmid\">4997822</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fuster</surname><given-names>JM</given-names></name></person-group><article-title>Unit activity in prefrontal cortex during delayed-response performance: neuronal correlates of transient memory</article-title><source>J. Neurophysiol.</source><year>1973</year><volume>36</volume><fpage>61</fpage><lpage>78</lpage><pub-id pub-id-type=\"pmid\">4196203</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kubota</surname><given-names>K</given-names></name></person-group><article-title>Prefrontal unit activity during delayed response and delayed response and delayed-alternation performances</article-title><source>Jpn. J. Physiol.</source><year>1975</year><volume>25</volume><fpage>481</fpage><lpage>493</lpage><pub-id pub-id-type=\"pmid\">813043</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Niki</surname><given-names>H</given-names></name></person-group><article-title>Differential activity of prefrontal units during right and left delayed response trials</article-title><source>Brain Res.</source><year>1974</year><volume>70</volume><fpage>346</fpage><lpage>349</lpage><pub-id pub-id-type=\"pmid\">4207718</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hasegawa</surname><given-names>R</given-names></name><name><surname>Sawaguchi</surname><given-names>T</given-names></name><name><surname>Kubota</surname><given-names>K</given-names></name></person-group><article-title>Monkey prefrontal neuronal activity coding forthcoming saccade an oculomotor delayed matching-to-sample task</article-title><source>J. Neurophysiol.</source><year>1998</year><volume>79</volume><fpage>322</fpage><lpage>333</lpage><pub-id pub-id-type=\"pmid\">9425201</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gottlieb</surname><given-names>Y</given-names></name><name><surname>Vaadia</surname><given-names>E</given-names></name><name><surname>Abeles</surname><given-names>M</given-names></name></person-group><article-title>Single unit activity in the auditory cortex of a monkey performing a short term memory task</article-title><source>Exp. Brain Res.</source><year>1989</year><volume>74</volume><fpage>139</fpage><lpage>148</lpage><pub-id pub-id-type=\"pmid\">2924831</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Baddeley</surname><given-names>A</given-names></name></person-group><article-title>Working memory</article-title><source>Science</source><year>1992</year><volume>255</volume><fpage>556</fpage><lpage>559</lpage><pub-id pub-id-type=\"pmid\">1736359</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Miller</surname><given-names>EK</given-names></name><name><surname>Erickson</surname><given-names>CA</given-names></name><name><surname>Desimone</surname><given-names>R</given-names></name></person-group><article-title>Neural mechanisms of visual working memory in prefrontal cortex of the macaque</article-title><source>J. Neurosci.</source><year>1996</year><volume>16</volume><fpage>5154</fpage><lpage>5167</lpage><pub-id pub-id-type=\"pmid\">8756444</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Funahashi</surname><given-names>S</given-names></name><name><surname>Bruce</surname><given-names>CJ</given-names></name><name><surname>Goldman-Rakic</surname><given-names>PS</given-names></name></person-group><article-title>Mnemonic coding of visual space in the monkey’s dorsolateral prefrontal cortex</article-title><source>J. Neurophysiol.</source><year>1989</year><volume>61</volume><fpage>331</fpage><lpage>349</lpage><pub-id pub-id-type=\"pmid\">2918358</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chafee</surname><given-names>MV</given-names></name><name><surname>Goldman-Rakic</surname><given-names>PS</given-names></name></person-group><article-title>Matching patterns of activity in primate prefrontal area 8a and parietal area 7ip neurons during a spatial working memorytask</article-title><source>J. Neurophysiol.</source><year>1998</year><volume>79</volume><fpage>2919</fpage><lpage>2940</lpage><pub-id pub-id-type=\"pmid\">9636098</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quintana</surname><given-names>J</given-names></name><name><surname>Fuster</surname><given-names>JM</given-names></name></person-group><article-title>From perception to action: temporal integrative functions of prefrontal and parietal neurons</article-title><source>Cereb. Cortex</source><year>1999</year><volume>9</volume><fpage>213</fpage><lpage>221</lpage><pub-id pub-id-type=\"pmid\">10355901</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Constantinidis</surname><given-names>C</given-names></name><etal/></person-group><article-title>Persistent spiking activity underlies working memory</article-title><source>J. Neurosci.</source><year>2018</year><volume>38</volume><fpage>7020</fpage><lpage>7028</lpage><pub-id pub-id-type=\"pmid\">30089641</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cavanagh</surname><given-names>SE</given-names></name><name><surname>Towers</surname><given-names>JP</given-names></name><name><surname>Wallis</surname><given-names>JD</given-names></name><name><surname>Hunt</surname><given-names>LT</given-names></name><name><surname>Kennerley</surname><given-names>SW</given-names></name></person-group><article-title>Reconciling persistent and dynamic hypotheses of working memory coding in prefrontal cortex</article-title><source>Nat. Commun.</source><year>2018</year><volume>9</volume><fpage>1</fpage><lpage>16</lpage><pub-id pub-id-type=\"pmid\">29317637</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sakai</surname><given-names>K</given-names></name><name><surname>Rowe</surname><given-names>JB</given-names></name><name><surname>Passingham</surname><given-names>RE</given-names></name></person-group><article-title>Active maintenance in prefrontal area 46 creates distractor-resistant memory</article-title><source>Nat. Neurosci.</source><year>2002</year><volume>5</volume><fpage>479</fpage><pub-id pub-id-type=\"pmid\">11953754</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Funahashi</surname><given-names>S</given-names></name><name><surname>Chafee</surname><given-names>MV</given-names></name><name><surname>Goldman-Rakic</surname><given-names>PS</given-names></name></person-group><article-title>Prefrontal neuronal activity in rhesus monkeys performing a delayed anti-saccade task</article-title><source>Nature</source><year>1993</year><volume>365</volume><fpage>753</fpage><pub-id pub-id-type=\"pmid\">8413653</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pessoa</surname><given-names>L</given-names></name><name><surname>Gutierrez</surname><given-names>E</given-names></name><name><surname>Bandettini</surname><given-names>PA</given-names></name><name><surname>Ungerleider</surname><given-names>LG</given-names></name></person-group><article-title>Neural correlates of visual working memory: fmri amplitude predicts task performance</article-title><source>Neuron</source><year>2002</year><volume>35</volume><fpage>975</fpage><lpage>987</lpage><pub-id pub-id-type=\"pmid\">12372290</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Murray</surname><given-names>JD</given-names></name><etal/></person-group><article-title>A hierarchy of intrinsic timescales across primate cortex</article-title><source>Nat. Neurosci.</source><year>2014</year><volume>17</volume><fpage>1661</fpage><pub-id pub-id-type=\"pmid\">25383900</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cavanagh</surname><given-names>SE</given-names></name><name><surname>Wallis</surname><given-names>JD</given-names></name><name><surname>Kennerley</surname><given-names>SW</given-names></name><name><surname>Hunt</surname><given-names>LT</given-names></name></person-group><article-title>Autocorrelation structure at rest predicts value correlates of single neurons during reward-guided choice</article-title><source>Elife</source><year>2016</year><volume>5</volume><fpage>e18937</fpage><pub-id pub-id-type=\"pmid\">27705742</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fascianelli</surname><given-names>V</given-names></name><name><surname>Tsujimoto</surname><given-names>S</given-names></name><name><surname>Marcos</surname><given-names>E</given-names></name><name><surname>Genovesio</surname><given-names>A</given-names></name></person-group><article-title>Autocorrelation structure in the macaque dorsolateral, but not orbital or polar, prefrontal cortex predicts response-coding strength in a visually cued strategy task</article-title><source>Cereb. Cortex</source><year>2019</year><volume>29</volume><fpage>230</fpage><lpage>241</lpage><pub-id pub-id-type=\"pmid\">29228110</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lu</surname><given-names>Z</given-names></name><name><surname>Williamson</surname><given-names>S</given-names></name><name><surname>Kaufman</surname><given-names>L</given-names></name></person-group><article-title>Behavioral lifetime of human auditory sensory memory predicted by physiological measures</article-title><source>Science</source><year>1992</year><volume>258</volume><fpage>1668</fpage><lpage>1670</lpage><pub-id pub-id-type=\"pmid\">1455246</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bigelow</surname><given-names>J</given-names></name><name><surname>Rossi</surname><given-names>B</given-names></name><name><surname>Poremba</surname><given-names>A</given-names></name></person-group><article-title>Neural correlates of short-term memory in primate auditory cortex</article-title><source>Front. Neurosci.</source><year>2014</year><volume>8</volume><fpage>250</fpage><pub-id pub-id-type=\"pmid\">25177266</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scott</surname><given-names>BH</given-names></name><name><surname>Mishkin</surname><given-names>M</given-names></name><name><surname>Yin</surname><given-names>P</given-names></name></person-group><article-title>Neural correlates of auditory short-term memory in rostral superior temporal cortex</article-title><source>Curr. Biol.</source><year>2014</year><volume>24</volume><issue>23</issue><fpage>2767</fpage><lpage>2775</lpage><pub-id pub-id-type=\"pmid\">25456448</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>Y</given-names></name><name><surname>Matysiak</surname><given-names>A</given-names></name><name><surname>Heil</surname><given-names>P</given-names></name><name><surname>König</surname><given-names>R</given-names></name><name><surname>Brosch</surname><given-names>M</given-names></name></person-group><article-title>Persistent neural activity in auditory cortex is related to auditory working memory in humans and nonhuman primates</article-title><source>Elife</source><year>2016</year><volume>5</volume><fpage>e15441</fpage><pub-id pub-id-type=\"pmid\">27438411</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rämä</surname><given-names>P</given-names></name><name><surname>Poremba</surname><given-names>A</given-names></name><name><surname>Sala</surname><given-names>JB</given-names></name><name><surname>Yee</surname><given-names>L</given-names></name><name><surname>Malloy</surname><given-names>M</given-names></name><name><surname>Mishkin</surname><given-names>M</given-names></name><name><surname>Courtney</surname><given-names>SM</given-names></name></person-group><article-title>Dissociable functional cortical topographies for working memory maintenance of voice identity and location</article-title><source>Cereb. Cortex</source><year>2004</year><volume>14</volume><issue>7</issue><fpage>768</fpage><lpage>780</lpage><pub-id pub-id-type=\"pmid\">15084491</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sakurai</surname><given-names>Y</given-names></name></person-group><article-title>Involvement of auditory cortical and hippocampal neurons in auditory working memory and reference memory in the rat</article-title><source>J. Neurosci.</source><year>1994</year><volume>14</volume><issue>5</issue><fpage>2606</fpage><lpage>2623</lpage><pub-id pub-id-type=\"pmid\">8182430</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shinba</surname><given-names>T</given-names></name><name><surname>Sumi</surname><given-names>M</given-names></name><name><surname>Iwanami</surname><given-names>A</given-names></name><name><surname>Ozawa</surname><given-names>N</given-names></name><name><surname>Yamamoto</surname><given-names>K</given-names></name></person-group><article-title>Increased neuronal firing in the rat auditory cortex associated with preparatory set</article-title><source>Brain Res. Bull.</source><year>1995</year><volume>37</volume><fpage>199</fpage><lpage>204</lpage><pub-id pub-id-type=\"pmid\">7606496</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quirk</surname><given-names>GJ</given-names></name><name><surname>Armony</surname><given-names>JL</given-names></name><name><surname>LeDoux</surname><given-names>JE</given-names></name></person-group><article-title>Fear conditioning enhances different temporal components of tone-evoked spike trains in auditory cortex and lateral amygdala</article-title><source>Neuron</source><year>1997</year><volume>19</volume><fpage>613</fpage><lpage>624</lpage><pub-id pub-id-type=\"pmid\">9331352</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Armony</surname><given-names>JL</given-names></name><name><surname>Quirk</surname><given-names>GJ</given-names></name><name><surname>LeDoux</surname><given-names>JE</given-names></name></person-group><article-title>Differential effects of amygdala lesions on early and late plastic components of auditory cortex spike trains during fear conditioning</article-title><source>J. Neurosci.</source><year>1998</year><volume>18</volume><fpage>2592</fpage><lpage>2601</lpage><pub-id pub-id-type=\"pmid\">9502818</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brosch</surname><given-names>M</given-names></name><name><surname>Schulz</surname><given-names>A</given-names></name><name><surname>Scheich</surname><given-names>H</given-names></name></person-group><article-title>Processing of sound sequences in macaque auditory cortex: response enhancement</article-title><source>J. Neurophysiol.</source><year>1999</year><volume>82</volume><issue>3</issue><fpage>1542</fpage><lpage>1559</lpage><pub-id pub-id-type=\"pmid\">10482768</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Brosch</surname><given-names>M</given-names></name><name><surname>Scheich</surname><given-names>H</given-names></name></person-group><person-group person-group-type=\"editor\"><name><surname>Heil</surname><given-names>P</given-names></name><name><surname>Scheich</surname><given-names>H</given-names></name><name><surname>Budinger</surname><given-names>E</given-names></name><name><surname>Konig</surname><given-names>R</given-names></name></person-group><article-title>Non-acoustic influence on neural activity in auditory cortex</article-title><source>The Auditory Cortex</source><year>2005</year><publisher-loc>New York</publisher-loc><publisher-name>Psychology Press</publisher-name><fpage>145</fpage><lpage>162</lpage></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brosch</surname><given-names>M</given-names></name><name><surname>Selezneva</surname><given-names>E</given-names></name><name><surname>Scheich</surname><given-names>H</given-names></name></person-group><article-title>Formation of associations in auditory cortex by slow changes of tonic firing</article-title><source>Hear. Res.</source><year>2011</year><volume>271</volume><issue>1–2</issue><fpage>66</fpage><lpage>73</lpage><pub-id pub-id-type=\"pmid\">20488230</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kumar</surname><given-names>S</given-names></name><name><surname>Joseph</surname><given-names>S</given-names></name><name><surname>Gander</surname><given-names>PE</given-names></name><name><surname>Barascud</surname><given-names>N</given-names></name><name><surname>Halpern</surname><given-names>AR</given-names></name><name><surname>Griffiths</surname><given-names>TD</given-names></name></person-group><article-title>A brain system for auditory working memory</article-title><source>J. Neurosci.</source><year>2016</year><volume>36</volume><issue>16</issue><fpage>4492</fpage><lpage>4505</lpage><pub-id pub-id-type=\"pmid\">27098693</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scholl</surname><given-names>B</given-names></name><name><surname>Gao</surname><given-names>X</given-names></name><name><surname>Wehr</surname><given-names>M</given-names></name></person-group><article-title>Nonoverlapping sets of synapses drive on responses and off responses in auditory cortex</article-title><source>Neuron</source><year>2010</year><volume>65</volume><fpage>412</fpage><lpage>421</lpage><pub-id pub-id-type=\"pmid\">20159453</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Qin</surname><given-names>L</given-names></name><name><surname>Chimoto</surname><given-names>S</given-names></name><name><surname>Sakai</surname><given-names>M</given-names></name><name><surname>Wang</surname><given-names>J</given-names></name><name><surname>Sato</surname><given-names>Y</given-names></name></person-group><article-title>Comparison between offset and onset responses of primary auditory cortex on–off neurons in awake cats</article-title><source>J. Neurophysiol.</source><year>2007</year><volume>97</volume><fpage>3421</fpage><lpage>3431</lpage><pub-id pub-id-type=\"pmid\">17360820</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Takahashi</surname><given-names>H</given-names></name><name><surname>Nakao</surname><given-names>M</given-names></name><name><surname>Kaga</surname><given-names>K</given-names></name></person-group><article-title>Cortical mapping of auditory-evoked offset responses in rats</article-title><source>NeuroReport</source><year>2004</year><volume>15</volume><fpage>1565</fpage><lpage>1569</lpage><pub-id pub-id-type=\"pmid\">15232284</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bendor</surname><given-names>D</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name></person-group><article-title>Neural response properties of primary, rostral, and rostrotemporal core fields in the auditory cortex of marmoset monkeys</article-title><source>J. Neurophysiol.</source><year>2008</year><volume>100</volume><fpage>888</fpage><lpage>906</lpage><pub-id pub-id-type=\"pmid\">18525020</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bendor</surname><given-names>D</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name></person-group><article-title>Neural coding of periodicity in marmoset auditory cortex</article-title><source>J. Neurophysiol.</source><year>2010</year><volume>103</volume><fpage>1809</fpage><lpage>1822</lpage><pub-id pub-id-type=\"pmid\">20147419</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bartlett</surname><given-names>EL</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name></person-group><article-title>Neural representations of temporally modulated signals in the auditory thalamus of awake primates</article-title><source>J. Neurophysiol.</source><year>2007</year><volume>97</volume><fpage>1005</fpage><lpage>1017</lpage><pub-id pub-id-type=\"pmid\">17050830</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bartlett</surname><given-names>EL</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name></person-group><article-title>Correlation of neural response properties with auditory thalamus subdivisions in the awake marmoset</article-title><source>J. Neurophysiol.</source><year>2011</year><volume>105</volume><fpage>2647</fpage><lpage>2667</lpage><pub-id pub-id-type=\"pmid\">21411564</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bartlett</surname><given-names>EL</given-names></name><name><surname>Sadagopan</surname><given-names>S</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name></person-group><article-title>Fine frequency tuning in monkey auditory cortex and thalamus</article-title><source>J. Neurophysiol.</source><year>2011</year><volume>106</volume><fpage>849</fpage><lpage>859</lpage><pub-id pub-id-type=\"pmid\">21613589</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rhode</surname><given-names>W</given-names></name><name><surname>Smith</surname><given-names>P</given-names></name><name><surname>Oertel</surname><given-names>D</given-names></name></person-group><article-title>Physiological response properties of cells labeled intracellularly with horseradish peroxidase in cat dorsal cochlear nucleus</article-title><source>J. Comp. Neurol.</source><year>1983</year><volume>213</volume><fpage>426</fpage><lpage>447</lpage><pub-id pub-id-type=\"pmid\">6300199</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parham</surname><given-names>K</given-names></name><name><surname>Kim</surname><given-names>D</given-names></name></person-group><article-title>Analysis of temporal discharge characteristics of dorsal cochlear nucleus neurons of unanesthetized decerebrate cats</article-title><source>J. Neurophysiol.</source><year>1992</year><volume>67</volume><fpage>1247</fpage><lpage>1263</lpage><pub-id pub-id-type=\"pmid\">1597710</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parham</surname><given-names>K</given-names></name><name><surname>Kim</surname><given-names>D</given-names></name></person-group><article-title>Discharge suppression in the silent interval preceding the tone burst in pause-build units of the dorsal cochlear nucleus of the unanesthetized decerebrate cat</article-title><source>J. Acoust. Soc. Am.</source><year>1993</year><volume>94</volume><fpage>3227</fpage><lpage>3231</lpage><pub-id pub-id-type=\"pmid\">8300957</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ono</surname><given-names>M</given-names></name><name><surname>Bishop</surname><given-names>DC</given-names></name><name><surname>Oliver</surname><given-names>DL</given-names></name></person-group><article-title>Long-lasting sound-evoked afterdischarge in the auditory midbrain</article-title><source>Sci. Rep.</source><year>2016</year><volume>6</volume><fpage>20757</fpage><pub-id pub-id-type=\"pmid\">26867811</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>X</given-names></name><name><surname>Lu</surname><given-names>T</given-names></name><name><surname>Snider</surname><given-names>RK</given-names></name><name><surname>Liang</surname><given-names>L</given-names></name></person-group><article-title>Sustained firing in auditory cortex evoked by preferred stimuli</article-title><source>Nature</source><year>2005</year><volume>435</volume><fpage>341</fpage><pub-id pub-id-type=\"pmid\">15902257</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Galloway</surname><given-names>EM</given-names></name><name><surname>Woo</surname><given-names>NH</given-names></name><name><surname>Lu</surname><given-names>B</given-names></name></person-group><article-title>Persistent neural activity in the prefrontal cortex: a mechanism by which bdnf regulates working memory?</article-title><source>Prog. Brain Res.</source><year>2008</year><volume>169</volume><fpage>251</fpage><lpage>266</lpage><pub-id pub-id-type=\"pmid\">18394479</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lester</surname><given-names>RA</given-names></name><name><surname>Clements</surname><given-names>JD</given-names></name><name><surname>Westbrook</surname><given-names>GL</given-names></name><name><surname>Jahr</surname><given-names>CE</given-names></name></person-group><article-title>Channel kinetics determine the time course of nmda receptor-mediated synaptic currents</article-title><source>Nature</source><year>1990</year><volume>346</volume><fpage>565</fpage><pub-id pub-id-type=\"pmid\">1974037</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>H</given-names></name><name><surname>Stradtman</surname><given-names>GG</given-names></name><name><surname>Wang</surname><given-names>XJ</given-names></name><name><surname>Gao</surname><given-names>WJ</given-names></name></person-group><article-title>A specialized NMDA receptor function in layer 5 recurrent microcircuitry of the adult rat prefrontal cortex</article-title><source>Proc. Natl. Acad. Sci.</source><year>2008</year><volume>105</volume><issue>43</issue><fpage>16791</fpage><lpage>16796</lpage><pub-id pub-id-type=\"pmid\">18922773</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Douglas</surname><given-names>RJ</given-names></name><name><surname>Koch</surname><given-names>C</given-names></name><name><surname>Mahowald</surname><given-names>M</given-names></name><name><surname>Martin</surname><given-names>K</given-names></name><name><surname>Suarez</surname><given-names>HH</given-names></name></person-group><article-title>Recurrent excitation in neocortical circuits</article-title><source>Science</source><year>1995</year><volume>269</volume><fpage>981</fpage><lpage>985</lpage><pub-id pub-id-type=\"pmid\">7638624</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>de Lorente</surname><given-names>NR</given-names></name></person-group><article-title>Vestibulo-ocular reflex arc</article-title><source>Arch. Neurol. Psychiatry</source><year>1933</year><volume>30</volume><fpage>245</fpage><lpage>291</lpage></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Hebb</surname><given-names>DO</given-names></name></person-group><source>The Organization of Behavior</source><year>2002</year><publisher-loc>New York</publisher-loc><publisher-name>Wiley</publisher-name></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McCulloch</surname><given-names>WS</given-names></name><name><surname>Pitts</surname><given-names>W</given-names></name></person-group><article-title>A logical calculus of the ideas immanent in nervous activity</article-title><source>Bull. Math. Biophys.</source><year>1943</year><volume>5</volume><fpage>115</fpage><lpage>133</lpage></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hopfield</surname><given-names>JJ</given-names></name></person-group><article-title>Neural networks and physical systems with emergent collective computational abilities</article-title><source>Proc. Natl. Acad. Sci.</source><year>1982</year><volume>79</volume><fpage>2554</fpage><lpage>2558</lpage><pub-id pub-id-type=\"pmid\">6953413</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>X-J</given-names></name></person-group><article-title>Synaptic basis of cortical persistent activity: the importance of nmda receptors to working memory</article-title><source>J. Neurosci.</source><year>1999</year><volume>19</volume><fpage>9587</fpage><lpage>9603</lpage><pub-id pub-id-type=\"pmid\">10531461</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>X-J</given-names></name></person-group><article-title>Synaptic reverberation underlying mnemonic persistent activity</article-title><source>Trends Neurosci.</source><year>2001</year><volume>24</volume><fpage>455</fpage><lpage>463</lpage><pub-id pub-id-type=\"pmid\">11476885</pub-id></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brody</surname><given-names>CD</given-names></name><name><surname>Romo</surname><given-names>R</given-names></name><name><surname>Kepecs</surname><given-names>A</given-names></name></person-group><article-title>Basic mechanisms for graded persistent activity: discrete attractors, continuous attractors, and dynamic representations</article-title><source>Curr. Opin. Neurobiol.</source><year>2003</year><volume>13</volume><fpage>204</fpage><lpage>211</lpage><pub-id pub-id-type=\"pmid\">12744975</pub-id></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Major</surname><given-names>G</given-names></name><name><surname>Tank</surname><given-names>D</given-names></name></person-group><article-title>Persistent neural activity: prevalence and mechanisms</article-title><source>Curr. opinion neurobiology</source><year>2004</year><volume>14</volume><fpage>675</fpage><lpage>684</lpage></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lau</surname><given-names>P-M</given-names></name><name><surname>Bi</surname><given-names>G-Q</given-names></name></person-group><article-title>Synaptic mechanisms of persistent reverberatory activity in neuronal networks</article-title><source>Proc. Natl. Acad. Sci.</source><year>2005</year><volume>102</volume><fpage>10333</fpage><lpage>10338</lpage><pub-id pub-id-type=\"pmid\">16006530</pub-id></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wehr</surname><given-names>M</given-names></name><name><surname>Zador</surname><given-names>AM</given-names></name></person-group><article-title>Synaptic mechanisms of forward suppression in rat auditory cortex</article-title><source>Neuron</source><year>2005</year><volume>47</volume><fpage>437</fpage><lpage>445</lpage><pub-id pub-id-type=\"pmid\">16055066</pub-id></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Heiss</surname><given-names>JE</given-names></name><name><surname>Katz</surname><given-names>Y</given-names></name><name><surname>Ganmor</surname><given-names>E</given-names></name><name><surname>Lampl</surname><given-names>I</given-names></name></person-group><article-title>Shift in the balance between excitation and inhibition during sensory adaptation of s1 neurons</article-title><source>J. Neurosci.</source><year>2008</year><volume>28</volume><fpage>13320</fpage><lpage>13330</lpage><pub-id pub-id-type=\"pmid\">19052224</pub-id></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>X-X</given-names></name><etal/></person-group><article-title>Characterization of rebound depolarization in neurons of the rat medial geniculate body in vitro</article-title><source>Neurosci. Bull.</source><year>2016</year><volume>32</volume><fpage>16</fpage><lpage>26</lpage><pub-id pub-id-type=\"pmid\">26781877</pub-id></element-citation></ref><ref id=\"CR63\"><label>63.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>Y-Q</given-names></name><name><surname>Xiong</surname><given-names>Y</given-names></name><name><surname>Chan</surname><given-names>Y-S</given-names></name><name><surname>He</surname><given-names>J</given-names></name></person-group><article-title>Corticofugal gating of auditory information in the thalamus: an in vivo intracellular recording study</article-title><source>J. Neurosci.</source><year>2004</year><volume>24</volume><fpage>3060</fpage><lpage>3069</lpage><pub-id pub-id-type=\"pmid\">15044545</pub-id></element-citation></ref><ref id=\"CR64\"><label>64.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Neisser</surname><given-names>U</given-names></name></person-group><source>Cognitive Psychology: Classic edition</source><year>2014</year><publisher-loc>New York</publisher-loc><publisher-name>Psychology Press</publisher-name></element-citation></ref><ref id=\"CR65\"><label>65.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Darwin</surname><given-names>CJ</given-names></name><name><surname>Turvey</surname><given-names>MT</given-names></name><name><surname>Crowder</surname><given-names>RG</given-names></name></person-group><article-title>An auditory analogue of the sperling partial report procedure: evidence for brief auditory storage</article-title><source>Cogn. Psychol.</source><year>1972</year><volume>3</volume><fpage>255</fpage><lpage>267</lpage></element-citation></ref><ref id=\"CR66\"><label>66.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Treisman</surname><given-names>A</given-names></name></person-group><article-title>Monitoring and storage of irrelevant messages in selective attention</article-title><source>J. Verbal Learn. Verbal Behav.</source><year>1964</year><volume>3</volume><fpage>449</fpage><lpage>459</lpage></element-citation></ref><ref id=\"CR67\"><label>67.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Norman</surname><given-names>DA</given-names></name></person-group><article-title>Memory while shadowing</article-title><source>Q. J. Exp. Psychol.</source><year>1969</year><volume>21</volume><fpage>85</fpage><lpage>93</lpage><pub-id pub-id-type=\"pmid\">5777987</pub-id></element-citation></ref><ref id=\"CR68\"><label>68.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wirtssohn</surname><given-names>S</given-names></name><name><surname>Ronacher</surname><given-names>B</given-names></name></person-group><article-title>Temporal integration at consecutive processing stages in the auditory pathway of the grasshopper</article-title><source>J. Neurophysiol.</source><year>2015</year><volume>113</volume><fpage>2280</fpage><lpage>2288</lpage><pub-id pub-id-type=\"pmid\">25609104</pub-id></element-citation></ref><ref id=\"CR69\"><label>69.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wallace</surname><given-names>AB</given-names></name><name><surname>Blumstein</surname><given-names>SE</given-names></name></person-group><article-title>Temporal integration in vowel perception</article-title><source>J. Acoust. Soc. Am.</source><year>2009</year><volume>125</volume><fpage>1704</fpage><lpage>1711</lpage><pub-id pub-id-type=\"pmid\">19275327</pub-id></element-citation></ref><ref id=\"CR70\"><label>70.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Musacchia</surname><given-names>G</given-names></name><name><surname>Schroeder</surname><given-names>CE</given-names></name></person-group><article-title>Neuronal mechanisms, response dynamics and perceptual functions of multisensory interactions in auditory cortex</article-title><source>Hear. Res.</source><year>2009</year><volume>258</volume><fpage>72</fpage><lpage>79</lpage><pub-id pub-id-type=\"pmid\">19595755</pub-id></element-citation></ref><ref id=\"CR71\"><label>71.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Curzon</surname><given-names>P</given-names></name><name><surname>Rustay</surname><given-names>NR</given-names></name><name><surname>Browman</surname><given-names>KE</given-names></name></person-group><person-group person-group-type=\"editor\"><name><surname>Buccafusco</surname><given-names>JJ</given-names></name></person-group><article-title>Cued and contextual fear conditioning for rodents</article-title><source>Methods of Behavior Analysis in Neuroscience</source><year>2009</year><edition>2</edition><publisher-loc>Boca Raton</publisher-loc><publisher-name>CRC Press/Taylor & Francis LCC</publisher-name><fpage>77</fpage><lpage>101</lpage></element-citation></ref><ref id=\"CR72\"><label>72.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lu</surname><given-names>T</given-names></name><name><surname>Liang</surname><given-names>L</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name></person-group><article-title>Neural representations of temporally asymmetric stimuli in the auditory cortex of awake primates</article-title><source>J. Neurophysiol.</source><year>2001</year><volume>85</volume><fpage>2364</fpage><lpage>2380</lpage><pub-id pub-id-type=\"pmid\">11387383</pub-id></element-citation></ref><ref id=\"CR73\"><label>73.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morel</surname><given-names>A</given-names></name><name><surname>Kaas</surname><given-names>JH</given-names></name></person-group><article-title>Subdivisions and connections of auditory cortex in owl monkeys</article-title><source>J. Comp. Neurol.</source><year>1992</year><volume>318</volume><fpage>27</fpage><lpage>63</lpage><pub-id pub-id-type=\"pmid\">1583155</pub-id></element-citation></ref><ref id=\"CR74\"><label>74.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morel</surname><given-names>A</given-names></name><name><surname>Garraghty</surname><given-names>P</given-names></name><name><surname>Kaas</surname><given-names>J</given-names></name></person-group><article-title>Tonotopic organization, architectonic fields, and connections of auditory cortex in macaque monkeys</article-title><source>J. Comp. Neurol.</source><year>1993</year><volume>335</volume><fpage>437</fpage><lpage>459</lpage><pub-id pub-id-type=\"pmid\">7693772</pub-id></element-citation></ref><ref id=\"CR75\"><label>75.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Petkov</surname><given-names>CI</given-names></name><name><surname>Kayser</surname><given-names>C</given-names></name><name><surname>Augath</surname><given-names>M</given-names></name><name><surname>Logothetis</surname><given-names>NK</given-names></name></person-group><article-title>Functional imaging reveals numerous fields in the monkey auditory cortex</article-title><source>PLoS Biol.</source><year>2006</year><volume>4</volume><fpage>e215</fpage><pub-id pub-id-type=\"pmid\">16774452</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807914</article-id><article-id pub-id-type=\"pmc\">PMC7431572</article-id><article-id pub-id-type=\"publisher-id\">70900</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70900-7</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Ionic liquid-assisted cellulose coating of chitosan hydrogel beads and their application as drug carriers</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Song</surname><given-names>Myung-Hee</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Pham</surname><given-names>Thi Phuong Thuy</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Yun</surname><given-names>Yeoung-Sang</given-names></name><address><email>ysyun@jbnu.ac.kr</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.411545.0</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0470 4320</institution-id><institution>School of Chemical Engineering, </institution><institution>Jeonbuk National University (formerly Chonbuk National University), </institution></institution-wrap>Baekje-daero, Jeonju-si, Jeollabuk-do 54896 Republic of Korea </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.491482.2</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 6041 6067</institution-id><institution>Faculty of Biotechnology, </institution><institution>Ho Chi Minh City University of Food Industry, </institution></institution-wrap>Ho Chi Minh City, Vietnam </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13905</elocation-id><history><date date-type=\"received\"><day>15</day><month>11</month><year>2019</year></date><date date-type=\"accepted\"><day>31</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">The present study proposes a simple yet effective method of cellulose coating onto chitosan (CS) hydrogel beads and application thereof as drug carriers. The beads were coated with cellulose dissolved in 1-ethyl-3-methylimidazolium acetate, an ionic liquid (IL) via a one-pot one-step process. Water molecules present in the CS beads diffused outward upon contact with the cellulose–IL mixture and acted as an anti-solvent. This allowed the surface of the beads to be coated with the regenerated cellulose. The regenerated cellulose was characterized by FE-SEM, FT-IR, and XRD analyses. To test potential application of the cellulose-coated CS hydrogel beads as a drug carrier, verapamil hydrochloride (VRP), used as a model drug, was impregnated into the beads. When the VRP-impregnated beads were immersed in the simulated gastric fluid (pH 1.2), the VRP was released in an almost ideal linear pattern. This easily fabricated cellulose-coated CS beads showed the possibility for application as carriers for drug release control.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Gels and hydrogels</kwd><kwd>Ionic liquids</kwd></kwd-group><funding-group><award-group><funding-source><institution>National Research Foundation of Korea</institution></funding-source><award-id>2020R1C1C1006369</award-id><award-id>2020R1A2C3009769</award-id><principal-award-recipient><name><surname>Song</surname><given-names>Myung-Hee</given-names></name><name><surname>Yun</surname><given-names>Yeoung-Sang</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Hydrogels are natural or synthetic hydrophilic structures capable of holding significant amounts of water or biological fluids in their three-dimensional structures<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. Hydrogels manufactured from renewable natural resources like polysaccharides being extensively used in the medical and pharmaceutical fields<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>–<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. Especially hydrogels made up of chitosan (CS), the cationic polysaccharide is of high priority owing to their impressive properties such as biodegradability, low toxicity, and biocompatibility<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>–<xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. However, the use of CS as drug carriers has been limited due to its weak physical properties and low acid resistance in gastric fluid when applied in oral delivery<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>–<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Therefore, in the present study, we used cellulose as a coating layer onto the surface of CS hydrogel beads to improve their stability in biomedical applications<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>.\n</p><p id=\"Par3\">Cellulose consists of β-(1 → 4)-linked glucose units and is one of the naturally abundant vital biomass components in the earth<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. Cellulose is a crucial ingredient for fabricating various products such as paper, textiles, membranes, biofuel, and value-added chemical products. However, due to the strong inter- and intra-molecular hydrogen bonding, cellulose possesses high order crystalline domains<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. Therefore, cellulose does not melt and is insoluble in conventional solvents such as water, dilute acids, alkali solutions, and the common organic solvents<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>, which limits the effective utilization of cellulose. Harsh conditions (high temperature and acid conditions) are usually required to disrupt the hydrogen bond network for the successful dissolution of the cellulose. These conventional methods require high energy consumption, and the use of toxic chemicals, thus, is not suitable for application in the biomedical field. As a result of thorough investigations, ionic liquids (ILs) have been considered a promising solvent that dissolves cellulose without any formation of derivatives<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>–<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>.</p><p id=\"Par4\">By definition, ILs are room temperature molten salts that consist of cations and anions with melting points of less than 100 °C<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. In recent years, ILs have been widely used to replace conventional organic solvents in various industrial applications. In 2002, Rogers et al.<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> reported a method to dissolve cellulose by using ILs under mild conditions without derivatization. The dissolved cellulose after that could be solidified by using anti-solvent such as water and ethanol and regenerated in various forms such as fiber<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup> film<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup> or membrane<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, according to the manufacturing conditions.</p><p id=\"Par5\">In this study, CS beads were coated with cellulose dissolved in IL using only water molecules contained in hydrogels (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>). The prepared cellulose coated CS beads were characterized by Field emission scanning electron microscope (FE-SEM), Fourier transform infrared spectrometer (FT-IR), and X-ray diffractometer (XRD), and applied to drug release experiments and discussed accordingly.</p></sec><sec id=\"Sec2\"><title>Results and discussion</title><sec id=\"Sec3\"><title>Morphology of cellulose-coated CS bead</title><p id=\"Par6\">The conceptual strategy for the cellulose coating on hydrogel is shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>. The strategy was to evenly coat wet CS hydrogel bead with dissolved cellulose via osmotic pressure difference. That is, cellulose was first dissolved in [Emim][Ac], which is a well-known cellulose processing IL<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. Then the wet CS bead, which was used as a model hydrogel, was brought into contact with the dissolved cellulose. Upon contact with the cellulose solution, the water molecules present inside of the CS bead diffused outwards due to osmotic pressure. Consequently, the diffused water molecules acted as anti-solvents to solidify the cellulose and formed cellulose coating on the surface of the hydrogel. It must be noted that control of the water content of the hydrogel bead was essential at this stage. It was observed that when the bead was too watery (having water on the outside), the cellulose coating formed was uneven, rough and tend to clump up. Moreover, when the bead was dried or had no water inside, it was impossible to form the cellulose coating due to the lack of water molecules to act as anti-solvents. Thus, the best condition for this idea to be realized was to control the moisture content of the bead by effectively wiping off surface water. For this purpose, the wet bead was placed on gauze to remove adhering water from the surface of the bead before use.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Schematic presentation of cellulose coating principle.</p></caption><graphic xlink:href=\"41598_2020_70900_Fig1_HTML\" id=\"MO1\"/></fig></p></sec><sec id=\"Sec4\"><title>FE-SEM analysis</title><p id=\"Par7\">To understand the texture, the cellulose-coated CS bead was cut in half, and the morphology was analyzed using FE-SEM, as shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>. The FE-SEM images showed the apparent boundary of CS and cellulose layers (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>a). The cross-sectional internal morphology (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>b) clearly showed that regenerated cellulose appeared as an outer layer (shell), and CS hydrogel was present as the core. Higher magnification of core and shell parts showed a connected three-dimensional network (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>). Both the core and shell parts possessed high porosities, with the shell part showing a broad range of pore sizes and the core part revealing smaller pore sizes. This porous network can be useful to encapsulate drugs or cells for biological applications. In addition, the results of FE-SEM analysis (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>A) and element mapping (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>B–D) of the cross-section of the cellulose-coated CS bead are shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>. Since carbon and oxygen are constituent elements of chitosan (CS) and cellulose, the elements C (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>B) and O (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>C) were evenly distributed throughout the cellulose-coated CS bead. However, it was confirmed that the elements of N (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>D) are concentrated in the core (chitosan part). Additionally, as a result of EDS mapping by separating the core part and the shell part, C, O, N, and Na were measured as 70.32, 25.71, 3.88, 0.09 wt% and 53.64, 46.28, 0.00, 0.08 wt%, respectively in the core and shell parts (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>).\nThat is, the N element was detected only in the core, the chitosan portion. Therefore, it was confirmed that the core is composed of chitosan and the shell is composed of cellulose. In addition, if some of the dissociated [Emim][Ac] remains in cellulose-coated CS bead, N, a constituent element of [Emim][Ac], must be detected not only in the core but also in the shell part. However, since the cellulose-coated CS beads were thoroughly washed, no ionic liquid residues were considered.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>FE-SEM images of freeze dried cellulose-coated CS bead (<bold>a</bold>) and intersection of CS and cellulose layers (<bold>b</bold>). Scale bars represent 1 mm and 20 µm respectively.</p></caption><graphic xlink:href=\"41598_2020_70900_Fig2_HTML\" id=\"MO2\"/></fig><fig id=\"Fig3\"><label>Figure 3</label><caption><p>FE-SEM images of cellulose-coated CS bead (<bold>A</bold>) and EDS mapping of C (<bold>B</bold>), O (<bold>C</bold>), N (<bold>D</bold>) elements.</p></caption><graphic xlink:href=\"41598_2020_70900_Fig3_HTML\" id=\"MO3\"/></fig><table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>The contents (wt%) of C, O, N, Na elements of chitosan and cellulose part of cellulose-coated CS bead analyzed by FE-SEM EDS mapping.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Element</th><th align=\"left\">Chitosan part (wt%)</th><th align=\"left\">Cellulose part (wt%)</th></tr></thead><tbody><tr><td align=\"left\">C</td><td char=\".\" align=\"char\">70.32</td><td char=\".\" align=\"char\">53.64</td></tr><tr><td align=\"left\">O</td><td char=\".\" align=\"char\">25.71</td><td char=\".\" align=\"char\">46.28</td></tr><tr><td align=\"left\"><bold>N</bold></td><td char=\".\" align=\"char\">3.88</td><td char=\".\" align=\"char\">0.00</td></tr><tr><td align=\"left\">Na</td><td char=\".\" align=\"char\">0.09</td><td char=\".\" align=\"char\">0.08</td></tr><tr><td align=\"left\">Total</td><td char=\".\" align=\"char\">100.00</td><td char=\".\" align=\"char\">100.00</td></tr></tbody></table></table-wrap></p></sec><sec id=\"Sec5\"><title>FT-IR analysis</title><p id=\"Par8\">Prior to FT-IR analysis, the cellulose-coated CS bead was separated into the shell (regenerated cellulose) and core (CS) part to confirm the functional groups of each section. FT-IR spectroscopic analysis of the original cellulose and regenerated cellulose on CS bead are shown in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>. The broad absorption band in the range of 3,300–3,400 cm<sup>−1</sup> was due to the intermolecular O–H stretching vibration of the cellulose molecule. The peak at 2,898 cm<sup>−1</sup> observed for native cellulose was due to the C–H stretching vibration of CH<sub>2</sub> and CH<sub>3</sub> groups. This band was not affected by changes of crystallinity; thus, there was no significant change of this band in the regenerated cellulose spectral data. The peak at 1,428 cm<sup>−1</sup> in the original cellulose was assigned to the crystallized cellulose I and amorphous cellulose, whereas in the case of the regenerated cellulose, this band shifted to 1,419 cm<sup>−1</sup>, representing cellulose II and amorphous cellulose<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref>,<xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. The peak at 1,428 cm<sup>−1</sup> in the cellulose spectra was assigned to symmetric CH<sub>2</sub> bending vibration. The peak at 1,103 cm<sup>−1</sup>, which appeared in the spectrum of the native cellulose was not observed for the regenerated cellulose. This further confirmed the prevalence of crystalline cellulose II<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. In the regenerated cellulose spectra, the new peak at 1,750 cm<sup>−1</sup> was attributed to the C=O in the acetate<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. This is good evidence that cellulose dissolved in [Emim] [Ac] had been regenerated. The band observed at around 895 cm<sup>−1</sup> was due to the stretching vibration of C–O bond in the amorphous regions of cellulose and regenerated cellulose.<fig id=\"Fig4\"><label>Figure 4</label><caption><p>FT-IR spectral data of original cellulose (<bold>a</bold>) and regenerated cellulose on CS bead (<bold>b</bold>). Original cellulose was in powder form and regenerated cellulose was separated from cellulose-coated CS bead.</p></caption><graphic xlink:href=\"41598_2020_70900_Fig4_HTML\" id=\"MO4\"/></fig></p></sec><sec id=\"Sec6\"><title>XRD analysis</title><p id=\"Par9\">The typical XRD profiles of the original cellulose and regenerated cellulose on CS bead are shown in Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>. The XRD pattern of original cellulose exhibits characteristic diffraction peaks at 2θ = 14.9°, 16.0°, 22.4°, and 34.5° corresponding to the planes (110), (110), (200) and (400). These diffraction patterns confirmed that the original cellulose was cellulose I. After dissolution and regeneration in [Emim][Ac] the regenerated cellulose exhibited characteristic diffraction patterns at 2θ 19°–20°, and these patterns were consistent with cellulose II. Similar results were reported with other IL solvents such as [Bmim]Cl, [Amim]Cl<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref>,<xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>. Due to the pronounced quantity of amorphous cellulose existing in the regenerated cellulose, it showed a lower crystallinity than the original cellulose. It could be explained by the fact that [Emim][Ac] broke the inter- and intramolecular hydrogen bonds and reduced the crystallinity of the original cellulose during the dissolution process.<fig id=\"Fig5\"><label>Figure 5</label><caption><p>XRD profiles of original cellulose (<bold>a</bold>) and regenerated cellulose on CS bead (<bold>b</bold>). Original cellulose was in powder form and regenerated cellulose was separated from cellulose-coated CS bead.</p></caption><graphic xlink:href=\"41598_2020_70900_Fig5_HTML\" id=\"MO5\"/></fig></p></sec><sec id=\"Sec7\"><title>In vitro release of VPR from CS beads and cellulose-coated CS beads</title><p id=\"Par10\">Since cellulose coatings may improve the physical and chemical stability of the hydrogels, cellulose-coated CS beads may have a wide range of applications. One of the attractive applications of the cellulose-coated hydrogels could be in the biomedical area because both cellulose and CS are biocompatible and biodegradable.</p><p id=\"Par11\">Hence we tested the cellulose-coated CS beads for in vitro verapamil hydrochloride (VRP) release applications. VRP is a calcium channel blocker used for the treatment of hypertension, angina and myocardial infarction<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. The release experiments were studied in different release fluids, including simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8). The release profiles corresponding to a formulation consisting of CS beads and cellulose-coated CS beads are displayed in Figs. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref> and <xref rid=\"Fig7\" ref-type=\"fig\">7</xref> for SGF and SIF, respectively. As shown in Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>, the release patterns of VRP from the cellulose-coated CS beads in SGF were significantly different from those of CS beads. This could be explained by the stability of the prepared CS hydrogels. In SGF, CS beads were dissolved only within 5 min, and no solid matter was left after 10 min in the system. Meanwhile, the cellulose-coated CS beads were stable and remained stable up to 24 h. The release of VRP from the cellulose-coated CS beads was almost linear, similar to the zero-order release.<fig id=\"Fig6\"><label>Figure 6</label><caption><p>Release patterns of VRP in SGF from CS beads and cellulose-coated CS beads. The in vitro release experiment was performed at 100 rpm and 37 ± 0.5 °C. The concentration of VRP was analyzed by taking a sample of 0.5 ml from 50 ml of SGF solution.</p></caption><graphic xlink:href=\"41598_2020_70900_Fig6_HTML\" id=\"MO6\"/></fig><fig id=\"Fig7\"><label>Figure 7</label><caption><p>Release patterns of VRP in SIF from CS beads and cellulose-coated CS beads. The in vitro release experiment was performed at 100 rpm and 37 ± 0.5 °C. The concentration of VRP was analyzed by taking a sample of 0.5 ml from 50 ml of SIF solution.</p></caption><graphic xlink:href=\"41598_2020_70900_Fig7_HTML\" id=\"MO7\"/></fig></p><p id=\"Par12\">Figure <xref rid=\"Fig7\" ref-type=\"fig\">7</xref> shows the VRP release profiles of the CS beads and cellulose-coated CS beads in SIF. The release of VRP from the CS beads was much faster than that from the cellulose-coated CS beads. After 120 min, 95 ± 0.5% and 65 ± 0.2% of VRP were released from the CS beads and cellulose-coated CS beads, respectively, indicating that the cellulose coating makes a significant change in VRP release profiles. The total amounts of drug released from both coated and uncoated CS beads after 300 min were nearly the same. The results of this study were tried to be applied to the well-explained drug release model Korsemeyer–Peppas model (supplementary information). The release exponent (<italic>n</italic>) for CS beads are smaller than 0.45 in SGF (0.0752) and SIF (0.3217), indicating that the release of VRP from the CS beads are a Quasi-Fickian diffusion mechanism (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>). This mechanism indicated that VRP diffuses partially through a swollen matrix or its pores in the chitosan hydrogels. Moreover, the <italic>n</italic> values of cellulose-coated CS beads in SGF and SIF were 0.7298 and 0.6628, respectively, which indicated the non-Fickian diffusion mechanism consisting of a combination of diffusion and polymer relaxation. By coating cellulose on CS beads, it was possible to alter the release pattern of the drugs in the CS beads. These results clearly demonstrated the potential application of the prepared cellulose-coated CS hydrogel beads in the biomedical area of drug delivery.</p></sec></sec><sec id=\"Sec8\"><title>Conclusions</title><p id=\"Par13\">Cellulose-coated CS beads were successfully fabricated via a facile one-pot one-step method. FE-SEM observation showed that the CS beads and the cellulose coating layer were separated and porous. The results of FT-IR and XRD also supported that the regenerated cellulose was well coated on the CS surface. The cellulose-coated CS beads exhibited sustained release patterns of VRP in SGF and SIF environments when applied as drug carriers. This simple cellulose coating method will be able to promote various applications of the hydrogels.</p></sec><sec id=\"Sec9\"><title>Experimental</title><sec id=\"Sec10\"><title>Chemicals and materials</title><p id=\"Par14\">Cellulose (powder) and verapamil hydrochloride (VRP) were purchased from Fluka and Sigma-Aldrich, respectively. CS (viscosity at 20 °C: 200–220 cP, deacetylation degree: 85.9%) was purchased from YBBio Co., Ltd. (Korea), at which CS was obtained on an industrial scale by the deacetylation of chitin from the red crab. The ILs 1-Ethyl-3-methylimidazolium acetate ([Emim][Ac], above 95%) was purchased from Ionic Liquids Technologies (Germany), and acetic acid (above 99.7%) was obtained from Junsei Chemical Co., Ltd. All of the other reagents used in this study were also of analytical grade. In addition, the plastic hub needle used to fabricate the CS beads were purchased from Teaha Co., Korea.</p></sec><sec id=\"Sec11\"><title>Dissolution of cellulose in ionic liquid</title><p id=\"Par15\">Cellulose/[Emim][Ac] solution was first prepared by dissolving 6 g of cellulose powder in 100 ml of [Emim][Ac] at 120 °C, and the obtained 6% (w/v) cellulose/[Emim][Ac] solution was used to manufacture the regenerated cellulose-coated CS beads.</p></sec><sec id=\"Sec12\"><title>Fabrication of CS beads</title><p id=\"Par16\">For the manufacture of the CS beads, CS powder (2 g) was dispersed in 100 ml of distilled water containing 2 ml of acetic acid solution at room temperature. The mixture was left overnight with continuous mechanical stirring to prepare completely dissolved CS solution. The as-obtained mixture was dropped through a plastic hub needle with a diameter of 310 µm into 1 M NaOH solution under gentle magnetic stirring at room temperature to obtain CS beads. After 2 h, the hydrogel beads were separated from the NaOH solution and washed with distilled water.</p></sec><sec id=\"Sec13\"><title>Fabrication of VRP-loaded CS beads</title><p id=\"Par17\">To fabricate the VRP-loaded CS beads, 1,000 mg of VRP was added to 10 ml of 2% CS solution and stirred for 30 min to obtain a homogeneous dispersion. The VRP-loaded CS beads were prepared in the same dropwise method, as described in the previous section.</p></sec><sec id=\"Sec14\"><title>Procedure for cellulose coating on CS beads</title><p id=\"Par18\">The wet CS beads were dropped into the 6% (w/v) cellulose dissolved in [Emim][Ac] solution and kept for 5 min, resulting in the formation of cellulose coat on the surface of CS beads. Then, the cellulose-coated CS beads were separated from the solution using gauze mesh and washed with deionized water three or more times.</p></sec><sec id=\"Sec15\"><title>Fabrication of VRP-loaded cellulose-coated CS beads</title><p id=\"Par19\">The VRP-loaded cellulose-coated CS beads were fabricated by first manufacturing the VRP-loaded CS beads and then coating it with cellulose. The verapamil encapsulation efficiency (EE) and loading capacity (LC) of cellulose-coated CS beads used in drug release experiments were 100 ± 0.0% and 8.62 ± 0.07%, respectively. Encapsulation efficiency (EE) and loading capacity (LC) were calculated as follows:<disp-formula id=\"Equa\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\begin{aligned} {\\text{EE }}\\left( {{\\% }} \\right) & = \\frac{weight\\,of\\,loaded\\,drug}{{weight\\,of\\,total\\,added\\,drug}} \\times 100 \\\\ {\\text{LC }}\\left( {{\\% }} \\right) & = \\frac{weight\\,of\\, loaded\\,drug}{{weight\\,of\\,drug\\,loaded\\,bead}} \\times 100. \\\\ \\end{aligned} $$\\end{document}</tex-math><mml:math id=\"M2\" display=\"block\"><mml:mrow><mml:mtable><mml:mtr><mml:mtd columnalign=\"right\"><mml:mrow><mml:mrow><mml:mtext>EE</mml:mtext><mml:mspace width=\"0.333333em\"/></mml:mrow><mml:mfenced close=\")\" open=\"(\"><mml:mo>%</mml:mo></mml:mfenced></mml:mrow></mml:mtd><mml:mtd columnalign=\"left\"><mml:mrow><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>w</mml:mi><mml:mi>e</mml:mi><mml:mi>i</mml:mi><mml:mi>g</mml:mi><mml:mi>h</mml:mi><mml:mi>t</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>o</mml:mi><mml:mi>f</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>l</mml:mi><mml:mi>o</mml:mi><mml:mi>a</mml:mi><mml:mi>d</mml:mi><mml:mi>e</mml:mi><mml:mi>d</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>d</mml:mi><mml:mi>r</mml:mi><mml:mi>u</mml:mi><mml:mi>g</mml:mi></mml:mrow><mml:mrow><mml:mi>w</mml:mi><mml:mi>e</mml:mi><mml:mi>i</mml:mi><mml:mi>g</mml:mi><mml:mi>h</mml:mi><mml:mi>t</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>o</mml:mi><mml:mi>f</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>t</mml:mi><mml:mi>o</mml:mi><mml:mi>t</mml:mi><mml:mi>a</mml:mi><mml:mi>l</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>a</mml:mi><mml:mi>d</mml:mi><mml:mi>d</mml:mi><mml:mi>e</mml:mi><mml:mi>d</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>d</mml:mi><mml:mi>r</mml:mi><mml:mi>u</mml:mi><mml:mi>g</mml:mi></mml:mrow></mml:mfrac><mml:mo>×</mml:mo><mml:mn>100</mml:mn></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd columnalign=\"right\"><mml:mrow><mml:mrow/><mml:mrow><mml:mtext>LC</mml:mtext><mml:mspace width=\"0.333333em\"/></mml:mrow><mml:mfenced close=\")\" open=\"(\"><mml:mo>%</mml:mo></mml:mfenced></mml:mrow></mml:mtd><mml:mtd columnalign=\"left\"><mml:mrow><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>w</mml:mi><mml:mi>e</mml:mi><mml:mi>i</mml:mi><mml:mi>g</mml:mi><mml:mi>h</mml:mi><mml:mi>t</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>o</mml:mi><mml:mi>f</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>l</mml:mi><mml:mi>o</mml:mi><mml:mi>a</mml:mi><mml:mi>d</mml:mi><mml:mi>e</mml:mi><mml:mi>d</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>d</mml:mi><mml:mi>r</mml:mi><mml:mi>u</mml:mi><mml:mi>g</mml:mi></mml:mrow><mml:mrow><mml:mi>w</mml:mi><mml:mi>e</mml:mi><mml:mi>i</mml:mi><mml:mi>g</mml:mi><mml:mi>h</mml:mi><mml:mi>t</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>o</mml:mi><mml:mi>f</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>d</mml:mi><mml:mi>r</mml:mi><mml:mi>u</mml:mi><mml:mi>g</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>l</mml:mi><mml:mi>o</mml:mi><mml:mi>a</mml:mi><mml:mi>d</mml:mi><mml:mi>e</mml:mi><mml:mi>d</mml:mi><mml:mspace width=\"0.166667em\"/><mml:mi>b</mml:mi><mml:mi>e</mml:mi><mml:mi>a</mml:mi><mml:mi>d</mml:mi></mml:mrow></mml:mfrac><mml:mo>×</mml:mo><mml:mn>100</mml:mn><mml:mo>.</mml:mo></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd columnalign=\"right\"><mml:mrow/></mml:mtd></mml:mtr></mml:mtable></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70900_Article_Equa.gif\" position=\"anchor\"/></alternatives></disp-formula></p></sec><sec id=\"Sec16\"><title>Drug release studies</title><p id=\"Par20\">The in vitro release studies were performed in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8). Ten beads were placed in plastic bottles containing 50 ml of the release medium. After the cellulose coating, the weight per bead was increased by cellulose coating, so the number of beads was used instead of the weight of beads. The drug release experiments were carried out at 100 rpm and 37 ± 0.5 °C in a shaker. At predetermined time intervals, samples of 0.5 ml were collected from the release medium and replaced with fresh SGF and SIF solution, respectively. The experiments were duplicated.</p><p id=\"Par21\">To estimate the initial concentration of VRP entrapped into the beads, the VRP concentration was measured after crushing and dissolving 10 VRP-loaded CS beads in 50 ml of medium, which is the same condition as the release experiment.<disp-formula id=\"Equb\"><alternatives><tex-math id=\"M3\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ \\% \\,{\\text{cumulative}}\\,{\\text{VRP}}\\,{\\text{release}} = { }\\frac{{{\\text{conc}}.{ }\\,{\\text{of}}\\,{\\text{VRP}}}}{{{\\text{initial}}\\,{\\text{conc}}.\\,{\\text{of}}\\,{\\text{VRP}}}}{ } \\times { }100. $$\\end{document}</tex-math><mml:math id=\"M4\" display=\"block\"><mml:mrow><mml:mo>%</mml:mo><mml:mspace width=\"0.166667em\"/><mml:mtext>cumulative</mml:mtext><mml:mspace width=\"0.166667em\"/><mml:mtext>VRP</mml:mtext><mml:mspace width=\"0.166667em\"/><mml:mtext>release</mml:mtext><mml:mo>=</mml:mo><mml:mrow/><mml:mfrac><mml:mrow><mml:mtext>conc</mml:mtext><mml:mo>.</mml:mo><mml:mrow/><mml:mspace width=\"0.166667em\"/><mml:mtext>of</mml:mtext><mml:mspace width=\"0.166667em\"/><mml:mtext>VRP</mml:mtext></mml:mrow><mml:mrow><mml:mtext>initial</mml:mtext><mml:mspace width=\"0.166667em\"/><mml:mtext>conc</mml:mtext><mml:mo>.</mml:mo><mml:mspace width=\"0.166667em\"/><mml:mtext>of</mml:mtext><mml:mspace width=\"0.166667em\"/><mml:mtext>VRP</mml:mtext></mml:mrow></mml:mfrac><mml:mrow/><mml:mo>×</mml:mo><mml:mrow/><mml:mn>100</mml:mn><mml:mo>.</mml:mo></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70900_Article_Equb.gif\" position=\"anchor\"/></alternatives></disp-formula></p></sec><sec id=\"Sec17\"><title>Instrumental details</title><p id=\"Par22\">In the dried cellulose-coated CS bead, the core and the shell part are easily separated. In this study, a cross-section was cut using a stationery knife and tweezers and then FE-SEM analysis was performed, and FT-IR and XRD analyses were performed by separating the core and shell. The morphology of the core (CS) and shell (cellulose) parts of the cellulose-coated CS beads was examined using an FE-SEM (Carl Zeiss, SUPRA 40VP, Germany) equipment. FT-IR (Spectrum GX, Perkin Elmer, USA) was used to obtain the functional groups present in the fabricated materials. Multipurpose high-performance X-ray diffractometer (XRD, X'pert Powder, PANalytical, Netherlands) was used to analyze the crystallographic nature of the samples. Furthermore, the concentration of drug in the solution was assayed by using high-performance liquid chromatography (HPLC, Shimadzu CBM-20A, Kyoto, Japan) at 278 nm. The mobile phase condition was 75% methanol and 25% buffer solution of 25 mM ammonium formate. The used column was Zorbax Eclipse XDB-C18 (Agilent, USA).</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec19\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70900_MOESM1_ESM.docx\"><caption><p>Supplementary Information.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70900-7.</p></sec><ack><title>Acknowledgements</title><p>This research was supported by the Korean Government through NRF (2020R1A2C3009769, 2020R1C1C1006369) grants.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>Dr. M.-H. Song performed the experiments and wrote the manuscript under cooperation and supervision of Dr. T. P. T. Pham and Dr. Y.-S. Yun. All authors reviewed and commented on the manuscript.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par24\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>L</given-names></name><name><surname>Ding</surname><given-names>J</given-names></name></person-group><article-title>Injectable hydrogels as unique biomedical materials</article-title><source>Chem. Soc. Rev.</source><year>2008</year><volume>37</volume><fpage>1473</fpage><lpage>1481</lpage><pub-id pub-id-type=\"doi\">10.1039/b713009k</pub-id><pub-id pub-id-type=\"pmid\">18648673</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Caló</surname><given-names>E</given-names></name><name><surname>Khutoryanskiy</surname><given-names>VV</given-names></name></person-group><article-title>Biomedical applications of hydrogels: a review of patents and commercial products</article-title><source>Eur. Polym. J.</source><year>2015</year><volume>65</volume><fpage>252</fpage><lpage>267</lpage><pub-id pub-id-type=\"doi\">10.1016/j.eurpolymj.2014.11.024</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sahranavard</surname><given-names>M</given-names></name><name><surname>Zamanian</surname><given-names>A</given-names></name><name><surname>Ghorbani</surname><given-names>F</given-names></name><name><surname>Shahrezaee</surname><given-names>MH</given-names></name></person-group><article-title>A critical review on three dimensional-printed chitosan hydrogels for development of tissue engineering</article-title><source>Bioprinting</source><year>2020</year><volume>17</volume><fpage>e00063</fpage><pub-id pub-id-type=\"doi\">10.1016/j.bprint.2019.e00063</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bhattarai</surname><given-names>N</given-names></name><name><surname>Gunn</surname><given-names>J</given-names></name><name><surname>Zhang</surname><given-names>M</given-names></name></person-group><article-title>Chitosan-based hydrogels for controlled, localized drug delivery</article-title><source>Adv. Drug Deliv. Rev.</source><year>2010</year><volume>62</volume><fpage>83</fpage><lpage>99</lpage><pub-id pub-id-type=\"doi\">10.1016/j.addr.2009.07.019</pub-id><pub-id pub-id-type=\"pmid\">19799949</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><mixed-citation publication-type=\"other\">Morin-Crini, N., Lichtfouse, E., Torri, G. & Crini, G. Applications of chitosan in food, pharmaceuticals, medicine, cosmetics, agriculture, textiles, pulp and paper, biotechnology, and environmental chemistry. <italic>Environ. Chem. Lett.</italic><bold>17</bold>, 1667–1692 (2019).</mixed-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shariatinia</surname><given-names>Z</given-names></name></person-group><article-title>Pharmaceutical applications of chitosan</article-title><source>Adv. Colloid Interface Sci.</source><year>2019</year><volume>263</volume><fpage>131</fpage><lpage>194</lpage><pub-id pub-id-type=\"doi\">10.1016/j.cis.2018.11.008</pub-id><pub-id pub-id-type=\"pmid\">30530176</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bernkop-Schnürch</surname><given-names>A</given-names></name><name><surname>Dünnhaupt</surname><given-names>S</given-names></name></person-group><article-title>Chitosan-based drug delivery systems</article-title><source>Eur. J. Pharm. Biopharm.</source><year>2012</year><volume>81</volume><fpage>463</fpage><lpage>469</lpage><pub-id pub-id-type=\"doi\">10.1016/j.ejpb.2012.04.007</pub-id><pub-id pub-id-type=\"pmid\">22561955</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martău</surname><given-names>GA</given-names></name><name><surname>Mihai</surname><given-names>M</given-names></name><name><surname>Vodnar</surname><given-names>DC</given-names></name></person-group><article-title>The use of chitosan, alginate, and pectin in the biomedical and food sector—biocompatibility, bioadhesiveness, and biodegradability</article-title><source>Polymers</source><year>2019</year><volume>11</volume><fpage>1837</fpage><pub-id pub-id-type=\"doi\">10.3390/polym11111837</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pellá</surname><given-names>MCG</given-names></name><etal/></person-group><article-title>Chitosan-based hydrogels: from preparation to biomedical applications</article-title><source>Carbohydr. Polym.</source><year>2018</year><volume>196</volume><fpage>233</fpage><lpage>245</lpage><pub-id pub-id-type=\"doi\">10.1016/j.carbpol.2018.05.033</pub-id><pub-id pub-id-type=\"pmid\">29891292</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Giri</surname><given-names>TK</given-names></name><name><surname>Thakur</surname><given-names>A</given-names></name><name><surname>Alexander</surname><given-names>A</given-names></name><name><surname>Badwaik</surname><given-names>H</given-names></name><name><surname>Tripathi</surname><given-names>DK</given-names></name></person-group><article-title>Modified chitosan hydrogels as drug delivery and tissue engineering systems: present status and applications</article-title><source>Acta Pharm. Sin B</source><year>2012</year><volume>2</volume><fpage>439</fpage><lpage>449</lpage><pub-id pub-id-type=\"doi\">10.1016/j.apsb.2012.07.004</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lopes</surname><given-names>M</given-names></name><etal/></person-group><article-title>Dual chitosan/albumin-coated alginate/dextran sulfate nanoparticles for enhanced oral delivery of insulin</article-title><source>J. Control. Release</source><year>2016</year><volume>232</volume><fpage>29</fpage><lpage>41</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jconrel.2016.04.012</pub-id><pub-id pub-id-type=\"pmid\">27074369</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Caddeo</surname><given-names>C</given-names></name><etal/></person-group><article-title>Physico-chemical characterization of succinyl chitosan-stabilized liposomes for the oral co-delivery of quercetin and resveratrol</article-title><source>Carbohydr. Polym.</source><year>2017</year><volume>157</volume><fpage>1853</fpage><lpage>1861</lpage><pub-id pub-id-type=\"doi\">10.1016/j.carbpol.2016.11.072</pub-id><pub-id pub-id-type=\"pmid\">27987905</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Munim</surname><given-names>SA</given-names></name><name><surname>Saddique</surname><given-names>MT</given-names></name><name><surname>Raza</surname><given-names>ZA</given-names></name><name><surname>Majeed</surname><given-names>MI</given-names></name></person-group><article-title>Fabrication of cellulose-mediated chitosan adsorbent beads and their surface chemical characterization</article-title><source>Polym. Bull.</source><year>2020</year><volume>77</volume><fpage>183</fpage><lpage>196</lpage><pub-id pub-id-type=\"doi\">10.1007/s00289-019-02711-4</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Duan</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>Y</given-names></name><name><surname>Wang</surname><given-names>Y</given-names></name><name><surname>Xiong</surname><given-names>X</given-names></name></person-group><article-title>Preparation and characterization of cellulose-coated chitosan beads with improved strength and acid resistivity</article-title><source>J. Appl. Polym. Sci.</source><year>2012</year><volume>126</volume><fpage>E173</fpage><lpage>E179</lpage><pub-id pub-id-type=\"doi\">10.1002/app.36914</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kurita</surname><given-names>K</given-names></name></person-group><article-title>Chitin and chitosan: functional biopolymers from marine crustaceans</article-title><source>Mar. Biotechnol.</source><year>2006</year><volume>8</volume><fpage>203</fpage><lpage>226</lpage><pub-id pub-id-type=\"doi\">10.1007/s10126-005-0097-5</pub-id><pub-id pub-id-type=\"pmid\">16532368</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fukaya</surname><given-names>Y</given-names></name><name><surname>Hayashi</surname><given-names>K</given-names></name><name><surname>Wada</surname><given-names>M</given-names></name><name><surname>Ohno</surname><given-names>H</given-names></name></person-group><article-title>Cellulose dissolution with polar ionic liquids under mild conditions: required factors for anions</article-title><source>Green Chem.</source><year>2008</year><volume>10</volume><fpage>44</fpage><lpage>46</lpage><pub-id pub-id-type=\"doi\">10.1039/B713289A</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>M-C</given-names></name><etal/></person-group><article-title>Cellulose nanoparticles: structure–morphology–rheology relationships</article-title><source>ACS Sustain. Chem. Eng.</source><year>2015</year><volume>3</volume><fpage>821</fpage><lpage>832</lpage><pub-id pub-id-type=\"doi\">10.1021/acssuschemeng.5b00144</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>C</given-names></name><etal/></person-group><article-title>Preparation of cellulose/graphene composite and its applications for triazine pesticides adsorption from water</article-title><source>ACS Sustain. Chem. Eng.</source><year>2015</year><volume>3</volume><fpage>396</fpage><lpage>405</lpage><pub-id pub-id-type=\"doi\">10.1021/sc500738k</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Keshk</surname><given-names>SM</given-names></name><name><surname>Bondock</surname><given-names>S</given-names></name><name><surname>El-Zahhar</surname><given-names>AA</given-names></name><name><surname>Haija</surname><given-names>MA</given-names></name></person-group><article-title>Synthesis and characterization of novel Schiff’s bases derived from dialdehyde cellulose-6-phosphate</article-title><source>Cellulose</source><year>2019</year><volume>26</volume><fpage>3703</fpage><lpage>3712</lpage><pub-id pub-id-type=\"doi\">10.1007/s10570-019-02360-w</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vancov</surname><given-names>T</given-names></name><name><surname>Alston</surname><given-names>A-S</given-names></name><name><surname>Brown</surname><given-names>T</given-names></name><name><surname>McIntosh</surname><given-names>S</given-names></name></person-group><article-title>Use of ionic liquids in converting lignocellulosic material to biofuels</article-title><source>Renew. Energy</source><year>2012</year><volume>45</volume><fpage>1</fpage><lpage>6</lpage><pub-id pub-id-type=\"doi\">10.1016/j.renene.2012.02.033</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><mixed-citation publication-type=\"other\">Mohd, N., Draman, S., Salleh, M. & Yusof, N. Dissolution of cellulose in ionic liquid: A review. <italic>AIP Conf. Proc.</italic><bold>1809</bold>, 020035. 10.1063/1.4975450 (2017).</mixed-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Minnick</surname><given-names>DL</given-names></name><name><surname>Flores</surname><given-names>RA</given-names></name><name><surname>DeStefano</surname><given-names>MR</given-names></name><name><surname>Scurto</surname><given-names>AM</given-names></name></person-group><article-title>Cellulose solubility in ionic liquid mixtures: temperature, cosolvent, and antisolvent effects</article-title><source>J. Phys. Chem. B</source><year>2016</year><volume>120</volume><fpage>7906</fpage><lpage>7919</lpage><pub-id pub-id-type=\"doi\">10.1021/acs.jpcb.6b04309</pub-id><pub-id pub-id-type=\"pmid\">27447741</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abe</surname><given-names>M</given-names></name><name><surname>Kuroda</surname><given-names>K</given-names></name><name><surname>Ohno</surname><given-names>H</given-names></name></person-group><article-title>Maintenance-free cellulose solvents based on onium hydroxides</article-title><source>ACS Sustain. Chem. Eng.</source><year>2015</year><volume>3</volume><fpage>1771</fpage><lpage>1776</lpage><pub-id pub-id-type=\"doi\">10.1021/acssuschemeng.5b00303</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Medronho</surname><given-names>B</given-names></name><name><surname>Lindman</surname><given-names>B</given-names></name></person-group><article-title>Brief overview on cellulose dissolution/regeneration interactions and mechanisms</article-title><source>Adv. Colloid Interface Sci.</source><year>2015</year><volume>222</volume><fpage>502</fpage><lpage>508</lpage><pub-id pub-id-type=\"doi\">10.1016/j.cis.2014.05.004</pub-id><pub-id pub-id-type=\"pmid\">24931119</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Swatloski</surname><given-names>RP</given-names></name><name><surname>Spear</surname><given-names>SK</given-names></name><name><surname>Holbrey</surname><given-names>JD</given-names></name><name><surname>Rogers</surname><given-names>RD</given-names></name></person-group><article-title>Dissolution of cellose with ionic liquids</article-title><source>J. Am. Chem. Soc.</source><year>2002</year><volume>124</volume><fpage>4974</fpage><lpage>4975</lpage><pub-id pub-id-type=\"doi\">10.1021/ja025790m</pub-id><pub-id pub-id-type=\"pmid\">11982358</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>H</given-names></name><etal/></person-group><article-title>Regenerated-cellulose/multiwalled-carbon-nanotube composite fibers with enhanced mechanical properties prepared with the ionic liquid 1-allyl-3-methylimidazolium chloride</article-title><source>Adv. Mater.</source><year>2007</year><volume>19</volume><fpage>698</fpage><lpage>704</lpage><pub-id pub-id-type=\"doi\">10.1002/adma.200600442</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mahmoudian</surname><given-names>S</given-names></name><name><surname>Wahit</surname><given-names>MU</given-names></name><name><surname>Ismail</surname><given-names>A</given-names></name><name><surname>Yussuf</surname><given-names>AJCP</given-names></name></person-group><article-title>Preparation of regenerated cellulose/montmorillonite nanocomposite films via ionic liquids</article-title><source>Carbohydr. Polym.</source><year>2012</year><volume>88</volume><fpage>1251</fpage><lpage>1257</lpage><pub-id pub-id-type=\"doi\">10.1016/j.carbpol.2012.01.088</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ma</surname><given-names>H</given-names></name><name><surname>Hsiao</surname><given-names>BS</given-names></name><name><surname>Chu</surname><given-names>B</given-names></name></person-group><article-title>Thin-film nanofibrous composite membranes containing cellulose or chitin barrier layers fabricated by ionic liquids</article-title><source>Polymer</source><year>2011</year><volume>52</volume><fpage>2594</fpage><lpage>2599</lpage><pub-id pub-id-type=\"doi\">10.1016/j.polymer.2011.03.051</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bian</surname><given-names>J</given-names></name><etal/></person-group><article-title>Effect of [Emim] Ac pretreatment on the structure and enzymatic hydrolysis of sugarcane bagasse cellulose</article-title><source>Carbohydr. Polym.</source><year>2014</year><volume>100</volume><fpage>211</fpage><lpage>217</lpage><pub-id pub-id-type=\"doi\">10.1016/j.carbpol.2013.02.059</pub-id><pub-id pub-id-type=\"pmid\">24188856</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carrillo</surname><given-names>F</given-names></name><name><surname>Colom</surname><given-names>X</given-names></name><name><surname>Sunol</surname><given-names>J</given-names></name><name><surname>Saurina</surname><given-names>J</given-names></name></person-group><article-title>Structural FTIR analysis and thermal characterisation of lyocell and viscose-type fibres</article-title><source>Eur. Polym. J.</source><year>2004</year><volume>40</volume><fpage>2229</fpage><lpage>2234</lpage><pub-id pub-id-type=\"doi\">10.1016/j.eurpolymj.2004.05.003</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fei</surname><given-names>P</given-names></name><name><surname>Liao</surname><given-names>L</given-names></name><name><surname>Cheng</surname><given-names>B</given-names></name><name><surname>Song</surname><given-names>J</given-names></name></person-group><article-title>Quantitative analysis of cellulose acetate with a high degree of substitution by FTIR and its application</article-title><source>Anal. Methods</source><year>2017</year><volume>9</volume><fpage>6194</fpage><lpage>6201</lpage><pub-id pub-id-type=\"doi\">10.1039/C7AY02165H</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>H</given-names></name><name><surname>Wu</surname><given-names>J</given-names></name><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>He</surname><given-names>J</given-names></name></person-group><article-title>1-Allyl-3-methylimidazolium chloride room temperature ionic liquid: a new and powerful nonderivatizing solvent for cellulose</article-title><source>Macromolecules</source><year>2005</year><volume>38</volume><fpage>8272</fpage><lpage>8277</lpage><pub-id pub-id-type=\"doi\">10.1021/ma0505676</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xiao</surname><given-names>W</given-names></name><name><surname>Yin</surname><given-names>W</given-names></name><name><surname>Xia</surname><given-names>S</given-names></name><name><surname>Ma</surname><given-names>P</given-names></name></person-group><article-title>The study of factors affecting the enzymatic hydrolysis of cellulose after ionic liquid pretreatment</article-title><source>Carbohydr. Polym.</source><year>2012</year><volume>87</volume><fpage>2019</fpage><lpage>2023</lpage><pub-id pub-id-type=\"doi\">10.1016/j.carbpol.2011.10.012</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Passerini</surname><given-names>N</given-names></name><etal/></person-group><article-title>Controlled release of verapamil hydrochloride from waxy microparticles prepared by spray congealing</article-title><source>J. Control. Release</source><year>2003</year><volume>88</volume><fpage>263</fpage><lpage>275</lpage><pub-id pub-id-type=\"doi\">10.1016/S0168-3659(03)00009-9</pub-id><pub-id pub-id-type=\"pmid\">12628333</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807825</article-id><article-id pub-id-type=\"pmc\">PMC7431573</article-id><article-id pub-id-type=\"publisher-id\">70769</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70769-6</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Plasmid-based complementation of large deletions in <italic>Phaeodactylum tricornutum</italic> biosynthetic genes generated by Cas9 editing</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Slattery</surname><given-names>Samuel S.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Helen</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Giguere</surname><given-names>Daniel J.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kocsis</surname><given-names>Csanad</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Urquhart</surname><given-names>Bradley L.</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Karas</surname><given-names>Bogumil J.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Edgell</surname><given-names>David R.</given-names></name><address><email>dedgell@uwo.ca</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.39381.30</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 8884</institution-id><institution>Department of Biochemistry, Schulich School of Medicine and Dentistry, </institution><institution>Western University, </institution></institution-wrap>London, Canada </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.39381.30</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 8884</institution-id><institution>Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, </institution><institution>Western University, </institution></institution-wrap>London, Canada </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13879</elocation-id><history><date date-type=\"received\"><day>20</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>27</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">The model diatom <italic>Phaeodactylum tricornutum</italic> is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of <italic>P. tricornutum</italic> would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Nanopore long-read sequencing indicates that editing events are characterized by the occurrence of large deletions of up to ~ 2.7 kb centered on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-fluoroorotic acid and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyltransferase activity. Our results expand the range of <italic>P. tricornutum</italic> auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered <italic>P. tricornutum</italic> strains.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Synthetic biology</kwd><kwd>Synthetic biology</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/501100000038</institution-id><institution>Natural Sciences and Engineering Research Council of Canada</institution></institution-wrap></funding-source><award-id>RGPIN-2018-06172</award-id><award-id>RPGIN-2015-04800</award-id><principal-award-recipient><name><surname>Karas</surname><given-names>Bogumil J.</given-names></name><name><surname>Edgell</surname><given-names>David R.</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Photoautotrophic microalgae and cyanobacteria are emerging as alternative platforms for synthetic biology applications<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. One microalgae species of interest is the diploid marine diatom <italic>Phaeodactylum tricornutum</italic>. A variety of plasmid-based genetic tools have been developed for <italic>P. tricornutum</italic> that facilitate basic molecular manipulations and expression of complex synthetic pathways<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>–<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. We, and others, have developed plasmid-based and DNA-free CRISPR (clustered regularly interspaced palindromic repeats) reagents for targeted chromosome editing in <italic>P. tricornutum</italic> and related diatoms using the Cas9 protein (CRISPR-associated protein 9)<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>–<xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. <italic>P. tricornutum</italic> is diploid, meaning that Cas9-edited cells must be carefully screened to determine if knockouts are monoallelic or biallelic and exhibit loss of heterozygosity. These plasmid-based tools and synthetic pathways are currently maintained by available antibiotic-based selections, including zeocin, phleomycin, nourseothricin, and blasticidin-S and their resistance genes, <italic>Sh ble</italic>, <italic>nat</italic>, and <italic>bsr</italic><sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>–<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. Antibiotic-based selections can be prohibitively expensive for maintaining large-scale cultures and are problematic for applications such as the biosynthesis of products intended for human consumption<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>–<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>.</p><p id=\"Par3\">A viable alternative to antibiotics is the use of auxotrophic selective markers which require a strain engineered to have a loss of function mutation in a key enzyme of an essential biosynthetic pathway. Examples of commonly used auxotrophic strains in industrial and academic labs include uracil, histidine, and tryptophan auxotrophs<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>–<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Two approaches have been taken to generate <italic>P. tricornutum</italic> auxotrophs. First, uracil-requiring mutants were generated by random mutagenesis that resulted in the identification of the bi-functional uridine monophosphate synthase (PtUMPS) gene predicted to catalyze the conversion of orotate into uridine monophosphate (UMP)<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Biolistic transformation and chromosomal integration of the PtUMPs gene rescued the uracil-requiring phenotype. Second, Cas9 was used to knockout the PtUMPS gene to create uracil auxotrophs and the PtAPT gene encoding a predicted adenine phosphoribosyl transferase to create adenine auxotrophs<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. However, direct selection of these auxotrophs via transformation with the corresponding complementation marker has not been explored and the generation of additional auxotrophic strains would facilitate development of new plasmid-based complementation markers.</p><p id=\"Par4\">Here, we used a plasmid-based editing strategy to generate knockouts in the uracil, histidine, and tryptophan biosynthesis pathways of <italic>P. tricornutum</italic> and show for the first time that plasmid-based copies of the intact PtUMPS and PtPRA-PH/CH genes can complement the uracil- and histidine-requiring phenotypes, respectively. Individual auxotrophic strains are characterized by loss of heterozygosity at the edited alleles, and Nanopore sequencing of the edited population reveals large, heterogeneous deletions up to <inline-formula id=\"IEq2\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim$$\\end{document}</tex-math><mml:math id=\"M2\"><mml:mo>∼</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq2.gif\"/></alternatives></inline-formula> 2.7 kb. The uracil and histidine auxotrophs and their respective complementation markers are a potential alternative to antibiotic-based selection of plasmids in <italic>P. tricornutum</italic>. Our results also suggest a simple methodology to cure plasmids from uracil auxotrophs to enable strain and genome engineering.</p></sec><sec id=\"Sec2\"><title>Results</title><sec id=\"Sec3\"><title>Identification of Cas9 targets in biosynthetic pathway genes</title><p id=\"Par5\">We examined the KEGG predictions<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> based on the genome sequence of <italic>P. tricornutum</italic> to identify genes in the uracil and histidine biosynthetic pathways for Cas9 editing. We focused on these two pathways as uracil and histidine auxotrophy, and counter-selection strategies are commonly used in other model organisms. This approach identified the previously described bi-functional PtUMPS gene that is predicted to catalyze two steps in the uracil pathway—conversion of orotate to orotidine monophosphate (OMP), and conversion of OMP to uridine monophosphate (UMP) (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>A, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Proteins that are orthologs of characterized enzymes involved in histidine biosynthesis were also identified (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>). The PHATR_3140 gene, hereafter called PtPRA-PH/CH, encodes a predicted bifunctional protein that shares sequence similarity with the bacterial protein HisIE, and its plant counterpart HISN2<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. These proteins possess two functional domains that are homologous to the phosphoribosyl-ATP pyrophosphohydrolase (PRA-PH) and phosphoribosyl-AMP cyclohydrolase (PRA-CH) enzymes, respectively. PRA-PH and PRA-CH, alone or as a bifunctional protein, are predicted to catalyze two successive steps that occur early in the histidine biosynthesis pathway (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>). The PtIGPS gene encoding imidazole glycerol phosphate synthase (a HIS3 homolog) was found to be a duplicated gene in the <italic>P. tricornutum</italic> genome assembly and thus not prioritized as a Cas9 target.</p><p id=\"Par6\">We also identified the PtI3GPS-PRAI gene as a potential target as it encodes a predicted bi-functional enzyme that is a fusion of indole-3-glycerol-phosphate synthase (I3GPS) and phosphoribosylanthranilate isomerase (PRAI), and would catalyze two successive steps in the tryptophan biosynthesis pathway (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>CRISPR-generated knockouts in the predicted <italic>P. tricornutum</italic> uracil biosynthesis pathway. (<bold>A</bold>) A portion of the predicted <italic>P. tricornutum</italic> biosynthesis pathway for conversion of carbonic acid to uracil and uridine triphosphate, with the PtUMPS enzyme highlighted in blue. The competitive inhibitor, 5-fluoroorotic acid (5-FOA), is shown in a dashed box at the position where it enters the pathway. Abbreviated names for molecules and enzymes are indicated in parentheses, and the predicted corresponding <italic>P. tricornutum</italic> gene names are indicated in square brackets. (<bold>B</bold>) Example image of T7EI editing assay to screen exconjugants for potential editing events in the PtUMPS gene. Substrate indicates PtUMPS gene fragments amplified by the PCR, while T7 product indicates exconjugants with evidence of Cas9 editing. WT, wild-type <italic>P. tricornutum</italic> genomic DNA used in the T7EI editing assay. M, 100 bp ladder with sizes indicated in basepairs (bp). This image was cropped from a larger image (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S10</xref>). (<bold>C</bold>) Example of phenotypic screening of one PtUMPS knockout strain (<inline-formula id=\"IEq3\"><alternatives><tex-math id=\"M3\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M4\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq3.gif\"/></alternatives></inline-formula>UMPS2) plated on L1 alone or L1 supplemented with uracil at the indicated dilution of initial concentration. (<bold>D</bold>) Example of screening for loss of the zeocin-resistant Cas9 editing plasmid in a <inline-formula id=\"IEq4\"><alternatives><tex-math id=\"M5\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M6\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq4.gif\"/></alternatives></inline-formula>UMPS2 knockout strain by plating on L1 supplemented with uracil or L1 supplemented with uracil and zeocin. (<bold>E</bold>) Sanger sequencing traces of characterized PtUMPS knockouts with the position (below trace) and type of insertion or deletion (above trace) indicated for each allele of the three strains. (<bold>F</bold>) Graphical map of the position and extent of indels for each of the three PtUMPS knockouts relative to the wild-type UMPS gene (shown at top). Red rectangles indicate nucleotide deletions, green triangles indicate nucleotide insertions, the yellow and blue rectangles on the WT gene indicate the position of the PtUMPS active sites (orotate phosphoribosyl transferase and orotidine-5’-phosphate decarboxylase), and the white rectangles with dashed lines represent introns.</p></caption><graphic xlink:href=\"41598_2020_70769_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par7\">To confirm the genomic target sites, we PCR-amplified and sequenced the PtUMPS and PtPRA-PH/CH genes of the <italic>P. tricornutum</italic> CCAP 1055/1 strain used in our laboratory. Two distinct alleles for both the PtUMPS and PtPRA-PH/CH genes were identified. Seven single-nucleotide polymorphisms (SNPs) in the PtUMPS alleles result in amino acid substitutions that differentiate the two alleles from each other and from the published <italic>P. tricornutum</italic> genome (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>). All substitutions are located in non-conserved regions of the PtUMPS protein (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S4</xref>). Similarly, an A to G mutation at base position 1205 in allele 2 of the PtPRA-PH/CH gene was identified (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>). This transversion converts a highly conserved glutamate to a glycine in the catalytic site of the PRA-PH domain. The impact of these substitutions on PtUMPS and PtPRA-PH/CH function is unknown.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>CRISPR-generated knockouts in the predicted <italic>P. tricornutum</italic> histidine biosynthesis pathway. (<bold>A</bold>) A portion of the predicted biosynthesis pathway for conversion of ribose-5-phosphate to <sc>l</sc>-histidine, with the bi-functional PtPRA-PH/CH enzyme highlighted in blue. Abbreviated names for each enzyme are indicated in parentheses, and the predicted corresponding <italic>P. tricornutum</italic> gene names are indicated in square brackets. (<bold>B</bold>) Example image of T7EI editing assay to screen exconjugants for potential editing events in the PtPRA-PH/CH gene. Substrate indicates PtPRA-PH/CH gene fragments amplified by the PCR, while T7 product indicates exconjugants with evidence of Cas9 editing. WT, wild-type <italic>P. tricornutum</italic> genomic DNA used in the T7EI editing assay. M, 1 kb ladder with sizes indicated in basepairs (bp). This image was cropped from a larger image (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S11</xref>). (<bold>C</bold>) Example of phenotypic screening of one PtPRA-PH/CH knockout strain (<inline-formula id=\"IEq5\"><alternatives><tex-math id=\"M7\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M8\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq5.gif\"/></alternatives></inline-formula>PtPRAPHCH1) transformed with or without the complementing PRA-PH/CH plasmid (pPtPRAPHCH) on L1 solid media alone or L1 supplemented with histidine at the indicated dilution of initial concentration. WT, wild-type <italic>P. tricornutum</italic> strain. (<bold>D</bold>) Sanger sequencing traces of characterized PtPRA-PH/CH knockouts with the position (below trace) and type of insertion or deletion (above trace) indicated for each allele. (<bold>E</bold>) Graphical map of the position and extent of indels for PtPRA-PH/CH knockout relative to the wild-type PtPRA-PH/CH gene (shown at top). Red rectangles indicate nucleotide deletions, while the yellow and blue rectangles on the WT gene indicated the position of the PRA-PH and PRA-CH active sites.</p></caption><graphic xlink:href=\"41598_2020_70769_Fig2_HTML\" id=\"MO2\"/></fig></p></sec><sec id=\"Sec4\"><title>Cas9 and TevCas9 editing of auxotrophic genes is characterized by loss of heterozygosity</title><p id=\"Par8\">To generate knockouts in uracil and histidine biosynthetic genes, we designed and individually cloned Cas9 and TevCas9 single guide RNAs (sgRNAs) against different sites in the PtUMPS, PtPRA-PH/CH, and PtI3GPS-PRAI genes (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S5</xref>–<xref rid=\"MOESM1\" ref-type=\"media\">S7</xref>). The TevCas9 nuclease is a dual nuclease that generates a 33–38 base pair deletion between the I-TevI (Tev) and Cas9 cut sites<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. The targeting requirements for a TevCas9 nuclease are an I-TevI 5<inline-formula id=\"IEq6\"><alternatives><tex-math id=\"M9\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$^\\prime$$\\end{document}</tex-math><mml:math id=\"M10\"><mml:msup><mml:mrow/><mml:mo>′</mml:mo></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq6.gif\"/></alternatives></inline-formula>-CNNNG-3<inline-formula id=\"IEq7\"><alternatives><tex-math id=\"M11\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$^\\prime$$\\end{document}</tex-math><mml:math id=\"M12\"><mml:msup><mml:mrow/><mml:mo>′</mml:mo></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq7.gif\"/></alternatives></inline-formula> cleavage motif positioned <inline-formula id=\"IEq8\"><alternatives><tex-math id=\"M13\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim$$\\end{document}</tex-math><mml:math id=\"M14\"><mml:mo>∼</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq8.gif\"/></alternatives></inline-formula> 15–18 base pairs upstream of the 5<inline-formula id=\"IEq9\"><alternatives><tex-math id=\"M15\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$^\\prime$$\\end{document}</tex-math><mml:math id=\"M16\"><mml:msup><mml:mrow/><mml:mo>′</mml:mo></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq9.gif\"/></alternatives></inline-formula> end of the sgRNA binding site. The Cas9 or TevCas9 editing plasmids were moved into <italic>P. tricornutum</italic> by bacterial conjugation and exconjugants selected on zeocin-containing media.<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Summary of sgRNAs used for Cas9 and TevCas9 editing.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Target</th><th align=\"left\">Platform</th><th align=\"left\">Guide RNA</th><th align=\"left\">Exconjugants edited / total screened (T7E1)</th><th align=\"left\">Number of subclones with auxotroph phenotype / screened</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"7\">UMPS</td><td align=\"left\" rowspan=\"4\">Cas9</td><td align=\"left\">sgRNA.UMPS.1944</td><td align=\"left\">0/10</td><td align=\"left\">N/A</td></tr><tr><td align=\"left\">sgRNA.UMPS.1646</td><td align=\"left\">0/10</td><td align=\"left\">N/A</td></tr><tr><td align=\"left\">sgRNA.UMPS.157</td><td align=\"left\">0/10</td><td align=\"left\">N/A</td></tr><tr><td align=\"left\">sgRNA.UMPS.311</td><td align=\"left\">4/10</td><td align=\"left\">1/35</td></tr><tr><td align=\"left\" rowspan=\"3\">TevCas9</td><td align=\"left\">sgRNA.UMPS.1944</td><td align=\"left\">0/10</td><td align=\"left\">N/A</td></tr><tr><td align=\"left\">sgRNA.UMPS.1646</td><td align=\"left\">4/10</td><td align=\"left\">2/35</td></tr><tr><td align=\"left\">sgRNA.UMPS.157</td><td align=\"left\">0/10</td><td align=\"left\">N/A</td></tr><tr><td align=\"left\" rowspan=\"5\">PRA-PH/CH</td><td align=\"left\" rowspan=\"3\">Cas9</td><td align=\"left\">sgRNA.PRAPHCH.929</td><td align=\"left\">2/6</td><td align=\"left\">1/28</td></tr><tr><td align=\"left\">sgRNA.PRAPHCH.120</td><td align=\"left\">0/6</td><td align=\"left\">N/A</td></tr><tr><td align=\"left\">sgRNA.PRAPHCH.1000</td><td align=\"left\">1/6</td><td align=\"left\">0/28</td></tr><tr><td align=\"left\" rowspan=\"2\">TevCas9</td><td align=\"left\">sgRNA.PRAPHCH.929</td><td align=\"left\">3/6</td><td align=\"left\">0/28</td></tr><tr><td align=\"left\">sgRNA.PRAPHCH.120</td><td align=\"left\">0/6</td><td align=\"left\">N/A</td></tr><tr><td align=\"left\" rowspan=\"2\">I3GPS-PRAI</td><td align=\"left\">Cas9</td><td align=\"left\">sgRNA.IGPSPRAI.244</td><td align=\"left\">0/10</td><td align=\"left\">N/A</td></tr><tr><td align=\"left\">TevCas9</td><td align=\"left\">sgRNA.IGPSPRAI.244</td><td align=\"left\">3/10</td><td align=\"left\">1/35</td></tr></tbody></table><table-wrap-foot><p>sgRNAs are named by the first nucleotide of the sgRNA binding site in the coding region of the target gene.</p></table-wrap-foot></table-wrap></p><p id=\"Par9\">We first assessed editing by screening <italic>P. tricornutum</italic> exconjugants by T7 endonuclease I (T7EI) mismatch cleavage assays on PCR products amplified from each target gene (Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B, <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>B, Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>). This assay identified 6 sgRNAs with detectable editing rates based on screening of exconjugants. Colonies that showed editing were diluted, plated to obtain subclones, and subsequently screened for the corresponding auxotrophic phenotype on solid media with and without auxotrophic supplement (uracil or histidine) (Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>C, <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>C). To cure the Cas9-editing plasmids, knockout strains were grown without zeocin selection for 1 week, and dilutions were plated to obtain single colonies. Colonies were streaked onto L1 plates with and without zeocin to screen for plasmid loss. A representative image demonstrating zeocin sensitivity due to loss of the Cas9-editing plasmid is shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>D. For knockout of the PtUMPS gene, we further characterized 3 subclones with a uracil-requiring phenotype to determine if the knockouts were monoallelic or biallelic. Because the two PtUMPS alleles of <italic>P. tricornutum</italic> possessed SNPs relative to each other, we were able to map allele-specific editing events (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>E,F). Two of the strains, <inline-formula id=\"IEq10\"><alternatives><tex-math id=\"M17\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M18\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq10.gif\"/></alternatives></inline-formula>UMPS1 and <inline-formula id=\"IEq11\"><alternatives><tex-math id=\"M19\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M20\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq11.gif\"/></alternatives></inline-formula>UMPS2, were biallelic and exhibited loss of heterozygosity with one allele possessing a small deletion (< 20 bps) and the other allele possessing a large deletion (> 610 bp). The third characterized subclone, <inline-formula id=\"IEq12\"><alternatives><tex-math id=\"M21\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M22\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq12.gif\"/></alternatives></inline-formula>UMPS3, was monoallelic and possessed a homozygous 1-bp insertion. For the PtPRA-PH/CH knockouts that generated a histidine-requiring phenotype (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>B,C), targeted sequencing of one subclone revealed a biallelic genotype with an 11-bp deletion in one allele and a 6-bp deletion in the second allele (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>D,E).<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Large deletions in edited <italic>P. tricornutum</italic> metabolic genes captured by Nanopore amplicon sequencing. For each (<bold>A</bold>–<bold>E</bold>), the name of the target gene as well as the editing enzyme are indicated. The <italic>leftmost</italic> plot shows normalized read coverage averaged over a 5-bp window for the edited sample (black dots) and the wild-type sample (orange dots) relative to the position in PCR amplicon. Numbering on the x-axis is relative to the ATG start codon for each gene, with sequence upstream indicated by a minus (−) symbol and sequence downstream indicated by a plus (+) symbol. The green vertical line indicates the Cas9 or TevCas9 cleavage site, while the shaded rectangle indicates the ORF. The <italic>middle</italic> plot is a density plot of deletions > 50-bp. The <italic>rightmost</italic> plot shows the length and position of deletions > 50-bp relative to their position in the PCR amplicon, with numbering of the x-axis as in the <italic>leftmost</italic> panel. Each horizontal line indicates a mapped deletion event. Deletions are ordered from longest to smallest. The green line indicates the Cas9 or TevCas9 cleavage site.</p></caption><graphic xlink:href=\"41598_2020_70769_Fig3_HTML\" id=\"MO3\"/></fig></p><p id=\"Par10\">The types of deletions observed in the uracil- and histidine-auxotrophs are consistent with heterogeneous editing events resulting in loss of heterozygosity<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>–<xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. To extend these observations, we used Nanopore sequencing to better assess the spectrum of large deletions that are often overlooked in Cas9-editing studies. In addition to the two sgRNAs that showed robust editing on the PtUMPS gene, we examined deletion events in exconjugants with sgRNAs targeted to the PtUREASE gene<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup> and the PtI3GPS-PRAI gene. For each experiment, <inline-formula id=\"IEq13\"><alternatives><tex-math id=\"M23\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim$$\\end{document}</tex-math><mml:math id=\"M24\"><mml:mo>∼</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq13.gif\"/></alternatives></inline-formula> 1,000 exconjugants were pooled and a <inline-formula id=\"IEq14\"><alternatives><tex-math id=\"M25\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim$$\\end{document}</tex-math><mml:math id=\"M26\"><mml:mo>∼</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq14.gif\"/></alternatives></inline-formula> 6 kb PCR product generated for each of the target genes with the predicted Cas9 or TevCas9 target sites in the middle of the amplicon. We focused our attention on deletions > 50 bp as these deletions are typically under-reported in targeted amplicon sequencing. We noted a drop in Nanopore read coverage centered around the predicted sgRNA target sites for products amplified from Cas9 and TevCas9 editing experiments (black dots) as compared to read coverage for control experiments (orange dots), consistent with editing at those sites (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>, <italic>left</italic> panels). Mapping the deletion start and end points revealed that most deletions were centered on the Cas9 or TevCas9 target site (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>, <italic>right</italic> panels), with deletions extending up to 2700 bp (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>, <italic>centre</italic> panel). The mean deletion length for editing events examined by Nanopore sequencing and > 50 bp was 1735±719 bp for Cas9 and 2006±633 bp for TevCas9.</p><p id=\"Par11\">Collectively, this data shows that Cas9 or TevCas9 editing of biosynthetic genes can readily generate <italic>P. tricornutum</italic> auxotrophs that can be identified by phenotypic or genetic screens. Moreover, our data agree with a growing body of evidence revealing that Cas9 editing (and TevCas9 editing here) generates large deletions that would typically be missed unless screening strategies are explicitly designed to look for loss of heterozygosity.<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Phenotypic and metabolomic characterization of PtUMPS knockouts. (<bold>A</bold>) Spot plating assays of wild type (WT), <inline-formula id=\"IEq15\"><alternatives><tex-math id=\"M27\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M28\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq15.gif\"/></alternatives></inline-formula>UMPS1 and <inline-formula id=\"IEq16\"><alternatives><tex-math id=\"M29\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M30\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq16.gif\"/></alternatives></inline-formula>UMPS2 strains on L1 solid media alone, L1 supplemented with uracil, L1 supplemented with 5-FOA, or L1 supplemented with both uracil and 5-FOA. Indicated dilutions are relative to the initial concentration. (<bold>B</bold>) Liquid growth curves of wild type (WT), <inline-formula id=\"IEq17\"><alternatives><tex-math id=\"M31\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M32\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq17.gif\"/></alternatives></inline-formula>UMPS1 and <inline-formula id=\"IEq18\"><alternatives><tex-math id=\"M33\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M34\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq18.gif\"/></alternatives></inline-formula>UMPS2 strains in L1 liquid media alone, or supplemented with uracil or 5-FOA or both. Data points are the mean of three independent replicates, with error bars representing the standard error of the mean. (<bold>C</bold>) Orotate concentrations were measured by LC-MS from cultures grown with and without uracil supplementation. Bars represent mean values and error bars represent standard deviation for three biological replicates. Individual data points are represented as colored dots. Statistical confidence level was calculated by one-sided t test. p < 0.001 is indicated by an asterisk. (<bold>D</bold>) Bar graph showing percent plasmid retention in the <inline-formula id=\"IEq19\"><alternatives><tex-math id=\"M35\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M36\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq19.gif\"/></alternatives></inline-formula>UMPS1 and <inline-formula id=\"IEq20\"><alternatives><tex-math id=\"M37\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M38\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq20.gif\"/></alternatives></inline-formula>UMPS2 strains harbouring various PtUMPS constructs after 14 days of outgrowth. Bars represent the mean ratio of colonies on selective L1 + nourseothricin versus non-selective L1 plates from three independent replicates, with error bars representing the standard error of the mean.</p></caption><graphic xlink:href=\"41598_2020_70769_Fig4_HTML\" id=\"MO4\"/></fig></p></sec><sec id=\"Sec5\"><title>Phenotypic and metabolomic characterization of the PtUMPS knockouts</title><p id=\"Par12\">Two uracil-requiring auxotrophs (<inline-formula id=\"IEq21\"><alternatives><tex-math id=\"M39\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M40\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq21.gif\"/></alternatives></inline-formula>UMPS1 and <inline-formula id=\"IEq22\"><alternatives><tex-math id=\"M41\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M42\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq22.gif\"/></alternatives></inline-formula>UMPS2) were selected for further characterization by first spot plating onto L1 media with and without uracil and 5-FOA (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>A). The PtUMPS knockout strains were only able to survive in the presence of uracil supplementation. Additionally, the knockouts survived on 5-FOA concentrations that fully inhibited the growth of wild-type <italic>P. tricornutum</italic> (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>A). This is consistent with phenotypes previously observed for <italic>P. tricornutum</italic> UMPS knockouts<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. There was a slight growth advantage of <inline-formula id=\"IEq23\"><alternatives><tex-math id=\"M43\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M44\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq23.gif\"/></alternatives></inline-formula>UMPS1 over <inline-formula id=\"IEq24\"><alternatives><tex-math id=\"M45\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M46\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq24.gif\"/></alternatives></inline-formula>UMPS2 on media supplemented with both 5-FOA and uracil, but not on media containing uracil alone. To compare if the observed phenotypes were consistent across solid and liquid media, we monitored the growth of these strains over 10 days in liquid media (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>B) and found that the growth rates were consistent with those observed on solid media, with one notable difference (Supplementary Figs. <xref rid=\"MOESM1\" ref-type=\"media\">S8</xref>, <xref rid=\"MOESM1\" ref-type=\"media\">S9</xref>, Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>). The growth advantage of <inline-formula id=\"IEq25\"><alternatives><tex-math id=\"M47\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M48\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq25.gif\"/></alternatives></inline-formula>UMPS1 over <inline-formula id=\"IEq26\"><alternatives><tex-math id=\"M49\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M50\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq26.gif\"/></alternatives></inline-formula>UMPS2 observed on solid media supplemented with both 5-FOA and uracil was not replicated in liquid media as the generation times for <inline-formula id=\"IEq27\"><alternatives><tex-math id=\"M51\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M52\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq27.gif\"/></alternatives></inline-formula>UMPS1 and <inline-formula id=\"IEq28\"><alternatives><tex-math id=\"M53\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M54\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq28.gif\"/></alternatives></inline-formula>UMPS2 were very similar (<inline-formula id=\"IEq29\"><alternatives><tex-math id=\"M55\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim$$\\end{document}</tex-math><mml:math id=\"M56\"><mml:mo>∼</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq29.gif\"/></alternatives></inline-formula> 24 and <inline-formula id=\"IEq30\"><alternatives><tex-math id=\"M57\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim$$\\end{document}</tex-math><mml:math id=\"M58\"><mml:mo>∼</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq30.gif\"/></alternatives></inline-formula> 22 h, respectively).</p><p id=\"Par13\">To investigate the impact of PtUMPS knockouts on uracil metabolism, we performed targeted metabolomics on the UMPS substrate orotate using LC–MS in wild-type and knockouts strains (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>C). We focused on characterizing the orotate intermediate in the uracil pathway (Figs. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A, <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>C) predicting that there should be an increase of orotate in knockout strains relative to wild type. We were unable to detect orotate in the <inline-formula id=\"IEq31\"><alternatives><tex-math id=\"M59\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M60\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq31.gif\"/></alternatives></inline-formula>UMPS1 strain in the absence of uracil supplementation (-uracil), or in the <inline-formula id=\"IEq32\"><alternatives><tex-math id=\"M61\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M62\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq32.gif\"/></alternatives></inline-formula>UMPS2 strain in either the -uracil or +uracil condition. A <inline-formula id=\"IEq33\"><alternatives><tex-math id=\"M63\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim$$\\end{document}</tex-math><mml:math id=\"M64\"><mml:mo>∼</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq33.gif\"/></alternatives></inline-formula> sixfold increase of cellular orotate levels was observed in the wild-type strain when L1 media was supplemented with uracil (+uracil) as compared to minimal L1 media (-uracil) (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>C). Interestingly, when the <inline-formula id=\"IEq34\"><alternatives><tex-math id=\"M65\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M66\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq34.gif\"/></alternatives></inline-formula>UMPS1 strain was grown with uracil supplementation we detected orotate at levels similar to those observed in the wild-type strain grown with uracil. This result suggests that allele 1 in the <inline-formula id=\"IEq35\"><alternatives><tex-math id=\"M67\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M68\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq35.gif\"/></alternatives></inline-formula>UMPS1 knockout strain (with an 18-bp in-frame deletion) retains UMPS activity that behaves similarly to the wild-type strain. In contrast, the <inline-formula id=\"IEq36\"><alternatives><tex-math id=\"M69\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M70\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq36.gif\"/></alternatives></inline-formula>UMPS2 strain has two out-of-frame deletions that likely abolish ODC and OPRT activity. We speculate that undetectable levels of orotate in the <inline-formula id=\"IEq37\"><alternatives><tex-math id=\"M71\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M72\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq37.gif\"/></alternatives></inline-formula>UMPS2 strain may be because it is diverted to another biosynthetic pathway.</p></sec><sec id=\"Sec6\"><title>Plasmid complementation of the uracil and histidine auxotrophs</title><p id=\"Par14\">Plasmid-based complementation of <italic>P. tricornutum</italic> auxotrophs would validate that the Cas9-editing event was the cause of the auxotrophic phenotype, as well as providing alternatives to antibiotic-based selection methods to maintain episomal vectors. We first examined complementation of the uracil-requiring phenotype by cloning both gDNA and cDNA versions of each PtUMPS allele with the native promoter and terminator into the nourseothricin-resistant pPtGE31 expression plasmid<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. These plasmids were designated pPtUMPSA1, pPtUMPSA2, pPtUMPScA1, and pPtUMPScA2 (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>) and moved into the <inline-formula id=\"IEq38\"><alternatives><tex-math id=\"M73\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M74\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq38.gif\"/></alternatives></inline-formula>UMPS1 and <inline-formula id=\"IEq39\"><alternatives><tex-math id=\"M75\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M76\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq39.gif\"/></alternatives></inline-formula>UMPS2 strains via conjugation. Exconjugants were spot-plated onto solid L1 media with and without uracil and 5-FOA supplementation (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>A). All complemented strains grew on minimal L1 media, while the uncomplemented knockouts did not, confirming expression of the UMPS gene from the pPtGE31 plasmid. No strain grew on 5-FOA alone. Unexpectedly, some of the complemented strains survived on plates supplemented with both 5-FOA and uracil. For example, when <inline-formula id=\"IEq40\"><alternatives><tex-math id=\"M77\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M78\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq40.gif\"/></alternatives></inline-formula>UMPS2 was transformed with either of the allele 1 complementation plasmids (pPtUMPSA1 and pPtUMPScA1), clear resistance to 5-FOA in the presence of uracil was observed. The phenotypes observed on solid media were consistent with those observed when the strains were grown in liquid media with similar media supplementation (Supplementary Figs. <xref rid=\"MOESM1\" ref-type=\"media\">S8</xref>, <xref rid=\"MOESM1\" ref-type=\"media\">S9</xref>).</p><p id=\"Par15\">The growth phenotype of the <inline-formula id=\"IEq41\"><alternatives><tex-math id=\"M79\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M80\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq41.gif\"/></alternatives></inline-formula>UMPS1 and <inline-formula id=\"IEq42\"><alternatives><tex-math id=\"M81\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M82\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq42.gif\"/></alternatives></inline-formula>UMPS2 strains in media supplemented with uracil and 5-FOA could be explained by counter-selection against the plasmid carrying an intact PtUMPS gene that would metabolize 5-FOA to a toxic intermediate. We thus tested for plasmid loss in the complemented strains by plating the <inline-formula id=\"IEq43\"><alternatives><tex-math id=\"M83\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M84\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq43.gif\"/></alternatives></inline-formula>UMPS1 and <inline-formula id=\"IEq44\"><alternatives><tex-math id=\"M85\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M86\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq44.gif\"/></alternatives></inline-formula>UMPS2 strains carrying different expression plasmids on solid L1 with and without nourseothricin after 14 days of growth. As shown in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>D, plasmid retention, as measured by the ratio of colonies on L1 plus nourseothricin versus L1 plates, was severely reduced in all strains, ranging from <inline-formula id=\"IEq45\"><alternatives><tex-math id=\"M87\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim$$\\end{document}</tex-math><mml:math id=\"M88\"><mml:mo>∼</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq45.gif\"/></alternatives></inline-formula> 1 to <inline-formula id=\"IEq46\"><alternatives><tex-math id=\"M89\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim$$\\end{document}</tex-math><mml:math id=\"M90\"><mml:mo>∼</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq46.gif\"/></alternatives></inline-formula> 33%. This observation could explain why colonies readily appeared on L1 media supplemented with 5-FOA and uracil and suggest that curing of plasmids carrying the PtUMPS gene from PtUMPS knockout strains is a simple matter of growth on the appropriate media.</p><p id=\"Par16\">Similarly, we were able to complement the histidine-requiring phenotype by cloning a wild-type copy of the PtPRA-PH/CH gene into an expression vector, and transforming the plasmid into the <inline-formula id=\"IEq47\"><alternatives><tex-math id=\"M91\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M92\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq47.gif\"/></alternatives></inline-formula>PRAPHCH1 strain by conjugation. The <inline-formula id=\"IEq48\"><alternatives><tex-math id=\"M93\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M94\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq48.gif\"/></alternatives></inline-formula>PRAPHCH1 strain with the complementing plasmid grew on both solid L1 media with and without histidine supplementation, whereas the <inline-formula id=\"IEq49\"><alternatives><tex-math id=\"M95\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M96\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq49.gif\"/></alternatives></inline-formula>PRAPHCH1 strain without the complementing plasmid only grew on L1 media with histidine supplementation (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>C).</p></sec></sec><sec id=\"Sec7\"><title>Discussion</title><p id=\"Par17\">The available tools for genetic manipulation of <italic>P. tricornutum</italic> and other diatoms have grown substantially in recent years, including the adaptation of TALEN and Cas9 genome-editing nucleases for targeted knockouts as well as plasmid-based and DNA-free methods to deliver the nucleases to cells<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>–<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. Applications of genome-editing nucleases in <italic>P. tricornutum</italic> at this point have mostly been to generate gene knockouts, with a few examples of reporter construct knockins. Generation of gene knockouts by Cas9 or other editing enzymes relies on non-homologous end-joining repair pathways<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, homologs of which are predicted to occur in the <italic>P. tricornutum</italic> genome<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. A recent study used antisense RNA to knockdown a predicted DNA ligase IV homolog (<italic>ligIV</italic>) in <italic>P. tricornutum</italic> resulting in an increased rate of homologous recombination of a reporter construct<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. What is not yet known for <italic>P. tricornutum</italic> is the balance between NHEJ and homology directed repair (HDR) pathways that process endonuclease-introduced double-strand breaks (see for example<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>). Examination of Cas9 or TALEN-edited sites in <italic>P. tricornutum</italic> revealed small nucleotide insertions or deletions localized near the editing site that are consistent with NHEJ repair events. It is becoming increasingly apparent that repair of Cas9-edited sites result in heterogenous alleles often characterized by both small and large deletions (for example, Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>D)<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. Repair events leading to large indels are often missed by experimental strategies that examine repair outcomes localized around the editing site. In contrast, large deletions visible by long-read sequencing methodologies and our Nanopore data indicate that Cas9 and TevCas9 editing events result in deletions up to <inline-formula id=\"IEq50\"><alternatives><tex-math id=\"M97\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim$$\\end{document}</tex-math><mml:math id=\"M98\"><mml:mo>∼</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq50.gif\"/></alternatives></inline-formula> 2.7 kb in length. Cas9 editing with a single sgRNA in <italic>P. tricornutum</italic> could achieve the same goal as the paired Cas9 nickase strategy to specifically introduce large deletions<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>, and may be complementary with recently developed methods to multiplex sgRNAs on Cas9-editing plasmids for <italic>P. tricornutum</italic><sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Regardless, a better understanding of DNA repair pathways that operate on Cas9-introduced double-strand breaks will better inform strategies to bias repair events depending on the experimental goal.</p><p id=\"Par18\">The creation of auxotrophic strains of <italic>P. tricornutum</italic> with plasmid based rather than chromosomally integrated complementation markers is critical for a number of reasons. Auxotrophic strains expand the available selection schemes beyond traditional antibiotic markers and provide a facile method for strain cataloging and validation. Antibiotic-free selection is also an advantage when <italic>P. tricornutum</italic> is used for production of human therapeutics. In the case of uracil auxotrophs, complementing plasmids can be cured (or counter selected) by simple inclusion of 5-FOA and uracil in the growth media. We have previously shown that plasmids are lost from <italic>P. tricornutum</italic> by passaging cultures over multiple days in the absence of antibiotic selection required for maintenance of the plasmid<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. However, the counter selection method by 5-FOA and uracil supplementation is more rapid and requires screening significantly fewer colonies to confirm plasmid loss. The ability to rapidly cure plasmids will be of tremendous value to prevent prolonged expression of Cas9 and possible toxicity issues during strain engineering, to cure incompatible plasmids, or to cure reporter or expression plasmids under distinct growth conditions. We also envision that rapid curing of plasmids would allow recycling of a limited number of selection markers for serial transformations needed for strain construction or genomic engineering.</p></sec><sec id=\"Sec8\"><title>Methods</title><sec id=\"Sec9\"><title>Microbial strains and growth conditions</title><p id=\"Par19\"><italic>Saccharomyces cerevisiae</italic> VL6-48 (ATCC MYA-3666: <italic>MAT</italic><inline-formula id=\"IEq51\"><alternatives><tex-math id=\"M99\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\alpha$$\\end{document}</tex-math><mml:math id=\"M100\"><mml:mi>α</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq51.gif\"/></alternatives></inline-formula><italic>his3</italic>-<inline-formula id=\"IEq52\"><alternatives><tex-math id=\"M101\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\delta$$\\end{document}</tex-math><mml:math id=\"M102\"><mml:mi>δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq52.gif\"/></alternatives></inline-formula>200 <italic>trp1</italic>-<inline-formula id=\"IEq53\"><alternatives><tex-math id=\"M103\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\delta$$\\end{document}</tex-math><mml:math id=\"M104\"><mml:mi>δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq53.gif\"/></alternatives></inline-formula>1 <italic>ura3</italic>-52 <italic>lys2</italic>\n<italic>ade2</italic>-1 <italic>met</italic>14 <inline-formula id=\"IEq54\"><alternatives><tex-math id=\"M105\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$^{\\circ }$$\\end{document}</tex-math><mml:math id=\"M106\"><mml:msup><mml:mrow/><mml:mo>∘</mml:mo></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq54.gif\"/></alternatives></inline-formula>) was grown in rich medium (YPD) or complete minimal medium lacking histidine (Teknova) supplemented with 60 mg <inline-formula id=\"IEq55\"><alternatives><tex-math id=\"M107\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M108\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq55.gif\"/></alternatives></inline-formula> adenine sulfate. Complete minimal media used for spheroplast transformation contained 1 M sorbitol. <italic>Escherichia coli</italic> (Epi300, Epicenter) was grown in Luria Broth (LB) supplemented with appropriate antibiotics (chloramphenicol 25 mg <inline-formula id=\"IEq56\"><alternatives><tex-math id=\"M109\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M110\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq56.gif\"/></alternatives></inline-formula> or kanamycin 50 mg <inline-formula id=\"IEq57\"><alternatives><tex-math id=\"M111\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M112\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq57.gif\"/></alternatives></inline-formula> or ampicillin 50 mg <inline-formula id=\"IEq58\"><alternatives><tex-math id=\"M113\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M114\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq58.gif\"/></alternatives></inline-formula> or gentamicin 20 mg <inline-formula id=\"IEq59\"><alternatives><tex-math id=\"M115\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M116\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq59.gif\"/></alternatives></inline-formula>). <italic>Phaeodactylum tricornutum</italic> (Culture Collection of Algae and Protozoa CCAP 1055/1) was grown in L1 medium without silica, with or without uracil (50 mg <inline-formula id=\"IEq60\"><alternatives><tex-math id=\"M117\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M118\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq60.gif\"/></alternatives></inline-formula>) or histidine (200 mg <inline-formula id=\"IEq61\"><alternatives><tex-math id=\"M119\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M120\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq61.gif\"/></alternatives></inline-formula>) or 5-FOA (100 mg <inline-formula id=\"IEq62\"><alternatives><tex-math id=\"M121\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M122\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq62.gif\"/></alternatives></inline-formula>), supplemented with appropriate antibiotics zeocin (50 mg <inline-formula id=\"IEq63\"><alternatives><tex-math id=\"M123\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M124\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq63.gif\"/></alternatives></inline-formula>) or nourseothricin (100 mg <inline-formula id=\"IEq64\"><alternatives><tex-math id=\"M125\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M126\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq64.gif\"/></alternatives></inline-formula>), at 18 °C under cool white fluorescent lights (75 μE <inline-formula id=\"IEq67\"><alternatives><tex-math id=\"M127\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {m}^{-2}$$\\end{document}</tex-math><mml:math id=\"M128\"><mml:msup><mml:mtext>m</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq67.gif\"/></alternatives></inline-formula><inline-formula id=\"IEq68\"><alternatives><tex-math id=\"M129\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {s}^{-1}$$\\end{document}</tex-math><mml:math id=\"M130\"><mml:msup><mml:mtext>s</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq68.gif\"/></alternatives></inline-formula>) and a photoperiod of 16 h light:8 h dark. L1 media supplemented with nourseothricin contained half the normal amount of aquil salts. <italic>P. tricornutum</italic> auxotroph genotypes are as follows. Mutations in PtUMPS are described in reference to the chromosome 6 sequence (GenBank: CM000609.1), and mutations for PtPRA-PH/CH are in reference to the chromosome 3 sequence (GenBank: CP001142.1). Mutations described for each gene are listed for allele 1 followed by allele 2, and numbered beginning from the first nucleotide of the start codon for simplicity. Genotypes of auxotroph strains generated in this study are listed in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S4</xref>.</p></sec><sec id=\"Sec10\"><title>Transfer of DNA to <italic>P. tricornutum</italic> via conjugation from <italic>E. coli</italic></title><p id=\"Par20\">Conjugations were performed as previously described<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. Briefly, liquid cultures (250 μL) of <italic>P. tricornutum</italic> were adjusted to a density of 1.0 × <inline-formula id=\"IEq71\"><alternatives><tex-math id=\"M131\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$10^{8}$$\\end{document}</tex-math><mml:math id=\"M132\"><mml:msup><mml:mn>10</mml:mn><mml:mn>8</mml:mn></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq71.gif\"/></alternatives></inline-formula> cells <inline-formula id=\"IEq72\"><alternatives><tex-math id=\"M133\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {mL}^{-1}$$\\end{document}</tex-math><mml:math id=\"M134\"><mml:msup><mml:mtext>mL</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq72.gif\"/></alternatives></inline-formula> using counts from a hemocytometer, plated on 1/2 x L1 1% agar plates and grown for four days. L1 media (1.5 mL) was added to the plate and cells were scraped and the concentration was adjusted to 5.0 <inline-formula id=\"IEq73\"><alternatives><tex-math id=\"M135\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\times$$\\end{document}</tex-math><mml:math id=\"M136\"><mml:mo>×</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq73.gif\"/></alternatives></inline-formula><inline-formula id=\"IEq74\"><alternatives><tex-math id=\"M137\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$10^{8}$$\\end{document}</tex-math><mml:math id=\"M138\"><mml:msup><mml:mn>10</mml:mn><mml:mn>8</mml:mn></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq74.gif\"/></alternatives></inline-formula> cells <inline-formula id=\"IEq75\"><alternatives><tex-math id=\"M139\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {mL}^{-1}$$\\end{document}</tex-math><mml:math id=\"M140\"><mml:msup><mml:mtext>mL</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq75.gif\"/></alternatives></inline-formula>. <italic>E. coli</italic> cultures (50 mL) were grown at 37 °C to A600 of 0.8–1.0, centrifuged for 10 min at 3,000<inline-formula id=\"IEq77\"><alternatives><tex-math id=\"M141\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\times$$\\end{document}</tex-math><mml:math id=\"M142\"><mml:mo>×</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq77.gif\"/></alternatives></inline-formula><italic>g</italic> and resuspended in 500 μL of SOC media. Conjugation was initiated by mixing 200 μL of <italic>P. tricornutum</italic> and 200 μL of <italic>E. coli</italic> cells. The cell mixture was plated on 1/2 x L1 5% LB 1% agar plates, incubated for 90 min at 30 °C in the dark, and then moved to 18 °C in the light and grown for 2 days. After 2 days, L1 media (1.5 mL) was added to the plates, the cells scraped, and 300 μL (20%) plated on 1/2 <inline-formula id=\"IEq84\"><alternatives><tex-math id=\"M143\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\times$$\\end{document}</tex-math><mml:math id=\"M144\"><mml:mo>×</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq84.gif\"/></alternatives></inline-formula> L1 1% agar plates supplemented with zeocin 50 mg <inline-formula id=\"IEq85\"><alternatives><tex-math id=\"M145\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M146\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq85.gif\"/></alternatives></inline-formula> or nourseothricin 200 mg <inline-formula id=\"IEq86\"><alternatives><tex-math id=\"M147\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M148\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq86.gif\"/></alternatives></inline-formula>. Colonies appeared after 7–14 days incubation at 18 °C with light.</p></sec><sec id=\"Sec11\"><title>Plasmid design and construction</title><p id=\"Par21\">All plasmids (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S5</xref>) were constructed using a modified yeast assembly protocol<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref>,<xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. Plasmids pPtUMPSA1 and pPtUMPSA2 were made from pPtGE31<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup> by replacing the URA3 element with a PCR fragment consisting of PtUMPS allele 1 or 2 with ~ 1 kb up- and down-stream of the PtUMPS ORF amplified from <italic>P. tricornutum</italic> genomic DNA (oligonucleotides are listed in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S6</xref>). Plasmids pPtUMPScA1 and pPtUMPScA2 were made from pPtUMPSA1 and pPtUMPSA2 by replacing the PtUMPS ORF with a PCR fragment consisting of PtUMPS allele 1 or 2 amplified from <italic>P. tricornutum</italic> cDNA. Plasmid pPtUMPS40S was made from pPtGE31 by replacing the URA3 element with a cassette consisting of PCR fragments of the 40SRPS8 promoter and terminator<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup> flanking a PCR fragment of the PtUMPS allele 1 ORF amplified from <italic>P. tricornutum</italic> genomic DNA. Plasmid pPtPRAPHCH was made from pPtGE31<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup> by replacing the URA3 element with a PCR fragment consisting of PtPRA-PH/CH with <inline-formula id=\"IEq89\"><alternatives><tex-math id=\"M149\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim$$\\end{document}</tex-math><mml:math id=\"M150\"><mml:mo>∼</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq89.gif\"/></alternatives></inline-formula> 1 kb up- and downstream of the PtPRA-PH/CH ORF, amplified from <italic>P. tricornutum</italic> genomic DNA. Using Golden Gate assembly, sgRNAs targeting different regions of the PtUMPS and PtPRA-PH/CH genes were cloned into the BsaI sites positioned between the <italic>P. tricornutum</italic> U6 promoter and terminator in pPtGE34 and pPtGE35. Plasmid constructs were confirmed by Sanger sequencing at the London Regional Genomics Facility.</p></sec><sec id=\"Sec12\"><title>Generation of PtUMPS and PtPRA-PH/CH knockouts using Cas9 and TevCas9</title><p id=\"Par22\">Plasmids pPtGE34 or pPtGE35, containing no guide RNA or sgRNA.UMPS.1944, sgRNA.UMPS.1646, sgRNA.UMPS.157, sgRNA.UMPS.311 for the PtUMPS gene, or sgRNA.PRAPHCH.929 or sgRNA.PRAPHCH.120 for the PtPRA-PH/CH gene, were conjugated from <italic>E. coli</italic> to <italic>P. tricornutum</italic> and exconjugants were selected on zeocin-containing media, supplemented with uracil or histidine as appropriate<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. Ten colonies from each conjugation were resuspended in TE buffer and flash frozen at − 80 °C followed by heating at 95 °C to lyse cells and extract genomic DNA. The genomic target site of each sgRNA in <italic>P. tricornutum</italic> was amplified by PCR and the products were analyzed by T7EI assay as follows; PCR products were denatured at 95 °C for 5 min, slowly cooled to 50 °C, and flash frozen at − 20 °C for 2 min. PCR products (250 ng) were incubated with 2U of T7EI (NEB) in 1 <inline-formula id=\"IEq95\"><alternatives><tex-math id=\"M151\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\times$$\\end{document}</tex-math><mml:math id=\"M152\"><mml:mo>×</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq95.gif\"/></alternatives></inline-formula> NEBuffer 2 for 15 min at 37 °C and analyzed by agarose gel electrophoresis. Colonies that showed editing by T7EI assay were grown in liquid culture supplemented with zeocin and uracil or histidine as appropriate for 2 weeks and serial dilutions were plated on selective media with uracil or histidine to isolate sub-clones. Sub-clones were then screened for homozygous PtUMPS or PtPRA-PH/CH knockout phenotypes by replica streaking on minimal L1 media and L1 media supplemented with uracil or histidine as appropriate. Streaks were grown for 5 days before visual identification of phenotypes. Sub-clones that were identified as phenotypic knockouts were resuspended in TE buffer and flash frozen at − 80 °C followed by heating at 95 °C to lyse cells and extract genomic DNA, then sgRNA target sites were PCR amplified. Sanger sequencing of PCR products was performed at the London Regional Genomics Facility to identify the type and length of indels generated. Stable bi-allelic PtUMPS or PtPRA-PH/CH knockout mutant lines were then grown in nonselective L1 media supplemented with uracil or histidine for 1 week to cure them of plasmids before plating to obtain single colonies. Resulting colonies were replica streaked onto nonselective and zeocin-containing media supplemented with uracil or histidine to identify colonies which had successfully been cured of the plasmid.</p></sec><sec id=\"Sec13\"><title>Spot plating <italic>P. tricornutum</italic></title><p id=\"Par23\">Cultures of <italic>P. tricornutum</italic> were adjusted to 1 <inline-formula id=\"IEq99\"><alternatives><tex-math id=\"M153\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\times$$\\end{document}</tex-math><mml:math id=\"M154\"><mml:mo>×</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq99.gif\"/></alternatives></inline-formula><inline-formula id=\"IEq100\"><alternatives><tex-math id=\"M155\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$10^{6}$$\\end{document}</tex-math><mml:math id=\"M156\"><mml:msup><mml:mn>10</mml:mn><mml:mn>6</mml:mn></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq100.gif\"/></alternatives></inline-formula> cells <inline-formula id=\"IEq101\"><alternatives><tex-math id=\"M157\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {mL}^{-1}$$\\end{document}</tex-math><mml:math id=\"M158\"><mml:msup><mml:mtext>mL</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq101.gif\"/></alternatives></inline-formula> and serially diluted 2 X three times. For uracil auxotrophs, 10 μL of each adjusted culture and dilutions were spot plated onto minimal L1 media and L1 media supplemented with uracil (50 mg <inline-formula id=\"IEq103\"><alternatives><tex-math id=\"M159\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M160\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq103.gif\"/></alternatives></inline-formula>), 5-FOA (100 mg <inline-formula id=\"IEq104\"><alternatives><tex-math id=\"M161\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M162\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq104.gif\"/></alternatives></inline-formula>), or both. For histidine auxotrophs, 10 μL of each adjusted culture and dilutions were spot plated onto minimal L1 media and L1 media supplemented with histidine (200 mg <inline-formula id=\"IEq106\"><alternatives><tex-math id=\"M163\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M164\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq106.gif\"/></alternatives></inline-formula>). Plates were incubated at 18 °C under cool white fluorescent lights (75 μE <inline-formula id=\"IEq109\"><alternatives><tex-math id=\"M165\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {m}^{-2}$$\\end{document}</tex-math><mml:math id=\"M166\"><mml:msup><mml:mtext>m</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq109.gif\"/></alternatives></inline-formula><inline-formula id=\"IEq110\"><alternatives><tex-math id=\"M167\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {s}^{-1}$$\\end{document}</tex-math><mml:math id=\"M168\"><mml:msup><mml:mtext>s</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq110.gif\"/></alternatives></inline-formula>) and a photoperiod of 16 h light:8 h dark for 7–10 days.</p></sec><sec id=\"Sec14\"><title>Measuring <italic>P. tricornutum</italic> growth rates</title><p id=\"Par24\">Growth was measured in a Multiskan Go microplate spectrophotometer. Cultures of each strain (WT, <inline-formula id=\"IEq111\"><alternatives><tex-math id=\"M169\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M170\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq111.gif\"/></alternatives></inline-formula>UMPS1, <inline-formula id=\"IEq112\"><alternatives><tex-math id=\"M171\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M172\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq112.gif\"/></alternatives></inline-formula>UMPS1 + pPtUMPS40S, <inline-formula id=\"IEq113\"><alternatives><tex-math id=\"M173\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M174\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq113.gif\"/></alternatives></inline-formula>UMPS1 + pPtUMPSA1, <inline-formula id=\"IEq114\"><alternatives><tex-math id=\"M175\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M176\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq114.gif\"/></alternatives></inline-formula>UMPS1 + pPtUMPSA2, <inline-formula id=\"IEq115\"><alternatives><tex-math id=\"M177\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M178\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq115.gif\"/></alternatives></inline-formula>UMPS1 + pPtUMPScA1, <inline-formula id=\"IEq116\"><alternatives><tex-math id=\"M179\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M180\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq116.gif\"/></alternatives></inline-formula>UMPS1 + pPtUMPScA2, <inline-formula id=\"IEq117\"><alternatives><tex-math id=\"M181\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M182\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq117.gif\"/></alternatives></inline-formula>UMPS2 + pPtUMPSA1, <inline-formula id=\"IEq118\"><alternatives><tex-math id=\"M183\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M184\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq118.gif\"/></alternatives></inline-formula>UMPS2 + pPtUMPSA2, <inline-formula id=\"IEq119\"><alternatives><tex-math id=\"M185\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M186\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq119.gif\"/></alternatives></inline-formula>UMPS2 + pPtUMPScA1, <inline-formula id=\"IEq120\"><alternatives><tex-math id=\"M187\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M188\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq120.gif\"/></alternatives></inline-formula>UMPS2 + pPtUMPScA2) were adjusted to 5 x <inline-formula id=\"IEq121\"><alternatives><tex-math id=\"M189\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$10^{5}$$\\end{document}</tex-math><mml:math id=\"M190\"><mml:msup><mml:mn>10</mml:mn><mml:mn>5</mml:mn></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq121.gif\"/></alternatives></inline-formula> cells <inline-formula id=\"IEq122\"><alternatives><tex-math id=\"M191\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {mL}^{-1}$$\\end{document}</tex-math><mml:math id=\"M192\"><mml:msup><mml:mtext>mL</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq122.gif\"/></alternatives></inline-formula> in L1 media with and without supplemented uracil (50 mg <inline-formula id=\"IEq123\"><alternatives><tex-math id=\"M193\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M194\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq123.gif\"/></alternatives></inline-formula>), 5-FOA (100 mg <inline-formula id=\"IEq124\"><alternatives><tex-math id=\"M195\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M196\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq124.gif\"/></alternatives></inline-formula>), or both. Two hundred microliters of each adjusted culture was added to three wells (technical replicates) of a 96-well microplate. The 96-well microplates were incubated at 18 °C under cool white fluorescent lights (75 μE <inline-formula id=\"IEq127\"><alternatives><tex-math id=\"M197\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {m}^{-2}$$\\end{document}</tex-math><mml:math id=\"M198\"><mml:msup><mml:mtext>m</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq127.gif\"/></alternatives></inline-formula><inline-formula id=\"IEq128\"><alternatives><tex-math id=\"M199\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {s}^{-1}$$\\end{document}</tex-math><mml:math id=\"M200\"><mml:msup><mml:mtext>s</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq128.gif\"/></alternatives></inline-formula>) and a photoperiod of 16 h light:8 h dark for 10 days, and absorbance at 670 nm (<inline-formula id=\"IEq129\"><alternatives><tex-math id=\"M201\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {A}_{670}$$\\end{document}</tex-math><mml:math id=\"M202\"><mml:msub><mml:mtext>A</mml:mtext><mml:mn>670</mml:mn></mml:msub></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq129.gif\"/></alternatives></inline-formula>) was measured every 24 h. The 96-well microplates were shaken briefly to resuspend any settled cells prior to absorbance measurements. Note that the <inline-formula id=\"IEq130\"><alternatives><tex-math id=\"M203\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {A}_{670}$$\\end{document}</tex-math><mml:math id=\"M204\"><mml:msub><mml:mtext>A</mml:mtext><mml:mn>670</mml:mn></mml:msub></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq130.gif\"/></alternatives></inline-formula> was not adjusted for path length and light scattering from the microplate lid and is therefore not directly comparable to optical density readings measured in a standard cuvette.</p></sec><sec id=\"Sec15\"><title><italic>P. tricornutum</italic> metabolite extraction</title><p id=\"Par25\">Cultures of <italic>P. tricornutum</italic> (Wild-type, <inline-formula id=\"IEq131\"><alternatives><tex-math id=\"M205\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M206\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq131.gif\"/></alternatives></inline-formula>UMPS1, and <inline-formula id=\"IEq132\"><alternatives><tex-math id=\"M207\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M208\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq132.gif\"/></alternatives></inline-formula>UMPS2) were grown with and without uracil supplementation and harvested during exponential phase as follows (Note: The <inline-formula id=\"IEq133\"><alternatives><tex-math id=\"M209\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M210\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq133.gif\"/></alternatives></inline-formula>UMPS1 and <inline-formula id=\"IEq134\"><alternatives><tex-math id=\"M211\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\Delta$$\\end{document}</tex-math><mml:math id=\"M212\"><mml:mi mathvariant=\"normal\">Δ</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq134.gif\"/></alternatives></inline-formula>UMPS2 cultures were first grown with uracil supplementation, then switched to minimal L1 media for 1 week prior to harvesting). Cultures (<inline-formula id=\"IEq135\"><alternatives><tex-math id=\"M213\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim 1\\times 10^{9}$$\\end{document}</tex-math><mml:math id=\"M214\"><mml:mrow><mml:mo>∼</mml:mo><mml:mn>1</mml:mn><mml:mo>×</mml:mo><mml:msup><mml:mn>10</mml:mn><mml:mn>9</mml:mn></mml:msup></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq135.gif\"/></alternatives></inline-formula> cells) were pelleted by centrifugation at 4000<inline-formula id=\"IEq136\"><alternatives><tex-math id=\"M215\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\times$$\\end{document}</tex-math><mml:math id=\"M216\"><mml:mo>×</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq136.gif\"/></alternatives></inline-formula><italic>g</italic> for 10 min and washed by resuspending in fresh L1 media. Cells were pelleted again, resuspended in a small volume (<inline-formula id=\"IEq137\"><alternatives><tex-math id=\"M217\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim$$\\end{document}</tex-math><mml:math id=\"M218\"><mml:mo>∼</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq137.gif\"/></alternatives></inline-formula> 5 mL) of L1 media, and transferred to a clean 10 mL syringe (without needle) with the exit plugged by parafilm. The syringe was placed, tip-down, into a clean 50 mL falcon tube and the cells were pelleted as above. The supernatant was removed and the pellet was slowly ejected from the syringe into a pre-chilled mortar containing liquid nitrogen. The frozen cells were ground to a fine powder and then transferred to a clean pre-weighed 1.5 mL Eppendorf tube, suspended half way in liquid nitrogen. Being careful to keep samples frozen, 50 mg of frozen ground powder was weighed out into a new clean 1.5 mL Eppendorf tube, pre-cooled in liquid nitrogen, and 250 μL of cold extraction buffer with internal standard (IS) (80% methanol in MilliQ water, 125 μM <inline-formula id=\"IEq140\"><alternatives><tex-math id=\"M219\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$^{15}\\hbox {N}_{2}-\\hbox {uracil}$$\\end{document}</tex-math><mml:math id=\"M220\"><mml:mrow><mml:msup><mml:mrow/><mml:mn>15</mml:mn></mml:msup><mml:msub><mml:mtext>N</mml:mtext><mml:mn>2</mml:mn></mml:msub><mml:mo>-</mml:mo><mml:mtext>uracil</mml:mtext></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq140.gif\"/></alternatives></inline-formula>) was added. The IS was added to the samples to compensate for losses that might occur during preparation of the samples and loss of sensitivity attributable to quenching of the signal by coeluting compounds. Samples were then homogenized by vigorous vortexing for 30 s in 10 second intervals, between which samples are kept on ice for <inline-formula id=\"IEq141\"><alternatives><tex-math id=\"M221\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim$$\\end{document}</tex-math><mml:math id=\"M222\"><mml:mo>∼</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq141.gif\"/></alternatives></inline-formula> 30 s. Homogenized samples were then spun down at 4 °C for 10 min at 20,000<inline-formula id=\"IEq143\"><alternatives><tex-math id=\"M223\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\times$$\\end{document}</tex-math><mml:math id=\"M224\"><mml:mo>×</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq143.gif\"/></alternatives></inline-formula><italic>g</italic>. The supernatant was transferred to a new clean 1.5 mL Eppendorf tube and spun down at 20,000<inline-formula id=\"IEq144\"><alternatives><tex-math id=\"M225\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\times$$\\end{document}</tex-math><mml:math id=\"M226\"><mml:mo>×</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq144.gif\"/></alternatives></inline-formula><italic>g</italic> for 5 min at 4 °C. The supernatant was again transferred to a new clean 1.5 mL Eppendorf tube and kept at 4<inline-formula id=\"IEq146\"><alternatives><tex-math id=\"M227\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$^{\\circ }$$\\end{document}</tex-math><mml:math id=\"M228\"><mml:msup><mml:mrow/><mml:mo>∘</mml:mo></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq146.gif\"/></alternatives></inline-formula>C overnight prior to LC–MS analysis.</p></sec><sec id=\"Sec16\"><title>Chromatographic separation and mass spectrometry</title><p id=\"Par26\">Metabolites were separated at 45 °C on a Waters Acquity HSS T3 column [2.1 <inline-formula id=\"IEq148\"><alternatives><tex-math id=\"M229\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\times$$\\end{document}</tex-math><mml:math id=\"M230\"><mml:mo>×</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq148.gif\"/></alternatives></inline-formula> 100 mm, 1.8 μm] in a Waters ACQUITY UPLC I-Class system (Waters, Milford, MA). Solvent A consisted of water and solvent B consisted of methanol, both containing 0.1% formic acid. Elution was performed by use of a linear gradient, at a flow rate of 0.3 mL/min, as follows: 0–2 min, 100% solvent A to 90% solvent B; 2.01 min, 100% solvent A to recondition the column. A Waters Xevo G2-S quadrupole time of flight mass spectrometer was operated in negative electrospray ionization (ESI) in resolution mode. The capillary voltage was set to 1.0 kV, the source temperature was 150 °C, desolvation temperature was 600 °C, the cone gas was 50 L/h and the desolvation gas was 1000 L/h. Leucine enkephalin was infused as the lock mass with a scan time of 0.3 seconds every 10 s, and three scans were averaged. Linearity and detection limits for each compound were established by injection of calibration mixtures with different concentrations (0, 1, 2, 4, 8, 16, 31.25, 62.5, 125, 250, and 500 μmol/L). Stable-isotope-labeled uracil (<inline-formula id=\"IEq153\"><alternatives><tex-math id=\"M231\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$^{15}\\hbox {N}^{2}-\\hbox {uracil}$$\\end{document}</tex-math><mml:math id=\"M232\"><mml:mrow><mml:msup><mml:mrow/><mml:mn>15</mml:mn></mml:msup><mml:msup><mml:mtext>N</mml:mtext><mml:mn>2</mml:mn></mml:msup><mml:mo>-</mml:mo><mml:mtext>uracil</mml:mtext></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq153.gif\"/></alternatives></inline-formula>) was used as the IS. The concentration of each analyte was determined by use of the slope and intercept of the calibration curve that was obtained from a least-squares regression for the analyte/IS peak-area ratio vs the concentration of the analyte in the calibration mixture.</p></sec><sec id=\"Sec17\"><title><italic>P. tricornutum</italic> DNA extraction and targeted long-read sequencing</title><p id=\"Par27\">Plasmids pPtGE34 or pPtGE35, containing sgRNAs targeting the PtUMPS, PtUrease, or PtI3GPS-PRAI gene were conjugated from <italic>E. coli</italic> to <italic>P. tricornutum</italic> and exconjugants were selected on zeocin-containing media, supplemented with uracil or tryptophan (100 mg <inline-formula id=\"IEq154\"><alternatives><tex-math id=\"M233\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {L}^{-1}$$\\end{document}</tex-math><mml:math id=\"M234\"><mml:msup><mml:mtext>L</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq154.gif\"/></alternatives></inline-formula>) as appropriate. For each transformation, colonies (<inline-formula id=\"IEq155\"><alternatives><tex-math id=\"M235\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim$$\\end{document}</tex-math><mml:math id=\"M236\"><mml:mo>∼</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq155.gif\"/></alternatives></inline-formula> 1,000) were scraped and pooled in liquid L1 media and genomic DNA was extracted using a modified akaline lysis protocol as follows: Cells were pelleted at 4,000<inline-formula id=\"IEq156\"><alternatives><tex-math id=\"M237\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\times$$\\end{document}</tex-math><mml:math id=\"M238\"><mml:mo>×</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq156.gif\"/></alternatives></inline-formula><italic>g</italic> for 5 min, and resuspended in 250 μL resuspension buffer consisting of 235 μL P1 (Qiagen), 5 μL hemicellulose 100 mg <inline-formula id=\"IEq160\"><alternatives><tex-math id=\"M239\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {mL}^{-1}$$\\end{document}</tex-math><mml:math id=\"M240\"><mml:msup><mml:mtext>mL</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq160.gif\"/></alternatives></inline-formula>, 5 μL of lysozyme 25 mg <inline-formula id=\"IEq162\"><alternatives><tex-math id=\"M241\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {mL}^{-1}$$\\end{document}</tex-math><mml:math id=\"M242\"><mml:msup><mml:mtext>mL</mml:mtext><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq162.gif\"/></alternatives></inline-formula>, and 5 μL zymolyase solution (200 mg zymolyase 20 T (USB), 9 mL <inline-formula id=\"IEq164\"><alternatives><tex-math id=\"M243\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\hbox {H}_{2}\\hbox {O}$$\\end{document}</tex-math><mml:math id=\"M244\"><mml:mrow><mml:msub><mml:mtext>H</mml:mtext><mml:mn>2</mml:mn></mml:msub><mml:mtext>O</mml:mtext></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq164.gif\"/></alternatives></inline-formula>, 1 mL 1 M Tris pH 7.5, 10 mL 50% glycerol) and incubated at 37°C for 30 min. Next, 250 μL of lysis buffer P2 (Qiagen) was added, followed by 250 μL of neutralization buffer P3 (Qiagen) and centrifugation at 16,000<inline-formula id=\"IEq168\"><alternatives><tex-math id=\"M245\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\times$$\\end{document}</tex-math><mml:math id=\"M246\"><mml:mo>×</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq168.gif\"/></alternatives></inline-formula><italic>g</italic> for 10 min. The supernatant was transferred to a clean tube, 750 μL isopropanol was added, and the samples centrifuged at 16,000<inline-formula id=\"IEq170\"><alternatives><tex-math id=\"M247\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\times$$\\end{document}</tex-math><mml:math id=\"M248\"><mml:mo>×</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq170.gif\"/></alternatives></inline-formula><italic>g</italic> for 10 min. A 70% EtOH wash was performed, centrifuged at 16,000<inline-formula id=\"IEq171\"><alternatives><tex-math id=\"M249\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\times$$\\end{document}</tex-math><mml:math id=\"M250\"><mml:mo>×</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq171.gif\"/></alternatives></inline-formula><italic>g</italic> for 5 min, and pellets briefly dried, resuspended in 50–100 μL of TE buffer, and incubated at 37 °C for 30–60 min.</p><p id=\"Par28\">The sgRNA target site regions were PCR amplified from sgRNA transformant genomic DNA samples, as well as a wild-type sample, with PrimeStar GXL polymerase (Takara) using primers positioned <inline-formula id=\"IEq174\"><alternatives><tex-math id=\"M251\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\sim$$\\end{document}</tex-math><mml:math id=\"M252\"><mml:mo>∼</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70769_Article_IEq174.gif\"/></alternatives></inline-formula> 3 kb up- and downstream of the target site (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S6</xref>). PCR products were purified and DNA libraries were prepared, barcoded, and pooled using an Oxford Nanopore Ligation Sequencing Kit (SQK LSK109) and Native Barcoding Expansion 1–12 (EXP-NBD104) kit according to manufacturers protocol with the following modification—all reactions were scaled down to half the recommended volume and the end prep incubation times were extended to 15 min at 20 °C and 15 min at 65 °C. The pooled library was then loaded on to a MinION R9.4.1 flowcell and sequenced.</p></sec><sec id=\"Sec18\"><title>Targeted long-read sequencing analysis</title><p id=\"Par29\">After sequencing on an R9.4.1 flowcell, base calling was performed using GPU Guppy with the high accuracy configuration file version 3.4.4 (<ext-link ext-link-type=\"uri\" xlink:href=\"https://community.nanoporetech.com\">https://community.nanoporetech.com</ext-link>). Reads in each barcode were filtered using NanoFilt<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup> for a minimum average read quality score of 10 and a minimum read length of 2,000, mapped using minimap2<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup> and filtered for reads that map to within 100 bases of each end of the reference sequence (the unedited 6 kb PCR product sequence) to remove short fragments. The filtered reads were mapped using minimap2 (parameters: -ax map-ont) and outputted in sam format, then converted to bam, sorted, and indexed using samtools<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. The per-base coverage depth for each barcode was calculated using Mosdepth<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. All plots were created in R using the ggplot2 package<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec19\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70769_MOESM1_ESM.pdf\"><caption><p>Supplementary Information.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70769-6.</p></sec><ack><title>Acknowledgements</title><p>This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants [RPGIN-2015-04800 to D.R.E. and RGPIN-2018-06172 to B.J.K.]. We thank Greg Gloor for advice on Nanopore sequencing and data analysis.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>S.S.S., D.J.G., B.L.U., B.J.K. and D.R.E. conceived the experiments, S.S.S., H.W., C.K., D.J.G. and B.L.U. performed the experiments, S.S.S, D.J.G., B.J.K. and D.R.E. analysed the results, S.S.S and D.R.E. wrote the manuscript. All authors reviewed the manuscript.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>The cDNA sequences of PtUMPS and PtPRA-PH/CH alleles have been deposited to GenBank under accession codes <bold>MN242208</bold>, <bold>MN242209</bold>, <bold>MN242210</bold>, and <bold>MN242211</bold>.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par32\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scaife</surname><given-names>MA</given-names></name><name><surname>Smith</surname><given-names>AG</given-names></name></person-group><article-title>Towards developing algal synthetic biology</article-title><source>Biochem. Soc. Trans.</source><year>2016</year><volume>44</volume><fpage>716</fpage><lpage>22</lpage><pub-id pub-id-type=\"pmid\">27284033</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>B</given-names></name><name><surname>Wang</surname><given-names>J</given-names></name><name><surname>Meldrum</surname><given-names>DR</given-names></name></person-group><article-title>Application of synthetic biology in cyanobacteria and algae</article-title><source>Front. Microbiol.</source><year>2012</year><volume>3</volume><fpage>1</fpage><lpage>15</lpage><pub-id pub-id-type=\"pmid\">22275914</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yongmanitchai</surname><given-names>W</given-names></name><name><surname>Ward</surname><given-names>OP</given-names></name></person-group><article-title>Growth of and omega-3 fatty acid production by Phaeodacytlum tricornutum under different culture conditions</article-title><source>Appl. Environ. Microbiol.</source><year>1991</year><volume>57</volume><fpage>419</fpage><lpage>425</lpage><pub-id pub-id-type=\"pmid\">2014989</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bozarth</surname><given-names>A</given-names></name><name><surname>Maier</surname><given-names>UG</given-names></name><name><surname>Zauner</surname><given-names>S</given-names></name></person-group><article-title>Diatoms in biotechnology: Modern tools and applications</article-title><source>Appl. Microbiol. Biotechnol.</source><year>2009</year><volume>82</volume><issue>2</issue><fpage>195</fpage><lpage>201</lpage><pub-id pub-id-type=\"pmid\">19082585</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Karas</surname><given-names>BJ</given-names></name><etal/></person-group><article-title>Designer diatom episomes delivered by bacterial conjugation</article-title><source>Nat. Commun.</source><year>2015</year><volume>6</volume><fpage>6925</fpage><pub-id pub-id-type=\"pmid\">25897682</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Slattery</surname><given-names>SS</given-names></name><etal/></person-group><article-title>an expanded plasmid-based genetic toolbox enables Cas9 genome editing and stable maintenance of synthetic pathways in Phaeodacytlum tricornutum</article-title><source>ACS Synth. Biol.</source><year>2018</year><volume>7</volume><fpage>328</fpage><lpage>338</lpage><pub-id pub-id-type=\"pmid\">29298053</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Serif</surname><given-names>M</given-names></name><etal/></person-group><article-title>One-step generation of multiple gene knock-outs in the diatom Phaeodacytlum tricornutum by dna-free genome editing</article-title><source>Nat. Commun.</source><year>2018</year><volume>9</volume><fpage>3924</fpage><pub-id pub-id-type=\"pmid\">30254261</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stukenberg</surname><given-names>DD</given-names></name><name><surname>Zauner</surname><given-names>SS</given-names></name><name><surname>Dell Aquila</surname><given-names>G</given-names></name><name><surname>Maier</surname><given-names>UG</given-names></name></person-group><article-title>Optimizing crispr/cas9 for the diatom Phaeodacytlum tricornutum</article-title><source>Front. Plant Sci.</source><year>2018</year><volume>9</volume><fpage>740</fpage><pub-id pub-id-type=\"pmid\">29928285</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sharma</surname><given-names>AK</given-names></name><name><surname>Nymark</surname><given-names>M</given-names></name><name><surname>Sparstad</surname><given-names>T</given-names></name><name><surname>Bones</surname><given-names>AM</given-names></name><name><surname>Winge</surname><given-names>P</given-names></name></person-group><article-title>Transgene-free genome editing in marine algae by bacterial conjugation-comparison with biolistic crispr/cas9 transformation</article-title><source>Sci. Rep.</source><year>2018</year><volume>8</volume><fpage>14401</fpage><pub-id pub-id-type=\"pmid\">30258061</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moosburner</surname><given-names>MA</given-names></name><etal/></person-group><article-title>Multiplexed knockouts in the model diatom phaeodactylum by episomal delivery of a selectable cas9</article-title><source>Front. Microbiol.</source><year>2020</year><pub-id pub-id-type=\"doi\">10.3389/fmicb.2020.00005</pub-id><pub-id pub-id-type=\"pmid\">32047486</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hopes</surname><given-names>A</given-names></name><name><surname>Nekrasov</surname><given-names>V</given-names></name><name><surname>Kamoun</surname><given-names>S</given-names></name><name><surname>Mock</surname><given-names>T</given-names></name></person-group><article-title>Editing of the urease gene by crispr-cas in the diatom Thalassiosira pseudonana</article-title><source>Plant Methods</source><year>2016</year><volume>12</volume><fpage>49</fpage><pub-id pub-id-type=\"pmid\">27904648</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gatignol</surname><given-names>A</given-names></name><name><surname>Durand</surname><given-names>H</given-names></name><name><surname>Tiraby</surname><given-names>G</given-names></name></person-group><article-title>Bleomycin resistance conferred by a drug-binding protein</article-title><source>FEBS Lett.</source><year>1988</year><volume>230</volume><fpage>171</fpage><lpage>5</lpage><pub-id pub-id-type=\"pmid\">2450783</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zaslavskaia</surname><given-names>LA</given-names></name><name><surname>Casey Lippmeier</surname><given-names>J</given-names></name><name><surname>Kroth</surname><given-names>PG</given-names></name><name><surname>Grossman</surname><given-names>AR</given-names></name><name><surname>Apt</surname><given-names>KE</given-names></name></person-group><article-title>Transformation of the diatom Phaeodacytlum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes</article-title><source>J. Phycol.</source><year>2000</year><volume>36</volume><fpage>379</fpage><lpage>386</lpage></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krügel</surname><given-names>H</given-names></name><name><surname>Fiedler</surname><given-names>G</given-names></name><name><surname>Smith</surname><given-names>C</given-names></name><name><surname>Baumberg</surname><given-names>S</given-names></name></person-group><article-title>Sequence and transcriptional analysis of the nourseothricin acetyltransferase-encoding gene nat1 from Streptomyces noursei</article-title><source>Gene</source><year>1993</year><volume>127</volume><fpage>127</fpage><lpage>131</lpage><pub-id pub-id-type=\"pmid\">8486278</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Buck</surname><given-names>JM</given-names></name><name><surname>Bártulos</surname><given-names>CR</given-names></name><name><surname>Gruber</surname><given-names>A</given-names></name><name><surname>Kroth</surname><given-names>PG</given-names></name></person-group><article-title>Blasticidin-s deaminase, a new selection marker for genetic transformation of the diatom Phaeodacytlum tricornutum</article-title><source>PeerJ</source><year>2018</year><volume>6</volume><fpage>e5884</fpage><pub-id pub-id-type=\"pmid\">30488015</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chassy</surname><given-names>BM</given-names></name></person-group><article-title>Food safety evaluation of crops produced through biotechnology</article-title><source>J. Am. Coll. Nutr.</source><year>2002</year><volume>21</volume><fpage>166S</fpage><lpage>173S</lpage><pub-id pub-id-type=\"pmid\">12071300</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><mixed-citation publication-type=\"other\">Romero, J., Feijoó, C. G. & Navarrete, P. Antibiotics in aquaculture-use, abuse and alternatives. <italic>Health Environ. Aquacult.</italic><bold>159</bold>, (2012).</mixed-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Peterbauer</surname><given-names>C</given-names></name><name><surname>Maischberger</surname><given-names>T</given-names></name><name><surname>Haltrich</surname><given-names>D</given-names></name></person-group><article-title>Food-grade gene expression in lactic acid bacteria</article-title><source>Biotechnol. J.</source><year>2011</year><volume>6</volume><fpage>1147</fpage><lpage>1161</lpage><pub-id pub-id-type=\"pmid\">21858927</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pronk</surname><given-names>JT</given-names></name></person-group><article-title>Auxotrophic yeast strains in fundamental and applied research</article-title><source>Appl. Environ. Microbiol.</source><year>2002</year><volume>68</volume><fpage>2095</fpage><lpage>2100</lpage><pub-id pub-id-type=\"pmid\">11976076</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Newman</surname><given-names>E</given-names></name><name><surname>Miller</surname><given-names>B</given-names></name><name><surname>Colebrook</surname><given-names>L</given-names></name><name><surname>Walker</surname><given-names>C</given-names></name></person-group><article-title>A mutation in escherichia coli k-12 results in a requirement for thiamine and a decrease in l-serine deaminase activity</article-title><source>J. Bacteriol.</source><year>1985</year><volume>161</volume><fpage>272</fpage><lpage>276</lpage><pub-id pub-id-type=\"pmid\">3918001</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Goldstein</surname><given-names>A</given-names></name><name><surname>Goldstein</surname><given-names>DB</given-names></name><name><surname>Brown</surname><given-names>BJ</given-names></name><name><surname>Chou</surname><given-names>S-C</given-names></name></person-group><article-title>Amino acid starvation in an escherichia coli auxotroph: I. effects on protein and nucleic acid synthesis and on cell division</article-title><source>Biochim. et Biophys. Acta</source><year>1959</year><volume>36</volume><fpage>163</fpage><lpage>172</lpage></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sakaguchi</surname><given-names>T</given-names></name><name><surname>Nakajima</surname><given-names>K</given-names></name><name><surname>Matsuda</surname><given-names>Y</given-names></name></person-group><article-title>Identification of the UMP Synthase Gene by Establishment of Uracil Auxotrophic Mutants and the Phenotypic Complementation System in the Marine Diatom Phaeodacytlum tricornutum</article-title><source>Plant Physiol.</source><year>2011</year><volume>156</volume><fpage>78</fpage><lpage>89</lpage><pub-id pub-id-type=\"pmid\">21367966</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kanehisa</surname><given-names>M</given-names></name><name><surname>Goto</surname><given-names>S</given-names></name></person-group><article-title>Kegg: kyoto encyclopedia of genes and genomes</article-title><source>Nucleic Acids Res.</source><year>2000</year><volume>28</volume><fpage>27</fpage><lpage>30</lpage><pub-id pub-id-type=\"pmid\">10592173</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kanehisa</surname><given-names>M</given-names></name><name><surname>Sato</surname><given-names>Y</given-names></name><name><surname>Furumichi</surname><given-names>M</given-names></name><name><surname>Morishima</surname><given-names>K</given-names></name><name><surname>Tanabe</surname><given-names>M</given-names></name></person-group><article-title>New approach for understanding genome variations in kegg</article-title><source>Nucleic Acids Res.</source><year>2019</year><volume>47</volume><fpage>D590</fpage><lpage>D595</lpage><pub-id pub-id-type=\"pmid\">30321428</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chiariotti</surname><given-names>L</given-names></name><name><surname>Alifano</surname><given-names>P</given-names></name><name><surname>Carlomagno</surname><given-names>MS</given-names></name><name><surname>Bruni</surname><given-names>CB</given-names></name></person-group><article-title>Nucleotide sequence of the escherichia coli hisd gene and of the escherichia coli and salmonella typhimurium hisie region</article-title><source>Molecular and General Genetics MGG</source><year>1986</year><volume>203</volume><fpage>382</fpage><lpage>388</lpage><pub-id pub-id-type=\"pmid\">3018428</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><mixed-citation publication-type=\"other\">Ingle, R. A. <italic>Histidine Biosynthesis. Arabidopsis Book</italic><bold>1–9</bold>, (2011).</mixed-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wolfs</surname><given-names>JM</given-names></name><etal/></person-group><article-title>Biasing genome-editing events toward precise length deletions with an RNA-guided TevCas9 dual nuclease</article-title><source>Proceedings of the National Academy of Sciences of the United States of America</source><year>2016</year><volume>113</volume><fpage>14988</fpage><lpage>14993</lpage><pub-id pub-id-type=\"pmid\">27956611</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Russo</surname><given-names>MT</given-names></name><name><surname>Cigliano</surname><given-names>RA</given-names></name><name><surname>Sanseverino</surname><given-names>W</given-names></name><name><surname>Ferrante</surname><given-names>MI</given-names></name></person-group><article-title>Assessment of genomic changes in a crispr/cas9 Phaeodacytlum tricornutum mutant through whole genome resequencing</article-title><source>PeerJ</source><year>2018</year><volume>6</volume><fpage>e5507</fpage><pub-id pub-id-type=\"pmid\">30310734</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kosicki</surname><given-names>M</given-names></name><name><surname>Tomberg</surname><given-names>K</given-names></name><name><surname>Bradley</surname><given-names>A</given-names></name></person-group><article-title>Repair of double-strand breaks induced by crispr-cas9 leads to large deletions and complex rearrangements</article-title><source>Nat. Biotechnol.</source><year>2018</year><volume>36</volume><fpage>765</fpage><lpage>771</lpage><pub-id pub-id-type=\"pmid\">30010673</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gorter de Vries</surname><given-names>AR</given-names></name><etal/></person-group><article-title>Allele-specific genome editing using crispr-cas9 is associated with loss of heterozygosity in diploid yeast</article-title><source>Nucleic Acids Res.</source><year>2019</year><volume>47</volume><fpage>1362</fpage><lpage>1372</lpage><pub-id pub-id-type=\"pmid\">30517747</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weyman</surname><given-names>PD</given-names></name><etal/></person-group><article-title>Inactivation of Phaeodacytlum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis</article-title><source>Plant Biotechnol. J.</source><year>2015</year><volume>13</volume><fpage>460</fpage><lpage>470</lpage><pub-id pub-id-type=\"pmid\">25302562</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Daboussi</surname><given-names>F</given-names></name><etal/></person-group><article-title>Genome engineering empowers the diatom Phaeodacytlum tricornutum for biotechnology</article-title><source>Nat. Commun.</source><year>2014</year><volume>5</volume><fpage>1</fpage><lpage>7</lpage></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yeh</surname><given-names>CD</given-names></name><name><surname>Richardson</surname><given-names>CD</given-names></name><name><surname>Corn</surname><given-names>JE</given-names></name></person-group><article-title>Advances in genome editing through control of dna repair pathways</article-title><source>Nat. Cell Biol.</source><year>2019</year><volume>1–11</volume><fpage>1468</fpage></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Angstenberger</surname><given-names>M</given-names></name><name><surname>Krischer</surname><given-names>J</given-names></name><name><surname>Aktaş</surname><given-names>O</given-names></name><name><surname>Bächel</surname><given-names>C</given-names></name></person-group><article-title>Knock-down of a ligiv homologue enables dna integration via homologous recombination in the marine diatom Phaeodacytlum tricornutum</article-title><source>ACS Synth. Biol.</source><year>2018</year><volume>8</volume><fpage>57</fpage><lpage>69</lpage><pub-id pub-id-type=\"pmid\">30525458</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Certo</surname><given-names>MT</given-names></name><etal/></person-group><article-title>Tracking genome engineering outcome at individual dna breakpoints</article-title><source>Nat. Methods</source><year>2011</year><volume>8</volume><fpage>671</fpage><pub-id pub-id-type=\"pmid\">21743461</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Serif</surname><given-names>M</given-names></name><name><surname>Lepetit</surname><given-names>B</given-names></name><name><surname>Weißert</surname><given-names>K</given-names></name><name><surname>Kroth</surname><given-names>P</given-names></name><name><surname>Rio Bartulos</surname><given-names>C</given-names></name></person-group><article-title>A fast and reliable strategy to generate TALEN-mediated gene knockouts in the diatom Phaeodacytlum tricornutum</article-title><source>Algal Res.</source><year>2017</year><volume>23</volume><fpage>186</fpage><lpage>195</lpage></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mali</surname><given-names>P</given-names></name><etal/></person-group><article-title>Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering</article-title><source>Nat. Biotechnol.</source><year>2013</year><volume>31</volume><fpage>833</fpage><pub-id pub-id-type=\"pmid\">23907171</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Karas</surname><given-names>BJ</given-names></name><etal/></person-group><article-title>Designer diatom episomes delivered by bacterial conjugation</article-title><source>Nat. Commun.</source><year>2015</year><volume>6</volume><fpage>1</fpage><lpage>10</lpage></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Noskov</surname><given-names>VN</given-names></name><etal/></person-group><article-title>Assembly of large, high G+C bacterial DNA fragments in yeast</article-title><source>ACS Synth. Biol.</source><year>2012</year><volume>1</volume><fpage>267</fpage><lpage>273</lpage><pub-id pub-id-type=\"pmid\">23651249</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gibson</surname><given-names>DG</given-names></name></person-group><article-title>Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides</article-title><source>Nucleic Acids Res.</source><year>2009</year><volume>37</volume><fpage>6984</fpage><lpage>6990</lpage><pub-id pub-id-type=\"pmid\">19745056</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>H</given-names></name><name><surname>Slattery</surname><given-names>S</given-names></name><name><surname>Karas</surname><given-names>B</given-names></name><name><surname>Edgell</surname><given-names>D</given-names></name></person-group><article-title>Delivery of the cas9 or tevcas9 system into Phaeodacytlum tricornutum via conjugation of plasmids from a bacterial donor</article-title><source>Bio-Protocol</source><year>2018</year><volume>8</volume><fpage>e2974</fpage></element-citation></ref><ref id=\"CR42\"><label>42.</label><mixed-citation publication-type=\"other\">Visualizing and processing long-read sequencing data. De Coster, W., D Hert, S., Schultz, D. T., Cruts, M. & Van Broeckhoven, C. Nanopack. Visualizing and processing long-read sequencing data. <italic>Bioinformatics</italic><bold>34</bold>, 2666–2669 (2018).</mixed-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>H</given-names></name></person-group><article-title>Minimap2: Pairwise alignment for nucleotide sequences</article-title><source>Bioinformatics</source><year>2018</year><volume>34</volume><fpage>3094</fpage><lpage>3100</lpage><pub-id pub-id-type=\"pmid\">29750242</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>H</given-names></name><etal/></person-group><article-title>The sequence alignment/map format and samtools</article-title><source>Bioinformatics</source><year>2009</year><volume>25</volume><fpage>2078</fpage><lpage>2079</lpage><pub-id pub-id-type=\"pmid\">19505943</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pedersen</surname><given-names>BS</given-names></name><name><surname>Quinlan</surname><given-names>AR</given-names></name></person-group><article-title>Mosdepth: Quick coverage calculation for genomes and exomes</article-title><source>Bioinformatics</source><year>2018</year><volume>34</volume><fpage>867</fpage><lpage>868</lpage><pub-id pub-id-type=\"pmid\">29096012</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Wickham</surname><given-names>H</given-names></name></person-group><source>ggplot2: Elegant Graphics for Data Analysis</source><year>2016</year><publisher-loc>New York</publisher-loc><publisher-name>Springer</publisher-name></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"brief-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Nat Commun</journal-id><journal-id journal-id-type=\"iso-abbrev\">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type=\"epub\">2041-1723</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807794</article-id><article-id pub-id-type=\"pmc\">PMC7431574</article-id><article-id pub-id-type=\"publisher-id\">17844</article-id><article-id pub-id-type=\"doi\">10.1038/s41467-020-17844-8</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Comment</subject></subj-group></article-categories><title-group><article-title>Machine learning for chemical discovery</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-1012-4854</contrib-id><name><surname>Tkatchenko</surname><given-names>Alexandre</given-names></name><address><email>alexandre.tkatchenko@uni.lu</email></address><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><aff id=\"Aff1\"><institution-wrap><institution-id institution-id-type=\"GRID\">grid.16008.3f</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2295 9843</institution-id><institution>Department of Physics and Materials Science, </institution><institution>University of Luxembourg, </institution></institution-wrap>L-1511 Luxembourg, Luxembourg </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>4125</elocation-id><history><date date-type=\"received\"><day>28</day><month>6</month><year>2020</year></date><date date-type=\"accepted\"><day>20</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\" abstract-type=\"Standfirst\"><p id=\"Par1\">Discovering chemicals with desired attributes is a long and painstaking process. Curated datasets containing reliable quantum-mechanical properties for millions of molecules are becoming increasingly available. The development of novel machine learning tools to obtain chemical knowledge from these datasets has the potential to revolutionize the process of chemical discovery. Here, I comment on recent breakthroughs in this emerging field and discuss the challenges for the years to come.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Chemistry</kwd><kwd>Materials science</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/100011199</institution-id><institution>EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: \"Ideas\" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))</institution></institution-wrap></funding-source><award-id>BeStMo</award-id><principal-award-recipient><name><surname>Tkatchenko</surname><given-names>Alexandre</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Toward chemical discovery revolution</title><p id=\"Par2\">Computational design and discovery of molecules and materials relies on the exploration of increasingly growing chemical spaces<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup> (see Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). The discovery and formulation of new drugs, antivirals, antibiotics, catalysts, battery materials, and in general chemicals with tailored properties, require a shift of paradigm to search in unchartered swaths of the vast chemical space<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. From the fundamental perspective of quantum mechanics (QM), this paradigm shift stems from the fact that molecular properties exhibit complex correlations<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>, which yields whole Pareto fronts of candidate molecules in multiproperty optimization algorithms, enabling “freedom of design”. As an example, taking data for more than 100,000 small drug-like molecules, it is found that their molecular electronic (highest occupied molecular orbital–lowest unoccupied molecular orbital) gap is not correlated at all with their polarizability<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>, in contrast to widely quoted chemical rules. This implies that it is possible to design highly conductive and weakly interacting molecules, or molecules that exhibit stability to dielectric breakdown and yet are strongly interacting.<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Schematic illustration of using machine learning in the process of chemical discovery.</title><p>Subsets of relevant chemical compound space (CCS) are sampled to create datasets of molecular structures. High-throughput quantum-mechanical (QM) calculations are subsequently used to construct QM molecular property datasets. Quantum machine learning (QML) algorithms are employed to enable interpolation and analysis of QM properties in CCS. QML model analysis is combined with chemical knowledge to extract insights into CCS, for example by constructing and analyzing Pareto fronts. Finally, the CCS can be further extended and explored with the accumulated knowledge from QML. The main applications of QML up to now cover CCS of small molecules and ordered extended solids. However, the applicability of QML should be further extended to biomolecular systems, nanostructures, surfaces, organic framework materials, supramolecular systems, and even quantum-mechanical model systems (see central panel).</p></caption><graphic xlink:href=\"41467_2020_17844_Fig1_HTML\" id=\"d30e235\"/></fig></p><p id=\"Par3\">Obviously, chemical discovery concerns not only with finding “this special molecule”, but also predicting reaction pathways and interactions between molecules, optimizing catalytic conditions, eliminating undesired side effects, among many other important degrees of freedom. Given this vast space of possibilities, a statistical view on chemical design and discovery is mandatory (see Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). This is the main reason behind the current rise of machine learning (ML) techiques applied to molecular and materials science. The current situation can be compared to the huge advances made by the sustained development of quantum chemistry and solid-state electronic structure codes for modeling molecules and materials during the 1980s and 1990s. The development of steadily more accurate quantum-mechanical approximations and increasingly efficient electronic-structure codes lead to the “chemical modeling revolution”. In a similar vein, the development of novel ML methods, combined with first principles of quantum and statistical mechanics, and fed with increasingly available molecular big data, could lead to the “chemical discovery revolution”.</p><p id=\"Par4\">Chemical discovery and ML are bound to evolve together, but achieving true synergy between them requires solving many outstanding challenges. The potential of using ML for increasing the accuracy and efficiency of molecular simulations has been established beyond any doubt<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>–<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. Data-driven high-throughput materials discovery has also been established as a field of its own<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. Physically inspired ML algorithms can identify new drug candidates<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>, find new phases in amorphous materials<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>, carry out molecular dynamics with essentially exact quantum forces<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>, and offer unprecedented statistical insights into chemical environments<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Up to now, most of these applications were done under idealized conditions. Future work should concentrate on enabling tighter embedding of molecular simulations and ML methods, combining QM and statistical mechanics via ML algorithms, developing universal ML approximations for covalent and non-covalent molecular interactions, and developing algorithms for targeted exploration of large chemical spaces. Obviously, all of these advances should be continuously assessed on growing community-curated datasets of microscopic and macroscopic molecular properties.</p></sec><sec id=\"Sec2\"><title>From molecular big data to chemical discovery</title><p id=\"Par5\">The quality and reliability of ML models in any scientific domain depends on the increasing availability of data. The first applications of ML to molecular and materials modeling in 2010–2012 relied on small datasets containing QM properties for 10<sup>2</sup>–10<sup>3</sup> systems. The development of physics-inspired ML models and sophisticated atomistic descriptors have been crucial for increasing the predictive power of ML models by at least two orders of magnitude in the past 8 years<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>—an incredible scientific progress. Today, advanced ML models are capable of achieving predictive accuracy in QM properties of large molecular datasets by learning from just 1 to 2% of the data<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. Such data efficiency and accuracy are essential for enabling in silico chemical discovery.</p><p id=\"Par6\">Recently, focus has been shifting towards constructing and exploring increasingly larger chemical spaces. Datasets such as QM9<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>, ANI-1x<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>, and QM7-X<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup> contain QM properties for up to 10<sup>7</sup> molecular structures and enable essentially complete coverage of the chemical space of small drug-like molecules. These data has been used in many applications, for exampling to construct fast-to-evaluate neural network potentials for small molecules<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>, develop improved semiempirical quantum methods<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>, and obtain new insights into partitioning of molecular quantum properties into atomic and fragment-based contributions<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>.</p><p id=\"Par7\">Another unique application of ML for molecular modeling is ML-driven molecular dynamics simulations. ML force fields are able to combine the accuracy of high-level QM with the efficiency of classical force fields. For example, the gradient-domain ML force fields enable MD simulations of small molecules with essentially exact quantum treatment of both electrons and nuclei<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>—a task which was considered unattainable just a few years ago. For elemental solids, Gaussian approximation potentials (GAP)<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup> are nowadays used to carry out MD simulations of unit cells with thousands of atoms and to obtain new insights into, for example, amorphous states of matter<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>.</p><p id=\"Par8\">Both wide exploration of chemical space and long time-scale MD simulations for single molecules are enabling tools for chemical discovery. Another important application of ML is inverse design of molecules with targeted properties. Ultimately, ML should also enable in silico guided discovery of novel molecules and materials and confirm such discoveries with experimental data. Indeed, successful ML-driven discoveries have been made in the search for organic light-emitting diodes<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>, redox-flow batteries<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>, and antibiotics<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>, among many other examples.</p><p id=\"Par9\">The most remarkable aspect of ML for chemical discovery is that the corresponding statistical view on chemical space often enables asking new questions and obtaining novel insights. The holistic analysis of large swaths of chemical space leads to discoveries of molecules with unexpected properties<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>, offers hints for new chemical reaction mechanisms<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>, or even suggests new physicochemical relations<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. Such novel discoveries are often made by interdisciplinary teams of researchers that are able to synergetically combine their knowledge of physical laws and constraints, chemical intuition, and sophisticated ML algorithms.</p></sec><sec id=\"Sec3\"><title>Future of ML for chemical discovery</title><p id=\"Par10\">Current successful applications of ML for chemical discovery have only scratched the surface of possibilities. There are many conceptual, theoretical, and practical challenges waiting to be solved to enable the “chemical discovery revolution”. Here I discuss the challenges that I consider to be the most pressing and interesting at this moment.</p><p id=\"Par11\">A universal ML approach should have the capacity to accurately predict both energetic and electronic properties of molecules. In addition, such an approach should uniformly describe compositional (chemical arrangement of atoms in a molecule) and configurational (physical arrangement of atoms in space) degrees of freedom on equal footing. Most existing ML approaches only describe a restricted subset of relevant degrees of freedom and physicochemical observables. Further progress in this field requires developing universal ML models for a diverse set of systems and physicochemical properties shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>.</p><p id=\"Par12\">From the perspective of atomic interactions, current ML representations are successful in describing local chemical bonding, but they completely miss long-range electrostatics, polarization, and van der Waals dispersion interactions. Combining intermolecular interaction theory with ML is an important direction for future progress towards studying complex molecular systems.</p><p id=\"Par13\">An emerging idea is to combine ML with approximate Hamiltonians for electronic interactions based on density-functional theory, tight-binding, molecular orbital techniques, or the many-body dispersion method. The ML approach is used to predict Hamiltonian parameters and the quantum-mechanical observables are calculated via diagonalization of the corresponding Hamiltonian. The challenge is to achieve tighter integration between ML and approximate Hamiltonians and to find an appropriate balance between prediction accuracy and computational efficiency.</p><p id=\"Par14\">Validation of ML predictions ultimately requires comparison to experimental observables, such as reaction rates, spectroscopic observations, solvation energies, melting temperatures, among other relevant quantities. Calculating these observables demands a tight integration of QM, statistical simulations, and fast ML predictions, all integrated in a comprehensive molecular simulations framework<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>.</p><p id=\"Par15\">Solving many of the challenges posed above will require coming up with creative interdisciplinary approaches combining quantum and statistical mechanics, chemical knowledge, and sophisticated ML tools, firmly based on growing datasets that cover increasingly broader domains of the vast chemical space.</p></sec></body><back><fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><ack><title>Acknowledgements</title><p>The author acknowledges the European Research Council (ERC-CoG grant BeStMo) and Dr. Leonardo Medrano-Sandonas for his help in preparing Fig. 1.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>The author has conceptualized the idea and wrote the paper.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par16\">The author declares no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kirkpatrick</surname><given-names>P</given-names></name><name><surname>Ellis</surname><given-names>C</given-names></name></person-group><article-title>Chemical space</article-title><source>Nature</source><year>2004</year><volume>432</volume><fpage>823</fpage><pub-id pub-id-type=\"doi\">10.1038/432823a</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reymond</surname><given-names>J-L</given-names></name></person-group><article-title>The chemical space project</article-title><source>Acc. Chem. Res.</source><year>2015</year><volume>48</volume><fpage>722</fpage><pub-id pub-id-type=\"doi\">10.1021/ar500432k</pub-id><pub-id pub-id-type=\"pmid\">25687211</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>von Lilienfeld</surname><given-names>OA</given-names></name><name><surname>Müller</surname><given-names>K-R</given-names></name><name><surname>Tkatchenko</surname><given-names>A</given-names></name></person-group><article-title>Exploring chemical compound space with quantum-based machine learning</article-title><source>Nat. Rev. Chem.</source><year>2020</year><volume>4</volume><fpage>347</fpage><pub-id pub-id-type=\"doi\">10.1038/s41570-020-0189-9</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bartók</surname><given-names>AP</given-names></name><etal/></person-group><article-title>Machine learning unifies the modeling of materials and molecules</article-title><source>Sci. Adv.</source><year>2017</year><volume>3</volume><fpage>e1701816</fpage><pub-id pub-id-type=\"doi\">10.1126/sciadv.1701816</pub-id><pub-id pub-id-type=\"pmid\">29242828</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Butler</surname><given-names>KT</given-names></name><name><surname>Davies</surname><given-names>DW</given-names></name><name><surname>Cartwright</surname><given-names>H</given-names></name><name><surname>Isayev</surname><given-names>O</given-names></name><name><surname>Walsh</surname><given-names>A</given-names></name></person-group><article-title>Machine learning for molecular and materials science</article-title><source>Nature</source><year>2018</year><volume>559</volume><fpage>547</fpage><pub-id pub-id-type=\"doi\">10.1038/s41586-018-0337-2</pub-id><pub-id pub-id-type=\"pmid\">30046072</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Noé</surname><given-names>F</given-names></name><name><surname>Tkatchenko</surname><given-names>A</given-names></name><name><surname>Müller</surname><given-names>K-R</given-names></name><name><surname>Clementi</surname><given-names>C</given-names></name></person-group><article-title>Machine learning for molecular simulation</article-title><source>Ann. Rev. Phys. Chem</source><year>2020</year><volume>71</volume><fpage>361</fpage><pub-id pub-id-type=\"doi\">10.1146/annurev-physchem-042018-052331</pub-id><pub-id pub-id-type=\"pmid\">32092281</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Curtarolo</surname><given-names>S</given-names></name><etal/></person-group><article-title>The high-throughput highway to computational materials design</article-title><source>Nat. Mater.</source><year>2013</year><volume>12</volume><fpage>191</fpage><pub-id pub-id-type=\"doi\">10.1038/nmat3568</pub-id><pub-id pub-id-type=\"pmid\">23422720</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>AA</given-names></name><name><surname>Brenner</surname><given-names>MP</given-names></name><name><surname>Colwell</surname><given-names>LJ</given-names></name></person-group><article-title>Predicting protein-ligand affinity with a random matrix framework</article-title><source>Proc. Natl Acad. Sci. USA</source><year>2016</year><volume>113</volume><fpage>13564</fpage><pub-id pub-id-type=\"doi\">10.1073/pnas.1611138113</pub-id><pub-id pub-id-type=\"pmid\">27856761</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Deringer</surname><given-names>VL</given-names></name><etal/></person-group><article-title>Realistic atomistic structure of amorphous silicon from machine-learning-driven molecular dynamics</article-title><source>J. Phys. Chem. Lett.</source><year>2018</year><volume>9</volume><fpage>2879</fpage><pub-id pub-id-type=\"doi\">10.1021/acs.jpclett.8b00902</pub-id><pub-id pub-id-type=\"pmid\">29754489</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chmiela</surname><given-names>S</given-names></name><name><surname>Sauceda</surname><given-names>HE</given-names></name><name><surname>Müller</surname><given-names>K-R</given-names></name><name><surname>Tkatchenko</surname><given-names>A</given-names></name></person-group><article-title>Towards exact molecular dynamics simulations with machine-learned force fields</article-title><source>Nat. Commun.</source><year>2018</year><volume>9</volume><fpage>3887</fpage><pub-id pub-id-type=\"doi\">10.1038/s41467-018-06169-2</pub-id><pub-id pub-id-type=\"pmid\">30250077</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schütt</surname><given-names>KT</given-names></name><name><surname>Arbabzadah</surname><given-names>F</given-names></name><name><surname>Chmiela</surname><given-names>S</given-names></name><name><surname>Müller</surname><given-names>KR</given-names></name><name><surname>Tkatchenko</surname><given-names>A</given-names></name></person-group><article-title>Quantum-chemical insights from deep tensor neural networks</article-title><source>Nat. Commun.</source><year>2017</year><volume>8</volume><fpage>13890</fpage><pub-id pub-id-type=\"doi\">10.1038/ncomms13890</pub-id><pub-id pub-id-type=\"pmid\">28067221</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zubatyuk</surname><given-names>R</given-names></name><name><surname>Smith</surname><given-names>JS</given-names></name><name><surname>Leszczynski</surname><given-names>J</given-names></name><name><surname>Isayev</surname><given-names>O</given-names></name></person-group><article-title>Accurate and transferable multitask prediction of chemical properties with an atoms-in-molecules neural network</article-title><source>Sci. Adv.</source><year>2019</year><volume>5</volume><fpage>eaav6490</fpage><pub-id pub-id-type=\"doi\">10.1126/sciadv.aav6490</pub-id><pub-id pub-id-type=\"pmid\">31448325</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ramakrishnan</surname><given-names>R</given-names></name><name><surname>Dral</surname><given-names>P</given-names></name><name><surname>Rupp</surname><given-names>M</given-names></name><name><surname>vonLilienfeld</surname><given-names>OA</given-names></name></person-group><article-title>Quantum chemistry structures and properties of 134 kilo molecules</article-title><source>Sci. Data</source><year>2014</year><volume>1</volume><fpage>140022</fpage><pub-id pub-id-type=\"doi\">10.1038/sdata.2014.22</pub-id><pub-id pub-id-type=\"pmid\">25977779</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Smith</surname><given-names>JS</given-names></name><etal/></person-group><article-title>The ANI-1ccx and ANI-1x data sets, coupled-cluster and density functional theory properties for molecules</article-title><source>Sci. Data</source><year>2020</year><volume>7</volume><fpage>134</fpage><pub-id pub-id-type=\"doi\">10.1038/s41597-020-0473-z</pub-id><pub-id pub-id-type=\"pmid\">32358545</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><mixed-citation publication-type=\"other\">Hoja, J. et al. QM7-X: A Comprehensive Dataset of Quantum-Mechanical Properties Spanning Equilibrium and Non-equilibrium Structures of Small Organic Molecules. <ext-link ext-link-type=\"uri\" xlink:href=\"http://arXiv.org/abs/2006.15139\">http://arXiv.org/abs/2006.15139</ext-link>.</mixed-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Smith</surname><given-names>JS</given-names></name><name><surname>Isayev</surname><given-names>O</given-names></name><name><surname>Roitberg</surname><given-names>AE</given-names></name></person-group><article-title>ANI-1: An extensible neural network potential with DFT accuracy at force field computational cost</article-title><source>Chem. Sci.</source><year>2017</year><volume>8</volume><fpage>3192</fpage><pub-id pub-id-type=\"doi\">10.1039/C6SC05720A</pub-id><pub-id pub-id-type=\"pmid\">28507695</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><mixed-citation publication-type=\"other\">Stöhr, M., Sandonas, L. M. & Tkatchenko, A. Accurate Many-Body Repulsive Potentials for Density-Functional Tight-Binding from Deep Tensor Neural Networks. <ext-link ext-link-type=\"uri\" xlink:href=\"http://arXiv.org/abs/2006.10429\">http://arXiv.org/abs/2006.10429</ext-link>; <italic>J. Phys. Chem. Lett.</italic>, in print (2020); 10.1021/acs.jpclett.0c01307.</mixed-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Panosetti</surname><given-names>C</given-names></name><name><surname>Engelmann</surname><given-names>A</given-names></name><name><surname>Nemec</surname><given-names>L</given-names></name><name><surname>Reuter</surname><given-names>K</given-names></name><name><surname>Margraf</surname><given-names>J</given-names></name></person-group><article-title>Learning to use the force: Fitting repulsive potentials in density-functional tight-binding with gaussian process regression</article-title><source>J. Chem. Theory Comput.</source><year>2020</year><volume>16</volume><fpage>2181</fpage><pub-id pub-id-type=\"doi\">10.1021/acs.jctc.9b00975</pub-id><pub-id pub-id-type=\"pmid\">32155065</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bartók</surname><given-names>AP</given-names></name><name><surname>Payne</surname><given-names>MC</given-names></name><name><surname>Kondor</surname><given-names>R</given-names></name><name><surname>Csányi</surname><given-names>G</given-names></name></person-group><article-title>Gaussian approximation potentials: the accuracy of quantum mechanics, without the electrons</article-title><source>Phys. Rev. Lett.</source><year>2010</year><volume>104</volume><fpage>136403</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevLett.104.136403</pub-id><pub-id pub-id-type=\"pmid\">20481899</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gómez-Bombarelli</surname><given-names>R</given-names></name><etal/></person-group><article-title>Design of efficient molecular organic light-emitting diodes by a high-throughput virtual screening and experimental approach</article-title><source>Nat. Mater.</source><year>2016</year><volume>15</volume><fpage>1120</fpage><pub-id pub-id-type=\"doi\">10.1038/nmat4717</pub-id><pub-id pub-id-type=\"pmid\">27500805</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Er</surname><given-names>S</given-names></name><name><surname>Suh</surname><given-names>C</given-names></name><name><surname>Marshak</surname><given-names>MP</given-names></name><name><surname>Aspuru-Guzik</surname><given-names>A</given-names></name></person-group><article-title>Computational design of molecules for an all-quinone redox flow battery</article-title><source>Chem. Sci.</source><year>2015</year><volume>6</volume><fpage>885</fpage><pub-id pub-id-type=\"doi\">10.1039/C4SC03030C</pub-id><pub-id pub-id-type=\"pmid\">29560173</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stokes</surname><given-names>JM</given-names></name><etal/></person-group><article-title>A deep learning approach to antibiotic discovery</article-title><source>Cell</source><year>2020</year><volume>180</volume><fpage>688</fpage><pub-id pub-id-type=\"doi\">10.1016/j.cell.2020.01.021</pub-id><pub-id pub-id-type=\"pmid\">32084340</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Segler</surname><given-names>MHS</given-names></name><name><surname>Preuss</surname><given-names>M</given-names></name><name><surname>Waller</surname><given-names>MP</given-names></name></person-group><article-title>Planning chemical syntheses with deep neural networks and symbolic AI</article-title><source>Nature</source><year>2018</year><volume>555</volume><fpage>604</fpage><pub-id pub-id-type=\"doi\">10.1038/nature25978</pub-id><pub-id pub-id-type=\"pmid\">29595767</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fedorov</surname><given-names>DV</given-names></name><name><surname>Sadhukhan</surname><given-names>M</given-names></name><name><surname>Stöhr</surname><given-names>M</given-names></name><name><surname>Tkatchenko</surname><given-names>A</given-names></name></person-group><article-title>Quantum-mechanical relation between atomic dipole polarizability and the van der Waals radius</article-title><source>Phys. Rev. Lett.</source><year>2018</year><volume>121</volume><fpage>183401</fpage><pub-id pub-id-type=\"doi\">10.1103/PhysRevLett.121.183401</pub-id><pub-id pub-id-type=\"pmid\">30444421</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wilkins</surname><given-names>DM</given-names></name><etal/></person-group><article-title>Accurate molecular polarizabilities with coupled cluster theory and machine learning</article-title><source>Proc. Natl Acad. Sci. USA</source><year>2019</year><volume>116</volume><fpage>3401</fpage><pub-id pub-id-type=\"doi\">10.1073/pnas.1816132116</pub-id><pub-id pub-id-type=\"pmid\">30733292</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807835</article-id><article-id pub-id-type=\"pmc\">PMC7431575</article-id><article-id pub-id-type=\"publisher-id\">70802</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70802-8</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Methylglyoxal inhibits nuclear division through alterations in vacuolar morphology and accumulation of Atg18 on the vacuolar membrane in <italic>Saccharomyces cerevisiae</italic></article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Nomura</surname><given-names>Wataru</given-names></name><address><email>nomura.wataru.4r@kyoto-u.ac.jp</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Aoki</surname><given-names>Miho</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Inoue</surname><given-names>Yoshiharu</given-names></name><address><email>y_inoue@kais.kyoto-u.ac.jp</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.258799.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0372 2033</institution-id><institution>Laboratory of Molecular Microbiology, Division of Applied Life Sciences, Graduate School of Agriculture, </institution><institution>Kyoto University, </institution></institution-wrap>Uji, Kyoto, 611-0011 Japan </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.258799.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0372 2033</institution-id><institution>Present Address: Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, </institution><institution>Kyoto University, </institution></institution-wrap>Uji, Kyoto, 611-0011 Japan </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13887</elocation-id><history><date date-type=\"received\"><day>7</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>3</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Methylglyoxal (MG) is a natural metabolite derived from glycolysis, and it inhibits the growth of cells in all kinds of organisms. We recently reported that MG inhibits nuclear division in <italic>Saccharomyces cerevisiae</italic>. However, the mechanism by which MG blocks nuclear division remains unclear. Here, we show that increase in the levels of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)<italic>P</italic><sub>2</sub>) is crucial for the inhibitory effects of MG on nuclear division, and the deletion of PtdIns(3,5)<italic>P</italic><sub>2</sub>-effector Atg18 alleviated the MG-mediated inhibitory effects. Previously, we reported that MG altered morphology of the vacuole to a single swelling form, where PtdIns(3,5)<italic>P</italic><sub>2</sub> accumulates. The changes in the vacuolar morphology were also needed by MG to exert its inhibitory effects on nuclear division. The known checkpoint machinery, including the spindle assembly checkpoint and morphological checkpoint, are not involved in the blockade of nuclear division by MG. Our results suggest that both the accumulation of Atg18 on the vacuolar membrane and alterations in vacuolar morphology are necessary for the MG-induced inhibition of nuclear division.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Cellular microbiology</kwd><kwd>Stress signalling</kwd></kwd-group><funding-group><award-group><funding-source><institution>JSPS KAKENHI</institution></funding-source><award-id>19K05949</award-id><award-id>18H02168</award-id><principal-award-recipient><name><surname>Nomura</surname><given-names>Wataru</given-names></name><name><surname>Inoue</surname><given-names>Yoshiharu</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>Lotte Shigemitsu Prize, Japan</institution></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par6\">The inheritance of chromosomes is a critical biological event for all organisms. Eukaryotic cells contain chromosomal DNA in the nucleus, an organelle enveloped in a double lipid bilayer (nuclear membrane). Unlike higher eukaryotes, the nuclear membrane in yeast cells is not degraded during mitosis (closed mitosis); therefore, the nucleus is transported to the daughter cell during the mitotic process<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. The budding yeast <italic>Saccharomyces cerevisiae</italic> is excellent for observing this process. A bud (daughter cell) begins to emerge from the mother cell at S phase and grows larger at M phase. The nuclear membrane is extended, a portion of the nucleus penetrates the bud, and then the nucleus is separated in the late M phase. Consequently, a set of chromosomes is inherited to the bud<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>.</p><p id=\"Par7\">Methylglyoxal (MG) is a typical 2-oxoaldehyde derived from glycolysis<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>–<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. Despite being a natural metabolite, MG at high concentrations inhibits the growth of cells in all types of organisms; however, precisely how it exerts its toxicity is unclear. Since MG enhances the frequency of sister chromatid exchange and endoreduplication in CHO-AUXBI cells<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>, it may have some effect on chromatin maintenance and inheritance. However, treatment with MG causes cell cycle arrest at G1 phase in human HL60 cells. Meanwhile, Kani et al.<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup> have reported that MG causes oxidative stress, thereby inducing G2/M arrest in HEK293 cells. However, whether treatment with MG can elicit oxidative stress depends on the cell lines used; therefore, it is unclear if MG per se is the cause of cell cycle arrest. We have reported that MG does not cause oxidative stress in <italic>S</italic>. <italic>cerevisiae</italic>, although at moderate concentrations it causes temporal growth arrest without decreasing the cell viability<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. In addition, we recently demonstrated that the depolarization of the actin cytoskeleton and blockade of the nuclear division by MG treatment are the reasons for MG toxicity<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. These phenotypes were also observed when a mutant defective in MG metabolizing enzymes was treated with dihydroxyacetone, which caused an increase in the intracellular levels of MG<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Therefore, intracellular MG seems to be involved in the inhibition of nuclear division; however, the mechanism by which MG blocks the transportation of nucleus to the daughter cells remains unknown.</p><p id=\"Par8\">Our recent study on the screening of MG-sensitive mutants showed that the mutants defective for the synthesis pathway of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)<italic>P</italic><sub>2</sub>), a minor species of phosphoinositide, exhibited higher susceptibility to MG<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. MG increases the levels of PtdIns(3,5)<italic>P</italic><sub>2</sub> and simultaneously induces morphological changes in the vacuoles in which PtdIns(3,5)<italic>P</italic><sub>2</sub> is predominantly distributed<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. In this study, we show that the alterations in the vacuolar morphology and increase in the levels of PtdIns(3,5)<italic>P</italic><sub>2</sub> are required for the MG-induced inhibition of nuclear division, and the PtdIns(3,5)<italic>P</italic><sub>2</sub> effector protein Atg18 is involved in the inhibitory machinery. The checkpoint machineries involved in the cell cycle arrest at G2/M in <italic>S. cerevisiae</italic>, i.e. the spindle assembly checkpoint and morphological checkpoint, were not crucial for the MG-induced inhibition of nuclear division. These results indicate that alterations in the vacuolar morphology and accumulation of Atg18 on the vacuolar membrane are necessary events for the MG-induced inhibition of nuclear division, which occurs independently of the activation of known checkpoints.</p></sec><sec id=\"Sec2\"><title>Results</title><sec id=\"Sec3\"><title>Effector of PtdIns(3,5)<italic>P</italic><sub>2</sub>, Atg18, is necessary for the MG-induced inhibition of nuclear division</title><p id=\"Par9\">MG blocks the nuclear division and induces an accumulation of undivided nuclei in the wild type cells<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B). To identify the mechanism involved in the inhibitory effects of MG on nuclear division, we focused on the involvement of PtdIns(3,5)<italic>P</italic><sub>2</sub> because its levels are increased by MG treatment. Although the total cellular abundance of PtdIns(3,5)<italic>P</italic><sub>2</sub> is low, it is predominantly distributed in the vacuolar membrane<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>, and is involved in the maintenance of vacuolar morphology and its functions<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>–<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. The synthesis of PtdIns(3,5)<italic>P</italic><sub>2</sub> is catalysed by PtdIns3<italic>P</italic> 5-kinase encoded by <italic>FAB1</italic>, and Fab1 interacts with some its regulators, Vac7, Vac14, and Fig4, which together form the Fab1 complex<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref>–<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup> (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>A). MG increases the levels of PtdIns(3,5)<italic>P</italic><sub>2</sub> at the vacuolar membrane in a Fab1 complex-dependent manner<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. We examined whether the increase in the levels of PtdIns(3,5)<italic>P</italic><sub>2</sub> is involved in the MG-induced inhibition of nuclear division. The results obtained showed that the nuclear division was hardly inhibited in the Fab1 complex mutants<italic>, vac14</italic>∆ and <italic>Fig4</italic>∆, in the presence of MG (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B), suggesting that the increase in the levels of PtdIns(3,5)<italic>P</italic><sub>2</sub> is required for the MG-induced inhibition of nuclear division.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Atg18 is involved in MG-induced inhibition of nuclear division. (<bold>A</bold>) Model of PtdIns(3,5)<italic>P</italic><sub><italic>2</italic></sub> synthesis on vacuolar membrane by Fab1 complex. MG activates the pathway of PtdIns(3,5)<italic>P</italic><sub><italic>2</italic></sub> synthesis. (<bold>B</bold>) Wild type, <italic>vac14</italic>∆, and <italic>fig4</italic>∆ cells were cultured in SD medium till <italic>A</italic><sub>610</sub> = 0.3–0.5, and were treated with 10 mM MG for 90 min. The inhibition rate of nuclear division was determined by evaluating the status of the nuclei (stained with Hoechst 33342) in cells having a large bud (bud diameter approximately two-thirds of that of the mother cell). Data are from three independent experiments (mean ± standard deviation), and more than 100 cells were counted for each experiment. (<bold>C</bold>) Wild type, <italic>atg18</italic>∆, <italic>atg21</italic>∆, <italic>hsv2</italic>∆, <italic>ent3</italic>∆<italic>ent5</italic>∆, and <italic>tup1</italic>∆ cells were cultured in SD medium until <italic>A</italic><sub>610</sub> = 0.3–0.5, and were treated with 10 mM MG for 90 min. The inhibition rate of nuclear division was determined as described in (<bold>B</bold>).</p></caption><graphic xlink:href=\"41598_2020_70802_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par10\">To date, 6 PtdIns(3,5)<italic>P</italic><sub>2</sub>-binding proteins (Atg18, Atg21, Hsv2, Ent3, Ent5, and Tup1) have been identified in <italic>S. cerevisiae</italic><sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref>–<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. Ent3 and Ent5 have redundant functions. Since an increase in the levels of PtdIns(3,5)<italic>P</italic><sub>2</sub> following treatment with MG appeared to be involved in the inhibition of nuclear division (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B), we determined whether these PtdIns(3,5)<italic>P</italic><sub>2</sub> effectors participated in the MG-induced accumulation of undivided nuclei. As shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>C, the deletion of <italic>ATG18</italic> suppressed the inhibitory effects of MG on nuclear division, whereas those of <italic>ATG21</italic>, <italic>HSV2</italic>, <italic>ENT3</italic>, <italic>ENT5</italic>, and <italic>TUP1</italic> did not.</p></sec><sec id=\"Sec4\"><title>Effect of mutated Atg18 localizing artificially at the vacuole on the blockade of nuclear division</title><p id=\"Par11\">Atg18 is well known as a core component for the vesicle formation during autophagy<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup> and its role in the regulation of vacuole fission<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. In the microscopy studies with GFP fusion protein, Atg18 was observed in the limited area of the vacuolar membrane as well as in the punctate structures<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>,<xref ref-type=\"bibr\" rid=\"CR29\">29</xref>,<xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. We have shown that MG enhanced the accumulation of Atg18 on the vacuolar membrane in accordance with the increase in the levels of PtdIns(3,5)<italic>P</italic><sub>2</sub>, and this effect was abrogated in <italic>vac14</italic>∆ cells<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup> (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A). These findings raised the possibility that the recruitment of Atg18 to the vacuolar membrane by an increasing concentration of PtdIns(3,5)<italic>P</italic><sub>2</sub> in the vacuolar membrane is the cause of MG-induced inhibition of nuclear division. Hence, the suppressive effects observed in the mutants of PtdIns(3,5)<italic>P</italic><sub>2</sub>, with decreased synthesis of PtdIns(3,5)<italic>P</italic><sub>2,</sub> on the inhibition of nuclear division might be abrogated when Atg18 can be artificially targeted to the vacuolar membrane. It is reported that a fusion protein GFP-Atg18-ALP is constitutively associated with the vacuole<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. We then examined whether the inhibition of nuclear division is induced by expressing GFP-Atg18-ALP in <italic>vac14</italic>∆ cells, where PtdIns(3,5)<italic>P</italic><sub>2</sub> is hardly detected. As shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>B, GFP-Atg18-ALP localized at the vacuolar membrane in <italic>vac14</italic>∆ cells, although the accumulation of GFP-Atg18 on the vacuolar membrane was not clearly observed. The vacuolar localization of GFP-Atg18-ALP was not changed following the treatment with MG (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>B). In <italic>vac14</italic>∆ cells expressing GFP-Atg18-ALP, the proportion of cells with undivided nuclei did not significantly increase in the presence of MG (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>C), indicating that the suppressive effects observed in the mutants of PtdIns(3,5)<italic>P</italic><sub>2</sub> synthesis on the inhibition of nuclear division were not abrogated by the artificial localization of Atg18 to the vacuolar membrane. Atg18 binds to phosphoinositides via the conserved FRRG motif, and mutation of the FRRG motif in Atg18, Atg18<sup>FTTG</sup>, abolishes the capability of binding to PtdIns(3,5)<italic>P</italic><sub>2</sub>, which loses its localization to the vacuolar membrane and punctate structures<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. We further verified whether the binding of Atg18 to PtdIns(3,5)<italic>P</italic><sub>2</sub> is necessary for its contribution to the MG-induced inhibition of nuclear division using an Atg18<sup>FTTG</sup> mutant. As shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>D, Atg18<sup>FTTG</sup> was not able to revert the suppressive effect of MG on the nuclear division in <italic>atg18</italic>∆ cells. These results suggest that the accumulation of Atg18 on the vacuolar membrane through the binding to PtdIns(3,5)<italic>P</italic><sub>2</sub> is necessary for the MG-induced inhibition of nuclear division.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Effect of mutated Atg18 localizing artificially at vacuole on the blockade of nuclear division. (<bold>A</bold>) Model showing that MG facilitates the accumulation of Atg18 on the vacuolar membrane and causes alterations in the vacuolar morphology. (<bold>B</bold>) <italic>vac14</italic>∆ cells carrying pRS415 (vector), pRS415-MET25p-GFP-Atg18 (GFP-Atg18), or pRS415-MET25p-GFP-Atg18-ALP (GFP-Atg18-ALP) were cultured in SD medium until <italic>A</italic><sub>610</sub> = 0.3–0.5. Atg18-GFP and the morphology of the vacuole (FM4-64) were observed using a fluorescence microscope. Bar, 5 µm. (<bold>C</bold>) <italic>vac14</italic>∆ cells carrying an empty vector, GFP-Atg18, or GFP-Atg18-ALP were cultured in SD medium until <italic>A</italic><sub>610</sub> = 0.3–0.5, and were treated with 10 mM MG for 90 min. The rate of nuclear division was determined as described in the legend for Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B. (<bold>D</bold>) <italic>atg18</italic>∆ cells carrying an pRS416 (vector), pRS416-<italic>ATG18</italic> or pRS416-<italic>ATG18</italic><sup><italic>FTTG</italic></sup> were cultured in SD medium until <italic>A</italic><sub>610</sub> = 0.3–0.5, and were treated with 10 mM MG for 90 min. The rate of nuclear division was determined as described in the legend for Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B. (<bold>E</bold>) Wild type, <italic>vps41</italic>∆, and <italic>vam3</italic>∆ cells were cultured in SD medium until <italic>A</italic><sub>610</sub> = 0.3–0.5, and were treated with 10 mM MG for 90 min. The inhibition rate of nuclear division was determined as described in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B.</p></caption><graphic xlink:href=\"41598_2020_70802_Fig2_HTML\" id=\"MO3\"/></fig></p></sec><sec id=\"Sec5\"><title>Changes in vacuolar morphology are involved in the MG-induced inhibition of nuclear division</title><p id=\"Par12\">Wild type cells predominantly have 2–4 fragmented vacuoles, while <italic>vac14</italic>∆ mutant cells have a single grossly enlarged vacuole<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. In Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>B, the expression of GFP-Atg18-ALP led to a striking fragmentation of the vacuole, and this phenotype was consistent with that shown in a previous study<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. We recently showed that MG changed the vacuolar morphology to a single swelling form<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup> (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A), and the MG-induced vacuolar swelling did not occur in <italic>vps41</italic>∆ and <italic>vam3</italic>∆ mutants, in which the vacuolar fusion is defective<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. These findings raise the possibility that the changes in the vacuolar morphology are also necessary for the MG-induced inhibition of nuclear division. Therefore, we investigated the effect of MG on nuclear division in these mutants. As shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>E, the proportion of cells which possess undivided nuclei did not significantly increase after treatment with MG in <italic>vps41</italic>∆ and <italic>vam3</italic>∆ cells, suggesting that the vacuolar swelling in addition to an increase in PtdIns(3,5)<italic>P</italic><sub>2</sub> and the vacuolar localization of Atg18 is a prerequisite for the MG-induced inhibition of nuclear division.</p></sec><sec id=\"Sec6\"><title>MG does not affect the formation of microtubules or duplication of spindle pole bodies</title><p id=\"Par13\">We recently reported that in association with the inhibition of nuclear division, the nuclear morphology changed from a globate shape to one with a central depression aligned with the mother-bud axis, which we refer as a “jellybean-like shape” of the nucleus, following treatment with MG<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. The jellybean-like shaped nucleus did not enter into the bud growing larger because MG seemed to have arrested the cell cycle at the G2/M transition with respect to the nuclear division<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Microtubules play a key role in the nuclear dynamics throughout the cell cycle<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. In <italic>S</italic>. <italic>cerevisiae</italic>, spindle pole bodies (SPBs) are the microtubule-organizing centres that are necessary for the nucleation and organization of microtubule arrays<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. Since <italic>S. cerevisiae</italic> undergoes closed mitosis, SPBs are embedded in the nuclear envelope throughout the cell cycle. SPBs are duplicated by a conservative mechanism at G1/S transition. Cells in preanaphase and anaphase contain two SPBs aligned with the mother-bud axis, which defines the direction of nuclear division. The nuclear microtubules (spindles) are organized toward the nucleus from SPBs. So, we determined the effects of MG on the formation of SPBs and microtubules. Cells in the early log phase were treated with nocodazole for 180 min to collapse the microtubules, and were then released to fresh medium with or without MG. We observed SPBs and microtubules using RFP (DsRed)-tagged Spc110, an inner plaque SPB component, and GFP-tagged Tub1, α-tubulin, respectively. As shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>A, SPBs were duplicated after 30 min of releasing cells to fresh medium without MG, and telophase spindle elongation was observed as the consequence of nuclear division. The duplication of SPBs and spindle formation and its orientation (aligned with the mother-bud axis) were normal in the presence of MG (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>A); however, the length of spindles was shorter because of the inhibition of nuclear division. These results suggest that MG is unlikely to affect the formation of spindles or duplication of SPBs.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Effect of MG on microtubule organization. (<bold>A</bold>) Cells (YPH250) carrying both <italic>TUB1-GFP</italic> and <italic>SPC110-RFP</italic> were cultured in SD medium until <italic>A</italic><sub>610</sub> = 0.3, harvested by centrifugation, and suspended in YPD medium containing 6 µg/ml nocodazole. After 180 min, cells were suspended in fresh SD medium, with or without 10 mM MG. After 30 min, SPB (Spc110-RFP) and microtubules (Tub1-GFP) were observed using a fluorescence microscope. Bar, 5 µm. (<bold>B</bold>) The wild-type (YPH250) and <italic>mad2</italic>∆ mutant cells were cultured in SD medium until <italic>A</italic><sub>610</sub> = 0.3–0.5, and were treated with 10 mM MG for 90 min. The inhibition rate of nuclear division was determined as described in the legend of Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B.</p></caption><graphic xlink:href=\"41598_2020_70802_Fig3_HTML\" id=\"MO4\"/></fig></p><p id=\"Par14\">The spindle assembly checkpoint ensures proper attachment between the spindles and kinetochores, and controls the fidelity of chromosome segregation<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. Hence, entry into anaphase is inhibited when spindles do not attach properly to the kinetochores. Mad2 is a component of the spindle assembly checkpoint complex that regulates entry of the cell cycle into anaphase<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>; therefore, cell cycle proceeds in <italic>mad2</italic>∆ cells even though spindles are not constructed adequately. As shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>B, the inhibition of nuclear division occurred following the treatment with MG, even in <italic>mad2</italic>∆ cells, suggesting that MG neither inhibits spindle formation nor activates the spindle assembly checkpoint.</p></sec><sec id=\"Sec7\"><title>The expression of constitutively active allele of <italic>PKC1</italic> or the disruption of <italic>SWE1</italic> alleviates the MG-induced inhibition of nuclear division</title><p id=\"Par15\"><italic>S. cerevisiae</italic> undergoes polarized growth, and the establishment of cell polarity that indicates the direction from the mother to the bud is crucial for it<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. Actin cytoskeleton is important for the establishment of cell polarity, and thereby a transportation of organelles to daughter cell during the polarized growth is warranted<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. A morphological checkpoint monitors actin organization<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>, and filamentous actin disorganization leading to the morphological checkpoint causes the phosphorylation of Tyr19 in Cdc28, a budding yeast homologue of the cyclin-dependent kinase Cdc2 that controls the timing of entry into mitosis<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>–<xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. Phosphorylation of Cdc28 at Tyr19 lowers the activity of the G2 cyclin-Cdc28 complex, which leads to the inhibition of entry into mitosis; consequently, the nuclear division is interrupted<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. Therefore, the activation of the morphological checkpoint induces G2/M cell cycle arrest<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>–<xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. We previously reported that MG induced the depolarization of actin patches, and the expression of a constitutively active allele of <italic>PKC1</italic> (<italic>PKC1</italic><sup><italic>R398P</italic></sup>) was partially able to repress the depolarization of the actin patches in cells treated with MG<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. As shown in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>A, an increase in the proportion of cells whose nuclei were neither divided nor transported into the bud following treatment with MG was suppressed when <italic>PKC1</italic><sup><italic>R398P</italic></sup> was introduced. We then explored the possibility that the morphological checkpoint participates in the MG-induced inhibition of nuclear division. To investigate this possibility, we determined the phosphorylation of Cdc28 at Tyr19 in cells treated with MG. As shown in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>B, phosphorylation occurred after 15 min of MG treatment. Hydroxyurea (HU) is well known to enhance the phosphorylation of Cdc28<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. The phosphorylation of Cdc28 is catalysed by the protein kinase Swe1, a homologue of <italic>S. cerevisiae</italic> Wee1<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. As expected, the phosphorylation of Cdc28 did not occur following treatment with MG in <italic>swe1</italic>∆ cells (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>C), indicating that MG causes the phosphorylation of Cdc28 at Tyr19 in a Swe1-dependent manner.<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Effect of MG on phosphorylation of Cdc28. (<bold>A</bold>) Cells (YPH250) carrying YCp50 (vector) or YCp50-<italic>PKC1</italic><sup><italic>R398P</italic></sup> were cultured in SD medium until <italic>A</italic><sub>610</sub> = 0.3, and were treated with 10 mM MG for 90 min. The inhibition rate of nuclear division was determined as described in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B. (<bold>B</bold>) Cells (DLY1) were cultured in SD medium until <italic>A</italic><sub>610</sub> = 0.3 and were treated with 10 mM MG for the prescribed time indicated in the figure. Phosphorylation levels of Cdc28 (p-Cdc28) and total protein expression levels of Cdc28 were determined using anti-phospho (Tyr19) Cdc28 antibodies and anti-Cdc2 antibodies, respectively. (<bold>C</bold>) Wild type (DLY1) and <italic>swe1</italic>∆ mutant (DLY1028) cells were cultured in SD medium until <italic>A</italic><sub>610</sub> = 0.3, and were treated with 10 mM MG for 30 min or 100 mM hydroxyurea (HU) for 120 min. The phosphorylation of Cdc28 was determined as described in (<bold>B</bold>). (<bold>D</bold>) Wild type (DLY1) and <italic>swe1</italic>∆ mutant (DLY1028) cells were cultured in SD medium until <italic>A</italic><sub>610</sub> = 0.3, and were treated with 10 mM MG for 90 min. The inhibition rate of nuclear division was determined as described in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B. (<bold>E</bold>) Wild type (W303-1B) and <italic>CDC28</italic><sup><italic>Y19F</italic></sup> mutant cells were cultured in SD medium until <italic>A</italic><sub>610</sub> = 0.3, and were treated with 10 mM MG for 90 min. The inhibition rate of nuclear division was determined as described in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B. (<bold>F</bold>) Cells (DLY1) carrying YCp50 (vector) or YCp50-<italic>PKC1</italic><sup><italic>R398P</italic></sup> at an early log-phase of growth were treated with 10 mM MG for the prescribed time as indicated in the figure, and the phosphorylation of Cdc28 was determined as described in (<bold>B</bold>). (<bold>G</bold>) <italic>swe1</italic>∆ cells (DLY1028) carrying YCp50 (vector), pKL2698, or YCp50-<italic>PKC1</italic><sup><italic>R398P</italic></sup> were treated with MG as described in (<bold>A</bold>). The inhibition rate of nuclear division was determined as described in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B. *<italic>p</italic> < 0.05; **<italic>p</italic> < 0.01.</p></caption><graphic xlink:href=\"41598_2020_70802_Fig4_HTML\" id=\"MO2\"/></fig></p><p id=\"Par16\">Next, we determined whether the disruption of <italic>SWE1</italic> alleviates the inhibitory effect of MG on nuclear division. As shown in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>D, the inhibitory effect of MG on the nuclear division in <italic>swe1</italic>∆ was slightly lower than that in wild type cells; however, the proportion of cells in which nuclear division was inhibited in the presence of MG did not decrease in cells carrying <italic>CDC28</italic><sup><italic>Y19F</italic></sup>, a non phosphorylatable allele of <italic>CDC28</italic> (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>E). The phosphorylation of Cdc28 occurred following the treatment with MG, even in cells introduced with <italic>PKC1</italic><sup><italic>R398P</italic></sup> (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>F). Furthermore, an additive effect between the expression of <italic>PKC1</italic><sup><italic>R398P</italic></sup> and the disruption of <italic>SWE1</italic> for the alleviation of MG-induced inhibition of nuclear division was observed (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>G). When the wild type allele of <italic>SWE1</italic> was reverted in the <italic>swe1</italic>∆ mutant, the proportion of cells with inhibited nuclear division was increased (<italic>swe1</italic>∆/<italic>SWE1</italic>: w/o MG, 22 ± 4%; w/ MG, 51 ± 3%) compared with that in <italic>swe1</italic>∆ cells carrying the vector alone (<italic>swe1</italic>∆/vector: w/o MG, 20 ± 4%; w/ MG, 37 ± 1%), and when <italic>PKC1</italic><sup><italic>R398P</italic></sup> was expressed in <italic>swe1</italic>∆ cells, the restoration of nuclear division that had been inhibited by MG occurred at a much greater extent compared with that in <italic>swe1</italic>∆ cells carrying the vector; i.e. the proportion of cells with inhibited nuclear division did not increase as much (<italic>swe1</italic>∆/<italic>PKC1</italic><sup><italic>R398P</italic></sup>: w/o MG, 18 ± 1%; w/ MG, 26 ± 1%) compared with the vector control. These results indicate that the phosphorylation of Cdc28 at Tyr19 does not commit to the MG-induced inhibition of nuclear division; the expression of <italic>PKC1</italic><sup><italic>R398P</italic></sup> and the disruption of <italic>SWE1</italic> independently contribute to the alleviation of inhibitory effect of MG on the nuclear division.</p></sec><sec id=\"Sec8\"><title>The expression of <italic>PKC1</italic><sup><italic>R398P</italic></sup> alleviates the MG-induced vacuolar swelling</title><p id=\"Par17\">MG-induced changes in the nuclear morphology were observed using nuclear membrane-located Nup116-GFP, a component of the nuclear pore complex, to the jellybean-like shape<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>A). In <italic>PKC1</italic><sup><italic>R398P</italic></sup>-expressing cells, we noticed that the emergence of the jellybean-like shaped nucleus following treatment with MG was partially repressed (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>A). The occurrence of the jellybean-like shaped nucleus was caused by MG-induced vacuolar swelling<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. We then observed the effects of the <italic>PKC1</italic><sup><italic>R398P</italic></sup> expression on the vacuolar morphology. The <italic>PKC1</italic><sup><italic>R398P</italic></sup>-expressing cells had fragmented vacuoles, and MG-induced vacuolar swelling in this strain was not so obvious as that in the wild type cells (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>B). The steady state levels of PtdIns(3,5)<italic>P</italic><sub>2</sub> were not affected by the expression of <italic>PKC1</italic><sup><italic>R398P</italic></sup>; however, the levels of which were slightly increased in the <italic>PKC1</italic><sup><italic>R398P</italic></sup>-expressed cells following the treatment with MG (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>). Consistent with this result, Atg18-GFP accumulated on the fragmented vacuolar membrane following treatment with MG in the <italic>PKC1</italic><sup><italic>R398P</italic></sup>-expressing cells (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>C). These findings indicate that the vacuolar fragmentation caused by the <italic>PKC1</italic><sup><italic>R398P</italic></sup> expression is involved in the suppression of the MG-induced inhibition of nuclear division for which the vacuolar swelling is a prerequisite.<fig id=\"Fig5\"><label>Figure 5</label><caption><p>Effect of the expression of <italic>PKC1</italic><sup><italic>R398P</italic></sup> on vacuolar morphology. (<bold>A</bold>) Cells (YPH250) carrying <italic>NUP116-GFP</italic> and either YCp50 (vector) or YCp50-<italic>PKC1</italic><sup><italic>R398P</italic></sup> were cultured in SD medium until <italic>A</italic><sub>610</sub> = 0.3 and were treated with 10 mM MG for 90 min. Nup116-GFP was observed using a fluorescence microscope. Bar, 5 µm. (<bold>B</bold>) Cells (YPH250) carrying YCp50 (vector) or YCp50-<italic>PKC1</italic><sup><italic>R398P</italic></sup> were cultured in SD medium until <italic>A</italic><sub>610</sub> = 0.3, and were treated with 10 mM MG for 90 min. The vacuolar membrane was stained with FM4-64. Bar, 5 µm. (<bold>C</bold>) Cells (YPH250) carrying <italic>ATG18-GFP</italic> and either pFL39 (vector) or pFL39-<italic>PKC1</italic><sup><italic>R398P</italic></sup> were cultured in SD medium until <italic>A</italic><sub>610</sub> = 0.3 and were treated with 10 mM MG for 90 min. Atg18-GFP and FM4-64 were observed using a fluorescence microscope. Bar, 5 µm.</p></caption><graphic xlink:href=\"41598_2020_70802_Fig5_HTML\" id=\"MO5\"/></fig><table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Levels of phosphatidylinositols in <italic>PKC</italic><sup><italic>R398P</italic></sup> cells.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\"/><th align=\"left\" colspan=\"2\"><bold>w/o MG</bold></th><th align=\"left\" colspan=\"2\"><bold>w/ MG</bold></th></tr><tr><th align=\"left\"><bold>vector</bold></th><th align=\"left\"><bold><italic>PKC1</italic></bold><sup><bold><italic>R398P</italic></bold></sup></th><th align=\"left\"><bold>vector</bold></th><th align=\"left\"><bold><italic>PKC1</italic></bold><sup><bold><italic>R398P</italic></bold></sup></th></tr></thead><tbody><tr><td align=\"left\">PtdIns3<italic>P</italic></td><td char=\".\" align=\"char\">0.97 ± 0.03</td><td char=\".\" align=\"char\">0.78 ± 0.05</td><td char=\".\" align=\"char\">0.96 ± 0.05</td><td char=\".\" align=\"char\">0.79 ± 0.03</td></tr><tr><td align=\"left\">PtdIns4<italic>P</italic></td><td char=\".\" align=\"char\">1.61 ± 0.05</td><td char=\".\" align=\"char\">1.57 ± 0.10</td><td char=\".\" align=\"char\">1.53 ± 0.04</td><td char=\".\" align=\"char\">1.39 ± 0.09</td></tr><tr><td align=\"left\">PtdIns(3,5)<italic>P</italic><sub>2</sub></td><td char=\".\" align=\"char\">0.023 ± 0.002</td><td char=\".\" align=\"char\">0.029 ± 0.014</td><td char=\".\" align=\"char\">0.055 ± 0.003</td><td char=\".\" align=\"char\">0.076 ± 0.02</td></tr><tr><td align=\"left\">PtdIns(4,5)<italic>P</italic><sub>2</sub></td><td char=\".\" align=\"char\">0.54 ± 0.07</td><td char=\".\" align=\"char\">0.54 ± 0.04</td><td char=\".\" align=\"char\">0.49 ± 0.08</td><td char=\".\" align=\"char\">0.48 ± 0.05</td></tr></tbody></table><table-wrap-foot><p>Wild type cells carrying YCp50 (vector) or YCp50-<italic>PKC1</italic><sup><italic>R398P</italic></sup> were treated with 10 mM MG for 60 min. The extraction and measurement of each phosphatidylinositol are described in the Methods section. The level of each phosphatidylinositol is shown as a percentage of the total levels of phosphatidylinositol (mean ± standard deviation, n = 3).</p></table-wrap-foot></table-wrap></p></sec></sec><sec id=\"Sec9\"><title>Discussion</title><p id=\"Par18\">MG inhibits the growth of cells in all organisms that have been examined so far; however, the underlying mechanism has not been fully elucidated. We recently reported that MG blocked the nuclear division in <italic>S. cerevisiae</italic>, implying that the blockade of nuclear division is one of the mechanisms by which MG arrests cell growth<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Even though the precise mechanism underlying the MG-induced inhibition of nuclear division has not been uncovered, we have identified that Atg18 is crucial for the inhibitory mechanism (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>C). In the present study, we have revealed that the inhibitory effect of MG on nuclear division was exerted through vacuolar swelling (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>E) and increase in the levels of PtdIns(3,5)<italic>P</italic><sub><italic>2</italic></sub> (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B), which facilitated the vacuolar localization of Atg18 (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>).<fig id=\"Fig6\"><label>Figure 6</label><caption><p>A model for the blockade of nuclear division by MG. Both the increase in levels of PtdIns(3,5)<italic>P</italic><sub>2</sub> and vacuolar swelling act as the primary signal for the MG-induced inhibition of nuclear division, and then Atg18 localized to the swollen vacuole through PtdIns(3,5)<italic>P</italic><sub>2</sub> commits to the blockage of nuclear division. Under that condition, the nuclear morphology changes to a jellybean-like shape. Nuc, nucleus. Vac, vacuole.</p></caption><graphic xlink:href=\"41598_2020_70802_Fig6_HTML\" id=\"MO6\"/></fig></p><p id=\"Par19\">Atg18 has a WD-40 repeat motif that shows binding affinity for PtdIns(3,5)<italic>P</italic><sub>2</sub><sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Previously, we reported that MG induces the accumulation of Atg18 at the vacuolar membrane through increased PtdIns(3,5)<italic>P</italic><sub>2</sub> levels on the vacuolar membrane<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. In Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>D, the blockade of nuclear division by MG was not observed in the <italic>atg18</italic>∆ cells expressing Atg18<sup>FTTG</sup> that is unable to bind PtdIns(3,5)<italic>P</italic><sub>2</sub> thereby losing its localization to the vacuolar membrane. It seems likely that the accumulation of Atg18 is sufficient for the inhibition of nuclear division; however, the expression of mutated Atg18 that artificially targeted at the vacuolar membrane in a PtdIns(3,5)<italic>P</italic><sub>2</sub>-independent manner did not enhance the inhibitory effect of nuclear division in <italic>vac14</italic>∆ cells (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>C), implying that the binding of Atg18 to PtdIns(3,5)<italic>P</italic><sub>2</sub> is a key to exert the inhibition of nuclear division. Meanwhile, the expression of mutated Atg18 caused morphological alterations in the vacuoles of <italic>vac14</italic>∆ cells, from a single large form to a fragmented form, which did not change to the swelling form by MG (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>B)<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. Therefore, non-significant effect of the expression of mutated Atg18 (Atg18-ALP) on the blockade of nuclear division may be due to the fragmentation of vacuoles, supporting our conclusion that both the accumulation of Atg18 at the vacuolar membrane and vacuolar swelling are necessary for the blockade of nuclear division by MG (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>). A recent study showed that Atg18 contributes to vacuolar fission<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, which may be the reason for the striking vacuolar fragmentation caused by the expression of vacuolar-localized mutant of Atg18. Atg18 is well known as a core component contributing to the vesicle formation during autophagy processes<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>; however, MG did not induce macroautophagy and micronucleophagy<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. Further investigations are needed to determine the molecular mechanism of Atg18-mediated inhibitory effects on nuclear division.</p><p id=\"Par20\">Upon MG-induced stress, cell cycle seems to be arrested at the G2/M phase in terms of nuclear division. Indeed, Tyr19 of Cdc28 was phosphorylated following the treatment with MG (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>B). Two checkpoints have so far been identified that can cause cell cycle arrest at the G2/M phase in <italic>S. cerevisiae</italic>: a spindle assembly checkpoint and a morphological checkpoint<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. Nocodazole activates the spindle assembly checkpoint through depolymerization of microtubules, thereby arresting the cell cycle at G2/M. In our study using the <italic>mad2</italic>∆ mutant, the spindle assembly checkpoint was not likely to be activated following treatment with MG (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>B). Meanwhile, when nocodazole-treated cells were released into fresh medium, the spindle was reorganized rapidly and immediately the cell cycle entered anaphase because DNA synthesis was completed during the period of nocodazole treatment. Consequently, cell cycle proceeded to telophase within 30 min after the release into the fresh medium (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>A). However, when nocodazole-treated cells were released into the MG-containing medium, cells in preanaphase, i. e. cells with G2-like short spindle and bud-bound SPB being not able to enter the bud, were accumulated even though the spindle exists at the bud neck, and the bud is large enough to accept the nucleus. Therefore, MG may influence some steps for the bud-bound SPB to cross the boundary of the bud neck.</p><p id=\"Par21\">Latrunculin A (Lat-A) activates the morphological checkpoint<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. When <italic>S. cerevisiae</italic> cells were treated with Lat-A, F actin was depolymerized within 15 min, which in turn served as a signal to phosphorylate Tyr19 of Cdc28 to arrest the cell cycle at G2/M<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. However, phosphorylation of Cdc28 occurred 120 min after treatment with Lat-A. Meanwhile, MG depolarized the actin patches from the buds in 30 min<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>; however, phosphorylation of Cdc28 at Tyr19 preceded (~ 15 min) the depolarization of the actin patches (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>B). The Lat-A-induced G2 arrest is dependent upon Mpk1 mitogen-activated protein (MAP) kinase cascade<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. We previously reported that MG induced the activation of Mpk1 MAP kinase cascade<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>, and the activation of Mpk1 MAP kinase cascade did not occur by MG derived from dihydroxyacetone, which increases the intracellular MG<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>, suggesting that the Mpk1 MAP kinase cascade is activated by extracellular MG. However, MG derived from dihydroxyacetone blocks nuclear division<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>, implying that the activation of Mpk1 MAP kinase cascade does not commit to the MG-induced inhibition of nuclear division. Since the mode of action of MG and Lat-A is different, i. e. MG depolarizes the actin cytoskeleton but does not depolymerize F actin whereas Lat-A depolymerizes the actin polymer, the morphological checkpoint may not be activated upon MG stress. In addition, the MG-induced inhibition of nuclear division occurred in <italic>CDC28</italic><sup><italic>Y19F</italic></sup> mutant cells in which the morphological checkpoint was bypassed (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>E). Therefore, MG-induced phosphorylation at Tyr19 of Cdc28 is not involved in the inhibition of nuclear division.</p><p id=\"Par22\">As an explanation for the attenuation of the blockade of nuclear division by MG in the <italic>PKC1</italic><sup><italic>R398P</italic></sup>-expressing cells, we found that MG could not cause vacuolar swelling in these cells (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>B). We reported earlier that the vacuolar swelling is necessary for the MG-induced morphological change in the nucleus to a jellybean-like shape<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. We found that the emergence of the jellybean-like shape of the nucleus following treatment with MG was partially repressed in <italic>PKC1</italic><sup><italic>R398P</italic></sup>-expressing cells (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>A), implying that the change in nuclear morphology to a jellybean-like shape contributes toward the blockade of nuclear division. Regarding the MG-induced inhibition of nuclear division, we would infer that it may not be a surveillance system but may imply the existence of “nuclear morphology checkpoint”. Further analysis of the MG-induced inhibition of nuclear division might lead to identification of the novel checkpoint mechanism.</p></sec><sec id=\"Sec10\"><title>Methods</title><sec id=\"Sec11\"><title>Media and reagents</title><p id=\"Par23\">The media used was SD (2% glucose, 0.67% yeast nitrogen base without amino acids) with appropriate amino acids and bases being added wherever necessary. MG was purchased from Sigma-Aldrich (MO, USA). When the <italic>A</italic><sub>610</sub> of the culture reached 0.3–0.5, 10 mM MG was added and the cells were incubated at 28ºC with reciprocal shaking. We verified that the cell growth was temporally arrested in the presence of 10 mM MG, while cell viability was maintained.</p></sec><sec id=\"Sec12\"><title>Strains</title><p id=\"Par24\">The yeast strains used in the present study are listed in Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>. The <italic>S. cerevisiae</italic> strains used had the YPH250, DLY1, and W303-1B backgrounds. Deletion mutants of <italic>ATG18</italic>, <italic>ATG21</italic>, <italic>HSV2</italic>, <italic>ENT3</italic>, <italic>ENT5</italic>, <italic>TUP1</italic>, and <italic>MAD2</italic> were constructed by PCR-based methods with <italic>KanMX</italic> or <italic>his5</italic><sup>+</sup> selection markers<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. Deletion constructs were amplified by PCR from BY4741-based deletion mutants (Invitrogen, Carlsbad, CA, USA).<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>List of <italic>Saccharomyces cerevisiae</italic> strains used in the present study.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Strain</th><th align=\"left\">Relevant genotype/description</th><th align=\"left\">Source/References</th></tr></thead><tbody><tr><td align=\"left\"><p>YPH250</p><p><italic>vac14</italic>∆</p><p><italic>Figure </italic><xref rid=\"Fig4\" ref-type=\"fig\"><italic>4</italic></xref>∆</p><p><italic>atg18</italic>∆</p><p><italic>atg21</italic>∆</p><p><italic>hsv2</italic>∆</p><p><italic>ent3</italic>∆<italic>ent5</italic>∆</p><p><italic>tup1</italic>∆</p><p><italic>mad2</italic>∆</p><p><italic>vps41</italic>∆</p><p><italic>vam3</italic>∆</p><p>Atg18-GFP</p><p>DLY1</p><p>DLY1028</p><p>W303-1B</p><p><italic>CDC28</italic><sup><italic>Y19F</italic></sup></p></td><td align=\"left\"><p><italic>MATa</italic>\n<italic>trp1</italic>-∆<italic>1 his3</italic>-∆<italic>200 leu2</italic>-∆<italic>1 lys2-801 ade2-101 ura3-52</italic></p><p>YPH250, <italic>vac14</italic>∆::<italic>his5</italic><sup>+</sup></p><p>YPH250, <italic>fig4</italic>∆::<italic>LEU2</italic></p><p>YPH250, <italic>atg18</italic>∆::<italic>his5</italic><sup>+</sup></p><p>YPH250, <italic>atg21</italic>∆::<italic>KanMX4</italic></p><p>YPH250, <italic>hsv2</italic>∆::<italic>KanMX4</italic></p><p>YPH250, <italic>ent3</italic>∆::<italic>his5</italic><sup>+</sup>\n<italic>ent5</italic>∆::<italic>KanMX4</italic></p><p>YPH250, <italic>tup1</italic>∆::<italic>KanMX4</italic></p><p>YPH250, <italic>mad2</italic>∆::<italic>KanMX4</italic></p><p>YPH250, <italic>vps41</italic>∆::<italic>KanMX4</italic></p><p>YPH250, <italic>vam3</italic>∆::<italic>KanMX4</italic></p><p>YPH250, <italic>ATG18-GFP</italic>::<italic>URA3</italic></p><p><italic>MATa</italic>\n<italic>ade1 his2 leu2-3</italic><italic>, </italic><italic>112 trp1-1</italic><sup><italic>a</italic></sup><italic> ura3ns bar1</italic></p><p>DLY1, <italic>swe1</italic>∆::<italic>LEU2</italic></p><p><italic>MAT</italic>α <italic>leu2</italic>-<italic>3</italic>, <italic>112 trp1</italic>-<italic>1 can1-100 ura3-1 ade2-1 his3</italic>-<italic>11</italic>, <italic>15</italic></p><p>W303-1B, <italic>CDC28</italic><sup><italic>Y19F</italic></sup>::<italic>HIS3</italic></p></td><td align=\"left\"><p>Lab stock</p><p><sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup></p><p><sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup></p><p>This study</p><p>This study</p><p>This study</p><p>This study</p><p>This study</p><p>This study</p><p><sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup></p><p><sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup></p><p><sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup></p><p><sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup></p><p><sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup></p><p><sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup></p><p>NBRP (BY22878)</p></td></tr></tbody></table></table-wrap></p><p id=\"Par25\">The allele <italic>SPC110</italic>::<italic>DsRed</italic>-<italic>KanMX</italic> of strain TYY115-2D<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup> was amplified by PCR using primers SPC110DsRed-F (5′-GATGATGAACTAGATCGTGATTACTACAAT-3′) and SPC110DsRed-R (5′-ATATACCACATACATAGATATACCCTACGT-3′). The PCR fragment was introduced into YPH250.</p><p id=\"Par26\">To construct the strains carrying <italic>TUB1</italic>::<italic>GFP</italic>-<italic>URA3</italic>, pAFS125<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup> was digested with StuI, and the linearized fragment was integrated into the locus of <italic>TUB1</italic>.</p><p id=\"Par27\">To add a GFP tag at the C terminus of Nup116, YIp-<italic>NUP116</italic>-GFP<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup> or pRS304-<italic>NUP116</italic>-GFP<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> was digested with EcoRI, and the linearized fragment was introduced into the locus of <italic>NUP116</italic>.</p></sec><sec id=\"Sec13\"><title>Plasmids</title><p id=\"Par28\">Plasmids used in this study are summarized in Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref>. The plasmids pRS415-<italic>GFP</italic>-<italic>ATG18</italic> and pRS415-<italic>GFP</italic>-<italic>ATG18</italic>-<italic>PHO8</italic> (ALP) were constructed as follows: the ORF of <italic>ATG18</italic> was amplified with the following primers: ATG18-F-SpeI (5′-TTTACTAGTATGTCTGATTCATCACCTACTATCAA-3′) and ATG18-R-XbaI-XhoI (5′-TTTCTCGAGTCATCTAGAATCCATCAAGATGGAAT-3′). The PCR product was digested with SpeI and XhoI, and the resultant fragment was introduced into the XbaI and XhoI sites of pRS415-<italic>MET25prom</italic>-<italic>GFP</italic> (laboratory stock), which is the plasmid for the expression of GFP-N-terminal tagged proteins under <italic>MET25</italic> promoter. The resultant plasmid (pRS415-<italic>GFP</italic>-<italic>ATG18</italic>) was digested with XbaI (the stop codon of <italic>ATG18</italic> was deleted), and the ORF of <italic>PHO8</italic>, which was amplified by PCR using primers PHO8-F-XbaI (5′-AAATCTAGAATGATGACTCACACATTACCAAGCGA-3′) and PHO8-R-XbaI (5′-AAATCTAGATCAGTTGGTCAACTCATGGTAGTATT-3′), was ligated in-frame into the XbaI site.<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>List of plasmids used in this study.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Plasmid</th><th align=\"left\">Description</th><th align=\"left\">Source/Reference</th></tr></thead><tbody><tr><td align=\"left\"><p>YCp50-<italic>PKC1</italic><sup><italic>R398P</italic></sup></p><p>pAFS125</p><p>YIp-<italic>NUP116</italic>-GFP</p><p>pRS304-<italic>NUP116</italic>-GFP</p><p>pKL2698</p><p>MET25p-GFP-<italic>ATG18</italic></p><p>MET25p-GFP-<italic>ATG18-ALP</italic></p><p>pRS416-<italic>ATG18</italic></p><p>pRS416-<italic>ATG18</italic><sup><italic>FTTG</italic></sup></p><p>pFL39-<italic>PKC1</italic><sup><italic>R398P</italic></sup></p></td><td align=\"left\"><p>YCp50 (CEN type, <italic>URA3</italic> marker) harboring <italic>PKC1</italic><sup><italic>R398P</italic></sup></p><p><italic>TUB1-GFP</italic> in an integrate-type, <italic>URA3</italic> marker plasmid</p><p><italic>NUP116-GFP</italic> in an integrate-type, <italic>URA3</italic> marker plasmid</p><p><italic>NUP116-GFP</italic> in an integrate-type, <italic>TRP1</italic> marker plasmid</p><p>pRS316 (CEN type, <italic>URA3</italic> marker) harboring <italic>SWE1</italic></p><p>pRS415 (CEN type, <italic>LEU2</italic> marker) harboring GFP-<italic>ATG18</italic></p><p>pRS415 harboring GFP-<italic>ATG18-ALP</italic></p><p>pRS416 (CEN type, <italic>URA3</italic> marker) harboring <italic>ATG18</italic></p><p>pRS416 harboring <italic>ATG18</italic><sup><italic>FTTG</italic></sup></p><p>pFL39 (CEN type, <italic>TRP1</italic> marker) harboring <italic>PKC1</italic><sup><italic>R398P</italic></sup></p></td><td align=\"left\"><p><sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup></p><p><sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup></p><p><sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup></p><p><sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup></p><p><sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup></p><p>This study</p><p>This study</p><p>This study</p><p>This study</p><p>This study</p></td></tr></tbody></table></table-wrap></p><p id=\"Par29\">The plasmids pRS416-<italic>ATG18</italic> was constructed as follows: the genomic fragment containing <italic>ATG18</italic> was amplified with the following primers: ATG18-F-XhoI (5′-TTGCTCGAGACCATCTGACACATGTACACAGTAAC-3′) and ATG18-R-SacI (5′-TTAGAGCTCCCTGATTCATCTATTAGCCGTATAGA-3′). The PCR product was digested with XhoI and SacI, and the resultant fragment was introduced into the XhoI and SacI sites of pRS416<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup>. pRS416-<italic>ATG18</italic><sup><italic>FTTG</italic></sup>, the substitution of two Arg residues (Arg285 and Arg286) with Thr, was constructed using a KOD -Plus- Mutagenesis Kit (TOYOBO, Osaka, Japan) with pRS416-<italic>ATG18</italic> as a template.</p><p id=\"Par30\">To construct pFL39-<italic>PKC1</italic><sup><italic>R398P</italic></sup>, YCp50-<italic>PKC1</italic><sup><italic>R398P</italic></sup><sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup> was digested with SphI. The DNA fragment containing a <italic>PKC1</italic><sup><italic>R398P</italic></sup> was cloned into the SphI site of pFL39.</p></sec><sec id=\"Sec14\"><title>Measurement of nuclear division</title><p id=\"Par31\">Cells were cultured in SD medium until the <italic>A</italic><sub>610</sub> reached 0.3–0.5, and 1 µg/ml Hoechst 33342 (Hoechst AG; Molecular Probes. Inc., Eugene, OR, USA) was added to stain the nucleus. To quantify the distribution of nuclei, cells with a large bud (bud diameter approximately two-thirds of that of the mother cell) containing the nucleus were counted after acquiring images with a fluorescence microscope, as reported previously<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Approximately 100–200 cells were counted for each experiment.</p></sec><sec id=\"Sec15\"><title>Vacuolar staining</title><p id=\"Par32\">Yeast vacuoles were visualized in vivo by labelling with FM4-64 (Molecular Probes. Inc., Eugene, OR, USA) as reported previously<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>.</p></sec><sec id=\"Sec16\"><title>Western blotting of Swe1</title><p id=\"Par33\">Preparation of total protein extracts using a Beads Smash 21 cell disrupter (Wakenyaku, Kyoto, Japan) were described previously<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. The phosphorylation status of Tyr19 of Cdc28 and the amount of Cdc28 protein were detected using anti-phospho-Cdc2 antibody (#9,111, Cell Signalling Technology, MA, USA) and anti-Cdc2 p34 antibody (sc-53, Santa Cruz Biotechnology, CA, USA), respectively. Immunoreactive bands were visualized using a peroxidase stain kit (Nacalai tesque, Kyoto, Japan).</p></sec><sec id=\"Sec17\"><title>Fluorescence microscopy</title><p id=\"Par34\">The fluorescence microscopes, BX51 and BX63 (Olympus equipped with the digital cameras DP70 (OLYMPUS, Tokyo, Japan) and ORCA-R2 (Hamamatsu Photonics, Shizuoka, Japan) were used.</p></sec><sec id=\"Sec18\"><title>Analysis of phosphatidylinositols</title><p id=\"Par35\">Phosphatidylinositols (PtdIns3<italic>P</italic><sub>,</sub> PtdIns4<italic>P</italic><sub>,</sub> PtdIns(3,5)<italic>P</italic><sub>2</sub>, and PtdIns(4,5)<italic>P</italic><sub>2</sub>) were separated with HPLC and were measured as reported previously<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>.</p></sec><sec id=\"Sec19\"><title>Statistical analysis</title><p id=\"Par36\">Data were presented as the means and standard deviation. The statistical significance of differences was evaluated using Student’s <italic>t</italic>-test. Differences with <italic>p</italic> values of < 0.05 were considered significant.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec20\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70802_MOESM1_ESM.pdf\"><caption><p>Supplementary file1.</p></caption></media></supplementary-material></p></sec></sec></body><back><glossary><title>Abbreviations</title><def-list><def-item><term>MG</term><def><p id=\"Par2\">Methylglyoxal</p></def></def-item><def-item><term>PtdIns(3,5)<italic>P</italic><sub>2</sub></term><def><p id=\"Par3\">Phosphatidylinositol 3,5-bisphosphate</p></def></def-item><def-item><term>SD</term><def><p id=\"Par4\">Synthetic dextrose</p></def></def-item><def-item><term>SPB</term><def><p id=\"Par5\">Spindle pole body</p></def></def-item></def-list></glossary><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70802-8.</p></sec><ack><title>Acknowledgements</title><p>We thank M. Hashimoto, Dr. S. Izawa, and Dr. K. Maeta for their technical support and helpful discussion. We are grateful to Drs. D. J. Lew, A. W. Murray, M. N. Hall, K. S. Lee, J. P. Hirsch, T. N. Davis, and the National Bio-Resource Project (NBRP), Japan, for providing the plasmids and yeast strains. This work was partly supported by JSPS KAKENHI, Grant Number: 19K05949 (to W.N.), JSPS KAKENHI, Grant Number: 18H02168 (to Y.I.), and Lotte Shigemitsu Prize, Japan (to W.N.).</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>W.N. and Y.I. designed the experiments, and W.N. and M.A. performed the experiments. W.N. and Y.I. analyzed the data and wrote the manuscript.</p></notes><notes notes-type=\"COI-statement\"><title>Competing interests</title><p>The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cavanaugh</surname><given-names>AM</given-names></name><name><surname>Jaspersen</surname><given-names>SL</given-names></name></person-group><article-title>Big lessons from little yeast: Budding and fission yeast centrosome structure, duplication, and function</article-title><source>Annu. Rev. Genet.</source><year>2017</year><volume>51</volume><fpage>361</fpage><lpage>383</lpage><pub-id pub-id-type=\"pmid\">28934593</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cid</surname><given-names>VJ</given-names></name><etal/></person-group><article-title>Orchestrating the cell cycle in yeast: sequential localization of key mitotic regulators at the spindle pole and the bud neck</article-title><source>Microbiology</source><year>2002</year><volume>148</volume><fpage>2647</fpage><lpage>2659</lpage><pub-id pub-id-type=\"pmid\">12213912</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Inoue</surname><given-names>Y</given-names></name><name><surname>Kimura</surname><given-names>A</given-names></name></person-group><article-title>Methylglyoxal and regulation of its metabolism in microorganisms</article-title><source>Adv. Microb. Physiol.</source><year>1995</year><volume>37</volume><fpage>177</fpage><lpage>227</lpage><pub-id pub-id-type=\"pmid\">8540421</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Inoue</surname><given-names>Y</given-names></name><name><surname>Maeta</surname><given-names>K</given-names></name><name><surname>Nomura</surname><given-names>W</given-names></name></person-group><article-title>Glyoxalase system in yeasts: structure, function, and physiology</article-title><source>Semin. Cell. Dev. Biol.</source><year>2011</year><volume>22</volume><fpage>278</fpage><lpage>284</lpage><pub-id pub-id-type=\"pmid\">21310260</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zemva</surname><given-names>J</given-names></name><etal/></person-group><article-title>Effects of the reactive metabolite methylglyoxal on cellular signalling, insulin action and metabolism—What we know in mammals and what we can learn from yeast</article-title><source>Exp. Clin. Endocrinol. Diabetes</source><year>2019</year><volume>127</volume><fpage>203</fpage><lpage>214</lpage><pub-id pub-id-type=\"pmid\">29421830</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tucker</surname><given-names>JD</given-names></name><name><surname>Taylor</surname><given-names>RT</given-names></name><name><surname>Christensen</surname><given-names>ML</given-names></name><name><surname>Strout</surname><given-names>CL</given-names></name><name><surname>Hanna</surname><given-names>ML</given-names></name><name><surname>Carrano</surname><given-names>AV</given-names></name></person-group><article-title>Cytogenetic response to 1,2-dicarbonyls and hydrogen peroxide in Chinese hamster ovary AUXB1 cells and human peripheral lymphocytes</article-title><source>Mutat. Res.</source><year>1989</year><volume>224</volume><fpage>269</fpage><lpage>279</lpage><pub-id pub-id-type=\"pmid\">2507914</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kani</surname><given-names>S</given-names></name><etal/></person-group><article-title>Chk2 kinase is required for methylglyoxal-induced G2/M cell-cycle checkpoint arrest: implication of cell-cycle checkpoint regulation in diabetic oxidative stress signaling</article-title><source>Genes Cells</source><year>2007</year><volume>12</volume><fpage>919</fpage><lpage>928</lpage><pub-id pub-id-type=\"pmid\">17663721</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maeta</surname><given-names>K</given-names></name><name><surname>Izawa</surname><given-names>S</given-names></name><name><surname>Inoue</surname><given-names>Y</given-names></name></person-group><article-title>Methylglyoxal, a metabolite derived from glycolysis, functions as a signal initiator of the high osmolarity glycerol—mitogen-activated protein kinase cascade and calcineurin/Crz1-mediated pathway in <italic>Saccharomyces cerevisiae</italic></article-title><source>J. Biol. Chem.</source><year>2005</year><volume>280</volume><fpage>253</fpage><lpage>260</lpage><pub-id pub-id-type=\"pmid\">15520007</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nomura</surname><given-names>W</given-names></name><name><surname>Inoue</surname><given-names>Y</given-names></name></person-group><article-title>Methylglyoxal activates the target of rapamycin complex 2-protein kinase C signaling pathway in <italic>Saccharomyces cerevisiae</italic></article-title><source>Mol. Cell. Biol.</source><year>2015</year><volume>35</volume><fpage>1269</fpage><lpage>1280</lpage><pub-id pub-id-type=\"pmid\">25624345</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nomura</surname><given-names>W</given-names></name><name><surname>Aoki</surname><given-names>M</given-names></name><name><surname>Inoue</surname><given-names>Y</given-names></name></person-group><article-title>Toxicity of dihydroxyacetone is exerted through the formation of methylglyoxal in <italic>Saccharomyces cerevisiae</italic>: effects on actin polarity and nuclear division</article-title><source>Biochem. J.</source><year>2018</year><volume>475</volume><fpage>2637</fpage><lpage>2652</lpage><pub-id pub-id-type=\"pmid\">30049894</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nomura</surname><given-names>W</given-names></name><name><surname>Maeta</surname><given-names>K</given-names></name><name><surname>Inoue</surname><given-names>Y</given-names></name></person-group><article-title>Phosphatidylinositol 3,5-bisphosphate is involved in methylglyoxal-induced activation of the Mpk1 mitogen-activated protein kinase cascade in <italic>Saccharomyces cerevisiae</italic></article-title><source>J. Biol. Chem.</source><year>2017</year><volume>292</volume><fpage>15039</fpage><lpage>15048</lpage><pub-id pub-id-type=\"pmid\">28743744</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McCartney</surname><given-names>AJ</given-names></name><name><surname>Zhang</surname><given-names>Y</given-names></name><name><surname>Weisman</surname><given-names>LS</given-names></name></person-group><article-title>Phosphatidylinositol 3,5-bisphosphate: low abundance, high significance</article-title><source>BioEssays</source><year>2014</year><volume>36</volume><fpage>52</fpage><lpage>64</lpage><pub-id pub-id-type=\"pmid\">24323921</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cooke</surname><given-names>FT</given-names></name><etal/></person-group><article-title>The stress-activated phosphatidylinositol 3-phosphate 5-kinase Fab1p is essential for vacuole function in S. cerevisiae</article-title><source>Curr. Biol.</source><year>1998</year><volume>8</volume><fpage>1219</fpage><lpage>1222</lpage><pub-id pub-id-type=\"pmid\">9811604</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gary</surname><given-names>JD</given-names></name><name><surname>Wurmser</surname><given-names>AE</given-names></name><name><surname>Bonangelino</surname><given-names>CJ</given-names></name><name><surname>Weisman</surname><given-names>LS</given-names></name><name><surname>Emr</surname><given-names>SD</given-names></name></person-group><article-title>Fab1p is essential for PtdIns(3)P 5-kinase activity and the maintenance of vacuolar size and membrane homeostasis</article-title><source>J. Cell Biol.</source><year>1998</year><volume>143</volume><fpage>65</fpage><lpage>79</lpage><pub-id pub-id-type=\"pmid\">9763421</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Efe</surname><given-names>JA</given-names></name><name><surname>Botelho</surname><given-names>RJ</given-names></name><name><surname>Emr</surname><given-names>SD</given-names></name></person-group><article-title>The Fab1 phosphatidylinositol kinase pathway in the regulation of vacuole morphology</article-title><source>Curr. Opin. Cell Biol.</source><year>2005</year><volume>17</volume><fpage>402</fpage><lpage>408</lpage><pub-id pub-id-type=\"pmid\">15975782</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>X</given-names></name><etal/></person-group><article-title>Genetically encoded fluorescent probe to visualize intracellular phosphatidylinositol 3,5-bisphosphate localization and dynamics</article-title><source>Proc. Natl. Acad. Sci. U. S. A.</source><year>2013</year><volume>110</volume><fpage>21165</fpage><lpage>21170</lpage><pub-id pub-id-type=\"pmid\">24324172</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dove</surname><given-names>SK</given-names></name><name><surname>Dong</surname><given-names>K</given-names></name><name><surname>Kobayashi</surname><given-names>T</given-names></name><name><surname>Williams</surname><given-names>FK</given-names></name><name><surname>Michell</surname><given-names>RH</given-names></name></person-group><article-title>Phosphatidylinositol 3,5-bisphosphate and Fab1p/PIKfyve underPPIn endo-lysosome function</article-title><source>Biochem. J.</source><year>2009</year><volume>419</volume><fpage>1</fpage><lpage>13</lpage><pub-id pub-id-type=\"pmid\">19272020</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bonangelino</surname><given-names>CJ</given-names></name><etal/></person-group><article-title>Osmotic stress-induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p</article-title><source>J. Cell Biol.</source><year>2002</year><volume>156</volume><fpage>1015</fpage><lpage>1028</lpage><pub-id pub-id-type=\"pmid\">11889142</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dove</surname><given-names>SK</given-names></name><etal/></person-group><article-title>Vac14 controls PtdIns(3,5)<italic>P</italic><sub>2</sub> synthesis and Fab1-dependent protein trafficking to the multivesicular body</article-title><source>Curr. Biol.</source><year>2002</year><volume>12</volume><fpage>885</fpage><lpage>893</lpage><pub-id pub-id-type=\"pmid\">12062051</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jin</surname><given-names>N</given-names></name><etal/></person-group><article-title>VAC14 nucleates a protein complex essential for the acute interconversion of PI3P and PI(3,5)P<sub>2</sub> in yeast and mouse</article-title><source>EMBO J.</source><year>2008</year><volume>27</volume><fpage>3221</fpage><lpage>3234</lpage><pub-id pub-id-type=\"pmid\">19037259</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Strahl</surname><given-names>T</given-names></name><name><surname>Thorner</surname><given-names>J</given-names></name></person-group><article-title>Synthesis and function of membrane phosphoinositides in budding yeast Saccharomyces cerevisiae</article-title><source>Biochim. Biophys. Acta</source><year>2007</year><volume>1771</volume><fpage>353</fpage><lpage>404</lpage><pub-id pub-id-type=\"pmid\">17382260</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dove</surname><given-names>SK</given-names></name><etal/></person-group><article-title>Svp1p defines a family of phosphatidylinositol 3,5-bisphosphate effectors</article-title><source>EMBO J.</source><year>2004</year><volume>23</volume><fpage>1922</fpage><lpage>1933</lpage><pub-id pub-id-type=\"pmid\">15103325</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Friant</surname><given-names>S</given-names></name><etal/></person-group><article-title>Ent3p Is a PtdIns(3,5)P<sub>2</sub> effector required for protein sorting to the multivesicular body</article-title><source>Dev. Cell</source><year>2003</year><volume>5</volume><fpage>499</fpage><lpage>511</lpage><pub-id pub-id-type=\"pmid\">12967568</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eugster</surname><given-names>A</given-names></name><name><surname>Pécheur</surname><given-names>EI</given-names></name><name><surname>Michel</surname><given-names>F</given-names></name><name><surname>Winsor</surname><given-names>B</given-names></name><name><surname>Letourneur</surname><given-names>F</given-names></name><name><surname>Friant</surname><given-names>S</given-names></name></person-group><article-title>Ent5p is required with Ent3p and Vps27p for ubiquitin-dependent protein sorting into the multivesicular body</article-title><source>Mol. Biol. Cell</source><year>2004</year><volume>15</volume><fpage>3031</fpage><lpage>3041</lpage><pub-id pub-id-type=\"pmid\">15107463</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Han</surname><given-names>B-K</given-names></name><name><surname>Emr</surname><given-names>SD</given-names></name></person-group><article-title>Phosphoinositide [PI(3,5)P<sub>2</sub>] lipid-dependent regulation of the general transcriptional regulator Tup1</article-title><source>Genes Dev.</source><year>2011</year><volume>25</volume><fpage>984</fpage><lpage>995</lpage><pub-id pub-id-type=\"pmid\">21536737</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Costaguta</surname><given-names>G</given-names></name><name><surname>Duncan</surname><given-names>MC</given-names></name><name><surname>Fernández</surname><given-names>GE</given-names></name><name><surname>Huang</surname><given-names>GH</given-names></name><name><surname>Payne</surname><given-names>GS</given-names></name></person-group><article-title>Distinct roles for TGN/endosome epsin-like adaptors Ent3p and Ent5p</article-title><source>Mol. Biol. Cell</source><year>2006</year><volume>17</volume><fpage>3907</fpage><lpage>3920</lpage><pub-id pub-id-type=\"pmid\">16790491</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Barth</surname><given-names>H</given-names></name><name><surname>Meiling-Wesse</surname><given-names>K</given-names></name><name><surname>Epple</surname><given-names>UD</given-names></name><name><surname>Thumm</surname><given-names>M</given-names></name></person-group><article-title>Autophagy and the cytoplasm to vacuole targeting pathway both require Aut10p</article-title><source>FEBS Lett.</source><year>2001</year><volume>508</volume><fpage>23</fpage><lpage>28</lpage><pub-id pub-id-type=\"pmid\">11707261</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gopaldass</surname><given-names>N</given-names></name><name><surname>Fauvet</surname><given-names>B</given-names></name><name><surname>Lashuel</surname><given-names>H</given-names></name><name><surname>Roux</surname><given-names>A</given-names></name><name><surname>Mayer</surname><given-names>A</given-names></name></person-group><article-title>Membrane scission driven by the PROPPIN Atg18</article-title><source>EMBO J.</source><year>2017</year><volume>36</volume><fpage>3274</fpage><lpage>3291</lpage><pub-id pub-id-type=\"pmid\">29030482</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Efe</surname><given-names>JA</given-names></name><name><surname>Botelho</surname><given-names>RJ</given-names></name><name><surname>Emr</surname><given-names>SD</given-names></name></person-group><article-title>Atg18 regulates organelle morphology and Fab1 kinase activity independent of its membrane recruitment by phosphatidylinositol 3,5-bisphosphate</article-title><source>Mol. Biol. Cell</source><year>2007</year><volume>18</volume><fpage>4232</fpage><lpage>4244</lpage><pub-id pub-id-type=\"pmid\">17699591</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krick</surname><given-names>R</given-names></name><name><surname>Henke</surname><given-names>S</given-names></name><name><surname>Tolstrup</surname><given-names>J</given-names></name><name><surname>Thumm</surname><given-names>M</given-names></name></person-group><article-title>Dissecting the localization and function of Atg18, Atg21 and Ygr223c</article-title><source>Autophagy</source><year>2008</year><volume>4</volume><fpage>896</fpage><lpage>910</lpage><pub-id pub-id-type=\"pmid\">18769150</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krick</surname><given-names>R</given-names></name><name><surname>Tolstrup</surname><given-names>J</given-names></name><name><surname>Appelles</surname><given-names>A</given-names></name><name><surname>Henke</surname><given-names>S</given-names></name><name><surname>Thumm</surname><given-names>M</given-names></name></person-group><article-title>The Relevance of the phosphatidylinositolphosphat-binding motif FRRGT of Atg18 and Atg21 for the Cvt pathway and autophagy</article-title><source>FEBS Lett.</source><year>2006</year><volume>580</volume><fpage>4632</fpage><lpage>4638</lpage><pub-id pub-id-type=\"pmid\">16876790</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Balderhaar</surname><given-names>HJ</given-names></name><name><surname>Ungermann</surname><given-names>C</given-names></name></person-group><article-title>CORVET and HOPS tethering complexes - coordinators of endosome and lysosome fusion</article-title><source>J. Cell Sci.</source><year>2013</year><volume>126</volume><fpage>1307</fpage><lpage>1316</lpage><pub-id pub-id-type=\"pmid\">23645161</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pearson</surname><given-names>CG</given-names></name><name><surname>Bloom</surname><given-names>K</given-names></name></person-group><article-title>Dynamic microtubules lead the way for spindle positioning</article-title><source>Nat. Rev. Mol. Cell. Biol.</source><year>2004</year><volume>5</volume><fpage>481</fpage><lpage>492</lpage><pub-id pub-id-type=\"pmid\">15173827</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huisman</surname><given-names>SM</given-names></name><name><surname>Segal</surname><given-names>M</given-names></name></person-group><article-title>Cortical capture of microtubules and spindle polarity in budding yeast: Where's the catch?</article-title><source>J. Cell Sci.</source><year>2005</year><volume>118</volume><fpage>463</fpage><lpage>471</lpage><pub-id pub-id-type=\"pmid\">15673685</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jaspersen</surname><given-names>SL</given-names></name><name><surname>Winey</surname><given-names>M</given-names></name></person-group><article-title>The budding yeast spindle pole body: structure, duplication, and function</article-title><source>Annu. Rev. Cell. Dev. Biol.</source><year>2004</year><volume>20</volume><fpage>1</fpage><lpage>28</lpage><pub-id pub-id-type=\"pmid\">15473833</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nasmyth</surname><given-names>K</given-names></name></person-group><article-title>How do so few control so many?</article-title><source>Cell</source><year>2005</year><volume>120</volume><fpage>739</fpage><lpage>746</lpage><pub-id pub-id-type=\"pmid\">15797376</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pruyne</surname><given-names>D</given-names></name><name><surname>Bretscher</surname><given-names>A</given-names></name></person-group><article-title>Polarization of cell growth in yeast</article-title><source>J. Cell Sci.</source><year>2000</year><volume>113</volume><fpage>571</fpage><lpage>585</lpage><pub-id pub-id-type=\"pmid\">10652251</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McMillan</surname><given-names>JN</given-names></name><name><surname>Sia</surname><given-names>RA</given-names></name><name><surname>Lew</surname><given-names>DJ</given-names></name></person-group><article-title>A morphogenesis checkpoint monitors the actin cytoskeleton in yeast</article-title><source>J. Cell. Biol.</source><year>1998</year><volume>142</volume><fpage>1487</fpage><lpage>1499</lpage><pub-id pub-id-type=\"pmid\">9744879</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McMillan</surname><given-names>JN</given-names></name><etal/></person-group><article-title>Phosphorylation-independent inhibition of Cdc28p by the tyrosine kinase Swe1p in the morphogenesis checkpoint</article-title><source>Mol. Cell. Biol.</source><year>1999</year><volume>19</volume><fpage>5981</fpage><lpage>5990</lpage><pub-id pub-id-type=\"pmid\">10454545</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Harrison</surname><given-names>JC</given-names></name><etal/></person-group><article-title>A role for the Pkc1p/Mpk1p kinase cascade in the morphogenesis checkpoint</article-title><source>Nat. Cell Biol.</source><year>2001</year><volume>3</volume><fpage>417</fpage><lpage>420</lpage><pub-id pub-id-type=\"pmid\">11283616</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nigg</surname><given-names>EA</given-names></name></person-group><article-title>Mitotic kinases as regulators of cell division and its checkpoints</article-title><source>Nat. Rev. Mol. Cell. Biol.</source><year>2001</year><volume>2</volume><fpage>21</fpage><lpage>32</lpage><pub-id pub-id-type=\"pmid\">11413462</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Amon</surname><given-names>A</given-names></name><name><surname>Surana</surname><given-names>U</given-names></name><name><surname>Muroff</surname><given-names>I</given-names></name><name><surname>Nasmyth</surname><given-names>K</given-names></name></person-group><article-title>Regulation of p34CDC28 tyrosine phosphorylation is not required for entry into mitosis in <italic>S. cerevisiae</italic></article-title><source>Nature</source><year>1992</year><volume>355</volume><fpage>368</fpage><lpage>371</lpage><pub-id pub-id-type=\"pmid\">1731251</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Booher</surname><given-names>RN</given-names></name><name><surname>Deshaies</surname><given-names>RJ</given-names></name><name><surname>Kirschner</surname><given-names>MW</given-names></name></person-group><article-title>Properties <italic>of Saccharomyces cerevisiae</italic> wee1 and its differential regulation of p34CDC28 in response to G1 and G2 cyclins</article-title><source>EMBO J.</source><year>1993</year><volume>12</volume><fpage>3417</fpage><lpage>3426</lpage><pub-id pub-id-type=\"pmid\">8253069</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nomura</surname><given-names>W</given-names></name><name><surname>Maeta</surname><given-names>K</given-names></name><name><surname>Kita</surname><given-names>K</given-names></name><name><surname>Izawa</surname><given-names>S</given-names></name><name><surname>Inoue</surname><given-names>Y</given-names></name></person-group><article-title>Methylglyoxal activates Gcn2 to phosphorylate eIF2alpha independently of the TOR pathway in <italic>Saccharomyces cerevisiae</italic></article-title><source>Appl. Microbiol. Biotechnol.</source><year>2010</year><volume>86</volume><fpage>1887</fpage><lpage>1894</lpage><pub-id pub-id-type=\"pmid\">20077113</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Longtine</surname><given-names>MS</given-names></name><etal/></person-group><article-title>Additional modules for versatile and economical PCR-based gene deletion and modification in <italic>Saccharomyces cerevisiae</italic></article-title><source>Yeast</source><year>1998</year><volume>14</volume><fpage>953</fpage><lpage>961</lpage><pub-id pub-id-type=\"pmid\">9717241</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yoder</surname><given-names>TJ</given-names></name><etal/></person-group><article-title>The <italic>Saccharomyces cerevisiae</italic> spindle pole body is a dynamic structure</article-title><source>Mol. Biol. Cell</source><year>2003</year><volume>14</volume><fpage>3494</fpage><lpage>3505</lpage><pub-id pub-id-type=\"pmid\">12925780</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Straight</surname><given-names>AF</given-names></name><etal/></person-group><article-title>Mitosis in living budding yeast: anaphase A but no metaphase plate</article-title><source>Science</source><year>1997</year><volume>277</volume><fpage>574</fpage><lpage>578</lpage><pub-id pub-id-type=\"pmid\">9228009</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Izawa</surname><given-names>S</given-names></name><name><surname>Takemura</surname><given-names>R</given-names></name><name><surname>Inoue</surname><given-names>Y</given-names></name></person-group><article-title>Gle2p is essential to induce adaptation of the export of bulk poly(A)<sup>+</sup> mRNA to heat shock in <italic>Saccharomyces cerevisiae</italic></article-title><source>J. Biol. Chem.</source><year>2004</year><volume>279</volume><fpage>35469</fpage><lpage>35478</lpage><pub-id pub-id-type=\"pmid\">15210706</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sikorski</surname><given-names>RS</given-names></name><name><surname>Hieter</surname><given-names>P</given-names></name></person-group><article-title>A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in <italic>Saccharomyces cerevisiae</italic></article-title><source>Genetics</source><year>1989</year><volume>122</volume><fpage>19</fpage><lpage>27</lpage><pub-id pub-id-type=\"pmid\">2659436</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Helliwell</surname><given-names>SB</given-names></name><etal/></person-group><article-title>The Rho1 effector Pkc1, but not Bni1, mediates signalling from Tor2 to the actin cytoskeleton</article-title><source>Curr. Biol.</source><year>1998</year><volume>8</volume><fpage>1211</fpage><lpage>1214</lpage><pub-id pub-id-type=\"pmid\">9811607</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vida</surname><given-names>TA</given-names></name><name><surname>Emr</surname><given-names>SD</given-names></name></person-group><article-title>A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast</article-title><source>J. Cell Biol.</source><year>1995</year><volume>128</volume><fpage>779</fpage><lpage>792</lpage><pub-id pub-id-type=\"pmid\">7533169</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hama</surname><given-names>H</given-names></name><name><surname>Takemoto</surname><given-names>JY</given-names></name><name><surname>DeWald</surname><given-names>DB</given-names></name></person-group><article-title>Analysis of phosphoinositides in protein trafficking</article-title><source>Methods</source><year>2000</year><volume>20</volume><fpage>465</fpage><lpage>473</lpage><pub-id pub-id-type=\"pmid\">10720467</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xue</surname><given-names>Y</given-names></name><name><surname>Batlle</surname><given-names>M</given-names></name><name><surname>Hirsch</surname><given-names>JP</given-names></name></person-group><article-title><italic>GPR1</italic> encodes a putative G protein-coupled receptor that associates with the Gpa2p Gα subunit and functions in a Ras-independent pathway</article-title><source>EMBO J.</source><year>1998</year><volume>17</volume><fpage>1996</fpage><lpage>2007</lpage><pub-id pub-id-type=\"pmid\">9524122</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sakchaisri</surname><given-names>K</given-names></name><etal/></person-group><article-title>Coupling morphogenesis to mitotic entry</article-title><source>Proc. Natl. Acad. Sci. U. S. A.</source><year>2004</year><volume>101</volume><fpage>4124</fpage><lpage>4129</lpage><pub-id pub-id-type=\"pmid\">15037762</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807829</article-id><article-id pub-id-type=\"pmc\">PMC7431576</article-id><article-id pub-id-type=\"publisher-id\">70968</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70968-1</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Dispersion-free extraction of In(III) from HCl solutions using a supported liquid membrane containing the HA324H<sup>+</sup>Cl<sup>−</sup> ionic liquid as the carrier</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Alguacil</surname><given-names>Francisco José</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>López</surname><given-names>Félix Antonio</given-names></name><address><email>f.lopez@csic.es</email></address><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><aff id=\"Aff1\"><institution-wrap><institution-id institution-id-type=\"GRID\">grid.4711.3</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2183 4846</institution-id><institution>National Center for Metallurgical Research (CENIM), </institution><institution>Spanish National Research Council (CSIC), </institution></institution-wrap>Avda. Gregorio del Amo, 8, 28040 Madrid, Spain </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13868</elocation-id><history><date date-type=\"received\"><day>27</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>4</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">By reaction of HCl and the tertiary amine HA324, an ionic liquid denoted HA324H<sup>+</sup>Cl<sup>−</sup> was generated and used in the transport of indium(III) from HCl solutions. Metal transport experiments were carried out with a supported liquid membrane, and several variables affecting the permeation of indium(III) across the membrane were tested: stirring speed, metal and acid concentrations in the feed solutions and the carrier concentration in the supported organic solution. The metal transport results were also compared with those obtained using different carriers in the solid support. A model that described indium(III) transport across the membrane was proposed, and the corresponding diffusional parameters were estimated.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Chemical engineering</kwd><kwd>Green chemistry</kwd></kwd-group><funding-group><award-group><funding-source><institution>The European Union’s Horizon 2020 research and innovation program under grant agreement No 776851 (CarEService).776851</institution></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Indium is classified by the European Commission as a critical raw material both for its economics and high supply risk, i.e., an expected increase in its demand over the next years. The above is a consequence of its special properties and wide range of industrial applications, i.e., liquid–crystal displays (LCDs), electronics, catalysts, etc. Thus, the recovery of this element from the above sources is an important target, and several technologies have been proposed to resolve this issue<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>, including ion-exchange<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>, adsorption<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>, cementation<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>, liquid–liquid extraction<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref>–<xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>, liquid membranes<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup> and a process that uses a sequence of steps: leaching-distillation-refluxing in SOCl<sub>2</sub><sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Moreover, the use of bioleaching with <italic>A. thiooxidans</italic> and <italic>A. ferrooxidans</italic> has been proposed in the treatment of LCD panels<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>; however, it is assumed that bioleaching processing has a higher environmental impact than some chemical processes due to its long duration and high electricity consumption<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. In addition, indium is considered a hazardous element due to its carcinogenic character<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. Its removal from residual aqueous solutions, i.e., that resulting from a number of the above processes, is of the foremost importance, and liquid membranes must be considered a technology suitable for the recovery of metals and other solutes present in wastewaters. Liquid membranes have presented a series of operational advantages over other separation technologies, e.g., electrophoresis. (i) The use of solvent extraction in the treatment of solutions containing metal at a few mg/L is not recommended; these concentrations are perfectly compatible with the use of liquid membranes. (ii) In liquid membranes, processing the extraction/stripping steps occurs simultaneously, whereas in solvent extraction, ion exchange with resins and adsorption processes, the above sequence occurs in two separate steps. Supported liquid membranes in various configurations, which are included in these liquid membrane operations, are useful for this task. Before the technology is scaled up in the form of hollow fibre modules, the investigation of a given system using a supported liquid membrane in a flat-sheet configuration is necessary to obtain information about the mass transfer processes involved in membrane operation. The key of the operation is the carrier used to transport the metals, and ionic liquids are such carriers. Ionic liquids are a group of chemicals whose properties identify them as <italic>green solvents</italic>, which among other uses<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>–<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>, makes them suitable for the removal of metals from aqueous solutions<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>–<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>.</p><p id=\"Par3\">As a part of the investigations carried out by our group related to the removal of indium(III) using liquid membrane technologies<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>, this work presented results for the removal of indium(III) from HCl solutions using a flat-sheet supported liquid membrane (FSSLM) containing the ionic liquid HA324H<sup>+</sup>Cl<sup>−</sup> as the carrier. Different hydrodynamic and chemical variables affecting the indium(III) transport process were investigated.</p></sec><sec id=\"Sec2\"><title>Results</title><sec id=\"Sec3\"><title>Influence of the stirring speed in the source phase on indium(III) permeation</title><p id=\"Par4\">In every separation technology, i.e., liquid membranes, ion exchange or adsorption, and when working in batch conditions, it is necessary to experimentally determine the best hydrodynamic conditions to ensure maximum solute transport, adsorption, etc. Thus, in the present system, the influence of the stirring speed in the source phase on metal permeation was investigated by the use of source phases containing 0.01 g/L In(III) in 2 M HCl and a receiving solution of 0.1 M HCl. The membrane phase was 0.23 M ionic liquid in Solvesso 100 solution supported in a Durapore GVHP4700 solid support. Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> shows the results from these experiments.<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Influence of stirring speed on the permeability (P) of indium(III).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Stirring speed (rpm)</th><th align=\"left\">P × 10<sup>3</sup> (cm/s)</th></tr></thead><tbody><tr><td align=\"left\">375</td><td char=\".\" align=\"char\">1.5</td></tr><tr><td align=\"left\">500</td><td char=\".\" align=\"char\">1.9</td></tr><tr><td align=\"left\">750</td><td char=\".\" align=\"char\">2.9</td></tr><tr><td align=\"left\">1000</td><td char=\".\" align=\"char\">1.5</td></tr><tr><td align=\"left\">1500</td><td char=\".\" align=\"char\">1.0</td></tr></tbody></table><table-wrap-foot><p><italic>P</italic> value, as in the rest of the work, calculated as in Eq. (<xref rid=\"Equ11\" ref-type=\"\">12</xref>).</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec4\"><title>Influence of the stirring speed in the receiving phase on In(III) permeation</title><p id=\"Par5\">In the present work, the receiving phase was 0.1 M HCl because previous data<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup> indicated that this solution was a good strippant for this system. Experiments carried out with this solution, as the receiving phase, with the same source and membrane phases as above and a 750 min<sup>-1</sup> stirring speed for the source phase, indicated that in the 500–750 min<sup>-1</sup> range of stirring speeds applied to the receiving phase, there was no appreciable difference in the transport of the metal. Thus, stirring speeds of 750 and 500 min<sup>-1</sup> in the source and receiving phases were selected for further experiments.</p></sec><sec id=\"Sec5\"><title>Influence of HCl concentration in the source phase on indium(III) transport</title><p id=\"Par6\">The variation of the HCl concentration in the source phase on In(III) permeation was investigated using the same receiving and membrane phases as above, with the source phase of 0.01 g/L In(III) in different HCl media. The results of the experiments are shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> by plotting ln ([In]<sub>s,t</sub>/[In]<sub>s,0</sub>) versus time, where [In]<sub>s,t</sub> and [In]<sub>s,0</sub> are the metal concentrations in the source phase at some time and the initial time, respectively.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Influence of HCl concentration in the source phase on In(III) transport. The stirring speeds of the source and receiving phases were 750 and 500 min-1, respectively.</p></caption><graphic xlink:href=\"41598_2020_70968_Fig1_HTML\" id=\"MO1\"/></fig></p></sec><sec id=\"Sec6\"><title>Influence of the carrier concentration on In(III) permeation</title><p id=\"Par7\">It was mentioned above that the carrier solution is the key for liquid membrane operation since a support with no carrier phase in it does not transport metal. Figure <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> shows the results when 0.01 g/L In(III) in a 1 M HCl source phase was transported across a membrane containing organic phases with different concentrations of the ionic liquid in Solvesso 100 and a 0.1 M HCl solution was used as the receiving phase.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Influence of the carrier concentration on In(III) transport. Source phase: 0.01 g/L In(III) in 1 M HCl. Membrane phase: different concentrations of the ionic liquid in Solvesso 100 in GVHP4700 supports. Receiving phase: 0.1 M HCl.</p></caption><graphic xlink:href=\"41598_2020_70968_Fig2_HTML\" id=\"MO2\"/></fig></p></sec><sec id=\"Sec7\"><title>Influence of the initial indium(III) concentration in the source phase on the metal permeation and flux</title><p id=\"Par8\">These results were obtained using membrane phases containing 0.23 M ionic liquid in Solvesso 100 that was supported in a GVHP4700 support and a receiving phase of 0.l M HCl. The source phases had various In(III) concentrations (0.01–0.1 g/L) in 1 M HCl. Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref> presents these results.<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Variation in the indium(III) permeation coefficient (P) and flux (J) with variation in the initial metal concentration in the source phase.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Initial In(III) concentration (g/L)</th><th align=\"left\">P × 10<sup>3</sup> (cm/s)</th><th align=\"left\">J × 10<sup>10</sup> (mol/cm<sup>2</sup> s)</th></tr></thead><tbody><tr><td char=\".\" align=\"char\">0.01</td><td char=\".\" align=\"char\">3.0</td><td char=\".\" align=\"char\">2.6</td></tr><tr><td char=\".\" align=\"char\">0.05</td><td char=\".\" align=\"char\">0.82</td><td char=\".\" align=\"char\">3.6</td></tr><tr><td char=\".\" align=\"char\">0.1</td><td char=\".\" align=\"char\">0.72</td><td char=\".\" align=\"char\">6.3</td></tr></tbody></table><table-wrap-foot><p>The stirring speeds in the source and receiving phases were 750 and 500 min<sup>-1</sup>, respectively.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec8\"><title>Influence of the support characteristics on indium(III) transport</title><p id=\"Par9\">The results derived with the GVHP4700 support were compared with those obtained when the HVHP4700 support was used as a solid support in this system, with all the experimental variables the same as in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>, except that in this case, the membrane phase was a 0.23 M carrier in Solvesso 100 solution that was supported in the HPVP and GVHP solid supports.</p></sec><sec id=\"Sec9\"><title>Indium(III) transport using different carriers</title><p id=\"Par10\">The transport of indium(III) was compared when different carriers were used. In this case, the source phase was 0.01 g/L In(III) in 1 M HCl, and the receiving phase was 0.1 M HCl. The membrane phases were 0.17 M carrier in Solvesso 100 solutions supported in the GVHP4700 solid support, and the stirring speeds applied to the source and receiving phases were 750 and 500 min<sup>-1</sup>, respectively.</p><p id=\"Par11\">These transport results, together with the indium(III) recovery in the receiving phase, are summarized in Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref>.<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>Results of In(III) transport and metal recoveries in the receiving phase using different carriers.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Carrier</th><th align=\"left\">P (cm/s)</th><th align=\"left\"><sup>b</sup>In recovery (%)</th></tr></thead><tbody><tr><td align=\"left\">Cyphos IL102</td><td char=\"×\" align=\"char\">1.2 × 10<sup>–3</sup></td><td align=\"left\">99</td></tr><tr><td align=\"left\">Cyphos IL101</td><td char=\"×\" align=\"char\">1.4 × 10<sup>–3</sup></td><td align=\"left\">99</td></tr><tr><td align=\"left\">Cyanex 923</td><td char=\"×\" align=\"char\">8.5 × 10<sup>–4</sup></td><td align=\"left\">99</td></tr><tr><td align=\"left\">Tributylphosphate</td><td char=\"×\" align=\"char\">4.1 × 10<sup>–4</sup></td><td align=\"left\">6</td></tr><tr><td align=\"left\"><sup>a</sup>Primene JMT</td><td char=\"×\" align=\"char\">3.1 × 10<sup>–4</sup></td><td align=\"left\">Nil</td></tr><tr><td align=\"left\">Aliquat 336</td><td char=\"×\" align=\"char\">2.9 × 10<sup>–4</sup></td><td align=\"left\">50</td></tr><tr><td align=\"left\"><sup>a</sup>Hostarex A324</td><td char=\"×\" align=\"char\">1.8 × 10<sup>–3</sup></td><td align=\"left\">83</td></tr><tr><td align=\"left\">2-Ethyl-1-hexanol</td><td char=\"×\" align=\"char\">4.2 × 10<sup>–7</sup></td><td align=\"left\">Nil</td></tr><tr><td align=\"left\">Isopentyl-methylketone</td><td char=\"×\" align=\"char\">3.9 × 10<sup>–4</sup></td><td align=\"left\">Nil</td></tr></tbody></table><table-wrap-foot><p><sup>a</sup>Acting as the corresponding quaternary ammonium salt.</p><p><sup>b</sup>After 3 h, and with respect to the metal transported from the source to the membrane phases.</p></table-wrap-foot></table-wrap></p></sec></sec><sec id=\"Sec10\"><title>Discussion</title><p id=\"Par12\">It was shown<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup> that the extraction of indium(III) from hydrochloric acid media by the ionic liquid HA324H<sup>+</sup>Cl<sup>−</sup> dissolved in Solvesso 100 (ExxonMobil ) occurs via the equilibrium<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$InCl_{{4_{aq} }}^{ - } + H324H^{ + } Cl_{org}^{ - } \\Leftrightarrow HA324H^{ + } InCl_{{4_{org} }}^{ - } + Cl_{aq}^{ - }$$\\end{document}</tex-math><mml:math id=\"M2\" display=\"block\"><mml:mrow><mml:mi>I</mml:mi><mml:mi>n</mml:mi><mml:mi>C</mml:mi><mml:msubsup><mml:mi>l</mml:mi><mml:mrow><mml:msub><mml:mn>4</mml:mn><mml:mrow><mml:mi mathvariant=\"italic\">aq</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>-</mml:mo></mml:msubsup><mml:mo>+</mml:mo><mml:mi>H</mml:mi><mml:mn>324</mml:mn><mml:msup><mml:mi>H</mml:mi><mml:mo>+</mml:mo></mml:msup><mml:mi>C</mml:mi><mml:msubsup><mml:mi>l</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow><mml:mo>-</mml:mo></mml:msubsup><mml:mo stretchy=\"false\">⇔</mml:mo><mml:mi>H</mml:mi><mml:mi>A</mml:mi><mml:mn>324</mml:mn><mml:msup><mml:mi>H</mml:mi><mml:mo>+</mml:mo></mml:msup><mml:mi>I</mml:mi><mml:mi>n</mml:mi><mml:mi>C</mml:mi><mml:msubsup><mml:mi>l</mml:mi><mml:mrow><mml:msub><mml:mn>4</mml:mn><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>-</mml:mo></mml:msubsup><mml:mo>+</mml:mo><mml:mi>C</mml:mi><mml:msubsup><mml:mi>l</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">aq</mml:mi></mml:mrow><mml:mo>-</mml:mo></mml:msubsup></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70968_Article_Equ1.gif\" position=\"anchor\"/></alternatives></disp-formula>where the subscripts org and aq refer to the organic and aqueous phases, respectively. Thus, extraction occurred when the equilibrium was shifted to the right, and metal stripping shifted the equilibrium to the left. It was also determined that the value of the equilibrium constant value for the above equilibrium was 10.96 in 1 M HCl medium.</p><p id=\"Par13\">Some authors<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup> considered that the extraction of indium(III) occurred via the extraction of InCl<sub>3</sub> species and the formation of R + InCl<sub>4</sub><sup>−</sup> species in the organic phase, where R + represents the cationic moiety of the ionic liquid (in the chloride form), that is:<disp-formula id=\"Equ2\"><label>2</label><alternatives><tex-math id=\"M3\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$InCl_{{3_{aq} }} + R^{ + } Cl_{org}^{ - } \\Leftrightarrow R^{ + } InCl_{{4_{org} }}^{ - }$$\\end{document}</tex-math><mml:math id=\"M4\" display=\"block\"><mml:mrow><mml:mi>I</mml:mi><mml:mi>n</mml:mi><mml:mi>C</mml:mi><mml:msub><mml:mi>l</mml:mi><mml:msub><mml:mn>3</mml:mn><mml:mrow><mml:mi mathvariant=\"italic\">aq</mml:mi></mml:mrow></mml:msub></mml:msub><mml:mo>+</mml:mo><mml:msup><mml:mi>R</mml:mi><mml:mo>+</mml:mo></mml:msup><mml:mi>C</mml:mi><mml:msubsup><mml:mi>l</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow><mml:mo>-</mml:mo></mml:msubsup><mml:mo stretchy=\"false\">⇔</mml:mo><mml:msup><mml:mi>R</mml:mi><mml:mo>+</mml:mo></mml:msup><mml:mi>I</mml:mi><mml:mi>n</mml:mi><mml:mi>C</mml:mi><mml:msubsup><mml:mi>l</mml:mi><mml:mrow><mml:msub><mml:mn>4</mml:mn><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>-</mml:mo></mml:msubsup></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70968_Article_Equ2.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p id=\"Par14\">However, these same authors also argued that the extraction of In(III) can be attributed to both reactions: ion exchange (as in Eq. (<xref rid=\"Equ1\" ref-type=\"\">1</xref>)) and extraction of InCl<sub>3</sub>, as mentioned above.</p><p id=\"Par15\">Accordingly with the data presented in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>, the indium(III) permeation increased from 375 to 750 min<sup>-1</sup> and then decreased, which is attributable to a continuous decrease in the source phase boundary layer thickness with increasing stirring speed in this phase. A limiting permeability value was obtained at 750 min<sup>-1</sup>. At this point, the system reached a minimum in the thickness of the aqueous source layer, and indium(III) transport was maximized; thus,<disp-formula id=\"Equ3\"><label>3</label><alternatives><tex-math id=\"M5\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$P_{\\lim } = \\frac{{D_{aq} }}{{d_{aq} }}$$\\end{document}</tex-math><mml:math id=\"M6\" display=\"block\"><mml:mrow><mml:msub><mml:mi>P</mml:mi><mml:mo movablelimits=\"true\">lim</mml:mo></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:msub><mml:mi>D</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">aq</mml:mi></mml:mrow></mml:msub><mml:msub><mml:mi>d</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">aq</mml:mi></mml:mrow></mml:msub></mml:mfrac></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70968_Article_Equ3.gif\" position=\"anchor\"/></alternatives></disp-formula>where D<sub>aq</sub> is the metal diffusion coefficient in the aqueous source phase (averaging a value of 10<sup>–5</sup> cm/s), d<sub>aq</sub> is the thickness of the aqueous source layer, and P<sub>lim</sub> is 2.9 × 10<sup>–3</sup> cm/s. The value of d<sub>aq</sub> is estimated to be 3.4 × 10<sup>–3</sup> cm. Thus, this value represented the minimum thickness of the stagnant source phase layer, considering the experimental conditions used in this work. It is also shown in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> that at stirring speeds above 750 min<sup>−1</sup>, the metal permeation decreased, which was caused by the turbulence caused by the stirring speed and the likely progressive instability of the liquid membrane.</p><p id=\"Par16\">Figure <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> shows that the indium permeation increased with increasing HCl concentration in the source solution up to 1–2 M and then decreased at higher HCl concentrations in this phase, which was probably attributable to the increase in the aqueous ionic strength and/or increases in the ion population in this phase (population growth effect), which often decreases metal permeation<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>.</p><p id=\"Par17\">Moreover, the percentage of indium(III) recovered in the receiving phase was, after three hours, approximately 90% when 1 or 2 M HCl solution was used.</p><p id=\"Par18\">From the results presented in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>, it was shown that the maximum metal permeability (3.0 × 10<sup>–3</sup> cm/s) was reached when a carrier concentration of 0.23 M was used in the membrane phase. The above was attributable to the fact that, at low carrier concentrations, diffusion of the indium(III)-carrier complex across the liquid membrane governed the rate, but at the maximum indium(III) permeation, diffusion of indium(III) across the source phase boundary layer is the rate-determining process. According to Eq. (<xref rid=\"Equ3\" ref-type=\"\">3</xref>), d<sub>aq</sub> was estimated as 3.3 × 10<sup>–3</sup> cm, a value that matched perfectly with that previously obtained with 2 M HCl concentration in the source phase.</p><p id=\"Par19\">When higher carrier concentrations, i.e., 0.46 and 0.92 M, were used in the membrane phase, increasing the organic phase viscosity decreased metal transport, which was attributable to an increase in the membrane resistance.</p><p id=\"Par20\">From results showed in Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>, it was observed that, first, the permeation coefficient, P, decreased as the initial metal concentration in the source phase increased, and second, the initial metal flux increased with increasing initial indium(III) concentration in this phase.</p><p id=\"Par21\">The decrease in metal permeation with the increasing indium(III) concentration may occur because the membrane pores become saturated with increasing metal concentrations, whereas the flux results were logical since the flux varies with metal concentration:<disp-formula id=\"Equ4\"><label>4</label><alternatives><tex-math id=\"M7\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$J = P\\left[ {In} \\right]_{s,0}$$\\end{document}</tex-math><mml:math id=\"M8\" display=\"block\"><mml:mrow><mml:mi>J</mml:mi><mml:mo>=</mml:mo><mml:mi>P</mml:mi><mml:msub><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi mathvariant=\"italic\">In</mml:mi></mml:mrow></mml:mfenced><mml:mrow><mml:mi>s</mml:mi><mml:mo>,</mml:mo><mml:mn>0</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70968_Article_Equ4.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p id=\"Par22\">Thus, the flux value increased with increasing metal concentration in the source phase. According to the flux values shown in Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>, in the range of indium(III) concentrations used in this work, the permeation process seemed to be controlled by the diffusion of the metal species.</p><p id=\"Par23\">The percentage of metal recovered in the receiving phase varied between 90 and 99% (after 3 h) with respect to the metal transported from the source to the membrane phase for the three initial indium(III) concentrations used in this work.</p><p id=\"Par24\">When using GVHP or HVHP supports, the initial fluxes (Eq. <xref rid=\"Equ4\" ref-type=\"\">4</xref>) obtained from these experiments were 2.6 × 10<sup>–10</sup> mol/cm<sup>2</sup> s and 1.7 × 10<sup>–10</sup> mol/cm<sup>2</sup> s for both supports, respectively; thus, all the characteristics but the pore size (2.2 × 10<sup>–3</sup> cm for GVHP versus 4.5 × 10<sup>–3</sup> cm for HVHP) were similar for both supports. This last characteristic dominated the metal transport, and apparently, the smaller the pore size was, the greater the flux value with respect to the indium(III) recovery in the receiving phase (91% for GVHP and 59% for HVHP supports after 3 h and, as above, with respect to the metal transported from the source to the membrane phases).</p><p id=\"Par25\">From the results presented in Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref> and under the present experimental conditions, the best transport results were obtained with the ionic liquid HA324H<sup>+</sup>Cl<sup>-</sup>, followed by the two ionic liquids Cyphos IL101 and Cyphos IL102, derived from a phosphonium salt. The worst transport result was obtained when 2-ethyl-hexanol was the carrier for indium transport. For the carriers derived from quaternary ammonium, the order found was tertiary amine (Hostarex A324) > Aliquat 336 > primary amine (Primene JMT); in the case of phosphorous derivatives, phosphonium salts > phosphine oxides > phosphoric ester; and for organics containing C–O bonds, ketone > alcohol. Apparently, indium(III) transport is closely related to liquid–liquid extraction using these same extractants. In fact, the liquid–liquid extraction of metal-chloride complexes is favoured when anion exchange equilibria are responsible for metal extraction, that is, as in the case of phosphonium salts and the tertiary amine. Neutral phosphorus derivatives, such as Cyanex 923 and TBP, extracted metals via the solvation of neutral metal species by donation of an electron pair from the oxygen of the P=O bond to the metal species, and in these particular cases, phosphine oxides had greater electron-donor properties than the phosphoric ester TBP. Alcohols and ketones also extracted metals via solvation of neutral metal species, and in these cases, it occurred via the donation of an electron pair (the oxygen atom in the C=O bond) in the case of the ketones or by bonding via the OH group of the alcohols and water molecules present in the metal complex.</p><p id=\"Par26\">The ranking of indium recovery in the receiving phase was not as clear; however, near quantitative In(III) recovery was achieved with Cyphos IL101, Cyphos IL102 and Cyanex 923, while worse recovery occurred with the HA324H<sup>+</sup>Cl<sup>-</sup> ionic liquid. However, greater percentages of In(III) recovery can be obtained in this phase (see \"<xref rid=\"Sec7\" ref-type=\"sec\">Influence of the initial indium(III) concentration in the source phase on the metal permeation and flux</xref>\" and \"<xref rid=\"Sec8\" ref-type=\"sec\">Influence of the support characteristics on indium(III) transport</xref>\" sections). The 0.1 M HCl solutions were not good receiving media for the primary amine (acting as the corresponding ammonium form), the ketone and the alcohol.</p><p id=\"Par27\">Under the present experimental conditions, the best transport results were obtained with the ionic liquid HA324H<sup>+</sup>Cl<sup>-</sup>, followed by the two ionic liquids Cyphos IL101 and Cyphos IL102, derived from a phosphonium salt. The worst transport result was obtained when 2-ethyl-hexanol was the carrier for indium transport. For the carriers derived from quaternary ammonium, the order found was tertiary amine (Hostarex A324) > Aliquat 336 > primary amine (Primene JMT); in the case of phosphorous derivatives, phosphonium salts > phosphine oxides > phosphoric ester; and for organics containing C–O bonds, ketone > alcohol. Apparently, indium(III) transport is closely related to liquid–liquid extraction using these same extractants. In fact, the liquid–liquid extraction of metal-chloride complexes is favoured when anion exchange equilibria are responsible for metal extraction, that is, as in the case of phosphonium salts and the tertiary amine. Neutral phosphorus derivatives, such as Cyanex 923 and TBP, extracted metals via the solvation of neutral metal species by donation of an electron pair from the oxygen of the P=O bond to the metal species, and in these particular cases, phosphine oxides had greater electron-donor properties than the phosphoric ester TBP. Alcohols and ketones also extracted metals via solvation of neutral metal species, and in these cases, it occurred via the donation of an electron pair (the oxygen atom in the C=O bond) in the case of the ketones or by bonding via the OH group of the alcohols and water molecules present in the metal complex.</p><p id=\"Par28\">The ranking of indium recovery in the receiving phase was not as clear; however, near quantitative In(III) recovery was achieved with Cyphos IL101, Cyphos IL102 and Cyanex 923, while worse recovery occurred with the HA324H<sup>+</sup>Cl<sup>-</sup> ionic liquid. However, greater percentages of In(III) recovery can be obtained in this phase (see see \"<xref rid=\"Sec7\" ref-type=\"sec\">Influence of the initial indium(III) concentration in the source phase on the metal permeation and flux</xref>\" and \"<xref rid=\"Sec8\" ref-type=\"sec\">Influence of the support characteristics on indium(III) transport</xref>\" sections). The 0.1 M HCl solutions were not good receiving media for the primary amine (acting as the corresponding ammonium form), the ketone and the alcohol.</p><p id=\"Par29\">The experimental results derived from this work, allowed to the estimation of the diffusional parameters involved in the present system. According to Eq. (<xref rid=\"Equ1\" ref-type=\"\">1</xref>), the equilibrium constant of the reaction can be written as:<disp-formula id=\"Equ5\"><label>5</label><alternatives><tex-math id=\"M9\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$K = \\frac{{\\left[ {HA324H^{ + } InCl_{4}^{ - } } \\right]_{org} \\left[ {Cl^{ - } } \\right]_{aq} }}{{\\left[ {InCl_{4}^{ - } } \\right]_{aq} \\left[ {HA324H^{ + } Cl^{ - } } \\right]_{org} }}$$\\end{document}</tex-math><mml:math id=\"M10\" display=\"block\"><mml:mrow><mml:mi>K</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi>H</mml:mi><mml:mi>A</mml:mi><mml:mn>324</mml:mn><mml:msup><mml:mi>H</mml:mi><mml:mo>+</mml:mo></mml:msup><mml:mi>I</mml:mi><mml:mi>n</mml:mi><mml:mi>C</mml:mi><mml:msubsup><mml:mi>l</mml:mi><mml:mrow><mml:mn>4</mml:mn></mml:mrow><mml:mo>-</mml:mo></mml:msubsup></mml:mrow></mml:mfenced><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub><mml:msub><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi>C</mml:mi><mml:msup><mml:mi>l</mml:mi><mml:mo>-</mml:mo></mml:msup></mml:mrow></mml:mfenced><mml:mrow><mml:mi mathvariant=\"italic\">aq</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:msub><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi>I</mml:mi><mml:mi>n</mml:mi><mml:mi>C</mml:mi><mml:msubsup><mml:mi>l</mml:mi><mml:mrow><mml:mn>4</mml:mn></mml:mrow><mml:mo>-</mml:mo></mml:msubsup></mml:mrow></mml:mfenced><mml:mrow><mml:mi mathvariant=\"italic\">aq</mml:mi></mml:mrow></mml:msub><mml:msub><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi>H</mml:mi><mml:mi>A</mml:mi><mml:mn>324</mml:mn><mml:msup><mml:mi>H</mml:mi><mml:mo>+</mml:mo></mml:msup><mml:mi>C</mml:mi><mml:msup><mml:mi>l</mml:mi><mml:mo>-</mml:mo></mml:msup></mml:mrow></mml:mfenced><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfrac></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70968_Article_Equ5.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p id=\"Par30\">Following the same reasoning published elsewhere<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>, an expression for the permeability coefficient can be written as:<disp-formula id=\"Equ6\"><label>6</label><alternatives><tex-math id=\"M11\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$P = \\frac{{K\\left[ {HA324H^{ + } Cl^{ - } } \\right]_{org} \\left[ {Cl^{ - } } \\right]_{aq}^{ - 1} }}{{\\Delta_{org} + \\Delta_{aq} \\left( {K\\left[ {Cl^{ - } } \\right]_{aq}^{ - 1} \\left[ {HA324H^{ + } Cl^{ - } } \\right]_{org} } \\right)}}$$\\end{document}</tex-math><mml:math id=\"M12\" display=\"block\"><mml:mrow><mml:mi>P</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>K</mml:mi><mml:msub><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi>H</mml:mi><mml:mi>A</mml:mi><mml:mn>324</mml:mn><mml:msup><mml:mi>H</mml:mi><mml:mo>+</mml:mo></mml:msup><mml:mi>C</mml:mi><mml:msup><mml:mi>l</mml:mi><mml:mo>-</mml:mo></mml:msup></mml:mrow></mml:mfenced><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub><mml:msubsup><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi>C</mml:mi><mml:msup><mml:mi>l</mml:mi><mml:mo>-</mml:mo></mml:msup></mml:mrow></mml:mfenced><mml:mrow><mml:mi mathvariant=\"italic\">aq</mml:mi></mml:mrow><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup></mml:mrow><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">aq</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>K</mml:mi><mml:msubsup><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi>C</mml:mi><mml:msup><mml:mi>l</mml:mi><mml:mo>-</mml:mo></mml:msup></mml:mrow></mml:mfenced><mml:mrow><mml:mi mathvariant=\"italic\">aq</mml:mi></mml:mrow><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:msub><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi>H</mml:mi><mml:mi>A</mml:mi><mml:mn>324</mml:mn><mml:msup><mml:mi>H</mml:mi><mml:mo>+</mml:mo></mml:msup><mml:mi>C</mml:mi><mml:msup><mml:mi>l</mml:mi><mml:mo>-</mml:mo></mml:msup></mml:mrow></mml:mfenced><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfenced></mml:mrow></mml:mfrac></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70968_Article_Equ6.gif\" position=\"anchor\"/></alternatives></disp-formula>where Δ<sub>aq</sub> and Δ<sub>org</sub> are the transport resistance related to diffusion in the source and membrane phases, respectively. This expression combines the diffusional and equilibrium parameters involved in the transport of indium(III) from HCl solutions across a membrane supporting the ionic liquid. Then,<disp-formula id=\"Equ7\"><label>7</label><alternatives><tex-math id=\"M13\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\frac{1}{P} = \\Delta_{aq} + \\Delta_{org} \\frac{1}{{K\\left[ {Cl^{ - } } \\right]_{aq}^{ - 1} \\left[ {HA324H^{ + } Cl^{ - } } \\right]_{org} }} = \\Delta_{aq} + \\Delta_{org} \\frac{1}{b}$$\\end{document}</tex-math><mml:math id=\"M14\" display=\"block\"><mml:mrow><mml:mfrac><mml:mn>1</mml:mn><mml:mi>P</mml:mi></mml:mfrac><mml:mo>=</mml:mo><mml:msub><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">aq</mml:mi></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:mi>K</mml:mi><mml:msubsup><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi>C</mml:mi><mml:msup><mml:mi>l</mml:mi><mml:mo>-</mml:mo></mml:msup></mml:mrow></mml:mfenced><mml:mrow><mml:mi mathvariant=\"italic\">aq</mml:mi></mml:mrow><mml:mrow><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msubsup><mml:msub><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi>H</mml:mi><mml:mi>A</mml:mi><mml:mn>324</mml:mn><mml:msup><mml:mi>H</mml:mi><mml:mo>+</mml:mo></mml:msup><mml:mi>C</mml:mi><mml:msup><mml:mi>l</mml:mi><mml:mo>-</mml:mo></mml:msup></mml:mrow></mml:mfenced><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:msub><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">aq</mml:mi></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub><mml:mfrac><mml:mn>1</mml:mn><mml:mi>b</mml:mi></mml:mfrac></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70968_Article_Equ7.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p id=\"Par31\">Thus, a plot of 1/P versus 1/b for various carrier concentrations in the membrane phase and 1 M Cl<sup>−</sup> concentration in the source solution may result in a straight line with an intercept and a slope that can be used to estimate the transport resistance in the source phase (Δaq = 166 s/cm) and in the membrane phase (Δorg = 0.84 s/cm), respectively. The estimated value of the membrane diffusion coefficient is:<disp-formula id=\"Equ8\"><label>8</label><alternatives><tex-math id=\"M15\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$D_{org} = \\frac{{d_{org} }}{{\\Delta_{org} }}$$\\end{document}</tex-math><mml:math id=\"M16\" display=\"block\"><mml:mrow><mml:msub><mml:mi>D</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:msub><mml:mi>d</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub><mml:msub><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub></mml:mfrac></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70968_Article_Equ8.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p id=\"Par32\">The membrane diffusion coefficient was 1.5 × 10<sup>–2</sup> cm<sup>2</sup>/s here. In the above expression, d<sub>org</sub> is the thickness of the membrane phase (see Sect. <xref rid=\"Sec10\" ref-type=\"sec\">3</xref>).</p><p id=\"Par33\">The diffusion coefficient of the indium(III)-ionic liquid complex in the bulk organic phase can be estimated by<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>:<disp-formula id=\"Equ9\"><label>9</label><alternatives><tex-math id=\"M17\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$D_{b,org} = D_{org} \\frac{{\\tau^{2} }}{\\varepsilon }$$\\end{document}</tex-math><mml:math id=\"M18\" display=\"block\"><mml:mrow><mml:msub><mml:mi>D</mml:mi><mml:mrow><mml:mi>b</mml:mi><mml:mo>,</mml:mo><mml:mi>o</mml:mi><mml:mi>r</mml:mi><mml:mi>g</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>D</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub><mml:mfrac><mml:msup><mml:mi>τ</mml:mi><mml:mn>2</mml:mn></mml:msup><mml:mi>ε</mml:mi></mml:mfrac></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70968_Article_Equ9.gif\" position=\"anchor\"/></alternatives></disp-formula>which gave the D<sub>b,org</sub> value 5.6 × 10<sup>–2</sup> cm<sup>2</sup>/s. D<sub>org</sub> had a lower value than D<sub>b,org</sub>, which may be caused by the diffusional resistance due to the thickness of the membrane between the source and receiving phases.</p><p id=\"Par34\">Moreover, an apparent diffusion coefficient for indium(III) can be estimated as:<disp-formula id=\"Equ10\"><label>10</label><alternatives><tex-math id=\"M19\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$D_{org}^{a} = \\frac{{Jd_{org} }}{{\\left[ {carrier} \\right]_{org} }}$$\\end{document}</tex-math><mml:math id=\"M20\" display=\"block\"><mml:mrow><mml:msubsup><mml:mi>D</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow><mml:mi>a</mml:mi></mml:msubsup><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>J</mml:mi><mml:msub><mml:mi>d</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:msub><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi mathvariant=\"italic\">carrier</mml:mi></mml:mrow></mml:mfenced><mml:mrow><mml:mi mathvariant=\"italic\">org</mml:mi></mml:mrow></mml:msub></mml:mfrac></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70968_Article_Equ10.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p id=\"Par35\">When the constant carrier concentration was assumed to be 0.23 M, the value of this coefficient was 1.3 × 10<sup>–8</sup> cm<sup>2</sup>/s.</p></sec><sec id=\"Sec11\"><title>Methods</title><p id=\"Par36\">The tertiary amine Hostarex A324 (Hoechst) has the active group tri-isooctyl amine and was used as the organic precursor of the ionic liquid. The inorganic moiety was hydrochloric acid, and the ionic liquid was generated by reaction of both<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. Other carriers used in this investigation had the composition shown in Table <xref rid=\"Tab4\" ref-type=\"table\">4</xref>. All were used without further purification. Other chemicals used in the work were G.R. quality, except for the organic diluent Solvesso 100 (99% aromatics), which was obtained from Exxon Chem., Iberia, and was also used without further purification. Though many authors claimed that they used ionic liquids without dilution, the truth was that in most, if not all, cases, an organic diluent was needed, and its use facilitated the liquid–liquid extraction operation because, among other reasons: <list list-type=\"simple\"><list-item><label>(i)</label><p id=\"Par37\">It decreased the high viscosity of the ionic liquid and, thus, facilitated phase disengagement in the settler, and.</p></list-item><list-item><label>(ii)</label><p id=\"Par38\">The range of carrier concentrations was adequate for any particular use. The use of an excess of carrier was avoided in the process, which gave the benefits of decreasing its financial cost and favouring metal transport, e.g., in this investigation, using an excess of carrier decreased indium(III) permeability.</p></list-item></list><table-wrap id=\"Tab4\"><label>Table 4</label><caption><p>Chemical compositions and sources of the carriers.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Carrier</th><th align=\"left\">Chemical composition</th><th align=\"left\">Source</th></tr></thead><tbody><tr><td align=\"left\">Cyphos IL102</td><td align=\"left\">Trihexyl tetradecylphosphonium bromide</td><td align=\"left\">Cytec Ind</td></tr><tr><td align=\"left\">Cyphos IL101</td><td align=\"left\">Trihexyl tetradecylphosphonium chloride</td><td align=\"left\">Cytec Ind</td></tr><tr><td align=\"left\">TBP</td><td align=\"left\">Tri-n-butyl phosphate</td><td align=\"left\">Fluka</td></tr><tr><td align=\"left\">Primene JMT</td><td align=\"left\">Mixture of t-alkyl primary amines</td><td align=\"left\">Rohm and Haas</td></tr><tr><td align=\"left\">Cyanex 923</td><td align=\"left\">Mixture of tri-n-alkyl phosphine oxides</td><td align=\"left\">Cytec Ind</td></tr><tr><td align=\"left\">Aliquat 336</td><td align=\"left\">Tri-octyl methylammonium chloride</td><td align=\"left\">Fluka</td></tr><tr><td align=\"left\">MIPK</td><td align=\"left\">Isopenthyl-methylketone</td><td align=\"left\">Fluka</td></tr><tr><td align=\"left\">2-Ethyl-1-hexanol</td><td align=\"left\">Alcohol</td><td align=\"left\">Fluka</td></tr></tbody></table></table-wrap></p><p id=\"Par39\">Transport experiments were carried out in a two-compartment cell, one compartment each for the (200 cm<sup>3</sup>) source and (200 cm<sup>3</sup>) receiving phases, with a membrane support separating the two aqueous phases. The source and receiving solutions were mechanically stirred by means of four blade impellers (11.5 cm diameter). Indium(III) permeability (P) was estimated by the use of the common relationship:<disp-formula id=\"Equ11\"><label>12</label><alternatives><tex-math id=\"M21\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\ln \\frac{{\\left[ {In} \\right]_{s,t} }}{{\\left[ {In} \\right]_{s,0} }} = - \\frac{A}{V}Pt$$\\end{document}</tex-math><mml:math id=\"M22\" display=\"block\"><mml:mrow><mml:mo>ln</mml:mo><mml:mfrac><mml:msub><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi mathvariant=\"italic\">In</mml:mi></mml:mrow></mml:mfenced><mml:mrow><mml:mi>s</mml:mi><mml:mo>,</mml:mo><mml:mi>t</mml:mi></mml:mrow></mml:msub><mml:msub><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi mathvariant=\"italic\">In</mml:mi></mml:mrow></mml:mfenced><mml:mrow><mml:mi>s</mml:mi><mml:mo>,</mml:mo><mml:mn>0</mml:mn></mml:mrow></mml:msub></mml:mfrac><mml:mo>=</mml:mo><mml:mo>-</mml:mo><mml:mfrac><mml:mi>A</mml:mi><mml:mi>V</mml:mi></mml:mfrac><mml:mi>P</mml:mi><mml:mi>t</mml:mi></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70968_Article_Equ11.gif\" position=\"anchor\"/></alternatives></disp-formula>where [In]<sub>s,t</sub> and [In]<sub>s,0</sub> are the indium concentrations in the source phase at a time during the experiment and at time zero, respectively; A is the effective membrane area (11.3 cm<sup>2</sup>); V is the volume of the source phase; and t is the elapsed time. The percentage of indium recovered in the receiving phase was calculated as:<disp-formula id=\"Equ12\"><label>13</label><alternatives><tex-math id=\"M23\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\% = \\frac{{\\left[ {In} \\right]_{s,0} - \\left[ {In} \\right]_{s,t} }}{{\\left[ {In} \\right]_{r,t} }}x100$$\\end{document}</tex-math><mml:math id=\"M24\" display=\"block\"><mml:mrow><mml:mo>%</mml:mo><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi mathvariant=\"italic\">In</mml:mi></mml:mrow></mml:mfenced><mml:mrow><mml:mi>s</mml:mi><mml:mo>,</mml:mo><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mo>-</mml:mo><mml:msub><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi mathvariant=\"italic\">In</mml:mi></mml:mrow></mml:mfenced><mml:mrow><mml:mi>s</mml:mi><mml:mo>,</mml:mo><mml:mi>t</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:msub><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi mathvariant=\"italic\">In</mml:mi></mml:mrow></mml:mfenced><mml:mrow><mml:mi>r</mml:mi><mml:mo>,</mml:mo><mml:mi>t</mml:mi></mml:mrow></mml:msub></mml:mfrac><mml:mi>x</mml:mi><mml:mn>100</mml:mn></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70968_Article_Equ12.gif\" position=\"anchor\"/></alternatives></disp-formula>where [In]<sub>r,t</sub> is the indium concentration in the receiving phase at a certain time, and [In]<sub>s,0</sub> and [In]<sub>s,t</sub> have the same meaning as above. Indium was analysed in the source and receiving phases by atomic absorption spectrometry. The In(III) concentration in the aqueous phases was found to be reproducible to ± 3%.</p><p id=\"Par40\">The membrane support used in the investigation was a Millipore Durapore GVHP4700, with 75% porosity (ε, defined as the ratio between the volume of the pores and the total volume of the membrane), 12.5 × 10<sup>–3</sup> cm thickness (d<sub>org</sub>), 1.67 tortuosity (τ, a property of the porous membrane support in relation to the turns in the pore channels) and 2.2 × 10<sup>–5</sup> cm pore size. The Millipore Durapore HVHP4700 support had the same specifications as above but with a 4.5 × 10<sup>–5</sup> cm pore size. Both were composed of polyvinylidene difluoride. The liquid membranes were prepared by immersion of the support in a carrier solution for 24 h and letting it drip for 20 s before being placed in the cell.</p></sec><sec id=\"Sec12\"><title>Conclusions</title><p id=\"Par41\">This investigation demonstrated that the ionic liquid HA324H<sup>+</sup>Cl<sup>-</sup> is efficient for the transport of indium(III) from hydrochloric acid solutions. It was experimentally found that the best conditions for indium(III) transport were 750 and 500 min<sup>-1</sup> stirring speeds in the source and receiving phases, respectively; 1–2 M HCl in the source phase and a carrier concentration of 0.23 M. In terms of metal transport, this carrier compares well with other carriers of various types, though indium(III) recovery in the receiving solution was not the best compared with that for other carriers, i.e., phosphonium salts and the phosphine oxide Cyanex 923. The kinetic model for indium(III) permeation showed that the permeation process is controlled by diffusion of the InCl4- species across the source phase layer and diffusion of the In(III)-carrier complex across the liquid membrane, with the former dominant when the carrier concentration in the membrane phase is low. The results derived from this work could be improved upon by the use of a smart membrane device, i.e., hollow fibre modules, and smart liquid membrane operation, i.e., pseudoemulsion-based hollow fibre strip dispersion technique.</p></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><ack><title>Acknowledgements</title><p>We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>F.A.L. funding acquisition; F.J.A. and F.A.L. methodology; F.J.A. formal analysis; F.J.A. and F.A.L. investigation and interpretation of data; F.J.A. writing-original draft; F.J.A. and F.A.L. writing-review & editing.</p></notes><notes notes-type=\"COI-statement\"><title>Competing interest</title><p>The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Amato</surname><given-names>A</given-names></name><etal/></person-group><article-title>End-of-life liquid crystal display recovery: toward a zero-waste approach</article-title><source>Appl. Sci.</source><year>2019</year><volume>9</volume><fpage>2985</fpage><pub-id pub-id-type=\"doi\">10.3390/app9152985</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Swain</surname><given-names>B</given-names></name><name><surname>Lee</surname><given-names>CG</given-names></name></person-group><article-title>Commercial indium recovery processes development from various e-(industry) waste through the insightful integration of valorization processes: a perspective</article-title><source>Waste Manag.</source><year>2019</year><volume>87</volume><fpage>597</fpage><lpage>611</lpage><pub-id pub-id-type=\"doi\">10.1016/j.wasman.2019.02.042</pub-id><pub-id pub-id-type=\"pmid\">31109560</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Amato</surname><given-names>A</given-names></name><name><surname>Rocchetti</surname><given-names>L</given-names></name><name><surname>Beolchini</surname><given-names>F</given-names></name></person-group><article-title>Environmental impact assessment of different end-of-life LCD management strategies</article-title><source>Waste Manag.</source><year>2017</year><volume>59</volume><fpage>432</fpage><lpage>441</lpage><pub-id pub-id-type=\"doi\">10.1016/j.wasman.2016.09.024</pub-id><pub-id pub-id-type=\"pmid\">27679968</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Amato</surname><given-names>A</given-names></name><name><surname>Beolchini</surname><given-names>F</given-names></name></person-group><article-title>End of life liquid crystal displays recycling: a patent review</article-title><source>J. Environ. Manag.</source><year>2018</year><volume>225</volume><fpage>1</fpage><lpage>9</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jenvman.2018.07.035</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Akcil</surname><given-names>A</given-names></name><name><surname>Agcasulu</surname><given-names>I</given-names></name><name><surname>Swain</surname><given-names>B</given-names></name></person-group><article-title>Valorization of waste LCD and recovery of critical raw material for circular economy: a review</article-title><source>Resour. Conserv. Recycl.</source><year>2019</year><volume>149</volume><fpage>622</fpage><lpage>637</lpage><pub-id pub-id-type=\"doi\">10.1016/j.resconrec.2019.06.031</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>López Díaz-Pavón</surname><given-names>A</given-names></name><name><surname>Cerpa</surname><given-names>A</given-names></name><name><surname>Alguacil</surname><given-names>FJ</given-names></name></person-group><article-title>Processing of indium (III) solutions via ion exchange with Lewatit K-2621 resin</article-title><source>Rev. Metal.</source><year>2014</year><volume>50</volume><fpage>e010</fpage><pub-id pub-id-type=\"doi\">10.3989/revmetalm.010</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alguacil</surname><given-names>FJ</given-names></name><name><surname>Lopez</surname><given-names>FA</given-names></name><name><surname>Rodriguez</surname><given-names>O</given-names></name><name><surname>Martinez-Ramirez</surname><given-names>S</given-names></name><name><surname>Garcia-Diaz</surname><given-names>I</given-names></name></person-group><article-title>Sorption of indium (III) onto carbon nanotubes</article-title><source>Ecotoxicol. Environ. Saf.</source><year>2016</year><volume>130</volume><fpage>81</fpage><lpage>86</lpage><pub-id pub-id-type=\"doi\">10.1016/j.ecoenv.2016.04.008</pub-id><pub-id pub-id-type=\"pmid\">27085001</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rocchetti</surname><given-names>L</given-names></name><name><surname>Amato</surname><given-names>A</given-names></name><name><surname>Beolchini</surname><given-names>F</given-names></name></person-group><article-title>Recovery of indium from liquid crystal displays</article-title><source>J. Clean. Prod.</source><year>2016</year><volume>116</volume><fpage>299</fpage><lpage>305</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jclepro.2015.12.080</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vostal</surname><given-names>R</given-names></name><name><surname>Reiber</surname><given-names>M</given-names></name><name><surname>Bertau</surname><given-names>M</given-names></name></person-group><article-title>Flüssig/flüssig-extraktion von indium aus sauren lösungen</article-title><source>Chem. Ing. Tech.</source><year>2019</year><volume>91</volume><fpage>1681</fpage><lpage>1687</lpage><pub-id pub-id-type=\"doi\">10.1002/cite.201900065</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alguacil</surname><given-names>FJ</given-names></name><name><surname>Garcia-Diaz</surname><given-names>I</given-names></name><name><surname>Escudero</surname><given-names>E</given-names></name></person-group><article-title>Extraction of indium(III) from sulphuric acid medium by the ionic liquid (PJMTH+HSO4−)</article-title><source>Sep. Purif. Technol.</source><year>2019</year><volume>211</volume><fpage>764</fpage><lpage>767</lpage><pub-id pub-id-type=\"doi\">10.1016/j.seppur.2018.10.051</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alguacil</surname><given-names>FJ</given-names></name><name><surname>Escudero</surname><given-names>E</given-names></name></person-group><article-title>Solvent extraction of indium(III) from HCl solutions by the ionic liquid (A324H+)(Cl−) dissolved in Solvesso 100</article-title><source>Hydrometallurgy</source><year>2019</year><volume>189</volume><fpage>105104</fpage><pub-id pub-id-type=\"doi\">10.1016/j.hydromet.2019.105104</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>García-Díaz</surname><given-names>I</given-names></name><name><surname>López</surname><given-names>FA</given-names></name><name><surname>Alguacil</surname><given-names>FJ</given-names></name></person-group><article-title>Transport of indium(III) using pseudo-emulsion based hollow fiber strip dispersion with ionic liquid RNH3+HSO4−</article-title><source>Chem. Eng. Res. Des.</source><year>2017</year><volume>126</volume><fpage>134</fpage><lpage>141</lpage><pub-id pub-id-type=\"doi\">10.1016/j.cherd.2017.08.012</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gu</surname><given-names>S</given-names></name><name><surname>Fu</surname><given-names>B</given-names></name><name><surname>Dodbiba</surname><given-names>G</given-names></name><name><surname>Fujita</surname><given-names>T</given-names></name><name><surname>Fang</surname><given-names>B</given-names></name></person-group><article-title>A sustainable approach to separate and recover indium and tin from spent indium–tin oxide targets</article-title><source>RSC Adv.</source><year>2017</year><volume>7</volume><fpage>52017</fpage><lpage>52023</lpage><pub-id pub-id-type=\"doi\">10.1039/C7RA11976C</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jowkar</surname><given-names>MJ</given-names></name><name><surname>Bahaloo-Horeh</surname><given-names>N</given-names></name><name><surname>Mousavi</surname><given-names>SM</given-names></name><name><surname>Pourhossein</surname><given-names>F</given-names></name></person-group><article-title>Bioleaching of indium from discarded liquid crystal displays</article-title><source>J. Clean. Prod.</source><year>2018</year><volume>180</volume><fpage>417</fpage><lpage>429</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jclepro.2018.01.136</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Willner</surname><given-names>J</given-names></name><name><surname>Fornalczyk</surname><given-names>A</given-names></name><name><surname>Gajda</surname><given-names>B</given-names></name><name><surname>Saternus</surname><given-names>M</given-names></name></person-group><article-title>Bioleaching of indium and tin from used LCD panels</article-title><source>Physicochem. Probl. Miner. Process.</source><year>2018</year><volume>54</volume><fpage>639</fpage><lpage>645</lpage></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Falke</surname><given-names>A</given-names></name><name><surname>Höck</surname><given-names>M</given-names></name></person-group><article-title>Ex-ante life cycle assessment of bioleaching in indium recovery from LCD-waste</article-title><source>MRS Adv.</source><year>2019</year><volume>4</volume><fpage>1949</fpage><lpage>1955</lpage><pub-id pub-id-type=\"doi\">10.1557/adv.2019.218</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luz</surname><given-names>AL</given-names></name><name><surname>Wu</surname><given-names>X</given-names></name><name><surname>Tokar</surname><given-names>EJ</given-names></name></person-group><article-title>Toxicology of inorganic carcinogens</article-title><source>Adv. Mol. Toxicol.</source><year>2018</year><volume>12</volume><fpage>1</fpage><lpage>46</lpage><pub-id pub-id-type=\"doi\">10.1016/B978-0-444-64199-1.00002-6</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chang</surname><given-names>A</given-names></name><name><surname>Li</surname><given-names>HY</given-names></name><name><surname>Chang</surname><given-names>IN</given-names></name><name><surname>Chu</surname><given-names>YH</given-names></name></person-group><article-title>Affinity ionic liquids for chemoselective gas sensing</article-title><source>Molecules (Basel, Switzerland)</source><year>2018</year><volume>23</volume><fpage>2380</fpage><pub-id pub-id-type=\"doi\">10.3390/molecules23092380</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Billard</surname><given-names>I</given-names></name></person-group><article-title>Green solvents in urban mining</article-title><source>Curr. Opin. Green Sustain. Chem.</source><year>2019</year><volume>18</volume><fpage>37</fpage><lpage>41</lpage><pub-id pub-id-type=\"doi\">10.1016/j.cogsc.2018.11.013</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fan</surname><given-names>Y</given-names></name><name><surname>Niu</surname><given-names>Z</given-names></name><name><surname>Xu</surname><given-names>C</given-names></name><name><surname>Yang</surname><given-names>L</given-names></name><name><surname>Yang</surname><given-names>T</given-names></name></person-group><article-title>Protic ionic liquids as efficient solvents in microwave-assisted extraction of rhein and emodin from <italic>Rheum palmatum</italic> L</article-title><source>Molecules (Basel, Switzerland)</source><year>2019</year><volume>24</volume><fpage>2770</fpage><pub-id pub-id-type=\"doi\">10.3390/molecules24152770</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Atanassova</surname><given-names>M</given-names></name><name><surname>Kurteva</surname><given-names>V</given-names></name></person-group><article-title>Synergism in the solvent extraction of Europium(III) with thenoyltrifluoroacetone and CMPO in methylimidazolium ionic liquids</article-title><source>J. Solut. Chem.</source><year>2019</year><volume>48</volume><fpage>15</fpage><lpage>30</lpage><pub-id pub-id-type=\"doi\">10.1007/s10953-019-00844-8</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Foltova</surname><given-names>SS</given-names></name><name><surname>Vander Hoogerstraete</surname><given-names>T</given-names></name><name><surname>Banerjee</surname><given-names>D</given-names></name><name><surname>Binnemans</surname><given-names>K</given-names></name></person-group><article-title>Samarium/cobalt separation by solvent extraction with undiluted quaternary ammonium ionic liquids</article-title><source>Sep. Purif. Technol.</source><year>2019</year><volume>210</volume><fpage>209</fpage><lpage>218</lpage><pub-id pub-id-type=\"doi\">10.1016/j.seppur.2018.07.069</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>S</given-names></name><name><surname>Chu</surname><given-names>N</given-names></name><name><surname>Li</surname><given-names>X</given-names></name><name><surname>Dong</surname><given-names>F</given-names></name><name><surname>Shen</surname><given-names>Y</given-names></name></person-group><article-title>Recovery of palladium from acidic nitrate media with triazole type extractants in ionic liquid</article-title><source>Hydrometallurgy</source><year>2019</year><volume>189</volume><fpage>105148</fpage><pub-id pub-id-type=\"doi\">10.1016/j.hydromet.2019.105148</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Merad</surname><given-names>NS</given-names></name><name><surname>Belkhouche</surname><given-names>NE</given-names></name></person-group><article-title>A comparative study of experimental optimization and taguchi design of Co(II) recovery by Aliquat 336 as ionic liquid carrier</article-title><source>J. Sustain. Metall.</source><year>2019</year><volume>5</volume><fpage>417</fpage><lpage>428</lpage><pub-id pub-id-type=\"doi\">10.1007/s40831-019-00232-y</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Svecova</surname><given-names>L</given-names></name><name><surname>Papaïconomou</surname><given-names>N</given-names></name><name><surname>Billard</surname><given-names>I</given-names></name></person-group><article-title>Rh(III) aqueous speciation with chloride as a driver for its extraction by phosphonium based ionic liquids</article-title><source>Molecules (Basel, Switzerland)</source><year>2019</year><volume>24</volume><fpage>1391</fpage><pub-id pub-id-type=\"doi\">10.3390/molecules24071391</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>N</given-names></name><name><surname>Wang</surname><given-names>Q</given-names></name><name><surname>Lu</surname><given-names>W</given-names></name><name><surname>Ru</surname><given-names>M</given-names></name><name><surname>Yang</surname><given-names>Y</given-names></name></person-group><article-title>Extraction and stripping of platinum (IV) from acidic chloride media using guanidinium ionic liquid</article-title><source>J. Mol. Liq.</source><year>2019</year><volume>293</volume><fpage>111040</fpage><pub-id pub-id-type=\"doi\">10.1016/j.molliq.2019.111040</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Deferm</surname><given-names>C</given-names></name><etal/></person-group><article-title>Speciation of indium(III) chloro complexes in the solvent extraction process from chloride aqueous solutions to ionic liquids</article-title><source>Dalton Trans.</source><year>2017</year><volume>46</volume><fpage>4412</fpage><lpage>4421</lpage><pub-id pub-id-type=\"doi\">10.1039/C7DT00618G</pub-id><pub-id pub-id-type=\"pmid\">28294262</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>de Gyves</surname><given-names>J</given-names></name><name><surname>de San</surname><given-names>Rodríguez</given-names></name><name><surname>Miguel</surname><given-names>E</given-names></name></person-group><article-title>Metal ion separations by supported liquid membranes</article-title><source>Ind. Eng. Chem. Res.</source><year>1999</year><volume>38</volume><fpage>2182</fpage><lpage>2202</lpage><pub-id pub-id-type=\"doi\">10.1021/ie980374p</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>El Aamrani</surname><given-names>FZ</given-names></name><name><surname>Kumar</surname><given-names>A</given-names></name><name><surname>Beyer</surname><given-names>L</given-names></name><name><surname>Florido</surname><given-names>A</given-names></name><name><surname>Sastre</surname><given-names>AM</given-names></name></person-group><article-title>Mechanistic study of active transport of silver(I) using sulfur containing novel carriers across a liquid membrane</article-title><source>J. Membr. Sci.</source><year>1999</year><volume>152</volume><fpage>263</fpage><lpage>275</lpage><pub-id pub-id-type=\"doi\">10.1016/S0376-7388(98)00241-5</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>TC</given-names></name><name><surname>Juang</surname><given-names>RS</given-names></name></person-group><article-title>Rate and mechanism of divalent metal transport through supported liquid membrane containing di(2-ethylhexyl) phosphoric acid as a mobile carrier</article-title><source>J. Chem. Technol. Biotechnol.</source><year>2007</year><volume>42</volume><fpage>3</fpage><lpage>17</lpage><pub-id pub-id-type=\"doi\">10.1002/jctb.280420103</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Nat Commun</journal-id><journal-id journal-id-type=\"iso-abbrev\">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type=\"epub\">2041-1723</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807782</article-id><article-id pub-id-type=\"pmc\">PMC7431577</article-id><article-id pub-id-type=\"publisher-id\">17933</article-id><article-id pub-id-type=\"doi\">10.1038/s41467-020-17933-8</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Cryo-EM structure of an activated VIP1 receptor-G protein complex revealed by a NanoBiT tethering strategy</article-title></title-group><contrib-group><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Duan</surname><given-names>Jia</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Shen</surname><given-names>Dan-dan</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Zhou</surname><given-names>X. Edward</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Bi</surname><given-names>Peng</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Liu</surname><given-names>Qiu-feng</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Tan</surname><given-names>Yang-xia</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhuang</surname><given-names>You-wen</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Hui-bing</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Xu</surname><given-names>Pei-yu</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Huang</surname><given-names>Si-Jie</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ma</surname><given-names>Shan-shan</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-7813-5480</contrib-id><name><surname>He</surname><given-names>Xin-heng</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-9125-4027</contrib-id><name><surname>Melcher</surname><given-names>Karsten</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-2189-0244</contrib-id><name><surname>Zhang</surname><given-names>Yan</given-names></name><address><email>zhang_yan@zju.edu.cn</email></address><xref ref-type=\"aff\" rid=\"Aff3\">3</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-6829-8144</contrib-id><name><surname>Xu</surname><given-names>H. Eric</given-names></name><address><email>eric.xu@simm.ac.cn</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-0723-1413</contrib-id><name><surname>Jiang</surname><given-names>Yi</given-names></name><address><email>yijiang@simm.ac.cn</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.9227.e</institution-id><institution-id institution-id-type=\"ISNI\">0000000119573309</institution-id><institution>The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, </institution><institution>Chinese Academy of Sciences, </institution></institution-wrap>Shanghai, 201203 China </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.410726.6</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1797 8419</institution-id><institution>University of Chinese Academy of Sciences, </institution></institution-wrap>100049 Beijing, China </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.13402.34</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1759 700X</institution-id><institution>Department of Pathology of Sir Run Run Shaw Hospital, </institution><institution>Zhejiang University School of Medicine, </institution></institution-wrap>Hangzhou, 310058 China </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.13402.34</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1759 700X</institution-id><institution>Department of Biophysics, </institution><institution>Zhejiang University School of Medicine, </institution></institution-wrap>Hangzhou, 310058 China </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.251017.0</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0406 2057</institution-id><institution>Center for Cancer and Cell Biology, Program for Structural Biology, </institution><institution>Van Andel Institute, </institution></institution-wrap>Grand Rapids, MI USA </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.440637.2</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 4657 8879</institution-id><institution>School of Life Science and Technology, </institution><institution>ShanghaiTech University, </institution></institution-wrap>Shanghai, 201210 China </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>4121</elocation-id><history><date date-type=\"received\"><day>30</day><month>1</month><year>2020</year></date><date date-type=\"accepted\"><day>17</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Vasoactive intestinal polypeptide receptor (VIP1R) is a widely expressed class B G protein-coupled receptor and a drug target for the treatment of neuronal, metabolic, and inflammatory diseases. However, our understanding of its mechanism of action and the potential of drug discovery targeting this receptor is limited by the lack of structural information of VIP1R. Here we report a cryo-electron microscopy structure of human VIP1R bound to PACAP27 and Gs heterotrimer, whose complex assembly is stabilized by a NanoBiT tethering strategy. Comparison with other class B GPCR structures reveals that PACAP27 engages VIP1R with its N-terminus inserting into the ligand binding pocket at the transmembrane bundle of the receptor, which subsequently couples to the G protein in a receptor-specific manner. This structure has provided insights into the molecular basis of PACAP27 binding and VIP receptor activation. The methodology of the NanoBiT tethering may help to provide structural information of unstable complexes.</p></abstract><abstract id=\"Abs2\" abstract-type=\"web-summary\"><p id=\"Par2\">Vasoactive intestinal polypeptide receptor (VIP1R) is a widely expressed class B G protein-coupled receptor and a drug target for the treatment of inflammatory diseases. Here authors report a cryoelectron microscopy structure of human VIP1R bound to PACAP27 and Gs heterotrimer, which provides insights into PACAP27 binding and VIP receptor activation.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>G protein-coupled receptors</kwd><kwd>Cryoelectron microscopy</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par3\">Vasoactive intestinal polypeptide receptors, also known as VIP receptors, including VIP1R and VIP2R, belong to the class B1 of G protein-coupled receptors. Upon activating by vasoactive intestinal peptide (VIP), an endogenous, 28-amino acid neuropeptide, a VIP receptor couples to Gs heterotrimer, resulting in the stimulation of adenylyl cyclase. In addition to VIP, VIP receptors also bind to other neuropeptides called pituitary adenylate cyclase-activating peptides (PACAPs) with similar affinity. Two forms of PACAP are known, the 27 amino acid long PACAP27 and the 38 amino acid long PACAP38, of which PACAP27 is a C terminally truncated variant of PACAP38, and shows particularly high homology (~68%) to VIP. The PACAP peptides have been in the spotlight of extensive basic and applied research, and have been linked to for over 40 different pathological conditions with clinical relevance<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>.</p><p id=\"Par4\">VIP1R is widely distributed in the CNS, most abundantly in the cerebral cortex and hippocampus<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>, where it plays diverse and important roles with functions in the control of circadian rhythms, learning, memory, anxiety and responses to stress, and brain injury. VIP1R is also expressed in a number of peripheral tissues, including liver, lung, and intestine<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>–<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>, and in T lymphocytes<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. The development of drugs acting on VIP receptors may lead to new treatments for sleep disorders, stroke, neurodegenerative disorders, and age-related memory impairment.</p><p id=\"Par5\">Extensive efforts have been made to discover the roles of the VIP1R system and to take advantage of VIP and PACAP analogs in therapeutic applications. Understanding the mechanism of peptide recognition and signal transduction by VIP1R has been aided by insights from several functional data from mutagenesis, photoaffinity labeling<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>, molecular modeling<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>, and limited structure information of VIP2R extracellular domain (ECD) (PDB code: 2X57) and VIP peptide<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Several of VIP and PACAP peptide analogs have been studied for their potential therapeutic applications<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. A high-resolution structure of a full-length VIP receptor is needed for both mechanistic research as well as drug discovery targeting this GPCR system.</p><p id=\"Par6\">The resolution revolution of cryo-EM has made a significant impact on GPCR structural biology<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. The atomic resolution or near-atomic resolution GPCR–G protein complex structures solved by cryo-EM have revealed structural details of ligand recognition and signal transduction by this superfamily of cell surface receptors. Various methods have been developed to improve the stability of GPCR-signal transducer complexes, such as the use of thermo-stabilizing mutations<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>, nanobodies, and antibody fragments<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>, to facilitate structural studies. However, poor sample stability remains the bottleneck in structural studies of GPCR complexes. In this work, we have developed a method to stabilize the interaction between VIP1R and the Gs heterotrimer by bringing the two proteins into close proximity through a NanoBiT tethering approach. This method greatly improved the stability and homogeneity of the PACAP27–VIP1R–Gs protein complex, allowing structural determination of human VIP1R in complex with PACAP27 and Gs heterotrimer. We also demonstrate that the NanoBiT tethering method can be applied to other GPCR–G protein complexes.</p></sec><sec id=\"Sec2\" sec-type=\"results\"><title>Results</title><sec id=\"Sec3\"><title>The tethered NanoBiT stabilize GPCR–G protein complexes</title><p id=\"Par7\">NanoBiT system is one of the protein-fragment complementation methods based on split luciferase, which is originally developed to monitor protein-protein interactions<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. When the NanoBiT is dissected between residues 156 and 157, it can be split into a large component containing 156 amino acid residues named large BiT (LgBiT), and a 13-amino acid peptide called small BiT (SmBiT, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1a</xref>). By engineering the sequence of SmBiT, a series of peptides with various equilibrium dissociation constants were created, among which peptide 86 (HiBiT) (VSGWRLFKKIS) has the most potent binding affinity, with five orders of magnitude (~1 nM to ~200 μM) greater than that of the wild-type (WT) peptide 114 (VTGYRLFEEIL) (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1b</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. The fragments of the LgBiT and SmBiT are genetically fused to a pair of interacting proteins. The interaction of fusion partners leads to structural complementation of LgBiT with SmBiT, generating a functional NanoBiT enzyme with a detectable luminescent signal.</p><p id=\"Par8\">Inspired by the complementation principle of NanoBiT, we fused the SmBiT peptide 86 at the C-terminus of the Gβ subunit to bind the LgBiT that was attached to the C-terminus of the truncated receptor (VIP1R 31–437), thus providing an additional linkage to stabilize the interface of helix 8 of VIP1R and the Gβ subunit of the G protein (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>; see “Methods”). The flexible C-terminus of VIP1R serves as the natural linker to connect LgBiT. The WT VIP1R and different lengths of C-terminus truncated VIP1R at L437, G424, and K417 were screened for assembly of the complex. When the receptor was truncated to L437, the components can be assembled into the VIP1R(31–437)–VIP1R–Gs complex with an equal proportion, suggesting a better assembly efficacy for the complex. Thus, unless otherwise specified, VIP1R refers to VIP1R(31–437), which is used in structure determination and functional analyses. Compared to WT VIP1R, the truncated receptor exhibited a comparable response to PACAP27-induced activation. The LgBiT fusion to the truncated VIP1R or cotransfection of the truncated receptor with Gβ-HiBiT does not affect PACAP27-induced VIP1R activation (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1c</xref>). Combined with Nb35, which is used to stabilize the complex between Gαs and Gβ<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>, the NanoBiT tethering method can enhance the stability of the VIP1R–Gs complex and facilitate the structure study of this GPCR complex.<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>The NanoBiT strategy for stabilization of PACAP27–VIP1R–Gs protein complex.</title><p><bold>a</bold> Schematic diagram of the NanoBiT aided assembly of the VIP1R–Gs complex. PACAP27 is colored in orange, VIP1R in green, Gαs in yellow, Gβ in blue, Gγ in purple, LgBiT in light blue, and HiBiT in red. <bold>b</bold> Respective size-exclusion chromatography elution profiles of the VIP1R–Gs and VIP1R-LgBiT-Gs-HiBiT complexes. <bold>c</bold> Dynamic light scattering (DLS) size distribution histograms of VIP1R–Gs and VIP1R-LgBiT-Gs-HiBiT complexes. Values of radius, % intensity of monomer, and ratio of monomer/aggregation (M/A) are listed. <bold>d</bold> Representative negative staining images of the corresponding complexes. The scale bar is 200 nm. Source data are provided as a <xref rid=\"MOESM4\" ref-type=\"media\">Source Data</xref> file.</p></caption><graphic xlink:href=\"41467_2020_17933_Fig1_HTML\" id=\"d30e524\"/></fig></p><p id=\"Par9\">To investigate the effect of the NanoBiT tethering method on stabilization of the PACAP27–VIP1R–Gs complex, SDS-PAGE analysis, gel filtration chromatography, dynamic light scattering (DLS), and negative staining was performed. The SDS-PAGE analysis showed that all components of the VIP1R–Gs complex were present with the NanoBiT tethering (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1d</xref>). Gel filtration chromatography also revealed that the complex with the NanoBiT tethering had a much more uniform distribution than the WT VIP1R–Gs complex, indicating that the NanoBiT tethering method contributed additional stability to the VIP1R–Gs complex (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref>).</p><p id=\"Par10\">We further used DLS to evaluate complex homogeneity and thermostability. A peak around a radius of ~10 nm corresponds to the monomeric complex of VIP1R–Gs complex, while the peak at ~100 nm represents protein aggregation. Our data show that the NanoBiT tethering improved the monodispersity of the VIP1R complex with a 3.3-fold increase of monomer/aggregation ratio (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1c</xref>). The relatively smaller radius size and narrower radius size distribution also suggest that the NanoBiT tethering complex was more compact and homogeneous than the WT complex, while the protein aggregation onset temperature (<italic>T</italic><sub>onset</sub>), a marked temperature point indicating protein denaturation and aggregation, remained unchanged (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1e</xref>). The negative staining images displayed that particle morphology and integrity of the NanoBiT-tethered complex have been improved relative to the less consistent particles of the WT complex, indicating improved homogeneity and integrity of the NanoBiT-tethered sample (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1d</xref>).</p><p id=\"Par11\">We further investigated whether NanoBiT tethering conferred a similar stabilization effect on other GPCR–G protein complexes. CCR7, a class A GPCR that couples to Gi protein, was chosen as a representative receptor. The NanoBiT tethering method significantly improved the homogeneity of the complex, leading to high homogeneity and integrity of negatively stained complex particles, which is in agreement with its effect on the VIP1R–Gs complex. The NanoBiT tethering also increased the thermostability of the CCR7-Gi complex, as evidenced by an increase of <italic>T</italic><sub>onset</sub> by ~10 °C (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>).</p><p id=\"Par12\">Taken together, we developed a strategy to stabilize the GPCR–G protein complex by direct linking of a GPCR with its G protein through NanoBiT protein-fragment complementation. Using this method, we were able to obtain a stable PACAP27–VIP1R–Gs complex for cryo-EM studies.</p></sec><sec id=\"Sec4\"><title>Structure determination of VIP1R bound to PACAP27 and Gs</title><p id=\"Par13\">The structure of the PACAP27–VIP1R–Gs complex was determined from 131,263 particles to a resolution of 3.2 Å (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4</xref> and Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>). The density is clear for the VIP1R TM bundle, the bound peptide PACAP27, the heterotrimeric Gs, and Nb35. Like many other GPCR–G protein complexes, density is missing for the α-helical domain of the Gαs. In addition, the ECD of VIP1R was not resolvable with this limited dataset, perhaps reflecting its highly dynamic and conformationally flexible property when bound to PACAP27. This is consistent with the highly dynamic nature of ECD in class B GPCRs when bound to activating ligands as the ECD structures were not well resolved in most of other active class B GPCR–G protein complexes<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>–<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. The complex structure of PACAP27–VIP1R–Gs was built with the recently published PTH1R–Gs complex structure (PDB: 6NBH)<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup> as an initial model. The final structure contains all residues of PACAP27 (residues 1–27), the Gαs Ras-like domain, Gβγ subunits, Nb35, and the VIP1R residues from A129<sup>1.26b</sup> to Q409<sup>8.64b</sup> (class B GPCR numbering in superscript<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>) (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). The majority of amino acid side chains were well resolved in the final model, which were refined against the EM density map (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>). Thus, the complex structure can provide detailed information on the interface between Gαs and the receptor, as well as the binding interface between PACAP27 and helix bundle core of the receptor.<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>The overall cryo-EM structure of PACAP27–VIP1R–Gs complex.</title><p><bold>a</bold> A cut-through view of the cryo-EM map of PACAP27–VIP1R–Gs complex with a disc-shaped micelle. <bold>b</bold> A cartoon representation of the PACAP27–VIP1R–Gs complex. <bold>c</bold> Extracellular view of the PACAP27–VIP1R–Gs complex structure. PACAP27 is colored in orange; VIP1R in green; Gαs Ras-like domain in yellow; Gβ subunit in blue; Gγ subunit in purple; Nb35 in gray; and lipid molecules in cyan.</p></caption><graphic xlink:href=\"41467_2020_17933_Fig2_HTML\" id=\"d30e618\"/></fig></p><p id=\"Par14\">The TMD of the VIP1R receptor is surrounded by an annular detergent micelle mimicking the natural phospholipid bilayer. Within the micelle, six cholesterol molecules are clearly visible in the cryo-EM density map (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>), which hydrophobically binds around the helix bundle of the receptor and may contribute to the stability of the receptor-ligand binding<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>.</p><p id=\"Par15\">Interestingly, the density of the NanoBiT is invisible in our structure. We suspect that the NanoBiT can increase the local concentration of Gs heterotrimer near VIP1R, and also make the Gs heterotrimer not easily dissociated from the receptor. Compared with the VIP1R–Gs protein complex, the NanoBiT is relatively flexible because of the existence of the linker between LgBiT and the receptor. The stabilization mode of NanoBiT is different from the antibodies that bind against the antigen directly and can be traced in a clear density map.</p></sec><sec id=\"Sec5\"><title>PACAP recognition by VIP1R and PAC1R</title><p id=\"Par16\">The activated VIP1R complex shows that PACAP27 adopts α-helical conformations and engages a V-shape binding pocket with a prominent open cleft at the extracellular part of the helix bundle. PACAP27 interacts with each of the TM helices except TM4, with the N-terminus of the peptide inserting deeply into the TMD core. ECL2 and ECL3 also mediate the interaction between peptide and receptor (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b, c</xref>).<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Comparison of the binding mode of PACAPs to VIP1R and PAC1R.</title><p><bold>a</bold> Sequence alignment of the VIP1R peptide ligands VIP, PACAP27, and PACAP38. <bold>b</bold> The binding mode of PACAP27 to VIP1R, showing that PACAP27 adopts α-helical conformation and interacts with all TM helices of VIP1R except TM4. <bold>c</bold> The cross-section view of the PACAP27 binding pocket in the TM bundle of VIP1R. Structural comparisons of PACAP binding pockets in VIP1R and PAC1R. Residues interact with peptide amino acids H1 and D3 (<bold>d</bold>), S2 (<bold>e</bold>), G4, I5, and F6 (<bold>f</bold>), as well as amino acids from T7 to R14 (<bold>g</bold>, <bold>h</bold>) are shown as sticks. The hydrogen bonds between PACAP27 and residues of VIP1R are marked as black dotted lines, and the hydrogen bonds between PACAP38 and residues of VIP1R are shown as red dotted lines. PACAP27 is colored in orange, and VIP1R in green. PACAP38 is shown in cyan, and PAC1R (PDB code: 6P9Y) in light blue.</p></caption><graphic xlink:href=\"41467_2020_17933_Fig3_HTML\" id=\"d30e672\"/></fig></p><p id=\"Par17\">Compared to other peptide ligands bound in the pockets of their cognate class B GPCRs<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref>–<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, PACAP27 shows different conformations primarily at its C-terminal end and is differently oriented in the ligand binding pocket of the receptor (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6a, b</xref>). In contrast, the N-terminal ends of all peptide ligands are well overlapped among different members of class B GPCRs (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6b</xref>). The orientation of a peptide ligand in the ligand binding pocket of a class B GPCR is determined by the specific interactions of the N-terminal portion of the peptide ligand with the TMD of the receptor, which keeps the peptide ligand in a specific position in the ligand binding pocket. The TMD peptide-binding pocket of VIP1R is similar to that of PAC1R with pocket volumes of 3261 and 3246 Å<sup>3</sup>, respectively, as these two receptors share peptidic ligand PACAP with similar affinity. Notably, the TMD peptide-binding pocket of VIP1R and PAC1R are smaller than those of class B GPCRs solved to date (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6c</xref> and Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>).</p><p id=\"Par18\">A comparison of the structures of PACAP27–VIP1R–Gs with the newly released PACAP38–PAC1R–Gs complex (PDB: 6P9Y)<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup> will help to clarify the recognition mechanism of VIP1R and PAC1R by PACAP peptide. For VIP1R, the first peptide residue H1 not only makes extensive hydrophobic contacts with several nonpolar residues from the ligand pocket (V226<sup>3.40b</sup>, F230<sup>3.44b</sup>, W294<sup>5.36b</sup>, I301<sup>5.43b</sup>, and the backbone of K298<sup>5.40b</sup>) but also forms a hydrogen bond with Q223<sup>3.37b</sup> (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3d</xref>, Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). Alanine mutations in Q223<sup>3.37b</sup> and W294<sup>5.36b</sup> reduced PACAP27-mediated VIP1R activation, supporting the fact that H1 of the peptide is critical for peptide-induced receptor activation (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>). For PAC1R, H1 interacts with highly conserved hydrophobic residues with VIP1R. However, the hydrogen bond interacts with residue at 3.37 (H234<sup>3.37</sup>) is absent (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3d</xref>, Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). Compared to H1, S2 faces a significant different residue environment in these two receptors. S2 additionally hydrogen-bonded with R199<sup>2.60</sup> and Y241<sup>3.44</sup> in PAC1R compared to VIP1R, making the PACAP38 inserted deeper into the TMD core of PAC1R (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3e</xref>, Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). D3 forms hydrogen bond with R188<sup>2.60</sup>, and also hydrophobic contacts with F222<sup>3.36</sup> and L374<sup>7.43</sup>. These interactions are highly conserved between VIP1R and PAC1R (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3d</xref>, Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). Mutations of the peptide residue D3 or the receptor residue R188<sup>2.60b</sup> and F222<sup>3.36</sup> impaired ligand-induced receptor activation<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup> (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>). G4, I5, and F6 from the peptide ligand are surrounded by hydrophobic pocket residues of VIP1R from TM1 (Y139<sup>1.36b</sup>, V142<sup>1.39b</sup>, Y146<sup>1.43b</sup>), TM2 (L199<sup>2.71b</sup>), TM5 (W294<sup>5.36b</sup>), and TM7 (M370<sup>7.39b</sup> and L374<sup>7.43b</sup>), as well as from ECL2 (I289<sup>ECL2</sup>) (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3f</xref>, Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). Mutations of hydrophobic residues Y146<sup>1.43b</sup>, L199<sup>2.71b</sup>, and W294<sup>5.36b</sup> to alanines significantly decreased the PACAP27-induced VIP1R activation, indicating that G4, I5, and F6 may be involved in VIP1R activation (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>). These interactions also highly conserved between PACAP38 and cognate residues in PAC1R (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>).</p><p id=\"Par19\">Compared to the six N-terminal residues of PACAPs, peptide residues from T7 to R14 exhibit different binding modes to VIP1R and PAC1R. Besides identical hydrogen bonds between S11 in PACAPs and D<sup>ECL2</sup> in two receptors, other polar interactions (D8 and I289<sup>ECL2</sup>, Y13 and D132<sup>1.29</sup>, as well as T136<sup>1.33</sup> for VIP1R, and S9 and K378<sup>7.35</sup>, S11 and Y211<sup>ECL1</sup>, R12 and D301<sup>ECL2</sup> for PAC1R) are unique (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3g, h</xref>, Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). Mutations of I289<sup>ECL2</sup> to Ala decreased PACAP27 potency in promoting VIP1R to couple with Gs, indicating a specific role of D8 for PACAP27 activity (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>). Thus, the N-terminus of PACAP27 engages within the helix bundle core in a receptor-specific manner.</p><p id=\"Par20\">The structural studies on VIP1R binding pocket also provide a clue on the potential recognition mechanism of VIP1R by VIP, a peptidic ligand shares highly conserved sequences and bound VIP1R with similar affinities compared to PACAP27<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. Although the peptide sequences from PACAP27 and VIP are highly conserved (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref>), these two peptides may interact with VIP1R in a peptide-specific mode. The previous alanine scanning analysis of VIP supported the fact that H1, D3, F6, R12, and R14, identical amino acids at cognate positions of PACAP27, are important for determining the affinity of VIP to VIP1R<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. Although H1, D3, F6, and R14 are also supposed to be involved in PACAP27-mediated activation of VIP1R, R12 of PACAP27 seems not to form any substantial interaction with residues in VIP1R binding pocket, indicating a distinct VIP1R binding mode for these two peptides.</p><p id=\"Par21\">The structural-based mutagenesis analysis also provides a potential explanation of VIP selectivity for VIP1R over PAC1R. Structurally, G4 in PACAP closely contacts W<sup>5.36b</sup> in VIP1R. When mutating G4 of PACAP to Ala, the cognate amino acid of VIP, a more significant steric constraint, is generated between the newly mutated A4 and W306<sup>5.36b</sup> of PAC1R compared to W296<sup>5.36b</sup> in VIP1R, which may restrict the binding of VIP to PAC1R and lead to a lower selectivity for VIP for PAC1R than VIP1R (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">7</xref>). This structure feature is coincident with the fact that when replacing A4-V5 dipeptide of VIP by G4-I5 in PACAP, the new VIP analog obtains the ability to bind and activate PAC1R. Similarly, PACAP27 abolished its propensity to bind PAC1R when its G4-I5 sequence was substituted for A4-V5 in VIP<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>.</p><p id=\"Par22\">Together, these observations provide a rationale for understanding VIP1R recognition by PACAP27 and VIP1R-targeted ligand discovery.</p></sec><sec id=\"Sec6\"><title>Activation of VIP1R by PACAP27</title><p id=\"Par23\">The structural hallmark of class B GPCR activation is the much more pronounced outward shift of TM6 than that in class A GPCRs, which is accompanied by the formation of a sharp kink in the middle of the TM6 induced and stabilized by ligand binding. The N-terminal residues H1 and S2 from PACAP27 pack directly against the C-terminus of TM6, and disrupt the helical conformation of the conserved PxxG motif (P348<sup>6.47b</sup>−L349<sup>6.48b</sup>−F350<sup>6.49b</sup>−G351<sup>6.50b</sup>), and create a ~90° sharp kink at the middle of TM6 (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4a</xref>). The kink conformation of TM6 is stabilized by polar interactions between P348<sup>6.47b</sup> and F350<sup>6.49b</sup> with the side chains of Q380<sup>7.49b</sup> and N308<sup>5.50b</sup>, respectively, (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4b</xref>). It is notable that Q380<sup>7.49b</sup> also forms polar interaction with Y354<sup>6.53b</sup>, a residue at the C-terminal end of the kink, suggesting its critical role in stabilizing the kink conformation of TM6 and the active state of the receptor (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4b</xref>, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">8</xref>). In addition, compared with conformations of L357<sup>6.48b</sup> and L358<sup>6.49b</sup> in the inactive GCGR structure, large conformational rotations of L349<sup>6.48b</sup> and F350<sup>6.49b</sup> were induced by the kink of TM6, creating extensive hydrophobic contacts with conserved residues in TM2 (H178<sup>2.50b</sup>), TM3 (L240<sup>3.54b</sup>), TM5 (F312<sup>5.54b</sup>, I315<sup>5.57b</sup>, and I316<sup>5.58b</sup>), TM6 (L346<sup>6.45b</sup>), and TM7 (Y388<sup>7.57b</sup>) to stabilize the kinked TM6 conformation (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4b</xref>).<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>Structure comparisons of active VIP1R with inactive GCGR.</title><p><bold>a</bold> The structural alignment of activated VIP1R with inactive GCGR showing the outward bending of the intracellular portion of TM6 of activated VIP1R, which results in a kink at the PxxG motif in TM6 and a ~90° angle between two portions of TM6 of the activated receptor. The TM6 kink in the active VIP1R structure is indicated by a dotted black line. The residues in the conserved PxxG motif in TM6 are shown in stick representation. <bold>b</bold> Polar and hydrophobic interactions that stabilize the kink at TM6 of activated VIP1R. The polar contacts are marked as black dotted lines. The positions of conserved polar residue networks located within VIP1R (green) and inactive GCGR (PDB code:4L6R, colored in salmon): central polar network (<bold>c</bold>), HETY network (<bold>d</bold>), and TM2–6–7–helix 8 network (<bold>e</bold>). Side chains of the residues are shown in stick representation.</p></caption><graphic xlink:href=\"41467_2020_17933_Fig4_HTML\" id=\"d30e1024\"/></fig></p><p id=\"Par24\">Compared with the conformation of the inactive GCGR structure<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>, the kink of TM6 and subsequent outward shift of its cytoplasmic end caused a rearrangement of three conserved polar interaction networks, including the central polar network (R188<sup>2.60b</sup>, N229<sup>3.43b</sup>, H353<sup>6.52b</sup>, and Q380<sup>7.49b</sup>), HETY (H178<sup>2.50b</sup>, E236<sup>3.50b</sup>, T343<sup>6.42b</sup>, and Y388<sup>7.57b</sup>), and TM2–6–7–helix 8 (R174<sup>2.46b</sup>, R338<sup>6.37b</sup>, N392<sup>7.61b</sup>, and E394<sup>8.49b</sup>) polar networks (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c–e</xref>). The residues from the central polar network are involved in peptide ligand binding by the receptor, suggesting that their conformational changes are required for the receptor to facilitate the peptide ligand binding and signal transduction (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">9</xref>). Previous experiments showed that point mutations of VIP1R residues R188<sup>2.60b</sup>, N229<sup>3.43b</sup>, and Q380<sup>7.49b</sup> severely affect the binding of VIP and VIP-mediated cAMP production<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>, in agreement with our structural data. Interestingly, in many published class B GPCR active structures, these polar network residues are not in close contact with peptide ligands, except for VIP1R, PAC1R, and PTH1R. We observed that residue R188<sup>2.60b</sup> of VIP1R forms a charge interaction with D3 of PACAP27. The corresponding residue in PAC1R, R199<sup>2.60b</sup>, forms direct polar interactions with N-terminal S2 and D3 of PACAP38. A similar interaction can also be observed between R233<sup>2.60b</sup> of PTH1R and E4 of LA-PTH (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">9</xref>). These polar interactions between the peptide and the receptor serve as the structural basis of ligand-induced receptor activation.</p><p id=\"Par25\">Our PACAP27-bound VIP1R–Gs complex structure also exhibits broken HETY and TM2–6–7–helix 8 polar networks, which are caused by the outward movement of the intracellular segment of TM6 that takes away TM6 residues T343<sup>6.42b</sup> and R338<sup>6.37b</sup>, respectively, from these two networks (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4d, e</xref>). VIP1R contains the conserved HETY motif, which is known to mediate inter-helix interactions of TM2–6–7–helix 8 polar networks in GCGR, PTH1R, and CRFR1. Disruption of this inter-helix interactions has resulted in constitutively active class B GPCRs receptors<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref>–<xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. We, therefore, speculate that TM2–3–6–7 polar networks may also be required for maintaining an inactive conformation, and the breakage of these polar networks may represent the active conformations of VIP1R. Indeed, mutations that disrupt this polar network in VIP1R have resulted in the constitutively active receptor<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>–<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>.</p><p id=\"Par26\">Taken together, despite the different sequence and physicochemical environment of VIP1R in ligand binding pocket, VIP1R shares a common activation mechanism with other class B GPCRs, which is characterized by a set of conserved residues involved in ligand-induced conformational changes in the receptor helix bundle as well as residues involved in G protein coupling. The polar networks in the helix bundle core, the central polar network, HETY, and TM2–6–7–helix 8 networks, required in maintaining the inactive conformation of the receptor, undergo ligand-induced conformational changes that rearrange the network residues to facilitate the ligand binding and to stabilize the active conformation of the receptor.</p></sec><sec id=\"Sec7\"><title>Gs heterotrimer coupling by VIP1R</title><p id=\"Par27\">The overall assembly of the receptor with Gs is remarkably similar to many other class B GPCRs solved to date, with several unique features of receptor-specific interactions with the Gs heterotrimer<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>–<xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref>–<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. The outward moved cytoplasmic end of TM6 and concomitantly shifted TM5 form a cytoplasmic cavity together with TM2, 3, and 7 to accommodate the α5 helix of Gαs. This interface serves as a crucial contact between the receptor and Gs heterotrimer. Additional contacts are observed between extended helix 8 of the receptor and the Gβ subunit of the Gs heterotrimer. ICL3, although invisible in our complex structure, also makes important contributions because residues I328-S331 in the central part of ICL3 are crucial for efficient binding of VIP1R to Gαs<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref>,<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. Structural alignment of our PACAP27–VIP1R–Gs complex with other class B GPCR–Gs protein complex structures solved to date by superimposing their receptor TM domains reveals different orientations of the Gs heterotrimers with rigid body rotations around the axis of the Gβ subunit (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">10a</xref>). The structural similarities in the Gs heterotrimer may be influenced by the use of Nb35, which has been used in all the structures of Gs-coupled receptor complexes reported thus far.</p><p id=\"Par28\">The VIP1R residues at the interface of the cytoplasmic cavity and the α5 helix of Gαs are highly conserved among class B GPCRs (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">8</xref>). The polar interactions mediated by these conserved residues on TM3, TM5, and TM6 can also be observed in the interface between VIP1R and Gαs, including an extensive hydrogen bond network formed between α5 helix and the cytoplasmic receptor cavity (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5a, b</xref>). The interface of ICL2 with α5 and αN-β1 junction of Gαs is primarily stabilized by hydrophobic contacts (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5c</xref>). Additional electrostatic contacts presented between K169<sup>ICL1</sup> and D312 of helix 8 together with hydrogen bonds between R405<sup>8.60b</sup> and the backbone oxygen of A309 and G310 may further stabilize the interface between helix 8 and Gβ (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5d</xref>).<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>The interactions between VIP1R and Gs heterotrimer.</title><p><bold>a</bold>, <bold>b</bold> The binding interface between the cavity on the intracellular side of VIP1R TMD (green) and α5 helix of the Gαs Ras-like domain (yellow). <bold>c</bold> The interface between ICL2 of VIP1R (green) and α5 and αN of the Gαs Ras-like domain (yellow). <bold>d</bold> The interface between helix 8 of VIP1R (green) and Gβ subunit (blue). Residues in VIP1R–Gs interfaces are shown in stick representation.</p></caption><graphic xlink:href=\"41467_2020_17933_Fig5_HTML\" id=\"d30e1202\"/></fig></p><p id=\"Par29\">Compared with other class B GPCR–Gs complex structures, the PACAP27–VIP1R–Gs complex shows different intermolecular interactions at the interface constituted of TM2 and TM3, and the TM7–helix 8 turn of the receptor and α5 of Gαs. Similar to the PTH1R–Gs complex, the VIP1R–Gs complex lacks several polar interactions that are present in other GPCR–Gs complexes between Q390/E392 on α5 and R<sup>2.46</sup>/N<sup>8.57</sup> as well as residues at 8.48 and 8.49 in the receptors (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">10b</xref>). The slightly outward movement of the turn between TM7 and helix 8 and the shift of α5 away from the TM7–helix 8 turn lead to a smaller Gαs-buried surface area of VIP1R than those of other class B GPCR–Gs complexes (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">10b</xref> and Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>). This is consistent with the fact that the VIP1R–Gs complex is not sufficiently stable for cryo-EM studies without NanoBiT tethering.</p></sec></sec><sec id=\"Sec8\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par30\">Here, we report a near-atomic resolution structure of PACAP27-bound VIP1R in complex with Gs, determined by cryo-EM. For successful structure determination, we stabilized the assembly between PACAP27-bound VIP1R and Gs heterotrimer using a developed NanoBiT tethering method. The structure has provided a rationale to understand how PACAP27 interacts with the transmembrane bundle of VIP1R and provides the basis of ligand binding specificity. Structural comparison with other class B GPCRs shed light on the basis of PACAP27 binding as well as a common mechanism of ligand-induced receptor activation and coupling to downstream Gs heterotrimer. As VIP receptors have been identified as potential therapeutic targets for metabolic, inflammatory, and neuronal diseases<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>, this structure presents key information for the rational design of peptides or small molecule compounds to target VIP receptors. In addition, we expect that NanoBiT tethering method can be used to stabilize not only GPCR–G protein complexes but also other unstable macromolecular complexes for structural determination.</p></sec><sec id=\"Sec9\"><title>Methods</title><sec id=\"Sec10\"><title>Constructs</title><p id=\"Par31\">Human VIP1R (residues 31–437) was cloned into pFastbac with an N-terminal FLAG tag followed by a His8 tag, as well as LgBiT at the C-terminus using homologous recombination (CloneExpress One Step Cloning Kit, Vazyme). The primers used in this study are shown in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>. The native signal peptide was replaced with the prolactin precursor sequence to increase the protein expression. A dominant-negative bovine Gαs (DNGαs) construct was generated by site-directed mutagenesis to incorporate mutations S54N, G226A, E268A, N271K, K274D, R280K, T284D, and I285T to decrease the affinity of nucleotide-binding and increase the stability of Gαβγ complex<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. Rat Gβ1 was cloned with an N-terminal His6 tag and a C-terminal SmBiT connected with a 15 residues linker. All three G protein components together with bovine Gγ2 were cloned into a pFastBac vector, respectively.</p></sec><sec id=\"Sec11\"><title>Insect cells expression</title><p id=\"Par32\">VIP1R(31–437)–LgBiT fusion, DNGαs, Gβ1–SmBiT fusion, and Gγ2 were coexpressed in <italic>Sf9</italic> insect cells (Invitrogen) using the Bac-to-Bac baculovirus expression system (Thermo Fisher). Cell cultures were grown in ESF 921 serum-free medium (Expression Systems) to a density of 3 × 10<sup>6</sup> cells mL<sup>−1</sup> and then infected with baculovirus expressing VIP1R(31–437)–LgBiT fusion, DNGαs, Gβ1–SmBiT fusion, and Gγ2, respectively, at the ratio of 1:1:1:1. The cells were collected by centrifugation at 1000 × <italic>g</italic> (Thermo Fisher, H12000) for 20 min after infection for 48 h, and kept frozen at −80 °C until use.</p></sec><sec id=\"Sec12\"><title>Expression and purification of Nb35</title><p id=\"Par33\">Nanobody-35 (Nb35) with a C-terminal His6 tag, was expressed in the periplasm of <italic>E. coli</italic> strain BL21<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. Cultures of 2 L cells were grown to OD600 = 1.0 at 37 °C in TB media containing 0.1% glucose, 2 mM MgCl<sub>2</sub>, and 100 μg mL<sup>−1</sup> ampicillin. Then, 1 mM IPTG was added to the medium to induce protein expression for another 4.5 h at 37 °C. Cells were harvested by centrifugation and lysed in ice-cold buffer (50 mM Tris pH 8.0, 12.5 mM EDTA, and 0.125 M sucrose), then centrifuged to remove cell debris. Nb35 was purified by nickel affinity chromatography, followed by size-exclusion chromatography using a HiLoad 16/600 Superdex 75 column, and finally spin concentrated to ~2.5 mg mL<sup>−1</sup>.</p></sec><sec id=\"Sec13\"><title>PACAP27–VIP1R–Gs complex formation and purification</title><p id=\"Par34\">Cell pellets from 2 L culture were thawed and lysed in 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 0.25 mM TCEP, 5 mM MgCl<sub>2</sub>, and 5 mM CaCl<sub>2</sub> supplemented with EDTA-Free Protease Inhibitor Cocktail (Selleck). The VIP1R–Gs complex was formed in membranes by the addition of 10 μM PACAP27 (Synpeptide), 10 μg mL<sup>−1</sup> Nb35, and 25 mU mL<sup>−1</sup> apyrase and incubation for 1.5 h at room temperature. Cell membranes were collected by ultracentrifugation at 64,000 × <italic>g</italic> for 35 min. The membranes were then resuspended and solubilized in buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 0.25 mM TCEP, 5 mM MgCl<sub>2</sub>, 5 mM CaCl<sub>2</sub>, and 0.5% (w/v) lauryl maltose neopentylglycol (LMNG, Anatrace), 0.1% (w/v) cholesteryl hemisuccinate TRIS salt (CHS, Anatrace), 5 µM PACAP27, and 25 mU mL<sup>−1</sup> apyrase for 3 h at 4 °C. The supernatant was collected by centrifugation at 80,000 × <italic>g</italic> for 40 min and then incubated with 3 mL pre-equilibrated Nickel-NTA resin for 2 h at 4 °C. After batch binding, the resin was loaded into a plastic gravity flow column and washed with ten column volumes of 20 mM HEPES, pH 7.4, 100 mM NaCl, 40 mM imidazole, 10% glycerol, 0.25 mM TCEP, 2 mM MgCl<sub>2</sub>, 2 mM CaCl<sub>2</sub>, 0.01% (w/v) LMNG, 0.01% glyco-diosgenin (GDN, Anatrace) and 0.002% (w/v) CHS, 5 μM PACAP27 and eluted with five column volumes of the same buffer plus 250 mM imidazole. The Ni-NTA-purified fraction was immobilized by batch binding to M1 anti-Flag affinity resin overnight at 4 °C. Next day, the M1 anti-Flag affinity resin was washed with five column volumes of 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 0.25 mM TCEP, 2 mM MgCl<sub>2</sub>, 2 mM CaCl<sub>2</sub>, 0.01% (w/v) LMNG, 0.01% GDN (Anatrace) and 0.002% (w/v) CHS, 5 μM PACAP27 and eluted with five column volumes of the same buffer plus 0.2 mg mL<sup>−1</sup> Flag peptide. The complex was then concentrated using an Amicon Ultra Centrifugal Filter (MWCO 100 kDa) and injected onto a Superdex 200 10/300 GL column (GE Healthcare) equilibrated in the buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM MgCl<sub>2</sub>, 2 mM CaCl<sub>2</sub>, 0.0015% (w/v) LMNG, 0.0005% GDN, 0.0003% (w/v) CHS, 5 μM PACAP27, and 100 μM TCEP. The complex fractions were collected and concentrated for electron microscopy experiments. The final yield of the purified complex was ~0.2 mg per liter of insect cell culture.</p></sec><sec id=\"Sec14\"><title>CCR7–Gi–scfv16 complex expression and purification</title><p id=\"Par35\">The cDNA of human WT CCR7 was cloned into pFastbac with an LgBiT inserted at the C-terminal of CCR7. The CCR7–LgBiT was followed by a C-terminal double MBP and His8 tag to facilitate purification. Receptor, human DNGαi (G203A, A326S), rat Gβ1, bovine Gγ2, and scfv16 were coexpressed and assembled in <italic>Sf9</italic> insect cells. The CCR7–Gi–scfv16 complex was purified substantially in the same way described above except for the MBP instead of M1 anti-Flag affinity purification.</p></sec><sec id=\"Sec15\"><title>Negative-stain electron microscopy screening</title><p id=\"Par36\">For preparing 0.75% uranyl formate solution, weigh out 37.5 mg of uranyl formate into a small beaker, add 5 mL of boiling water and stir for 5 min in the dark, add 10 μL of 5 M NaOH, continue stirring for 5 min, and finally filter the solution using a syringe filter<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. 300-mesh copper grids with carbon film (Electron Microscopy Sciences) were glow-discharged (PELCO easiGlow™ Glow Discharge Cleaning System) for 1 min at 25 mA before 3.5 µL purified complex was applied to the grids and incubated for 30 s. After blotting the sample using filter paper, the grid surface was touched on two drops of 40 µL 0.75% uranyl formate, and then the grids were stained on the third drop of uranyl formate with gentle stirring for 40 s. Stained grids were blotted to remove excess stain. Negative-stain data collection was carried out on a Tecnai G2 Spirit transmission electron microscopy (Thermo FEI) operating at 120 kV. Images were collected at a nominal magnification of 105,000 (3.1 Å pixel size) within a −0.5 to −2.5 µm defocus range.</p></sec><sec id=\"Sec16\"><title>Cryo-EM data acquisition</title><p id=\"Par37\">The purified PACAP27–VIP1R–Gs complex (3.0 μL) at a concentration of 4–5 mg mL<sup>−1</sup> was applied to glow-discharged holey carbon grids (Quantifoil R1.2/1.3, 200 mesh), and subsequently vitrified using a Vitrobot Mark IV (Thermo Fisher Scientific). Cryo-EM images were collected on a Titan Krios equipped with a Gatan K2 Summit direct electron detector. The microscope was operated at 300 kV accelerating voltage, at a nominal magnification of ×29,000 in counting mode, corresponding to a pixel size of 1.014 Å. In total, 4215 image stacks were obtained at the dose rate of about eight electrons per Å<sup>2</sup> per second with a defocus range of −1.5 to −2.3 μm. The total exposure time was set to 8 s with intermediate frames recorded every 0.2 s, resulting in an accumulated dose of 64 electrons per Å<sup>2</sup>.</p></sec><sec id=\"Sec17\"><title>Image processing and 3D reconstruction</title><p id=\"Par38\">Dose-fractionated image stacks were subjected to beam-induced motion correction and dose-weighting using MotionCor2.1<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. A sum of all frames, filtered according to the exposure dose, in each image stack was used for further processing. Contrast transfer function parameters for each micrograph were determined by Gctf v1.06<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. The further data processing was performed in RELION-3.0-beta2<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. Particle selection, two-dimensional classification and the first round of three-dimensional classification were performed on a binned dataset with a pixel size of 2.028 Å. Auto-picking yielded 2,547,930 particle projections that were sequentially subjected to reference-free two-dimensional classification and produced 2,460,220 projections for further processing. This step barely discard false-positive particles or particles categorized in poorly defined classes, indicating the complex stability of the sample generated using NanoBiT tethering method developed in this study. This subset of particle projections was subjected to consecutive rounds of 3D classifications with a pixel size of 2.028 Å. A selected subset containing 131,263 projections was used to obtain the final map using a pixel size of 1.014 Å. After the last round of refinement, the final map has an indicated global resolution of 3.2 Å at a Fourier shell correlation of 0.143. Local resolution was determined using the Bsoft package with half maps as input maps<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>.</p></sec><sec id=\"Sec18\"><title>Model building and refinement</title><p id=\"Par39\">The cryo-EM structure of PTH1R–Gs–Nb35 complex (PDB code 6NBF) was used as the start for model rebuilding and refinement against the electron microscopy map. The model was docked into the electron microscopy density map using Chimera<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>, followed by iterative manual adjustment and rebuilding in COOT<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. Real space refinement was performed using phenix.real_space_refine from Phenix program package<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>. The model statistics were validated using MolProbity<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>. Structural figures were prepared in Chimera and PyMOL (<ext-link ext-link-type=\"uri\" xlink:href=\"https://pymol.org/2/\">https://pymol.org/2/</ext-link>). The final refinement statistics are provided in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>.</p></sec><sec id=\"Sec19\"><title>cAMP accumulation assay</title><p id=\"Par40\">The full-length VIP1R(31–457) and VIP1R mutants was cloned into pcDNA6.0 vector (Invitrogen) with a FLAG tag at its N-terminus (see Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">5</xref> for a list of primers used in this study). CHO-K1 cells (ATCC, #CCL-61) were cultured in Ham’s F-12 Nutrient Mix (Gibco) supplemented with 10% (w/v) fetal bovine serum. Cells were maintained at 37 °C in a 5% CO<sub>2</sub> incubator with 100,000 cells per well in a 12-well plate. Cells were grown overnight and then transfected with 1 μg VIP1R constructs by FuGENE<sup>®</sup> HD transfection reagent (DNA/FuGENE<sup>®</sup> HD ratio of 1:3) in each well. After 24 h, the transfected cells were seeded onto 384-well microtiter plates (3000 cells per well). cAMP accumulation was measured using the LANCE cAMP kit (PerkinElmer) according to the manufacturer’s instructions with different concentrations of peptides. Fluorescence signals were then measured at 620 and 665 nm by an Envision multilabel plate reader (PerkinElmer). Data presented are means ± SEM of at least three independent experiments.</p></sec><sec id=\"Sec20\"><title>Detection of surface expression of VIP1R mutants</title><p id=\"Par41\">The VIP1R mutants were cloned into pcDNA6.0 vector (Invitrogen) with a FLAG tag at its N-terminus. The cell seeding and transfection follow the same method as cAMP accumulation assay. After 24 h of transfection, cells were washed once with PBS and digested with 0.2% (w/v) EDTA in PBS. Cells were blocked with PBS containing 5% (w/v) BSA for 15 min at room temperature and then incubated with primary anti-Flag antibody (diluted with PBS containing 5% BSA at a ratio of 1:300, Sigma) for 1 h at room temperature. Thereafter, cells were washed three times with PBS containing 1% (w/v) BSA before incubating with anti-mouse Alexa-488-conjugated secondary antibody (diluted with PBS containing 5% BSA at a ratio of 1:1000, Invitrogen) at 4 °C in the dark for 1 h. After another three times wash, cells were resuspended, and fluorescence intensity was quantified in a BD Accuri C6 flow cytometer system (BD Biosciences) at excitation 488 nm and emission 519 nm. Approximately 10,000 cellular events per sample were collected and data were normalized to WT.</p></sec><sec id=\"Sec21\"><title>Dynamic light scattering</title><p id=\"Par42\">DLS sample was prepared at about 0.2–1.0 mg mL<sup>−1</sup> and equilibrated for 5 min before loading 10 μL onto the DynaPro NanoStar (Wyatt Technology). For thermostability assay, the intensity was read with a thermal ramp from 25 to 75 °C with a ramp rate of 2 °C min<sup>−1</sup>. All data acquisition and analysis were performed by the Dynamics software.</p></sec><sec id=\"Sec22\"><title>Reporting summary</title><p id=\"Par43\">Further information on research design is available in the <xref rid=\"MOESM3\" ref-type=\"media\">Nature Research Reporting Summary</xref> linked to this article.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec23\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41467_2020_17933_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41467_2020_17933_MOESM2_ESM.docx\"><caption><p>Peer Review File</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM3\"><media xlink:href=\"41467_2020_17933_MOESM3_ESM.pdf\"><caption><p>Reporting Summary</p></caption></media></supplementary-material></sec></sec></body><back><app-group><app id=\"App1\"><sec id=\"Sec24\"><title>Source data</title><p id=\"Par46\"><media position=\"anchor\" xlink:href=\"41467_2020_17933_MOESM4_ESM.xlsx\" id=\"MOESM4\"><caption><p>Source Data</p></caption></media></p></sec></app></app-group><fn-group><fn><p><bold>Peer review information</bold>\n<italic>Nature Communications</italic> thanks Roger Sunahara, Asuka Inoue and other, anonymous, reviewers for their contributions to the peer review of this work. Peer review reports are available.</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Jia Duan, Dan-dan Shen, X. Edward Zhou, Peng Bi.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary information</bold> is available for this paper at 10.1038/s41467-020-17933-8.</p></sec><ack><title>Acknowledgements</title><p>The cryo-EM data were collected at the Center of Cryo-Electron Microscopy, Zhejiang University. This work was partially supported by the National Natural Science Foundation of China (31770796 to Y.J. and 81922071 to Y.Z.); the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (2018ZX09711002 to Y.J.); Shanghai Municipal Science and Technology Major Project (2019SHZDZX02 to H.E.X.); CAS Strategic Priority Research Program (XDB37030103 to H.E.X.); Ministry of Science and Technology (China) grant (2018YFA0507002 to H.E.X.); National Key Basic Research Program of China (2019YFA0508800 to Y.Z.); Zhejiang Province Science Fund for Distinguished Young Scholars (LR19H310001 to Y.Z.); Fundamental Research Funds for the Central Universities (2019XZZX001-01-06 to Y.Z.); Shanghai Sailing Program (19YF1457600 to Q.f.-L.); the K.C. Wong Education Foundation (to Y.J.); and the Van Andel Research Institute (K.M.).</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>J.D. designed the expression constructs, purified the PACAP27–VIP1R–Gs complex, prepared the final samples for negative stain and data collection toward the structures, and participated in figure and manuscript preparation; D.-d.S. and P.B. performed specimen screening by negative-stain EM, cryo-EM grid preparation, cryo-EM data collection, and map calculations; X.E.Z. built and refined the structure models, analyzed the structures, and wrote the manuscript; Q.f.-L., Y.-x.T., H.-b.Z., P.-y.X., S.-J.H., S.-s.M., Y.-w.Z., and X.-h.H. performed the experiments; K.M. analyzed the structures, and wrote the manuscript; Y.J. prepared the bulk of figures, performed the structural analysis, and wrote the manuscript; Y.J. and H.E.X. conceived the project, initiated collaborations with Y.Z., and supervised J.D.; and Y.Z. supervised the EM studies.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>Data supporting the findings of this manuscript are available from the corresponding authors upon reasonable request. A reporting summary for this article is available as a <xref rid=\"MOESM1\" ref-type=\"media\">Supplementary Information</xref> file. Source data are provided with this paper. Density map and structure coordinate have been deposited to the Electron Microscopy Database and the Protein Data Bank with the accession number of EMD-21249, <ext-link ext-link-type=\"uri\" xlink:href=\"http://dx.doi.org/10.2210/pdb6VN7/pdb\">PDB6VN7</ext-link> for the PACAP27–VIP1R–Gs complex.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par44\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Denes</surname><given-names>V</given-names></name><name><surname>Geck</surname><given-names>P</given-names></name><name><surname>Mester</surname><given-names>A</given-names></name><name><surname>Gabriel</surname><given-names>R</given-names></name></person-group><article-title>Pituitary adenylate cyclase-activating polypeptide: 30 years in research spotlight and 600 million years in service</article-title><source>J. Clin. Med.</source><year>2019</year><volume>8</volume><fpage>1488</fpage></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ishihara</surname><given-names>T</given-names></name><name><surname>Shigemoto</surname><given-names>R</given-names></name><name><surname>Mori</surname><given-names>K</given-names></name><name><surname>Takahashi</surname><given-names>K</given-names></name><name><surname>Nagata</surname><given-names>S</given-names></name></person-group><article-title>Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide</article-title><source>Neuron</source><year>1992</year><volume>8</volume><fpage>811</fpage><lpage>819</lpage><pub-id pub-id-type=\"pmid\">1314625</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Usdin</surname><given-names>TB</given-names></name><name><surname>Bonner</surname><given-names>TI</given-names></name><name><surname>Mezey</surname><given-names>E</given-names></name></person-group><article-title>Two receptors for vasoactive intestinal polypeptide with similar specificity and complementary distributions</article-title><source>Endocrinology</source><year>1994</year><volume>135</volume><fpage>2662</fpage><lpage>2680</lpage><pub-id pub-id-type=\"pmid\">7988457</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ichikawa</surname><given-names>S</given-names></name><name><surname>Sreedharan</surname><given-names>SP</given-names></name><name><surname>Owen</surname><given-names>RL</given-names></name><name><surname>Goetzl</surname><given-names>EJ</given-names></name></person-group><article-title>Immunochemical localization of type I VIP receptor and NK-1-type substance P receptor in rat lung</article-title><source>Am. J. Physiol.</source><year>1995</year><volume>268</volume><fpage>L584</fpage><lpage>L588</lpage><pub-id pub-id-type=\"pmid\">7537460</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sreedharan</surname><given-names>SP</given-names></name><name><surname>Huang</surname><given-names>JX</given-names></name><name><surname>Cheung</surname><given-names>MC</given-names></name><name><surname>Goetzl</surname><given-names>EJ</given-names></name></person-group><article-title>Structure, expression, and chromosomal localization of the type I human vasoactive intestinal peptide receptor gene</article-title><source>Proc. Natl Acad. Sci. USA</source><year>1995</year><volume>92</volume><fpage>2939</fpage><lpage>2943</lpage><pub-id pub-id-type=\"pmid\">7708752</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kaltreider</surname><given-names>HB</given-names></name><etal/></person-group><article-title>Upregulation of neuropeptides and neuropeptide receptors in a murine model of immune inflammation in lung parenchyma</article-title><source>Am. J. Respir. Cell Mol. Biol.</source><year>1997</year><volume>16</volume><fpage>133</fpage><lpage>144</lpage><pub-id pub-id-type=\"pmid\">9032120</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reubi</surname><given-names>JC</given-names></name></person-group><article-title>In vitro evaluation of VIP/PACAP receptors in healthy and diseased human tissues. Clinical implications</article-title><source>Ann. N. Y. Acad. Sci.</source><year>2000</year><volume>921</volume><fpage>1</fpage><lpage>25</lpage><pub-id pub-id-type=\"pmid\">11193811</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reubi</surname><given-names>JC</given-names></name><etal/></person-group><article-title>Vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor subtypes in human tumors and their tissues of origin</article-title><source>Cancer Res.</source><year>2000</year><volume>60</volume><fpage>3105</fpage><lpage>3112</lpage><pub-id pub-id-type=\"pmid\">10850463</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Delgado</surname><given-names>M</given-names></name><name><surname>Martinez</surname><given-names>C</given-names></name><name><surname>Johnson</surname><given-names>MC</given-names></name><name><surname>Gomariz</surname><given-names>RP</given-names></name><name><surname>Ganea</surname><given-names>D</given-names></name></person-group><article-title>Differential expression of vasoactive intestinal peptide receptors 1 and 2 (VIP-R1 and VIP-R2) mRNA in murine lymphocytes</article-title><source>J. Neuroimmunol.</source><year>1996</year><volume>68</volume><fpage>27</fpage><lpage>38</lpage><pub-id pub-id-type=\"pmid\">8784257</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Langer</surname><given-names>I</given-names></name></person-group><article-title>Mechanisms involved in VPAC receptors activation and regulation: lessons from pharmacological and mutagenesis studies</article-title><source>Front. Endocrinol.</source><year>2012</year><volume>3</volume><fpage>129</fpage></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sarkar</surname><given-names>A</given-names></name><name><surname>Sen</surname><given-names>S</given-names></name></person-group><article-title>3D structure prediction of VAPC1 and identification of dual natural inhibitors for VPAC1 and EGFR</article-title><source>J. Bioenerg. Biomembr.</source><year>2019</year><volume>51</volume><fpage>89</fpage><lpage>102</lpage><pub-id pub-id-type=\"pmid\">30810849</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Latek</surname><given-names>D</given-names></name><name><surname>Langer</surname><given-names>I</given-names></name><name><surname>Krzysko</surname><given-names>K</given-names></name><name><surname>Charzynski</surname><given-names>L</given-names></name></person-group><article-title>A molecular dynamics study of vasoactive intestinal peptide receptor 1 and the basis of its therapeutic antagonism</article-title><source>Int. J. Mol. Sci.</source><year>2019</year><volume>20</volume><fpage>4348</fpage></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Umetsu</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Structural difference of vasoactive intestinal peptide in two distinct membrane-mimicking environments</article-title><source>Biochim. et Biophys. Acta</source><year>2011</year><volume>1814</volume><fpage>724</fpage><lpage>730</lpage></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Safdari</surname><given-names>HA</given-names></name><name><surname>Pandey</surname><given-names>S</given-names></name><name><surname>Shukla</surname><given-names>AK</given-names></name><name><surname>Dutta</surname><given-names>S</given-names></name></person-group><article-title>Illuminating GPCR signaling by cryo-EM</article-title><source>Trends Cell Biol.</source><year>2018</year><volume>28</volume><fpage>591</fpage><lpage>594</lpage><pub-id pub-id-type=\"pmid\">29945844</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vaidehi</surname><given-names>N</given-names></name><name><surname>Grisshammer</surname><given-names>R</given-names></name><name><surname>Tate</surname><given-names>CG</given-names></name></person-group><article-title>How can mutations thermostabilize G-protein-coupled receptors?</article-title><source>Trends Pharmacol. Sci.</source><year>2016</year><volume>37</volume><fpage>37</fpage><lpage>46</lpage><pub-id pub-id-type=\"pmid\">26547284</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maeda</surname><given-names>S</given-names></name><etal/></person-group><article-title>Development of an antibody fragment that stabilizes GPCR/G-protein complexes</article-title><source>Nat. Commun.</source><year>2018</year><volume>9</volume><fpage>3712</fpage><pub-id pub-id-type=\"pmid\">30213947</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manglik</surname><given-names>A</given-names></name><name><surname>Kobilka</surname><given-names>BK</given-names></name><name><surname>Steyaert</surname><given-names>J</given-names></name></person-group><article-title>Nanobodies to study G protein-coupled receptor structure and function</article-title><source>Annu. Rev. Pharmacol. Toxicol.</source><year>2017</year><volume>57</volume><fpage>19</fpage><lpage>37</lpage><pub-id pub-id-type=\"pmid\">27959623</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dixon</surname><given-names>AS</given-names></name><etal/></person-group><article-title>NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells</article-title><source>ACS Chem. Biol.</source><year>2016</year><volume>11</volume><fpage>400</fpage><lpage>408</lpage><pub-id pub-id-type=\"pmid\">26569370</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rasmussen</surname><given-names>SG</given-names></name><etal/></person-group><article-title>Crystal structure of the beta2 adrenergic receptor-Gs protein complex</article-title><source>Nature</source><year>2011</year><volume>477</volume><fpage>549</fpage><lpage>555</lpage><pub-id pub-id-type=\"pmid\">21772288</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dal Maso</surname><given-names>E</given-names></name><etal/></person-group><article-title>The molecular control of calcitonin receptor signaling</article-title><source>ACS Pharm. Transl. Sci.</source><year>2019</year><volume>2</volume><fpage>31</fpage><lpage>51</lpage></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liang</surname><given-names>YL</given-names></name><etal/></person-group><article-title>Toward a structural understanding of class B GPCR peptide binding and activation</article-title><source>Mol. Cell</source><year>2020</year><volume>77</volume><fpage>656</fpage><lpage>668 e655</lpage><pub-id pub-id-type=\"pmid\">32004469</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ma</surname><given-names>S</given-names></name><etal/></person-group><article-title>Molecular basis for hormone recognition and activation of corticotropin-releasing factor receptors</article-title><source>Mol. Cell</source><year>2020</year><volume>77</volume><fpage>669</fpage><lpage>680 e664</lpage><pub-id pub-id-type=\"pmid\">32004470</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhao</surname><given-names>LH</given-names></name><etal/></person-group><article-title>Structure and dynamics of the active human parathyroid hormone receptor-1</article-title><source>Science</source><year>2019</year><volume>364</volume><fpage>148</fpage><lpage>153</lpage><pub-id pub-id-type=\"pmid\">30975883</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wootten</surname><given-names>D</given-names></name><name><surname>Simms</surname><given-names>J</given-names></name><name><surname>Miller</surname><given-names>LJ</given-names></name><name><surname>Christopoulos</surname><given-names>A</given-names></name><name><surname>Sexton</surname><given-names>PM</given-names></name></person-group><article-title>Polar transmembrane interactions drive formation of ligand-specific and signal pathway-biased family B G protein-coupled receptor conformations</article-title><source>Proc. Natl Acad. Sci. USA</source><year>2013</year><volume>110</volume><fpage>5211</fpage><lpage>5216</lpage><pub-id pub-id-type=\"pmid\">23479653</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liang</surname><given-names>YL</given-names></name><etal/></person-group><article-title>Phase-plate cryo-EM structure of a class B GPCR-G-protein complex</article-title><source>Nature</source><year>2017</year><volume>546</volume><fpage>118</fpage><lpage>123</lpage><pub-id pub-id-type=\"pmid\">28437792</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein</article-title><source>Nature</source><year>2017</year><volume>546</volume><fpage>248</fpage><lpage>253</lpage><pub-id pub-id-type=\"pmid\">28538729</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liang</surname><given-names>YL</given-names></name><etal/></person-group><article-title>Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor</article-title><source>Nature</source><year>2018</year><volume>561</volume><fpage>492</fpage><lpage>497</lpage><pub-id pub-id-type=\"pmid\">30209400</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liang</surname><given-names>YL</given-names></name><etal/></person-group><article-title>Phase-plate cryo-EM structure of a biased agonist-bound human GLP-1 receptor-Gs complex</article-title><source>Nature</source><year>2018</year><volume>555</volume><fpage>121</fpage><lpage>125</lpage><pub-id pub-id-type=\"pmid\">29466332</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Solano</surname><given-names>RM</given-names></name><etal/></person-group><article-title>Two basic residues of the h-VPAC1 receptor second transmembrane helix are essential for ligand binding and signal transduction</article-title><source>J. Biol. Chem.</source><year>2001</year><volume>276</volume><fpage>1084</fpage><lpage>1088</lpage><pub-id pub-id-type=\"pmid\">11013258</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Igarashi</surname><given-names>H</given-names></name><etal/></person-group><article-title>Elucidation of vasoactive intestinal peptide pharmacophore for VPAC(1) receptors in human, rat, and guinea pig</article-title><source>J. Pharm. Exp. Ther.</source><year>2002</year><volume>301</volume><fpage>37</fpage><lpage>50</lpage></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Siu</surname><given-names>FY</given-names></name><etal/></person-group><article-title>Structure of the human glucagon class B G-protein-coupled receptor</article-title><source>Nature</source><year>2013</year><volume>499</volume><fpage>444</fpage><lpage>449</lpage><pub-id pub-id-type=\"pmid\">23863937</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chugunov</surname><given-names>AO</given-names></name><etal/></person-group><article-title>Evidence that interaction between conserved residues in transmembrane helices 2, 3, and 7 are crucial for human VPAC1 receptor activation</article-title><source>Mol. Pharmacol.</source><year>2010</year><volume>78</volume><fpage>394</fpage><lpage>401</lpage><pub-id pub-id-type=\"pmid\">20573782</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hjorth</surname><given-names>SA</given-names></name><name><surname>Orskov</surname><given-names>C</given-names></name><name><surname>Schwartz</surname><given-names>TW</given-names></name></person-group><article-title>Constitutive activity of glucagon receptor mutants</article-title><source>Mol. Endocrinol.</source><year>1998</year><volume>12</volume><fpage>78</fpage><lpage>86</lpage><pub-id pub-id-type=\"pmid\">9440812</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schipani</surname><given-names>E</given-names></name><name><surname>Kruse</surname><given-names>K</given-names></name><name><surname>Juppner</surname><given-names>H</given-names></name></person-group><article-title>A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia</article-title><source>Science</source><year>1995</year><volume>268</volume><fpage>98</fpage><lpage>100</lpage><pub-id pub-id-type=\"pmid\">7701349</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yin</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors</article-title><source>J. Biol. Chem.</source><year>2017</year><volume>292</volume><fpage>9865</fpage><lpage>9881</lpage><pub-id pub-id-type=\"pmid\">28356352</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Langer</surname><given-names>I</given-names></name><name><surname>Vertongen</surname><given-names>P</given-names></name><name><surname>Perret</surname><given-names>J</given-names></name><name><surname>Waelbroeck</surname><given-names>M</given-names></name><name><surname>Robberecht</surname><given-names>P</given-names></name></person-group><article-title>A small sequence in the third intracellular loop of the VPAC(1) receptor is responsible for its efficient coupling to the calcium effector</article-title><source>Biochem Soc. Trans.</source><year>2002</year><volume>30</volume><fpage>447</fpage><lpage>450</lpage><pub-id pub-id-type=\"pmid\">12196112</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Langer</surname><given-names>I</given-names></name><name><surname>Robberecht</surname><given-names>P</given-names></name></person-group><article-title>Mutations in the carboxy-terminus of the third intracellular loop of the human recombinant VPAC1 receptor impair VIP-stimulated [Ca2+]i increase but not adenylate cyclase stimulation</article-title><source>Cell Signal</source><year>2005</year><volume>17</volume><fpage>17</fpage><lpage>24</lpage><pub-id pub-id-type=\"pmid\">15451021</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dickson</surname><given-names>L</given-names></name><name><surname>Finlayson</surname><given-names>K</given-names></name></person-group><article-title>VPAC and PAC receptors: from ligands to function</article-title><source>Pharmacol. Ther.</source><year>2009</year><volume>121</volume><fpage>294</fpage><lpage>316</lpage><pub-id pub-id-type=\"pmid\">19109992</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Booth</surname><given-names>DS</given-names></name><name><surname>Avila-Sakar</surname><given-names>A</given-names></name><name><surname>Cheng</surname><given-names>Y</given-names></name></person-group><article-title>Visualizing proteins and macromolecular complexes by negative stain EM: from grid preparation to image acquisition</article-title><source>J. Vis. Exp.</source><year>2011</year><volume>58</volume><fpage>3227</fpage></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zheng</surname><given-names>SQ</given-names></name><etal/></person-group><article-title>MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy</article-title><source>Nat. Methods</source><year>2017</year><volume>14</volume><fpage>331</fpage><lpage>332</lpage><pub-id pub-id-type=\"pmid\">28250466</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>K</given-names></name></person-group><article-title>Gctf: real-time CTF determination and correction</article-title><source>J. Struct. Biol.</source><year>2016</year><volume>193</volume><fpage>1</fpage><lpage>12</lpage><pub-id pub-id-type=\"pmid\">26592709</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scheres</surname><given-names>SH</given-names></name></person-group><article-title>RELION: implementation of a Bayesian approach to cryo-EM structure determination</article-title><source>J. Struct. Biol.</source><year>2012</year><volume>180</volume><fpage>519</fpage><lpage>530</lpage><pub-id pub-id-type=\"pmid\">23000701</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Heymann</surname><given-names>JB</given-names></name></person-group><article-title>Guidelines for using Bsoft for high resolution reconstruction and validation of biomolecular structures from electron micrographs</article-title><source>Protein Sci.</source><year>2018</year><volume>27</volume><fpage>159</fpage><lpage>171</lpage><pub-id pub-id-type=\"pmid\">28891250</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pettersen</surname><given-names>EF</given-names></name><etal/></person-group><article-title>UCSF chimera–a visualization system for exploratory research and analysis</article-title><source>J. Comput. Chem.</source><year>2004</year><volume>25</volume><fpage>1605</fpage><lpage>1612</lpage><pub-id pub-id-type=\"pmid\">15264254</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Emsley</surname><given-names>P</given-names></name><name><surname>Cowtan</surname><given-names>K</given-names></name></person-group><article-title>Coot: model-building tools for molecular graphics</article-title><source>Acta Crystallogr. Sect. D, Biol. Crystallogr.</source><year>2004</year><volume>60</volume><fpage>2126</fpage><lpage>2132</lpage><pub-id pub-id-type=\"pmid\">15572765</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Adams</surname><given-names>PD</given-names></name><etal/></person-group><article-title>PHENIX: a comprehensive Python-based system for macromolecular structure solution</article-title><source>Acta Crystallogr. Sect. D, Biol. Crystallogr.</source><year>2010</year><volume>66</volume><fpage>213</fpage><lpage>221</lpage><pub-id pub-id-type=\"pmid\">20124702</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>VB</given-names></name><etal/></person-group><article-title>MolProbity: all-atom structure validation for macromolecular crystallography</article-title><source>Acta Crystallogr. Sect. D, Biol. Crystallogr.</source><year>2010</year><volume>66</volume><fpage>12</fpage><lpage>21</lpage><pub-id pub-id-type=\"pmid\">20057044</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Nat Commun</journal-id><journal-id journal-id-type=\"iso-abbrev\">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type=\"epub\">2041-1723</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807787</article-id><article-id pub-id-type=\"pmc\">PMC7431578</article-id><article-id pub-id-type=\"publisher-id\">17958</article-id><article-id pub-id-type=\"doi\">10.1038/s41467-020-17958-z</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Enhancing chemotherapy response through augmented synthetic lethality by co-targeting nucleotide excision repair and cell-cycle checkpoints</article-title></title-group><contrib-group><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Kong</surname><given-names>Yi Wen</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Dreaden</surname><given-names>Erik C.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref><xref ref-type=\"aff\" rid=\"Aff9\">9</xref></contrib><contrib contrib-type=\"author\"><name><surname>Morandell</surname><given-names>Sandra</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff10\">10</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhou</surname><given-names>Wen</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-3906-5923</contrib-id><name><surname>Dhara</surname><given-names>Sanjeev S.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-1651-3607</contrib-id><name><surname>Sriram</surname><given-names>Ganapathy</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-9801-5037</contrib-id><name><surname>Lam</surname><given-names>Fred C.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>Patterson</surname><given-names>Jesse C.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Quadir</surname><given-names>Mohiuddin</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref><xref ref-type=\"aff\" rid=\"Aff11\">11</xref></contrib><contrib contrib-type=\"author\"><name><surname>Dinh</surname><given-names>Anh</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Shopsowitz</surname><given-names>Kevin E.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Varmeh</surname><given-names>Shohreh</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-7577-4612</contrib-id><name><surname>Yilmaz</surname><given-names>Ömer H.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lippard</surname><given-names>Stephen J.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Reinhardt</surname><given-names>H. Christian</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff12\">12</xref><xref ref-type=\"aff\" rid=\"Aff13\">13</xref><xref ref-type=\"aff\" rid=\"Aff14\">14</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hemann</surname><given-names>Michael T.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-9835-192X</contrib-id><name><surname>Hammond</surname><given-names>Paula T.</given-names></name><address><email>hammond@mit.edu</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-9547-3251</contrib-id><name><surname>Yaffe</surname><given-names>Michael B.</given-names></name><address><email>myaffe@mit.edu</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref><xref ref-type=\"aff\" rid=\"Aff7\">7</xref><xref ref-type=\"aff\" rid=\"Aff8\">8</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.116068.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2341 2786</institution-id><institution>David H. Koch Institute for Integrative Cancer Research, </institution><institution>Massachusetts Institute of Technology, </institution></institution-wrap>Cambridge, MA 02139 USA </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.116068.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2341 2786</institution-id><institution>Center for Precision Cancer Medicine, </institution><institution>Massachusetts Institute of Technology, </institution></institution-wrap>Cambridge, MA 02139 USA </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.116068.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2341 2786</institution-id><institution>Department of Chemical Engineering, </institution><institution>Massachusetts Institute of Technology, </institution></institution-wrap>Cambridge, MA 02139 USA </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.116068.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2341 2786</institution-id><institution>Department of Chemistry, </institution><institution>Massachusetts Institute of Technology, </institution></institution-wrap>Cambridge, MA 02139 USA </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.25073.33</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 8227</institution-id><institution>Division of Neurosurgery, Hamilton General Hospital, </institution><institution>McMaster University Faculty of Health Sciences, </institution></institution-wrap>Hamilton, ON L8L 2X2 Canada </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.116068.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2341 2786</institution-id><institution>Department of Biology, </institution><institution>Massachusetts Institute of Technology, </institution></institution-wrap>Cambridge, MA 02139 USA </aff><aff id=\"Aff7\"><label>7</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.116068.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2341 2786</institution-id><institution>Department of Biological Engineering, </institution><institution>Massachusetts Institute of Technology, </institution></institution-wrap>Cambridge, MA 02139 USA </aff><aff id=\"Aff8\"><label>8</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.38142.3c</institution-id><institution-id institution-id-type=\"ISNI\">000000041936754X</institution-id><institution>Divisions of Surgical Oncology, Trauma, and Surgical Critical Care, Department of Surgery, Beth Israel Deaconess Medical Center, </institution><institution>Harvard Medical School, </institution></institution-wrap>Boston, MA 02215 USA </aff><aff id=\"Aff9\"><label>9</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.189967.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0941 6502</institution-id><institution>Present Address: Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, </institution><institution>Emory University School of Medicine, </institution></institution-wrap>Atlanta, GA 30322 USA </aff><aff id=\"Aff10\"><label>10</label>Present Address: Molecular Health GmbH, 69115 Heidelberg, Germany </aff><aff id=\"Aff11\"><label>11</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.261055.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2293 4611</institution-id><institution>Present Address: Department of Coatings and Polymeric Materials, </institution><institution>North Dakota State University, </institution></institution-wrap>Fargo, USA </aff><aff id=\"Aff12\"><label>12</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.411097.a</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 8852 305X</institution-id><institution>Present Address: Clinic I of Internal Medicine, </institution><institution>University Hospital Cologne, </institution></institution-wrap>Cologne, Germany </aff><aff id=\"Aff13\"><label>13</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.6190.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 8580 3777</institution-id><institution>Present Address: Cologne Excellence Cluster in Cellular Stress Response in Aging-Associated Disorders (CECAD), </institution><institution>University of Cologne, </institution></institution-wrap>Cologne, Germany </aff><aff id=\"Aff14\"><label>14</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.6190.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 8580 3777</institution-id><institution>Present Address: Center for Molecular Medicine Cologne, </institution><institution>University of Cologne, </institution></institution-wrap>Cologne, Germany </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>4124</elocation-id><history><date date-type=\"received\"><day>16</day><month>10</month><year>2018</year></date><date date-type=\"accepted\"><day>22</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.</p></abstract><abstract id=\"Abs2\" abstract-type=\"web-summary\"><p id=\"Par2\">Cell cycle checkpoint kinase, MK2, is in synthetic relationship with p53 in the DNA damage response to chemotherapeutic agents. Here, the authors report XPA as a third gene in which simultaneous targeting of MK2 and XPA further enhances sensitivity to cisplatin in p53-deficient tumours.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Kinases</kwd><kwd>DNA damage and repair</kwd><kwd>RNAi</kwd><kwd>Cancer</kwd><kwd>Nanomedicine</kwd></kwd-group><funding-group><award-group><funding-source><institution>Misrock Foundation, MIT Center for Precision Cancer Medicine</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/100000070</institution-id><institution>U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)</institution></institution-wrap></funding-source><award-id>F32-EB017614</award-id><principal-award-recipient><name><surname>Dreaden</surname><given-names>Erik C.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>Mazumdar-Shaw International Oncology Fellowship</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/100000054</institution-id><institution>U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)</institution></institution-wrap></funding-source><award-id>CA034992</award-id><award-id>R01-CA226898</award-id><principal-award-recipient><name><surname>Lippard</surname><given-names>Stephen J.</given-names></name><name><surname>Yaffe</surname><given-names>Michael B.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/100000090</institution-id><institution>United States Department of Defense | United States Army | Army Medical Command | Congressionally Directed Medical Research Programs (CDMRP)</institution></institution-wrap></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>Ovarian Cancer Research Foundation, Breast Cancer Alliance</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/100000066</institution-id><institution>U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)</institution></institution-wrap></funding-source><award-id>R35-ES028374</award-id><award-id>R01-ES015339</award-id><principal-award-recipient><name><surname>Yaffe</surname><given-names>Michael B.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/100000057</institution-id><institution>U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)</institution></institution-wrap></funding-source><award-id>R01-GM104047</award-id><principal-award-recipient><name><surname>Yaffe</surname><given-names>Michael B.</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)</institution></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution>Charles & Marjorie Halloway Foundation, MIT Center for Precision Cancer Medicine, STARR Cancer Consortium,</institution></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par3\">Although there have been significant advances in the treatment of certain types of cancers with molecularly targeted therapies against driver oncogenes such as <italic>EGFR</italic>, <italic>BRAF</italic>, and <italic>ALK</italic><sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>, for most tumor types, treatment with cytotoxic chemotherapeutic regimens, often containing platinum-based compounds, remains the frontline therapy<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. Platinum drugs exert their effects by causing DNA damage, leading to the activation of a complex signaling network that induces cell cycle arrest and recruits DNA repair machinery to sites of damage<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>–<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. If the damage exceeds the cells’ ability for repair, cell death ensues, typically via apoptosis<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>–<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. Thus, to enhance the effectiveness of DNA damaging chemotherapy for tumor cell killing, two general parallel approaches have been pursued: either disruption of cell cycle checkpoints by targeting critical effector kinases, or interfering with the process of DNA repair itself<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>.</p><p id=\"Par4\">The concept of synthetic lethality (SL) holds great promise for the treatment of human cancers, best exemplified by the now widespread use of PARP inhibitors in BRCA mutant cancers. SL originally described a relationship between two genes, where alteration of either gene alone results in viable cells, but alteration (mutation, loss, or inhibition) of both genes simultaneously was lethal. The concept has now been extended to embrace synthetic lethal drug sensitivity, such as that observed with PARP inhibitors in combination with DNA-damaging chemotherapy in a variety of BRCA defective tumors<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Because BRCA mutations are observed in fewer than 10% of cancer patients (cBioPortal: 6.7%)<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>–<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup> the identification of additional genes that share synthetic lethal sensitivity relationships with mutated oncogenes or tumor suppressors would greatly enhance the implementation of tumor cell-specific synthetic lethal sensitivity to improve an anticancer therapeutic response. We recently identified one such SL sensitivity relationship between loss or mutation of the tumor suppressor p53 and the cell cycle checkpoint effector kinase mitogen-activated protein kinase activated protein kinase-2 (MAPKAP kinase 2, MAPKAPK2, or MK2) in the context of DNA damaging chemotherapy<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>–<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. Cancer cells that are defective in p53 function are deficient in their ability to transcriptionally upregulate the CDK inhibitor p21 after genotoxic stress. Therefore, compared to normal p53-proficient cells, p53-defective cells are more reliant on MK2 activity, which drives an alternative cell cycle checkpoint pathway that stabilizes the CKI inhibitors p27<sup>Kip1</sup> and Gadd45α in order to maintain G<sub>1</sub>/S and G<sub>2</sub>/M arrest after certain types of DNA damage<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. Because most tumors are deficient in one or more aspects of the function of the p53 tumor suppressor, either as a consequence of mutations within p53, or impairment of upstream and downstream modulators of p53 activity<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>, targeting MK2 has the potential to selectively enhance tumor cell killing without increasing the genotoxic effects of chemotherapy on normal p53-wild type tissues. Approximately, 50% of non-small cell lung cancers (NSCLC), for example, contain mutations, deletions, or truncations of p53 (cBioPortal: 55.5% of NSCLC and 45.7% of all tumor types)<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>–<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Genetic deletion of MK2 prior to tumor development dramatically enhances subsequent tumor killing by DNA-damaging chemotherapy selectively in tumor cells that lack functional p53, both in vitro and in vivo<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. However, whether targeting of MK2 after the development of well-established tumors in vivo would also enhance the response to chemotherapy is unknown.</p><p id=\"Par5\">Platinum-containing compounds like cisplatin and carboplatin are amongst the most widely used cytotoxic chemotherapeutics for many tumor types, including ovarian cancer and lung adenocarcinoma. The major (>90%) adducts of cisplatin and carboplatin on DNA are 1,2-intrastrand d(GpG) and d(ApG) cross-links, which are repaired by the nucleotide excision repair (NER) pathway<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Tumor responses to cisplatin or carboplatin therefore depends on the levels of platinum–DNA adducts that are formed and the DNA repair capacity of the cell<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>, as exemplified by the fact that testicular cancers, many of which have deficiencies in the NER pathway, respond extremely well to cisplatin with cure rates reaching ~95%<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>–<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. However, in many other common tumor types such as NSCLC—the leading cause of cancer related death in the United States—the therapeutic efficacy of platinum-based DNA damaging agents is limited, with only about one third of patients receiving benefit<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. It is well-established that tumor cells in culture can be sensitized to cisplatin through the inhibition of the NER pathway; however, utilizing this information for therapeutic gain in patients is limited by the fact that systemic inhibition of NER is highly toxic to host tissues<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>.</p><p id=\"Par6\">Here, we describe the first example of an augmented synthetic lethal (ASL) sensitivity relationship between p53, MK2, and the DNA repair enzyme XPA. Simultaneous loss of MK2 and XPA increased tumor cell killing by cisplatin in p53-defective lung adenocarcinoma, the major subtype of NSCLC, cells relative to targeting either pathway alone due to hyperactivation of MK2 signaling in XPA-deficient cells. To leverage this ASL relationship for therapeutic gain, we utilized our recently developed RNAi delivery system to simultaneously co-target MK2 and XPA in p53-defective tumors in vivo, revealing superior tumor control with ASL targeting vs. SL targeting, and demonstrating a dramatic improvement in long-term survival. Together, these results highlight the utility of targeting an augmented SL relationship between cell cycle checkpoints and DNA repair to improve response to frontline chemotherapy in aggressive mouse models of human cancer.</p></sec><sec id=\"Sec2\" sec-type=\"results\"><title>Results</title><sec id=\"Sec3\"><title>Cells defective in DNA repair by NER hyperactivate MK2 signaling</title><p id=\"Par7\">Cisplatin-induced DNA damage primarily results from platinum (Pt)-mediated 1,2-intrastrand cross-links, and at a lower frequency, interstrand adducts. The former lesions are primarily repaired by the NER pathway, which includes both global genome repair (GG-NER) and transcription-coupled repair (TC-NER) (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>, and the latter by NER, translesion synthesis (TLS), homologous recombination (HR)<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>, and the Fanconi anemia (FA) pathway<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. Cells genetically lacking key NER proteins involved in GG-NER have reduced ability to repair certain types of Pt-induced lesions and related types of DNA cross-links<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, and have persistent DNA damage and error-prone repair, resulting in the human cancer-prone syndrome Xeroderma pigmentosum (XP)<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. Defects in the CSB protein, which is involved in TC-NER, base-excision repair, and transcription, results in Cockayne syndrome (CS), characterized by mitochondrial dysfunction, premature aging, and neurodegeneration<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref>,<xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. We hypothesized that defects in NER and the resulting persistent DNA damage accumulation, might further augment the activation of the MK2 signaling pathway in response to genotoxic stress. To investigate this, fibroblasts from XP or CS patients defective for individual proteins involved in GG-NER and/or TC-NER, and from a healthy control, were treated with increasing doses of cisplatin for 24 h, lysed, and probed for MK2 pathway activation by immunoblotting for the active, phosphorylated form of MK2<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. As shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1A</xref>, cells deficient in the NER proteins XPA, XPG, XPC, or CSB showed enhanced activation of MK2 at low doses (2.5 μM) of cisplatin compared to controls. Since XPA functions as a critical common scaffold required for both the TC-NER and GG-NER branches of the NER pathway (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>), we examined changes in DNA damage signaling in more detail using XPA-deficient cells (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1c</xref>, XPA−). In addition, we used the same XPA-deficient cell line in which XPA function had been restored by transfection with the wild-type XPA gene (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1c</xref>, XPA+). Hyperactivation of both p38 (the upstream MK2 activating kinase) and MK2 observed in response to low doses of cisplatin in XPA− cells was extinguished when XPA activity was restored, indicating that increased MK2 signaling in these cells is a direct result of impaired NER activity. Interestingly, no difference in Chk1 signaling after cisplatin treatment was observed in these experiments, suggesting that these effects may be specific to MK2, rather than applicable to all checkpoint kinase signaling pathways per se (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1c</xref>).<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Cells defective in repair by NER hyperactivate MK2 signaling.</title><p><bold>a</bold> Nucleotide excision repair pathways involved in cisplatin-induced DNA damage. <bold>b</bold> MK2 activity was assessed by Western blot analysis 24 h after vehicle or cisplatin treatment in control and XPA-deficient fibroblasts. Data are representative of two independent experiments. Note the slight activation of MK2 in the XPA deficient cells even in the absence of cisplatin treatment. <bold>c</bold> Western blot of MK2, Chk1, and p38 activation 24 h after cisplatin treatment in XPA-deficient fibroblasts transfected with vector control (−) or with restoration of XPA (+). <bold>d</bold> Western blot of MK2, Chk1, and p38 activation, and γH2AX levels, 6 h after vehicle (Veh) or cisplatin (Cis) treatment (25 µM) in XPA-proficient or deficient KP7B cells. Data representative of three independent experiments. The prominent doublet banding pattern of MK2 arises from use of alternative translation start sites in the mRNA<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>. <bold>e</bold> Quantification of phospho-MK2 in XPA-depleted KP7B cells. <italic>n</italic> = 3 independent experiments; **<italic>p</italic> = 0.007; two-tailed unpaired <italic>t</italic> test. Error bars represent mean ± SEM.</p></caption><graphic xlink:href=\"41467_2020_17958_Fig1_HTML\" id=\"d30e866\"/></fig></p><p id=\"Par8\">To explore the generality of this finding, we performed similar experiments in murine <italic>K-Ras</italic><sup><italic>G12D</italic><italic>/+</italic></sup>; <italic>p53</italic><sup><italic>−/−</italic></sup> (KP7B) lung adenocarcinoma tumor cells<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. Importantly, these cells can be used in vivo with an established transplantable model of aggressive lung cancer in fully immunocompetent hosts (see below). Consistent with our XPA-MK2 findings in human fibroblasts, knockdown of XPA using siRNA in murine KP7B lung adenocarcinoma cells resulted in a similar increase in both p38 and MK2 at 6 h after treatment with cisplatin, indicating enhanced reliance on MK2 signaling in these tumor cells when the NER pathway was impaired (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1d</xref>). Quantification of phospho-MK2 levels showed an approximately two-fold increase in the siXPA cisplatin-treated KP7B cells compared to control cisplatin-treated KP7B cells (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1e</xref>; c. f. red bars). Of note, in the lung adenocarcinoma cells in which XPA was knocked down, we observed elevated levels of γH2AX, phospho-p38, phospho-MK2, and phospho-Chk1 even in the absence of cisplatin treatment, consistent with elevated basal levels of endogenous DNA damage in tumor cells compared to non-tumorigenic fibroblasts (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1d</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>.</p></sec><sec id=\"Sec4\"><title>Co-targeting NER and MK2 enhances cisplatin lethality</title><p id=\"Par9\">The finding that NER-defective cells hyperactivate MK2 signaling, particularly in response to cisplatin-induced DNA damage suggested that these cells may have an increased reliance on MK2 to survive platinum-based chemotherapy. We therefore investigated whether co-targeting both pathways in KP7B lung adenocarcinoma cells could further enhance tumor cell death. Individual or combined knockdowns were used to determine whether loss of XPA was additive or synergistic with MK2 inhibition for tumor cell killing by cisplatin in culture. Cells depleted of either MK2 or XPA alone showed modestly increased sensitivity to cisplatin (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a</xref>, compare blue and red lines to black line), consistent with our prior data implicating MK2 in cell cycle arrest following DNA damage<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>–<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>, and the ability of other DNA repair pathways such as TLS, FA, and HR to partially compensate for defective NER. Combined depletion of MK2 and XPA, however, revealed a dramatic synergistic killing of KP7B cells in response to cisplatin treatment (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a</xref>, compare green and purple lines). Additive or greater than additive killing by cisplatin after combined MK2 and XPA knockdown, compared to individual knockdowns, was also observed in three p53 deficient NSCLC tumor cell lines (H2009, H1299, and KP7B), but not observed in a p53+ cell line (H1563) (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2A–C</xref>). To verify that this enhanced killing was not dependent on the siRNA sequence used, a second set of siRNAs was used to co-target MK2 and XPA in KP7B with similar results (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2D</xref>). Furthermore, to demonstrate that this effect was strictly p53-dependent, and not the result of other genetic abnormalities in the various cell lines, we next used isogenic HCT116 p53 wild type and p53 null colon cancer cells. As shown in Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2E, F</xref>, cisplatin treatment resulted in markedly reduced cell viability following combined MK2 and XPA knockdown, relative to the individual knock downs, only in the p53 null cells.<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Co-targeting NER and MK2 enhances cisplatin lethality in cells.</title><p><bold>a</bold> KP7B cells were depleted of MK2, XPA, or both using siRNA, and treated with the indicated concentrations of cisplatin. Cell viability was measured 72 h later using the CellTiter-Glo luminescence assay. The expected viability following combined MK2 and XPA knockdown was calculated assuming a Bliss independence model of additivity (see section “Methods”). <bold>b</bold> KP7B cells were treated with 25 µM cisplatin for 5 h. The drug-containing media was then replaced with drug-free media, and DNA repair allowed to occur for 12 h. Residual cisplatin adducts were then quantified by atomic absorption spectroscopy (left graph; siXPA + cis vs. siCon + cis, *<italic>p</italic> = 0.0280; siMK2 + siXPA + cis vs. siCon + cis, *<italic>p</italic> = 0.0144; two-tailed unpaired <italic>t</italic> test. <italic>n</italic> = 3 experiments. Error bars represent mean ± SEM) and by immunofluorescence using an antibody against cisplatin–DNA adducts (right graph; siXPA + cis vs. siCon + cis, ****<italic>p</italic> ≤ 0.0001; siMK2 + siXPA + cis vs. siCon + cis, ****<italic>p</italic> ≤ 0.0001; siMK2 + siXPA + cis vs. siMK2 + cis, ****<italic>p</italic> ≤ 0.0001; siMK2 + siXPA + cis vs. siXPA + cis, ****<italic>p</italic> ≤ 0.0001; two-tailed unpaired <italic>t</italic> test. <italic>n</italic> = 3 separate samples <italic>n</italic> > 70 cells per condition). <bold>c</bold> Representative immunofluorescence images of KP7B cells depleted of MK2, XPA, or both proteins treated with 25 μM cisplatin for 24 h, then fixed and stained with an antibody against γH2AX. <bold>d</bold> Quantification of the number of γH2AX foci in the KP7B cells treated as in Fig. 2c for each treatment in <italic>n</italic> = 3 separate samples. (siMK2 + cis vs siCon + cis, ****<italic>p</italic> < 0.0001; siMK2 + siXPA + cis vs. siCon + cis, ****<italic>p</italic> < 0.0001; siMK2 + siXPA + cis vs. siMK2 + cis, **<italic>p</italic> ≤ 0.002; siMK2 + siXPA + cis vs. siXPA + cis, **<italic>p</italic> < 0.0001; two-tailed unpaired <italic>t</italic> test. <italic>n</italic> = 3 separate samples and <italic>n</italic> > 90 cells per condition). Width in violin plot indicates frequency for each observed value from maximum to minimum, with dotted line indicating median.</p></caption><graphic xlink:href=\"41467_2020_17958_Fig2_HTML\" id=\"d30e1019\"/></fig></p><p id=\"Par10\">To investigate the underlying mechanism responsible for these effects, we measured platinum–DNA adducts by both atomic absorption spectroscopy on purified DNA, and by immunofluorescence of cells stained for platinum adducts. In both assays, co-depletion of MK2 and XPA greatly enhanced the number of cisplatin-DNA adducts over either knockdown alone (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2b</xref>). In addition, co-depletion of MK2 and XPA showed increased DNA damage as measured by the intensity of γH2AX foci (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2c, d</xref>), suggesting that co-targeting both MK2 and XPA in tumor cells results in a further abrogation of cisplatin–DNA adduct repair.</p><p id=\"Par11\">Together, these data suggest that MK2 and XPA have an augmented synthetic lethal relationship in p53-defective cells, and that simultaneous targeting of MK2 and XPA enhances cisplatin lethality owing to persistent DNA damage.</p></sec><sec id=\"Sec5\"><title>siRNA–peptide nanoplex delivery to lung tumors in vivo</title><p id=\"Par12\">Despite the therapeutic potential of MK2 inhibition to enhance the antitumor response to DNA-damaging chemotherapy, currently available small molecule MK2 inhibitors are sub-optimal and nonspecific due to the presence of a shallow ATP-binding pocket in MK2. This results in cross-reactivity with MK3 and MK5, as well as several other kinases<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. Furthermore, MK2 also plays an important role in the host innate immune response<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>, raising the potential for systemic toxicity in response to generalized inhibition. In an effort to specifically target MK2 and co-target both MK2 and XPA within tumors, we extended a polypeptide-based nanoscale complexation approach that we have recently developed for the delivery of individual small interfering RNAs (siRNAs) to tumors<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>, in order to deliver siRNAs against MK2 (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref>) or to simultaneously co-deliver siRNAs against both MK2 and XPA (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4a</xref>). These nanoparticle delivery vehicles (RNA–peptide nanoplexes) are constructed from three unique lipid-like polypeptides containing a hydrophilic headgroup attached to a long hydrophobic alpha helical tail comprising poly(benzyl-<sc>l</sc>-aspartate) (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref>; Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3A</xref>; “Methods”)<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. Nanoplexes containing fluorescently tagged siRNAs form punctate foci in cells at early time points, consistent with endosomal/lysosomal cellular uptake (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3B</xref>). These steps are followed by gradual endosomal escape and release of siRNA to the cytoplasm<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. When the nanoplexes were used to deliver siRNA to cells in vitro, single and combined target knockdown efficiencies at least as effective as those obtained using conventional cationic lipids were observed (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b, c</xref>), with little indiscriminant toxicity at varying siRNA and nanoplex concentrations and ratios (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3C</xref>). Nanoplexes efficiently delivered siRNA against MK2 and XPA and knocked down both genes at the single cell level, as shown by immunofluorescence staining in KP7B cells treated with nanoplex–siMK2, nanoplex–siXPA, and nanoplex–siMK2/siXPA (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3D–F</xref>). We found using immunofluorescence that >95% of the KP7B cells treated with nanoplex–siMK2/siXPA show dual MK2 and XPA knockdown (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3F</xref>). Interestingly, using an ex vivo flow cytometry based assay of tumor cells cocultured with splenocytes, we observed that the siRNA nanoplexes were preferentially taken up by the tumor cells, compared to macrophages or T-cells (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3G</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>, suggesting that this nanoparticle approach might limit the effect of MK2 inhibition in immune cells.<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>siRNA nanoplexes targeting MK2 improves tumor response to Pt.</title><p><bold>a</bold> Lipid-like polypeptide nanocarriers and chemical structure of peptide-based nanoplex components. <bold>b</bold> Efficiency of siRNA delivery of nanoplexes in KP7B cells measured by RT-qPCR for <italic>MK2</italic> mRNA and <bold>c</bold> Western blotting for MK2 protein. Cells were harvested 72 h after lipofectamine transfection or nanoplex treatment and analyzed. Data representative of <italic>n</italic> = 3 experiments. <bold>d</bold> Schematic of nanoplex treatment in a recalcitrant syngeneic orthotopic lung adenocarcinoma mouse model. Luciferase-GFP-expressing KP7B cells form lung tumors in recipient mice, and are then treated with siRNA-loaded nanplexes 2–3 weeks later. <bold>e</bold> Timeline of nanoplex–siRNA and cisplatin treatment of mice with tumors. Blue arrows indicate time of nanoplex–siRNA treatment. Red arrows indicate time of cisplatin treatment. <bold>f</bold> Mice were sacrificed at day 36 and knockdown of MK2 mRNA measured by RT-qPCR in tumors. Data show mRNA levels as fold-change vs. nanoplex-siControl (<italic>n</italic> = 3 animals per group; 1 mg kg<sup>−1</sup> siRNA encapsulated with 200 mg kg<sup>−1</sup> nanoplexes; *<italic>p</italic> = 0.0366; two-tailed unpaired <italic>t</italic> test). <bold>g</bold> Western blot of lung tumors harvested on day 36 from 2 representative mice treated with nanoplex–siMK2 vs. nanoplexes-siCon for MK2. <bold>h</bold> Quantification of MK2 protein levels in tumors at day 36. Data show MK2 protein levels normalized to vinculin as fold-change vs. nanoplex–siControl. <italic>n</italic> = 3 animals per group, 1 mg kg<sup>−1</sup> nanoplex–siRNA; *<italic>p</italic> = 0.0215; two-tailed unpaired <italic>t</italic> test. <bold>i</bold> Representative bioluminescence images before and after nanoplex–siRNA and saline or cisplatin treatment. <bold>j</bold> Quantification of lung bioluminescence following first, second, and third treatments after tumor induction, shown as fold-change compared to pre-treatment (day 15) (nanoplex–siCon <italic>n</italic> = 4 animals, nanoplex–siMK2 <italic>n</italic> = 3 animals, nanoplex–siCon + cisplatin <italic>n</italic> = 5 animals, nanoplex–siMK2 + cisplatin <italic>n</italic> = 5 animals; *<italic>p</italic> ≤ 0.0143; two-tailed unpaired <italic>t</italic> test, post third treatment). <bold>k</bold> Representative H&E and Ki-67 staining of lungs at the end of three rounds of treatment. <bold>l</bold> Quantification of tumor burden as a percentage of lung area. <italic>n</italic> = 3 animals per condition; *<italic>p</italic> = 0.0422; two-tailed unpaired <italic>t</italic> test. <bold>m</bold> Quantification of Ki-67 as a percentage of positive cells. <italic>n</italic> = 3 animals per condition; ****<italic>p</italic> ≤ 0.0001; two-tailed unpaired <italic>t</italic> test. Data shown as violin plots as in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2d</xref>. <bold>n</bold> Kaplan–Meier survival analysis of tumor-bearing mice treated with nanoplex–siMK2 or nanoplex–siCon and cisplatin. <italic>n</italic> = 3 animals per condition; *<italic>p</italic> = 0.0035, calculated using the log-rank test. Error bars in panels <bold>b</bold>, <bold>f</bold>, <bold>h</bold>, <bold>j</bold>, and <bold>l</bold> represent mean ± SEM.</p></caption><graphic xlink:href=\"41467_2020_17958_Fig3_HTML\" id=\"d30e1249\"/></fig><fig id=\"Fig4\"><label>Fig. 4</label><caption><title>Augmented synthetic lethality for Pt by co-targeting XPA and MK2 in vivo.</title><p><bold>a</bold> Schematic of dual-targeting peptide-based nanoplexes. <bold>b</bold> MK2 and XPA knockdown efficiency of nanoplex–siMK2/siXPA compared to lipofectamine RNAiMax–siMK2/siXPA measured by Western blotting for MK2 and XPA. Data representative of two independent experiments. <bold>c</bold> Representative bioluminescence images before and after indicated siRNA and cisplatin treatment on days 22, 29, 36, and 43. <bold>d</bold> Quantification of lung bioluminescence at 43 days after tumor implantation (<italic>n</italic> = 3 animals were used for each condition; only two mice remained alive in the nanoplex–siControl + cisplatin at day 43, and they died by day 50). Error bars represent mean ± SEM. <bold>e</bold> Representative H&E and Ki67 lung staining at the end of three rounds of the indicated treatments. Three animals were used for each condition. <bold>f</bold> Kaplan–Meier survival analysis of tumor-bearing mice treated with the indicated nanoplex–siRNA in combination with cisplatin treatment (nanoplex–siControl + cisplatin <italic>n</italic> = 6 animals, nanoplex–siMK2 + cisplatin <italic>n</italic> = 3 animals, nanoplex–siXPA + cisplatin <italic>n</italic> = 5 animals, and nanoplex–siMK2/siXPA + cisplatin <italic>n</italic> = 5 animals. *<italic>p</italic> ≤ 0.05 and **<italic>p</italic> ≤ 0.01 calculated using the log-rank test). <bold>g</bold> Model illustrating crosstalk between the MK2 signaling pathway and nucleotide excision repair in p53-defective cells. Co-targeting these pathways in established tumors prolongs spontaneous survival and potently enhances the antitumor response to cisplatin treatment.</p></caption><graphic xlink:href=\"41467_2020_17958_Fig4_HTML\" id=\"d30e1303\"/></fig></p><p id=\"Par13\">To determine whether the nanoplexes could be used to deliver single or dual-targeting siRNAs to lung adenocarcinomas in vivo, we took advantage of an aggressive transplantable model where tumors are generated in immunocompetent, syngeneic recipient animals by tail vein injection of <italic>K-Ras</italic><sup><italic>G12D/+</italic></sup>; <italic>p53</italic><sup><italic>−/−</italic></sup> (KP7B) tumor cells<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>, followed by subsequent tumor seeding in the lung (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3d</xref>). We choose to use this model for our experiments because the transplanted cells give rise to tumors in mice with fully functional immune systems, at the correct anatomical location, and result in murine tumors that are pathologically and molecularly similar to the tumors from which they were derived<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>, as well as their human tumor counterparts<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref>,<xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>.</p><p id=\"Par14\">As a control for nanoparticle delivery of siRNAs to lung adenocarcinomas in vivo, nanoplexes containing a fluorescently tagged control non-targeting siRNA were administered intraperitoneally to mice with luciferase/GFP-expressing lung tumors (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3d</xref>; Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4A</xref>). The siRNA nanoplexes localized to the vicinity of the tumors and delivered ~5% of the initial siRNA injected dose as determined by co-registry of lung tumor bioluminescence with thoracic tomograms of fluorescently labeled siRNA (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4B, C</xref>). By comparison, small molecule inhibitors typically deliver <1% of the initial drug dose to the tumor<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref>,<xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. Removal of nanoparticles from the body typically involves a combination of hepatic and renal clearance<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. We observed no hepatic or renal dysfunction as determined by serum biochemical markers 24 h after treatment (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4D–I</xref>). To ensure that the nanoplexes that we engineered are not immunogenic in this fully immunocompetent mouse model, serum cytokine profiles were measured 24 h after nanoplex/siRNA delivery. There were minimal changes in the levels of serum cytokines in nanoplex-treated animals compared to controls (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4J</xref>). In particular, the levels of IL-6 and TNFα, classic indicators of an innate immunogenic response, were unchanged. These data suggest that our nanoplex carriers can efficiently deliver siRNAs to lung adenocarcinoma tumors in vivo.</p></sec><sec id=\"Sec6\"><title>siRNA nanoplex targeting of MK2 improves Pt response in NSCLC</title><p id=\"Par15\">We next used the nanoplexes to specifically target MK2 alone in vivo in pre-existing lung tumors using the aforementioned immunocompetent transplant model. As shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3e</xref>, tumors were allowed to form for 14 days prior to treatment in order to assess therapeutic response in established tumors. Mice were then treated with non-targeting (control) or MK2-targeting siRNA-loaded nanoplexes (1 mg kg<sup>−1</sup>) twice weekly on days 1 and 4, followed by a single dose of cisplatin (7 mg kg<sup>−1</sup>) on day 3 of each week for a total of 3 weekly cycles (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3e</xref>). The administration of systemic cisplatin was chosen to correspond with the time when maximal mRNA and protein inhibition was observed in in vitro pilot studies (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>). A second nanoplex–siRNA dose was given at day 4 to ensure sustained knockdown. Analysis of MK2 mRNA and protein levels from tumors excised at day 36, performed at the conclusion of the experiment, validated that the MK2 siRNA nanoplexes had significantly reduced the levels of MK2 mRNA and protein in the tumors by 70% (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3f</xref>) and 65% (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3g, h</xref>), respectively, in vivo.</p><p id=\"Par16\">Both control and MK2 siRNA nanoplex-treated animals showed a similar level of tumor bioluminescence in the absence of DNA damaging chemotherapy (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3i, j</xref>, compare black and dark blue bars), indicating that depletion of MK2 alone had no significant effect on tumor growth in this model. Following systemic administration (intraperitoneal injection, IP) of cisplatin, control tumors continued to grow rapidly despite platinum treatment, (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3i, j</xref>, compare black and gray bars). In contrast, cisplatin treatment markedly inhibited the growth of MK2-depleted tumors (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3i, j</xref>, compare gray and cyan bars). By the conclusion of the experiment at day 36, the tumor burden in mice with established tumors treated in vivo with siMK2–nanoplexes in combination with cisplatin was 3.5-fold lower than that of the controls (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3j</xref>). These data demonstrate, for the first time, superior responses to cisplatin-based chemotherapy in established MK2-proficient tumors subsequently depleted of MK2 by a drug-like modality. The increased efficacy of cisplatin in lung tumors depleted of MK2 was further validated by histology and immunohistochemistry (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3k</xref>). Quantification of H&E staining of lung sections from cisplatin-treated animals who received nanoplex-siMK2 demonstrated a six-fold reduction in tumor burden and Ki67 staining compared to control siRNA nanoplex-treated animals (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3l, m</xref>), indicating reduced proliferation in tumors treated with the combination of nanoplex–siMK2 and cisplatin. The observed increase in platinum response seen in established lung tumors upon MK2 knockdown with nanoplex-targeted siRNA in vivo further resulted in a modest but significant extension of lifespan following platinum therapy (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3n</xref>). We confirmed that our nanoplex–siMK2 results were due to inhibition of tumor cell MK2, and not due to off-target effects, by examining the response of an independent set of mice. These animals bore lung tumors derived from KP7B cells expressing a MK2-targetting shRNA that targets an alternative sequence within the MK2 mRNA with remarkably similar results (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6A–F</xref>). Together, these data provide compelling evidence that inhibition of MK2 by therapeutic RNAi nanoparticles can markedly augment the response of established, aggressive tumors to frontline platinum-based chemotherapy and improve survival.</p></sec><sec id=\"Sec7\"><title>Loss of XPA synergizes with MK2 inhibition in NSCLC tumors</title><p id=\"Par17\">Although MK2 depletion clearly enhanced tumor cell responses to cisplatin (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3i–m</xref>; Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6D, E</xref>), the overall increase in survival was modest (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3n</xref>; Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6F</xref>). We next investigated whether we could exploit the ASL relationship between XPA and MK2 (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>) for treatment of murine lung adenocarcinomas using our nanoplexes to co-deliver siRNAs against both MK2 and XPA in vivo. Dual targeting nanoplex–siRNA delivery vehicles against MK2 and XPA were synthesized and validated for their ability to simultaneously knockdown both MK2 and XPA (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4a, b</xref>). Using the immunocompetent murine lung adenocarcinoma transplant model described in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3d</xref>, tumors were allowed to form, but treatment was withheld until 21 days post transplantation (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">7A</xref>). This period is 1-week longer than that shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>, in order to allow the tumors to grow even larger, thus raising the bar for therapeutic intervention. Mice were then treated with non-targeting nanoplex–siRNA (control), nanoplex–siRNA against MK2 alone or XPA alone, or with dual-targeted nanoplex–siRNA directed against both MK2 and XPA, on a twice weekly schedule, along with administration of a single dose of cisplatin each week for 3 weeks. Of note, both tumor-bearing and nontumor-bearing mice treated with nanoplex-siRNA against MK2 and XPA in combination with cisplatin did not have excessive weight loss compared to control mice treated with cisplatin alone, consistent with minimal direct toxicity of the nanoparticle formulation even when administered in combination with a platinum agent (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">7B, C</xref>). Cisplatin treatment markedly suppressed the growth of MK2, XPA, or MK2/XPA-depleted tumors compared to controls (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c, d</xref>). At day 36, the tumor burden of mice treated with nanoplex-siXPA was 2.8-fold less (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">8A</xref>, red vs. black post-treatment bars), and nanoplex–siMK2/siXPA was 20-fold less, than those of the controls treated with cisplatin alone (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">8A</xref>, purple vs. black bars). Furthermore, the tumor burden of mice treated with dual-targeted nanoplex–siMK2/siXPA was threefold less than that of the animals treated with nanoplex–siMK2 alone (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">8A</xref>, purple vs. blue post treatment bars) at this same time point. By day 43 (22 days post initial treatment), we noted that mice receiving nanoplex-siMK2/siXPA targeted combination continued to sustain lower tumor burden even after treatment cessation on day 33 (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4d</xref>). Both H&E and Ki67 staining performed on lung sections from mice that either received nanoplex–siMK2, nanoplex–siXPA, or the nanoplex–siMK2/siXPA combination with cisplatin further confirmed reductions in tumor burden and proliferation (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4e</xref>), consistent with the results of whole animal bioluminescence imaging (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c, d</xref>). Together, these data indicate that, whereas MK2 and XPA inhibition alone sensitizes established refractory lung adenocarcinoma tumors to cisplatin, (with MK2 inhibition being superior to that of XPA inhibition alone, based on residual tumor burden), co-targeting of XPA and MK2 within the same tumor further reduces tumor burden with a notably increased efficiency (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c–e</xref>; Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">8A, B</xref>).</p><p id=\"Par18\">To determine whether the observed response to cisplatin in tumors co-depleted of MK2/XPA translated into a significant overall therapeutic benefit to the animals compared to MK2 depletion alone, we analyzed long-term animal survival. Mice that received either single nanoplex–siXPA or nanoplex–siMK2 treatment in combination with cisplatin displayed a significant but modest survival benefit (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4f</xref>; median survival 32 and 30 days, respectively, compared to 17 days for nanoplex–siControl). However, co-depletion of both XPA and MK2 using dual targeted XPA/MK2 siRNA nanoplexes in combination with cisplatin had a strikingly profound effect, increasing the median survival to 50 days, and more than doubling the survival of longest-lived animals in any of the other cohorts (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4f</xref>). These data indicate establishment of ASL in vivo by combined MK2/XPA depletion significantly increases the overall therapeutic benefit of cisplatin and is superior compared to depletion of MK2 or XPA alone.</p><p id=\"Par19\">Remarkably, we also observed an unexpected significant survival benefit from long-term simultaneous co-targeting of XPA and MK2 within tumors even in the absence of cisplatin treatment (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">8C</xref>), consistent with the previously observed increase in spontaneous DNA damage signaling observed in XPA depleted cells in culture (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b–d</xref>). The addition of cisplatin, however, further extended the median survival of animals treated with combination MK2/XPA siRNA nanoplexes by an additional 15 days (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">8C</xref>). Taken together, these data indicate that the combined targeting of DNA damage-induced cell cycle checkpoint pathways and DNA repair via co-inhibition of MK2 and XPA, respectively using polypeptide-based nanocarriers results in spontaneous DNA damage and antitumor responses, that can be further enhanced by the addition of cisplatin to improve therapeutic outcomes in established cisplatin-resistant lung tumors in vivo (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4g</xref>).</p></sec></sec><sec id=\"Sec8\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par20\">Manipulation of the DNA-damage response, either by disrupting cell cycle checkpoints, or by interfering with DNA repair, is emerging as a promising approach to promote tumor cell killing and enhance the response to DNA-damaging chemotherapeutic drugs<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. Three checkpoint effector kinases, Chk1, Chk2, and MK2, play critical roles in initiating or maintaining cell cycle arrest after genotoxic damage<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. Chk1 activity in the nucleus is required for the initiation of cell cycle arrest after DNA damage<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>, and small molecule inhibitors for systemic administration are currently under investigation in Phase 1 and 2 clinical trials<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. Although promising, their use has been complicated by significant dose-limiting toxicity<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>. Since Chk1 function is critical during normal DNA replication, homozygous genetic deletion of <italic>Chk1</italic> results in embryonic lethality, while heterozygous <italic>Chk1</italic><sup><italic>+/−</italic></sup> animals have a significantly increased propensity to develop cancer<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>. Prolonged inhibition of Chk1 function in non-tumorigenic tissues during cancer therapy therefore also inadvertently increase the incidence of secondary malignancies.</p><p id=\"Par21\">In contrast, mice with homozygous and heterozygous loss of <italic>MK2</italic> are fully viable<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>, and the essential function of MK2 as a checkpoint kinase become prominently unmasked, in a synthetic lethal manner, when p53 function is lost, making it an ideal target for certain anticancer therapies<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. MK2, when activated downstream of ATM and ATR, acts within the cytoplasm, independently of CHK1, to maintain G1/S, and intra-S cell cycle checkpoints through p27, and the G2/M checkpoint through Gadd45<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>. To date, however, there are no MK2 inhibitors approved for clinical use<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>, and the small molecule inhibitors that are currently available for laboratory and animal use are both sub-optimal and non-specific, owing to the shallow MK2 ATP-binding pocket<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. In addition, MK2 has a well-established role in innate immunity and inflammatory signaling<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>, similar to DNA-PK, raising the possibility of off-target side effects from systemic inhibition. To overcome these limitations, we investigated a tumor-targeting nanoparticle that delivers siRNAs to the site of pre-existing MK2-containing tumors and efficiently depletes both its target RNA and protein.</p><p id=\"Par22\">In addition to targeting cell cycle checkpoint pathways, the specific targeting of DNA repair pathways to improve the efficacy of genotoxic chemotherapy may be particularly important in NSCLC, since evidence suggests that these tumors have a pre-existing increase in intrinsic DNA repair activity, possibly as a consequence of long-time adaptation to DNA lesions induced by cigarette smoking<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref>,<xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>. We focused on the NER pathway, which is required for the repair of cisplatin adducts. XPA depletion alone resulted in elevated levels of MK2 signaling which could be further enhanced by exogenous genotoxic injury with cisplatin. Furthermore, this combined MK2 and XPA loss resulted in increased platinum-induced DNA lesions. These results indicate that, in the absence of a functional NER pathway, MK2 appears to be hyperactivated to assist in cell cycle arrest and damage repair. Loss of XPA synergistically augmented the pre-existing dependency of p53-defective lung adenocarcinoma cells on MK2 to survive cisplatin treatment in vitro, revealing an augmented synthetic lethal relationship between p53, MK2, and XPA. This mammalian cell trigenic interaction which we unmasked upon cisplatin treatment, bears some conceptual similarity to a trigenic mutant screening and colony size scoring approach performed in budding yeast by Kuzmin et al.<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>, though in that work extrinsic perturbations such as genotoxic stress, were not used.</p><p id=\"Par23\">To implement this ASL relationship between p53, MK2, and XPA in the setting of platinum-induced DNA damage in vivo, we co-encapsulated siRNAs against MK2 and XPA into the same nanoplex particles. These were then administered alone or in conjunction with systemic cisplatin treatment, resulting in a markedly improved anti-tumor response of established lung tumors in vivo upon platinum treatment that was associated with a dramatically prolonged survival. In vivo, tumor cell-enriched nanoparticle uptake is likely to be further exploited by the preferential targeting of the nanoparticles to tumors as a consequence of so-called “passive” targeting<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>. Nanoparticles are able to exploit the cancer’s distinct vascular and lymphatic pathology (i.e., leaky vasculature and defective lymphatic drainage) to accumulate in the tumor. Here we utilize the enhanced tumor cell uptake and passive tumor targeting properties to formulate nanoparticles that are able to pass through the leaky vascular junctions in tumors resulting in preferential accumulation at the tumor site over time, as shown in Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>. The fact that not all of the tumor cells were killed in vivo suggests that not all tumor cells may have internalized the nanoparticles, consistent with the fractional uptake that we observed in vitro. Design strategies to improve uptake of the nanoplexes within tumors might result in even better tumor cell killing.</p><p id=\"Par24\">To our knowledge, this study is the first to simultaneously target both a DNA repair pathway and a cell cycle checkpoint pathway with a drug-like modality in vivo, both alone and in the context of cytotoxic chemotherapy treatment. Taken together, our data indicate that: (1) cross-talk exists between the NER and p38/MK2 pathways, which coordinates DNA repair and cell fate after DNA damage, and that (2) this synergistic interplay between two key biological pathways for DNA damage response can result in highly effective targeted combination therapies for cancer treatment. Overall these data establish the paradigm that identification and therapeutic targeting of augmented synthetic lethal relationships can produce a safe and highly effective therapy by ‘re-wiring’ multiple DNA damage response pathways, the systemic inhibition might otherwise be toxic. Our nanoplexes are highly modular, and consequently they facilitate the encapsulation of any combination of siRNAs, leading to rapid translation of defined SL or ASL interactions from discovery to therapeutic targeting in vivo.</p></sec><sec id=\"Sec9\"><title>Methods</title><sec id=\"Sec10\"><title>Cell culture</title><p id=\"Par25\">All human cell lines were purchased from ATCC (American Type Culture Collection) or Coriell Institute. HCT116 p53 Null cells were a gift from the Vogelstein laboratory. H1299 and H1563 cells were grown in RPMI supplemented with 10% fetal bovine serum (FBS) and 2 mM <sc>l</sc>-Glutamine. 293T, H2009 and KP7B cells were grown in DMEM supplemented with 10% FBS and 2 mM <sc>l</sc>-Glutamine. KP7B cells were described previously by Doles et al.<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>, and were a gift from Tyler Jacks’ laboratory. HCT116 cells were grown in McCoy’s 5a supplemented with 10% FBS and 2 mM <sc>l</sc>-Glutamine. Human fibroblast cell lines GM15876A and GM04312 were grown in DMEM supplemented with 10% FBS and 2 mM <sc>l</sc>-Glutamine. Human non-transformed fibroblast cell lines GM16684, GM00739, and GM16181 were grown in DMEM supplemented with 15% FBS and 2 mM <sc>l</sc>-Glutamine. All cell lines were cultured in a 37 °C humidified incubator with 5% CO<sub>2</sub>, maintained subconfluently and used for no more than 20 passages.</p></sec><sec id=\"Sec11\"><title>Antibodies and chemicals</title><p id=\"Par26\">Antibodies against MK2/MAPKAPK2 (#3042, 1:1000), Phospho-Thr-334 MAPKAPK-2 (#3041), Phospho-Ser345 Chk1 (#2348, 1:1000), Phospho-Thr-180/Tyr-182 p38 (#9211, 1:1000), Phospho-Ser-139 Histone H2AX (#9718, 1:1000), and Histone H2AX (D17A3, #7631, 1:1000), were purchased from Cell Signaling Technologies. Antibodies for Chk1 (#8408, 1:1000), p38 (A-12, sc-7972, 1:1000) and XPA (B-1 sc-28353, 1:500) were purchased from Santa Cruz biotechnology. Ki-67 (ab16667, 1:1000), and anti-cisplatin modified DNA (CP9/19; Abcam ab103261, 1:500) antibodies were purchased from Abcam. Antibodies against β-Actin (A5441, 1:2000), GAPDH (G8795, 1:5000) and vinculin (V4505, 1:5000), as well as doxorubicin and cisplatin were purchased from Sigma Aldrich. All chemicals were used at the indicated doses. Antibodies for flow cytometry, CD45-PECy7 (Clone: 30-F11, #25-0451-82, 1:100), CD11b-FITC (Clone: M1/70, #11-0112082, 1:100), and F480-PE (Clone: BM8, #12-4801-82, 1:100) were from Thermo (eBioscience) while CD3e-APC-Cy7 (Clone: 145-2C11, #100330, 1:100) was from BioLegend.</p></sec><sec id=\"Sec12\"><title>Retro-virus production</title><p id=\"Par27\">For VSVG-pseudotyped virus production, 293T cells were transfected using the calcium phosphate method (Clontech) using either pMLS-tomato (for shRNAs in KP7B cells) along with packaging and structural vectors VSVG and GAG/POL. Supernatants containing virus were then used to transduce target cells in the presence of 8 µg/mL polybrene for three rounds of infection. Successfully transduced cells were sorted for tdTomato expression by flow cytometry for pMLS-tomato infected cells. GFP-Luciferase was used to infect KP7B cells for bioluminescent imaging and was a kind gift from Bonnie Huang (MIT).</p></sec><sec id=\"Sec13\"><title>shRNA sequences</title><p id=\"Par28\">All shRNAs were designed using the Cold Spring Harbor web portal (<ext-link ext-link-type=\"uri\" xlink:href=\"http://katahdin.cshl.org/siRNA/RNAi.cgi?type=shRNA\">http://katahdin.cshl.org/siRNA/RNAi.cgi?type=shRNA</ext-link>) and 97mer oligonucleotides (see below for sequence, underlined sequences are gene-specific) were used as templates for PCR using miR-30 shRNA amplification primers (see below). PCR products were digested with XhoI and EcoRI and ligated into pMLS-Tomato.</p><p id=\"Par29\">shMK2 mmu</p><p id=\"Par30\">TGCTGTTGACAGTGAGCG<underline>ATCCTTGGGTGTCATCATGTAT</underline>TAGTGAAGCCACAGATGTA<underline>ATACATGATGACACCCAAGGAC</underline>TGCCTACTGCCTCGGA</p><p id=\"Par31\">miR-30 cloning XhoI Fwd primer</p><p id=\"Par32\">CAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCG</p><p id=\"Par33\">miR-30 cloning EcoRI Rev primer</p><p id=\"Par34\">CTAAAGTAGCCCCTTGAATTCCGAGGCAGTAGGCA</p></sec><sec id=\"Sec14\"><title>siRNA oligonucleotides and siRNA transfection</title><p id=\"Par35\">MK2, XPA siRNA, and nontargeting control siRNA (Silencer negative control No. 1 siRNA) were purchased from Ambion. siRNA transfection was performed using Lipofectamine RNAiMAX as per manufacturer’s instruction (Invitrogen) using a final concentration of 5 nM siRNA in H1299 and H2009 and 50 nM in KP7B cells unless otherwise stated.</p><p id=\"Par36\">Sequences of Silencer select siRNA are as follows: Murine MAPKAPK2 (s201671) sense: ACAGAAUUCAUGAACCACCTT, antisense: GGUGGUUCAUGAAUUCUGUGA; murine MAPKAPK2-2 (s201670) sense: GAACGAUGGGAGGAUGUCATT, antisense: UGACAUCCUCCCAUCGUUCCT; murine XPA (s76138) sense: GCUUAUAACCAAGACAGAATT, antisense: UUCUGUCCUUGGUUAUAAGCTT; murine XPA-2 (s76140) sense: CCAAAAUGAUUGACACCAATT, antisense: UUGGUGUCAAUCAUUUUGGGA; human MK2 (s569) sense: GGAUCAUGCAAUCAACAAATT, antisense: UUUGUUGAUUGCAUGAUCCAA; and human XPA (s14925) sense: GAAGAUGACAUGUACCGUATT, antisense: UACGGUACAUGUCAUCUUCTA. Fluorescently labeled siRNA was purchased from Qiagen (AllStars Negative Control siRNA, Alexa Fluor 647; Qiagen).</p></sec><sec id=\"Sec15\"><title>Electron microscopy</title><p id=\"Par37\">TEM was performed using a JEOL 2100 FEG instrument equipped with a Gatan 626 Single Tilt Liquid Nitrogen Cryo Transfer Holder. Samples were prepared on QUANTIFOIL Holey Carbon Films (Electron Microscopy Sciences) using a Gatan Cryo-Plunge3 system.</p></sec><sec id=\"Sec16\"><title>Survival assays</title><p id=\"Par38\">Survival assays were performed on either 96-well (2500 cells) or 384-well (500 cells) plates. Cells were transfected with siRNA as described above and treated with various doses of cisplatin 48 h after transfection. Cell viability was measured 72 h after cisplatin treatment using the CellTiter-Glo luminescent cell viability assay as per manufacturer’s instructions (Promega) using the Tecan Infinite 200 PRO plate reader. Expected values were calculated based on the Bliss independence model of additivity following methods outlined in Foucquier and Guedj<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>, (siRNA1 viability × siRNA2 viability × cisplatin viability).</p></sec><sec id=\"Sec17\"><title>In vivo biodistribution</title><p id=\"Par39\">Tumor accumulation was approximated using a Xenogen IVIS Imaging System (Caliper). Tumor-bearing mice (<italic>n</italic> = 3) were intraperitoneally injected with nanoparticles (1 mg kg<sup>−1</sup>, AllStars Negative Control siRNA, Alexa Fluor 647; Qiagen) dispersed in phosphate-buffered saline (PBS). After 24 h, mice were anesthetized with isoflurane and imaged (640/700 nm ex/em) using aperture-limited transillumination fluorescence tomography. The signal in tomographic images was spatially limited to that from the upper abdomen. Biodistribution measurements were taken from whole animal epifluorescence images. Recovered fluorescence from the lungs and whole animal were quantified using the region-of-interest analysis package in Living Image (Perkin Elmer).</p></sec><sec id=\"Sec18\"><title>Analysis of differential siRNA uptake by tumor and immune cells using splenocyte–tumor cell coculture</title><p id=\"Par40\">Spleens were isolated from C57BL6/Jx129-JAE mice and mashed through a 40 μm filter. Red blood cells were lysed after incubation with ACK lysis buffer for 5 min and splenocytes were washed with complete growth media (RPMI, 10% FBS, 20 mM HEPES, 1 mM sodium pyruvate, 0.055 mM 2-mercaptoethanol, 2 mM <sc>l</sc>-glutamine, 1× nonessential amino acids and antibiotics). 500,000 splenocytes were co-cultured with 500,000 KP7B tumor cells per replicate per condition and treated for 24 h with AF647-siRNA-nanoparticles as indicated. Viable cells were assessed by flow cytometry for uptake of fluorescent siRNA by specific immune populations by co-staining with fluorophore conjugated antibodies for CD45, CD11b, F480, and CD3. CD45+ CD11b+ AF647+ cells were scored as siRNA+ macrophages, CD45+ CD3+ AF647+ cells were scored as siRNA+ T-cells and CD45-AF647+ cells were scored as siRNA+ tumor cells. The gating strategy is shown in Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">9</xref>.</p></sec><sec id=\"Sec19\"><title>Fluorescence microscopy</title><p id=\"Par41\">Live-cell fluorescence imaging was performed using a Nikon 1AR Ultra-Fast Spectral Scanning Confocal Microscope equipped with an environmental chamber providing temperature control. KP7B cells were passaged onto 35 mm glass-bottom culture dishes (MatTek). Adherent cells were washed with DPBS and concurrently incubated with nanoparticles (50 nM, BLOCK-iT Red control siRNA, Thermo Fisher), LysoTracker Deep Red (50 nM, Thermo Fisher), and Hoechst 34580 (10 ug/mL, Thermo Fisher) in Opti-MEM media at 37 °C for 1 h. Cell monolayers were then washed in DPBS and imaged in HEPES buffer (10 mM, pH 7.4). Immunofluorescence was performed by seeding cells onto poly-<sc>l</sc>-Lysine coated glass coverslips in 12-well plates and treated with cisplatin. Cells were washed in PBS, and then fixed with a 4% paraformaldehyde solution for 20 min at room temperature. Cells were blocked in 16.6% goat serum, 0.3% Triton X-100, 20 mM sodium phosphate, and 0.45 M sodium chloride prior to incubation in primary antibody overnight followed by secondary antibodies conjugated with Alexa Fluor (Invitrogen). Coverslips were then mounted using ProLong Gold Antifade Mountant (Invitrogen P36934) and imaged on Nikon Eclipse 800i fluorescence microscope and Applied Precision DeltaVision Ultimate Focus Micropscope with TIRF Module. CellProfiler 3.0.0 (developed by the Broad Institute of MIT and Harvard’s Imaging Platform and available at <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.cellprofiler.org\">http://www.cellprofiler.org</ext-link>) was used to quantify the integrated intensity of cells stained with γH2AX and cisplatin-DNA adduct antibodies. Each image was broken down into its component grayscale images for analysis using a CellProfiler pipeline. An illumination function module was utilized to uniformly reduce and smoothen background staining on the entire image set. The cisplatin–DNA adduct antibody images were further processed with a module to reduce background signal outside the nucleus. Cell nuclei were defined as primary objects using the DAPI grayscale images. The integrated intensity within the primary objects was then quantified for cisplatin adducts and γH2AX foci. Quantification of cells staining positively for MK2 and XPA, using a DAPI overlay, was performed using ImageJ Fuji 1.0. The same thresholding values were applied to all images.</p></sec><sec id=\"Sec20\"><title>Cell toxicity studies</title><p id=\"Par42\">Cell toxicity studies were performed at various N:P ratios (the ratio of positively charged polymer amine to negatively charged nucleic acid (siRNA) and assayed using the CellTiter-Glo luminescent cell viability assay (Promega).</p></sec><sec id=\"Sec21\"><title>In vivo cytokine and toxicity studies</title><p id=\"Par43\">Serum and plasma from mice treated with nanoplex-siRNA (200 mg kg<sup>−1</sup> nanoplexes and 1 mg kg<sup>−1</sup> siRNA) obtained at 24 h following treatment. Albumin, blood urea nitrogen (BUN), and creatinine (Cr) levels were measured by Charles River Laboratories from serum samples obtained via cardiac puncture 24 h following nanoplex–siRNA administration in 8- to 10-week-old male <italic>C57BL6/Jx129-JAE</italic> mice. Cytokine levels in plasma were measured by Eve Technologies and visualized using TIGR MeV version 4.9.</p></sec><sec id=\"Sec22\"><title>Histology</title><p id=\"Par44\">Tumor samples were obtained at 72 h following final nanoplex-siRNA administration, then formalin-fixed, paraffin-embedded, stained (H&E and Ki67), and scanned (Aperio slide scanner, Leica Biosystems).</p></sec><sec id=\"Sec23\"><title>qRT-PCR</title><p id=\"Par45\">For qRT-PCR analysis RNA was extracted from mouse tumor or cells using TRIzol reagent (Ambion) according to the manufacturer’s instructions and 1 μg of total RNA was used for reverse transcription using the superscript III first-strand synthesis kit (Invitrogen) as per the manufacturer’s instructions. For qPCR cDNA was amplified using SYBR green PCR mastermix (Applied Biosystems) according to the manufacturer’s cycling conditions for 40 cycles on a Bio-Rad C1000 Thermal Cycler. Data were analyzed using the delta-delta Ct method and plotted as fold change vs. control<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. Primers were ordered from Invitrogen and/or IDT: Actin <italic>mmu</italic> Fwd TGTTACCAACTGGGACGACA, Actin <italic>mmu</italic> Rev GGGGTGTTGAAGGTCTCAAA, Gapdh <italic>mmu</italic> Fwd GGGAAATTCAACGGCACAGT, Gapdh <italic>mmu</italic> Rev AGATGGTGATGGGCTTCCC, MK2 <italic>mmu</italic> Fwd CTTCCAAAAGGCCCAATGCC, MK2 <italic>mmu</italic> Rev GGACTTCCGGAGCCACATAG, XPA <italic>mmu</italic> Fwd ACTGCTTCTTATTGCTCGCC, and XPA <italic>mmu</italic> Rev AGCTCTGGAAGATGCAAAGG.</p></sec><sec id=\"Sec24\"><title>Immunoblot analysis</title><p id=\"Par46\">Cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitor (Roche). Protein concentration was measured using BCA (Pierce). Cell extracts containing the same amount of protein in every condition were mixed with 6× reducing sample buffer and boiled at 95 °C for 5 min, and subjected to electrophoresis using the standard sodium dodecyl sulfate polyacrylamide gel electrophoresis method. For LICOR-based blotting, proteins were transferred to nitrocellulose membranes (Biorad; Catalog# 162-0115) and blocked with Odyssey blocking buffer for 1 h. Primary antibodies were then incubated overnight at 4 °C followed by secondary antibodies conjugated with LICOR fluorophores. Samples were scanned with a LICOR/Odyssey infrared imaging system (LICOR Biosciences) and band densitometries were quantified using ImageStudio. For enhanced chemiluminescence (ECL)-based blotting, proteins were transferred to methanol-activated PVDF membranes (Biorad; Catalog# 162-0177) and blocked with 5% nonfat dried milk for 1 h. Primary antibodies were then incubated overnight at 4 °C followed by secondary antibodies conjugated with HRP for developing with ECL (Perkin Elmer). Uncropped versions of blots are included in Supplementary Figs. <xref rid=\"MOESM1\" ref-type=\"media\">10</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">11</xref>, and also provided as Source Data File.</p></sec><sec id=\"Sec25\"><title>Murine lung adenocarcinoma transplant model</title><p id=\"Par47\">KP7B cells (5 × 10<sup>4</sup>), labeled with GFP-Luciferase, were transplanted into 10- to 12-week-old syngeneic <italic>C57BL6/Jx129-JAE</italic> male recipient mice 6 h after 5 Gy whole body irradiation<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. Tumors were allowed to form for 2-3 weeks and tumor growth was measured by bioluminescent imaging. Mice were injected IP with 165 mg kg<sup>−1</sup> luciferin 10 min prior to bioluminescent imaging on an IVIS Spectrum-bioluminescent and fluorescent imaging system (Xenogen Corporation). For all imaging procedures animals were pre-anesthetized with isoflourane. For siRNA treatment, nanoplex-siRNA (200 mg kg<sup>−1</sup> nanoplexes, 1 mg kg<sup>−1</sup> siRNA) was injected twice weekly for 3 weeks. For drug treatments, cisplatin was dissolved in saline and injected IP at 15 mg kg<sup>−1</sup> for single high dose treatment, or with 7 mg kg<sup>−1</sup> once weekly for a total of three weeks for low dose treatment. Mice were sacrificed when moribund or when they had lost 20% of their initial body weight, whichever occurred sooner, according to MIT Committee on Animal Care guidelines. All mouse studies were approved by the MIT Institutional Committee for Animal Care (CAC), and conducted in compliance with the Animal Welfare Act Regulations and other federal statutes relating to animals and experiments involving animals and adheres to the principles set forth in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996 (Institutional Animal Welfare Assurance #A-3125-01).</p></sec><sec id=\"Sec26\"><title>Nanoplex formulation</title><p id=\"Par48\">Peptide amphiphiles were synthesized<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> by <italic>N</italic>-carboxy-anhydride ring-opening polymerization<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref>–<xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup> of benzyl <sc>l</sc>-aspartate NCA and subsequent deprotection or methylation. Nanoplexes containing siRNA were prepared using a modified thin-film hydration<sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup> and ethanol injection<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup> method. Peptide amphiphiles were dried under a stream of nitrogen and reconstituted with siRNA under sonication (96:2:2 mol% cation:helper:PEG; N:P = 1). The resulting mixture was then diluted using 50 v/v% ethanol:water with sonication and subsequent polyplexes were purified by dialysis against ultrapure water and diluted in isotonic saline immediately prior to injection.</p></sec><sec id=\"Sec27\"><title>DNA platination assay</title><p id=\"Par49\">KP7B cells were seeded on 6 well plate and incubated for 24 h at 37 °C. These cells were then treated with cisplatin (25 μM), and subsequently incubated for 5 h at 37 °C. Afterward, fresh medium was added, followed by an additional 12 h of incubation at 37 °C. The medium was then removed and the cells were washed twice with PBS (1 mL), harvested by trypsinization (1 mL), and washed twice with 0.5 mL PBS. Solutions containing cells were centrifuged at 400×<italic>g</italic> for 5 min at 4 °C. The cell pellet was suspended in DNAzol (1 mL, genomic DNA isolation reagent, MRC). The DNA was precipitated with ethanol (0.5 mL), washed with 75% ethanol (0.75 mL × 3), and redissolved in 1 mL of 8 mM NaOH. The DNA concentration was determined by UV–vis spectroscopy and the platinum content was quantified by graphite furnace atomic absorption.</p></sec><sec id=\"Sec28\"><title>Statistical analysis</title><p id=\"Par50\">All <italic>p</italic> values were calculated using a two-tailed student’s <italic>t</italic> test in Graphpad Prism unless otherwise specified. *, **, ***, and **** denotes <italic>p</italic> ≤ 0.05, <italic>p</italic> ≤ 0.01, <italic>p</italic> ≤ 0.001, and <italic>p</italic> ≤ 0.0001, respectively. All error bars shown in Figures indicate standard error of the mean.</p></sec><sec id=\"Sec29\"><title>Reporting summary</title><p id=\"Par51\">Further information on research design is available in the <xref rid=\"MOESM3\" ref-type=\"media\">Nature Research Reporting Summary</xref> linked to this article.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec30\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41467_2020_17958_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41467_2020_17958_MOESM2_ESM.pdf\"><caption><p>Peer Review File</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM3\"><media xlink:href=\"41467_2020_17958_MOESM3_ESM.pdf\"><caption><p>Reporting Summary</p></caption></media></supplementary-material></sec></sec></body><back><app-group><app id=\"App1\"><sec id=\"Sec31\"><title>Source data</title><p id=\"Par54\"><media position=\"anchor\" xlink:href=\"41467_2020_17958_MOESM4_ESM.xlsx\" id=\"MOESM4\"><caption><p>Source Data</p></caption></media></p></sec></app></app-group><fn-group><fn><p><bold>Peer review information</bold>\n<italic>Nature Communications</italic> thanks Dan Peer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Yi Wen Kong, Erik C. Dreaden.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary information</bold> is available for this paper at 10.1038/s41467-020-17958-z.</p></sec><ack><title>Acknowledgements</title><p>We wish to thank Drs. Ian Cannell, Mun Kyung Hwang, Karl Merrick, Leny Gocheva, and all members of the Yaffe, Hammond and Hemann laboratories for helpful discussions. We thank the Robert A. Swanson (1969) Biotechnology Center, especially the Preclinical Modeling, Imaging & Testing Facility, the Flow Cytometry Facility, the Hope Babette Tang (1983) Histology Facility, Microscopy, and the Peterson (1957) Nanotechnology Materials Core Facility at the Koch Institute/MIT. This work was supported by grants from the National Institutes of Health (R01-ES015339, R35-ES028374, R01-CA226898, and R01-GM104047 to M.B.Y., NIBIB 1F32EB017614 to E.C.D., CA034992 to S.J.L. and O.H.Y., AG045144, CA211184 to O.H.Y.), the Ovarian Cancer Research Foundation (M.B.Y. and P.T.H.), the Breast Cancer Alliance (M.B.Y. and P.T.H.), US Department of Defense Congressionally Directed Medical Research Ovarian Cancer Research Program (P.T.H.; OCRP Teal Innovator Award; W81XWH-13-1-0151), the Charles and Marjorie Holloway Foundation (M.B.Y.), the STARR Cancer Consortium (M.B.Y. and M.T.H.), the Misrock Foundation (Y.W.K.), the MIT Center for Precision Cancer Medicine (M.B.Y., M.T.H., Y.W.K., and F.C.L.), and the Mazumdar-Shaw International Oncology Fellowship (G.S.). Support was provided in part by the Koch Institute Support Grant (P30-CA14051) from the National Cancer Institute, and the MIT MRSEC Shared Experimental Facilities Grant (DMR-0819762) from the National Science Foundation.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>Conceptualization: Y.W.K., E.C.D., P.T.H., and M.B.Y.; methodology: Y.W.K., E.C.D., P.T.H., and M.B.Y.; acquisition of data, Y.W.K., E.C.D., S.M., S.S.D., J.C.P., M.Q., A.D., F.C.L., G.S., K.E.S., S.V., H.C.R., and W.Z.; analysis and interpretation of data, Y.W.K., E.C.D., J.C.P., F.C.L., G.S., P.T.H., and M.B.Y.; writing, review, and/or revision of the manuscript, Y.W.K., E.C.D., O.H.Y., S.J.L., M.T.H., P.T.H., and M.B.Y.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>All data are available from the corresponding authors upon reasonable request. Source data are provided as a Source Data file. Source data are provided with this paper.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par52\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fleuren</surname><given-names>ED</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Wu</surname><given-names>J</given-names></name><name><surname>Daly</surname><given-names>RJ</given-names></name></person-group><article-title>The kinome ‘at large’ in cancer</article-title><source>Nat. Rev. Cancer</source><year>2016</year><volume>16</volume><fpage>83</fpage><lpage>98</lpage><pub-id pub-id-type=\"pmid\">26822576</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Socinski</surname><given-names>MA</given-names></name></person-group><article-title>Clinical issues in the management of non-small-cell lung cancer and the role of platinum-based therapy</article-title><source>Clin. Lung Cancer</source><year>2004</year><volume>5</volume><fpage>274</fpage><lpage>289</lpage><pub-id pub-id-type=\"pmid\">15086966</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ciccia</surname><given-names>A</given-names></name><name><surname>Elledge</surname><given-names>SJ</given-names></name></person-group><article-title>The DNA damage response: making it safe to play with knives</article-title><source>Mol. Cell</source><year>2010</year><volume>40</volume><fpage>179</fpage><lpage>204</lpage><pub-id pub-id-type=\"pmid\">20965415</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Goldstein</surname><given-names>M</given-names></name><name><surname>Kastan</surname><given-names>MB</given-names></name></person-group><article-title>The DNA damage response: implications for tumor responses to radiation and chemotherapy</article-title><source>Annu. Rev. Med.</source><year>2015</year><volume>66</volume><fpage>129</fpage><lpage>143</lpage><pub-id pub-id-type=\"pmid\">25423595</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>D</given-names></name><name><surname>Lippard</surname><given-names>SJ</given-names></name></person-group><article-title>Cellular processing of platinum anticancer drugs</article-title><source>Nat. Rev. Drug Discov.</source><year>2005</year><volume>4</volume><fpage>307</fpage><lpage>320</lpage><pub-id pub-id-type=\"pmid\">15789122</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jamieson</surname><given-names>ER</given-names></name><name><surname>Lippard</surname><given-names>SJ</given-names></name></person-group><article-title>Structure, recognition, and processing of cisplatin-DNA adducts</article-title><source>Chem. Rev.</source><year>1999</year><volume>99</volume><fpage>2467</fpage><lpage>2498</lpage><pub-id pub-id-type=\"pmid\">11749487</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Galluzzi</surname><given-names>L</given-names></name><name><surname>Vitale</surname><given-names>I</given-names></name><name><surname>Vacchelli</surname><given-names>E</given-names></name><name><surname>Kroemer</surname><given-names>G</given-names></name></person-group><article-title>Cell death signaling and anticancer therapy</article-title><source>Front Oncol.</source><year>2011</year><volume>1</volume><fpage>5</fpage><pub-id pub-id-type=\"pmid\">22655227</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Barry</surname><given-names>MA</given-names></name><name><surname>Behnke</surname><given-names>CA</given-names></name><name><surname>Eastman</surname><given-names>A</given-names></name></person-group><article-title>Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia</article-title><source>Biochem. Pharm.</source><year>1990</year><volume>40</volume><fpage>2353</fpage><lpage>2362</lpage><pub-id pub-id-type=\"pmid\">2244936</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bouwman</surname><given-names>P</given-names></name><name><surname>Jonkers</surname><given-names>J</given-names></name></person-group><article-title>The effects of deregulated DNA damage signalling on cancer chemotherapy response and resistance</article-title><source>Nat. Rev. Cancer</source><year>2012</year><volume>12</volume><fpage>587</fpage><lpage>598</lpage><pub-id pub-id-type=\"pmid\">22918414</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oza</surname><given-names>AM</given-names></name><etal/></person-group><article-title>Olaparib combined with chemotherapy for recurrent platinum-sensitive ovarian cancer: a randomised phase 2 trial</article-title><source>Lancet Oncol.</source><year>2015</year><volume>16</volume><fpage>87</fpage><lpage>97</lpage><pub-id pub-id-type=\"pmid\">25481791</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cerami</surname><given-names>E</given-names></name><etal/></person-group><article-title>The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data</article-title><source>Cancer Discov.</source><year>2012</year><volume>2</volume><fpage>401</fpage><lpage>404</lpage><pub-id pub-id-type=\"pmid\">22588877</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gao</surname><given-names>J</given-names></name><etal/></person-group><article-title>Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal</article-title><source>Sci. Signal.</source><year>2013</year><volume>6</volume><fpage>pl1</fpage><pub-id pub-id-type=\"pmid\">23550210</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zehir</surname><given-names>A</given-names></name><etal/></person-group><article-title>Erratum: Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients</article-title><source>Nat. Med.</source><year>2017</year><volume>23</volume><fpage>1004</fpage><pub-id pub-id-type=\"pmid\">28777785</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reinhardt</surname><given-names>HC</given-names></name><name><surname>Aslanian</surname><given-names>AS</given-names></name><name><surname>Lees</surname><given-names>JA</given-names></name><name><surname>Yaffe</surname><given-names>MB</given-names></name></person-group><article-title>p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage</article-title><source>Cancer Cell</source><year>2007</year><volume>11</volume><fpage>175</fpage><lpage>189</lpage><pub-id pub-id-type=\"pmid\">17292828</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cannell</surname><given-names>IG</given-names></name><etal/></person-group><article-title>p38 MAPK/MK2-mediated induction of miR-34c following DNA damage prevents Myc-dependent DNA replication</article-title><source>Proc. Natl Acad. Sci. USA</source><year>2010</year><volume>107</volume><fpage>5375</fpage><lpage>5380</lpage><pub-id pub-id-type=\"pmid\">20212154</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reinhardt</surname><given-names>HC</given-names></name><etal/></person-group><article-title>DNA damage activates a spatially distinct late cytoplasmic cell-cycle checkpoint network controlled by MK2-mediated RNA stabilization</article-title><source>Mol. Cell</source><year>2010</year><volume>40</volume><fpage>34</fpage><lpage>49</lpage><pub-id pub-id-type=\"pmid\">20932473</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morandell</surname><given-names>S</given-names></name><etal/></person-group><article-title>A reversible gene-targeting strategy identifies synthetic lethal interactions between MK2 and p53 in the DNA damage response in vivo</article-title><source>Cell Rep.</source><year>2013</year><volume>5</volume><fpage>868</fpage><lpage>877</lpage><pub-id pub-id-type=\"pmid\">24239348</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cannell</surname><given-names>IG</given-names></name><etal/></person-group><article-title>A pleiotropic RNA-binding protein controls distinct cell cycle checkpoints to drive resistance of p53-defective tumors to chemotherapy</article-title><source>Cancer Cell</source><year>2015</year><volume>28</volume><fpage>623</fpage><lpage>637</lpage><pub-id pub-id-type=\"pmid\">26602816</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Muller</surname><given-names>PA</given-names></name><name><surname>Vousden</surname><given-names>KH</given-names></name></person-group><article-title>p53 mutations in cancer</article-title><source>Nat. Cell Biol.</source><year>2013</year><volume>15</volume><fpage>2</fpage><lpage>8</lpage><pub-id pub-id-type=\"pmid\">23263379</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kelland</surname><given-names>L</given-names></name></person-group><article-title>The resurgence of platinum-based cancer chemotherapy</article-title><source>Nat. Rev. Cancer</source><year>2007</year><volume>7</volume><fpage>573</fpage><lpage>584</lpage><pub-id pub-id-type=\"pmid\">17625587</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bowden</surname><given-names>NA</given-names></name></person-group><article-title>Nucleotide excision repair: why is it not used to predict response to platinum-based chemotherapy?</article-title><source>Cancer Lett.</source><year>2014</year><volume>346</volume><fpage>163</fpage><lpage>171</lpage><pub-id pub-id-type=\"pmid\">24462818</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Koberle</surname><given-names>B</given-names></name><name><surname>Masters</surname><given-names>JR</given-names></name><name><surname>Hartley</surname><given-names>JA</given-names></name><name><surname>Wood</surname><given-names>RD</given-names></name></person-group><article-title>Defective repair of cisplatin-induced DNA damage caused by reduced XPA protein in testicular germ cell tumours</article-title><source>Curr. Biol.</source><year>1999</year><volume>9</volume><fpage>273</fpage><lpage>276</lpage><pub-id pub-id-type=\"pmid\">10074455</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Welsh</surname><given-names>C</given-names></name><etal/></person-group><article-title>Reduced levels of XPA, ERCC1 and XPF DNA repair proteins in testis tumor cell lines</article-title><source>Int J. Cancer</source><year>2004</year><volume>110</volume><fpage>352</fpage><lpage>361</lpage><pub-id pub-id-type=\"pmid\">15095299</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shuck</surname><given-names>SC</given-names></name><name><surname>Short</surname><given-names>EA</given-names></name><name><surname>Turchi</surname><given-names>JJ</given-names></name></person-group><article-title>Eukaryotic nucleotide excision repair: from understanding mechanisms to influencing biology</article-title><source>Cell Res.</source><year>2008</year><volume>18</volume><fpage>64</fpage><lpage>72</lpage><pub-id pub-id-type=\"pmid\">18166981</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sirohi</surname><given-names>B</given-names></name><etal/></person-group><article-title>Early response to platinum-based first-line chemotherapy in non-small cell lung cancer may predict survival</article-title><source>J. Thorac. Oncol.</source><year>2007</year><volume>2</volume><fpage>735</fpage><lpage>740</lpage><pub-id pub-id-type=\"pmid\">17762340</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Deans</surname><given-names>AJ</given-names></name><name><surname>West</surname><given-names>SC</given-names></name></person-group><article-title>DNA interstrand crosslink repair and cancer</article-title><source>Nat. Rev. Cancer</source><year>2011</year><volume>11</volume><fpage>467</fpage><lpage>480</lpage><pub-id pub-id-type=\"pmid\">21701511</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Andreassen</surname><given-names>PR</given-names></name><name><surname>Ren</surname><given-names>K</given-names></name></person-group><article-title>Fanconi anemia proteins, DNA interstrand crosslink repair pathways, and cancer therapy</article-title><source>Curr. Cancer Drug Targets</source><year>2009</year><volume>9</volume><fpage>101</fpage><lpage>117</lpage><pub-id pub-id-type=\"pmid\">19200054</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wood</surname><given-names>RD</given-names></name></person-group><article-title>Mammalian nucleotide excision repair proteins and interstrand crosslink repair</article-title><source>Environ. Mol. Mutagen</source><year>2010</year><volume>51</volume><fpage>520</fpage><lpage>526</lpage><pub-id pub-id-type=\"pmid\">20658645</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scheibye-Knudsen</surname><given-names>M</given-names></name><name><surname>Croteau</surname><given-names>DL</given-names></name><name><surname>Bohr</surname><given-names>VA</given-names></name></person-group><article-title>Mitochondrial deficiency in Cockayne syndrome</article-title><source>Mech. Ageing Dev.</source><year>2013</year><volume>134</volume><fpage>275</fpage><lpage>283</lpage><pub-id pub-id-type=\"pmid\">23435289</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Niedernhofer</surname><given-names>LJ</given-names></name><name><surname>Bohr</surname><given-names>VA</given-names></name><name><surname>Sander</surname><given-names>M</given-names></name><name><surname>Kraemer</surname><given-names>KH</given-names></name></person-group><article-title>Xeroderma pigmentosum and other diseases of human premature aging and DNA repair: molecules to patients</article-title><source>Mech. Ageing Dev.</source><year>2011</year><volume>132</volume><fpage>340</fpage><lpage>347</lpage><pub-id pub-id-type=\"pmid\">21708183</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Doles</surname><given-names>J</given-names></name><etal/></person-group><article-title>Suppression of Rev3, the catalytic subunit of Pol{zeta}, sensitizes drug-resistant lung tumors to chemotherapy</article-title><source>Proc. Natl Acad. Sci. USA</source><year>2010</year><volume>107</volume><fpage>20786</fpage><lpage>20791</lpage><pub-id pub-id-type=\"pmid\">21068376</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>DuPage</surname><given-names>M</given-names></name><name><surname>Dooley</surname><given-names>AL</given-names></name><name><surname>Jacks</surname><given-names>T</given-names></name></person-group><article-title>Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase</article-title><source>Nat. Protoc.</source><year>2009</year><volume>4</volume><fpage>1064</fpage><lpage>1072</lpage><pub-id pub-id-type=\"pmid\">19561589</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bartkova</surname><given-names>J</given-names></name><etal/></person-group><article-title>DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis</article-title><source>Nature</source><year>2005</year><volume>434</volume><fpage>864</fpage><lpage>870</lpage><pub-id pub-id-type=\"pmid\">15829956</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Macheret</surname><given-names>M</given-names></name><name><surname>Halazonetis</surname><given-names>TD</given-names></name></person-group><article-title>DNA replication stress as a hallmark of cancer</article-title><source>Annu Rev. Pathol.</source><year>2015</year><volume>10</volume><fpage>425</fpage><lpage>448</lpage><pub-id pub-id-type=\"pmid\">25621662</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mourey</surname><given-names>RJ</given-names></name><etal/></person-group><article-title>A benzothiophene inhibitor of mitogen-activated protein kinase-activated protein kinase 2 inhibits tumor necrosis factor alpha production and has oral anti-inflammatory efficacy in acute and chronic models of inflammation</article-title><source>J. Pharm. Exp. Ther.</source><year>2010</year><volume>333</volume><fpage>797</fpage><lpage>807</lpage></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ronkina</surname><given-names>N</given-names></name><name><surname>Kotlyarov</surname><given-names>A</given-names></name><name><surname>Gaestel</surname><given-names>M</given-names></name></person-group><article-title>MK2 and MK3–a pair of isoenzymes?</article-title><source>Front Biosci.</source><year>2008</year><volume>13</volume><fpage>5511</fpage><lpage>5521</lpage><pub-id pub-id-type=\"pmid\">18508601</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gaestel</surname><given-names>M</given-names></name></person-group><article-title>MAPKAP kinases—MKs—two’s company, three’s a crowd</article-title><source>Nat. Rev. Mol. Cell Biol.</source><year>2006</year><volume>7</volume><fpage>120</fpage><lpage>130</lpage><pub-id pub-id-type=\"pmid\">16421520</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dreaden</surname><given-names>EC</given-names></name><etal/></person-group><article-title>RNA-Peptide nanoplexes drug DNA damage pathways in high-grade serous ovarian tumors</article-title><source>Bioeng. Transl. Med.</source><year>2018</year><volume>3</volume><fpage>26</fpage><lpage>36</lpage><pub-id pub-id-type=\"pmid\">29376131</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nyland</surname><given-names>JF</given-names></name><name><surname>Bai</surname><given-names>JJ</given-names></name><name><surname>Katz</surname><given-names>HE</given-names></name><name><surname>Silbergeld</surname><given-names>EK</given-names></name></person-group><article-title>In vitro interactions between splenocytes and dansylamide dye-embedded nanoparticles detected by flow cytometry</article-title><source>Nanomedicine</source><year>2009</year><volume>5</volume><fpage>298</fpage><lpage>304</lpage><pub-id pub-id-type=\"pmid\">19523425</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jackson</surname><given-names>EL</given-names></name><etal/></person-group><article-title>The differential effects of mutant p53 alleles on advanced murine lung cancer</article-title><source>Cancer Res.</source><year>2005</year><volume>65</volume><fpage>10280</fpage><lpage>10288</lpage><pub-id pub-id-type=\"pmid\">16288016</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sweet-Cordero</surname><given-names>A</given-names></name><etal/></person-group><article-title>An oncogenic KRAS2 expression signature identified by cross-species gene-expression analysis</article-title><source>Nat. Genet.</source><year>2005</year><volume>37</volume><fpage>48</fpage><lpage>55</lpage><pub-id pub-id-type=\"pmid\">15608639</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jandial</surname><given-names>DD</given-names></name><name><surname>Messer</surname><given-names>K</given-names></name><name><surname>Farshchi-Heydari</surname><given-names>S</given-names></name><name><surname>Pu</surname><given-names>M</given-names></name><name><surname>Howell</surname><given-names>SB</given-names></name></person-group><article-title>Tumor platinum concentration following intraperitoneal administration of cisplatin versus carboplatin in an ovarian cancer model</article-title><source>Gynecol. Oncol.</source><year>2009</year><volume>115</volume><fpage>362</fpage><lpage>366</lpage><pub-id pub-id-type=\"pmid\">19775736</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Minchinton</surname><given-names>AI</given-names></name><name><surname>Tannock</surname><given-names>IF</given-names></name></person-group><article-title>Drug penetration in solid tumours</article-title><source>Nat. Rev. Cancer</source><year>2006</year><volume>6</volume><fpage>583</fpage><lpage>592</lpage><pub-id pub-id-type=\"pmid\">16862189</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wilhelm</surname><given-names>S</given-names></name><etal/></person-group><article-title>Analysis of nanoparticle delivery to tumours</article-title><source>Nat. Rev. Mater.</source><year>2016</year><volume>1</volume><fpage>16014</fpage></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reinhardt</surname><given-names>HC</given-names></name><name><surname>Yaffe</surname><given-names>MB</given-names></name></person-group><article-title>Kinases that control the cell cycle in response to DNA damage: Chk1, Chk2, and MK2</article-title><source>Curr. Opin. Cell Biol.</source><year>2009</year><volume>21</volume><fpage>245</fpage><lpage>255</lpage><pub-id pub-id-type=\"pmid\">19230643</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sausville</surname><given-names>E</given-names></name><etal/></person-group><article-title>Phase I dose-escalation study of AZD7762, a checkpoint kinase inhibitor, in combination with gemcitabine in US patients with advanced solid tumors</article-title><source>Cancer Chemother. Pharm.</source><year>2014</year><volume>73</volume><fpage>539</fpage><lpage>549</lpage></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lam</surname><given-names>MH</given-names></name><name><surname>Liu</surname><given-names>Q</given-names></name><name><surname>Elledge</surname><given-names>SJ</given-names></name><name><surname>Rosen</surname><given-names>JM</given-names></name></person-group><article-title>Chk1 is haploinsufficient for multiple functions critical to tumor suppression</article-title><source>Cancer Cell</source><year>2004</year><volume>6</volume><fpage>45</fpage><lpage>59</lpage><pub-id pub-id-type=\"pmid\">15261141</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manke</surname><given-names>IA</given-names></name><etal/></person-group><article-title>MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation</article-title><source>Mol. Cell</source><year>2005</year><volume>17</volume><fpage>37</fpage><lpage>48</lpage><pub-id pub-id-type=\"pmid\">15629715</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coate</surname><given-names>LE</given-names></name><name><surname>John</surname><given-names>T</given-names></name><name><surname>Tsao</surname><given-names>MS</given-names></name><name><surname>Shepherd</surname><given-names>FA</given-names></name></person-group><article-title>Molecular predictive and prognostic markers in non-small-cell lung cancer</article-title><source>Lancet Oncol.</source><year>2009</year><volume>10</volume><fpage>1001</fpage><lpage>1010</lpage><pub-id pub-id-type=\"pmid\">19796752</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Spitz</surname><given-names>MR</given-names></name><name><surname>Wei</surname><given-names>Q</given-names></name><name><surname>Dong</surname><given-names>Q</given-names></name><name><surname>Amos</surname><given-names>CI</given-names></name><name><surname>Wu</surname><given-names>X</given-names></name></person-group><article-title>Genetic susceptibility to lung cancer: the role of DNA damage and repair</article-title><source>Cancer Epidemiol. Biomark. Prev.</source><year>2003</year><volume>12</volume><fpage>689</fpage><lpage>698</lpage></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuzmin</surname><given-names>E</given-names></name><etal/></person-group><article-title>Systematic analysis of complex genetic interactions</article-title><source>Science</source><year>2018</year><volume>360</volume><fpage>eaao1729</fpage><pub-id pub-id-type=\"pmid\">29674565</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Harrington</surname><given-names>KJ</given-names></name><etal/></person-group><article-title>Effective targeting of solid tumors in patients with locally advanced cancers by radiolabeled pegylated liposomes</article-title><source>Clin. Cancer Res.</source><year>2001</year><volume>7</volume><fpage>243</fpage><lpage>254</lpage><pub-id pub-id-type=\"pmid\">11234875</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Foucquier</surname><given-names>J</given-names></name><name><surname>Guedj</surname><given-names>M</given-names></name></person-group><article-title>Analysis of drug combinations: current methodological landscape</article-title><source>Pharm. Res. Perspect.</source><year>2015</year><volume>3</volume><fpage>e00149</fpage></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Engler</surname><given-names>AC</given-names></name><name><surname>Lee H-i</surname></name><name><surname>Hammond</surname><given-names>PT</given-names></name></person-group><article-title>Highly efficient “Grafting onto” a polypeptide backbone using click chemistry</article-title><source>Angew. Chem. Int. Ed.</source><year>2009</year><volume>48</volume><fpage>9334</fpage><lpage>9338</lpage></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Engler</surname><given-names>AC</given-names></name><name><surname>Bonner</surname><given-names>DK</given-names></name><name><surname>Buss</surname><given-names>HG</given-names></name><name><surname>Cheung</surname><given-names>EY</given-names></name><name><surname>Hammond</surname><given-names>PT</given-names></name></person-group><article-title>The synthetic tuning of clickable pH responsive cationic polypeptides and block copolypeptides</article-title><source>Soft Matter</source><year>2011</year><volume>7</volume><fpage>5627</fpage><lpage>5637</lpage></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhao</surname><given-names>X</given-names></name><name><surname>Poon</surname><given-names>Z</given-names></name><name><surname>Engler</surname><given-names>AC</given-names></name><name><surname>Bonner</surname><given-names>DK</given-names></name><name><surname>Hammond</surname><given-names>PT</given-names></name></person-group><article-title>Enhanced stability of polymeric micelles based on postfunctionalized poly(ethylene glycol)-b-poly(gamma-propargyl L-glutamate): the substituent effect</article-title><source>Biomacromolecules</source><year>2012</year><volume>13</volume><fpage>1315</fpage><lpage>1322</lpage><pub-id pub-id-type=\"pmid\">22376183</pub-id></element-citation></ref><ref id=\"CR57\"><label>57.</label><mixed-citation publication-type=\"other\">Bangham A. D., Hill M. W., Miller N. G. A. Preparation and use of liposomes as models of biological membranes. in <italic>Methods in Membrane Biology: Volume 1</italic> (ed Korn E. D.). (Springer, US, 1974).</mixed-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Batzri</surname><given-names>S</given-names></name><name><surname>Korn</surname><given-names>ED</given-names></name></person-group><article-title>Single bilayer liposomes prepared without sonication</article-title><source>Biochem. Biophys. Acta</source><year>1973</year><volume>298</volume><fpage>1015</fpage><lpage>1019</lpage><pub-id pub-id-type=\"pmid\">4738145</pub-id></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Trulley</surname><given-names>P</given-names></name><etal/></person-group><article-title>Alternative translation initiation generates a functionally distinct isoform of the stress-activated protein kinase MK2</article-title><source>Cell Rep.</source><year>2019</year><volume>27</volume><fpage>2859</fpage><lpage>2870.e2856</lpage><pub-id pub-id-type=\"pmid\">31167133</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Nat Commun</journal-id><journal-id journal-id-type=\"iso-abbrev\">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type=\"epub\">2041-1723</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807778</article-id><article-id pub-id-type=\"pmc\">PMC7431579</article-id><article-id pub-id-type=\"publisher-id\">17946</article-id><article-id pub-id-type=\"doi\">10.1038/s41467-020-17946-3</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Discovery of flat seismic reflections in the mantle beneath the young Juan de Fuca Plate</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-6265-0493</contrib-id><name><surname>Qin</surname><given-names>Yanfang</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-4594-801X</contrib-id><name><surname>Singh</surname><given-names>Satish C.</given-names></name><address><email>singh@ipgp.fr</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-6807-604X</contrib-id><name><surname>Grevemeyer</surname><given-names>Ingo</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Marjanović</surname><given-names>Milena</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Roger Buck</surname><given-names>W.</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.9489.c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0675 8101</institution-id><institution>Institut de Physique de Globe de Paris, </institution></institution-wrap>1 rue Jussieu, 75238 Paris, France </aff><aff id=\"Aff2\"><label>2</label>Now at Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Showa-machi 3173-25, Kanazawa-ku, Yokohama, 236-0001 Japan </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.15649.3f</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9056 9663</institution-id><institution>GEOMAR, </institution><institution>Helmholtz Centre for Ocean Research Kiel, </institution></institution-wrap>Wischhofstr 1-3, 24148 Kiel, Germany </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.21729.3f</institution-id><institution-id institution-id-type=\"ISNI\">0000000419368729</institution-id><institution>Lamont-Doherty Earth Observatory, </institution><institution>Columbia University, </institution></institution-wrap>61 Route 9W, Palisades, NY 10964-1000 USA </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>4122</elocation-id><history><date date-type=\"received\"><day>30</day><month>7</month><year>2019</year></date><date date-type=\"accepted\"><day>24</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Crustal properties of young oceanic lithosphere have been examined extensively, but the nature of the mantle lithosphere underneath remains elusive. Using a novel wide-angle seismic imaging technique, here we show the presence of two sub-horizontal reflections at ∼11 and ∼14.5 km below the seafloor over the 0.51–2.67 Ma old Juan de Fuca Plate. We find that the observed reflectors originate from 300–600-m-thick layers, with an ∼7–8% drop in P-wave velocity. They could be explained either by the presence of partially molten sills or frozen gabbroic sills. If partially molten, the shallower sill would define the base of a thin lithosphere with the constant thickness (11 km), requiring the presence of a mantle thermal anomaly extending up to 2.67 Ma. In contrast, if these reflections were frozen melt sills, they would imply the presence of thick young oceanic lithosphere (20–25 km), and extremely heterogeneous upper mantle.</p></abstract><abstract id=\"Abs2\" abstract-type=\"web-summary\"><p id=\"Par2\">Applying seismic imaging methods on ocean bottom hydrophone data, the authors here describe a horizontal, flat lithosphere base plus lithosphere-asthenosphere boundary beneath the young (0.51 to 2.67 Ma) Juan de Fuca plate.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Geophysics</kwd><kwd>Tectonics</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/100010663</institution-id><institution>EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)</institution></institution-wrap></funding-source><award-id>339442_TransAtlanticILAB</award-id><principal-award-recipient><name><surname>Singh</surname><given-names>Satish C.</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par3\">The plate tectonic theory requires the presence of a rigid lithosphere floating over a ductile asthenosphere. In the oceanic domain, the plate-cooling model suggests that the lithosphere should be thin near the ridge axis and thicken as a plate moves away from the ridge axis<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. However, as the crustal accretion<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup> and active hydrothermal circulation processes occurring near the ridge axis modify the thermal regime significantly<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>, the thickness of the lithosphere of a young oceanic plate should be higher than that predicted by the plate cooling model<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>.</p><p id=\"Par4\">The thickness of the lithosphere has been traditionally estimated using surface wave studies<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>–<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup> but as long period (>20 s) surface waves with wavelengths >80–100 km<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup> are generally used, imaging the base of a young, thin lithosphere has proven challenging<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>.</p><p id=\"Par5\">The lithosphere–asthenosphere boundary (LAB) has also been imaged using receiver function methods, indicating a sharp S-wave velocity contrast at the base of the lithosphere<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>–<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. A single ocean-bottom seismometer (OBS) receiver function study near the Juan de Fuca Ridge indicated the presence of an 8–15-km-thick high-velocity mantle over a low-velocity layer, suggesting that lithosphere beneath the OBS could be 14–21-km thick<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. However, in order explain the receiver function signal, a 26–30% of anisotropy in 8–15-km-thick layer was required, requiring unacceptably large velocity variations (e.g. a P-wave velocity 8.0 ± 1.2 km s<sup>−1</sup>) below the Moho. Another receiver function study covering 0–8 Ma of the Juan de Fuca (JdF) Plate provided a phase converted S-wave sub-horizontal image at ∼30-km depth beneath the sea surface. However, this receiver function image spans in a wide depth range (±10 km)<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>, indicating that the receiver function imaging methods have a limited resolution to precisely decipher the thickness of an young lithosphere.</p><p id=\"Par6\">Recently, seismic reflection methods have been used to image the LAB for the old oceanic lithosphere with much more precision<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>, indicating that the LAB is also associated with a sharp P-wave velocity contrast. These results indicated two reflections, associated with the top and bottom of the LAB, arguing that the LAB represents a melt channel at the base of the oceanic lithosphere. A magnetotelluric study conducted in the Middle America Trench imaged a thin, high conductivity anomaly, which is attributed to the presence of a partial melt channel, corroborating the results of the controlled source seismic studies<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. In addition, a wide-angle seismic method revealed mid-lithospheric discontinuities between 37 and 59 km depth over the 128–148 Ma old Pacific Plate, which are interpreted as frozen melt sills<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. However, the nature of the LAB near the ridge axis for a young oceanic plate remains largely unidentified. Here, we present the P-wave image of a young (0.51–2.67 Ma) oceanic lithosphere using a novel approach of wide-angle seismic reflection imaging.</p><p id=\"Par7\">Our study area lies on the JdF Plate, covering the 0.51–2.67 Ma old oceanic crust formed along the Endeavour segment of the intermediate-spreading JdF Ridge (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). This ∼90-km-long ridge segment is bounded by the Endeavour–West Valley overlapping spreading centre (OSC) in the north and the Cobb OSC in the south. On the western flank, this segment is characterized by the prominent Heckle seamount chain (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>), resulting from the presence of a small-scale mantle thermal anomaly and the north-westward advance of the JdF Ridge (30 mm yr<sup>−1</sup>) in the fixed hotspot reference frame<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>–<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. An enhanced crustal production since 0.71 Myr and the presence of an ∼40-km wide and 300-m high plateau are linked to this mantle thermal anomaly<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. In contrast to the seamount dominated west flank, the seafloor on the east flank is flat and covered with thick sediments<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>.<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Study area and the young Juan de Fuca (JdF) Plate.</title><p><bold>a</bold> Regional map of the JdF Plate and Cascadia subduction zone. The inset globe shows the global location of the area marked with the yellow star. Our study area is outlined by a black box (close-up is shown in panel <bold>b</bold>). The locations of the seismic reflection profiles collected in 2002 (refs. <sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>) crossing the Endeavour and Northern Symmetric segments are shown in white lines; the locations of the reflection seismic profiles collected in 2012<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup> are shown in white-black lines. Coloured areas bounded by black lines represent the magnetic anomalies with the corresponding age shown in the time scale below the panel<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. Grey shaded zones outline the extent of the propagator wakes<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. The red dots represent epicentres along plate boundaries (June 1995–June 2019, United States Advanced National Seismic System and Canadian National Seismic Network catalogues). <bold>b</bold> The close-up of the study area. Around the ridge axis, dashed and grey transparent contours outline zones with lower (∼0%) and higher (3%) melt fraction at 7.8-km depth<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. The remaining symbols are shown in Legend.</p></caption><graphic xlink:href=\"41467_2020_17946_Fig1_HTML\" id=\"d30e427\"/></fig></p><p id=\"Par8\">The tectonic history of the Endeavour segment is dominated by several episodes of ridge propagation starting from the northward propagation of the Cobb OSC at ∼4.5 Myr ago<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. Coincident with the onset of the activity of the Heckle melt anomaly ∼2 Myr, the Cobb OSC propagated southward for about 35 km<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. For young crustal ages (<200 ka), a small eastward jump of the Endeavour segment has been indicated due to the readjustment of the plate boundary, and the influence of the Heckle melt anomaly<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Finally, numerical modelling of the tectonic fabric suggested that the northward propagation of the Cobb OSC has been restored in the last 100 ka<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. This change in the direction of the ridge propagation could be associated with the diminished melt supply to the ridge axis due to the transfer of the Heckle melt anomaly to the east flank<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Recently, active-source seismic studies showed that there is a lateral offset between the mantle and the crustal magmatic systems<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup> (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref>), which is attributed to the difference in mechanisms of heat transfer that operate in the crust (advection) and mantle (conduction)<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. Interestingly, the most prominent low-velocity zones lie directly beneath the OSCs. In addition to the tectono-magmatic processes, the Endeavour segment is highly influenced by hydrothermal processes. The rift valley in the central part of the Endeavour segment hosts five long-lived vent fields underlain by the existence of intra-crustal melt lenses at ∼2.6 km below the seafloor<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>.</p><p id=\"Par9\">Four ridge-parallel wide-angle seismic profiles (RFR96-01, -03, -05, and -08) were acquired aboard R/V Sonne in 1996 (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref>). Along each of the profiles, six ocean-bottom hydrophones (OBHs) were deployed at 4 km interval. The details of the acquisition parameters are given in the “Methods” section. The OBH data contain strong crustal arrivals (Pg) and reflections from the Mohorovičić discontinuity—Moho (PmP). In addition, we observe wide-angle reflections that seem to originate in the mantle at a source-receiver offset range of 7–20 km, ~1–2 s after Pg arrivals (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>).<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Wide-angle seismic data recorded on OBH 502 along profile RFR96-01.</title><p><bold>a</bold> The primary seismic phases are direct water wave (P<sub>0</sub>, magenta), crustal P-wave refractions (Pg, green) and crust-mantle boundary (Moho) P-wave reflections (PmP, orange). The presence of multiples is shown in a dashed green line. The mantle reflection events, the focus of this study, are marked in blue and red arrows. In green arrows, we mark P-to-S converted energy. The black boxes (<bold>b</bold>) and (<bold>c</bold>) show the close-up regions on the right focusing on the mantle reflection events (blue and red arrows). Yellow filled red curves indicate seismic waveforms for Pg and mantle reflected waves demonstrating the polarities of the mantle reflections. The bottom panel shows seafloor bathymetry along the line with the location of OBH indicated.</p></caption><graphic xlink:href=\"41467_2020_17946_Fig2_HTML\" id=\"d30e501\"/></fig></p><p id=\"Par10\">We apply an advanced imaging technique to these wide-angle reflection data. We find two sub-horizontal reflections at ∼11 and 14.5 km below the seafloor in the mantle over 0.5–2.67 Ma JdF Plate and suggest that they could either represent the LAB or frozen melt sills in the lithospheric mantle, requiring the presence of steady-state melt sills beneath the ridge axis.</p></sec><sec id=\"Sec2\" sec-type=\"results\"><title>Results</title><sec id=\"Sec3\"><title>Velocity model</title><p id=\"Par11\">For constraining the crustal thickness and velocity structure sampled by the profiles, we first performed ray-based travel time tomography of Pg and PmP arrivals (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>; see the “Methods” section). The observed crustal thickness is consistent with that observed along the orthogonal seismic reflection profile<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>,<xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. In the absence of any velocity constraints from the mantle turning ray arrivals (Pn), we used a gradually increasing age (temperature) dependent one-dimensional velocity function below the Moho. The velocity just below the Moho varied from 7.65 to 7.8 km s<sup>−1</sup> for the youngest to the oldest profile, respectively (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). We then employed travel time tomography to estimate the depth of the mantle reflections (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). A synthetic seismogram modelling tests (Methods section) indicates that these arrivals are not multiples or artefacts, but real reflection arrivals from the mantle (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>).</p></sec><sec id=\"Sec4\"><title>Wide-angle seismic image</title><p id=\"Par12\">To obtain a seismic reflection equivalent image, we performed a pre-stack depth migration<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup> of the reflected part of the wide-angle seismic data by using the above velocity models (Methods section; Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). Prior to the migration the data were downward continued near the seafloor (only the source-side), so that both sources and receivers are at approximately the same depth. A wave equation datuming method was used for the downward continuation<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. To avoid migration artefacts, the Pg and P-to-S converted waves, and water bottom multiples were muted. A Kirchhoff pre-stack depth migration technique<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup> was applied to the resulting downward continued OBH data to map the seismic reflection events to the appropriate depth and distance locations in the sub-surface. The velocity model obtained by the tomography for profile RFR96-01 was used to compute the Green’s function for the migration. The final migrated gathers were summed, and the seismic images are displayed in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>. In order to make sure that our pre-stack depth migration procedure is accurate, we performed a pre-stack depth migration of the synthetic data after performing all the pre-processing steps that were applied to the real data. The final image shows that the synthetic pre-stack depth migrated image contains Moho and two mantle reflections accurately (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6</xref>). To verify that the migrated images are consistent with reflection arrivals, we also present the post-stack time migrated image (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">7</xref>). The similarities between the pre-stack and post-stack migrated images confirm that they are real images originating from the mantle. The depth obtained using travel time modelling of these reflections is consistent with the migrated images (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">8</xref>).<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Pre-stack depth migrated images of the OBH profiles.</title><p>Prominent mantle reflections are indicated in blue (shallower) and red (deeper) arrows. <bold>a</bold> Profile RFR96-05 sampling crustal age ∼0.9 Ma is shown. <bold>b</bold> Profile RFR96-03, sampling crustal age of ∼1.65 Ma on average (please note that the line is crossing a propagator’s wake spanning crustal ages from 0.78 to 1.86 Ma). <bold>c</bold> Profile RFR96-01 sampling almost uniform crustal age ∼2.67 Ma. The blue and red arrows mark the locations of the two reflectors. The OBH instruments, numbered (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>), are indicated at the top of each panel. For profile RFR96-08 that samples ∼0.51 Ma old crust, weak and interrupted events in the mantle are observed (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>).</p></caption><graphic xlink:href=\"41467_2020_17946_Fig3_HTML\" id=\"d30e605\"/></fig></p><p id=\"Par13\">Uncertainties in the depth of these reflections are primarily due to the uncertainties in the mantle velocity structure. Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">8</xref> indicates that the upper reflection lies at 11 ± 0.5 km and the lower reflection at 14.5 ± 0.5 km. To assess uncertainty in the velocity model, we performed a pre-stack depth migration of OBH data along profile RFR96-01 using three different velocity models: (1) lower limit of the tomography velocity model, (2) the tomographic velocity model and (3) upper limit of the tomographic velocity model. Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">9</xref> shows that the image obtained using the lower velocity model is similar to that with the tomographic velocity model, but the higher velocity model produces a lower quality image. The best image is obtained using the tomographic velocity model, suggesting that the velocity model used for the migration is satisfactory, and the image is a truthful representation of the sub-surface.</p><p id=\"Par14\">It should be noted that as the downward continued data were muted to remove the crustal turning rays, i.e., Pg and converted S-wave energy (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">7</xref>), the first 8 km of the migrated signals are reverberations and are not interpreted.</p></sec><sec id=\"Sec5\"><title>Deep mantle reflections</title><p id=\"Par15\">The depth migrated seismic images consistently show a bright reflection at an average 10.5 km below the seafloor for line RFR96-01 over the 2.67 Ma old crust (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>). The other profiles show similar features, with small depth variations. For profile RFR96-05, the top reflection varies from about 10.2 to 11.5 km from north to south, while in the other two profiles, this event occurs at around 10.5 km. Overall, the top reflection is observed along all profiles; it can be followed for >20–25 km along each line and for ∼65 km across the ridge axis approximately at a depth of 11 ± 0.5 km below the seafloor (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">8</xref>).</p><p id=\"Par16\">At about 3.5 km below this first reflector, i.e., at 14.5 ± 0.5 km below the seafloor, a second reflection can be observed along the older profiles for the 1.65 and 2.67 Ma old lithosphere (RFR96-01, RFR96-03, respectively). One could also see small segments of the deeper reflections in profiles RFR96-05 and RFR96-08 that sample ∼0.9 and 0.51 Ma old lithosphere, respectively (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref>, Supplementary Figs. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">7</xref>). In addition, there are several small reflections in between these reflections that can be identified in all of the profiles.</p></sec><sec id=\"Sec6\"><title>Nature of the mantle reflections</title><p id=\"Par17\">The quantitative nature of these mantle reflections could be obtained using a combination of (a) polarity analysis of the seismic signal, (b) synthetic seismogram modelling and (c) amplitude versus offset analysis. Although the low signal-to-noise ratio present in the data and the interference of different arrivals, the signal of the top reflection shows a reversed polarity with respect to the Pg arrivals (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a</xref>) at certain locations, suggesting that it could be produced by a decrease in velocity at an interface.</p><p id=\"Par18\">The observed wide-angle reflections are limited in the 7 and 20 km source-receiver offset range. As we do not observe any large amplitude critical angle reflections, these reflections cannot originate from thick high-velocity layers. However, they could be produced either by thin or thick, low-velocity layers or thin, high-velocity layers. To assess the different possibilities, we carried out synthetic seismogram modelling for a series of one-dimensional models by varying both thickness and velocity of a layer embedded in the mantle peridotite. Previous studies indicated that the mantle reflections are produced by a 6–8.5% change in the P-wave velocity<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>–<xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>, and therefore, we used ±7% as an average value. We found that an increase in velocity of +7% in a 1200-m-thick layer would produce two reflection arrivals, one from the top and the other from the bottom of each layer. The pair of top and bottom reflectors is not observed in the data (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">10</xref>). However, a 300-m-thick high velocity does produce reflections that resemble the observed data, but its polarity is positive (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4F</xref>). We then tested a −7% decrease in velocity, where the thickness of the layers varied from 300 to 1200 m (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">11</xref>). For the layers thicker than 600 m, there would be two reflection arrivals originating from the top and the bottom of the layer, suggesting that the layer thickness should be around 300 m. Finally, we compared the modelling results obtained by varying the velocity in a 300-m-thick layer by −7% (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">11c</xref>), −15% (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4d</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">12a</xref>) and −30% (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4e</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">12b</xref>). A −7% decrease in velocity could either correspond to the presence of a frozen gabbroic sill<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup> or partially molten sill<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup> within mantle peridotite, whereas a −30% decrease could be due to the presence of a large amount of melt<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. We find that a decrease in velocity of −30% in a 300-m-thick layer would produce a strong reflection that is not observed in our data. We also tested the presence of a velocity gradient layer, where the velocity first decreases and then increases in a 600-m-thick layer, and the results are shown in Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">13</xref>. The observed reversed polarity of the event, combined with the modelling results, lead us to suggest that the first mantle reflection is related to a negative velocity contrast, or a negative velocity gradient within a thin zone (<1/4 of wavelength). Similarly, the deeper reflections require the presence of a thin, low-velocity layer. Four different models and their associated synthetic seismograms are shown in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>.<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>Synthetic seismograms.</title><p><bold>a</bold> The observed data recorded by the instrument OBH 501, <bold>b</bold> a template for 1D velocity model where the upper mantle layer is 300-m thick with a velocity decrease of −7% and a 1200-m-thick lower layer with an increase in velocity of +7%. The black rectangle indicates the blow of the velocities shown in subsequent panels. <bold>c</bold> Synthetic seismograms for the velocity model shown in (<bold>b</bold>). <bold>d</bold> Synthetic seismogram for velocity in the upper layer of −15%, <bold>e</bold> −30% and <bold>f</bold> +7%.</p></caption><graphic xlink:href=\"41467_2020_17946_Fig4_HTML\" id=\"d30e748\"/></fig></p><p id=\"Par19\">A relatively poor signal-to-noise ratio does not permit to carry out thorough amplitude versus offset analysis, but we did compute the relative amplitude versus offset variations for the upper reflection (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>) and those for the best-fitting synthetic models (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>). The relative amplitude decreases very slowly with offset (from −1 to −0.95) for a velocity decrease of −30% in a 300-m-thick layer in the 8.5–17 km offset range whereas that more rapidly for −7% (from −1 to −0.65). An increase in the velocity of +7% produces a similar relative amplitude with offset, but it varies from +1 to +0.6 (i.e., the polarity does not match the observations), and therefore we can rule out an increase in velocity in the 300-m-thick layer. Taken together, a 300-m-thick layer with a decrease in velocity of −7% fits best the observed amplitude versus offset (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>).<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>Relative amplitude versus offset variation.</title><p><bold>a</bold> Observed OBH 501 gather with a reduced velocity of 6 km/s highlighting the part of reflection event (black arrows) used for the amplitude versus offset (AVO) analyses. <bold>b</bold> Normal moveout corrected gather used for the AVO study. Black arrows highlight the mantle reflection. <bold>c</bold> Relative amplitude versus offset curves for real data (thick black) and synthetic data with different velocities (−7, −15, −30, +7%) in a 300-m-thick layer for the upper layer. Note that for the increase in velocity, the variation is from +1 to +0.5 and for the decrease in velocity the variation is from −1 to −0.5.</p></caption><graphic xlink:href=\"41467_2020_17946_Fig5_HTML\" id=\"d30e778\"/></fig></p></sec><sec id=\"Sec7\"><title>Frozen melt or partial melt sills</title><p id=\"Par20\">The characteristics of the wide-angle reflection events, together with the synthetic test we conducted, suggest that the observed events could be either produced by the presence of frozen or partially molten sills in the mantle. A frozen gabbroic sill at 11–14-km depth would have a P-wave velocity of ∼7 km s<sup>−1</sup>, whereas the surrounding mantle peridotite would have a P-wave velocity of ∼7.6–7.8 km s<sup>−1</sup>, producing a velocity contrast of −8–10%, consistent with our observations. Frozen gabbroic sills have been suggested to occur in at mid-lithospheric depths (39–59 km) for older lithosphere (128–148 Ma) in the Pacific Ocean<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>.</p><p id=\"Par21\">On the other hand, a melt channel has been proposed to occur at the base of 40–70-Ma old lithosphere in the Atlantic Ocean at 72–88-km depth<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. The top of the melt channel, marked by a reflection with negative velocity contrast (−8.5%), is associated with 1260 °C isotherm, and is considered as a freezing boundary with solid lithosphere above and partially molten channel below. The bottom of the melt channel, defined by a deeper reflection with positive velocity contrast (+8.5%), corresponds to 1355 °C, and marks the top of the mantle asthenosphere. Although the top of the melt channel (base of the lithosphere) deepens with age following the 1260 °C isotherm, the thickness of the channel decreases with age, from 17 km at 40 Ma to 11 km at 70 Ma, indicating that the thickness of the melt channel should thicken towards the ridge axis<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. One possibility is that the base of melt channel might correspond to base of the melting zone near the ridge axis, where the temperature must be higher<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. Furthermore, based on these two positive/negative velocity contrasts, it was suggested that an average of 1.4% of melt might be present in the melt channel<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. However, given the limitation of seismic reflection technique, it would be difficult to say with confidence if the top reflection corresponds to a thin melt sill or a thick melt channel. Therefore, the top reflection we image might be a partially molten sill with ∼1% of melt at the base of the lithosphere and the deeper reflections might be partially molten sills within the melt channel as the base of melt channel would be much deeper (>70 km).</p><p id=\"Par22\">In conclusion, these reflections could either be partially molten or frozen sills, depending upon the temperature. As the solidus temperature of basaltic melt is ∼1200 °C<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>, we could use the 1200 °C isotherm to demarcate the boundary between the solid lithosphere above the melt channel.</p></sec></sec><sec id=\"Sec8\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par23\">Thermal regime near a ridge axis is influenced by several factors that include (a) asthenosphere upwelling beneath the ridge axis, (b) mantle flow stresses due to plate spreading, (c) melt segregation and associated mantle compaction, (d) horizontal extensional stresses in thickening lithosphere<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup> and (e) hydrothermal circulation<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. All these effects would tend to steepen the isotherms in the vicinity of the ridge axis and flatten them off axis, leading to a thick and nearly sub-horizontal base of the lithosphere near the ridge axis. As we move far away from the ridge, vertical thermal conduction will dominate, and the isotherm would follow the plate-cooling model and the lithosphere will thicken with age. The presence of nearly flat reflectors at 11 ± 0.5 and 14.5 ± 0.5 km below the seafloor spanning the lithosphere ages from 0.51 to 2.67 Ma seems to indicate that the ridge axis processes control the thermal structure up to at least 2.67 Ma.</p><p id=\"Par24\">On the intermediate-spreading JdF Ridge, an axial melt lens is observed at ∼2–3 km below the seafloor<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>, where the temperature should be ∼1200 °C, and hence the lithosphere just beneath the ridge axis would be 2–3-km thick. For purely a plate-cooling model (see the Methods section), the 1200 °C isotherm will cross the upper reflector at 1.25 Ma (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6a</xref>). On the other hand, if we take hydrothermal circulation into consideration<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup> (see the Methods section) the 1200 °C will be deeper, and the upper reflector will lie between 800 and 900 °C isotherms, and the lower reflector between 900 and 1200 °C isotherms (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6b</xref>). So there are two possibilities:<list list-type=\"order\"><list-item><p id=\"Par25\">The top reflection at 11 ± 0.5 km represents the base of the lithosphere, and the other deeper reflections could be melt sills within the melt channel (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6a</xref>), where shallowing and flattening of the lithosphere could be due to factors like mantle thermal anomaly, volatiles and sediment blanketing.</p></list-item><list-item><p id=\"Par26\">These reflections are produced by frozen melt sills in the lithospheric mantle, and the base of the lithosphere is deeper corresponding the 1200 °C isotherm, resulting in a thick young lithosphere (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6b</xref>).</p></list-item></list><fig id=\"Fig6\"><label>Fig. 6</label><caption><title>Thin lithosphere with flat lithosphere–asthenosphere boundary (LAB) versus thick lithosphere.</title><p>A schematic diagram showing two different models of the LAB. <bold>a</bold> Thin lithosphere and flat LAB: The top reflector imaged in the seismic sections (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>) is interpreted as the top of the LAB (blue). The deeper reflectors are interpreted as a presence of melt pockets (red ellipses). Pre-stack migrated traces at the crossing of three seismic profiles with the ridge perpendicular seismic reflection profile (black-white dashed line in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>) are shown. The red dotted lines represent isotherms calculated using the plate-cooling model<sup><xref ref-type=\"bibr\" rid=\"CR62\">62</xref></sup>. Bathymetry is shown at an angle to add ‘depth’ to the two-dimensional illustration. The yellow triangles indicate hydrothermal vent fields. <bold>b</bold> Thick and heterogeneous lithosphere: The isotherms are represented by grey dashed lines obtained by the approach<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup> described in Methods. The thick blue line marks the base of the thick and heterogeneous lithosphere. The red dashed curve is the 1200 °C isotherm based on the plate-cooling model in (<bold>a</bold>).</p></caption><graphic xlink:href=\"41467_2020_17946_Fig6_HTML\" id=\"d30e900\"/></fig></p><p id=\"Par27\">The thinning of the lithosphere due to thermal mantle anomalies have been observed in the Pacific and Atlantic Oceans due to the presence Hawaiian Plume<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup> and Cameroon Plume<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>, respectively. In our study, the existence of several seamount chains on the west flank<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>–<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>, the skewed N–S mantle low-velocity anomaly connecting the two OSCs<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup> and the axis-centred 40-km-wide plateau associated with anomalous crustal thickening<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup> indicate the presence of thermal anomaly near the ridge axis. These observations, and in particular the latter two suggest that this thermal anomaly may extend farther eastward from the ridge axis, and the flat reflection we observed up to 2.67 Ma old lithosphere might be due to this extended thermal anomaly. A subsidence analysis due to sediment loading using information from reflection seismic data<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup> was done for the transect that crosses our survey area (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>). It indicates an excess basement uplift of about 200 m extending up to 140 km (up to 3.7 Ma) from the ridge axis (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">14</xref>), which may indicate the presence of this thermal anomaly with a temperature ∼30 °C higher than the surrounding mantle (assuming ∼100-km-thick sub-plate asthenospheric channel and using the equation for estimating the regional uplift<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>). Therefore, a possible mantle thermal anomaly could be produced by a combination of hotspots on the Pacific Plate and north-westward migration of the JdF Ridge<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>.</p><p id=\"Par28\">In order to explain the presence of melt channel below the lithosphere at 1260 °C in the Atlantic Ocean, the existence of water was invoked to reduce the solidus temperature<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. If water is the main volatile in the melt channel at the base the lithosphere, for the upper reflections at ∼11.0-km depth (∼13.6 km below sea surface) at 1.5 and 2.5 Ma, a water content of 25 and 700 parts per million (ppm) would be required to reduce the solidus temperature, respectively (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">15</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>. As the deeper reflection at 14.5-km depth is close to the 1200 °C isotherm, only at 2.5 Ma, some water (∼100 ppm) would be required to maintain molten sills at these depths (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">15</xref>).</p><p id=\"Par29\">A significant part of the Endeavour segment and the young JdF Plate are covered with low-permeable terrigenous sediments<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>, which can act as a thermal blanket, effectively limiting the hydrothermal circulation, and changing the thermal structure of the underlying lithosphere<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup>. Drilling results from our study area<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup> (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref>) suggest that the basement temperature is up to 60 °C higher with respect to a non-sedimented young oceanic lithosphere. The presence of sediments would have tendency to flatten the isotherm away from the ridge axis. Taken together, these factors could produce a thin lithosphere and a flat base of the lithosphere.</p><p id=\"Par30\">The S-wave receiver function results show the presence of a sub-horizontal converted S-wave receiver function image at ∼30 km below the sea surface<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup> underlying the JdF Plate between 0 and 8 Ma old lithosphere, suggesting that the flat base of the lithosphere might be a ubiquitous feature in this region. Furthermore, a S-wave tomography study suggests that the S-wave velocity is higher than expected for a plate-cooling model, indicating the presence of a colder and thicker lithosphere<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. Assuming an S-wave velocity contour of 4.4 km s<sup>−1</sup> for the base of the lithosphere at 48°N on the JdF Plate, tomographic results indicated that the lithosphere should be ∼28-km thick near the ridge axis, increasing rapidly to ∼38 km thickness farther from the ridge axis and then remain constant up to the subduction front<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>, further supporting the idea of a flat but thick lithosphere. An excessively thick young ocean lithosphere has also been detected in a magnetotelluric study east of the Mohns Ridge in the northern Atlantic Ocean<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>.</p><p id=\"Par31\">The presence of the extended frozen melt sills over a 25 km by 65 km area in a 0.5–2.65 Ma old lithosphere requires the formation of these sills to be very close to the ridge axis. As the thermal gradient near the ridge axis is very high (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6b</xref>), melt sills present at 11 ± 0.5 km, and 14.5 ± 0.5 km depth beneath the ridge axis could freeze across the 1200 °C isotherm very rapidly, forming extended frozen melt sills in the upper lithosphere. Recent studies have suggested that the LAB is controlled by an impermeable boundary related to melt crystallization preventing melt migrating upward, representing a freezing boundary<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR52\">52</xref>,<xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. Therefore, in order to have frozen melt sills extending over 25 km along the axis and more than 65 km away from the ridge axis, the molten melt sills must be in a steady state at 11 ± 0.5 and 14.5 ± 0.5 km depths along a significant part of the spreading centre.</p><p id=\"Par32\">The presence of frozen basaltic melt sills would make the mantle lithosphere chemically heterogeneous. The chemical heterogeneity of the mid-ocean ridge basalt (MORB) has been known for a long time<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>. It has been suggested that about 10% of recycled MORB are present in the bulk mantle. Furthermore, the mantle source comprises a small amount of (10%) eclogites or pyroxenite hosted in ambient peridotite mantle<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup> that would be more fusible than the host peridotite. The heterogeneous mantle rich in fusible components will melt easily and will create a magmatic channel that could extend up to the base of the lithosphere and freeze as the lithosphere cools<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>, forming the frozen melt sills we observe.</p><p id=\"Par33\">Surface wave tomography results indicate the S-wave velocity is higher than normal east of the JdF Ridge, indicating the absence of a thermal anomaly in our study area<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. Furthermore, a recent high-resolution surface wave study<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup> indicates a 28-km-thick lithosphere near the ridge axis. Taken together, all these results support the idea that the reflections we have observed are frozen melt sills, and the lithosphere is heterogeneous and thick. If oceanic lithosphere is heterogeneous, one should be able to image frozen melt sills elsewhere in ocean basins using existing wide-angle data.</p><p id=\"Par34\">However, the presence of a thermal anomaly in the mantle and a thin lithosphere could not be ruled out. Surface wave tomography results west of the JdF Ridge show a low S-wave velocity anomaly, indicating the presence of thermal anomaly in the mantle, supported by the existence of the chain of seamounts. The thickened crust near the ridge axis and the high attenuation near the ridge axis<sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>, support the idea of the mantle thermal anomaly.</p><p id=\"Par35\">The thermal evolution of thin and thick lithosphere with age would be very different, and especially their roles in the subduction process at the Cascadia subduction zone. A thin and warm slab would be buoyant, and hence would lead to a shallow dipping slab, confirmed by the low dip (10–12°) of the subducting plate observed along the Washington segment of the Cascadia subduction<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>. In contrast, a thick heterogeneous lithosphere would be cold and hence would subduct easily.</p></sec><sec id=\"Sec9\"><title>Methods</title><sec id=\"Sec10\"><title>Seismic data acquisition</title><p id=\"Par36\">Twenty-four ocean-bottom hydrophones (OBH) were deployed along four ridge-parallel profiles. Along each profile, six OBHs were deployed at a 4-km interval over 20-km distance (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). OBH525 did not provide any data along profile RER96-05, and OBH534 was offline of profile RER96-08, and hence was not used in the data analyses. The shots were fired along ~50-km long profiles, providing maximum offsets of ∼35 km (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref>).</p><p id=\"Par37\">Except for profile RFR96-05, the data were acquired using a single BOLT Inc. airgun of 32 l, fired at 140 bar, towed at 15-m water depth. The dominant frequency of the source was 6 Hz. For profile RFR96-05, a PS100 Sleeve gun (60 l) was used instead. The shot spacing was 40 s (equivalent to ∼90 m). OBH 501–506 were deployed along profile RFR96-01, OBH 511–516 along profile 96-03, OBH 521–526 along profile 96-05 and OBH 531–536 along profile 96-08.</p><p id=\"Par38\">The data were band-pass filtered between 2 and 15 Hz. A predictive deconvolution was performed to enhance the deep crustal and mantle arrivals. Strong crustal arrivals (Pg) are observed to an offset of 22.5 km, which are followed by wide-angle reflections from the Moho (PmP). As the maximum offset is 35 km, no mantle arrivals (Pn) are observed in the data. Representative examples of OBH data are shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>. Apart from the crustal arrivals, one can also observe wide-angle reflection arrivals, originating within the mantle on most of the OBH gathers, on both sides of the OBH. This reflection generally cuts across P-to-S converted energy.</p></sec><sec id=\"Sec11\"><title>Tomography</title><p id=\"Par39\">Travel times of first arrival P-waves have been hand-picked with picking uncertainties of 20–30 ms for short-offset P-waves (Pg) and 40–60 ms for secondary wide-angle reflected arrivals (PmP and sub-Moho mantle reflection). We applied a joint first arrival refraction and reflection tomography using a hybrid ray-tracing scheme combining the graph method with further refinements utilizing ray bending with a local conjugate gradients method for inversion<sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup>. Smoothing and damping constraints regularize the iterative inversion.</p><p id=\"Par40\">For the starting model, we used a 1D velocity for the oceanic crust characterizing the area<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup> and hung it below the basement defined by coincident seismic reflection data<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref>,<xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup>. The inversion was carried out using a top-down approach, inverting first for the shallow structure and adding additional arrivals while inverting for crustal thickness and velocity structure. Later, we inverted for the depth to the upper mantle reflector. In general, the inversion results provided smooth velocity models with a root-mean square misfit of 35–50 ms.</p><p id=\"Par41\">We carried out a nonlinear Monte Carlo type error analysis to derive model uncertainties<sup><xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup>. The mean of the model gives the average velocity and the variance deviation from the average. The best-fitting model is shown in Supplementary Figs <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>, with the uncertainty and derivative weight sum. The final model had root-mean-square misfits of 40–60 ms and Chi-square <1.</p><p id=\"Par42\">In addition, we performed tomographic inversion to fit the deep-seated wide-angle reflections, as shown in Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>. Here, we assumed that the upper mantle velocity increases with age as has been observed along the East Pacific Rise<sup><xref ref-type=\"bibr\" rid=\"CR62\">62</xref></sup> and included the mantle discontinuity as a floating reflector, inverting the geometry and depth of the reflector.</p></sec><sec id=\"Sec12\"><title>Pre-stack depth migration</title><p id=\"Par43\">The pre-processing of the data includes a predictive deconvolution and band-pass filtering (2–15 Hz), followed by a spherical divergence compensation. The OBH geometry places the shots and receivers on a different datum. To relocate the shots and have them at approximately the same datum level as the receivers, we applied a wave equation datuming<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR34\">34</xref>,<xref ref-type=\"bibr\" rid=\"CR63\">63</xref>,<xref ref-type=\"bibr\" rid=\"CR64\">64</xref></sup> to a constant depth, which is the shallowest point of the seafloor depth along a profile. Finally, to compensate for the small elevation differences between sources and receivers, we applied static corrections.</p><p id=\"Par44\">The spatial aliasing caused by the downward continuation was removed by dip filtering (5–6°). Similarly, the Pg and P–S-converted waves were removed as they could affect the migration of mantle reflection signals. The water bottom multiples were also muted. The resulting receiver gathers were migrated using the Kirchhoff pre-stack depth migration technique<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. Travel times for the migration were calculated by ray tracing through the velocity model obtained by the tomography for profile RFR96-01. The velocity model was sampled on a 30 m × 30 m grid for the migration. The final migrated gathers were summed, and the seismic images are displayed in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>. To make sure that the migrated images are real, we also stacked the normal moveout (NMO) corrected downward continued data, and post-stack time migrated them (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">7</xref>). The NMO velocity was estimated from the tomographic velocity model. The correspondence between the post-stack migrated image and the NMO corrected OBH gathers (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">7</xref>) confirms that they are real images from the mantle.</p></sec><sec id=\"Sec13\"><title>Synthetic seismogram modelling</title><p id=\"Par45\">In order to make sure that the deep reflection arrivals are real, not multiples, and to quantify the nature of the reflection interface, we computed synthetic seismograms (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>). Based on the travel time tomography and velocity model obtained for profile RFR96-01 (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3d</xref>), we extrapolated the velocity model down to 20-km depth. To simulate the mantle reflectors, we inserted low- and high-velocity layers in the mantle of different thicknesses and velocity contrasts.</p><p id=\"Par46\">We computed the synthetic seismograms by solving the 2-D isotropic elastic wave equation by fourth-order finite difference in space and second-order in time using the staggered grid method<sup><xref ref-type=\"bibr\" rid=\"CR65\">65</xref></sup>. The first model is a 4-km-thick low-velocity zone where the velocity is decreased by −7%. The modelling results confirm that these reflections are real, not multiples (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>).</p><p id=\"Par47\">The next step was to determine if the reflection is due to high or low thin velocity layers. We included 300- and 600-m-thick low- and high-velocity layers with a velocity contrast of ±7% for the upper layer (Supplementary Figs. <xref rid=\"MOESM1\" ref-type=\"media\">10</xref>–<xref rid=\"MOESM1\" ref-type=\"media\">12</xref>).</p><p id=\"Par48\">Extensive synthetic seismogram modelling was carried out by varying the thickness of the upper layer from 300 to 1200 m (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">10</xref>), the velocity variations from ±7 to −30% (Supplementary Figs. <xref rid=\"MOESM1\" ref-type=\"media\">11</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">12</xref>), and also allowing a gradient in the velocity (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">13</xref>).</p><p id=\"Par49\">These results suggest that the first mantle reflection is related to a negative velocity contrast, or a negative velocity gradient within a thin zone (<1/4 of wavelength). Similarly, the deeper reflections require the presence of thin low-velocity layers.</p></sec><sec id=\"Sec14\"><title>Amplitude versus offset</title><p id=\"Par50\">We computed the amplitude versus offset variation for real as well synthetically computed data for different velocities in a 300-m-thick layer (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>). A curve was fitted to obtain a smooth amplitude versus offset curve. In order to compare different amplitude versus offset variations for different cases, the amplitude versus offset curve was normalised by the amplitudes at 8.5-km offset for each case, creating a relative amplitude versus offset plot (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>).</p></sec><sec id=\"Sec15\"><title>Computation of isotherms</title><p id=\"Par51\">The isotherm in Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6a</xref> were computed using the plate-cooling model<sup><xref ref-type=\"bibr\" rid=\"CR66\">66</xref>,<xref ref-type=\"bibr\" rid=\"CR67\">67</xref></sup> for a spreading rate of 60 mm yr<sup>−1</sup>, and a mantle temperature of 1350 °C at the base of the lithosphere at 106 km and thermal diffusivity κ = 1 mm<sup>2</sup> s<sup>−1</sup>.</p><p id=\"Par52\">The isotherms in Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6b</xref> were computed using the approach that incorporates extensive hydrothermal circulation near the ridge axis and the plate-cooling model away from the ridge axis<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. A key element of this simplified model is that the crust cooled near the axis has to be heated from below before near-surface temperature gradients. As a result, the isotherms will deepen rapidly near the spreading axis and flatten away from the ridge axis. This model treats only the vertical conduction of heat through the lithosphere as it moves away from a spreading centre. The effect of hydrothermal convection near the spreading axis is approximated by the enhancement of thermal diffusivity near the axis. The enhancement factor, termed the Nusselt number, gives the ratio of the steady-state average vertical heat transport through a convecting layer to the heat transported by conduction alone.</p><p id=\"Par53\">An explicit finite-difference approximation of the one-dimensional, time-dependent equation for vertical, conductive heat transport is used to solve lithospheric temperatures as functions of time <italic>t</italic> since the formation of the lithosphere and depth <italic>z</italic> below the seafloor. We assume the initial temperature of the crust and mantle at the ridge axis, to be 1200 and 1350 °C, respectively. Latent heat is liberated as the crust cools between its liquidus temperature <italic>T</italic><sub>L</sub> = 1150 °C and its solidus temperature <italic>T</italic><sub>S</sub> = 1100 °C. The total latent heat <italic>L</italic> released over this temperature range is set to 400 kJ kg<sup>−1</sup> (ref. <sup><xref ref-type=\"bibr\" rid=\"CR66\">66</xref></sup>) and the assumed value of the specific heat <italic>c</italic> of 1.1 kJ kg<sup>−1</sup> °K<sup>−1</sup>, consistent with measurements for basalt<sup><xref ref-type=\"bibr\" rid=\"CR68\">68</xref></sup>. The resulting value of <italic>L/c</italic> = 360 °K, which is the change in sensible temperature that would produce the same release of heat as through the latent heat release.</p><p id=\"Par54\">The thermal diffusivity is set to an effective value of <italic>Nu</italic> times <italic>κ,</italic> where <italic>κ</italic> = 5 × 10<sup>−7</sup> m<sup>2</sup> s<sup>−1</sup> appropriate for basalt<sup><xref ref-type=\"bibr\" rid=\"CR69\">69</xref></sup> or olivine at temperatures above ∼500 °C<sup><xref ref-type=\"bibr\" rid=\"CR70\">70</xref></sup>. From time <italic>t</italic> = 0 to <italic>t</italic> = <italic>t</italic><sub>OFF</sub> and from <italic>T</italic> = 0 to <italic>T</italic> = <italic>T</italic><sub>OFF</sub> the <italic>Nu</italic> is set to the on-axis value of <italic>Nu</italic><sub>ON</sub>. The idea behind setting a maximum temperature for hydrothermal flow effects is that ductile flow should close cracks above a rheologically controlled temperature. For <italic>t</italic> > <italic>t</italic><sub>OFF</sub> or <italic>T</italic> > <italic>T</italic><sub>OFF</sub> then <italic>Nu</italic> = 1. We obtain temperature changes by solving a finite-difference version of:<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\frac{{\\partial T(z,t)}}{{\\partial t}} = \\frac{\\partial }{{\\partial z}}\\left( {Nu\\,{\\upkappa}\\frac{{\\partial T(z,t)}}{{\\partial z}}} \\right) - H_{\\mathrm{L}}(T)$$\\end{document}</tex-math><mml:math id=\"M2\"><mml:mfrac><mml:mrow><mml:mi>∂</mml:mi><mml:mi>T</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>z</mml:mi><mml:mo>,</mml:mo><mml:mi>t</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:mi>∂</mml:mi><mml:mi>t</mml:mi></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>∂</mml:mi></mml:mrow><mml:mrow><mml:mi>∂</mml:mi><mml:mi>z</mml:mi></mml:mrow></mml:mfrac><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>N</mml:mi><mml:mi>u</mml:mi><mml:mspace width=\"0.25em\"/><mml:mi mathvariant=\"normal\">κ</mml:mi><mml:mfrac><mml:mrow><mml:mi>∂</mml:mi><mml:mi>T</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>z</mml:mi><mml:mo>,</mml:mo><mml:mi>t</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:mi>∂</mml:mi><mml:mi>z</mml:mi></mml:mrow></mml:mfrac></mml:mrow></mml:mfenced><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>H</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">L</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>T</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:math><graphic xlink:href=\"41467_2020_17946_Article_Equ1.gif\" position=\"anchor\"/></alternatives></disp-formula>where<disp-formula id=\"Equ2\"><label>2</label><alternatives><tex-math id=\"M3\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$H_{\\mathrm{L}}\\left( T \\right) = 0\\,{\\mathrm{for}}\\,T \\,> \\, T_{\\mathrm{L}}$$\\end{document}</tex-math><mml:math id=\"M4\"><mml:msub><mml:mrow><mml:mi>H</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">L</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>T</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mn>0</mml:mn><mml:mspace width=\"0.25em\"/><mml:mi mathvariant=\"normal\">for</mml:mi><mml:mspace width=\"0.25em\"/><mml:mi>T</mml:mi><mml:mspace width=\"0.25em\"/><mml:mo>></mml:mo><mml:mspace width=\"0.25em\"/><mml:msub><mml:mrow><mml:mi>T</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">L</mml:mi></mml:mrow></mml:msub></mml:math><graphic xlink:href=\"41467_2020_17946_Article_Equ2.gif\" position=\"anchor\"/></alternatives></disp-formula><disp-formula id=\"Equ3\"><label>3</label><alternatives><tex-math id=\"M5\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$H_{\\mathrm{L}}\\left( T \\right) = \\frac{L}{{c\\left( {T_{\\mathrm{L}} - T_{\\mathrm{S}}} \\right)}}\\,{\\mathrm{for}}\\,T_{\\mathrm{S}} \\,<\\, T \\,<\\, T_{\\mathrm{L}}$$\\end{document}</tex-math><mml:math id=\"M6\"><mml:msub><mml:mrow><mml:mi>H</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">L</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>T</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>L</mml:mi></mml:mrow><mml:mrow><mml:mi>c</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:msub><mml:mrow><mml:mi>T</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">L</mml:mi></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>T</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">S</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfenced></mml:mrow></mml:mfrac><mml:mspace width=\"0.25em\"/><mml:mi mathvariant=\"normal\">for</mml:mi><mml:mspace width=\"0.25em\"/><mml:msub><mml:mrow><mml:mi>T</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">S</mml:mi></mml:mrow></mml:msub><mml:mspace width=\"0.25em\"/><mml:mo><</mml:mo><mml:mspace width=\"0.25em\"/><mml:mi>T</mml:mi><mml:mspace width=\"0.25em\"/><mml:mo><</mml:mo><mml:mspace width=\"0.25em\"/><mml:msub><mml:mrow><mml:mi>T</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">L</mml:mi></mml:mrow></mml:msub></mml:math><graphic xlink:href=\"41467_2020_17946_Article_Equ3.gif\" position=\"anchor\"/></alternatives></disp-formula><disp-formula id=\"Equ4\"><label>4</label><alternatives><tex-math id=\"M7\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$H_{\\mathrm{L}}\\left( T \\right) = 0\\,{\\mathrm{for}}\\,T \\,<\\, T_{\\mathrm{S}}$$\\end{document}</tex-math><mml:math id=\"M8\"><mml:msub><mml:mrow><mml:mi>H</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">L</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>T</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mn>0</mml:mn><mml:mspace width=\"0.25em\"/><mml:mi mathvariant=\"normal\">for</mml:mi><mml:mspace width=\"0.25em\"/><mml:mi>T</mml:mi><mml:mspace width=\"0.25em\"/><mml:mo><</mml:mo><mml:mspace width=\"0.25em\"/><mml:msub><mml:mrow><mml:mi>T</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">S</mml:mi></mml:mrow></mml:msub></mml:math><graphic xlink:href=\"41467_2020_17946_Article_Equ4.gif\" position=\"anchor\"/></alternatives></disp-formula>relates to the release of latent heat. Temperatures are laterally advected at the plate velocity <italic>V</italic><sub>P</sub> so that the horizontal position <italic>x</italic> can be related to time as <italic>x</italic> <italic>=</italic> <italic>V</italic><sub>P</sub><italic>t</italic>. The top boundary was kept at 0 °C and the bottom, at 60-km depth was kept at 1350 °C. A grid size of 200 m was used.</p><p id=\"Par55\">We varied <italic>Nu</italic><sub>ON</sub>, <italic>t</italic><sub>OFF</sub> and <italic>T</italic><sub>OFF</sub> to see if we could produce a fairly flat isotherm at ∼11 km out to ∼3 Ma as indicated by the seismic data for the flank of the Juan de Fuca Ridge. The case shown in Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6b</xref> assumes the rather extreme values of <italic>Nu</italic><sub>ON</sub> = 100, <italic>t</italic><sub>OFF</sub> = 0.3 Ma and <italic>T</italic><sub>OFF</sub> = 100 °C but decreasing <italic>Nu</italic><sub>ON</sub> and increasing <italic>T</italic><sub>OFF</sub> = 100 °C gives similar lithospheric thickening with time.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec16\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41467_2020_17946_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41467_2020_17946_MOESM2_ESM.pdf\"><caption><p>Peer Review File</p></caption></media></supplementary-material></sec></sec></body><back><fn-group><fn><p><bold>Peer review information</bold>\n<italic>Nature Communications</italic> thanks Samer Naif, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary information</bold> is available for this paper at 10.1038/s41467-020-17946-3.</p></sec><ack><title>Acknowledgements</title><p>We thank Heinrich Villinger and Fares Mehouachi for helpful discussions and valuable suggestions that substantially improved the paper. We are grateful to Heinrich Villinger, chief scientist of the R/V Sonne cruise SO111 in 1996. We would like to thank Tim Stern and two anonymous reviewers for their constructive reviews, which improved the paper significantly. Funding from the Germany Science Foundation (DFG grant GR1964/2-1) and German Federal Government (BMBF grant 03G0111A) is acknowledged. The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC Advance Grant agreement n° 339442_TransAtlanticILAB. The work is IPGP contribution number 4160.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>Y.Q. processed and analysed the seismic data and participated in the writing of the paper. S.C.S. supervised the project and wrote the paper. I.G. recognized the deep-seated mantle reflections in deconvolved record sections performed seismic tomographic inversions and contributed in the writing of the paper. M.M. participated in the writing of the paper. R.B. performed the thermal modelling.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>The following figures are based on the raw data used in this study: (1) Figures <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>, <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> and <xref rid=\"Fig5\" ref-type=\"fig\">5</xref> in the main text. (2) Supplementary Figs. <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>, <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>, <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>, <xref rid=\"MOESM1\" ref-type=\"media\">5</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">7</xref>. The raw ocean-bottom seismometer data are available on the <ext-link ext-link-type=\"uri\" xlink:href=\"https://doi.pangaea.de/10.1594/PANGAEA.919664\">German PANGAEA database</ext-link>. The processed data shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>, Supplementary Figs. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>, <xref rid=\"MOESM1\" ref-type=\"media\">7</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">8</xref> the <ext-link ext-link-type=\"uri\" xlink:href=\"https://doi.pangaea.de/10.1594/PANGAEA.920682\">German PANGAEA database</ext-link>.</p></notes><notes notes-type=\"data-availability\"><title>Code availability</title><p>We have used two types of codes: (1) to model synthetic seismograms shown in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>, Supplementary Figs. <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>, <xref rid=\"MOESM1\" ref-type=\"media\">9</xref>, <xref rid=\"MOESM1\" ref-type=\"media\">10</xref>, <xref rid=\"MOESM1\" ref-type=\"media\">11</xref>, <xref rid=\"MOESM1\" ref-type=\"media\">12</xref> and (2) to compute isotherms in Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6b</xref>. These codes are propriety code but will be available on request from Singh (synthetic seismogram modelling code) and Buck (isotherm computation code).</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par56\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parsons</surname><given-names>B</given-names></name><name><surname>Sclater</surname><given-names>JG</given-names></name></person-group><article-title>An analysis of the variation of ocean floor bathymetry and heat flow with age</article-title><source>J. Geophys. Res.</source><year>1977</year><volume>82</volume><fpage>803</fpage><lpage>827</lpage></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Phipps Morgan</surname><given-names>J</given-names></name><name><surname>Parmentier</surname><given-names>EM</given-names></name><name><surname>Lin</surname><given-names>J</given-names></name></person-group><article-title>Mechanisms for the origin of Mid-Ocean Ridge Axial Topography: implications for thermal and mechanical structure of accreting plate boundaries</article-title><source>J. Geophys. Res.</source><year>1987</year><volume>92</volume><fpage>823</fpage><lpage>836</lpage></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cochran</surname><given-names>JR</given-names></name><name><surname>Buck</surname><given-names>WR</given-names></name></person-group><article-title>Near-axis subsidence rates, hydrothermal circulation, and thermal structure of mid-ocean ridge crests</article-title><source>J. Geophys. Res.</source><year>2001</year><volume>106</volume><fpage>19233</fpage><lpage>19258</lpage></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dubinin</surname><given-names>EP</given-names></name><etal/></person-group><article-title>Hot and cold zones of the Southeast Indian Ridge and their influence on the peculiarities of its structure and magmatism (Numerical and Physical Modelling)</article-title><source>Geotectonics</source><year>2017</year><volume>51</volume><fpage>209</fpage></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Priestley</surname><given-names>K</given-names></name><name><surname>McKenzie</surname><given-names>D</given-names></name></person-group><article-title>The thermal structure of the lithosphere from shear wave velocities</article-title><source>Earth Planet. Sci. Lett.</source><year>2006</year><volume>244</volume><fpage>285</fpage><lpage>301</lpage></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fishwick</surname><given-names>S</given-names></name></person-group><article-title>Surface wave tomography: imaging of the lithosphere–asthenosphere boundary beneath central and southern Africa?</article-title><source>Lithos</source><year>2010</year><volume>120</volume><fpage>63</fpage><lpage>73</lpage></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Burgos</surname><given-names>G</given-names></name><etal/></person-group><article-title>Oceanic lithosphere-asthenosphere boundary from surface wave dispersion data</article-title><source>J. Geophys. Res.</source><year>2014</year><volume>119</volume><fpage>1079</fpage><lpage>1093</lpage></element-citation></ref><ref id=\"CR8\"><label>8.</label><mixed-citation publication-type=\"other\">Priestley, K., McKenzie, D. & Ho, T. A lithosphere–asthenosphere boundary—a global model derived from multimode surface-wave tomography and petrology. <italic>Geophys. Monogr. Series</italic> 111–123, 10.1002/9781119249740.ch6 (2018).</mixed-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bell</surname><given-names>S</given-names></name><name><surname>Ruan</surname><given-names>Y</given-names></name><name><surname>Forsyth</surname><given-names>DW</given-names></name></person-group><article-title>Ridge asymmetry and deep aqueous alteration at the trench observed from Rayleigh wave tomography of the Juan de Fuca plate</article-title><source>J. Geophys. Res. Solid Earth</source><year>2016</year><volume>121</volume><fpage>7298</fpage><lpage>7321</lpage></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oreshin</surname><given-names>S</given-names></name><name><surname>Vinnik</surname><given-names>L</given-names></name><name><surname>Peregoudov</surname><given-names>D</given-names></name><name><surname>Roecker</surname><given-names>S</given-names></name></person-group><article-title>Lithosphere and asthenosphere of the Tien Shan imaged by S receiver functions</article-title><source>Geophys. Res. Lett.</source><year>2002</year><volume>29</volume><fpage>1191</fpage></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kawakatsu</surname><given-names>H</given-names></name><etal/></person-group><article-title>Seismic evidence for sharp lithosphere–asthenosphere boundaries of oceanic plates</article-title><source>Science</source><year>2009</year><volume>324</volume><fpage>499</fpage><lpage>502</lpage><pub-id pub-id-type=\"pmid\">19390042</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kumar</surname><given-names>P</given-names></name><name><surname>Kind</surname><given-names>R</given-names></name><name><surname>Yuan</surname><given-names>X</given-names></name><name><surname>Mechie</surname><given-names>J</given-names></name></person-group><article-title>USArray receiver function images of the lithosphere-asthenosphere boundary</article-title><source>Seismological Res. Lett.</source><year>2012</year><volume>83</volume><fpage>486</fpage><lpage>491</lpage></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Audet</surname><given-names>P</given-names></name></person-group><article-title>Receiver function using OBS data: promises and limitations from numerical modelling and examples from the Cascadia Initiative</article-title><source>Geophys. J. Int.</source><year>2016</year><volume>205</volume><fpage>1740</fpage><lpage>1755</lpage></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rychert</surname><given-names>CA</given-names></name><name><surname>Harmon</surname><given-names>N</given-names></name><name><surname>Tharimena</surname><given-names>S</given-names></name></person-group><article-title>Scattered wave imaging of the oceanic plate in Cascadia</article-title><source>Sci. Adv.</source><year>2018</year><volume>4</volume><fpage>eaao1908</fpage><pub-id pub-id-type=\"pmid\">29457132</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stern</surname><given-names>TA</given-names></name><etal/></person-group><article-title>A seismic reflection image for the base of a tectonic plate</article-title><source>Nature</source><year>2015</year><volume>518</volume><fpage>85</fpage><pub-id pub-id-type=\"pmid\">25653000</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mehouachi</surname><given-names>F</given-names></name><name><surname>Singh</surname><given-names>SC</given-names></name></person-group><article-title>Water-rich sublithospheric melt channel in the equatorial Atlantic Ocean</article-title><source>Nat. Geosci.</source><year>2018</year><volume>11</volume><fpage>65</fpage><lpage>69</lpage></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Naif</surname><given-names>S</given-names></name><name><surname>Key</surname><given-names>K</given-names></name><name><surname>Constable</surname><given-names>S</given-names></name><name><surname>Evans</surname><given-names>RL</given-names></name></person-group><article-title>Melt-rich channel observed at the lithosphere-asthenosphere boundary</article-title><source>Nature</source><year>2013</year><volume>495</volume><fpage>356</fpage><lpage>359</lpage><pub-id pub-id-type=\"pmid\">23518564</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ohira</surname><given-names>A</given-names></name><etal/></person-group><article-title>Evidence for frozen melts in the mid-lithosphere detected from active source seismic data</article-title><source>Sci. Rep.</source><year>2017</year><volume>7</volume><fpage>15770</fpage><pub-id pub-id-type=\"pmid\">29150652</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Davis</surname><given-names>EE</given-names></name><name><surname>Karsten</surname><given-names>JL</given-names></name></person-group><article-title>On the cause of the asymmetric distribution of seamounts about the Juan de Fuca Ridge: Ridge-crest migration over a heterogeneous asthenosphere</article-title><source>Earth Planet. Sci. Lett.</source><year>1986</year><volume>79</volume><fpage>385</fpage><lpage>396</lpage></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Karsten</surname><given-names>JL</given-names></name><name><surname>Delaney</surname><given-names>J</given-names></name></person-group><article-title>Hotspot-ridge crest convergence in the northeast Pacific</article-title><source>J. Geophys. Res.</source><year>1989</year><volume>94</volume><fpage>700</fpage><lpage>712</lpage></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Desonie</surname><given-names>DL</given-names></name><name><surname>Duncan</surname><given-names>RA</given-names></name></person-group><article-title>The Cobb-Eickelberg seamount chain: hotspot volcanism with mid-ocean ridge basalt affinity</article-title><source>J. Geophys. Res.</source><year>1990</year><volume>95</volume><fpage>697</fpage><lpage>12,711</lpage></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carbotte</surname><given-names>SM</given-names></name><etal/></person-group><article-title>Variable crustal structure along the Juan de Fuca Ridge: influence of on-axis hotspots and absolute plate motions</article-title><source>Geochem. Geophys. Geosyst.</source><year>2008</year><volume>9</volume><fpage>Q08001</fpage></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carbotte</surname><given-names>SM</given-names></name><etal/></person-group><article-title>Rift topography linked to magmatism at the intermediate spreading Juan de Fuca Ridge</article-title><source>Geology</source><year>2006</year><volume>34</volume><fpage>209</fpage><lpage>212</lpage></element-citation></ref><ref id=\"CR24\"><label>24.</label><mixed-citation publication-type=\"other\">Davis, E. E. & Villinger, H. Tectonic and thermal structure of the Middle Valley sedimented rift, northern Juan de Fuca Ridge. in <italic>Proc. ODP, Init. Repts</italic>. 139 (Ocean Drilling Program, College Station, TX, 1992).</mixed-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Davis</surname><given-names>EE</given-names></name><name><surname>Currie</surname><given-names>RG</given-names></name></person-group><article-title>Geophysical observations of the northern Juan de Fuca Ridge system: lessons in seafloor spreading</article-title><source>Can. J. Earth Sci.</source><year>1993</year><volume>30</volume><fpage>278</fpage><lpage>300</lpage></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wilson</surname><given-names>DS</given-names></name></person-group><article-title>Confidence intervals for motion and deformation of the Juan de Fuca plate</article-title><source>J. Geophys. Res.</source><year>1993</year><volume>98</volume><fpage>053</fpage><lpage>16,071</lpage></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shoberg</surname><given-names>T</given-names></name><name><surname>Stein</surname><given-names>S</given-names></name><name><surname>Karsten</surname><given-names>J</given-names></name></person-group><article-title>Constraints on rift propagation history at the Cobb Offset, Juan de Fuca Ridge, from numerical modeling of tectonic fabric</article-title><source>Tectonophysics</source><year>1991</year><volume>197</volume><fpage>295</fpage><lpage>308</lpage></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>VanderBeek</surname><given-names>BP</given-names></name><name><surname>Toomey</surname><given-names>DR</given-names></name><name><surname>Hooft</surname><given-names>EE</given-names></name><name><surname>Wilcock</surname><given-names>WS</given-names></name></person-group><article-title>Segmentation of mid-ocean ridges attributed to oblique mantle divergence</article-title><source>Nat. Geosci.</source><year>2016</year><volume>9</volume><fpage>636</fpage></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arnoux</surname><given-names>GM</given-names></name><name><surname>Toomey</surname><given-names>DR</given-names></name><name><surname>Hooft</surname><given-names>EEE</given-names></name><name><surname>Wilcock</surname><given-names>WSD</given-names></name></person-group><article-title>Seismic imaging and physical properties of the Endeavour segment: evidence that skew between mantle and crustal magmatic systems governs spreading center processes</article-title><source>Geochem. Geophys. Geosyst.</source><year>2019</year><volume>20</volume><fpage>1319</fpage><lpage>1339</lpage></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Marjanović</surname><given-names>M</given-names></name><name><surname>Carbotte</surname><given-names>SM</given-names></name><name><surname>Nedimović</surname><given-names>MR</given-names></name><name><surname>Canales</surname><given-names>JP</given-names></name></person-group><article-title>Gravity and seismic study of crustal structure along the Juan de Fuca Ridge axis and across pseudofaults on the ridge flanks</article-title><source>Geochem. Geophys. Geosyst.</source><year>2011</year><volume>12</volume><fpage>Q05008</fpage></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gazdag</surname><given-names>J</given-names></name><name><surname>Sguazzero</surname><given-names>P</given-names></name></person-group><article-title>Migration of seismic data</article-title><source>Proc. Ieeee.</source><year>1984</year><volume>72</volume><fpage>1302</fpage><lpage>1315</lpage></element-citation></ref><ref id=\"CR32\"><label>32.</label><mixed-citation publication-type=\"other\">Yilmaz, O. <italic>Seismic Data Processing</italic> (Society of Exploration Geophysicists, Tulsa, 1987).</mixed-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Berryhill</surname><given-names>JR</given-names></name></person-group><article-title>Wave equation datuming</article-title><source>Geophysics</source><year>1979</year><volume>44</volume><fpage>1329</fpage><lpage>1344</lpage></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arnulf</surname><given-names>AF</given-names></name><name><surname>Singh</surname><given-names>SC</given-names></name><name><surname>Harding</surname><given-names>AJ</given-names></name><name><surname>Kent</surname><given-names>GM</given-names></name><name><surname>Crawford</surname><given-names>W</given-names></name></person-group><article-title>Strong seismic heterogeneity in layer 2A near hydrothermal vents at the mid-Atlantic ridge</article-title><source>Geophys. Res. Lett.</source><year>2011</year><volume>38</volume><fpage>L13320</fpage></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gaherty</surname><given-names>JB</given-names></name><name><surname>Jordan</surname><given-names>TH</given-names></name><name><surname>Gee</surname><given-names>LS</given-names></name></person-group><article-title>Seismic structure of the upper mantle in a central Pacific corridor</article-title><source>J. Geophys. Res.</source><year>1996</year><volume>101</volume><fpage>22291</fpage><lpage>22309</lpage></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gaherty</surname><given-names>JB</given-names></name><name><surname>Kato</surname><given-names>M</given-names></name><name><surname>Jordan</surname><given-names>TH</given-names></name></person-group><article-title>Seismological structure of the upper mantle: a regional comparison of seismic layering</article-title><source>Phys. Earth Planet. Inter.</source><year>1999</year><volume>110</volume><fpage>21</fpage><lpage>41</lpage></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Taylor</surname><given-names>M</given-names></name><name><surname>Singh</surname><given-names>SC</given-names></name></person-group><article-title>Composition and microstructure of magma bodies from effective medium theory</article-title><source>Geophys. J. Int.</source><year>2002</year><volume>149</volume><fpage>15</fpage><lpage>21</lpage></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Singh</surname><given-names>SC</given-names></name><name><surname>Kent</surname><given-names>GM</given-names></name><name><surname>Collier</surname><given-names>JS</given-names></name><name><surname>Harding</surname><given-names>AJ</given-names></name><name><surname>Orcutt</surname><given-names>JA</given-names></name></person-group><article-title>Melt to mush variations in crustal magma properties along the ridge crest at the southern East Pacific Rise</article-title><source>Nature</source><year>1998</year><volume>394</volume><fpage>874</fpage><lpage>878</lpage></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Katz</surname><given-names>R</given-names></name></person-group><article-title>Porosity-driven convection and asymmetry beneath mid-ocean ridges</article-title><source>Geochem. Geophys. Geosyst.</source><year>2010</year><volume>10</volume><fpage>Q0AC07</fpage></element-citation></ref><ref id=\"CR40\"><label>40.</label><mixed-citation publication-type=\"other\">Langmuir, C. H., Klein, E. M. & Plank, T. in <italic>Mantle Flow and Melt Generation at Mid-Ocean Ridges</italic> (eds Morgan, J. P., Blackman, D. K. & Sinton, J. M.) 183–280 (American Geophysical Union, Washington, 1993).</mixed-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Baker</surname><given-names>ET</given-names></name><name><surname>Chen</surname><given-names>YJ</given-names></name><name><surname>Morgan</surname><given-names>JP</given-names></name></person-group><article-title>The relationship between near-axis hydrothermal cooling and the spreading rate of mid-ocean ridges</article-title><source>Earth Planet. Sc. Lett.</source><year>1996</year><volume>142</volume><fpage>137</fpage><lpage>145</lpage></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>X</given-names></name><etal/></person-group><article-title>Rejuvenation of the lithosphere by the Hawaiian plume</article-title><source>Nature</source><year>2004</year><volume>427</volume><fpage>827</fpage><lpage>829</lpage><pub-id pub-id-type=\"pmid\">14985758</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><mixed-citation publication-type=\"other\">Singh, S. C. & Audhkhasi, P. Continuous seismic reflection image of the Lithosphere-Asthenosphere Boundary (LAB) from 2–75 Ma on the African plate in the Equatorial Atlantic Ocean. <italic>EGU General Assembly</italic><bold>2020</bold>, EGU2020–EGU9622 (2020).</mixed-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Han</surname><given-names>S</given-names></name><etal/></person-group><article-title>Seismic reflection imaging of the Juan de Fuca plate from ridge to trench: New constraints on the distribution of faulting and evolution of the crust prior to subduction</article-title><source>J. Geophys. Res. Solid Earth</source><year>2016</year><volume>121</volume><fpage>1849</fpage><lpage>1872</lpage></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hartley</surname><given-names>RA</given-names></name><name><surname>Roberts</surname><given-names>GG</given-names></name><name><surname>White</surname><given-names>N</given-names></name><name><surname>Richardson</surname><given-names>C</given-names></name></person-group><article-title>Transient convective uplift of an ancient buried landscape</article-title><source>Nat. Geosci.</source><year>2011</year><volume>4</volume><fpage>562</fpage><lpage>565</lpage></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carbotte</surname><given-names>SM</given-names></name><name><surname>Small</surname><given-names>C</given-names></name><name><surname>Donnelly</surname><given-names>K</given-names></name></person-group><article-title>The influence of ridge migration on the magmatic segmentation of mid-ocean ridges</article-title><source>Nature</source><year>2004</year><volume>429</volume><fpage>743</fpage><lpage>746</lpage><pub-id pub-id-type=\"pmid\">15201906</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Katz</surname><given-names>RF</given-names></name><name><surname>Spiegelman</surname><given-names>M</given-names></name><name><surname>Langmuir</surname><given-names>CH</given-names></name></person-group><article-title>A new parameterization of hydrous mantle melting</article-title><source>Geochem. Geophys. Geosyst.</source><year>2003</year><volume>4</volume><fpage>1073</fpage></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fisher</surname><given-names>AT</given-names></name><name><surname>Fischer</surname><given-names>K</given-names></name><name><surname>Lavoie</surname><given-names>D</given-names></name><name><surname>Langseth</surname><given-names>M</given-names></name><name><surname>Xu</surname><given-names>J</given-names></name></person-group><article-title>Geotechnical and hydrological properties of sediments from Middle Valley, northern, Juan de Fuca Ridge</article-title><source>Proc. Ocean Drill. Program Sci. Results</source><year>1994</year><volume>139</volume><fpage>627</fpage><lpage>647</lpage></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jourdain</surname><given-names>A</given-names></name><etal/></person-group><article-title>Crustal accretion at a sedimented spreading center in the Andaman Sea</article-title><source>Geology</source><year>2016</year><volume>44</volume><fpage>351</fpage><lpage>354</lpage></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Davis</surname><given-names>EE</given-names></name><etal/></person-group><article-title>Regional heat flow variations across the sedimented Juan de Fuca Ridge eastern flank: Constraints on lithospheric cooling and lateral hydrothermal heat transport</article-title><source>J. Geophys. Res.</source><year>1999</year><volume>104</volume><fpage>17675</fpage><lpage>17688</lpage></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gao</surname><given-names>H</given-names></name></person-group><article-title>Seismic velocity structure of the Juan de Fuca and Gorda plates revealed by a joint inversion of ambient noise and regional earthquakes</article-title><source>Geophys. Res. Lett.</source><year>2016</year><volume>43</volume><fpage>5194</fpage><lpage>5201</lpage></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Johansen</surname></name><etal/></person-group><article-title>B. Deep electrical imaging of the ultraslow-spreading Mohns Ridge</article-title><source>Nature</source><year>2019</year><volume>567</volume><fpage>379</fpage><lpage>383</lpage><pub-id pub-id-type=\"pmid\">30894724</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tharimena</surname><given-names>S</given-names></name><name><surname>Rychert</surname><given-names>C</given-names></name><name><surname>Harmon</surname><given-names>N</given-names></name></person-group><article-title>A unified continental thickness from seismology and diamonds suggests a melt-defined plate</article-title><source>Science</source><year>2017</year><volume>357</volume><fpage>580</fpage><lpage>583</lpage><pub-id pub-id-type=\"pmid\">28798127</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weatherley</surname><given-names>SM</given-names></name><name><surname>Katz</surname><given-names>RF</given-names></name></person-group><article-title>Melting and channelized magmatic flow in chemically heterogeneous, upwelling mantle</article-title><source>Geochem. Geophys. Geosyst.</source><year>2012</year><volume>13</volume><fpage>Q0AC18</fpage></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eilon</surname><given-names>ZC</given-names></name><name><surname>Abers</surname><given-names>GA</given-names></name></person-group><article-title>High seismic attenuation at a mid-ocean ridge reveals the distribution of deep melt</article-title><source>Sci. Adv.</source><year>2017</year><volume>3</volume><fpage>e1602829</fpage><pub-id pub-id-type=\"pmid\">28560338</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Creager</surname><given-names>KC</given-names></name><etal/></person-group><article-title>Three-dimensional reflection image of the subducting Juan de Fuca plate, The Cascadia Subduction Zone and Related Subduction Systems-Seismic</article-title><source>Struct., Intraslab Earthq. Process., Earthq. Hazards</source><year>2002</year><volume>2</volume><fpage>37</fpage></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Korenaga</surname><given-names>J</given-names></name><etal/></person-group><article-title>Crustal structure of the southeast Greenland margin from joint refraction and reflection seismic tomography</article-title><source>J. Geophys. Res.</source><year>2000</year><volume>105</volume><fpage>21591</fpage><lpage>21614</lpage></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Horning</surname><given-names>G</given-names></name><etal/></person-group><article-title>A 2-D tomographic model of the Juan de Fuca plate from accretion at axial seamount to subduction at the Cascadia margin from an active source ocean bottom seismometer survey</article-title><source>J. Geophys. Res.</source><year>2016</year><volume>121</volume><fpage>5859</fpage><lpage>5879</lpage></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nedimović</surname><given-names>MR</given-names></name><etal/></person-group><article-title>Upper crustal evolution across the Juan de Fuca ridge flanks</article-title><source>Geochem. Geophys. Geosyst.</source><year>2008</year><volume>9</volume><fpage>Q09006</fpage></element-citation></ref><ref id=\"CR60\"><label>60.</label><mixed-citation publication-type=\"other\">Rosenberger, A., Davis, E. E. & Villinger, H. Data report: HYDROCELL-95 and -96 single-channel seismic data on the eastern Juan de Fuca Ridge Flank. in <italic>Proc. ODP, Sci. Results</italic> Vol. 168 (eds Davis, E. E., Fisher, A. T. & Firth, J. V.) (Ocean Drill. Program, College Station, TX, 1999).</mixed-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grevemeyer</surname><given-names>I</given-names></name><etal/></person-group><article-title>Episodic magmatism and serpentinized mantle exhumation at an ultraslow-spreading centre</article-title><source>Nat. Geosci.</source><year>2018</year><volume>11</volume><fpage>444</fpage><lpage>448</lpage></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grevemeyer</surname><given-names>I</given-names></name><name><surname>Weigel</surname><given-names>W</given-names></name><name><surname>Jennrich</surname><given-names>C</given-names></name></person-group><article-title>Structure and ageing of oceanic crust at 14°S on the East Pacific Rise</article-title><source>Geophys. J. Int.</source><year>1998</year><volume>135</volume><fpage>573</fpage><lpage>584</lpage></element-citation></ref><ref id=\"CR63\"><label>63.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Qin</surname><given-names>Y</given-names></name><name><surname>Singh</surname><given-names>SC</given-names></name></person-group><article-title>Detailed seismic velocity of the incoming subducting sediments in the 2004 great Sumatra earthquake rupture zone from full waveform inversion of long offset seismic data</article-title><source>Geophys. Res. Lett.</source><year>2017</year><volume>44</volume><fpage>3090</fpage><lpage>3099</lpage></element-citation></ref><ref id=\"CR64\"><label>64.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Qin</surname><given-names>Y</given-names></name><name><surname>Singh</surname><given-names>SC</given-names></name></person-group><article-title>Insight into frontal seismogenic zone in the Mentawai locked region from seismic full waveform inversion of ultralong offset streamer data</article-title><source>Geochem. Geophys. Geosyst.</source><year>2018</year><volume>19</volume><fpage>4342</fpage><lpage>4365</lpage></element-citation></ref><ref id=\"CR65\"><label>65.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Levander</surname><given-names>A</given-names></name></person-group><article-title>Fourth-order finite-difference P-SV seismograms</article-title><source>Geophysics</source><year>1988</year><volume>53</volume><fpage>1425</fpage><lpage>1436</lpage></element-citation></ref><ref id=\"CR66\"><label>66.</label><mixed-citation publication-type=\"other\">Turcotte, D. L. & Schubert, G. <italic>Geodynamics: Applications of Continuum Physics to Geological Problems</italic>, 2nd edn, 161 (John Wiley & Sons, New York, 2002).</mixed-citation></ref><ref id=\"CR67\"><label>67.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hasterok</surname><given-names>D</given-names></name></person-group><article-title>A heat flow-based cooling model for tectonic plates</article-title><source>Earth Planet. Sci. Lett.</source><year>2013</year><volume>361</volume><fpage>34</fpage><lpage>43</lpage></element-citation></ref><ref id=\"CR68\"><label>68.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kojitani</surname><given-names>H</given-names></name><name><surname>Akaogi</surname><given-names>A</given-names></name></person-group><article-title>Melting enthalpies of mantle peridotite: calorimetric determinations in the system CaO-MgO-Al<sub>2</sub>O<sub>3</sub>-SiO<sub>2</sub> and application to magma generation</article-title><source>Earth Planet. Sci. Lett.</source><year>1997</year><volume>153</volume><fpage>209</fpage><lpage>222</lpage></element-citation></ref><ref id=\"CR69\"><label>69.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hartlieb</surname><given-names>P</given-names></name><name><surname>Toifl</surname><given-names>M</given-names></name><name><surname>Kuchar</surname><given-names>F</given-names></name><name><surname>Meisels</surname><given-names>R</given-names></name><name><surname>Antretter</surname><given-names>T</given-names></name></person-group><article-title>Thermo-physical properties of selected hard rocks and their relation to microwave-assisted communication</article-title><source>Miner. Eng.</source><year>2016</year><volume>91</volume><fpage>34</fpage><lpage>41</lpage></element-citation></ref><ref id=\"CR70\"><label>70.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gibert</surname><given-names>B</given-names></name><name><surname>Schilling</surname><given-names>FR</given-names></name><name><surname>Tommasi</surname><given-names>A</given-names></name><name><surname>Mainprice</surname><given-names>D</given-names></name></person-group><article-title>Thermal diffusivity of olivine single‐crystals and polycrystalline aggregates at ambient conditions—a comparison</article-title><source>Geophys. Res. Lett.</source><year>2003</year><volume>30</volume><fpage>2172</fpage></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Nat Commun</journal-id><journal-id journal-id-type=\"iso-abbrev\">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type=\"epub\">2041-1723</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807780</article-id><article-id pub-id-type=\"pmc\">PMC7431580</article-id><article-id pub-id-type=\"publisher-id\">17998</article-id><article-id pub-id-type=\"doi\">10.1038/s41467-020-17998-5</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Measuring the Hubble constant with a sample of kilonovae</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-8262-2924</contrib-id><name><surname>Coughlin</surname><given-names>Michael W.</given-names></name><address><email>cough052@umn.edu</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Antier</surname><given-names>Sarah</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Dietrich</surname><given-names>Tim</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>Foley</surname><given-names>Ryan J.</given-names></name><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Heinzel</surname><given-names>Jack</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref><xref ref-type=\"aff\" rid=\"Aff8\">8</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-8255-5127</contrib-id><name><surname>Bulla</surname><given-names>Mattia</given-names></name><xref ref-type=\"aff\" rid=\"Aff9\">9</xref></contrib><contrib contrib-type=\"author\"><name><surname>Christensen</surname><given-names>Nelson</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref><xref ref-type=\"aff\" rid=\"Aff8\">8</xref></contrib><contrib contrib-type=\"author\"><name><surname>Coulter</surname><given-names>David A.</given-names></name><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Issa</surname><given-names>Lina</given-names></name><xref ref-type=\"aff\" rid=\"Aff9\">9</xref><xref ref-type=\"aff\" rid=\"Aff10\">10</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-2720-8904</contrib-id><name><surname>Khetan</surname><given-names>Nandita</given-names></name><xref ref-type=\"aff\" rid=\"Aff11\">11</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.17635.36</institution-id><institution-id institution-id-type=\"ISNI\">0000000419368657</institution-id><institution>School of Physics and Astronomy, </institution><institution>University of Minnesota, </institution></institution-wrap>Minneapolis, MN 55455 USA </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.20861.3d</institution-id><institution-id institution-id-type=\"ISNI\">0000000107068890</institution-id><institution>Division of Physics, Math, and Astronomy, </institution><institution>California Institute of Technology, </institution></institution-wrap>Pasadena, CA 91125 USA </aff><aff id=\"Aff3\"><label>3</label>APC, UMR 7164, 10 rue Alice Domon et Léonie Duquet, 75205 Paris, France </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.11348.3f</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0942 1117</institution-id><institution>Institut für Physik und Astronomie, </institution><institution>Universität Potsdam, </institution></institution-wrap>Haus 28, Karl-Liebknecht-Str. 24/25, 14476 Potsdam, Germany </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.420012.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0646 2193</institution-id><institution>Nikhef, </institution></institution-wrap>Science Park 105, 1098 XG Amsterdam, The Netherlands </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.205975.c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0740 6917</institution-id><institution>Department of Astronomy and Astrophysics, </institution><institution>University of California, </institution></institution-wrap>Santa Cruz, CA 95064 USA </aff><aff id=\"Aff7\"><label>7</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.4444.0</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2112 9282</institution-id><institution>Artemis, Université Côte d’Azur, Observatoire Côte d’Azur, CNRS, CS 34229, </institution></institution-wrap>F-06304 Nice Cedex 4, France </aff><aff id=\"Aff8\"><label>8</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.253692.9</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0445 5969</institution-id><institution>Physics and Astronomy, </institution><institution>Carleton College, </institution></institution-wrap>Northfield, MN 55057 USA </aff><aff id=\"Aff9\"><label>9</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.10548.38</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 9377</institution-id><institution>Nordita, KTH Royal Institute of Technology and Stockholm University, </institution></institution-wrap>Roslagstullsbacken 23, SE-106 91 Stockholm, Sweden </aff><aff id=\"Aff10\"><label>10</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.460789.4</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 4910 6535</institution-id><institution>Département de Phyisque, </institution><institution>Université Paris-Saclay, ENS Paris-Saclay, </institution></institution-wrap>91190 Gif-sur-Yvette, France </aff><aff id=\"Aff11\"><label>11</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.466750.6</institution-id><institution>Gran Sasso Science Institute (GSSI), </institution></institution-wrap>I-67100 L’Aquila, Italy </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>4129</elocation-id><history><date date-type=\"received\"><day>31</day><month>1</month><year>2020</year></date><date date-type=\"accepted\"><day>29</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Kilonovae produced by the coalescence of compact binaries with at least one neutron star are promising standard sirens for an independent measurement of the Hubble constant (<italic>H</italic><sub>0</sub>). Through their detection via follow-up of gravitational-wave (GW), short gamma-ray bursts (sGRBs) or optical surveys, a large sample of kilonovae (even without GW data) can be used for <italic>H</italic><sub>0</sub> contraints. Here, we show measurement of <italic>H</italic><sub>0</sub> using light curves associated with four sGRBs, assuming these are attributable to kilonovae, combined with GW170817. Including a systematic uncertainty on the models that is as large as the statistical ones, we find <inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${H}_{0}=73.{8}_{-5.8}^{+6.3}\\ {\\rm{km}}\\ {{\\rm{s}}}^{-1}\\ {{\\rm{Mpc}}}^{-1}$$\\end{document}</tex-math><mml:math id=\"M2\"><mml:msub><mml:mrow><mml:mi>H</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>73</mml:mn><mml:mo>.</mml:mo><mml:msubsup><mml:mrow><mml:mn>8</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>5.8</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>6.3</mml:mn></mml:mrow></mml:msubsup><mml:mspace width=\"0.33em\"/><mml:mi mathvariant=\"normal\">km</mml:mi><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">Mpc</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq1.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq2\"><alternatives><tex-math id=\"M3\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${H}_{0}=71.{2}_{-3.1}^{+3.2}\\ {\\rm{km}}\\ {{\\rm{s}}}^{-1}\\ {{\\rm{Mpc}}}^{-1}$$\\end{document}</tex-math><mml:math id=\"M4\"><mml:msub><mml:mrow><mml:mi>H</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>71</mml:mn><mml:mo>.</mml:mo><mml:msubsup><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>3.1</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>3.2</mml:mn></mml:mrow></mml:msubsup><mml:mspace width=\"0.33em\"/><mml:mi mathvariant=\"normal\">km</mml:mi><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">Mpc</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq2.gif\"/></alternatives></inline-formula> for two different kilonova models that are consistent with the local and inverse-distance ladder measurements. For a given model, this measurement is about a factor of 2-3 more precise than the standard-siren measurement for GW170817 using only GWs.</p></abstract><abstract id=\"Abs2\" abstract-type=\"web-summary\"><p id=\"Par2\">Kilonovae observations can be used to out constraints on the Hubble constant (H0). Here, the authors show H0 measurements by combining light curves of four short gamma-ray burts with GW170817 are about a factor of 2-3 more precise than the standard-siren measurements using only gravitational-waves.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>General relativity and gravity</kwd><kwd>High-energy astrophysics</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par3\">Since the discovery of the accelerating expansion rate of the universe<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>, cosmology surveys have tried to measure the properties of dark energy. One of the most common metrics, type Ia supernovae (SNe), which are standardizable, have been an important tool in this endeavour, with the particular benefit of being detectable throughout a large portion of cosmic time. It has been previously found that the cosmic microwave background (CMB) is consistent with <italic>Λ</italic><sub><italic>C</italic><italic>D</italic><italic>M</italic></sub> cosmology, but predicts a value for <italic>H</italic><sub>0</sub> in direct tension with other measurements<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. The redshifts of type Ia SNe in hosts with distances, already determined according to Cepheid variables<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>, were used in combination with Hubble Space Telescope imaging<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> to obtain a value 4.4<italic>σ</italic> distinct from the Planck Collaboration measurement. It is not yet clear whether this tension is due to the experimental procedures themselves—perhaps rooted in some hidden systematic error—or if it indicates a more exotic physics; additional independent measurements are necessary to assess the true source of the tension.</p><p id=\"Par4\">One of the possible independent measurement methods for <italic>H</italic><sub>0</sub> connects to the multi-messenger observation of compact binary mergers in which at least one neutron star is present. This approach has been vitalized by the recent combined detection of the neutron star merger (BNS) GW170817<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>, GRB 170817A<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>, and the optical transient AT2017gfo<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>, found in the galaxy NGC 4993 12 h after the GWs and GRB. In addition to the resulting insight into the equation of state (EOS) of neutron stars<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> and the formation of heavy elements<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>, one of the most exciting results was that of the <italic>H</italic><sub>0</sub> measurement<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. This measurement is particularly powerful because GWs are standard sirens<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>, which do not rely on a cosmic distance ladder and do not assume any cosmological model as a prior (outside of assuming general relativity is correct). The combination of the distance measurement by the GWs and redshift from the electromagnetic counterpart makes constraints on <italic>H</italic><sub>0</sub> possible<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. The distance ladder independent measurement using GWs and the host redshift was <inline-formula id=\"IEq3\"><alternatives><tex-math id=\"M5\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${H}_{0}=6{8}_{-8}^{+18}$$\\end{document}</tex-math><mml:math id=\"M6\"><mml:msub><mml:mrow><mml:mi>H</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>8</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>8</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>18</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq3.gif\"/></alternatives></inline-formula> km/s/Mpc (68.3% highest density posterior interval with a flat-in-log prior)<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>; inclusion of all O2 events reduced this uncertainty to <inline-formula id=\"IEq4\"><alternatives><tex-math id=\"M7\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${H}_{0}=6{8}_{-7}^{+14}$$\\end{document}</tex-math><mml:math id=\"M8\"><mml:msub><mml:mrow><mml:mi>H</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>8</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>7</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>14</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq4.gif\"/></alternatives></inline-formula> km/s/Mpc<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. Improvements on this measurement using more electromagnetic information, such as high angular resolution imaging of the radio counterparts<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup> or information about the internal composition of the NSs<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>, are also possible.</p><p id=\"Par5\">Here, we show that the electromagnetic evolution of kilonovae—particularly their decay rate and color evolution—can be compared to theoretical models to determine their intrinsic luminosity, making kilonovae standardizable candles<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. Along with the measured brightnesses, kilonovae can be used to measure cosmological distances. We apply two kilonovae models to sGRB light curves to constrain <italic>H</italic><sub>0</sub> to <inline-formula id=\"IEq5\"><alternatives><tex-math id=\"M9\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${H}_{0}=73.{8}_{-5.8}^{+6.3}\\ {\\rm{km}}\\ {{\\rm{s}}}^{-1}\\ {{\\rm{Mpc}}}^{-1}$$\\end{document}</tex-math><mml:math id=\"M10\"><mml:msub><mml:mrow><mml:mi>H</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>73</mml:mn><mml:mo>.</mml:mo><mml:msubsup><mml:mrow><mml:mn>8</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>5.8</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>6.3</mml:mn></mml:mrow></mml:msubsup><mml:mspace width=\"0.33em\"/><mml:mi mathvariant=\"normal\">km</mml:mi><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">Mpc</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq5.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq6\"><alternatives><tex-math id=\"M11\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${H}_{0}=71.{2}_{-3.1}^{+3.2}\\ {\\rm{km}}\\ {{\\rm{s}}}^{-1}\\ {{\\rm{Mpc}}}^{-1}$$\\end{document}</tex-math><mml:math id=\"M12\"><mml:msub><mml:mrow><mml:mi>H</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>71</mml:mn><mml:mo>.</mml:mo><mml:msubsup><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>3.1</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>3.2</mml:mn></mml:mrow></mml:msubsup><mml:mspace width=\"0.33em\"/><mml:mi mathvariant=\"normal\">km</mml:mi><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">Mpc</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq6.gif\"/></alternatives></inline-formula> for the two models, improving on what has been achieved so far with GWs alone by about a factor of 2-3.</p></sec><sec id=\"Sec2\" sec-type=\"results\"><title>Results</title><sec id=\"Sec3\"><title>Measuring <italic>H</italic><sub>0</sub> using kilonovae</title><p id=\"Par6\">Here, we focus on the kilonova observation happening in coincidence with sGRBs. This type of analysis is particularly prescient given the difficulty of searches for GW counterparts during Advanced LIGO and Advanced Virgo’s third observing run (O3)<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. AT2017gfo, synthesized by the radioactive decay of r-process elements in neutron-rich matter ejected during the merger<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>, is certainly the best sampled kilonova observation to date. Significant theoretical modeling prior to and after GW170817 has made it possible to study AT2017gfo in great detail, including measurements of the masses, velocities, and compositions of the different ejecta types. These measurements rely on models employing both simplified semi-analytical descriptions of the observational signatures<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup> and modelling using full-radiative transfer simulations<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>.</p><p id=\"Par7\">In addition to the observation of AT2017gfo, GW170817 was associated with GRB 170817A, which proved that at least some of the observed sGRBs are produced during the merger of compact binaries. This multi-messenger observation revealed the possible connection between kilonovae and sGRBs. For both cases, the GRB is then followed by an afterglow visible in X-rays, optical, and radio for days to months after the initial prompt <italic>γ</italic>-ray emission derived from the shock of the jet with the external medium. Our sGRB/kilonova sample follows ref. <sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>, which combined state-of-the-art afterglow and kilonova models, jointly fitting the observational data to determine whether there was any excess light from a kilonova. The analysis showed light curves consistent with kilonovae in the cases of GRB 150101B<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>, GRB 050709<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, GRB 160821B<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>, and GRB 060614<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. Naturally, the error bars on the kilonova parameters are larger for these objects than for GW170817, which have light curves with potentially significant contamination from the afterglow. We refer the reader to ref. <sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup> for extensive discussions of the photometric data quality and light-curve parameters and modeling. On top of these GRB observations, we will also include measurements from GW170817 (GRB 170817A)<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. While no spectra of the kilonova excesses exist and X-ray excesses may point to shock heating driving these near-infrared emission<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>, kilonovae are one possible (if not even the most likely) interpretation of the excesses. We point out that for the purpose of this article, we assume that the light curves are solely caused by a kilonova emission and neglect the possible contamination due to the sGRB afterglow. While this assumption leads to possible biases if strong sGRB afterglows would contaminate the observed data, adding an sGRB afterglow model on top of the kilonova light curves would increase the dimensionality of the analysis significantly and thus no (or only limited) constraints could be obtained.</p><p id=\"Par8\">The idea is to use techniques borrowed from the type-Ia SNe community to measure distance moduli based on kilonova light curves. We use the light-curve flux and color evolution, which do not depend on the overall luminosity, compared to kilonova models, to predict the luminosity; when combined with the measured brightness, the distance is constrained (see Methods). Here, we develop a model for the intrinsic luminosity of kilonovae based on observables, such that the luminosity can be standardized. Given the potential of multiple components and the change in color depending on the lanthanide fraction, it is useful to use kilonova models to perform the standardization. While it may be possible to standardize the kilonova luminosities based on measured properties, as is done for SN Ia cosmology measurements, in this analysis, we assume that we can use quantities inferred from the light-curve models<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>; this assumption will be testable when a sufficiently large sample of high-quality kilonovae observations are available. In this analysis, we use models from Kasen et al.<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> and Bulla<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. We note that this analysis uses some of the sampling techniques in the kilonova hypothesis testing and parameter estimation as demonstrated in refs. <sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>, but this is a fundamentally orthogonal exercise to the use of observations in one particular band to standardize the light curves based on measurements from theoretical models. The use of one of the kilonovae such as GW170817 to inform standardization could be used however with unknown systematic errors. While other models for kilonovae exist at this point, e.g., ref. <sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>, they have been shown to give similar light-curve fits to GW170817, and so the expectation is they would give similar results to those here.</p><p id=\"Par9\">We measure the distances to both GW170817 and the sGRBs in our sample using the posteriors for model parameters and the distributions for the measured parameters from the fit. For the sGRBs, we use two distance estimates based on GW170817 to inform the standardization; we use GW170817’s distance combined with the difference between the computed distance moduli to extract the distance moduli for the sGRBs (see Methods). One powerful aspect of this is that for GW170817 in particular, surface brightness fluctuations (SBF) of the host galaxy NGC 4993 (blue)<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup> pin the distance to within 1 Mpc. This requirement is similar to SN Ia measurements, where local distance ladders are required to calibrate the measurement. We also perform a comparison where we use the GW-derived posteriors to anchor the distance distribution, which results in broader but consistent posteriors (see Methods). From there, the distance modulus for each sGRB is solved for, resulting in a distribution of distances.</p></sec><sec id=\"Sec4\"><title><italic>H</italic><sub>0</sub> constraints</title><p id=\"Par10\">In addition to the study of GW170817/GRB 170817A/AT2017gfo<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>, we compute the corresponding values of <italic>H</italic><sub>0</sub> for the sGRBs<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. As described above, we use the Kasen et al.<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> and Bulla<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> models, and we assume systematic error bars with 0.1 mag and 0.25 mag errors for comparison; these are chosen to be similar to photometric errors (0.1 mag) and twice as large (0.2 mag) to establish robustness. This broadens the posterior distributions on the ejecta parameters and these systematic errors also reweight the dependence of the eventual <italic>H</italic><sub>0</sub> measurement on individual objects. Results for all kilonovae and the combined analysis are listed in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> and Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>. To determine the posterior distribution for <italic>H</italic><sub>0</sub>, we perform a simultaneous fit for two cosmology models. The first is an empirical model taken to be the following<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup><disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M13\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$D=\\frac{z\\times c}{{H}_{0}}\\left(1+\\frac{1}{2}(1-{q}_{0})z-\\frac{1}{6}(1-{q}_{0}-3{q}_{0}^{2}+{j}_{0}){z}^{2}+O({z}^{3})\\right).$$\\end{document}</tex-math><mml:math id=\"M14\"><mml:mi>D</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>z</mml:mi><mml:mo>×</mml:mo><mml:mi>c</mml:mi></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mi>H</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub></mml:mrow></mml:mfrac><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>+</mml:mo><mml:mfrac><mml:mrow><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:mfrac><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>q</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mi>z</mml:mi><mml:mo>−</mml:mo><mml:mfrac><mml:mrow><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mn>6</mml:mn></mml:mrow></mml:mfrac><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>q</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:mn>3</mml:mn><mml:msubsup><mml:mrow><mml:mi>q</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msubsup><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mi>j</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:msup><mml:mrow><mml:mi>z</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msup><mml:mo>+</mml:mo><mml:mi>O</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msup><mml:mrow><mml:mi>z</mml:mi></mml:mrow><mml:mrow><mml:mn>3</mml:mn></mml:mrow></mml:msup></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:mfenced><mml:mo>.</mml:mo></mml:math><graphic xlink:href=\"41467_2020_17998_Article_Equ1.gif\" position=\"anchor\"/></alternatives></disp-formula>The second is <italic>Λ</italic><sub>CDM</sub>, which depends on <italic>H</italic><sub>0</sub>, <italic>Ω</italic><sub><italic>m</italic></sub>, and <italic>Ω</italic><sub><italic>Λ</italic></sub>. We checked that both analyses give similar constraints on <italic>H</italic><sub>0</sub>, but do not significantly constrain the other model parameters. The systematics from using a particular kilonova model will remain, but the idea is that a sufficient sample of kilonovae will average out variations in the kilonovae. However, the relative consistency between the results of the two models<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> yields some confidence that we are still statistics dominated. We caution that the systematic uncertainty could still be significantly larger than what is assumed here, but the expectation is that the difference between the models should be resolved with future observations. The final, combined <italic>H</italic><sub>0</sub> measurement of our analysis is <inline-formula id=\"IEq7\"><alternatives><tex-math id=\"M15\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${H}_{0}=73.{8}_{-5.8}^{+6.3}\\ {\\rm{km}}\\ {{\\rm{s}}}^{-1}\\ {{\\rm{Mpc}}}^{-1}$$\\end{document}</tex-math><mml:math id=\"M16\"><mml:msub><mml:mrow><mml:mi>H</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>73</mml:mn><mml:mo>.</mml:mo><mml:msubsup><mml:mrow><mml:mn>8</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>5.8</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>6.3</mml:mn></mml:mrow></mml:msubsup><mml:mspace width=\"0.33em\"/><mml:mi mathvariant=\"normal\">km</mml:mi><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">Mpc</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq7.gif\"/></alternatives></inline-formula> for the Kasen model and <inline-formula id=\"IEq8\"><alternatives><tex-math id=\"M17\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${H}_{0}=71.{2}_{-3.1}^{+3.2}\\ {\\rm{km}}\\ {{\\rm{s}}}^{-1}\\ {{\\rm{Mpc}}}^{-1}$$\\end{document}</tex-math><mml:math id=\"M18\"><mml:msub><mml:mrow><mml:mi>H</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>71</mml:mn><mml:mo>.</mml:mo><mml:msubsup><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>3.1</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>3.2</mml:mn></mml:mrow></mml:msubsup><mml:mspace width=\"0.33em\"/><mml:mi mathvariant=\"normal\">km</mml:mi><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">Mpc</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq8.gif\"/></alternatives></inline-formula> for the Bulla model. These improve on what has been achieved so far with GWs alone by about a factor of 2–3 (see left panel of Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>); the results are consistent with both Planck CMB<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup> and SHoES Cepheid-SN distance ladder surveys analyses<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. We also performed the same analysis without GW170817, due to possible systematic uncertainties from the peculiar velocity of the host; this analysis resulted in both larger error bars than the analysis with GW170817, while still being consistent with it (see Methods).<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Summary of <italic>H</italic><sub>0</sub> results.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Kilonova</th><th>Kasen—0.25</th><th>Kasen—0.1</th><th>Bulla—0.25</th><th>Bulla—0.1</th></tr></thead><tbody><tr><td>GW170817</td><td><inline-formula id=\"IEq9\"><alternatives><tex-math id=\"M19\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$7{5}_{-8}^{+9}$$\\end{document}</tex-math><mml:math id=\"M20\"><mml:mn>7</mml:mn><mml:msubsup><mml:mrow><mml:mn>5</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>8</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>9</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq9.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq10\"><alternatives><tex-math id=\"M21\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$7{8}_{-8}^{+9}$$\\end{document}</tex-math><mml:math id=\"M22\"><mml:mn>7</mml:mn><mml:msubsup><mml:mrow><mml:mn>8</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>8</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>9</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq10.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq11\"><alternatives><tex-math id=\"M23\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$7{5}_{-7}^{+7}$$\\end{document}</tex-math><mml:math id=\"M24\"><mml:mn>7</mml:mn><mml:msubsup><mml:mrow><mml:mn>5</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>7</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>7</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq11.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq12\"><alternatives><tex-math id=\"M25\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$7{5}_{-6}^{+6}$$\\end{document}</tex-math><mml:math id=\"M26\"><mml:mn>7</mml:mn><mml:msubsup><mml:mrow><mml:mn>5</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>6</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>6</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq12.gif\"/></alternatives></inline-formula></td></tr><tr><td>GRB 060614</td><td><inline-formula id=\"IEq13\"><alternatives><tex-math id=\"M27\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$6{8}_{-16}^{+22}$$\\end{document}</tex-math><mml:math id=\"M28\"><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>8</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>16</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>22</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq13.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq14\"><alternatives><tex-math id=\"M29\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$6{6}_{-12}^{+13}$$\\end{document}</tex-math><mml:math id=\"M30\"><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>6</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>12</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>13</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq14.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq15\"><alternatives><tex-math id=\"M31\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$7{1}_{-20}^{+33}$$\\end{document}</tex-math><mml:math id=\"M32\"><mml:mn>7</mml:mn><mml:msubsup><mml:mrow><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>20</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>33</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq15.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq16\"><alternatives><tex-math id=\"M33\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$6{6}_{-11}^{+16}$$\\end{document}</tex-math><mml:math id=\"M34\"><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>6</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>11</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>16</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq16.gif\"/></alternatives></inline-formula></td></tr><tr><td>GRB 150101B</td><td><inline-formula id=\"IEq17\"><alternatives><tex-math id=\"M35\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$6{6}_{-20}^{+40}$$\\end{document}</tex-math><mml:math id=\"M36\"><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>6</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>20</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>40</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq17.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq18\"><alternatives><tex-math id=\"M37\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$7{6}_{-20}^{+39}$$\\end{document}</tex-math><mml:math id=\"M38\"><mml:mn>7</mml:mn><mml:msubsup><mml:mrow><mml:mn>6</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>20</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>39</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq18.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq19\"><alternatives><tex-math id=\"M39\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$8{9}_{-33}^{+54}$$\\end{document}</tex-math><mml:math id=\"M40\"><mml:mn>8</mml:mn><mml:msubsup><mml:mrow><mml:mn>9</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>33</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>54</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq19.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq20\"><alternatives><tex-math id=\"M41\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$9{6}_{-38}^{+51}$$\\end{document}</tex-math><mml:math id=\"M42\"><mml:mn>9</mml:mn><mml:msubsup><mml:mrow><mml:mn>6</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>38</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>51</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq20.gif\"/></alternatives></inline-formula></td></tr><tr><td>GRB 050709</td><td><inline-formula id=\"IEq21\"><alternatives><tex-math id=\"M43\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$7{8}_{-19}^{+29}$$\\end{document}</tex-math><mml:math id=\"M44\"><mml:mn>7</mml:mn><mml:msubsup><mml:mrow><mml:mn>8</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>19</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>29</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq21.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq22\"><alternatives><tex-math id=\"M45\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$6{3}_{-11}^{+14}$$\\end{document}</tex-math><mml:math id=\"M46\"><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>3</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>11</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>14</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq22.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq23\"><alternatives><tex-math id=\"M47\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$6{2}_{-7}^{+9}$$\\end{document}</tex-math><mml:math id=\"M48\"><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>7</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>9</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq23.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq24\"><alternatives><tex-math id=\"M49\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$6{1}_{-6}^{+8}$$\\end{document}</tex-math><mml:math id=\"M50\"><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>6</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>8</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq24.gif\"/></alternatives></inline-formula></td></tr><tr><td>GRB 160821B</td><td><inline-formula id=\"IEq25\"><alternatives><tex-math id=\"M51\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$6{4}_{-10}^{+11}$$\\end{document}</tex-math><mml:math id=\"M52\"><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>4</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>10</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>11</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq25.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq26\"><alternatives><tex-math id=\"M53\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$6{3}_{-9}^{+12}$$\\end{document}</tex-math><mml:math id=\"M54\"><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>3</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>9</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>12</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq26.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq27\"><alternatives><tex-math id=\"M55\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$6{2}_{-6}^{+8}$$\\end{document}</tex-math><mml:math id=\"M56\"><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>6</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>8</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq27.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq28\"><alternatives><tex-math id=\"M57\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$6{2}_{-6}^{+8}$$\\end{document}</tex-math><mml:math id=\"M58\"><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>6</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>8</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq28.gif\"/></alternatives></inline-formula></td></tr><tr><td>Combined</td><td><inline-formula id=\"IEq29\"><alternatives><tex-math id=\"M59\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$71.{9}_{-5.5}^{+6.4}$$\\end{document}</tex-math><mml:math id=\"M60\"><mml:mn>71</mml:mn><mml:mo>.</mml:mo><mml:msubsup><mml:mrow><mml:mn>9</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>5.5</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>6.4</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq29.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq30\"><alternatives><tex-math id=\"M61\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$73.{8}_{-5.8}^{+6.3}$$\\end{document}</tex-math><mml:math id=\"M62\"><mml:mn>73</mml:mn><mml:mo>.</mml:mo><mml:msubsup><mml:mrow><mml:mn>8</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>5.8</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>6.3</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq30.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq31\"><alternatives><tex-math id=\"M63\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$69.{9}_{-3.7}^{+3.6}$$\\end{document}</tex-math><mml:math id=\"M64\"><mml:mn>69</mml:mn><mml:mo>.</mml:mo><mml:msubsup><mml:mrow><mml:mn>9</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>3.7</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>3.6</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq31.gif\"/></alternatives></inline-formula></td><td><inline-formula id=\"IEq32\"><alternatives><tex-math id=\"M65\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$71.{2}_{-3.1}^{+3.2}$$\\end{document}</tex-math><mml:math id=\"M66\"><mml:mn>71</mml:mn><mml:mo>.</mml:mo><mml:msubsup><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>3.1</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>3.2</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq32.gif\"/></alternatives></inline-formula></td></tr></tbody></table><table-wrap-foot><p>We use units of km s<sup>−1</sup> Mpc<sup>−1</sup>. Individual rows refer to the GRB/GW observations and individual columns to the Kasen et al. and Bulla et al. model assuming a 0.25 and 0.1 mag systematic uncertainty. We note that the GRB individual <italic>H</italic><sub>0</sub> measurements use the SBF of the host galaxy NGC 4993<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup> to pin the distance.</p></table-wrap-foot></table-wrap><fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Posterior distributions for <italic>H</italic><sub>0</sub> for individual events.</title><p>We show GW170817, GRB 060614, GRB 150101B, GRB 160821B, GRB 050709, and their combined posteriors. Fig. <bold>a</bold> is the Kasen<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> model with 0.1 mag errors, <bold>b</bold> is the Bulla<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> model with 0.1 mag errors, <bold>c</bold> is is the Kasen model with 0.25 mag errors, and <bold>d</bold> is is the Bulla model with 0.25 mag errors. The 1- and 2-<italic>σ</italic> regions determined by the superluminal motion measurement from the radio counterpart (blue)<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>, Planck CMB (TT,TE,EE+lowP+lensing) (green)<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup> and SHoES Cepheid-SN distance ladder surveys (orange)<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> are also depicted as vertical bands.</p></caption><graphic xlink:href=\"41467_2020_17998_Fig1_HTML\" id=\"d30e2069\"/></fig><fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Summary of posterior distributions for <italic>H</italic><sub>0</sub>.</title><p>In <bold>a</bold>, we show the GW-only analysis for GW170817, in addition to the Kasen<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> and Bulla<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> model analyses with 0.1 mag and 0.25 mag errors from this letter. In addition, the 1- and 2-<italic>σ</italic> regions determined by the superluminal motion measurement from the radio counterpart (blue)<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>, Planck CMB (TT,TE,EE+lowP+lensing) (green)<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>, and SHoES Cepheid-SN distance ladder surveys (orange)<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> are also depicted as vertical bands. In <bold>b</bold>, we use GRB 060614 and the Kasen<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> model with 0.1 mag errors. In addition to the standard SBF measurement in the letter, we show an analysis where we systematically add 0.1 <italic>M</italic><sub>⊙</sub> to <italic>M</italic><sub>ej</sub> and 0.5 decades to <italic>X</italic><sub>lan</sub>. Finally, we show a distribution where we replace the standard SBF measurement with the distance measurement from the GW170817 high spin posteriors.</p></caption><graphic xlink:href=\"41467_2020_17998_Fig2_HTML\" id=\"d30e2135\"/></fig></p><p id=\"Par11\">We can use our results to construct a so-called Hubble–Lemai^tre Diagram (see Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>), where we plot distance modulus vs. redshift for the observed kilonovae. The main benefit of plotting distance modulus instead of apparent magnitude is that it is independent of the source. For comparison, the green dashed line shows <italic>Λ</italic><sub>CDM</sub>, showing the consistency in the results. We include a Hubble–Lemai^tre residual panel to show the error bars. The error bars are, of course, large relative to SNe samples, where <italic>σ</italic> ~ 0.1 mag.<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Hubble–Lemai^tre Diagram for the kilonova analysis.</title><p>We plot distance modulus vs. redshift. The error bars indicate the extent of the posterior samples. The green dashed line shows <italic>Λ</italic><sub>CDM</sub>. We include a Hubble–Lemai^tre residual panel to show the residuals.</p></caption><graphic xlink:href=\"41467_2020_17998_Fig3_HTML\" id=\"d30e2164\"/></fig></p></sec></sec><sec id=\"Sec5\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par12\">We perform a few different tests to assess the systematics of the analysis (see right panel of Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). The first is where we systematically change the estimated values for the ejecta mass and lanthanide fractions from the light-curve analysis to assess the dependence on those values. Based on ref. <sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>, we take values of 0.1 <italic>M</italic><sub>⊙</sub> to add to the ejecta mass and 0.5 decades to add to <italic>X</italic><sub>lan</sub>, corresponding to the approximate size of the error bars on those parameters. We show these curves along with the original analysis of GRB 060614 on the right of Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>. While the shift in the derived distribution is clear, in particular for <italic>X</italic><sub>lan</sub>, the distributions still remain consistent with one another; we find distributions of <inline-formula id=\"IEq33\"><alternatives><tex-math id=\"M67\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$6{7}_{-11}^{+13}$$\\end{document}</tex-math><mml:math id=\"M68\"><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>7</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>11</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>13</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq33.gif\"/></alternatives></inline-formula> for SBF, <inline-formula id=\"IEq34\"><alternatives><tex-math id=\"M69\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$6{7}_{-15}^{+19}$$\\end{document}</tex-math><mml:math id=\"M70\"><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>7</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>15</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>19</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq34.gif\"/></alternatives></inline-formula> for SBF—<italic>M</italic><sub>ej</sub>, and <inline-formula id=\"IEq35\"><alternatives><tex-math id=\"M71\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$6{2}_{-11}^{+13}$$\\end{document}</tex-math><mml:math id=\"M72\"><mml:mn>6</mml:mn><mml:msubsup><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>11</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>13</mml:mn></mml:mrow></mml:msubsup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq35.gif\"/></alternatives></inline-formula> for SBF—<italic>X</italic><sub>lan</sub>. This indicates that we are still dominated by statistical errors. As a further test, we show a distribution where we replace the standard SBF measurement with the distance measurement from the high spin posteriors presented in ref. <sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. Given the wide posterior distributions, this leads to a broadening of the <italic>H</italic><sub>0</sub> measurement, but again, the distributions are consistent with one another. In the following, we will use the SBF measurement as the distance anchor for the analysis, but in the future, the GW based distributions may be appropriate to minimize systematics.</p><p id=\"Par13\">These results indicate that continued searches for and the analyses of sGRB afterglows have significant science benefits. In particular, our analysis shows that <italic>H</italic><sub>0</sub> measurements may be improved given more detections of sGRB afterglows and their peak luminosities, requiring detections as early as possible to be most usable. These objects also have observational challenges, at least those identified by the Fermi Gamma-ray Burst Monitor<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>, given the large sky areas that require coverage and the candidate vetting that follows<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. Observations of more kilonovae will significantly constrain the kilonova peak luminosity distribution<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>, which is clearly a driving force of this analysis. This will likely depend on both whether the original system producing a kilonova is a binary neutron star or neutron star-black hole, in addition to the inclination angle influence on the kilonova characteristics. Kilonovae, in particular, are less bright than SNe Ia, and could be more useful for constraining other distance indicators rather than directly as cosmological probes.</p></sec><sec id=\"Sec6\"><title>Methods</title><sec id=\"Sec7\"><title>Kilonova analysis</title><p id=\"Par14\">In addition to the photometric error bars arising from the measured signal to noise, the modeling also has associated errors, which we will add in quadrature. Building upon the first analysis<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>, where we employed a Gaussian Process Regression (GPR) based interpolation<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup> to create a surrogate model of the model of Kasen et al.<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> for arbitrary ejecta properties<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>, we also use the model of Bulla<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> for comparison. The idea is that we can use these models to derive constraints on possible kilonova light curves. We chose to have large prior boundaries that allows to describe both, kilonova produced by BNSs and by BHNS systems. For the Kasen et al. model, each light curve depends on the ejecta mass <italic>M</italic><sub>ej</sub>, the mass fraction of lanthanides <italic>X</italic><sub>lan</sub>, and the ejecta velocity <italic>v</italic><sub>ej</sub>. We use flat priors for each parameter covering: <inline-formula id=\"IEq36\"><alternatives><tex-math id=\"M73\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-3\\le {\\mathrm{log}\\,}_{10}({M}_{{\\rm{ej}}}/{M}_{\\odot })\\le -\\!\\!1$$\\end{document}</tex-math><mml:math id=\"M74\"><mml:mo>−</mml:mo><mml:mn>3</mml:mn><mml:mo>≤</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi></mml:mrow></mml:msub><mml:mo>/</mml:mo><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mo>⊙</mml:mo></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>≤</mml:mo><mml:mo>−</mml:mo><mml:mspace width=\"0.3em\"/><mml:mspace width=\"0.3em\"/><mml:mn>1</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq36.gif\"/></alternatives></inline-formula>, 0 ≤ <italic>v</italic><sub>ej</sub> ≤ 0.3 <italic>c</italic>, and <inline-formula id=\"IEq37\"><alternatives><tex-math id=\"M75\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-9\\le {\\mathrm{log}\\,}_{10}({X}_{{\\rm{lan}}})\\le -\\!\\!1$$\\end{document}</tex-math><mml:math id=\"M76\"><mml:mo>−</mml:mo><mml:mn>9</mml:mn><mml:mo>≤</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>X</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">lan</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>≤</mml:mo><mml:mo>−</mml:mo><mml:mspace width=\"0.3em\"/><mml:mspace width=\"0.3em\"/><mml:mn>1</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq37.gif\"/></alternatives></inline-formula>. For the 2D<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> model, each light curve depends on the ejecta mass <italic>M</italic><sub>ej</sub>, the half-opening angle of the lanthanide-rich component Φ (with Φ = 0 and Φ = 90° corresponding to one-component lanthanide-free and lanthanide-rich models, respectively) and the observer viewing angle <italic>θ</italic><sub>obs</sub> (with <inline-formula id=\"IEq38\"><alternatives><tex-math id=\"M77\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\cos {\\theta }_{{\\rm{obs}}}=0$$\\end{document}</tex-math><mml:math id=\"M78\"><mml:mi>cos</mml:mi><mml:msub><mml:mrow><mml:mi>θ</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">obs</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>0</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq38.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq39\"><alternatives><tex-math id=\"M79\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\cos {\\theta }_{{\\rm{obs}}}=1$$\\end{document}</tex-math><mml:math id=\"M80\"><mml:mi>cos</mml:mi><mml:msub><mml:mrow><mml:mi>θ</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">obs</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq39.gif\"/></alternatives></inline-formula> corresponding to a system viewed edge-on and face-on, respectively). We again use flat priors for each parameter covering: <inline-formula id=\"IEq40\"><alternatives><tex-math id=\"M81\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-3\\le {\\mathrm{log}\\,}_{10}({M}_{{\\rm{ej}}}/{M}_{\\odot })\\le -\\!\\!1$$\\end{document}</tex-math><mml:math id=\"M82\"><mml:mo>−</mml:mo><mml:mn>3</mml:mn><mml:mo>≤</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi></mml:mrow></mml:msub><mml:mo>/</mml:mo><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mo>⊙</mml:mo></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>≤</mml:mo><mml:mo>−</mml:mo><mml:mspace width=\"0.3em\"/><mml:mspace width=\"0.3em\"/><mml:mn>1</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq40.gif\"/></alternatives></inline-formula>, 15° ≤ Φ ≤ 30°, and 0 ≤ <italic>θ</italic><sub>obs</sub> ≤ 15. We restrict 0° ≤ <italic>θ</italic><sub>obs</sub> ≤ 15° for the sGRB analysis because the viewing angle is much closer to the polar axis than for GW170817<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. By observing the sGRB, we can assume that we are near or within the opening angle of the sGRB jet, which is taken to be less than 15°<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref>–<xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. Because the distribution of the viewing angles of kilonovae from sGRBs are likely quite anisotropic, we would expect this to create an appearance of changing lanthanide fractions as the viewing angle changed for spherical geometries, such as in the model of Kasen et al.<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>; this could cause a bias in the Hubble Constant measurements using spherical models. Asymmetric models such as that of Bulla<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> overcome this potential issue.</p><p id=\"Par15\">Compared to the models presented in ref. <sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>, those used here adopt thermalization efficiencies from ref. <sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup> and estimate the temperature at each time from the mean intensity of the radiation field in each region of the ejecta. In addition, we assume that no mass is located below <inline-formula id=\"IEq41\"><alternatives><tex-math id=\"M83\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${v}_{\\min }=0.025$$\\end{document}</tex-math><mml:math id=\"M84\"><mml:msub><mml:mrow><mml:mi>v</mml:mi></mml:mrow><mml:mrow><mml:mi>min</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>0.025</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq41.gif\"/></alternatives></inline-formula> c (where <italic>c</italic> is the speed of light) following ref. <sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>. Studying the predictions of two independent models with physical assumptions allows us to estimate the systematic uncertainties of our analysis. More specifically, some of these physical assumptions (e.g., spherical or axial symmetry) may give the false impression of special kilonovae properties. Doing this study with multiple different models is therefore critical to reveal such systematics.</p><p id=\"Par16\">In addition to the attempt of providing a measure of the systematic uncertainty, our parameter ranges for both models are very agnostic in the sense that we do not restrict us to particular parameter ranges that are predicted by numerical-relativity simulations<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>, in fact, the proposed method works for an even larger parameter space, thus, it seems to be very general approach that can be employed for a variety of future events.</p></sec><sec id=\"Sec8\"><title>Light curves</title><p id=\"Par17\">All data presented here were compiled from public sources and collated as presented in ref. <sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> for GW170817 and ref. <sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup> for the remaining SGRBs. For GRB 150101B, data can be found in refs. <sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>, for GRB 050709, data can be found in refs. <sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR48\">48</xref>–<xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>, for GRB 160821B, data can be found in ref. <sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref>,<xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>, and in GRB 060614, data can be found in refs. <sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR52\">52</xref>,<xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. We remind the reader that for the SGRB analyses, we take the peak <italic>r</italic>-band observation for comparison, as opposed to the <italic>K</italic>-band as used in the GW170817 analysis due to the sparsity of available light curves in that band. We perform the parameter fits using the entire light curves, assuming the light curves are the result of kilonovae. While GW170817 remains the only completely unambiguous kilonova detection, ref. <sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup> and other analyses, such as refs. <sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref>,<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup> for GRB 160821B, have indicated that these SGRBs are consistent with having kilonovae present. On the other hand, GRB 060614 in particular has a number of possible interpretations, including a tidal disruption event<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>, a WD-NS system<sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup> or a long GRB<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup>. We include it for completeness, but further analysis may require its removal in future analysis.</p></sec><sec id=\"Sec9\"><title><italic>H</italic><sub>0</sub> analysis</title><p id=\"Par18\">The underpinning assumption for this paper is that kilonovae can be standardized using models for their luminosity and color evolution, which was first explored in ref. <sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. While in principle this method could be used for other transient types which are more numerous, including core collapse or superluminous supernovae, models for their emission properties are not nearly developed as those for kilonovae. This requires, of course, believable models that we expect encode the evolution of the kilonovae we measure. Metzger<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup> showed that the semi-analytic methods of Arnett<sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup> already reproduce much of the expected physics for these systems, and these models are sufficient for predicting the GW170817 lightcurves (e.g., ref. <sup><xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup>). For radioactivity models<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>, it assumes a power term taken to be <italic>P</italic> ∝ <italic>t</italic><sup><italic>β</italic></sup>, and is also dependent on the energy in the system (related to the velocity <italic>v</italic>), the ejecta mass <italic>M</italic>, and the opacity <italic>κ</italic>. Under these assumptions, the luminosity as a function of time evolves as <inline-formula id=\"IEq42\"><alternatives><tex-math id=\"M85\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\mathrm{log}\\,(L(t)/{L}_{0})\\propto -t/\\tau$$\\end{document}</tex-math><mml:math id=\"M86\"><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>L</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>t</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>/</mml:mo><mml:msub><mml:mrow><mml:mi>L</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>∝</mml:mo><mml:mo>−</mml:mo><mml:mi>t</mml:mi><mml:mo>/</mml:mo><mml:mi>τ</mml:mi></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq42.gif\"/></alternatives></inline-formula>, where <italic>τ</italic> is the diffusion timescale <inline-formula id=\"IEq43\"><alternatives><tex-math id=\"M87\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\tau \\propto {\\left(\\frac{\\kappa M}{v}\\right)}^{1/2}$$\\end{document}</tex-math><mml:math id=\"M88\"><mml:mi>τ</mml:mi><mml:mo>∝</mml:mo><mml:msup><mml:mrow><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mfrac><mml:mrow><mml:mi>κ</mml:mi><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi>v</mml:mi></mml:mrow></mml:mfrac></mml:mrow></mml:mfenced></mml:mrow><mml:mrow><mml:mn>1</mml:mn><mml:mo>/</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq43.gif\"/></alternatives></inline-formula><sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. Numerical models build upon these semi-analytic models with line-based opacities, radiative transport, thermalization efficiency and other parameters to make these models further realistic, although the key point that luminosity depends on these measurable parameters remains.</p><p id=\"Par19\">We now explain explicitly how to derive the standardization. For each model in the simulation set, each of which corresponds to a specific set of intrinsic parameters, we compute the peak magnitude in each passband. We choose <italic>K</italic>-band for the GW170817 analysis when analyzed on its own, given that the transient was observable for the longest in the near-infrared; we chose <italic>r</italic>-band for the sGRB analyses, including in the standardization of GW170817 in the magnitude differences computed below, as it was the band most commonly imaged between the various sGRBs. In this way, we have for both the Kasen et al.<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> model and the Bulla<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> model, a grid of the intrinsic parameters and the peak magnitude associated with the simulation. To map the intrinsic parameters to a peak magnitude, we use a GPR based interpolation (similar to the light-curve interpolation described above)<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>.</p><p id=\"Par20\">For the Kasen et al.<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> model, the equation takes the form<disp-formula id=\"Equ2\"><label>2</label><alternatives><tex-math id=\"M89\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${M}_{{\\rm{r}} = {{\\rm{r}}}_{\\max }}=f({\\mathrm{log}\\,}_{10}{M}_{{\\rm{ej}}},{v}_{{\\rm{ej}}},{\\mathrm{log}\\,}_{10}{X}_{{\\rm{lan}}})$$\\end{document}</tex-math><mml:math id=\"M90\"><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">r</mml:mi><mml:mo>=</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">r</mml:mi></mml:mrow><mml:mrow><mml:mi>max</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mi>f</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi>v</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>X</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">lan</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:math><graphic xlink:href=\"41467_2020_17998_Article_Equ2.gif\" position=\"anchor\"/></alternatives></disp-formula>and for the Bulla<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> model,<disp-formula id=\"Equ3\"><label>3</label><alternatives><tex-math id=\"M91\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${M}_{{\\rm{r}} = {{\\rm{r}}}_{\\max }}=f({\\mathrm{log}\\,}_{10}{M}_{{\\rm{ej}}},\\Phi ,{\\theta }_{{\\rm{obs}}})$$\\end{document}</tex-math><mml:math id=\"M92\"><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">r</mml:mi><mml:mo>=</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">r</mml:mi></mml:mrow><mml:mrow><mml:mi>max</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mi>f</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:mi mathvariant=\"normal\">Φ</mml:mi><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi>θ</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">obs</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:math><graphic xlink:href=\"41467_2020_17998_Article_Equ3.gif\" position=\"anchor\"/></alternatives></disp-formula>where <italic>f</italic> is a GPR based interpolation and the parameters are inferred quantities based on the light-curve fits. The idea is that these magnitude fits are related to the (observed) apparent magnitudes by the distance modulus <inline-formula id=\"IEq44\"><alternatives><tex-math id=\"M93\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\mu =5{\\mathrm{log}\\,}_{10}(\\frac{D}{10{\\rm{pc}}})$$\\end{document}</tex-math><mml:math id=\"M94\"><mml:mi>μ</mml:mi><mml:mo>=</mml:mo><mml:mn>5</mml:mn><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:mi>D</mml:mi></mml:mrow><mml:mrow><mml:mn>10</mml:mn><mml:mi mathvariant=\"normal\">pc</mml:mi></mml:mrow></mml:mfrac></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq44.gif\"/></alternatives></inline-formula>.<disp-formula id=\"Equ4\"><label>4</label><alternatives><tex-math id=\"M95\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$M=m-\\mu .$$\\end{document}</tex-math><mml:math id=\"M96\"><mml:mi>M</mml:mi><mml:mo>=</mml:mo><mml:mi>m</mml:mi><mml:mo>−</mml:mo><mml:mi>μ</mml:mi><mml:mo>.</mml:mo></mml:math><graphic xlink:href=\"41467_2020_17998_Article_Equ4.gif\" position=\"anchor\"/></alternatives></disp-formula>We can evaluate the performance of these fits by comparing the peak K-band magnitudes to those predicted as a function of ejecta mass. Figure <xref rid=\"Fig4\" ref-type=\"fig\">4</xref> uses the simulation set for the Bulla<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> model, showing the performance as a function of viewing and opening angle. While the performance tends to be worst for the most extreme viewing and opening angles, the fact that the Gaussian Process estimates errors, which change across the parameter space help to sufficiently reproduce the behavior.<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>Fit of Eq. (<xref rid=\"Equ3\" ref-type=\"\">3</xref>) for the models in ref. <sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>.</title><p>We vary the opening and viewing angles of the employed simulation set in <italic>r</italic>-band. The black points are the fits (with the measured error bar from the Chi-squared) while the blue points are computed directly from the models in ref. <sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>.</p></caption><graphic xlink:href=\"41467_2020_17998_Fig4_HTML\" id=\"d30e3118\"/></fig></p><p id=\"Par21\">We use the fit of Eqs. (<xref rid=\"Equ2\" ref-type=\"\">2</xref>) and (<xref rid=\"Equ3\" ref-type=\"\">3</xref>) and apply it to the posteriors on the model parameters, as were derived previously for GW170817<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup> and the sGRBs<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. Applying directly to the GW170817 posteriors, we show the estimated distances for both models in Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>, consistent with other measurements of the host galaxy, e.g., refs. <sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR62\">62</xref>,<xref ref-type=\"bibr\" rid=\"CR63\">63</xref></sup>, and the GW posteriors<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>.<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>Posterior distributions for distance to GW170817.</title><p>We show the results of the GW-only analyses (high spin)<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>, the Kasen et al.<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> and the Bulla<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> kilonova analysis for both systematic errors assumed. The 1- and 2-<italic>σ</italic> regions determined by the surface brightness fluctuations (SBF) of NGC 4993 (blue)<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup> and the Fundamental Plane (FP) of E and S0 galaxies (red)<sup><xref ref-type=\"bibr\" rid=\"CR62\">62</xref></sup> are also depicted as vertical bands.</p></caption><graphic xlink:href=\"41467_2020_17998_Fig5_HTML\" id=\"d30e3190\"/></fig></p><p id=\"Par22\">To apply the model to the sGRB light curves, we combine Eqs. (<xref rid=\"Equ2\" ref-type=\"\">2</xref>) and (<xref rid=\"Equ4\" ref-type=\"\">4</xref>) as<disp-formula id=\"Equ5\"><label>5</label><alternatives><tex-math id=\"M97\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\mu =m-f({\\mathrm{log}\\,}_{10}{M}_{{\\rm{ej}}},{v}_{{\\rm{ej}}},{\\mathrm{log}\\,}_{10}{X}_{{\\rm{lan}}})$$\\end{document}</tex-math><mml:math id=\"M98\"><mml:mi>μ</mml:mi><mml:mo>=</mml:mo><mml:mi>m</mml:mi><mml:mo>−</mml:mo><mml:mi>f</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi>v</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>X</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">lan</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:math><graphic xlink:href=\"41467_2020_17998_Article_Equ5.gif\" position=\"anchor\"/></alternatives></disp-formula>and Eqs. (<xref rid=\"Equ3\" ref-type=\"\">3</xref>) and (<xref rid=\"Equ4\" ref-type=\"\">4</xref>) as<disp-formula id=\"Equ6\"><label>6</label><alternatives><tex-math id=\"M99\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\mu =m-f({\\mathrm{log}\\,}_{10}{M}_{{\\rm{ej}}},\\Phi ,{\\theta }_{{\\rm{obs}}}).$$\\end{document}</tex-math><mml:math id=\"M100\"><mml:mi>μ</mml:mi><mml:mo>=</mml:mo><mml:mi>m</mml:mi><mml:mo>−</mml:mo><mml:mi>f</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:mi mathvariant=\"normal\">Φ</mml:mi><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi>θ</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">obs</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>.</mml:mo></mml:math><graphic xlink:href=\"41467_2020_17998_Article_Equ6.gif\" position=\"anchor\"/></alternatives></disp-formula>We then take the difference between two observations,<disp-formula id=\"Equ7\"><label>7</label><alternatives><tex-math id=\"M101\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\begin{array}{ccc}{\\mu }_{1}-{\\mu }_{2}&=&{m}_{1}-{m}_{2}+f({\\mathrm{log}\\,}_{10}{M}_{{\\rm{ej}},1},{v}_{{\\rm{ej}},1},{\\mathrm{log}\\,}_{10}{X}_{{\\rm{lan}},1})\\\\ &&-f({\\mathrm{log}\\,}_{10}{M}_{{\\rm{ej}},2},{v}_{{\\rm{ej}},2},{\\mathrm{log}\\,}_{10}{X}_{{\\rm{lan}},2}).\\end{array}$$\\end{document}</tex-math><mml:math id=\"M102\"><mml:mtable><mml:mtr><mml:mtd columnalign=\"center\"><mml:msub><mml:mrow><mml:mi>μ</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>μ</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msub></mml:mtd><mml:mtd columnalign=\"center\"><mml:mo>=</mml:mo></mml:mtd><mml:mtd columnalign=\"center\"><mml:msub><mml:mrow><mml:mi>m</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>m</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:mi>f</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi><mml:mo>,</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi>v</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi><mml:mo>,</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>X</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">lan</mml:mi><mml:mo>,</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd columnalign=\"center\"/><mml:mtd columnalign=\"center\"/><mml:mtd columnalign=\"center\"><mml:mo>−</mml:mo><mml:mi>f</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi><mml:mo>,</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi>v</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi><mml:mo>,</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>X</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">lan</mml:mi><mml:mo>,</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>.</mml:mo></mml:mtd></mml:mtr></mml:mtable></mml:math><graphic xlink:href=\"41467_2020_17998_Article_Equ7.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p id=\"Par23\">and<disp-formula id=\"Equ8\"><label>8</label><alternatives><tex-math id=\"M103\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\begin{array}{ccc}{\\mu }_{1}-{\\mu }_{2}&=&{m}_{1}-{m}_{2}+f({\\mathrm{log}\\,}_{10}{M}_{{\\rm{ej}},1},{\\Phi }_{{\\rm{1}}},{\\theta }_{{\\rm{obs}},1})\\\\ &&-f({\\mathrm{log}\\,}_{10}{M}_{{\\rm{ej}},2},{\\Phi }_{{\\rm{2}}},{\\theta }_{{\\rm{obs}},2}).\\end{array}$$\\end{document}</tex-math><mml:math id=\"M104\"><mml:mtable><mml:mtr><mml:mtd columnalign=\"center\"><mml:msub><mml:mrow><mml:mi>μ</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>μ</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msub></mml:mtd><mml:mtd columnalign=\"center\"><mml:mo>=</mml:mo></mml:mtd><mml:mtd columnalign=\"center\"><mml:msub><mml:mrow><mml:mi>m</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>m</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:mi>f</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi><mml:mo>,</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">Φ</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">1</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi>θ</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">obs</mml:mi><mml:mo>,</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd columnalign=\"center\"/><mml:mtd columnalign=\"center\"/><mml:mtd columnalign=\"center\"><mml:mo>−</mml:mo><mml:mi>f</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi><mml:mo>,</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">Φ</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">2</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo><mml:msub><mml:mrow><mml:mi>θ</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">obs</mml:mi><mml:mo>,</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>.</mml:mo></mml:mtd></mml:mtr></mml:mtable></mml:math><graphic xlink:href=\"41467_2020_17998_Article_Equ8.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p id=\"Par24\">Combined with either a SBF-based measurement of host galaxy NGC 4993<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup> or the GW-derived posteriors to anchor the distance distribution for GW170817, this equation is used to measure the sGRB distance moduli (see Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref> for GRB 060614 as an example). We note that the overall luminosity of the light curve is allowed to vary arbitrarily, which adds a linear offset. But, this offset does not affect the color evolution, which is mostly determining the intrinsic parameter distributions. Therefore, we are able to extract both, the intrinsic parameters and the distance to the source, which is a crucial prerequisite for our study.<fig id=\"Fig6\"><label>Fig. 6</label><caption><title>Corner plots for the model parameters.</title><p>We show them for both Kasen et al.<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> model (<bold>a</bold>) and the Bulla<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> model (<bold>b</bold>) for GRB 060614. We also show the inferred distance modulus in the last column.</p></caption><graphic xlink:href=\"41467_2020_17998_Fig6_HTML\" id=\"d30e3700\"/></fig></p></sec><sec id=\"Sec10\"><title><italic>H</italic><sub>0</sub> analysis without GW170817</title><p id=\"Par25\">In the main text, we included GW170817 when combining the posterior distributions for the Hubble constant. Although the combined analysis is more constraining, the inclusion of GW170817 increases the systematic uncertainties as its H0 measurement depends on the peculiar velocity of the host. This problem will remain for all close-by kilonovae. Due to their distance, the sGRB analysis is not affected nearly as much by the peculiar motion of local galaxies. Removing GW170817’s Hubble Constant constraints yields measurements of <inline-formula id=\"IEq45\"><alternatives><tex-math id=\"M105\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${H}_{0}=71.{9}_{-7.7}^{+8.2}\\ {\\rm{km}}\\ {{\\rm{s}}}^{-1}\\ {{\\rm{Mpc}}}^{-1}$$\\end{document}</tex-math><mml:math id=\"M106\"><mml:msub><mml:mrow><mml:mi>H</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>71</mml:mn><mml:mo>.</mml:mo><mml:msubsup><mml:mrow><mml:mn>9</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>7.7</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>8.2</mml:mn></mml:mrow></mml:msubsup><mml:mspace width=\"0.33em\"/><mml:mi mathvariant=\"normal\">km</mml:mi><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">Mpc</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq45.gif\"/></alternatives></inline-formula> for the Kasen model and <inline-formula id=\"IEq46\"><alternatives><tex-math id=\"M107\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${H}_{0}=68.{2}_{-4.3}^{+4.6}\\ {\\rm{km}}\\ {{\\rm{s}}}^{-1}\\ {{\\rm{Mpc}}}^{-1}$$\\end{document}</tex-math><mml:math id=\"M108\"><mml:msub><mml:mrow><mml:mi>H</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mn>68</mml:mn><mml:mo>.</mml:mo><mml:msubsup><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>4.3</mml:mn></mml:mrow><mml:mrow><mml:mo>+</mml:mo><mml:mn>4.6</mml:mn></mml:mrow></mml:msubsup><mml:mspace width=\"0.33em\"/><mml:mi mathvariant=\"normal\">km</mml:mi><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">s</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup><mml:mspace width=\"0.33em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">Mpc</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msup></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq46.gif\"/></alternatives></inline-formula> for the Bulla model (see Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>); these measurements both have larger error bars than the analysis with GW170817, while still being consistent with it.<fig id=\"Fig7\"><label>Fig. 7</label><caption><title>Posterior distributions for <italic>H</italic><sub>0</sub> for individual events without GW170817.</title><p>We show them for GRB 060614, GRB 150101B, GRB 160821B, GRB 050709, and their combined posteriors are also shown. Fig. <bold>a</bold> is the Kasen<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> model with 0.1 mag errors, <bold>b</bold> is the Bulla<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> model with 0.1 mag errors, <bold>c</bold> is is the Kasen model with 0.25 mag errors, and <bold>d</bold> is is the Bulla model with 0.25 mag errors. The 1- and 2-<italic>σ</italic> regions determined by the superluminal motion measurement from the radio counterpart (blue)<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>, Planck CMB (TT,TE,EE+lowP+lensing) (green)<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup> and SHoES Cepheid-SN distance ladder surveys (orange)<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> are also depicted as vertical bands.</p></caption><graphic xlink:href=\"41467_2020_17998_Fig7_HTML\" id=\"d30e3880\"/></fig></p><p id=\"Par26\">We discussed our prior choices in Section 5. In particular, we choose large prior boundaries that allows to describe both, kilonova produced by BNSs and by BHNS systems, and in particular for ejecta mass, <inline-formula id=\"IEq47\"><alternatives><tex-math id=\"M109\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-3\\le {\\mathrm{log}\\,}_{10}({M}_{{\\rm{ej}}}/{M}_{\\odot })\\le -\\!\\!1$$\\end{document}</tex-math><mml:math id=\"M110\"><mml:mo>−</mml:mo><mml:mn>3</mml:mn><mml:mo>≤</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi></mml:mrow></mml:msub><mml:mo>/</mml:mo><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mo>⊙</mml:mo></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>≤</mml:mo><mml:mo>−</mml:mo><mml:mspace width=\"0.3em\"/><mml:mspace width=\"0.3em\"/><mml:mn>1</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq47.gif\"/></alternatives></inline-formula>. We can compare this choice to a few other distributions. For example, taking the fit of ref. <sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup> and applying them to the posteriors of GW170817<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup> and GW190425<sup><xref ref-type=\"bibr\" rid=\"CR64\">64</xref></sup>, we see a bi-modal distribution, the former peaked near to <inline-formula id=\"IEq48\"><alternatives><tex-math id=\"M111\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathrm{log}\\,}_{10}({M}_{ej}/{M}_{\\odot })=-1.3$$\\end{document}</tex-math><mml:math id=\"M112\"><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi>e</mml:mi><mml:mi>j</mml:mi></mml:mrow></mml:msub><mml:mo>/</mml:mo><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mo>⊙</mml:mo></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:mo>−</mml:mo><mml:mn>1.3</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq48.gif\"/></alternatives></inline-formula> and the latter near to <inline-formula id=\"IEq49\"><alternatives><tex-math id=\"M113\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathrm{log}\\,}_{10}({M}_{ej}/{M}_{\\odot })=-2.2$$\\end{document}</tex-math><mml:math id=\"M114\"><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi>e</mml:mi><mml:mi>j</mml:mi></mml:mrow></mml:msub><mml:mo>/</mml:mo><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mo>⊙</mml:mo></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:mo>−</mml:mo><mml:mn>2.2</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq49.gif\"/></alternatives></inline-formula>; cf. Fig. <xref rid=\"Fig8\" ref-type=\"fig\">8</xref>. We also include distributions flat in the component masses with 100 nonparametric EOSs consistent with GW170817 as provided in ref. <sup><xref ref-type=\"bibr\" rid=\"CR65\">65</xref></sup>. This analysis shows that our flat priors cover both of the confirmed events so far, and in this sense, applying flat priors has the significant advantage that a larger range of the parameter space is covered. Further, it reduces systematic uncertainties since no numerical-relativity inferred relations have been applied.<fig id=\"Fig8\"><label>Fig. 8</label><caption><title>Potential prior distributions for <italic>M</italic><sub><italic>e</italic><italic>j</italic></sub>.</title><p>In addition to the flat prior used in this analysis, we show ejecta mass distributions for GW170817<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup> and GW190425<sup><xref ref-type=\"bibr\" rid=\"CR64\">64</xref></sup> based on the fit of ref. <sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. We also include distributions flat in the component masses with 100 nonparametric EOSs consistent with GW170817 from ref. <sup><xref ref-type=\"bibr\" rid=\"CR65\">65</xref></sup>.</p></caption><graphic xlink:href=\"41467_2020_17998_Fig8_HTML\" id=\"d30e4081\"/></fig></p></sec><sec id=\"Sec11\"><title>Choice of ejecta mass priors</title><p id=\"Par27\">When performing the fits, we rely on the assumption that the standardization is equally valid across the parameter space. Until there are further unambiguous kilonova detections, in particular those that also have GW counterparts, it will be difficult to create more strongly informed priors; for now, doing so would require assumptions on both the distribution of BNS and BHNS masses and an estimate of the NS EOS. It is not so obvious what unphysical regions there are yet, outside of likely very high ejecta masses, which are already excluded by our priors. To evaluate the effect this may have, Figure <xref rid=\"Fig9\" ref-type=\"fig\">9</xref> shows the performance of the fit of Eqs. (<xref rid=\"Equ3\" ref-type=\"\">3</xref>) compared to the Bulla<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> model for both Broad and Realistic priors, i.e., those used in this analysis. Here, the Broad prior values are derived from the entire available grid, covering a range <inline-formula id=\"IEq50\"><alternatives><tex-math id=\"M115\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-6\\le {\\mathrm{log}\\,}_{10}({M}_{{\\rm{ej}}}/{M}_{\\odot })\\le -\\!0$$\\end{document}</tex-math><mml:math id=\"M116\"><mml:mo>−</mml:mo><mml:mn>6</mml:mn><mml:mo>≤</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi></mml:mrow></mml:msub><mml:mo>/</mml:mo><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mo>⊙</mml:mo></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>≤</mml:mo><mml:mo>−</mml:mo><mml:mspace width=\"0.3em\"/><mml:mn>0</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq50.gif\"/></alternatives></inline-formula>, 15° ≤ Φ ≤ 75°, and 0 ≤ <italic>θ</italic><sub>obs</sub> ≤ 90. The Realistic prior values are derived from the prior range used in the sampling, <inline-formula id=\"IEq51\"><alternatives><tex-math id=\"M117\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-3\\le {\\mathrm{log}\\,}_{10}({M}_{{\\rm{ej}}}/{M}_{\\odot })\\le -\\!\\!1$$\\end{document}</tex-math><mml:math id=\"M118\"><mml:mo>−</mml:mo><mml:mn>3</mml:mn><mml:mo>≤</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi></mml:mrow></mml:msub><mml:mo>/</mml:mo><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mo>⊙</mml:mo></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>≤</mml:mo><mml:mo>−</mml:mo><mml:mspace width=\"0.3em\"/><mml:mspace width=\"0.3em\"/><mml:mn>1</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq51.gif\"/></alternatives></inline-formula>, 15° ≤ Φ ≤ 30°, and 0° ≤ <italic>θ</italic><sub>obs</sub> ≤ 15°, which was tuned for the sGRBs. In general, the estimated values from both versions of the fit are consistent with one another and the measured values from the model, and therefore, the effect on the standardization from this perspective is minimal. In the future, if there are regions which are deemed to be disfavored, the priors should be updated to reflect this.<fig id=\"Fig9\"><label>Fig. 9</label><caption><title>Fundamental plane plot.</title><p>The top panel shows the fit of Eq. (<xref rid=\"Equ3\" ref-type=\"\">3</xref>) for the models in Bulla<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> model. The Broad Priors values are derived from the entire available grid, covering a range <inline-formula id=\"IEq52\"><alternatives><tex-math id=\"M119\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-6\\le {\\mathrm{log}\\,}_{10}({M}_{{\\rm{ej}}}/{M}_{\\odot })\\le -0$$\\end{document}</tex-math><mml:math id=\"M120\"><mml:mo>−</mml:mo><mml:mn>6</mml:mn><mml:mo>≤</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi></mml:mrow></mml:msub><mml:mo>/</mml:mo><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mo>⊙</mml:mo></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>≤</mml:mo><mml:mo>−</mml:mo><mml:mn>0</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq52.gif\"/></alternatives></inline-formula>, 15° ≤ Φ ≤ 75°, and 0° ≤ <italic>θ</italic><sub>obs</sub> ≤ 90°. The Realistic Priors values are derived from a fraction of the grid drawn from the sGRB informed priors, covering a range <inline-formula id=\"IEq53\"><alternatives><tex-math id=\"M121\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$-3\\le {\\mathrm{log}\\,}_{10}({M}_{{\\rm{ej}}}/{M}_{\\odot })\\le -1$$\\end{document}</tex-math><mml:math id=\"M122\"><mml:mo>−</mml:mo><mml:mn>3</mml:mn><mml:mo>≤</mml:mo><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/></mml:mrow><mml:mrow><mml:mn>10</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">ej</mml:mi></mml:mrow></mml:msub><mml:mo>/</mml:mo><mml:msub><mml:mrow><mml:mi>M</mml:mi></mml:mrow><mml:mrow><mml:mo>⊙</mml:mo></mml:mrow></mml:msub></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>≤</mml:mo><mml:mo>−</mml:mo><mml:mn>1</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17998_Article_IEq53.gif\"/></alternatives></inline-formula>, 15° ≤ Φ ≤ 30°, and 0° ≤ <italic>θ</italic><sub>obs</sub> ≤ 15°. The bottom panel shows the difference between the computed values from the model and the fits for the simulations analyzed here. The 1 − <italic>σ</italic> error bars correspond to those assigned by the Gaussian Process Regression.</p></caption><graphic xlink:href=\"41467_2020_17998_Fig9_HTML\" id=\"d30e4342\"/></fig></p><p id=\"Par28\">As for assumptions about the ejecta geometry, the current version is generic enough such that it is applicable to both BNS and BHNS cases. However, an improved geometry would likely need to be split into a BNS and BHNS case, which would mean that the standardization for BNS systems could be different from the one for BHNS systems. However, it is currently difficult to assess what regions of the parameter space are favoured for each system and whether these are distinct or overlap. The detection of more kilonovae in the future will help pin down the ejecta geometry and ejecta mass ratio for BNS and BHNS, allowing us to update our priors on <italic>ϕ</italic> and <italic>M</italic><sub>ej</sub> and investigate the possibility of different standardizations.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec12\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41467_2020_17998_MOESM1_ESM.pdf\"><caption><p>Peer Review File</p></caption></media></supplementary-material></sec></sec></body><back><fn-group><fn><p><bold>Peer review information</bold>\n<italic>Nature Communications</italic> thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary information</bold> is available for this paper at 10.1038/s41467-020-17998-5.</p></sec><ack><title>Acknowledgements</title><p>M.W.C. acknowledges support from the National Science Foundation with grant number PHY-2010970. T.D. acknowledges support by the European Union’s Horizon 2020 research and innovation program under grant agreement No 749145, BNSmergers. N.C. and J.H. acknowledge support from the National Science Foundation with grant number PHY-1806990. S.A. is supported by the CNES Postdoctoral Fellowship at Laboratoire Astroparticle et Cosmologie. N.C., M.C., and J.H. gratefully acknowledge support from the Observatoire Côte d’Azur, including hospitality for M.C. and J.H. in Summer 2019. The UCSC team is supported in part by NASA grant NNG17PX03C, NSF grant AST-1911206, the Gordon & Betty Moore Foundation, the Heising-Simons Foundation, and by fellowships from the David and Lucile Packard Foundation to R.J.F. D.A.C. acknowledges support from the National Science Foundation Graduate Research Fellowship under Grant DGE1339067.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>M.W.C. conducted the light-curve analysis and was the primary author of the manuscript. M.W.C., S.A., T.D., R.J.F., J.H., M.B., N.C., D.C., L.I., and N.A. contributed to the data analysis procedures and edits to the manuscript.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>Upon request, the first author will provide posterior samples from these analyses. All photometric data used in this analysis are publically available from a variety of sources, specified in Methods Section 6 and compiled in https://github.com/mcoughlin/gwemlightcurves/tree/master/lightcurves. Spectral energy distributions for the grid used here will be made available at https://github.com/mbulla/kilonova_models.</p></notes><notes notes-type=\"data-availability\"><title>Code availability</title><p>The light-curve fitting and Hubble Constant code is available at: https://github.com/mcoughlin/gwemlightcurves.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par29\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Riess</surname><given-names>AG</given-names></name><etal/></person-group><article-title>Observational evidence from supernovae for an accelerating universe and a cosmological constant</article-title><source>Astron. J.</source><year>1998</year><volume>116</volume><fpage>1009</fpage><lpage>1038</lpage></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Perlmutter</surname><given-names>S</given-names></name><etal/></person-group><article-title>Measurements of <italic>Ω</italic> and <italic>Λ</italic> from 42 high-redshift Supernovae</article-title><source>Astrophys. J.</source><year>1999</year><volume>517</volume><fpage>565</fpage><lpage>586</lpage></element-citation></ref><ref id=\"CR3\"><label>3.</label><mixed-citation publication-type=\"other\">Planck Collaboration et al. Planck 2015 results. XIII. Cosmological parameters. <italic>Astron. Astrophys</italic>. <bold>594</bold>, A13 (2016).</mixed-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Riess</surname><given-names>AG</given-names></name><etal/></person-group><article-title>A 2.4% determination of the local value of the Hubble constant</article-title><source>Astrophys. J.</source><year>2016</year><volume>826</volume><fpage>56</fpage></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Riess</surname><given-names>AG</given-names></name><name><surname>Casertano</surname><given-names>S</given-names></name><name><surname>Yuan</surname><given-names>W</given-names></name><name><surname>Macri</surname><given-names>LM</given-names></name><name><surname>Scolnic</surname><given-names>D</given-names></name></person-group><article-title>Large magellanic cloud Cepheid standards provide a 1% foundation for the determination of the Hubble constant and stronger evidence for physics beyond <italic>Λ</italic>CDM</article-title><source>Astrophys. J.</source><year>2019</year><volume>876</volume><fpage>85</fpage></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abbott</surname><given-names>BP</given-names></name><etal/></person-group><article-title>GW170817: observation of gravitational waves from a binary neutron star inspiral</article-title><source>Phys. Rev. Lett.</source><year>2017</year><volume>119</volume><fpage>161101</fpage><pub-id pub-id-type=\"pmid\">29099225</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abbott</surname><given-names>BP</given-names></name><etal/></person-group><article-title>Gravitational waves and gamma-rays from a binary neutron star merger: GW170817 and GRB 170817A</article-title><source>Astrophys. J. Lett.</source><year>2017</year><volume>848</volume><fpage>L13</fpage></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Goldstein</surname><given-names>A</given-names></name><etal/></person-group><article-title>An ordinary short gamma-ray burst with extraordinary implications: Fermi-GBM detection of GRB 170817a</article-title><source>Astrophys. J.</source><year>2017</year><volume>848</volume><fpage>L14</fpage></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coulter</surname><given-names>DA</given-names></name><etal/></person-group><article-title>Swope Supernova Survey 2017a (SSS17a), the optical counterpart to a gravitational wave source</article-title><source>Science</source><year>2017</year><volume>358</volume><fpage>1556</fpage><lpage>1558</lpage><pub-id pub-id-type=\"pmid\">29038368</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coughlin</surname><given-names>MW</given-names></name><etal/></person-group><article-title>Constraints on the neutron star equation of state from AT2017gfo using radiative transfer simulations</article-title><source>Mon. Not. R. Astron. Soc.</source><year>2018</year><volume>480</volume><fpage>3871</fpage><lpage>3878</lpage></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abbott</surname><given-names>BP</given-names></name><etal/></person-group><article-title>Estimating the contribution of dynamical ejecta in the kilonova associated with GW170817</article-title><source>Astrophys. J. Lett.</source><year>2017</year><volume>850</volume><fpage>L39</fpage></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abbott</surname><given-names>BP</given-names></name><etal/></person-group><article-title>A gravitational-wave standard siren measurement of the Hubble constant</article-title><source>Nature</source><year>2017</year><volume>551</volume><fpage>85</fpage><lpage>88</lpage><pub-id pub-id-type=\"pmid\">29094696</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schutz</surname><given-names>BF</given-names></name></person-group><article-title>Determining the Hubble constant from gravitational wave observations</article-title><source>Nature</source><year>1986</year><volume>323</volume><fpage>310</fpage></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Doctor</surname><given-names>Z</given-names></name></person-group><article-title>Thunder and lightning: using neutron-star mergers as simultaneous standard candles and sirens to measure cosmological parameters</article-title><source>Astrophys. J.</source><year>2020</year><volume>892</volume><fpage>L16</fpage></element-citation></ref><ref id=\"CR15\"><label>15.</label><mixed-citation publication-type=\"other\">Abbott, B. P. et al. A gravitational-wave measurement of the Hubble constant following the second observing run of Advanced LIGO and Virgo. Preprint at <ext-link ext-link-type=\"uri\" xlink:href=\"https://arxiv.org/abs/1908.06060\">https://arxiv.org/abs/1908.06060</ext-link> (2019).</mixed-citation></ref><ref id=\"CR16\"><label>16.</label><mixed-citation publication-type=\"other\">Hotokezaka, K. et al. A hubble constant measurement from superluminal motion of the jet in GW170817. <italic>Nat. Astron</italic>. <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.nature.com/articles/s41550-019-0820-1?error=cookies_not_supported&code=e98ffae6-00e6-4882-a9a6-3443f30d831b\">https://www.nature.com/articles/s41550-019-0820-1?error=cookies_not_supported&code=e98ffae6-00e6-4882-a9a6-3443f30d831b</ext-link>. (2019).</mixed-citation></ref><ref id=\"CR17\"><label>17.</label><mixed-citation publication-type=\"other\">Dietrich, T. et al. New constraints on the supranuclear equation of state and the Hubble constant from nuclear physics—multi-messenger astronomy. Preprint at <ext-link ext-link-type=\"uri\" xlink:href=\"https://arxiv.org/abs/2002.11355\">https://arxiv.org/abs/2002.11355</ext-link> (2020).</mixed-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coughlin</surname><given-names>MW</given-names></name><etal/></person-group><article-title>Standardizing kilonovae and their use as standard candles to measure the Hubble constant</article-title><source>Phys. Rev. Res.</source><year>2020</year><volume>2</volume><fpage>022006</fpage></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kashyap</surname><given-names>R</given-names></name><name><surname>Raman</surname><given-names>G</given-names></name><name><surname>Ajith</surname><given-names>P</given-names></name></person-group><article-title>Can kilonova light curves be standardized?</article-title><source>Astrophys. J.</source><year>2019</year><volume>886</volume><fpage>L19</fpage></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coughlin</surname><given-names>MW</given-names></name><etal/></person-group><article-title>GROWTH on S190425z: searching thousands of square degrees to identify an optical or infrared counterpart to a binary neutron star merger with the zwicky transient facility and palomar gattini-IR</article-title><source>Astrophys. J.</source><year>2019</year><volume>885</volume><fpage>L19</fpage></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lattimer</surname><given-names>JM</given-names></name><name><surname>Schramm</surname><given-names>DN</given-names></name></person-group><article-title>Black-hole-neutron-star collisions</article-title><source>Astrophys. J. Lett.</source><year>1974</year><volume>192</volume><fpage>L145</fpage><lpage>L147</lpage></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>L-X</given-names></name><name><surname>Paczynski</surname><given-names>B</given-names></name></person-group><article-title>Transient events from neutron star mergers</article-title><source>Astrophys. J. Lett.</source><year>1998</year><volume>507</volume><fpage>L59</fpage></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Metzger</surname><given-names>BD</given-names></name></person-group><article-title>Kilonovae</article-title><source>Living Rev. Rel.</source><year>2017</year><volume>20</volume><fpage>3</fpage></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kasen</surname><given-names>D</given-names></name><name><surname>Metzger</surname><given-names>B</given-names></name><name><surname>Barnes</surname><given-names>J</given-names></name><name><surname>Quataert</surname><given-names>E</given-names></name><name><surname>Ramirez-Ruiz</surname><given-names>E</given-names></name></person-group><article-title>Origin of the heavy elements in binary neutron-star mergers from a gravitational-wave event</article-title><source>Nature</source><year>2017</year><volume>551</volume><fpage>80 EP –</fpage><pub-id pub-id-type=\"pmid\">29094687</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bulla</surname><given-names>M</given-names></name></person-group><article-title>POSSIS: predicting spectra, light curves, and polarization for multidimensional models of supernovae and kilonovae</article-title><source>Mon. Not. R. Astron. Soc.</source><year>2019</year><volume>489</volume><fpage>5037</fpage><lpage>5045</lpage></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ascenzi</surname><given-names>S</given-names></name><etal/></person-group><article-title>A luminosity distribution for kilonovae based on short gamma-ray burst afterglows</article-title><source>Mon. Not. R. Astron. Soc.</source><year>2019</year><volume>486</volume><fpage>672</fpage><lpage>690</lpage></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fong</surname><given-names>W</given-names></name><etal/></person-group><article-title>The afterglow and early-type host galaxy of the short GRB 150101b at z = 0.1343</article-title><source>Astrophys. J.</source><year>2016</year><volume>833</volume><fpage>151</fpage></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fox</surname><given-names>DB</given-names></name><etal/></person-group><article-title>The afterglow of GRB 050709 and the nature of the short-hard <italic>γ</italic>-ray bursts</article-title><source>Nature</source><year>2005</year><volume>437</volume><fpage>845 EP –</fpage><pub-id pub-id-type=\"pmid\">16208362</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kasliwal</surname><given-names>MM</given-names></name><name><surname>Korobkin</surname><given-names>O</given-names></name><name><surname>Lau</surname><given-names>RM</given-names></name><name><surname>Wollaeger</surname><given-names>R</given-names></name><name><surname>Fryer</surname><given-names>CL</given-names></name></person-group><article-title>Infrared emission from Kilonovae: the case of the nearby short hard burst GRB 160821B</article-title><source>Astrophys. J. Lett.</source><year>2017</year><volume>843</volume><fpage>L34</fpage></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>B</given-names></name><etal/></person-group><article-title>Making a short gamma-ray burst from a long one: implications for the nature of GRB 060614</article-title><source>Astrophys. J. Lett.</source><year>2007</year><volume>655</volume><fpage>L25</fpage></element-citation></ref><ref id=\"CR31\"><label>31.</label><mixed-citation publication-type=\"other\">Coughlin, M. W. et al. Implications of the search for optical counterparts during the first six months of the Advanced LIGO’s and Advanced Virgo’s third observing run: possible limits on the ejecta mass and binary properties. <italic>Mon. Not. R. Astron. Soc</italic>. <ext-link ext-link-type=\"uri\" xlink:href=\"https://academic.oup.com/mnras/article-abstract/492/1/863/5672639?redirectedFrom=fulltext\">https://academic.oup.com/mnras/article-abstract/492/1/863/5672639?redirectedFrom=fulltext</ext-link>. (2019).</mixed-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hotokezaka</surname><given-names>K</given-names></name><name><surname>Nakar</surname><given-names>E</given-names></name></person-group><article-title>Radioactive heating rate of r-process elements and macronova light curve</article-title><source>Astrophys. J.</source><year>2020</year><volume>891</volume><fpage>152</fpage></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cantiello</surname><given-names>M</given-names></name><etal/></person-group><article-title>A precise distance to the host galaxy of the binary neutron star merger GW170817 using surface brightness fluctuations</article-title><source>Astrophys. J.</source><year>2018</year><volume>854</volume><fpage>L31</fpage></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ade</surname><given-names>PAR</given-names></name><etal/></person-group><article-title>Planck 2015 results. XIII. Cosmological parameters</article-title><source>Astron. Astrophys.</source><year>2016</year><volume>594</volume><fpage>A13</fpage></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coughlin</surname><given-names>MW</given-names></name><etal/></person-group><article-title>Multimessenger Bayesian parameter inference of a binary neutron star merger</article-title><source>Mon. Not. R. Astron. Soc. Lett.</source><year>2019</year><volume>489</volume><fpage>L91</fpage><lpage>L96</lpage></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abbott</surname><given-names>BP</given-names></name><etal/></person-group><article-title>Properties of the binary neutron star merger GW170817</article-title><source>Phys. Rev.</source><year>2019</year><volume>X9</volume><fpage>011001</fpage></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Meegan</surname><given-names>C</given-names></name><etal/></person-group><article-title>The fermi gamma-ray burst monitor</article-title><source>Astrophys. J.</source><year>2009</year><volume>702</volume><fpage>791–804</fpage></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coughlin</surname><given-names>MW</given-names></name><etal/></person-group><article-title>2900 square degree search for the optical counterpart of short gamma-ray burst GRB 180523b with the Zwicky Transient Facility</article-title><source>Publ. Astron. Soc. Pac.</source><year>2019</year><volume>131</volume><fpage>048001</fpage></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Doctor</surname><given-names>Z</given-names></name><name><surname>Farr</surname><given-names>B</given-names></name><name><surname>Holz</surname><given-names>DE</given-names></name><name><surname>Pürrer</surname><given-names>M</given-names></name></person-group><article-title>Statistical gravitational waveform models: what to simulate next?</article-title><source>Phys. Rev.</source><year>2017</year><volume>D96</volume><fpage>123011</fpage></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Troja</surname><given-names>E</given-names></name><etal/></person-group><article-title>A luminous blue kilonova and an off-axis jet from a compact binary merger at z=0.1341</article-title><source>Nat. Commun.</source><year>2018</year><volume>9</volume><fpage>4089</fpage><pub-id pub-id-type=\"pmid\">30327476</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fong</surname><given-names>W</given-names></name><etal/></person-group><article-title>Short GRB 130603B: discovery of a jet break in the optical and radio afterglows, and a mysterious late-time X-ray excess</article-title><source>Astrophys. J.</source><year>2014</year><volume>780</volume><fpage>118</fpage></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>D’Avanzo</surname><given-names>P</given-names></name></person-group><article-title>Short gamma-ray bursts: a review</article-title><source>J. High. Energy Phys.</source><year>2015</year><volume>7</volume><fpage>73</fpage><lpage>80</lpage></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Saleem</surname><given-names>M</given-names></name></person-group><article-title>Prospects of joint detections of neutron star mergers and short GRBs with Gaussian structured jets</article-title><source>Mon. Not. R. Astron. Soc.</source><year>2020</year><volume>493</volume><fpage>1633</fpage><lpage>1639</lpage></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Beniamini</surname><given-names>P</given-names></name><name><surname>Petropoulou</surname><given-names>M</given-names></name><name><surname>Barniol Duran</surname><given-names>R</given-names></name><name><surname>Giannios</surname><given-names>D</given-names></name></person-group><article-title>A lesson from GW170817: most neutron star mergers result in tightly collimated successful GRB jets</article-title><source>Mon. Not. R. Astron. Soc.</source><year>2018</year><volume>483</volume><fpage>840</fpage><lpage>851</lpage></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Barnes</surname><given-names>J</given-names></name><name><surname>Kasen</surname><given-names>D</given-names></name><name><surname>Wu</surname><given-names>M-R</given-names></name><name><surname>Martínez-Pinedo</surname><given-names>G</given-names></name></person-group><article-title>Radioactivity and thermalization in the ejecta of compact object mergers and their impact on kilonova light curves</article-title><source>Astrophys. J.</source><year>2016</year><volume>829</volume><fpage>110</fpage></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kawaguchi</surname><given-names>K</given-names></name><name><surname>Shibata</surname><given-names>M</given-names></name><name><surname>Tanaka</surname><given-names>M</given-names></name></person-group><article-title>Radiative transfer simulation for the optical and near-infrared electromagnetic counterparts to GW170817</article-title><source>Astrophys. J. Lett.</source><year>2018</year><volume>865</volume><fpage>L21</fpage></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Radice</surname><given-names>D</given-names></name><etal/></person-group><article-title>Binary neutron star mergers: mass ejection, electromagnetic counterparts and nucleosynthesis</article-title><source>Astrophys. J.</source><year>2018</year><volume>869</volume><fpage>130</fpage></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hjorth</surname><given-names>J</given-names></name><etal/></person-group><article-title>The optical afterglow of the short <italic>γ</italic>-ray burst GRB 050709</article-title><source>Nature</source><year>2005</year><volume>437</volume><fpage>859</fpage><lpage>861</lpage><pub-id pub-id-type=\"pmid\">16208365</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Covino</surname><given-names>S</given-names></name><etal/></person-group><article-title>Optical emission from GRB 050709: a short/hard GRB in a star-forming galaxy</article-title><source>Astron. Astrophys.</source><year>2006</year><volume>447</volume><fpage>L5</fpage><lpage>L8</lpage></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jin</surname><given-names>Z-P</given-names></name><etal/></person-group><article-title>The Macronova in GRB 050709 and the GRB-macronova connection</article-title><source>Nat. Commun.</source><year>2016</year><volume>7</volume><fpage>12898</fpage><pub-id pub-id-type=\"pmid\">27659791</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jin</surname><given-names>Z-P</given-names></name><etal/></person-group><article-title>Short GRBs: opening angles, local neutron star merger rate, and off-axis events for GRB/GW Association</article-title><source>Astrophys. J.</source><year>2018</year><volume>857</volume><fpage>128</fpage></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jin</surname><given-names>Z-P</given-names></name><etal/></person-group><article-title>The light curve of the macronova associated with the long-short burst GRB 060614</article-title><source>Astrophys. J. Lett.</source><year>2015</year><volume>811</volume><fpage>L22</fpage></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>B</given-names></name><etal/></person-group><article-title>A possible macronova in the late afterglow of the long-short burst GRB 060614</article-title><source>Nat. Commun.</source><year>2015</year><volume>6</volume><fpage>7323</fpage><pub-id pub-id-type=\"pmid\">26065563</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lamb</surname><given-names>GP</given-names></name><etal/></person-group><article-title>Short GRB 160821b: a reverse shock, a refreshed shock, and a well-sampled kilonova</article-title><source>Astrophys. J.</source><year>2019</year><volume>883</volume><fpage>48</fpage></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rossi</surname><given-names>A</given-names></name><etal/></person-group><article-title>A comparison between short GRB afterglows and kilonova AT2017gfo: shedding light on kilonovae properties</article-title><source>Mon. Not. Roy. Astron. Soc.</source><year>2020</year><volume>493</volume><fpage>3379</fpage><lpage>3397</lpage></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lu</surname><given-names>Y</given-names></name><name><surname>Huang</surname><given-names>YF</given-names></name><name><surname>Zhang</surname><given-names>SN</given-names></name></person-group><article-title>A tidal disruption model for the gamma-ray burst of GRB 060614</article-title><source>Astrophys. J.</source><year>2008</year><volume>684</volume><fpage>1330</fpage><lpage>1335</lpage></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Caito</surname><given-names>L</given-names></name><etal/></person-group><article-title>GRB060614: a “fake” short GRB from a merging binary system</article-title><source>Astron. Astrophys.</source><year>2009</year><volume>498</volume><fpage>501</fpage><lpage>507</lpage></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xu</surname><given-names>D</given-names></name><etal/></person-group><article-title>In search of progenitors for supernovaless gamma-ray bursts 060505 and 060614: re-examination of their afterglows</article-title><source>Astrophys. J.</source><year>2009</year><volume>696</volume><fpage>971</fpage><lpage>979</lpage></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Metzger</surname><given-names>BD</given-names></name></person-group><article-title>Kilonovae</article-title><source>Living Rev. Relativ.</source><year>2017</year><volume>20</volume><fpage>3</fpage><pub-id pub-id-type=\"pmid\">28579916</pub-id></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arnett</surname><given-names>WD</given-names></name></person-group><article-title>Type I supernovae. I—ānalytic solutions for the early part of the light curve</article-title><source>Astrophys. J.</source><year>1982</year><volume>253</volume><fpage>785</fpage><lpage>797</lpage></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Smartt</surname><given-names>SJ</given-names></name><etal/></person-group><article-title>A kilonova as the electromagnetic counterpart to a gravitational-wave source</article-title><source>Nature</source><year>2017</year><volume>551</volume><fpage>75 EP –</fpage><pub-id pub-id-type=\"pmid\">29094693</pub-id></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hjorth</surname><given-names>J</given-names></name><etal/></person-group><article-title>The distance to NGC 4993: the host galaxy of the gravitational-wave event GW170817</article-title><source>Astrophys. J.</source><year>2017</year><volume>848</volume><fpage>L31</fpage></element-citation></ref><ref id=\"CR63\"><label>63.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Levan</surname><given-names>AJ</given-names></name><etal/></person-group><article-title>The environment of the binary neutron star merger GW170817</article-title><source>Astrophys. J.</source><year>2017</year><volume>848</volume><fpage>L28</fpage></element-citation></ref><ref id=\"CR64\"><label>64.</label><mixed-citation publication-type=\"other\">Abbott, B. P. et al. GW190425: observation of a compact binary coalescence with total mass  ~ 3.4<italic>m</italic><sub>⊙</sub>. <italic>arXiv</italic><ext-link ext-link-type=\"uri\" xlink:href=\"https://iopscience.iop.org/article/10.3847/2041-8213/ab75f5\">https://iopscience.iop.org/article/10.3847/2041-8213/ab75f5</ext-link> (2020).</mixed-citation></ref><ref id=\"CR65\"><label>65.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Landry</surname><given-names>P</given-names></name><name><surname>Essick</surname><given-names>R</given-names></name></person-group><article-title>Nonparametric inference of the neutron star equation of state from gravitational wave observations</article-title><source>Phys. Rev. D.</source><year>2019</year><volume>99</volume><fpage>084049</fpage></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807815</article-id><article-id pub-id-type=\"pmc\">PMC7431581</article-id><article-id pub-id-type=\"publisher-id\">70814</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70814-4</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Development and validation of a sample entropy-based method to identify complex patient-ventilator interactions during mechanical ventilation</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\" equal-contrib=\"yes\"><name><surname>Sarlabous</surname><given-names>Leonardo</given-names></name><address><email>lsarlabous@tauli.cat</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Aquino-Esperanza</surname><given-names>José</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Magrans</surname><given-names>Rudys</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>de Haro</surname><given-names>Candelaria</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>López-Aguilar</surname><given-names>Josefina</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Subirà</surname><given-names>Carles</given-names></name><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Batlle</surname><given-names>Montserrat</given-names></name><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rué</surname><given-names>Montserrat</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><name><surname>Gomà</surname><given-names>Gemma</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Ochagavia</surname><given-names>Ana</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Fernández</surname><given-names>Rafael</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Blanch</surname><given-names>Lluís</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.7080.f</institution-id><institution>Critical Care Center, Hospital Universitari Parc Taulí, Institut d’Investigació i Innovació Parc Taulí I3PT, </institution><institution>Universitat Autònoma de Barcelona, </institution></institution-wrap>Parc Taulí 1, 08208 Sabadell, Barcelona Spain </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.413448.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9314 1427</institution-id><institution>Biomedical Research Networking Center in Respiratory Disease (CIBERES), </institution><institution>Instituto de Salud Carlos III, </institution></institution-wrap>Madrid, Spain </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5841.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1937 0247</institution-id><institution>Facultat de Medicina, </institution><institution>Universitat de Barcelona, </institution></institution-wrap>Barcelona, Spain </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.413448.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9314 1427</institution-id><institution>Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), </institution><institution>Instituto de Salud Carlos III, </institution></institution-wrap>Madrid, Spain </aff><aff id=\"Aff5\"><label>5</label>BetterCare S.L, Sabadell, Spain </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.410675.1</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2325 3084</institution-id><institution>Department of Intensive Care, Fundació Althaia, </institution><institution>Universitat Internacional de Catalunya , </institution></institution-wrap>Manresa, Spain </aff><aff id=\"Aff7\"><label>7</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.15043.33</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2163 1432</institution-id><institution>Department of Basic Medical Sciences, </institution><institution>Universitat de Lleida-IRBLLEIDA, </institution></institution-wrap>Lleida, Spain </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13911</elocation-id><history><date date-type=\"received\"><day>17</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>5</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Patient-ventilator asynchronies can be detected by close monitoring of ventilator screens by clinicians or through automated algorithms. However, detecting complex patient-ventilator interactions (CP-VI), consisting of changes in the respiratory rate and/or clusters of asynchronies, is a challenge. Sample Entropy (<italic>SE</italic>) of airway flow (<italic>SE</italic>-Flow) and airway pressure (<italic>SE</italic>-Paw) waveforms obtained from 27 critically ill patients was used to develop and validate an automated algorithm for detecting CP-VI. The algorithm’s performance was compared versus the gold standard (the ventilator’s waveform recordings for CP-VI were scored visually by three experts; Fleiss’ kappa = 0.90 (0.87–0.93)). A repeated holdout cross-validation procedure using the Matthews correlation coefficient (MCC) as a measure of effectiveness was used for optimization of different combinations of <italic>SE</italic> settings (embedding dimension, <italic>m</italic>, and tolerance value, <italic>r</italic>), derived <italic>SE</italic> features (mean and maximum values), and the thresholds of change (<italic>Th</italic>) from patient’s own baseline <italic>SE</italic> value. The most accurate results were obtained using the maximum values of <italic>SE</italic>-Flow (<italic>m</italic> = 2, <italic>r</italic> = 0.2, <italic>Th</italic> = 25%) and <italic>SE</italic>-Paw (<italic>m</italic> = 4, <italic>r</italic> = 0.2, <italic>Th</italic> = 30%) which report MCCs of 0.85 (0.78–0.86) and 0.78 (0.78–0.85), and accuracies of 0.93 (0.89–0.93) and 0.89 (0.89–0.93), respectively. This approach promises an improvement in the accurate detection of CP-VI, and future study of their clinical implications.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Biomarkers</kwd><kwd>Translational research</kwd><kwd>Biomarkers</kwd><kwd>Engineering</kwd><kwd>Biomedical engineering</kwd><kwd>Scientific data</kwd><kwd>Statistics</kwd><kwd>Data acquisition</kwd><kwd>Data processing</kwd><kwd>Databases</kwd><kwd>Machine learning</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Invasive mechanical ventilation (MV) is a life-support measure administered to patients who cannot breathe on their own. Patient-ventilator asynchronies occur when there is a mismatch between the ventilator’s setting and patient’s breathing pattern. Recent studies have emphasized the impact of asynchronies upon clinical outcomes<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>, focusing on the incidence of specific subtypes of asynchronies or on the asynchrony index, and also on their distribution over time given that they occur in clusters within prolonged uneventful periods<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. Importantly, in most of these studies ventilator’s waveforms were analysed visually<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>; only a few analyses have been based on automated algorithms<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> or, more recently, on machine learning algorithms incorporating not only ventilator waveforms but also clinical data<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>.</p><p id=\"Par3\">Asynchronies are difficult to define when supported only by visual assessment carried out by inexperienced personnel, since different types may develop in a short time period or may even overlap with each other. Furthermore, asynchronies, which are by nature time-limited and transient, lead to patient distress, impede the ventilator’s effectiveness in decreasing the work of breathing, increase the time on mechanical ventilation and have a negative impact on outcome<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Additionally, sometimes patient’s drive only becomes evident due to an increase in the respiratory rate itself<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>–<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>, which, given its irregular and complex behaviour, may be overestimated by visual observation or dedicated algorithms. Therefore, it would be extremely useful to have access to a method for assessing irregularity and complexity which could detect Complex Patient-Ventilator interactions (CP-VI), including not just asynchronies of any kind but also changes in the respiratory rate, in an automated, non-invasive and personalized fashion.</p><p id=\"Par4\">Normal physiological data are non-linear<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. The complex behavior of a non-linear system cannot be characterized by the sum of its inputs, and the study of these systems requires methods that take into account the non-linear physiological response to a given stimulus. These methods could provide insights into organ-system interconnectivity, regulatory control, and complexity in time series during disease<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>–<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>.</p><p id=\"Par5\">Entropy is a non-linear method derived from the theory of complex systems which measures the randomness and predictability of stochastic processes. Various types of entropy have been used in clinical monitoring<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>–<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Sample Entropy (<italic>SE</italic>) is a measure of complexity and regularity, defined as the negative natural logarithm of the conditional probability that two sequences similar for <italic>m</italic> points will remain similar at the next point, where self-matching is not included<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Thus, a lower <italic>SE</italic> value indicates more self-similarity in a time series.</p><p id=\"Par6\"><italic>SE</italic> has proved to be an effective tool for investigating different types of time series data derived from various biological conditions in the human body. Examples of these conditions include the activation of inspiratory muscles in COPD patients<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>, the analysis of atrial fibrillation on electrocardiograms<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>, background electroencephalograms in Alzheimer’s patients<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>, heart rate variability<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>, human postural sway<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup> and seizure termination during electroconvulsive therapy<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>.</p><p id=\"Par7\">Interestingly, only a few entropy approaches have been applied in the respiratory system to study breath-to-breath variability and its components as predictors of successful separation from MV during spontaneous breathing trials (SBT)<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR31\">31</xref>–<xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. Breath-to-breath approaches suggest that increased irregularity of the respiratory system may be a marker of pulmonary health<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup> and may serve as a weaning predictor<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref>–<xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>, opening up the possibility that a certain degree of irregularity may be normal<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. However, these studies rely on the detection of the appropriate respiratory cycle. Hence, the performance of automated algorithms in breathing cycle detection may be jeopardized when transient asynchronies occur during patient-ventilator interaction or even overlap with each other. In this respect, other authors have applied the <italic>SE</italic> to the entire signal, as is the case of Sá et al.<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup> who evaluated the respiratory changes by applying <italic>SE</italic> upon the entire airway flow signal providing an early and sensitive functional indicator of interstitial asbestosis.</p><p id=\"Par8\">We hypothesized that analyzing transient complexity of CP-VI may provide clinically relevant information during MV. Therefore, we sought to develop and validate a non-invasive method based on <italic>SE</italic> measurement using the entire airway pressure (Paw) and airway flow (Flow) waveforms to detect CP-VI, defined as the occurrence of asynchronies and changes in the respiratory rate.</p></sec><sec id=\"Sec2\"><title>Methods</title><sec id=\"Sec3\"><title>Defining complex patient ventilator interactions</title><p id=\"Par9\">We defined CP-VI as a > 50% change in the respiratory rate<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup> and/or > 30% asynchronous breaths of any type (ineffective expiratory efforts, double cycling, premature cycling, prolonged cycling, or reverse triggering) over a 3-min period. A recent study found that 38% of mechanically ventilated patients had clusters of ≥ 30 ineffective expiratory efforts in a 3-min period (i.e., ≥ 50% of all breaths in a patient with a respiratory rate of 20 breaths per minute), and that the median duration of these clusters was 20 min<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. Another study found that 59.7% of patients had clusters in which > 10% of all breaths in a 3-min period were double cycled, with a mean cluster duration of 15.5 min<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. Figure <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> shows a representative example of different CP-VIs consisting of increased respiratory rate, asynchronies, or a combination of these phenomena.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Tracing of Flow and Paw from three different patients. (<bold>a</bold>) Continuous Positive Airway Pressure (CPAP) of 6 cmH<sub>2</sub>O. (<bold>b</bold>) Pressure assist-control ventilation (PCV) with pressure of 10 cmH<sub>2</sub>O, (<bold>c</bold>) PSV with a pressure support of 10 cmH<sub>2</sub>O and PEEP of 8 cmH<sub>2</sub>O. In (<bold>a1</bold>) and (<bold>a2</bold>), Complex Patient-Ventilator Interactions (CP-VI) consists of an increase in respiratory rate > 50%; in (<bold>b1)</bold> and (<bold>b2</bold>), it consists of > 30% asynchronies (ineffective expiratory effort, double cycling, premature cycling, prolonged cycling, and/or reverse triggering) in the 3-min period; and in (<bold>c1</bold>) and (<bold>c2</bold>) it consists of a combination of change in the respiratory rate and asynchronies.</p></caption><graphic xlink:href=\"41598_2020_70814_Fig1_HTML\" id=\"MO1\"/></fig></p></sec><sec id=\"Sec4\"><title>Data acquisition and data analysis</title><p id=\"Par10\">The Better Care system (Better Care, Barcelona, Spain. US patent No. 12/538,940) continuously records Paw and Flow signals at a sample frequency of 200 Hz from intubation to liberation from MV<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. Better Care uses drivers specifically designed to interact with output signals from mechanical ventilators and bedside monitors rather than directly with patients, synchronizing recorded signals and storing them for further analysis. We used MATLAB (The MathWorks, Inc., vR2018b, Natick, MA, USA) for signal processing, data analysis, and visual assessment. Signals were decimated at a sampling rate of 40 Hz before entropy calculation.</p></sec><sec id=\"Sec5\"><title>Study population</title><p id=\"Par11\">The findings presented in this paper represents an ancillary analysis on an ongoing clinical study (ENTROPY-ICU, ClincalTrials.gov NCT04128124) designed to assess the feasibility of using <italic>SE</italic> to identify CP-VI during MV. Data from 27 patients were obtained from an ongoing database at two centers in Spain. The database was constructed prospectively for the development of a connectivity platform (Better Care) to interoperate signals from different ventilators and monitors and subsequently compute algorithms for diagnosing patient-ventilator asynchronies (ClinicalTrial.gov, NCT03451461). The Comitè d’Ètica d’Investigació amb medicaments at the Corporació Sanitària Parc Taulí and the Clinical Research Ethics Committee of Fundació Unió Catalana d’Hospitals approved the database and the study protocol. The need for informed consent was waived because the current study was an ancillary analysis with anonymized data. The guidelines followed in this study were according to the applicable Spanish regulations (Biomedical Research Law 14/2007). This type of study must be evaluated and approved by at least one Institutional Review Board (IRB). Parc Taulí’s IRB approved this study to be carried out in all participating centers. The IRB approved the study allowing it to be carried out without the explicit request of informed consent from each participant given that it is a study with retrospective data. Spanish regulations allow studies to be carried out with this condition as long as they are approved by an IRB.</p><p id=\"Par12\">The <italic>SE</italic> analysis was performed on the complete set of Flow and Paw data collected during the two hours before self-extubation. Self-extubations, defined as extubations performed by the patient himself, are included in unplanned extubations but its mechanisms differ from accidental extubations<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. Clinical and demographic data were obtained from medical charts (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>).<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Patient characteristics. APACHE II: Acute Physiology and Chronic Health Evaluation.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" colspan=\"2\">Clinical and demographic data of patient</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"2\">Sex</td></tr><tr><td align=\"left\"> Male, n (%)</td><td align=\"left\">22 (81.5%)</td></tr><tr><td align=\"left\"> Female, n (%)</td><td align=\"left\">5 (18.5%)</td></tr><tr><td align=\"left\">Mean age (range), in years</td><td align=\"left\">63.8 (57–72)</td></tr><tr><td align=\"left\">APACHE II at admission</td><td align=\"left\">16.7 (9–22)</td></tr><tr><td align=\"left\">Mean ICU–LOS (range), in days</td><td align=\"left\">18.7 (7.5–27)</td></tr><tr><td align=\"left\">Mean hospital–LOS (range), in days</td><td align=\"left\">34 (15.5–41)</td></tr><tr><td align=\"left\" colspan=\"2\">Reason for MV, n (%)</td></tr><tr><td align=\"left\"> Respiratory insufficiency</td><td align=\"left\">9 (33.3%)</td></tr><tr><td align=\"left\"> Sepsis/septic shock</td><td align=\"left\">10 (37%)</td></tr><tr><td align=\"left\"> Altered consciousness</td><td align=\"left\">3 (11.1%)</td></tr><tr><td align=\"left\"> Others</td><td align=\"left\">7 (25.9%)</td></tr><tr><td align=\"left\">Use of sedatives (%)</td><td align=\"left\">71.4%</td></tr><tr><td align=\"left\">RASS</td><td align=\"left\">0.6 ± 1.7</td></tr></tbody></table><table-wrap-foot><p><italic>ICU</italic> intensive care unit, <italic>LOS</italic> length of stay, <italic>MV</italic> mechanical ventilation, <italic>RASS</italic> Richmond Agitation-Sedation Scale.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec6\"><title>Visual validation of CP-VI</title><p id=\"Par13\">Experts’ visual assessment was considered the gold standard. Three critical care physicians with extensive experience in analyzing ventilator waveforms visually reviewed 92 15-min-long segments of Flow and Paw recordings from the two-hour period immediately before self-extubation. The 15-min window was selected based on two previous studies evaluating clusters of asynchronies, in which mean cluster duration was 15.5 and 20 min respectively<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. An expert in MV selected the segments to ensure a balanced proportion of different ventilation modes (grouped into pressure support ventilation (PSV) or assist-control ventilation (ACV) modes, comprising volume assist-control and pressure assist-control ventilation) and of segments with and without CP-VIs. Every patient contributed both CP-VI and non-CP-VI segments with at least one 15-min segment of each type; however, some patients contributed more segments than others. In order to ensure that the most valuable CP-VI events were not missed, all the 15-min segments immediately preceding self-extubation were included. To ensure masking of the scorers, Flow and Paw tracings were randomly ordered in MATLAB prior to visual analysis. To standardize scoring criteria, scorers were provided with written descriptions of the characteristics of CP-VI before visual analysis. Scorers were asked to determine whether CP-VI were present in each segment. No time limitations were imposed.</p></sec><sec id=\"Sec7\"><title>Sample entropy</title><p id=\"Par14\"><italic>SE</italic> is a non-linear technique that measures the randomness of a series of data<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Compared to other approaches, <italic>SE</italic>’s main advantage is that it provides consistent results even in short and noisy medical time series<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. To calculate <italic>SE</italic>, three parameters are necessary: the embedding dimension, <italic>m</italic> (a positive integer); the tolerance value or similarity criterion, <italic>r</italic> (a positive real number); and the total length of the series, <italic>N</italic>. Briefly, <italic>SE</italic> is defined as the negative logarithm of the conditional probability that two sequences of patterns of <italic>m</italic> consecutive samples that are similar to each other within a tolerance <italic>r</italic> will remain similar when one consecutive sample is added (<inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$m + 1$$\\end{document}</tex-math><mml:math id=\"M2\"><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq1.gif\"/></alternatives></inline-formula>), excluding self-matches. <italic>SE</italic> is calculated as follows<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>:</p><p id=\"Par15\">Given a time series of N samples <inline-formula id=\"IEq2\"><alternatives><tex-math id=\"M3\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\left\\{ {x\\left( n \\right) = x\\left( 1 \\right),x\\left( 2 \\right), \\ldots ,x\\left( N \\right)} \\right\\}$$\\end{document}</tex-math><mml:math id=\"M4\"><mml:mfenced close=\"}\" open=\"{\"><mml:mrow><mml:mi>x</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mi>n</mml:mi></mml:mfenced><mml:mo>=</mml:mo><mml:mi>x</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mn>1</mml:mn></mml:mfenced><mml:mo>,</mml:mo><mml:mi>x</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mn>2</mml:mn></mml:mfenced><mml:mo>,</mml:mo><mml:mo>…</mml:mo><mml:mo>,</mml:mo><mml:mi>x</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mi>N</mml:mi></mml:mfenced></mml:mrow></mml:mfenced></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq2.gif\"/></alternatives></inline-formula>, a subset of <inline-formula id=\"IEq3\"><alternatives><tex-math id=\"M5\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$N - m + 1$$\\end{document}</tex-math><mml:math id=\"M6\"><mml:mrow><mml:mi>N</mml:mi><mml:mo>-</mml:mo><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq3.gif\"/></alternatives></inline-formula>, overlapping vectors <inline-formula id=\"IEq4\"><alternatives><tex-math id=\"M7\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$X_{m} \\left( i \\right)$$\\end{document}</tex-math><mml:math id=\"M8\"><mml:mrow><mml:msub><mml:mi>X</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>i</mml:mi></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq4.gif\"/></alternatives></inline-formula> of length <inline-formula id=\"IEq5\"><alternatives><tex-math id=\"M9\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$m$$\\end{document}</tex-math><mml:math id=\"M10\"><mml:mi>m</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq5.gif\"/></alternatives></inline-formula> are defined:</p><p><list list-type=\"order\"><list-item><p id=\"Par16\"> Form <italic>m</italic> vectors defined by <inline-formula id=\"IEq6\"><alternatives><tex-math id=\"M11\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$X_{m} \\left( i \\right) = \\left[ {x\\left( i \\right),x\\left( {i + 1} \\right), \\ldots ,x\\left( {i + m - 1} \\right)} \\right],{ }i = 1,2, \\ldots ,N - m + 1$$\\end{document}</tex-math><mml:math id=\"M12\"><mml:mrow><mml:msub><mml:mi>X</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>i</mml:mi></mml:mfenced><mml:mo>=</mml:mo><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mi>x</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mi>i</mml:mi></mml:mfenced><mml:mo>,</mml:mo><mml:mi>x</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>i</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfenced><mml:mo>,</mml:mo><mml:mo>…</mml:mo><mml:mo>,</mml:mo><mml:mi>x</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>i</mml:mi><mml:mo>+</mml:mo><mml:mi>m</mml:mi><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfenced></mml:mrow></mml:mfenced><mml:mo>,</mml:mo><mml:mrow/><mml:mi>i</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn><mml:mo>,</mml:mo><mml:mn>2</mml:mn><mml:mo>,</mml:mo><mml:mo>…</mml:mo><mml:mo>,</mml:mo><mml:mi>N</mml:mi><mml:mo>-</mml:mo><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq6.gif\"/></alternatives></inline-formula>. These represent <inline-formula id=\"IEq7\"><alternatives><tex-math id=\"M13\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$m$$\\end{document}</tex-math><mml:math id=\"M14\"><mml:mi>m</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq7.gif\"/></alternatives></inline-formula> consecutive <inline-formula id=\"IEq8\"><alternatives><tex-math id=\"M15\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$x$$\\end{document}</tex-math><mml:math id=\"M16\"><mml:mi>x</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq8.gif\"/></alternatives></inline-formula> values.</p></list-item><list-item><p id=\"Par17\"> Then, define Chebyshev distance between vectors <inline-formula id=\"IEq9\"><alternatives><tex-math id=\"M17\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${ }X_{m} \\left( i \\right)$$\\end{document}</tex-math><mml:math id=\"M18\"><mml:mrow><mml:mrow/><mml:msub><mml:mi>X</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>i</mml:mi></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq9.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq10\"><alternatives><tex-math id=\"M19\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$X_{m} \\left( j \\right)$$\\end{document}</tex-math><mml:math id=\"M20\"><mml:mrow><mml:msub><mml:mi>X</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>j</mml:mi></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq10.gif\"/></alternatives></inline-formula>, i.e., the maximum absolute difference between their scalar components:<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M21\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$d\\left[ {X_{m} \\left( i \\right),X_{m} \\left( j \\right)} \\right] = \\begin{array}{*{20}c} {max} \\\\ {k = 0, \\ldots ,m - 1} \\\\ \\end{array} \\left[ {\\left| {x\\left( {i + k} \\right) - x\\left( {j + k} \\right)} \\right|} \\right].$$\\end{document}</tex-math><mml:math id=\"M22\" display=\"block\"><mml:mrow><mml:mi>d</mml:mi><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:msub><mml:mi>X</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>i</mml:mi></mml:mfenced><mml:mo>,</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>j</mml:mi></mml:mfenced></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mrow><mml:mtable><mml:mtr><mml:mtd><mml:mrow><mml:mi mathvariant=\"italic\">max</mml:mi></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mrow><mml:mrow/><mml:mrow><mml:mi>k</mml:mi><mml:mo>=</mml:mo><mml:mn>0</mml:mn><mml:mo>,</mml:mo><mml:mo>…</mml:mo><mml:mo>,</mml:mo><mml:mi>m</mml:mi><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mrow></mml:mtd></mml:mtr></mml:mtable></mml:mrow><mml:mfenced close=\"]\" open=\"[\"><mml:mfenced close=\"|\" open=\"|\"><mml:mrow><mml:mi>x</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>i</mml:mi><mml:mo>+</mml:mo><mml:mi>k</mml:mi></mml:mrow></mml:mfenced><mml:mo>-</mml:mo><mml:mi>x</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>j</mml:mi><mml:mo>+</mml:mo><mml:mi>k</mml:mi></mml:mrow></mml:mfenced></mml:mrow></mml:mfenced></mml:mfenced><mml:mo>.</mml:mo></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70814_Article_Equ1.gif\" position=\"anchor\"/></alternatives></disp-formula></p></list-item><list-item><p id=\"Par18\"> For a given <inline-formula id=\"IEq11\"><alternatives><tex-math id=\"M23\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$X_{m} \\left( i \\right)$$\\end{document}</tex-math><mml:math id=\"M24\"><mml:mrow><mml:msub><mml:mi>X</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>i</mml:mi></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq11.gif\"/></alternatives></inline-formula>, count the number of j <inline-formula id=\"IEq12\"><alternatives><tex-math id=\"M25\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\left( {1 \\le j \\ge N - m,i \\ne j} \\right)$$\\end{document}</tex-math><mml:math id=\"M26\"><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>≤</mml:mo><mml:mi>j</mml:mi><mml:mo>≥</mml:mo><mml:mi>N</mml:mi><mml:mo>-</mml:mo><mml:mi>m</mml:mi><mml:mo>,</mml:mo><mml:mi>i</mml:mi><mml:mo>≠</mml:mo><mml:mi>j</mml:mi></mml:mrow></mml:mfenced></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq12.gif\"/></alternatives></inline-formula>, denoted as <inline-formula id=\"IEq13\"><alternatives><tex-math id=\"M27\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$B_{i} \\left( r \\right)$$\\end{document}</tex-math><mml:math id=\"M28\"><mml:mrow><mml:msub><mml:mi>B</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq13.gif\"/></alternatives></inline-formula>, such that the distance between <inline-formula id=\"IEq14\"><alternatives><tex-math id=\"M29\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$X_{m} \\left( i \\right)$$\\end{document}</tex-math><mml:math id=\"M30\"><mml:mrow><mml:msub><mml:mi>X</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>i</mml:mi></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq14.gif\"/></alternatives></inline-formula> and <inline-formula id=\"IEq15\"><alternatives><tex-math id=\"M31\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$X_{m} \\left( j \\right)$$\\end{document}</tex-math><mml:math id=\"M32\"><mml:mrow><mml:msub><mml:mi>X</mml:mi><mml:mi>m</mml:mi></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>j</mml:mi></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq15.gif\"/></alternatives></inline-formula> is less than or equal to a threshold <inline-formula id=\"IEq16\"><alternatives><tex-math id=\"M33\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$r$$\\end{document}</tex-math><mml:math id=\"M34\"><mml:mi>r</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq16.gif\"/></alternatives></inline-formula>.<disp-formula id=\"Equ2\"><label>2</label><alternatives><tex-math id=\"M35\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\begin{gathered} \\hfill \\\\ B_{i}^{m} \\left( r \\right) = \\frac{{B_{i} \\left( r \\right)}}{N - m - 1}, \\\\ \\text{Then, for}\\, 1 \\le i \\ge N - m, \\hfill \\\\ \\end{gathered}$$\\end{document}</tex-math><mml:math id=\"M36\" display=\"block\"><mml:mrow><mml:mtable><mml:mtr><mml:mtd/></mml:mtr><mml:mtr><mml:mtd><mml:mrow><mml:mrow/><mml:msubsup><mml:mi>B</mml:mi><mml:mrow><mml:mi>i</mml:mi></mml:mrow><mml:mi>m</mml:mi></mml:msubsup><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mi>B</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced></mml:mrow><mml:mrow><mml:mi>N</mml:mi><mml:mo>-</mml:mo><mml:mi>m</mml:mi><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfrac><mml:mo>,</mml:mo></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mrow><mml:mrow/><mml:mtext>Then, for</mml:mtext><mml:mspace width=\"0.166667em\"/><mml:mn>1</mml:mn><mml:mo>≤</mml:mo><mml:mi>i</mml:mi><mml:mo>≥</mml:mo><mml:mi>N</mml:mi><mml:mo>-</mml:mo><mml:mi>m</mml:mi><mml:mo>,</mml:mo></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mrow/></mml:mtd></mml:mtr></mml:mtable></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70814_Article_Equ2.gif\" position=\"anchor\"/></alternatives></disp-formula></p></list-item><list-item><p id=\"Par19\"> Defined <inline-formula id=\"IEq17\"><alternatives><tex-math id=\"M37\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$B^{m} \\left( r \\right)$$\\end{document}</tex-math><mml:math id=\"M38\"><mml:mrow><mml:msup><mml:mi>B</mml:mi><mml:mi>m</mml:mi></mml:msup><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq17.gif\"/></alternatives></inline-formula> as<disp-formula id=\"Equ3\"><label>3</label><alternatives><tex-math id=\"M39\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$B^{m} = \\frac{1}{N - m}\\mathop \\sum \\limits_{i = 1}^{N - m} B_{i}^{m} \\left( r \\right)$$\\end{document}</tex-math><mml:math id=\"M40\" display=\"block\"><mml:mrow><mml:msup><mml:mi>B</mml:mi><mml:mi>m</mml:mi></mml:msup><mml:mo>=</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:mi>N</mml:mi><mml:mo>-</mml:mo><mml:mi>m</mml:mi></mml:mrow></mml:mfrac><mml:munderover><mml:mo movablelimits=\"false\">∑</mml:mo><mml:mrow><mml:mi>i</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>N</mml:mi><mml:mo>-</mml:mo><mml:mi>m</mml:mi></mml:mrow></mml:munderover><mml:msubsup><mml:mi>B</mml:mi><mml:mrow><mml:mi>i</mml:mi></mml:mrow><mml:mi>m</mml:mi></mml:msubsup><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70814_Article_Equ3.gif\" position=\"anchor\"/></alternatives></disp-formula></p></list-item><list-item><p id=\"Par20\"> This previous procedure is repeated, increasing the dimension to <inline-formula id=\"IEq18\"><alternatives><tex-math id=\"M41\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$m + 1$$\\end{document}</tex-math><mml:math id=\"M42\"><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq18.gif\"/></alternatives></inline-formula> to calculate <inline-formula id=\"IEq19\"><alternatives><tex-math id=\"M43\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$A_{i} \\left( r \\right)$$\\end{document}</tex-math><mml:math id=\"M44\"><mml:mrow><mml:msub><mml:mi>A</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq19.gif\"/></alternatives></inline-formula> as the number of <inline-formula id=\"IEq20\"><alternatives><tex-math id=\"M45\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$X_{m + 1} \\left( i \\right)$$\\end{document}</tex-math><mml:math id=\"M46\"><mml:mrow><mml:msub><mml:mi>X</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>i</mml:mi></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq20.gif\"/></alternatives></inline-formula> within <inline-formula id=\"IEq21\"><alternatives><tex-math id=\"M47\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$r$$\\end{document}</tex-math><mml:math id=\"M48\"><mml:mi>r</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq21.gif\"/></alternatives></inline-formula> of <inline-formula id=\"IEq22\"><alternatives><tex-math id=\"M49\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$X_{m + 1} \\left( j \\right)$$\\end{document}</tex-math><mml:math id=\"M50\"><mml:mrow><mml:msub><mml:mi>X</mml:mi><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>j</mml:mi></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq22.gif\"/></alternatives></inline-formula>, where <inline-formula id=\"IEq23\"><alternatives><tex-math id=\"M51\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$j$$\\end{document}</tex-math><mml:math id=\"M52\"><mml:mi>j</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq23.gif\"/></alternatives></inline-formula> ranges from 1 to <inline-formula id=\"IEq24\"><alternatives><tex-math id=\"M53\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$N - m\\left( {i \\ne j} \\right)$$\\end{document}</tex-math><mml:math id=\"M54\"><mml:mrow><mml:mi>N</mml:mi><mml:mo>-</mml:mo><mml:mi>m</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>i</mml:mi><mml:mo>≠</mml:mo><mml:mi>j</mml:mi></mml:mrow></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq24.gif\"/></alternatives></inline-formula>. Then,<inline-formula id=\"IEq25\"><alternatives><tex-math id=\"M55\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${ }A_{i}^{m} \\left( r \\right)$$\\end{document}</tex-math><mml:math id=\"M56\"><mml:mrow><mml:mrow/><mml:msubsup><mml:mi>A</mml:mi><mml:mrow><mml:mi>i</mml:mi></mml:mrow><mml:mi>m</mml:mi></mml:msubsup><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq25.gif\"/></alternatives></inline-formula> is defined as:<disp-formula id=\"Equ4\"><label>4</label><alternatives><tex-math id=\"M57\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$A_{i}^{m} \\left( r \\right) = \\frac{{A_{i} \\left( r \\right)}}{N - m - 1}$$\\end{document}</tex-math><mml:math id=\"M58\" display=\"block\"><mml:mrow><mml:msubsup><mml:mi>A</mml:mi><mml:mrow><mml:mi>i</mml:mi></mml:mrow><mml:mi>m</mml:mi></mml:msubsup><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mi>A</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced></mml:mrow><mml:mrow><mml:mi>N</mml:mi><mml:mo>-</mml:mo><mml:mi>m</mml:mi><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:mfrac></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70814_Article_Equ4.gif\" position=\"anchor\"/></alternatives></disp-formula></p></list-item><list-item><p id=\"Par21\"> Set <inline-formula id=\"IEq26\"><alternatives><tex-math id=\"M59\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$A^{m} \\left( r \\right)$$\\end{document}</tex-math><mml:math id=\"M60\"><mml:mrow><mml:msup><mml:mi>A</mml:mi><mml:mi>m</mml:mi></mml:msup><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq26.gif\"/></alternatives></inline-formula> as<disp-formula id=\"Equ5\"><label>5</label><alternatives><tex-math id=\"M61\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$A^{m} = \\frac{1}{N - m}\\mathop \\sum \\limits_{i = 1}^{N - m} A_{i}^{m} \\left( r \\right)$$\\end{document}</tex-math><mml:math id=\"M62\" display=\"block\"><mml:mrow><mml:msup><mml:mi>A</mml:mi><mml:mi>m</mml:mi></mml:msup><mml:mo>=</mml:mo><mml:mfrac><mml:mn>1</mml:mn><mml:mrow><mml:mi>N</mml:mi><mml:mo>-</mml:mo><mml:mi>m</mml:mi></mml:mrow></mml:mfrac><mml:munderover><mml:mo movablelimits=\"false\">∑</mml:mo><mml:mrow><mml:mi>i</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>N</mml:mi><mml:mo>-</mml:mo><mml:mi>m</mml:mi></mml:mrow></mml:munderover><mml:msubsup><mml:mi>A</mml:mi><mml:mrow><mml:mi>i</mml:mi></mml:mrow><mml:mi>m</mml:mi></mml:msubsup><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70814_Article_Equ5.gif\" position=\"anchor\"/></alternatives></disp-formula></p></list-item></list></p><p id=\"Par22\">Thus, <inline-formula id=\"IEq27\"><alternatives><tex-math id=\"M63\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$B^{m} \\left( r \\right)$$\\end{document}</tex-math><mml:math id=\"M64\"><mml:mrow><mml:msup><mml:mi>B</mml:mi><mml:mi>m</mml:mi></mml:msup><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq27.gif\"/></alternatives></inline-formula> is the probability that two sequences will match for <inline-formula id=\"IEq28\"><alternatives><tex-math id=\"M65\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$m$$\\end{document}</tex-math><mml:math id=\"M66\"><mml:mi>m</mml:mi></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq28.gif\"/></alternatives></inline-formula> samples, whereas <inline-formula id=\"IEq29\"><alternatives><tex-math id=\"M67\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$A^{m} \\left( r \\right)$$\\end{document}</tex-math><mml:math id=\"M68\"><mml:mrow><mml:msup><mml:mi>A</mml:mi><mml:mi>m</mml:mi></mml:msup><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq29.gif\"/></alternatives></inline-formula> is the probability that two sequences will match for <inline-formula id=\"IEq30\"><alternatives><tex-math id=\"M69\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$m + 1$$\\end{document}</tex-math><mml:math id=\"M70\"><mml:mrow><mml:mi>m</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:math><inline-graphic xlink:href=\"41598_2020_70814_Article_IEq30.gif\"/></alternatives></inline-formula> samples. Finally, sample entropy is then defined as<disp-formula id=\"Equ6\"><label>6</label><alternatives><tex-math id=\"M71\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$SE\\left( {m,{ }r} \\right) = \\mathop {\\lim }\\limits_{N \\to \\infty } \\left\\{ { - ln\\left[ {\\frac{{A^{m} \\left( r \\right)}}{{B^{m} \\left( r \\right)}}} \\right]} \\right\\}{ }$$\\end{document}</tex-math><mml:math id=\"M72\" display=\"block\"><mml:mrow><mml:mi>S</mml:mi><mml:mi>E</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>m</mml:mi><mml:mo>,</mml:mo><mml:mrow/><mml:mi>r</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:munder><mml:mo movablelimits=\"false\">lim</mml:mo><mml:mrow><mml:mi>N</mml:mi><mml:mo stretchy=\"false\">→</mml:mo><mml:mi>∞</mml:mi></mml:mrow></mml:munder><mml:mfenced close=\"}\" open=\"{\"><mml:mrow><mml:mo>-</mml:mo><mml:mi>l</mml:mi><mml:mi>n</mml:mi><mml:mfenced close=\"]\" open=\"[\"><mml:mfrac><mml:mrow><mml:msup><mml:mi>A</mml:mi><mml:mi>m</mml:mi></mml:msup><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced></mml:mrow><mml:mrow><mml:msup><mml:mi>B</mml:mi><mml:mi>m</mml:mi></mml:msup><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced></mml:mrow></mml:mfrac></mml:mfenced></mml:mrow></mml:mfenced><mml:mrow/></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70814_Article_Equ6.gif\" position=\"anchor\"/></alternatives></disp-formula>\nwhich is estimated by the statistic:<disp-formula id=\"Equ7\"><label>7</label><alternatives><tex-math id=\"M73\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$SE\\left( {m, r, N} \\right) = - ln\\left( {\\frac{{A^{m} \\left( r \\right)}}{{B^{m} \\left( r \\right)}}} \\right)$$\\end{document}</tex-math><mml:math id=\"M74\" display=\"block\"><mml:mrow><mml:mi>S</mml:mi><mml:mi>E</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>m</mml:mi><mml:mo>,</mml:mo><mml:mi>r</mml:mi><mml:mo>,</mml:mo><mml:mi>N</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mo>-</mml:mo><mml:mi>l</mml:mi><mml:mi>n</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mfrac><mml:mrow><mml:msup><mml:mi>A</mml:mi><mml:mi>m</mml:mi></mml:msup><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced></mml:mrow><mml:mrow><mml:msup><mml:mi>B</mml:mi><mml:mi>m</mml:mi></mml:msup><mml:mfenced close=\")\" open=\"(\"><mml:mi>r</mml:mi></mml:mfenced></mml:mrow></mml:mfrac></mml:mfenced></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70814_Article_Equ7.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p id=\"Par24\">The <italic>m</italic> parameter is generally taken as 2, while the <italic>r</italic> parameter normally ranges between 0.1 and 0.25 times the standard deviation (SD) of the segment analyzed of length <italic>N</italic>. In this study, <italic>SE</italic> was calculated over the Flow (<italic>SE</italic>-Flow) and Paw (<italic>SE</italic>-Paw) signals using a 30-s sliding window (<italic>N</italic> = 1,200 samples) with 50% overlap. <italic>SE</italic> was explored using <italic>m</italic> from 1 to 20 and with <italic>r</italic> values equal to 0.1, 0.2, 0.3, and 0.4 times the SD of each sliding window. To reduce noise and to increase the consistency of the results, we applied an 8-period-long exponential moving average filter to the <italic>SE</italic> series.</p></sec><sec id=\"Sec8\"><title>Automatic CP-VI detection</title><p id=\"Par25\">We devised an automated algorithm based on <italic>SE</italic> to detect CP-VI events (European patent application number EP19383116). Figure <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> summarizes the algorithm in a flowchart. Detection of a CP-VI depends on whether the percentage of change (<italic>PC</italic>) in <italic>SE</italic> with respect to the patient's own <italic>SE</italic> baseline value during the 15-min period is greater than a predefined threshold of change (<italic>Th)</italic>. We calculated <italic>PC</italic> for <italic>SE</italic>-Flow and <italic>SE</italic>-Paw in each 15-min period in two ways, using the following derived features (the mean <italic>SE</italic> value [<italic>SE</italic>-Flow<sub>mean</sub> and <italic>SE</italic>-Paw<sub>mean</sub>], and the maximum <italic>SE</italic> value [<italic>SE</italic>-Flow<sub>max</sub> and <italic>SE</italic>-Paw<sub>max</sub>]), applying different values of <italic>Th</italic> (15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50%). We hypothesized that <italic>SE</italic> values would be higher in periods with CP-VI than in periods with regular patient-ventilator interactions. Periods were considered to contain a CP-VI event when <italic>PC</italic> exceeded the <italic>Th</italic>. The optimal <italic>Th</italic> for CP-VI detection was selected during the <italic>SE</italic> setting optimization procedure (explained below).<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Automatic CP-VI detection. Respiratory signals (Flow and Paw) are decimated at a sampling rate of 40 Hz. Sample entropy (<italic>SE</italic>) is calculated for different values of the embedding dimension (<italic>m</italic>) and tolerance (<italic>r</italic>). An 8-period-long exponential moving average is used to reduce noise and to increase the consistency of the <italic>SE</italic> results. Two <italic>SE</italic> features are determined for each 15-min period: the mean value and the maximum value. The percentage of change (<italic>PC</italic>) from the patient's own baseline value is calculated for each <italic>SE</italic> setting. When <italic>PC</italic> exceeds a determined threshold (<italic>Th</italic>), the period is considered to contain a CP-VI event. An optimization procedure is required to select the values of <italic>m</italic>, <italic>r</italic>, <italic>Th</italic>, respiratory signal, and <italic>SE</italic> feature that yield the most robust estimations of CP-VI.</p></caption><graphic xlink:href=\"41598_2020_70814_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par26\">Keim-Malpas<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup> recently proposed that alert thresholds derived from continuous analytic monitoring should be based on the degree of change from the patient’s own baseline, rather than on general cutoff thresholds. In our study there was no single baseline value common to all patients; each patient had their own baseline.</p><p id=\"Par27\">The baseline value of each <italic>SE</italic> feature was initialized with the value calculated in the first 15-min period. This value was updated with each new 15-min segment if the <italic>SE</italic> feature of the new one was lower than the current baseline.</p></sec><sec id=\"Sec9\"><title>Statistical analysis</title><p id=\"Par28\">Fleiss’ kappa coefficient was used to assess the reliability of agreement among scorers for visual assessment<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. The automated CP-VI detection algorithm was applied over the <italic>SE</italic> series derived from the same Flow and Paw tracings previously used for visual assessment. To evaluate the performance of the automated algorithm with respect to the gold standard visual assessment, we calculated sensitivity, specificity, positive and negative predictive values (PPV and NPV respectively), accuracy, and the Matthews correlation coefficient (MCC)<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. Widely used in biomedical research, the MCC is considered a balanced measure of the confusion matrix of true and false positives and negatives<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref>–<xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>. Calculation of the MCC is based on all four elements of the confusion matrix: true positive (TP), true negative (TN), false positive (FP), and false negative (FN) values, as follows:<disp-formula id=\"Equ8\"><label>8</label><alternatives><tex-math id=\"M75\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$MCC = \\frac{{TP{*}TN - FP{*}FN}}{{\\sqrt {\\left( {TP + FP} \\right){*}\\left( {TP + FN} \\right){*}\\left( {TN + FP} \\right){*}\\left( {TN + FN} \\right)} }}$$\\end{document}</tex-math><mml:math id=\"M76\" display=\"block\"><mml:mrow><mml:mi>M</mml:mi><mml:mi>C</mml:mi><mml:mi>C</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>T</mml:mi><mml:mi>P</mml:mi><mml:mrow><mml:mrow/><mml:mo>∗</mml:mo></mml:mrow><mml:mi>T</mml:mi><mml:mi>N</mml:mi><mml:mo>-</mml:mo><mml:mi>F</mml:mi><mml:mi>P</mml:mi><mml:mrow><mml:mrow/><mml:mo>∗</mml:mo></mml:mrow><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:mrow><mml:msqrt><mml:mrow><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>T</mml:mi><mml:mi>P</mml:mi><mml:mo>+</mml:mo><mml:mi>F</mml:mi><mml:mi>P</mml:mi></mml:mrow></mml:mfenced><mml:mrow><mml:mrow/><mml:mo>∗</mml:mo></mml:mrow><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>T</mml:mi><mml:mi>P</mml:mi><mml:mo>+</mml:mo><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:mrow></mml:mfenced><mml:mrow><mml:mrow/><mml:mo>∗</mml:mo></mml:mrow><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>T</mml:mi><mml:mi>N</mml:mi><mml:mo>+</mml:mo><mml:mi>F</mml:mi><mml:mi>P</mml:mi></mml:mrow></mml:mfenced><mml:mrow><mml:mrow/><mml:mo>∗</mml:mo></mml:mrow><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>T</mml:mi><mml:mi>N</mml:mi><mml:mo>+</mml:mo><mml:mi>F</mml:mi><mml:mi>N</mml:mi></mml:mrow></mml:mfenced></mml:mrow></mml:msqrt></mml:mfrac></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70814_Article_Equ8.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p id=\"Par29\">MCC values can range from − 1 to + 1. An MCC value of − 1 suggests perfect disagreement between the predictions and the gold standard, and a value of 1 suggests perfect agreement between the predictions and the gold standard; a value of 0 indicates that the prediction is no better than random. The MCC index was used as the measure of effectiveness during the process to optimize <italic>SE</italic> settings so as to achieve the most robust CP-VI estimation.</p></sec><sec id=\"Sec10\"><title>Optimization procedure (selection of <italic>m</italic>, <italic>r</italic>, and <italic>Th</italic>)</title><p id=\"Par30\">In entropy studies, determining the optimal settings to robustly extract the randomness of a series of data is an important step<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref>,<xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>. To select the optimal settings for the <italic>SE</italic> parameters <italic>m</italic> and <italic>r</italic> and the optimal <italic>Th</italic> for estimating CP-VI, we used a repeated holdout cross-validation method with the MCC as a measure of effectiveness.</p><p id=\"Par31\">Figure <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> depicts the steps involved in the optimization and the validation procedure. Once the experts had visually validated the set of 92 observations, it was randomly divided into two subsets: 70% of the data for optimization and the remaining 30% of the data for validation. This optimization procedure was repeated a total of 15 times using different subsets (randomly selected each time) to capture as much relevant information as possible and to minimize the potential bias resulting from fitting the settings on a single partition. The MCC metric was computed for all combinations of <italic>m</italic>, <italic>r</italic>, and <italic>Th</italic> for each repetition. Finally, the maximum mean MCC value determined the optimal combination of <italic>SE</italic> settings and <italic>Th</italic> among all possible combinations. The optimization procedure was individually applied to the features derived from <italic>SE</italic>-Flow (<italic>SE</italic>-Flow<sub>mean</sub>, <italic>SE</italic>-Flow<sub>max</sub>) and <italic>SE</italic>-Paw (<italic>SE</italic>-Paw<sub>mean</sub>, <italic>SE</italic>-Paw<sub>max</sub>) in order to determine the respiratory signal and features that best reflect CP-VI.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Flowchart for the optimization procedure and validation. Procedure to select the optimal sample entropy (<italic>SE</italic>) settings (<italic>m</italic> and <italic>r</italic>) and the threshold of change (<italic>Th</italic>) for each <italic>SE</italic> airway flow (Flow) and airway pressure (Paw) features (<italic>SE</italic>-Flow<sub>mean</sub>, <italic>SE</italic>-Flow<sub>max</sub>, <italic>SE</italic>-Paw<sub>mean</sub>, and <italic>SE</italic>-Paw<sub>max</sub>). The dataset visually validated by the experts was randomly divided into two subsets: optimization (Opt.) and validation (Val.). The optimization procedure was repeated a total of 15 times using different subsets (randomly selected each time). The global maximum mean value of the Matthews correlation coefficient (MCC) determined the optimal values of <italic>m*</italic>, <italic>r*</italic>, and <italic>Th*</italic> among all possible combinations and all <italic>SE</italic>-derived features. Finally, the mean values of the measures of accuracy were computed for the optimal combination of parameters in both the optimization and the validation subsets.</p></caption><graphic xlink:href=\"41598_2020_70814_Fig3_HTML\" id=\"MO3\"/></fig></p><p id=\"Par32\">In addition, a sensitivity analysis by using a small grid search of <italic>r</italic> values (step = 0.01) around the optimal value in the best features derived from <italic>SE</italic>-Flow and <italic>SE</italic>-Paw was performed to compare regions of confidence and to investigate whether the selected <italic>r</italic> value is a robust local maximum.</p><p id=\"Par33\">To assess the robustness of the optimization procedure, we computed the medians and interquartile ranges of all measures of performance (MCC, sensitivity, specificity, accuracy, PPV, and NPV) considering the optimal combination for both the optimization and validation subsets.</p></sec></sec><sec id=\"Sec11\"><title>Results</title><sec id=\"Sec12\"><title>Visual CP-VI analysis by experts</title><p id=\"Par34\">The experts visually assessed a total of 92 periods: 45 periods of PSV (22 with CP-VI and 23 without) and 47 periods of ACV (24 with CP-VI and 23 without). Fleiss’ kappa for inter-rater agreement was 0.90 (0.87–0.93), indicating almost perfect agreement.</p></sec><sec id=\"Sec13\"><title>Detecting CP-VI with <italic>SE</italic></title><p id=\"Par35\">The exponential moving average filter reduced the noise in <italic>SE</italic> series and generated a smoothed <italic>SE</italic> version suitable for detecting CP-VI (see Supplementary Methods and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>). Figure <xref rid=\"Fig4\" ref-type=\"fig\">4</xref> shows representative examples of respiratory signal tracings with the corresponding <italic>SE</italic>-Flow (<italic>m</italic> = 2 and <italic>r</italic> = 0.2) and <italic>SE</italic>-Paw (<italic>m</italic> = 4 and <italic>r</italic> = 0.2) tracings. <italic>SE</italic> was highly sensitive to changes in the irregularity of the respiratory pattern occurring during ventilation.<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Representative examples of ventilator signals for airway flow (Flow) (<bold>a1</bold>–<bold>a3</bold>) and airway pressure (Paw) (<bold>c1</bold>–<bold>c3</bold>) recorded over 15-min periods in the two hours prior to self-extubation, together with the sample entropy (<italic>SE</italic>) tracings derived from Flow (<bold>b1</bold>–<bold>b3</bold>) and Paw (<bold>d1</bold>–<bold>d3</bold>). Both, <italic>SE</italic>-Flow and <italic>SE</italic>-Paw were calculated with <italic>r</italic> = 0.2 × SD of each overlapping 30-s-long sliding window, by using different values of <italic>m</italic> equal to 2 and 4, respectively. Three 15-min periods are represented, corresponding to (1) no occurrence of CP-VI (left panel), (2) occurrence of CP-VI that returned to baseline values (middle panel), and (3) progressive increase in CP-VI leading to self-extubation. <italic>SE</italic> is highly sensitive to changes in irregularity during MV.</p></caption><graphic xlink:href=\"41598_2020_70814_Fig4_HTML\" id=\"MO4\"/></fig></p></sec><sec id=\"Sec14\"><title>Optimization of <italic>SE</italic> settings, <italic>Th</italic> detection using a repeated holdout cross-validation procedure</title><p id=\"Par36\">Figure <xref rid=\"Fig5\" ref-type=\"fig\">5</xref> shows the procedure used to optimize <italic>SE</italic> settings and <italic>Th</italic> for CP-VI detection. We calculated the mean MCC value for each combination of <italic>m</italic>, <italic>r</italic>, and <italic>Th</italic> for all derived features analyzed (<italic>SE</italic>-Flow<sub>mean</sub>, <italic>SE</italic>-Flow<sub>max</sub>, <italic>SE</italic>-Paw<sub>mean</sub>, and <italic>SE</italic>-Paw<sub>max</sub>). In general, <italic>SE</italic>-Paw features exhibit much less sensitivity to <italic>m</italic> parameter selection than <italic>SE</italic>-Flow features. <italic>SE</italic>-Flow<sub>max</sub> and <italic>SE</italic>-Paw<sub>max</sub> features yielded the highest mean MCC values. The highest MCC values for <italic>SE</italic>-Flow<sub>max</sub> were found for values of <italic>m</italic> = 2, <italic>r</italic> equal to 0.2 and 0.3, and <italic>Th</italic> between 20 and 35%, whereas for <italic>SE</italic>-Paw<sub>max</sub> were found for values of <italic>m</italic> equal to 3 and 4, <italic>r</italic> = 0.2, and <italic>Th</italic> between 25 and 30%. The optimal <italic>SE</italic> settings for <italic>SE</italic>-Flow<sub>max</sub> were <italic>m</italic> = 2, <italic>r</italic> = 0.2, and <italic>Th</italic> = 25%, and for <italic>SE</italic>-Paw<sub>max</sub>\n<italic>m</italic> = 4, <italic>r</italic> = 0.2, and <italic>Th</italic> = 30%. As regards the optimal <italic>SE</italic> settings, <italic>SE</italic>-Flow<sub>max</sub> at <italic>Th</italic> = 25% (<italic>SE</italic>-Flow<sub>max</sub>25) yielded the highest mean MCC value (0.84) and <italic>SE</italic>-Paw<sub>max</sub> at <italic>Th</italic> = 30% (<italic>SE</italic>-Flow<sub>max</sub>30) yielded the highest mean MCC value (0.86). Both <italic>SE</italic>-Flow<sub>max</sub>25 and <italic>SE</italic>-Paw<sub>max</sub>30 yielded their highest MCC values in 13 of the 15 repetitions. The sensitivity analysis conducted for the <italic>SE</italic>-Paw<sub>max</sub> and <italic>SE</italic>-Flow<sub>max</sub> features around the optimal value of <italic>r</italic> = 0.2 is shown in Supplementary Figure <xref rid=\"MOESM1\" ref-type=\"media\">S3</xref>. Once we had determined the settings that best detected CP-VI, we evaluated the performance of the algorithm in the 15 repetitions of the cross-validation procedure. Figure <xref rid=\"Fig6\" ref-type=\"fig\">6</xref> displays the algorithm’s performance statistics. The median values of all the parameters observed in the optimization subset were slightly higher than those observed in the validation subset (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>); this is a common consequence of the repeated holdout cross-validation process. The performance of <italic>SE</italic>-Flow<sub>max</sub>25 and <italic>SE</italic>-Paw<sub>max</sub>30 stratified by ventilator modality (grouped into pressure support ventilation and assist-control ventilation modes) is shown in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>.<fig id=\"Fig5\"><label>Figure 5</label><caption><p>Repeated holdout cross-validation process for optimization of sample entropy (<italic>SE</italic>) settings and threshold (<italic>Th</italic>) for the detection of complex patient-ventilator interactions. We calculated the mean Matthews correlation coefficient (MCC) for each derived feature analyzed and each combination of <italic>m</italic> (1 to 20), <italic>r</italic> (0.1, 0.2, 0.3, and 0.4 times the SD of each sliding window), and <italic>Th</italic> (15%, 20%, 25%, 30%, 35%, 40%, 45%, and 50%) a total of 15 times using different randomly selected subsets. The upper panels show the results of the optimization process for <italic>SE</italic>-airway flow (<italic>SE</italic>-Flow), and the lower panels show the results for <italic>SE</italic>-airway pressure (<italic>SE</italic>-Paw). The color bar in each subplot shows the mean MCC scale, where values near 1 indicate more robust and consistent results. The MCC was positive in all cases. The black dot in each subplot indicates the combination that yielded the maximum mean MCC.</p></caption><graphic xlink:href=\"41598_2020_70814_Fig5_HTML\" id=\"MO5\"/></fig><fig id=\"Fig6\"><label>Figure 6</label><caption><p>Performance statistics for <italic>SE</italic>-Flow<sub>max</sub>25 (<italic>m</italic> = 2 and <italic>r</italic> = 0.2) and <italic>SE</italic>-Paw<sub>max</sub>30 (<italic>m</italic> = 4 and <italic>r</italic> = 0.2) for detecting CP-VI. Boxplots of (<bold>a</bold>) Matthews correlation coefficient (MCC), (<bold>b</bold>) sensitivity (Se), (<bold>c</bold>) specificity (Sp), (<bold>d</bold>) accuracy (Acc), (<bold>e</bold>) positive predictive value (PPV), and (<bold>f</bold>) negative predictive value (NPV) from 15 repetitions during optimization (white) and 15 repetitions during validation (gray). The red dot represents the mean value.</p></caption><graphic xlink:href=\"41598_2020_70814_Fig6_HTML\" id=\"MO6\"/></fig></p><p id=\"Par37\">For comparative purposes, we also carried out the procedure for optimizing <italic>SE</italic> settings and <italic>Th</italic> over the unfiltered <italic>SE</italic> series. The Supplementary Methods and the Supplementary Figure <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref> show the results obtained in this case.</p></sec></sec><sec id=\"Sec15\"><title>Discussion</title><p id=\"Par38\">Our automatic algorithm for detecting CP-VI from ventilator signals proved highly sensitive and specific in individual patients. Using non-linear analysis of <italic>SE</italic> to measure irregularity and randomness in the entire set of physiological Flow and Paw signals, the algorithm compared data from different periods in each patient’s interaction with the ventilator to detect CP-VI. In our analyses the maximum changes of <italic>SE</italic> in both Flow and Paw signals yielded the most accurate results at different thresholds and settings. The most accurate results for <italic>SE</italic>-Flow<sub>max</sub> were obtained with a threshold of change of 25% with <italic>m</italic> = 2, <italic>r</italic> = 0.2, and for <italic>SE</italic>-Paw<sub>max</sub> with a threshold of change of 30% with <italic>m</italic> = 4 and <italic>r</italic> = 0.2.</p><p id=\"Par40\">The recognition of the hidden information contained in physiological time series draws attention to the extraordinary complexity of physiological systems<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup>. Several non-linear techniques have been developed to study the irregularity and complexity of these physiomarkers<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR50\">50</xref>–<xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. Previous studies have used methods based on approximate entropy and sample entropy using breath-to-breath variability and derived indices<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR32\">32</xref>–<xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>, which relies on the detection of the appropriate respiratory cycle.</p><p id=\"Par41\">The main advantage of our approach is that it does not require the detection of each single breathing cycle to measure irregularity in Flow and Paw waveforms and thus identify the development of a CP-VI. This approach makes a fundamentally different assumption about where complexity occurs in the physical signal, focusing on transient Flow and Paw complexity rather than breath-to-breath complexity in order to accurately identify changes in the respiratory rate and asynchronies which by their nature are transient and time-limited.</p><p id=\"Par42\">To our knowledge, no recommendations are currently available for the estimation of respiratory dynamics by applying an entropy approach to the entire dataset of Flow and Paw tracings during MV<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>. Recently, Sá et al.<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>, used <italic>SE</italic> estimation upon entire Flow signal without optimized parameters. Thus, one important contribution of our study is the description of a set of optimization and validation procedures based on a repeated holdout cross-validation method used in machine-learning models, which we used to obtain the optimal <italic>m</italic>, <italic>r</italic> and <italic>Th</italic> values. Ensuring the robustness of the validation procedure.</p><p id=\"Par43\">Our study also applied a personalized threshold to determine the occurrence of a CP-VI event based on a proportional change from the patient’s own baseline value, which is continuously updated. Continuous predictive analytics monitoring achieves early detection of changes in status over time in previously stable patients. Keim-Malpas et al.<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup> recently suggested that an absolute threshold of change from baseline values may not be clinically significant in real-world settings and could lead to a high rate of false-positives in patients with high baseline values<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>. In our study, thresholds of change of 25% and 30% from <italic>SE</italic>-Flow<sub>max</sub> and <italic>SE</italic>-Paw<sub>max</sub> respectively, proved to be the most accurate for CP-VI detection. The optimization procedure found that <italic>r</italic> = 0.2 is suitable for detecting CP-VI events using <italic>SE</italic>-Flow<sub>max</sub> (<italic>m</italic> = 2, <italic>Th</italic> = 25%) or <italic>SE</italic>-Paw<sub>max</sub> (<italic>m</italic> = 4, <italic>Th</italic> = 30%) features. Additionally, the sensitivity analysis indicates that <italic>r</italic> = 0.2 proved to be a more robust local maximum for <italic>SE</italic>-Flow<sub>max</sub> feature. This might suggest that the algorithm predictions seems to be not influenced by small changes in underlying unknown parameters (i.e., different dataset, different measurement equipment or ventilator waveforms) when using <italic>SE</italic>-Flow<sub>max</sub> (<italic>m</italic> = 2, <italic>r</italic> = 0.2, <italic>Th</italic> = 25%), and therefore, could be a more suitable feature than <italic>SE</italic>-Paw<sub>max</sub> (<italic>m</italic> = 4, <italic>r</italic> = 0.2, <italic>Th</italic> = 30%).</p><p id=\"Par44\">Interestingly, both <italic>SE</italic>-Flow<sub>max</sub>25 and <italic>SE</italic>-Paw<sub>max</sub>30 performed well in detecting CP-VI in Assist-Control Ventilation, while <italic>SE</italic>-Flow<sub>max</sub>25 performed slightly better than <italic>SE</italic>-Paw<sub>max</sub>30 in Pressure Support Ventilation mode. The reason for the latter finding may be that during PSV the pressure is constant, and it is the flow waveform that exhibits more changes in accordance with patient’s demand and the mechanical properties of the diseased lung. However, due to the small sample size these sub-analysis results should be interpreted with care, and further research is needed.</p><p id=\"Par45\">Our study has several limitations. First, our algorithm responds to changes in the respiratory rate based on transient changes of Flow and Paw waveforms detected by <italic>SE</italic>, but not on inspiratory effort. This means that respiratory drive, the intensity of the neural output from the respiratory center that regulates the magnitude of inspiratory effort<sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>, may not have been fully assessed<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR56\">56</xref>,<xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup>. Unfortunately, although many techniques have been proposed<sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref>,<xref ref-type=\"bibr\" rid=\"CR58\">58</xref>–<xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup> none have been implemented at the bedside to monitor drive and effort. Our proposed algorithm does not include measurements of effort; nevertheless, whenever a diaphragmatic contraction occurs unassisted by the ventilator, and an asynchrony develops our algorithm is able to detect it.</p><p id=\"Par46\">Second, although our method does not rely on the detection of breathing cycles to measure irregularity and is based on changes in <italic>SE</italic> of Flow and Paw waveforms, none of the features deriving from breath-to-breath variability were considered. Therefore, their potential importance in detecting CP-VI is yet to be assessed.</p><p id=\"Par47\">Third, while the dataset used for the repeated hold out cross-validation method was paired between segments with and without CP-VI, most of them were from tracings of patients who self-extubate, in whom the occurrence of events of poor patient-ventilator interactions is highly unpredictable. For that reason, the clinical meaning of CP-VI in critically ill patients is yet to be determined and requires more research. Additionally, in the current study we have only examined <italic>SE</italic>, and other promising measures of entropy may also provide adequate diagnostic tool. For instance, multiscale entropy analysis<sup><xref ref-type=\"bibr\" rid=\"CR61\">61</xref>,<xref ref-type=\"bibr\" rid=\"CR62\">62</xref></sup>, Fuzzy approximate entropy<sup><xref ref-type=\"bibr\" rid=\"CR63\">63</xref></sup>, conditional entropy<sup><xref ref-type=\"bibr\" rid=\"CR64\">64</xref></sup> and distribution entropy<sup><xref ref-type=\"bibr\" rid=\"CR65\">65</xref></sup> could be others potentially useful entropy measures to be investigated.</p><p id=\"Par48\">Finally, we did not analyze data from proportional modes of MV. Thus, although it is tempting to speculate that ventilatory modes that adapt to patients’ efforts and variability might induce higher changes in <italic>SE</italic>, the performance of our algorithm in patients ventilated in these modes may differ substantially, and it should not be implemented in these modes until validated by future research.</p></sec><sec id=\"Sec16\"><title>Conclusion</title><p id=\"Par49\">Our non-invasive method based on <italic>SE</italic> measurement of Paw and Flow is able to detect CP-VI, defined as the occurrence of transient asynchronies and changes in the respiratory rate, with high accuracy. Clinical relevance and usefulness of identifying Complex Patient-Ventilator Interactions in different clinical scenarios deserves to be explored.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec17\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70814_MOESM1_ESM.docx\"><caption><p>Supplementary Information.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Leonardo Sarlabous and José Aquino-Esperanza.</p></fn></fn-group><sec><title>Supplementary information</title><p> is available for this paper at 10.1038/s41598-020-70814-4.</p></sec><ack><p>This work was funded by projects PI16/01606, integrated in the Plan Nacional de R+D+I and co-funded by the ISCIII- Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER). RTC-2017-6193-1 (AEI/FEDER UE). CIBER Enfermedades Respiratorias, and Fundació Parc Taulí.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>Study concept and design: L.S., J.A.E., R.M., C.d.H., and L.B. Data acquisition: L.S., J.A.E., C.d.H., C.S., M.B., G.G., A.O., and R.F. Data processing and interpretation: L.S., J.A.E., R.M., J.L.A., R.F., and L.B. Statistical analysis: L.S., R.M., and M.R. Figure preparation: L.S., J.A.E. and R.M. Drafting of the manuscript: L.S., J.A.E., and R.M. Revision of manuscript for important intellectual content: L.S., J.A.E., R.M., C.d.H., J.L.A., R.F., and L.B. Study supervision: L.S., J.A.E., R.M., C.d.H., A.O., R.F., and L.B. Data access and responsibility: L.B. had full access to all of the data in the study and takes full responsibility for the integrity of the data and the accuracy of the data analysis. L.S. and J.A.E. contributed equally to the study. All authors reviewed the manuscript.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>The datasets generated and analyzed in the current study are available from the corresponding author on reasonable request.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par50\">L.S., J.A.E., R.M., C.d.H., J.L.A, and L.B. have been named in a provisional European patent application number EP19383116 owned by Corporació Sanitària Parc Taulí: “A device and method for respiratory monitoring in mechanically ventilated patients”. L.B. is inventor of a US patent owned by Corporació Sanitària Parc Taulí: “Method and system for managed related patient parameters provided by a monitoring device”, US Patent No. 12/538,940. L.B. own stock options of BetterCare S.L., a research and development spinoff of Corporació Sanitària Parc Taulí. The remaining authors have no conflicts of interest.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Blanch</surname><given-names>L</given-names></name><etal/></person-group><article-title>Asynchronies during mechanical ventilation are associated with mortality</article-title><source>Intensive Care Med.</source><year>2015</year><volume>41</volume><fpage>633</fpage><lpage>641</lpage><pub-id pub-id-type=\"pmid\">25693449</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thille</surname><given-names>AW</given-names></name><name><surname>Rodriguez</surname><given-names>P</given-names></name><name><surname>Cabello</surname><given-names>B</given-names></name><name><surname>Lellouche</surname><given-names>F</given-names></name><name><surname>Brochard</surname><given-names>L</given-names></name></person-group><article-title>Patient-ventilator asynchrony during assisted mechanical ventilation</article-title><source>Intensive Care Med.</source><year>2006</year><volume>32</volume><fpage>1515</fpage><lpage>1522</lpage><pub-id pub-id-type=\"pmid\">16896854</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rué</surname><given-names>M</given-names></name><etal/></person-group><article-title>Bayesian joint modeling of bivariate longitudinal and competing risks data: an application to study patient-ventilator asynchronies in critical care patients</article-title><source>Biom. J.</source><year>2017</year><volume>59</volume><fpage>1184</fpage><lpage>1203</lpage><pub-id pub-id-type=\"pmid\">28799274</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vaporidi</surname><given-names>K</given-names></name><etal/></person-group><article-title>Clusters of ineffective efforts during mechanical ventilation: impact on outcome</article-title><source>Intensive Care Med.</source><year>2017</year><volume>43</volume><fpage>184</fpage><lpage>191</lpage><pub-id pub-id-type=\"pmid\">27778044</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Beitler</surname><given-names>JR</given-names></name><etal/></person-group><article-title>Quantifying unintended exposure to high tidal volumes from breath stacking dyssynchrony in ARDS: the BREATHE criteria</article-title><source>Intensive Care Med.</source><year>2016</year><volume>42</volume><fpage>1427</fpage><lpage>1436</lpage><pub-id pub-id-type=\"pmid\">27342819</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>de Haro</surname><given-names>C</given-names></name><etal/></person-group><article-title>Double cycling during mechanical ventilation: frequency, mechanisms, and physiological implications</article-title><source>Crit. Care Med.</source><year>2018</year><volume>46</volume><fpage>1385</fpage><lpage>1392</lpage><pub-id pub-id-type=\"pmid\">29985211</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Wit</surname><given-names>M</given-names></name><etal/></person-group><article-title>Ineffective triggering predicts increased duration of mechanical ventilation</article-title><source>Crit. Care Med.</source><year>2009</year><volume>37</volume><fpage>2740</fpage><lpage>2745</lpage><pub-id pub-id-type=\"pmid\">19886000</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wysocki</surname><given-names>M</given-names></name><etal/></person-group><article-title>Reduced breathing variability as a predictor of unsuccessful patient separation from mechanical ventilation</article-title><source>Crit Care Med</source><year>2006</year><volume>34</volume><fpage>2076</fpage><lpage>2083</lpage><pub-id pub-id-type=\"pmid\">16755257</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Blanch</surname><given-names>L</given-names></name><etal/></person-group><article-title>Validation of the Better Care® system to detect ineffective efforts during expiration in mechanically ventilated patients: a pilot study</article-title><source>Intensive Care Med.</source><year>2012</year><volume>38</volume><fpage>772</fpage><lpage>780</lpage><pub-id pub-id-type=\"pmid\">22297667</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Marchuk</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Predicting patient-ventilator asynchronies with hidden Markov models</article-title><source>Sci. Rep.</source><year>2018</year><volume>8</volume><fpage>1</fpage><lpage>7</lpage><pub-id pub-id-type=\"pmid\">29311619</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sottile</surname><given-names>PD</given-names></name><name><surname>Albers</surname><given-names>D</given-names></name><name><surname>Higgins</surname><given-names>C</given-names></name><name><surname>Mckeehan</surname><given-names>J</given-names></name><name><surname>Moss</surname><given-names>MM</given-names></name></person-group><article-title>The association between ventilator dyssynchrony, delivered tidal volume, and sedation using a novel automated ventilator dyssynchrony detection algorithm</article-title><source>Crit. Care Med.</source><year>2018</year><volume>46</volume><fpage>e151</fpage><lpage>e157</lpage><pub-id pub-id-type=\"pmid\">29337804</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><mixed-citation publication-type=\"other\">Tobin, M. J., Alex, C. G. & Fahey, P. J. Fighting the ventilator. in <italic>Principles and Practice of Mechanical Ventialtion</italic> (ed. Tobin, M. J.) 1121–1136 (2006).</mixed-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tobin</surname><given-names>MJ</given-names></name><etal/></person-group><article-title>The pattern of breathing during successful and unsuccessful trials of weaning from mechanical ventilation</article-title><source>Am. Rev. Respir. Dis.</source><year>1986</year><volume>134</volume><fpage>1111</fpage><lpage>1118</lpage><pub-id pub-id-type=\"pmid\">3789513</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tobin</surname><given-names>MJ</given-names></name><name><surname>Perez</surname><given-names>W</given-names></name><name><surname>Guenther</surname><given-names>SM</given-names></name><name><surname>D’Alonzo</surname><given-names>G</given-names></name><name><surname>Dantzker</surname><given-names>DR</given-names></name></person-group><article-title>Breathing pattern and metabolic behavior during anticipation of exercise</article-title><source>J. Appl. Physiol.</source><year>1986</year><volume>60</volume><fpage>1306</fpage><lpage>1312</lpage><pub-id pub-id-type=\"pmid\">3700308</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tobin</surname><given-names>M</given-names></name><etal/></person-group><article-title>Variability and timing of resting respiratory in healthy subjects drive</article-title><source>J. Appl. Physiol.</source><year>1988</year><volume>65</volume><fpage>309</fpage><lpage>317</lpage><pub-id pub-id-type=\"pmid\">3403474</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Benchetrit</surname><given-names>G</given-names></name></person-group><article-title>Breathing pattern in humans: diversity and individuality</article-title><source>Respir. Physiol.</source><year>2000</year><volume>122</volume><fpage>123</fpage><lpage>129</lpage><pub-id pub-id-type=\"pmid\">10967339</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Godin</surname><given-names>P</given-names></name><name><surname>Buchman</surname><given-names>T</given-names></name></person-group><article-title>Uncoupling of biological oscillators: a complementary hypothesis concerning the pathogenesis of multiple organ dysfunction syndrome</article-title><source>Crit. Care Med.</source><year>1996</year><volume>24</volume><fpage>1107</fpage><lpage>1116</lpage><pub-id pub-id-type=\"pmid\">8674321</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pincus</surname><given-names>SM</given-names></name></person-group><article-title>Greater signal regularity may indicate increased system isolation</article-title><source>Math. Biosci.</source><year>1994</year><volume>122</volume><fpage>161</fpage><lpage>181</lpage><pub-id pub-id-type=\"pmid\">7919665</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>White</surname><given-names>CE</given-names></name><etal/></person-group><article-title>Lower interbreath interval complexity is associated with extubation failure in mechanically ventilated patients during spontaneous breathing trials</article-title><source>J. Trauma</source><year>2010</year><volume>68</volume><fpage>1310</fpage><lpage>1316</lpage><pub-id pub-id-type=\"pmid\">20539175</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dong</surname><given-names>X</given-names></name><etal/></person-group><article-title>An improved method of handling missing values in the analysis of sample entropy for continuous monitoring of physiological signals</article-title><source>Entropy</source><year>2019</year><volume>21</volume><fpage>274</fpage></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martínez-Cagigal</surname><given-names>V</given-names></name><name><surname>Santamaría-Vázquez</surname><given-names>E</given-names></name><name><surname>Hornero</surname><given-names>R</given-names></name></person-group><article-title>Asynchronous control of P300-based brain–computer interfaces using sample entropy</article-title><source>Entropy</source><year>2019</year><volume>21</volume><fpage>230</fpage></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Su</surname><given-names>C</given-names></name><etal/></person-group><article-title>A comparison of multiscale permutation entropy measures in on-line depth of anesthesia monitoring</article-title><source>PLoS ONE</source><year>2016</year><volume>11</volume><fpage>1</fpage><lpage>22</lpage></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Richman</surname><given-names>JS</given-names></name><name><surname>Moorman</surname><given-names>R</given-names></name></person-group><article-title>Physiological time-series analysis using approximate entropy and sample entropy</article-title><source>Am. J. Physiol. Hear. Circ. Physiol</source><year>2000</year><volume>278</volume><fpage>2039</fpage><lpage>2049</lpage></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sarlabous</surname><given-names>L</given-names></name><etal/></person-group><article-title>Efficiency of mechanical activation of inspiratory muscles in COPD using sample entropy</article-title><source>Eur. Respir. J.</source><year>2015</year><volume>46</volume><fpage>1808</fpage><lpage>1811</lpage><pub-id pub-id-type=\"pmid\">26493808</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sarlabous</surname><given-names>L</given-names></name><etal/></person-group><article-title>Electromyography-based respiratory onset detection in COPD patients on non-invasive mechanival ventilation</article-title><source>Entropy</source><year>2019</year><volume>21</volume><fpage>258</fpage></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alcaraz</surname><given-names>R</given-names></name><name><surname>Rieta</surname><given-names>JJ</given-names></name></person-group><article-title>A review on sample entropy applications for the non-invasive analysis of atrial fibrillation electrocardiograms</article-title><source>Biomed. Signal. Process. Control</source><year>2010</year><volume>5</volume><fpage>1</fpage><lpage>14</lpage></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abásolo</surname><given-names>D</given-names></name><name><surname>Hornero</surname><given-names>R</given-names></name><name><surname>Espino</surname><given-names>P</given-names></name><name><surname>Álvarez</surname><given-names>D</given-names></name><name><surname>Poza</surname><given-names>J</given-names></name></person-group><article-title>Entropy analysis of the EEG background activity in Alzheimer’s disease patients</article-title><source>Physiol. Meas.</source><year>2006</year><volume>27</volume><fpage>241</fpage><lpage>253</lpage><pub-id pub-id-type=\"pmid\">16462011</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Al-angari</surname><given-names>HM</given-names></name><name><surname>Sahakian</surname><given-names>AV</given-names></name></person-group><article-title>Use of sample entropy approach to study heart rate variability in obstructive sleep apnea syndrome</article-title><source>IEEE Trans. Biomed. Eng.</source><year>2007</year><volume>54</volume><fpage>1900</fpage><lpage>1904</lpage><pub-id pub-id-type=\"pmid\">17926691</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lake</surname><given-names>DE</given-names></name><etal/></person-group><article-title>Sample entropy analysis of neonatal heart rate variability</article-title><source>Am. J. Physiol. Regul. Integr. Comp. Physiol.</source><year>2002</year><volume>283</volume><fpage>789</fpage><lpage>797</lpage></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yoo</surname><given-names>CS</given-names></name><etal/></person-group><article-title>Automatic detection of seizure termination during electroconvulsive therapy using sample entropy of the electroencephalogram</article-title><source>Psychiatry Res.</source><year>2012</year><volume>195</volume><fpage>76</fpage><lpage>82</lpage><pub-id pub-id-type=\"pmid\">21831451</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>El-Khatib</surname><given-names>M</given-names></name><name><surname>Jamaleddine</surname><given-names>G</given-names></name><name><surname>Soubra</surname><given-names>R</given-names></name><name><surname>Muallem</surname><given-names>M</given-names></name></person-group><article-title>Pattern of spontaneous breathing: potential marker for weaning outcome: Spontaneous breathing pattern and weaning from mechanical ventilation</article-title><source>Intensive Care Med.</source><year>2001</year><volume>27</volume><fpage>52</fpage><lpage>58</lpage><pub-id pub-id-type=\"pmid\">11280673</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Engoren</surname><given-names>M</given-names></name></person-group><article-title>Approximate entropy of respiratory rate and tidal volume during weaning from mechanical ventilation</article-title><source>Crit. Care Med.</source><year>1998</year><volume>26</volume><fpage>1817</fpage><lpage>1823</lpage><pub-id pub-id-type=\"pmid\">9824073</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Papaioannou</surname><given-names>VE</given-names></name><name><surname>Chouvarda</surname><given-names>IG</given-names></name><name><surname>Maglaveras</surname><given-names>NK</given-names></name><name><surname>Pneumatikos</surname><given-names>IA</given-names></name></person-group><article-title>Study of multiparameter respiratory pattern complexity in surgical critically ill patients during weaning trials</article-title><source>BMC Physiol.</source><year>2011</year><volume>11</volume><fpage>2</fpage><pub-id pub-id-type=\"pmid\">21255420</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Papaioannou</surname><given-names>VE</given-names></name><name><surname>Chouvarda</surname><given-names>I</given-names></name><name><surname>Maglaveras</surname><given-names>N</given-names></name><name><surname>Dragoumanis</surname><given-names>C</given-names></name><name><surname>Pneumatikos</surname><given-names>I</given-names></name></person-group><article-title>Changes of heart and respiratory rate dynamics during weaning from mechanical ventilation: a study of physiologic complexity in surgical critically ill patients</article-title><source>J. Crit. Care</source><year>2011</year><volume>26</volume><fpage>262</fpage><lpage>272</lpage><pub-id pub-id-type=\"pmid\">20869842</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bien</surname><given-names>MY</given-names></name><etal/></person-group><article-title>Breathing pattern variability: a weaning predictor in postoperative patients recovering from systemic inflammatory response syndrome</article-title><source>Intensive Care Med.</source><year>2004</year><volume>30</volume><fpage>241</fpage><lpage>247</lpage><pub-id pub-id-type=\"pmid\">14647889</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brochard</surname><given-names>L</given-names></name></person-group><article-title>Breathing: does regular mean normal?</article-title><source>Crit. Care Med.</source><year>1998</year><volume>26</volume><fpage>1773</fpage><lpage>1774</lpage><pub-id pub-id-type=\"pmid\">9824058</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sá</surname><given-names>PM</given-names></name><name><surname>Castro</surname><given-names>HA</given-names></name><name><surname>Lopes</surname><given-names>AJ</given-names></name><name><surname>Melo</surname><given-names>PL</given-names></name></person-group><article-title>Entropy analysis for the evaluation of respiratory changes due to asbestos exposure and associated smoking</article-title><source>Entropy</source><year>2019</year><volume>21</volume><fpage>225</fpage></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tobin</surname><given-names>MJ</given-names></name></person-group><article-title>Advances in mechanical ventilation</article-title><source>N. Engl. J. Med.</source><year>2001</year><volume>344</volume><fpage>1986</fpage><lpage>1996</lpage><pub-id pub-id-type=\"pmid\">11430329</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cohen</surname><given-names>CA</given-names></name><name><surname>Zagelbaum</surname><given-names>G</given-names></name><name><surname>Gross</surname><given-names>D</given-names></name><name><surname>Ph</surname><given-names>D</given-names></name></person-group><article-title>Clinical manifestations of lnspiratory muscle fatigue</article-title><source>Am. J. Med.</source><year>1982</year><volume>73</volume><fpage>308</fpage><lpage>316</lpage><pub-id pub-id-type=\"pmid\">6812417</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Epstein</surname><given-names>SK</given-names></name><name><surname>Nevins</surname><given-names>ML</given-names></name><name><surname>Chung</surname><given-names>J</given-names></name></person-group><article-title>Effect of unplanned extubation on outcome of mechanical ventilation</article-title><source>Am. J. Respir. Crit. Care Med.</source><year>2000</year><volume>161</volume><fpage>1912</fpage><lpage>1916</lpage><pub-id pub-id-type=\"pmid\">10852766</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><mixed-citation publication-type=\"other\">Keim-Malpass, J., Clark, M. T., Lake, D. E. & Moorman, J. R. Towards development of alert thresholds for clinical deterioration using continuous predictive analytics monitoring. <italic>J. Clin. Monit. Comput.</italic> (2019).</mixed-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fleiss</surname><given-names>JL</given-names></name><name><surname>Cohen</surname><given-names>J</given-names></name><name><surname>Everitt</surname><given-names>B</given-names></name></person-group><article-title>Large sample standard errors of Kappa and weighted Kappa</article-title><source>Psychol. Bull.</source><year>1969</year><volume>72</volume><fpage>323</fpage><lpage>327</lpage></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Matthews</surname><given-names>BW</given-names></name></person-group><article-title>Comparison of the predicted and observed secondary struccture of T4 phagel lysozyme</article-title><source>Biochim. Biophys. Acta</source><year>1975</year><volume>405</volume><fpage>442</fpage><lpage>451</lpage><pub-id pub-id-type=\"pmid\">1180967</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chaudhary</surname><given-names>K</given-names></name><name><surname>Nagpal</surname><given-names>G</given-names></name><name><surname>Dhanda</surname><given-names>SK</given-names></name><name><surname>Raghava</surname><given-names>GPS</given-names></name></person-group><article-title>Prediction of Immunomodulatory potential of an RNA sequence for designing non-toxic siRNAs and RNA-based vaccine adjuvants</article-title><source>Sci. Rep.</source><year>2016</year><volume>6</volume><fpage>1</fpage><lpage>11</lpage><pub-id pub-id-type=\"pmid\">28442746</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Johnstone</surname><given-names>D</given-names></name><name><surname>Milward</surname><given-names>EA</given-names></name><name><surname>Berretta</surname><given-names>R</given-names></name><name><surname>Moscato</surname><given-names>P</given-names></name></person-group><article-title>Multivariate protein signatures of pre-clinical Alzheimer’s disease in the Alzheimer’s disease neuroimaging initiative (ADNI) plasma proteome dataset</article-title><source>PLoS ONE</source><year>2012</year><volume>7</volume><fpage>e34341</fpage><pub-id pub-id-type=\"pmid\">22485168</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boughorbel</surname><given-names>S</given-names></name><name><surname>Jarray</surname><given-names>F</given-names></name><name><surname>El-anbari</surname><given-names>M</given-names></name></person-group><article-title>Optimal classifier for imbalanced data using Matthews correlation coefficient metric</article-title><source>PLoS ONE</source><year>2017</year><volume>12</volume><fpage>1</fpage><lpage>17</lpage></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Estrada</surname><given-names>L</given-names></name><name><surname>Torres</surname><given-names>A</given-names></name><name><surname>Sarlabous</surname><given-names>L</given-names></name><name><surname>Jan</surname><given-names>R</given-names></name></person-group><article-title>Improvement in neural respiratory drive estimation from diaphragm electromyographic signals using fixed sample entropy</article-title><source>IEEE J. Biomed. Heal. Informatics</source><year>2016</year><volume>20</volume><fpage>476</fpage><lpage>485</lpage></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Estrada</surname><given-names>L</given-names></name><name><surname>Torres</surname><given-names>A</given-names></name><name><surname>Sarlabous</surname><given-names>L</given-names></name><name><surname>Jané</surname><given-names>R</given-names></name></person-group><article-title>Influence of parameter selection in fixed sample entropy of surface diaphragm electromyography for estimating respiratory activity</article-title><source>Entropy</source><year>2017</year><volume>19</volume><fpage>460</fpage></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Buchman</surname><given-names>TG</given-names></name></person-group><article-title>The community of the self</article-title><source>Nature</source><year>2002</year><volume>420</volume><fpage>246</fpage><lpage>251</lpage><pub-id pub-id-type=\"pmid\">12432410</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pincus</surname><given-names>SM</given-names></name></person-group><article-title>Approximate entropy as a measure of system complexity</article-title><source>Proc. Natl. Acad. Sci U. S. A.</source><year>1991</year><volume>88</volume><fpage>2297</fpage><lpage>2301</lpage><pub-id pub-id-type=\"pmid\">11607165</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pincus</surname><given-names>S</given-names></name></person-group><article-title>Approximate entropy (ApEn) as a complexity measure</article-title><source>Chaos</source><year>1995</year><volume>5</volume><fpage>110</fpage><lpage>117</lpage><pub-id pub-id-type=\"pmid\">12780163</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Suki</surname><given-names>B</given-names></name><name><surname>Bates</surname><given-names>JHT</given-names></name><name><surname>Frey</surname><given-names>U</given-names></name></person-group><article-title>Complexity and emergent phenomena</article-title><source>Compr. Physiol.</source><year>2011</year><volume>1</volume><fpage>995</fpage><lpage>1029</lpage><pub-id pub-id-type=\"pmid\">23737210</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Seely</surname><given-names>AJE</given-names></name><etal/></person-group><article-title>Proceedings from the Montebello round table discussion. Second annual conference on complexity and variability discusses research that brings innovation to the bedside</article-title><source>J. Crit. Care</source><year>2011</year><volume>26</volume><fpage>325</fpage><lpage>327</lpage><pub-id pub-id-type=\"pmid\">21616298</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sullivan</surname><given-names>BA</given-names></name><etal/></person-group><article-title>Early heart rate characteristics predict death and morbidities in preterm infants</article-title><source>J. Pediatr.</source><year>2016</year><volume>174</volume><fpage>1</fpage><lpage>6</lpage></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vaporidi</surname><given-names>K</given-names></name><etal/></person-group><article-title>Respiratory drive in critically Ill patients: pathophysiology and clinical implications</article-title><source>Am. J. Respir. Crit. Care Med.</source><year>2019</year><volume>201</volume><fpage>20</fpage><lpage>32</lpage></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Georgopoulos</surname><given-names>D</given-names></name><name><surname>Roussos</surname><given-names>C</given-names></name></person-group><article-title>Control of breathing in mechanically ventilated patients</article-title><source>Eur. Respir. J.</source><year>1996</year><volume>9</volume><fpage>2151</fpage><lpage>2160</lpage><pub-id pub-id-type=\"pmid\">8902482</pub-id></element-citation></ref><ref id=\"CR57\"><label>57.</label><mixed-citation publication-type=\"other\">Georgopoulos, D. Effects of mechanical ventilation on control of breathing. in <italic>Principles and Practice of Mechanical Ventialtion</italic> (ed. Tobin, M. J.) 805–820 (2013).</mixed-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Laghi</surname><given-names>F</given-names></name></person-group><article-title>Assessment of respiratory output in mechanically ventilated patients</article-title><source>Respir. Care Clin. N. Am.</source><year>2005</year><volume>11</volume><fpage>173</fpage><lpage>199</lpage><pub-id pub-id-type=\"pmid\">15936689</pub-id></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tobin</surname><given-names>MJ</given-names></name><name><surname>Laghi</surname><given-names>F</given-names></name><name><surname>Jubran</surname><given-names>A</given-names></name></person-group><article-title>Ventilatory failure, ventilator support, and ventilator weaning</article-title><source>Compr. Physiol.</source><year>2012</year><volume>2</volume><fpage>2871</fpage><lpage>2921</lpage><pub-id pub-id-type=\"pmid\">23720268</pub-id></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bertoni</surname><given-names>M</given-names></name><etal/></person-group><article-title>A novel non-invasive method to detect excessively high respiratory effort and dynamic transpulmonary driving pressure during mechanical ventilation</article-title><source>Crit. Care</source><year>2019</year><volume>23</volume><fpage>1</fpage><lpage>10</lpage><pub-id pub-id-type=\"pmid\">30606235</pub-id></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Raoufy</surname><given-names>RM</given-names></name><name><surname>Ghafari</surname><given-names>T</given-names></name><name><surname>Mani</surname><given-names>AR</given-names></name></person-group><article-title>Complexity analysis of respiratory dynamics Mohammad</article-title><source>Am. J. Respir. Crit. Care Med.</source><year>2017</year><volume>196</volume><fpage>247</fpage><lpage>248</lpage><pub-id pub-id-type=\"pmid\">28222267</pub-id></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Costa</surname><given-names>MD</given-names></name><name><surname>Goldberger</surname><given-names>AL</given-names></name></person-group><article-title>Generalized multiscale entropy analysis: Application to quantifying the complex volatility of human heartbeat time series</article-title><source>Entropy</source><year>2015</year><volume>17</volume><fpage>1197</fpage><lpage>1203</lpage><pub-id pub-id-type=\"pmid\">27099455</pub-id></element-citation></ref><ref id=\"CR63\"><label>63.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>W</given-names></name><name><surname>Zhuang</surname><given-names>J</given-names></name><name><surname>Yu</surname><given-names>W</given-names></name><name><surname>Wang</surname><given-names>Z</given-names></name></person-group><article-title>Measuring complexity using FuzzyEn, ApEn, and SampEn</article-title><source>Med. Eng. Phys.</source><year>2009</year><volume>31</volume><fpage>61</fpage><lpage>68</lpage><pub-id pub-id-type=\"pmid\">18538625</pub-id></element-citation></ref><ref id=\"CR64\"><label>64.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Porta</surname><given-names>A</given-names></name><etal/></person-group><article-title>Measuring regularity by means of a corrected conditional entropy in sympathetic outflow</article-title><source>Biol. Cybern.</source><year>1998</year><volume>78</volume><fpage>71</fpage><lpage>78</lpage><pub-id pub-id-type=\"pmid\">9485587</pub-id></element-citation></ref><ref id=\"CR65\"><label>65.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>P</given-names></name><etal/></person-group><article-title>Assessing the complexity of short-term heartbeat interval series by distribution entropy</article-title><source>Med. Biol. Eng. Comput.</source><year>2015</year><volume>53</volume><fpage>77</fpage><lpage>87</lpage><pub-id pub-id-type=\"pmid\">25351477</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Nat Commun</journal-id><journal-id journal-id-type=\"iso-abbrev\">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type=\"epub\">2041-1723</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807770</article-id><article-id pub-id-type=\"pmc\">PMC7431582</article-id><article-id pub-id-type=\"publisher-id\">17904</article-id><article-id pub-id-type=\"doi\">10.1038/s41467-020-17904-z</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Dynamic evolution and reversibility of single-atom Ni(II) active site in 1T-MoS<sub>2</sub> electrocatalysts for hydrogen evolution</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Pattengale</surname><given-names>Brian</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-2320-2919</contrib-id><name><surname>Huang</surname><given-names>Yichao</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-7991-4849</contrib-id><name><surname>Yan</surname><given-names>Xingxu</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yang</surname><given-names>Sizhuo</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Younan</surname><given-names>Sabrina</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hu</surname><given-names>Wenhui</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Zhida</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-1425-9852</contrib-id><name><surname>Lee</surname><given-names>Sungsik</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>Pan</surname><given-names>Xiaoqing</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Gu</surname><given-names>Jing</given-names></name><address><email>jgu@sdsu.edu</email></address><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Huang</surname><given-names>Jier</given-names></name><address><email>jier.huang@marquette.edu</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.259670.f</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2369 3143</institution-id><institution>Department of Chemistry, </institution><institution>Marquette University, </institution></institution-wrap>Milwaukee, WI 53201 USA </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.263081.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0790 1491</institution-id><institution>Department of Chemistry and Biochemistry, </institution><institution>San Diego State University, </institution></institution-wrap>San Diego, CA 92181 USA </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.12527.33</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0662 3178</institution-id><institution>Key Lab of Organic Optoelectronics & Molecular Engineering of Ministry of Education, Department of Chemistry, </institution><institution>Tsinghua University, </institution></institution-wrap>100084 Beijing, PR China </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.266093.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0668 7243</institution-id><institution>Department of Materials Science and Engineering, </institution><institution>University of California, </institution></institution-wrap>Irvine, CA 92697 USA </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.187073.a</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 1939 4845</institution-id><institution>X-ray Science Division, </institution><institution>Argonne National Laboratory, </institution></institution-wrap>Argonne, IL 60349 USA </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.266093.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0668 7243</institution-id><institution>Department of Physics and Astronomy, </institution><institution>University of California, </institution></institution-wrap>Irvine, CA 92697 USA </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>4114</elocation-id><history><date date-type=\"received\"><day>21</day><month>2</month><year>2020</year></date><date date-type=\"accepted\"><day>24</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">1T-MoS<sub>2</sub> and single-atom modified analogues represent a highly promising class of low-cost catalysts for hydrogen evolution reaction (HER). However, the role of single atoms, either as active species or promoters, remains vague despite its essentiality toward more efficient HER. In this work, we report the unambiguous identification of Ni single atom as key active sites in the basal plane of 1T-MoS<sub>2</sub> (Ni@1T-MoS<sub>2</sub>) that result in efficient HER performance. The intermediate structure of this Ni active site under catalytic conditions was captured by in situ X-ray absorption spectroscopy, where a reversible metallic Ni species (Ni<sup>0</sup>) is observed in alkaline conditions whereas Ni remains in its local structure under acidic conditions. These insights provide crucial mechanistic understanding of Ni@1T-MoS<sub>2</sub> HER electrocatalysts and suggest that the understanding gained from such in situ studies is necessary toward the development of highly efficient single-atom decorated 1T-MoS<sub>2</sub> electrocatalysts.</p></abstract><abstract id=\"Abs2\" abstract-type=\"web-summary\"><p id=\"Par2\">While single atom catalysis combines heterogeneous materials with molecular understanding, the role of the single atoms remains vague. Here, authors examine single Ni on MoS<sub>2</sub> via in situ X-ray absorption spectroscopy to reveal the intermediate and catalytically active species.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Catalytic mechanisms</kwd><kwd>Energy</kwd><kwd>Electrocatalysis</kwd><kwd>Nanoscale materials</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/100000015</institution-id><institution>U.S. Department of Energy (DOE)</institution></institution-wrap></funding-source><award-id>DE-AC02-06CH11357</award-id><principal-award-recipient><name><surname>Huang</surname><given-names>Jier</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/100000001</institution-id><institution>National Science Foundation (NSF)</institution></institution-wrap></funding-source><award-id>DMR-1654140</award-id><principal-award-recipient><name><surname>Huang</surname><given-names>Jier</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par3\">As the world continues to transition toward the usage of carbon neutral energy technologies, there remains a need to develop efficient and cost-effective hydrogen evolution reaction (HER) catalysts to support the development of a hydrogen economy<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>. A very promising HER catalyst that meets both the technical and economical requirements is MoS<sub>2</sub><sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>–<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. Tremendous efforts have been made to clarify the catalytic mechanism with various morphological and compositional MoS<sub>2</sub>, such as chemical exfoliated MoS<sub>2</sub><sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>, nanostructured particles<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref>–<xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>, heterostructures<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>, amorphous<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>–<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>, and doped MoS<sub>2</sub><sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>–<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup> etc. Both experimental and computational studies demonstrated that the edge sites of the crystalline MoS<sub>2</sub> are catalytically active, while its basal plane is inert<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. As a result, one desirable approach to further improving the activity of MoS<sub>2</sub> is to activate the inert basal plane.</p><p id=\"Par4\">1T-MoS<sub>2</sub>, which features a well-defined octahedral symmetry in contrast to the traditional trigonal prismatic 2H-phase MoS<sub>2</sub><sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>, represents an emerging platform for further improving the HER performance of MoS<sub>2</sub> owing to their great potential in activating basal planes. This is not only theoretically predicted that the metallic 1T-MoS<sub>2</sub> may have basal plane active sites<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> but also justified by recent experiment where MoS<sub>2</sub> tethered with single-atom catalyst at basal plane can effectively enhance the catalytic activity of 1T-MoS<sub>2</sub><sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref>–<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup><sub>.</sub> However, whether the single atoms in the basal edge act as active sites and what the exact function of these single atoms in 1T-MoS<sub>2</sub> remain unclear. It is thus essential to seek direct evidence for the origins of active sites due to the single-atom modification from an experimental aspect of view.</p><p id=\"Par5\">In this work, we report the direct observation of single-atom Ni replacing Mo and S as active sites on basal edge of the Ni@1T-MoS<sub>2</sub> HER electrocatalyst in the acidic condition. The Mo and Ni atoms both adopt octahedral structure in the 1T phase of MoS<sub>2</sub>. Using in situ X-ray absorption spectroscopy (XAS), we show that the dominant active site for HER is the Ni single atom in its intrinsic environment in an acidic electrolyte, while, in alkaline media, Ni single atoms reconstruct into an S-supported NiO species and reversibly forms a metallic active species under applied potential. These findings provide important insight into the dynamic evolution of basal plane Ni active sites in a state-of-the-art 1T-MoS<sub>2</sub> HER electrocatalyst, thereby revealing key intermediate and active species that dictate the catalytic function.</p></sec><sec id=\"Sec2\" sec-type=\"results\"><title>Results</title><sec id=\"Sec3\"><title>Synthesis, characterization, and electrocatalysis</title><p id=\"Par6\">The synthesis of Ni@1T-MoS<sub>2</sub> HER electrocatalyst follows the previously published protocols<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. The synthesis procedure was conducted as follows. First, a Anderson-type POM nanocluster precursors, [(NH<sub>4</sub>)<sub>4</sub>[NiH<sub>6</sub>Mo<sub>6</sub>O<sub>24</sub>]·5H<sub>2</sub>O (NiMo<sub>6</sub>) were prepared. It contains well-defined, 1:6 heteropolyanion clusters, composed of a single metal heteroatom NiO<sub>6</sub> octahedron with six edge-sharing MoO<sub>6</sub> octahedrons. The as-prepared NiMo<sub>6</sub> precursors react with thioacetamide at 180 °C for 24 h in the presence of carbon fiber paper (CFP, 1 × 2 cm<sup>2</sup>) to give rise to the corresponding Ni@1T-MoS<sub>2</sub>/CFP electrocatalyst. The loading amount of Ni@1T-MoS<sub>2</sub> on CFP is about 1 mg cm<sup>−2</sup>. The facile sulfuration reaction incompletely replaces O atoms with S atoms and generates 1T-MoS<sub>2</sub> nanosheets randomly decorated with Ni single atoms (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>.<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Ni@1T-MoS<sub>2</sub> structure.</title><p>Segment of 1T-MoS<sub>2</sub> structure with basal plane dopant NiO moiety shown in the center.</p></caption><graphic xlink:href=\"41467_2020_17904_Fig1_HTML\" id=\"d30e622\"/></fig></p><p id=\"Par7\">The as-prepared Ni@1T-MoS<sub>2</sub> catalyst was extensively characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive X-rays spectroscopy (EDX), Raman spectrum, and powder X-ray diffraction (XRD) (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>). SEM image shows that ultrathin Ni@MoS<sub>2</sub> coated evenly throughout the CFP (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1a</xref>). More detailed structures of Ni@MoS<sub>2</sub> were further demonstrated by TEM images (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1b</xref>). The XRD patterns of Ni@1T-MoS<sub>2</sub> electrocatalyst show clear characteristic diffraction peaks at around 10.35°, 32.84°, and 35.70° (red plot in Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1c</xref>), which correspond to the (002), (100), and (102) planes of hexagonal MoS<sub>2</sub> (PDF#75-1539), respectively. However, the peak of Ni@1T-MoS<sub>2</sub> at 2θ = 10.25° represents a stacked, multilayered structure, which is lower than that of MoS<sub>2</sub> (PDF#75-1539) at 14.13°, indicating that this multilayered spacing of 1T-MoS<sub>2</sub> is larger than that of the bulk 2H-MoS<sub>2</sub> (6.3 Å, PDF#75-1539). Previous publications also verified analogous XRD patterns for 1T-MoS<sub>2</sub> or few-layered 2H-MoS<sub>2</sub><sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. The Raman peaks of Ni@1T-MoS<sub>2</sub> resemble that of 1T-MoS<sub>2</sub>, which is consistent with their similar structures. The characteristic Raman peaks at 147, 214, 236, 283, and 335 cm<sup>−1</sup> (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1f</xref>) can be assigned to the phonon modes in 1T-MoS<sub>2</sub>, which suggests the formation of a pure 1T-MoS<sub>2</sub> nanosheet. These results together confirmed the phase of MoS<sub>2</sub> to be 1T, similar to previous reported 1T-MoS<sub>2</sub> work (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1c, f</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref>–<xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. Furthermore, the Ni@1T-MoS<sub>2</sub> showed similar structure as pristine 1T-MoS<sub>2</sub>, indicating that the addition of Ni did not change the structure of 1T-MoS<sub>2</sub>. EDX analysis confirms that Mo, Ni, S, and O atoms are uniformly distributed in Ni@1T-MoS<sub>2</sub> electrocatalyst (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1d, e</xref>). The uniform distribution of oxygen (O) element indicates that sulfur atom (S) only partially replaced O in the POM precursor, which is similar to the previous study<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref>,<xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. Furthermore, STEM (scanning TEM) confirmed that the single-layer 1T-MoS<sub>2</sub> consists of symmetric hexagon units, which corresponds to the 1T-MoS<sub>2</sub> structure with intensity variation, i.e., Ni atoms are shown as darker dots and Mo atoms are shown as brighter dots (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a</xref>). In the annular dark-field (ADF) STEM images, the intensity of ADF signal is proportional to the atomic number (<italic>Z</italic>) of observed materials and scales as <italic>Z</italic><sup>1.6–2</sup><sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. Therefore, the STEM intensity of single doped Ni atom (<italic>Z</italic><sub>Ni</sub> = 28) should be dimmer than that of Mo atom (<italic>Z</italic><sub>Mo</sub> = 42) in MoS<sub>2</sub> lattice. The simulated spectrum intensity matches with the experimental intensity, indicating that the bright spots are Mo and dark spots are Ni, rather than a vacant site in STEM images (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2b</xref>). Different from most single-atom modified MoS<sub>2</sub>, where heavier single atoms are usually shown as brighter spots on the substrate<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>, here the intensity variation confirms that Ni has successfully replaced Mo. In addition, the HAADF experimental and simulated intensity profiles further show that the dark and bright spots are corresponding to Ni sites and Mo sites (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>), respectively.<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>STEM characterization of Ni@1T-MoS<sub>2</sub> sample.</title><p><bold>a</bold> Atomic resolution ADF-STEM image of monolayer sample at 60 keV. The length of the scale bar is 5 Å. <bold>b</bold> Intensity profiles taken along two adjacent lines indicated by blue and red rectangles in (<bold>a</bold>). The red arrow points to the location of Ni single atom.</p></caption><graphic xlink:href=\"41467_2020_17904_Fig2_HTML\" id=\"d30e809\"/></fig></p><p id=\"Par8\">The catalytic activity of the synthesized Ni@1T-MoS<sub>2</sub> was examined by linear sweep voltammetry (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>), which shows significantly enhanced catalytic activity in Ni@1T-MoS<sub>2</sub> compared to pristine 1T-MoS<sub>2</sub> in both acidic and alkaline electrolyte, with a more prominent difference in alkaline electrolyte. Ni@1T-MoS<sub>2</sub> showed ~80 mV more positive onset potential in 0.5 M H<sub>2</sub>SO<sub>4</sub> while its onset potential shifted ~300 mV catholically in 1M NaOH. The HER stability of the as-prepared Ni@1T-MoS<sub>2</sub> in terms of chronoamperometry was also examined, which exhibited excellent stability in both acidic and basic conditions at the applied −0.76 V vs. RHE, which is the potential later used for the in situ XAS analysis (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>).</p></sec><sec id=\"Sec4\"><title>In situ XAS under acidic and alkaline conditions</title><p id=\"Par9\">In situ XAS was used to directly measure the local bonding and electronic structures at Ni and Mo centers under the standard catalytic conditions in both acidic and alkaline media. In both acidic and alkaline media, 0 V vs. RHE represents a pre-catalytic potential and −0.76 V vs. RHE represents a proceeding catalytic potential for HER. All potentials reported in this work are with respect to RHE if not indicated. In addition to these potentials, samples were measured as-prepared (dry sample) and with no applied potential, representing a sample that is immersed in electrolyte with N<sub>2</sub> purging but has not been externally connected to a potentiostat.</p><p id=\"Par10\">Figure <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref> shows the X-ray absorption near-edge structure (XANES) region of the XAS spectrum collected at each condition at the Mo K-edge. In both acidic and alkaline media, the XANES spectra at Mo K-edge, corresponding to the 1s–5p transition, show very little change in either shape or edge position as shown by the first-derivative spectra (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b</xref>) as a function of applied potential, suggesting negligible structure or oxidation state changes. The extended X-ray absorption fine structure (EXAFS) spectra (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>) are plotted in R-space in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3c</xref>.<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>In situ XAS at Mo K-edge.</title><p><bold>a</bold> XANES spectra, <bold>b</bold> first-derivative spectra, and <bold>c</bold> Fourier-transformed R-space spectra (open points) and fits (solid lines) of Ni@1T-MoS<sub>2</sub>.</p></caption><graphic xlink:href=\"41467_2020_17904_Fig3_HTML\" id=\"d30e883\"/></fig></p><p id=\"Par11\">The EXAFS data were analyzed using FEFF fitting via the Demeter suite of programs<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup> to extract quantitative Mo local structure information. The published 1T-MoS<sub>2</sub> crystal structure was used to build FEFF models<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>. The EXAFS spectra in R-space and k-space, as well as the fitting results, are presented in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3c</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6</xref>, respectively, where their corresponding fitting parameters are listed in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>. Further explanation of the model is given in the Experimental section and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">7</xref>. Two relatively minor changes are observed at the Mo K-edge via in situ XAS. The first is an increase in coordination number (CN) with little change in the correlated Debye–Waller factor (σ<sup>2</sup>) after immersion in electrolyte, implying solvent coordination to undercoordinated Mo centers. The second is the shortening of Mo–S distance under catalytic applied potential (i.e., −0.76 V) in both acidic and alkaline conditions. Beyond this slight change, the Mo centers under applied catalytic potential appear to have largely similar local structure to the as-prepared sample and the small changes observed are insufficient for us to draw decisive conclusions. This result is different from a previous report on amorphous MoS<sub>x</sub>, where significant structural changes at Mo center were observed<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. It is unsurprising, however, as Ni in this work was shown to be the active catalyst site in Ni@1T-MoS<sub>2</sub>. One additional activation mechanism that needs to be considered is lattice structure changes, namely a phase change from the 1T phase to the 2H phase. Based on our previous work<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, and published work<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>–<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>, the 2H phase gives a clear second-shell Mo–Mo peak while 1T-MoS<sub>2</sub> does not have a clear second-shell peak. Because no such feature is observed under any of the measured conditions in situ, we conclude that the lattice does not undergo a full transition to the 2H phase and can also rule out a 1T-2H polymorphic state as the catalytically active state. Therefore, the 1T phase is shown to be the catalytically active phase in both acidic and alkaline conditions with minimal changes at the Mo center and discussion hereafter focuses on the major changes observed at the Ni K edge.</p><p id=\"Par12\">The Ni–K edge EXAFS spectra (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5b</xref>) were measured under the same conditions as Mo K-edge data. The XANES spectra (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4a</xref>) show that significant local structure changes occur throughout the catalysis process. Beginning from the dry sample, a weak white line feature is observed on-edge (1s–4p transition, 8.350 keV). Such a feature is typically observed for Ni<sup>2+</sup> with unsaturated coordination and is sensitive to the identity of coordinating atom<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>–<xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>, suggesting that single-atom Ni preserves an oxidation state of 2+ after replacing Mo atoms. The oxidation state is further supported by Ni reference spectra (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">8</xref>). Moreover, no peak is observed at 2.10 Å in the R-space EXAFS spectra (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c</xref>) where Ni–Ni coordination (first shell) is, correlating well with the previous conclusion that Ni exists as single atom in 1T-MoS<sub>2</sub>. Upon immersing in acidic electrolyte (No potential), it is observed that the shape of the spectrum at above-edge region has little change; however, a shoulder (~8.340 keV) is observed on the rising part of the edge along with a reduced white line intensity. When the first-derivative spectra are compared (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4b</xref>), it is apparent that the edge shifts by approximately 3 eV to a lower energy. While this is consistent with reduction of the Ni metal center without applied potential<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref>–<xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>, structural and electronic factors could contribute and are examined in more detail via EXAFS fitting (<italic>vide infra</italic>). Further shifting of the edge toward lower energies is observed with the application of pre-catalytic (0 V) and catalytic (−0.76 V) potentials.<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>In situ XAS at Ni K-edge.</title><p><bold>a</bold> XANES spectra, <bold>b</bold> first-derivative spectra, and <bold>c</bold> Fourier-transformed R-space spectra (open points) and fits (solid lines) of Ni@1T-MoS<sub>2</sub>.</p></caption><graphic xlink:href=\"41467_2020_17904_Fig4_HTML\" id=\"d30e1000\"/></fig></p><p id=\"Par13\">Immersion in alkaline electrolyte yields strikingly different results compared to the acidic electrolyte. Without applied potential, a strengthening in the intensity of the 1s–4p white line transition is observed (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4a</xref>), which occurs without an apparent change in oxidation state (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4b</xref>). The XANES spectrum for no potential, alkaline appears to resemble NiO (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">9a, b</xref>), however the local structure revealed by EXAFS suggests a very different species (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">9c, d</xref>) still associated with 1T-MoS<sub>2</sub>. The application of a pre-catalytic potential (0 V) results in little change in the spectrum and yields no change in edge position. Applying a catalytic potential (−0.76 V) causes a significant change in the XANES spectrum. A shoulder at the base of the spectrum (8.334 keV) appears to grow while above-edge oscillations >8.350 keV are changed and dampened. The first-derivative spectrum (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4b</xref>) shows that a new edge feature consistent with the position of the shoulder is observed at 8.333 keV. Evidently, this peak is lower in energy than the species observed in acidic media and is consistent with the formation of a Ni<sup>0</sup> species, as the Ni metal edge position is 8.333 keV<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>.</p><p id=\"Par14\">The Fourier-transformed R-space spectra (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c</xref>) were fit to further extract quantitative information about species formed at the Ni center. The K-space spectra and fits are shown in Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">10</xref>. Beginning with acidic conditions, no significant changes are observed in the quantified local structure FEFF parameters (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>) as a function of applied potential in the first coordination shell. However, a shortening of the Ni–Mo second-shell interaction is observed upon immersion in acidic electrolyte and further shortening due to applied potential. Compared to the Mo K edge results, this result does appear to be consistent with the shortening of first shell Mo–S first shell interactions observed under an applied bias of −0.76 V. At the no potential, acidic and 0 V vs. RHE conditions, where changes were not observed at the Mo K-edge, a shortening of Ni–Mo in the first shell is still observed. This is likely correlated with the oxidation state changes observed only at Ni that result in local structure changes, whereas the Mo K-edge probes an average of all Mo atoms irrespective of their proximity to Ni sites. Overall, however, these results suggest that the intrinsic Ni site in Ni@1T-MoS<sub>2</sub> is catalytically active and does not undergo significant transformation into a different active species in acidic conditions.<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>FEFF fitting parameters for Ni–K edge in situ XAS data.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th>Condition</th><th>Vector</th><th>CN<sup>a</sup></th><th>σ<sup>2</sup> (Å)<sup>2b</sup></th><th>Δ<italic>E</italic><sub>0</sub> (eV)</th><th><italic>R</italic> (Å)<sup>c</sup></th></tr></thead><tbody><tr><td rowspan=\"3\">As-prepared</td><td>Ni–S</td><td>3.6</td><td>0.006</td><td>−8.01</td><td>2.20</td></tr><tr><td>Ni–O</td><td>1.0</td><td>0.006</td><td>−1.03</td><td>2.37</td></tr><tr><td>Ni–Mo</td><td>0.8</td><td>0.010</td><td>−15.44</td><td>3.10</td></tr><tr><td rowspan=\"3\">No potential, acidic</td><td>Ni–S</td><td>3.5</td><td>0.007</td><td>−5.52</td><td>2.21</td></tr><tr><td>Ni–O</td><td>1.0</td><td>0.007</td><td>−5.48</td><td>2.41</td></tr><tr><td>Ni–Mo</td><td>1.3</td><td>0.010</td><td>−13.22</td><td>3.02</td></tr><tr><td rowspan=\"3\">0 V vs. RHE, acidic</td><td>Ni–S</td><td>3.6</td><td>0.006</td><td>−2.52</td><td>2.21</td></tr><tr><td>Ni–O</td><td>1.0</td><td>0.006</td><td>−1.39</td><td>2.37</td></tr><tr><td>Ni–Mo</td><td>1.0</td><td>0.012</td><td>−16.37</td><td>2.96</td></tr><tr><td rowspan=\"3\">−0.76 V vs. RHE, acidic</td><td>Ni–S</td><td>3.6</td><td>0.006</td><td>0.35</td><td>2.22</td></tr><tr><td>Ni–O</td><td>1.0</td><td>0.006</td><td>6.53</td><td>2.38</td></tr><tr><td>Ni–Mo</td><td>2.8</td><td>0.010</td><td>−20.75</td><td>2.97</td></tr><tr><td rowspan=\"3\">No potential, alkaline</td><td>Ni–S</td><td>1.7</td><td>0.010</td><td>0.58</td><td>2.21</td></tr><tr><td>Ni–O</td><td>4.2</td><td>0.006</td><td>0.58</td><td>2.05</td></tr><tr><td>Ni–Ni</td><td>4.9</td><td>0.008</td><td>6.13</td><td>3.12</td></tr><tr><td rowspan=\"3\">0 V vs. RHE, alkaline</td><td>Ni–S</td><td>1.9</td><td>0.008</td><td>−0.25</td><td>2.20</td></tr><tr><td>Ni–O</td><td>3.8</td><td>0.007</td><td>−0.25</td><td>2.04</td></tr><tr><td>Ni–Ni</td><td>3.9</td><td>0.008</td><td>1.26</td><td>3.08</td></tr><tr><td rowspan=\"3\">−0.76 V vs. RHE, alkaline</td><td>Ni–S</td><td>1.0</td><td>0.001</td><td>4.65</td><td>2.20</td></tr><tr><td>Ni–O</td><td>2.1</td><td>0.009</td><td>−0.79</td><td>2.07</td></tr><tr><td>Ni–Ni</td><td>2.1</td><td>0.005</td><td>5.53</td><td>2.53</td></tr></tbody></table><table-wrap-foot><p><sup>a</sup>Coordination number, uncertainty ± 0.5.</p><p><sup>b</sup>Uncertainty ± 0.001 Å<sup>2</sup>.</p><p><sup>c</sup>Uncertainty ± 0.02 Å.</p></table-wrap-foot></table-wrap></p><p id=\"Par15\">The R-space spectra (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c</xref>) under alkaline conditions, however, suggest that the active species is not the intrinsic Ni species in as-prepared Ni@1T-MoS<sub>2</sub>. Upon immersion in alkaline electrolyte, a significantly different second-shell scattering feature is observed, which is accompanied by an obvious shortening in the first-shell peak distance. In the first shell, such a shortening is consistent with enhanced Ni–O coordination and reduced Ni–S coordination. As discussed in the XANES results, the local structure is not consistent with a pure NiO phase but rather a phase still associated with 1T-MoS<sub>2</sub>. The FEFF model therefore incorporated Ni–O first shell and Ni–S first shell scattering, where it was observed that Ni is predominantly coordinated to O with a smaller contribution of S compared to the as-prepared dry sample. As this species is not NiO, we term the species NiS<sub>x</sub>O<sub>y</sub>, where <italic>x</italic> and <italic>y</italic> represent the CNs of their respectively labeled atoms, as suggested by EXAFS analysis (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>) and not the stoichiometric amounts of the atoms in the entire catalyst material. The newly observed second-shell feature is intense relative to the first shell peak and indicates the presence of a large scattering atom in the second shell of Ni. In Ni@1T-MoS<sub>2</sub>, such a scattering atom could be either Ni or Mo. If the second-shell scattering was Ni–Mo, then second-shell scattering should be also observed from the Mo K-edge. Because no significant second-shell scattering was observed under identical conditions in the Mo K edge data (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3c</xref>), it is more likely due to Ni–Ni second-shell scattering. Evidently, alkaline conditions even in the absence of applied potential impart lability to Ni and leads to the formation of Ni–O–Ni moieties in the structure. Based on the FEFF fit results in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>, the Ni–O–Ni moieties are still anchored by direct coordination to S atoms within the 1T-MoS<sub>2</sub> structure, i.e., an NiS<sub>x</sub>O<sub>y</sub> structure.</p><p id=\"Par16\">Application of a pre-catalytic potential (0 V) does not significantly change the Ni local structure compared to the no potential alkaline electrolyte measurement. When a catalytic potential (−0.76 V) is applied in alkaline electrolyte, the R-space spectrum becomes to be a very broad feature spanning the entire first-shell region of the spectrum (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c</xref>). Combined with the observation of Ni<sup>0</sup> species in the XANES spectra, we assigned the new long-distance first shell feature to the formation of Ni–Ni coordination concurrent with reduction of Ni to Ni<sup>0</sup>. We can exclude the formation of Ni–Mo bonds because no changes were observed at the Mo K-edge under the same conditions. The optimum FEFF model therefore incorporated Ni–S, Ni–O, and Ni–Ni paths, giving a species that is partly metallic in nature and is coordinated to the 1T-MoS<sub>2</sub> scaffold via O and S bonds. To confirm the validity of this claim, we performed FEFF fitting of Ni foil (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">11</xref> and Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>) where it was observed that the fit Ni–Ni distance matches the catalytic alkaline condition within uncertainty.</p></sec><sec id=\"Sec5\"><title>Dynamic evolution and reversibility of Ni species</title><p id=\"Par17\">Given the nature of the active site in alkaline conditions, we performed post-catalysis ex situ XAS on Mo K-edge and Ni K-edge Mo K-edge (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">12</xref>) and Ni K-edge (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>). The Mo K-edge shows a slight shortening in Mo–S distance, however, the FEFF model adequately describes the local structure owing to the stability of the catalyst. The Ni K-edge gives a Fourier-transformed R-space spectrum very similar to the no potential and 0 V spectra in alkaline conditions, i.e., the NiS<sub>x</sub>O<sub>y</sub> structure. Indeed, FEFF fitting parameters confirm that these species are indeed the same, within uncertainty. In full, this implies that the metallic Ni phase that forms as a result of catalytic potentials is indeed reversible, i.e., Ni returns to its original oxidation state and local structure rather than remaining in a metallic phase under applied bias. The insights gained in this study are limited by the inability to directly observe atom migration in situ to explain the migration of Ni atoms that are initially well-dispersed in the structure without Ni neighbors<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. However, surface reconstruction in 2D materials<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR46\">46</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup> and other materials<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup> has been reported before. Relatedly, defect and vacancy sites have been shown to be important in MoS<sub>2</sub>-catalyzed HER<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref>,<xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>. Furthermore, electrolyte dependent performance and electrolyte-induced dopant local structure changes have been observed in MoS<sub>2</sub><sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref>,<xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>. We therefore posit that alkalinity induces Ni atom lability within the basal plane allowing the formation of Ni–O–Ni moieties, and leads to the formation of Ni<sup>0</sup> under applied catalytic potential.<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>Ex situ XAS after alkaline electrocatalysis.</title><p><bold>a</bold> Mo K edge K-space spectra and <bold>b</bold> R-space spectra and <bold>c</bold> Ni K edge K-space spectra and <bold>d</bold> R-space spectra ex situ EXAFS results, which are compared to the applicable results in this work.</p></caption><graphic xlink:href=\"41467_2020_17904_Fig5_HTML\" id=\"d30e1615\"/></fig></p><p id=\"Par18\">The active species as suggested by this work are depicted in Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>. In both acidic and alkaline electrolyte, the Mo K-edge results indicate that the 1T phase is maintained and harbours the active Ni species that was investigated by probing the Ni K edge under identical conditions. Under acidic conditions, Ni was shown to remain coordinated by predominantly S atoms and underwent reduction under applied catalytic potential. In alkaline conditions, the active species was determined to be Ni<sup>0</sup> in nature and Ni–Ni coordination was observed directly via EXAFS. In addition to Ni–Ni coordination, it was observed that Ni–O and Ni–S coordination was still present, suggesting that the active species is still anchored within the basal plane of 1T-MoS<sub>2</sub>.<fig id=\"Fig6\"><label>Fig. 6</label><caption><title>The proposed Ni@1T-MoS<sub>2</sub> active species.</title><p>The active species under acidic (left) and alkaline (right) conditions. Under alkaline conditions, the structure shown is representative, and the Ni<sup>0</sup> cluster size is further discussed in the text.</p></caption><graphic xlink:href=\"41467_2020_17904_Fig6_HTML\" id=\"d30e1643\"/></fig></p><p id=\"Par19\">The CN of Ni–Ni in the alkaline active species was found to be ~2. If we consider that Ni<sup>0</sup> atoms form a cluster within the 2D 1T-MoS<sub>2</sub> sheet that is bound on its periphery by the lattice, an average CN of 2 suggests that the size of Ni<sup>0</sup> clusters is small such that some Ni atoms contain >2 neighbors while others contain <2 neighbors. We therefore estimate that the cluster size is on the order of 10 Ni atoms or less. Furthermore, clear Ni–Ni second-shell interactions were not observed in the data for the catalytically active species, suggesting that a pure Ni metal phase is not observed. The lattice-contained small Ni cluster proposed here, however, supports the lack of a second-shell Ni–Ni feature, as a small, disordered cluster would inherently not give such a feature due to disorder.</p><p id=\"Par20\">Further, from the comparison in pre- and post-electrolysis XPS spectra and ICP-MS solution analysis, we have found that Ni@MoS<sub>2</sub> is quite stable under acidic conditions, correlating well with the XAS results. To further confirm that Mo does not go to Mo (0), as shown in Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">13</xref>, the high-resolution (HR) Mo 3d spectra were mainly deconvoluted into two peaks in the original and acid-operated samples. These two characteristic peaks of Mo 3d<sub>5/2</sub> (~228.5 eV) and Mo 3d<sub>3/2</sub> (231.6 eV) suggest the dominating oxidation state of Mo is +4 in Ni@1-TMoS<sub>2</sub>)<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. Interestingly, the Mo 3d peaks in basic condition shows a slight shift of ~0.3 eV to higher binding energy compared to those of original and acidity ones. Moreover, a higher Mo<sup>6+</sup> peak is generated after electrolysis under basic condition, indicating that Ni@1T-MoS<sub>2</sub> catalyst probably undergoes a surface oxidation in the electrolysis process. Combining XAS and XPS results, we believe that Mo does not go to Mo (0) in HER. However, the catalysts are oxidized in alkaline conditions, reflected by the increased binding energies in Mo3d, Ni 2p, and S 2p peaks (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">13</xref>). This is consistent with the XAS result that Ni–O coordination increases in Ni@1T-MoS when exposed to alkaline electrolyte. Further ex situ characterization was performed via Raman spectroscopy. Raman spectra commonly detect at a much deeper material depth (μm range) than XPS (several nm), and are therefore more sensitive to the bulk structure of the catalyst. The Raman spectra (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">14</xref>) indicate that the bulk material is stable after 30 h of electrolysis. This is in good agreement with EXAFS results, where a 1T to 2H phase transition is not observed, supporting that the active catalyst remains in the 1T phase. ICP-MS was further applied to detect leaching into the electrolyte after electrolysis (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). In acidic conditions, no detectable leaching was observed. In alkaline conditions, no leaching of the catalytically active Ni species was observed, and 0.47 µg mL<sup>−1</sup> Mo was observed, suggesting that, as further suggested by XPS (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">13</xref>), oxidized Mo species gradually leach during long catalysis runs in alkaline electrolyte. We note that these leached Mo species were not observed in EXAFS measurements likely due to their low concentration in solution relative to the Mo concentration on the electrode.</p><p id=\"Par21\">Because of the observed reversibility, the second-shell Ni–Ni scattering in the alkaline sample without applied potential should also be informative toward the size of Ni–O clusters in the pre-catalytic states. A fit CN of ~4 was obtained in both the in situ (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>) and ex situ (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>) data for the second-shell Ni–Ni path. This implies that all Ni atoms in second-shell proximity do not become associated in the first shell under applied catalytic potential. This is likely due to the mixed coordination with the 1T-MoS<sub>2</sub> lattice as observed in the alkaline −0.76 V in situ XAS data. This result is significant since it clarifies the central role of single-atom Ni, replacing Mo and S, as the main active species rather than as promoters in both acidic and alkaline condition. Moreover, under alkaline condition, single-atom Ni undergoes a structure evolution from forming Ni–O, followed by Ni–Ni species. Interestingly, without bias, the reduced species possibly can be regenerated by oxidation from oxygen and return back to Ni–O clusters.</p><p id=\"Par22\">In summary, we performed in situ XAS to investigate the active site structure of the high-performance Ni@1T-MoS<sub>2</sub> HER electrocatalyst at both Mo and Ni k-edge. It was discovered that Mo sites undergo very little change during the process of immersion into electrolyte and consequent potential-driven electrocatalysis. At the active Ni single-atom sites, however, significant changes are observed. In acidic electrolyte, the active site structure of Ni center experiences a net reduction in oxidation state without change of intrinsic structure during catalysis. In contrast, under alkaline conditions, coordination changes are observed from immersion in electrolyte, where NiS<sub>x</sub>O<sub>y</sub> species form, and the application of a catalytic potential reversibly forms a metallic Ni species as the active site. These findings provided direct evidence that single-atom Ni(II) can itself perform as active species at the interface of Ni@1T-MoS<sub>2</sub> in acidic conditions while it undergoes structural reconstruction in the alkaline medium to form a NiS<sub>x</sub>O<sub>y</sub> species that reversibly forms a catalytically active Ni<sup>0</sup> species under applied potential. Moreover, since most Ni single atoms are placed on the basal edge of 1T-MoS<sub>2</sub>, these results also support the fact that Ni sites in the basal plane of Ni@1T-MoS<sub>2</sub> are active towards HER under acidic conditions and sheds light on the function and evolution of Ni sites in 1T-MoS<sub>2</sub> under alkaline conditions.</p></sec></sec><sec id=\"Sec6\"><title>Methods</title><sec id=\"Sec7\"><title>Preparation of NiMo<sub>6</sub> precursor</title><p id=\"Par23\">All chemicals were obtained as reagent grade chemicals from Alfa Aesar<sup>®</sup> unless noted. The (NH<sub>4</sub>)<sub>4</sub>[NiH<sub>6</sub>Mo<sub>6</sub>O<sub>24</sub>]·5H<sub>2</sub>O (NiMo<sub>6</sub>) precursor was prepared according to a modified published procedure. (NH<sub>4</sub>)<sub>6</sub>Mo<sub>7</sub>O<sub>24</sub>·4H<sub>2</sub>O (denoted Mo7, 5.19 g, 4.2 mmol, 99%) was dissolved in DI water (80 mL) and then heated to 100 °C. Ni(NO<sub>3</sub>)<sub>2</sub>·6H<sub>2</sub>O (1.16 g, 4 mmol, 99.99%) was dissolved in water (20 mL), which was added to the above solution with stirring. The mixture was kept heating and stirring to give rise to a deep green solution. The crude product (5.4 g) was isolated with evaporation and filteration. The green targeted product (4.6 g, 79.1% yield based on Mo) was obtained by recrystallization in hot water (80 °C) two times, then dried under vacuum. Elemental analysis calcd (%) for H32N4O29NiMo6 (M = 1186.60 gmol<sup>−1</sup>): H, 2.72; N, 4.72; Mo, 48.51; Found: H, 2.70; N, 4.66; Mo, 48.62. IR (KBr pellet, major absorbances, cm<sup>−1</sup>): 3402 (ν<sub>as</sub>OH, m), 3152 (ν<sub>as</sub>NH, m), 1627 (δOH, m), 1402 (δNH, s), 929 (νMo = O, vs), 876 (νMo = O, vs), 635 (νMo–O–Mo, vs), 577 (νNi–O–Mo, w).</p></sec><sec id=\"Sec8\"><title>Preparation of Ni@1T-MoS<sub>2</sub>/CFP</title><p id=\"Par24\">The Ni@1T-MoS<sub>2</sub> catalyst was prepared according to our previous work<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. The as-prepared NiMo6 (50 mg, 0.042 mmol) precursors, thioacetamide (TAA, 80 mg, 1.065 mmol, 98%) and CFP (1 × 2 cm<sup>2</sup>, Toray carbon paper, TGP-H-60) were mixed in 10 mL H<sub>2</sub>O, transferred into a 20 mL Teflon autoclave, and heated at 180 °C for 24 h to give rise to the corresponding Ni@1T-MoS<sub>2</sub>/CFP electrocatalyst. The loading amount of Ni@1T-MoS<sub>2</sub> on CFP is about 1 mg cm<sup>−2</sup>. (Note: CFP represents conductive CFP, which can serve as substrate to enable the loading of Ni@1T-MoS<sub>2</sub> catalyst, forming Ni@1T-MoS<sub>2</sub>/CFP).</p></sec><sec id=\"Sec9\"><title>Preparation of 1T-MoS<sub>2</sub></title><p id=\"Par25\">80 mg thioacetamide and 50 mg (NH<sub>4</sub>)<sub>6</sub>Mo<sub>7</sub>O<sub>24</sub>·4H<sub>2</sub>O (Alfa Aesar) were dissolved in 10 mL DI water under sonication treatment for 20 min to form homogeneous solution. Afterwards the solution was transferred into a 25 mL Teflon autoclave, and CFP (1 × 2 cm<sup>2</sup>) was placed onto the bottom. The Teflon autoclave was heated at 180 °C for 24 h to give rise to the in situ growth of 1T-MoS<sub>2</sub> on CFP substrate. In addition to the 1T-MoS<sub>2</sub> grown on the CFP, the remained free 1T-MoS<sub>2</sub> was collected by centrifugation and then was washed with DI water, ethanol and acetone (each for two times). The purified 1T-MoS<sub>2</sub> was dried in thermal oven for overnight and ground into fine powders. Loading amount of 1T-MoS<sub>2</sub> on CFP is quantified by 0.5 mg/cm<sup>2</sup> by comparing the mass difference before and after hydrothermal growth.</p></sec><sec id=\"Sec10\"><title>Material characterization</title><p id=\"Par26\">The morphology and size of the nanostructured materials were characterized by a HITACHI H-7700 TEM with an accelerating voltage of 100 kV, and a FEI Tecnai G2 F20 S-Twin HR TEM, operating at 200 kV on a HITACHI S-5500. Aberration-corrected STEM imaging was performed by Nion UltraSTEM 200 at UC-Irvine, equipped with C3/C5 corrector and high-energy resolution monochromated EELS system (HERMES). The instrument was operated at accelerating voltage of 60 kV with convergence semi-angle of 38 mrad and with a beam current of ~10 pA to reach atomic resolution. For STEM imaging, the inner and outer collection semi-angles of ADF detector were 70 and 210 mrad respectively. SEM with energy dispersive X-ray spectroscopy equipment was conducted on a LEO 1530. Raman spectra were recorded using a HORIBA JY HR800 confocal Raman microscope, employing an Ar-ion laser operating at 532 nm. The Raman spectra of Ni@1T-MoS<sub>2</sub> show distinct peaks from 2H-MoS<sub>2</sub> (379, 404, and 454 cm<sup>−1</sup>)<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>, as well as the new peaks between 100 and 375 cm<sup>−1</sup> (i.e., 147, 198, 240, 283, and 345 cm<sup>−1</sup>), corresponding to the phonon modes in 1T-MoS<sub>2</sub>. Powder XRD characterization was performed on a Bruker D8 Advance X-ray diffractometer using Cu-Kα radiation (<italic>λ</italic> = 1.5418 Å).</p></sec><sec id=\"Sec11\"><title>In situ XAS measurements</title><p id=\"Par27\">In situ XAS was performed at beamline 12-BM at the Advanced Photon Source, Argonne National Laboratory. Fluorescence mode detection was used for all samples using a 13-element germanium solid state detector (Canberra). Reference metal foil spectra were collected in transmission mode with ion chambers for energy calibration.</p><p id=\"Par28\">The dry sample spectra were collected in air, while all other samples were collected in a customized 3-electrode in situ electrochemical cell (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">15</xref>). For the acidic condition, 0.5M H<sub>2</sub>SO<sub>4</sub> was used as the electrolyte whereas the alkaline condition used 1M KOH. The electrode is positioned in front of the Kapton window to give the minimum possible path length through electrolyte. The cell was purged with N<sub>2</sub> for 15 min before beginning experiments. The cell contains four inlets, one for working electrode clamp which was connected to the sample on CFP, one reference electrode (SCE), and a graphite rod counter electrode. The other inlet is a small hole for a Teflon tubing for purging. Finally, another small hole acts as the purge outlet to exhaust purge gas as well as gas produced during electrocatalysis reactions.</p><p id=\"Par29\">Potentials were applied during in situ experiments using an EC Epsilon potentiostat and controlled potential electrolysis experiment. Prior to applying the 0 V potential, a linear sweep voltammetry experiment was performed to confirm function of the catalyst. The applied potentials were determined using the PH value of 1 for acidic condition and PH value of 14 for the alkaline condition. The correct potential applied during in situ experiments was calculated against the SCE reference via Eq. <xref rid=\"Equ1\" ref-type=\"\">1</xref>, using a value of +0.241 V vs. RHE for E<sup>0</sup>SCE:<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathrm{E}}\\left( {{\\mathrm{RHE}}} \\right) \\,=\\, {\\mathrm{E}}\\left( {{\\mathrm{SCE}}} \\right) + 0.059 \\, {\\mathrm{pH}} \\,+\\, {\\mathrm{E}}^0{\\mathrm{SCE}}.$$\\end{document}</tex-math><mml:math id=\"M2\"><mml:mi mathvariant=\"normal\">E</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi mathvariant=\"normal\">RHE</mml:mi></mml:mrow></mml:mfenced><mml:mspace width=\"0.25em\"/><mml:mo>=</mml:mo><mml:mspace width=\"0.25em\"/><mml:mi mathvariant=\"normal\">E</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi mathvariant=\"normal\">SCE</mml:mi></mml:mrow></mml:mfenced><mml:mo>+</mml:mo><mml:mn>0.059</mml:mn><mml:mspace width=\"0.25em\"/><mml:mi mathvariant=\"normal\">pH</mml:mi><mml:mspace width=\"0.25em\"/><mml:mo>+</mml:mo><mml:mspace width=\"0.25em\"/><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">E</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msup><mml:mi mathvariant=\"normal\">SCE</mml:mi><mml:mo>.</mml:mo></mml:math><graphic xlink:href=\"41467_2020_17904_Article_Equ1.gif\" position=\"anchor\"/></alternatives></disp-formula></p></sec><sec id=\"Sec12\"><title>XAS data analysis</title><p id=\"Par30\">XAS data were analyzed using the Demeter suite of programs<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>. Raw spectra were averaged, calibrated according to the metal foil reference, and normalized in Athena. FEFF fitting was performed using Artemis. FEFF models were constructed from the published crystal structure of 1T-MoS<sub>2</sub>. For the Mo K-edge, no second-shell scattering was observed, so only the first shell was fit with the addition of a single-scattering Mo–O path. Based on previous ICP-MS measurements<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, it was shown that the Ni@1T-MoS<sub>2</sub> structure is doped with both Ni and a single O atom associating with each Ni dopant. However, from FEFF fitting, it is not possible to fit first shell Mo–O scattering due to the much larger amplitude of Mo–S scattering in the first shell. Because of this, EXAFS fitting using a Mo–O path with variable CN is not feasible and results in indefinite results. Furthermore, due to the doping percentage of the sample, the CN of Mo–O in the first shell is expected to be <<1 and was therefore not included. There is, however, second-shell Mo–O scattering that was discussed previously<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. The model utilized is further demonstrated in the calculated FEFF paths (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">7</xref>). The CN of both the first and second-shell vectors were allowed to vary along with the Debye–Waller factor (σ<sup>2</sup>), Δ<italic>E</italic><sub>0</sub>, and Δ<italic>R</italic>. For simplicity, the value of <italic>R</italic> is reported rather than Δ<italic>R</italic> which is relative to the input model.</p><p id=\"Par31\">For the Ni K-edge, Mo was substituted for Ni in the 1T-MoS<sub>2</sub> structure and used directly for fitting. For acidic conditions, a fixed CN of 1 was used for the first shell Ni–O path. For alkaline conditions, Ni–O first shell scattering was accounted for by replacing a S for O in the model and then allowing the CN of each vector to fit. For the catalytic alkaline condition, a Ni atom was added to the model.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec13\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41467_2020_17904_MOESM1_ESM.pdf\"><caption><p>Supporting Information</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41467_2020_17904_MOESM2_ESM.docx\"><caption><p>Peer Review File</p></caption></media></supplementary-material></sec></sec></body><back><fn-group><fn><p><bold>Peer review information</bold>\n<italic>Nature Communications</italic> thanks Dibyendu Bhattacharyya, Song Jin, and other anonymous reviewers for their contributions to the peer review of this work. Peer review reports are available.</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary information</bold> is available for this paper at 10.1038/s41467-020-17904-z.</p></sec><ack><title>Acknowledgements</title><p>This work was supported by National Science Foundation (DMR-1654140). Use of the Advanced Photon Source at Argonne National Laboratory was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Award No. DE-AC02-06CH11357. J.G. wants to acknowledge NSF award CEBT-1704992 to support this research.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>J.H. and J.G. led this research. B.P. designed and performed the in situ XAS experiments and analyzed the EXAFS data. S. Yang, W.H., and S.L. assisted with performing the in situ XAS experiments. S. Younan and Z.L. synthesized and characterized the material. Y.H. conducted all electrochemical measurements. X.Y. and X.P. did all STEM characterization and analysis. B.P., J.H., and J.G. wrote the paper with inputs from all the authors.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par32\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Turner</surname><given-names>JA</given-names></name></person-group><article-title>Sustainable hydrogen production</article-title><source>Science</source><year>2004</year><volume>305</volume><fpage>972</fpage><lpage>974</lpage><pub-id pub-id-type=\"pmid\">15310892</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dresselhaus</surname><given-names>MS</given-names></name><name><surname>Thomas</surname><given-names>IL</given-names></name></person-group><article-title>Alternative energy technologies</article-title><source>Nature</source><year>2001</year><volume>414</volume><fpage>332</fpage><lpage>337</lpage><pub-id pub-id-type=\"pmid\">11713539</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jaramillo</surname><given-names>TF</given-names></name><etal/></person-group><article-title>Identification of active edge sites for electrochemical H<sub>2</sub> evolution from MoS<sub>2</sub> nanocatalysts</article-title><source>Science</source><year>2007</year><volume>317</volume><fpage>100</fpage><lpage>102</lpage><pub-id pub-id-type=\"pmid\">17615351</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Laursen</surname><given-names>AB</given-names></name><name><surname>Kegnaes</surname><given-names>S</given-names></name><name><surname>Dahl</surname><given-names>S</given-names></name><name><surname>Chorkendorff</surname><given-names>I</given-names></name></person-group><article-title>Molybdenum sulfides-efficient and viable materials for electro- and photoelectrocatalytic hydrogen evolution</article-title><source>Energy Environ. Sci.</source><year>2012</year><volume>5</volume><fpage>5577</fpage><lpage>5591</lpage></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>GQ</given-names></name><etal/></person-group><article-title>All the catalytic active sites of MoS<sub>2</sub> for hydrogen evolution</article-title><source>J. Am. Chem. Soc.</source><year>2016</year><volume>138</volume><fpage>16632</fpage><lpage>16638</lpage><pub-id pub-id-type=\"pmid\">27977198</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Voiry</surname><given-names>D</given-names></name><etal/></person-group><article-title>Conducting MoS<sub>2</sub> nanosheets as catalysts for hydrogen evolution reaction</article-title><source>Nano Lett.</source><year>2013</year><volume>13</volume><fpage>6222</fpage><lpage>6227</lpage><pub-id pub-id-type=\"pmid\">24251828</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lukowski</surname><given-names>MA</given-names></name><etal/></person-group><article-title>Enhanced hydrogen evolution catalysis from chemically exfoliated metallic MoS<sub>2</sub> nanosheets</article-title><source>J. Am. Chem. Soc.</source><year>2013</year><volume>135</volume><fpage>10274</fpage><lpage>10277</lpage><pub-id pub-id-type=\"pmid\">23790049</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>J</given-names></name><name><surname>Shin</surname><given-names>HS</given-names></name></person-group><article-title>Recent advances in layered transition metal dichalcogenides for hydrogen evolution reaction</article-title><source>J. Mater. Chem. A</source><year>2014</year><volume>2</volume><fpage>5979</fpage><lpage>5985</lpage></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hinnemann</surname><given-names>B</given-names></name><etal/></person-group><article-title>Biomimetic hydrogen evolution: MoS<sub>2</sub> nanoparticles as catalyst for hydrogen evolution</article-title><source>J. Am. Chem. Soc.</source><year>2005</year><volume>127</volume><fpage>5308</fpage><lpage>5309</lpage><pub-id pub-id-type=\"pmid\">15826154</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tang</surname><given-names>ML</given-names></name><etal/></person-group><article-title>Structural and electronic study of an amorphous MoS<sub>3</sub> hydrogen-generation catalyst on a quantum-controlled photosensitizer</article-title><source>Angew. Chem. Int. Ed.</source><year>2011</year><volume>50</volume><fpage>10203</fpage><lpage>10207</lpage></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>YG</given-names></name><etal/></person-group><article-title>MoS<sub>2</sub> nanoparticles grown on graphene: an advanced catalyst for the hydrogen evolution reaction</article-title><source>J. Am. Chem. Soc.</source><year>2011</year><volume>133</volume><fpage>7296</fpage><lpage>7299</lpage><pub-id pub-id-type=\"pmid\">21510646</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>X</given-names></name><etal/></person-group><article-title>Solution-phase epitaxial growth of noble metal nanostructures on dispersible single-layer molybdenum disulfide nanosheets</article-title><source>Nat. Commun.</source><year>2013</year><volume>4</volume><fpage>1</fpage><lpage>8</lpage></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lassalle-Kaiser</surname><given-names>B</given-names></name><etal/></person-group><article-title>Evidence from in situ X-ray absorption spectroscopy for the involvement of terminal disulfide in the reduction of protons by an amorphous molybdenum sulfide electrocatalyst</article-title><source>J. Am. Chem. Soc.</source><year>2015</year><volume>137</volume><fpage>314</fpage><lpage>321</lpage><pub-id pub-id-type=\"pmid\">25427231</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Laursen</surname><given-names>AB</given-names></name><name><surname>Vesborg</surname><given-names>PCK</given-names></name><name><surname>Chorkendorff</surname><given-names>I</given-names></name></person-group><article-title>A high-porosity carbon molybdenum sulphide composite with enhanced electrochemical hydrogen evolution and stability</article-title><source>Chem. Commun.</source><year>2013</year><volume>49</volume><fpage>4965</fpage><lpage>4967</lpage></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Benck</surname><given-names>JD</given-names></name><name><surname>Chen</surname><given-names>ZB</given-names></name><name><surname>Kuritzky</surname><given-names>LY</given-names></name><name><surname>Forman</surname><given-names>AJ</given-names></name><name><surname>Jaramillo</surname><given-names>TF</given-names></name></person-group><article-title>Amorphous molybdenum sulfide catalysts for electrochemical hydrogen production: insights into the origin of their catalytic activity</article-title><source>ACS Catal.</source><year>2012</year><volume>2</volume><fpage>1916</fpage><lpage>1923</lpage></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chang</surname><given-names>YH</given-names></name><etal/></person-group><article-title>Highly efficient electrocatalytic hydrogen production by MoSx grown on graphene-protected 3D Ni foams</article-title><source>Adv. Mater.</source><year>2013</year><volume>25</volume><fpage>756</fpage><lpage>760</lpage><pub-id pub-id-type=\"pmid\">23060076</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Merki</surname><given-names>D</given-names></name><name><surname>Fierro</surname><given-names>S</given-names></name><name><surname>Vrubel</surname><given-names>H</given-names></name><name><surname>Hu</surname><given-names>XL</given-names></name></person-group><article-title>Amorphous molybdenum sulfide films as catalysts for electrochemical hydrogen production in water</article-title><source>Chem. Sci.</source><year>2011</year><volume>2</volume><fpage>1262</fpage><lpage>1267</lpage></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>SZ</given-names></name><etal/></person-group><article-title>Rhenium-doped and stabilized MoS<sub>2</sub> atomic layers with basal-plane catalytic activity</article-title><source>Adv. Mater.</source><year>2018</year><volume>30</volume><fpage>1803477</fpage></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>HT</given-names></name><etal/></person-group><article-title>Transition-metal doped edge sites in vertically aligned MoS<sub>2</sub> catalysts for enhanced hydrogen evolution</article-title><source>Nano Res.</source><year>2015</year><volume>8</volume><fpage>566</fpage><lpage>575</lpage></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lauritsen</surname><given-names>JV</given-names></name><name><surname>Besenbacher</surname><given-names>F</given-names></name></person-group><article-title>Atom-resolved scanning tunneling microscopy investigations of molecular adsorption on MoS<sub>2</sub> and CoMoS hydrodesulfurization catalysts</article-title><source>J. Catal.</source><year>2015</year><volume>328</volume><fpage>49</fpage><lpage>58</lpage></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>L</given-names></name><name><surname>Liu</surname><given-names>P</given-names></name><name><surname>Li</surname><given-names>J</given-names></name><name><surname>Xiang</surname><given-names>B</given-names></name></person-group><article-title>Two-dimensional material molybdenum disulfides as electrocatalysts for hydrogen evolution</article-title><source>Catalysts</source><year>2017</year><volume>7</volume><fpage>285</fpage></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ding</surname><given-names>Q</given-names></name><etal/></person-group><article-title>Basal-plane ligand functionalization on semiconducting 2H-MoS<sub>2</sub> monolayers</article-title><source>ACS Appl. Mater. Interfaces</source><year>2017</year><volume>9</volume><fpage>12734</fpage><lpage>12742</lpage><pub-id pub-id-type=\"pmid\">28332817</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ding</surname><given-names>Q</given-names></name><name><surname>Song</surname><given-names>B</given-names></name><name><surname>Xu</surname><given-names>P</given-names></name><name><surname>Jin</surname><given-names>S</given-names></name></person-group><article-title>Efficient electrocatalytic and photoelectrochemical hydrogen generation using MoS<sub>2</sub> and related compounds</article-title><source>Chem</source><year>2016</year><volume>1</volume><fpage>699</fpage><lpage>726</lpage></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maitra</surname><given-names>U</given-names></name><etal/></person-group><article-title>Highly effective visible-light-induced H<sub>2</sub> generation by single-layer 1T-MoS<sub>2</sub> and a nanocomposite of few-layer 2H-MoS<sub>2</sub> with heavily nitrogenated graphene</article-title><source>Angew. Chem. Int. Ed.</source><year>2013</year><volume>52</volume><fpage>13057</fpage><lpage>13061</lpage></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tang</surname><given-names>Q</given-names></name><name><surname>Jiang</surname><given-names>D-e</given-names></name></person-group><article-title>Mechanism of hydrogen evolution reaction on 1T-MoS<sub>2</sub> from first principles</article-title><source>ACS Catal.</source><year>2016</year><volume>6</volume><fpage>4953</fpage><lpage>4961</lpage></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Qi</surname><given-names>K</given-names></name><etal/></person-group><article-title>Single-atom cobalt array bound to distorted 1T MoS<sub>2</sub> with ensemble effect for hydrogen evolution catalysis</article-title><source>Nat. Commun.</source><year>2019</year><volume>10</volume><fpage>5231</fpage><pub-id pub-id-type=\"pmid\">31745074</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ji</surname><given-names>L</given-names></name><etal/></person-group><article-title>One-pot synthesis of porous 1T-phase MoS<sub>2</sub> integrated with single-atom Cu doping for enhancing electrocatalytic hydrogen evolution reaction</article-title><source>Appl. Catal. B</source><year>2019</year><volume>251</volume><fpage>87</fpage><lpage>93</lpage></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>YC</given-names></name><etal/></person-group><article-title>Atomically engineering activation sites onto metallic 1T-MoS<sub>2</sub> catalysts for enhanced electrochemical hydrogen evolution</article-title><source>Nat. Commun.</source><year>2019</year><volume>10</volume><fpage>1</fpage><lpage>11</lpage><pub-id pub-id-type=\"pmid\">30602773</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Geng</surname><given-names>X</given-names></name><etal/></person-group><article-title>Pure and stable metallic phase molybdenum disulfide nanosheets for hydrogen evolution reaction</article-title><source>Nat. Commun.</source><year>2016</year><volume>7</volume><fpage>10672</fpage><pub-id pub-id-type=\"pmid\">26861766</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Guo</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Probing the dynamics of the metallic-to-semiconducting structural phase transformation in MoS<sub>2</sub> crystals</article-title><source>Nano Lett.</source><year>2015</year><volume>15</volume><fpage>5081</fpage><lpage>5088</lpage><pub-id pub-id-type=\"pmid\">26134736</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krivanek</surname><given-names>OL</given-names></name><etal/></person-group><article-title>Atom-by-atom structural and chemical analysis by annular dark-field electron microscopy</article-title><source>Nature</source><year>2010</year><volume>464</volume><fpage>571</fpage><lpage>574</lpage><pub-id pub-id-type=\"pmid\">20336141</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Deng</surname><given-names>J</given-names></name><etal/></person-group><article-title>Triggering the electrocatalytic hydrogen evolution activity of the inert two-dimensional MoS<sub>2</sub> surface via single-atom metal doping</article-title><source>Energy Environ. Sci.</source><year>2015</year><volume>8</volume><fpage>1594</fpage><lpage>1601</lpage></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ravel</surname><given-names>B</given-names></name><name><surname>Newville</surname><given-names>M</given-names></name></person-group><article-title>ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT</article-title><source>J. Synchrotron Radiat.</source><year>2005</year><volume>12</volume><fpage>537</fpage><lpage>541</lpage><pub-id pub-id-type=\"pmid\">15968136</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fang</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Structure re-determination and superconductivity observation of bulk 1T MoS<sub>2</sub></article-title><source>Angew. Chem. Int. Ed.</source><year>2018</year><volume>57</volume><fpage>1232</fpage><lpage>1235</lpage></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chung</surname><given-names>DY</given-names></name><etal/></person-group><article-title>Edge-exposed MoS<sub>2</sub> nano-assembled structures as efficient electrocatalysts for hydrogen evolution reaction</article-title><source>Nanoscale</source><year>2014</year><volume>6</volume><fpage>2131</fpage><lpage>2136</lpage><pub-id pub-id-type=\"pmid\">24310105</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lince</surname><given-names>JR</given-names></name><name><surname>Hilton</surname><given-names>MR</given-names></name><name><surname>Bommannavar</surname><given-names>AS</given-names></name></person-group><article-title>EXAFS of sputter-deposited MoS<sub>2</sub> films</article-title><source>Thin Solid Films</source><year>1995</year><volume>264</volume><fpage>120</fpage><lpage>134</lpage></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>Q</given-names></name><etal/></person-group><article-title>Gram-scale aqueous synthesis of stable few-layered 1T-MoS<sub>2</sub>: applications for visible-light-driven photocatalytic hydrogen evolution</article-title><source>Small</source><year>2015</year><volume>11</volume><fpage>5556</fpage><lpage>5564</lpage><pub-id pub-id-type=\"pmid\">26332270</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pandya</surname><given-names>KI</given-names></name><name><surname>Hoffman</surname><given-names>RW</given-names></name><name><surname>McBreen</surname><given-names>J</given-names></name><name><surname>O’Grady</surname><given-names>WE</given-names></name></person-group><article-title>In situ X-ray absorption spectroscopic studies of nickel oxide electrodes</article-title><source>J. Electrochem. Soc.</source><year>1990</year><volume>137</volume><fpage>383</fpage><lpage>388</lpage></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>D’Angelo</surname><given-names>P</given-names></name><name><surname>Benfatto</surname><given-names>M</given-names></name><name><surname>Della Longa</surname><given-names>S</given-names></name><name><surname>Pavel</surname><given-names>NV</given-names></name></person-group><article-title>Combined XANES and EXAFS analysis of Co<sup>2+</sup>, Ni<sup>2+</sup>, and Zn<sup>2+</sup> in aqueous solutions</article-title><source>Phys. Rev. B</source><year>2002</year><volume>66</volume><fpage>064209</fpage></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Colpas</surname><given-names>GJ</given-names></name><etal/></person-group><article-title>X-ray spectroscopic studies of nickel complexes, with application to the structure of nickel sites in hydrogenases</article-title><source>Inorg. Chem.</source><year>1991</year><volume>30</volume><fpage>920</fpage><lpage>928</lpage></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Strathmann</surname><given-names>TJ</given-names></name><name><surname>Myneni</surname><given-names>SCB</given-names></name></person-group><article-title>Speciation of aqueous Ni(II)-carboxylate and Ni(II)-fulvic acid solutions: combined ATR-FTIR and XAFS analysis</article-title><source>Geochim. Cosmochim. Acta</source><year>2004</year><volume>68</volume><fpage>3441</fpage><lpage>3458</lpage></element-citation></ref><ref id=\"CR42\"><label>42.</label><mixed-citation publication-type=\"other\">Crespin, M., Levitz, P. & Gatineau, L. In <italic>EXAFS and Near Edge Structure,</italic> (eds Bianconi, A., Incoccia, L., & Stipcich, S.) 228–230 (Springer, Berlin Heidelberg, 1983).</mixed-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mansour</surname><given-names>AN</given-names></name><name><surname>Melendres</surname><given-names>CA</given-names></name><name><surname>Pankuch</surname><given-names>M</given-names></name><name><surname>Brizzolara</surname><given-names>RA</given-names></name></person-group><article-title>X-ray absorption fine structure spectra and the oxidation state of nickel in some of its oxycompounds</article-title><source>J. Electrochem. Soc.</source><year>1994</year><volume>141</volume><fpage>L69</fpage><lpage>L71</lpage></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Woolley</surname><given-names>RJ</given-names></name><name><surname>Illy</surname><given-names>BN</given-names></name><name><surname>Ryan</surname><given-names>MP</given-names></name><name><surname>Skinner</surname><given-names>SJ</given-names></name></person-group><article-title>In situ determination of the nickel oxidation state in La<sub>2</sub>NiO<sub>4+δ</sub> and La<sub>4</sub>Ni3O<sub>10−δ</sub> using X-ray absorption near-edge structure</article-title><source>J. Mater. Chem.</source><year>2011</year><volume>21</volume><fpage>18592</fpage><lpage>18596</lpage></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bearden</surname><given-names>JA</given-names></name><name><surname>Burr</surname><given-names>AF</given-names></name></person-group><article-title>Reevaluation of X-ray atomic energy levels</article-title><source>Rev. Mod. Phys.</source><year>1967</year><volume>39</volume><fpage>125</fpage><lpage>142</lpage></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nakatsu</surname><given-names>K</given-names></name><etal/></person-group><article-title>Controlled synthesis of carbon-supported Co catalysts from single-sites to nanoparticles: characterization of the structural transformation and investigation of their oxidation catalysis</article-title><source>Phys. Chem. Chem. Phys.</source><year>2017</year><volume>19</volume><fpage>4967</fpage><lpage>4974</lpage><pub-id pub-id-type=\"pmid\">27841416</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>X</given-names></name><etal/></person-group><article-title>In situ/operando techniques for characterization of single-atom catalysts</article-title><source>ACS Catal.</source><year>2019</year><volume>9</volume><fpage>2521</fpage><lpage>2531</lpage></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bergmann</surname><given-names>A</given-names></name><etal/></person-group><article-title>Reversible amorphization and the catalytically active state of crystalline Co<sub>3</sub>O<sub>4</sub> during oxygen evolution</article-title><source>Nat. Commun.</source><year>2015</year><volume>6</volume><fpage>8625</fpage><pub-id pub-id-type=\"pmid\">26456525</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yin</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Contributions of phase, sulfur vacancies, and edges to the hydrogen evolution reaction catalytic activity of porous molybdenum disulfide nanosheets</article-title><source>J. Am. Chem. Soc.</source><year>2016</year><volume>138</volume><fpage>7965</fpage><lpage>7972</lpage><pub-id pub-id-type=\"pmid\">27269185</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>G</given-names></name><etal/></person-group><article-title>All the catalytic active sites of MoS<sub>2</sub> for hydrogen evolution</article-title><source>J. Am. Chem. Soc.</source><year>2016</year><volume>138</volume><fpage>16632</fpage><lpage>16638</lpage><pub-id pub-id-type=\"pmid\">27977198</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wiensch</surname><given-names>JD</given-names></name><etal/></person-group><article-title>Comparative study in acidic and alkaline media of the effects of pH and crystallinity on the hydrogen-evolution reaction on MoS<sub>2</sub> and MoSe<sub>2</sub></article-title><source>ACS Energy Lett.</source><year>2017</year><volume>2</volume><fpage>2234</fpage><lpage>2238</lpage></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Park</surname><given-names>S</given-names></name><etal/></person-group><article-title>Enhancing catalytic activity of MoS<sub>2</sub> basal plane S-vacancy by Co cluster addition</article-title><source>ACS Energy Lett.</source><year>2018</year><volume>3</volume><fpage>2685</fpage><lpage>2693</lpage></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>Y</given-names></name><etal/></person-group><article-title>High phase-purity 1T′-MoS<sub>2</sub>- and 1T′-MoSe<sub>2</sub>-layered crystals</article-title><source>Nat. Chem.</source><year>2018</year><volume>10</volume><fpage>638</fpage><lpage>643</lpage><pub-id pub-id-type=\"pmid\">29610461</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yao</surname><given-names>Y</given-names></name><name><surname>Ao</surname><given-names>K</given-names></name><name><surname>Lv</surname><given-names>P</given-names></name><name><surname>Wei</surname><given-names>Q</given-names></name></person-group><article-title>MoS<sub>2</sub> coexisting in 1T and 2H phases synthesized by common hydrothermal method for hydrogen evolution reaction</article-title><source>Nanomaterials</source><year>2019</year><volume>9</volume><fpage>844</fpage></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807821</article-id><article-id pub-id-type=\"pmc\">PMC7431583</article-id><article-id pub-id-type=\"publisher-id\">70874</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70874-6</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>A decrease in NAMPT activity impairs basal PARP-1 activity in cytidine deaminase deficient-cells, independently of NAD<sup>+</sup></article-title></title-group><contrib-group><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Silveira</surname><given-names>Sandra Cunha</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Buhagiar-Labarchède</surname><given-names>Géraldine</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Onclercq-Delic</surname><given-names>Rosine</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Gemble</surname><given-names>Simon</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Bou Samra</surname><given-names>Elias</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mameri</surname><given-names>Hamza</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Duchambon</surname><given-names>Patricia</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>Machon</surname><given-names>Christelle</given-names></name><xref ref-type=\"aff\" rid=\"Aff6\">6</xref><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><name><surname>Guitton</surname><given-names>Jérôme</given-names></name><xref ref-type=\"aff\" rid=\"Aff6\">6</xref><xref ref-type=\"aff\" rid=\"Aff8\">8</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Amor-Guéret</surname><given-names>Mounira</given-names></name><address><email>mounira.amor@curie.fr</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.440907.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1784 3645</institution-id><institution>Institut Curie, UMR 3348, </institution><institution>PSL Research University, </institution></institution-wrap>91405 Orsay, France </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5842.b</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2171 2558</institution-id><institution>CNRS UMR 3348, Centre Universitaire, </institution></institution-wrap>91405 Orsay, France </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5842.b</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2171 2558</institution-id><institution>Université Paris Sud, Université Paris-Saclay, Centre Universitaire, UMR 3348, </institution></institution-wrap>91405 Orsay, France </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.440907.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1784 3645</institution-id><institution>Protein Expression and Purification Core Facility, Institut Curie, </institution><institution>PSL Research University, </institution></institution-wrap>75248 Paris, France </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5842.b</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2171 2558</institution-id><institution>Université Paris Sud, Université Paris-Saclay, Centre Universitaire, UMR 9187 - INSERM U1196, </institution></institution-wrap>91405 Orsay, France </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.411430.3</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0288 2594</institution-id><institution>Laboratoire de Biochimie et Toxicologie, </institution><institution>Centre Hospitalier Lyon-Sud, Hospices Civils de Lyon, </institution></institution-wrap>Pierre-Bénite, France </aff><aff id=\"Aff7\"><label>7</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.25697.3f</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2172 4233</institution-id><institution>Laboratoire de Chimie Analytique, ISPB, Faculté de Pharmacie, </institution><institution>Université Lyon 1, Université de Lyon, </institution></institution-wrap>Lyon, France </aff><aff id=\"Aff8\"><label>8</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.25697.3f</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2172 4233</institution-id><institution>Laboratoire de Toxicologie, ISPB, Faculté de Pharmacie, </institution><institution>Université Lyon 1, Université de Lyon, </institution></institution-wrap>Lyon, France </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13907</elocation-id><history><date date-type=\"received\"><day>29</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>3</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Cytidine deaminase (CDA) deficiency causes pyrimidine pool disequilibrium. We previously reported that the excess cellular dC and dCTP resulting from CDA deficiency jeopardizes genome stability, decreasing basal poly(ADP-ribose) polymerase 1 (PARP-1) activity and increasing ultrafine anaphase bridge (UFB) formation. Here, we investigated the mechanism underlying the decrease in PARP-1 activity in CDA-deficient cells. PARP-1 activity is dependent on intracellular NAD<sup>+</sup> concentration. We therefore hypothesized that defects of the NAD<sup>+</sup> salvage pathway might result in decreases in PARP-1 activity. We found that the inhibition or depletion of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD<sup>+</sup> salvage biosynthesis pathway, mimicked CDA deficiency, resulting in a decrease in basal PARP-1 activity, regardless of NAD<sup>+</sup> levels. Furthermore, the expression of exogenous wild-type NAMPT fully restored basal PARP-1 activity and prevented the increase in UFB frequency in CDA-deficient cells. No such effect was observed with the catalytic mutant. Our findings demonstrate that (1) the inhibition of NAMPT activity in CDA-proficient cells lowers basal PARP-1 activity, and (2) the expression of exogenous wild-type NAMPT, but not of the catalytic mutant, fully restores basal PARP-1 activity in CDA-deficient cells; these results strongly suggest that basal PARP-1 activity in CDA-deficient cells decreases due to a reduction of NAMPT activity.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Cancer</kwd><kwd>Cell biology</kwd><kwd>Molecular biology</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/501100010463</institution-id><institution>Institut Curie</institution></institution-wrap></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/501100009517</institution-id><institution>Université de Recherche Paris Sciences et Lettres</institution></institution-wrap></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/501100004794</institution-id><institution>Centre National de la Recherche Scientifique</institution></institution-wrap></funding-source></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">http://dx.doi.org/10.13039/501100004099</institution-id><institution>Ligue Contre le Cancer</institution></institution-wrap></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">The maintenance of genome stability is crucial for preventing various aging-related diseases, including cancer<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. We have shown that cytidine deaminase (CDA) plays an essential role in maintaining genome integrity. CDA is an enzyme of the pyrimidine salvage pathway catalyzing the hydrolytic deamination of cytidine (C) and deoxycytidine (dC) to uridine (U) and deoxyuridine (dU), respectively<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>.</p><p id=\"Par3\">CDA activity has been widely studied in the context of the inactivation of nucleoside analogs widely used in chemotherapy. The determination of CDA expression status within cancer cells and tissues is opening up new possibilities for cancer treatment<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>–<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. We have shown that CDA expression is downregulated in about 60% of cancer cells and tissues, mostly due to DNA methylation<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. We have also demonstrated the existence of a causal link between the expression of CDA and other genes, in particular the gene encoding the microtubule-associated protein Tau (MAPT), highlighting the importance of analyzing CDA expression levels alone or together with the expression of other genes, as a relevant and predictive marker of susceptibility to antitumor drugs<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. The pyrimidine pool disequilibrium resulting from CDA deficiency also contributes to the genetic instability of cells, particularly in Bloom syndrome (BS)<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. Indeed, we have reported that the resulting intracellular accumulation of dC and dCTP decreases PARP-1 activity in basal conditions and in response to genotoxic stress, leading to the accumulation of unreplicated DNA during mitosis and, thus, to high levels of UFB formation<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>–<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. However, based on in vitro data that were not consistent with the direct inhibition of PARP-1 by dC or dCTP in vivo, we hypothesized that the intracellular accumulation of dC and dCTP might impair PARP-1 activity indirectly<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. We therefore investigated the mechanism responsible for decreasing basal PARP-1 activity in CDA-deficient cells. PARP-1 is a multifunctional enzyme that mediates several aspects of the DNA damage response through its poly(ADP-ribosyl)ation (PARylation) activity, involving the transfer of PAR units from nicotinamide-adenine-dinucleotide (NAD<sup>+</sup>) to diverse acceptor proteins, including histones<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. Thus, PARP-1 plays a key role in preventing genetic instability, and its activity is dependent on intracellular NAD<sup>+</sup> concentration. NAD<sup>+</sup> is mostly synthesized from the precursor nicotinamide (NAM), via the salvage pathway. NAMPT is the rate-limiting enzyme in NAD<sup>+</sup> biosynthesis via this pathway<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. It catalyzes the transfer of a phosphoribosyl group from 5-phosphoribosyl-pyrophosphate (PRPP) to NAM, generating the NAD<sup>+</sup> intermediate nicotinamide mononucleotide (NMN), which is converted to NAD<sup>+</sup> by nicotinamide mononucleotide adenylyltransferase (NMNAT)<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. Through this function in NAD<sup>+</sup> biosynthesis, NAMPT activity is crucial for regulation of the activity of NAD<sup>+</sup>-dependent enzymes, such as sirtuins and PARPs<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>.</p><p id=\"Par4\">In this study, we investigated the mechanism responsible for decreasing basal PARP-1 activity in CDA-deficient cells. We found that NAMPT inhibition or depletion reproduced the cellular phenotype associated with CDA deficiency, lowering basal PARP-1 activity and increasing UFB frequency to levels similar to those in CDA-deficient cells. We also showed that the decrease in basal PARP-1 activity was independent of NAD<sup>+</sup> levels. Finally, we demonstrated that the expression of exogenous wild-type NAMPT fully restored basal PARP-1 activity, thereby preventing the increase in UFB frequency in CDA-deficient cells, whereas no such effect was observed with the catalytic mutant. These results provide the first evidence of a link between the pyrimidine and NAD<sup>+</sup> salvage pathways.</p></sec><sec id=\"Sec2\"><title>Results</title><sec id=\"Sec3\"><title>NAMPT inhibition decreases basal PARP-1 activity, mimicking CDA deficiency</title><p id=\"Par5\">We explored the potential link between the decrease in basal PARP-1 activity in CDA-deficient cells and NAD<sup>+</sup> metabolism, by investigating the effect of inhibiting NAMPT activity on basal PARP-1 activity. We used two pairs of isogenic cellular models of CDA deficiency displaying decreases in basal PARP-1 activity: the GM8505B-derived BS cell line, which has a mutated <italic>BLM</italic> gene and strongly downregulated CDA expression (BS-Ctrl<sub>(BLM)</sub>; BLM<sup>-</sup>/CDA<sup>-</sup>), and its counterpart stably expressing an exogenous GFP-BLM construct restoring the expression of both BLM and CDA (BS-BLM; BLM<sup>+</sup>/CDA<sup>+</sup>); and a HeLa cell line stably expressing an adenoviral short hairpin RNA (shRNA) specific for CDA, displaying strong CDA downregulation (HeLa-shCDA: BLM<sup>+</sup>/CDA<sup>-</sup>), and its control counterpart expressing CDA (HeLa-Ctrl<sub>(CDA)</sub>; BLM<sup>+</sup>/CDA<sup>+</sup>)<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. As expected, both CDA-deficient cell lines contained significantly larger amounts of cytidine and smaller amounts of uridine than their control counterparts (Figure <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>a and <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>b). We treated the cells with the NAMPT inhibitor FK866. FK866 treatment did not affect the levels of the PARP-1, NAMPT or CDA proteins (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>a and <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>c), and did not lead to an inhibition of recombinant PARP-1 protein activity (Figure <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>d). We then performed immunofluorescence assays to assess the basal cellular levels of PARylation, by measuring the relative number of PAR foci as readout of cellular PARP-1 activity (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b). FK866 treatment significantly decreased the prevalence of PAR foci in both CDA-proficient HeLa and BS-BLM cells, to the levels observed in CDA-deficient cells (Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>c and <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>e). FK866 treatment also decreased significantly the frequency of PAR foci in both cell lines lacking CDA (Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>c and <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>e). We previously reported that decreases in basal PARP-1 activity lead to an increase in the frequency of UFB formation<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. We therefore analyzed the frequency of UFBs in these cells, by staining them with antibodies specific for the helicase-like protein PICH (Plk1-interaction checkpoint “helicase”). This is the only way to detect the total UFB population<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup> (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>d). FK866 treatment increased UFB frequency in CDA-expressing cells to levels similar to those in CDA-deficient cells, but had no effect on UFB frequency in CDA-deficient cells (Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>e and <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>f.).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>NAMPT inhibition or depletion impairs basal PARP-1 activity. (<bold>a</bold>) PARP-1, NAMPT and CDA protein levels assessed by immunoblotting in HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA cell lines left untreated or treated with 1 μM FK866 for 10 h. Actin was used as protein loading control. (<bold>b</bold>) Representative immunofluorescence deconvoluted <italic>z</italic>-projection images of HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA cells showing PAR foci in interphase cells. Scale bar: 5 µm. (<bold>c</bold>) Analysis of PAR foci number in HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA cell lines left untreated or treated with 1 μM FK866 for 10 h. The data shown are the means ± SD from four independent experiments (> 350 cells per condition). (<bold>d</bold>) Representative immunofluorescence deconvoluted <italic>z</italic>-projection images of PICH-positive UFBs in HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA anaphase cells. DNA was visualized by DAPI staining (blue). UFBs were stained with PICH antibody (in green, Alexa Fluor 555). Scale bar: 5 µm. (<bold>e</bold>) Mean number of UFBs per anaphase cell, for HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA cell lines left untreated or treated with 1 μM FK866 for 10 h. The data shown are the means <inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\pm$$\\end{document}</tex-math><mml:math id=\"M2\"><mml:mo>±</mml:mo></mml:math><inline-graphic xlink:href=\"41598_2020_70874_Article_IEq1.gif\"/></alternatives></inline-formula> SD from three independent experiments (> 80 anaphase cells per condition). (<bold>f</bold>) PARP-1, NAMPT and CDA protein levels assessed by immunoblotting in HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA cell lines transiently transfected with the indicated siRNAs twice successively for a total of 144 h (96 h + 48 h). HSP90 was used as protein loading control. (<bold>g</bold>) Analysis of PAR foci number in HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA cell lines transiently transfected with the indicated siRNAs. The data shown are the means ± SD from four independent experiments (> 350 cells per condition). (<bold>h</bold>) Mean number of UFBs per anaphase cell, for HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA cell lines transiently transfected with the indicated siRNAs. The data shown are means ± SD from three independent experiments (> 120 anaphase cells per condition). The significance of differences was assessed with Student’s <italic>t</italic>-test.</p></caption><graphic xlink:href=\"41598_2020_70874_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par6\">For confirmation of these results, we induced a transient depletion of NAMPT in the two pairs of isogenic cell lines, by transfecting them with a pool of four NAMPT-targeting siRNAs. We then assessed changes in NAMPT protein levels (Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>f and <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>g) and in the frequencies of PAR foci and UFBs (Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>g, h, <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>h and <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>i). NAMPT depletion had no impact on PAR foci or UFB frequencies in CDA-deficient cells (Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>g,h, <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>h and <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>i). However, it decreased PAR focus frequency (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>g and <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>h) and increased UFB frequency (Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>h and <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>i) in CDA-proficient cells, to levels similar to those observed in CDA-deficient cells. Thus, NAMPT inhibition or depletion in CDA-proficient cells decreases the basal activity of PARP-1 and increases the frequency of UFBs to levels similar to those in CDA-deficient cells, mimicking CDA deficiency. Moreover, no additive effect of CDA deficiency and NAMPT inhibition or depletion was observed on UFB frequency, suggesting that CDA and NAMPT probably prevent UFB formation by acting on the same pathway.</p></sec><sec id=\"Sec4\"><title>The decrease in basal PARP-1 activity resulting from NAMPT inhibition is independent of NAM accumulation and NAD<sup>+</sup> levels</title><p id=\"Par7\">As NAMPT converts NAM to NMN, which is in turn converted to NAD<sup>+</sup> by NMNAT (Figure <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>a), NAMPT defects would be expected to result in higher NAM and lower NAD<sup>+</sup> levels. We then analyzed NAM levels by LC-HRMS (liquid chromatography-high-resolution mass spectrometry) in cells left untreated or treated with FK866. Surprisingly, FK866 treatment did not increase NAM levels in any of the four cell lines. Moreover, NAM levels were slightly but significantly lower in CDA-deficient cells than in control cells (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>a and b), disabling us to conclude about the possible contribution of unbalanced NAM levels to the decrease in basal PARP-1 activity in CDA-deficient cells.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>The decrease in basal PARP-1 activity resulting from NAMPT inhibition is independent of NAM accumulation and NAD<sup>+</sup> levels. (<bold>a</bold>) and (<bold>b</bold>) Analysis of intracellular NAM levels in HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA cell lines (<bold>a</bold>) or in BS-BLM and BS-Ctrl<sub>(BLM)</sub> cell lines (<bold>b</bold>) left untreated or treated with 1 μM FK866 for 10 h. The data shown are means ± SD from four (<bold>a</bold>) or three (<bold>b</bold>) independent experiments, respectively. (<bold>c</bold>) and (<bold>d</bold>) Analysis of intracellular NAD<sup>+</sup> levels (luciferase assay) in HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA cell lines (<bold>c</bold>) or in BS-BLM and BS-Ctrl<sub>(BLM)</sub> (<bold>d</bold>) left untreated or treated with 1 μM FK866 for 10 h and/or with 500 μM NMN for 24 h. The data shown are means ± SD from five (<bold>c</bold>) or from three (<bold>d</bold>) independent experiments, respectively. (<bold>e</bold>) and (<bold>f</bold>) Analysis of PAR foci number in HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA cell lines (<bold>e</bold>) or in BS-BLM and BS-Ctrl<sub>(BLM)</sub> cell lines (<bold>f</bold>) left untreated or treated with 1 μM FK866 for 10 h and/or with 500 μM NMN for 24 h. The data shown are means ± SD from four independent experiments (> 400 cells per condition) (<bold>e</bold>) or from three independent experiments (> 400 cells per condition) (<bold>f</bold>), respectively. (<bold>g</bold>) and (<bold>h</bold>) Mean number of UFBs per anaphase cell, for HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA cell lines (<bold>g</bold>) or for BS-BLM and BS-Ctrl<sub>(BLM)</sub> cell lines (<bold>h</bold>) left untreated or treated with 1 μM FK866 for 10 h and/or with 500 μM NMN for 24 h. The data shown are means ± SD from three independent experiments (> 100 anaphase cells per condition) (<bold>g</bold>) or from three independent experiments (> 120 anaphase cells per condition) (<bold>h</bold>). The significance of differences was assessed with Student’s <italic>t</italic>-test.</p></caption><graphic xlink:href=\"41598_2020_70874_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par8\">We then analyzed NAD<sup>+</sup> levels by LC-HRMS or with a luciferase assay. NAD<sup>+</sup> levels were significantly higher in CDA-depleted HeLa cells than in control cells, whereas no significant difference was observed between the BS-Ctrl<sub>(BLM)</sub> and BS-BLM cell lines, suggesting that the decrease in PARP-1 activity in CDA-deficient cells was independent of NAD<sup>+</sup> levels (Figures <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>b, <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>c, <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>d and <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>e).</p><p id=\"Par9\">For confirmation of these results, we evaluated NAD<sup>+</sup> levels, PAR foci and UFB frequencies after treatment with NMN and/or FK866 (Figure <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>a). We observed either no change or a significant increase in NAD<sup>+</sup> levels in the four cell lines in response to NMN treatment (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>c,d). Several studies have reported that the blockade of NAD<sup>+</sup> resynthesis by inhibition of the rate-limiting enzyme NAMPT can be circumvented by alternative metabolic pathways leading to NAD<sup>+</sup> formation<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. Thus, these results strongly suggest that, in the context of CDA deficiency, the partial inhibition of NAMPT is circumvented for NAD<sup>+</sup> synthesis.</p><p id=\"Par10\">By contrast, NAD<sup>+</sup> levels were significantly lower in these cells after FK866 treatment, as expected (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>c,d). Moreover, NMN treatment partly rescued NAD<sup>+</sup> levels in the four FK866-treated cell lines (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>c,d). Importantly, although the addition of NMN made it possible to maintain high levels of NAD<sup>+</sup> in cells treated with FK866, it did not prevent the decrease in PAR focus frequency and the subsequent increase in UFB frequency resulting from NAMPT inhibition by FK866 in CDA-proficient HeLa-Ctrl<sub>(CDA)</sub> or BS-BLM cells (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>e–h). Together, these results indicate that NAD<sup>+</sup> levels do not reflect NAMPT activity in the context of CDA deficiency, and demonstrate that the decrease in basal PARP-1 activity resulting from NAMPT inhibition is completely independent of cellular NAD<sup>+</sup> levels.</p></sec><sec id=\"Sec5\"><title>The decrease in basal PARP-1 activity in CDA-deficient cells is rescued by the expression of an exogenous wild-type NAMPT, but not by a mutated NAMPT</title><p id=\"Par11\">For formal confirmation of the involvement of the decrease in NAMPT enzymatic activity in the low basal levels of PARP-1 activity observed in CDA-deficient cells, we investigated whether overexpressing an exogenous NAMPT could restore the levels of PARP-1 activity in CDA-deficient cells. We used a construct expressing either a wild-type (WT) NAMPT or the NAMPT protein with a mutated catalytic site (H247A)<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. In parallel, two recombinant proteins — WT and H247A-mutated NAMPT proteins—were produced and their activity was assessed by LC-HRMS or in an assay measuring the conversion of <sup>14</sup>C<sup>-</sup>NAM to <sup>14</sup>C-NMN. The wild-type NAMPT was highly active, whereas the mutated protein had no activity, as expected (Figures S3a and S3b). Similar numbers of CDA-deficient and CDA-proficient HeLa cells were transfected with the wild-type and mutated NAMPT constructs (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a–c). The levels of exogenous wild-type and mutated NAMPT proteins (NAMPT-His and the upper NAMPT band) expressed were lower in CDA-deficient cells than in control cells, and transfection with any NAMPT-expressing construct decreased PARP-1 and CDA protein levels (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>c). However, the expression of exogenous wild-type or mutated NAMPT had no effect on the frequencies of PAR foci or UFBs in CDA-proficient HeLa cells, whereas the expression of wild-type NAMPT, but not of the mutated NAMPT, fully restored the frequency of PAR foci and, consequently, UFB frequency to normal levels in CDA-deficient HeLa cells (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>d,e). Similarly, the expression of exogenous wild-type NAMPT in CDA-deficient BS cells led to a significant increase in the frequency of PAR foci and a significant decrease in UFB frequency (Figures S3c, S3d and S3e), whereas the mutated NAMPT had no such effect. Thus, basal PARP-1 activity was fully restored in both BS and CDA-deficient HeLa cells by the expression of an exogenous wild-type NAMPT, whereas the inactive NAMPT mutant had no effect in these cells. These data indicate that the lower basal PARP-1 activity in CDA-deficient cells results from a lower level of NAMPT activity in these cells.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>The low levels of PARP-1 activity in CDA-deficient cells are rescued by the overexpression of wild-type NAMPT (<bold>a</bold>) Representative immunofluorescence deconvoluted <italic>z</italic>-projection images showing DAPI and His-tag staining in HeLa-Ctrl<sub>(CDA)</sub> cells not transfected (NT) or transiently transfected with pPM-C-His empty vector (EV), or with a pPM-C-His construct expressing wild-type NAMPT (NAMPT WT) or mutated NAMPT (NAMPT H247A). Nuclei were visualized by DAPI staining (blue) and the His-tag was visualized with Alexa Fluor 555 (red). Scale bar: 5 µm. (<bold>b</bold>) Percentage of His-tag-positive cells among HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA cells transiently transfected with EV, NAMPT WT or NAMPT H247A. The data shown are means ± SD from four independent experiments. (<bold>c</bold>) PARP-1, NAMPT, NAMPT-HIS and CDA proteins levels assessed by immunoblotting in HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA cells transiently transfected with EV, NAMPT WT or NAMPT H247A. (<bold>d</bold>) Relative number of PAR foci in HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA) cells transiently transfected with EV, NAMPT WT or NAMPT H247A. The data shown are means ± SD from four independent experiments (> 800 cells per condition). (<bold>e</bold>) Mean number of UFBs per anaphase cell, for HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA cell lines transiently transfected with EV, NAMPT WT or NAMPT H247A. Error bars represent means ± SD from three independent experiments (> 120 anaphase cells per condition). The significance of differences was assessed in Student’s <italic>t</italic>-tests. (<bold>f</bold>) (1) CDA deficiency leads to (2) intracellular dC/dCTP accumulation that (3) decreases NAMPT activity, directly or indirectly, leading to the (4) intracellular accumulation of an as yet unidentified factor X (5) lowering basal PARP-1 activity, causing (6) excess UFB formation.</p></caption><graphic xlink:href=\"41598_2020_70874_Fig3_HTML\" id=\"MO3\"/></fig></p></sec></sec><sec id=\"Sec6\"><title>Discussion</title><p id=\"Par12\">We report here, for the first time, an unexpected link between CDA and NAMPT, CDA deficiency probably being associated with a decrease in NAMPT activity. Indeed, we found that NAMPT inhibition or depletion in CDA-expressing cells reproduced the main feature of CDA deficiency: a decrease in basal PARP-1 activity leading to an increase in UFB frequency.</p><p id=\"Par13\">We previously showed that the pyrimidine pool disequilibrium resulting from CDA deficiency decreases basal PARP-1 activity, leading to UFB formation. Increasing the size of the intracellular dC and dCTP pools by culturing CDA-expressing cells in the presence of dC was sufficient to decrease PARP-1 activity. However, our in vitro data were not consistent with the direct inhibition of PARP-1 by dC or dCTP<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. We report here that the inhibition or depletion of NAMPT activity decreases PARP-1 activity. These findings strongly suggest that the lower levels of basal PARP-1 activity in CDA-deficient cells result from the partial inhibition of NAMPT activity by the excess dC and dCTP. We tested this hypothesis by performing several experiments with cell extracts prepared under various conditions, or by studying the recombinant NAMPT protein in vitro in the presence of various concentrations of dC or dCTP, with an assay measuring the conversion of <sup>14</sup>C<sup>-</sup>NAM to <sup>14</sup>C-NMN, or with an LC-HRMS approach for the selective detection of NAM and NMN. Neither of these experimental approaches yielded reproducible results and, despite our best efforts, we were unable to identify the cause of this lack of reproducibility. It probably resulted, at least in part, from the unusually high affinity of NAMPT for NAM<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>, making it very difficult to detect its inhibition by less effective substrates. We cannot, therefore, exclude the possibility that the decrease in NAMPT activity was a direct or indirect consequence of the excess cellular dC and/or dCTP.</p><p id=\"Par14\">Another hypothesis based on our results showing that NAMPT inhibition leads to a decrease in basal PARP-1 activity independently of NAD<sup>+</sup> levels is that the excess NAM resulting from NAMPT inhibition inhibits PARP-1. However, we detected no excess of NAM in CDA-deficient cells. Indeed, we found a small but significant decrease in NAM levels in these cells. Excess NAM that is not recycled is known to be methylated by nicotinamide N-methyltransferase to generate MNAM (methylated NAM) for excretion from the body<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. However, some of the excess NAM may be captured by PARP-1 in CDA-deficient cells, before NAM clearance, decreasing its activity.</p><p id=\"Par15\">We propose a model in which an as yet unidentified factor resulting from the decrease in NAMPT activity impairs PARP-1 activity (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>f).</p><p id=\"Par16\">Our data also reveal that cellular NAD<sup>+</sup> levels were unchanged or increased in CDA-deficient cells, and that the decrease in PARP-1 activity resulting from the inhibition of NAMPT activity was independent of cellular NAD<sup>+</sup> levels. These results indicate that NAD<sup>+</sup> levels are maintained in cells, despite NAMPT inhibition, suggesting that, in CDA-deficient cells, the partial inhibition of NAMPT is circumvented for NAD<sup>+</sup> synthesis, as proposed to explain inconclusive attempts to reduce NAD<sup>+</sup> production in patients with cancer<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. This point is of particular interest, because cancer cells require high levels of NAD<sup>+</sup>, and the mechanisms underlying the maintenance of these high levels may be involved in resistance to anticancer treatments, particularly those targeting NAMPT<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Activation of the de novo NAD<sup>+</sup> biosynthesis pathway by the upregulation of quinolinate phosphoribosyl transferase (QPRT) has been reported to confer resistance to NAMPT inhibition<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. However, PARP-1 activity is partially inhibited in CDA-deficient cells, and would therefore be expected to consume less NAD<sup>+</sup>, thereby contributing to the maintenance of cellular NAD<sup>+</sup> levels.</p><p id=\"Par17\">In conclusion, our results are consistent with a decrease in NAMPT activity in CDA-deficient cells, leading to a decrease in basal PARP-1 activity. In several preclinical models, combinations of NAMPT and PARP-1 inhibitors have been shown to induce cancer cell death and tumor regression, revealing a synthetic lethal interaction between NAMPT and PARP-1 deficiencies<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>–<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. Our study suggests that the concomitant reduction of NAMPT and PARP-1 activities in CDA-deficient tumor cells may be associated with a better prognosis.</p></sec><sec id=\"Sec7\"><title>Materials and methods</title><sec id=\"Sec8\"><title>Cell culture and drug treatments</title><p id=\"Par18\">BS GM08505B and HeLa cells were purchased from the Coriell Institute and ATCC, respectively. Cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS.</p><p id=\"Par19\">BS-Ctrl<sub>(BLM)</sub> and BS-BLM cells, and HeLa-Ctrl<sub>(CDA)</sub> and HeLa-shCDA cells, were obtained as previously reported<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup></p><p id=\"Par20\">All cell lines were routinely checked for mycoplasma infection. Authenticity was assessed by comparing the short tandem repeat profile generated with the profiles present in the Deutsche Sammlung von Mikroorganismen und Zellkulturen.</p><p id=\"Par21\">Nicotinamide mononucleotide was purchased from Sigma (NMN #N3501), and FK866 was purchased from Calbiochem (#481908). Drugs were added to the cell culture medium at the following concentrations and for the following amounts of time: FK866, 1 µM for 10 h; and NMN, 500 µM for 24 h.</p></sec><sec id=\"Sec9\"><title>Transfection with siRNA</title><p id=\"Par22\">Cells were transfected with a pool of four siRNAs specific for NAMPT (ON-TARGETplus SMART-pool, Dharmacon) or negative control siRNAs (ON-TARGETplus siCONTROL Non Targeting Pool, Dharmacon), in the presence of DharmaFECT 1 (Dharmacon). We used a standard siRNA concentration of 100 nM. The sequences of the siRNAs are provided in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>.</p></sec><sec id=\"Sec10\"><title>NAD<sup>+</sup> quantification in a luciferase assay</title><p id=\"Par23\">NAD<sup>+</sup> levels were quantified with a luciferase assay provided in the NAD<sup>+</sup>/NADH Glo Assay kit (#G9071, Promega), used according to the manufacturer’s instructions. NAD<sup>+</sup> levels were normalized against cell viability in a CellTiter-Glo Luminescent Cell Viability Assay (#G7570, Promega), according to the manufacturer’s instructions. Luminescence was read on a Tristar2 multimode microplate reader (Berthold Technologies).</p></sec><sec id=\"Sec11\"><title>Quantification of intracellular NAD<sup>+</sup>, NAM, NMN, cytidine and uridine by LC-HRMS</title><p id=\"Par24\">Intracellular NAM, NMN, NAD<sup>+</sup>, cytidine and uridine were extracted with a mixture of cold methanol/water/formic acid (70/27/3, v/v/v). We added <sup>13</sup>C<sub>6</sub>NAM, thioNAD and <sup>13</sup>C<sub>5</sub>Uridine as internal standards, and the supernatant of the extract was evaporated off to dryness. The residue was then resuspended in 200 µl of mobile phase and 10 µl was injected into the HPLC-HRMS system. Chromatographic separation was achieved on a Ultimate 3,000 HPLC system (Thermo) with an Atlantis dC 18 Waters column (150 × 2.1 mm, 3 µm) and a gradient from 0.1% formic acid in water to 0.1% formic acid in methanol. NAM, NMN, NAD<sup>+</sup>, cytidine, uridine and thioNAD were detected with a HRMS Q Exactive Plus mass spectrometer (Thermo), after positive or negative electrospray ionization. The m/z values in positive mode were 123.05528, 335.06390, 664.11639, 129.07542, 680.09355 and 244.09280 for NAM, NMN, NAD<sup>+</sup>, <sup>13</sup>C<sub>6</sub>NAM, thioNAD and cytidine, respectively. The values of m/z in negative mode were 243.06226 and 248.07903 for uridine and <sup>13</sup>C<sub>5</sub>uridine, respectively. Results are expressed as (NAM area)/(<sup>13</sup>C<sub>6</sub>NAM area), (NAD<sup>+</sup> area)/(thioNAD area), (cytidine area)/(<sup>13</sup>C<sub>5</sub>uridine area) and (uridine area)/(<sup>13</sup>C<sub>5</sub>uridine area) ratios normalized according to the number of cells.</p></sec><sec id=\"Sec12\"><title>Immunoblotting</title><p id=\"Par25\">Immunoblotting was performed as previously reported<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. The following antibodies were used for detection: rabbit anti-NAMPT (#A300-372A, Bethyl Laboratories, Inc., 1:20,000), rabbit anti-PARP-1 (#ALX-210-302-R100, Enzo Life Sciences, 1:4000), rabbit anti-CDA (#ab56053, Abcam, dilution 1:500), rabbit anti-BLM (#ab2179, Abcam, dilution 1:5000), rabbit anti-β-actin (#A2066, Sigma-Aldrich, 1:5000), rabbit anti-HSP90 (#ab2928, Abcam, 1:5000), rabbit anti-His-tag (#66,005-I, Proteintech, dilution 1:1000), mouse anti-GAPDH (#G8795 1:1000), horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#A9169, Sigma-Aldrich, 1:5000), and HRP-conjugated goat anti-mouse IgG (#A3682, Sigma-Aldrich, 1:5000). Bands were visualized by chemiluminescence (Clarity Western ECL Substrate, Bio-Rad), with a ChemiDoc XRS+ Molecular Imager and Image Lab Software (Bio-Rad).</p></sec><sec id=\"Sec13\"><title>Immunofluorescence microscopy</title><p id=\"Par26\">Immunofluorescence staining and analysis were performed as previously described<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. Primary and secondary antibodies were used at the following concentrations: rabbit anti-PICH antibody (H00054821-D01P, Abnova, dilution 1:150), and goat anti-rabbit Alexa Fluor 555 (#A21429, Life Technologies, dilution 1:500). Cell images were acquired with a 3-D deconvolution imaging system consisting of a Leica DM RXA microscope equipped with a piezoelectric translator (PIFOC; PI) placed at the base of a 63× PlanApo N.A. 1.4 objective, and a CoolSNAP HQ interline CCD camera (Photometrics). Stacks of conventional fluorescence images were collected automatically at a <italic>Z</italic>-distance of 0.2 micrometer (Metamorph software; Molecular Devices). NH<sub>4</sub>Cl incubation was not performed for His-tag staining. The primary and secondary antibodies for His-tag staining were used at the following concentrations: rabbit anti-His-tag (#66005-I, Proteintech, dilution 1:500), and goat anti-mouse Alexa Fluor 555 (#A21050, Life Technologies, dilution 1:500).</p></sec><sec id=\"Sec14\"><title>Poly(ADP)-ribose immunofluorescence</title><p id=\"Par27\">Poly(ADP)-ribose immunofluorescence was analyzed as previously described<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>.</p></sec><sec id=\"Sec15\"><title>Colorimetric PARP assay kit candidate inhibitor screening</title><p id=\"Par28\">PARP-1 inhibition by FK866 was assessed with the HT universal colorimetric PARP assay kit, with histone-coated strip wells (4677-096-K from Trevigen), according to the manufacturer’s instructions.</p></sec><sec id=\"Sec16\"><title>Cloning, site-directed mutagenesis and vector transfection</title><p id=\"Par29\">Site-directed mutagenesis was performed with the pPM-C-His vector carrying the full-length cDNA for human NAMPT (#314140210600, Applied Biological Materials, abmgood) (GenBank accession no. BC072439). The His codon in position 247 of NAMPT was mutated into an Ala (H27A) codon with the QuikChange XL site-directed mutagenesis kit (#200251 Agilent), according to the manufacturer’s instructions. The primer sequences are provided in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>. Cells were transfected by incubation with NAMPT WT, NAMPT H247A or empty vector in the presence of JetPrime (Polyplus Transfection) for 48 h.</p></sec><sec id=\"Sec17\"><title>Protein production and purification</title><p id=\"Par30\">A pPM-C-His plasmid carrying the full-length cDNA for human NAMPT was purchased from Applied Biological Materials (abmgood). The NAMPT cDNA was amplified by PCR with primers containing <italic>Nhe</italic>I and <italic>Xho</italic>I sites. The amplified DNA fragment was inserted between the <italic>Nhe</italic>I and <italic>Xho</italic>I sites of pET24a(+)(#69749–3), Novagen), resulting in the attachment of a his<sub>6</sub> tag at the C-terminus of the protein. The H247A mutation was generated with the QuikChange XL site-directed mutagenesis kit (#200251, Agilent) according to the manufacturer’s instructions. The primer sequences are provided in Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>. Both constructs were overexpressed in BL21 Dsbc plSO in 1 L of Terrific Broth. Expression was induced by incubation with 0.5% arabinose and 1 mM IPTG at 20 °C overnight, with shaking at 160 rpm. The cells were harvested by centrifugation at 4000×<italic>g</italic> for 15 min at 4 °C.</p><p id=\"Par31\">The cell pellets were suspended in lysis buffer (20 mM Tris–HCl pH 8, 500 mM NaCl, 10% glycerol, 1 mM TCEP and 1× Complete EDTA-free protease inhibitor cocktail (Roche)). The cell suspension was disrupted by passage through a T75 cell disruptor (Constant Systems). The resulting cell lysate was centrifuged at 43,000×<italic>g</italic> for 1 h at 4 °C. The supernatant was applied to a HisTrap HP column (GE Healthcare), washed thoroughly and the proteins were eluted in elution buffer (20 mM Tris–HCl pH 8, 500 mM NaCl, 10% glycerol, 0.5 mM TCEP, 400 mM imidazole). After overnight dialysis against 20 mM Tris–HCl pH 8, 100 mM NaCl, 10% glycerol, 0.5 mM TCEP, the eluate was loaded onto a Capto Q ImpRes ion exchange column (GE Healthcare) for elution with a continuous gradient NaCl (0.1–1 M) in the same buffer. Fractions containing NAMPT were dialyzed against 20 mM Tris–HCl pH 8, 500 mM NaCl, 10% glycerol, 0.5 mM TCEP, and loaded on a HisTrap HP column. NAMPT was eluted with an imidazole gradient. NAMPT-containing fractions were pooled and dialyzed against 20 mM Tris–HCl pH 8, 100 mM NaCl, 10% glycerol, 0.5 mM TCEP. The protein was visualized by SDS-PAGE in a 4–20% acrylamide gel and protein concentration was determined by measuring absorbance at 280 nm. The mutant protein was purified with the same protocol as the wild-type protein.</p></sec><sec id=\"Sec18\"><title>NAMPT enzyme assay measuring the conversion of <sup>14</sup>C<sup>-</sup>NAM to <sup>14</sup>C-NMN</title><p id=\"Par32\">NAMPT enzymatic activity was measured as previously described<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. We diluted 1 µg of wild-type or mutated recombinant NAMPTs in assay buffer without substrate (10 µl in total), which was then mixed with 50 µl reaction mixture (20 mmol/l Tris–HCl pH7.4; 2.5 mmol/l ATP; 50 mmol/l NaCl; 12,5 mmol/l MgCl<sub>2</sub>; 2 mmol/l DTT; 0.5 mmol/l PRPP; 13 µmol/l <sup>14</sup>C-nicotinamide) and incubated at 37 °C for 1 h. Each point was tested in triplicate. The reaction was stopped by adding 1.8 ml acetone to each tube. The mixture was then pipetted onto acetone-presoaked glass microfiber filters (GF/A diameter 24 mm). The filters were rinsed twice, with 2 ml acetone each, air-dried and transferred to vials with 2 ml scintillation cocktail. We quantified <sup>14</sup>C-NMN radioactivity in a liquid scintillation counter, as the number of disintegrations per minute (dpm), with a Tri-Carb 2910 TR machine (PerkinElmer). We subtracted the values for the buffer as background, and the mean number of dpm obtained per 1 µg of enzyme was compared. The histogram shows the normalized results for wt and mutated NAMPT activities. Each experiment was performed three times.</p></sec><sec id=\"Sec19\"><title>NAMPT enzyme assay by HPLC-HRMS</title><p id=\"Par33\">NAMPT activity was studied by monitoring the decrease in NAM levels following incubation in a mixture with a final volume of 60 µL. The incubation mixture contained Tris–HCl (pH7.4, 17 mM), ATP (2.1 mM), NaCl (83 mM), MgCl<sub>2</sub> (10.5 mM), DTT (2 mM) and PRPP (0.2 mM). Preincubation with NAMPT (0.5 µg) was performed for 10 min at 37 °C. For some samples, dCTP (10 µM) was added at the same time as NAMPT. We then added NAM (5 µM) and incubated for 45 min, after which, 25 µl of the mixture was transferred to 300 µl of a cold methanol/water mixture (70/30, v/v) to stop the reaction. <sup>13</sup>C<sub>6</sub>NAM was added as an internal standard, and the supernatant of the extract was evaporated to dryness. The residue was resuspended in 200 µl of mobile phase and 10 µl was injected into the HPLC-HRMS system. Chromatographic separation and mass spectrometry detection were performed as described above for the determination of intracellular NAM, NMN and NAD<sup>+</sup>.</p></sec><sec id=\"Sec20\"><title>Statistical analysis</title><p id=\"Par34\">At least three independent experiments were carried out to generate each data set. The statistical significance of differences was calculated with two-tailed unpaired Student’s <italic>t</italic>-tests.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec21\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70874_MOESM1_ESM.pdf\"><caption><p>Supplementary file1.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Sandra Cunha Silveira, Géraldine Buhagiar-Labarchède and Rosine Onclercq-Delic.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70874-6.</p></sec><ack><title>Acknowledgements</title><p>This work was supported by Institut Curie (<ext-link ext-link-type=\"uri\" xlink:href=\"https://curie.fr/\">https://curie.fr/</ext-link>), Centre National de la Recherche Scientifique (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.cnrs.fr/\">https://www.cnrs.fr/</ext-link>), Ligue contre le Cancer, Comité de l’Essonne (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ligue-cancer.net/\">https://www.ligue-cancer.net/</ext-link>) and PSL Research University (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.psl.eu\">https://www.psl.eu</ext-link>). We thank S. Lambert, M. Gratia, V. Pennaneach and L. Mouawad for stimulating discussions. We thank V. Schreiber and JC Amé for providing us with anti-PAR antibodies and for advice about PAR detection. We thank A. El-Marjou from the Recombinant Protein Platform (Institut Curie, Paris) for helping to construct the NAMPT expression vector for the production of recombinant protein. We acknowledge the assistance of the PICT-IBISA, Institut Curie, Orsay.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>S.C.S., G.B.-L., R.O.-D. performed most of the experiments, participated in the design of the experiments and data analyses and generated the figures. S.G. performed some experiments, participated in data analyses and generated figures. E.B.S. and H.M. contributed to data analyses. P.D. designed and performed some experiments. C.M. and J.G. designed and performed some experiments, contributed to data analyses and generated some figures. M.A.-G. supervised the study, analyzed the data and wrote the manuscript.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par35\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Veith</surname><given-names>S</given-names></name><name><surname>Mangerich</surname><given-names>A</given-names></name></person-group><article-title>RecQ helicases and PARP1 team up in maintaining genome integrity</article-title><source>Ageing Res. Rev.</source><year>2015</year><volume>23</volume><fpage>12</fpage><lpage>28</lpage><pub-id pub-id-type=\"doi\">10.1016/j.arr.2014.12.006</pub-id><pub-id pub-id-type=\"pmid\">25555679</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Nygaard</surname><given-names>P</given-names></name></person-group><person-group person-group-type=\"editor\"><name><surname>Nyhan</surname><given-names>WL</given-names></name><name><surname>Thompson</surname><given-names>LF</given-names></name><name><surname>Watts</surname><given-names>RWE</given-names></name></person-group><article-title>On the role of cytidine deaminase in cellular metabolism</article-title><source>Purine and Pyrimidine Metabolism in Man V: Part B: Basic Science Aspects</source><year>1986</year><publisher-loc>Boston</publisher-loc><publisher-name>Springer</publisher-name><fpage>415</fpage><lpage>420</lpage></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mameri</surname><given-names>H</given-names></name><etal/></person-group><article-title>Cytidine deaminase deficiency reveals new therapeutic opportunities against cancer</article-title><source>Clin. Cancer Res.</source><year>2017</year><volume>23</volume><fpage>2116</fpage><lpage>2126</lpage><pub-id pub-id-type=\"doi\">10.1158/1078-0432.CCR-16-0626</pub-id><pub-id pub-id-type=\"pmid\">27601591</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ye</surname><given-names>F-G</given-names></name><etal/></person-group><article-title>Cytidine deaminase axis modulated by miR-484 differentially regulates cell proliferation and chemoresistance in breast cancer</article-title><source>Cancer Res.</source><year>2015</year><volume>75</volume><fpage>1504</fpage><lpage>1515</lpage><pub-id pub-id-type=\"doi\">10.1158/0008-5472.CAN-14-2341</pub-id><pub-id pub-id-type=\"pmid\">25643696</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zauri</surname><given-names>M</given-names></name><etal/></person-group><article-title>CDA directs metabolism of epigenetic nucleosides revealing a therapeutic window in cancer</article-title><source>Nature</source><year>2015</year><volume>524</volume><fpage>114</fpage><pub-id pub-id-type=\"doi\">10.1038/nature14948</pub-id><pub-id pub-id-type=\"pmid\">26200337</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bou Samra</surname><given-names>E</given-names></name><etal/></person-group><article-title>A role for Tau protein in maintaining ribosomal DNA stability and cytidine deaminase-deficient cell survival</article-title><source>Nat. Commun.</source><year>2017</year><volume>8</volume><fpage>693</fpage><pub-id pub-id-type=\"doi\">10.1038/s41467-017-00633-1</pub-id><pub-id pub-id-type=\"pmid\">28947735</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chabosseau</surname><given-names>P</given-names></name><etal/></person-group><article-title>Pyrimidine pool imbalance induced by BLM helicase deficiency contributes to genetic instability in Bloom syndrome</article-title><source>Nat. Commun.</source><year>2011</year><volume>2</volume><fpage>368</fpage><pub-id pub-id-type=\"doi\">10.1038/ncomms1363</pub-id><pub-id pub-id-type=\"pmid\">21712816</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gemble</surname><given-names>S</given-names></name><etal/></person-group><article-title>A balanced pyrimidine pool is required for optimal Chk1 activation to prevent ultrafine anaphase bridge formation</article-title><source>J. Cell Sci.</source><year>2016</year><volume>129</volume><fpage>3167</fpage><lpage>3177</lpage><pub-id pub-id-type=\"doi\">10.1242/jcs.187781</pub-id><pub-id pub-id-type=\"pmid\">27383768</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gemble</surname><given-names>S</given-names></name><name><surname>Buhagiar-Labarchède</surname><given-names>G</given-names></name><name><surname>Onclercq-Delic</surname><given-names>R</given-names></name><name><surname>Jaulin</surname><given-names>C</given-names></name><name><surname>Amor-Guéret</surname><given-names>M</given-names></name></person-group><article-title>Cytidine deaminase deficiency impairs sister chromatid disjunction by decreasing PARP-1 activity</article-title><source>Cell Cycle</source><year>2017</year><volume>16</volume><fpage>1128</fpage><lpage>1135</lpage><pub-id pub-id-type=\"doi\">10.1080/15384101.2017.1317413</pub-id><pub-id pub-id-type=\"pmid\">28463527</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gemble</surname><given-names>S</given-names></name><etal/></person-group><article-title>Pyrimidine pool disequilibrium induced by a cytidine deaminase deficiency inhibits PARP-1 activity, leading to the under replication of DNA</article-title><source>PLoS Genet.</source><year>2015</year><volume>11</volume><fpage>e1005384</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pgen.1005384</pub-id><pub-id pub-id-type=\"pmid\">26181065</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ray Chaudhuri</surname><given-names>A</given-names></name><name><surname>Nussenzweig</surname><given-names>A</given-names></name></person-group><article-title>The multifaceted roles of PARP1 in DNA repair and chromatin remodelling</article-title><source>Nat. Rev. Mol. Cell Biol.</source><year>2017</year><volume>18</volume><fpage>610</fpage><pub-id pub-id-type=\"doi\">10.1038/nrm.2017.53</pub-id><pub-id pub-id-type=\"pmid\">28676700</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Revollo</surname><given-names>JR</given-names></name><name><surname>Grimm</surname><given-names>AA</given-names></name><name><surname>Imai</surname><given-names>S-I</given-names></name></person-group><article-title>The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells</article-title><source>J. Biol. Chem.</source><year>2004</year><volume>279</volume><fpage>50754</fpage><lpage>50763</lpage><pub-id pub-id-type=\"doi\">10.1074/jbc.M408388200</pub-id><pub-id pub-id-type=\"pmid\">15381699</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yaku</surname><given-names>K</given-names></name><etal/></person-group><article-title>Metabolism and biochemical properties of nicotinamide adenine dinucleotide (NAD) analogs, nicotinamide guanine dinucleotide (NGD) and nicotinamide hypoxanthine dinucleotide (NHD)</article-title><source>Sci. Rep.</source><year>2019</year><volume>9</volume><fpage>13102</fpage><pub-id pub-id-type=\"doi\">10.1038/s41598-019-49547-6</pub-id><pub-id pub-id-type=\"pmid\">31511627</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Garten</surname><given-names>A</given-names></name><name><surname>Petzold</surname><given-names>S</given-names></name><name><surname>Körner</surname><given-names>A</given-names></name><name><surname>Imai</surname><given-names>S-I</given-names></name><name><surname>Kiess</surname><given-names>W</given-names></name></person-group><article-title>Nampt: linking NAD biology, metabolism and cancer</article-title><source>Trends Endocrinol. Metab.</source><year>2009</year><volume>20</volume><fpage>130</fpage><lpage>138</lpage><pub-id pub-id-type=\"doi\">10.1016/j.tem.2008.10.004</pub-id><pub-id pub-id-type=\"pmid\">19109034</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sommer</surname><given-names>G</given-names></name><etal/></person-group><article-title>Visfatin/PBEF/Nampt: structure, regulation and potential function of a novel adipokine</article-title><source>Clin. Sci.</source><year>2008</year><volume>115</volume><fpage>13</fpage><lpage>23</lpage><pub-id pub-id-type=\"doi\">10.1042/CS20070226</pub-id><pub-id pub-id-type=\"pmid\">19016657</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Garten</surname><given-names>A</given-names></name><etal/></person-group><article-title>Physiological and pathophysiological roles of NAMPT and NAD metabolism</article-title><source>Nat. Rev. Endocrinol.</source><year>2015</year><volume>11</volume><fpage>535</fpage><pub-id pub-id-type=\"doi\">10.1038/nrendo.2015.117</pub-id><pub-id pub-id-type=\"pmid\">26215259</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Baumann</surname><given-names>C</given-names></name><name><surname>Körner</surname><given-names>R</given-names></name><name><surname>Hofmann</surname><given-names>K</given-names></name><name><surname>Nigg</surname><given-names>EA</given-names></name></person-group><article-title>PICH, a Centromere-associated SNF2 family ATPase, is regulated by Plk1 and required for the spindle checkpoint</article-title><source>Cell</source><year>2007</year><volume>128</volume><fpage>101</fpage><lpage>114</lpage><pub-id pub-id-type=\"doi\">10.1016/j.cell.2006.11.041</pub-id><pub-id pub-id-type=\"pmid\">17218258</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Buonvicino</surname><given-names>D</given-names></name><etal/></person-group><article-title>Identification of the nicotinamide salvage pathway as a new toxification route for antimetabolites</article-title><source>Cell Chem. Biol.</source><year>2018</year><volume>25</volume><fpage>471</fpage><lpage>482.e477</lpage><pub-id pub-id-type=\"doi\">10.1016/j.chembiol.2018.01.012</pub-id><pub-id pub-id-type=\"pmid\">29478906</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Piacente</surname><given-names>F</given-names></name><etal/></person-group><article-title>Nicotinic acid phosphoribosyltransferase regulates cancer cell metabolism, susceptibility to NAMPT inhibitors, and DNA repair</article-title><source>Cancer Res.</source><year>2017</year><volume>77</volume><fpage>3857</fpage><lpage>3869</lpage><pub-id pub-id-type=\"doi\">10.1158/0008-5472.CAN-16-3079</pub-id><pub-id pub-id-type=\"pmid\">28507103</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>T</given-names></name><etal/></person-group><article-title>Structure of Nampt/PBEF/visfatin, a mammalian NAD+ biosynthetic enzyme</article-title><source>Nat. Struct. Mol. Biol.</source><year>2006</year><volume>13</volume><fpage>661</fpage><pub-id pub-id-type=\"doi\">10.1038/nsmb1114</pub-id><pub-id pub-id-type=\"pmid\">16783373</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bockwoldt</surname><given-names>M</given-names></name><etal/></person-group><article-title>Identification of evolutionary and kinetic drivers of NAD-dependent signaling</article-title><source>Proc. Natl. Acad. Sci.</source><year>2019</year><volume>116</volume><fpage>15957</fpage><lpage>15966</lpage><pub-id pub-id-type=\"doi\">10.1073/pnas.1902346116</pub-id><pub-id pub-id-type=\"pmid\">31341085</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pissios</surname><given-names>P</given-names></name></person-group><article-title>Nicotinamide N-methyltransferase: more than a vitamin B3 clearance enzyme</article-title><source>Trends Endocrinol. Metab.</source><year>2017</year><volume>28</volume><fpage>340</fpage><lpage>353</lpage><pub-id pub-id-type=\"doi\">10.1016/j.tem.2017.02.004</pub-id><pub-id pub-id-type=\"pmid\">28291578</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Guo</surname><given-names>J</given-names></name><etal/></person-group><article-title>Identification of novel resistance mechanisms to NAMPT inhibition via the de novo NAD+ biosynthesis pathway and NAMPT mutation</article-title><source>Biochem. Biophys. Res. Commun.</source><year>2017</year><volume>491</volume><fpage>681</fpage><lpage>686</lpage><pub-id pub-id-type=\"doi\">10.1016/j.bbrc.2017.07.143</pub-id><pub-id pub-id-type=\"pmid\">28756225</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bajrami</surname><given-names>I</given-names></name><etal/></person-group><article-title>Synthetic lethality of PARP and NAMPT inhibition in triple-negative breast cancer cells</article-title><source>EMBO Mol. Med.</source><year>2012</year><volume>4</volume><fpage>1087</fpage><lpage>1096</lpage><pub-id pub-id-type=\"doi\">10.1002/emmm.201201250</pub-id><pub-id pub-id-type=\"pmid\">22933245</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Heske</surname><given-names>CM</given-names></name><etal/></person-group><article-title>Matrix screen identifies synergistic combination of PARP inhibitors and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors in Ewing Sarcoma</article-title><source>Clin. Cancer Res.</source><year>2017</year><volume>23</volume><fpage>7301</fpage><lpage>7311</lpage><pub-id pub-id-type=\"doi\">10.1158/1078-0432.CCR-17-1121</pub-id><pub-id pub-id-type=\"pmid\">28899971</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Heske</surname><given-names>CM</given-names></name></person-group><article-title>Beyond Energy Metabolism: Exploiting the Additional Roles of NAMPT for Cancer Therapy</article-title><source>Front. Oncol.</source><year>2020</year><volume>9</volume><fpage>1514</fpage><pub-id pub-id-type=\"doi\">10.3389/fonc.2019.01514</pub-id><pub-id pub-id-type=\"pmid\">32010616</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rouzeau</surname><given-names>S</given-names></name><etal/></person-group><article-title>Bloom's syndrome and PICH helicases cooperate with topoisomerase IIalpha in centromere disjunction before anaphase</article-title><source>PLoS ONE</source><year>2012</year><volume>7</volume><fpage>e33905</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0033905</pub-id><pub-id pub-id-type=\"pmid\">22563370</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Elliott</surname><given-names>GC</given-names></name><name><surname>Ajioka</surname><given-names>J</given-names></name><name><surname>Okada</surname><given-names>CY</given-names></name></person-group><article-title>A rapid procedure for assaying nicotinamide phosphoribosyltransferase</article-title><source>Anal. Biochem.</source><year>1980</year><volume>107</volume><fpage>199</fpage><lpage>205</lpage><pub-id pub-id-type=\"doi\">10.1016/0003-2697(80)90512-6</pub-id><pub-id pub-id-type=\"pmid\">6449162</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807833</article-id><article-id pub-id-type=\"pmc\">PMC7431584</article-id><article-id pub-id-type=\"publisher-id\">70296</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70296-4</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>A comparison of the metabolic effects of sustained strenuous activity in polar environments on men and women</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\" equal-contrib=\"yes\"><name><surname>Hattersley</surname><given-names>John</given-names></name><address><email>john.hattersley@uhcw.nhs.uk</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Wilson</surname><given-names>Adrian J.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-6248-6400</contrib-id><name><surname>Gifford</surname><given-names>Rob</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><name><surname>Facer-Childs</surname><given-names>Jamie</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-7933-9805</contrib-id><name><surname>Stoten</surname><given-names>Oliver</given-names></name><xref ref-type=\"aff\" rid=\"Aff8\">8</xref></contrib><contrib contrib-type=\"author\"><name><surname>Cobb</surname><given-names>Rinn</given-names></name><xref ref-type=\"aff\" rid=\"Aff9\">9</xref></contrib><contrib contrib-type=\"author\"><name><surname>Thake</surname><given-names>C. Doug</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Reynolds</surname><given-names>Rebecca M.</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>Woods</surname><given-names>David</given-names></name><xref ref-type=\"aff\" rid=\"Aff6\">6</xref><xref ref-type=\"aff\" rid=\"Aff10\">10</xref><xref ref-type=\"aff\" rid=\"Aff11\">11</xref></contrib><contrib contrib-type=\"author\"><name><surname>Imray</surname><given-names>Chris</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref><xref ref-type=\"aff\" rid=\"Aff12\">12</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.15628.38</institution-id><institution>Coventry NIHR CRF Human Metabolic Research Unit, </institution><institution>University Hospitals Coventry and Warwickshire NHS Trust, </institution></institution-wrap>Coventry, CV2 2DX UK </aff><aff id=\"Aff2\"><label>2</label>School of Engineering, University of Warwick, Coventry, CV4 7AL USA </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.8096.7</institution-id><institution-id institution-id-type=\"ISNI\">0000000106754565</institution-id><institution>Faculty of Health and Life Sciences, </institution><institution>Coventry University, </institution></institution-wrap>Coventry, UK </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.7372.1</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 8809 1613</institution-id><institution>Department of Physics, </institution><institution>University of Warwick, </institution></institution-wrap>Coventry, CV4 7AL UK </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.4305.2</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 7988</institution-id><institution>University/British Heart Foundation Centre for Cardiovascular Science, Queen’s Medical Research Institute, </institution><institution>University of Edinburgh, </institution></institution-wrap>Edinburgh, UK </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.415490.d</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2177 007X</institution-id><institution>Research and Clinical Innovation, </institution><institution>Royal Centre for Defence Medicine, </institution></institution-wrap>Birmingham, UK </aff><aff id=\"Aff7\"><label>7</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.83440.3b</institution-id><institution-id institution-id-type=\"ISNI\">0000000121901201</institution-id><institution>Institute of Child Health, </institution><institution>University College London, </institution></institution-wrap>London, WC1N 1EH UK </aff><aff id=\"Aff8\"><label>8</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.416098.2</institution-id><institution-id institution-id-type=\"ISNI\">0000 0000 9910 8169</institution-id><institution>Emergency Department, </institution><institution>Royal Bournemouth Hospital, </institution></institution-wrap>Bournemouth, BH7 7DW UK </aff><aff id=\"Aff9\"><label>9</label>Performance, Nutrition and Dietetic Consulting, pnd consulting.co.uk, Middlesbrough, UK </aff><aff id=\"Aff10\"><label>10</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.10346.30</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0745 8880</institution-id><institution>Research Institute for Sport, Physical Activity and Leisure, </institution><institution>Leeds Beckett University, </institution></institution-wrap>Leeds, UK </aff><aff id=\"Aff11\"><label>11</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.419334.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0641 3236</institution-id><institution>Northumbria and Newcastle NHS Trusts, </institution><institution>Wansbeck General and Royal Victoria Infirmary, </institution></institution-wrap>Newcastle, UK </aff><aff id=\"Aff12\"><label>12</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.15628.38</institution-id><institution>Department of Vascular and Renal Transplant Surgery, </institution><institution>University Hospitals Coventry and Warwickshire NHS Trust, </institution></institution-wrap>Coventry, CV2 2DX UK </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13912</elocation-id><history><date date-type=\"received\"><day>9</day><month>1</month><year>2020</year></date><date date-type=\"accepted\"><day>24</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">This study investigates differences in pre- to post-expedition energy expenditure, substrate utilisation and body composition, between the all-male Spear17 (SP-17) and all-female Ice Maiden (IM) transantarctic expeditions (IM: N = 6, 61 days, 1700 km; SP-17: N = 5, 67 days, 1750 km). Energy expenditure and substrate utilisation were measured by a standardised 36 h calorimetry protocol; body composition was determined using air displacement plethysmography. Energy balance calculation were used to assess the physical challenge. There was difference in the daily energy expenditure (IM: 4,939 kcal day<sup>−1</sup>; SP-17: 6,461 kcal day<sup>−1</sup>, <italic>p</italic> = 0.004); differences related to physical activity were small, but statistically significant (IM = 2,282 kcal day<sup>−1</sup>; SP-17 = 3,174 kcal day<sup>−1</sup>; <italic>p</italic> = 0.004). Bodyweight loss was modest (IM = 7.8%, SP-17 = 6.5%; <italic>p</italic> > 0.05) as was fat loss (IM = 30.4%, SP-17 = 40.4%; <italic>p</italic> > 0.05). Lean tissue weight change was statistically significant (IM = − 2.5%, SP-17 = + 1.0%; <italic>p</italic> = 0.05). No difference was found in resting or sleeping energy expenditure, normalised to lean tissue weight (<italic>p</italic> > 0.05); nor in energy expenditure when exercising at 80, 100 and 120 steps min<sup>−1</sup>, normalised to body weight (<italic>p</italic> > 0.05). Similarly, no difference was found in the change in normalised substrate utilisation for any of the activities (<italic>p</italic> > 0.05). Analysis suggested that higher daily energy expenditures for the men in Spear-17 was the result of higher physical demands resulting in a reduced demand for energy to thermoregulate compared to the women in Ice Maiden. The lack of differences between men and women in the change in energy expenditure and substrate utilisation, suggests no sex difference in response to exposure to extreme environments.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Endocrinology</kwd><kwd>Medical research</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">There is an increasing involvement of women in extreme activities often in adverse environmental conditions that are characterised by a deficit in Energy Availability (EA), the difference between the calorific intake and the energy expended. These activities, which include extreme sports<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>, expeditionary travel and military combat training<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup>, have traditionally been undertaken by men with the result that the majority of the research looking at physiological adaptation and responses, particularly during expeditionary travel in polar regions and to altitude, has been on men. Therefore, there is a paucity of knowledge regarding the similarity and differences in the adaption of women to high levels of physical activity in extreme environments.</p><p id=\"Par3\">Antarctica is the coldest and highest continent on earth with summer temperatures down to − 70 °C and an average elevation above the sea of 2,400 m (maximum elevation is Mount Vinson at 4,892 m) and therefore presents multiple challenges to those exploring it. Whilst the elevations in Antarctica are modest when considered against the Himalayan or Karakoram peaks, some studies<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup> but not all<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> have shown a prioritised utilisation of glucose over fatty acids in male subjects exposed to the altitudes encountered in Antarctica that were not found in female subjects exposed to similar altitudes<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. The primary impact of the low temperatures in Antarctica is to increase the basal metabolic rate (BMR) to maintain core temperature. A non-expedition study containing both male and female participants which modelled the components of the metabolic rate showed that the increase in BMR required for thermoregulation in high ambient temperatures was less than that required in low temperatures<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. Unfortunately, these data were not partitioned on sex and therefore it is not possible to determine whether the changes were the same for male and female participants. Isotope techniques can be used to measure time-averaged non-protein metabolism<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup> and time-averaged protein metabolism<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup> during expeditions<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>–<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. From earlier expeditionary studies the measured energy expenditure was found to be much higher than that predicted from activity scores<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup> and a 60% increase was found in the BMR<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup> which was attributed to increased thyroid activity. This increase in BMR is consistent with small levels of weight loss seen in men undertaking low levels of physical work whilst over-wintering in Antarctica<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. In a recent analysis of energy expenditure and substrate utilisation on an all-male transantarctic expedition (Spear-17), we found that this 60% increase in BMR was consistent with an increase in the energy required for thermoregulation<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. The same analysis on data from participants in an all-female transantarctic expedition of similar length and duration (Ice Maiden) did not give a similar difference in BMR<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>.</p><p id=\"Par4\">Participants in supported polar expeditions have traditionally experienced modest levels of negative energy availability<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>–<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup> whilst those undertaking unsupported polar expeditions have experienced high levels of negative energy availability as evidenced by up to 25% loss of body weight<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Our previous work has suggested only modest levels of negative energy availability in participants in both the Spear-17<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup> and Ice Maiden<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup> expeditions. Negative energy availability has been postulated as a possible cause of the hypothalamic pituitary gonad (HPG) axis suppression (termed the female triad) in female participants in extreme physical activities, including military combat training<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Whilst women appear to have a higher susceptibility to HPG suppression during extreme physical activity<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>, HPG suppression is also seen in men<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. However, the impact of HPG axis suppression on energy expenditure is unknown.</p><p id=\"Par5\">It has previously been suggested<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> that there are potential differences in the way male and female participants respond to extreme physical and physiological demands. We have recently had the opportunity to study energy expenditure and substrate utilisation pre- and post-expedition on participant from two independent polar expeditions (one all male—Spear-17; one all-female—Ice Maiden):<list list-type=\"bullet\"><list-item><p id=\"Par6\"><italic>Spear-17</italic>—during the Antarctic summer of 2016/17 a team of six male British Army Reservists, including an experienced polar traveller who was their leader, undertook an unassisted crossing of Antarctica on ski (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.forces.net/news/feature/spear-17-team-complete-mammoth-antarctic-expedition\">https://www.forces.net/news/feature/spear-17-team-complete-mammoth-antarctic-expedition</ext-link>). The 67 day, 1,750 km journey took them from the Hercules Inlet (altitude 244 m) to the South Pole (altitude 2840 m), where the single resupply took place, then down the Shackleton Glacier, finally finishing on the Ross Sea Ice (altitude < 50 m). During the journey, they experienced temperatures as low as – 57 C whilst pulling sledges weighing up to 120 kg to an altitude of 3350 m. Expedition participants were provided with individually pre-packaged daily food packs which had a mean nutritional value of 27.2 MJ day<sup>−1</sup> (6,500 kcal day<sup>−1</sup>) with a macronutrient breakdown of 35% carbohydrate, 55% fat and 8% protein<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. The estimated average consumption was 92%<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. Neither the calorific value of the diet, nor its composition was not part of the study but were determined by the expeditionary team.</p></list-item><list-item><p id=\"Par7\"><italic>Ice Maiden</italic>—during the Antarctic summer of 2017/2018, six women participated in a 1700 km unassisted ski traverse of the Antarctic continent from Leverett Glacier (altitude 244 m) along the McMurdo Route to the South Pole (altitude 2840 m), where the first of two resupplies took place, to Hercules Inlet (altitude 767 m) via the Thiel Mountains, where the second resupply occurred. The team was hauling sledges weighing up to 80 kg. The expedition lasted 61 days with two resupplies. This was the first all-female team to use muscle power alone to ski coast-to-coast across (<ext-link ext-link-type=\"uri\" xlink:href=\"https://exicemaiden.com\">https://exicemaiden.com</ext-link>). The six participants in this British Army expedition were selected from 250 volunteers, all serving members of the British armed forces, following 2 years of selection and training activities<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. During the expedition, participants were provided with individually pre-packaged daily food packs which had a mean nutritional value of 20.9 MJ day<sup>−1</sup> (5,000 kcal day<sup>−1</sup>) with a macronutrient breakdown of 45% carbohydrate, 45% fat and 10% protein with an estimated average consumption of 85%. Neither the caloric value nor the composition of the diet formed part of the study on energy expenditure and substrate utilisation, but were determined by one of the authors (RC) in collaboration with the expeditionary team.</p></list-item></list></p><p id=\"Par8\">We have previously reported on the energy expenditure and substrate utilisation on participants from each expedition<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. These studies were carried out in a whole-body calorimeter to a standard protocol and present a rare opportunity to gain an insight into differences in the physiological response of men and women undertaking expeditionary polar travel. To allow for changes in body weight and body composition between men and women, in this paper we compare: the fractional change in body composition; the normalised change in energy expenditure and normalised substrate utilisation measured pre- and post-expedition between participants in the two expeditions.</p></sec><sec id=\"Sec2\"><title>Materials and methods</title><sec id=\"Sec3\"><title>Study participants</title><p id=\"Par9\">There were six male participants in the Spear-17 expedition (age 26–40 years) and six female participants (age 28–36 years) in the Ice Maiden expedition. Participants were invited to participate in a scientific study of the impact of the expedition on their metabolism. For both expeditions, participation in the scientific study was voluntary and independent of their participation in the expedition itself. All six male participants in the Spear-17 expedition and all six female participants in the Ice Maiden expedition volunteered to be participants in the scientific studies and gave written consent prior to any data collection. Only five members of Spear-17 completed the expedition, therefore data from the sixth participant was excluded from the analysis presented in this paper. Ethics approval for the Spear-17 study was obtained from the National Health Authority Research Ethics committee, West Midlands—Solihull (ID: 13/WM/0327), Ministry of Defence—Research Ethics Committee and University Hospitals Coventry and Warwickshire Research and Development Governance Committee, under the GAFREC framework (REF: GF0121). Ethics approval for the Ice Maiden study was obtained from the Ministry of Defence Research Ethics Committee (827MoDREC/17). Both studies were carried out in compliance with the Ethical Principles for Medical Research on Human Subjects set down in the Declaration of Helsinki by the World Medical Association. All study protocols and objectives were fully explained to all participants before securing informed written consent.</p></sec><sec id=\"Sec4\"><title>Measurements</title><p id=\"Par10\">A common protocol was used to study the energy expenditure of participants from each of the two expeditions on two separate occasions: the first in the two weeks before departure from the UK (2 weeks prior to the start of the expedition) and the second within two weeks following their return to the UK (2 weeks after the expedition successfully concluded).</p><p id=\"Par11\">Full details of the protocol have been published previously<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup> but will be summarised here for completeness. Each participant spent 36 h in a dual whole-body calorimeters (Maastricht Instruments, Netherlands<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>). The measurement period incorporated an hour supine rest to measure the resting metabolic rate (RMR), four meals, three exercise sessions and two sleeping periods. Calorimeters allow accurate measurements of oxygen and carbon dioxide, and with the collection of three consecutive 12-h urine samples for urinary protein estimates<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. The energy expenditure and substrate utilisation were calculated on a minute resolution using standard formulae<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>–<xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. During the period in the calorimeters the food intake of the participants was isocaloric based on lean tissue mass with a composition typical of a western diet (50% carbohydrate, 35% fat and 15% protein).</p><p id=\"Par12\">To maintain thermal neutrality, the environment within the chamber was controlled at a relative humidity of 57 ± 5% at a temperature of 24 ± 0.5 °C during the day and 22 ± 0.5 °C during the night.</p><p id=\"Par13\">Before arriving for the start of the measurement session at around 6 pm, participants were asked to refrain from eating and drinking tea or coffee from lunchtime onwards. On arrival, the participants had their height and weight measured (Seca 799, Seca, UK) and then their body composition determined by Air Displacement Plethysmography (ADP) using a BodPod 2000A (Cosmed Inc., USA).</p></sec><sec id=\"Sec5\"><title>Data analysis</title><p id=\"Par14\">The metabolic rate was determined on a minute-by-minute basis from the continuous measurements of the difference in concentration of O<sub>2</sub> and CO<sub>2</sub> in the gases entering and leaving the room. This, together with the data from the movement sensors, gave a profile of the energy expenditure and activity for the participant over the study period as shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>The raw data obtained from the whole body calorimeter studies annotated with the events analysed in this paper and the times when the participants were fed. The (non-protein) energy expenditure calculated from the difference in the concentrations of O<sub>2</sub> and CO<sub>2</sub> entering and leaving the room is shown in the lower pane, the protein energy expenditure from the 12-h urine sample analysis in the middle pane and the subject activity from the ultrasound movement sensors, scaled 0–1 is shown in the upper pane.</p></caption><graphic xlink:href=\"41598_2020_70296_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par15\">Figure <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> also shows the energy due to protein metabolism where expenditure was estimated from the analysed creatinine and urea in the 12-h urine samples<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. The total energy expenditure was the sum of the energy expenditure from the O<sub>2</sub>/CO<sub>2</sub> data and the protein energy expenditure from the urine nitrogen analysis.</p><p id=\"Par16\">In this study we aim to determine differences in the pre- to post-expedition measurements of body composition, energy expenditure and substrate utilisation to determine whether there are any differences between the participants in the all-male (Spear-17) and all-female (Ice Maiden) expeditions. The two expeditions were not identical and therefore to gain an insight into the challenge the expeditions presented, a method of calculating the energy expenditure during the expedition is also described.</p></sec><sec id=\"Sec6\"><title>Change in body composition</title><p id=\"Par17\">The change in total body, lean tissue and body fat weight were determined from the pre- and post-expedition measurements. These were expressed as a percentage of pre-expedition values in order to make comparisons between participants in the Spear-17 and Ice Maiden expeditions.</p></sec><sec id=\"Sec7\"><title>Change in sleeping metabolic rate (ΔSMR-1, ΔSMR-2)</title><p id=\"Par18\">The metabolic rate during sleep (calculated between 00:00 and 06:00) was determined by the amount of metabolically active tissue in the body. Therefore, energy expenditure and substrate utilisation values were divided by lean tissue weight to give normalised measures and the difference between these pre- and post-expedition for the two sleep periods were used to compare participants from the Spear-17 and Ice Maiden expeditions.</p></sec><sec id=\"Sec8\"><title>Change in resting metabolic rate (ΔRMR)</title><p id=\"Par19\">The resting metabolic rate was measured between 07:00 and 08:00 after waking at 06:30 following the first sleep period. The subject was asked to lie quietly on the bed undertaking no activity and not going to sleep. The RMR was calculated as the average metabolic rate for the period 07:20–07:50 when participants were least restless. As with the sleeping metabolic rate, the values would be expected to vary with the weight of lean tissue and so the difference between pre- and post-expedition energy expenditure values normalised to lean tissue weight were used to compare participants from the Spear-17 and Ice-Maiden expeditions.</p></sec><sec id=\"Sec9\"><title>Change in metabolic rate during exercise (ΔEMR-80, ΔEMR-100, ΔEMR-120)</title><p id=\"Par20\">Three 30 min periods of stepping exercise were performed using a standard height exercise step (Reebok Aerobic Step, height 150 mm, Reebok, UK) at step rates of 80, 100 and 120 steps min<sup>−1</sup>, with the step rate controlled by a simple metronome (Tempo Perfect, <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.nch.com.au/metronome\">https://www.nch.com.au/metronome</ext-link>). The exercising metabolic rate was calculated as the mean energy expenditure throughout the 30-min exercise period and denoted EMR80, EMR100 and EMR120 for the three different step rates. The stepping exercise is a cyclical change in potential energy and the energy expenditure will depend on the total weight of the subject. Therefore the change in values normalised to the total body weight from the pre- and post-expedition measurements, denoted ΔEMR-80, ΔEMR-100 and ΔEMR-120, were used to compare participants in the Spear-17 and Ice Maiden expeditions.</p></sec><sec id=\"Sec10\"><title>Energy expenditure during the expedition</title><p id=\"Par21\">Ignoring diet induced thermogenesis (DIT) which is small<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, the energy expenditure for a short period of time (e.g. the minute-by-minute values shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>) can be thought of as the sum of the energy to deliver physical activity, the energy for non-shivering thermogenesis and the energy to maintain body functions, the basal metabolic rate (BMR). A 60% increase in BMR during exposure to the Antarctic environment had previously been reported<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. Assuming this fractional increase in the BMR is the additional energy required to maintain organ function and achieve thermoregulation at rest in a polar region, then the daily energy expenditure due to activity, <italic>E</italic><sub><italic>act</italic></sub>, during the expedition can be estimated from:<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ E_{act} = E_{tot} - BMR_{temp} (1 + T_{f} ) $$\\end{document}</tex-math><mml:math id=\"M2\" display=\"block\"><mml:mrow><mml:msub><mml:mi>E</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">act</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>E</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">tot</mml:mi></mml:mrow></mml:msub><mml:mo>-</mml:mo><mml:mi>B</mml:mi><mml:mi>M</mml:mi><mml:msub><mml:mi>R</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">temp</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mn>1</mml:mn><mml:mo>+</mml:mo><mml:msub><mml:mi>T</mml:mi><mml:mi>f</mml:mi></mml:msub><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70296_Article_Equ1.gif\" position=\"anchor\"/></alternatives></disp-formula>where <italic>E</italic><sub><italic>tot</italic></sub> is the total daily energy expenditure during the expedition, <italic>BMR</italic><sub><italic>temp</italic></sub> is the BMR measured in a temperate environment and <italic>T</italic><sub><italic>f</italic></sub> is the time-averaged fractional increase in the BMR as a result of exposure to the polar environment measured over 24 h. Values of <italic>BMR</italic><sub><italic>temp</italic></sub> were based on the values of RMR measured at 24 °C during the whole-body calorimeter studies on all participants both pre- and post-expedition. To obtain a value of <italic>E</italic><sub><italic>tot</italic></sub> the conservation of energy is applied to the energy obtained from the food ingested and the loss of body weight. Assuming a linear loss of lean and fat weight during the expedition and 100% absorption of the food consumed, <italic>E</italic><sub><italic>tot</italic></sub>, in Eq. (<xref rid=\"Equ1\" ref-type=\"\">1</xref>) can be estimated from:<disp-formula id=\"Equ2\"><label>2</label><alternatives><tex-math id=\"M3\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ E_{tot} = f_{c} E_{nutr} + \\frac{{{\\Delta }_{fat} ED_{fat} }}{{Ex_{dur} }} + \\frac{{{\\Delta }_{lean} ED_{lean} }}{{Ex_{dur} }} $$\\end{document}</tex-math><mml:math id=\"M4\" display=\"block\"><mml:mrow><mml:msub><mml:mi>E</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">tot</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:msub><mml:mi>f</mml:mi><mml:mi>c</mml:mi></mml:msub><mml:msub><mml:mi>E</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">nutr</mml:mi></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">fat</mml:mi></mml:mrow></mml:msub><mml:mi>E</mml:mi><mml:msub><mml:mi>D</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">fat</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:mi>E</mml:mi><mml:msub><mml:mi>x</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">dur</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfrac><mml:mo>+</mml:mo><mml:mfrac><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">lean</mml:mi></mml:mrow></mml:msub><mml:mi>E</mml:mi><mml:msub><mml:mi>D</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">lean</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:mi>E</mml:mi><mml:msub><mml:mi>x</mml:mi><mml:mrow><mml:mi mathvariant=\"italic\">dur</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfrac></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70296_Article_Equ2.gif\" position=\"anchor\"/></alternatives></disp-formula>where <italic>E</italic><sub><italic>nutr</italic></sub> is the energy available from the supplied nutrition, <italic>f</italic><sub><italic>c</italic></sub> is the fraction of the supplied nutrition consumed, Δ<sub><italic>fat</italic></sub> is the change in the weight of body fat determined from body composition measurements before and after the expedition, <italic>ED</italic><sub><italic>fat</italic></sub> is the metabolisable energy density of human body fat, Δ<sub><italic>lean</italic></sub> is the change in the weight of lean body tissue determined from body composition measurements before and after the expedition, <italic>ED</italic><sub><italic>lean</italic></sub> is the metabolisable energy density of human lean tissue and <italic>Ex</italic><sub><italic>dur</italic></sub> is the duration of the expedition in days. Using Eqs. (<xref rid=\"Equ1\" ref-type=\"\">1</xref>) and (<xref rid=\"Equ2\" ref-type=\"\">2</xref>) the total daily energy expenditure, <italic>E</italic><sub><italic>tot</italic></sub>, and the daily energy expenditure due to activity, <italic>E</italic><sub><italic>act</italic></sub>, were compared for participants in the two expeditions.</p></sec><sec id=\"Sec11\"><title>Statistical analysis</title><p id=\"Par22\">As the number of participants was small it was not possible to robustly determine the normality of the two sets of data. Therefore summarised values for the group of participants are presented as median, lower (Q1) and upper quartile (Q3) of the values. Statistical comparisons between participants in the Spear-17 and Ice Maiden data have used the non-parametric Mann–Whitney U-test for very small samples<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup> with statistical significance taken as <italic>p</italic> ≤ 0.05. We have also analysed the changes for individual participants and how these varied across the two expedition groups.</p></sec><sec id=\"Sec12\"><title>Ethics approval</title><p id=\"Par23\">Ethics approval for the Spear-17 study was obtained from the National Health Authority Research Ethics committee, West Midlands—Solihull (ID: 13/WM/0327), Ministry of Defence—Research Ethics Committee and University Hospitals Coventry and Warwickshire Research and Development Governance Committee, under the GAFREC framework (REF: GF0121). Ethics approval for the Ice Maiden study was obtained from the Ministry of Defence Research Ethics Committee (827MoDREC/17). Both studies were carried out in compliance with the Ethical Principles for Medical Research on Human Subjects set down in the Declaration of Helsinki by the World Medical Association. Informed consent was obtained for each participant, for pre and post-expedition measurements.</p></sec></sec><sec id=\"Sec13\"><title>Results</title><p id=\"Par24\">For the Spear-17 expedition, five of the six reservists, including their leader, completed the traverse of Antarctica from the Hercules Inlet to the Ross Sea Ice. The sixth reservist left the expedition at the South Pole due to extreme fatigue, and thus their data was excluded from the analysis. All six participants in the Ice Maiden expedition completed the Antarctic crossing from the Leverett Glacier to the Hercules Inlet.</p><p id=\"Par25\">The results for pre- to post-expedition differences energy expenditure and substrate utilisation during sleeping, resting and exercise, and the determination of the energy expenditure during the expedition are given in the following sections. The relationship between these different measures will form part of the Discussion.</p><sec id=\"Sec14\"><title>Change in body composition</title><p id=\"Par26\">The median and quartile (Q1; Q3) values for the pre- to post-expedition difference in composition for the Spear-17 expedition were: total body weight [− 5.6 kg (− 7.6 kg; − 4.6 kg)], lean weight [+ 1.0 kg (0.4 kg; 1.1 kg)] and fat weight [− 6.5 kg (− 8.3 kg; − 2.4 kg)]; For the Ice Maiden expedition: total body weight [− 5.8 kg (− 6.7 kg; − 5.0 kg)], lean weight [− 1.9 kg (− 2.0 kg; − 0.9 kg)] and fat tissue weight [− 4.3 kg (− 5.3 kg; − 3.5 kg)]. The median and quartile values for the pre- to post-expedition differences in body composition expressed as a percentage of the pre-expedition values are given in the upper section of Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>. The changes in body composition were similar between the two expeditions; the only statistically significant difference being between the small gain in lean tissue weight for Spear-17 participants and the loss of lean tissue weight by Ice Maiden participants (<italic>p</italic> < 0.05).<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>The median, lower and upper quartiles (Q1; Q3) for the changes between pre- and post-expedition measurements in: body composition, energy expenditure during sleep (ΔSMR-1 and ΔSMR-2) and rest (ΔRMR); and during exercise at 80 steps min<sup>−1</sup> (ΔEMR-80), 100 steps min<sup>−1</sup> (ΔEMR-100) and 120 steps min<sup>−1</sup> (ΔEMR-120).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Factor</th><th align=\"left\">Spear-17</th><th align=\"left\">Ice Maiden</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"3\"><bold>Change in body composition</bold></td></tr><tr><td align=\"left\">ΔBody weight (%)</td><td char=\"(\" align=\"char\">− 6.5 (− 8.9; − 5.3)</td><td char=\"(\" align=\"char\">− 7.8 (− 9.5; − 6.9)</td></tr><tr><td align=\"left\">ΔLean tissue weight (%)</td><td char=\"(\" align=\"char\">1.0 (0.5; 1.5)</td><td char=\"(\" align=\"char\">− 2.5 (− 3.6; − 1.7)*</td></tr><tr><td align=\"left\">ΔFat weight (%)</td><td char=\"(\" align=\"char\">− 40.4 (− 43.7; -32.1)</td><td char=\"(\" align=\"char\">− 30.4 (− 32.3; − 22.6)</td></tr><tr><td align=\"left\" colspan=\"3\"><bold>Change in energy expenditure during sleep and rest</bold></td></tr><tr><td align=\"left\">ΔSMR-1/cal min<sup>−1</sup> kg<sup>−1</sup> lean tissue weight</td><td char=\"(\" align=\"char\">0.2 (− 0.5; 1.0)</td><td char=\"(\" align=\"char\">0.3 (− 0.7; 2.7)</td></tr><tr><td align=\"left\">ΔSMR-2/cal min<sup>−1</sup> kg<sup>−1</sup> lean tissue weight</td><td char=\"(\" align=\"char\">0.0 (− 0.8; 0.3)</td><td char=\"(\" align=\"char\">0.2 (− 0.6; 1.7)</td></tr><tr><td align=\"left\">ΔRMR/cal min<sup>−1</sup> kg<sup>−1</sup> lean tissue weight</td><td char=\"(\" align=\"char\">− 0.4 (− 0.7; 0.1)</td><td char=\"(\" align=\"char\">− 0.3 (− 0.8; 0.5)</td></tr><tr><td align=\"left\" colspan=\"3\"><bold>Change in energy expenditure during exercise</bold></td></tr><tr><td align=\"left\">ΔEMR-80/cal min<sup>−1</sup> kg<sup>−1</sup> body weight</td><td char=\"(\" align=\"char\">0.7 (− 2.4; 1.9)</td><td char=\"(\" align=\"char\">3.3 (0.2; 6.3)</td></tr><tr><td align=\"left\">ΔEMR-100/cal min<sup>−1</sup> kg<sup>−1</sup> body weight</td><td char=\"(\" align=\"char\">0.5 (0.0; 0.7)</td><td char=\"(\" align=\"char\">0.7 (0.2; 1.2)</td></tr><tr><td align=\"left\">ΔEMR-120/cal min<sup>−1</sup> kg<sup>−1</sup> body weight</td><td char=\"(\" align=\"char\">− 2.5 (− 3.2; 3.0)</td><td char=\"(\" align=\"char\">1.1 (− 0.3; 2.5)</td></tr></tbody></table><table-wrap-foot><p>*Statistical significance between the data sets (<italic>p</italic> ≤ 0.05). It should be noted that the units of energy expenditure are cal min<sup>−1</sup> kg<sup>−1</sup> rather than the more usual kcal min<sup>−1</sup> kg<sup>−1</sup>.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec15\"><title>Change in sleeping (ΔSMR1, ΔSMR2) and resting (ΔRMR) metabolic rate</title><p id=\"Par27\">From the middle section of Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> it can be seen that the changes in energy expenditure during sleep and resting were small and not statistically significant, but that the range of values was large (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>). It should be noted that the values are given in units of cal min<sup>−1</sup> kg<sup>−1</sup> rather than the more usual kcal min<sup>−1</sup> kg<sup>−1</sup>.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>The percentage change in body composition between the pre- and post-expedition measurements for the individual participants in the two expeditions.</p></caption><graphic xlink:href=\"41598_2020_70296_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par28\">The upper two sections of Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref> shows the median and quartile (Q1; Q3) values for the difference in substrate utilisation for the two expeditions with individual values shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>. From these it can be seen there was little difference in the average value or spread of values for protein and lipid utilisation across the two sleep periods and the rest period or between the participants in the two expeditions. The modest change in carbohydrate utilisation between the pre- and post-expedition measurements for both expeditions was characterised all three measures (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>) but a much larger range of values than found for either the protein or lipid utilisations for all three measures (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>).<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>The median, lower and upper quartiles (Q1; Q3) for the changes between pre- and post-expedition measurements of substrate utilisation during sleep (ΔSMR-1 and ΔSMR-2), rest (ΔRMR) and exercise (ΔEMR-80, ΔEMR-100 and ΔEMR-120).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Factor</th><th align=\"left\" colspan=\"3\">Spear-17</th><th align=\"left\" colspan=\"3\">Ice Maiden</th></tr><tr><th align=\"left\">Protein</th><th align=\"left\">Carbohydrate</th><th align=\"left\">Lipid</th><th align=\"left\">Protein</th><th align=\"left\">Carbohydrate</th><th align=\"left\">Lipid</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"7\"><bold>Sleep</bold></td></tr><tr><td align=\"left\">ΔSMR-1/g day<sup>−1</sup> kg<sup>−1</sup> lean tissue</td><td char=\"(\" align=\"char\">0.1 (− 0.1; 0.3)</td><td char=\"(\" align=\"char\">0.5 (− 0.1; 1.0)</td><td char=\"(\" align=\"char\">− 0.2 (− 0.4; 0.0)</td><td char=\"(\" align=\"char\">0.2 (0.1; 0.6)</td><td char=\"(\" align=\"char\">0.9 (0.3; 1.5)</td><td char=\"(\" align=\"char\">− 0.4 (− 0.6; − 0.1)</td></tr><tr><td align=\"left\">ΔSMR-2/g day<sup>−1</sup> kg<sup>−1</sup> lean tissue</td><td char=\"(\" align=\"char\">− 0.2 (− 0.2; 0.1)</td><td char=\"(\" align=\"char\">− 0.4 (− 0.9; − 0.2)</td><td char=\"(\" align=\"char\">0.2 (0.1; 0.4)</td><td char=\"(\" align=\"char\">0.0 (− 0.1; 0.3)</td><td char=\"(\" align=\"char\">0.5 (0.1; 1.1)</td><td char=\"(\" align=\"char\">− 0.3 (− 0.3; − 0.1)</td></tr><tr><td align=\"left\" colspan=\"7\"><bold>Rest</bold></td></tr><tr><td align=\"left\">ΔRMR/g day<sup>−1</sup> kg<sup>−1</sup> lean tissue</td><td char=\"(\" align=\"char\">0.0 (− 0.1; 0.1)</td><td char=\"(\" align=\"char\">0.7 (− 0.6; 0.8)</td><td char=\"(\" align=\"char\">− 0.3 (− 0.3; − 0.2)</td><td char=\"(\" align=\"char\">0.0 (0.0; 0.4)</td><td char=\"(\" align=\"char\">0.1 (− 0.2; 1.1)</td><td char=\"(\" align=\"char\">− 0.1 (− 0.5; 0.0)</td></tr><tr><td align=\"left\" colspan=\"7\"><bold>Exercise</bold></td></tr><tr><td align=\"left\">ΔEMR-80/g day<sup>−1</sup> kg body weight</td><td char=\"(\" align=\"char\">0.1 (− 0.4; 0.1)</td><td char=\"(\" align=\"char\">0.8 (0.8; 1.5)</td><td char=\"(\" align=\"char\">− 0.5 (− 0.8; − 0.4)</td><td char=\"(\" align=\"char\">− 0.1 (− 0.1; 0.1)</td><td char=\"(\" align=\"char\">2.2 (1.0; 5.4)</td><td char=\"(\" align=\"char\">− 0.4 (− 1.3; 0.8)</td></tr><tr><td align=\"left\">ΔEMR-100/g day<sup>−1</sup> kg body weight</td><td char=\"(\" align=\"char\">0.1 (− 0.4; 0.1)</td><td char=\"(\" align=\"char\">1.6 (0.2; 3.2)</td><td char=\"(\" align=\"char\">− 0.6 (− 1.0; 0.2)</td><td char=\"(\" align=\"char\">− 0.1 (− 0.1; 0.1)</td><td char=\"(\" align=\"char\">0.0 (− 1.0; 3.3)</td><td char=\"(\" align=\"char\">0.2 (− 1.4; 0.6)</td></tr><tr><td align=\"left\">ΔEMR-120/g day<sup>−1</sup> kg body weight</td><td char=\"(\" align=\"char\">0.1 (− 0.4; 0.1)</td><td char=\"(\" align=\"char\">2.5 (− 0.8; 3.5)</td><td char=\"(\" align=\"char\">0.1 (− 1.6; 0.2)</td><td char=\"(\" align=\"char\">− 0.1 (− 0.1; 0.1)</td><td char=\"(\" align=\"char\">1.3 (0.3; 2.3)</td><td char=\"(\" align=\"char\">− 0.4 (− 0.8; 0.1)</td></tr></tbody></table></table-wrap><fig id=\"Fig3\"><label>Figure 3</label><caption><p>The change in substrate utilisation between the pre- and post-expedition measurements of the rest (ΔRMR) and two sleep periods (ΔSMR-1 and ΔSMR-2) for individual participants in the two expeditions.</p></caption><graphic xlink:href=\"41598_2020_70296_Fig3_HTML\" id=\"MO3\"/></fig></p><p id=\"Par29\">For the change in carbohydrate utilisation between the pre- and post-expedition measurements of sleep (ΔSMR-1 and ΔSMR-2) it should be noticed that: (a) whilst modest, the median value for participants in the Spear-17 expedition was positive for the first sleep period (ΔSMR-1) and negative for the second sleep period (ΔSMR-2) whilst that for participants in the Ice Maiden expedition was positive for both sleep periods; and (b) participants from both expeditions showed a reduction for the second sleep period (ΔSMR-2) when compared with the first sleep period (ΔSMR-1). A negative value indicates an absolute reduction in utilisation post-expedition when compared with the pre-expedition value. For the carbohydrate utilisation normalised to lean tissue weight there was no separation of the participants from the two expeditions (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>). When participants in the two expeditions were considered together with the median and quartile values (Q1; Q3) for the difference between the pre- and post-expedition measurements of carbohydrate utilisation during the two sleep periods were 0.7 g day<sup>−1</sup> kg<sup>−1</sup> of lean tissue (0.0; 1.3 g day<sup>−1</sup> kg<sup>−1</sup> of lean tissue) and 0.1 g day<sup>−1</sup> kg<sup>−1</sup> of lean tissue (− 0.5; 0.8 g day<sup>−1</sup> kg<sup>−1</sup> of lean tissue) for ΔSMR-1 and ΔSMR-2 respectively. Similarly, the difference in carbohydrate utilisation for all participants during rest (ΔRMR) was 0.2 g day<sup>−1</sup> kg<sup>−1</sup> lean tissue (− 0.4; 1.1 g day<sup>−1</sup> kg<sup>−1</sup> lean tissue). Thus, when participants from the two expeditions were considered together there was a larger carbohydrate utilisation in the post-expedition measurements for both sleep periods and during rest but the difference was largest for the first sleep period.</p></sec><sec id=\"Sec16\"><title>Change in metabolic rate during exercise (ΔEMR-80, ΔEMR-100 and ΔEMR-120)</title><p id=\"Par30\">The median and quartile (Q1; Q3) values for the difference in energy expenditure between the pre- and post-expedition measurements during the three exercise intensities are given in the lower section of Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>. As with the ΔSMR and ΔRMR values, the units are cal min<sup>−1</sup> kg<sup>−1</sup>. For the 80 steps min<sup>−1</sup> exercise, the average difference between the pre- and post-expedition measurements for the female participants was greater than that for the male participants but the difference was not statistically significant. The plot of the values for individual participants, Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>, shows that one of the female participants had a much higher change in energy expenditure between the pre- and post-expedition measurements than the remaining participants. However, since non-parametric measures were used this single value will have minimal effect on the estimate of the average value or the determination of the U value for the Mann–Whitney test, there was no statistically significant difference.<fig id=\"Fig4\"><label>Figure 4</label><caption><p>The percentage change in energy expenditure between the pre- and post-expedition measurements of the exercising metabolic rate at 3 intensities: 80 steps min<sup>−1</sup> (ΔEMR-80); 100 steps min<sup>−1</sup> (ΔEMR-100) and 120 steps min<sup>−1</sup> (ΔEMR-120) for the individual participants in the two expeditions.</p></caption><graphic xlink:href=\"41598_2020_70296_Fig4_HTML\" id=\"MO4\"/></fig></p><p id=\"Par31\">From Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref> it can be seen that the range of value for the difference in energy expenditure between the pre- and post-expedition measurements for participants in the two expeditions is very similar if the single high value for the Ice Maiden participant at 80 steps min<sup>−1</sup> is excluded. There was little difference in the values from the two expeditions at 100 steps min<sup>−1</sup>, but at 120 steps min<sup>−1</sup> participants in the Spear-17 expedition had a non-statistically significant reduction in energy expenditure compared with participants in the Ice Maiden expedition. The lower part of Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref> shows the average values for the difference in substrate utilisation between the pre- and post-expedition measurements from participants in the two expeditions. It should be noted that the difference in protein utilisation determined from 12-h urine samples, was the same for all exercise intensities, but values for each exercise intensity have been included in results tables and graphs for completeness. From Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref> it can be seen that the pre- to post-expedition differences in protein and lipid utilisation were similar for the two expeditions but there was a larger difference in the carbohydrate utilisation. At 80 steps min<sup>−1</sup> Ice Maiden participants had a higher difference in carbohydrate utilisation between the pre- and post-expedition measurements than the Spear-17 participants. However, this difference between expeditions was reversed for the 100 steps min<sup>−1</sup> and 120 steps min<sup>−1</sup> exercise intensities. None of the differences were statistically significant. A plot of the substrate utilisation for individual participants from the two expeditions for the different exercise intensities is (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>) shows a trend towards participants from both expeditions having a higher difference in carbohydrate utilisation between the pre- and post-expedition measurements at 80 steps min<sup>−1</sup> that reduces as the exercise intensity increases.<fig id=\"Fig5\"><label>Figure 5</label><caption><p>The change in substrate utilisation between the pre- and post-expedition during measurements of the exercising metabolic rate at three intensities: 80 steps min<sup>−1</sup> (ΔEMR-80); 100 steps min<sup>−1</sup> (ΔEMR-100); and 120 steps min<sup>−1</sup> (ΔEMR-120) for individual participants in the two expeditions. Extreme values are shown at the edge of the plot together with their numerical values.</p></caption><graphic xlink:href=\"41598_2020_70296_Fig5_HTML\" id=\"MO5\"/></fig></p><p id=\"Par32\">This trend was not seen in the difference in lipid utilisation between the pre- and post-expedition measurements. There was a large overlap in the difference in carbohydrate utilisation between the pre- and post-expedition measurements for participants in the two expeditions for all exercise intensities. The distribution of these values for the 80 step min<sup>−1</sup> intensity (ΔEMR-80, Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>) was very similar to that for the first sleep period (ΔSMR-1, Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>) and the resting metabolic rate (ΔRMR, Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>). The median and quartile (Q1; Q3) values for the difference in carbohydrate utilisation between the pre- and post-expedition measurements for all participants studied for the three exercise intensities studied was 1.5 g day<sup>−1</sup> kg<sup>−1</sup> of lean tissue (0.8; 4.2 g day<sup>−1</sup> kg<sup>−1</sup> of lean tissue), 0.2 g day<sup>−1</sup> kg<sup>−1</sup> of lean tissue (− 0.7; 3.6 g day<sup>−1</sup> kg<sup>−1</sup> of lean tissue) and 2.1 g day<sup>−1</sup> kg<sup>−1</sup> of lean tissue (− 0.3; 3.0 g day<sup>−1</sup> kg<sup>−1</sup> of lean tissue) for ΔEMR-80, ΔEMR-100 and ΔEMR-120 respectively.</p></sec><sec id=\"Sec17\"><title>Energy expenditure during the expedition</title><p id=\"Par33\">For both expeditions, the difference in the RMR measured before and after the expedition was small and not statistically significant<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>,<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. Therefore the values for <italic>BMR</italic><sub><italic>temp</italic></sub> used in Eq. (<xref rid=\"Equ1\" ref-type=\"\">1</xref>) were the average of the RMR values measured pre- and post-expedition. Summarised values for <italic>BMR</italic><sub><italic>temp</italic></sub> and the change in lean and fat weight for the two expeditions are shown in Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref> together with the expedition data required for Eqs. (<xref rid=\"Equ1\" ref-type=\"\">1</xref>) and (<xref rid=\"Equ2\" ref-type=\"\">2</xref>).<table-wrap id=\"Tab3\"><label>Table 3</label><caption><p>The expedition and measured values to allow the total energy and the energy due to activity during the expeditions to be determined from Eqs. (<xref rid=\"Equ1\" ref-type=\"\">1</xref>) and (<xref rid=\"Equ2\" ref-type=\"\">2</xref>).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">Spear-17</th><th align=\"left\">Ice Maiden</th></tr></thead><tbody><tr><td align=\"left\">Expedition duration (days)</td><td align=\"left\">67</td><td align=\"left\">61</td></tr><tr><td align=\"left\">Nutritional energy available (E<sub>nutr</sub>) (kcal day<sup>−1</sup>)</td><td align=\"left\">6,500</td><td align=\"left\">5,000</td></tr><tr><td align=\"left\">Fraction of nutritional energy consumed (%)</td><td align=\"left\">92%</td><td align=\"left\">85%</td></tr><tr><td align=\"left\">Median (Q1; Q3) BMR<sub>temp</sub> (kcal day<sup>−1</sup>)</td><td align=\"left\">2054 (2000; 2,106)</td><td align=\"left\">1793 (1629; 1865)</td></tr><tr><td align=\"left\">Median (Q1; Q3) change in fat weight (Δfat) (kg)</td><td align=\"left\">− 2.7 (− 8.3; − 2.4)</td><td align=\"left\">− 4.4 (− 5.3; − 3.5)</td></tr><tr><td align=\"left\">Median (Q1; Q3) change in lean weight (Δlean) (kg)</td><td align=\"left\">0.7 (0.4; 1.1)</td><td align=\"left\">− 1.4 (− 2.0; − 0.9)</td></tr></tbody></table></table-wrap></p><p id=\"Par34\">The values of energy density used in calculating substrate utilisation from the O<sub>2</sub>/CO<sub>2</sub> measurements in the whole body calorimeter were 9.461 kcal g<sup>−1</sup> for fat, <italic>ED</italic><sub><italic>fat</italic></sub>, and 4.316 kcal g<sup>−1</sup> for protein, <italic>ED</italic><sub><italic>lean</italic></sub>,<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup> Substituting theses values and the data from Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref> into Eq. (<xref rid=\"Equ2\" ref-type=\"\">2</xref>) gave median and quartile (Q1; Q3) values for the total energy expenditure during the expedition, E<sub>tot</sub>, of 6,461 kcal day<sup>−1</sup> (6,335; 7,107 kcal day<sup>−1</sup>) for participants in the Spear-17 expedition and 4,939 kcal day<sup>−1</sup> (4,803; 5,088 kcal day<sup>−1</sup>) for participants in the Ice Maiden expedition, a difference that was statistically significant (<italic>p</italic> = 0.004). The only value available for the time-averaged fractional increase in BMR for thermoregulation, <italic>T</italic><sub><italic>f</italic></sub>, is the 60% measured by Stroud<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. Substituting this together with the values for E<sub>tot</sub> into Eq. (<xref rid=\"Equ2\" ref-type=\"\">2</xref>) gave an energy expenditure on activity during the expedition, <italic>E</italic><sub><italic>act</italic></sub> of 3,174 kcal day<sup>−1</sup> (3,136; 4,117 kcal day<sup>−1</sup>) for participants in the Spear-17 expedition and 2,282 kcal day<sup>−1</sup> (1,801; 2,559 kcal day<sup>−1</sup>) for participants in the Ice Maiden expedition, a statistically significant difference (<italic>p</italic> = 0.004). The graph of total energy expenditure, <italic>E</italic><sub><italic>tot</italic></sub>, and the energy expenditure on activity, <italic>E</italic><sub><italic>act</italic></sub>, during the expedition for individual participants (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>) shows that values for participants in the Ice Maiden expedition were clustered together beneath a cluster of three of the five participants in the Spear-17 expedition. There were two outliers, both from the Spear-17 expedition, with values much greater than those from the other participants: one with values for <italic>E</italic><sub><italic>to</italic>t</sub> and <italic>E</italic><sub><italic>act</italic></sub> of 8,470 and 7,107 kcal day<sup>−1</sup>, respectively and the other with values for <italic>E</italic><sub><italic>to</italic>t</sub> and <italic>E</italic><sub><italic>act</italic></sub> of 5,054 and 4,117 kcal day<sup>−1</sup>, respectively.<fig id=\"Fig6\"><label>Figure 6</label><caption><p>The total daily energy expenditure during the expedition (E<sub>tot</sub>) estimated using Eq. (<xref rid=\"Equ2\" ref-type=\"\">2</xref>) and the energy expended on activity during the expedition (E<sub>act</sub>) estimated using Eq. (<xref rid=\"Equ1\" ref-type=\"\">1</xref>) with <italic>T</italic><sub><italic>f</italic></sub> = 60% for individual participants in the Spear-17 and Ice Maiden expeditions.</p></caption><graphic xlink:href=\"41598_2020_70296_Fig6_HTML\" id=\"MO6\"/></fig></p><p id=\"Par35\">The data in Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref> were calculated using a value for <italic>T</italic><sub><italic>f</italic></sub> measured at rest<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. In practice, this may not be a good estimate of the time-averaged value measured throughout the day. To investigate how the values of <italic>T</italic><sub><italic>f</italic></sub> affected the calculated energy expenditure due to activity the median values of this were plotted as <italic>T</italic><sub><italic>f</italic></sub> was varied from 0 to 100% (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>).<fig id=\"Fig7\"><label>Figure 7</label><caption><p>A plot of the median values for the energy expenditure on activity, <italic>E</italic><sub><italic>act</italic></sub>, as the time-averaged fractional increase in RMR, <italic>T</italic><sub><italic>f</italic></sub>, in Eq. (<xref rid=\"Equ1\" ref-type=\"\">1</xref>) was varied from 0–100%.</p></caption><graphic xlink:href=\"41598_2020_70296_Fig7_HTML\" id=\"MO7\"/></fig></p><p id=\"Par36\">As predicted by Eq. (<xref rid=\"Equ1\" ref-type=\"\">1</xref>), Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref> shows the energy expenditure due to activity decreases as the time-averaged percentage increase in RMR, <italic>T</italic><sub><italic>f</italic></sub>, increases. From the figure, it can also be seen that the difference in the median energy expenditure between the two expeditions decreases as <italic>T</italic><sub><italic>f</italic></sub> increases.</p></sec></sec><sec id=\"Sec18\"><title>Discussion</title><p id=\"Par37\">We have previously reported a detailed analysis of the energy expenditure and substrate utilisation from participants in the Ice Maiden<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup> and Spear-17<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup> expeditions. Whilst these expeditions were of similar duration and length, comparing energy expenditure and substrate utilisation across the two expeditions is challenging because differences in body size and composition will affect the measurements. A common approach is to normalise rest and sleep data to lean weight on the assumption that fat is metabolically inactive<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>–<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup> However, there are metabolic processes involving fat that are dependent on the amount present<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. A more robust normalising factor is undoubtedly obtained if this metabolising fat contribution is accounted for, but to do that in a study population requires covariance<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup> or correlation<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup> techniques. In this study we would need to do this for Spear-17 and Ice Maiden participants independently both before and after their expeditions to avoid discontinuities; the number of participants (5 and 6 respectively) is not sufficient to give robust results. As a result of this together with participants not being obese, we have followed the recommendations of Arch et al.<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>, if in the absence of more robust methods, as a minimum, normalised non-exercise measures to lean weight. The stepping exercises involve a cyclical change in potential energy determined by body weight and therefore the energy expenditure and substrate utilisation values have been normalised to body weight. Other metabolic measures traditionally cited include diet induced thermogenesis (DIT) and Total Energy Expenditure (TEE) during the 24-h measurement period. During much of the daytime component of the 24-h measurement period there is undocumented very low levels of physical activity for which the energy expenditure will be an unknown function of both lean tissue weight and body weight. Therefore the measure has not been included in the analysis reported in this paper. Similarly, DIT has been omitted from the current analysis as it is not directly measured but rather was determined using the intercept method<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup> where the effect of data normalisation is unknown.</p><p id=\"Par38\">The modest percentage change in body weight suggests that the composition and quantity of the diet was appropriate to both the environment and the physical effort required to pull the sledges. The available diet for Spear-17 participants delivered 6,500 kcal day<sup>−1</sup> whilst that for Ice Maiden participants delivered 5,000 kcal day<sup>−1</sup>. Importantly, even though not all the available calories were consumed, there was minimal loss of lean tissue weight from participants in the Ice Maiden expedition whilst participants in the Spear-17 expedition gained a small amount of lean tissue weight; a difference that gave the only statistically significant difference between the two expeditions in the factors studied. Taken together the results for the change in body composition for both expeditions suggests that whilst there was negative Energy Availability (EA), this was modest.</p><p id=\"Par39\">The median of the total energy expenditure during the expedition for participants in the Spear-17 expedition (6,461 kcal day<sup>−1</sup>) was consistent with an activity estimate of 6,100 kcal day<sup>−1</sup><sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Noting the two outliers, with energy expenditure of 7,107 and 8,470 kcal day<sup>−1</sup>, the total energy expenditure by participants in the Spear-17 expedition was also consistent with the average of 7,390 kcal day<sup>−1</sup> measured using doubly labelled water (DLW) on two male subjects during polar expeditions<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. It is interesting to note that in both Arctic<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup> and Antarctic<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> expeditions, the older of the two participants had a substantially higher daily energy expenditure than the younger. The second highest value from participants in the Spear-17 expedition was for the oldest member who had the smallest lean tissue weight from the body composition measures and, as a result, the smallest RMR values and hence the smallest <italic>BMR</italic><sub><italic>temp</italic></sub> value. There is anecdotal evidence from the expedition team that the Spear-17 participant with the highest value for total energy expenditure spent periods of the expedition walking rather than skiing due to a technical issue with a ski; walking in such conditions would require much greater energy expenditure. The median total energy expenditure for participants in the Ice Maiden expedition, 4,939 kcal day<sup>−1</sup>, was much lower than that for participants in the Spear-17 expedition and inconsistent with both activity based estimates from previously measured values<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Despite being statistically significant, the difference in the median energy due to activity, <italic>E</italic><sub><italic>act</italic></sub>, between the two expeditions was surprisingly small (892 kcal day<sup>−1</sup>) given two key differences between the expeditions that would suggest much greater energy expenditure from participants in the Spear-17 expedition. Firstly, due to the single versus dual resupply strategy, the sledges on the Spear-17 expedition weighed a maximum of 120 kg; whilst those on the Ice Maiden expedition weighed a maximum of 80 kg. Secondly, whilst both expeditions crossed un-traversed ice, the initial section of the Ice Maiden expedition (Leverett Glacier to the South-pole) used a semi-prepared supply track. To put the difference in energy expenditure due to activity during the expeditions into perspective, it is 43% and 50% of the RMR measured in a temperate climate for the Spear-17 and Ice Maiden participants respectively (Table <xref rid=\"Tab3\" ref-type=\"table\">3</xref>). The values of <italic>E</italic><sub><italic>tot</italic></sub> for participants in the all-male Spear-17 expedition were consistent with measurements made on male participants in previous expeditions whereas those for participants in the all-female Ice Maiden expedition were not. This suggests the calculated energy expenditure on activity, <italic>E</italic><sub><italic>act</italic></sub>, by participants in the Ice Maiden expedition is too high. Whilst the equation for determining the total daily energy expenditure, <italic>E</italic><sub><italic>tot</italic></sub>, from Eq. (<xref rid=\"Equ2\" ref-type=\"\">2</xref>) must be treated with some caution because of the assumptions inherent in it, we can postulate that the too high value for the energy due to activity, <italic>E</italic><sub><italic>act</italic></sub>, is the result of a too low value for the energy expenditure on thermoregulation through non-shivering thermogenesis (<italic>T</italic><sub><italic>f</italic></sub> × <italic>BMR</italic><sub><italic>temp</italic></sub>) in Eq. (<xref rid=\"Equ1\" ref-type=\"\">1</xref>). Since <italic>BMR</italic><sub><italic>temp</italic></sub> was taken from measured values of the RMR, it follows that the estimate for the time-averaged fractional increase in BMR due to the low temperatures, <italic>T</italic><sub><italic>f</italic></sub>, of 60% for the Ice Maiden expedition was too low. The energy for thermoregulation is higher in men when compared with women as a result of the greater lean tissue weight and body surface area<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>; but a larger value of <italic>T</italic><sub><italic>f</italic></sub> for the Spear-17 participants would tend to reduce the difference in the energy available for activities between the two expeditions, rather than increase it (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>). In addition, we have previously shown through comparisons with other studies that a value of 60% for <italic>T</italic><sub><italic>f</italic></sub> is consistent with the energy for thermoregulation for participants in the Spear-17 expedition. Both expeditions were equipped with clothing appropriate to the Antarctic environment leaving only the face exposed. Experimentally, facial cooling has been shown to cause a less than 5% increase in the metabolic rate in men when exercising<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup> so this was unlikely to explain the increase in the energy for thermoregulation in the Ice Maiden participants. A possible explanation for the higher energy for thermoregulation by participants in the Ice Maiden expedition stems from the greater effort required by participants in the Spear-17 expedition to travel over previously un-traversed ice. The higher muscle activity of the Spear-17 participants would increase the energy due to activity, <italic>E</italic><sub><italic>act</italic></sub>, whilst also increasing the heat generated due to the inefficiency of muscle activity resulting in reduced energy for non-shivering thermogenesis and hence a smaller value for <italic>T</italic><sub><italic>f</italic></sub> in Eq. (<xref rid=\"Equ1\" ref-type=\"\">1</xref>) compared with the value for participants in the Ice Maiden expedition.</p><p id=\"Par40\">The value for <italic>T</italic><sub><italic>f</italic></sub> is a time-averaged value will depend on the intensity and duration of physical activity. Without additional measurements made during the expedition it is impossible to determine what the numerical value for the Ice Maiden expedition should be, only that it is greater than 60%. The current analysis has used a single value of <italic>T</italic><sub><italic>f</italic></sub> for all participants in an expedition. However, the wide variation in energy expenditure, particularly amongst participants in the Spear-17 expedition, suggests that values for individual participants may be more appropriate.</p><p id=\"Par41\">It is recognised that measurements to establish personalised values is challenging, made more challenging under expedition conditions. Isotope techniques, which have been used on previous expeditions<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>, could have given a measurement of the total daily energy expenditure, <italic>E</italic><sub><italic>tot</italic></sub>, replacing the estimate obtained from Eq. (<xref rid=\"Equ2\" ref-type=\"\">2</xref>) but approvals and logistics constraints prevented this. A non-laboratory study on thermoregulation where participants undertook strenuous work in hot and cold environments<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup> used a modelling and qualitative scoring system to determine the energy required for thermoregulation<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. This system, which was designed for short periods of well-defined activity, could potentially be adapted for use on polar expeditionary journeys but the time required by participants for measurement and record-keeping to obtain robust values may be prohibitive. A more realistic approach to determining the energy expenditure on activity and thermoregulation during expeditions may be the next generation of body-worn biosensors<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>.</p><p id=\"Par42\">The differences between the pre- and post-expedition energy expenditure during sleeping, resting and exercise for the participants in the two expeditions were small. There was no statistically significant difference in these measures between participants from the two expeditions. If energy expenditure is considered a proxy for metabolic activity then these results suggest that there was little metabolic consequence to the participants for either of the expeditions studied. Importantly, there was no difference in the metabolic consequence of men and women undertaking expeditionary travel when appropriately trained and with an appropriate diet. The participant from Spear-17 who left the expedition at the South Pole did so because of extreme exhaustion.</p><p id=\"Par43\">The changes in protein and lipid utilisation between the pre- and post-expedition measurements were small for participants from both expeditions and there was no statistically significant difference between participants in the two expeditions. The range of values for the change in carbohydrate utilisation between the pre- and post-expedition measurements was much higher than the other substrates for participants from both expeditions, but once again there was no statistically significant difference between participants in the two expeditions. The first sleep period occurs before the 24-h measurement period and measurements may be affected by activities and diet prior to arrival for the start of the study. Within the 24-h measurement period, the upper quartile measured across participants in both studies for the pre- to post-expedition change in carbohydrate utilisation was higher during resting (ΔRMR) when compared with the second sleeping period (ΔSMR-2) and higher during the lowest intensity exercise (ΔEMR-80) when compared with higher intensity exercise (ΔEMR-100 and ΔEMR-120). The measurements used to determine the values for ΔRMR and ΔEMR-80 were both performed when participants were fasted whilst those for ΔEMR-100 and ΔEMR-120 are performed when participants were fed. We have previously reported that some, but not all participants in the Spear-17 expedition had increased carbohydrate utilisation when fasted which decreased when fed in the post-expedition measurements, a decrease that became absolute during sleep, rest and very low levels of physical activity<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. These changes were not seen in the female participants in the Ice Maiden expedition<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. However, the number of participants in each study was small and the spread of values large with a large overlap of values between participants in the two expeditions. In a study where lightly clad men and women were exposed to air temperatures at 5 °C, a higher carbohydrate utilisation was found in men when compared to women, but the spread of values for individual participants was large<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. The analysis reported in this paper show that the difference between pre- and post-expedition carbohydrate utilisation is larger in a subset of participants when they have fasted; a difference which reduces when they are fed. This subset includes participants from both expeditions. However, the impact of the environmental and physical activity on diurnal substrate utilisation, particularly in the fasted state, should be the focus of further investigation.</p><p id=\"Par44\">The same energy density values used in the determination of substrate utilisation from the measured O<sub>2</sub> and CO<sub>2</sub> in the whole body calorimeter were used for the determination of energy expenditure during the expeditions from Eqs. (<xref rid=\"Equ1\" ref-type=\"\">1</xref>) and (<xref rid=\"Equ2\" ref-type=\"\">2</xref>). Livesey and Elia<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup> showed that the chemical composition of substrates affects their energy density and the authors compare values for the energy density of fat and protein obtained from different studies. Using these data, the variation in energy density for fat across the different studies was about 2% and for protein was much higher at about 14%. A simple sensitivity analysis on our data showed that a ± 2.5% change in the energy density value of fat produced a maximum change in the calculated energy values during the expedition of < 40 kcal day<sup>−1</sup> (< 1.2%) and a ± 15% change in the median energy density of protein produced a maximum change < 125 kcal day<sup>−1</sup> (< 2%) in the median calculated energy values.</p><p id=\"Par45\">Perhaps the most important finding from this current work is that no difference was found between participants in the two expeditions for any of the energetics measures studies and therefore between male and female participants undertaking sustained expeditionary polar travel. The large range of values and the extensive overlap in values from participants in the two expeditions suggest that the differences have their origins in individual adaptation to an extreme environment rather than a systematic difference between men and women. This is consistent with findings at altitude where some studies have reported increased carbohydrate utilisation in men<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup> whilst other studies have failed to find an increase in men<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> or women<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>.</p><p id=\"Par46\">Previous research on healthy volunteers undertaking activities in extreme environments has generated new medical and physiological knowledge, including insights into the human skeleton, energetics<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref>,<xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>, cardiac function<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>, cerebral blood flow<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>, treatment of lung disease<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup> and the pre-hospital treatment of emergencies<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>. The work reported in this paper demonstrates the importance of appropriate physical and nutritional preparation before and appropriate nutrition during physical and physiological challenges. In a healthcare context, this includes the planning and post treatment care of patients undergoing surgery, and the planning intra- and post-treatment care of patients undergoing radiotherapy and chemotherapy. This is an area for research that we believe has received inadequate attention in the past.</p></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: John Hattersley and Adrian J. Wilson.</p></fn></fn-group><ack><title>Acknowledgements</title><p>We gratefully acknowledge the time and dedication that all members of the Spear-17 and Ice Maiden expeditions put into the projects. We also thank Research Nurse Alison Campbell and her nursing team for the data collection and David Dixon for technical support.</p></ack><notes notes-type=\"author-contribution\"><title>Authors contribution</title><p>J.H., A.W., R.G., J.F.-C., O.S., R.C., C.D.T., R.R., D.W. and C.I. were responsible for the conception and design of the ‘Spear-17’ and ‘Ice Maiden’ studies; J.H. and A.W. designed the chamber studies, performed the data analysis and interpretation. J.H. performed scientific management of data collection. The manuscript was prepared by J.H., A.W. (contributing equally as first author) and C.I.; J.H., A.W., R.G., J.F.-C., O.S., R.C., C.D.T., R.R., D.W. and C.I. reviewed, suggested improvements and approved the document.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par47\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mountjoy</surname><given-names>M</given-names></name><etal/></person-group><article-title>The IOC consensus statement: beyond the Female Athlete Triad-Relative Energy Deficiency in Sport (RED-S)</article-title><source>Br. J. Sports Med.</source><year>2014</year><volume>48</volume><fpage>491</fpage><lpage>497</lpage><pub-id pub-id-type=\"pmid\">24620037</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McGraw</surname><given-names>K</given-names></name><name><surname>Koehlmops</surname><given-names>TP</given-names></name><name><surname>Ritchie</surname><given-names>R</given-names></name></person-group><article-title>Women in combat: framing the issues of health and health research for America’s servicewomen</article-title><source>Mil. Med.</source><year>2016</year><volume>181</volume><issue>Suppl 1</issue><fpage>7</fpage><lpage>11</lpage><pub-id pub-id-type=\"pmid\">26741896</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cartee</surname><given-names>DG</given-names></name><name><surname>Douen</surname><given-names>AG</given-names></name><name><surname>Ramlal</surname><given-names>T</given-names></name><name><surname>Klip</surname><given-names>A</given-names></name><name><surname>Holloszy</surname><given-names>JO</given-names></name></person-group><article-title>Stimulation of glucose transport in skeletal muscle by hypoxia</article-title><source>J. Appl. Physiol.</source><year>1991</year><volume>70</volume><fpage>1593</fpage><lpage>1600</lpage><pub-id pub-id-type=\"pmid\">2055841</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Hochachka</surname><given-names>PW</given-names></name></person-group><person-group person-group-type=\"editor\"><name><surname>Gilles</surname><given-names>R</given-names></name></person-group><article-title>Exercise limitations at high altitude: the metabolic problem and search for its solution</article-title><source>Circulation, Respiration, and Metabolism</source><year>1985</year><publisher-loc>Berlin</publisher-loc><publisher-name>Springer</publisher-name><fpage>240</fpage><lpage>249</lpage></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>O’Hara</surname><given-names>JP</given-names></name><etal/></person-group><article-title>A comparison of substrate oxidation during prolonged exercise in men at terrestrial altitude and normobaric normoxia following the coingestion of 13C glucose and 13C fructose</article-title><source>Physiol. Rep.</source><year>2017</year><volume>5</volume><fpage>1</fpage></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Braun</surname><given-names>B</given-names></name><etal/></person-group><article-title>Women at altitude: carbohydrate utilisation during exercise at 4,300m</article-title><source>J. Appl. Physiol.</source><year>2000</year><volume>88</volume><fpage>246</fpage><lpage>256</lpage><pub-id pub-id-type=\"pmid\">10642387</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ocobock</surname><given-names>C</given-names></name></person-group><article-title>Human energy expenditure, allocation, and interactions in natural temperate, hot, and cold environments?</article-title><source>Am. J. Phys Anthr.</source><year>2016</year><volume>161</volume><fpage>667</fpage><lpage>675</lpage></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schoeller</surname><given-names>DA</given-names></name><etal/></person-group><article-title>Energy expenditure by doubly labelled water: validation in humans and proposed calculation</article-title><source>Am. J. Physiol.</source><year>1986</year><volume>250</volume><fpage>R823</fpage><lpage>R830</lpage><pub-id pub-id-type=\"pmid\">3085521</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fern</surname><given-names>EB</given-names></name><name><surname>Garlick</surname><given-names>PJ</given-names></name><name><surname>Waterlow</surname><given-names>JC</given-names></name></person-group><article-title>The concept of the single body pool of metabolic Nitrogen in determining the rate of whole body nitrogen turnover</article-title><source>Hum. Nutr. Clin Nutr.</source><year>1985</year><volume>39</volume><fpage>85</fpage><lpage>89</lpage><pub-id pub-id-type=\"pmid\">3894301</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stroud</surname><given-names>MA</given-names></name><etal/></person-group><article-title>Energy Expenditure using isotope-labelled water (<sup>2</sup>H<sub>2</sub><sup>18</sup>O ), exercise performance, skeletal muscle enzyme activities and plasma biochemical parameters in humans during 95 days of endurance exercise with inadequate energy intake</article-title><source>Eur. J. Appl. Physiol.</source><year>1997</year><volume>76</volume><fpage>243</fpage><lpage>252</lpage></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stroud</surname><given-names>MA</given-names></name><name><surname>Jackson</surname><given-names>AA</given-names></name><name><surname>Waterlow</surname><given-names>JC</given-names></name></person-group><article-title>Protein turnover rates of two human subjects during an unassisted crossing of Antarctica</article-title><source>Brit. J. Nutr.</source><year>1996</year><volume>76</volume><fpage>165</fpage><lpage>174</lpage><pub-id pub-id-type=\"pmid\">8813892</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stroud</surname><given-names>MA</given-names></name><name><surname>Coward</surname><given-names>WA</given-names></name><name><surname>Sawyer</surname><given-names>MB</given-names></name></person-group><article-title>Measurement of energy expenditure using isotope-labelled water (<sup>2</sup>H<sub>2</sub><sup>18</sup>O) during an Arctic expedition</article-title><source>Eur. J. Appl. Physiol.</source><year>1993</year><volume>67</volume><fpage>375</fpage><lpage>379</lpage></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Junghans</surname><given-names>P</given-names></name><etal/></person-group><article-title>Studies of protein and the energy metabolism in man during a wintering in Antarctica</article-title><source>Isot. Environ. Health Stud.</source><year>2012</year><volume>48</volume><fpage>208</fpage><lpage>225</lpage></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stroud</surname><given-names>M</given-names></name></person-group><article-title>Effects on energy expenditure of facial cooling during exercise</article-title><source>Eur. J Appl. Physiol.</source><year>1991</year><volume>63</volume><fpage>376</fpage><lpage>380</lpage></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stroud</surname><given-names>MA</given-names></name></person-group><article-title>Did Scott simply starve? Lessons from The footsteps Scott Antarct</article-title><source>Exped. Proc. Nutr. Soc.</source><year>1987</year><volume>46</volume><fpage>2</fpage></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>O’Brien</surname><given-names>KA</given-names></name><etal/></person-group><article-title>Human physiological and metabolic responses to an attempted winter crossing of Antarctica: the effects of prolonged hypobaric hypoxia</article-title><source>Physiol. Rep.</source><year>2018</year><volume>6</volume><fpage>5</fpage></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hattersley</surname><given-names>J</given-names></name><etal/></person-group><article-title>Metabolic rate and substrate utilisation resilience in men undertaking polar expeditionary travel</article-title><source>PLoS ONE</source><year>2019</year><volume>14</volume><fpage>8</fpage></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hattersley</surname><given-names>J</given-names></name><etal/></person-group><article-title>Pre- to post-expedition changes in the energy usage of women undertaking sustained expeditionary polar travel</article-title><source>J. Appl. Physiol.</source><year>2019</year><volume>126</volume><fpage>681</fpage><lpage>690</lpage><pub-id pub-id-type=\"pmid\">30571278</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Frykman</surname><given-names>PN</given-names></name><etal/></person-group><article-title>Effects of a 3-month endurance event on physical performance and body composition: the G2 trans-Greenland expedition</article-title><source>Wilderness Environ.</source><year>2003</year><volume>14</volume><fpage>240</fpage><lpage>248</lpage></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>O’Brien</surname><given-names>KA</given-names></name><etal/></person-group><article-title>Metabolomic and lipidomic changes in human participants ascending to Everest base camp</article-title><source>Sci. Rep.</source><year>2019</year><volume>9</volume><fpage>2292</fpage><pub-id pub-id-type=\"pmid\">30783162</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Siervo</surname><given-names>M</given-names></name><etal/></person-group><article-title>Effects of prolonged exposure to hypobaric hypoxia on oxidative stress, inflammation and gluco-insular regulation: the not-so-sweet price for good regulation</article-title><source>PLoS ONE</source><year>2014</year><volume>9</volume><fpage>4</fpage></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gagnon</surname><given-names>DD</given-names></name><name><surname>Pullinen</surname><given-names>T</given-names></name><name><surname>Karinen</surname><given-names>H</given-names></name><name><surname>Rintamaki</surname><given-names>H</given-names></name><name><surname>Kryolainen</surname><given-names>H</given-names></name></person-group><article-title>Recovery of hormonal, blood lipid and haematological profiles from a north pole expedition</article-title><source>Aviat. Space Environ. Med.</source><year>2011</year><volume>82</volume><fpage>1110</fpage><lpage>1117</lpage><pub-id pub-id-type=\"pmid\">22195391</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gifford</surname><given-names>RM</given-names></name><name><surname>Reynolds</surname><given-names>RM</given-names></name><name><surname>Greeves</surname><given-names>J</given-names></name><name><surname>Anderson</surname><given-names>RA</given-names></name><name><surname>Woods</surname><given-names>DR</given-names></name></person-group><article-title>Reproductive dysfunction and associated pathology in women undergoing military training</article-title><source>J. R. Army Med. Corps</source><year>2017</year><volume>163</volume><fpage>301</fpage><lpage>310</lpage><pub-id pub-id-type=\"pmid\">28213614</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gibbs</surname><given-names>JC</given-names></name><name><surname>Williams</surname><given-names>NI</given-names></name><name><surname>De Souza</surname><given-names>MJ</given-names></name></person-group><article-title>Prevalence of individual and combined components of the female athlete triad</article-title><source>Med. Sci. Sports Exerc.</source><year>2013</year><volume>45</volume><fpage>985</fpage><lpage>996</lpage><pub-id pub-id-type=\"pmid\">23247706</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pettit</surname><given-names>SE</given-names></name><name><surname>Marchand</surname><given-names>I</given-names></name><name><surname>Graham</surname><given-names>T</given-names></name></person-group><article-title>Gender differences in the cardiovascular and catecholamine responses to cold-air exposure at rest</article-title><source>Can. J. Appl. Physiol</source><year>1999</year><volume>24</volume><fpage>131</fpage><lpage>147</lpage><pub-id pub-id-type=\"pmid\">10198139</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><mixed-citation publication-type=\"other\">Taylor, N. <italic>et al.</italic> Experience from the selection and nutritional preparation for Expedition ICE MAIDEN: the first successful all-female unassisted Antarctic traverse. <italic>J. R. Army Med. Corps</italic><bold>15</bold> (2019).</mixed-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schoffelen</surname><given-names>PFM</given-names></name><name><surname>Westererp</surname><given-names>KR</given-names></name><name><surname>Saris</surname><given-names>WHM</given-names></name><name><surname>ten Hoor</surname><given-names>F</given-names></name></person-group><article-title>A dual chamber respiration system for automated calibration</article-title><source>J. Appl. Physiol.</source><year>1997</year><volume>83</volume><fpage>2064</fpage><lpage>2072</lpage><pub-id pub-id-type=\"pmid\">9390982</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hattersley</surname><given-names>J</given-names></name><name><surname>Schoffelen</surname><given-names>P</given-names></name><name><surname>Arnoldussen</surname><given-names>E</given-names></name><name><surname>Wilson</surname><given-names>A</given-names></name></person-group><article-title>Impact of improved environment control and sample conditioning on the accuracy of respiratory rooms</article-title><source>Proc. RACMEM (Maastricht, Netherlands)</source><year>2011</year><volume>2011</volume><fpage>2</fpage><lpage>4</lpage></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bingham</surname><given-names>SA</given-names></name><name><surname>Williams</surname><given-names>R</given-names></name><name><surname>Cole</surname><given-names>TJ</given-names></name><name><surname>Price</surname><given-names>CP</given-names></name><name><surname>Cummings</surname><given-names>JH</given-names></name></person-group><article-title>Reference values for analytes of 24-h urine collections known to be complete</article-title><source>Ann. Clin. Biochem.</source><year>1988</year><volume>25</volume><fpage>610</fpage><lpage>619</lpage><pub-id pub-id-type=\"pmid\">3267076</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weir</surname><given-names>JBV</given-names></name></person-group><article-title>New methods for calculating metabolic rate with special reference to protein metabolism</article-title><source>J. Physiol.</source><year>1949</year><volume>109</volume><fpage>1</fpage><lpage>9</lpage><pub-id pub-id-type=\"pmid\">15394301</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brouwer</surname><given-names>E</given-names></name></person-group><article-title>On simple formulae for calculating the heat expenditure and the quantities of carbohydrate and fat oxidised in metabolism of men and animals, from gaseous exchange (oxygen intake and carbonic acid output) and urine-n</article-title><source>Acta Physiol. Pharmacol. Neerl.</source><year>1957</year><volume>6</volume><fpage>795</fpage><lpage>802</lpage><pub-id pub-id-type=\"pmid\">13487422</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Frayn</surname><given-names>KN</given-names></name></person-group><article-title>Calculations of substrate oxidation rates in vivo from gaseous exchange</article-title><source>J. Appl. Physiol.</source><year>1983</year><volume>55</volume><fpage>628</fpage><lpage>634</lpage><pub-id pub-id-type=\"pmid\">6618956</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Westerterp</surname><given-names>KR</given-names></name></person-group><article-title>Diet induced thermogenesis</article-title><source>Nutr. Metab.</source><year>2004</year><volume>1</volume><fpage>5</fpage></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Siegel</surname><given-names>S</given-names></name></person-group><source>Non-parametric Statistics for the Behavioural Sciences</source><year>1956</year><publisher-loc>New York</publisher-loc><publisher-name>McGarw Hill</publisher-name></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arch</surname><given-names>JRS</given-names></name><name><surname>Hislop</surname><given-names>D</given-names></name><name><surname>Wang</surname><given-names>SJY</given-names></name><name><surname>Speakman</surname><given-names>JR</given-names></name></person-group><article-title>Some mathematical and technical issues in the measurement and interpretation of open-circuit indirect calorimetry in small animals</article-title><source>Int. J. Obes.</source><year>2006</year><volume>30</volume><fpage>1322</fpage><lpage>1331</lpage></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kaiyala</surname><given-names>KJ</given-names></name><name><surname>Schwartz</surname><given-names>MW</given-names></name></person-group><article-title>Toward a more complete (and less controversial) understanding of energy expenditure and its role in obesity pathogenesis</article-title><source>Diabetes</source><year>2011</year><volume>60</volume><fpage>17</fpage><lpage>23</lpage><pub-id pub-id-type=\"pmid\">21193735</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bosy-Westphal</surname><given-names>A</given-names></name><name><surname>Braun</surname><given-names>W</given-names></name><name><surname>Schautz</surname><given-names>B</given-names></name><name><surname>Müller</surname><given-names>MJ</given-names></name></person-group><article-title>Issues in characterizing resting energy expenditure in obesity and after weight loss</article-title><source>Physiol. Front.</source><year>2013</year><pub-id pub-id-type=\"doi\">10.3389/fphys.2013.00047</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ravussin</surname><given-names>E</given-names></name><name><surname>Lillioja</surname><given-names>S</given-names></name><name><surname>Anderson</surname><given-names>TE</given-names></name><name><surname>Christin</surname><given-names>L</given-names></name><name><surname>Bogardus</surname><given-names>C</given-names></name></person-group><article-title>Determinants of 24-hour energy expenditure in man: methods and results using a respiratory chamber</article-title><source>J. Clin. Invest.</source><year>1986</year><volume>78</volume><fpage>1568</fpage><lpage>1578</lpage><pub-id pub-id-type=\"pmid\">3782471</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tikuisis</surname><given-names>P</given-names></name><name><surname>Jacobs</surname><given-names>I</given-names></name><name><surname>Moroz</surname><given-names>D</given-names></name><name><surname>Vallerand</surname><given-names>AL</given-names></name><name><surname>Martineau</surname><given-names>L</given-names></name></person-group><article-title>Comparison of thermoregulatory responses between mean and women immersed in cold water</article-title><source>J. Appl. Physiol.</source><year>2000</year><volume>89</volume><fpage>1403</fpage><lpage>1411</lpage><pub-id pub-id-type=\"pmid\">11007575</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kenny</surname><given-names>NA</given-names></name><name><surname>Warland</surname><given-names>JS</given-names></name><name><surname>Brown</surname><given-names>RD</given-names></name><name><surname>Gillespie</surname><given-names>G</given-names></name></person-group><article-title>Part A: assessing the performance of the COMFA outdoor thermal comfort model on subjects performing physical activity</article-title><source>Int. J. Biometeoerol.</source><year>2009</year><volume>53</volume><fpage>415</fpage><lpage>428</lpage></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gao</surname><given-names>W</given-names></name><name><surname>Brooks</surname><given-names>GA</given-names></name><name><surname>Klonoff</surname><given-names>DC</given-names></name></person-group><article-title>Wearable physiological systems and technologies for metabolic monitoring</article-title><source>J. Appl. Physiol.</source><year>2018</year><volume>124</volume><fpage>548</fpage><lpage>556</lpage><pub-id pub-id-type=\"pmid\">28970200</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Livesey</surname><given-names>G</given-names></name><name><surname>Elia</surname><given-names>M</given-names></name></person-group><article-title>Estimation of energy expenditure, net carbohydrate utilization, and net fat oxidation and synthesis by indirect calorimetry: evaluation of errors with special reference to the detailed composition of fuels</article-title><source>Am. J. Clin. Nutr.</source><year>1988</year><volume>47</volume><fpage>608</fpage><lpage>628</lpage><pub-id pub-id-type=\"pmid\">3281434</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><mixed-citation publication-type=\"other\">Edwards, L. M. <italic>et al.</italic> The effect of high-altitude on human skeletal muscle energetics: P-MRS results from the Caudwell Xtreme Everest expedition. <italic>PLoS ONE</italic><bold>5</bold> (2010).</mixed-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Levett</surname><given-names>DZ</given-names></name><etal/></person-group><article-title>Acclimatization of skeletal muscle mitochondria to high-altitude hypoxia during an ascent of Everest</article-title><source>J. FASEB’14</source><year>2012</year><volume>26</volume><fpage>1431</fpage></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Holloway</surname><given-names>CJ</given-names></name><etal/></person-group><article-title>Cardiac response to hypobaric hypoxia: persistent changes in cardiac mass, function, and energy metabolism after a trek to Mt. Everest Base Camp</article-title><source>J. FASEB’96</source><year>2011</year><volume>25</volume><fpage>792</fpage></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wilson</surname><given-names>MH</given-names></name><etal/></person-group><article-title>Cerebral artery dilatation maintains cerebral oxygenation at extreme altitude and in acute hypoxia an ultrasound and MRI study</article-title><source>J. Cereb. Blood Flow Metab.</source><year>2011</year><volume>31</volume><fpage>2019</fpage><pub-id pub-id-type=\"pmid\">21654697</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Imray</surname><given-names>CH</given-names></name><name><surname>Grocott</surname><given-names>MP</given-names></name><name><surname>Wilson</surname><given-names>MH</given-names></name><name><surname>Hughes</surname><given-names>A</given-names></name><name><surname>Auerbach</surname><given-names>PS</given-names></name></person-group><article-title>Extreme, expedition and wilderness medicine</article-title><source>Lancet</source><year>2015</year><volume>386</volume><fpage>2520</fpage><lpage>2525</lpage><pub-id pub-id-type=\"pmid\">26738718</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807850</article-id><article-id pub-id-type=\"pmc\">PMC7431585</article-id><article-id pub-id-type=\"publisher-id\">70004</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70004-2</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>A dual mechanism underlying retroactive shifts of auditory spatial attention: dissociating target- and distractor-related modulations of alpha lateralization</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Klatt</surname><given-names>Laura-Isabelle</given-names></name><address><email>klatt@ifado.de</email></address><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><contrib contrib-type=\"author\"><name><surname>Getzmann</surname><given-names>Stephan</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><contrib contrib-type=\"author\"><name><surname>Begau</surname><given-names>Alexandra</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><contrib contrib-type=\"author\"><name><surname>Schneider</surname><given-names>Daniel</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\"/></contrib><aff id=\"Aff1\"><institution-wrap><institution-id institution-id-type=\"GRID\">grid.419241.b</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2285 956X</institution-id><institution>Leibniz Research Centre for Working Environment and Human Factors, </institution></institution-wrap>Ardeystraße 67, 44139 Dortmund, Germany </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13860</elocation-id><history><date date-type=\"received\"><day>27</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>21</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Attention can be allocated to mental representations to select information from working memory. To date, it remains ambiguous whether such retroactive shifts of attention involve the inhibition of irrelevant information or the prioritization of relevant information. Investigating asymmetries in posterior alpha-band oscillations during an auditory retroactive cueing task, we aimed at differentiating those mechanisms. Participants were cued to attend two out of three sounds in an upcoming sound array. Importantly, the resulting working memory representation contained one laterally and one centrally presented item. A centrally presented retro-cue then indicated the lateral, the central, or both items as further relevant for the task (comparing the cued item(s) to a memory probe). Time–frequency analysis revealed opposing patterns of alpha lateralization depending on target eccentricity: A contralateral decrease in alpha power in <italic>target lateral</italic> trials indicated the involvement of target prioritization. A contralateral increase in alpha power when the central item remained relevant (<italic>distractor lateral</italic> trials) suggested the de-prioritization of irrelevant information. No lateralization was observed when both items remained relevant, supporting the notion that auditory alpha lateralization is restricted to situations in which spatial information is task-relevant. Altogether, the data demonstrate that retroactive attentional deployment involves excitatory and inhibitory control mechanisms.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Working memory</kwd><kwd>Cognitive neuroscience</kwd><kwd>Attention</kwd><kwd>Neuroscience</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">In everyday life, we frequently rely on selective attention in order to focus on information that is relevant while ignoring behaviorally irrelevant sensory input. Without such an attentional filter, we would be overwhelmed by the sheer abundance of sensory information. Analogously, selective attention can operate on working memory contents that are no longer physically present in the environment. Such retroactive shifts of attention are critical in order to adapt to changing task demands and allow for an efficient allocation of limited mental storage resources. The deployment of covert spatial attention to one side in mnemonic (or perceptual) space has been linked to spatially-specific modulations of alpha oscillations<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. Typically, there is a relative decrease in alpha power over posterior scalp sites contralateral to the attended location, while alpha power increases contralateral to the unattended location. Based on the gating-by-inhibition framework by Jensen and Mazaheri<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>, low alpha power has been proposed to reflect a state of high excitability in the respective neural areas, whereas high alpha power reflects the functional inhibition of task-irrelevant regions.</p><p id=\"Par3\">Analogously, two mechanisms could underlie the selection of information from working memory: shifting attention within working memory may either <italic>facilitate</italic> or <italic>strengthen</italic> the relevant information, or, on the other hand, the no longer relevant contents may be <italic>inhibited</italic> and thereby dropped from the focus of attention within working memory. Although many studies investigating alpha lateralization interpret their findings in terms of an inhibition account, very few have been successful in actually dissociating those two mechanisms<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. That is largely due to the fact that the majority of studies has used lateralized stimulus displays, in which targets and distractors are presented in opposite hemifields. Thus, a lateralization of alpha power in response to a shift of attention towards a left-sided target can be likewise due to a contralateral (i.e., right-hemispheric) decrease in alpha power (reflecting target prioritization) or to an ipsilateral (i.e., left-hemispheric) increase in alpha power (reflecting distractor inhibition). Notably, the same debate and issues apply to the perceptual domain, that is, to the focusing of attention on relevant aspects of the external environment (for a review, see<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>).</p><p id=\"Par4\">Here, we aimed at distinguishing those mechanisms using an auditory working memory paradigm. In an auditory retroactive cueing task (design adapted from a previous experiment in the visual modality: Schneider et al.<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>), participants were initially cued to attend two out of three sounds in an upcoming sound array. In any case, the resulting working memory representation contained one laterally (left or right) and one centrally presented item. A retroactive cue (retro-cue) then indicated either one (i.e., selective retro-cue) or both (i.e., neutral retro-cue) of those items as further relevant for the task, which required participants to compare the cued item(s) to a centrally presented probe stimulus. Participants were instructed to indicate whether the probe stimulus was equal to the retro-cued item(s) or not. The probe stimulus could be either a new sound (i.e., a sound that never appeared in the given trial; no response), the cued sound (i.e., the sound indicated as relevant by the retro-cue; yes response), or the non-cued sound (i.e., the sound indicated as irrelevant by the retro-cue; no response).</p><p id=\"Par5\">This design (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>) entails two major strengths that differ from previous (predominantly visual) retro-cueing studies. First, the retro-cue (as well as the pre-cue) was presented from a central position behind the participants’ head, eliminating the risk that associated EEG asymmetries reflect the processing of the cue rather than the attentional selection or inhibition of working memory items. Second, the key aspect of this design was that only either the target (i.e., the cued item) or the distractor (i.e., the non-cued item) was lateralized. This spatial arrangement of stimuli in the sound array was essential to make sure that hemispheric asymmetries in the alpha frequency-band following the retro-cue could be unambiguously linked to either the processing of the target or the distractor. The reasoning behind this design is based on the organization of afferent auditory connections and the resulting implications for hemispheric differences in processing: It is known that the contralateral projections, transmitting auditory input, are stronger and more preponderant than the ipsilateral projections<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>. Thus, the contralateral hemisphere should predominately process the attended stimulus, whereas the ipsilateral hemisphere should predominantly process the unattended stimulus. A centrally presented sound, however, should be equally represented in both hemispheres. According to this, if the selection of information from auditory working memory involves the spatially-specific inhibition of irrelevant information at a lateralized position, a contralateral increase in alpha power should be evident when the central working memory item remains relevant (i.e., when the lateral, non-cued item becomes irrelevant, <italic>distractor lateral condition</italic>). Here, the non-cued item is the only lateralized stimulus; hence, alpha lateralization should be unambiguously related to the processing (i.e., inhibition) of the distractor. If, however, retroactive shifts of auditory attention involve the prioritization of relevant information, a contralateral decrease in alpha power should be evident when the lateral working memory item remains relevant (i.e., when the central, non-cued item becomes irrelevant; <italic>target lateral condition</italic>). Again, since the cued item is the only lateralized stimulus in that case, any hemispheric asymmetry in the alpha frequency band can solely be related to the processing of the target. Obviously, those two possibilities are not mutually exclusive and may as well both contribute to successful selection of information. Yet, the experimental design allows us to distinguish the two from one another. A third neutral retro-cue condition, in which both items remained relevant, served as a control condition. A neutral cue did neither require a re-orienting of attention within working memory nor the access to the stored spatial position of the memorized sounds. Instead, once the neutral retro-cue appeared, it was sufficient to maintain the sounds’ identities. Thus, in line with previous results, showing that alpha lateralization is limited to situations in which spatial position is a task-relevant feature<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>, we did not expect an asymmetry following neutral retro-cues.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Schematic illustration of the task design. The three front loudspeakers were located at azimuthal positions of − 90°, 0°, 90° in the horizontal plane. The back loudspeaker, through which pre-cue, retro-cue, and probe were presented, was located right behind the participant’s head at an approximate distance of 30 cm. <italic>ISI</italic> inter-stimulus-interval.</p></caption><graphic xlink:href=\"41598_2020_70004_Fig1_HTML\" id=\"MO1\"/></fig></p></sec><sec id=\"Sec2\"><title>Results</title><sec id=\"Sec3\"><title>Behavioral results</title><p id=\"Par6\">To investigate whether a selective retro-cue, allowing for working memory updating and a reduction in working memory load, led to an improvement in task performance compared to neutral retro-cues, we conducted paired sample <italic>t</italic>-tests for response times and accuracy. Participants performed significantly faster (<italic>t</italic><sub>(19)</sub> = − 7.84, <italic>p</italic> < 0.001, <italic>p</italic><sub>adj</sub> < 0.001, <italic>g</italic> = − 0.65, BF > 1,000) when only one item remained relevant (i.e., selective retro-cue trials) than when both items remained relevant (i.e., neutral retro-cue trials). Accuracy did not differ between the two conditions (<italic>t</italic><sub>(19)</sub> = 0.72, <italic>p</italic> = 0.481, <italic>p</italic><sub>adj</sub> = 0.721, <italic>g</italic> = 0.09, BF = 0.29).</p><p id=\"Par7\">In addition, to differentiate the effects of different probe types (cued, non-cued, new), we performed a one-way repeated-measures analysis of variance (rANOVA) for response times and a Friedman’s ANOVA for accuracy. This analysis allows us to shed light on the fate of the non-cued information; that is, it demonstrates whether or not the non-cued item (which was indicated as <italic>irrelevant</italic> by the retro-cue) was completely removed (i.e., successfully inhibited) from working memory. Critically, although non-cued items could be probed, the retro-cue was always 100% valid (i.e., knowing the cued item was always sufficient for solving the task). That is, there was no incentive for participants to retain the non-cued item in working memory. We expected selective retro-cue trials probing the non-cued item to result in slower and less accurate responses compared to trials in which the probe had never appeared in the current trial (i.e., new probe). That is, due to the interference from previously relevant information (i.e., the non-cued item), performance was expected to decline. For this comparison to work properly, neutral trials were excluded from the rANOVA, since there was no “non-cued item” when both items remained relevant.</p><p id=\"Par8\">The rANOVA revealed a significant effect of probe type on response times (<italic>F</italic><sub>(2,38)</sub> = 39.56, <italic>p</italic> < 0.001, <italic>p</italic><sub>adj</sub> < 0.001, ηp<sup>2</sup> = 0.68, ε = 0.93) as well as on accuracy (χ<sup>2</sup><sub>(2)</sub> = 27.1, <italic>p</italic> < 0.001, <italic>p</italic><sub>adj</sub> < 0.001). In line with our hypotheses, post-hoc comparisons revealed that participants responded slower (<italic>t</italic><sub>(19)</sub> = 2.59, <italic>p</italic> = 0.018, <italic>p</italic><sub>adj</sub> = 0.033, <italic>g</italic> = 0.18, BF = 3.17) as well as less accurate (<italic>t</italic><sub>(19)</sub> = − 10.32, <italic>p</italic> < 0.001, <italic>p</italic><sub>adj</sub> < 0.001, <italic>g</italic> = − 1.63, BF > 1,000) in trials with non-cued compared to new probes. In addition, participants responded significantly faster (<italic>t</italic><sub>(19)</sub> = − 5.50, p < 0.001, <italic>p</italic><sub>adj</sub> < 0.001, <italic>g</italic> = − 0.49, BF = 917.85) as well as less accurate (<italic>t</italic><sub>(19)</sub> =  − 7.05, <italic>p</italic> < 0.001, <italic>p</italic><sub>adj</sub> < 0.001, <italic>g</italic> = − 1.95, BF > 1,000) when the cued item was probed as opposed to a new item. When contrasting trials with cued versus non-cued probes, post-hoc tests showed a significant difference in response times (<italic>t </italic><sub>(19)</sub> = − 9.38, <italic>p</italic> < 0.001, <italic>p</italic><sub>adj</sub> < 0.001, <italic>g</italic> = − 0.68, BF > 1,000), but not in accuracy (<italic>t</italic><sub>(19)</sub> = 0.17, <italic>p</italic> = 0.871, <italic>p</italic><sub>adj</sub> = 1.60, <italic>g</italic> = 0.04, BF = 0.24). The described behavioral results are illustrated in Figs. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> and <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Accuracy (<bold>a</bold>) and response times (<bold>b</bold>), depending on retro-cue type and probe type. Boxplots illustrate the interquartile range and the median. Whiskers extend to 1.5 times the interquartile range. The dots indicate individual participants’ mean scores per condition. A black cross denotes the mean values across subjects for each condition. Note that in neutral retro-cue trials both items remained relevant and thus, a “cued probe” could be either one of those two sounds.</p></caption><graphic xlink:href=\"41598_2020_70004_Fig2_HTML\" id=\"MO2\"/></fig><fig id=\"Fig3\"><label>Figure 3</label><caption><p>Accuracy (<bold>a</bold>,<bold>c</bold>) and response times (<bold>b</bold>,<bold>d</bold>), depending on the comparisons considered for statistical analyses. To test for benefits of selective retro-cues, performance in selective and neutral retro-cues was compared, using paired sample <italic>t</italic>-tests. Because in neutral retro-cue trials, it was not possible to probe a non-cued item, the data depicted for selective retro-cues do not include non-cued probe trials (<bold>a</bold>,<bold>b</bold>). Accuracy (<bold>c</bold>) and response times (<bold>d</bold>), depending on probe type, refer exclusively to selective retro-cue trials because neutral retro-cue trials did not allow for the main comparison of interest between non-cued and new items. A repeated-measured ANOVA was conducted to test for effects of probe type. For further details on statistical analyses see the methods section. Boxplots illustrate the interquartile range and the median. Whiskers extend to 1.5 times the interquartile range. The dots indicate individual participants’ mean scores per condition. A black cross denotes the mean values across subjects for each condition.</p></caption><graphic xlink:href=\"41598_2020_70004_Fig3_HTML\" id=\"MO3\"/></fig></p></sec><sec id=\"Sec4\"><title>Alpha lateralization as a correlate of target prioritization versus Inhibition</title><p id=\"Par9\">To differentiate whether attentional selection within auditory working memory is associated with target prioritization or distractor inhibition, we calculated the Alpha Lateralization Index (ALI) for target lateral, distractor lateral, as well as neutral retro-cue trials. The ALI indicates the ratio of ipsilateral minus contralateral alpha power and the total power across both hemispheres [ALI = ipsilateral − contralateral alpha power / ipsilateral + contralateral alpha power]. Ipsilateral and contralateral refer to the hemispheres (or electrode positions) relative to the lateralized item. Thus, positive ALI values indicate a contralateral decrease of alpha power (i.e., the processing of the lateralized information) and negative ALI values indicate a contralateral increase of alpha power (i.e., the suppression of the lateralized information).</p><p id=\"Par10\">As illustrated in Figs. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref> and <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>, opposite patterns of lateralization were observed when comparing target lateral (Figs. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>a, <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>a) and distractor lateral trials (Figs. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>b, <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>b). That is, target lateral trials resulted in a contralateral decrease of alpha power, whereas distractor lateral trials resulted in a contralateral increase of alpha power. A cluster-based permutation analysis confirmed this impression, revealing a significant difference between conditions in the alpha frequency band following the retro-cue (cluster size > 95th percentile of null-distribution and <italic>p</italic> < 0.05, Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>c). Follow-up analyses were performed on mean alpha power (8–13 Hz) in an approximate time-window derived from this comparison, ranging from 700 to 1,300 ms post retro-cue onset: Paired-sample <italic>t</italic>-tests revealed a significant difference between neutral and target lateral trials (<italic>t</italic><sub>(19)</sub> = − 3.45, <italic>p</italic> = 0.003, <italic>p</italic><sub>adj</sub> = 0.016, <italic>g</italic> = − 0.98, BF = 15.46), whereas the difference between neutral trials (Figs. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>d, <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>c) and distractor lateral trials failed to reach statistical significance (<italic>t</italic><sub>(19)</sub> = − 1.71, <italic>p</italic> = 0.104, <italic>p</italic><sub>adj</sub> = 0.297, <italic>g</italic> = − 0.52, BF = 0.80). Consistent with the reported <italic>p </italic>values, the BF provided strong support for the alternative hypothesis in the neutral versus target lateral comparison, whereas it was rather inconclusive for the neutral versus distractor lateral comparison.<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Alpha lateralization results for target lateral, distractor lateral, and neutral trials. Time–frequency plots (<bold>a</bold>,<bold>b</bold>,<bold>d</bold>) show the average lateralization indices at posterior electrodes (PO7/8, P7/8, P5/6, and PO3/4) per condition. The black dashed line indicates retro-cue onset. Target lateral and distractor lateral trials were contrasted using cluster permutation statistics. The resulting significant cluster (<italic>p</italic> < .05 and size > 95th percentile of the null distribution) is framed by a black line in the target lateral minus distractor lateral difference plot (<bold>c</bold>). Corresponding topographies are based on the normalized difference between ipsilateral and contralateral alpha power following the retro-cue (<bold>e</bold>).</p></caption><graphic xlink:href=\"41598_2020_70004_Fig4_HTML\" id=\"MO4\"/></fig><fig id=\"Fig5\"><label>Figure 5</label><caption><p>Alpha lateralization results depending on condition. Line plots for the target lateral (<bold>a</bold>), distractor lateral (<bold>b</bold>), and neutral condition (<bold>c</bold>) illustrate the contralateral and ipsilateral portion of alpha power (raw power values) for a posterior cluster of electrodes (PO7/8, P7/8, P5/6, and PO3/4). The bottom, right plot (<bold>d</bold>) shows the Alpha Lateralization Index (ALI) values for all three conditions. The analysis time window is highlighted in grey.</p></caption><graphic xlink:href=\"41598_2020_70004_Fig5_HTML\" id=\"MO5\"/></fig></p><p id=\"Par11\">In order to verify that the observed hemispheric asymmetries within conditions were significantly different from zero, one-sample <italic>t</italic>-tests were conducted: The results suggested that there was a reliable lateralization of alpha power in the target lateral (<italic>t</italic><sub>(19)</sub> = 2.85, <italic>p</italic> = 0.010, <italic>p</italic><sub>adj</sub> = 0.039, <italic>g</italic><sub><italic>1</italic></sub> = 0.64, BF = 5.07) as well as the distractor lateral condition (<italic>t</italic><sub>(19)</sub> = − 3.58, <italic>p</italic> = 0.002, <italic>p</italic><sub>adj</sub> = 0.016, <italic>g</italic><sub><italic>1</italic></sub> = − 0.80, BF = 20.20). With BFs greater than 3 and 10, respectively, those tests provide moderate and strong support for the alternative hypothesis (i.e., the lateralization is significantly different from zero). In contrast, there was no evidence for a lateralization of alpha power in the neutral retro-cue condition (<italic>t</italic><sub>(19)</sub> = − 1.54, <italic>p</italic> = 0.139, <italic>p</italic><sub>adj</sub> = 0.317, <italic>g</italic><sub><italic>1</italic></sub> = − 0.35, BF = 0.64). The corresponding BF did neither support the null nor the alternative hypothesis. Scalp topographies corresponding to the alpha lateralization indices are depicted in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>e. Related studies from the visual domain have previously raised concerns that posterior alpha power asymmetries might be confounded by lateral saccadic eye movement artifacts. See the supplementary material (Appendix A1) for a control analysis, invalidating such confounding effects.</p><p id=\"Par12\">In addition to alpha lateralization, the line plots in Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>a–c clearly illustrate that there is a bilateral suppression of alpha power following retro-cue onset. For an analysis of non-lateralized alpha power modulations, we refer the reader to the supplementary material (Appendix A2).</p></sec></sec><sec id=\"Sec5\"><title>Discussion</title><p id=\"Par13\">The present study investigated the mechanisms underlying retroactive attentional orienting within auditory working memory. We demonstrated that signatures of target-related and distractor-related alpha lateralization can be dissociated. Specifically, by manipulating the spatial arrangement of targets and distractors such that only one of them appeared in a lateralized position, we were able to show that a retro-cue induced shift of attention towards a <italic>lateralized target</italic> resulted in a contralateral <italic>decrease</italic> in alpha power; in contrast, shifting attention to a centrally presented target and away from a <italic>lateralized distractor</italic> resulted in a contralateral <italic>increase</italic> in alpha power. The findings support the interpretation of alpha lateralization as a dual mechanism underlying the deployment of attention within working memory.</p><sec id=\"Sec6\"><title>Retro-cue benefits: effects on accuracy and response times</title><p id=\"Par14\">On the behavioral level, participants clearly benefited from selective retro-cues, reducing the amount of relevant information to be maintained in working memory. While participants were initially required to maintain two sound stimuli and their respective locations, following a selective retro-cue, only one item remained relevant. This resulted in a behavioral improvement in terms of faster response times compared to neutral retro-cue trials, in which both the lateral and the central item remained relevant. A large body of literature has investigated the mechanisms underlying such “<italic>retro-cue benefits</italic>” (for a review, see<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>), showing that focusing attention within working memory results in the <italic>strengthening</italic> of attended working memory representations<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup> and the <italic>removal</italic> of non-cued information from a state of active maintenance (i.e., through persistent neural activity) in working memory<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref>,<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>, thereby releasing resources for further processing<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. When looking specifically at selective retro-cue trials and the different types of probe items, we found that participants responded the fastest when the probe matched the current working memory content (i.e., the cued item, yes response). However, in comparison to new probe items (no response), this speed benefit for cued items was associated with higher error rates. Given that those two conditions required different types of responses (yes vs. no), this can be most likely attributed to a speed-accuracy trade off.</p><p id=\"Par15\">Critically, we found increased response times and decreased accuracy in non-cued compared to new probe trials, suggesting a proactive interference effect in non-cued probe trials. It should be noted that although non-cued items were probed in a subset of trials, the retro-cue was always 100% valid. That is, when participants were asked to judge whether the probe item matched the cued item, it made no difference whether the probe was a new item or the previously non-cued item. As long as they remembered the (validly) cued item, they would always be able to respond correctly. Hence, the question emerges, if the non-cued item is <italic>removed</italic> from working memory, why does it still cause interference when presented as a probe item? If the non-cued item had been completely removed from short-term storage, responses should not have differed from those to new probe items. Instead, the present results suggest that the non-cued item was still maintained outside the focus of attention. This is in line with a previous visual retro-cueing study, similarly showing an interference effect, that is, slower response times for non-cued compared to new probe items<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. By contrasting the ERP response to non-cued and new probes, Schneider et al.<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup> demonstrated that an additional recollection process was involved in the rejection of the non-cued item. Critically, the ERP response to the probe also indicated that participants always focused their attention onto the cued item in all probe conditions, rather than shifting attention back towards the non-cued item, yet, the related contralateral negativity was more sustained in non-cued probe trials. The authors thus argue that while new probes can be easily rejected based on a fast familiarity process, non-cued probes require a more time-demanding comparison process through prolonged focusing on the cued item. Given the very comparable design of our auditory retro-cueing task, we assume similar processes might be involved in the present study.</p><p id=\"Par16\">The presence of a “residual” representation of non-cued information is also in line with retro-cue studies incorporating invalidly cued trials, which consistently suggest that non-cued items are not irreversibly lost. Those studies reliably report a decline in performance when the non-cued item was invalidly probed; however, performance never dropped to chance level<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. In addition, double-cueing paradigms suggest that the information that was initially dropped from working memory (in response to a first cue) can be restored to some extent by a second retro-cue<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. Such findings corroborate a recently proposed framework of “activity-silent” working memory<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>, suggesting that persistent neural firing is not a necessary prerequisite for successful short-term memory maintenance. For instance, using multivariate pattern analysis, it could be convincingly demonstrated that the active neural signature representing currently maintained information disappears once they become temporarily irrelevant. Critically, their neural signature is restored once those items are again cued to be relevant for the task<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. Accordingly, these studies suggest that non-cued items are <italic>removed from the focus of attention</italic> and from a state of persistent neural activity, but are still available for reactivation by re-storing their neural signature (see<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup> for alternative accounts regarding the storage of un-prioritized items). However, the reactivation of information from activity-silent representations was originally only investigated for information that remained task-relevant (e.g., at a later point in time). In contrast, when a retro-cue indicated the to-be-probed item with 100% validity, Wolff and colleagues were not able to decode the irrelevant (non-cued) item from a hidden state in the EEG signature<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. This contradicts the present findings as well as those previously reported by Schneider et al., both of which used perfectly valid retro-cues. Yet, Bae and Luck<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup> recently reported that the reactivation of task-irrelevant information from the previous trial in a working memory task was possible.</p><p id=\"Par17\">Taken together, we argue that in the present study, the non-cued information was removed from the focus of attention, but remained in a state of alternative, potentially passive storage, which caused a conflict in non-cued probe trials and thus, might have been reactivated by the current retrieval context (i.e., the probe)<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. Due to this conflict, non-cued probe trials required a more time-consuming and more error-prone comparison process to correctly reject the probe item. After all, although the residual representation of the non-cued item clearly caused a performance decline (cf. intrusion costs in a modified Sternberg paradigm<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>), we need to acknowledge that we cannot rule out that participants strategically held on to the non-cued item in an altered form, simply because they figured it would sometimes appear as a probe stimulus. We will further discuss the underlying mechanisms of such “imperfect” removal below in the context of the obtained time–frequency results.</p></sec><sec id=\"Sec7\"><title>Alpha lateralization as a proxy of target prioritization and distractor inhibition?</title><p id=\"Par18\">On a neural level, shifts of attention in response to the retro-cue were reflected by a hemispheric lateralization of posterior alpha lateralization. Critically, a contralateral decrease in alpha power was evident in target lateral trials, whereas a contralateral increase in alpha power was evident in distractor lateral trials. Broadly speaking, this is consistent with a growing body of literature showing that attentional modulations of alpha oscillations are comparable for shifts of attention within perceptual space and within working memory<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. More specifically, this pattern of results replicates previous findings from an analogous visual retro-cueing study<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>, revealing dissociable modulations of target- and distractor-related alpha power when shifting attention within visual working memory representations.</p><p id=\"Par19\">Given that it is relatively undisputed that alpha lateralization tracks the attended location within working memory<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>–<xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>, the observed spatially-specific modulations clearly indicate a re-orienting of spatial attention towards relevant items (and away from irrelevant items). Consequentially, the relevant item is moved into a prioritized state, commonly referred to as the focus of attention<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>, which renders the selected information more accessible for future cognitive operations. In line with that, because the cued information is already selected and ready to be acted on, cued probes result in faster response times compared to non-cued and new probes. Consistently, decreases in alpha power have been previously associated with increased neural firing<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup> and improved behavioral performance<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref>–<xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, suggesting that the observed target-related alpha power decrease reflects a signature of target prioritization, that allows for a fast adaption to current task demands. More specifically, it has been hypothesized that such a prioritization may result from the attentional strengthening of relevant items to their (spatial) context<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>.</p><p id=\"Par20\">Whether in addition to a prioritization mechanism, (active) inhibition is also involved in the attentional selection from working memory remains a matter of debate. The majority of previous studies, using completely lateralized stimulus displays, do not allow for a clear distinction between an inhibition or a prioritization account<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. Here, we demonstrated a relative distractor-related alpha power increase when participants were cued to re-orient their attention to a non-lateralized (i.e., central) working memory item, or—in other words—when shifting their attention away from a lateralized distractor. Increases in alpha power have been linked to decreased neuronal firing rates<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup> and have been shown to occur over task-irrelevant cortical areas<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup> as well as in anticipation of distractors<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. Hence, interpreting the observed alpha power increase in terms of an inhibition mechanism appears plausible. Yet, the exact nature of such an inhibitory attentional signature still remains elusive. First of all, we need to acknowledge that we cannot be certain that the inhibitory process, as indicated by the distractor-related alpha modulations, is independent from the attentional template for the target. Thus, it remains possible that distractor inhibition is an automatic consequence of target prioritization. Further, if we do conceptualize distractor inhibition as a top-down control mechanism that deteriorates the respective representations<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>, the question brought forth above likewise applies: If non-cued items are inhibited, why do non-cued items still cause interference when presented as a probe stimulus? Accordingly, both behavioral as well as neural evidence question the notion that an (active) inhibition mechanism completely deteriorates the non-cued item: On a behavioral level, using a series of cues, it has been shown that originally cued and then defocused items remain strengthened and result in faster and more accurate performance compared to trials in which a previously un-cued item (and thus “un-strengthened” item) is probed<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. In addition, a number of EEG studies has cast doubt on whether alpha lateralization actually mediates potential distractor exclusion effects<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>: For instance, Noonan and colleagues failed to find a lateralization of alpha power in response to a pre-cue indicating the location of an upcoming distractor, although, on the behavioral level, clear response time and accuracy improvements provide evidence for effective distractor exclusion (see also de Vries et al.<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>). However, it should be considered that the latter findings refer to anticipatory suppression of distractors in the visual domain, whereas the present study deals with the inhibition of previously relevant information within auditory working memory that subsequently becomes irrelevant.</p><p id=\"Par21\">Taken together, it remains possible that alpha enhancement contralateral to the distractor may be related to the temporal suppression of maintenance of information in a state of persistent activity (i.e., within the focus of attention), which does not necessarily alter the quality of unattended working memory contents<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. Alternatively, we could conceptualize distractor inhibition as a mechanism that ultimately aims to permanently remove irrelevant items from working memory. Such a permanent removal has been conceptualized to operate through an un-binding mechanism, that unties the present item-context bindings<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>, rather than affecting the representation of the item itself. This is in line with recent evidence, demonstrating that alpha oscillations carry information about the maintained location (e.g., the “context” in the present case) but <italic>not</italic> about location-unrelated stimulus-features (e.g., the sound’s identity)<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. Given that the present behavioral results argue against a complete removal of the un-cued item, such a mechanism would be expected to unfold gradually, resulting in weakened but not entirely removed representations or item-context bindings.</p></sec><sec id=\"Sec8\"><title>A dual mechanism underlying alpha lateralization</title><p id=\"Par22\">Overall, although we can only speculate on the exact nature of an inhibitory attentional control mechanism within auditory working memory, it is clear that we can dissociate distinct target- and distractor-related modulations of posterior alpha power. This supports the notion that working memory is a highly-flexible system that allows for the dynamic adaption to changing task demands by means of a dual-control mechanism. Alpha oscillations may serve as the underlying substrate that allows for flexible control over the state of current working memory representations, switching between prioritized and deprioritized representations depending on their current relevance for the task<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>.</p><p id=\"Par23\">That the flexible prioritization and de-prioritization of working memory representation may, in fact, be mediated by two independent mechanisms, has been supported by a number of recent studies: Schneider et al.<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup> independently manipulated the meaning of a retro-cue such that it either indicated the to-be-remembered (remember-cue) or the to-be-forgotten (forget-cue) item(s). Their results indicated that both types of retro-cues elicited a target-related decrease in alpha power as well as a distractor-related increase in alpha power. Interestingly, the “cued process” (i.e., inhibition in forget-cue trials and target prioritization in remember-cue trials) always emerged first and was only later followed by its complementary counterpart (see also Poch et al.<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>). In addition to those studies revealing latency effects, Wöstmann and colleagues<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup> were able to demonstrate that alpha lateralization for target selection and distractor suppression were both different in strength and source origin as well as statistically uncorrelated. Evidence for the assumption that the strengthening of target representations can in fact occur independently from the de-prioritization of irrelevant information also comes from behavioral findings, illustrating how strategic considerations may affect the underlying control mechanisms<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>: When a retro-cue indicated the to-be-probed item with low validity (i.e. valid cue in 50% of trials), invalid-cue costs were largely absent, while participants still showed a clear benefit for validly cued trials. In line with the strengthening hypothesis, such a pattern of results suggests that it is, in fact, possible to prioritize or strengthen the relevant information without affecting the non-cued, irrelevant information. However, under conditions of high cue validity (i.e., valid cue in 80% of trials), high invalid-cueing costs were observed, suggesting that in addition to a target prioritization mechanism, non-cued items might have been “removed” in order to free working memory resources. Interestingly, in a separate study, Günseli et al. found that cue validity (50% vs. 80%) only affected contralateral, but not ipsilateral alpha power (relative to the cued item)<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>, which corroborates the above-mentioned claim that the decision to remove or inhibit an item from working memory appears to be separate from the decision to prioritize the relevant information. Contrasting this finding to the present results, pointing to both a target- and a distractor-related modulation of alpha power, one may speculate that the incentive to (actively) inhibit the irrelevant information increases with unambiguous, perfectly reliable retro-cues.</p><p id=\"Par24\">Finally, considering the broad consensus that there is a large overlap between attentional selection in the perceptual environment (i.e., external attention) and attentional selection within working memory (i.e., internal attention; see<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref>–<xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup> for elaborate reviews considering both domains), the question emerges to what extent the mechanisms examined in the present study may be comparable to respective processes in the perceptual domain. Similar to the working memory literature, facilitative effects of attention are well-characterized and widely accepted in perception, whereas it remains a matter of debate to what extent inhibition acts as an active, top-down controlled mechanism that is independent from the former (reviewed by van Moorselaar et al.<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>). In fact, a number of studies have demonstrated that shifts of external<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref>,<xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup> and internal attention<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup> yield roughly comparable signatures of alpha-band modulations, suggesting the reliance on a common underlying control mechanism. Surprisingly, despite the common notion that alpha oscillations reflect functional inhibition (see e.g., the gating by inhibition framework by Jensen and Mazaheri<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>), the empirical evidence associating anticipatory modulations of alpha oscillations with top-down inhibition still remains scarce (reviewed by<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>,<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>).</p></sec><sec id=\"Sec9\"><title>Alpha lateralization is absent when spatial information becomes irrelevant</title><p id=\"Par25\">We did not observe any lateralization of alpha power following a neutral retro-cue, in which case both items maintained in working memory remained relevant. This appears plausible, considering that the neutral retro-cue did not require a re-orienting of spatial attention. In addition, this is consistent with the notion that auditory alpha lateralization is limited to situations in which spatial information is task-relevant<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>. Accordingly, once the neutral retro-cue appeared, the spatial location of the sounds became irrelevant to solving the task, as it was sufficient to know the sounds’ identities. In turn, this also means that alpha lateralization in response to a selective retro-cue reflects the spatially-specific access to the contents of working memory. This further corroborates the above-mentioned claim that alpha suppression indicates the strengthening of item-context bindings in working memory. In the present case, spatial location represents the item’s context, which is required to successfully distinguish cued from non-cued items. In contrast, in neutral retro-cue trials, knowledge of the spatial context is not required to solve the task.</p><p id=\"Par26\">Finally, the absence of alpha lateralization in neutral retro-cue trials points toward a critical difference between the auditory and the visual modality. Notably, Schneider et al.<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>, using a related design, found significant alpha lateralization following neutral retro-cues. This can be most likely attributed to the fact that the organization of the visual system is fundamentally different in that it is inherently spatial. Consistently, it has been shown that spatial locations are actively coded and maintained by population-level alpha-band activity throughout a non-spatial visual working memory task<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>.</p></sec></sec><sec id=\"Sec10\"><title>Conclusion</title><p id=\"Par27\">Taken together, using an auditory retro-active cueing design, we demonstrate that it is possible to unambiguously dissociate target- and distractor related modulations of alpha power oscillations. The results indicate that both excitatory and inhibitory attentional control mechanisms contribute to the selection of information from working memory. Accordingly, shifts of attention toward a lateralized target resulted in a contralateral decrease in alpha power, whereas shifting attention away from a lateralized distractor resulted in a contralateral increase in alpha power. The pattern of results support the notion that alpha lateralization mediates the task-dependent prioritization and de-prioritization of items within working memory<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. In addition, we strengthen the previous claim that auditory alpha lateralization is absent when spatial information is task-irrelevant<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>.</p></sec><sec id=\"Sec11\"><title>Methods</title><sec id=\"Sec12\"><title>Participants</title><p id=\"Par28\">Twenty-eight volunteers were paid to participate in this study. Four participants had to be excluded due to technical problems with their EEG recordings. Four additional participants showed excessive eye-movement artefacts (containing excessive lateral eye movements on more than 1/3rd of all trials) and were therefore excluded from further analyses. The remaining twenty participants (12 female) were aged between 19 and 28 years (mean age: 23.4 years) and right-handed as assessed using the Edinburgh Handedness Inventory<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. All participants reported normal or normal-to corrected vision, no history of or current neurological or psychiatric disorders. Hearing acuity was assessed using an audiometry, including eleven pure-tone frequencies (0.125–8 kHz; Oscilla USB 100, Inmedico, Lystrup, Denmark). Hearing thresholds indicated normal hearing (≤ 25 dB hearing level). The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethical Committee of the Leibniz Research Centre for Working Environment and Human Factors. Written informed consent was given by all participants prior to the beginning of the experimental procedure.</p></sec><sec id=\"Sec13\"><title>Experimental setup and stimuli</title><p id=\"Par29\">The experiment took place in a dimly lit, echo-attenuated, sound-proof room. Participants were seated in front of a semi-circular array of eight broad-band loudspeakers (SC5.9; Visaton, Haan, Germany; housing volume 340 cm<sup>3</sup>) mounted in the horizontal plane. Three of those loudspeakers, located at azimuthal positions of − 90°, 0°, 90°, were used for sound presentation in the present study. One additional loudspeaker was located right behind the participant’s head at a distance of approximately 30 cm. A red light-emitting diode, attached below the central loudspeaker (diameter 3 mm, turned off) served as a central fixation point. The participants’ head was kept at a constant position using a chin rest.</p><p id=\"Par30\">Eight familiar animal sounds, chosen from an online sound archive<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>, served as experimental stimuli. The original sounds (‘birds chirping’, ‘dog barking’, frog croaking’, ‘sheep baaing’, ‘cat meowing’, ‘duck quacking’, ‘cow mooing’, ‘rooster crowing’) were cut to a constant duration of 600 ms (10 ms on/off ramp), while leaving the spectro-temporal characteristics unchanged. In addition, syllable sounds served as cue-stimuli, indicating a certain subset of sound positions as relevant in a given trial. That is, the first two or three letters of the German words for right (i.e. “rechts”), left (i.e. “links”), middle (i.e. “mitte”), and both (i.e. “beide”) were used to construct the six cue-stimuli (“<italic>li</italic>”, “<italic>re</italic>”, “<italic>mi</italic>”, “bei”, “<italic>mi-li</italic>”, “<italic>mi-re</italic>”). The words were spoken by a female speaker (mean pitch 199 Hz). All cues had a duration of 400 ms. The overall sound pressure level of the sound arrays (presented at frontal loud speakers) was about 65 dB(A), whereas single animal sounds and the speech cue-stimuli (presented from behind participants’ heads) were presented at a sound pressure level of 60 dB(A).</p></sec><sec id=\"Sec14\"><title>Procedure and task</title><p id=\"Par31\">The present experiment is a modified and simplified version of a retroactive cueing paradigm previously used in an investigation of visual retroactive attentional orienting<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. Each trial comprised a sequence of four acoustic stimulus events, consisting of pre-cue, sound array, retro-cue, and probe. A trial started with the presentation of a pre-cue (400 ms), instructing participants to attend either the central and the left loudspeaker (“<italic>mi-li</italic>”) or the central and the right loudspeaker (“<italic>mi-re</italic>”). Thus, in the subsequently upcoming sound array, containing three animal vocalizations, one lateral sound was always known to be completely irrelevant for the rest of the trial. The sound array appeared 1,000 ms after the pre-cue and was presented for 600 ms. Rather than just presenting two sounds to begin with, we incorporated a pre-cue and a three-sound array to warrant that both hemispheres were equally activated during the sensory processing of the sound array. While in the visual domain, this can be easily implemented by displaying an irrelevant grey bar (cf. the original design by Schneider et al.<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>), it required an additional pre-cue in the present study. Following a short delay (1,000 ms), a retroactive cue (400 ms) indicated either one (i.e., selective retro-cue) or both (i.e., neutral retro-cue; “<italic>bei</italic>”) of the items currently maintained in working memory as further relevant for the task. Selective retro-cue trials can be further subdivided into target lateral trials (i.e., the retro-cue indicated the lateral item as further relevant, “<italic>li</italic>” or “<italic>re</italic>”) or distractor lateral trials (i.e., the retro-cue indicated the central item as relevant whereas the lateral item becomes irrelevant, “<italic>mi</italic>”). Each of the four retro-cue types was presented equally often (25% of trials), to enforce that none of the sound positions were favored irrespective of cue-type. That resulted in a higher number of trials for target lateral retro-cues (“li” and “re”; 50% of trials) as opposed to distractor lateral (“mi”, 25% of trials) and neutral (“bei”, 25% of trials) retro-cues. Finally, 1,000 ms subsequent to the retro-cue, a probe stimulus was presented. The probe stimulus was either the item that became irrelevant following the retro-cue (“non-cued probe”), the retro-cued item (“cued probe”), or a sound stimulus that never appeared in a given trial before (“new probe). Note that following a neutral retro-cue, the “cued probe” could be either one of the two still relevant items. The animal vocalization which was initially presented at the third, irrelevant position in the sound array (as indicated by the pre-cue) never appeared as a probe to ensure that participants would always ignore it. Because non-cued and new probes required a NO response they constituted 25% of all trials, respectively, whereas cued probes, requiring a YES response, constituted 50% of all trials. Participants were instructed to indicate whether the probe item matched (one of) the retro-cued item(s) by giving a YES (50% of trials) vs. NO (50% of trials) response. Participants responded by pressing one out of two vertically aligned keys, using the index finger and thumb of their right hand. The assignment of response alternatives (yes vs. no) to keys was counterbalanced across participants. Responses had to be given within 1,500 ms following probe offset. All probe stimuli, as well as pre- and retro-cues, were presented from a loudspeaker located in the median plane behind the participant’s head. The experiment consistent of a total of 30 practice trials and 800 experimental trials. The latter were divided into eight task blocks of 100 trials each. In-between blocks, short self-paced breaks served the prevention of fatigue in the course of the experiment. All participants were presented with the same, pseudo-random sequence of trials.</p></sec><sec id=\"Sec15\"><title>Data analysis</title><sec id=\"Sec16\"><title>Behavioural data</title><p id=\"Par32\">Response times and percentage of correct responses served as measures of behavioural performance. Trials with responses that occurred after the pre-defined response period (i.e., within the inter-trial interval), pre-mature (i.e., < 200 ms) and missing responses (i.e., no button press) were considered as erroneous responses. For analysis of response times, only correctly answered trials were included. In order to verify that the paradigm resulted in a retro-cue effect<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>, we performed paired sample <italic>t</italic>-tests, contrasting selective versus neutral retro-cues, for response times and accuracy data, respectively. Note, that for this comparison the non-cued probe trials (which were not present for neutral retro-cues) were excluded to avoid an imbalance between conditions. In addition, to test for the hypothesized interference effect in non-cued probe types, we contrasted the different probe types within the selective retro-cue condition by means of a repeated-measures analysis of variance (rANOVA) for response times and a Friedman’s ANOVA for accuracy, both of which included the factor <italic>probe type</italic>. Since for both the response time data as well as the accuracy data, we conducted a total of two analyses on the same data set (i.e., test for retro-cue type as well as for probe type), <italic>p</italic>-values were FDR-corrected for multiple comparisons across those two sets of analyses. In addition, post-hoc paired-sample <italic>t</italic>-tests (or their non-parametric alternative, i.e., Wilcoxon signed-rank test) were conducted and corrected for multiple comparisons. Note that adjusted <italic>p</italic>-values can be greater than 1.</p></sec><sec id=\"Sec17\"><title>EEG recording and processing</title><p id=\"Par33\">The EEG was recorded with a sampling rate of 1,000 Hz from 64 Ag/AgCl passive electrodes (Easycap GmbH, Herrsching, Germany), using a QuickAmp-72 amplifier (Brain products, Gilching, Germany). The electrodes were arranged across the scalp according to the extended 10/20 scalp configuration. AFz served as the ground electrode. The average of all channels constituted the online-reference. Impedances were kept below 10 kΩ during recording. Further pre-processing of the data was run in MATLAB (R2018b) and EEGLAB<sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>. First, a Hamming windowed sinc FIR high-pass (0.5 Hz, 0.25 Hz cut-off frequency, 0.5 Hz transition band width, filter order: 6,600) and low-pass filter (30 Hz, 30.25 Hz cut-off frequency, 0.5 Hz transition band width, filter order 440) were applied. Then, channels with a normalized kurtosis (20% trimming before normalization) exceeding 5 standard deviations of the mean were rejected, using the automated channel rejection procedure implemented in EEGLAB (M = 3.25 channels, range = 1–5). Anterior lateral channels (Fp1/2, AF7/8, AF3/4, F9/10) were excluded from channel rejection to ensure reliable identification of eye movements. Data were re-referenced to the average of all non-rejected channels. Epochs ranging from − 1,000 to 7,500 ms relative to the onset of the pre-cue were generated. A rank-reduced independent component analysis (ICA) was run on a subset of the data, down-sampled to 200 Hz and including every second epoch. To detect and remove independent components (ICs) reflecting eye blinks, vertical eye movements, and generic discontinuities, the EEGLAB plugin ADJUST<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup> was applied. In addition, for each IC, a single-equivalent current dipole model was estimated by means of a spherical four-shell head model<sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup>. Any components with a dipole solution exceeding a threshold of 40% residual variance were also removed. Taken together, on average 20.75 ICs (range = 7–33) were rejected. This was followed by an automatic trial rejection procedure, detecting and removing data epochs, containing data values exceeding a threshold of 5 standard deviations in an iterative procedure (threshold limit: 1000 μV, maximum % of trials rejected per iteration: 5%). On average, 163.3 (20.41%) trials (range 0–309) were rejected in the course of this procedure. Finally, data from channels that were originally rejected were replaced using spherical interpolation. For faster processing of time–frequency analyses (cf. below), the data were down-sampled to 500 Hz.</p></sec><sec id=\"Sec18\"><title>Time–frequency analysis</title><p id=\"Par34\">In order to extract spectral power, the epoched EEG data was convoluted with a complex Morlet wavelet. The frequencies of the wavelets ranged from 4 to 30 Hz, increasing logarithmically in 52 steps. Convolution began with a 3-cycle wavelet for the lowest frequency and increased linearly as a function of frequency with a factor of 0.5, resulting in a 11.25-cycle wavelet for the highest frequency. No spectral baseline-correction was applied, since the calculation of alpha lateralization indices requires raw power input. The resulting event-related spectral perturbation (ERSP) epochs ranged from − 582 to 7,080 ms relative to pre-cue onset. Lateralized effects in posterior alpha band power (8–13 Hz) in response to the retro-cue were assessed as a measure of retroactive attentional orienting. As a robust measure of lateralization, a lateralization index<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup> was calculated as follows:<disp-formula id=\"Equa\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$ lateralization\\; index = \\frac{ipsilateral \\;power - contralateral \\;power}{{ipsilateral + contralateral \\;power}} $$\\end{document}</tex-math><mml:math id=\"M2\" display=\"block\"><mml:mrow><mml:mi>l</mml:mi><mml:mi>a</mml:mi><mml:mi>t</mml:mi><mml:mi>e</mml:mi><mml:mi>r</mml:mi><mml:mi>a</mml:mi><mml:mi>l</mml:mi><mml:mi>i</mml:mi><mml:mi>z</mml:mi><mml:mi>a</mml:mi><mml:mi>t</mml:mi><mml:mi>i</mml:mi><mml:mi>o</mml:mi><mml:mi>n</mml:mi><mml:mspace width=\"0.277778em\"/><mml:mi>i</mml:mi><mml:mi>n</mml:mi><mml:mi>d</mml:mi><mml:mi>e</mml:mi><mml:mi>x</mml:mi><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi>i</mml:mi><mml:mi>p</mml:mi><mml:mi>s</mml:mi><mml:mi>i</mml:mi><mml:mi>l</mml:mi><mml:mi>a</mml:mi><mml:mi>t</mml:mi><mml:mi>e</mml:mi><mml:mi>r</mml:mi><mml:mi>a</mml:mi><mml:mi>l</mml:mi><mml:mspace width=\"0.277778em\"/><mml:mi>p</mml:mi><mml:mi>o</mml:mi><mml:mi>w</mml:mi><mml:mi>e</mml:mi><mml:mi>r</mml:mi><mml:mo>-</mml:mo><mml:mi>c</mml:mi><mml:mi>o</mml:mi><mml:mi>n</mml:mi><mml:mi>t</mml:mi><mml:mi>r</mml:mi><mml:mi>a</mml:mi><mml:mi>l</mml:mi><mml:mi>a</mml:mi><mml:mi>t</mml:mi><mml:mi>e</mml:mi><mml:mi>r</mml:mi><mml:mi>a</mml:mi><mml:mi>l</mml:mi><mml:mspace width=\"0.277778em\"/><mml:mi>p</mml:mi><mml:mi>o</mml:mi><mml:mi>w</mml:mi><mml:mi>e</mml:mi><mml:mi>r</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi><mml:mi>p</mml:mi><mml:mi>s</mml:mi><mml:mi>i</mml:mi><mml:mi>l</mml:mi><mml:mi>a</mml:mi><mml:mi>t</mml:mi><mml:mi>e</mml:mi><mml:mi>r</mml:mi><mml:mi>a</mml:mi><mml:mi>l</mml:mi><mml:mo>+</mml:mo><mml:mi>c</mml:mi><mml:mi>o</mml:mi><mml:mi>n</mml:mi><mml:mi>t</mml:mi><mml:mi>r</mml:mi><mml:mi>a</mml:mi><mml:mi>l</mml:mi><mml:mi>a</mml:mi><mml:mi>t</mml:mi><mml:mi>e</mml:mi><mml:mi>r</mml:mi><mml:mi>a</mml:mi><mml:mi>l</mml:mi><mml:mspace width=\"0.277778em\"/><mml:mi>p</mml:mi><mml:mi>o</mml:mi><mml:mi>w</mml:mi><mml:mi>e</mml:mi><mml:mi>r</mml:mi></mml:mrow></mml:mfrac></mml:mrow></mml:math><graphic xlink:href=\"41598_2020_70004_Article_Equa.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p id=\"Par35\">Mean power across trials was extracted for each subject ipsilateral and contralateral relative to the lateralized item in a given trial (i.e. the target in target lateral and neutral trials or the distractor in distractor lateral trials). Based on the electrode cluster used by Schneider et al.<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>, whose design this study was based on, power was averaged across the following electrodes of interest: PO7/8, P7/8, P5/6, and PO3/4. Because we had a clear hypothesis regarding an expected difference between the target lateral and the distractor lateral conditions, we first contrasted those two conditions using a cluster-based permutation approach: In a first step, the original data from the two conditions were contrasted by means of paired <italic>t</italic>-tests, comparing the lateralization index at each time-point (i.e., 200) and each frequency (i.e., 52). Note that no space dimension exists here, because power was already averaged across the electrodes mentioned above. This resulted in a time by frequency matrix of <italic>p</italic>-values. In a second step, the condition labels (i.e., target lateral vs. distractor lateral) were randomly assigned to the actual data points and again, a paired <italic>t</italic>-test was run for each time–frequency point. This procedure was iterated 1,000 times and thus, resulted in a 52 × 200 × 1,000 matrix of <italic>p</italic>-values. For each iteration, the size of the largest cluster of time–frequency points with a <italic>p</italic>-value below 0.05 was determined, resulting in a distribution of maximum cluster sizes to be expected under the null hypothesis. The 95th percentile of this distribution served as a cut-off value against which the clusters from the true data were compared. That is, only clusters of time–frequency points with a <italic>p</italic>-value below 0.05 that were larger than the 95th percentile of the permutation-based distribution of maximum cluster sizes were considered significant.</p><p id=\"Par36\">Based on the approximate cluster time-window in the alpha frequency range (8–13 Hz) derived from this comparison (700 to 1,300 ms post retro-cue onset), we compared the neutral retro-cue condition to the target lateral as well as the distractor lateral condition by means of paired-sample <italic>t</italic>-tests. Note that time-windows determined based on a cluster-permutation approach should not be interpreted in terms of an exact on- or offset of an effect<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>. In addition, we tested for significant alpha lateralization within conditions (i.e., one-sample <italic>t</italic>-tests against zero). <italic>P</italic> values were corrected for multiple comparisons including all five post-hoc comparisons (i.e., 3 one sample-<italic>t</italic>-tests within conditions and 2 paired sample <italic>t</italic>-test comparing conditions).</p></sec><sec id=\"Sec19\"><title>Inferential statistics and effect sizes</title><p id=\"Par37\">All statistical analyses of the data were conducted in MATLAB (R2018b). Parametric tests were applied when the data met the normality assumption (<italic>p</italic> > 0.05 in Lilliefors test). Otherwise, appropriate non-parametric tests, including Friedman’s test and Wilcoxon signed rank test, were conducted. Mauchly’s test for sphericity was performed for all repeated-measures ANOVA models. In case of significant violations of sphericity (<italic>p</italic> < 0.05), Greenhouse Geisser correction was applied. The significance of effects was assessed at an alpha level of 0.05. Reported <italic>p</italic>-values associated with tests based on the <italic>F</italic>-distribution are directional, given that the <italic>F</italic>-distribution is not symmetrical. All conducted (non-)parametric <italic>t</italic>-tests were two-tailed. As measures of effect size, partial eta-squared (ηp<sup>2</sup>) is provided for repeated-measures ANOVA. Hedges’s <italic>g</italic> and <italic>g</italic><sub>1</sub> served as an effect size measure for paired and one-sample <italic>t</italic>-tests, respectively. Hedges’s <italic>g</italic>, and <italic>g</italic>1 were calculated using the MATLAB Toolbox ‘Measures of Effect Size’<sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup>. To correct for multiple comparisons, false discovery rate (FDR) correction was applied<sup><xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup>. Corrected <italic>p</italic>-values are denoted as <italic>p</italic><sub>adj</sub>.</p><p id=\"Par38\">To further facilitate the interpretation of results, we additionally report the Bayes factor (BF). While classical null hypothesis significance testing only allows conclusions on whether we can disprove the null hypothesis, the BF also allows for an assessment of whether the data favors the null hypothesis compared to an alternative hypothesis (see<sup><xref ref-type=\"bibr\" rid=\"CR62\">62</xref>,<xref ref-type=\"bibr\" rid=\"CR63\">63</xref></sup> for a general introduction to Bayesian hypothesis testing). A BF greater than 3, 10, 30, and 100 provides moderate, strong, very strong, and extreme support for the alternative hypothesis<sup><xref ref-type=\"bibr\" rid=\"CR64\">64</xref></sup>. Values in-between 0.33 and 3 are usually interpreted as anecdotal evidence, whereas values lower than 0.33, 0.1, 0.03, and 0.01 indicate moderate, strong, very strong, and extreme evidence in favor of the null hypothesis<sup><xref ref-type=\"bibr\" rid=\"CR62\">62</xref></sup>. BFs were calculated using the MATLAB BF functions and default priors implemented by Krekelberg<sup><xref ref-type=\"bibr\" rid=\"CR64\">64</xref></sup>.</p></sec></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec20\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70004_MOESM1_ESM.pdf\"><caption><p>Supplementary file 1</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70004-2.</p></sec><ack><title>Acknowledgements</title><p>The authors would like to thank Kimberly Freytag and Stefan Weber for their contribution to data collection. Open access funding provided by Projekt DEAL.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>L.I.K., D.S. and S.G. designed the study. Data collection was performed by L.I.K. and A.B. L.I.K. performed the data analysis and interpretation under supervision of D.S. and S.G. L.I.K. drafted the manuscript. All authors contributed to the revision of the manuscript and approved the final version of the manuscript for submissions.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>The datasets generated in the course of present study are stored in the Leibniz Research Centre repository and are available from the corresponding author upon request.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par39\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hakim</surname><given-names>N</given-names></name><name><surname>Adam</surname><given-names>KCS</given-names></name><name><surname>Günseli</surname><given-names>E</given-names></name><name><surname>Awh</surname><given-names>E</given-names></name><name><surname>Vogel</surname><given-names>EK</given-names></name></person-group><article-title>Dissecting the neural focus of attention reveals distinct processes for spatial attention and object-based storage in visual working memory</article-title><source>Psychol. Sci.</source><year>2019</year><volume>30</volume><fpage>526</fpage><lpage>540</lpage><pub-id pub-id-type=\"pmid\">30817220</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Myers</surname><given-names>NE</given-names></name><name><surname>Walther</surname><given-names>L</given-names></name><name><surname>Wallis</surname><given-names>G</given-names></name><name><surname>Stokes</surname><given-names>MG</given-names></name><name><surname>Nobre</surname><given-names>AC</given-names></name></person-group><article-title>Temporal dynamics of attention during encoding vs. maintenance of working memory: Complementary views from event-related potentials and alpha-band oscillations</article-title><source>J. Cogn. Neurosci.</source><year>2015</year><volume>27</volume><fpage>492</fpage><lpage>508</lpage><pub-id pub-id-type=\"pmid\">25244118</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schneider</surname><given-names>D</given-names></name><name><surname>Mertes</surname><given-names>C</given-names></name><name><surname>Wascher</surname><given-names>E</given-names></name></person-group><article-title>On the fate of non-cued mental representations in visuo-spatial working memory: Evidence by a retro-cuing paradigm</article-title><source>Behav. Brain Res.</source><year>2015</year><volume>293</volume><fpage>114</fpage><lpage>124</lpage><pub-id pub-id-type=\"pmid\">26196953</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jensen</surname><given-names>O</given-names></name><name><surname>Mazaheri</surname><given-names>A</given-names></name></person-group><article-title>Shaping functional architecture by oscillatory alpha activity: Gating by inhibition</article-title><source>Front. Hum. Neurosci.</source><year>2010</year><volume>4</volume><fpage>1</fpage><lpage>8</lpage><pub-id pub-id-type=\"pmid\">20204154</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Foster</surname><given-names>JJ</given-names></name><name><surname>Awh</surname><given-names>E</given-names></name></person-group><article-title>The role of alpha oscillations in spatial attention: Limited evidence for a suppression account</article-title><source>Curr. Opin. Psychol.</source><year>2019</year><volume>29</volume><fpage>34</fpage><lpage>40</lpage><pub-id pub-id-type=\"pmid\">30472541</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Van Moorselaar</surname><given-names>D</given-names></name><name><surname>Slagter</surname><given-names>HA</given-names></name></person-group><article-title>Inhibition in selective attention</article-title><source>Ann. N. Y. Acad. Sci.</source><year>2020</year><volume>1464</volume><fpage>204</fpage><lpage>221</lpage><pub-id pub-id-type=\"pmid\">31951294</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schneider</surname><given-names>D</given-names></name><name><surname>Göddertz</surname><given-names>A</given-names></name><name><surname>Haase</surname><given-names>H</given-names></name><name><surname>Hickey</surname><given-names>C</given-names></name><name><surname>Wascher</surname><given-names>E</given-names></name></person-group><article-title>Hemispheric asymmetries in EEG alpha oscillations indicate active inhibition during attentional orienting within working</article-title><source>Behav. Brain Res.</source><year>2019</year><volume>359</volume><fpage>38</fpage><lpage>46</lpage><pub-id pub-id-type=\"pmid\">30339872</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tervaniemi</surname><given-names>M</given-names></name><name><surname>Hugdahl</surname><given-names>K</given-names></name></person-group><article-title>Lateralization of auditory-cortex functions</article-title><source>Brain Res. Rev.</source><year>2003</year><volume>43</volume><fpage>231</fpage><lpage>246</lpage><pub-id pub-id-type=\"pmid\">14629926</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Klatt</surname><given-names>L-I</given-names></name><name><surname>Getzmann</surname><given-names>S</given-names></name><name><surname>Wascher</surname><given-names>E</given-names></name><name><surname>Schneider</surname><given-names>D</given-names></name></person-group><article-title>Searching for auditory targets in external space and in working memory: Electrophysiological mechanisms underlying perceptual and retroactive spatial attention</article-title><source>Behav. Brain Res.</source><year>2018</year><volume>353</volume><fpage>98</fpage><lpage>107</lpage><pub-id pub-id-type=\"pmid\">29958962</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Souza</surname><given-names>AS</given-names></name><name><surname>Oberauer</surname><given-names>K</given-names></name></person-group><article-title>In search of the focus of attention in working memory: 13 years of the retro-cue effect</article-title><source>Atten. Percept. Psychophys.</source><year>2016</year><volume>78</volume><fpage>1839</fpage><lpage>1860</lpage><pub-id pub-id-type=\"pmid\">27098647</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rerko</surname><given-names>L</given-names></name><name><surname>Oberauer</surname><given-names>K</given-names></name></person-group><article-title>Focused, unfocused, and defocused information in working memory</article-title><source>J. Exp. Psychol. Learn. Mem. Cogn.</source><year>2013</year><volume>39</volume><fpage>1075</fpage><lpage>1096</lpage><pub-id pub-id-type=\"pmid\">23421511</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Souza</surname><given-names>AS</given-names></name><name><surname>Rerko</surname><given-names>L</given-names></name><name><surname>Oberauer</surname><given-names>K</given-names></name></person-group><article-title>Refreshing memory traces: Thinking of an item improves retrieval from visual working memory</article-title><source>Ann. N. Y. Acad. Sci.</source><year>2015</year><volume>1339</volume><fpage>20</fpage><lpage>31</lpage><pub-id pub-id-type=\"pmid\">25557544</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuo</surname><given-names>B-C</given-names></name><name><surname>Stokes</surname><given-names>MG</given-names></name><name><surname>Nobre</surname><given-names>AC</given-names></name></person-group><article-title>Attention modulates maintenance of representations in visual short-term memory</article-title><source>J. Cogn. Neurosci.</source><year>2012</year><volume>24</volume><fpage>51</fpage><lpage>60</lpage><pub-id pub-id-type=\"pmid\">21736457</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Souza</surname><given-names>AS</given-names></name><name><surname>Rerko</surname><given-names>L</given-names></name><name><surname>Oberauer</surname><given-names>K</given-names></name></person-group><article-title>Unloading and reloading working memory: Attending to one item frees capacity</article-title><source>J. Exp. Psychol. Hum. Percept. Perform.</source><year>2014</year><volume>40</volume><fpage>1237</fpage><lpage>1256</lpage><pub-id pub-id-type=\"pmid\">24730737</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Griffin</surname><given-names>IC</given-names></name><name><surname>Nobre</surname><given-names>AC</given-names></name></person-group><article-title>Orienting attention to locations in internal representations</article-title><source>J. Cogn. Neurosci.</source><year>2003</year><volume>15</volume><fpage>1176</fpage><lpage>1194</lpage><pub-id pub-id-type=\"pmid\">14709235</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Matsukura</surname><given-names>M</given-names></name><name><surname>Luck</surname><given-names>SJ</given-names></name><name><surname>Vecera</surname><given-names>S</given-names></name></person-group><article-title>Attention effects during visual short-term memory maintenance: Protection or prioritization?</article-title><source>Percept. Psychophys.</source><year>2007</year><volume>69</volume><fpage>1422</fpage><lpage>1434</lpage><pub-id pub-id-type=\"pmid\">18078232</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van Moorselaar</surname><given-names>D</given-names></name><name><surname>Olivers</surname><given-names>CNL</given-names></name><name><surname>Lamme</surname><given-names>VAF</given-names></name><name><surname>Theeuwes</surname><given-names>J</given-names></name><name><surname>Sligte</surname><given-names>IG</given-names></name></person-group><article-title>Forgotten but not gone: Retro-cue costs and benefits in a double-cueing paradigm suggest multiple states in visual short-term memory</article-title><source>J. Exp. Psychol. Learn. Mem. Cogn.</source><year>2015</year><volume>41</volume><fpage>1755</fpage><lpage>1763</lpage><pub-id pub-id-type=\"pmid\">25867613</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stokes</surname><given-names>MG</given-names></name></person-group><article-title>‘Activity-silent’ working memory in prefrontal cortex: A dynamic coding framework</article-title><source>Trends Cogn. Sci.</source><year>2015</year><volume>19</volume><fpage>394</fpage><lpage>405</lpage><pub-id pub-id-type=\"pmid\">26051384</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lewis-Peacock</surname><given-names>JA</given-names></name><name><surname>Drysdale</surname><given-names>AT</given-names></name><name><surname>Oberauer</surname><given-names>K</given-names></name><name><surname>Postle</surname><given-names>BR</given-names></name></person-group><article-title>Neural evidence for a distinction between short-term memory and the focus of attention</article-title><source>J. Cogn. Neurosci.</source><year>2012</year><volume>24</volume><fpage>61</fpage><lpage>79</lpage><pub-id pub-id-type=\"pmid\">21955164</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>LaRocque</surname><given-names>JJ</given-names></name><name><surname>Lewis-Peacock</surname><given-names>JA</given-names></name><name><surname>Drysdale</surname><given-names>AT</given-names></name><name><surname>Oberauer</surname><given-names>K</given-names></name><name><surname>Postle</surname><given-names>BR</given-names></name></person-group><article-title>Decoding attended information in short-term memory: An EEG study</article-title><source>J. Cogn. Neurosci.</source><year>2013</year><volume>25</volume><fpage>127</fpage><lpage>142</lpage><pub-id pub-id-type=\"pmid\">23198894</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Christophel</surname><given-names>TB</given-names></name><name><surname>Iamshchinina</surname><given-names>P</given-names></name><name><surname>Yan</surname><given-names>C</given-names></name><name><surname>Allefeld</surname><given-names>C</given-names></name><name><surname>Haynes</surname><given-names>JD</given-names></name></person-group><article-title>Cortical specialization for attended versus unattended working memory</article-title><source>Nat. Neurosci.</source><year>2018</year><volume>21</volume><fpage>494</fpage><lpage>496</lpage><pub-id pub-id-type=\"pmid\">29507410</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><mixed-citation publication-type=\"other\">Yu, Q., Teng, C. & Postle, B. R. Different states of priority recruit different neural representations in visual working memory. <italic>PLoS Biol</italic><bold>18</bold>, e3000769. 10.1371/journal.pbio.3000769 (2020).</mixed-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wolff</surname><given-names>MJ</given-names></name><name><surname>Jochim</surname><given-names>J</given-names></name><name><surname>Akyürek</surname><given-names>EG</given-names></name><name><surname>Stokes</surname><given-names>MG</given-names></name></person-group><article-title>Dynamic hidden states underlying working-memory-guided behavior</article-title><source>Nat. Neurosci.</source><year>2017</year><volume>20</volume><fpage>864</fpage><lpage>871</lpage><pub-id pub-id-type=\"pmid\">28414333</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bae</surname><given-names>GY</given-names></name><name><surname>Luck</surname><given-names>SJ</given-names></name></person-group><article-title>Reactivation of previous experiences in a working memory task</article-title><source>Psychol. Sci.</source><year>2019</year><volume>30</volume><fpage>587</fpage><lpage>595</lpage><pub-id pub-id-type=\"pmid\">30817224</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oberauer</surname><given-names>K</given-names></name></person-group><article-title>Binding and inhibition in working memory: Individual and age differences in short-term recognition</article-title><source>J. Exp. Psychol. Gen.</source><year>2005</year><volume>134</volume><fpage>368</fpage><lpage>387</lpage><pub-id pub-id-type=\"pmid\">16131269</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Poch</surname><given-names>C</given-names></name><name><surname>Campo</surname><given-names>P</given-names></name><name><surname>Barnes</surname><given-names>GR</given-names></name></person-group><article-title>Modulation of alpha and gamma oscillations related to retrospectively orienting attention within working memory</article-title><source>Eur. J. Neurosci.</source><year>2014</year><volume>40</volume><fpage>2399</fpage><lpage>2405</lpage><pub-id pub-id-type=\"pmid\">24750388</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bae</surname><given-names>G-Y</given-names></name><name><surname>Luck</surname><given-names>SJ</given-names></name></person-group><article-title>Dissociable decoding of spatial attention and working memory from EEG oscillations and sustained potentials</article-title><source>J. Neurosci.</source><year>2018</year><volume>38</volume><fpage>409</fpage><lpage>422</lpage><pub-id pub-id-type=\"pmid\">29167407</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Foster</surname><given-names>JJ</given-names></name><name><surname>Sutterer</surname><given-names>DW</given-names></name><name><surname>Serences</surname><given-names>JT</given-names></name><name><surname>Vogel</surname><given-names>EK</given-names></name><name><surname>Awh</surname><given-names>E</given-names></name></person-group><article-title>The topography of alpha-band activity tracks the content of spatial working memory</article-title><source>J. Neurophysiol.</source><year>2016</year><volume>115</volume><fpage>168</fpage><lpage>177</lpage><pub-id pub-id-type=\"pmid\">26467522</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Foster</surname><given-names>JJ</given-names></name><name><surname>Bsales</surname><given-names>EM</given-names></name><name><surname>Jaffe</surname><given-names>RJ</given-names></name><name><surname>Awh</surname><given-names>E</given-names></name></person-group><article-title>Alpha-band activity reveals spontaneous representations of spatial position in visual working memory</article-title><source>Curr. Biol.</source><year>2017</year><volume>27</volume><fpage>3216</fpage><lpage>3223</lpage><pub-id pub-id-type=\"pmid\">29033335</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oberauer</surname><given-names>K</given-names></name></person-group><article-title>Access to information in working memory: Exploring the focus of attention</article-title><source>J. Exp. Psychol. Learn. Mem. Cogn.</source><year>2002</year><volume>28</volume><fpage>411</fpage><lpage>421</lpage><pub-id pub-id-type=\"pmid\">12018494</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haegens</surname><given-names>S</given-names></name><name><surname>Nácher</surname><given-names>V</given-names></name><name><surname>Luna</surname><given-names>R</given-names></name><name><surname>Romo</surname><given-names>R</given-names></name><name><surname>Jensen</surname><given-names>O</given-names></name></person-group><article-title>α-Oscillations in the monkey sensorimotor network influence discrimination performance by rhythmical inhibition of neuronal spiking</article-title><source>Proc. Natl. Acad. Sci. USA</source><year>2011</year><volume>108</volume><fpage>19377</fpage><lpage>19382</lpage><pub-id pub-id-type=\"pmid\">22084106</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kelly</surname><given-names>SP</given-names></name><name><surname>Gomez-Ramirez</surname><given-names>M</given-names></name><name><surname>Foxe</surname><given-names>JJ</given-names></name></person-group><article-title>The strength of anticipatory spatial biasing predicts target discrimination at attended locations: A high-density EEG study</article-title><source>Eur. J. Neurosci.</source><year>2009</year><volume>30</volume><fpage>2224</fpage><lpage>2234</lpage><pub-id pub-id-type=\"pmid\">19930401</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gould</surname><given-names>IC</given-names></name><name><surname>Rushworth</surname><given-names>MF</given-names></name><name><surname>Nobre</surname><given-names>AC</given-names></name></person-group><article-title>Indexing the graded allocation of visuospatial attention using anticipatory alpha oscillations</article-title><source>J. Neurophysiol.</source><year>2011</year><volume>105</volume><fpage>1318</fpage><lpage>1326</lpage><pub-id pub-id-type=\"pmid\">21228304</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Snyder</surname><given-names>AC</given-names></name><name><surname>Foxe</surname><given-names>JJ</given-names></name></person-group><article-title>Anticipatory attentional suppression of visual features indexed by oscillatory alpha-band power increases: A high-density electrical mapping study</article-title><source>J. Neurosci.</source><year>2010</year><volume>30</volume><fpage>4024</fpage><lpage>4032</lpage><pub-id pub-id-type=\"pmid\">20237273</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bonnefond</surname><given-names>M</given-names></name><name><surname>Jensen</surname><given-names>O</given-names></name></person-group><article-title>Alpha oscillations serve to protect working memory maintenance against anticipated distracters</article-title><source>Curr. Biol.</source><year>2012</year><volume>22</volume><fpage>1969</fpage><lpage>1974</lpage><pub-id pub-id-type=\"pmid\">23041197</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rösner</surname><given-names>M</given-names></name><name><surname>Arnau</surname><given-names>S</given-names></name><name><surname>Wascher</surname><given-names>E</given-names></name><name><surname>Schneider</surname><given-names>D</given-names></name></person-group><article-title>Hemispheric asymmetries in posterior alpha power reflect the selection and inhibition of spatial context information in working memory</article-title><source>bioRxiv</source><year>2019</year><pub-id pub-id-type=\"doi\">10.1101/681031</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Noonan</surname><given-names>MP</given-names></name><etal/></person-group><article-title>Distinct mechanisms for distractor suppression and target facilitation</article-title><source>J. Neurosci.</source><year>2016</year><volume>36</volume><fpage>1797</fpage><lpage>1807</lpage><pub-id pub-id-type=\"pmid\">26865606</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>de Vries</surname><given-names>IEJ</given-names></name><name><surname>De Slagter</surname><given-names>HA</given-names></name><name><surname>Olivers.</surname><given-names>CNL</given-names></name></person-group><article-title>Oscillatory control over representational states in working memory</article-title><source>Trends Cogn. Sci.</source><year>2019</year><pub-id pub-id-type=\"doi\">10.1016/j.tics.2019.11.006</pub-id><pub-id pub-id-type=\"pmid\">31791896</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>de Vries</surname><given-names>IEJ</given-names></name><name><surname>Savran</surname><given-names>E</given-names></name><name><surname>van Driel</surname><given-names>J</given-names></name><name><surname>Olivers</surname><given-names>CNL</given-names></name></person-group><article-title>Oscillatory mechanisms of preparing for visual distraction</article-title><source>J. Cogn. Neurosci.</source><year>2019</year><volume>31</volume><fpage>1873</fpage><lpage>1894</lpage><pub-id pub-id-type=\"pmid\">31418334</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lewis-Peacock</surname><given-names>JA</given-names></name><name><surname>Postle</surname><given-names>BR</given-names></name></person-group><article-title>Decoding the internal focus of attention</article-title><source>Neuropsychologia</source><year>2012</year><volume>50</volume><fpage>470</fpage><lpage>478</lpage><pub-id pub-id-type=\"pmid\">22108440</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lewis-Peacock</surname><given-names>JA</given-names></name><name><surname>Kessler</surname><given-names>Y</given-names></name><name><surname>Oberauer</surname><given-names>K</given-names></name></person-group><article-title>The removal of information from working memory</article-title><source>Ann. N. Y. Acad. Sci.</source><year>2018</year><volume>1424</volume><fpage>33</fpage><lpage>44</lpage><pub-id pub-id-type=\"pmid\">29741212</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Poch</surname><given-names>C</given-names></name><name><surname>Carretie</surname><given-names>L</given-names></name><name><surname>Campo</surname><given-names>P</given-names></name></person-group><article-title>A dual mechanism underlying alpha lateralization in attentional orienting to mental representation</article-title><source>Biol. Psychol.</source><year>2017</year><volume>128</volume><fpage>63</fpage><lpage>70</lpage><pub-id pub-id-type=\"pmid\">28746832</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wöstmann</surname><given-names>M</given-names></name><name><surname>Alavash</surname><given-names>M</given-names></name><name><surname>Obleser</surname><given-names>J</given-names></name></person-group><article-title>Alpha oscillations in the human brain implement distractor suppression independent of target selection</article-title><source>J. Neurosci.</source><year>2019</year><volume>39</volume><fpage>9797</fpage><lpage>9805</lpage><pub-id pub-id-type=\"pmid\">31641052</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Günseli</surname><given-names>E</given-names></name><name><surname>van Moorselaar</surname><given-names>D</given-names></name><name><surname>Meeter</surname><given-names>M</given-names></name><name><surname>Olivers</surname><given-names>CNL</given-names></name></person-group><article-title>The reliability of retro-cues determines the fate of noncued visual working memory representations</article-title><source>Psychon. Bull. Rev.</source><year>2015</year><volume>22</volume><fpage>1334</fpage><lpage>1341</lpage><pub-id pub-id-type=\"pmid\">25563713</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Günseli</surname><given-names>E</given-names></name><etal/></person-group><article-title>EEG dynamics reveal a dissociation between storage and selective attention within working memory</article-title><source>Sci. Rep.</source><year>2019</year><volume>9</volume><fpage>1</fpage><lpage>13</lpage><pub-id pub-id-type=\"pmid\">30626917</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oberauer</surname><given-names>K</given-names></name></person-group><article-title>Working memory and attention—A conceptual analysis and review</article-title><source>J. Cogn.</source><year>2019</year><volume>2</volume><fpage>1</fpage><lpage>23</lpage><pub-id pub-id-type=\"pmid\">31517219</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kiyonaga</surname><given-names>A</given-names></name><name><surname>Egner</surname><given-names>T</given-names></name></person-group><article-title>Working memory as internal attention: Toward an integrative account of internal and external selection processes</article-title><source>Psychon. Bull. Rev.</source><year>2013</year><volume>20</volume><fpage>228</fpage><lpage>242</lpage><pub-id pub-id-type=\"pmid\">23233157</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zimmermann</surname><given-names>JF</given-names></name><name><surname>Moscovitch</surname><given-names>M</given-names></name><name><surname>Alain</surname><given-names>C</given-names></name></person-group><article-title>Attending to auditory memory</article-title><source>Brain Res.</source><year>2016</year><volume>1640</volume><fpage>208</fpage><lpage>221</lpage><pub-id pub-id-type=\"pmid\">26638836</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Worden</surname><given-names>MS</given-names></name><name><surname>Foxe</surname><given-names>JJ</given-names></name><name><surname>Wang</surname><given-names>N</given-names></name><name><surname>Simpson</surname><given-names>GV</given-names></name></person-group><article-title>Anticipatory biasing of visuospatial attention indexed by retinotopically specific α-band electroencephalography increases over occipital cortex</article-title><source>J. Neurosci.</source><year>2000</year><volume>20</volume><fpage>RC63</fpage><pub-id pub-id-type=\"pmid\">10704517</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Klatt</surname><given-names>L-I</given-names></name><name><surname>Getzmann</surname><given-names>S</given-names></name><name><surname>Wascher</surname><given-names>E</given-names></name><name><surname>Schneider</surname><given-names>D</given-names></name></person-group><article-title>The contribution of selective spatial attention to sound detection and sound localization: Evidence from event-related potentials and lateralized alpha oscillations</article-title><source>Biol. Psychol.</source><year>2018</year><volume>138</volume><fpage>133</fpage><lpage>145</lpage><pub-id pub-id-type=\"pmid\">30165081</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jensen</surname><given-names>O</given-names></name><name><surname>Mazaheri</surname><given-names>A</given-names></name></person-group><article-title>Shaping functional architecture by oscillatory alpha activity: gating by inhibition</article-title><source>Front. Hum. Neurosci.</source><year>2010</year><volume>4</volume><fpage>186</fpage><pub-id pub-id-type=\"pmid\">21119777</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rösner</surname><given-names>M</given-names></name><name><surname>Arnau</surname><given-names>S</given-names></name><name><surname>Skiba</surname><given-names>I</given-names></name><name><surname>Wascher</surname><given-names>E</given-names></name><name><surname>Schneider</surname><given-names>D</given-names></name></person-group><article-title>The spatial orienting of the focus of attention in working memory makes use of inhibition: Evidence by hemispheric asymmetries in posterior alpha oscillations</article-title><source>Neuropsychologia</source><year>2020</year><volume>142</volume><fpage>107442</fpage><pub-id pub-id-type=\"pmid\">32205083</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oldfield</surname><given-names>RC</given-names></name></person-group><article-title>The assessment and analysis of handedness: The Edinburgh inventory</article-title><source>Neuropsychologia</source><year>1971</year><volume>9</volume><fpage>97</fpage><lpage>113</lpage><pub-id pub-id-type=\"pmid\">5146491</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Marcell</surname><given-names>MM</given-names></name><name><surname>Borella</surname><given-names>D</given-names></name><name><surname>Greene</surname><given-names>M</given-names></name><name><surname>Kerr</surname><given-names>E</given-names></name><name><surname>Rogers</surname><given-names>S</given-names></name></person-group><article-title>Confrontation naming of environmental sounds</article-title><source>J. Clin. Exp. Neuropsychol.</source><year>2000</year><volume>22</volume><fpage>830</fpage><lpage>864</lpage><pub-id pub-id-type=\"pmid\">11320440</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Delorme</surname><given-names>A</given-names></name><name><surname>Makeig</surname><given-names>S</given-names></name></person-group><article-title>EEGLAB: An open source toolbox for analysis of single-trial EEG dynamics including independent component analysis</article-title><source>J. Neurosci. Methods</source><year>2004</year><volume>134</volume><fpage>9</fpage><lpage>21</lpage><pub-id pub-id-type=\"pmid\">15102499</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mognon</surname><given-names>A</given-names></name><name><surname>Jovicich</surname><given-names>J</given-names></name><name><surname>Bruzzone</surname><given-names>L</given-names></name><name><surname>Buiatti</surname><given-names>M</given-names></name></person-group><article-title>ADJUST: An automatic EEG artifact detector based on the joint use of spatial and temporal features</article-title><source>Psychphysiology</source><year>2011</year><volume>48</volume><fpage>229</fpage><lpage>240</lpage></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kavanagk</surname><given-names>RN</given-names></name><name><surname>Darcey</surname><given-names>TM</given-names></name><name><surname>Lehmann</surname><given-names>D</given-names></name><name><surname>Fender</surname><given-names>DH</given-names></name></person-group><article-title>Evaluation of methods for three-dimensional localization of electrical sources in the human brain</article-title><source>IEEE Trans. Biomed. Eng.</source><year>1978</year><volume>BME-25</volume><fpage>421</fpage><lpage>429</lpage></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haegens</surname><given-names>S</given-names></name><name><surname>Handel</surname><given-names>BF</given-names></name><name><surname>Jensen</surname><given-names>O</given-names></name></person-group><article-title>Top-down controlled alpha band activity in somatosensory areas determines behavioral performance in a discrimination task</article-title><source>J. Neurosci.</source><year>2011</year><volume>31</volume><fpage>5197</fpage><lpage>5204</lpage><pub-id pub-id-type=\"pmid\">21471354</pub-id></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sassenhagen</surname><given-names>J</given-names></name><name><surname>Draschkow</surname><given-names>D</given-names></name></person-group><article-title>Cluster-based permutation tests of MEG/EEG data do not establish significance of effect latency or location</article-title><source>Psychophysiology</source><year>2019</year><volume>56</volume><fpage>e13335</fpage><pub-id pub-id-type=\"pmid\">30657176</pub-id></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hentschke</surname><given-names>H</given-names></name><name><surname>Stüttgen</surname><given-names>MC</given-names></name></person-group><article-title>Computation of measures of effect size for neuroscience data sets</article-title><source>Eur. J. Neurosci.</source><year>2011</year><volume>34</volume><fpage>1887</fpage><lpage>1894</lpage><pub-id pub-id-type=\"pmid\">22082031</pub-id></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Benjamini</surname><given-names>Y</given-names></name><name><surname>Yekutieli</surname><given-names>D</given-names></name></person-group><article-title>The control of the false discovery rate in multiple testing under dependency</article-title><source>Ann. Stat.</source><year>2001</year><volume>29</volume><fpage>1165</fpage><lpage>1188</lpage></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quintana</surname><given-names>DS</given-names></name><name><surname>Williams</surname><given-names>DR</given-names></name></person-group><article-title>Bayesian alternatives for common null-hypothesis significance tests in psychiatry: A non-technical guide using JASP</article-title><source>BMC Psychiatry</source><year>2018</year><volume>18</volume><fpage>1</fpage><lpage>8</lpage><pub-id pub-id-type=\"pmid\">29304757</pub-id></element-citation></ref><ref id=\"CR63\"><label>63.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wagenmakers</surname><given-names>EJ</given-names></name><etal/></person-group><article-title>Bayesian inference for psychology. Part I: Theoretical advantages and practical ramifications</article-title><source>Psychon. Bull. Rev.</source><year>2018</year><volume>25</volume><fpage>35</fpage><lpage>57</lpage><pub-id pub-id-type=\"pmid\">28779455</pub-id></element-citation></ref><ref id=\"CR64\"><label>64.</label><mixed-citation publication-type=\"other\">Krekelberg, B. (2019). bayesFactor: A Matlab package for Bayes factor statistical analysis. <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/klabhub/bayesFactor\">https://github.com/klabhub/bayesFactor</ext-link>. Accessed 20 Mar 2019.</mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Nat Commun</journal-id><journal-id journal-id-type=\"iso-abbrev\">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type=\"epub\">2041-1723</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807786</article-id><article-id pub-id-type=\"pmc\">PMC7431586</article-id><article-id pub-id-type=\"publisher-id\">17563</article-id><article-id pub-id-type=\"doi\">10.1038/s41467-020-17563-0</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Exciton-driven change of phonon modes causes strong temperature dependent bandgap shift in nanoclusters</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Muckel</surname><given-names>Franziska</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Lorenz</surname><given-names>Severin</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yang</surname><given-names>Jiwoong</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Nugraha</surname><given-names>Taufik Adi</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-1465-0939</contrib-id><name><surname>Scalise</surname><given-names>Emilio</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-5959-6257</contrib-id><name><surname>Hyeon</surname><given-names>Taeghwan</given-names></name><address><email>thyeon@snu.ac.kr</email></address><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-9680-2128</contrib-id><name><surname>Wippermann</surname><given-names>Stefan</given-names></name><address><email>s.wippermann@mpie.de</email></address><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-8419-2158</contrib-id><name><surname>Bacher</surname><given-names>Gerd</given-names></name><address><email>gerd.bacher@uni-due.de</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5718.b</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2187 5445</institution-id><institution>Werkstoffe der Elektrotechnik and CENIDE, </institution><institution>Universität Duisburg-Essen, </institution></institution-wrap>Bismarckstraße 81, 47057 Duisburg, Germany </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.410720.0</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1784 4496</institution-id><institution>Center for Nanoparticle Research, </institution><institution>Institute for Basic Science (IBS), </institution></institution-wrap>Seoul, 08826 Republic of Korea </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.417736.0</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0438 6721</institution-id><institution>Department of Energy Science & Engineering, </institution><institution>Daegu Gyeongbuk Institute of Science & Technology (DGIST), </institution></institution-wrap>Daegu, 42988 Republic of Korea </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.13829.31</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0491 378X</institution-id><institution>Max-Planck-Institut für Eisenforschung, </institution></institution-wrap>Max-Planck-Strasse 1, 40237 Düsseldorf, Germany </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.31501.36</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0470 5905</institution-id><institution>School of Chemical and Biological Engineering, and Institute of Chemical Processes, </institution><institution>Seoul National University, </institution></institution-wrap>Seoul, 08826 Republic of Korea </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>4127</elocation-id><history><date date-type=\"received\"><day>4</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>2</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">The fundamental bandgap <italic>E</italic><sub>g</sub> of a semiconductor—often determined by means of optical spectroscopy—represents its characteristic fingerprint and changes distinctively with temperature. Here, we demonstrate that in magic sized II-VI clusters containing only 26 atoms, a pronounced weakening of the bonds occurs upon optical excitation, which results in a strong exciton-driven shift of the phonon spectrum. As a consequence, a drastic increase of d<italic>E</italic><sub>g</sub>/d<italic>T</italic> (up to a factor of 2) with respect to bulk material or nanocrystals of typical size is found. We are able to describe our experimental data with excellent quantitative agreement from first principles deriving the bandgap shift with temperature as the vibrational entropy contribution to the free energy difference between the ground and optically excited states. Our work demonstrates how in small nanoparticles, photons as the probe medium affect the bandgap—a fundamental semiconductor property.</p></abstract><abstract id=\"Abs2\" abstract-type=\"web-summary\"><p id=\"Par2\">The bandgap of nanostructures usually follows the bulk value upon temperature change. Here, the authors find that in small nanocrystals a weakening of the bonds due to optical excitation causes a pronounced phonon shift, leading to a drastic enhancement of the bandgap’s temperature dependence.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Electronic materials</kwd><kwd>Quantum dots</kwd><kwd>Electronic properties and materials</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100001659</institution-id><institution>Deutsche Forschungsgemeinschaft (German Research Foundation)</institution></institution-wrap></funding-source><award-id>BA 1422/13-2</award-id><principal-award-recipient><name><surname>Bacher</surname><given-names>Gerd</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100001655</institution-id><institution>Deutscher Akademischer Austauschdienst (German Academic Exchange Service)</institution></institution-wrap></funding-source><award-id>605728</award-id><principal-award-recipient><name><surname>Bacher</surname><given-names>Gerd</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100010571</institution-id><institution>Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Federal Ministry for Education, Science, Research and Technology)</institution></institution-wrap></funding-source><award-id>13N12972</award-id><principal-award-recipient><name><surname>Bacher</surname><given-names>Gerd</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100002790</institution-id><institution>Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada (NSERC Canadian Network for Research and Innovation in Machining Technology)</institution></institution-wrap></funding-source><award-id>35687</award-id><principal-award-recipient><name><surname>Bacher</surname><given-names>Gerd</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par3\">The tunability of the fundamental bandgap, <italic>E</italic><sub><italic>g</italic></sub>, through size control on the nanometer scale is a key attribute of semiconductor nanocrystals, enabling applications<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>,<xref ref-type=\"bibr\" rid=\"CR2\">2</xref></sup> spanning from biomedical imaging<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup> to optoelectronic devices such as light emitting diodes<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> and photodetectors<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. The bandgap of semiconductors with reduced dimensions can usually be derived from the bulk value, correcting for electronic perturbations<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>, such as charge carrier quantization and Coulomb interaction. Although the electronic structure of semiconductor nanocrystals is changed by quantum confinement, the temperature dependence of the bandgap usually appears to be dominated by bulk mechanisms in both epitaxial<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref>,<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> as well as most colloidal quantum dots (QDs)<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>–<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. However, there are few examples<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>–<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup> that reported conflicting results on the temperature dependence of the bandgap in quantum dots, opening room for debate. For bulk, it is widely accepted that the temperature dependence of the bandgap results from a superposition of electron–phonon interactions and thermal lattice expansion<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Besides widely used empirical equations<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>, the AHC approach<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref>–<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> (after Allen, Heine and Cardona), which is based on second-order perturbation theory, approaches the temperature-induced bandgap shift as the change in energy levels when phonons are thermally populated. This has been used to obtain the temperature dependent bandgap computationally for a number of bulk semiconductors<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. Following semi-empirical approaches, AHC was subsequently combined with density functional theory (DFT) or density functional perturbation theory (DFPT)<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>–<xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. For nanocrystals, the temperature dependence of the bandgap has been derived either (i) as time average of the bandgap with first principle molecular dynamic (MD) calculations<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref>–<xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, or (ii) via the frozen-phonon approach<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref>–<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>, summing over the Bose–Einstein weighted changes in eigenenergies due to atomic displacements along the different phonon modes.</p><p id=\"Par4\">Here, we demonstrate that the impact of temperature on the optical bandgap is fundamentally altered in materials at the border between solids and molecules. In magic sized clusters<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref>–<xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup> (MSC) containing a defined number of 26 atoms, a pronounced exciton-induced weakening of the crystal bonds occurs, resulting in a giant shift of the phonon spectrum driven by the exciton. As a consequence, a drastic increase of d<italic>E</italic><sub>g</sub>/d<italic>T</italic> (up to a factor of 2) with respect to bulk material or nanocrystals of typical size is found. Using a first principles approach, we computed the exciton-induced change of the phonon density of states from molecular dynamics simulations on the excited state potential energy surface. This enabled us to quantitatively describe our experimental data by deriving the bandgap shift with temperature as the vibrational entropy contribution to the free energy difference between the ground and optically excited states.</p></sec><sec id=\"Sec2\" sec-type=\"results\"><title>Results</title><p id=\"Par5\">Our study benefits from the spectrally narrow absorption and emission resonances of high quality II–VI MSCs that consist of a well-defined number of atoms and vanishing size distribution. This allows us to trace the bandgap energy shift between 5 and 300 K with various optical techniques, as summarized in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> for (CdSe)<sub>13</sub> MSCs. The absorption of (CdSe)<sub>13</sub> MSCs, which is dominated by multiple resonances affiliated to the fine structure of the lowest excitonic transition<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>, exhibits a redshift with increasing temperature for the whole collectivity of peaks of about 160 meV (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>) between cryogenic and room temperature. The short-time photoluminescence (PL) from the bandgap states (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref>), which is obtained by integrating the emission within the first 20 ps after laser excitation, is found to coincide with the low energy absorption feature of the band edge fine structure, with virtually no Stokes shift within our resolution (compare Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1b</xref>). This is in distinct contrast to what is observed in typical QDs with Stokes shifts of up to 100 meV for diameters below 2 nm<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>, and enables us to trace the temperature dependence of an individual state (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref>). In agreement with absorption, an energy shift of 160 meV is observed between cryogenic and room temperature.<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Temperature dependent bandgap shift in undoped as well as doped CdSe magic sized cluster (MSC).</title><p><bold>a</bold> Absorption and <bold>b</bold> short-time photoluminescence (PL), measured between 5 K and 300 K. <bold>c</bold>, <bold>d</bold> Temperature dependence of the bandgap in Mn<sup>2+</sup>-doped (CdSe)<sub>13</sub> MSCs using optical signatures introduced by the magnetic dopants. <bold>c</bold> Magnetic circular dichroism (MCD) spectra of Mn:(CdSe)<sub>13</sub> MSCs at 1.6 T between 5.2 and 300 K. The signals allow tracking of the bandgap shift with temperature by monitoring the energetic position of the first zero-crossing. d, Photoluminescence excitation (PLE) spectra probed at the <sup>4</sup>T<sub>1</sub> → <sup>6</sup>A<sub>1</sub> Mn<sup>2+</sup> internal emission between 5.2 and 300 K. The inset depicts the time integrated PL signal of Mn<sup>2+</sup>-doped (CdSe)<sub>13</sub> MSCs revealing the specific orange Mn<sup>2+</sup> luminescence, used for probing bandgap states in PLE experiments.</p></caption><graphic xlink:href=\"41467_2020_17563_Fig1_HTML\" id=\"d30e569\"/></fig></p><p id=\"Par6\">Transition metal doping with manganese (Mn<sup>2+</sup>)<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup> offers additional possibilities to track the bandgap shift for specific fine structure states. The sp-d exchange interactions, which persist up to room temperature in these clusters<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>, generate a pronounced magneto-optical response at the bandgap, representing a powerful tool to distinguish between magneto-optically active and inactive states via magnetic circular dichroism (MCD) spectroscopy (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1c</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. For MSCs with <italic>x</italic><sub>Mn</sub> = 2% (average Mn<sup>2+</sup> content among the cations in all clusters of the measured ensemble), the shift of the bandgap slightly exceeds that of the undoped clusters (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1c</xref>), while the mean energy shift between cryogenic and room temperature among a concentration series (<italic>x</italic><sub>Mn</sub> = 2–10%, average out of 5 data sets) accounts for 164 ± 16 meV (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>).</p><p id=\"Par7\">In addition, Mn<sup>2+</sup> ions modify the emission of the MSCs, leading to a characteristic orange luminescence originated in the internal Mn<sup>2+ 4</sup>T<sub>1</sub> → <sup>6</sup>A<sub>1</sub> transition. Monitoring this emission in photoluminescence excitation (PLE) spectroscopy reveals a shift of about 200 meV of the bandgap absorption between 5 and 300 K (see Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1d</xref>, 174 ± 23 meV in average among three doped samples). Mn<sup>2+</sup>-doped (ZnSe)<sub>13</sub> MSCs<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup> prepared for comparison exhibit a similarly enhanced temperature dependence as the CdSe based MSCs (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>). As a reference, we synthesized CdSe QDs covered with amine ligands or a ZnSe shell, which exhibit significantly smaller bandgap shifts with temperature in this regime (average shift of both samples is 93 ± 1 meV in agreement with literature, see Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> and Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). This demonstrates that our findings are related to the small size of the clusters, irrespective of the material system.<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Giant temperature dependent shift of the bandgap in magic sized cluster (MSC).</title><p><bold>a</bold>, <bold>b</bold> Comparison of the shift in transition energies from cryogenic to room temperature (<bold>Δ</bold><bold><italic>E</italic></bold><sub><bold><italic>g</italic></bold></sub>) for undoped (<bold>a</bold>) and Mn<sup>2+</sup>-doped (CdSe)<sub>13</sub> MSCs (<bold>b</bold>) as extracted from different experimental approaches. Data for conventional CdSe (light grey) and CdSe/ZnS (dark grey) quantum dots (QDs) are displayed as dots for comparison (see Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref> for the spectra). The peak positions for absorption, photoluminescence (PL) and photoluminescence excitation spectroscopy (PLE) signals are extracted via Gaussian fits from the spectra. The peak positions for the energetically lowest magneto-optically active transition are taken from MCD zero-crossings (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1c</xref>). Solid lines represent guides to the eye. <bold>c</bold> Comparison of the temperature dependence of the band edge transitions for the entity of (CdSe)<sub>13</sub> (blue area) and (ZnSe)<sub>13</sub> MSCs (black symbols) analyzed within this study to some of the most important group IV, III–V, and II–VI semiconductors (data sets are plotted using parametrization following ref. <sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>). The bandgap shifts for Mn<sup>2+</sup>-doped (ZnSe)<sub>13</sub> MSCs are extracted from MCD zero-crossings (black circles) and absorption (black stars) (spectra shown in Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>).</p></caption><graphic xlink:href=\"41467_2020_17563_Fig2_HTML\" id=\"d30e716\"/></fig></p><p id=\"Par8\">The bandgap shift with temperature as derived by different techniques for various doped and undoped MSCs are summed up in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>. The entity of evaluated data in the MSCs exhibits drastically enhanced values of d<italic>E</italic><sub>g</sub>/d<italic>T</italic> in the high temperature regime as compared to typical CdSe QDs or the most common bulk semiconductors (ref. <sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup> and references therein). For (CdSe)<sub>13</sub> MSCs, our data reveals a mean increase of 89 % compared to bulk CdSe (wurtzite) for the high temperature limit of d<italic>E</italic><sub>g</sub>/d<italic>T</italic> (between 150 and 300 K) among 14 data sets (Supplementary Table <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>).</p><p id=\"Par9\">To reconstruct the impact of electron–phonon coupling on the temperature dependent bandgap shift, it is instructive to interpret the bandgap energy as the standard Gibbs free energy for the formation of an electron-hole pair<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. As first suggested in the context of Brook’s theorem<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>, the bandgap change with temperature due to electron–phonon interactions can be expressed as the change in the vibronic free energy caused by the excitation of an electron from a bonding state in the valence band into a non-bonding state in the conduction band<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>. The limited number of atoms in our material allows us to directly calculate the free energy difference for (CdSe)<sub>13</sub> MSCs between the ground and the optically excited states as a function of temperature using constrained DFT. We performed the same set of calculations for the two most prominent structural models for the (CdSe)<sub>13</sub> MSCs<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>: the so-called sliced-wurtzite MSC without ligands (see Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>) and a core/cage MSC with methylamine ligands (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref>). In harmonic approximation, the free energy of the phonons is given by<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$F_{{\\mathrm{vib}}}(T) = \\int {\\mathrm{d}}\\omega \\,g(\\omega ,T)\\left( {\\frac{1}{2}{\\hbar {\\omega}} + k_{\\mathrm{B}}T{\\mathrm{ln}}[1 - {\\mathrm{e}}^{ - (\\frac{\\hbar {\\omega}}{{k_{\\mathrm{B}}T}})}]} \\right),$$\\end{document}</tex-math><mml:math id=\"M2\"><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">vib</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>T</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:mo>∫</mml:mo><mml:mi mathvariant=\"normal\">d</mml:mi><mml:mi>ω</mml:mi><mml:mspace width=\"0.25em\"/><mml:mi>g</mml:mi><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>ω</mml:mi><mml:mo>,</mml:mo><mml:mi>T</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mfrac><mml:mrow><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:mfrac><mml:mi>ħ</mml:mi><mml:mi>ω</mml:mi><mml:mo>+</mml:mo><mml:msub><mml:mrow><mml:mi>k</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">B</mml:mi></mml:mrow></mml:msub><mml:mi>T</mml:mi><mml:mi mathvariant=\"normal\">ln</mml:mi><mml:mrow><mml:mo>[</mml:mo><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">e</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mfrac><mml:mrow><mml:mi>ħ</mml:mi><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mi>k</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">B</mml:mi></mml:mrow></mml:msub><mml:mi>T</mml:mi></mml:mrow></mml:mfrac></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:msup></mml:mrow><mml:mo>]</mml:mo></mml:mrow></mml:mrow></mml:mfenced><mml:mo>,</mml:mo></mml:math><graphic xlink:href=\"41467_2020_17563_Article_Equ1.gif\" position=\"anchor\"/></alternatives></disp-formula>where <italic>g</italic>(<italic>ω</italic>, <italic>T</italic>) is the phonon density of states (PDOS). The first optically bright transition in the (CdSe)<sub>13</sub> MSC is the HOMO→LUMO+1 transition (highest occupied/lowest unoccupied molecular orbital, note that the LUMO describes a mid-gap state, which is related to the surface and frequently observed in CdSe nanoclusters<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref>–<xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>, see Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6</xref>).<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Calculated phonon density of states (PDOS) and bandgap relaxation with temperature.</title><p><bold>a</bold> Relaxed structural model of a core/cage (CdSe)<sub>13</sub> magic sized cluster (MSC) with methylamine (MA) ligands in the ground state electronic configuration. <bold>b</bold> Normalized PDOS for core/cage (CdSe)<sub>13</sub> MSC as shown in <bold>a</bold> in the ground and HOMO→LUMO+1 excited state electronic configurations, respectively, used to calculate Δ<italic>E</italic><sub><italic>g</italic></sub>(<italic>T</italic>, <italic>g</italic><sub>corecage</sub>(<italic>ω</italic>, <italic>T</italic> = 0 K)) (blue straight line in <bold>c</bold>). Note, that beside the modes inherent to the CdSe cluster, the PDOS also contains vibrational and rotational modes stemming from the MA ligands. <bold>c</bold> Temperature-dependent bandgap shift of (CdSe)<sub>13</sub> MSCs calculated using phonon frequencies obtained in harmonic approximation <italic>g</italic>(<italic>ω</italic>, <italic>T</italic> = 0 K)) (blue), including anharmonicities <italic>g</italic>(<italic>ω</italic>, <italic>T</italic> = 300 K) (red) and with full temperature dependence <italic>g</italic>(<italic>ω</italic>, <italic>T)</italic> (green), in comparison to experiment (grey area). Solid and dashed lines denote the core/cage MSC with MA ligands and the sliced-wurtzite MSC without ligands, respectively.</p></caption><graphic xlink:href=\"41467_2020_17563_Fig3_HTML\" id=\"d30e1004\"/></fig></p><p id=\"Par10\">To obtain the excited state PDOS, we constrained the electron occupations of the orbitals, so that one electron from the HOMO is transferred to the LUMO+1 state. We calculated the phonon frequencies, <italic>ω</italic><sub><italic>i</italic></sub>, for the cluster in the ground and the excited state from the force constants. Compared to the MSC in the ground state electronic configuration, the presence of the excited electron weakens the bonds and causes a red-shift of the average phonon frequency of 4.2% for the sliced-wurtzite structure (116.0 cm<sup>−1</sup>→111.1 cm<sup>−1</sup>) (individual phonon frequencies in Supplementary Tables <xref rid=\"MOESM1\" ref-type=\"media\">2</xref> and <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>). This shift in the mean frequency is almost two orders of magnitude larger than calculated for bulk Si (<0.1% at the melting temperature)<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. Inserting the obtained phonon frequencies, the term for the free energy is reduced to a summation. Neglecting the explicit temperature and volume dependence of the PDOS (<italic>g</italic>(<italic>ω</italic>, <italic>T</italic>) ≈ <italic>g</italic>(<italic>ω</italic>, <italic>T</italic> = 0 K)), we obtain the bandgap shift with temperature<disp-formula id=\"Equ2\"><label>2</label><alternatives><tex-math id=\"M3\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathrm{\\Delta }}E_{\\mathrm{g}}\\left( T \\right) = E_{\\mathrm{g}}\\left( T \\right) - E_{\\mathrm{g}}\\left( {0\\,{\\mathrm{K}}} \\right)$$\\end{document}</tex-math><mml:math id=\"M4\"><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:msub><mml:mrow><mml:mi>E</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>T</mml:mi></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:msub><mml:mrow><mml:mi>E</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>T</mml:mi></mml:mrow></mml:mfenced><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>E</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mn>0</mml:mn><mml:mspace width=\"0.25em\"/><mml:mi mathvariant=\"normal\">K</mml:mi></mml:mrow></mml:mfenced></mml:math><graphic xlink:href=\"41467_2020_17563_Article_Equ2.gif\" position=\"anchor\"/></alternatives></disp-formula>as the free energy difference<disp-formula id=\"Equ3\"><label>3</label><alternatives><tex-math id=\"M5\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${\\mathrm{\\Delta }}E_{\\mathrm{g}}(T) = F_{{\\mathrm{vib}},{\\mathrm{exc}}}\\left( T \\right) - F_{{\\mathrm{vib}},{\\mathrm{gs}}}\\left( T \\right) - \\left( {F_{{\\mathrm{vib}},{\\mathrm{exc}}}\\left( {0\\,{\\mathrm{K}}} \\right) - F_{{\\mathrm{vib}},{\\mathrm{gs}}}\\left( {0\\,{\\mathrm{K}}} \\right)} \\right)$$\\end{document}</tex-math><mml:math id=\"M6\"><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:msub><mml:mrow><mml:mi>E</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>T</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">vib</mml:mi><mml:mo>,</mml:mo><mml:mi mathvariant=\"normal\">exc</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>T</mml:mi></mml:mrow></mml:mfenced><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">vib</mml:mi><mml:mo>,</mml:mo><mml:mi mathvariant=\"normal\">gs</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>T</mml:mi></mml:mrow></mml:mfenced><mml:mo>−</mml:mo><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">vib</mml:mi><mml:mo>,</mml:mo><mml:mi mathvariant=\"normal\">exc</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mn>0</mml:mn><mml:mspace width=\"0.25em\"/><mml:mi mathvariant=\"normal\">K</mml:mi></mml:mrow></mml:mfenced><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>F</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">vib</mml:mi><mml:mo>,</mml:mo><mml:mi mathvariant=\"normal\">gs</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mn>0</mml:mn><mml:mspace width=\"0.25em\"/><mml:mi mathvariant=\"normal\">K</mml:mi></mml:mrow></mml:mfenced></mml:mrow></mml:mfenced></mml:math><graphic xlink:href=\"41467_2020_17563_Article_Equ3.gif\" position=\"anchor\"/></alternatives></disp-formula>between the ground and excited state electronic configurations (blue lines in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3c</xref>). For <italic>T</italic> < 150 K, the measured and calculated temperature-dependence of Δ<italic>E</italic><sub><italic>g</italic></sub> agree very closely. At higher temperatures, the experimental data deviate slightly from a perfectly linear behavior, indicating anharmonic contributions to the free energy.</p><p id=\"Par11\">In order to explicitly account for the temperature-dependence of the phonon frequencies, we used the same constrained DFT approach to perform ab initio MD calculations to obtain the PDOS at 300 K (see Methods). Inserting the ground and excited state PDOSs for <italic>T</italic> = 300 K, we obtained a slight overestimation for the temperature-dependence of the bandgap (red lines in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3c</xref>). In the case of the sliced-wurtzite (CdSe)<sub>13</sub> MSCs, we then approximated the temperature-dependent PDOS <italic>g</italic>(<italic>ω, T</italic>) using Gaussian functions as a basis set and interpolated between the PDOSs at <italic>T</italic> = 0 K and <italic>T</italic> = 300 K (see “Methods”). As shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3c</xref>, the bandgap calculated ab initio from the fully temperature-dependent <italic>g</italic>(<italic>ω, T</italic>) (green dashed line) is in excellent agreement with the experiments within the whole measured temperature range.</p></sec><sec id=\"Sec3\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par12\">We now discuss our results in connection to current literature. Previous DFT calculations for nanoclusters determined the temperature dependent bandgap either (i) as the mean HOMO-LUMO energy difference in a MD<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref>–<xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, which distributes the atomic velocities according to classical Maxwell-Boltzmann statistics instead of Bose–Einstein occupation of the vibrational modes, and thus inherently overestimates d<italic>E</italic><sub>g</sub>/d<italic>T</italic> at low temperatures, or (ii) by summing over Bose–Einstein weighted eigenenergy changes induced by frozen-phonon modes<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref>–<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. The latter is known to severely overestimate the temperature dependence of the bandgap in nanocrystals<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>, often attributed to overrated contributions of surface and ligand modes. Indeed, we obtain an overestimation by almost an order of magnitude, calculating the bandgap shift with temperature based on a frozen-phonon approach (compare Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>). The failure of the commonly used frozen-phonon approach, which is very successful in the bulk, indicates that there is something fundamentally different about the temperature-dependence of the band gap in NCs. The approach presented in this work, which explicitly incorporates the effect of photon absorption on phonon modes, is able to successfully describe d<italic>E</italic><sub>g</sub>/d<italic>T</italic> over the entire temperature range between 5  and 300 K with quantitative accuracy and without any free parameters. Brook’s theorem<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup> states that the temperature-dependence of the bandgap can be obtained equally from either the changes in the eigenenergies induced by the phonons or the changes in the vibrational frequencies induced by the optical excitations. Clearly, our results demonstrate that Brook’s theorem is invalid for small nanoclusters and they highlight the importance of taking excitonic effects into account explicitly.</p><p id=\"Par13\">For high temperatures (ℏ<italic>ω</italic> ≪ <italic>k</italic><sub>B</sub><italic>T</italic>), the slope of the temperature-induced band gap shift can be estimated as<disp-formula id=\"Equ4\"><label>4</label><alternatives><tex-math id=\"M7\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$\\frac{{{\\mathrm{d}}E_{\\mathrm{g}}}}{{{\\mathrm{dT}}}} \\propto \\ln \\left( {\\frac{{\\omega _{{\\mathrm{exc}}}}}{{\\omega _{{\\mathrm{gs}}}}}} \\right) = \\ln \\left( {1 + \\frac{{\\Delta \\omega }}{{\\omega _{{\\mathrm{gs}}}}}} \\right),$$\\end{document}</tex-math><mml:math id=\"M8\"><mml:mfrac><mml:mrow><mml:mi mathvariant=\"normal\">d</mml:mi><mml:msub><mml:mrow><mml:mi>E</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">g</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">dT</mml:mi></mml:mrow></mml:mfrac><mml:mo>∝</mml:mo><mml:mo>ln</mml:mo><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">exc</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">gs</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfrac></mml:mrow></mml:mfenced><mml:mo>=</mml:mo><mml:mo>ln</mml:mo><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mn>1</mml:mn><mml:mo>+</mml:mo><mml:mfrac><mml:mrow><mml:mi mathvariant=\"normal\">Δ</mml:mi><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:msub><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">gs</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfrac></mml:mrow></mml:mfenced><mml:mo>,</mml:mo></mml:math><graphic xlink:href=\"41467_2020_17563_Article_Equ4.gif\" position=\"anchor\"/></alternatives></disp-formula>disclosing two factors which might enhance d<italic>E</italic><sub>g</sub>/d<italic>T</italic> in nanoclusters compared to bulk: (i) Softer phonon modes in general compared to bulk, e.g. due to surface related acoustic modes, and (ii) an enhanced change Δ<italic>ω</italic> in the phonon energies due to the absorption process. The former has been predicted for PbS<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup> and Si<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>, while for CdSe nanoparticles theoretically<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref>,<xref ref-type=\"bibr\" rid=\"CR54\">54</xref>,<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup> and experimentally<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup> a blueshift of the acoustic surface modes has been observed. Our calculations reveal a distinct redshift in the acoustic modes for the core-cage nanocluster model compared to bulk, while the data exhibit no clear shift for the sliced-wurtzite structure (see Supplementary Figure 4c). Both structures reveal enhanced temperature-induced bandgap shifts (red lines in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3c</xref>), indicating that the main origin lies in the significant change in the PDOS of clusters in the presence of an exciton. Due to the small size of the clusters, the exciton is distributed over a minimal number of 40–50 bonds. Note that one exciton per cluster corresponds to a photo-generated charge carrier density in the range of 10<sup>21</sup> cm<sup>−3</sup>, representing the regime of particularly high optical pumping in conventional semiconductors.</p><p id=\"Par14\">Our results not only highlight how the absorption of one photon as the probe medium influences the observed bandgap, but also visibly demonstrate how the temperature dependence of the bandgap immediately depends on the phonon dispersion of a material. We expect these findings to be relevant in materials that are either quantum-confined, strongly correlated or exhibit strong electron–phonon coupling and thus optical excitations significantly alter the vibrational free energies and in turn derived properties.</p></sec><sec id=\"Sec4\"><title>Methods</title><sec id=\"Sec5\"><title>Sample preparation</title><p id=\"Par15\">Nanocrystals and MSCs used in this study were synthesized using standard Schlenk techniques under an argon atmosphere. The MSCs were obtained from a Lewis acid-base reaction between metal-ammine halide complexes (CdCl<sub>2</sub>(octylamine)<sub>2</sub>, ZnCl<sub>2</sub>(octylamine)<sub>2</sub>, and MnCl<sub>2</sub>(octylamine)<sub>2</sub>) and octylammonium selenocarbamate (0.67 M) in <italic>n</italic>-octylamine (Aldrich, 99%). For Mn<sup>2+</sup>:(CdSe)<sub>13</sub> MSCs, 1.0 mmol of CdCl<sub>2</sub> and 0.1 mmol of MnCl<sub>2</sub> were heated in 7 ml of n-octylamine at 120 °C for 2 h. The average doping concentration is changed by adjusting the initial precursor ratio (MnCl<sub>2</sub>/CdCl<sub>2</sub>). Octylammonium selenocarbamate was prepared by bubbling CO gas into 3 ml of <italic>n</italic>-octylamine containing 2.0 mmol Se powder for 1 h at room temperature. This solution was injected into the as-prepared precursor mixture containing metal-ammine halide complexes and kept at room temperature for more than 24 h. The MSCs were precipitated by adding ethanol containing trioctylphosphine followed by washing several times with ethanol<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. For the synthesis of Mn<sup>2+</sup>:(ZnSe)<sub>13</sub> MSCs, ZnCl<sub>2</sub> was used instead of CdCl<sub>2</sub>. The average doping concentration of the MSCs was measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES, Shimadzu ICPS-7500). To enhance the dispersibility of MSCs, which results in minimization of light scattering, the initial octylamine ligands were exchanged with long-chain oleylamine ligands<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. CdSe QDs and CdSe/ZnS QDs were synthesized by conventional hot injection method using Cd(oleate)<sub>2</sub> and trioctylphosphine selenide (TOPSe) as precursors<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>. In all, 1.2 mmol CdO were added to a mixture of 1.5 mL of oleic acid and 20 mL of 1-octadecene and heated under vacuum for 2 h at 120 °C to form Cd(oleate)<sub>2</sub> complexes. The mixture was then heated to 300 °C under Ar and TOPSe (1 M) was rapidly injected to obtain CdSe QDs. For the growth of ZnS shells, 4.8 mmol of Zn(oleate)<sub>2</sub> and 4.8 mmol of tributylphosphine sulfide were additionally introduced to the as-prepared solution containing CdSe QDs and the reaction mixture was kept at 300 °C for 20 min. For CdSe QDs and CdSe/ZnS QDs, we also performed a surface modification with oleylamine to neglect the effect of the surface ligands.</p></sec><sec id=\"Sec6\"><title>Optical characterization</title><p id=\"Par16\">For absorption, photoluminescence excitation spectroscopy (PLE) and magnetic circular dichroism (MCD) measurements the MSCs passivated by oleylamine were prepared as thin films between two quartz glass substrates, and for PL measurements between a silicon and a quartz glass substrate. MCD spectroscopy was conducted on a homemade setup consisting of a 75 W Xenon lamp (Lot-Oriel) equipped with a monochromator (omni-λ 150, Lot-Oriel) and a photomultiplier (R928, Hamamatsu). The excitation light was modulated using a photoelastic modulator (PEM-90, Hinds Instruments). The sample was placed in a helium vapor cryostat (ST-300, Janis) between two poles of an electromagnet (EM4-HVA, Lake Shore) in Faraday geometry. Absorption measurements were either extracted from the DC signal during MCD measurements or collected with an UV-VIS spectrophotometer (Shimadzu 2550) equipped with a helium vapor cryostat (ST-300, Janis). PLE measurements were conducted with a helium vapor cryostat (ST-300, Janis) placed in a Fluorolog-3 (FL3-22, Horiba Scientific). For time resolved analysis of the PL signal the samples placed in a helium vapor cryostat (ST-300, Janis) were excited with a frequency tripled Ti:sapphire laser (Mira 900, Coherent, 270 nm) pumped with an Nd:YAG laser (Verdi-V10, Coherent) with 100 fs pulses at a repetition rate of 76 MHz. The signal was detected with a streak camera system consisting of a monochromator (250IS, Bruker Optics) and a Synchroscan Streak camera (C5680-24 c, Hamamatsu), providing a temporal resolution of 4 ps.</p></sec><sec id=\"Sec7\"><title>DFT calculations</title><p id=\"Par17\">We carried out first principles calculations with density functional theory and plane-wave basis sets, within the Perdew-Burke-Ernzerhof (PBE)<sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref>,<xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup> approximation as implemented in the Quantum Espresso package<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>. We used norm-conserving pseudopotentials and a wavefunction energy cutoff of 45 Ry. The molecular dynamics (MD) calculations were performed at 300 K, using a time step of 2 fs and the Berendsen<sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref></sup> thermostat with a rise time of 10 time steps. The clusters were equilibrated for 25 ps and subsequently data was sampled for 100 ps. Excited states were described by pair excitations<sup><xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup> within constrained DFT, where the Kohn-Sham energy is minimized under the constraint that the occupation of the highest occupied molecular orbital (HOMO) is reduced by 1 e<sup>−</sup>, compared to the ground state, and the occupation of the lowest energy state directly above the lowest unoccupied molecular orbital (LUMO + 1) is increased by 1 e<sup>−</sup>. To prevent convergence issues within the electronic loop due to level crossings and concurrent discontinuous changes in the charge density, we implemented a smearing scheme into Quantum Espresso, where a Gaussian smearing is applied individually to the electron and the hole with a smearing of <italic>σ</italic> = 0.001 Ry. At <italic>T</italic> = 0 K, phonon eigenfrequencies and eigenvectors are calculated from finite differences. At <italic>T</italic> = 300 K, phonon densities of states (PDOS) are computed from the MD trajectories using the maximum entropy method (MEM)<sup><xref ref-type=\"bibr\" rid=\"CR62\">62</xref>,<xref ref-type=\"bibr\" rid=\"CR63\">63</xref></sup>, the same numerical setup as above and 4096 poles in the frequency interval from 0 cm<sup>−1</sup> to 300 cm<sup>−1</sup>. The spectra are renormalized so that the integrals ∫d<italic>ωg</italic>(<italic>ω</italic>) are equal to the number of phonon modes. To calculate <italic>g</italic>(<italic>ω</italic>, <italic>T</italic>), we approximate the set of discrete phonon frequencies for <italic>T</italic> = 0 K by Gaussians with a very small but finite width <italic>σ</italic> = 2 cm<sup>−1</sup> according to:<disp-formula id=\"Equ5\"><label>5</label><alternatives><tex-math id=\"M9\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$g\\left( {\\omega ,\\,T = 0} \\right) = \\Sigma _i\\frac{1}{{\\sqrt {2{\\uppi}\\sigma ^2} }}{\\mathrm{e}}^{ - \\frac{{\\left( {\\omega - \\omega _i} \\right)^2}}{{2\\sigma ^2}}}$$\\end{document}</tex-math><mml:math id=\"M10\"><mml:mi>g</mml:mi><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>ω</mml:mi><mml:mo>,</mml:mo><mml:mspace width=\"0.25em\"/><mml:mi>T</mml:mi><mml:mo>=</mml:mo><mml:mn>0</mml:mn></mml:mrow></mml:mfenced><mml:msub><mml:mrow><mml:mo>=</mml:mo><mml:mi mathvariant=\"normal\">Σ</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi></mml:mrow></mml:msub><mml:mfrac><mml:mrow><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:msqrt><mml:mrow><mml:mn>2</mml:mn><mml:mi mathvariant=\"normal\">π</mml:mi><mml:msup><mml:mrow><mml:mi>σ</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:msqrt></mml:mrow></mml:mfrac><mml:msup><mml:mrow><mml:mi mathvariant=\"normal\">e</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mfrac><mml:mrow><mml:msup><mml:mrow><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>ω</mml:mi><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>ω</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:mfenced></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:mrow><mml:mrow><mml:mn>2</mml:mn><mml:msup><mml:mrow><mml:mi>σ</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:mfrac></mml:mrow></mml:msup></mml:math><graphic xlink:href=\"41467_2020_17563_Article_Equ5.gif\" position=\"anchor\"/></alternatives></disp-formula></p><p id=\"Par18\">Subsequently. <italic>g</italic>(<italic>ω</italic>, <italic>T</italic> <italic>=</italic> 300 K) is expanded into the same number of Gaussians, albeit with modified positions and widths. All resulting PDOS within the Gaussian basis set have been confirmed to yield the same free energies as before. We then linearly interpolate the widths and positions of the Gaussians as a function of temperature to obtain <italic>g</italic>(<italic>ω</italic>, <italic>T</italic>).</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec8\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41467_2020_17563_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41467_2020_17563_MOESM2_ESM.pdf\"><caption><p>Peer Review File</p></caption></media></supplementary-material></sec></sec></body><back><app-group><app id=\"App1\"><sec id=\"Sec9\"><title>Source data</title><p id=\"Par21\"><media position=\"anchor\" xlink:href=\"41467_2020_17563_MOESM3_ESM.xlsx\" id=\"MOESM3\"><caption><p>Source Data</p></caption></media></p></sec></app></app-group><fn-group><fn><p><bold>Peer review information</bold>\n<italic>Nature Communications</italic> thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary information</bold> is available for this paper at 10.1038/s41467-020-17563-0.</p></sec><ack><title>Acknowledgements</title><p>F.M., S.L., and G.B. gratefully acknowledge financial support from the German Research Foundation DFG under contract Ba 1422/13-2. F.M. in addition was supported by the German Academic Exchange Service (DAAD) with funds from the German Federal Ministry of Education and Research (BMBF) and the European Union (FP7-PEOPLE-2013-COFUND - grant agreement n° 605728). T.A.N., E.S., and S.W. were supported by the German Federal Ministry of Education and Research (BMBF) within the NanoMatFutur programme, grant no. 13N12972. Supercomputer time provided by NERSC (project no. 35687) and the Max-Planck Computing and Data Facility, Garching, is gratefully acknowledged. T.H. acknowledges financial support by the Research Center Program of the Institute for Basic Science (IBS) in Korea (IBS-R006-D1). J.Y. acknowledges the financial support by the DGIST Start-up Fund Program of the Ministry of Science, ICT and Future Planning (2019070014).</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>F.M. and G.B. conceived and designed the experiments, led the research and wrote the paper. J.Y. and T.H. designed and prepared the materials. S.L. contributed to the optical measurements, data analysis and interpretation. S.W. conceived and designed the DFT calculations, T.A.N. and E.S. conducted the DFT calculations. All authors discussed the results and assisted in paper preparation.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>The source data underlying Figs. <xref rid=\"MOESM3\" ref-type=\"media\">1</xref>–<xref rid=\"MOESM3\" ref-type=\"media\">3b</xref>, c) are provided in a source data file. All other relevant data are available from G.B. on request. Source data are provided with this paper. </p></notes><notes notes-type=\"data-availability\"><title>Code availability</title><p>The computer code used for the DFT and MD calculations during the study is available from S.W. on request.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par19\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kovalenko</surname><given-names>MV</given-names></name><etal/></person-group><article-title>Prospects of Nanoscience with Nanocrystals</article-title><source>ACS Nano</source><year>2015</year><volume>9</volume><fpage>1012</fpage><lpage>1057</lpage><pub-id pub-id-type=\"pmid\">25608730</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Akkerman</surname><given-names>QA</given-names></name><name><surname>Rainò</surname><given-names>G</given-names></name><name><surname>Kovalenko</surname><given-names>MV</given-names></name><name><surname>Manna</surname><given-names>L</given-names></name></person-group><article-title>Genesis, challenges and opportunities for colloidal lead halide perovskite nanocrystals</article-title><source>Nat. Mater.</source><year>2018</year><volume>17</volume><fpage>394</fpage><lpage>405</lpage><pub-id pub-id-type=\"pmid\">29459748</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Medintz</surname><given-names>IL</given-names></name><name><surname>Uyeda</surname><given-names>HT</given-names></name><name><surname>Goldman</surname><given-names>ER</given-names></name><name><surname>Mattoussi</surname><given-names>H</given-names></name></person-group><article-title>Quantum dot bioconjugates for imaging, labelling and sensing</article-title><source>Nat. Mater.</source><year>2005</year><volume>4</volume><fpage>435</fpage><lpage>446</lpage><pub-id pub-id-type=\"pmid\">15928695</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Choi</surname><given-names>MK</given-names></name><name><surname>Yang</surname><given-names>J</given-names></name><name><surname>Hyeon</surname><given-names>T</given-names></name><name><surname>Kim</surname><given-names>D-H</given-names></name></person-group><article-title>Flexible quantum dot light-emitting diodes for next-generation displays</article-title><source>npj Flex. Electron.</source><year>2018</year><volume>2</volume><fpage>10</fpage></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shirasaki</surname><given-names>Y</given-names></name><name><surname>Supran</surname><given-names>GJ</given-names></name><name><surname>Bawendi</surname><given-names>MG</given-names></name><name><surname>Bulović</surname><given-names>V</given-names></name></person-group><article-title>Emergence of colloidal quantum-dot light-emitting technologies</article-title><source>Nat. Photonics</source><year>2013</year><volume>7</volume><fpage>13</fpage><lpage>23</lpage></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>García de Arquer</surname><given-names>FP</given-names></name><name><surname>Armin</surname><given-names>A</given-names></name><name><surname>Meredith</surname><given-names>P</given-names></name><name><surname>Sargent</surname><given-names>EH</given-names></name></person-group><article-title>Solution-processed semiconductors for next-generation photodetectors</article-title><source>Nat. Rev. Mater.</source><year>2017</year><volume>2</volume><fpage>16100</fpage></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Efros</surname><given-names>A</given-names></name><etal/></person-group><article-title>Band-edge exciton in quantum dots of semiconductors with a degenerate valence band: dark and bright exciton states</article-title><source>Phys. Rev. B</source><year>1996</year><volume>54</volume><fpage>4843</fpage><lpage>4856</lpage></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Norris</surname><given-names>D</given-names></name><name><surname>Efros</surname><given-names>A</given-names></name><name><surname>Rosen</surname><given-names>M</given-names></name><name><surname>Bawendi</surname><given-names>M</given-names></name></person-group><article-title>Size dependence of exciton fine structure in CdSe quantum dots</article-title><source>Phys. Rev. B. Condens. Matter</source><year>1996</year><volume>53</volume><fpage>16347</fpage><lpage>16354</lpage><pub-id pub-id-type=\"pmid\">9983473</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ortner</surname><given-names>G</given-names></name><etal/></person-group><article-title>Temperature dependence of the excitonic band gap in In<sub>x</sub>Ga<sub>1−x</sub>As/GaAs self-assembled quantum dots</article-title><source>Phys. Rev. B</source><year>2005</year><volume>72</volume><fpage>085328</fpage></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sanguinetti</surname><given-names>S</given-names></name><etal/></person-group><article-title>Electron-phonon interaction in individual strain-free GaAs∕Al<sub>0.3</sub>Ga<sub>0.7</sub>As quantum dots</article-title><source>Phys. Rev. B</source><year>2006</year><volume>73</volume><fpage>1</fpage><lpage>7</lpage></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liptay</surname><given-names>TJ</given-names></name><name><surname>Ram</surname><given-names>RJ</given-names></name></person-group><article-title>Temperature dependence of the exciton transition in semiconductor quantum dots</article-title><source>Appl. Phys. Lett.</source><year>2006</year><volume>89</volume><fpage>223132</fpage></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Joshi</surname><given-names>A</given-names></name><name><surname>Narsingi</surname><given-names>KY</given-names></name><name><surname>Manasreh</surname><given-names>MO</given-names></name><name><surname>Davis</surname><given-names>EA</given-names></name><name><surname>Weaver</surname><given-names>BD</given-names></name></person-group><article-title>Temperature dependence of the band gap of colloidal CdSe/ZnS core/shell nanocrystals embedded into an ultraviolet curable resin</article-title><source>Appl. Phys. Lett.</source><year>2006</year><volume>89</volume><fpage>89</fpage><lpage>92</lpage></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Valerini</surname><given-names>D</given-names></name><etal/></person-group><article-title>Temperature dependence of the photoluminescence properties of colloidal CdSe∕ZnS core/shell quantum dots embedded in a polystyrene matrix</article-title><source>Phys. Rev. B</source><year>2005</year><volume>71</volume><fpage>1</fpage><lpage>6</lpage></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jing</surname><given-names>P</given-names></name><etal/></person-group><article-title>Temperature-dependent photoluminescence of CdSe-Core CdS/CdZnS/ZnS-multishell quantum dots</article-title><source>J. Phys. Chem. C.</source><year>2009</year><volume>113</volume><fpage>13545</fpage><lpage>13550</lpage></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nomura</surname><given-names>S</given-names></name><name><surname>Kobayashi</surname><given-names>T</given-names></name></person-group><article-title>Exciton–LO-phonon couplings in spherical semiconductor microcrystallites</article-title><source>Phys. Rev. B</source><year>1992</year><volume>45</volume><fpage>1305</fpage><lpage>1316</lpage></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Korsunska</surname><given-names>NE</given-names></name><name><surname>Dybiec</surname><given-names>M</given-names></name><name><surname>Zhukov</surname><given-names>L</given-names></name><name><surname>Ostapenko</surname><given-names>S</given-names></name><name><surname>Zhukov</surname><given-names>T</given-names></name></person-group><article-title>Reversible and non-reversible photo-enhanced luminescence in CdSe/ZnS quantum dots</article-title><source>Semicond. Sci. Technol.</source><year>2005</year><volume>20</volume><fpage>876</fpage><lpage>881</lpage></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Olkhovets</surname><given-names>A</given-names></name><name><surname>Hsu</surname><given-names>R-C</given-names></name><name><surname>Lipovskii</surname><given-names>A</given-names></name><name><surname>Wise</surname><given-names>F</given-names></name></person-group><article-title>Size-dependent temperature variation of the energy gap in lead-salt quantum dots</article-title><source>Phys. Rev. Lett.</source><year>1998</year><volume>81</volume><fpage>3539</fpage><lpage>3542</lpage></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wise</surname><given-names>FW</given-names></name></person-group><article-title>Lead salt quantum dots: the limit of strong quantum confinement</article-title><source>Acc. Chem. Res.</source><year>2000</year><volume>33</volume><fpage>773</fpage><lpage>780</lpage><pub-id pub-id-type=\"pmid\">11087314</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vossmeyer</surname><given-names>T</given-names></name><etal/></person-group><article-title>CdS nanoclusters: synthesis, characterization, size dependent oscillator strength, temperature shift of the excitonic transition energy, and reversible absorbance shift</article-title><source>J. Phys. Chem.</source><year>1994</year><volume>98</volume><fpage>7665</fpage><lpage>7673</lpage></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>O’Donnell</surname><given-names>KP</given-names></name><name><surname>Chen</surname><given-names>X</given-names></name></person-group><article-title>Temperature dependence of semiconductor band gaps</article-title><source>Appl. Phys. Lett.</source><year>1991</year><volume>58</volume><fpage>2924</fpage><lpage>2926</lpage></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pässler</surname><given-names>R</given-names></name></person-group><article-title>Dispersion-related description of temperature dependencies of band gaps in semiconductors</article-title><source>Phys. Rev. B</source><year>2002</year><volume>66</volume><fpage>085201</fpage></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Varshni</surname><given-names>YP</given-names></name></person-group><article-title>Temperature dependence of the energy gap in semiconductors</article-title><source>Physica</source><year>1967</year><volume>34</volume><fpage>149</fpage><lpage>154</lpage></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Allen</surname><given-names>PB</given-names></name><name><surname>Heine</surname><given-names>V</given-names></name></person-group><article-title>Theory of the temperature dependence of electronic band structures</article-title><source>J. Phys. C. Solid State Phys.</source><year>1976</year><volume>9</volume><fpage>2305</fpage><lpage>2312</lpage></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Heine</surname><given-names>V</given-names></name><name><surname>Van Vechten</surname><given-names>JA</given-names></name></person-group><article-title>Effect of electron-hole pairs on phonon frequencies in Si related to temperature dependence of band gaps</article-title><source>Phys. Rev. B</source><year>1976</year><volume>13</volume><fpage>1622</fpage><lpage>1626</lpage></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Allen</surname><given-names>PB</given-names></name><name><surname>Cardona</surname><given-names>M</given-names></name></person-group><article-title>Temperature dependence of the direct gap of Si and Ge</article-title><source>Phys. Rev. B</source><year>1983</year><volume>27</volume><fpage>4760</fpage><lpage>4769</lpage></element-citation></ref><ref id=\"CR26\"><label>26.</label><mixed-citation publication-type=\"other\">Ridley, B. K. <italic>Quantum Process in Semiconductors</italic>. (Oxford University Press, 1993).</mixed-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Giustino</surname><given-names>F</given-names></name><name><surname>Louie</surname><given-names>SG</given-names></name><name><surname>Cohen</surname><given-names>ML</given-names></name></person-group><article-title>Electron-phonon renormalization of the direct band gap of diamond</article-title><source>Phys. Rev. Lett.</source><year>2010</year><volume>105</volume><fpage>1</fpage><lpage>4</lpage></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Antonius</surname><given-names>G</given-names></name><name><surname>Poncé</surname><given-names>S</given-names></name><name><surname>Boulanger</surname><given-names>P</given-names></name><name><surname>Côté</surname><given-names>M</given-names></name><name><surname>Gonze</surname><given-names>X</given-names></name></person-group><article-title>Many-body effects on the zero-point renormalization of the band structure</article-title><source>Phys. Rev. Lett.</source><year>2014</year><volume>112</volume><fpage>1</fpage><lpage>5</lpage></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Saidi</surname><given-names>WA</given-names></name><name><surname>Poncé</surname><given-names>S</given-names></name><name><surname>Monserrat</surname><given-names>B</given-names></name></person-group><article-title>Temperature dependence of the energy levels of methylammonium lead iodide perovskite from first principles</article-title><source>J. Phys. Chem. Lett.</source><year>2016</year><volume>7</volume><fpage>5247</fpage><lpage>5252</lpage><pub-id pub-id-type=\"pmid\">27973908</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Poncé</surname><given-names>S</given-names></name><etal/></person-group><article-title>Temperature dependence of the electronic structure of semiconductors and insulators</article-title><source>J. Chem. Phys.</source><year>2015</year><volume>143</volume><fpage>102813</fpage><pub-id pub-id-type=\"pmid\">26374006</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Franceschetti</surname><given-names>A</given-names></name></person-group><article-title>First-principles calculations of the temperature dependence of the band gap of Si nanocrystals</article-title><source>Phys. Rev. B - Condens. Matter Mater. Phys.</source><year>2007</year><volume>76</volume><fpage>16</fpage><lpage>18</lpage></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kamisaka</surname><given-names>H</given-names></name><name><surname>Kilina</surname><given-names>SV</given-names></name><name><surname>Yamashita</surname><given-names>K</given-names></name><name><surname>Prezhdo</surname><given-names>OV</given-names></name></person-group><article-title>Ab initio study of temperature and pressure dependence of energy and phonon-induced dephasing of electronic excitations in CdSe and PbSe quantum dots †</article-title><source>J. Phys. Chem. C.</source><year>2008</year><volume>112</volume><fpage>7800</fpage><lpage>7808</lpage></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ibrahim</surname><given-names>ZA</given-names></name><etal/></person-group><article-title>Temperature dependence of the optical response: application to bulk GaAs using first-principles molecular dynamics simulations</article-title><source>Phys. Rev. B</source><year>2008</year><volume>77</volume><fpage>125218</fpage></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Capaz</surname><given-names>RB</given-names></name><name><surname>Spataru</surname><given-names>CD</given-names></name><name><surname>Tangney</surname><given-names>P</given-names></name><name><surname>Cohen</surname><given-names>ML</given-names></name><name><surname>Louie</surname><given-names>SG</given-names></name></person-group><article-title>Temperature dependence of the band gap of semiconducting carbon nanotubes</article-title><source>Phys. Rev. Lett.</source><year>2005</year><volume>94</volume><fpage>1</fpage><lpage>4</lpage></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Han</surname><given-names>P</given-names></name><name><surname>Bester</surname><given-names>G</given-names></name></person-group><article-title>Large nuclear zero-point motion effect in semiconductor nanoclusters</article-title><source>Phys. Rev. B</source><year>2013</year><volume>88</volume><fpage>165311</fpage></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Han</surname><given-names>P</given-names></name><name><surname>Bester</surname><given-names>G</given-names></name></person-group><article-title>Fundamental difference between measured and calculated exciton-phonon coupling in nanostructures</article-title><source>Phys. Rev. B</source><year>2019</year><volume>99</volume><fpage>100302</fpage></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kasuya</surname><given-names>A</given-names></name><etal/></person-group><article-title>Ultra-stable nanoparticles of CdSe revealed from mass spectrometry</article-title><source>Nat. Mater.</source><year>2004</year><volume>3</volume><fpage>99</fpage><lpage>102</lpage><pub-id pub-id-type=\"pmid\">14743211</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>JH</given-names></name><etal/></person-group><article-title>Giant Zeeman splitting in nucleation-controlled doped CdSe:Mn<sup>2+</sup> quantum nanoribbons</article-title><source>Nat. Mater.</source><year>2010</year><volume>9</volume><fpage>47</fpage><lpage>53</lpage><pub-id pub-id-type=\"pmid\">19915554</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cossairt</surname><given-names>BM</given-names></name><name><surname>Owen</surname><given-names>JS</given-names></name></person-group><article-title>CdSe clusters: at the interface of small molecules and quantum dots</article-title><source>Chem. Mater.</source><year>2011</year><volume>23</volume><fpage>3114</fpage><lpage>3119</lpage></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bowers</surname><given-names>MJ</given-names></name><name><surname>McBride</surname><given-names>JR</given-names></name><name><surname>Rosenthal</surname><given-names>SJ</given-names></name></person-group><article-title>White-light emission from magic-sized cadmium selenide nanocrystals</article-title><source>J. Am. Chem. Soc.</source><year>2005</year><volume>127</volume><fpage>15378</fpage><lpage>15379</lpage><pub-id pub-id-type=\"pmid\">16262395</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>J</given-names></name><etal/></person-group><article-title>Route to the smallest doped semiconductor: Mn+2-Doped (CdSe)<sub>13</sub> clusters</article-title><source>J. Am. Chem. Soc.</source><year>2015</year><volume>137</volume><fpage>12776</fpage><lpage>12779</lpage><pub-id pub-id-type=\"pmid\">26431472</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuno</surname><given-names>M</given-names></name><name><surname>Lee</surname><given-names>JK</given-names></name><name><surname>Dabbousi</surname><given-names>BO</given-names></name><name><surname>Mikulec</surname><given-names>FV</given-names></name><name><surname>Bawendi</surname><given-names>MG</given-names></name></person-group><article-title>The band edge luminescence of surface modified CdSe nanocrystallites: probing the luminescing state</article-title><source>J. Chem. Phys.</source><year>1997</year><volume>106</volume><fpage>9869</fpage><lpage>9882</lpage></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Muckel</surname><given-names>F</given-names></name><etal/></person-group><article-title>Digital doping in magic-sized CdSe clusters</article-title><source>ACS Nano</source><year>2016</year><volume>10</volume><fpage>7135</fpage><lpage>7141</lpage><pub-id pub-id-type=\"pmid\">27420556</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>J</given-names></name><etal/></person-group><article-title>Chemical synthesis, doping, and transformation of magic-sized semiconductor alloy nanoclusters</article-title><source>J. Am. Chem. Soc.</source><year>2017</year><volume>139</volume><fpage>6761</fpage><lpage>6770</lpage><pub-id pub-id-type=\"pmid\">28481516</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thurmond</surname><given-names>CD</given-names></name></person-group><article-title>The standard thermodynamic functions for the formation of electrons and holes in Ge, Si, GaAs, and GaP</article-title><source>J. Electrochem. Soc.</source><year>1975</year><volume>122</volume><fpage>1133</fpage></element-citation></ref><ref id=\"CR46\"><label>46.</label><mixed-citation publication-type=\"other\">Brooks, H. Theory of the electrical properties of germanium and silicon. <italic>Adv. Electr</italic>. <bold>7</bold>, 85–182 (1955).</mixed-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>King-Smith</surname><given-names>RD</given-names></name><name><surname>Needs</surname><given-names>RJ</given-names></name><name><surname>Heine</surname><given-names>V</given-names></name><name><surname>Hodgson</surname><given-names>MJ</given-names></name></person-group><article-title>A first-principle calculation of the temperature dependence of the indirect band gap of silicon</article-title><source>Epl</source><year>1989</year><volume>10</volume><fpage>569</fpage><lpage>574</lpage></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Azpiroz</surname><given-names>JM</given-names></name><name><surname>Matxain</surname><given-names>JM</given-names></name><name><surname>Infante</surname><given-names>I</given-names></name><name><surname>Lopez</surname><given-names>X</given-names></name><name><surname>Ugalde</surname><given-names>JM</given-names></name></person-group><article-title>A DFT/TDDFT study on the optoelectronic properties of the amine-capped magic (CdSe)13 nanocluster</article-title><source>Phys. Chem. Chem. Phys.</source><year>2013</year><volume>15</volume><fpage>10996</fpage><pub-id pub-id-type=\"pmid\">23712668</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bawendi</surname><given-names>MG</given-names></name><name><surname>Carroll</surname><given-names>PJ</given-names></name><name><surname>Wilson</surname><given-names>WL</given-names></name><name><surname>Brus</surname><given-names>LE</given-names></name></person-group><article-title>Luminescence properties of CdSe quantum crystallites: resonance between interior and surface localized states</article-title><source>J. Chem. Phys.</source><year>1992</year><volume>96</volume><fpage>946</fpage><lpage>954</lpage></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Landes</surname><given-names>CF</given-names></name><name><surname>Braun</surname><given-names>M</given-names></name><name><surname>El-Sayed</surname><given-names>MA</given-names></name></person-group><article-title>On the nanoparticle to molecular size transition: fluorescence quenching studies</article-title><source>J. Phys. Chem. B</source><year>2001</year><volume>105</volume><fpage>10554</fpage><lpage>10558</lpage></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Puzder</surname><given-names>A</given-names></name><name><surname>Williamson</surname><given-names>AJ</given-names></name><name><surname>Gygi</surname><given-names>F</given-names></name><name><surname>Galli</surname><given-names>G</given-names></name></person-group><article-title>Self-healing of CdSe nanocrystals: first-principles calculations</article-title><source>Phys. Rev. Lett.</source><year>2004</year><volume>92</volume><fpage>1</fpage><lpage>4</lpage></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vörös</surname><given-names>M</given-names></name><name><surname>Galli</surname><given-names>G</given-names></name><name><surname>Zimanyi</surname><given-names>GT</given-names></name></person-group><article-title>Colloidal nanoparticles for intermediate band solar cells</article-title><source>ACS Nano</source><year>2015</year><volume>9</volume><fpage>6882</fpage><lpage>6890</lpage><pub-id pub-id-type=\"pmid\">26042468</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bozyigit</surname><given-names>D</given-names></name><etal/></person-group><article-title>Soft surfaces of nanomaterials enable strong phonon interactions</article-title><source>Nature</source><year>2016</year><volume>531</volume><fpage>618</fpage><lpage>622</lpage><pub-id pub-id-type=\"pmid\">26958836</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shi</surname><given-names>C</given-names></name><etal/></person-group><article-title>Size-dependent lattice dynamics of atomically precise cadmium selenide quantum dots</article-title><source>Phys. Rev. Lett.</source><year>2019</year><volume>122</volume><fpage>26101</fpage></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Han</surname><given-names>P</given-names></name><name><surname>Bester</surname><given-names>G</given-names></name></person-group><article-title>Insights about the surface of colloidal nanoclusters from their vibrational and thermodynamic properties</article-title><source>J. Phys. Chem. C.</source><year>2012</year><volume>116</volume><fpage>10790</fpage><lpage>10795</lpage></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Choi</surname><given-names>MK</given-names></name><etal/></person-group><article-title>Extremely vivid, highly transparent, and ultrathin quantum dot light-emitting diodes</article-title><source>Adv. Mater.</source><year>2018</year><volume>30</volume><fpage>1703279</fpage></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Perdew</surname><given-names>JP</given-names></name><name><surname>Burke</surname><given-names>K</given-names></name><name><surname>Ernzerhof</surname><given-names>M</given-names></name></person-group><article-title>Generalized gradient approximation made simple</article-title><source>Phys. Rev. Lett.</source><year>1996</year><volume>77</volume><fpage>3865</fpage><lpage>3868</lpage><pub-id pub-id-type=\"pmid\">10062328</pub-id></element-citation></ref><ref id=\"CR58\"><label>58.</label><mixed-citation publication-type=\"other\">Perdew, J. P., Burke, K. & Ernzerhof, M. Generalized gradient approximation made simple. <italic>Phys. Rev. Lett</italic>. <bold>77</bold>, 3865 (1996); erratum <bold>78</bold>, 1396 (1997).</mixed-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Giannozzi</surname><given-names>P</given-names></name><etal/></person-group><article-title>QUANTUM ESPRESSO: a modular and open-source software project for quantum simulations of materials</article-title><source>J. Phys. Condens. Matter</source><year>2009</year><volume>21</volume><fpage>395502</fpage><pub-id pub-id-type=\"pmid\">21832390</pub-id></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Berendsen</surname><given-names>HJC</given-names></name><name><surname>Postma</surname><given-names>JPM</given-names></name><name><surname>van Gunsteren</surname><given-names>WF</given-names></name><name><surname>DiNola</surname><given-names>A</given-names></name><name><surname>Haak</surname><given-names>JR</given-names></name></person-group><article-title>Molecular dynamics with coupling to an external bath</article-title><source>J. Chem. Phys.</source><year>1984</year><volume>81</volume><fpage>3684</fpage><lpage>3690</lpage></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weissker</surname><given-names>H-C</given-names></name><name><surname>Furthmüller</surname><given-names>J</given-names></name><name><surname>Bechstedt</surname><given-names>F</given-names></name></person-group><article-title>Structure- and spin-dependent excitation energies and lifetimes of Si and Ge nanocrystals from ab initio calculations</article-title><source>Phys. Rev. B</source><year>2004</year><volume>69</volume><fpage>115310</fpage></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jaynes</surname><given-names>ET</given-names></name></person-group><article-title>Information theory and statistical mechanics</article-title><source>Phys. Rev.</source><year>1957</year><volume>106</volume><fpage>620</fpage><lpage>630</lpage></element-citation></ref><ref id=\"CR63\"><label>63.</label><mixed-citation publication-type=\"other\">Press, W., Teukolsky, S., Vetterling, W. & Flannery, B. <italic>Numerical Recipes in C</italic>. (Cambridge University Press, 2002)</mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807800</article-id><article-id pub-id-type=\"pmc\">PMC7431587</article-id><article-id pub-id-type=\"publisher-id\">70375</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70375-6</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>A common partitivirus infection in United States and Czech Republic isolates of bat white-nose syndrome fungal pathogen <italic>Pseudogymnoascus destructans</italic></article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Ren</surname><given-names>Ping</given-names></name><address><email>piren@utmb.edu</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rajkumar</surname><given-names>Sunanda S.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff10\">10</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Tao</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sui</surname><given-names>Haixin</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\"><name><surname>Masters</surname><given-names>Paul S.</given-names></name><xref ref-type=\"aff\" rid=\"Aff5\">5</xref><xref ref-type=\"aff\" rid=\"Aff6\">6</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-4556-4363</contrib-id><name><surname>Martinkova</surname><given-names>Natalia</given-names></name><xref ref-type=\"aff\" rid=\"Aff7\">7</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-2837-1087</contrib-id><name><surname>Kubátová</surname><given-names>Alena</given-names></name><xref ref-type=\"aff\" rid=\"Aff8\">8</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-8747-9365</contrib-id><name><surname>Pikula</surname><given-names>Jiri</given-names></name><xref ref-type=\"aff\" rid=\"Aff9\">9</xref></contrib><contrib contrib-type=\"author\"><name><surname>Chaturvedi</surname><given-names>Sudha</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-3922-9676</contrib-id><name><surname>Chaturvedi</surname><given-names>Vishnu</given-names></name><address><email>vishnu.chaturvedi@health.ny.gov</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.238491.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0367 6866</institution-id><institution>Mycology Laboratory, Wadsworth Center, </institution><institution>New York State Department of Health, </institution></institution-wrap>Albany, NY USA </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.176731.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 1547 9964</institution-id><institution>Present Address: Department of Pathology, </institution><institution>University of Texas Medical Branch, </institution></institution-wrap>Galveston, TX USA </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.506261.6</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0706 7839</institution-id><institution>Institute of Medicinal Biotechnology, </institution><institution>Chinese Academy of Medical Sciences and Peking Union Medical College, </institution></institution-wrap>Beijing, People’s Republic of China </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.238491.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0367 6866</institution-id><institution>Cellular and Molecular Basis of Diseases Laboratory, Wadsworth Center, </institution><institution>New York State Department of Health, </institution></institution-wrap>Albany, NY USA </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.265850.c</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2151 7947</institution-id><institution>Department of Biomedical Sciences, </institution><institution>University of Albany School of Public Health, </institution></institution-wrap>Albany, NY USA </aff><aff id=\"Aff6\"><label>6</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.238491.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0367 6866</institution-id><institution>Viral Replication and Vector Biology Laboratory, Wadsworth Center, </institution><institution>New York State Department of Health, </institution></institution-wrap>Albany, NY USA </aff><aff id=\"Aff7\"><label>7</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.418095.1</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 1015 3316</institution-id><institution>Institute of Vertebrate Biology, </institution><institution>Czech Academy of Sciences, </institution></institution-wrap>Brno, Czech Republic </aff><aff id=\"Aff8\"><label>8</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.4491.8</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1937 116X</institution-id><institution>Department of Botany, Faculty of Science, </institution><institution>Charles University in Prague, </institution></institution-wrap>Praha, Czech Republic </aff><aff id=\"Aff9\"><label>9</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.412968.0</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 1009 2154</institution-id><institution>Department of Ecology and Diseases of Zoo Animals, Game, Fish and Bees, </institution><institution>University of Veterinary and Pharmaceutical Sciences, </institution></institution-wrap>Brno, Czech Republic </aff><aff id=\"Aff10\"><label>10</label>Present Address: ICMR Medical Research Institute, Puducherry, India </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13893</elocation-id><history><date date-type=\"received\"><day>27</day><month>5</month><year>2019</year></date><date date-type=\"accepted\"><day>24</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">The psychrophilic (cold-loving) fungus <italic>Pseudogymnoascus destructans</italic> was discovered more than a decade ago to be the pathogen responsible for white-nose syndrome, an emerging disease of North American bats causing unprecedented population declines. The same species of fungus is found in Europe but without associated mortality in bats. We found <italic>P. destructans</italic> was infected with a mycovirus [named Pseudogymnoascus destructans partitivirus 1 (PdPV-1)]. The virus is bipartite, containing two double-stranded RNA (dsRNA) segments designated as dsRNA1 and dsRNA2. The cDNA sequences revealed that dsRNA1 dsRNA is 1,683 bp in length with an open reading frame (ORF) that encodes 539 amino acids (molecular mass of 62.7 kDa); dsRNA2 dsRNA is 1,524 bp in length with an ORF that encodes 434 amino acids (molecular mass of 46.9 kDa). The dsRNA1 ORF contains motifs representative of RNA-dependent RNA polymerase (RdRp), whereas the dsRNA2 ORF sequence showed homology with the putative capsid proteins (CPs) of mycoviruses. Phylogenetic analyses with PdPV-1 RdRp and CP sequences indicated that both segments constitute the genome of a novel virus in the family <italic>Partitiviridae</italic>. The purified virions were isometric with an estimated diameter of 33 nm. Reverse transcription PCR (RT-PCR) and sequencing revealed that all US isolates and a subset of Czech Republic isolates of <italic>P. destructans</italic> were infected with PdPV-1. However, PdPV-1 appears to be not widely dispersed in the fungal genus <italic>Pseudogymnoascus</italic>, as non-pathogenic fungi <italic>P. appendiculatus</italic> (1 isolate) and <italic>P. roseus</italic> (6 isolates) tested negative. <italic>P. destructans</italic> PdPV-1 could be a valuable tool to investigate fungal biogeography and the host–pathogen interactions in bat WNS.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Fungi</kwd><kwd>Viral epidemiology</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">The fungus <italic>Pseudogymnoascus destructans</italic> (previously named as <italic>Geomyces destructans</italic>) is a psychrophilic (cold-loving) fungus responsible for the white-nose syndrome (WNS) in bat populations in North America<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. This newly discovered pathogen has directly or indirectly caused the death of more than 5.7 million bats since 2006<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. <italic>P. destructans</italic> was found to infect at least eleven species of bats, of which seven species exhibited disease symptoms upon infection. The infected bat species include the endangered Indiana bat (<italic>Myotis sodalis</italic>) and the grey bat (<italic>Myotis grisescens</italic>) <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.whitenosesyndrome.org/about/bats-affected-wns\">https://www.whitenosesyndrome.org/about/bats-affected-wns</ext-link><sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>.</p><p id=\"Par3\"><italic>P. destructans</italic> grows as an opportunistic pathogen on bat skin during bat hibernation in caves. It also persists in the cave environment as a saprotroph<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>–<xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. The optimal temperature of growth for this fungus is around 15 °C. It produces brown and grey colonies, secretes a brownish pigment, and reproduces asexually via asymmetrically curved conidia when cultured on Sabouraud dextrose agar (SAB)<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. Although <italic>P. destructans</italic> isolates analyzed to date from the US and Canada represent a clonal population, variations were reported among isolates<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>–<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Despite apparent genetic homogeneity, phenotypic variations were reported among North American <italic>P. destructans</italic> isolates, specifically in the mycelial growth rate, exudate production, pigment formation, and diffusion into agar media<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. The underlying cause(s) for the phenotypic variations remain to be discovered. Viral infection could be a potential reason for the variation in fungal phenotypic expression<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>,<xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>.</p><p id=\"Par4\">Viruses that infect fungi (mycoviruses) are rather common and mostly latent<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. The majority of mycoviruses are dsRNA-containing isometric particles, although ssRNA and ssDNA mycoviruses have also been recognized. Three well-studied mycovirus systems include (i) yeast killer toxins in <italic>Saccharomyces cerevisiae</italic> and non-conventional yeasts<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>, (ii) hypovirulence in <italic>Cryphonectria parasitica</italic> and <italic>Sclerotinia sclerotiorum</italic>, the causal agents of Chestnut blight and white mold diseases on hundreds of plant species, respectively<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>–<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>, and (iii) a symbiotic role in conferring heat tolerance to fungal endophytes of grasses<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. There are reports linking mycovirus infection to phenotypic changes in the fungal host<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>.</p><p id=\"Par5\">Recently, dsRNA mycoviruses in the human pathogens <italic>Aspergillus fumigatus</italic> and <italic>Talaromyces marneffei</italic> were found to cause hypervirulence<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. Mycoviruses could also provide a ‘phylogenomics window’ as their evolution showed a strong co-divergence with their fungal hosts<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. We hypothesized that the origin, evolution, and virulence of <italic>P. destructans</italic> could be investigated by focusing on mycoviruses. The current study summarizes the molecular characterization of a virus [named Pseudogymnoascus destructans partitivirus 1 (PdPV-1)] that infects <italic>P. destructans</italic>. All US and a few Czech Republic isolates tested positive for PdPV-1. PdPV-1 appears to be host-specific to <italic>P. destructans,</italic> as closely related non-pathogenic fungi <italic>P. appendiculatus</italic> and <italic>P. roseus</italic><sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup> tested negative for the mycovirus. The results indicated that PdPV-1 represents a common feature among all US and some Czech Republic isolates of <italic>P. destructans</italic> and could be used to further investigate fungal biogeography and the host–pathogen interactions in bat WNS.</p></sec><sec id=\"Sec2\"><title>Materials and methods</title><sec id=\"Sec3\"><title>Fungal isolates</title><p id=\"Par6\"><italic>P. destructans</italic> isolate (MYC80251) has been described previously<sup><xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. <italic>P. destructans</italic> isolates and other <italic>Pseudogymnoascus</italic> species listed in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>, were maintained on SAB agar.<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Fungal isolates used in this study.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Fungal isolate</th><th align=\"left\" rowspan=\"2\">Location</th><th align=\"left\" rowspan=\"2\">Source</th><th align=\"left\" rowspan=\"2\">Viral RNA detection</th><th align=\"left\" colspan=\"2\">GenBank accession#</th></tr><tr><th align=\"left\">RdRp</th><th align=\"left\">Capsid</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"6\"><bold><italic>Pseudogymnoascus destructans</italic></bold></td></tr><tr><td align=\"left\">MYC80251</td><td align=\"left\">Albany, New York</td><td align=\"left\"><italic>Myotis lucifugus</italic></td><td align=\"left\">Yes</td><td align=\"left\"><p>KP128044</p><p>MK789667*</p></td><td align=\"left\"><p>KP128045</p><p>MK789674*</p></td></tr><tr><td align=\"left\">PESU14</td><td align=\"left\">Avery, North Carolina</td><td align=\"left\"><italic>Perimyotis subflavus</italic></td><td align=\"left\">Yes</td><td align=\"left\">MN990689</td><td align=\"left\">MN990699</td></tr><tr><td align=\"left\">LBB17</td><td align=\"left\">Lawrence, Ohio</td><td align=\"left\"><italic>Myotis lucifugus</italic></td><td align=\"left\">Yes</td><td align=\"left\">MN990690</td><td align=\"left\">MN990700</td></tr><tr><td align=\"left\">PESU8</td><td align=\"left\">Greenbrier, West Virginia</td><td align=\"left\"><italic>Perimyotis subflavus</italic></td><td align=\"left\">Yes</td><td align=\"left\">MN990691</td><td align=\"left\">MN990701</td></tr><tr><td align=\"left\">LBB11</td><td align=\"left\">Woodward, Pennsylvania</td><td align=\"left\"><italic>Myotis lucifugus</italic></td><td align=\"left\">Yes</td><td align=\"left\">MN990692</td><td align=\"left\">MN990702</td></tr><tr><td align=\"left\">M2335</td><td align=\"left\">Tompkins, New York</td><td align=\"left\"><italic>Myotis lucifugus</italic></td><td align=\"left\">Yes</td><td align=\"left\">MN990693</td><td align=\"left\">MN990703</td></tr><tr><td align=\"left\">M2337</td><td align=\"left\">Erie, New York</td><td align=\"left\"><italic>Myotis lucifugus</italic></td><td align=\"left\">Yes</td><td align=\"left\">MN990694</td><td align=\"left\">MN990704</td></tr><tr><td align=\"left\">M2339</td><td align=\"left\">Ulster, New York</td><td align=\"left\"><italic>Myotis lucifugus</italic></td><td align=\"left\">Yes</td><td align=\"left\">MN990695</td><td align=\"left\">MN990705</td></tr><tr><td align=\"left\">HA-8-2</td><td align=\"left\">Hailes Cave, New York</td><td align=\"left\">Cave wall swab</td><td align=\"left\">Yes</td><td align=\"left\">MN990696</td><td align=\"left\">MN990706</td></tr><tr><td align=\"left\">VTG1-5-1</td><td align=\"left\">Greely Mine, Vermont</td><td align=\"left\">Soil</td><td align=\"left\">Yes</td><td align=\"left\">MN990697</td><td align=\"left\">MN990707</td></tr><tr><td align=\"left\">AC-3-3</td><td align=\"left\">Aelous Cave, Vermont</td><td align=\"left\">Soil</td><td align=\"left\">Yes</td><td align=\"left\">MN990698</td><td align=\"left\">MN990708</td></tr><tr><td align=\"left\">CCF3937</td><td align=\"left\">Malá Amerika mine, Czech Republic</td><td align=\"left\"><italic>Myotis myotis</italic></td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">CCF3938</td><td align=\"left\">Solenice tunnel, Czech Republic</td><td align=\"left\"><italic>Myotis myotis</italic></td><td align=\"left\">Yes</td><td align=\"left\"><p>KY609331</p><p>MK789668*</p></td><td align=\"left\"><p>KY609337</p><p>MK789675*</p></td></tr><tr><td align=\"left\">CCF3939</td><td align=\"left\">Solenice tunnel, Czech Republic</td><td align=\"left\"><italic>Myotis myotis</italic></td><td align=\"left\">Yes</td><td align=\"left\">MK789669*</td><td align=\"left\">MK789676*</td></tr><tr><td align=\"left\">CCF3941</td><td align=\"left\">Malá Amerika mine, Czech Republic</td><td align=\"left\"><italic>Myotis myotis</italic></td><td align=\"left\">Yes</td><td align=\"left\"><p>KY609332</p><p>MK789670*</p></td><td align=\"left\"><p>KY609338</p><p>MK789677*</p></td></tr><tr><td align=\"left\">CCF3944</td><td align=\"left\">Nový Knín, Czech Republic</td><td align=\"left\"><italic>Myotis myotis</italic></td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">CCF4103</td><td align=\"left\">Herlíkovice tunnel, Czech Republic</td><td align=\"left\"><italic>Myotis myotis</italic></td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">CCF4124</td><td align=\"left\">Albeřická/Bischofova cave, Czech Republic</td><td align=\"left\"><italic>Plecotus auritus</italic></td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">CCF4125</td><td align=\"left\">Albeřická/Bischofova cave, Czech Republic</td><td align=\"left\"><italic>Myotis myotis</italic></td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">CCF4126</td><td align=\"left\">Portál tunnel, Czech Republic</td><td align=\"left\"><italic>Myotis myotis</italic></td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">CCF4127</td><td align=\"left\">Herlíkovice tunnel, Czech Republic</td><td align=\"left\"><italic>Myotis myotis</italic></td><td align=\"left\">Yes</td><td align=\"left\"><p>KY609329</p><p>MK789671*</p></td><td align=\"left\"><p>KY609335</p><p>MK789678*</p></td></tr><tr><td align=\"left\">CCF4129</td><td align=\"left\">Pístov cellar, Czech Republic</td><td align=\"left\"><italic>Myotis myotis</italic></td><td align=\"left\">Yes</td><td align=\"left\">MK789672*</td><td align=\"left\">MK789679*</td></tr><tr><td align=\"left\">CCF4130</td><td align=\"left\">Fučná-Otov tunnel, Czech Republic</td><td align=\"left\"><italic>Myotis myotis</italic></td><td align=\"left\">Yes</td><td align=\"left\"><p>KY609328</p><p>MK789673*</p></td><td align=\"left\"><p>KY609334</p><p>MK789680*</p></td></tr><tr><td align=\"left\">M3695</td><td align=\"left\">Malá Morávka mine, Czech Republic</td><td align=\"left\"><italic>Myotis myotis</italic></td><td align=\"left\">Yes</td><td align=\"left\">KY609330</td><td align=\"left\">KY609336</td></tr><tr><td align=\"left\">M3696</td><td align=\"left\">Kateřinská cave, Czech Republic</td><td align=\"left\"><italic>Myotis bechsteinii</italic></td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">M3697</td><td align=\"left\">Malá Morávka mine, Czech Republic</td><td align=\"left\"><italic>Myotis emarginatus</italic></td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">M3698</td><td align=\"left\">Kateřinská cave, Czech Republic</td><td align=\"left\"><italic>Myotis nattereri</italic></td><td align=\"left\">Yes</td><td align=\"left\">KY609333</td><td align=\"left\">KY609339</td></tr><tr><td align=\"left\">M3699</td><td align=\"left\">Malá Morávka mine, Czech Republic</td><td align=\"left\"><italic>Myotis daubentonii</italic></td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">M3701</td><td align=\"left\">Sloupsko-Šošůvské cave, Czech Republic</td><td align=\"left\"><italic>Myotis myotis</italic></td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"6\"><bold><italic>Pseudogymnoascus roseus</italic></bold></td></tr><tr><td align=\"left\">3-VT-5</td><td align=\"left\">Vermont</td><td align=\"left\">Hibernacular soil</td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">5-NY-6</td><td align=\"left\">New York</td><td align=\"left\">Hibernacular soil</td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">5-NY-8</td><td align=\"left\">New York</td><td align=\"left\">Hibernacular soil</td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">5-NY-9</td><td align=\"left\">New York</td><td align=\"left\">Hibernacular soil</td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">WSF-3629</td><td align=\"left\">Wisconsin</td><td align=\"left\">Amorphus peat</td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\">UAMH1658</td><td align=\"left\">Canada</td><td align=\"left\">Sphagnum bog</td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr><tr><td align=\"left\" colspan=\"6\"><bold><italic>Pseudogymnoascus appendiculatus</italic></bold></td></tr><tr><td align=\"left\">UAMH10510</td><td align=\"left\">Canada</td><td align=\"left\">Wood bait block, Sphagnum bog</td><td align=\"left\">No</td><td align=\"left\"/><td align=\"left\"/></tr></tbody></table><table-wrap-foot><p>*GenBank accession numbers for sequences that were determined in the laboratory in Beijing, China; all other accession numbers are for sequences determined in the Albany, NY laboratory.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec4\"><title>dsRNA extraction</title><p id=\"Par7\"><italic>P. destructans</italic> was grown in a stationary culture in potato dextrose broth (PDB) at 15 °C for 1–2 months in 500-ml flasks. About 1 g wet weight mycelium was harvested and ground to powder under liquid nitrogen with a mortar and pestle. The powder was collected and suspended in 1 ml extraction buffer (150 mM sodium acetate, pH 5.0, 100 mM LiCl, 4% sodium dodecyl sulfate, 10 mM EDTA, pH 8.0, and 20 mM β-mercaptoethanol) and incubated on ice for 10 min. Total nucleic acids were obtained by standard phenol–chloroform extraction followed by precipitation with LiCl and isopropanol. ssRNA and DNA were removed by treatment with S1 nuclease (Life Technologies; Carlsbad, CA) in buffer containing 30 mM sodium acetate (pH 4.6), 50 mM NaCl, 1 mM zinc acetate, 0.5 mg/ml heat-denatured DNA, and 5% (v/v) glycerol at 37 °C for 10 min, followed by incubation with DNase I (Epicentre; Madison, WI) at 37 °C for 30 min. The reaction was extracted with an equal volume of Tris–EDTA–saturated phenol–chloroform–isoamyl alcohol (25:24:1), followed by extraction with an equal volume of chloroform–isoamyl alcohol (24:1). Undigested nucleic acid was precipitated with 2 volumes of absolute ethanol at − 20 °C overnight and recovered by centrifugation for 15 min at 10,000×<italic>g</italic>. Then the pellet was rinsed with 70% ethanol, air-dried and resuspended in 40 µl of RNase-free water. The extracted material was electrophoresed on a 1% agarose gel, stained with ethidium bromide and visualized by UV transillumination. To confirm the dsRNA nature of the remaining material, a 15-µg sample was treated with RNase III and RNase A (Life Technologies) at 37 °C for 1 h. Denaturing polyacrylamide gel electrophoresis of the purified dsRNA was used to determine the size of the two RNA bands.</p></sec><sec id=\"Sec5\"><title>cDNA synthesis and sequence analysis</title><p id=\"Par8\">Purified dsRNA fractions (1–5 µg) containing 2 segments were denatured in 90% dimethyl sulfoxide (DMSO) at 65 °C for 20 min in the presence of random hexadeoxynucleotide and quickly chilled on ice. The RNA-primer mixture was precipitated with ethanol and resuspended in 11 µl RNase-free water. First-strand cDNA was synthesized using M-MLV reverse transcriptase (Life Technologies), based on the manufacturer’s instructions. Briefly, the denatured dsRNA with the random hexadeoxynucleotide were mixed with dNTPs, 5 × First-Strand Buffer, 0.1 M DTT, and RNaseOUT Recombinant Ribonuclease Inhibitor (40 units/µl) and incubated at 37 °C for 2 min. After adding 1 µl (200 units) of M-MLV RT, the mixture was incubated at 25 °C for 10 min, followed by 37 °C for 50 min. The reaction was inactivated by heating at 70 °C for 15 min. The resulting cDNA was amplified with random primers and the variously sized PCR products were cloned in TOPO TA cloning vector. A series of overlapping cDNA clones were obtained from the sequencing of the positive clones (<xref rid=\"MOESM1\" ref-type=\"media\">S1</xref> Table). Determination of the ends of each dsRNA was done using FirstChoice<sup>®</sup> RLM-RACE Kit (Life Technologies). All the sequence contigs were assembled by Sequencher 4.8 (Gene Codes Co.; Ann Arbor, MI). Conserved sequences in GenBank were identified by searches using the tblastx program. Multiple sequence alignments for the two dsRNA segments were carried out using ClustalW analysis by MacVector 7.2 (Accelrys Inc.; Cary, NC). The maximum likelihood phylogenetic trees were generated by MEGA 6.0<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup> (<xref rid=\"MOESM1\" ref-type=\"media\">S2</xref> Table).</p></sec><sec id=\"Sec6\"><title>Virus purification</title><p id=\"Par9\">Approximately 60 g wet weight fungal mycelium were collected and ground to powder as described above. The homogenate was mixed with extraction buffer (0.1 M sodium phosphate, pH 7.4, containing 0.1% β-mercaptoethanol) at a volume of 5 ml/g of wet mycelium. Following the addition of an equal volume of chloroform, the suspension was vortexed extensively, and the resulting emulsion was broken by centrifugation in a Sorvall GSA rotor at 10,000×<italic>g</italic> for 20 min. The upper aqueous layer was then mixed thoroughly with 0.5 volumes of 30% polyethylene glycol 8000 (PEG) in 0.85% NaCl and held on ice for 1 h. The PEG precipitate was pelleted in a Sorvall GSA rotor at 16,000×<italic>g</italic> at 4 °C for 30 min and resuspended in 0.1 M sodium phosphate, pH 7.4. After centrifugation at 23,000×<italic>g</italic> at 4 °C for 20 min to remove unsuspended debris, virus was collected by ultracentrifugation of the supernatant in a Beckman SW41 rotor at 76,000×<italic>g</italic>, 4 °C for 2 h. The pelleted virus was resuspended in a total of 4 ml 0.1 M sodium phosphate buffer and purified by loading the viral suspension onto pre-formed gradients of 10–50% (w/v) sucrose in 0.1 M sodium phosphate buffer and centrifuging at 76,000×<italic>g</italic>, 4 °C overnight. The collected virus fractions were diluted in 0.1 M sodium phosphate buffer and preserved at 4 °C for immediate electron microscopy<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref>–<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>.</p></sec><sec id=\"Sec7\"><title>Negatively staining electron microscopy</title><p id=\"Par10\">Two µl of the virus solution was placed on a glow-discharged copper grid covered with a continuous carbon film. After 1 min of adsorption, the grid was washed with pure water for several seconds and stained with 3 µl of 2% (w/v) uranyl acetate solution for 1 min. The staining solution was blotted away with Whatman No. 1 filter paper. The grid was air-dried completely before it was examined in a JEOL JEM-1400 electron microscope operating at 120 keV. The micrographs were recorded at various magnifications using a 4K × 4K CMOS camera (TVIPS F-416).</p></sec><sec id=\"Sec8\"><title>Reverse transcription PCR (RT-PCR) assay</title><p id=\"Par11\">First-strand cDNA synthesis was performed as described earlier and followed by PCR using primer pairs V2085/V2090 (40 cycles of 94 °C for 30 s, 57.5 °C for 30 s, and 72 °C for 1 min) and V2168/V2164 (40 cycles of 94 °C for 30 s, 56.5 °C for 30 s, and 72 °C for 1 min) to amplify partial dsRNA1 and dsRNA2 segments with expected fragment lengths of 828 bp and 613 bp, respectively (<xref rid=\"MOESM1\" ref-type=\"media\">S1</xref> Table). PCR products were sequenced using the same primers and aligned to the prototype PdPV-1 sequence from MYC80251 to verify the identities of the amplicons obtained.</p></sec><sec id=\"Sec9\"><title>Independent laboratory confirmatory analysis</title><p id=\"Par12\">In order to further confirm our findings, one of us (TZ) imported four of the original six positive Czech <italic>P. destructans</italic> isolates, as well as two isolates not previously tested in the US laboratory, directly from the Czech Republic to China. Additionally, the MYC80251 isolate was imported from the US to China. RNA extraction, RT-PCR, and sequencing analysis of these isolates were carried out with primers, probes, and fresh reagents in a laboratory in Beijing, China that had no prior history of <italic>P. destructans</italic> investigations.</p></sec></sec><sec id=\"Sec10\"><title>Results</title><sec id=\"Sec11\"><title>Isolation and characterization of dsRNA from <italic>P. destructans</italic></title><p id=\"Par13\">Total nucleic acids extracted from <italic>P. destructans</italic> isolate MYC80251 migrated as multiple bands when analyzed by native agarose gel electrophoresis (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>, lane 1). The most slowly migrating set of bands was found to be removed by treatment with DNase I, showing that these corresponded to fungal genomic DNA (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>, lane 2). Conversely, only the high molecular weight fungal genomic DNA bands remained following treatment of total <italic>P. destructans</italic> nucleic acids with a combination of RNase III, a dsRNA-specific endoribonuclease, and RNase A, a pancreatic ribonuclease that cleaves ssRNA (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>, lane 3). This demonstrated that all of the other bands were RNA species. The two dominant RNA bands were most likely fungal 28S and 18S ribosomal RNAs. Migrating more slowly than the rRNAs were two prominent discrete bands of unknown identities (labeled by an asterisk in lane 1). To determine the nature of these bands, total extracted nucleic acids were digested with a combination of S1 nuclease (a single-strand-specific endonuclease) and DNase I. This treatment abolished all RNA and DNA species except the unknown doublet of bands (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>, lane 4). Collectively, these results strongly indicated that the unknown bands were double-stranded RNA (dsRNA), most likely derived from a fungal virus. Because the analysis in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> was carried out in native agarose gels, the mobilities of RNA species were compared to those of dsDNA size markers. To more accurately gauge the sizes of the two dsRNA bands, they were analyzed by denaturing polyacrylamide gel electrophoresis in comparison to ssRNA size markers. In this type of gel, the denatured dsRNAs were almost totally masked by the presence of the 18S rRNA band in a sample of total <italic>P. destructans</italic> RNA (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>, lane 1). However, removal of rRNAs by RNase A treatment of total RNA prior to reisolation, denaturation and electrophoresis allowed us to estimate that the dsRNA species fell in the size range of 1.5–2.0 kb (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>, lane 2, labeled by asterisk).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Analysis of nucleic acids extracted from <italic>P. destructans</italic> by differential endonuclease sensitivity or resistance. Samples were digested with the indicated enzymes, and undigested species were separated by electrophoresis in 1% native agarose gels. Lane 1: total <italic>P. destructans</italic> nucleic acids; the 28 S and 18 S ribosomal RNAs are indicated, and the two unknown, more slowly migrating bands are labeled with an asterisk. Lane 2: total nucleic acids following digestion with DNase I. Lane 3: total nucleic acids following digestion with both RNase III and RNase A. Lane 4: total nucleic acids following digestion with both S1 nuclease and DNase I. Size markers: lanes M1, lambda DNA-HindIII digest; lane M2, 2-log DNA ladder (New England BioLabs).</p></caption><graphic xlink:href=\"41598_2020_70375_Fig1_HTML\" id=\"MO1\"/></fig><fig id=\"Fig2\"><label>Figure 2</label><caption><p>Estimation of the size of the dsRNA species in nucleic acids extracted from <italic>P. destructans</italic>. Samples were analyzed by electrophoresis in denaturing polyacrylamide gels. Lane 1: total RNA; the 28 S and 18 S ribosomal RNAs are indicated. Lane 2: total RNA digested with RNase A prior to reisolation and denaturation. Size marker (lanes M): ssRNA ladder (in nucleotides).</p></caption><graphic xlink:href=\"41598_2020_70375_Fig2_HTML\" id=\"MO2\"/></fig></p></sec><sec id=\"Sec12\"><title>Electron microscopy (EM)</title><p id=\"Par14\">The presence of viral particles in <italic>P. destructans</italic> was confirmed by transmission EM. Viral particles (named as PdPV-1) were purified from mycelia of <italic>P. destructans</italic> MYC80251 by homogenization, PEG precipitation, and sucrose density gradient centrifugation. Large amounts of isometric viral particles were observed under EM. The diameter of the virions was estimated to be about 33 nm (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>).<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Electron microscopy of purified PdPV-1. Isometric viral particles with an estimated diameter of 33 nm were observed. The bar represents 100 nm.</p></caption><graphic xlink:href=\"41598_2020_70375_Fig3_HTML\" id=\"MO3\"/></fig></p></sec><sec id=\"Sec13\"><title>Sequence and phylogenetic analysis</title><p id=\"Par15\">The complete sequences of the two dsRNA genome segments of PdPV-1 were obtained by sequencing of a library of cDNA clones, gap-filling reverse transcription PCR, and RLM-RACE. The large segment dsRNA1 comprised 1,683 bp with 46% GC content. The dsRNA1 segment contains an open reading frame (ORF) that encodes 539 amino acids with a molecular mass of approximately 62.7 kDa (GenBank Accession number: KP128044). The small segment dsRNA2 comprised 1,524 bp with 49% GC content. The dsRNA2 segment contains an ORF that encodes 434 amino acids with a molecular mass of approximately 46.9 kDa (GenBank Accession number: KP128045). Both ORFs were identified on the positive strand of each dsRNA segment. The negative strands did not contain any significant ORFs that are longer than 82 amino acids. The search for similar deduced amino-acid sequences in GenBank revealed that the ORFs of dsRNA dsRNA1 and dsRNA2 have significant similarities to the putative RNA-dependent RNA polymerase (RdRp) and the capsid protein (CP), respectively, of viruses from the family <italic>Partitiviridae</italic> genus <italic>Gammapartitivirus</italic><sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup><italic>.</italic> These include Penicillium stoloniferum virus S (PsV-S), Aspergillus fumigatus partitivirus-1 (AfuPV-1), Aspergillus ochraceus virus (AoV), Botryotinia fuckeliana partitivirus 1 (BfPV1), Discula destructiva virus 2 (DdV2), Fusarium solani virus 1 (FusoV), Gremmeniella abietina virus MS1 (GaV-MS1), Ophiostoma partitivirus (OPV1), Ustilaginoidea virens partitivirus (UvPV-1), and Verticillium dahliae partitivirus 1 (VdPV1) (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref> and Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>). Specifically, the RdRp of PdPV-1 showed 76% identity with PsV-S RdRp, whereas the CP of PdPV-1 showed 67% identity with that of PsV-S. Homologies (amino acid identity in red and consensus match in blue) were much higher within the core motif regions of the RdRp (amino acids 174–491) of PdPV-1, than elsewhere in the molecule (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>). Phylogenetic analyses were performed by using the RdRp and CP sequences of the various mycoviruses such as alphapartitiviruses, betapartitivirues, deltapartitiviruses, gammapartitiviruses, totiviruses, and chrysoviruses. Phylogenetic trees derived from both RdRp and CP sequences exhibited three major branches and suggested that PdPV-1 is a member of the genus <italic>Gammapartitivirus</italic> in the family <italic>Partitiviridae</italic> (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref> and Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>). In addition, phylogenetic analyses of the putative RdRp and CP of PdPV-1 showed that PdPV-1 was most closely related to the gammapartitivirus PsV-S.<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Comparison of the amino acid sequences of putative RdRp of the Pseudogymnoascus destructans virus (PdPV-1), Penicillium stoloniferum virus S (PsV-S), Gremmeniella abietina virus MS1 (GaV-MS1), Aspergillus ochraceus virus (AoV), Botryotinia fuckeliana partitivirus-1 (BfPV1), Aspergillus fumigatus partitivirus-1 (AfuPV-1), Ustilaginoidea virens partitivirus 1 (UvPV-1), Verticillium dahliae partitivirus 1 (VdPV1), Ophiostoma partitivirus (OPV1), Discula destructiva virus 2 (DdV2), and Fusarium solani virus 1 (FusoV). Red: 100% identity; Blue: consensus match; Green: mismatch.</p></caption><graphic xlink:href=\"41598_2020_70375_Fig4_HTML\" id=\"MO4\"/></fig><fig id=\"Fig5\"><label>Figure 5</label><caption><p>Phylogenetic analysis of PdPV-1. Maximum likelihood phylogenetic tree based on RdRp amino acid sequences of representative members of the families <italic>Partitiviridae </italic>(including genera <italic>Alphapartitivirus, Betapartitivirus, Deltapartitivirus,</italic> and <italic>Gammapartitivirus</italic>),<italic> Totiviridae,</italic> and <italic>Chrysoviridae</italic> were constructed using the program MEGA 6 (GenBank accession numbers are in the Table <xref rid=\"MOESM1\" ref-type=\"media\">S2</xref>).</p></caption><graphic xlink:href=\"41598_2020_70375_Fig5_HTML\" id=\"MO5\"/></fig></p><p id=\"Par16\">We found that the 5′ untranslated regions (UTRs) of the plus strands of segments dsRNA1 and dsRNA2 were 8 and 60 nucleotides, respectively; whereas the 3′ UTRs of the plus strands of segments dsRNA1 and dsRNA2 were 54 and 159 nucleotides, respectively. In contrast, a separate study of PdPV-1<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup> obtained longer RLM-RACE reads than ours and was able to identify conserved 5′-terminal nucleotide sequence motifs in both genome segments, a feature that is common to the majority of partitiviruses<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref>,<xref ref-type=\"bibr\" rid=\"CR34\">34</xref>–<xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. Additionally, as a consequence of the observed longer 5′ end of the dsRNA2 sequence, the deduced CP amino-acid sequence in that study<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup> had a 35-residue amino-terminal extension relative to the CP ORF that we determined. If confirmed, such an extension would make the PdPV-1 CP unique among the partitiviruses (Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>). This apparent discrepancy remains to be resolved in the future by the direct characterization of the CP. Very recently, a PdPV-1 sequence was also assembled in a metagenomic analysis of fungal transcriptomic datasets<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. Notably, other than the amino-terminal CP extension<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>, the PdPV-1 sequences obtained in all three studies were 100% identical in the dsRNA1 and dsRNA2 coding regions.</p></sec><sec id=\"Sec14\"><title>Reverse transcription PCR (RT-PCR) screening of <italic>P. destructans</italic> isolates for the presence of PdPV-1</title><p id=\"Par17\">RT-PCR was performed to examine whether PdPV-1 exists in various <italic>P. destructans</italic> isolates from diverse sources. <italic>P. destructans</italic> isolates listed in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> include those collected from fungus-infected bats in the caves from various geographic locations in the US and the Czech Republic, as well as those that were purified from environmental samples collected from caves or mines where bats with WNS hibernated. Closely related strains of <italic>Pseudogymnoascus roseus</italic> that were isolated from soil samples collected in bat hibernacula were also tested. <italic>P. roseus</italic> WSF-3629 was collected from Amorphus peat in Wisconsin<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. <italic>P. roseus</italic> UAMH1658 and <italic>P. appendiculatus</italic> UAMH10510 were obtained from the University of Alberta Microfungus Collection and Herbarium. All these bat tissue samples and environmental isolates were subjected to total RNA extraction, followed by virus-specific RT-PCR detection and sequencing of RT-PCR product fragments. Both dsRNA1 and dsRNA2 segments of PdPV-1 were detected in RNA extracts of all <italic>P. destructans</italic> isolates from the US, irrespective of where they were collected, and their partial sequences (GenBank accession numbers: MN990689–MN990708) were 100% identical to those of our original PdPV-1 strain from MYC80251. Six out of sixteen <italic>P. destructans</italic> isolates collected in the Czech Republic were also found to be PdPV-1 positive. dsRNA1 and dsRNA2 segments of these six PdPV-1 positive isolates were partially sequenced (GenBank accession numbers: KY609328–KY609339), and we found that some of these PdPV-1 strains had polymorphisms in both their dsRNA1 and dsRNA2 segments, while others were identical to the sequences of PdPV-1 from the US <italic>P. destructans</italic> isolates. Additionally, examination of the closely related fungi <italic>P. roseus</italic> and <italic>P. appendiculatus</italic> showed no detectable dsRNA1 or dsRNA2 segment of PdPV-1 dsRNA (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>).</p></sec><sec id=\"Sec15\"><title>Independent analysis of <italic>P. destructans</italic> isolates from the Czech Republic</title><p id=\"Par18\">To obtain independent verification of some of these findings, four of the Czech Republic isolates (CCF3938, CCF3941, CCF4127, and CCF4130) that had been found to be positive for PdPV-1 by testing in the US were imported directly from the Czech Republic to China and were tested in a laboratory in Beijing that had no prior history of work with <italic>P. destructans</italic>. These isolates, plus two previously untested Czech isolates (CCF3939 and CCF4129), were confirmed by RT-PCR and sequencing to harbor PdPV-1 (GenBank accession numbers: MK789668–MK789673 and MK789675–MK789680) (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>).The laboratory in Beijing also confirmed the presence of PdPV-1 in the US <italic>P. destructans</italic> isolate MYC80251 (GenBank accession numbers: MK789667 and MK789674) (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>).</p></sec></sec><sec id=\"Sec16\"><title>Discussion</title><p id=\"Par19\">Bat WNS continues to devastate the bat populations in the United States and Canada and therefore, there remains an urgent need to understand the origin and spread of this disease. In this study, we characterized mycovirus PdPV-1 infecting the WNS etiological fungus <italic>P. destructans</italic>. The identity of PdPV-1 was confirmed by (i) demonstration of its dsRNA content by differential sensitivity or resistance to DNA and RNA endonucleases; (ii) complete viral genome sequencing; (iii) nucleotide similarities of the dsRNA viral genome segments and phylogenetic alignment of their encoded proteins with mycoviruses from the family <italic>Partitiviridae</italic> genus <italic>Gammapartitivirus</italic>; (iv) TEM confirmation of isometric viral particles, and (v) host specificity of PdPV-1 for <italic>P. destructans</italic> and no detection of this virus in closely related <italic>Pseudogymnoascus</italic> species. Overall, PdPV-1 exhibited size, morphology, and nucleotide sequences typical of the fungal viruses in the family <italic>Partitiviridae</italic> genus <italic>Gammapartitivirus</italic><sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>.</p><p id=\"Par20\">These findings in our study (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.biorxiv.org/content/10.1101/059709v1\">https://www.biorxiv.org/content/10.1101/059709v1</ext-link>) were obtained independently from and prior to a report by Thapa et al<italic>.</italic><sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>. In contrast to results contained in that publication, we found that some of the Czech Republic <italic>P. destructans</italic> isolates were infected with PdPV-1. To eliminate the possibility that these results were false-positives, virus testing was repeated separately using four of the original six positive isolates that were shipped directly to China from the Czech Republic. Testing in the laboratory in Beijing verified that these four isolates contained PdPV-1, as did two other isolates not previously tested in the US (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>). This confirmation of our results strongly supports our conclusion that PdPV-1 infection is not unique to the North American isolates of <italic>P. destructans</italic>. We do not know the basis for the discrepancy between the results of our laboratories and those of the other report<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup>; notably, dsRNA1 (GenBank Accession number KP128044 and KY207543) and dsRNA2 (GenBank Accession number KP128045 and KY207544) from the two laboratories have 100% nucleotide identity, respectively. It is possible that it may be attributable to differences in the methods used for dsRNA extraction. Also, we note that the Czech <italic>P. destructans</italic> isolates that we tested were obtained from laboratories in the Czech Republic, whereas those analyzed by Thapa et al<italic>.</italic><sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup> were acquired from the Center for Forest Mycology Research in Madison, Wisconsin.</p><p id=\"Par21\">Both <italic>P. destructans</italic> and its closely related species of <italic>P. roseus</italic> have been isolated from the soil of WNS affected caves and mines<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. PdPV-1 was only detected from the randomly selected samples of <italic>P. destructans</italic>, but not from <italic>P. roseus</italic>. The result showed that all US <italic>P. destructans</italic> isolates were infected with PdPV-1, no matter whether the samples were isolated from the bats with WNS or its environment in the US. Virus-free fungal isolates of <italic>P. roseus</italic> were not found on the bats with WNS although this fungal species was found in the same environmental reservoir as the WNS pathogen. The absence of PdPV-1 infection in other <italic>Pseudogymnoascus</italic> species, collected from the same bat hibernacula in the US, suggested a close viral association with <italic>P. destructans</italic>. The observed host-specificity of PdPV-1 was consistent with the narrow host ranges of mycoviruses due to severe bottlenecks in their horizontal transmission<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. The above results raise two questions: (i) If PdPV-1 can infect <italic>P. roseus</italic>, will <italic>P. roseus</italic> with virus cause WNS? (ii) Can virus-free isolates of <italic>P. destructans</italic> infect bats to cause WNS? Additional studies are needed to address these questions.</p><p id=\"Par22\">The discovery of a dsRNA mycovirus in <italic>P. destructans</italic> is consistent with the wide occurrence of mycoviruses in fungi<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. Mycovirus infection of fungi is usually asymptomatic<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. However, mycovirus infection could alter the ability of plant-pathogenic fungi to cause diseases<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. In plant-pathogenic fungi, mycovirus infections generally reduce fungal yield, attenuate mycelial growth, abolish female fertility in sexual crosses, decrease asexual sporulation, alter colony morphologies, modulate pigment production, and diminish the accumulation of specific metabolites. These effects lead to hypovirulence, an attenuation of the pathogenic outcome of fungal infection on the plant host<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. The harmful effects of mycoviruses on fungal growth were exploited as beneficial functions in phytopathogen control<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>,<xref ref-type=\"bibr\" rid=\"CR43\">43</xref>,<xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>. On the contrary, virus infections of animal pathogenic fungi and protozoan pathogens showed hypervirulence<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. A recent publication suggested that partitivirus infections of the dimorphic human fungal pathogen <italic>Talaromyces marneffei</italic> caused aberrant gene expression and hypervirulence in an animal model<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. More in-depth experiments will be needed to discern the outcome of PdPV-1 infection on <italic>P. destructans</italic> phenotype and virulence. These investigations will be facilitated by the recent availability of a molecular tool kit and experimental infection model of <italic>P. destructans</italic><sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref>–<xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>.</p><p id=\"Par23\">In conclusion, mycoviruses are ubiquitous in fungi, while the connection between fungal phenotype and mycovirus presence is not always straightforward. In this study, we discovered the existence of the partitivirus PdPV-1 in <italic>P. destructans</italic> isolated from various species of bats with WNS or from their living environment in both the US and the Czech Republic. <italic>P. destructans</italic> PdPV-1 could be a valuable tool to investigate fungal biogeography and the host–pathogen interactions in bat WNS.</p></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec18\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70375_MOESM1_ESM.docx\"><caption><p>Supplementary Information.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-70375-6.</p></sec><ack><title>Acknowledgements</title><p>This study was funded in part by the US Fish and Wildlife Service (F12AP01167), National Science Foundation (1203528), National Natural Science Foundation of China (31872617), and the Fundamental Research Funds for the Central Universities, China (3332018097). DNA sequencing in the US laboratory was performed at the Wadsworth Center Advance Genomics Technologies Core. EM was carried out at the Wadsworth Center EM Core.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>V.C. conceived the study, obtained initial funding, and supervised experiments. P.R. and S.S.R. carried out experiments described in Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>, <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>, <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>, and <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>. P.R., H.S., and P.S.M. collaborated on Figs. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref> and <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>. S.C. advised on the design and interpretation of data in Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>, <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>, <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>, Fig. <xref rid=\"MOESM1\" ref-type=\"media\">S1</xref>, and Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>. N.M., A.K., and J.P. contributed samples and details of isolates from the Czech Republic. T.Z. carried out independent confirmation of major findings and obtained partial funding. V.C., with contributions from P.R., wrote the draft manuscript. P.R., P.S.M., and S.C. edited the draft manuscript. All authors reviewed the final manuscript.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>All nucleotide sequences generated in this study are deposited in the NCBI database.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par25\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Blehert</surname><given-names>DS</given-names></name><etal/></person-group><article-title>Bat white-nose syndrome: an emerging fungal pathogen?</article-title><source>Science</source><year>2009</year><volume>323</volume><fpage>227</fpage><pub-id pub-id-type=\"pmid\">18974316</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Frick</surname><given-names>WF</given-names></name><etal/></person-group><article-title>An emerging disease causes regional population collapse of a common North American bat species</article-title><source>Science</source><year>2010</year><volume>329</volume><fpage>679</fpage><lpage>682</lpage><pub-id pub-id-type=\"doi\">10.1126/science.1188594</pub-id><pub-id pub-id-type=\"pmid\">20689016</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chaturvedi</surname><given-names>V</given-names></name><etal/></person-group><article-title>Morphological and molecular characterizations of psychrophilic fungus <italic>Geomyces destructans</italic> from New York bats with white nose syndrome (WNS)</article-title><source>PLoS ONE</source><year>2010</year><volume>5</volume><fpage>e10783</fpage><pub-id pub-id-type=\"pmid\">20520731</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Froschauer</surname><given-names>A</given-names></name><name><surname>Coleman</surname><given-names>J</given-names></name></person-group><source>North American Bat Death Toll Exceeds 5.5 Million from White-Nose Syndrome</source><year>2012</year><publisher-loc>Arlington</publisher-loc><publisher-name>US Fish and Wildlife Service</publisher-name><fpage>1</fpage><lpage>2</lpage></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Warnecke</surname><given-names>L</given-names></name><etal/></person-group><article-title>Inoculation of bats with European <italic>Geomyces destructans</italic> supports the novel pathogen hypothesis for the origin of white-nose syndrome</article-title><source>Proc. Natl. Acad. Sci. U.S.A.</source><year>2012</year><volume>109</volume><fpage>6999</fpage><lpage>7003</lpage><pub-id pub-id-type=\"pmid\">22493237</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>O'Shea</surname><given-names>TJ</given-names></name><name><surname>Cryan</surname><given-names>PM</given-names></name><name><surname>Hayman</surname><given-names>DTS</given-names></name><name><surname>Plowright</surname><given-names>RK</given-names></name><name><surname>Streicker</surname><given-names>DG</given-names></name></person-group><article-title>Multiple mortality events in bats: a global review</article-title><source>Mammal Rev.</source><year>2016</year><volume>46</volume><fpage>175</fpage><lpage>190</lpage></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lorch</surname><given-names>JM</given-names></name><etal/></person-group><article-title>A culture-based survey of fungi in soil from bat hibernacula in the eastern United States and its implications for detection of <italic>Geomyces destructans</italic>, the causal agent of bat white-nose syndrome</article-title><source>Mycologia</source><year>2013</year><volume>105</volume><fpage>237</fpage><lpage>252</lpage><pub-id pub-id-type=\"pmid\">23074174</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Raudabaugh</surname><given-names>DB</given-names></name><name><surname>Miller</surname><given-names>AN</given-names></name></person-group><article-title>Nutritional capability of and substrate suitability for <italic>Pseudogymnoascus destructans</italic>, the causal agent of bat white-nose syndrome</article-title><source>PLoS ONE</source><year>2013</year><volume>8</volume><fpage>e78300</fpage><pub-id pub-id-type=\"pmid\">24205191</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reynolds</surname><given-names>HT</given-names></name><name><surname>Barton</surname><given-names>HA</given-names></name></person-group><article-title>Comparison of the white-nose syndrome agent <italic>Pseudogymnoascus destructans</italic> to cave-dwelling relatives suggests reduced saprotrophic enzyme activity</article-title><source>PLoS ONE</source><year>2014</year><volume>9</volume><fpage>e86437</fpage><pub-id pub-id-type=\"pmid\">24466096</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rajkumar</surname><given-names>SS</given-names></name><etal/></person-group><article-title>Clonal genotype of <italic>Geomyces destructans</italic> among bats with white nose syndrome, New York, USA</article-title><source>Emerg. Infect. Dis.</source><year>2011</year><volume>17</volume><fpage>1273</fpage><lpage>1276</lpage><pub-id pub-id-type=\"pmid\">21762585</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ren</surname><given-names>P</given-names></name><etal/></person-group><article-title>Clonal Spread of <italic>Geomyces destructans</italic> among Bats, Midwestern and Southern United States</article-title><source>Emerg. Infect. Dis.</source><year>2012</year><volume>18</volume><fpage>883</fpage><lpage>885</lpage><pub-id pub-id-type=\"pmid\">22516471</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Khankhet</surname><given-names>J</given-names></name><etal/></person-group><article-title>Clonal expansion of the <italic>Pseudogymnoascus destructans</italic> genotype in North America is accompanied by significant variation in phenotypic expression</article-title><source>PLoS ONE</source><year>2014</year><volume>9</volume><fpage>e104684</fpage><pub-id pub-id-type=\"pmid\">25122221</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>KM</given-names></name><etal/></person-group><article-title>A comparison of transcriptional patterns and mycological phenotypes following infection of <italic>Fusarium graminearum</italic> by four mycoviruses</article-title><source>PLoS ONE</source><year>2014</year><volume>9</volume><fpage>e100989</fpage><pub-id pub-id-type=\"pmid\">24964178</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Deng</surname><given-names>F</given-names></name><name><surname>Allen</surname><given-names>TD</given-names></name><name><surname>Hillman</surname><given-names>BI</given-names></name><name><surname>Nuss</surname><given-names>DL</given-names></name></person-group><article-title>Comparative analysis of alterations in host phenotype and transcript accumulation following hypovirus and mycoreovirus infections of the chestnut blight fungus <italic>Cryphonectria parasitica</italic></article-title><source>Eukaryot. Cell</source><year>2007</year><volume>6</volume><fpage>1286</fpage><lpage>1298</lpage><pub-id pub-id-type=\"pmid\">17557883</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ghabrial</surname><given-names>SA</given-names></name><name><surname>Castón</surname><given-names>JR</given-names></name><name><surname>Jiang</surname><given-names>D</given-names></name><name><surname>Nibert</surname><given-names>ML</given-names></name><name><surname>Suzuki</surname><given-names>N</given-names></name></person-group><article-title>50-plus years of fungal viruses</article-title><source>Virology</source><year>2015</year><volume>479</volume><fpage>356</fpage><lpage>368</lpage><pub-id pub-id-type=\"pmid\">25771805</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Son</surname><given-names>M</given-names></name><name><surname>Yu</surname><given-names>J</given-names></name><name><surname>Kim</surname><given-names>K-H</given-names></name></person-group><article-title>Five questions about mycoviruses</article-title><source>PLoS Pathog.</source><year>2015</year><volume>11</volume><fpage>e1005172</fpage><pub-id pub-id-type=\"pmid\">26539725</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schmitt</surname><given-names>MJ</given-names></name><name><surname>Breinig</surname><given-names>F</given-names></name></person-group><article-title>The viral killer system in yeast: from molecular biology to application</article-title><source>FEMS Microbiol. Rev.</source><year>2002</year><volume>26</volume><fpage>257</fpage><lpage>276</lpage><pub-id pub-id-type=\"pmid\">12165427</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Anagnostakis</surname><given-names>SL</given-names></name></person-group><article-title>Biological control of chestnut blight</article-title><source>Science</source><year>1982</year><volume>215</volume><fpage>466</fpage><lpage>471</lpage><pub-id pub-id-type=\"pmid\">17771259</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rogers</surname><given-names>H</given-names></name><name><surname>Buck</surname><given-names>K</given-names></name><name><surname>Brasier</surname><given-names>C</given-names></name></person-group><article-title>A mitochondrial target for double-stranded RNA in diseased isolates of the fungus that causes Dutch elm disease</article-title><source>Nature</source><year>1987</year><volume>329</volume><fpage>558</fpage><lpage>560</lpage></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nuss</surname><given-names>DL</given-names></name></person-group><article-title>Hypovirulence: mycoviruses at the fungal–plant interface</article-title><source>Nat. Rev. Microbiol.</source><year>2005</year><volume>3</volume><fpage>632</fpage><lpage>642</lpage><pub-id pub-id-type=\"pmid\">16064055</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>X</given-names></name><etal/></person-group><article-title>A geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic fungus</article-title><source>Proc. Natl. Acad. Sci.</source><year>2010</year><volume>107</volume><fpage>8387</fpage><lpage>8392</lpage><pub-id pub-id-type=\"pmid\">20404139</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Márquez</surname><given-names>LM</given-names></name><name><surname>Redman</surname><given-names>RS</given-names></name><name><surname>Rodriguez</surname><given-names>RJ</given-names></name><name><surname>Roossinck</surname><given-names>MJ</given-names></name></person-group><article-title>A virus in a fungus in a plant: three-way symbiosis required for thermal tolerance</article-title><source>Science</source><year>2007</year><volume>315</volume><fpage>513</fpage><lpage>515</lpage><pub-id pub-id-type=\"pmid\">17255511</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pearson</surname><given-names>MN</given-names></name><name><surname>Beever</surname><given-names>RE</given-names></name><name><surname>Boine</surname><given-names>B</given-names></name><name><surname>Arthur</surname><given-names>K</given-names></name></person-group><article-title>Mycoviruses of filamentous fungi and their relevance to plant pathology</article-title><source>Mol. Plant Pathol.</source><year>2009</year><volume>10</volume><fpage>115</fpage><lpage>128</lpage><pub-id pub-id-type=\"pmid\">19161358</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bhatti</surname><given-names>MF</given-names></name><etal/></person-group><article-title>The effects of dsRNA mycoviruses on growth and murine virulence of <italic>Aspergillus fumigatus</italic></article-title><source>Fungal Genet. Biol.</source><year>2011</year><volume>48</volume><fpage>1071</fpage><lpage>1075</lpage><pub-id pub-id-type=\"pmid\">21840413</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lau</surname><given-names>SK</given-names></name><etal/></person-group><article-title>Novel partitivirus enhances virulence of and causes aberrant gene expression in <italic>Talaromyces marneffei</italic></article-title><source>mBio</source><year>2018</year><volume>9</volume><fpage>e00947–00918</fpage><pub-id pub-id-type=\"pmid\">29895639</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Goker</surname><given-names>M</given-names></name><name><surname>Scheuner</surname><given-names>C</given-names></name><name><surname>Klenk</surname><given-names>HP</given-names></name><name><surname>Stielow</surname><given-names>JB</given-names></name><name><surname>Menzel</surname><given-names>W</given-names></name></person-group><article-title>Codivergence of mycoviruses with their hosts</article-title><source>PLoS ONE</source><year>2011</year><volume>6</volume><fpage>e22252</fpage><pub-id pub-id-type=\"pmid\">21829452</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Minnis</surname><given-names>AM</given-names></name><name><surname>Lindner</surname><given-names>DL</given-names></name></person-group><article-title>Phylogenetic evaluation of <italic>Geomyces </italic>and allies reveals no close relatives of <italic>Pseudogymnoascus</italic><italic>destructans</italic>, comb. nov., in bat hibernacula of eastern North America</article-title><source>Fungal Biol.</source><year>2013</year><volume>117</volume><fpage>638</fpage><lpage>649</lpage><pub-id pub-id-type=\"pmid\">24012303</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tamura</surname><given-names>K</given-names></name><name><surname>Filipski</surname><given-names>A</given-names></name><name><surname>Peterson</surname><given-names>D</given-names></name><name><surname>Stecher</surname><given-names>G</given-names></name><name><surname>Kumar</surname><given-names>S</given-names></name></person-group><article-title>MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0</article-title><source>Mol. Biol. Evol.</source><year>2013</year><volume>30</volume><fpage>2725</fpage><lpage>2729</lpage><pub-id pub-id-type=\"pmid\">24132122</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Crawford</surname><given-names>LJ</given-names></name><etal/></person-group><article-title>Molecular characterization of a partitivirus from <italic>Ophiostoma himal-ulmi</italic></article-title><source>Virus Genes</source><year>2006</year><volume>33</volume><fpage>33</fpage><lpage>39</lpage><pub-id pub-id-type=\"pmid\">16791416</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lin</surname><given-names>YH</given-names></name><etal/></person-group><article-title>A novel quadripartite dsRNA virus isolated from a phytopathogenic filamentous fungus, <italic>Rosellinia necatrix</italic></article-title><source>Virology</source><year>2012</year><volume>426</volume><fpage>42</fpage><lpage>50</lpage><pub-id pub-id-type=\"pmid\">22321722</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ochoa</surname><given-names>WF</given-names></name><etal/></person-group><article-title>Partitivirus structure reveals a 120-subunit, helix-rich capsid with distinctive surface arches formed by quasisymmetric coat-protein dimers</article-title><source>Structure</source><year>2008</year><volume>16</volume><fpage>776</fpage><lpage>786</lpage><pub-id pub-id-type=\"pmid\">18462682</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vainio</surname><given-names>EJ</given-names></name><etal/></person-group><article-title>ICTV virus taxonomy profile: partitiviridae</article-title><source>J. Gen. Virol.</source><year>2018</year><volume>99</volume><fpage>17</fpage><lpage>18</lpage><pub-id pub-id-type=\"pmid\">29214972</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thapa</surname><given-names>V</given-names></name><etal/></person-group><article-title>Using a novel partitivirus in <italic>Pseudogymnoascus destructans</italic> to understand the epidemiology of white-nose syndrome</article-title><source>PLoS Pathog.</source><year>2016</year><volume>12</volume><fpage>e1006076</fpage><pub-id pub-id-type=\"pmid\">28027325</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hacker</surname><given-names>CV</given-names></name><name><surname>Brasier</surname><given-names>CM</given-names></name><name><surname>Buck</surname><given-names>KW</given-names></name></person-group><article-title>A double-stranded RNA from a <italic>Phytophthora</italic> species is related to the plant endornaviruses and contains a putative UDP glycosyltransferase gene</article-title><source>J. Gen. Virol.</source><year>2005</year><volume>86</volume><fpage>1561</fpage><lpage>1570</lpage><pub-id pub-id-type=\"pmid\">15831970</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>W</given-names></name><name><surname>Duns</surname><given-names>G</given-names></name><name><surname>Chen</surname><given-names>J</given-names></name></person-group><article-title>Genomic characterization of a novel partitivirus infecting <italic>Aspergillus ochraceus</italic></article-title><source>Virus Genes</source><year>2008</year><volume>37</volume><fpage>322</fpage><lpage>327</lpage><pub-id pub-id-type=\"pmid\">18770020</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tuomivirta</surname><given-names>T</given-names></name><name><surname>Hantula</surname><given-names>J</given-names></name></person-group><article-title>Two unrelated double-stranded RNA molecule patterns in <italic>Gremmeniella abietina</italic> type A code for putative viruses of the families <italic>Totiviridae</italic> and <italic>Partitiviridae</italic></article-title><source>Adv. Virol.</source><year>2003</year><volume>148</volume><fpage>2293</fpage><lpage>2305</lpage></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gilbert</surname><given-names>KB</given-names></name><name><surname>Holcomb</surname><given-names>EE</given-names></name><name><surname>Allscheid</surname><given-names>RL</given-names></name><name><surname>Carrington</surname><given-names>JC</given-names></name></person-group><article-title>Hiding in plain sight: new virus genomes discovered via a systematic analysis of fungal public transcriptomes</article-title><source>PLoS ONE</source><year>2019</year><volume>14</volume><fpage>e0219207</fpage><pub-id pub-id-type=\"pmid\">31339899</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nibert</surname><given-names>ML</given-names></name><etal/></person-group><article-title>Taxonomic reorganization of family <italic>Partitiviridae</italic> and other recent progress in partitivirus research</article-title><source>Virus Res.</source><year>2014</year><volume>188</volume><fpage>128</fpage><lpage>141</lpage><pub-id pub-id-type=\"pmid\">24768846</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>T</given-names></name><etal/></person-group><article-title>Mycobiome of the bat white nose syndrome affected caves and mines reveals diversity of fungi and local adaptation by the fungal pathogen <italic>Pseudogymnoascus</italic> (<italic>Geomyces</italic>) <italic>destructans</italic></article-title><source>PLoS ONE</source><year>2014</year><volume>9</volume><fpage>e108714</fpage><pub-id pub-id-type=\"pmid\">25264864</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bozarth</surname><given-names>RF</given-names></name></person-group><article-title>Mycoviruses: a new dimension in microbiology</article-title><source>Environ. Health Perspect.</source><year>1972</year><volume>2</volume><fpage>23</fpage><lpage>39</lpage><pub-id pub-id-type=\"pmid\">4628853</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Buck</surname><given-names>KW</given-names></name></person-group><article-title>From interferon induction to fungal viruses</article-title><source>Eur. J. Epidemiol.</source><year>1988</year><volume>4</volume><fpage>395</fpage><lpage>399</lpage><pub-id pub-id-type=\"pmid\">2462507</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ghabrial</surname><given-names>SA</given-names></name><name><surname>Suzuki</surname><given-names>N</given-names></name></person-group><article-title>Viruses of plant pathogenic fungi</article-title><source>Annu. Rev. Phytopathol.</source><year>2009</year><volume>47</volume><fpage>353</fpage><lpage>384</lpage><pub-id pub-id-type=\"pmid\">19400634</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ro</surname><given-names>HS</given-names></name><etal/></person-group><article-title>Isolation and characterization of a novel mycovirus, PeSV, in <italic>Pleurotus eryngii</italic> and the development of a diagnostic system for it</article-title><source>Biotechnol. Lett.</source><year>2007</year><volume>29</volume><fpage>129</fpage><lpage>135</lpage><pub-id pub-id-type=\"pmid\">17136571</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>HJ</given-names></name><name><surname>Lim</surname><given-names>D</given-names></name><name><surname>Lee</surname><given-names>HS</given-names></name></person-group><article-title>Characterization of a novel single-stranded RNA mycovirus in <italic>Pleurotus</italic><italic>ostreatus</italic></article-title><source>Virology</source><year>2003</year><volume>314</volume><fpage>9</fpage><lpage>15</lpage><pub-id pub-id-type=\"pmid\">14517055</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ives</surname><given-names>A</given-names></name><etal/></person-group><article-title><italic>Leishmania</italic> RNA virus controls the severity of mucocutaneous leishmaniasis</article-title><source>Science</source><year>2011</year><volume>331</volume><fpage>775</fpage><lpage>778</lpage><pub-id pub-id-type=\"pmid\">21311023</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>T</given-names></name><name><surname>Ren</surname><given-names>P</given-names></name><name><surname>De Jesus</surname><given-names>M</given-names></name><name><surname>Chaturvedi</surname><given-names>V</given-names></name><name><surname>Chaturvedi</surname><given-names>S</given-names></name></person-group><article-title>Green fluorescent protein expression in <italic>Pseudogymnoascus destructans</italic> to study its abiotic and biotic lifestyles</article-title><source>Mycopathologia</source><year>2018</year><volume>183</volume><fpage>805</fpage><lpage>814</lpage><pub-id pub-id-type=\"pmid\">29987576</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Drees</surname><given-names>KP</given-names></name><etal/></person-group><article-title>Use of multiple sequencing technologies to produce a high-quality genome of the fungus <italic>Pseudogymnoascus destructans</italic>, the causative agent of bat white-nose syndrome</article-title><source>Genome Announc.</source><year>2016</year><volume>4</volume><fpage>e00445-00416</fpage><pub-id pub-id-type=\"pmid\">27365344</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>T</given-names></name><name><surname>Ren</surname><given-names>P</given-names></name><name><surname>Chaturvedi</surname><given-names>V</given-names></name><name><surname>Chaturvedi</surname><given-names>S</given-names></name></person-group><article-title>Development of an Agrobacterium-mediated transformation system for the cold-adapted fungi <italic>Pseudogymnoascus</italic><italic>destructans</italic> and <italic>P. pannorum</italic></article-title><source>Fungal Genet. Biol.</source><year>2015</year><volume>81</volume><fpage>73</fpage><lpage>81</lpage><pub-id pub-id-type=\"pmid\">26051491</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chaturvedi</surname><given-names>V</given-names></name><name><surname>DeFiglio</surname><given-names>H</given-names></name><name><surname>Chaturvedi</surname><given-names>S</given-names></name></person-group><article-title>Phenotype profiling of white-nose syndrome pathogen <italic>Pseudogymnoascus destructans</italic> and closely-related <italic>Pseudogymnoascus pannorum</italic> reveals metabolic differences underlying fungal lifestyles</article-title><source>F1000Res</source><year>2018</year><volume>7</volume><fpage>665</fpage><pub-id pub-id-type=\"pmid\">30026932</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fuchs</surname><given-names>BB</given-names></name><etal/></person-group><article-title><italic>Galleria mellonella</italic> experimental model for bat fungal pathogen <italic>Pseudogymnoascus destructans</italic> and human fungal pathogen <italic>Pseudogymnoascus pannorum</italic></article-title><source>Virulence</source><year>2018</year><volume>9</volume><fpage>1539</fpage><lpage>1547</lpage><pub-id pub-id-type=\"pmid\">30289352</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Beekman</surname><given-names>CN</given-names></name><name><surname>Meckler</surname><given-names>L</given-names></name><name><surname>Kim</surname><given-names>E</given-names></name><name><surname>Bennett</surname><given-names>RJ</given-names></name></person-group><article-title><italic>Galleria</italic><italic>mellonella</italic> as an insect model for <italic>P. destructans</italic>, the cause of white-nose syndrome in bats</article-title><source>PLoS ONE</source><year>2018</year><volume>13</volume><fpage>e0201915</fpage><pub-id pub-id-type=\"pmid\">30183704</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807808</article-id><article-id pub-id-type=\"pmc\">PMC7431588</article-id><article-id pub-id-type=\"publisher-id\">70636</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70636-4</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>TMPRSS13 promotes cell survival, invasion, and resistance to drug-induced apoptosis in colorectal cancer</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-5675-4405</contrib-id><name><surname>Varela</surname><given-names>Fausto A.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Foust</surname><given-names>Victoria L.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hyland</surname><given-names>Thomas E.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Sala-Hamrick</surname><given-names>Kimberley E.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Mackinder</surname><given-names>Jacob R.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Martin</surname><given-names>Carly E.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-2899-4970</contrib-id><name><surname>Murray</surname><given-names>Andrew S.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Todi</surname><given-names>Sokol V.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>List</surname><given-names>Karin</given-names></name><address><email>klist@med.wayne.edu</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.254444.7</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 1456 7807</institution-id><institution>Department of Pharmacology, </institution><institution>Wayne State University School of Medicine, </institution></institution-wrap>Detroit, 48201 MI USA </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.254444.7</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 1456 7807</institution-id><institution>Department of Oncology, </institution><institution>Wayne State University School of Medicine, </institution></institution-wrap>Detroit, 48201 MI USA </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.254444.7</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 1456 7807</institution-id><institution>Department of Neurology, </institution><institution>Wayne State University School of Medicine, </institution></institution-wrap>Detroit, 48201 MI USA </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13896</elocation-id><history><date date-type=\"received\"><day>5</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>9</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Cancer progression is often accompanied by increased levels of extracellular proteases capable of remodeling the extracellular matrix and promoting pro-cancerous signaling pathways by activating growth factors and receptors. The type II transmembrane serine protease (TTSP) family encompasses several proteases that play critical roles in cancer progression; however, the expression or function of the TTSP TMPRSS13 in carcinogenesis has not been examined. In the present study, we found TMPRSS13 to be differentially expressed at both the transcript and protein levels in human colorectal cancer (CRC). Immunohistochemical analyses revealed consistent high expression of TMPRSS13 protein on the cancer cell surface in CRC patient samples; in contrast, the majority of normal colon samples displayed no detectable expression. On a functional level, TMPRSS13 silencing in CRC cell lines increased apoptosis and impaired invasive potential. Importantly, transgenic overexpression of TMPRSS13 in CRC cell lines increased tolerance to apoptosis-inducing agents, including paclitaxel and HA14-1. Conversely, TMPRSS13 silencing rendered CRC cells more sensitive to these agents. Together, our findings suggest that TMPRSS13 plays an important role in CRC cell survival and in promoting resistance to drug-induced apoptosis; we also identify TMPRSS13 as a potential new target for monotherapy or combination therapy with established chemotherapeutics to improve treatment outcomes in CRC patients.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Cancer</kwd><kwd>Cell biology</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">According to the American Cancer Society, colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related deaths in both genders in the United States<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. Early diagnosis of disease can lead to successful treatment through surgical interventions, although the prognosis for advanced and metastatic CRC is poor due to limited medical treatment options. Fluorouracil (5-FU) and its pro-drug form capecitabine are currently the most frequently used agents, alone or in combination with drugs such as oxaliplatin and irinotecan<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>–<xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. Although targeted therapies have been successful in the treatment of some types of cancers, such as breast cancer, they have limited efficacy in adjuvant treatment of colorectal cancer (i.e., cetuximab, panitumumab, bevacizumab, ramucirumab, ziv-aflibercept, and regorafenib) and add relatively small survival benefits for those with advanced disease<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref>–<xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. Therefore, there is an urgent need to develop novel drug regimens for patients suffering from advanced CRC. To this end, understanding the molecular mechanisms driving CRC represents a critical step toward the development of novel targeted therapeutics for this particularly deadly type of cancer.</p><p id=\"Par3\">Proteolysis is a tightly regulated process under normal physiological conditions and proteolytic dysregulation constitutes both a hallmark of cancer and a contributing factor. The type II transmembrane serine protease (TTSP) subfamily is a relatively new classification of membrane-anchored serine proteases; many TTSPs play key roles in processes exploited by cancer, such as tissue remodeling, cellular migration and invasion, and metastasis<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>–<xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Cancer-focused studies using cell culture and animal models have identified the pro-oncogenic properties of several TTSPs<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>–<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>.</p><p id=\"Par4\">TMPRSS13 (transmembrane protease, serine 13; also known as mosaic serine protease large-form, or MSPL) is expressed in several epithelial tissues, such as the epithelia of the oral cavity, esophagus, bladder, stomach, and skin<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. TMPRSS13-deficiency causes abnormal development of the epidermal stratum corneum in newborn mice and leads to mildly compromised barrier function, detectable as an increased rate of trans-epidermal fluid loss in neonates<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. This phenotype is transient, with the aberrant epidermal stratum corneum of the newborn mice not observed in adults<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. Long-term studies (up to 12 months of age) show that TMPRSS13-deficient mice are outwardly healthy and present no detectable histological tissue abnormalities. TMPRSS13 is also expressed in mouse and human respiratory epithelium and several studies show a role for TMPRSS13 in influenza infection by proteolytically modifying the viral protein hemagglutinin, which is necessary for virus infectivity<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref>–<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. To date, two mammalian substrates, the pro-form of hepatocyte growth factor (HGF) and the epithelial sodium channel (ENaC), have been shown to be activated by TMPRSS13 in vitro<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>.</p><p id=\"Par5\">In this study, we identify TMPRSS13 as a differentially expressed TTSP in human CRC. Functionally, we present a role for TMPRSS13 in CRC cell survival, invasiveness, and resistance to apoptosis-inducing agents.</p></sec><sec id=\"Sec2\"><title>Results</title><sec id=\"Sec3\"><title>TMPRSS13 is upregulated in human colorectal cancer</title><p id=\"Par6\">As part of an ongoing effort to determine the expression and function of the TTSP family in healthy colon tissue and CRC, we performed a systematic expression analysis of TTSPs in cancer through in silico data mining using the Oncomine™ microarray database. TMPRSS13 transcripts were found to be significantly upregulated in human colon adenocarcinomas compared to normal human colon<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup> (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>A). TMPRSS13 expression in the normal colon has previously been reported to be low or undetectable in both human and mouse tissue<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. To confirm the elevated levels of TMPRSS13 at the protein level in human CRC, we performed an immunohistochemical (IHC) analysis on human colon tissue arrays. Low protein expression levels of TMPRSS13 were detected in colon crypt epithelial cells in some normal samples (~ 2%) (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B, lower right panel); however, the majority of normal colon samples displayed no detectable expression. Strong expression of TMPRSS13 was detected in epithelial-derived colon adenocarcinoma (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B, lower left panel). TMPRSS13 protein localized on the cell surface of the epithelial cells and no nuclear staining was observed. This cell surface localization is in agreement with the expected membrane-anchored structure of TMPRSS13. No significant staining was observed when primary antibodies were substituted with non-immune rabbit IgG in serial sections of all samples (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>B, upper panels). Antibody specificity was verified using IHC of known low-expressing versus high-expressing normal human tissues and western blot analysis of TMPRSS13-expressing HEK293T cells (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>).<fig id=\"Fig1\"><label>Figure 1</label><caption><p>TMPRSS13 transcript and protein are upregulated in human colorectal cancer. (<bold>A</bold>) Box-and-whisker plot showing TMPRSS13 mRNA expression data in normal and colon adenocarcinoma tissue samples (The Cancer Genome Atlas (TCGA))<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR71\">71</xref></sup>. TMPRSS13 gene expression values in normal colon (N = 19, black) and colon adenocarcinoma tissue (N = 102, purple) are shown. Box-and-whisker plots represent interquartile ranges with the median indicated as the horizontal line inside boxes (*p = 2.96 × 10<sup>−26</sup>, fold change = 4.918). The results are based upon data generated by the TCGA Research Network: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.cancer.gov/tcga\">https://www.cancer.gov/tcga</ext-link>. (<bold>B</bold>) Representative samples from tissue array IHC analysis of TMPRSS13 protein expression in normal human colon (right panels) and colon adenocarcinoma (left panels) samples. Primary, rabbit anti-TMPRSS13 antibody was substituted with non-immune rabbit IgG in serial sections of all samples and no significant staining was observed (upper panels). Open arrowheads indicate epithelial cells with undetectable or weak TMPRSS13 staining in normal colon (lower right panel) compared to strong TMPRSS13 staining in epithelial cells of a grade II colon adenocarcinoma (lower left panel). The lamina propria is indicated with black arrowheads. Scale bars = 50 µm.</p></caption><graphic xlink:href=\"41598_2020_70636_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par7\">Evaluation of differential expression of TMPRSS13 in CRC was conducted using normal and cancerous tissue samples with grades ranging from I to III (Normal colon, N = 14; colorectal adenocarcinomas Grade I, N = 16; Grade II, N = 65; and Grade III, N = 23). The CRC tissue arrays were incubated with anti-TMPRSS13 rabbit antibody (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A, representative samples of each grade shown) and non-immune rabbit IgG as a negative control (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A, upper right panel). Staining intensity was scored on a scale from 0 to 3 (see “<xref rid=\"Sec9\" ref-type=\"sec\">Methods</xref>”). The majority of normal colon samples (11/14) displayed no detectable TMPRSS13 expression while three samples showed low expression (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>B). In contrast, all CRC samples were scored positive for TMPRSS13 staining (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>B). Well-differentiated, low-grade carcinomas (Grade I) showed low to moderate cell surface staining (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A, upper right panel), while the majority of moderately differentiated carcinomas (Grade II) displayed moderate to strong cell surface staining (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A, lower left panel). In poorly differentiated carcinomas (grade III), TMPRSS13 expression mainly localized to the cell surface, with some areas displaying dispersed staining (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A, lower right panel) and the majority of samples showing low to moderate staining. Statistical analyses showed a significant increase in staining intensity in all CRC grades (I–III) in comparison to normal tissue (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>B).<fig id=\"Fig2\"><label>Figure 2</label><caption><p>TMPRSS13 protein expression is upregulated in human colorectal cancer. (<bold>A</bold>) Representative samples from tissue array IHC analysis of TMPRSS13 protein expression in Grade I (upper left panel), Grade II (lower left panel), and Grade III (lower right panel) colorectal adenocarcinomas. Primary, rabbit anti-TMPRSS13 antibody was substituted with non-immune rabbit IgG in serial sections of all samples and no significant staining was observed (example in upper right panel). Open arrowheads indicate epithelial cells with weak or moderate TMPRSS13 staining in Grade I (upper left panel) compared to strong TMPRSS13 staining in epithelial cells of a Grade II (lower left panel), and weak or moderate TMPRSS13 staining in Grade III colon adenocarcinoma (lower right panel). The lamina propria is indicated with black arrowheads. Scale bars = 100 µm. (<bold>B</bold>) Staining intensities were determined as described in “<xref rid=\"Sec9\" ref-type=\"sec\">Methods</xref>” and presented in a scatterplot categorized by cancer grade. Each circle represents one individual patient. Normal colon (N = 14), Grade I (N = 16), Grade II (N = 65), and Grade III (N = 23) colorectal adenocarcinoma. (**<italic>p</italic> < 0.01; ****<italic>p</italic> < 0.0001; determined by Dunn’s test posthoc, following Kruskal–Wallis ANOVA).</p></caption><graphic xlink:href=\"41598_2020_70636_Fig2_HTML\" id=\"MO3\"/></fig></p><p id=\"Par8\">Together, these findings demonstrate differential TMPRSS13 protein expression in CRC, validating increased levels of transcripts as being accompanied by increased protein levels, and indicating a proteolytic imbalance in the colorectal tumor microenvironment.</p></sec><sec id=\"Sec4\"><title>Loss of TMPRSS13 induces apoptosis in CRC cells</title><p id=\"Par9\">Based on our observations that TMPRSS13 is upregulated in human CRC on the transcript and protein levels, we set out to determine the role of TMPRSS13 in pro-oncogenic cellular processes by using two different human CRC cell lines. The DLD-1 (high TMPRSS13 expression) and HCT116 (low TMPRSS13 expression) cell lines are both derived from colorectal adenocarcinomas. DLD-1 cells harbor mutations in the <italic>KRAS, PIK3CA, APC,</italic> and <italic>TP53</italic> genes. HCT116 cells harbor mutated <italic>KRAS</italic> and <italic>PIK3CA,</italic> and wildtype <italic>APC</italic> and <italic>TP53</italic> genes<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. Both cell lines grow primary tumors upon orthotopic microinjection in nude mice with dissemination of cancer cells to local and distant sites<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup>. To assess the effects of TMPRSS13 loss-of-function on cell survival, two non-overlapping siRNAs targeting TMPRSS13 were used and cells were counted at different time points after transfection. A significant decrease in the number of viable TMPRSS13-silenced cells was observed beginning three days post-siRNA transfection in HCT116 cells and five days post-siRNA transfection in DLD-1 cells compared to cells transfected with a scrambled %GC matched control siRNA (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>A). TMPRSS13-silencing was confirmed in DLD-1 cells by western blotting (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>B), whereas qRT-PCR analysis was used to verify silencing of TMPRSS13 in HCT116 (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>C) due to markedly lower baseline expression levels in this cell line, which led to unreliable detection of TMPRSS13 by western blotting (See Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>, empty vector lanes; other supportive data not shown). The multiple bands (~ 65–75 kDa) observed by western blot analysis in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>B may represent different isoforms of TMPRSS13, as five isoforms produced by alternative splicing have been reported<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup> and/or differential glycosylation of one or more of these isoforms. The size differences between MSPL, isoform 1, and isoform 4 are predicted to result in marginal migration differences (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">6</xref> and Supplementary Table). We have previously reported that TMPRSS13 is subject to post-translational modification by glycosylation and phosphorylation<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. The dominant TMPRSS13 form detected at ~ 70 kDa represents a glycosylated full-length form of TMPRSS13 and the species detected as a band of ~ 90 kDa represents a glycosylated, phosphorylated form of TMPRSS13 (TMPRSS13-(P))<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. We previously identified these forms in multiple cancer cell lines, including DLD-1<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Silencing of TMPRSS13 decreases cell survival and leads to increased apoptosis in colorectal carcinoma cells. (<bold>A</bold>) TMPRSS13 was silenced using two non-overlapping synthetic RNA duplexes (siRNA 1 and siRNA 2) in the human colorectal carcinoma cell lines DLD-1 (top panel) and HCT116 (bottom panel) and cells were counted on day 3, day 5, and day 7 following siRNA treatment. A %GC-matched non-targeting RNA duplex was used as a negative control (Scramble). The number of viable cells counted was plotted for each time point. Error bars indicate SD (***<italic>p</italic> < 0.001; ****<italic>p</italic> < 0.0001, determined by Tukey’s posthoc test, following two-way ANOVA). (<bold>B</bold>) Verification of TMPRSS13 knockdown in DLD-1 cells was performed by western blot analysis. The high molecular weight band labeled TMPRSS13-(P) represents the phosphorylated form of TMPRSS13. The lower molecular weight bands (at or below the 72 kDa marker) labeled TMPRSS13, likely represent different glycosylation forms or isoforms of full-length TMPRSS13. Apoptosis was assessed by probing for cleaved PARP (middle panel) and anti-ß-actin was used as a control for equal loading (bottom panel). Dashed lines indicate cropped lanes. (<bold>C</bold>) Verification of TMPRSS13 knockdown in HCT116 cells was performed by qRT-PCR analysis using fold-change analysis normalized to HPRT1. Error bars indicate SEM (****<italic>p</italic> < 0.0001, determined using a one-way ANOVA with Tukey posthoc tests). (<bold>D</bold>) Apoptosis following TMPRSS13 knockdown was assessed by detection of cleaved PARP in HCT116 cells by western blot analysis (top panel). Anti-ß-actin was used as a control for equal loading (bottom panel). Dashed lines indicate cropped lanes.</p></caption><graphic xlink:href=\"41598_2020_70636_Fig3_HTML\" id=\"MO4\"/></fig></p><p id=\"Par10\">To further characterize the cellular effects of TMPRSS13 knockdown, we assessed the level of cleaved Poly (ADP-ribose) polymerase (PARP) by western blotting as a marker for cells undergoing apoptosis in cell lysates. Robust increases in cleaved PARP levels were observed at all three time points (days 3, 5, and 7) upon TMPRSS13 silencing in both DLD-1 cells (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>B) and HCT116 cells (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>D). For a detailed comparative analysis of TMPRSS13-deficient cell populations versus TMPRSS13-sufficient cell populations, cells were stained with Annexin V-AlexaFluor™ 488 conjugate (AV488) in conjunction with the vital dye propidium iodide (PI) for analysis by flow cytometry to identify early- and late-stage apoptotic cells. The flow cytometric AV488/PI analysis data shows that a significantly higher proportion of TMPRSS13-silenced HCT116 cells underwent apoptosis compared to control cells (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>A, B left panels). The largest relative difference was observed in early apoptotic cells (AV488-positive/PI-negative) following TMPRSS13-silencing (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>B, right panels). The relative fractions of early apoptotic cells were: 22% for siRNA1 and 25% for siRNA2 versus 9% for control (day 4) and 25% for siRNA1 and 16% for siRNA2 versus 5% for control (day 5) (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>B, right panels). Silencing of TMPRSS13 in HCT116 was verified by qRT-PCR (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>C). DLD-1 cells were not amenable to the AV488/PI assay due to poor annexin-V staining, which is in line with previous reports<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. Together, the decrease in viable cells, increased PARP-cleavage, and a higher relative apoptotic cell population revealed by flow cytometry analysis following TMPRSS13-silencing suggests an important role for TMPRSS13 in CRC cell survival and apoptosis.<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Flow cytometry analysis indicates increased apoptosis in colorectal carcinoma cells upon TMPRSS13 silencing. (<bold>A</bold>) Apoptosis in HCT116 cells was quantified by flow cytometry using an Annexin V-Alexa Fluor-488 conjugate (AV488) and propidium iodide (PI) at 4 days and 5 days post-siRNA treatment. Dot plots summarizing populations stained with AV488 and PI staining are shown for two non-overlapping synthetic RNA duplexes (siRNA 1 and siRNA 2). A %GC-matched non-targeting RNA duplex was used as a negative control (Scramble). Unstained cells are in Q4, with early-apoptotic AV488-positive stained cells being in Q3, late-apoptotic AV488/PI-positive stained cells captured in Q2, and necrotic PI-stained cells in Q1. (<bold>B</bold>) Analysis of AV488-positive populations, encompassing both early- and late-apoptotic cells (AV488+/PI + plus AV488+/PI-, left panels) and early apoptotic cells alone (AV488+/PI-, right panels), shows a significant increase in apoptotic cells among TMPRSS13 siRNA-treated cells. Error bars indicate SEM. (**<italic>p</italic> < 0.01; ***<italic>p</italic> < 0.001; ****<italic>p</italic> < 0.0001, compared to scramble control, determined by Tukey posthoc test, following one-way ANOVA). (<bold>C</bold>) qRT-PCR analysis of TMPRSS13 expression normalized to HPRT1 in HCT116 cells was used to confirm gene silencing. Error bars indicate SEM (****<italic>p</italic> < 0.0001).</p></caption><graphic xlink:href=\"41598_2020_70636_Fig4_HTML\" id=\"MO6\"/></fig></p></sec><sec id=\"Sec5\"><title>Increased TMPRSS13 expression promotes resistance to drug-induced apoptosis in CRC cells</title><p id=\"Par11\">Spurred by the finding that TMPRSS13 expression is elevated in CRC and that loss of expression results in an apoptotic response, we performed gain-of-function experiments to determine whether overexpression of the protease is sufficient for cells to acquire protective properties against apoptotic stimuli (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>). For this purpose, we utilized a panel of drugs including the chemotherapeutic drugs paclitaxel, carboplatin, and 5-FU; the broad-spectrum kinase inhibitor staurosporine; and the small-molecule Bcl-2 inhibitor HA14-1. These drugs cause cytotoxicity in a variety of cells through different mechanisms, but with apoptosis as the major outcome<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref>–<xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. Full-length human TMPRSS13 (isoform 1; see “<xref rid=\"Sec9\" ref-type=\"sec\">Methods</xref>”) was overexpressed in DLD-1 and HCT116 cells using transient plasmid transfection and confirmed by western blot (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>A,B, upper panels). The responses to drug treatment were assessed by western blot detection of caspase-3 cleavage (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>A,B, middle panels). Overexpression of TMPRSS13 led to a profound decrease in detected cleaved caspase-3 in CRC cells in response to HA14-1 in both cell lines. Thus, both DLD-1 cells, which have a relatively high level of endogenous TMPRSS13, and HCT116 cells with relatively low endogenous TMPRSS13 levels (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>A,B) were protected from HA14-1-induced apoptosis upon increased TMPRSS13 expression. Both cell lines also displayed TMPRSS13-mediated protection from apoptosis upon paclitaxel treatment. TMPRSS13 overexpression had no detectable effect on caspase-3 cleavage upon treatment with carboplatin, 5-FU, or staurosporine. The observed lack of response to 5-FU, which is widely used in advanced CRC, emphasizes a major clinical obstacle posed by acquired resistance to this and other chemotherapy drugs, which occurs in 90% of patients with metastatic cancer<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>.<fig id=\"Fig5\"><label>Figure 5</label><caption><p>Overexpression of TMPRSS13 confers resistance to drug-induced apoptosis in colorectal cancer cells. (<bold>A</bold>) DLD-1 cells were transfected with a mammalian expression vector containing full-length, human TMPRSS13 (T13) cDNA (GenBank accession no. AAI14929.1). Full-length TMPRSS13 is shown, with and without phosphorylation (as indicated by (P)). As a control, cells were transfected with the same expression vector without the TMPRSS13 cDNA insert (EV). Twenty-four hours after transfection, cells were treated for 48 h with 50 µM carboplatin, 10 µM paclitaxel, or 100 µM 5-fluorouracil (5-FU); 10 µM HA14-1 for 1.5 h; or 1 µM staurosporine (STS) for 4 h. VEH = vehicle control cells. Cell lysates were collected 72 h post-transfection. TMPRSS13 overexpression was verified by western blotting (top panel). Apoptosis was examined by the detection of cleaved caspase-3 (middle panel) and anti-ß-actin was used as a control for equal loading (bottom panel). The break with the vertical heavy dashed line separates images from different blots. (<bold>B</bold>) HCT116 cells were transfected as described for DLD-1 cells to express TMPRSS13 and treated with 10 µM paclitaxel for 48 h, or 60 µM HA14-1 for 1 h. Cellular lysates were collected 72 h post-transfection and analyzed for TMPRSS13 expression and the presence of cleaved caspase-3 by western blot analysis. Anti-ß-actin was used as a control for equal loading (bottom panels). Light dashed vertical lines indicate cropping within one membrane.</p></caption><graphic xlink:href=\"41598_2020_70636_Fig5_HTML\" id=\"MO2\"/></fig></p></sec><sec id=\"Sec6\"><title>Loss of TMPRSS13 renders CRC cells more sensitive to drug-induced apoptosis</title><p id=\"Par12\">To further investigate the role of TMPRSS13 in drug-induced apoptosis, we investigated whether silencing of TMPRSS13 would increase sensitivity to this process, thereby exploring the potential of a novel combination treatment strategy using TMPRSS13 inhibitors together with chemotherapy to improve treatment efficacy (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>). HA14-1 and paclitaxel were selected based on the protective effect that TMPRSS13 overexpression had against these two apoptosis-inducing agents (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>).<fig id=\"Fig6\"><label>Figure 6</label><caption><p>TMPRSS13 silencing sensitizes colorectal cancer cells to drug-induced apoptosis. Whole-cell lysates from DLD-1 (<bold>A</bold>, <bold>B</bold>) and HCT116 cells (<bold>C</bold>) were collected 96 h following siRNA treatment using two non-overlapping synthetic RNA duplexes (siRNA 1 and siRNA 2 = siR-1 and siR-2) targeting TMPRSS13. A %GC-matched RNA duplex was used as a negative control (scr). Before lysis, cells were subject to treatment with HA14-1 for 1 h (30 or 60 µM) or paclitaxel for 48 h (10 µM) or vehicle (VEH) as indicated. (<bold>A</bold>, <bold>B</bold>) Western blot analysis was used to verify TMPRSS13 knockdown in DLD-1 cells (top panels) and cleaved PARP and cleaved caspase-3 were used as markers of apoptosis in DLD-1 (middle panels). The high molecular weight band labeled TMPRSS13-(P) represents the phosphorylated form of TMPRSS13. The lower molecular weight bands (at or below the 72 kDa marker) labeled TMPRSS13, likely represent different glycosylation forms/isoforms of full-length TMPRSS13. Anti-ß-actin was used as a control for loading (bottom panels). Dashed lines indicate cropped membranes. (<bold>C</bold>) HCT116 cells treated as described above with HA14-1 or vehicle, respectively (<bold>D</bold>) qRT-PCR analysis of TMPRSS13 expression normalized to HPRT1 was used to verify gene silencing in HCT116 cells. Error bars indicate SEM (****<italic>p</italic> < 0.0001, determined using a one-way ANOVA with Tukey posthoc tests).</p></caption><graphic xlink:href=\"41598_2020_70636_Fig6_HTML\" id=\"MO5\"/></fig></p><p id=\"Par13\">HA14-1 or paclitaxel was added to TMPRSS13-silenced cells and levels of cleaved PARP and caspase-3 were detected by western blotting (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>A–C, middle panels). TMPRSS13 knockdown was confirmed by western blotting for DLD-1 (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>A,B, top panels) and by qRT-PCR for HCT116 cells (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>D). As expected, silencing of TMPRSS13 alone induced apoptosis in DLD-1 cells (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>A,B) and HCT116 cells (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>C). Importantly, HA14-1 treatment in combination with TMPRSS13 silencing further increased the level of apoptotic markers, indicating that the combination elicits a stronger apoptotic response than targeting either Bcl-2 (by HA14-1 treatment) or TMPRSS13 individually. The apoptotic response in DLD-1 cells to paclitaxel treatment was also enhanced in TMPRSS13-siRNA treated cells, although the potentiation by combination targeting compared to single targeting was less pronounced (Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>B).</p></sec><sec id=\"Sec7\"><title>Loss of TMPRSS13 reduces invasive potential of CRC cells</title><p id=\"Par14\">For progression from localized to advanced/metastatic CRC to occur, cancer cells acquire properties consistent with a propensity to invade into surrounding tissues and distal organs. The role of TMPRSS13 for the invasive potential of CRC cells was assessed using a transwell assay in which cells were seeded on top of an extracellular matrix hydrogel in serum-free media and allowed to invade overnight towards full-serum media in the bottom chamber (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>). Upon silencing of TMPRSS13, a significant decrease in invasive potential was observed in DLD-1 cells (Fig. <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>). Importantly, the experiment was carried out on day 2 after siRNA transfection when no difference in cell number is observed between control and TMPRSS13-silenced DLD-1 cells (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A), minimizing interference from differences in cell proliferation/survival. These data suggest that TMPRSS13, in addition to anti-apoptotic functions, also promotes cellular invasion, two key pro-oncogenic functional properties involved in primary tumor growth and metastasis.<fig id=\"Fig7\"><label>Figure 7</label><caption><p>TMPRSS13 silencing reduces the invasive potential of colorectal cancer cells. (<bold>A</bold>) Invasion assays were performed in siRNA-treated DLD-1 cells using two non-overlapping synthetic RNA duplexes (siRNA 1 and siRNA 2) targeting TMPRSS13 and a %GC-matched RNA duplex as a negative control (Scramble). 48 h following siRNA treatment, DLD-1 cells were seeded in serum-free media onto transwell inserts coated with 1 mg/mL Cultrex® basement membrane gel, inserted into 24-well plates with serum-containing media. Cells were incubated for 16 h and invading cells were fixed and stained. Representative images of Cultrex®-coated transwell membranes containing invading cells are shown. (<bold>B</bold>) Invading cells were counted, and numbers analyzed by ANOVA, with Tukey’s posthoc test for multiple comparisons. Error bars indicate SEM (n = 3, ***<italic>p</italic> < 0.001).</p></caption><graphic xlink:href=\"41598_2020_70636_Fig7_HTML\" id=\"MO7\"/></fig></p></sec></sec><sec id=\"Sec8\"><title>Discussion</title><p id=\"Par15\">After seeking to identify uncharacterized TTSPs dysregulated in cancers, we discovered that the TMPRSS13 transcript is upregulated in CRC. Immunohistochemistry indicated increased TMPRSS13 protein levels in both well-differentiated and poorly differentiated cancers, suggesting that this protease is a potential promoter of CRC progression. Through further characterization of TMPRSS13 in cell culture models, we found that TMPRSS13 expression promoted cellular survival and invasive potential in CRC cell lines. Flow cytometric analysis revealed a significant increase in Annexin V-positive/PI-negative TMPRSS13-silenced cells, indicative of early apoptotic stages. In colon epithelia, apoptosis is a tightly regulated process crucial to the maintenance of epithelial integrity<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. Suppression of apoptosis in cancer is critical for tumor progression and various pathways are altered to facilitate escape from cell death<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. Our findings suggest that TMPRSS13 is a key player in promoting resistance to apoptosis in CRC cells. These properties of TMPRSS13 have not previously been described in any cancer type. A recent study demonstrated that expression of another member of the Hepsin/TMPRSS subfamily of TTSPs, TMPRSS4, correlates with colorectal cancer pathological stage<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. Furthermore, RNA-mediated silencing experiments in HCT116 cells revealed that TMPRSS4 is involved in the regulation of cell proliferation, apoptosis, and invasion<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. It is plausible that the two closely related proteases both promote CRC progression.</p><p id=\"Par16\">Identifying novel key players in CRC progression and their functions on the cellular level is a critical step towards the development of potential new targeted therapies. On the mechanistic level, a major challenge in current protease research is determining their mechanism of action, including the identification of physiologically relevant substrates. Many proteases, including TTSPs, are capable of cleaving multiple substrates in vitro<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>–<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. Approaches to identify critical substrates for TTSPs and other extracellular/pericellular proteases include biased experiments where the effect of genetic ablation or inhibition of the protease in question on potential substrate cleavage is assessed. Alternatively, mass spectrometry-based approaches for determining the proteolytic events in complex biological samples (degradomics) have been used to discover substrates and potential cleavage sites<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. However, understanding the physiological relevance of these results remains a significant challenge, and substrate candidates identified in these experiments must still be rigorously validated in cell-based assays or animal models<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. Because the biochemical and cell biological features of TMPRSS13 are still relatively uncharacterized and have not been studied in the context of cancer, the only two candidate mammalian substrates for TMPRSS13 reported to date are pro-HGF and ENaC<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. HGF is biosynthesized as the single-chain zymogen-like inactive precursor, pro-HGF, and it has been shown that TMPRSS13 can proteolytically process pro-HGF into its two-chain form in a cell-free system<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. HGF is a pleiotropic growth factor and a key mediator of cell migration/invasion, proliferation, and survival/apoptosis in cancer cells via activation of its receptor, c-Met<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. It has previously been reported that matriptase is the main activator of pro-oncogenic HGF/c-Met in in vivo models of breast and squamous cell carcinoma<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref>,<xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. Furthermore, pertaining specifically to CRC, inhibition or silencing of matriptase in DLD1 cells efficiently impaired the conversion of pro-HGF into active HGF at the cell surface and inhibited cell scattering upon pro-HGF stimulation<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>. Matriptase is highly expressed in CRC patient samples and in CRC cell lines, including those used for this study<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref>,<xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>, which makes it plausible that matriptase is the major activator of pro-HGF in this cancer type. However, it cannot be ruled out that other proteases, including TMPRSS13, contribute to pro-HGF activation in CRC under certain physiological or pathological conditions. The other candidate substrate, human ENaC, was shown to be activated by TMPRSS13 in a <italic>Xenopus laevis</italic> cellular assay<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup> and activation of ENaC in cancer cells has been implicated in regulation of cellular survival/apoptosis (see further discussion below)<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>.</p><p id=\"Par17\">Despite advances in systemic therapies, the five-year survival rate for metastatic CRC remains below 15%<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup>, making novel approaches to combat late-stage disease necessary, including the development of novel targeted therapies. This prompted us to test whether TMPRSS13 contributes to a drug-resistant phenotype in CRC cells. Indeed, upon overexpression of TMPRSS13, CRC cells exhibited resistance to treatment with the apoptosis-inducing drugs HA14-1 and paclitaxel. Conversely, TMPRSS13-silenced cells exhibited increased sensitivity to cell death induced by HA14-1 and, to a lesser extent, paclitaxel. Taxanes, including paclitaxel, have failed to demonstrate significant clinical benefit in phase II trials in CRC and are not used as standard-of-care<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref>,<xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>. In tissue culture experiments using SW480 and DLD-1 cells, paclitaxel-induced apoptosis can be enhanced by simultaneous inhibition of the mitogen-activated protein kinase (MAPK) pathway in CRC<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>. Thus, the treatment of SW480 and DLD-1 cells with paclitaxel resulted in increased activation of the MAPK pathway. In both cell lines, MAPK attenuation by PD98059, a MEK inhibitor, led to an enhancement of paclitaxel-induced apoptosis<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup>. Synergistic inhibition of colon cancer cell growth with paclitaxel and the PI3K/mTOR dual inhibitor BEZ235 through apoptosis in HCT116 and HT-29 colon cancer cells has also been reported<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>. Further studies are needed to determine whether paclitaxel in combination with other drugs, including TMPRSS13 inhibitors, could be a viable therapeutic strategy in CRC.</p><p id=\"Par18\">In our studies, we observed the most profound apoptotic effect when combining TMPRSS13 silencing with HA14-1 treatment, which provided a rational starting point to explore potential mechanisms. Based on the selective action of HA14-1 on Bcl-2<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>, we investigated whether Bcl-2 levels were affected upon TMPRSS13 silencing. While decreases in Bcl-2 expression upon TMPRSS13 silencing were frequently observed, this phenomenon was not consistently detected under all cell culture conditions; therefore, we could not definitively confirm a functional relationship between TMPRSS13 and Bcl-2 (data not shown). Crosstalk between TMPRSS13 and Bcl-2 associated pathways may contribute to the increased apoptotic effect we observed; the implications of such a connection are significant, as Bcl-2 is often upregulated in cancer, including CRC<sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>. Bcl-2 promotes Akt signaling independent of mitochondrial involvement<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>; however, as a cell-surface protease, a point of entry for TMPRSS13 into this pathway remains elusive. In this context, it should be mentioned that TMPRSS13 differs from other TTSPs by having a large intracellular domain with tandem repeat phosphorylation motifs of various protein kinases<sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup>, and it has been demonstrated that TMPRSS13 is phosphorylated in cancer cells, including DLD1 cells<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. The roles of TMPRSS13 phosphorylation in pro-oncogenic signaling, including regulation of apoptosis, are currently under investigation.</p><p id=\"Par19\">It is noteworthy that inhibitors targeting the Bcl-2 protein have gained attention in recent years. In 2016 the FDA approved venetoclax, a selective small-molecule inhibitor of Bcl-2, for treatment of chronic lymphocytic leukemia (CLL). Particularly in hematological malignancies, multiple clinical trials are assessing the possible use of venetoclax, alone or in combination with other chemotherapies<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup>. It remains to be seen whether the positive results observed in diseases like CLL using anti-apoptotic inhibition will be mirrored in solid tumors, including CRC, that are commonly more difficult to treat in the clinic. Promising results have recently been reported on venetoclax when combined with tamoxifen in ER/Bcl-2-positive metastatic breast cancer with an overall response rate of 61% and a 72% clinical benefit rate<sup><xref ref-type=\"bibr\" rid=\"CR59\">59</xref></sup>, which underscores the importance of testing new combination therapies.</p><p id=\"Par20\">Ion channels, including calcium and sodium channels, play a critical role in cancer by regulating cell survival and apoptosis<sup><xref ref-type=\"bibr\" rid=\"CR60\">60</xref>,<xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup>. HA14-1 and paclitaxel can both facilitate increased intracellular calcium levels, albeit by different mechanisms, which trigger mitochondria-mediated and ER stress-associated apoptotic cascades. In our present study, these two drugs enhanced the cellular death observed in combination with TMPRSS13 knockdown<sup><xref ref-type=\"bibr\" rid=\"CR62\">62</xref>–<xref ref-type=\"bibr\" rid=\"CR67\">67</xref></sup>. With both HA14-1 and paclitaxel increasing intracellular calcium levels, ion imbalances may potentiate the cellular death associated with TMPRSS13 silencing. One potential mechanism by which TMPRSS13 might affect intracellular ion levels is by proteolytic activation of ENaC. It was previously reported that increased ENaC-mediated sodium currents were measured in <italic>Xenopus</italic> oocytes injected with human ENaC and TMPRSS13 mRNA<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. In cancer, it has been proposed that ENaC-mediated Na<sup>+</sup> influx promotes survival, proliferation, and invasion of cancer cells, whereas blockade of Na<sup>+</sup> influx via ENaC induces cell cycle arrest and apoptosis<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref>,<xref ref-type=\"bibr\" rid=\"CR61\">61</xref></sup>. It can be speculated that TMPRSS13-mediated ENaC activation in CRC (ENaC is expressed in colon epithelial cells and datasets in Oncomine™ report high expression of ENaC in both DLD1 and HCT116 cells<sup><xref ref-type=\"bibr\" rid=\"CR68\">68</xref>,<xref ref-type=\"bibr\" rid=\"CR69\">69</xref></sup>) could contribute to survival and invasion, whereas silencing of the protease may decrease ENaC activation, leading to increased apoptosis and enhanced sensitivity to HA14-1 and paclitaxel.</p><p id=\"Par21\">Our studies indicate a role for TMPRSS13 in conferring cells with invasive potential and protecting cells from apoptosis, suggesting that TMPRSS13 is a potential therapeutic target in CRC. One factor to consider is the potential side effects of TMPRSS13 inhibition in vivo. TMPRSS13 is an attractive target because of its low baseline expression levels in the healthy colon, potentially limiting the adverse effects of TMPRSS13 inhibition. Furthermore, previous studies in mouse genetic loss-of-function models demonstrate that TMPRSS13 deficiency has no discernible consequences for adult mice<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. Still, since the function of TMPRSS13 under challenged conditions remains uncharacterized, the impact of TMPRSS13 inhibition on the function and homeostasis of the colon and other tissues requires additional examination.</p><p id=\"Par22\">In summary, this study represents a comprehensive characterization of TMPRSS13 in CRC, highlighting cancer-associated dysregulation of this TTSP and underscoring it as a critical component of CRC cell survival and protection from drug-induced apoptosis. These findings lay the foundation for continuing studies to further decipher the molecular mechanisms by which TMPRSS13 exerts its pro-oncogenic functions, to promote the development of TMPRSS13 inhibitors, and to study their potential as targeted therapeutic drugs in CRC.</p></sec><sec id=\"Sec9\"><title>Materials and methods</title><sec id=\"Sec10\"><title>In silico analysis</title><p id=\"Par23\">The Oncomine™ online platform (<ext-link ext-link-type=\"uri\" xlink:href=\"https://www.oncomine.org\">https://www.oncomine.org</ext-link>) was used to perform a meta-analysis of TMPRSS13 expression, comparing colorectal cancer samples to normal colon across multiple transcriptome-wide studies. Relevant datasets were identified utilizing the differential analysis; cancer vs. normal analysis; and TMPRSS13 gene filters. The results shown were based upon data generated by the TCGA Research Network: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.cancer.gov/tcga\">https://www.cancer.gov/tcga</ext-link>.</p></sec><sec id=\"Sec11\"><title>Tissue samples, immunohistochemistry, and evaluation of staining</title><p id=\"Par24\">Colorectal (CO1921) tissue arrays containing both cancer and normal samples were obtained from US Biomax, Inc. Analysis of TMPRSS13 protein in normal human tissue for antibody validation was performed using human tissue arrays UNC241 and OR301 (US Biomax Inc.). Paraffin-embedded arrays were cleared in xylene (Fisher Scientific) and rehydrated in a graded series of ethanol solutions. For antigen retrieval, tissue arrays were boiled in reduced pH citrate antigen retrieval buffer (Vector Laboratories) for 10 min. Endogenous peroxidase activity was quenched by incubating tissue arrays in 3% H<sub>2</sub>O<sub>2</sub> for 15 min. Arrays were blocked with 2.5% bovine serum albumin (Sigma Aldrich) in PBS for 1 h at room temperature and incubated overnight in anti-TMPRSS13 antibody (1:150, PA5-30935, Thermo Fisher Scientific) at 4 °C in a humidified chamber. All washing steps were performed with PBS. Non-immune rabbit IgG (Neomarkers) was used as a negative control. Visualization of bound primary antibody was performed using a biotinylated anti-rabbit secondary antibody and conjugated horseradish peroxidase H contained in the VECTASTAIN ABC kit (Vector Laboratories). Enzymatic visualization was carried out with 3,3-diaminobenzidine (DAB) substrate (Sigma-Aldrich) and arrays were subsequently counterstained with hematoxylin. Stained slides were washed and dehydrated in a series of graded ethanol solutions followed by xylene and mounted with glass coverslips using Permount (Thermo Fisher Scientific). Microscopic images were acquired on a Zeiss Axio Scope. A1 using digital imaging.</p><p id=\"Par25\">To evaluate staining intensity in colon tissue arrays, samples were assessed microscopically. Epithelial staining of TMPRSS13 was rated based on intensity in 20 × microscopic fields on a scale of 0 to 3, where: 0 = no epithelial staining; 1 = majority weakly stained epithelial cells OR few moderately stained epithelial cells among a majority of non-stained cells; 2 = majority moderately stained epithelial cells OR few strongly stained among a majority of weakly or non-stained cells; 3 = majority strongly stained epithelial cells.</p></sec><sec id=\"Sec12\"><title>Cell lines and culture conditions</title><p id=\"Par26\">HCT116 cells (ATCC) were cultured in minimal essential media (MEM) (Gibco/Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals), 10 units/mL penicillin and 10 µg/mL streptomycin (Gibco/Thermo Fisher Scientific), and 1 × non-essential amino acids (Gibco/Thermo Fisher Scientific). DLD-1 cells (ATCC) were cultured with RPMI-1640 media supplemented with 10% FBS and 10 units/mL Penicillin and 10 µg/mL Streptomycin (Gibco/Thermo Fisher Scientific). Cells were maintained in a humidified incubator at 37 °C with an atmosphere of 5% CO<sub>2</sub>.</p></sec><sec id=\"Sec13\"><title>RNAi-mediated gene silencing</title><p id=\"Par27\">For TMPRSS13 knockdown in DLD-1 and HCT116 colon cancer cells, two independent Stealth RNAi™ siRNA duplexes (Invitrogen/Thermo Fisher Scientific) targeting TMPRSS13 (siRNA 1 = HSS130531, siRNA 2 = HSS130532) were used. A matched %GC negative scramble control (12,935,300) was included in all experiments. Reverse transfections, in which siRNA-lipid complexes were added to wells before seeding, were performed using Lipofectamine RNAiMAX (Invitrogen/Thermo Fisher Scientific) following manufacturer instructions. Cells were transfected in 6-well plates for all experiments except for cell-counting experiments and flow cytometric analyses, in which 12-well plates were used and reagent levels adjusted according to transfection reagent manufacturer guidelines. Media was replaced every 48 h during siRNA treatment for all experiments. Cellular lysates were collected 3, 5, and 7 days post-transfection for proliferation experiments, 4 days post-transfection for drug treatment experiments, and 4 to 5 days post-transfection for flow cytometry experiments.</p></sec><sec id=\"Sec14\"><title>Cell counting</title><p id=\"Par28\">DLD-1 and HCT116 cells were reverse transfected with siRNAs targeting TMPRSS13 and seeded onto 12-well plates at 50,000 cells/well for DLD-1 and 250,000 cells/well for HCT116. Cells were trypsinized 3, 5, and 7 days post-transfection, pelleted by centrifugation at 200×<italic>g</italic> for 5 min, and resuspended in media. Samples were mixed 1:1 with 0.4% trypan blue stain (Gibco/Thermo Fisher Scientific) to distinguish viable and dead cells. Counting was performed using a hemocytometer.</p></sec><sec id=\"Sec15\"><title>Western blot analyses</title><p id=\"Par29\">Cultured DLD-1 or HCT116 cells were washed 3 times with ice-cold PBS and lysed in- well using ice-cold RIPA buffer (150 mM NaCl; 50 mM Tris/HCl, pH 7.4; 0.1% SDS; 1% NP-40) with protease inhibitor cocktail (Sigma Aldrich); and phosphatase inhibitor cocktail (Sigma Aldrich) and cleared by centrifugation at 12,000×<italic>g</italic> at 4 °C. Quantification of cell lysate protein concentrations was performed using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Lysates were prepared with SDS lysis buffer containing a reducing agent (50 mM Tris–HCl, pH 6.8, 0.25% bromophenol blue, 5% glycerol, 1.5% SDS, and 100 mM dithiothreitol) and boiled for 5 min before loading onto 4–15% Mini-Protean® or Criterion® TGX gels (Bio-Rad) for SDS-PAGE, followed by blotting onto 0.2 µm Immun-Blot® polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked with 5% (w/v) dry milk powder in TBS-T (Tris-buffered saline, 0.1% Tween-20) for 1 h at room temperature and subsequently incubated overnight at 4 °C in primary antibodies diluted in 5% dry milk powder/TBS-T. Primary antibodies used for western blotting included rabbit anti-TMPRSS13 (1:2000, ab59862, Abcam), rabbit anti-cleaved caspase-3 (1:500, 9661, Cell Signaling Technology), rabbit anti-PARP (1:1000, 9532, Cell Signaling Technology), rabbit anti-cleaved PARP (1:1000, 5625, Cell Signaling Technology), and mouse anti-β-actin (1:10,000, NB600-501, Novus Biologicals). Goat anti-rabbit and goat anti-mouse (Millipore) HRP-conjugated antibodies were used as secondary antibodies. Detection of antibodies was performed using ECL Western Blotting substrate or Super-Signal West Femto Chemiluminescent Substrate (Pierce, Thermo Fisher Scientific).</p></sec><sec id=\"Sec16\"><title>Transient transfections with TMPRSS13 expression vector</title><p id=\"Par30\">Transient expression of TMPRSS13 in DLD-1 and HCT116 cells was performed in 6-well plates through reverse transfection with 500 ng of plasmid using Lipofectamine LTX (Invitrogen) according to manufacturer instructions. The pcDNA3.1 (Invitrogen/Thermo Fisher Scientific) plasmid vector lacking the coding sequence was used as an empty vector control, as well as pcDNA3.1-TMPRSS13 (human full-length)<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>, a plasmid containing the coding sequence for TMPRSS13 (GenBank accession no. AAI14929.1; see Supplementary Table for isoform comparison). This clone is representative of TMPRSS13 isoform 1 with a Q83-A87 (QASPA) deletion. The Catalogue of Somatic Mutations in Cancer (COSMIC) reports that in screens targeting TMPRSS13, nearly 13% of cancer samples are positive for a Q83-Q87 deletion of TMPRSS13. Notably, colorectal cancer makes up 41% of cases positive for this deletion<sup><xref ref-type=\"bibr\" rid=\"CR70\">70</xref></sup>. Media was replaced 24 h following transfection and cells were subjected to drug treatment as described below. Cellular lysates were collected 3 days post-transfection in drug treatment experiments.</p></sec><sec id=\"Sec17\"><title>Drugs and drug treatments</title><p id=\"Par31\">For drug treatment experiments, stock solutions of paclitaxel (Sigma Aldrich), 5-FU (Sigma Aldrich), HA14-1 (Cayman Chemical), and staurosporine (STS) (Cell Signaling Technology) were diluted in DMSO, whereas water was used to dilute a stock solution of carboplatin (Sigma-Aldrich). For the treatment of cells overexpressing TMPRSS13, DLD-1 cells were subjected to 48-h treatments with paclitaxel (10 µM), carboplatin (50 µM), or 5-FU (100 µM) starting 24 h after transient transfection with plasmid vectors. Twenty-four hours after transient transfection with an expression vector, DLD-1 and HCT116 cells were subjected to treatment with HA14-1 or STS. The duration of HA14-1 treatment for DLD-1 cells was 1.5 h at a concentration of 10 µM and a duration of 4 h for STS treatment at a concentration of 1 µM. In HCT116 cells, HA14-1 treatment lasted 1 h at 60 µM. All drug treatment conditions were concluded by lysate collection. For drug treatment of TMPRSS13-silenced cells, siRNA-treated DLD-1 cells were subjected to a 48-h treatment of paclitaxel at a final concentration of 10 µM (48 h post-transfection) and lysates were collected four days post-transfection. Four days post-transfection, siRNA-treated DLD-1 and HCT116 cells were subjected to a 1-h treatment of HA14-1 at final concentrations of 30 and 60 µM.</p></sec><sec id=\"Sec18\"><title>Quantitative real-time polymerase chain reaction (qRT-PCR)</title><p id=\"Par32\">Total RNA was isolated from cultured cells using the RNeasy Plus Kit (Qiagen) according to manufacturer instructions. Reverse transcription of RNA isolates was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The qPCR reactions were performed with probes for TMPRSS13 (Hs00361060_m1, TaqMan®, Applied Biosystems) and expression levels were analyzed using the -2<sup>ΔΔCt</sup> method and normalized to HPRT1 or GAPDH (Hs02800695_m1 and Hs02758991_g1, TaqMan®, Applied Biosystems).</p></sec><sec id=\"Sec19\"><title>Analysis of apoptosis by Annexin V/propidium staining and flow cytometry</title><p id=\"Par33\">Flow cytometry analysis was performed in the Wayne State University Flow Cytometry Core. Labeling of live HCT116 cells was performed using the Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Molecular Probes) according to manufacturer instructions. Specifically, siRNA-treated HCT116 cells were cultured in 12-well plates, with media replaced every 48 h. At four- and five-days post-transfection, cells were trypsinized with 0.25% Trypsin–EDTA (Gibco, Thermo Fisher Scientific), centrifuged at 200×<italic>g</italic> for 5 min, and cell pellets washed twice with ice-cold PBS. Following washes, 100 ul of cellular suspension from each biological sample was combined with 400 µL of 5 × annexin binding buffer and gently mixed. Cells were analyzed within 30 min of staining with an LSR II (Becton Dickinson) cytometer. Cytometric data were plotted and analyzed with FlowJo software.</p></sec><sec id=\"Sec20\"><title>Invasion assay</title><p id=\"Par34\">Two days following siRNA-mediated TMPRSS13 silencing, DLD-1 cells were serum-starved for 5 h, and then seeded in serum-free media onto 1 mg/mL Cultrex®-coated (Trevigen, Gaithersburg, MD) permeable support inserts (8.0 µm pore size, Falcon). Inserts were placed in 24-well plates with serum-containing media as a chemoattractant and cells were cultured on inserts for 24 h, after which invading cells were fixed using Z-fix (Anatech, Battle Creek, MI) and stained using Diff-Quik (Siemens, Deerfield, IL). Images of inserts were acquired using an EZ4D Stereo Zoom microscope with an integrated digital camera (Leica Microsystems, Buffalo Grove, IL), and invading cells quantified from images using ImageJ software.</p></sec><sec id=\"Sec21\"><title>Statistical analyses</title><p id=\"Par35\">All statistical analyses were performed using GraphPad Prism software. For immunohistochemical staining, differences in staining scores between cancer grade groups were analyzed using the non-parametric Kruskal–Wallis ANOVA test with posthoc comparisons performed with Dunn’s test. For proliferation experiments, differences between siRNA treatment groups were analyzed using the two-way ANOVA test, with siRNA treatment and time point as independent factors. Posthoc comparisons were performed between siRNA treatments for each time point using Tukey’s multiple comparisons test. One-way ANOVA tests with Tukey’s multiple comparisons posthoc tests were used for comparison of siRNA treatment groups in flow cytometry, invasion, and qRT-PCR experiments.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec22\"><p>\n<supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41598_2020_70636_MOESM1_ESM.pdf\"><caption><p>Supplementary Information.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p> is available for this paper at 10.1038/s41598-020-70636-4.</p></sec><ack><title>Acknowledgements</title><p>This work was supported by NIH/NCI R01CA160565 grant (K.L.), NIH/NCI R01CA160565-04S grant (K.L., F.A.V.), NIH/NCI R01CA222359 (K.L), NIH/NCI F31CA217148 (F.A.V), NIGMS/NIH grant R25 GM 058905-15 (F.A.V.) NIH Ruth L. Kirschstein National Research Service Award T32-CA009531 (A.S.M. and C.E.M) and The DeRoy Testamentary Foundation (A.S.M.), and R01NS086778 from NINDS (S. V. T.). We thank Dr. Jessica Back and Mr. Eric Van Buren from the Wayne State University Flow Cytometry Core for their assistance and Dr. John Reiners for advice regarding apoptosis assays.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>Conception and design: F.A.V., K.L.; Development of methodology: F.A.V., A.S.M., K.L.; Acquisition of data (cell culture models, immunohistochemical analysis, flow cytometry, biochemical analysis): F.A.V., V.L.F., T.E.H., K.E.S.-H., J.R.M., C.E.M., A.S.M.; Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): F.A.V., K.L.; Writing, review, and/or revision of the manuscript: F.A.V., K.L. (writing), V.L.F., T.E.H., K.E.S.-H., J.R.M., C.E.M., A.S.M., S.V.T. (reviewing); Administrative, technical, or material support (i.e., reporting or organizing data): F.A.V., V.L.F., T.E.H., K.E.S.-H., J.R.M., K.L., C.E.M., A.S.M., K.L.; Study supervision: S.V.T., K.L.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par36\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><mixed-citation publication-type=\"other\">American Cancer Society. <italic>Cancer Facts and Figures 2018</italic>. <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.cancer.org/content/dam/cancer-org/research/cancer-facts-and-statistics/annual-cancer-facts-and-figures/2018/cancer-facts-and-figures-2018.pdf\">https://www.cancer.org/content/dam/cancer-org/research/cancer-facts-and-statistics/annual-cancer-facts-and-figures/2018/cancer-facts-and-figures-2018.pdf</ext-link> (2018).</mixed-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gill</surname><given-names>S</given-names></name><etal/></person-group><article-title>Pooled analysis of fluorouracil-based adjuvant therapy for stage II and III colon cancer: who benefits and by how much?</article-title><source>J. Clin. Oncol.</source><year>2004</year><volume>22</volume><fpage>1797</fpage><lpage>1806</lpage><pub-id pub-id-type=\"doi\">10.1200/JCO.2004.09.059</pub-id><pub-id pub-id-type=\"pmid\">15067028</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Erlichman</surname><given-names>C</given-names></name><etal/></person-group><article-title>Efficacy of adjuvant fluorouracil and folinic acid in B2 colon cancer</article-title><source>J. Clin. Oncol.</source><year>1999</year><volume>17</volume><fpage>1356</fpage><lpage>1363</lpage><pub-id pub-id-type=\"doi\">10.1200/JCO.1999.17.5.1356</pub-id><pub-id pub-id-type=\"pmid\">10334519</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mamounas</surname><given-names>E</given-names></name><etal/></person-group><article-title>Comparative efficacy of adjuvant chemotherapy in patients with Dukes' B versus Dukes' C colon cancer: results from four National Surgical Adjuvant Breast and Bowel Project adjuvant studies (C-01, C-02, C-03, and C-04)</article-title><source>J. Clin. Oncol.</source><year>1999</year><volume>17</volume><fpage>1349</fpage><lpage>1355</lpage><pub-id pub-id-type=\"doi\">10.1200/JCO.1999.17.5.1349</pub-id><pub-id pub-id-type=\"pmid\">10334518</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Roock</surname><given-names>W</given-names></name><etal/></person-group><article-title>KRAS wild-type state predicts survival and is associated to early radiological response in metastatic colorectal cancer treated with cetuximab</article-title><source>Ann. Oncol.</source><year>2008</year><volume>19</volume><fpage>508</fpage><lpage>515</lpage><pub-id pub-id-type=\"doi\">10.1093/annonc/mdm496</pub-id><pub-id pub-id-type=\"pmid\">17998284</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brenner</surname><given-names>H</given-names></name><name><surname>Kloor</surname><given-names>M</given-names></name><name><surname>Pox</surname><given-names>CP</given-names></name></person-group><article-title>Colorectal cancer</article-title><source>Lancet (London, England)</source><year>2014</year><volume>383</volume><fpage>1490</fpage><lpage>1502</lpage><pub-id pub-id-type=\"doi\">10.1016/S0140-6736(13)61649-9</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mody</surname><given-names>K</given-names></name><name><surname>Baldeo</surname><given-names>C</given-names></name><name><surname>Bekaii-Saab</surname><given-names>T</given-names></name></person-group><article-title>Antiangiogenic therapy in colorectal cancer</article-title><source>Cancer J. (Sudbury, Mass)</source><year>2018</year><volume>24</volume><fpage>165</fpage><lpage>170</lpage><pub-id pub-id-type=\"doi\">10.1097/PPO.0000000000000328</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Netzel-Arnett</surname><given-names>S</given-names></name><etal/></person-group><article-title>Membrane anchored serine proteases: a rapidly expanding group of cell surface proteolytic enzymes with potential roles in cancer</article-title><source>Cancer Metastasis Rev.</source><year>2003</year><volume>22</volume><fpage>237</fpage><lpage>258</lpage><pub-id pub-id-type=\"doi\">10.1023/A:1023003616848</pub-id><pub-id pub-id-type=\"pmid\">12784999</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Antalis</surname><given-names>TM</given-names></name><name><surname>Bugge</surname><given-names>TH</given-names></name><name><surname>Wu</surname><given-names>Q</given-names></name></person-group><article-title>Membrane-anchored serine proteases in health and disease</article-title><source>Prog. Mol. Biol. Transl. Sci</source><year>2011</year><volume>99</volume><fpage>1</fpage><lpage>50</lpage><pub-id pub-id-type=\"doi\">10.1016/B978-0-12-385504-6.00001-4</pub-id><pub-id pub-id-type=\"pmid\">21238933</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bugge</surname><given-names>TH</given-names></name><name><surname>Antalis</surname><given-names>TM</given-names></name><name><surname>Wu</surname><given-names>Q</given-names></name></person-group><article-title>Type II transmembrane serine proteases</article-title><source>J. Biol. Chem.</source><year>2009</year><volume>284</volume><fpage>23177</fpage><lpage>23181</lpage><pub-id pub-id-type=\"doi\">10.1074/jbc.R109.021006</pub-id><pub-id pub-id-type=\"pmid\">19487698</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sevenich</surname><given-names>L</given-names></name><name><surname>Joyce</surname><given-names>JA</given-names></name></person-group><article-title>Pericellular proteolysis in cancer</article-title><source>Genes Dev.</source><year>2014</year><volume>28</volume><fpage>2331</fpage><lpage>2347</lpage><pub-id pub-id-type=\"doi\">10.1101/gad.250647.114</pub-id><pub-id pub-id-type=\"pmid\">25367033</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hooper</surname><given-names>JD</given-names></name><name><surname>Clements</surname><given-names>JA</given-names></name><name><surname>Quigley</surname><given-names>JP</given-names></name><name><surname>Antalis</surname><given-names>TM</given-names></name></person-group><article-title>Type II transmembrane serine proteases. Insights into an emerging class of cell surface proteolytic enzymes</article-title><source>J. Biol. Chem.</source><year>2001</year><volume>276</volume><fpage>857</fpage><lpage>860</lpage><pub-id pub-id-type=\"doi\">10.1074/jbc.R000020200</pub-id><pub-id pub-id-type=\"pmid\">11060317</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Szabo</surname><given-names>R</given-names></name><name><surname>Bugge</surname><given-names>TH</given-names></name></person-group><article-title>Membrane-anchored serine proteases in vertebrate cell and developmental biology</article-title><source>Annu. Rev. Cell. Dev. Biol.</source><year>2011</year><volume>27</volume><fpage>213</fpage><lpage>235</lpage><pub-id pub-id-type=\"doi\">10.1146/annurev-cellbio-092910-154247</pub-id><pub-id pub-id-type=\"pmid\">21721945</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Webb</surname><given-names>SL</given-names></name><name><surname>Sanders</surname><given-names>AJ</given-names></name><name><surname>Mason</surname><given-names>MD</given-names></name><name><surname>Jiang</surname><given-names>WG</given-names></name></person-group><article-title>Type II transmembrane serine protease (TTSP) deregulation in cancer</article-title><source>Front. Biosci.</source><year>2011</year><volume>16</volume><fpage>539</fpage><lpage>552</lpage><pub-id pub-id-type=\"doi\">10.1007/978-1-4684-7612-5_2</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tanabe</surname><given-names>LM</given-names></name><name><surname>List</surname><given-names>K</given-names></name></person-group><article-title>The role of type II transmembrane serine protease-mediated signaling in cancer</article-title><source>FEBS J.</source><year>2017</year><volume>284</volume><fpage>1421</fpage><lpage>1436</lpage><pub-id pub-id-type=\"doi\">10.1111/febs.13971</pub-id><pub-id pub-id-type=\"pmid\">27870503</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>List</surname><given-names>K</given-names></name></person-group><article-title>Matriptase: a culprit in cancer?</article-title><source>Future Oncol.</source><year>2009</year><volume>5</volume><fpage>97</fpage><lpage>104</lpage><pub-id pub-id-type=\"doi\">10.2217/14796694.5.1.97</pub-id><pub-id pub-id-type=\"pmid\">19243302</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Murray</surname><given-names>AS</given-names></name><name><surname>Varela</surname><given-names>FA</given-names></name><name><surname>List</surname><given-names>K</given-names></name></person-group><article-title>Type II transmembrane serine proteases as potential targets for cancer therapy</article-title><source>Biol. Chem.</source><year>2016</year><volume>397</volume><fpage>815</fpage><lpage>826</lpage><pub-id pub-id-type=\"doi\">10.1515/hsz-2016-0131</pub-id><pub-id pub-id-type=\"pmid\">27078673</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martin</surname><given-names>CE</given-names></name><name><surname>List</surname><given-names>K</given-names></name></person-group><article-title>Cell surface-anchored serine proteases in cancer progression and metastasis</article-title><source>Cancer Metastasis Rev..</source><year>2019</year><pub-id pub-id-type=\"doi\">10.1007/s10555-019-09811-7</pub-id><pub-id pub-id-type=\"pmid\">31529338</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Madsen</surname><given-names>DH</given-names></name><name><surname>Szabo</surname><given-names>R</given-names></name><name><surname>Molinolo</surname><given-names>AA</given-names></name><name><surname>Bugge</surname><given-names>TH</given-names></name></person-group><article-title>TMPRSS13 deficiency impairs stratum corneum formation and epidermal barrier acquisition</article-title><source>Biochem. J.</source><year>2014</year><volume>461</volume><fpage>487</fpage><lpage>495</lpage><pub-id pub-id-type=\"doi\">10.1042/BJ20140337</pub-id><pub-id pub-id-type=\"pmid\">24832573</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kido</surname><given-names>H</given-names></name><name><surname>Okumura</surname><given-names>Y</given-names></name></person-group><article-title>MSPL/TMPRSS13</article-title><source>Front. Biosci. J. Virtual Library</source><year>2008</year><volume>13</volume><fpage>754</fpage><lpage>758</lpage><pub-id pub-id-type=\"doi\">10.2741/2717</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zmora</surname><given-names>P</given-names></name><etal/></person-group><article-title>DESC1 and MSPL activate influenza A viruses and emerging coronaviruses for host cell entry</article-title><source>J. Virol.</source><year>2014</year><volume>88</volume><fpage>12087</fpage><lpage>12097</lpage><pub-id pub-id-type=\"doi\">10.1128/jvi.01427-14</pub-id><pub-id pub-id-type=\"pmid\">25122802</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Choi</surname><given-names>SY</given-names></name><name><surname>Bertram</surname><given-names>S</given-names></name><name><surname>Glowacka</surname><given-names>I</given-names></name><name><surname>Park</surname><given-names>YW</given-names></name><name><surname>Pohlmann</surname><given-names>S</given-names></name></person-group><article-title>Type II transmembrane serine proteases in cancer and viral infections</article-title><source>Trends Mol. Med.</source><year>2009</year><volume>15</volume><fpage>303</fpage><lpage>312</lpage><pub-id pub-id-type=\"doi\">10.1016/j.molmed.2009.05.003</pub-id><pub-id pub-id-type=\"pmid\">19581128</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kido</surname><given-names>H</given-names></name><etal/></person-group><article-title>Host envelope glycoprotein processing proteases are indispensable for entry into human cells by seasonal and highly pathogenic avian influenza viruses</article-title><source>J. Mol. Genet. Med.</source><year>2008</year><volume>3</volume><fpage>167</fpage><lpage>175</lpage><pub-id pub-id-type=\"pmid\">19565019</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Okumura</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Novel type II transmembrane serine proteases, MSPL and TMPRSS13, proteolytically activate membrane fusion activity of the hemagglutinin of highly pathogenic avian influenza viruses and induce their multicycle replication</article-title><source>J. Virol.</source><year>2010</year><volume>84</volume><fpage>5089</fpage><lpage>5096</lpage><pub-id pub-id-type=\"doi\">10.1128/JVI.02605-09</pub-id><pub-id pub-id-type=\"pmid\">20219906</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hashimoto</surname><given-names>T</given-names></name><name><surname>Kato</surname><given-names>M</given-names></name><name><surname>Shimomura</surname><given-names>T</given-names></name><name><surname>Kitamura</surname><given-names>N</given-names></name></person-group><article-title>TMPRSS13, a type II transmembrane serine protease, is inhibited by hepatocyte growth factor activator inhibitor type 1 and activates pro-hepatocyte growth factor</article-title><source>FEBS J.</source><year>2010</year><volume>277</volume><fpage>4888</fpage><lpage>4900</lpage><pub-id pub-id-type=\"doi\">10.1111/j.1742-4658.2010.07894.x</pub-id><pub-id pub-id-type=\"pmid\">20977675</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Faller</surname><given-names>N</given-names></name><name><surname>Gautschi</surname><given-names>I</given-names></name><name><surname>Schild</surname><given-names>L</given-names></name></person-group><article-title>Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea</article-title><source>PLoS ONE</source><year>2014</year><volume>9</volume><fpage>e94267</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0094267</pub-id><pub-id pub-id-type=\"pmid\">24722141</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Atlas</surname><given-names>TCG</given-names></name></person-group><article-title>Comprehensive molecular characterization of human colon and rectal cancer</article-title><source>Nature</source><year>2012</year><volume>487</volume><fpage>330</fpage><lpage>337</lpage><pub-id pub-id-type=\"doi\">10.1038/nature11252</pub-id><pub-id pub-id-type=\"pmid\">22810696</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Faller</surname><given-names>N</given-names></name><name><surname>Gautschi</surname><given-names>I</given-names></name><name><surname>Schild</surname><given-names>L</given-names></name></person-group><article-title>Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea</article-title><source>PLoS ONE.</source><year>2014</year><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0094267</pub-id><pub-id pub-id-type=\"pmid\">25333710</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ahmed</surname><given-names>D</given-names></name><etal/></person-group><article-title>Epigenetic and genetic features of 24 colon cancer cell lines</article-title><source>Oncogenesis</source><year>2013</year><volume>2</volume><fpage>e71</fpage><pub-id pub-id-type=\"doi\">10.1038/oncsis.2013.35</pub-id><pub-id pub-id-type=\"pmid\">24042735</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cespedes</surname><given-names>MV</given-names></name><etal/></person-group><article-title>Orthotopic microinjection of human colon cancer cells in nude mice induces tumor foci in all clinically relevant metastatic sites</article-title><source>Am. J. Pathol.</source><year>2007</year><volume>170</volume><fpage>1077</fpage><lpage>1085</lpage><pub-id pub-id-type=\"doi\">10.2353/ajpath.2007.060773</pub-id><pub-id pub-id-type=\"pmid\">17322390</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Murray</surname><given-names>AS</given-names></name><etal/></person-group><article-title>Phosphorylation of the type II transmembrane serine protease, TMPRSS13, in hepatocyte growth factor activator inhibitor-1 and -2-mediated cell-surface localization</article-title><source>J. Biol. Chem.</source><year>2017</year><volume>292</volume><fpage>14867</fpage><lpage>14884</lpage><pub-id pub-id-type=\"doi\">10.1074/jbc.M117.775999</pub-id><pub-id pub-id-type=\"pmid\">28710277</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dicker</surname><given-names>DT</given-names></name><name><surname>Kim</surname><given-names>SH</given-names></name><name><surname>Jin</surname><given-names>Z</given-names></name><name><surname>El-Deiry</surname><given-names>WS</given-names></name></person-group><article-title>Heterogeneity in non-invasive detection of apoptosis among human tumor cell lines using annexin-V tagged with EGFP or Qdot-705</article-title><source>Cancer Biol. Ther.</source><year>2005</year><volume>4</volume><fpage>1014</fpage><lpage>1017</lpage><pub-id pub-id-type=\"doi\">10.4161/cbt.4.9.2150</pub-id><pub-id pub-id-type=\"pmid\">16222122</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hassan</surname><given-names>M</given-names></name><name><surname>Watari</surname><given-names>H</given-names></name><name><surname>AbuAlmaaty</surname><given-names>A</given-names></name><name><surname>Ohba</surname><given-names>Y</given-names></name><name><surname>Sakuragi</surname><given-names>N</given-names></name></person-group><article-title>Apoptosis and molecular targeting therapy in cancer</article-title><source>Biomed. Res. Int.</source><year>2014</year><volume>2014</volume><fpage>150845</fpage><pub-id pub-id-type=\"doi\">10.1155/2014/150845</pub-id><pub-id pub-id-type=\"pmid\">25013758</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Couldwell</surname><given-names>WT</given-names></name><etal/></person-group><article-title>Protein kinase C inhibitors induce apoptosis in human malignant glioma cell lines</article-title><source>FEBS Lett.</source><year>1994</year><volume>345</volume><fpage>43</fpage><lpage>46</lpage><pub-id pub-id-type=\"doi\">10.1016/0014-5793(94)00415-3</pub-id><pub-id pub-id-type=\"pmid\">8194597</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kessel</surname><given-names>D</given-names></name><name><surname>Reiners</surname><given-names>JJ</given-names></name></person-group><article-title>Initiation of apoptosis and autophagy by the Bcl-2 antagonist HA14-1</article-title><source>Cancer Lett.</source><year>2007</year><volume>249</volume><fpage>294</fpage><lpage>299</lpage><pub-id pub-id-type=\"doi\">10.1016/j.canlet.2006.09.009</pub-id><pub-id pub-id-type=\"pmid\">17055152</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Long</surname><given-names>BH</given-names></name><name><surname>Fairchild</surname><given-names>CR</given-names></name></person-group><article-title>Paclitaxel inhibits progression of mitotic cells to G1 phase by interference with spindle formation without affecting other microtubule functions during anaphase and telephase</article-title><source>Can. Res.</source><year>1994</year><volume>54</volume><fpage>4355</fpage><lpage>4361</lpage></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Longley</surname><given-names>DB</given-names></name><name><surname>Harkin</surname><given-names>DP</given-names></name><name><surname>Johnston</surname><given-names>PG</given-names></name></person-group><article-title>5-fluorouracil: mechanisms of action and clinical strategies</article-title><source>Nat. Rev. Cancer</source><year>2003</year><volume>3</volume><fpage>330</fpage><lpage>338</lpage><pub-id pub-id-type=\"doi\">10.1038/nrc1074</pub-id><pub-id pub-id-type=\"pmid\">12724731</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hammond</surname><given-names>WA</given-names></name><name><surname>Swaika</surname><given-names>A</given-names></name><name><surname>Mody</surname><given-names>K</given-names></name></person-group><article-title>Pharmacologic resistance in colorectal cancer: a review</article-title><source>Ther. Adv. Med. Oncol.</source><year>2016</year><volume>8</volume><fpage>57</fpage><lpage>84</lpage><pub-id pub-id-type=\"doi\">10.1177/1758834015614530</pub-id><pub-id pub-id-type=\"pmid\">26753006</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Delgado</surname><given-names>ME</given-names></name><name><surname>Grabinger</surname><given-names>T</given-names></name><name><surname>Brunner</surname><given-names>T</given-names></name></person-group><article-title>Cell death at the intestinal epithelial front line</article-title><source>FEBS J.</source><year>2016</year><volume>283</volume><fpage>2701</fpage><lpage>2719</lpage><pub-id pub-id-type=\"doi\">10.1111/febs.13575</pub-id><pub-id pub-id-type=\"pmid\">26499289</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mohammad</surname><given-names>RM</given-names></name><etal/></person-group><article-title>Broad targeting of resistance to apoptosis in cancer</article-title><source>Semin. Cancer Biol.</source><year>2015</year><volume>35</volume><fpage>S78</fpage><lpage>S103</lpage><pub-id pub-id-type=\"doi\">10.1016/j.semcancer.2015.03.001</pub-id><pub-id pub-id-type=\"pmid\">25936818</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>A</given-names></name><etal/></person-group><article-title>TMPRSS4 correlates with colorectal cancer pathological stage and regulates cell proliferation and self-renewal ability</article-title><source>Cancer Biol. Ther.</source><year>2014</year><volume>15</volume><fpage>297</fpage><lpage>304</lpage><pub-id pub-id-type=\"doi\">10.4161/cbt.27308</pub-id><pub-id pub-id-type=\"pmid\">24335200</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vizovisek</surname><given-names>M</given-names></name><name><surname>Vidmar</surname><given-names>R</given-names></name><name><surname>Fonovic</surname><given-names>M</given-names></name><name><surname>Turk</surname><given-names>B</given-names></name></person-group><article-title>Current trends and challenges in proteomic identification of protease substrates</article-title><source>Biochimie</source><year>2016</year><volume>122</volume><fpage>77</fpage><lpage>87</lpage><pub-id pub-id-type=\"doi\">10.1016/j.biochi.2015.10.017</pub-id><pub-id pub-id-type=\"pmid\">26514758</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gherardi</surname><given-names>E</given-names></name><name><surname>Birchmeier</surname><given-names>W</given-names></name><name><surname>Birchmeier</surname><given-names>C</given-names></name><name><surname>Vande Woude</surname><given-names>G</given-names></name></person-group><article-title>Targeting MET in cancer: rationale and progress</article-title><source>Nat. Rev. Cancer</source><year>2012</year><volume>12</volume><fpage>89</fpage><lpage>103</lpage><pub-id pub-id-type=\"doi\">10.1038/nrc3205</pub-id><pub-id pub-id-type=\"pmid\">22270953</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zoratti</surname><given-names>GL</given-names></name><etal/></person-group><article-title>Targeting matriptase in breast cancer abrogates tumour progression via impairment of stromal-epithelial growth factor signalling</article-title><source>Nat. Commun.</source><year>2015</year><volume>6</volume><fpage>6776</fpage><pub-id pub-id-type=\"doi\">10.1038/ncomms7776</pub-id><pub-id pub-id-type=\"pmid\">25873032</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Szabo</surname><given-names>R</given-names></name><etal/></person-group><article-title>c-Met-induced epithelial carcinogenesis is initiated by the serine protease matriptase</article-title><source>Oncogene</source><year>2011</year><volume>30</volume><fpage>2003</fpage><lpage>2016</lpage><pub-id pub-id-type=\"doi\">10.1038/onc.2010.586</pub-id><pub-id pub-id-type=\"pmid\">21217780</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Forbs</surname><given-names>D</given-names></name><etal/></person-group><article-title>In vitro inhibition of matriptase prevents invasive growth of cell lines of prostate and colon carcinoma</article-title><source>Int. J. Oncol.</source><year>2005</year><volume>27</volume><fpage>1061</fpage><lpage>1070</lpage><pub-id pub-id-type=\"pmid\">16142324</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lanchec</surname><given-names>E</given-names></name><etal/></person-group><article-title>The type II transmembrane serine protease matriptase cleaves the amyloid precursor protein and reduces its processing to beta-amyloid peptide</article-title><source>J. Biol. Chem.</source><year>2017</year><volume>292</volume><fpage>20669</fpage><lpage>20682</lpage><pub-id pub-id-type=\"doi\">10.1074/jbc.M117.792911</pub-id><pub-id pub-id-type=\"pmid\">29054928</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>C</given-names></name><name><surname>Zhu</surname><given-names>LL</given-names></name><name><surname>Xu</surname><given-names>SG</given-names></name><name><surname>Ji</surname><given-names>HL</given-names></name><name><surname>Li</surname><given-names>XM</given-names></name></person-group><article-title>ENaC/DEG in tumor development and progression</article-title><source>J. Cancer</source><year>2016</year><volume>7</volume><fpage>1888</fpage><lpage>1891</lpage><pub-id pub-id-type=\"doi\">10.7150/jca.15693</pub-id><pub-id pub-id-type=\"pmid\">27698929</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Siegel</surname><given-names>RL</given-names></name><etal/></person-group><article-title>Colorectal cancer statistics, 2017</article-title><source>CA Cancer J. Clin.</source><year>2017</year><volume>67</volume><fpage>177</fpage><lpage>193</lpage><pub-id pub-id-type=\"doi\">10.3322/caac.21395</pub-id><pub-id pub-id-type=\"pmid\">28248415</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Swanton</surname><given-names>C</given-names></name><name><surname>Tomlinson</surname><given-names>I</given-names></name><name><surname>Downward</surname><given-names>J</given-names></name></person-group><article-title>Chromosomal instability, colorectal cancer and taxane resistance</article-title><source>Cell Cycle</source><year>2006</year><volume>5</volume><fpage>818</fpage><lpage>823</lpage><pub-id pub-id-type=\"doi\">10.4161/cc.5.8.2682</pub-id><pub-id pub-id-type=\"pmid\">16628000</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Einzig</surname><given-names>AI</given-names></name><etal/></person-group><article-title>Phase II trial of paclitaxel in patients with advanced colon cancer previously untreated with cytotoxic chemotherapy: an Eastern Cooperative Oncology Group Trial (PA286)</article-title><source>Am. J .Ther.</source><year>1996</year><volume>3</volume><fpage>750</fpage><lpage>754</lpage><pub-id pub-id-type=\"doi\">10.1097/00045391-199611000-00003</pub-id><pub-id pub-id-type=\"pmid\">11862233</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xu</surname><given-names>R</given-names></name><etal/></person-group><article-title>Enhancement of paclitaxel-induced apoptosis by inhibition of mitogen-activated protein kinase pathway in colon cancer cells</article-title><source>Anticancer Res.</source><year>2009</year><volume>29</volume><fpage>261</fpage><lpage>270</lpage><pub-id pub-id-type=\"pmid\">19331159</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zou</surname><given-names>H</given-names></name><etal/></person-group><article-title>Synergistic inhibition of colon cancer cell growth with nanoemulsion-loaded paclitaxel and PI3K/mTOR dual inhibitor BEZ235 through apoptosis</article-title><source>Int. J. Nanomed.</source><year>2016</year><volume>11</volume><fpage>1947</fpage><lpage>1958</lpage><pub-id pub-id-type=\"doi\">10.2147/ijn.s100744</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>J-L</given-names></name><etal/></person-group><article-title>Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells</article-title><source>Proc. Natl. Acad. Sci.</source><year>2000</year><volume>97</volume><fpage>7124</fpage><lpage>7129</lpage><pub-id pub-id-type=\"doi\">10.1073/pnas.97.13.7124</pub-id><pub-id pub-id-type=\"pmid\">10860979</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Violette</surname><given-names>S</given-names></name><etal/></person-group><article-title>Resistance of colon cancer cells to long-term 5-fluorouracil exposure is correlated to the relative level of Bcl-2 and Bcl-X(L) in addition to Bax and p53 status</article-title><source>Int. J. Cancer</source><year>2002</year><volume>98</volume><fpage>498</fpage><lpage>504</lpage><pub-id pub-id-type=\"doi\">10.1002/ijc.10146</pub-id><pub-id pub-id-type=\"pmid\">11920608</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mortenson</surname><given-names>MM</given-names></name><etal/></person-group><article-title>BCL-2 functions as an activator of the AKT signaling pathway in pancreatic cancer</article-title><source>J. Cell. Biochem.</source><year>2007</year><volume>102</volume><fpage>1171</fpage><lpage>1179</lpage><pub-id pub-id-type=\"doi\">10.1002/jcb.21343</pub-id><pub-id pub-id-type=\"pmid\">17960583</pub-id></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kido</surname><given-names>H</given-names></name><name><surname>Okumura</surname><given-names>Y</given-names></name></person-group><article-title>MSPL/TMPRSS13</article-title><source>Front. Biosci.</source><year>2008</year><volume>13</volume><fpage>754</fpage><lpage>758</lpage><pub-id pub-id-type=\"doi\">10.2741/2717</pub-id><pub-id pub-id-type=\"pmid\">17981585</pub-id></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Montero</surname><given-names>J</given-names></name><name><surname>Letai</surname><given-names>A</given-names></name></person-group><article-title>Why do BCL-2 inhibitors work and where should we use them in the clinic?</article-title><source>Cell Death Differ.</source><year>2018</year><volume>25</volume><fpage>56</fpage><lpage>64</lpage><pub-id pub-id-type=\"doi\">10.1038/cdd.2017.183</pub-id><pub-id pub-id-type=\"pmid\">29077093</pub-id></element-citation></ref><ref id=\"CR59\"><label>59.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lindeman</surname><given-names>GJ</given-names></name><etal/></person-group><article-title>A phase 1b dose-escalation and expansion study of the BCL-2 inhibitor venetoclax combined with tamoxifen in ER and BCL-2-positive metastatic breast cancer</article-title><source>Cancer Discov.</source><year>2018</year><pub-id pub-id-type=\"doi\">10.1158/2159-8290.cd-18-1151</pub-id><pub-id pub-id-type=\"pmid\">30518523</pub-id></element-citation></ref><ref id=\"CR60\"><label>60.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bortner</surname><given-names>CD</given-names></name><name><surname>Cidlowski</surname><given-names>JA</given-names></name></person-group><article-title>Ion channels and apoptosis in cancer</article-title><source>Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci.</source><year>2014</year><volume>369</volume><fpage>20130104</fpage><pub-id pub-id-type=\"doi\">10.1098/rstb.2013.0104</pub-id><pub-id pub-id-type=\"pmid\">24493752</pub-id></element-citation></ref><ref id=\"CR61\"><label>61.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xu</surname><given-names>S</given-names></name><name><surname>Liu</surname><given-names>C</given-names></name><name><surname>Ma</surname><given-names>Y</given-names></name><name><surname>Ji</surname><given-names>HL</given-names></name><name><surname>Li</surname><given-names>X</given-names></name></person-group><article-title>Potential roles of amiloride-sensitive sodium channels in cancer development</article-title><source>Biomed. Res. Int.</source><year>2016</year><volume>2016</volume><fpage>2190216</fpage><pub-id pub-id-type=\"doi\">10.1155/2016/2190216</pub-id><pub-id pub-id-type=\"pmid\">27403419</pub-id></element-citation></ref><ref id=\"CR62\"><label>62.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liao</surname><given-names>PC</given-names></name><name><surname>Tan</surname><given-names>SK</given-names></name><name><surname>Lieu</surname><given-names>CH</given-names></name><name><surname>Jung</surname><given-names>HK</given-names></name></person-group><article-title>Involvement of endoplasmic reticulum in paclitaxel-induced apoptosis</article-title><source>J. Cell. Biochem.</source><year>2008</year><volume>104</volume><fpage>1509</fpage><lpage>1523</lpage><pub-id pub-id-type=\"doi\">10.1002/jcb.21730</pub-id><pub-id pub-id-type=\"pmid\">18452161</pub-id></element-citation></ref><ref id=\"CR63\"><label>63.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Marchi</surname><given-names>S</given-names></name><etal/></person-group><article-title>Mitochondrial and endoplasmic reticulum calcium homeostasis and cell death</article-title><source>Cell Calcium</source><year>2018</year><volume>69</volume><fpage>62</fpage><lpage>72</lpage><pub-id pub-id-type=\"doi\">10.1016/j.ceca.2017.05.003</pub-id><pub-id pub-id-type=\"pmid\">28515000</pub-id></element-citation></ref><ref id=\"CR64\"><label>64.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hermanson</surname><given-names>D</given-names></name><etal/></person-group><article-title>Dual mechanisms of sHA 14–1 in inducing cell death through endoplasmic reticulum and mitochondria</article-title><source>Mol. Pharmacol.</source><year>2009</year><volume>76</volume><fpage>667</fpage><lpage>678</lpage><pub-id pub-id-type=\"doi\">10.1124/mol.109.055830</pub-id><pub-id pub-id-type=\"pmid\">19561125</pub-id></element-citation></ref><ref id=\"CR65\"><label>65.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Akl</surname><given-names>H</given-names></name><etal/></person-group><article-title>HA14-1, but not the BH3 mimetic ABT-737, causes Ca2+ dysregulation in platelets and human cell lines</article-title><source>Haematologica</source><year>2013</year><volume>98</volume><fpage>e49</fpage><lpage>51</lpage><pub-id pub-id-type=\"doi\">10.3324/haematol.2012.080598</pub-id><pub-id pub-id-type=\"pmid\">23403318</pub-id></element-citation></ref><ref id=\"CR66\"><label>66.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kidd</surname><given-names>JF</given-names></name><etal/></person-group><article-title>Paclitaxel affects cytosolic calcium signals by opening the mitochondrial permeability transition pore</article-title><source>J. Biol. Chem.</source><year>2002</year><volume>277</volume><fpage>6504</fpage><lpage>6510</lpage><pub-id pub-id-type=\"doi\">10.1074/jbc.M106802200</pub-id><pub-id pub-id-type=\"pmid\">11724773</pub-id></element-citation></ref><ref id=\"CR67\"><label>67.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pan</surname><given-names>Z</given-names></name><name><surname>Gollahon</surname><given-names>L</given-names></name></person-group><article-title>Taxol directly induces endoplasmic reticulum-associated calcium changes that promote apoptosis in breast cancer cells</article-title><source>Breast J.</source><year>2011</year><volume>17</volume><fpage>56</fpage><lpage>70</lpage><pub-id pub-id-type=\"doi\">10.1111/j.1524-4741.2010.00988.x</pub-id><pub-id pub-id-type=\"pmid\">21073601</pub-id></element-citation></ref><ref id=\"CR68\"><label>68.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hanukoglu</surname><given-names>I</given-names></name><name><surname>Hanukoglu</surname><given-names>A</given-names></name></person-group><article-title>Epithelial sodium channel (ENaC) family: phylogeny, structure-function, tissue distribution, and associated inherited diseases</article-title><source>Gene</source><year>2016</year><volume>579</volume><fpage>95</fpage><lpage>132</lpage><pub-id pub-id-type=\"doi\">10.1016/j.gene.2015.12.061</pub-id><pub-id pub-id-type=\"pmid\">26772908</pub-id></element-citation></ref><ref id=\"CR69\"><label>69.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Barretina</surname><given-names>J</given-names></name><etal/></person-group><article-title>The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity</article-title><source>Nature</source><year>2012</year><volume>483</volume><fpage>603</fpage><lpage>607</lpage><pub-id pub-id-type=\"doi\">10.1038/nature11003</pub-id><pub-id pub-id-type=\"pmid\">22460905</pub-id></element-citation></ref><ref id=\"CR70\"><label>70.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tate</surname><given-names>JG</given-names></name><etal/></person-group><article-title>COSMIC: the catalogue of somatic mutations in cancer</article-title><source>Nucl. Acids Res.</source><year>2019</year><volume>47</volume><fpage>D941</fpage><lpage>d947</lpage><pub-id pub-id-type=\"doi\">10.1093/nar/gky1015</pub-id><pub-id pub-id-type=\"pmid\">30371878</pub-id></element-citation></ref><ref id=\"CR71\"><label>71.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><collab>Nature The Cancer Genome Atlas Network</collab></person-group><article-title>The Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer</article-title><source>Nature</source><year>2012</year><volume>487</volume><fpage>330–337.https://doi.org/10.1038/nature11252</fpage><pub-id pub-id-type=\"doi\">10.1038/nature11252</pub-id><pub-id pub-id-type=\"pmid\">22810696</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807828</article-id><article-id pub-id-type=\"pmc\">PMC7431589</article-id><article-id pub-id-type=\"publisher-id\">70873</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70873-7</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Prepregnancy smoking and the risk of gestational diabetes requiring insulin therapy</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Kim</surname><given-names>Mee Kyoung</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Han</surname><given-names>Kyungdo</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>You</surname><given-names>Sang Youn</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Kwon</surname><given-names>Hyuk-Sang</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Yoon</surname><given-names>Kun-Ho</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Lee</surname><given-names>Seung-Hwan</given-names></name><address><email>hwanx2@catholic.ac.kr</email></address><xref ref-type=\"aff\" rid=\"Aff4\">4</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.411947.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0470 4224</institution-id><institution>Division of Endocrinology and Metabolism, Department of Internal Medicine, Yeouido St. Mary’s Hospital, College of Medicine, </institution><institution>The Catholic University of Korea, </institution></institution-wrap>Seoul, 07345 South Korea </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.263765.3</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0533 3568</institution-id><institution>Department of Statistics and Actuarial Science, </institution><institution>Soongsil University, </institution></institution-wrap>Seoul, 06978 South Korea </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.411947.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0470 4224</institution-id><institution>College of Medicine, </institution><institution>The Catholic University of Korea, </institution></institution-wrap>Seoul, 06591 South Korea </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.411947.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0470 4224</institution-id><institution>Division of Endocrinology and Metabolism, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, </institution><institution>The Catholic University of Korea, </institution></institution-wrap>#222 Banpo-daero, Seocho-gu,, Seoul, 06591 South Korea </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.411947.e</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0470 4224</institution-id><institution>Department of Medical Informatics, College of Medicine, </institution><institution>The Catholic University of Korea, </institution></institution-wrap>Seoul, 06591 South Korea </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13901</elocation-id><history><date date-type=\"received\"><day>28</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>3</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">The relationship between maternal smoking and gestational diabetes mellitus (GDM) is inconclusive. We investigated whether prepregnancy smoking is a risk factor for insulin-requiring GDM in Korean women. Using the National Health Insurance Service database, 325,297 women who delivered between 2011 and 2015 and who received a health examination within 52 weeks before pregnancy were included. Insulin-requiring GDM was defined as no claims for diabetes mellitus and a fasting blood glucose level of < 126 mg/dL before pregnancy, and initiation of insulin treatment during pregnancy. Smoking status was identified in a self-reported questionnaire completed during the health examination. There were 2,114 women (0.65%) with GDM who required insulin therapy. Compared with nonsmokers, the fully adjusted odd ratios (ORs) of former smokers and current smokers for insulin-requiring GDM were 1.55 (95% confidence interval [CI] 1.27–1.90) and 1.73 (1.42–2.09), respectively. The ORs (95% CIs) of insulin-requiring GDM among women who reported ≤ 2, 2–≤ 4, 4–≤ 6, 6–≤ 8, 8–≤ 10, and > 10 pack-years of smoking were 1.50 (1.22–1.84), 1.71 (1.31–2.22), 1.60 (1.13–2.26), 1.97 (1.14–3.40), 2.34 (1.22–4.51), and 2.29 (1.25–4.22), respectively, compared with nonsmokers (<italic>P</italic> for trend < 0.001). This association was similar in women with or without obesity and abdominal obesity. In conclusions, women who smoke have a significantly higher risk of GDM requiring insulin therapy, which may be proportional to the cumulative exposure to smoking. Cessation of smoking should be emphasized in women of childbearing age for the prevention of GDM.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Gestational diabetes</kwd><kwd>Epidemiology</kwd></kwd-group><funding-group><award-group><funding-source><institution>Seoul St.Mary's Hospital</institution></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Gestational diabetes mellitus (GDM) is a common medical condition during pregnancy and represents a leading cause of adverse pregnancy outcomes worldwide. The number of women being diagnosed with GDM has increased in the past decades, and this increase is largely attributable to increased obesity and age of pregnant women<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. Behavioral risk factors for GDM are not well understood, and the relationship between prepregnancy smoking and GDM is inconclusive<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>–<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. Some of the previous population-based cohort studies have found a positive relationship between cigarette smoking during pregnancy and GDM<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>, whereas other cohort and cross-sectional studies have not<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>.\n</p><p id=\"Par3\">The smoking rate has increased among adolescent and young women worldwide<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. During the past 25 years, the prevalence of smoking in Korean men decreased in all age groups but increased from 1.6 to 4.0% in Korean women aged 19–34 years<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. From the public health perspective, recognizing behavioral risk factors is especially important because these factors may be modifiable through appropriate interventions. Having more precise information about how smoking influences GDM will provide a basis for tailoring lifestyle advice for those at higher risk of developing GDM.</p><p id=\"Par4\">The severity of GDM is associated with maternal glucose level that present a positive and direct correlation with the risk of fetal involvement<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. The need for insulin therapy might be a starting point for the characterization of patients with more severe GDM related to greater difficulty in achieving glycemic control<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. Therefore, it is important to identify and mitigate any potentially avoidable risks for insulin-requiring GDM. The present study used a nationwide population-based cohort from the National Health Insurance Service (NHIS) database of Korea to examine the relationship between smoking and insulin-requiring GDM.</p></sec><sec id=\"Sec2\"><title>Methods</title><sec id=\"Sec3\"><title>Data source and study population</title><p id=\"Par5\">Details of the NHIS cohort design, methods, and validity of the records are available in previous studies<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>–<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. Briefly, the NHIS is managed by the government and is the single insurer for health-care services; it has a coverage rate of ~ 97% in the Republic of Korea. The NHIS database is available for population-based cohort studies, and information on demographics, national health screening data, diagnosis statements defined by the International Classification of Disease 10th revision (ICD-10) codes, medical treatment, and drug prescription is routinely collected and undergoes quality control before being released for research purpose. As part of the health screening examination, participants provide blood samples and their lifestyle patterns, past medical and family history via a self‐reported questionnaire, and anthropometric measurements such as body weight, waist circumference (WC), and blood pressure are recorded. Enrollees in the NHIS are recommended to undergo a standardized medical examination at least every 2 years.</p><p id=\"Par6\">We used the NHIS database to identify women who had delivered between 2011 and 2015. In our study, 280 days before the delivery date was regarded as the date of conception. A total of 329,675 women who had had received a health checkup during the 52 weeks before conception were selected (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>). Women taking a glucose-lowering agent before pregnancy (n = 2,187), having a fasting glucose level ≥ 126 mg/dL at the health checkup (n = 1,346), or with missing data (n = 845) were excluded. Finally, 325,297 women without diabetes were included in this study. All procedures performed in studies involving human participants were in accordance with the ethical standards of the Helsinki Declaration. This study was approved by the Institutional Review Board (IRB) of Seoul St. Mary’s Hospital, The Catholic University of Korea (No. KC19ZESI0586). Informed consent was waived by IRB because anonymous and deidentified information was used for the analysis.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Timeline for the study data collection.</p></caption><graphic xlink:href=\"41598_2020_70873_Fig1_HTML\" id=\"MO1\"/></fig></p></sec><sec id=\"Sec4\"><title>Smoking status</title><p id=\"Par7\">At the health checkup, the participants completed a self-reported health survey questionnaire<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Smoking status was classified as follows: current smoker, defined as those who had smoked > 5 packs (a total of 100 cigarettes) throughout their lifetime and continued to smoke; former smoker, defined as those who had smoked > 5 packs throughout their lifetime but had quit smoking; and nonsmoker, defined as those who had smoked ≤ 5 packs. Both former smokers and current smokers recorded the total duration of smoking (years) and the average number of cigarettes smoked daily. The cumulative lifetime smoking exposure was reported as pack-years by multiplying the average cigarette consumption per day (packs) and the smoking period (years).</p></sec><sec id=\"Sec5\"><title>Measurements and definitions</title><p id=\"Par8\">Body mass index (BMI) was calculated by dividing the weight in kilograms by height in meters squared. Obesity was defined as a BMI ≥ 25.0 kg/m<sup>2</sup> according to the World Health Organization Western Pacific Region guideline<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>. Abdominal obesity was defined as a WC ≥ 85 cm according to the criterion defined by the Korean Society for the Study of Obesity<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref></sup>. Information on alcohol consumption (heavy alcohol consumption defined as ≥ 30 g/day) was obtained from the questionnaire. Regular exercise was defined as > 30 min of moderate physical activity performed ≥ 5 times per week or > 20 min of strenuous physical activity performed ≥ 3 times per week. Household income level was dichotomized at the lowest 25%. Blood samples were drawn after an overnight fast, and the serum fasting blood glucose (FBG), lipid, and creatinine levels were measured. People having FBG levels of 100–125 mg/dL were defined as impaired fasting glucose (IFG). Estimated glomerular filtration rate (eGFR) was calculated using the equation from the Modification of Diet in Renal Disease study: eGFR = 175 × serum creatinine<sup>–1.154</sup> × age<sup>–0.203</sup> × 0.742. Hospitals that performed these health examinations were certified by the NHIS and were subject to regular quality control.</p><p id=\"Par9\">GDM requiring insulin therapy was defined as having no history of previous diabetes and receiving a prescription of insulin during the pregnancy period. Participants with non-GDM or GDM without insulin treatment were regarded as the control group.</p></sec><sec id=\"Sec6\"><title>Statistical analysis</title><p id=\"Par10\">The data are presented as mean ± standard deviation (SD), median (25–75%), or n (%). Student’s <italic>t</italic> test or chi-squared test was used to compare differences in clinical and biochemical characteristics between the insulin-requiring GDM group and control group. Odds ratios (ORs) and 95% confidence intervals (CIs) for GDM were obtained using multiple logistic regression analysis. Multivariable-adjusted analysis was performed to control for the confounding effects of known risk factors for GDM. Model 1 was adjusted for age, alcohol consumption, regular exercise, and income status. Model 2 was adjusted further for baseline BMI, fasting glucose level, family history of diabetes, and dyslipidemia. Potential effect modifications by general obesity, abdominal obesity and baseline glucose tolerance status were identified using stratified analysis and interaction testing using the likelihood-ratio test. Statistical analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA), and a <italic>P</italic> value < 0.05 was considered to indicate significance.</p></sec><sec id=\"Sec7\"><title>Prior presentation</title><p id=\"Par11\">This study was presented in abstract form at the Korean Diabetes Association’s 33rd spring scientific congress, Korea, 8–9 May 2020.</p></sec></sec><sec id=\"Sec8\"><title>Results</title><sec id=\"Sec9\"><title>Clinical characteristics according to the presence of insulin-requiring GDM</title><p id=\"Par12\">There were 2,114 women (0.65%) with GDM who received insulin therapy. These women were older, more obese, had a higher prevalence of hypertension and metabolic syndrome, and were more likely to have a family history of diabetes mellitus (DM) compared with the control group. Women with GDM who received insulin therapy also had higher levels of FBG, total cholesterol, triglycerides, and low-density lipoprotein cholesterol, and lower high-density lipoprotein (HDL) cholesterol level. The percentage of former or current smokers was significantly higher in the insulin-requiring GDM group than in the control group (Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>).<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Clinical characteristics of the study subjects before pregnancy.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">Control</th><th align=\"left\">GDM requiring insulin therapy</th><th align=\"left\"><italic>P</italic> value</th></tr></thead><tbody><tr><td align=\"left\">N</td><td align=\"left\">323,183</td><td align=\"left\">2,114</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"><bold>Age (years)</bold></td><td align=\"left\">29.6 ± 3.6</td><td align=\"left\">32.1 ± 4.3</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> < 35 years</td><td align=\"left\">295,315 (91.4)</td><td align=\"left\">1,590 (75.2)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"> ≥ 35 years</td><td align=\"left\">27,868 (8.6)</td><td align=\"left\">524 (24.8)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\"><bold>Smoking</bold></td><td align=\"left\"/><td align=\"left\"/><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">Non</td><td align=\"left\">298,331 (92.3)</td><td align=\"left\">1795 (84.9)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Former smoker</td><td align=\"left\">12,031 (3.7)</td><td align=\"left\">143 (6.8)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Current smoker</td><td align=\"left\">12,821 (4.0)</td><td align=\"left\">176 (8.3)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Heavy alcohol consumption</td><td align=\"left\">6,950 (2.2)</td><td align=\"left\">47 (2.2)</td><td char=\".\" align=\"char\">0.274</td></tr><tr><td align=\"left\">Regular exercise</td><td align=\"left\">34,029 (10.5)</td><td align=\"left\">248 (11.7)</td><td char=\".\" align=\"char\">0.075</td></tr><tr><td align=\"left\">Income (lower 25%)</td><td align=\"left\">64,921 (20.1)</td><td align=\"left\">442 (20.9)</td><td char=\".\" align=\"char\">0.348</td></tr><tr><td align=\"left\">Family history of DM</td><td align=\"left\">30,687 (13.2)</td><td align=\"left\">477 (29.2)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\"><bold>BMI (kg/m</bold><sup><bold>2</bold></sup><bold>)</bold></td><td align=\"left\">20.8 ± 2.7</td><td align=\"left\">23.4 ± 4.1</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">< 18.5</td><td align=\"left\">55,441 (17.2)</td><td align=\"left\">127 (6.0)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">18.5–22.9</td><td align=\"left\">214,553 (66.4)</td><td align=\"left\">1,037 (49.1)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">23–24.9</td><td align=\"left\">30,163 (9.3)</td><td align=\"left\">329 (15.6)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">25–29.9</td><td align=\"left\">19,629 (6.1)</td><td align=\"left\">465 (22.0)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">≥ 30</td><td align=\"left\">3,397 (1.1)</td><td align=\"left\">156 (7.4)</td><td char=\".\" align=\"char\"/></tr><tr><td align=\"left\">Waist circumferences (cm)</td><td align=\"left\">69.3 ± 7.0</td><td align=\"left\">75.5 ± 9.7</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">FBG (mg/dL)</td><td align=\"left\">87.3 ± 9.1</td><td align=\"left\">95.5 ± 12.1</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">TC (mg/dL)</td><td align=\"left\">177.0 ± 28.8</td><td align=\"left\">190.2 ± 34.5</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">HDL cholesterol (mg/dL)</td><td align=\"left\">63.7 ± 14.3</td><td align=\"left\">57.9 ± 13.4</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">LDL cholesterol (mg/dL)</td><td align=\"left\">98.5 ± 32.1</td><td align=\"left\">111 ± 38.6</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">Triglyceride (mg/dL)</td><td align=\"left\">65 (50–88)</td><td align=\"left\">90 (64–132)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">eGFR (ml/min/1.73 m<sup>2</sup>)</td><td align=\"left\">101.0 ± 33.6</td><td align=\"left\">100.7 ± 38.2</td><td char=\".\" align=\"char\">0.766</td></tr><tr><td align=\"left\">Systolic BP (mmHg)</td><td align=\"left\">110.0 ± 10.8</td><td align=\"left\">113.7 ± 12.3</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">Diastolic BP (mmHg)</td><td align=\"left\">69.2 ± 8.1</td><td align=\"left\">71.8 ± 8.9</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">Hypertension</td><td align=\"left\">4,258 (1.3)</td><td align=\"left\">93 (4.4)</td><td char=\".\" align=\"char\"> < 0.001</td></tr><tr><td align=\"left\">Metabolic syndrome</td><td align=\"left\">7,865 (2.4)</td><td align=\"left\">414 (19.6)</td><td char=\".\" align=\"char\"> < 0.001</td></tr></tbody></table><table-wrap-foot><p>Data are expressed as the mean ± SD, median (25–75%), or n (%).</p><p>BMI, body mass index; BP, blood pressure; DM, diabetes mellitus; eGFR, estimated glomerular filtration rate; GDM, gestational diabetes mellitus; FBG, fasting blood glucose; HDL, high-density lipoprotein; LDL, low-density lipoprotein; TC, total cholesterol.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec10\"><title>Risk of insulin-requiring GDM according to the duration and amount of smoking</title><p id=\"Par13\">Using the nonsmokers as the reference group, the multivariable-adjusted ORs (95% CIs) of former and current smokers for insulin-requiring GDM were 1.55 (1.27–1.90) and 1.73 (1.42–2.09), respectively. In both former and current smokers, more amount of smoking was associated with a higher incidence rate and OR of insulin-requiring GDM. Compared with nonsmokers, the ORs of insulin-requiring GDM were higher in former and current smokers in women who smoked ≥ 15 cigarettes/day than in women who smoked < 15 cigarettes/day. The ORs (95% CIs) were 2.42 (1.36–4.31) in former smokers and 2.35 (1.46–3.79) in current smokers who smoked ≥ 15 cigarettes/day and 1.49 (1.21–1.84) in former smokers and 1.65 (1.35–2.03) in current smokers who smoked < 15 cigarettes/day. In current smokers, a long duration of smoking was associated with a higher OR of insulin-requiring GDM; the ORs (95% CIs) were 1.38 (0.82–2.30) and 1.78 (1.45–2.19) for women who smoked cigarettes for < 5 years and ≥ 5 years, respectively (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>).<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Adjusted odd ratios and 95% confidence intervals of gestational diabetes mellitus requiring insulin therapy by smoking status, amount and duration.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\"/><th align=\"left\">n (%)</th><th align=\"left\">Events (n)</th><th align=\"left\">Incidence rate<sup>a</sup></th><th align=\"left\">Model 1</th><th align=\"left\">Model 2</th></tr></thead><tbody><tr><td align=\"left\" colspan=\"6\"><bold>Smoking status</bold></td></tr><tr><td align=\"left\">Nonsmoker</td><td char=\"(\" align=\"char\">300,126 (92.3)</td><td char=\".\" align=\"char\">1,795</td><td char=\".\" align=\"char\">6.0</td><td char=\"(\" align=\"char\">1 (ref.)</td><td char=\"(\" align=\"char\">1 (ref.)</td></tr><tr><td align=\"left\">Former smoker</td><td char=\"(\" align=\"char\">12,174 (3.7)</td><td char=\".\" align=\"char\">143</td><td char=\".\" align=\"char\">11.7</td><td char=\"(\" align=\"char\">1.83 (1.54, 2.18)</td><td char=\"(\" align=\"char\">1.55 (1.27, 1.90)</td></tr><tr><td align=\"left\">Current smoker</td><td char=\"(\" align=\"char\">12,997 (4.0)</td><td char=\".\" align=\"char\">176</td><td char=\".\" align=\"char\">13.5</td><td char=\"(\" align=\"char\">2.36 (2.01, 2.77)</td><td char=\"(\" align=\"char\">1.73 (1.42, 2.09)</td></tr><tr><td align=\"left\" colspan=\"6\"><bold>Amount of smoking (no. of cigarettes smoked per day)</bold></td></tr><tr><td align=\"left\">Nonsmoker</td><td char=\"(\" align=\"char\">300,126 (92.3)</td><td char=\".\" align=\"char\">1,795</td><td char=\".\" align=\"char\">6.0</td><td char=\"(\" align=\"char\">1 (ref.)</td><td char=\"(\" align=\"char\">1 (ref.)</td></tr><tr><td align=\"left\" colspan=\"6\">Former smoker</td></tr><tr><td align=\"left\"> < 15</td><td char=\"(\" align=\"char\">11,454 (3.5)</td><td char=\".\" align=\"char\">127</td><td char=\".\" align=\"char\">11.1</td><td char=\"(\" align=\"char\">1.75 (1.46, 2.11)</td><td char=\"(\" align=\"char\">1.49 (1.21, 1.84)</td></tr><tr><td align=\"left\"> ≥ 15</td><td char=\"(\" align=\"char\">720 (0.2)</td><td char=\".\" align=\"char\">16</td><td char=\".\" align=\"char\">22.2</td><td char=\"(\" align=\"char\">2.93 (1.77, 4.85)</td><td char=\"(\" align=\"char\">2.42 (1.36, 4.31)</td></tr><tr><td align=\"left\" colspan=\"6\">Current smoker</td></tr><tr><td align=\"left\"> < 15</td><td char=\"(\" align=\"char\">11,736 (3.6)</td><td char=\".\" align=\"char\">150</td><td char=\".\" align=\"char\">12.8</td><td char=\"(\" align=\"char\">2.26 (1.90, 2.68)</td><td char=\"(\" align=\"char\">1.65 (1.35, 2.03)</td></tr><tr><td align=\"left\"> ≥ 15</td><td char=\"(\" align=\"char\">1,261 (0.4)</td><td char=\".\" align=\"char\">26</td><td char=\".\" align=\"char\">20.6</td><td char=\"(\" align=\"char\">3.27 (2.19, 4.88)</td><td char=\"(\" align=\"char\">2.35 (1.46, 3.79)</td></tr><tr><td align=\"left\" colspan=\"6\"><bold>Duration of smoking (years)</bold></td></tr><tr><td align=\"left\">Nonsmoker</td><td char=\"(\" align=\"char\">300,268 (92.3)</td><td char=\".\" align=\"char\">1,797</td><td char=\".\" align=\"char\">6.0</td><td char=\"(\" align=\"char\">1 (ref.)</td><td char=\"(\" align=\"char\">1 (ref.)</td></tr><tr><td align=\"left\" colspan=\"6\">Former smoker</td></tr><tr><td align=\"left\"> < 5</td><td char=\"(\" align=\"char\">4,816 (1.5)</td><td char=\".\" align=\"char\">50</td><td char=\".\" align=\"char\">10.4</td><td char=\"(\" align=\"char\">1.93 (1.45, 2.56)</td><td char=\"(\" align=\"char\">1.60 (1.14, 2.23)</td></tr><tr><td align=\"left\"> ≥ 5</td><td char=\"(\" align=\"char\">7,265 (2.2)</td><td char=\".\" align=\"char\">92</td><td char=\".\" align=\"char\">12.7</td><td char=\"(\" align=\"char\">1.78 (1.43, 2.21)</td><td char=\"(\" align=\"char\">1.53 (1.20, 1.95)</td></tr><tr><td align=\"left\" colspan=\"6\">Current smoker</td></tr><tr><td align=\"left\"> < 5</td><td char=\"(\" align=\"char\">2,347 (0.7)</td><td char=\".\" align=\"char\">19</td><td char=\".\" align=\"char\">8.1</td><td char=\"(\" align=\"char\">1.84 (1.16, 2.90)</td><td char=\"(\" align=\"char\">1.38 (0.82, 2.30)</td></tr><tr><td align=\"left\"> ≥ 5</td><td char=\"(\" align=\"char\">10,601 (3.3)</td><td char=\".\" align=\"char\">156</td><td char=\".\" align=\"char\">14.7</td><td char=\"(\" align=\"char\">2.43 (2.05, 2.89)</td><td char=\"(\" align=\"char\">1.78 (1.45, 2.19)</td></tr><tr><td align=\"left\" colspan=\"6\"><bold>Amount × duration of smoking (pack-years)</bold></td></tr><tr><td align=\"left\">Nonsmoker</td><td char=\"(\" align=\"char\">300,126 (92.3)</td><td char=\".\" align=\"char\">1,795</td><td char=\".\" align=\"char\">6.0</td><td char=\"(\" align=\"char\">1 (ref.)</td><td char=\"(\" align=\"char\">1 (ref.)</td></tr><tr><td align=\"left\">≤ 2</td><td char=\"(\" align=\"char\">13,625 (4.2)</td><td char=\".\" align=\"char\">139</td><td char=\".\" align=\"char\">10.2</td><td char=\"(\" align=\"char\">1.86 (1.56, 2.21)</td><td char=\"(\" align=\"char\">1.50 (1.22, 1.84)</td></tr><tr><td align=\"left\">2 <, ≤ 4</td><td char=\"(\" align=\"char\">6,210 (1.9)</td><td char=\".\" align=\"char\">81</td><td char=\".\" align=\"char\">13.0</td><td char=\"(\" align=\"char\">2.21 (1.76, 2.77)</td><td char=\"(\" align=\"char\">1.71 (1.31, 2.22)</td></tr><tr><td align=\"left\">4 <, ≤ 6</td><td char=\"(\" align=\"char\">3,222 (2.0)</td><td char=\".\" align=\"char\">49</td><td char=\".\" align=\"char\">15.2</td><td char=\"(\" align=\"char\">2.23 (1.67, 2.98)</td><td char=\"(\" align=\"char\">1.60 (1.13, 2.26)</td></tr><tr><td align=\"left\">6 <, ≤ 8</td><td char=\"(\" align=\"char\">1,085 (0.3)</td><td char=\".\" align=\"char\">18</td><td char=\".\" align=\"char\">16.6</td><td char=\"(\" align=\"char\">2.20 (1.37, 3.53)</td><td char=\"(\" align=\"char\">1.97 (1.14, 3.40)</td></tr><tr><td align=\"left\">8 <, ≤ 10</td><td char=\"(\" align=\"char\">568 (0.2)</td><td char=\".\" align=\"char\">16</td><td char=\".\" align=\"char\">28.2</td><td char=\"(\" align=\"char\">2.97 (1.76, 5.00)</td><td char=\"(\" align=\"char\">2.34 (1.22, 4.51)</td></tr><tr><td align=\"left\">> 10</td><td char=\"(\" align=\"char\">461 (0.1)</td><td char=\".\" align=\"char\">16</td><td char=\".\" align=\"char\">34.7</td><td char=\"(\" align=\"char\">3.27 (1.96, 5.45)</td><td char=\"(\" align=\"char\">2.29 (1.25, 4.22)</td></tr></tbody></table><table-wrap-foot><p>Model 1: Adjusted for age, alcohol drinking, regular exercise, and income status.</p><p>Model 2: Adjusted for model 1 + body mass index, fasting blood glucose, family history of diabetes, and dyslipidemia.</p><p><sup>a</sup>Per 1,000 person-years.</p></table-wrap-foot></table-wrap></p><p id=\"Par14\">Analysis of both the amount and duration of smoking showed a dose–response relationship between pack-years of smoking and the incidence rate or the risk of insulin-requiring GDM. Even ≤ 2 pack-years of smoking was significantly associated with an increased risk of insulin-requiring GDM. In the multivariable-adjusted model, the ORs (95% CIs) of insulin-requiring GDM among women who smoked ≤ 2, 2–≤ 4, 4–≤ 6, 6–≤ 8, 8–≤ 10, and > 10 pack-years were 1.50 (1.22–1.84), 1.71 (1.31–2.22), 1.60 (1.13–2.26), 1.97 (1.14–3.40), 2.34(1.22–4.51), and 2.29 (1.25–4.22), respectively, compared with nonsmokers (<italic>P</italic> for trend < 0.001) (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>).</p></sec><sec id=\"Sec11\"><title>Effect of smoking on the risk of insulin-requiring GDM according to obesity and glucose tolerance status</title><p id=\"Par15\">The interaction between smoking and obesity on the risk of insulin-requiring GDM was tested. The effect of smoking did not differ according to general obesity (<italic>P</italic> for interaction = 0.050) and abdominal obesity (<italic>P</italic> for interaction = 0.129). However, the ORs for insulin-requiring GDM were higher in current smokers with general obesity (2.19 [1.64–2.93]) or abdominal obesity (2.14 [1.60–2.86]) compared with other groups (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>A,B).<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Adjusted odd ratios and 95% confidence intervals for gestational diabetes requiring insulin therapy according to smoking status and prepregnancy body mass index (<bold>A</bold>), waist circumferences (<bold>B</bold>), and baseline glucose tolerance status (<bold>C</bold>). Adjusted for age, alcohol consumption, regular exercise, income status, baseline fasting blood glucose level, family history of diabetes, and dyslipidemia. BMI, body mass index; IFG, impaired fasting glucose; NGT, normal glucose tolerance; WC, waist circumference.</p></caption><graphic xlink:href=\"41598_2020_70873_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par16\">The effect of smoking did not differ according to the presence of IFG (<italic>P</italic> for interaction = 0.393; Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>C). Regardless of the presence of pre-pregnancy IFG, former or current smoking was significantly associated with an increased risk of insulin-requiring GDM.</p></sec></sec><sec id=\"Sec12\"><title>Discussion</title><p id=\"Par17\">In this study, we found that smoking before pregnancy was associated with an increased risk of GDM requiring insulin therapy. We also found a dose–response relationship between the lifetime amount of smoking and risk of GDM requiring insulin therapy. This association was found consistently in both nonobese and obese women. Although previous studies have reported inconsistent results, our data suggest that prepregnancy smoking should be considered as a significant contributor to insulin-requiring GDM.</p><p id=\"Par18\">Epidemiological studies have tested associations between smoking and risk of DM<sup><xref ref-type=\"bibr\" rid=\"CR15\">15</xref>–<xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>, and some have also investigated the mechanisms underlying these associations<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>–<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. Smoking may trigger inflammatory responses, oxidative stress, and insulin resistance<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. For example, an analysis of skeletal muscle biopsy specimens revealed that smokers had decreased expression of peroxisome proliferator-activated receptor-gamma and greater Ser636 phosphorylation of insulin receptor substrate-1 compared with nonsmokers<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. Smoking is also implicated as a risk factor for metabolic syndrome<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>, and metabolic abnormalities may be modulated by the direct negative effect of smoking on insulin resistance. Of the components of metabolic syndrome, high triglyceride and low HDL-cholesterol levels, and abdominal obesity are thought to be the main contributors to this association. A dose-dependent relationship between the number of cigarettes smoked and decreased HDL-cholesterol level and increased triglyceride level has been consistently reported<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. Although smoking is often linked to reduced body weight, several studies suggest that there are deleterious changes in body composition<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. Other population-based studies have reported a positive association between the amount of smoking and abdominal obesity in current smokers<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Greater life-time smoking was significantly associated with higher waist-to-hip ratio and visceral-to-subcutaneous adipose ratio in a population-based cross-sectional study of relatively lean Japanese men<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>. These findings support the general notion that smoking is linked to adverse fat distribution that leads to metabolically unhealthy status. Cigarette smoking also increases exposure to various chemicals and heavy metals such as nicotine, lead, arsenic, and cadmium<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. This might alter glucose homeostasis and increase the risk of DM<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>.</p><p id=\"Par19\">In a study of US postmenopausal women, those who smoked an average of 16 cigarettes per day had a 1.28-fold higher risk of new diabetes. This risk was mitigated in those who had stopped smoking, and 10 years after smoking cessation, the risk of diabetes became equivalent to that of never-smokers<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. Our study included young women of childbearing age, and the duration of smoking cessation is probably shorter and, therefore, the risk of GDM in former smokers was similar to or slightly lower than that of current smokers. We found that the total amount of smoking was associated with the risk of GDM requiring insulin therapy, regardless of current or former smoking status.</p><p id=\"Par20\">The prevalence of GDM in Korean women was 5.7% in 2009, 7.8% in 2010, and 9.5% in 2011<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. About 5% of women with GDM took medication during pregnancy, and more than 98% of the Korean patients with GDM administered insulin<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>. In our study, women with GDM being treated with insulin accounted for 0.6% of all pregnant women, which is similar to the statistics for GDM in Korea. The debate about GDM screening and diagnostic methods persists in global academic circles and professional societies. It is important to identify factors related to the development of severe GDM and pay more attention to them. In our study, we used the administration of insulin to control blood glucose level as an indicator of severe GDM, and we found that the amount of smoking was an independent risk factor for severe GDM.</p><p id=\"Par21\">The key strengths of this study include the enrollment of the large study population of > 300,000 deliveries. Because of national health insurance coverage, almost all pregnant women in Korea undergo GDM screening and treatment during pregnancy. Therefore, our study included almost all women who underwent a health examination within 1 year of conception. The dose–response relationship between smoking and GDM was explained using detailed smoking indices. A dose–response relationship is usually considered to be an evidence in support of causality. However, we acknowledge some limitations. Because smoking status was obtained from self-administered questionnaires, we cannot exclude the possibility that misreporting led to some individuals being misclassified with regard to their smoking status. Considering the large number of participants, we believe that misclassification had only very limited influence on the results obtained; earlier studies have also reported low misclassification rates of smoking status<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. Second, our reported relationship between smoking and GDM requiring insulin therapy was likely to have been attenuated by the inclusion of women with mild GDM who were treated with diet and exercise in the control group. The higher risk of severe GDM among pregnant women who smoke could be mediated through the same pathophysiologic mechanisms that underlie the higher risk of diabetes in people who smoke, which includes insulin resistance and impaired glucose homeostasis. Therefore, we can assume that the same correlation may be seen in women with mild GDM who were treated with diet and exercise. Recently, it has been reported that prenatal smoking is associated with a higher risk of GDM, independent of the treatment modality of GDM<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. Third, we had no data on changes in smoking status during pregnancy. The prevalence of active smoking among pregnant women may be even lower because women smokers may stop during pregnancy. In our study, increasing the amount of smoking, regardless of being a former or current smoker before pregnancy, influenced the risk of GDM requiring insulin therapy. Lastly, data on passive smoking were not available in the NHIS database. Exposure to secondhand smoke has been reported to be associated with a higher risk of several types of cancer and cardiovascular disease<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. It would be interesting to examine the effect of passive smoking on the risk of GDM.</p><p id=\"Par22\">In conclusion, in a cohort of 325,297 women in Korea, former and current smokers had a significantly increased risk for GDM requiring insulin therapy and the risk increased with the number of cigarettes smoked, regardless of the current smoking status. The risk of insulin-requiring GDM was 2.3 times higher in women who smoked > 10 pack-years than in nonsmokers. Of note, even ≤ 2 pack-years of smoking increased the risk by 50%. Cessation of smoking should be emphasized in women of childbearing age because cumulative lifetime smoking is a major contributing factor to insulin-requiring GDM.</p></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><ack><title>Acknowledgements</title><p>This study was performed using the database from the National Health Insurance System (NHIS-2020-1-113), and the results do not necessarily represent the opinion of the National Health Insurance Corporation. This study was supported by the research fund of Seoul St.Mary’s Hospital, The Catholic University of Korea.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>M.K.K., K.H., and S.-H.L. designed the study. K.H. performed statistical analysis. M.K.K., S.Y.Y., H.-S.K., and S.-H.L., interpreted the data. H.-S.K., and K.-H.Y contributed to discussion. M.K.K., and S.-H.L. wrote the manuscript.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par23\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>MH</given-names></name><etal/></person-group><article-title>Pregnancy outcomes of women additionally diagnosed as gestational diabetes by the International Association of the Diabetes and Pregnancy Study Groups criteria</article-title><source>Diabetes. Metab. J.</source><year>2019</year><volume>43</volume><fpage>766</fpage><lpage>775</lpage><pub-id pub-id-type=\"doi\">10.4093/dmj.2018.0192</pub-id><pub-id pub-id-type=\"pmid\">30877713</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>England</surname><given-names>LJ</given-names></name><etal/></person-group><article-title>Glucose tolerance and risk of gestational diabetes mellitus in nulliparous women who smoke during pregnancy</article-title><source>Am. J. Epidemiol.</source><year>2004</year><volume>160</volume><fpage>1205</fpage><lpage>1213</lpage><pub-id pub-id-type=\"doi\">10.1093/aje/kwh340</pub-id><pub-id pub-id-type=\"pmid\">15583373</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Solomon</surname><given-names>CG</given-names></name><etal/></person-group><article-title>A prospective study of pregravid determinants of gestational diabetes mellitus</article-title><source>JAMA</source><year>1997</year><volume>278</volume><fpage>1078</fpage><lpage>1083</lpage><pub-id pub-id-type=\"doi\">10.1001/jama.1997.03550130052036</pub-id><pub-id pub-id-type=\"pmid\">9315766</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thériault</surname><given-names>S</given-names></name><name><surname>Giguère</surname><given-names>Y</given-names></name><name><surname>Massé</surname><given-names>J</given-names></name><name><surname>Girouard</surname><given-names>J</given-names></name><name><surname>Forest</surname><given-names>JC</given-names></name></person-group><article-title>Early prediction of gestational diabetes: A practical model combining clinical and biochemical markers</article-title><source>Clin. Chem. Lab. Med.</source><year>2016</year><volume>54</volume><fpage>509</fpage><lpage>518</lpage><pub-id pub-id-type=\"doi\">10.1515/cclm-2015-0537</pub-id><pub-id pub-id-type=\"pmid\">26351946</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hosler</surname><given-names>AS</given-names></name><name><surname>Nayak</surname><given-names>SG</given-names></name><name><surname>Radigan</surname><given-names>AM</given-names></name></person-group><article-title>Stressful events, smoking exposure and other maternal risk factors associated with gestational diabetes mellitus</article-title><source>Paediatr. Perinat. Epidemiol.</source><year>2011</year><volume>25</volume><fpage>566</fpage><lpage>574</lpage><pub-id pub-id-type=\"doi\">10.1111/j.1365-3016.2011.01221.x</pub-id><pub-id pub-id-type=\"pmid\">21980946</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chang</surname><given-names>Y</given-names></name><name><surname>Kang</surname><given-names>HY</given-names></name><name><surname>Lim</surname><given-names>D</given-names></name><name><surname>Cho</surname><given-names>HJ</given-names></name><name><surname>Khang</surname><given-names>YH</given-names></name></person-group><article-title>Long-term trends in smoking prevalence and its socioeconomic inequalities in Korea, 1992–2016</article-title><source>Int. J. Equity. Health.</source><year>2019</year><volume>18</volume><fpage>148</fpage><pub-id pub-id-type=\"doi\">10.1186/s12939-019-1051-x</pub-id><pub-id pub-id-type=\"pmid\">31533732</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sapienza</surname><given-names>AD</given-names></name><name><surname>Francisco</surname><given-names>RP</given-names></name><name><surname>Trindade</surname><given-names>TC</given-names></name><name><surname>Zugaib</surname><given-names>M</given-names></name></person-group><article-title>Factors predicting the need for insulin therapy in patients with gestational diabetes mellitus</article-title><source>Diabetes. Res. Clin. Pract.</source><year>2010</year><volume>88</volume><fpage>81</fpage><lpage>86</lpage><pub-id pub-id-type=\"doi\">10.1016/j.diabres.2009.12.023</pub-id><pub-id pub-id-type=\"pmid\">20071050</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ko</surname><given-names>SH</given-names></name><etal/></person-group><article-title>Past and current status of adult type 2 diabetes mellitus management in Korea: A National Health Insurance Service Database Analysis</article-title><source>Diabetes. Metab. J.</source><year>2018</year><volume>42</volume><fpage>93</fpage><lpage>100</lpage><pub-id pub-id-type=\"doi\">10.4093/dmj.2018.42.2.93</pub-id><pub-id pub-id-type=\"pmid\">29676539</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>MK</given-names></name><etal/></person-group><article-title>Associations of variability in blood pressure, glucose and cholesterol concentrations, and body mass index with mortality and cardiovascular outcomes in the general population</article-title><source>Circulation</source><year>2018</year><volume>138</volume><fpage>2627</fpage><lpage>2637</lpage><pub-id pub-id-type=\"doi\">10.1161/CIRCULATIONAHA.118.034978</pub-id><pub-id pub-id-type=\"pmid\">30571256</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>JA</given-names></name><etal/></person-group><article-title>Impact of the dynamic change of metabolic health status on the incident type 2 diabetes: A Nationwide Population-Based Cohort Study</article-title><source>Endocrinol. Metab.</source><year>2019</year><volume>34</volume><fpage>406</fpage><lpage>414</lpage><pub-id pub-id-type=\"doi\">10.3803/EnM.2019.34.4.406</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Choi</surname><given-names>HS</given-names></name><etal/></person-group><article-title>Smoking and risk of incident end-stage kidney disease in general population: A Nationwide Population-based Cohort Study from Korea</article-title><source>Sci. Rep.</source><year>2019</year><volume>9</volume><fpage>19511</fpage><pub-id pub-id-type=\"doi\">10.1038/s41598-019-56113-7</pub-id><pub-id pub-id-type=\"pmid\">31862942</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>K</given-names></name><name><surname>Park</surname><given-names>SM</given-names></name><name><surname>Lee</surname><given-names>K</given-names></name></person-group><article-title>Weight gain after smoking cessation does not modify its protective effect on myocardial infarction and stroke: Evidence from a cohort study of men</article-title><source>Eur. Heart. J.</source><year>2018</year><volume>39</volume><fpage>1523</fpage><lpage>1531</lpage><pub-id pub-id-type=\"doi\">10.1093/eurheartj/ehx761</pub-id><pub-id pub-id-type=\"pmid\">29324990</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>MK</given-names></name><etal/></person-group><article-title>2019 Clinical practice guidelines for type 2 diabetes mellitus in Korea</article-title><source>Diabetes. Metab. J.</source><year>2019</year><volume>43</volume><fpage>398</fpage><lpage>406</lpage><pub-id pub-id-type=\"doi\">10.4093/dmj.2019.0137</pub-id><pub-id pub-id-type=\"pmid\">31441247</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>SY</given-names></name><etal/></person-group><article-title>Appropriate waist circumference cutoff points for central obesity in Korean adults</article-title><source>Diabetes. Res. Clin. Pract.</source><year>2007</year><volume>75</volume><fpage>72</fpage><lpage>80</lpage><pub-id pub-id-type=\"doi\">10.1016/j.diabres.2006.04.013</pub-id><pub-id pub-id-type=\"pmid\">16735075</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Willi</surname><given-names>C</given-names></name><name><surname>Bodenmann</surname><given-names>P</given-names></name><name><surname>Ghali</surname><given-names>WA</given-names></name><name><surname>Faris</surname><given-names>PD</given-names></name><name><surname>Cornuz</surname><given-names>J</given-names></name></person-group><article-title>Active smoking and the risk of type 2 diabetes: A systematic review and meta-analysis</article-title><source>JAMA</source><year>2007</year><volume>298</volume><fpage>2654</fpage><lpage>2664</lpage><pub-id pub-id-type=\"doi\">10.1001/jama.298.22.2654</pub-id><pub-id pub-id-type=\"pmid\">18073361</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jee</surname><given-names>SH</given-names></name><name><surname>Foong</surname><given-names>AW</given-names></name><name><surname>Hur</surname><given-names>NW</given-names></name><name><surname>Samet</surname><given-names>JM</given-names></name></person-group><article-title>Smoking and risk for diabetes incidence and mortality in Korean men and women</article-title><source>Diabetes Care</source><year>2010</year><volume>33</volume><fpage>2567</fpage><lpage>2572</lpage><pub-id pub-id-type=\"doi\">10.2337/dc10-0261</pub-id><pub-id pub-id-type=\"pmid\">20823342</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cho</surname><given-names>NH</given-names></name><name><surname>Chan</surname><given-names>JC</given-names></name><name><surname>Jang</surname><given-names>HC</given-names></name><name><surname>Lim</surname><given-names>S</given-names></name><name><surname>Kim</surname><given-names>HL</given-names></name><name><surname>Choi</surname><given-names>SH</given-names></name></person-group><article-title>Cigarette smoking is an independent risk factor for type 2 diabetes: A four-year community-based prospective study</article-title><source>Clin. Endocrinol.</source><year>2009</year><volume>71</volume><fpage>679</fpage><lpage>685</lpage><pub-id pub-id-type=\"doi\">10.1111/j.1365-2265.2009.03586.x</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yeh</surname><given-names>HC</given-names></name><name><surname>Duncan</surname><given-names>BB</given-names></name><name><surname>Schmidt</surname><given-names>MI</given-names></name><name><surname>Wang</surname><given-names>NY</given-names></name><name><surname>Brancati</surname><given-names>FL</given-names></name></person-group><article-title>Smoking, smoking cessation, and risk for type 2 diabetes mellitus: A cohort study</article-title><source>Ann. Intern. Med.</source><year>2010</year><volume>152</volume><fpage>10</fpage><lpage>17</lpage><pub-id pub-id-type=\"doi\">10.7326/0003-4819-152-1-201001050-00005</pub-id><pub-id pub-id-type=\"pmid\">20048267</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maddatu</surname><given-names>J</given-names></name><name><surname>Anderson-Baucum</surname><given-names>E</given-names></name><name><surname>Evans-Molina</surname><given-names>C</given-names></name></person-group><article-title>Smoking and the risk of type 2 diabetes</article-title><source>Transl. Res.</source><year>2017</year><volume>184</volume><fpage>101</fpage><lpage>107</lpage><pub-id pub-id-type=\"doi\">10.1016/j.trsl.2017.02.004</pub-id><pub-id pub-id-type=\"pmid\">28336465</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bergman</surname><given-names>BC</given-names></name><name><surname>Perreault</surname><given-names>L</given-names></name><name><surname>Hunerdosse</surname><given-names>DM</given-names></name><name><surname>Koehler</surname><given-names>MC</given-names></name><name><surname>Samek</surname><given-names>AM</given-names></name><name><surname>Eckel</surname><given-names>RH</given-names></name></person-group><article-title>Intramuscular lipid metabolism in the insulin resistance of smoking</article-title><source>Diabetes</source><year>2009</year><volume>58</volume><fpage>2220</fpage><lpage>2227</lpage><pub-id pub-id-type=\"doi\">10.2337/db09-0481</pub-id><pub-id pub-id-type=\"pmid\">19581421</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rhee</surname><given-names>SY</given-names></name><name><surname>Hwang</surname><given-names>YC</given-names></name><name><surname>Woo</surname><given-names>JT</given-names></name><name><surname>Chin</surname><given-names>SO</given-names></name><name><surname>Chon</surname><given-names>S</given-names></name><name><surname>Kim</surname><given-names>YS</given-names></name></person-group><article-title>Arsenic exposure and prevalence of diabetes mellitus in Korean adults</article-title><source>J. Korean. Med. Sci.</source><year>2013</year><volume>28</volume><fpage>861</fpage><lpage>868</lpage><pub-id pub-id-type=\"doi\">10.3346/jkms.2013.28.6.861</pub-id><pub-id pub-id-type=\"pmid\">23772150</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Slagter</surname><given-names>SN</given-names></name><etal/></person-group><article-title>Associations between smoking, components of metabolic syndrome and lipoprotein particle size</article-title><source>BMC. Med.</source><year>2013</year><volume>11</volume><fpage>195</fpage><pub-id pub-id-type=\"doi\">10.1186/1741-7015-11-195</pub-id><pub-id pub-id-type=\"pmid\">24228807</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>JH</given-names></name><name><surname>Shim</surname><given-names>KW</given-names></name><name><surname>Yoon</surname><given-names>YS</given-names></name><name><surname>Lee</surname><given-names>SY</given-names></name><name><surname>Kim</surname><given-names>SS</given-names></name><name><surname>Oh</surname><given-names>SW</given-names></name></person-group><article-title>Cigarette smoking increases abdominal and visceral obesity but not overall fatness: An observational study</article-title><source>PLoS ONE</source><year>2012</year><volume>7</volume><fpage>e45815</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0045815</pub-id><pub-id pub-id-type=\"pmid\">23029258</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fujiyoshi</surname><given-names>A</given-names></name><etal/></person-group><article-title>Lifetime cigarette smoking is associated with abdominal obesity in a community-based sample of Japanese men: The Shiga Epidemiological Study of Subclinical Atherosclerosis (SESSA)</article-title><source>Prev. Med. Rep.</source><year>2016</year><volume>4</volume><fpage>225</fpage><lpage>232</lpage><pub-id pub-id-type=\"doi\">10.1016/j.pmedr.2016.06.013</pub-id><pub-id pub-id-type=\"pmid\">27413686</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luo</surname><given-names>J</given-names></name><etal/></person-group><article-title>Smoking and diabetes: Does the increased risk ever go away?</article-title><source>Am. J. Epidemiol.</source><year>2013</year><volume>178</volume><fpage>937</fpage><lpage>945</lpage><pub-id pub-id-type=\"doi\">10.1093/aje/kwt071</pub-id><pub-id pub-id-type=\"pmid\">23817918</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Koo</surname><given-names>BK</given-names></name><name><surname>Lee</surname><given-names>JH</given-names></name><name><surname>Kim</surname><given-names>J</given-names></name><name><surname>Jang</surname><given-names>EJ</given-names></name><name><surname>Lee</surname><given-names>CH</given-names></name></person-group><article-title>Prevalence of gestational diabetes mellitus in Korea: A National Health Insurance Database Study</article-title><source>PLoS ONE</source><year>2016</year><volume>11</volume><fpage>e0153107</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0153107</pub-id><pub-id pub-id-type=\"pmid\">27046149</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wagenknecht</surname><given-names>LE</given-names></name><name><surname>Burke</surname><given-names>GL</given-names></name><name><surname>Perkins</surname><given-names>LL</given-names></name><name><surname>Haley</surname><given-names>NJ</given-names></name><name><surname>Friedman</surname><given-names>GD</given-names></name></person-group><article-title>Misclassification of smoking status in the CARDIA study: A comparison of self-report with serum cotinine levels</article-title><source>Am. J. Public. Health.</source><year>1992</year><volume>82</volume><fpage>33</fpage><lpage>36</lpage><pub-id pub-id-type=\"doi\">10.2105/AJPH.82.1.33</pub-id><pub-id pub-id-type=\"pmid\">1536331</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bar-Zeev</surname><given-names>Y</given-names></name><name><surname>Haile</surname><given-names>ZT</given-names></name><name><surname>Chertok</surname><given-names>IA</given-names></name></person-group><article-title>Association between prenatal smoking and gestational diabetes mellitus</article-title><source>Obstet. Gynecol.</source><year>2020</year><volume>135</volume><fpage>91</fpage><lpage>99</lpage><pub-id pub-id-type=\"doi\">10.1097/AOG.0000000000003602</pub-id><pub-id pub-id-type=\"pmid\">31809434</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>He</surname><given-names>J</given-names></name><name><surname>Vupputuri</surname><given-names>S</given-names></name><name><surname>Allen</surname><given-names>K</given-names></name><name><surname>Prerost</surname><given-names>MR</given-names></name><name><surname>Hughes</surname><given-names>J</given-names></name><name><surname>Whelton</surname><given-names>PK</given-names></name></person-group><article-title>Passive smoking and the risk of coronary heart disease—A meta-analysis of epidemiologic studies</article-title><source>N. Engl. J. Med.</source><year>1999</year><volume>340</volume><fpage>920</fpage><lpage>926</lpage><pub-id pub-id-type=\"doi\">10.1056/NEJM199903253401204</pub-id><pub-id pub-id-type=\"pmid\">10089185</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Nat Commun</journal-id><journal-id journal-id-type=\"iso-abbrev\">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type=\"epub\">2041-1723</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807779</article-id><article-id pub-id-type=\"pmc\">PMC7431590</article-id><article-id pub-id-type=\"publisher-id\">17974</article-id><article-id pub-id-type=\"doi\">10.1038/s41467-020-17974-z</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Human NMD ensues independently of stable ribosome stalling</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-0390-0181</contrib-id><name><surname>Karousis</surname><given-names>Evangelos D.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-1463-2207</contrib-id><name><surname>Gurzeler</surname><given-names>Lukas-Adrian</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Annibaldis</surname><given-names>Giuditta</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Dreos</surname><given-names>René</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-0657-1368</contrib-id><name><surname>Mühlemann</surname><given-names>Oliver</given-names></name><address><email>oliver.muehlemann@dcb.unibe.ch</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5734.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0726 5157</institution-id><institution>Department of Chemistry and Biochemistry, </institution><institution>University of Bern, </institution></institution-wrap>CH-3012 Bern, Switzerland </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5734.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 0726 5157</institution-id><institution>Graduate School for Cellular and Biomedical Sciences, </institution><institution>University of Bern, </institution></institution-wrap>CH-3012 Bern, Switzerland </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.9851.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2165 4204</institution-id><institution>Center for Integrative Genomics, </institution><institution>Université de Lausanne, </institution></institution-wrap>CH-1015 Lausanne, Switzerland </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>4134</elocation-id><history><date date-type=\"received\"><day>13</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>20</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway that is important for the elimination of faulty, and the regulation of normal, mRNAs. The molecular details of the early steps in NMD are not fully understood but previous work suggests that NMD activation occurs as a consequence of ribosome stalling at the termination codon (TC). To test this hypothesis, we established an in vitro translation-coupled toeprinting assay based on lysates from human cells that allows monitoring of ribosome occupancy at the TC of reporter mRNAs. In contrast to the prevailing NMD model, our in vitro system reveals similar ribosomal occupancy at the stop codons of NMD-sensitive and NMD-insensitive reporter mRNAs. Moreover, ribosome profiling reveals a similar density of ribosomes at the TC of endogenous NMD-sensitive and NMD-insensitive mRNAs in vivo. Together, these data show that NMD activation is not accompanied by stable stalling of ribosomes at TCs.</p></abstract><abstract id=\"Abs2\" abstract-type=\"web-summary\"><p id=\"Par2\">Nonsense-mediated mRNA decay (NMD) was thought to ensue when ribosomes fail to terminate translation properly. However, the authors observe similar ribosome occupancy at stop codons of NMD sensitive and insensitive mRNAs, showing that human NMD is not activated by stable ribosome stalling as previously suggested.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>RNA</kwd><kwd>RNA metabolism</kwd><kwd>Translation</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/501100001711</institution-id><institution>Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)</institution></institution-wrap></funding-source><award-id>31003A-162986</award-id><award-id>310030B-182831</award-id><principal-award-recipient><name><surname>Mühlemann</surname><given-names>Oliver</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>Swiss National Science Foundation National Center of Competence in Research RNA and disease (51NF40-141735, 51NF40-182880) Canton of Bern, Switzerland (intramural funds)</institution></funding-source></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par3\">Nonsense-mediated mRNA decay (NMD) is a translation-dependent mRNA surveillance pathway that targets physiological as well as aberrant mRNAs for degradation. Initially NMD was considered to be a quality control pathway that degrades mRNAs harboring a premature termination codon (PTC) within their open reading frame (ORF). These PTCs could originate from mutations as well as transcriptional or splicing errors<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. The fact that NMD targets endogenous mRNAs that encode functional proteins revealed an important role of NMD as a post-transcriptional gene expression regulation mechanism, affecting a series of biological functions ranging from tissue differentiation to protection of host cells from RNA viruses (reviewed in refs. <sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>–<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>).</p><p id=\"Par4\">Since NMD is crucial for a wide range of biological functions, an accurate and highly specific recognition mechanism of mRNAs that need to be engaged in the pathway is essential. Many studies have addressed the early events of NMD activation and overall it seems that the NMD machinery can “sense” many different cues from each mRNP. Τhe position of splicing sites relative to the termination codon (TC) through the corresponding exon junction complexes (EJCs), the length of the 3′UTR, the presence of specific protein factors and the dynamics of translation termination are features that have been directly linked to the sensitivity of mRNAs to NMD. This surveillance mechanism is orchestrated through the omnipresent RNA helicase UPF1, a universal NMD factor that is crucial for NMD activation through phosphorylation (reviewed in refs. <sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref>,<xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>).</p><p id=\"Par5\">An important feature of NMD is that it is dependent on translation, either on the first or a later round<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref>–<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Evidence supporting the formation of complexes containing NMD and translation termination factors suggested a functional connection between the two processes<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref>–<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. In view of this data, a currently prevailing working model for NMD activation suggests that NMD ensues when translation termination is aberrant, either because it requires additional factors for ribosome release or because it is not fast enough. Based on evidence from <italic>S.cerevisiae</italic> extracts and rabbit reticulocyte lysate (RRL), it was proposed that ribosomes stall at NMD-triggering TCs, suggesting a kinetically slower translation termination that may be attributed to the absence of the poly(A)-binding protein (PABPC1 in mammals, Pab1 in yeast) in the vicinity of the TC<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. PABPC1 can antagonize NMD when tethered on NMD-sensitive mRNA reporters and in vitro, PABPC1 competes with UPF1 for binding eRF3<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. These data entertain the hypothesis that the difference between productive and aberrant, NMD-eliciting translation termination might rely on whether PABPC1 or UPF1 interacts with eRF3 at the terminating ribosome, thereby either promoting or inhibiting translation termination<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. It should be noted, however, that PABP has been shown to stimulate translation termination only under non-physiological, limiting concentrations of release factors using a eukaryotic reconstituted system<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. Using a similar approach, it was suggested that under decreased concentrations of release factors, the conserved NMD factor UPF3B can delay translation termination and dissociate post-termination ribosomal complexes that are devoid of a nascent peptide<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. This finding and additional evidence documenting that the eRF3-interacting C-terminal domain of PABPC1 is not required for its NMD antagonizing capacity<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>,<xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup> suggest that the “UPF1 versus PABPC1 competition model” is oversimplified.</p><p id=\"Par6\">A better understanding of the dynamics of translation termination in the context of NMD-sensitive mRNAs is important to comprehend the activation of the NMD pathway. Previous approaches in measuring ribosomal density at the TC of reconstituted mammalian in vitro translation systems have offered important insights into the roles of crucial translation termination factors<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. However, the fact that they are developed based on short open reading frames and that the reconstituted systems lack essential NMD factors limits their application in understanding translation termination in the context of NMD.</p><p id=\"Par7\">Here we present the development of an in vitro biochemical approach that allows the detection and comparison of ribosomal density at the TC using human cell lysates and in vitro transcribed mRNAs. We show that in this system, the occupancy of ribosomes at the TC is similar on both NMD-sensitive and insensitive reporter mRNAs, as well as in the presence or absence of a poly(A) tail. We complement these results by comparing the ribosomal density at the TC of mRNAs in vivo by ribosome profiling, which also revealed a similar occupancy of ribosomes at TCs, independently of their sensitivity to NMD.</p></sec><sec id=\"Sec2\" sec-type=\"results\"><title>Results</title><sec id=\"Sec3\"><title>Assessment of ribosomal density at the TC of in vitro translated mRNAs in human cell lysates</title><p id=\"Par8\">Previous work has suggested that prolonged ribosomal occupancy at the TC is a characteristic feature and maybe even the trigger for an mRNA to be subjected to the NMD pathway<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. In order to assess ribosomal density at the TC, we developed an in vitro assay that allowed us to reproducibly identify terminating ribosomes on in vitro transcribed reporter mRNAs, based on previous protocols for the production of translation-competent cell lysates<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>. We opted for an approach that would allow all stages of translation of different mRNAs to occur using HeLa cell lysates followed by toeprinting assays. To this end, Micrococcal nuclease (MNase)-treated HeLa cell extracts were incubated with in vitro transcribed and capped mRNA reporters that harbor a humanized <italic>Renilla</italic> luciferase (Rluc) ORF followed by a 200 nucleotide long 3′UTR and an 80 nucleotides long poly(A) tail (reporter 200+ pA) (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>). After optimization of different parameters such as mRNA concentration, incubation time, and titration of magnesium concentration to ensure efficient translation activity (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>), we utilized our in vitro translation-competent lysates to assess ribosome occupancy at the TC of reporter mRNAs by toeprinting assay. After a 50-min incubation at 33 °C, robust Rluc activity was measured, documenting the translation competence of the lysate (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref>). No luminescence, and hence translation activity, was detected in the presence of the translation inhibitor puromycin. The in vitro translation reaction was followed by a primer extension reaction (toeprint assay) using a 5′ <sup>32</sup>P-labeled oligonucleotide that binds the reporter mRNA 66 nucleotides downstream of the Rluc TC, which was optimized to yield signals originating from Rluc TC region (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1a</xref>, red arrow). After purification, the cDNA molecules were analysed by denaturing polyacrylamide gel electrophoresis and autoradiography. A puromycin-treated control reaction was used to distinguish between translation-dependent toeprints and translation-independent break-offs of the reverse transcriptase. To observe reproducible translation-dependent toeprints originating from ribosomes at TCs, we titrated a broad range of biochemical parameters. Among these, the appropriate Mg<sup>2+</sup> concentration turned out to be crucial to stabilize ribosomes on mRNA after translation, in agreement with previous observations<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>. The optimization of the Mg<sup>2+</sup> concentration improved the sensitivity of the translation-dependent toeprints and enabled the use of our protocol with lysates produced from smaller scale cell cultures (<4 × 10<sup>7</sup> cells), which are more challenging to generate due to their small volume (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>). This technical improvement allowed us, for example, to produce lysates from cells with an siRNA-mediated knockdown. As shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1c</xref>, a translation-dependent band appeared 18 nucleotides downstream of the first nucleotide of the TC in transcript 200+ pA. To distinguish whether translation-dependent bands correspond to ribosomes preventing the reverse transcriptase from passing or whether they originate from mRNA molecules cleaved during translation, we phenol-extracted the mRNA molecules prior to the primer extension step. Phenol extraction or heating to 95 °C of the reactions after translation leads to the disappearance of translation-dependent bands that originate from ribosomes, whereas bands originating from cleaved mRNAs will persist. While such cleaved mRNA fragments were detected in a series of toeprint experiments performed in rabbit reticulocyte lysates (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>), the +18 translation-specific band disappeared under denaturing conditions in HeLa lysates, excluding the possibility that this band represents a co-translational RNA cleavage event (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>). In order to further verify that the translation-dependent +18 bands indeed derived from ribosomes residing at the TC, we designed reporter transcripts in which we moved the position of the TC. Moving the TC 6 or 15 nucleotides downstream of the original position resulted in a corresponding shift of the translation-dependent bands, confirming that our assay reliably detects toeprints originating from ribosomes at stop codons (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1E</xref>).<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Toeprint analysis allows the detection of ribosomes at the termination codon of in vitro translated reporter mRNAs.</title><p><bold>a</bold> Schematic representation of the in vitro synthesized capped (black dot) and polyadenylated reporter mRNA coding for humanized <italic>Renilla</italic> Luciferase (hRluc). The red arrow denotes the radiolabeled toeprint primer binding 66 nts downstream of the TC (orange dot). The 200 nt-long 3′UTR is followed by an 80 nts long template-encoded poly(A) tail depicted as A(80). <bold>b</bold> Rluc activity measurements of in vitro translation reactions. Luminescence is depicted as arbitrary units (AU), mean values ± standard deviations of 3 technical replicates are shown, values of the individual measurements are indicated by dots. <bold>c</bold> Toeprint analysis with the 200 + pA reporter transcript shown in <bold>a</bold>. Translation was performed in HeLa lysates for 50 min in the presence or absence of puromycin. To locate the positions of the toeprints, a Sanger sequencing reaction was run in parallel (G, T, C, A) using the same primer. The positions of termination codon (TC) and the toeprint band 18 nts downstream of the first nucleotide of the TC (+18), which originates from ribosomes located at the TC, are indicated. <bold>d</bold> Schematic representation of in vitro synthesized Rluc reporter transcripts A (=200 + pA), B and C, which differ with regards to the position of the termination codon (TC1, TC2, and TC3, respectively). The expected position of toeprints originating from ribosomes at the corresponding TC relative to the original position of TC1 is shown (+18, +24, and +33, respectively). <bold>e</bold> Toeprint analysis with the reporter transcripts shown in <bold>d</bold>. The translation-dependent bands corresponding to toeprints from ribosomes at the respective TCs are marked with dots following color code of <bold>d</bold>. In vitro translation and toeprint analysis were performed as in <bold>c</bold>. Source data are provided as a Source Data File.</p></caption><graphic xlink:href=\"41467_2020_17974_Fig1_HTML\" id=\"d30e473\"/></fig></p></sec><sec id=\"Sec4\"><title>Detection of stable ribosome stalling using toeprint assays</title><p id=\"Par9\">To further assess whether our toeprint assay is suitable to monitor changes of ribosomal occupancy at the TC, we introduced the regulatory peptide of the human cytomegalovirus (hCMV) gp48 upstream open reading frame 2 (uORF2)<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup> into the 200 + pA reporter construct. Translation of the hCMV uORF2 peptide inhibits termination at the TC, because the presence of a Pro-tRNA at the P site and a TC at the A site cause ribosome stalling<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. We in vitro transcribed two versions of the modified 200 + pA reporter construct, one appending the hCMV stalling peptide sequence to the Rluc ORF and the other harboring a mutation that leads to the recruitment of an Ala-tRNA (GCG codon) instead of a Pro-tRNA (CCU codon) at the P-site of the terminating ribosome (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2a</xref>). The second reporter transcript served as a control, as it does not lead to ribosome stalling. The two reporter mRNAs were translated in HeLa cell lysates and were subjected to toeprint analysis as described before. While no (or only an extremely faint) toeprint at the +18 position could be detected with the non-stalling control transcript (CTR + pA), the transcript with the stalling peptide (SP + pA) showed a robust translation-dependent +18 band, demonstrating that our assay is suitable to measure differences in ribosomal occupancy at TCs (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2b</xref>). Since the C-terminal extension of the Rluc ORF by the hCMV ORF2 sequence inactivated the luciferase enzyme, translation of the reporter transcripts had to be verified by western blot for this experiment (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2c</xref>). Overall, the above results verified that our system can reproducibly detect terminating ribosomes as well as differences in ribosomal density at the termination codon.<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Stable ribosome stalling caused by the addition of the hCMV uORF2 stalling peptide downstream of the Rluc ORF.</title><p><bold>a</bold> Schematic representation of modified RLuc reporter mRNA constructs containing the hCMV uORF2 stalling peptide (SP + pA) or a non-stalling control sequence (CTR + pA). The red arrow denotes the radiolabeled toeprint primer and the orange dot the termination codon. The 200-nt long 3′UTR is followed by an 80 nt-long poly(A) tail depicted as A(80). <bold>b</bold> Toeprint analysis of ribosome occupancy at the TC of the hCMV uORF2 was performed after in vitro translation in HeLa cell lysates for 50 min at 33 °C of equimolar amounts of the two reporter mRNAs described in <bold>a</bold>. Sanger sequencing reactions were run in parallel (G, T, C, A) to locate the positions of the toeprints. The position of the TC and the toeprint band corresponding to the ribosomes at the TC (+18) are indicated. <bold>c</bold> Representative western blot analysis of an aliquot of the translation reactions analyzed in <bold>b</bold>. Equal amounts of the translation reactions were separated on a 12% SDS-PAGE, transferred onto a nitrocellulose membrane and probed for tyrosine-tubulin (Tyr-Tub) and Rluc protein. The band corresponding to full-length Rluc protein is denoted by an arrow. The experiment was repeated three times. Source data are provided as a Source Data File.</p></caption><graphic xlink:href=\"41467_2020_17974_Fig2_HTML\" id=\"d30e521\"/></fig></p></sec><sec id=\"Sec5\"><title>Depletion of the recycling factor ABCE1 leads to increased ribosome occupancy at the TC</title><p id=\"Par10\">Next, we wanted to assess whether our toeprint assay is also able to identify changes of ribosome density at the TC under aberrant translation termination conditions. A previous study showed that reduction of the eukaryotic recycling factor ABCE1 can lead to prolonged ribosome occupancy at the TC<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. In order to address whether we can recapitulate this event in our assay, we prepared lysates from siRNA-mediated control and ABCE1 knockdown cells (ABCE1 KD) and compared ribosomal density by toeprint assays. The efficacy of the ABCE1 knockdown was documented by probing equal amounts of lysates by western blotting (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3a</xref>). The higher intensity of the +18 band in the ABCE1-depleted lysate indicates an increased ribosome occupancy at the TC in the absence of the recycling factor (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3b</xref>). This confirms that our in vitro system has the capacity to monitor differences in ribosome occupancy at the TC after depleting translation termination-related factors.<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Increased ribosome occupancy at the TC of in vitro translated mRNAs under decreased concentrations of the recycling factor ABCE1.</title><p><bold>a</bold> Western blot analysis of ABCE1 depletion in HeLa lysates. Cell lysates equivalent to 4 × 10<sup>5</sup> cells treated with a CTR or ABCE1 siRNAs were loaded on a 10% SDS-PAGE, transferred onto a nitrocellulose membrane and probed for ABCE1 and beta-actin. <bold>b</bold> Toeprint analysis of ribosome occupancy at the TC under ABCE1 depletion was performed after in vitro translation of the 200 + pA construct for 50 min at 33 °C in lysates from cells treated with CTR or ABCE1 siRNAs. Sanger sequencing reactions were run in parallel (G, T, C, A) to locate the positions of the toeprints. The position of the TC and the toeprint band corresponding to the ribosomes at the TC (+18) are indicated. <bold>c</bold> Relative quantification of the translation-dependent +18 band under ABCE1 conditions of 4 independent toeprinting experiments, normalized to the translation-independent mRNA signals and to the values of the Ctr KD conditions. Mean values ± standard deviation are shown and the values of the individual experiments are depicted by dots (Unpaired, two-sided statistical <italic>t</italic>-test: Ctr/ABCE1 <italic>p</italic> value: 0,0014). Source data are provided as a Source Data File.</p></caption><graphic xlink:href=\"41467_2020_17974_Fig3_HTML\" id=\"d30e564\"/></fig></p></sec><sec id=\"Sec6\"><title>NMD sensitivity of reporters with different 3′UTR lengths</title><p id=\"Par11\">To directly test the prevailing NMD model by assessing whether NMD-sensitive mRNAs induce ribosome stalling at the TC, we designed a series of reporter mRNAs that differ in the length of the 3′UTR and portray different sensitivities to NMD. The reporters were designed in a way that allows the comparison of ribosome occupancy at the TC in toeprint assays using the same radiolabeled primer. All reporters share an identical 5′UTR, a humanized Rluc ORF and an identical 200 nucleotide long 3′UTR (construct 200 + pA). In addition to this sequence, a second construct has the 3′UTR extended to 1400 nucleotides (1400 + pA; Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4a</xref>). The two constructs were transiently transfected into HeLa cells that additionally express a TCR-β NMD reporter and to test if they were targeted by NMD, we knocked down the key NMD factor UPF1. The efficacy of the UPF1 knockdown was monitored by western blotting (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4b</xref>) and the steady-state mRNA levels of the Rluc reporters 200 + pA and 1400 + pA were assessed by RT-qPCR (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4c</xref>). While the relative abundance of the 200 + pA transcript remained almost unchanged upon UPF1 depletion, the level of the 1400 + pA increased 4-fold, indicating that the transcript with the long 3′UTR (1400 + pA) was a target for NMD, whereas the one with the short 3′UTR (200 + pA) was not. This observation is in agreement with previous reports showing that long 3′UTRs can render mRNAs sensitive to NMD<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref>–<xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. The mRNA levels of the TCR-β NMD reporter and the endogenous NMD-sensitive Retinitis Pigmentosa 9 Pseudogene transcript (RP9P)<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup> confirmed that NMD activity was reduced in all UPF1 KD samples (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>).<fig id=\"Fig4\"><label>Fig. 4</label><caption><title>Extension of the 3′UTR renders the Rluc reporter mRNA NMD-sensitive in vivo.</title><p><bold>a</bold> Schematic representation of Rluc reporter constructs with either a 200 nts long (200 + pA) or a 1400 nt-long 3′UTR (1400 + pA), modified for expression in human cells. The first 200 nts of the 3′UTR (green line) are identical in both constructs, the additional 1200 nts (red line) of the 1400 + pA 3′UTR correspond to a head-to-tail duplicated sequence originating from the ampicillin resistance gene. The constructs were cloned into pcDNA3.1(−) expression plasmid, where their expression is controlled by a CMV promoter and a bovine growth hormone polyadenylation signal. <bold>b</bold> Western blot analysis to monitor UPF1 knockdown efficacy in HeLa cells transiently expressing either 200 + pA or 1400 + pA Rluc reporter mRNA. Cell lysates equivalent to 2 × 10<sup>5</sup> cells were loaded on a 10% SDS-PAGE, transferred onto a nitrocellulose membrane and probed for UPF1 and beta-actin. <bold>c</bold> Relative abundance of 200 + pA and 1400 + pA mRNAs in cells depleted for UPF1 (UPF1 KD) normalized to cells with a control knockdown (CTR KD) were measured by RT-qPCR. Mean values ± standard deviations of 3 biological replicates are shown, values of the individual experiments are indicated by dots. (Unpaired, two-sided statistical <italic>t</italic>-test: 1400 + pA vs 200 + pA <italic>p</italic> value: 0.0039). Source data are provided as a Source Data File.</p></caption><graphic xlink:href=\"41467_2020_17974_Fig4_HTML\" id=\"d30e621\"/></fig></p></sec><sec id=\"Sec7\"><title>Similar ribosome density at NMD-sensitive and insensitive TC</title><p id=\"Par12\">It has been proposed that a long physical distance between the TC and the poly(A) binding protein (PABP) may hinder translation termination by causing prolonged ribosomal occupancy at the TC<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. Previous reports have also suggested that PABP can facilitate translation termination under decreased release factors concentrations in vitro<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup> and we therefore wanted to assess in our toeprint assay whether ribosome occupancy at the TC is affected by the presence or absence of a poly(A) tail, or by its physical distance from the TC. For this reason, the toeprint assay was performed using both NMD-sensitive (with the 1400 nucleotides long 3′UTR) and insensitive reporter mRNAs (with 200 nucleotides long 3′UTR) in two variants, either containing an 80 nucleotides long poly(A) tail or lacking it (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5a</xref>). To this end, equimolar amounts of the in vitro transcribed, capped and purified reporter mRNAs were used for translation in HeLa lysates as described above, followed by primer extension reactions. Again, puromycin-treated samples were run alongside to unambiguously identify the translation-dependent toeprints at position +18 relative to the TC (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5b</xref>, lanes 2, 4, 6, and 8). The intensity of the +18 band was similar for the 200 + pA and the 1400 + pA transcripts, indicating that ribosome occupancy at the TC was not affected by the 3′UTR length (Compare lanes 1 and 5). Furthermore, we could also not detect a significant difference in the intensity of the +18 band depending on whether or not the transcripts contained a poly(A) tail, suggesting that, at least in this in vitro system, the presence of a poly(A) tail in the vicinity of the TC has no termination-promoting effect (Compare lanes 1 with 3 and 5 with 7). Altogether, this result is inconsistent with the prevailing NMD model proposing prolonged stalling at NMD-eliciting TCs. However, it is also possible that our in vitro toeprinting assay may simply not fully recapitulate the situation in vivo.<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>Similar ribosome occupancy at the termination codon of NMD-sensitive and NMD-insensitive mRNAs.</title><p><bold>a</bold> Scheme of in vitro synthesized RLuc reporter mRNAs. with (+pA) or without (−pA) a 80 nts long poly(A) tail. TC: orange dot. <bold>b</bold> Toeprint analysis with reporter mRNAs depicted in <bold>a</bold>. In vitro translation was performed in the presence (+) or absence (−) of puromycin. Sanger sequencing reactions were run in parallel (G,T,C,A). The TC position and the toeprint band corresponding to the ribosomes at the TC (+18) are indicated. <bold>c</bold> Relative quantification of +18 translation-dependent band from three individual experiments versus translation-independent mRNA signals normalized to 200 + pA conditions. Bars denote average values, dots depict the measurements of the three independent experiments, bars represent SD (Unpaired, two-sided statistical <italic>t</italic>-tests: 200-pA/p200 + A <italic>p</italic> value = 0.983, 1400-pA/1400 + pA <italic>p</italic> value = 0.701 1400 + pA/200 + pA <italic>p</italic> value = 0,078). <bold>d</bold> (Top) Metagene analysis of ribosome-protected footprints from HeLa cells from three independent ribosome profiling experiments. Transcripts were aligned to the stop codon (0) and the mapped reads from 300 nucleotides upstream to 100 nucleotides downstream of the stop codon are shown. (Bottom) Heatmap of the ribosome-protected reads of all 60’000 transcripts considered for the metagene analysis. Transcripts were ordered according to their ribosome occupancy at the termination codon (transcripts with the highest number of reads at the stop codon at the top). NMD-sensitive transcripts are shown on the left of the heatmap panels. Each pixel line corresponds to the average of 10 transcripts. <bold>e</bold> Analysis of mean ribosome occupancy at the stop codon relative to mean ribosome occupancy in CDS, performed for NMD targets (identified as in <bold>d</bold>) and all other transcripts (Others). Violin plots showing the counts distribution of ribosome-derived reads mapping at the stop codon relative to ribosome-derived reads aligning to the CDS as an average of 3 biological replicates. Boxplots (middle) indicate the percentiles (5,25,50,75, and 95) of the ribosome occupancy values distribution with dots representing outliers. Percentage of reads relative to total reads are shown on the y-axis. Anova statistic test, <italic>p</italic> value = 0.65. Source data are provided as a Source Data File.</p></caption><graphic xlink:href=\"41467_2020_17974_Fig5_HTML\" id=\"d30e690\"/></fig></p><p id=\"Par13\">To test in vivo whether ribosomes show an increased occupancy at TCs of NMD-triggering mRNAs, we analysed ribosome profiling data to assess ribosome occupancy at the TC between NMD-sensitive and NMD-insensitive transcripts originating from previous work in the lab that was also performed in HeLa cells<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. The ribosome profiling protocol was modified to assess specifically ribosome occupancy at the TC of endogenous mRNAs by omitting cycloheximide and applying instead snap-freezing of the cell lysates in liquid nitrogen<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. Metagene analysis of ribosome-associated footprints from three independent experiments showed that under these conditions, ribosomes tend to reside longer on average at the TC than at a codon within the ORF (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5d</xref>, top). A list of 678 NMD-sensitive transcripts was compiled by including the most abundant isoform for each of previously identified NMD-sensitive genes that is stabilized under inhibited NMD (UPF1 KD) conditions<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. When all 60’000 transcripts were ordered according to their ribosome occupancy at the TC (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5d</xref>, heatmap) and then labeled if they belong to the set endogenous NMD-sensitive transcripts (NMD targets), it became apparent that NMD-sensitive transcripts were not enriched among the transcripts with high ribosome occupancy at the termination codon. To normalize for the overall translation efficiency of a given mRNA, we determined the relative ribosome occupancy at TCs compared to CDS for each transcript by defining the ratio of the total counts of ribosome-derived reads aligning to the TC relative to the average of ribosome reads mapping at the CDS. In order to assess whether ribosomes reside longer at the TCs of NMD-sensitive mRNAs, we compared this relative stop codon ribosome occupancy between NMD-sensitive and insensitive mRNAs (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5e</xref>) and found no statistically significant difference between NMD-sensitive and insensitive mRNAs. This result corroborates our in vitro data and altogether challenges the view that ribosome stalling at the TC is a hallmark of NMD-sensitive mRNAs.</p></sec></sec><sec id=\"Sec8\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par14\">We report here the development of an in vitro assay to examine the ribosome occupancy at the TC of NMD-sensitive and NMD-insensitive mRNAs using translation-competent lysates from human cells. In contrast to previous evidence originating from in vitro studies performed with yeast extracts and rabbit reticulocyte lysate<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>, we found a similar ribosome occupancy at the TC of reporter mRNAs, independently of whether they contained a long 3′UTR that renders them sensitive to NMD in vivo. In addition, omitting a poly(A) tail from our reporter constructs did not affect the ribosome occupancy at the TC. These results from our in vitro toeprinting system are corroborated by our ribosome profiling experiments, in which normalized ribosome occupancy at TCs of mRNAs did not correlate with whether an mRNA was a target for the NMD pathway or not. Thus, our in vitro and in vivo data are in disagreement with a suggested working model for NMD, which posits that inefficient or aberrant translation termination that can be detected as ribosome stalling at the TC is the signal for NMD to ensue<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>.</p><p id=\"Par15\">In vitro translation systems have significantly contributed to assess a broad range of translation-related mechanisms<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref>–<xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. However, the study of translation termination in vitro using mammalian systems has remained technically challenging and has only recently gained broader attention. The cryo-electron microscopic analysis of ribosomal complexes isolated from in vitro translation reactions in rabbit reticulocyte lysate yielded structural information about distinct steps of translation<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>, and fully reconstituted eukaryotic systems allowed a detailed, stepwise functional interrogation of translation termination complexes and of the roles of release factors<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref>,<xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. In this context, reconstituted translation is performed with isolated ribosomes that are supplemented with purified translation factors and aminoacylated tRNAs. The separate addition of each component allows tight control of the system and its modulation (e.g. introducing nucleotide analogs or mutant versions of translation factors). On the other side, translation factor concentrations are far from physiological conditions and there is a requirement of intermediate purification steps of ribosome complexes, which limits the biological relevance of reconstituted translation systems. While they are well suited to study mechanistic aspects of translation, they will fail to recapitulate many steps of translation regulation and processes more accessorily connected to translation. Since we are mainly interested in investigating the connection between translation and NMD, we opted for the development of a more physiologically relevant human-based in vitro system that does not require the isolation of specific ribosomal sub-complexes. This system allows all steps of translation of the in vitro transcribed reporter mRNA in the lysates to occur before toeprinting and detection of ribosomes at the TC. The system can be modulated, for example by knocking down specific factors or by the addition of recombinant proteins. We based our in vitro system on HeLa cells, because the majority of biochemical data available concerning human NMD derives from studies in this system. The development of an assay that allows monitoring of ribosome occupancy at the TC was an important achievement to test the idea that prolonged ribosome stalling at the TC is indeed the trigger for NMD.</p><p id=\"Par16\">Our in vitro toeprinting approach allows a direct comparison of ribosome occupancy at TCs between different reporter mRNAs translated in lysates of human cells. Extensive titration and optimization of different biochemical parameters (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>) finally permitted that all steps of translation occur in the cell lysate, which was verified by the production of enzymatically active Rluc (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1b</xref>). Optimization of our system was focused on yielding reproducible translation-dependent bands in toeprint assays (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1c</xref>). To this end, a limited treatment of the lysates with micrococcal nuclease to reduce the number of ribosomes engaged in translating endogenous mRNAs, the titration of the primer distance from the TC and the optimization of Mg<sup>2+</sup> concentration to stabilize the ribosomes on the mRNA were crucial steps in establishing a successful protocol (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>). Toeprint assays under the established conditions allow the reproducible detection of ribosomes at TCs and changes in the ribosome occupancy induced by stalling peptide sequences (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>) or depletion of translation termination factors (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>). The fact that our assay can detect ribosomes at the TC rather than at codons further upstream is in agreement with our ribosome profiling data that also showed an overall higher occupancy of ribosomes at the TC compared to the ORF. These two results agree with the notion that translational pauses at the termination codon is a common feature of translation<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>.</p><p id=\"Par17\">As aforementioned, comparison between NMD-sensitive and NMD-insensitive mRNA reporters revealed a similar occupancy of ribosomes at the TC both in our in vitro toeprinting assay as well as by in vivo ribosome profiling (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>), suggesting that the 3′UTR length of an NMD-sensitive mRNA is not per se causing ribosome stalling at the TC. This data contrasts with two previous reports according to which increased ribosome density was observed on PTC-containing transcripts in yeast extracts and in rabbit reticulocyte lysates<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup>. While it is conceivable that the difference between the yeast system and the human system reflects indeed a species difference, we speculate that the difference between the rabbit reticulocyte lysate and the human system might be attributed to toeprints originating from RNA cleavage in the reticulocyte lysate. We had initially also developed our toeprint assay using rabbit reticulocyte lysate and found that the toeprints we detected in this system almost exclusively represented co-translational RNA cleavages next to the ribosome (Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>), a feature that has been previously reported in reticulocyte lysates<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. For this reason, we performed an additional control to monitor whether toeprints originate from ribosomes or RNA cleavage events by re-purifying the RNA after translation and prior to primer extension. A side-by-side comparison between these phenol-purified and untreated samples reveals whether toeprints derive from ribosomes or from cleaved RNAs.</p><p id=\"Par18\">The extent of increased ribosome occupancy at the termination codon may vary and stimulate different effects on mRNAs that are monitored by translation-dependent pathways. In cases of faulty mRNAs that are degraded due to a lack of a TC (nonstop decay) or due to a strong secondary structure or the absence of a cognate tRNA (no-go decay), the activating signal is the ribosome stalling during elongation. In this scenario, ribosome stalling marks a dead-end event and the rescue of an otherwise trapped ribosome is crucial (reviewed in refs. <sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref>,<xref ref-type=\"bibr\" rid=\"CR44\">44</xref>,<xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>). The term “stalling” is used broadly to describe increased ribosome density, without distinguishing between transient pauses and stable stalls. Even though a clear distinction between the two is still technically difficult, stalling as well as ribosome pausing have clearly been detectable by means of ribosome profiling or toeprinting<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref>–<xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup> and both could be reproduced in our in vitro system. While we cannot exclude the possibility that NMD is associated with transient delays of translation at the termination codon that are too brief to be captured by means of toeprinting or ribosome profiling, our data clearly argue against the occurrence of stable ribosome stalling at TCs of NMD-sensitive mRNAs.</p><p id=\"Par19\">There is a growing body of evidence that NMD occurs stochastically depending on highly variable interactions that finally lead to the degradation of the mRNA, opposed to the traditional view of a linear, ordered, and irreversible pathway that leads to decay<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. In yeast, NMD substrates were found to have an increased rate of out-of-frame translation, accompanied by an overall decreased codon optimality or stretches of non-optimal codons compared to NMD-insensitive mRNAs<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref></sup>. This observation led to the idea that NMD is a constantly active mRNA surveillance pathway that monitors every mRNA throughout its life cycle<sup><xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>. The very recent observation that only a relatively small subset of termination events results in NMD using single-molecule kinetics of NMD-sensitive mRNA reporters<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup> further supports the emerging view of a more complex mechanism of NMD activation and is inconsistent with stable ribosome stalling constituting the activation step of NMD. Collectively, these data and our results described herein warrant a critical revisiting and further testing of the prevailing NMD model that postulates ribosome stalling as the NMD-triggering signal.</p></sec><sec id=\"Sec9\"><title>Methods</title><sec id=\"Sec10\"><title>Plasmids</title><p id=\"Par20\">To create reporter constructs that differ in the length of the 3′UTR, a synthetic version of <italic>Renilla</italic> luciferase (Rluc) from the phRG-TK vector (Promega) was cloned into the pTRE-Tight vector (Clontech) via <italic>Hind</italic>III and <italic>Xba</italic>I, yielding pTRE-Tight-hRluc-200bp. For cloning of pTRE-Tight-hRluc-800bp 3′UTR, a 600 bp PCR product of the ampicillin resistance gene located in phRG-TK was generated using the primers 5′-AAT TTC TAG AAT TGT TGC CGG GAA GCT AGA GTA AG-3′ and 5′-AAT TTC TAG ATG AGT ATT CAA CAT TTC CGT GTC G-3′, cut with <italic>Xba</italic>I and inserted into the <italic>Xba</italic>I-linearized pTRE-Tight-hRluc-200bp 3′UTR vector. A further elongation of the 3′UTR to 1400 bp (pTRE-TightRluc-1400bp 3′UTR) was achieved by a partial digestion of pTRE-Tight-hRluc-800bp 3′UTR with XbaI and inserting the <italic>Xba</italic>I-digested 600 bp long PCR product a second time. The pTRE-Tight-hRluc constructs were subcloned into into pCRII-TOPO (Invitrogen) according to the guidelines of the ‘TOPO TA Cloning Kit Dual Promoter’ (Invitrogen), resulting in pCRII-hRluc constructs with various 3′UTR lengths (pCRII-hRluc-200bp 3′UTR, pCRII-hRluc-800bp 3′UTR, pCRII-hRluc-1400bp 3′UTR). The reporter plasmids A, B and C are derivatives of pCRII-hRluc-800bp 3′UTR and were generated by site-directed mutagenesis using the primers 5′-CAA ATG TGG TAT GGC TGA TTA GAT CCT CTA GAA TTC CTG CTC-3′, 5′-CAA ATG TGG TAT GGC TGA TTA GAT CCT CAA GAA TTC CTG CTC-3′ and 5′-AAA TGT GGT ATG GCT GAT TGG ATC CTC AAG AAT TCC TGC-3′, respectively. We introduced the regulatory peptide of the human cytomegalovirus (hCMV) gp48 upstream open reading frame 2<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup> into our 200 + pA reporter construct by fusion PCR yielding pCRII-hRLuc-200bp-hCMV SP-(A)80 and pCRII-hRLuc-200bp-hCMV CTRL-(A)80.</p><p id=\"Par21\">For expression tests in HeLa cells, we created the eukaryotic construct expressing the 200 + pA mRNA by amplifying the ORF of Rluc omitting the poly(A) signal that is harbored in the SV40-derived 3′UTR using the primers 5′-GGG CCC ATG GCT TCC AAG GTG TAC GA-3′ and 5′-GGT ACC AAC AAC AAC AAT TGC ATT CA-3′ and cut with <italic>Apa</italic>I and <italic>Kpn</italic>I to be inserted to equally treated pcDNA 3.1(−) vector yielding p200eu. To improve translation potential, the Kozak sequence<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup> was optimized by site-directed mutagenesis using 5′-CTG GCT AGC GTT TAA ACG CCA CCA TGG CTT CCA AGG TGT-3′, yielding p200eukoz. For creating the eukaryotic construct for the expression of p1400 + pA, two regions of pCRII-hRluc_SV40_amp1200 (A)80 were amplified by fusion PCR using the primer pairs 5′-GGG CCC ATG GCT TCC AAG GTG TAC GA-3′/5′-TGG CGA TGA GAA CAA CAA CAA TTG CAT TCA-3′ and 5′-TGT TGT TGT TCT CAT CGC CAA TTG TTG CC-3′/5′-GGT ACC CTA GAT GAG TAT TC-3′. The fused product was then TOPO TA cloned into a pCRII-TOPO vector (Invitrogen) and ligated into the eukaryotic expression vector pcDNA 3.1 (−) using the restriction sites <italic>Apa</italic>I and <italic>Kpn</italic>I. The plasmids used for KD and rescue of UPF1 are described elsewhere<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>.</p></sec><sec id=\"Sec11\"><title>Cell lines and cell culture</title><p id=\"Par22\">Lysate preparation and transfection experiments were performed using HeLa Tet-Off TCR-β ter 68 cells expressing a stably integrated TCR-β PTC+ (at position 68) minigene with an upstream tetracycline responsive element (TRE) and a minimal CMV promoter<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup>. TRE promoter is regulated by a constitutively expressed Tet-Off advanced transactivator deriving from the parental cell line HeLa tTA-advanced clone 9 (Hela tetR clone 9)<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>. HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with FCS, Penicillin and Streptomycin (DMEM +/+) at 37 °C under 5% carbon dioxide atmosphere. The cells were grown in tissue culture flasks of variable sizes and kept in culture for no longer than one month. Passaging of cells was carried out by detachment adding Trypsin/EDTA solution (usually 1:10 (v/v) of the initial culture volume. The cell density was quantified by trypan staining and automated cell counting (Countess<sup>®</sup> Automated Cell Counter, Thermo Fisher scientific). HeLa cells were obtained from ATCC (CCL2).</p></sec><sec id=\"Sec12\"><title>Preparation of translation-competent HeLa lysates</title><p id=\"Par23\">Translation-competent HeLa lysates were prepared from approximately 80% confluent HeLa cell cultures ranging from 1 × 10<sup>7</sup> cells to 5 × 10<sup>8</sup> cells. Cells were washed with PBS pH 7.4 at room temperature (RT) and detached by trypsinization, resuspended in full growth medium, counted and pelleted (200 <italic>g</italic>, 4 °C for 5 min). The cell pellet was washed three times with ice cold PBS pH 7.4 and finally resuspended in ice-cold hypotonic lysis buffer [10 mM HEPES pH 7.3, 10 mM K-acetate, 500 μM Mg-acetate, 5 mM DTT and 1x protease inhibitor cocktail (biotool.com)] at a final concentration of 2 × 10<sup>8</sup> cells/ml. The suspension was incubated on ice for 10 min and cells were lysed by syringe treatment with a 1 ml syringe and a 27-gauge needle at 4 °C (cold room). The lysis process was monitored by trypan stain until more than 95% of the cells were lysed. The lysate was centrifuged at 13’000 <italic>g</italic>, 4 °C for 10 min and the supernatant was complemented to a final concentration of 1 mM CaCl<sub>2</sub> and 0.8 u/µl Micrococcal Nuclease (Thermo Fisher Scientific). The mixture was incubated at 20 °C for 10 min and then transferred on ice. The enzyme activity was quenched by addition of EGTA to a final concentration of 10 mM. Finally, the nuclease-treated HeLa lysate was aliquoted, snap frozen, and stored at −80 °C.</p></sec><sec id=\"Sec13\"><title>In vitro transcription of reporter mRNAs</title><p id=\"Par24\">A total of 4 μg of linearized pCRII vectors encoding the desired reporter mRNA downstream of a T7 promoter were mixed to yield an in vitro transcription reaction in 1x OPTIZYME™ Transcription Buffer (Thermo Fisher Scientific) to a final concentration of 40 ng/µl. The mixture further contained 1 mM of each ribonucleotide (rNTPs, New England Biolabs), 0.4 u/µl NxGen RNase inhibitor (Lucigen), 0.001 u/µl Pyrophosphatase (Thermo Fisher Scientific) and 5% (v/v) T7-RNA-polymerase (custom-made). The reaction was incubated at 37 °C for 1 h and then an equal quantity of T7-RNA polymerase was added for another 30 min. The mixture was then supplemented with TURBO DNase (Thermo Fisher Scientific) to a final concentration of 0.15 u/µl at 37 °C for 30 min. The transcribed mRNA was purified from the reaction using an acidic phenol-chloroform-isoamylalcohol (P.C.I) mixture followed by ethanol precipitation and two washes with 70% ethanol. The product was dissolved in disodium citrate buffer, pH 6.5, quantified by NanoDrop measurement and quality was assessed by agarose gel electrophoresis. Prior to capping, the RNA was incubated at 65 °C for 5 min and supplemented accordingly to yield a 100 μl reaction that consisted of 250 ng/µl RNA, 0.1 mM guanosine triphosphate (GTP, New England Biolabs), 0.1 mM S-adenosylmethionine (SAM, New England Biolabs), 2 u/µl NxGen RNase inhibitor (Lucigen), 0.1 u/µl vaccinia capping enzyme (VCE, New England Biolabs) in 1x Capping buffer (New England Biolabs). The capping reaction was carried out at 37 °C for 1 h and quenched by the addition of acidic P.C.I, followed by RNA purification. Capped mRNAs were quantified by NanoDrop, 0.5 μg of RNA was analysed by agarose gel electrophoresis and aliquots were stored at −80 °C until use.</p></sec><sec id=\"Sec14\"><title>In vitro translation</title><p id=\"Par25\">For a typical in vitro translation reaction, an amount of lysate corresponding to 1.11 × 10<sup>6</sup> cell equivalents was used at a concentration of 8.88 × 10<sup>7</sup> cell equivalents/ml. The reaction was supplemented to a final concentration of 15 mM HEPES, pH 7.3, 0.3 mM MgCl<sub>2</sub>, 24 mM KCl, 28 mM K-acetate, 6 mM creatine phosphate (Roche), 102 ng/µl creatine kinase (Roche), 0.4 mM amino acid mixture (Promega) and 1 u/µl NxGen RNase inhibitor (Lucigen). Control reactions contained 320 µg/ml puromycin (Santa Cruz Biotechnology). Before addition of mRNA, the supplemented lysate was incubated at 33 °C for 5 min. In vitro transcribed and capped mRNAs were pre-incubated at 65 °C for 5 min and cooled down on ice before addition to the pre-incubated translation reaction mixtures at a final concentration of 40 fmol/μl. Translation was performed at 33 °C for 50 min. To monitor the protein synthesis output, samples corresponding to 4.44 × 10<sup>5</sup> cell equivalents of the translation reaction were put on ice and mixed with 1x <italic>Renilla</italic>-Glo substrate (Promega) in <italic>Renilla</italic>-Glo (Promega) assay buffer on a white bottom 96 well plate. The plate was incubated at 30 °C for 10 min and the luminescence signal was measured three times using the TECAN infinite M100 Pro plate reader. Experiments using nuclease-treated Rabbit Reticulocyte Lysates (Promega) were performed by supplementing the reactions as above at 37 °C for 30 min following the manufacturer guidelines.</p></sec><sec id=\"Sec15\"><title>Labeling of the toeprint primer and Sanger sequencing</title><p id=\"Par26\">A labeling reaction contained 0.2 µM PAGE-purified primer (5′-TCA GGT TCA GGG GGA GGT G-3′), 0.39 u/μl T4 PNK (Thermo Scientific) and 0.59 µM [γ-<sup>32</sup>Ρ] ΑΤΡ (Hartmann analytics). The mixture was incubated at 37 °C for 1 h and quenched by incubation at 68 °C for 10 min. The radiolabeled primer was separated from the free nucleotides using the Microspin G-25 Columns (GE Healthcare) according to the manufacturer’s descriptions. Using the radiolabeled primer, a sequencing reaction was performed for each of the four nucleotides (G, T, C, A) using the same plasmid DNA templates that were used for in vitro transcription of the reporter mRNAs. The procedure was carried out using the USB Sequenase Version 2.0 DNA Sequencing Kit (Affymetrix) following the manufacturers guidelines.</p></sec><sec id=\"Sec16\"><title>Toeprint assay</title><p id=\"Par27\">After completion, translation reaction was incubated at 52 °C for 70 s and then immediately cooled down on ice. 2.66 × 10<sup>5</sup> cell equivalents of the stopped translation reaction were diluted to 2.72 × 10<sup>7</sup> cell equivalents/ml in a pre-cooled toeprint buffer containing (final concentrations given including the reverse transcriptase added at a later step) 37.0 mM Tris-HCl pH 7.3, 55.5 mM KCl, 5.0 mM MgCl2, 7.2 mM DTT, 0.2 mM dNTPs, 0.6 u/μl NxGen RNase inhibitor (Lucigen) and 20.3 nM radiolabeled primer. The mixture was pre-incubated at 37 °C for 5 min and AffinityScript Multiple Temperature Reverse Transcriptase (Agilent Technologies) was added to each toeprinting reaction at a final content of 10.2% (v/v, no unit definition available). Primer extension was carried out at 37 °C for 30 min. After completion, each reaction was diluted 1:11 in H<sub>2</sub>O and placed on ice. The cDNA products were purified using the ChIP DNA Clean and concentrator (Zymo Research) according to the product manual. cDNA reactions were loaded on a 6% (v/v) polycrylamide gel containing 6.67 M urea in 1x TBE. Along with the samples, four Sanger sequencing reactions were loaded whereby the volumes were adjusted according to the radioactive counts of the cDNA samples (equal to counts). The gel was then run in 0.5x TBE at 27 W, fixed, dried and exposed overnight. The screen was scanned using the Typhoon FLA 9500 Laser scanner (pixel size 100 micrometer, sensitivity 100 V). Quantification of the toeprints was performed using ImageJ 1.52p where band intensities of translation-dependent toeprints were normalized to the overall intensity of translation-independent bands of the corresponding lane (background).</p></sec><sec id=\"Sec17\"><title>siRNA-mediated ABCE1 depletion in HeLa cells</title><p id=\"Par28\">The transfection mix added to the cells (grown to 40–60% confluency) consisted of 1:10 (v/v) of the culture medium and contained 25 nM siRNA (ABCE1 siRNA, 5′-GAG GAG AGU UGC AGA GAU UU dTdT-3′ or Negative Control siRNA, 5′-AGG UAG UGU AAU CGC CUU G dTdT-3′, Microsynth) and 0.25% (v/v) Lullaby transfection reagent (OZBiosciences) dissolved in Opti-MEM (Thermofisher). Before addition to the cells, the mix was incubated at RT for 20 min to allow complex formation. After 24 h of incubation, the cells were split to a higher format including a PBS pH 7.4 washing step. On the next day, the transfection was repeated under the same conditions and 24 h later cells were harvested and immediately processed to yield translation-competent lysates.</p></sec><sec id=\"Sec18\"><title>Expression of Luc reporters in HeLa cells and NMD inhibition</title><p id=\"Par29\">In order to express luciferase NMD reporters in vivo, HeLa cells (grown to 60–80% confluency) were transfected with the reporter constructs in pcDNA 3.1 (+) vectors (described above). Short hairpin RNA (shRNA)-mediated RNA interference (RNAi) degradation<sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup> was applied to knockdown UPF1 as described previously<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref>,<xref ref-type=\"bibr\" rid=\"CR53\">53</xref>,<xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>. The transfection mix consisted of 20 ng/µl reporter plasmid, 20 ng/µl pSUPuro plasmid (1:1 mixture of pSUPuro UPF1 against two target sequences<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>) in Opti-MEM containing 3% (v/v) Dogtor (OZ Biosciences) transfection reagent. As a negative control, a pSUPuro plasmid containing a randomized target sequence was used (pSUPuro Scr). After 12 h the medium was replaced by DMEM +/+ containing 1.5 µg/ml Puromycin (Santa Cruz Biotechnology). The antibiotic selection was carried out for 48 h until the medium was replaced with DMEM +/+ to let the cells recover for approx. 24 h. To obtain a list of NMD-sensitive RNAs 3 × 10<sup>5</sup> HeLa cells per well were seeded in 6-well plates. Twenty-four hours later, the cells were transfected with 52 pmol of siRNA using Lullaby reagent (OZ Biosciences). After 48 h, the cells were re-transfected as before. Protein and total RNA were isolated after one additional day. The siRNA sequence 5′-GAUGCAGUUCCGCUCCAUU-3′ was used for targeting UPF1.</p></sec><sec id=\"Sec19\"><title>Western blot</title><p id=\"Par30\">To examine whether UPF1 or ABCE1 were depleted from HeLa cells and whether Rluc reporters are efficiently transfected or expressed after in vitro translation, cell lysates corresponding to 2 × 10<sup>5</sup> cells in the case of in vivo experiments or 4 × 10<sup>5</sup> per sample in the case of nuclease-treated lysates were analyzed by electrophoresis on 10 or 12% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (GE Healthcare Life Science). The proteins of interest were probed with antibodies against the following proteins: UPF1 (Bethyl A300-038A, 1:1000), ABCE1 (Abcam, ab185548, 1:1000), beta-actin (Sigma Aldrich A5060, 1:2000), Rluc (Thermo Fisher PA5-32210, 1:600), Tyr-Tubulin (Sigma T9028, 1:5000).</p></sec><sec id=\"Sec20\"><title>RT-qPCR</title><p id=\"Par31\">Approximately 2.7 × 10<sup>6</sup> HeLa cells were resuspended in 900 μl TRI reagent and total RNA was isolated with isopropanol as precipitation agent. The purified RNA was diluted in disodium citrate buffer, pH 6.5 and DNA was degraded using TURBO DNA-free™ Kit (Ambion, Thermo Fisher Scientific) following the manufacturers guidelines.</p><p id=\"Par32\">Reverse transcription reactions contained 50 ng/µl RNA, 15 ng/µl random hexamers, 1x AffinityScript RT buffer, 10 mM DTT, 0.4 mM dNTP mix (each), 1 u/µl NxGen RNase inhibitor (Lucigen) and 5% (v/v, no unit definition available) AffinityScript Multiple Temperature Reverse Transcriptase (Agilent). RNA was incubated at 65 °C for 5 min and was then left at RT for 10 min for primer annealing. The cDNA synthesis was carried out at 50 °C for 60 min and inactivated at 70 °C for 15 min. The cDNA was diluted with water to a final concentration of 8 ng/µl. For qPCR, each reaction consisted of 1.6 ng/µl cDNA and 0.5 µM of each primer specific for beta-actin (5′-TCC ATC ATG AAG TGT GAC GT-3′ and 5′-TAC TCC TGC TTG CTG ATC CAC-3′), Mini-TCRβ reporter (5′-AGT TGG CTT CCC TTT CTC AG-3′ 5′-CTT GGG TGG AGT CAC ATT TC-3′), Retinitis Pigmentosa 9 Pseudogene (RP9P) (5′-CAA GCG CCT GGA GTC CTT AA-3′ and 5′-AGG AGG TTT TTC ATA ACT CGT GAT CT-3′) or humanized Renilla luciferase (5′-CCC CGA GCA ACG CAA AC-3′ and 5′-GCA CGT TCA TTT GCT TGC A-3′) and in 1x Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix (Agilent Technologies). The reaction and fluorescence readout were performed in Rotor-Gene 6200 (Corbett Life Science) real-time system. Using the Rotor-Gene 6200 software (Corbett, version 1.7) threshold cycle values (ct-values) were set manually and the relative mRNA levels were subsequently calculated using the comparative CT method.</p></sec><sec id=\"Sec21\"><title>Ribosome profiling</title><p id=\"Par33\">Ribosome profiling was performed as in ref. <sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>. Briefly, HeLa cells at 80% confluency were washed with ice-cold phosphate buffered saline (PBS) and flash-frozen in liquid nitrogen. Subsequently, cells were scraped and lysed in lysis buffer (20 mM Tris HCL pH7.4, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 1 mM DTT, 25 U/uL Turbo DNase, Turbo DNase buffer) on ice. Cells were then triturated ten times through a 27-gauge needle of a syringe and clarified by centrifugation. For ribosome profiling, lysates (about 4 U260) were subsequently treated with 200 U RNase I (Ambion) for 10 min at 23 °C and shaking at 300 rpm. The digestion was stopped by addition of 100 U SUPERase In RNase inhibitor (Ambion). Monosomes were separated on Illustra Micro-Spin S-400 HR gel filtration columns (GE Healthcare Life Science) as previously described<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>. TriReagent was added immediately to the eluates and samples were stored at −80 °C until further processing. RNA was isolated according to the TriReagent protocol and separated on 15% Novex polyacrylamide gels (Invitrogen). Ribosome footprints were excised between 26 and 34 nucleotide RNA size markers. After RNA isolation and purification, rRNAs were removed using the RiboZero kit (Illumina) according to manufacturer’s datasheet. RNA was isolated from cleared lysates by addition of TriReagent as for the ribosome-protected fragments. Total RNA was used for library generation with the TruSeq Stranded mRNA Library Prep Kit (Illumina) according to the manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq2500 generating 100 nt single-end reads. For compiling a list of NMD-sensitive transcripts we included the most abundantly expressed isoform under UPF1 KD treatment that originates from a previously identified list of NMD-sensitive genes<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. Metagene analysis of ribosome-protected footprints from HeLa cells was performed from three independent ribosome profiling experiments. 639 NMD-sensitive transcripts were defined as the most abundant isoforms of previously identified NMD-sensitive genes<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup> under UPF1 KD conditions. Mean ribosome occupancy at the stop codon was calculated relative to mean ribosome occupancy in CDS, performed for NMD targets (identified as in d) and all other transcripts (Others). Total counts of ribosome-derived reads mapping at the stop codon are plotted relative to the average of ribosome-derived reads aligning to the CDS for each biological replicate.</p></sec><sec id=\"Sec22\"><title>Reporting summary</title><p id=\"Par34\">Further information on research design is available in the <xref rid=\"MOESM3\" ref-type=\"media\">Nature Research Reporting Summary</xref> linked to this article.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec23\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41467_2020_17974_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM2\"><media xlink:href=\"41467_2020_17974_MOESM2_ESM.pdf\"><caption><p>Peer Review File</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"MOESM3\"><media xlink:href=\"41467_2020_17974_MOESM3_ESM.pdf\"><caption><p>Reporting Summary</p></caption></media></supplementary-material></sec></sec></body><back><app-group><app id=\"App1\"><sec id=\"Sec24\"><title>Source data</title><p id=\"Par37\"><media position=\"anchor\" xlink:href=\"41467_2020_17974_MOESM4_ESM.xlsx\" id=\"MOESM4\"><caption><p>Source Data</p></caption></media></p></sec></app></app-group><fn-group><fn><p><bold>Peer review information</bold>\n<italic>Nature Communications</italic> thanks the anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary information</bold> is available for this paper at 10.1038/s41467-020-17974-z.</p></sec><ack><title>Acknowledgements</title><p>We are grateful to Roland Beckmann (LMU Munich, Germany) and to Stefanie Metze for providing plasmids, to Lara Contu for proofreading of the manuscript, and to Asimina Gkratsou and Andrea Eberle for valuable discussions and advice. This work has been supported by the National Center of Competence in Research (NCCR) on RNA & Disease funded by the Swiss National Science Foundation (SNSF), by SNSF grants 31003A-162986 and 310030B-182831 to O.M., and by the canton of Bern (University intramural funding to O.M.).</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>O.M. and E.D.K. conceived the project and designed the experiments. E.D.K., L.A.G., and O.M. designed translation experiments, E.D.K. and L.G. were involved in cloning and E.D.K. performed in vitro translation and toeprint reactions with the help of L.A.G. L.A.G. and E.D.K. performed in vivo experiments and prepared lysates. G.A. conducted the ribosome profiling and R.D. performed the bioinformatics analysis. All authors contributed to the final version of the manuscript.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>Ribo-sequencing data that support the findings of this study have been deposited in the Gene Expression Ominibus (GEO) under accession numbers: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM4256659\">GSM4256659</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM4256660\">GSM4256660</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM4256661\">GSM4256661</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM4256665\">GSM4256665</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM4256666\">GSM4256666</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM4256667\">GSM4256667</ext-link> and total RNA sequencing data under UPF1 KD under accession numbers: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM4407914\">GSM4407914</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM4407915\">GSM4407915</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM4407916\">GSM4407916</ext-link>. All data supporting the findings of this study are available within the paper and its <xref rid=\"MOESM1\" ref-type=\"media\">Supplementary Information</xref> files. Source data for Figs. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b, c, e, <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>b, c, <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a–c, <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>b, c, <xref rid=\"Fig5\" ref-type=\"fig\">5b</xref>, Supplementary Fig. <xref rid=\"MOESM1\" ref-type=\"media\">1b, c, d</xref>, Supplementary Figs. <xref rid=\"MOESM1\" ref-type=\"media\">2a</xref>, b, <xref rid=\"MOESM1\" ref-type=\"media\">3</xref> were provided with the paper. All data is available from the corresponding author upon reasonable request. Source data are provided with this paper.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par35\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Peltz</surname><given-names>SW</given-names></name><name><surname>Brown</surname><given-names>aH</given-names></name><name><surname>Jacobson</surname><given-names>A</given-names></name></person-group><article-title>mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor</article-title><source>Genes Dev.</source><year>1993</year><volume>7</volume><fpage>1737</fpage><lpage>1754</lpage><pub-id pub-id-type=\"pmid\">8370523</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nasif</surname><given-names>S</given-names></name><name><surname>Contu</surname><given-names>L</given-names></name><name><surname>Mühlemann</surname><given-names>O</given-names></name></person-group><article-title>Beyond quality control: the role of nonsense-mediated mRNA decay (NMD) in regulating gene expression</article-title><source>Semin. Cell Dev. Biol.</source><year>2018</year><volume>75</volume><fpage>78</fpage><lpage>87</lpage><pub-id pub-id-type=\"pmid\">28866327</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lykke-Andersen</surname><given-names>S</given-names></name><name><surname>Jensen</surname><given-names>TH</given-names></name></person-group><article-title>Nonsense-mediated mRNA decay: an intricate machinery that shapes transcriptomes</article-title><source>Nat. Rev. Mol. Cell Biol.</source><year>2015</year><volume>16</volume><fpage>665</fpage><lpage>677</lpage><pub-id pub-id-type=\"pmid\">26397022</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Celik</surname><given-names>A</given-names></name><name><surname>Kervestin</surname><given-names>S</given-names></name><name><surname>Jacobson</surname><given-names>A</given-names></name></person-group><article-title>NMD: At the crossroads between translation termination and ribosome recycling</article-title><source>Biochimie</source><year>2015</year><volume>114</volume><fpage>2</fpage><lpage>9</lpage><pub-id pub-id-type=\"pmid\">25446649</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fernandes</surname><given-names>R</given-names></name><name><surname>Nogueira</surname><given-names>G</given-names></name><name><surname>da Costa</surname><given-names>PJ</given-names></name><name><surname>Pinto</surname><given-names>F</given-names></name><name><surname>Romão</surname><given-names>L</given-names></name></person-group><article-title>The mRNA metabolism in human disease</article-title><source>Adv. Exp. Med. Biol.</source><year>2019</year><volume>1157</volume><fpage>41</fpage><lpage>83</lpage><pub-id pub-id-type=\"pmid\">31342437</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kurosaki</surname><given-names>T</given-names></name><name><surname>Popp</surname><given-names>MW</given-names></name><name><surname>Maquat</surname><given-names>LE</given-names></name></person-group><article-title>Quality and quantity control of gene expression by nonsense-mediated mRNA decay</article-title><source>Nat. Rev. Mol. Cell Biol.</source><year>2019</year><volume>20</volume><fpage>406</fpage><lpage>420</lpage><pub-id pub-id-type=\"pmid\">30992545</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kishor</surname><given-names>A</given-names></name><name><surname>Fritz</surname><given-names>SE</given-names></name><name><surname>Hogg</surname><given-names>JR</given-names></name></person-group><article-title>Nonsense‐mediated mRNA decay: the challenge of telling right from wrong in a complex transcriptome</article-title><source>Wiley Interdiscip. Rev. RNA</source><year>2019</year><volume>10</volume><fpage>e1548</fpage><pub-id pub-id-type=\"pmid\">31131562</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>YKI</given-names></name><name><surname>Maquat</surname><given-names>LE</given-names></name></person-group><article-title>UPFront and center in RNA decay: UPF1 in nonsense-mediated mRNA decay and beyond</article-title><source>RNA</source><year>2019</year><volume>25</volume><fpage>407</fpage><lpage>422</lpage><pub-id pub-id-type=\"pmid\">30655309</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Durand</surname><given-names>S</given-names></name><name><surname>Lykke-Andersen</surname><given-names>J</given-names></name></person-group><article-title>Nonsense-mediated mRNA decay occurs during eIF4F-dependent translation in human cells</article-title><source>Nat. Struct. Mol. Biol.</source><year>2013</year><volume>20</volume><fpage>702</fpage><lpage>709</lpage><pub-id pub-id-type=\"pmid\">23665580</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rufener</surname><given-names>SC</given-names></name><name><surname>Mühlemann</surname><given-names>O</given-names></name></person-group><article-title>eIF4E-bound mRNPs are substrates for nonsense-mediated mRNA decay in mammalian cells</article-title><source>Nat. Struct. Mol. Biol.</source><year>2013</year><volume>20</volume><fpage>710</fpage><lpage>717</lpage><pub-id pub-id-type=\"pmid\">23665581</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morisaki</surname><given-names>T</given-names></name><etal/></person-group><article-title>Real-time quantification of single RNA translation dynamics in living cells</article-title><source>Sci.</source><year>2016</year><volume>352</volume><fpage>1425</fpage><lpage>1429</lpage></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hoek</surname><given-names>TA</given-names></name><etal/></person-group><article-title>Single-Molecule imaging uncovers rules governing nonsense-mediated mRNA decay</article-title><source>Mol. Cell</source><year>2019</year><volume>75</volume><fpage>324</fpage><lpage>339</lpage><pub-id pub-id-type=\"pmid\">31155380</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lopez-Perrote</surname><given-names>A</given-names></name><etal/></person-group><article-title>Human nonsense-mediated mRNA decay factor UPF2 interacts directly with eRF3 and the SURF complex</article-title><source>Nucleic Acids Res</source><year>2016</year><volume>44</volume><fpage>1909</fpage><lpage>1923</lpage><pub-id pub-id-type=\"pmid\">26740584</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kashima</surname><given-names>I</given-names></name><etal/></person-group><article-title>Binding of a novel SMG-1 – Upf1 – eRF1 – eRF3 complex (SURF) to the exon junction complex triggers Upf1 phosphorylation and nonsense-mediated mRNA</article-title><source>Genes Dev</source><year>2006</year><volume>20</volume><fpage>355</fpage><lpage>367</lpage><pub-id pub-id-type=\"pmid\">16452507</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Karousis</surname><given-names>ED</given-names></name><name><surname>Mühlemann</surname><given-names>O</given-names></name></person-group><article-title>Nonsense-mediated mRNA decay begins where translation ends</article-title><source>Cold Spring Harb. Perspect. Biol.</source><year>2019</year><volume>11</volume><fpage>a032862</fpage><pub-id pub-id-type=\"pmid\">29891560</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Neu‐Yilik</surname><given-names>G</given-names></name><etal/></person-group><article-title>Dual function of UPF3B in early and late translation termination</article-title><source>EMBO J.</source><year>2017</year><volume>36</volume><fpage>2968</fpage><lpage>2986</lpage><pub-id pub-id-type=\"pmid\">28899899</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schuller</surname><given-names>AP</given-names></name><name><surname>Green</surname><given-names>R</given-names></name></person-group><article-title>Roadblocks and resolutions in eukaryotic translation</article-title><source>Nat. Rev. Mol. Cell Biol.</source><year>2018</year><volume>19</volume><fpage>526</fpage><lpage>541</lpage><pub-id pub-id-type=\"pmid\">29760421</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Amrani</surname><given-names>N</given-names></name><etal/></person-group><article-title>A faux 3′-UTR promotes aberrant termination and triggers nonsense-mediated mRNA decay</article-title><source>Nature</source><year>2004</year><volume>432</volume><fpage>112</fpage><lpage>118</lpage><pub-id pub-id-type=\"pmid\">15525991</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Peixeiro</surname><given-names>I</given-names></name><etal/></person-group><article-title>Interaction of PABPC1 with the translation initiation complex is critical to the NMD resistance of AUG-proximal nonsense mutations</article-title><source>Nucleic Acids Res</source><year>2012</year><volume>40</volume><fpage>1160</fpage><lpage>1173</lpage><pub-id pub-id-type=\"pmid\">21989405</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Singh</surname><given-names>G</given-names></name><name><surname>Rebbapragada</surname><given-names>I</given-names></name><name><surname>Lykke-Andersen</surname><given-names>J</given-names></name></person-group><article-title>A competition between stimulators and antagonists of Upf complex recruitment governs human nonsense-mediated mRNA decay</article-title><source>PLoS Biol.</source><year>2008</year><volume>6</volume><fpage>e111</fpage><pub-id pub-id-type=\"pmid\">18447585</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eberle</surname><given-names>AB</given-names></name><name><surname>Stalder</surname><given-names>L</given-names></name><name><surname>Mathys</surname><given-names>H</given-names></name><name><surname>Orozco</surname><given-names>RZ</given-names></name><name><surname>Mühlemann</surname><given-names>O</given-names></name></person-group><article-title>Posttranscriptional gene regulation by spatial rearrangement of the 3′ untranslated region</article-title><source>PLoS Biol.</source><year>2008</year><volume>6</volume><fpage>e92</fpage><pub-id pub-id-type=\"pmid\">18447580</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mühlemann</surname><given-names>O</given-names></name><name><surname>Jensen</surname><given-names>TH</given-names></name></person-group><article-title>mRNP quality control goes regulatory</article-title><source>Trends Genet.</source><year>2012</year><volume>28</volume><fpage>70</fpage><lpage>77</lpage><pub-id pub-id-type=\"pmid\">22154474</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ivanov</surname><given-names>A</given-names></name><etal/></person-group><article-title>PABP enhances release factor recruitment and stop codon recognition during translation termination</article-title><source>Nucleic Acids Res</source><year>2016</year><volume>44</volume><fpage>7766</fpage><lpage>7776</lpage><pub-id pub-id-type=\"pmid\">27418677</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fatscher</surname><given-names>T</given-names></name><name><surname>Boehm</surname><given-names>V</given-names></name><name><surname>Weiche</surname><given-names>B</given-names></name><name><surname>Gehring</surname><given-names>NH</given-names></name></person-group><article-title>The interaction of cytoplasmic poly(A)-binding protein with eukaryotic initiation factor 4G suppresses nonsense-mediated mRNA decay</article-title><source>RNA</source><year>2014</year><volume>20</volume><fpage>1579</fpage><lpage>1592</lpage><pub-id pub-id-type=\"pmid\">25147240</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Joncourt</surname><given-names>R</given-names></name><name><surname>Eberle</surname><given-names>AB</given-names></name><name><surname>Rufener</surname><given-names>SC</given-names></name><name><surname>Mühlemann</surname><given-names>O</given-names></name></person-group><article-title>Eukaryotic initiation factor 4G suppresses nonsense-mediated mRNA decay by two genetically separable mechanisms</article-title><source>PLoS One</source><year>2014</year><volume>9</volume><fpage>e104391</fpage><pub-id pub-id-type=\"pmid\">25148142</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alkalaeva</surname><given-names>EZ</given-names></name><name><surname>Pisarev</surname><given-names>AV</given-names></name><name><surname>Frolova</surname><given-names>LY</given-names></name><name><surname>Kisselev</surname><given-names>LL</given-names></name><name><surname>Pestova</surname><given-names>TV</given-names></name></person-group><article-title>In vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3</article-title><source>Cell</source><year>2006</year><volume>125</volume><fpage>1125</fpage><lpage>1136</lpage><pub-id pub-id-type=\"pmid\">16777602</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pisarev</surname><given-names>AV</given-names></name><etal/></person-group><article-title>The role of ABCE1 in eukaryotic posttermination ribosomal recycling</article-title><source>Mol. Cell</source><year>2010</year><volume>37</volume><fpage>196</fpage><lpage>210</lpage><pub-id pub-id-type=\"pmid\">20122402</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rakotondrafara</surname><given-names>AM</given-names></name><name><surname>Hentze</surname><given-names>MW</given-names></name></person-group><article-title>An efficient factor-depleted mammalian in vitro translation system</article-title><source>Nat. Protoc.</source><year>2011</year><volume>6</volume><fpage>563</fpage><lpage>571</lpage><pub-id pub-id-type=\"pmid\">21527914</pub-id></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Heyer</surname><given-names>EE</given-names></name><name><surname>Moore</surname><given-names>MJ</given-names></name></person-group><article-title>Redefining the translational status of 80S monosomes</article-title><source>Cell</source><year>2016</year><volume>164</volume><fpage>757</fpage><lpage>769</lpage><pub-id pub-id-type=\"pmid\">26871635</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Degnin</surname><given-names>CR</given-names></name><name><surname>Schleiss</surname><given-names>MR</given-names></name><name><surname>Cao</surname><given-names>J</given-names></name><name><surname>Geballe</surname><given-names>AP</given-names></name></person-group><article-title>Translational inhibition mediated by a short upstream open reading frame in the human cytomegalovirus gpUL4 (gp48) transcript</article-title><source>J. Virol.</source><year>1993</year><volume>67</volume><fpage>5514</fpage><lpage>5521</lpage><pub-id pub-id-type=\"pmid\">8394459</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bhushan</surname><given-names>S</given-names></name><etal/></person-group><article-title>Structural basis for translational stalling by human cytomegalovirus and fungal arginine attenuator peptide</article-title><source>Mol. Cell</source><year>2010</year><volume>40</volume><fpage>138</fpage><lpage>146</lpage><pub-id pub-id-type=\"pmid\">20932481</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pulak</surname><given-names>R</given-names></name><name><surname>Anderson</surname><given-names>P</given-names></name></person-group><article-title>mRNA surveillance by the Caenorhabditis elegans smg genes</article-title><source>Genes Dev.</source><year>1993</year><volume>7</volume><fpage>1885</fpage><lpage>1897</lpage><pub-id pub-id-type=\"pmid\">8104846</pub-id></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hogg</surname><given-names>JR</given-names></name><name><surname>Goff</surname><given-names>SP</given-names></name></person-group><article-title>Upf1 senses 3′UTR length to potentiate mRNA decay</article-title><source>Cell</source><year>2010</year><volume>143</volume><fpage>379</fpage><lpage>389</lpage><pub-id pub-id-type=\"pmid\">21029861</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bühler</surname><given-names>M</given-names></name><name><surname>Steiner</surname><given-names>S</given-names></name><name><surname>Mohn</surname><given-names>F</given-names></name><name><surname>Paillusson</surname><given-names>A</given-names></name><name><surname>Mühlemann</surname><given-names>O</given-names></name></person-group><article-title>EJC-independent degradation of nonsense immunoglobulin-μ mRNA depends on 3′ UTR length</article-title><source>Nat. Struct. Mol. Biol.</source><year>2006</year><volume>13</volume><fpage>462</fpage><lpage>464</lpage><pub-id pub-id-type=\"pmid\">16622410</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Colombo</surname><given-names>M</given-names></name><name><surname>Karousis</surname><given-names>ED</given-names></name><name><surname>Bourquin</surname><given-names>J</given-names></name><name><surname>Bruggmann</surname><given-names>R</given-names></name><name><surname>Mühlemann</surname><given-names>O</given-names></name></person-group><article-title>Transcriptome-wide identification of NMD-targeted human mRNAs reveals extensive redundancy between SMG6- and SMG7-mediated degradation pathways</article-title><source>RNA</source><year>2017</year><volume>23</volume><fpage>189</fpage><lpage>201</lpage><pub-id pub-id-type=\"pmid\">27864472</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><mixed-citation publication-type=\"other\">Annibaldis, G., Dreos, R., Domanski, M., Carl, S. & Muhlemann, O. Ribosome recycling factor ABCE1 depletion inhibits nonsense-mediated mRNA decay by promoting stop codon readthrough. Preprint at 10.1101/870097 (2019).</mixed-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ingolia</surname><given-names>NT</given-names></name><name><surname>Lareau</surname><given-names>LF</given-names></name><name><surname>Weissman</surname><given-names>JS</given-names></name></person-group><article-title>Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes</article-title><source>Cell</source><year>2011</year><volume>147</volume><fpage>789</fpage><lpage>802</lpage><pub-id pub-id-type=\"pmid\">22056041</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Amrani</surname><given-names>N</given-names></name><name><surname>Sachs</surname><given-names>MS</given-names></name><name><surname>Jacobson</surname><given-names>A</given-names></name></person-group><article-title>Early nonsense: mRNA decay solves a translational problem</article-title><source>Nat. Rev. Mol. Cell Biol.</source><year>2006</year><volume>7</volume><fpage>415</fpage><lpage>425</lpage><pub-id pub-id-type=\"pmid\">16723977</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fritz</surname><given-names>SE</given-names></name><name><surname>Haque</surname><given-names>N</given-names></name><name><surname>Hogg</surname><given-names>JR</given-names></name></person-group><article-title>Highly efficient in vitro translation of authentic affinity-purified messenger ribonucleoprotein complexes</article-title><source>RNA</source><year>2018</year><volume>24</volume><fpage>982</fpage><lpage>989</lpage><pub-id pub-id-type=\"pmid\">29724884</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chong</surname><given-names>S</given-names></name></person-group><article-title>Overview of cell‐free protein synthesis: historic landmarks, commercial systems, and expanding applications</article-title><source>Curr. Protoc. Mol. Biol.</source><year>2014</year><volume>108</volume><fpage>16.30.1</fpage><lpage>16.30.11</lpage></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Silverman</surname><given-names>AD</given-names></name><name><surname>Karim</surname><given-names>AS</given-names></name><name><surname>Jewett</surname><given-names>MC</given-names></name></person-group><article-title>Cell-free gene expression: an expanded repertoire of applications</article-title><source>Nat. Rev. Genet.</source><year>2020</year><volume>21</volume><fpage>151</fpage><lpage>170</lpage><pub-id pub-id-type=\"pmid\">31780816</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brown</surname><given-names>A</given-names></name><name><surname>Shao</surname><given-names>S</given-names></name><name><surname>Murray</surname><given-names>J</given-names></name><name><surname>Hegde</surname><given-names>RS</given-names></name><name><surname>Ramakrishnan</surname><given-names>V</given-names></name></person-group><article-title>Structural basis for stop codon recognition in eukaryotes</article-title><source>Nature</source><year>2015</year><volume>524</volume><fpage>493</fpage><lpage>496</lpage><pub-id pub-id-type=\"pmid\">26245381</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pisareva</surname><given-names>VP</given-names></name><name><surname>Muslimov</surname><given-names>IA</given-names></name><name><surname>Tcherepanov</surname><given-names>A</given-names></name><name><surname>Pisarev</surname><given-names>AV</given-names></name></person-group><article-title>Characterization of novel ribosome-associated endoribonuclease SLFN14 from rabbit reticulocytes</article-title><source>Biochemistry</source><year>2015</year><volume>54</volume><fpage>3286</fpage><lpage>3301</lpage><pub-id pub-id-type=\"pmid\">25996083</pub-id></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Joazeiro</surname><given-names>CAP</given-names></name></person-group><article-title>Ribosomal stalling during translation: providing substrates for ribosome-associated protein quality control</article-title><source>Annu. Rev. Cell Dev. Biol.</source><year>2017</year><volume>33</volume><fpage>343</fpage><lpage>368</lpage><pub-id pub-id-type=\"pmid\">28715909</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><mixed-citation publication-type=\"other\">Heck, A. M. & Wilusz, J. The interplay between the RNA decay and translation machinery in eukaryotes. <italic>Cold Spring Harb. Perspect. Biol.</italic><bold>10</bold>, 263–282 (2018).</mixed-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shirokikh</surname><given-names>NE</given-names></name><etal/></person-group><article-title>Quantitative analysis of ribosome-mRNA complexes at different translation stages</article-title><source>Nucleic Acids Res</source><year>2010</year><volume>38</volume><fpage>e15</fpage><lpage>e15</lpage><pub-id pub-id-type=\"pmid\">19910372</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sachs</surname><given-names>MS</given-names></name><etal/></person-group><article-title>Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs</article-title><source>Methods</source><year>2002</year><volume>26</volume><fpage>105</fpage><lpage>114</lpage><pub-id pub-id-type=\"pmid\">12054887</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Presnyak</surname><given-names>V</given-names></name><etal/></person-group><article-title>Codon optimality is a major determinant of mRNA stability</article-title><source>Cell</source><year>2015</year><volume>160</volume><fpage>1111</fpage><lpage>1124</lpage><pub-id pub-id-type=\"pmid\">25768907</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Colin Chih-Chien</surname><given-names>Wu,A</given-names></name><etal/></person-group><article-title>High-Resolution Ribosome Profiling Defines Discrete Ribosome Elongation States and Translational Regulation during Cellular Stress</article-title><source>Mol. Cell</source><year>2019</year><volume>73</volume><fpage>959</fpage><lpage>970.e5</lpage><pub-id pub-id-type=\"pmid\">30686592</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Celik</surname><given-names>A</given-names></name><name><surname>Baker</surname><given-names>R</given-names></name><name><surname>He</surname><given-names>F</given-names></name><name><surname>Jacobson</surname><given-names>A</given-names></name></person-group><article-title>High-resolution profiling of NMD targets in yeast reveals translational fidelity as a basis for substrate selection</article-title><source>RNA</source><year>2017</year><volume>23</volume><fpage>735</fpage><lpage>748</lpage><pub-id pub-id-type=\"pmid\">28209632</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Celik</surname><given-names>A</given-names></name><name><surname>He</surname><given-names>F</given-names></name><name><surname>Jacobson</surname><given-names>A</given-names></name></person-group><article-title>NMD monitors translational fidelity 24/7</article-title><source>Curr. Genet.</source><year>2017</year><volume>63</volume><fpage>1007</fpage><lpage>1010</lpage><pub-id pub-id-type=\"pmid\">28536849</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kozak</surname><given-names>M</given-names></name></person-group><article-title>The scanning model for translation: an update</article-title><source>J. Cell Biol.</source><year>1989</year><volume>108</volume><fpage>229</fpage><lpage>241</lpage><pub-id pub-id-type=\"pmid\">2645293</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Paillusson</surname><given-names>A</given-names></name><name><surname>Hirschi</surname><given-names>N</given-names></name><name><surname>Vallan</surname><given-names>C</given-names></name><name><surname>Azzalin</surname><given-names>CM</given-names></name><name><surname>Mühlemann</surname><given-names>O</given-names></name></person-group><article-title>A GFP-based reporter system to monitor nonsense-mediated mRNA decay</article-title><source>Nucleic Acids Res.</source><year>2005</year><volume>33</volume><fpage>1</fpage><lpage>12</lpage><pub-id pub-id-type=\"pmid\">15640442</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eberle</surname><given-names>AB</given-names></name><name><surname>Stalder</surname><given-names>L</given-names></name><name><surname>Mathys</surname><given-names>H</given-names></name><name><surname>Orozco</surname><given-names>RZ</given-names></name><name><surname>Mühlemann</surname><given-names>O</given-names></name></person-group><article-title>Posttranscriptional gene regulation by spatial rearrangement of the 3′ untranslated region</article-title><source>PLoS Biol.</source><year>2008</year><volume>6</volume><fpage>e92</fpage><pub-id pub-id-type=\"pmid\">18447580</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><mixed-citation publication-type=\"other\">Nicholson, P., Joncourt, R. & Mühlemann, O. Analysis of nonsense-mediated mRNA decay in mammalian cells. <italic>Current Protocols in Cell Biology</italic><bold>27</bold> 1–61 (2012).</mixed-citation></ref><ref id=\"CR56\"><label>56.</label><mixed-citation publication-type=\"other\">Aeschimann, F et al. Transcriptome-wide measurement of ribosomal occupancy by ribosome profiling. <italic>Methods</italic><bold>85</bold>, 75–89 (2015) </mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Nat Commun</journal-id><journal-id journal-id-type=\"iso-abbrev\">Nat Commun</journal-id><journal-title-group><journal-title>Nature Communications</journal-title></journal-title-group><issn pub-type=\"epub\">2041-1723</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807796</article-id><article-id pub-id-type=\"pmc\">PMC7431591</article-id><article-id pub-id-type=\"publisher-id\">17919</article-id><article-id pub-id-type=\"doi\">10.1038/s41467-020-17919-6</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Demonstration of chip-based coupled degenerate optical parametric oscillators for realizing a nanophotonic spin-glass</article-title></title-group><contrib-group><contrib contrib-type=\"author\" equal-contrib=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-9639-0549</contrib-id><name><surname>Okawachi</surname><given-names>Yoshitomo</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\" equal-contrib=\"yes\"><name><surname>Yu</surname><given-names>Mengjie</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Jang</surname><given-names>Jae K.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-0284-0818</contrib-id><name><surname>Ji</surname><given-names>Xingchen</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhao</surname><given-names>Yun</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0002-9891-6206</contrib-id><name><surname>Kim</surname><given-names>Bok Young</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0003-2903-3765</contrib-id><name><surname>Lipson</surname><given-names>Michal</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><contrib-id contrib-id-type=\"orcid\">http://orcid.org/0000-0001-6877-7316</contrib-id><name><surname>Gaeta</surname><given-names>Alexander L.</given-names></name><address><email>a.gaeta@columbia.edu</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.21729.3f</institution-id><institution-id institution-id-type=\"ISNI\">0000000419368729</institution-id><institution>Department of Applied Physics and Applied Mathematics, </institution><institution>Columbia University, </institution></institution-wrap>New York, NY 10027 USA </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.5386.8</institution-id><institution-id institution-id-type=\"ISNI\">000000041936877X</institution-id><institution>School of Electrical and Computer Engineering, </institution><institution>Cornell University, </institution></institution-wrap>Ithaca, NY 14853 USA </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.21729.3f</institution-id><institution-id institution-id-type=\"ISNI\">0000000419368729</institution-id><institution>Department of Electrical Engineering, </institution><institution>Columbia University, </institution></institution-wrap>New York, NY 10027 USA </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>4119</elocation-id><history><date date-type=\"received\"><day>6</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>22</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">The need for solving optimization problems is prevalent in various physical applications, including neuroscience, network design, biological systems, socio-economics, and chemical reactions. Many of these are classified as non-deterministic polynomial-time hard and thus become intractable to solve as the system scales to a large number of elements. Recent research advances in photonics have sparked interest in using a network of coupled degenerate optical parametric oscillators (DOPOs) to effectively find the ground state of the Ising Hamiltonian, which can be used to solve other combinatorial optimization problems through polynomial-time mapping. Here, using the nanophotonic silicon-nitride platform, we demonstrate a spatial-multiplexed DOPO system using continuous-wave pumping. We experimentally demonstrate the generation and coupling of two microresonator-based DOPOs on a single chip. Through a reconfigurable phase link, we achieve both in-phase and out-of-phase operation, which can be deterministically achieved at a fast regeneration speed of 400 kHz with a large phase tolerance.</p></abstract><abstract id=\"Abs2\" abstract-type=\"web-summary\"><p id=\"Par2\">The use of a photonic network of coupled degenerate optical parametric oscillators for solving complex optimisation problems would require scalable integration capabilities. Here, the authors exploit <italic>χ</italic> (3) nonlinearity in SiN to demonstrate on-chip phase-tunable coupling between two DOPO based Ising nodes.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Integrated optics</kwd><kwd>Nonlinear optics</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type=\"FundRef\">https://doi.org/10.13039/100000181</institution-id><institution>United States Department of Defense | United States Air Force | AFMC | Air Force Office of Scientific Research (AF Office of Scientific Research)</institution></institution-wrap></funding-source><award-id>FA9550-15-1-0303</award-id><principal-award-recipient><name><surname>Kim</surname><given-names>Bok Young</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\" sec-type=\"introduction\"><title>Introduction</title><p id=\"Par3\">The processing speed of modern computers is limited by the fact that program memory and data memory share the same bus. While processors have become faster, the overall speed is limited by the data transfer rate, known as the von Neumann bottleneck<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref></sup>. There has been an increase in demand for solving certain classes of computation problems that scale exponentially in time and energy for the current von Neumann architecture. Many combinatorial optimization problems fall under this category and are classified as non-deterministic polynomial-time (NP) hard and have applications in areas including finance, scheduling, trajectory planning, and artificial intelligence. The ability to solve these problems efficiently has motivated the development of computing accelerators based on digital and physical systems<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>–<xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>.</p><p id=\"Par4\">Recently, there has been considerable interest in using photonic processors to realize a novel form of coherent computing by simulating the Ising model<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR20\">20</xref>–<xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. The Ising model was developed for modeling ferromagnetism and is governed by a Hamiltonian that couples discrete variables that represent spin glasses<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. Solving for the ground state of such a system corresponds to solving an NP-hard computational problem and can provide an architecture for solving other NP-complete problems through polynomial-time mapping to the Ising model<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>. The Ising Hamiltonian with <italic>N</italic> spins and no external field is given by <inline-formula id=\"IEq1\"><alternatives><tex-math id=\"M1\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$$H=-\\mathop{\\sum }\\nolimits_{ij}^{N}{J}_{ij}{\\sigma }_{i}{\\sigma }_{j},$$\\end{document}</tex-math><mml:math id=\"M2\"><mml:mi>H</mml:mi><mml:mo>=</mml:mo><mml:mo>−</mml:mo><mml:msubsup><mml:mrow><mml:mo>∑</mml:mo></mml:mrow><mml:mrow><mml:mi>i</mml:mi><mml:mi>j</mml:mi></mml:mrow><mml:mrow><mml:mi>N</mml:mi></mml:mrow></mml:msubsup><mml:msub><mml:mrow><mml:mi>J</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi><mml:mi>j</mml:mi></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>σ</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi></mml:mrow></mml:msub><mml:msub><mml:mrow><mml:mi>σ</mml:mi></mml:mrow><mml:mrow><mml:mi>j</mml:mi></mml:mrow></mml:msub><mml:mo>,</mml:mo></mml:math><inline-graphic xlink:href=\"41467_2020_17919_Article_IEq1.gif\"/></alternatives></inline-formula> where <italic>J</italic><sub><italic>i</italic><italic>j</italic></sub> is the coupling coefficient and <italic>σ</italic><sub><italic>i</italic></sub> corresponds to the projection of the <italic>i</italic>th spin along the <italic>z</italic>-axis that can have two states  ±1. The physical realization of an Ising machine requires binary degrees of freedom (i.e.,  spins <italic>σ</italic><sub><italic>i</italic></sub>) and reconfigurable coupling (i.e., <italic>J</italic><sub><italic>i</italic><italic>j</italic></sub>), and initially was studied using a network of injection-locked lasers<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>–<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. More recently, investigations have shown that a network of coupled degenerate optical parametric oscillators (DOPOs) based on the <italic>χ</italic><sup>(2)</sup> nonlinearity can be used to realize a hybrid temporally multiplexed coherent Ising machine<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>–<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>, which includes a recent demonstration of a system of 2000 spins<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. The nonlinearity is based on the nonequilibrium phase transition that occurs at the parametric oscillation threshold, resulting in two possible phase states of the DOPO offset by <italic>π</italic>, and the couplings between the DOPOs are implemented via measurement feedback or optical delay lines. By controlling the coupling between these DOPOs, it is possible to achieve more complex, phase-locked output states that encode the ground state of an Ising model. These demonstrations have utilized a time-multiplexed DOPO system using a 1-km-long fiber ring cavity to simulate the ground state of the Ising model<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>,<xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup>. In addition, extensive experimental and theoretical analysis has been done to characterize the potential performance of such systems<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR33\">33</xref>–<xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. Furthermore, alternative approaches towards a coherent Ising machine, such as opto-electronic oscillators with self-feedback<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>–<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup> and spatial light modulation<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, has been demonstrated and an approach using a dispersive optical bistability has been proposed<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup>.</p><p id=\"Par5\">An alternative approach to realize a DOPO is to use the <italic>χ</italic><sup>(3)</sup> nonlinearity in which a frequency degenerate signal/idler pair is generated via parametric four-wave mixing (FWM)<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref>–<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. Such a scheme has been implemented using silicon nitride (SiN, Si<sub>3</sub>N<sub>4</sub>) microresonators and bi-phase state generation has been achieved enabling quantum random-number generation in a chip-scale device (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref>,<xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. The SiN platform is ideally suited for scalability to an all-photonic network of coupled DOPOs since it is CMOS (complementary metal–oxide–semiconductor) process compatible, has low losses in the near infrared, and allows for dispersion engineering, which is crucial for efficient phase-matched nonlinear processes<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. Unlike the <italic>χ</italic><sup>(2)</sup> process, the wavelengths of the pump and degenerate signal are spectrally close, allowing for phase matching of the signal through dispersion engineering of the fundamental waveguide mode. In addition, unlike traditional FWM, which requires operation in the anomalous group-velocity dispersion (GVD) regime, the DOPO requires normal GVD for phase matching<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>, which is more readily accessible across a wider range of photonic platforms. Furthermore, the microresonator-based DOPO system allows for simultaneous oscillation of all DOPOs, enables continuous-wave (cw) operation, and does not rely on long cavity lengths as in the time-multiplexing scheme, which requires phase stabilization to support multiple trains of femtosecond pulses, offering faster computational speeds with lower power consumption in a compact footprint. A recent preliminary study has also reported on numerical simulation of coupled Lugiato–Lefever equations to tackle the MAX-CUT problem, further demonstrating the potential capability of SiN platform for optical computing<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>.<fig id=\"Fig1\"><label>Fig. 1</label><caption><title>Conceptual schematic of a network of coupled degenerate optical parametric oscillators.</title><p>Two-frequency nondegenerate pumps are injected into the network of coupled degenerate optical parametric oscillators (DOPOs) based on silicon nitride (SiN) microresonators. The pumps are split on-chip using multimode interference (MMI) splitters. The DOPO signal is phase matched and generated via four-wave mixing parametric oscillation. Phase-matching conditions result in a bi-phase state for the generated signal. Coupling between DOPOs can be performed using a matrix of reconfigurable Mach–Zehnder interferometers (MZIs).</p></caption><graphic xlink:href=\"41467_2020_17919_Fig1_HTML\" id=\"d30e550\"/></fig></p><p id=\"Par6\">In this paper, we demonstrate an integrated photonic circuit that consists of spatial-multiplexed DOPOs on a single SiN chip and present the first demonstration of a coupled DOPO system using cw pumping. We experimentally show reconfigurability of the coupling phase between the two DOPOs through thermal control of the coupling waveguide between the DOPOs and show interference measurements between the DOPOs indicating in-phase and out-of-phase operation. In our system, we solve at a 400-kHz rate with a convergence time of  <310 ns. In addition, we numerically model the coupled DOPO system and confirm the behavior for in-phase and out-of-phase operation and explore the transition region between the two phase states. A distinct transition region between two states is revealed both numerically and experimentally, suggesting a tremendous phase tolerance of such parametric process. We also theoretically investigate our coupled DOPO system using coupled Lugiato–Lefever equations and describe the scalability of such a system to a large number of oscillators on-chip and the challenges in achieving a large-scale photonic coherent Ising machine.</p></sec><sec id=\"Sec2\" sec-type=\"results\"><title>Results</title><sec id=\"Sec3\"><title>Device characterization and experimental setup</title><p id=\"Par7\">The key components for realizing a network of coupled DOPOs on-chip are, (1) power splitters for routing the pump fields to the microresonators, (2) nonlinear microresonators designed for DOPO generation, and (3) the <italic>N</italic> × <italic>N</italic> photonic coupling system between the different microresonators. A microscope image of the SiN device is shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>a and is fabricated using techniques similar to those reported in Luke et al.<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. For routing the two-frequency nondegenerate pumps to the microresonators, we employ an on-chip power splitter using multimode interference (MMI)<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>, where the dimensions of the MMI are designed to allow for 50/50 power splitting ratio. The insertion loss of the MMI splitter is 2 dB. The two SiN microresonators (DOPO<sub>1</sub> and DOPO<sub>2</sub>) have a radius of 45.84 μm, which corresponds to a free spectral range (FSR) of 500 GHz. The loaded quality (<italic>Q</italic>) factors of the microresonators are 630,000. The condition <italic>L</italic><sub>D</sub> > <italic>L</italic><sub>NL</sub>, where <italic>L</italic><sub>D</sub> = 1/<italic>δ</italic><sup>2</sup>∣<italic>β</italic><sub>2</sub>∣ is the dispersion length and <italic>L</italic><sub>NL</sub> = 1/2<italic>γ</italic><italic>P</italic> is the nonlinear length, is critical for achieving maximum gain at the frequency degeneracy point and enabling pure DOPO generation (see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">1</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. Here, <italic>β</italic><sub>2</sub> is the GVD parameter, <italic>γ</italic> is the nonlinear parameter, <italic>P</italic> is the power of each pump, and <italic>δ</italic> is the pump frequency offset. Based on simulations using a finite-element mode solver, we use a waveguide cross section of 730 × 1050 nm<sup>2</sup> such that the two pumps are placed in the normal GVD regime for the fundamental transverse electric (TE) mode to allow for efficient phase matching and maximum gain at the frequency degeneracy point<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref></sup>. The simulated GVD is shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>b, and the corresponding gain for 1 W of pump power is shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>c. The cavity resonance for each microresonator is thermally controlled using integrated platinum resistive microheaters. The coupling between the microresonator and the bus waveguide is designed to have near-critical coupling, where the extinction ratio of the resonances is ~92%. To compensate for the difference in the resonance frequencies of the two resonators due to the fabrication tolerances in microresonator geometry, we use microheaters above each resonator that allow for electrical control of the resonances via the thermo-optic effect<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref>,<xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>. We set the electrical power to the heaters such that the cavity resonances for the two microresonators corresponding to both pump frequencies overlap. We implement reconfigurable unidirectional coupling between the two DOPOs by using a coupling waveguide that directs a fraction of the DOPO<sub>1</sub> output field to the input of DOPO<sub>2</sub>. The coupling strength of the coupling waveguide is adjusted by designing the separation between the bus waveguide and coupling waveguide. In our device, the ratio between the coupled field from DOPO<sub>1</sub> and the DOPO<sub>2</sub> field is 0.048 (see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">2</xref>). In order to minimize reflections, we have implemented a taper design on the end facets. We tune the phase coupling between the two DOPOs from in-phase to out-of-phase by thermally tuning the path length using microheaters.<fig id=\"Fig2\"><label>Fig. 2</label><caption><title>Experimental schematic of coupled DOPOs.</title><p><bold>a</bold> Microscope image of the device. The pump waves are split on-chip and sent to DOPO<sub>1</sub> (bottom) and DOPO<sub>2</sub> (top). A coupling waveguide after DOPO<sub>1</sub> is used to send a fraction of the DOPO<sub>1</sub> field to DOPO<sub>2</sub>. <bold>b</bold> Simulated group-velocity dispersion (GVD) of the SiN microresonator for the fundamental transverse electric (TE) mode. The waveguide cross section is 730 × 1050 nm<sup>2</sup>. The region of normal GVD is shaded, and the pump wavelengths are indicated with vertical lines. <bold>c</bold> Calculated parametric gain for 1 W of combined pump power. The pump waves are each located two free spectral ranges (FSRs) from the degeneracy point. <bold>d</bold> Experimental setup for measurement of coupled DOPO system. Two-frequency nondegenerate pumps are sent into the SiN chip. The output is collected using an aspheric lens and sent to a free-space interferometer to measure the interference signal. EDFA erbium-doped fiber amplifier, BPF bandpass filter, AOM acousto-optic modulator, FPC fiber polarization controller, MMI multimode interference splitter, OSA optical spectrum analyzer.</p></caption><graphic xlink:href=\"41467_2020_17919_Fig2_HTML\" id=\"d30e724\"/></fig></p><p id=\"Par8\">  Figure <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>d shows the experimental setup for generation and detection of the coupled DOPO signal. The SiN chip is pumped using two-frequency nondegenerate cw pump lasers that are offset by  ±2 FSRs from the degeneracy point at wavelengths of 1543 and 1559.1 nm. In order to ensure that the DOPO builds up from noise for each initiation, we modulate one of the pumps using an acousto-optic modulator (AOM) with 310-ns pulses at a repetition rate of 400 kHz. For simultaneous degenerate oscillation for both DOPOs, we use 72 mW of combined pump power in each bus waveguide and set the electrical power to the heater in DOPO<sub>1</sub> to 13.5 mW such that the resonances of the two microresonators are spectrally overlapped. The two DOPO outputs from the chip are collimated using an aspheric lens and sent to our detection setup [dashed red box in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>a].</p></sec><sec id=\"Sec4\"><title>Phase characterization of coupled DOPOs</title><p id=\"Par9\">The readout of the coherent phase states of the coupled DOPO system is implemented by directly measuring the interference between the two DOPOs by coupling both outputs into a single fiber collimator and detecting the combined signal on a fast photodiode. The collimated outputs from the DOPOs are combined using a 50/50 beamsplitter and fiber coupled using a collimator. Before combining the beams, a 50/50 beamsplitter is used in each output arm to collect the individual DOPO signals. The signals are each fiber coupled and a 90/10 coupler is used to monitor the time trace and the optical spectrum. The generated DOPO spectra from the microresonators are shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>. In our interference measurement, we manipulate the coupling phase <italic>ϕ</italic><sub>c</sub> between the DOPOs below threshold by controlling the electrical power sent to the integrated heater while monitoring the time trace. Figure <xref rid=\"Fig4\" ref-type=\"fig\">4</xref> shows the measured temporal interference signal (green) along with DOPO<sub>1</sub> (red) and DOPO<sub>2</sub> (blue), for three different heater powers. At 48.3 mW, we observe constructive interference between the two DOPOs, corresponding to in-phase operation (<italic>ϕ</italic><sub>out</sub> = 0) [Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref> (top)]. For 54.2 mW of heater power, we observe destructive interference [Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref> (middle)] corresponding to out-of-phase operation (<italic>ϕ</italic><sub>out</sub> = <italic>π</italic>). The transition from in-phase to out-of-phase operation occurs at 50.2 mW of heater power, and we observe both constructive and destructive interference [Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref> (bottom)]. Moreover, we observe in-phase operation (<italic>ϕ</italic><sub>out</sub> = 0) for heater powers below 48.3 mW and out-of-phase operation (<italic>ϕ</italic><sub>out</sub> = <italic>π</italic>) for powers above 54.2 mW, indicating that the DOPOs above threshold operate in-phase or out-of-phase for a continuous range of coupling phases <italic>ϕ</italic><sub>c</sub>. The convergence time of the DOPO is well within the pump pulse duration of 310 ns. For comparison, we perform interference measurements on a similar device with no coupling waveguide between DOPO<sub>1</sub> and DOPO<sub>2</sub> and verify that there is no phase correlation between the two signals (see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">3</xref>).<fig id=\"Fig3\"><label>Fig. 3</label><caption><title>Optical spectra of two DOPO signals.</title><p>The measured spectra of the primary DOPO (top, DOPO<sub>1</sub>) and the secondary DOPO (bottom, DOPO<sub>2</sub>).</p></caption><graphic xlink:href=\"41467_2020_17919_Fig3_HTML\" id=\"d30e826\"/></fig><fig id=\"Fig4\"><label>Fig. 4</label><caption><title>Phase characterization of coupled DOPOs.</title><p>Temporal ensemble of interference measurements of the combined DOPOs (green) along with measurements of DOPO<sub>1</sub> (red) and DOPO<sub>2</sub> (blue). Inset shows the phase chart of the coupling phase <italic>ϕ</italic><sub>c</sub>. We indicate the coupled DOPO operation regime for in-phase (pink), out-of-phase (blue), and transition regime (gray). <bold>a</bold> Constructive interference (in-phase, <italic>ϕ</italic><sub>out</sub> = 0) is observed for a heater power of 48.3 mW. <bold>b</bold> Destructive interference (out-of-phase <italic>ϕ</italic><sub>out</sub> = <italic>π</italic>) is observed for a heater power of 54.2 mW. <bold>c</bold> Transition from in-phase to out-of-phase operation for heater power of 50.2 mW. For clarity, DOPO<sub>2</sub> and the interference are offset from DOPO<sub>1</sub> in power by 40 and 80, respectively.</p></caption><graphic xlink:href=\"41467_2020_17919_Fig4_HTML\" id=\"d30e878\"/></fig></p></sec><sec id=\"Sec5\"><title>Theoretical analysis of coupled DOPO system</title><p id=\"Par10\">We theoretically investigate this coupled DOPO system using coupled Lugiato–Lefever equations with two pump waves<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref>,<xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>,<disp-formula id=\"Equ1\"><label>1</label><alternatives><tex-math id=\"M3\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${t}_{{\\rm{R}}}\\frac{\\partial {E}_{1}}{\\partial t}\\,=\t\\, \\left[-\\alpha -i{\\delta }_{0}-{\\delta }_{1}\\frac{\\partial }{\\partial \\tau }+iL{\\mathop {\\sum}\\limits_{k\\ge 2}}\\frac{{\\beta }_{k}}{k!}{\\left(i\\frac{\\partial }{\\partial \\tau }\\right)}^{k}+i\\gamma L| {E}_{1}(t,\\tau ){| }^{2}\\right]{E}_{1}(t,\\tau )\\\\ \t+\\sqrt{\\theta }{A}_{{\\rm{in}}}\\left({e}^{-i{\\Omega }_{0}\\tau }+{e}^{i{\\Omega }_{0}\\tau }\\right),$$\\end{document}</tex-math><mml:math id=\"M4\"><mml:msub><mml:mrow><mml:mi>t</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">R</mml:mi></mml:mrow></mml:msub><mml:mfrac><mml:mrow><mml:mi>∂</mml:mi><mml:msub><mml:mrow><mml:mi>E</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:mi>∂</mml:mi><mml:mi>t</mml:mi></mml:mrow></mml:mfrac><mml:mspace width=\"0.25em\"/><mml:mo>=</mml:mo><mml:mspace width=\"0.25em\"/><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mo>−</mml:mo><mml:mi>α</mml:mi><mml:mo>−</mml:mo><mml:mi>i</mml:mi><mml:msub><mml:mrow><mml:mi>δ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>δ</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mfrac><mml:mrow><mml:mi>∂</mml:mi></mml:mrow><mml:mrow><mml:mi>∂</mml:mi><mml:mi>τ</mml:mi></mml:mrow></mml:mfrac><mml:mo>+</mml:mo><mml:mi>i</mml:mi><mml:mi>L</mml:mi><mml:munder><mml:mrow><mml:mo>∑</mml:mo></mml:mrow><mml:mrow><mml:mi>k</mml:mi><mml:mo>≥</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:munder><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mi>β</mml:mi></mml:mrow><mml:mrow><mml:mi>k</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:mi>k</mml:mi><mml:mo>!</mml:mo></mml:mrow></mml:mfrac><mml:msup><mml:mrow><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>i</mml:mi><mml:mfrac><mml:mrow><mml:mi>∂</mml:mi></mml:mrow><mml:mrow><mml:mi>∂</mml:mi><mml:mi>τ</mml:mi></mml:mrow></mml:mfrac></mml:mrow></mml:mfenced></mml:mrow><mml:mrow><mml:mi>k</mml:mi></mml:mrow></mml:msup><mml:mo>+</mml:mo><mml:mi>i</mml:mi><mml:mi>γ</mml:mi><mml:mi>L</mml:mi><mml:mo>∣</mml:mo><mml:msub><mml:mrow><mml:mi>E</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>t</mml:mi><mml:mo>,</mml:mo><mml:mi>τ</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:msup><mml:mrow><mml:mo>∣</mml:mo></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:mfenced><mml:msub><mml:mrow><mml:mi>E</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>t</mml:mi><mml:mo>,</mml:mo><mml:mi>τ</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>+</mml:mo><mml:msqrt><mml:mrow><mml:mi>θ</mml:mi></mml:mrow></mml:msqrt><mml:msub><mml:mrow><mml:mi>A</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">in</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:msup><mml:mrow><mml:mi>e</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mi>i</mml:mi><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">Ω</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mi>τ</mml:mi></mml:mrow></mml:msup><mml:mo>+</mml:mo><mml:msup><mml:mrow><mml:mi>e</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">Ω</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mi>τ</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:mfenced><mml:mo>,</mml:mo></mml:math><graphic xlink:href=\"41467_2020_17919_Article_Equ1.gif\" position=\"anchor\"/></alternatives></disp-formula><disp-formula id=\"Equ2\"><label>2</label><alternatives><tex-math id=\"M5\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${t}_{{\\rm{R}}}\\frac{\\partial {E}_{2}}{\\partial t}\\,=\t\\, \\left[-\\alpha -i{\\delta }_{0}-{\\delta }_{1}\\frac{\\partial }{\\partial \\tau }+iL{\\mathop {\\sum}\\limits_{k\\ge 2}}\\frac{{\\beta }_{k}}{k!}{\\left(i\\frac{\\partial }{\\partial \\tau }\\right)}^{k}+i\\gamma L| {E}_{2}(t,\\tau ){| }^{2}\\right]{E}_{2}(t,\\tau )\\\\ \t+\\sqrt{\\theta }{A}_{{\\rm{in}}}\\left({e}^{-i{\\Omega }_{0}\\tau }+{e}^{i{\\Omega }_{0}\\tau }\\right) \\ +\\kappa {E}_{1}(t,\\tau ),$$\\end{document}</tex-math><mml:math id=\"M6\"><mml:msub><mml:mrow><mml:mi>t</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">R</mml:mi></mml:mrow></mml:msub><mml:mfrac><mml:mrow><mml:mi>∂</mml:mi><mml:msub><mml:mrow><mml:mi>E</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:mi>∂</mml:mi><mml:mi>t</mml:mi></mml:mrow></mml:mfrac><mml:mspace width=\"0.25em\"/><mml:mo>=</mml:mo><mml:mspace width=\"0.25em\"/><mml:mfenced close=\"]\" open=\"[\"><mml:mrow><mml:mo>−</mml:mo><mml:mi>α</mml:mi><mml:mo>−</mml:mo><mml:mi>i</mml:mi><mml:msub><mml:mrow><mml:mi>δ</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mo>−</mml:mo><mml:msub><mml:mrow><mml:mi>δ</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mfrac><mml:mrow><mml:mi>∂</mml:mi></mml:mrow><mml:mrow><mml:mi>∂</mml:mi><mml:mi>τ</mml:mi></mml:mrow></mml:mfrac><mml:mo>+</mml:mo><mml:mi>i</mml:mi><mml:mi>L</mml:mi><mml:munder><mml:mrow><mml:mo>∑</mml:mo></mml:mrow><mml:mrow><mml:mi>k</mml:mi><mml:mo>≥</mml:mo><mml:mn>2</mml:mn></mml:mrow></mml:munder><mml:mfrac><mml:mrow><mml:msub><mml:mrow><mml:mi>β</mml:mi></mml:mrow><mml:mrow><mml:mi>k</mml:mi></mml:mrow></mml:msub></mml:mrow><mml:mrow><mml:mi>k</mml:mi><mml:mo>!</mml:mo></mml:mrow></mml:mfrac><mml:msup><mml:mrow><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:mi>i</mml:mi><mml:mfrac><mml:mrow><mml:mi>∂</mml:mi></mml:mrow><mml:mrow><mml:mi>∂</mml:mi><mml:mi>τ</mml:mi></mml:mrow></mml:mfrac></mml:mrow></mml:mfenced></mml:mrow><mml:mrow><mml:mi>k</mml:mi></mml:mrow></mml:msup><mml:mo>+</mml:mo><mml:mi>i</mml:mi><mml:mi>γ</mml:mi><mml:mi>L</mml:mi><mml:mo>∣</mml:mo><mml:msub><mml:mrow><mml:mi>E</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>t</mml:mi><mml:mo>,</mml:mo><mml:mi>τ</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:msup><mml:mrow><mml:mo>∣</mml:mo></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msup></mml:mrow></mml:mfenced><mml:msub><mml:mrow><mml:mi>E</mml:mi></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>t</mml:mi><mml:mo>,</mml:mo><mml:mi>τ</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>+</mml:mo><mml:msqrt><mml:mrow><mml:mi>θ</mml:mi></mml:mrow></mml:msqrt><mml:msub><mml:mrow><mml:mi>A</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">in</mml:mi></mml:mrow></mml:msub><mml:mfenced close=\")\" open=\"(\"><mml:mrow><mml:msup><mml:mrow><mml:mi>e</mml:mi></mml:mrow><mml:mrow><mml:mo>−</mml:mo><mml:mi>i</mml:mi><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">Ω</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mi>τ</mml:mi></mml:mrow></mml:msup><mml:mo>+</mml:mo><mml:msup><mml:mrow><mml:mi>e</mml:mi></mml:mrow><mml:mrow><mml:mi>i</mml:mi><mml:msub><mml:mrow><mml:mi mathvariant=\"normal\">Ω</mml:mi></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msub><mml:mi>τ</mml:mi></mml:mrow></mml:msup></mml:mrow></mml:mfenced><mml:mspace width=\"0.33em\"/><mml:mo>+</mml:mo><mml:mi>κ</mml:mi><mml:msub><mml:mrow><mml:mi>E</mml:mi></mml:mrow><mml:mrow><mml:mn>1</mml:mn></mml:mrow></mml:msub><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mi>t</mml:mi><mml:mo>,</mml:mo><mml:mi>τ</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow><mml:mo>,</mml:mo></mml:math><graphic xlink:href=\"41467_2020_17919_Article_Equ2.gif\" position=\"anchor\"/></alternatives></disp-formula>where <italic>t</italic><sub>R</sub> is the roundtrip time in the resonator, <italic>α</italic> is the total roundtrip loss, <italic>δ</italic><sub>0</sub> is the effective phase detuning, <italic>δ</italic><sub>1</sub> is the mode-dependent detuning, Ω<sub>0</sub> corresponds to the pump offset frequency from the degeneracy point, <italic>θ</italic> is the transmission coefficient between the resonator and the bus waveguide, <italic>L</italic> is the cavity length, <italic>γ</italic> is the nonlinear parameter, and <italic>β</italic><sub><italic>k</italic></sub> corresponds to the <italic>k</italic>th-order dispersion coefficients of the Taylor expansion of the propagation constant. Here, <italic>τ</italic> represents the temporal coordinate within the time scale of a single round trip and <italic>t</italic> represents the long-time-scale evolution over many round trips. The term with <italic>A</italic><sub>in</sub> describes the bichromatic pump waves, and <italic>κ</italic> represents the complex coupling coefficient from the primary DOPO (DOPO<sub>1</sub>) to the secondary DOPO (DOPO<sub>2</sub>) (see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">4</xref>). We add a noise of one photon per spectral mode with random phase onto the pump<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>. The simulation parameters are similar to that of our experiment, including the coupling between the DOPOs being unidirectional. Our simulations (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>) show oscillation behavior for three different values of the coupling phase <italic>ϕ</italic><sub>c</sub>. For <italic>ϕ</italic><sub>c</sub> = 0°, constructive interference between the DOPOs is favored [Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref> (top)], indicating that the DOPOs oscillate in-phase. We observe similar in-phase behavior for  −70° < <italic>ϕ</italic><sub>c</sub> < 70°. In contrast, for <italic>ϕ</italic><sub>c</sub> = 180°, we observe destructive interference [Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref> (middle)] from the DOPOs oscillating <italic>π</italic> out-of-phase. Likewise, similar out-of-phase behavior is observed for 110° < <italic>ϕ</italic><sub>c</sub> < 250°. These predictions are consistent with our experimental results. In addition, we have numerically explored the transition region between in-phase and out-of-phase operation. Figure <xref rid=\"Fig5\" ref-type=\"fig\">5</xref> (bottom) shows the simulated interference for <italic>ϕ</italic><sub>c</sub> = 90°. Here, we observe that the secondary DOPO no longer oscillates out-of-phase with respect to the primary DOPO and becomes frustrated with the oscillation becoming uncorrelated, which is also consistent with our experimental observations for the transition region (heater power of 50.2 mW). Work is ongoing to determine the degree of phase tolerance as the number of DOPOs is increased.<fig id=\"Fig5\"><label>Fig. 5</label><caption><title>Numerical modeling of coupled DOPO system.</title><p>Simulated temporal interference between two DOPOs with unidirectional coupling. The plot shows the combined DOPO (green) and the individual DOPOs, DOPO<sub>1</sub> (red) and DOPO<sub>2</sub> (blue) for coupling phase <italic>ϕ</italic><sub>c</sub> of 0° (in-phase, <italic>ϕ</italic><sub>out</sub> = 0), 180° (out-of-phase, <italic>ϕ</italic><sub>out</sub> = <italic>π</italic>), and 90° (transition from in-phase to out-of-phase) from top to bottom.</p></caption><graphic xlink:href=\"41467_2020_17919_Fig5_HTML\" id=\"d30e1528\"/></fig></p></sec><sec id=\"Sec6\"><title>Scalability of coupled DOPO system</title><p id=\"Par11\">Lastly, we discuss the potential of a large-scale DOPO photonic system in light of power consumption and computing speed. Figure <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>a shows the combined pump power required for a single microresonator-based DOPO to oscillate as a function of the intrinsic <italic>Q</italic>-factor of the SiN microresonator for critical coupling. The measured values for the current device and a similar single DOPO device are denoted with a diamond (see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">5</xref>). For a <italic>Q</italic>-factor of 10 million, oscillation can occur with 1  mW of combined pump power, implying that 1000 DOPOs can be pumped simultaneously with an on-chip optical power of 1 W, offering promise for scaling to a large number of DOPOs (see Supplementary Note 6). Here, since the power scales as 1/<italic>Q</italic><sup>2</sup> and the lifetime scales as <italic>Q</italic>, the energy required for the entire computation scales as 1/<italic>Q</italic>. With reduction of surface roughness, <italic>Q</italic>-factors of 37 million have been achieved in high-confinement SiN microresonators<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>, which could further reduce the pump power to 80 μW per DOPO (labeled with a triangle in Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>a), at the expense of computing rate. In addition, Fig. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>b, c shows the simulated DOPO oscillation time for two different <italic>Q</italic>-factors at near-threshold pump powers. For a single DOPO in a microresonator with an intrinsic <italic>Q</italic> of 1.26 million (our experiment), we observe a convergence time of 174 ns with a cavity lifetime of 0.52 ns based on our numerical model. For a microresonator with an intrinsic <italic>Q</italic> of 10 million, we observe convergence times of 540 ns. Experimentally in our 2-DOPO system, we achieve a convergence time <310 ns with a computing rate of 400 kHz. Furthermore, we have performed numerical modeling of a <italic>N</italic> = 100 DOPO system and observe an annealing time <italic>T</italic><sub>ann</sub> = 0.2 μs with a 35% ground-state success probability (see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">7</xref>). We calculate a time to solution, <inline-formula id=\"IEq2\"><alternatives><tex-math id=\"M7\">\\documentclass[12pt]{minimal}\n\t\t\t\t\\usepackage{amsmath}\n\t\t\t\t\\usepackage{wasysym} \n\t\t\t\t\\usepackage{amsfonts} \n\t\t\t\t\\usepackage{amssymb} \n\t\t\t\t\\usepackage{amsbsy}\n\t\t\t\t\\usepackage{mathrsfs}\n\t\t\t\t\\usepackage{upgreek}\n\t\t\t\t\\setlength{\\oddsidemargin}{-69pt}\n\t\t\t\t\\begin{document}$${T}_{{\\rm{sol}}}=\\frac{\\mathrm{log}\\,(0.01)}{\\mathrm{log}\\,(1-P)}=2.1$$\\end{document}</tex-math><mml:math id=\"M8\"><mml:msub><mml:mrow><mml:mi>T</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">sol</mml:mi></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mfrac><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mn>0.01</mml:mn></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow><mml:mrow><mml:mi mathvariant=\"normal\">log</mml:mi><mml:mspace width=\"0.25em\"/><mml:mrow><mml:mo>(</mml:mo><mml:mrow><mml:mn>1</mml:mn><mml:mo>−</mml:mo><mml:mi>P</mml:mi></mml:mrow><mml:mo>)</mml:mo></mml:mrow></mml:mrow></mml:mfrac><mml:mo>=</mml:mo><mml:mn>2.1</mml:mn></mml:math><inline-graphic xlink:href=\"41467_2020_17919_Article_IEq2.gif\"/></alternatives></inline-formula> μs<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref>,<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>. As a comparison, the fiber-based DOPO system has a <italic>T</italic><sub>sol</sub> = 3.3 ms for solving cubic MAX-CUT problems and <italic>T</italic><sub>sol</sub> = 2.3 ms for dense MAX-CUT with <italic>N</italic> = 100<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup>, suggesting that the spatial-multiplexing approach offers promise for accelerating the computing time of an Ising model. As the number of DOPOs in the network increases, the anneal time that corresponds to the pump turn-on time must be controlled to slow down the DOPO dynamics to prevent freeze-out effects that prevent the system from reaching the ground-state solution<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref>,<xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>. More specifically for our system of microresonator-based DOPOs, it has been reported elsewhere that the rate at which the DOPOs are tuned into resonance is a decisive parameter that determines the success probability of finding the ground-state solution (see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">8</xref>)<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. As the pump-to-resonance detuning dictates the power build-up within the microresonator, this observation is consistent with previous studies where the pump power was ramped up at a controlled rate to improve the performance of their systems<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. In other words, the detuning is the more natural control parameter for our microresonator-based DOPOs, which must be carefully tuned. This fact will be subject to a more comprehensive investigation and reported elsewhere. We also observe that the oscillation threshold is reduced for a 2-DOPO system for both in-phase and out-of-phase couplings<sup><xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>, which leads to apparent faster convergence times as compared to a single DOPO. Investigations are ongoing for the optimal turn-on time as the problem size further increases for our spatial-multiplexed DOPO system. Finally, we have developed a preliminary 4-DOPO system and have observed DOPO generation in a single microresonator as shown in Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">9</xref>. Future work will implement reconfigurable phase coupling between the DOPOs via integrated heaters.<fig id=\"Fig6\"><label>Fig. 6</label><caption><title>Scaling to larger number of coupled DOPOs.</title><p><bold>a</bold> Combined pump power required for single DOPO as a function of the intrinsic <italic>Q</italic>-factor of the microresonator. The two different pump powers (and the corresponding <italic>Q</italic>) used in our experiments is denoted with diamonds. Triangle (green) denotes pump power based on the <italic>Q</italic> of state-of-the-art SiN microresonators<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref></sup>. Simulated DOPO convergence for <bold>b</bold>\n<italic>Q</italic><sub>int</sub> = 1.26 × 10<sup>6</sup>, which corresponds to a cavity lifetime <italic>τ</italic><sub>p</sub> = 0.52 ns, and <bold>c</bold>\n<italic>Q</italic><sub>int</sub> = 10 × 10<sup>6</sup>, which corresponds to <italic>τ</italic><sub>p</sub> = 4.1 ns, for critical coupling.</p></caption><graphic xlink:href=\"41467_2020_17919_Fig6_HTML\" id=\"d30e1753\"/></fig></p></sec></sec><sec id=\"Sec7\" sec-type=\"discussion\"><title>Discussion</title><p id=\"Par12\">In conclusion, we demonstrate phase reconfigurable all-optical coupling between microresonator-based DOPOs in an integrated silicon nitride platform. We experimentally observe that the system is highly tolerant to the coupling phase between the DOPOs, offering flexibility in setting up the system when scaling to larger number of DOPOs. Since the system does not rely on time multiplexing, and the computation time is comparable to the build-up time of a single DOPO, it is possible to rapidly test the fidelity of the final state. To enable reconfigurable coupling between arbitrary DOPOs, a fraction of the DOPO power will be sent to a matrix of 2<italic>N</italic> × 2<italic>N</italic> Mach–Zehnder interferometers (MZIs) to perform <italic>N</italic> × <italic>N</italic> arbitrary linear transformations<sup><xref ref-type=\"bibr\" rid=\"CR49\">49</xref>–<xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>. To achieve this on a single device layer, intersections between waveguides are required. Our transmission measurements (see Supplementary Note <xref rid=\"MOESM1\" ref-type=\"media\">10</xref>) indicate 0.45 dB loss per intersection, which can be further optimized. In addition, we are investigating a multilayer design<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref></sup> where the coupling matrix based on MZIs is located in a different material plane, which uses silicon for the waveguides. Due to the larger thermo-optic effect in silicon, the MZIs can be further miniaturized such that each MZI element resides on a footprint of 100 μm × 100 μm (0.01 mm<sup>2</sup>)<sup><xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>, which is significantly smaller than that required for a SiN MZI (0.7 mm<sup>2</sup>)<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup>. Such a design would enable all-to-all coupling among the DOPOs with arbitrary weightings and the resulting system should in principle allow the implementation of an arbitrary Hamiltonian. Our results provide the initial building blocks for the realization of a large-scale DOPO network for studying nontrivial coupled oscillator dynamics, offering potential towards the realization of a chip-based photonic Ising machine.</p></sec><sec id=\"Sec8\"><title>Methods</title><sec id=\"Sec9\"><title>Experimental setup</title><p id=\"Par13\">The setup is shown in Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>a. Two tunable cw lasers (New Focus Velocity) are amplified using an erbium-doped fiber amplifier (EDFA) to use as pumps. The high-wavelength pump is modulated using an AOM, where the modulation depth and frequency are carefully chosen such that the DOPO signal reaches the noise level each time the AOM turned off. The pulse duration, repetition rate, and the extinction ratio are largely dependent on the quality factor of the microresonator<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup>. For our system, with our pulse parameters we use an extinction ratio of 5:1 for our AOM. This extinction ratio is sufficient to reduce the pump power to a level below the oscillation threshold and, this together with our choice of pulse duration and repetition rate, allows for the DOPO signal to decay to a level below the noise before the initiation of the next period. In order to suppress the amplified spontaneous emission from the EDFA at the DOPO wavelength, we use 9-nm-wide bandpass filters after each EDFA. Fiber polarization controllers are used in each arm to set the input polarization to TE. For the chip output, the individual DOPO arms and the interferometer output are collected using a fiber collimator and the DOPO wavelength is filtered using a tunable bandpass filter with a 0.8-nm bandwidth and sent to an InGaAs amplifier photodetector (150-MHz bandwidth) and a 1-GHz real-time oscilloscope.</p></sec></sec><sec sec-type=\"supplementary-material\"><title>Supplementary information</title><sec id=\"Sec10\"><supplementary-material content-type=\"local-data\" id=\"MOESM1\"><media xlink:href=\"41467_2020_17919_MOESM1_ESM.pdf\"><caption><p>Supplementary Information</p></caption></media></supplementary-material></sec></sec></body><back><fn-group><fn><p><bold>Peer review information</bold>\n<italic>Nature Communications</italic> thanks the anonymous reviewer(s) for their contribution to the peer review of this work.</p></fn><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>These authors contributed equally: Yoshitomo Okawachi and Mengjie Yu.</p></fn></fn-group><sec><title>Supplementary information</title><p><bold>Supplementary information</bold> is available for this paper at 10.1038/s41467-020-17919-6.</p></sec><ack><title>Acknowledgements</title><p>This work was supported by Army Research Office (ARO) (grant W911NF-17-1-0016), National Science Foundation (NSF) (grant CCF-1640108), Semiconductor Research Corporation (SRC) (grant SRS 2016-EP-2693-A), and Air Force Office of Scientific Research (AFOSR) (grant FA9550-15-1-0303). This work was performed in part at the Cornell Nano-Scale Facility, which is a member of the National Nanotechnology Infrastructure Network, supported by the NSF, and at the CUNY Advanced Science Research Center NanoFabrication Facility. We also acknowledge useful discussions with A. Farsi, C. Joshi, C. Joshi, A. Mohanty, S. Ramelow, and L. Shao.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>Y.O. and M.Y. designed the devices and experiment. Y.O., M.Y., and Y.Z. performed the experiments. J.K.J. performed numerical modeling. X.J. fabricated the devices. Y.O., M.Y., Y.Z., and B.Y.K. performed characterization of the DOPO devices. Y.O. and M.Y. analyzed the data. Y.O. prepared the manuscript in discussion with all authors. M.L. and A.L.G. supervised the project.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.</p></notes><notes notes-type=\"data-availability\"><title>Code availability</title><p>The modeling is described in the Supplementary information and the code is available from the corresponding author upon reasonable request.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par14\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Backus</surname><given-names>J</given-names></name></person-group><article-title>Can programming be liberated from the von Neumann style?: a functional style and its algebra of programs</article-title><source>Commun. ACM</source><year>1978</year><volume>21</volume><fpage>613</fpage></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Johnson</surname><given-names>MW</given-names></name><etal/></person-group><article-title>Quantum annealing with manufactured spins</article-title><source>Nature</source><year>2011</year><volume>473</volume><fpage>194</fpage><lpage>198</lpage><pub-id pub-id-type=\"pmid\">21562559</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bojnordi</surname><given-names>MN</given-names></name><name><surname>Ipek</surname><given-names>E</given-names></name></person-group><article-title>The memristive Boltzmann machines</article-title><source>IEEE Micro</source><year>2017</year><volume>37</volume><fpage>22</fpage><lpage>29</lpage></element-citation></ref><ref id=\"CR4\"><label>4.</label><mixed-citation publication-type=\"other\">Shin, J. H., Jeong, Y. J., Zidan, M. A., Wang, Q. & Lu, W. D., Hardware acceleration of simulated annealing of spin glass by RRAM crossbar array. In <italic>2018 IEEE International Electron Devices Meeting (IEDM)</italic>, 3.3.1–3.3.4 (2018).</mixed-citation></ref><ref id=\"CR5\"><label>5.</label><mixed-citation publication-type=\"other\">King, A. D., Bernoudy, W., King, J., Berkley, A. J., & Lanting, T., Emulating the coherent Ising machine with a mean-field algorithm. Preprint at <ext-link ext-link-type=\"uri\" xlink:href=\"https://arxiv.org/abs/1806.08422\">https://arxiv.org/abs/1806.08422</ext-link> (2018).</mixed-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aramon</surname><given-names>M</given-names></name><etal/></person-group><article-title>Physics-inspired optimization for quadratic unconstrained problems using a digital annealer</article-title><source>Front. Phys.</source><year>2019</year><volume>7</volume><fpage>48</fpage></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cai</surname><given-names>F</given-names></name><etal/></person-group><article-title>Power-efficient combinatorial optimization using intrinsic noise in memristor Hopfield neural networks</article-title><source>Nat. Electron.</source><year>2020</year><volume>3</volume><fpage>409</fpage><lpage>418</lpage></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mahmoodi</surname><given-names>MR</given-names></name><name><surname>Prezioso</surname><given-names>M</given-names></name><name><surname>Strukov</surname><given-names>DB</given-names></name></person-group><article-title>Versatile stochastic dot product circuits based on nonvolatile memories for high performance neurocomputing and neurooptimization</article-title><source>Nat. Commun.</source><year>2019</year><volume>10</volume><fpage>5113</fpage><pub-id pub-id-type=\"pmid\">31704925</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yamamoto</surname><given-names>Y</given-names></name><name><surname>Takata</surname><given-names>K</given-names></name><name><surname>Utsunomiya</surname><given-names>S</given-names></name></person-group><article-title>Quantum computing vs. coherent computing</article-title><source>N. Gener. Comput.</source><year>2012</year><volume>30</volume><fpage>327</fpage></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Utsunomiya</surname><given-names>S</given-names></name><name><surname>Takata</surname><given-names>K</given-names></name><name><surname>Yamamoto</surname><given-names>Y</given-names></name></person-group><article-title>Mapping of Ising models onto injection-locked laser systems</article-title><source>Opt. Express</source><year>2011</year><volume>19</volume><fpage>18091</fpage><pub-id pub-id-type=\"pmid\">21935175</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Takata</surname><given-names>K</given-names></name><name><surname>Utsunomiya</surname><given-names>S</given-names></name><name><surname>Yamamoto</surname><given-names>Y</given-names></name></person-group><article-title>Transient time of an Ising machine based on injection-locked laser network</article-title><source>N. J. Phys.</source><year>2012</year><volume>14</volume><fpage>013052</fpage></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Takata</surname><given-names>K</given-names></name><name><surname>Yamamoto</surname><given-names>Y</given-names></name></person-group><article-title>Data search by a coherent Ising machine based on an injection-locked laser network with gradual pumping or coupling</article-title><source>Phys. Rev. A</source><year>2014</year><volume>89</volume><fpage>032319</fpage></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Utsunomiya</surname><given-names>S</given-names></name><etal/></person-group><article-title>Binary phase oscillation of two mutually coupled semiconductor lasers</article-title><source>Opt. Express</source><year>2015</year><volume>23</volume><fpage>6029</fpage><pub-id pub-id-type=\"pmid\">25836827</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><mixed-citation publication-type=\"other\">Tradonsky, C. et al. Rapid laser solver for the phase retrieval problem. <italic>Sci. Adv.</italic><bold>5</bold>, eaax4530 (2019).</mixed-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Babaeian</surname><given-names>M</given-names></name><etal/></person-group><article-title>A single shot coherent Ising machine based on a network of injection-locked multicore fiber lasers</article-title><source>Nat. Commun.</source><year>2019</year><volume>10</volume><fpage>3516</fpage><pub-id pub-id-type=\"pmid\">31388011</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yamamoto</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Coherent Ising machines—optical neural networks operating at the quantum limit</article-title><source>npj Quantum Inf.</source><year>2017</year><volume>3</volume><fpage>49</fpage></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Marandi</surname><given-names>A</given-names></name><name><surname>Wang</surname><given-names>Z</given-names></name><name><surname>Takata</surname><given-names>K</given-names></name><name><surname>Byer</surname><given-names>RL</given-names></name><name><surname>Yamamoto</surname><given-names>Y</given-names></name></person-group><article-title>Network of time-multiplexed optical parametric oscillators as a coherent Ising machine</article-title><source>Nat. Photonics</source><year>2014</year><volume>8</volume><fpage>937</fpage></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Inagaki</surname><given-names>T</given-names></name><etal/></person-group><article-title>Large-scale Ising spin network based on degenerate optical parametric oscillators</article-title><source>Nat. Photonics</source><year>2016</year><volume>10</volume><fpage>415</fpage></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>Z</given-names></name><name><surname>Marandi</surname><given-names>A</given-names></name><name><surname>Wen</surname><given-names>K</given-names></name><name><surname>Byer</surname><given-names>RL</given-names></name><name><surname>Yamamoto</surname><given-names>Y</given-names></name></person-group><article-title>Coherent Ising machine based on degenerate optical parametric oscillators</article-title><source>Phys. Rev. A</source><year>2013</year><volume>88</volume><fpage>063853</fpage></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Inagaki</surname><given-names>T</given-names></name><etal/></person-group><article-title>A coherent Ising machine for 2000-node optimization problems</article-title><source>Science</source><year>2016</year><volume>354</volume><fpage>603</fpage><pub-id pub-id-type=\"pmid\">27811271</pub-id></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McMahon</surname><given-names>PL</given-names></name><etal/></person-group><article-title>A fully programmable 100-spin coherent Ising machine with all-to-all connections</article-title><source>Science</source><year>2016</year><volume>354</volume><fpage>614</fpage><pub-id pub-id-type=\"pmid\">27811274</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Guo</surname><given-names>Y</given-names></name><name><surname>Kroeze</surname><given-names>RM</given-names></name><name><surname>Vaidya</surname><given-names>VD</given-names></name><name><surname>Keeling</surname><given-names>J</given-names></name><name><surname>Lev</surname><given-names>BL</given-names></name></person-group><article-title>Sign-changing photon-mediated atom interactions in multimode cavity quantum electrodynamics</article-title><source>Phys. Rev. Lett.</source><year>2019</year><volume>122</volume><fpage>193601</fpage><pub-id pub-id-type=\"pmid\">31144918</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hamerly</surname><given-names>R</given-names></name><etal/></person-group><article-title>Experimental investigation of performance differences between coherent Ising machines and a quantum annealer</article-title><source>Sci. Adv.</source><year>2019</year><volume>5</volume><fpage>eaau0823</fpage><pub-id pub-id-type=\"pmid\">31139743</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Böhm</surname><given-names>F</given-names></name><name><surname>Verschaffelt</surname><given-names>G</given-names></name><name><surname>Van der Sande</surname><given-names>G</given-names></name></person-group><article-title>A poor man’s coherent Ising machine based on opto-electronic feedback systems for solving optimization problems</article-title><source>Nat. Commun.</source><year>2019</year><volume>10</volume><fpage>3538</fpage><pub-id pub-id-type=\"pmid\">31395872</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Prabhu</surname><given-names>M</given-names></name><etal/></person-group><article-title>Accelerating recurrent Ising machines in photonic integrated circuits</article-title><source>Optica</source><year>2020</year><volume>7</volume><fpage>551</fpage><lpage>558</lpage></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Roques-Carmes</surname><given-names>C</given-names></name><etal/></person-group><article-title>Heuristic recurrent algorithms for photonic Ising machines</article-title><source>Nat. Commun.</source><year>2020</year><volume>11</volume><fpage>249</fpage><pub-id pub-id-type=\"pmid\">31937776</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pierangeli</surname><given-names>D</given-names></name><name><surname>Marcucci</surname><given-names>G</given-names></name><name><surname>Conti</surname><given-names>C</given-names></name></person-group><article-title>Large-scale photonic Ising machine by spatial light modulation</article-title><source>Phys. Rev. Lett.</source><year>2019</year><volume>122</volume><fpage>213902</fpage><pub-id pub-id-type=\"pmid\">31283311</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kumar</surname><given-names>S</given-names></name><name><surname>Zhang</surname><given-names>H</given-names></name><name><surname>Huang</surname><given-names>Y-P</given-names></name></person-group><article-title>Large-scale Ising emulation with four body interaction and all-to-all connections</article-title><source>Commun. Phys.</source><year>2020</year><volume>3</volume><fpage>108</fpage></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tezak</surname><given-names>N</given-names></name><etal/></person-group><article-title>Integrated coherent Ising machines based on self-phase modulation in microring resonators</article-title><source>IEEE J. Sel. Top. Quantum Electron.</source><year>2020</year><volume>26</volume><fpage>1</fpage><lpage>15</lpage></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ising</surname><given-names>E</given-names></name></person-group><article-title>Beitrag zur theorie des ferromagnetismus</article-title><source>Z. Phys.</source><year>1925</year><volume>31</volume><fpage>253</fpage></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Binder</surname><given-names>K</given-names></name><name><surname>Young</surname><given-names>AP</given-names></name></person-group><article-title>Spin glasses: experimental facts, theoretical concepts, and open questions</article-title><source>Rev. Mod. Phys.</source><year>1986</year><volume>58</volume><fpage>801</fpage></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lucas</surname><given-names>A</given-names></name></person-group><article-title>Ising formulations of many NP problems</article-title><source>Front. Phys.</source><year>2014</year><volume>2</volume><fpage>5</fpage></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yamamura</surname><given-names>A</given-names></name><name><surname>Aihara</surname><given-names>K</given-names></name><name><surname>Yamamoto</surname><given-names>Y</given-names></name></person-group><article-title>Quantum model for coherent Ising machines: discrete-time measurement feedback formulation</article-title><source>Phys. Rev. A</source><year>2017</year><volume>96</volume><fpage>053834</fpage></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haribara</surname><given-names>Y</given-names></name><name><surname>Ishikawa</surname><given-names>H</given-names></name><name><surname>Utsunomiya</surname><given-names>S</given-names></name><name><surname>Aihara</surname><given-names>K</given-names></name><name><surname>Yamamoto</surname><given-names>Y</given-names></name></person-group><article-title>Performance evaluation of coherent Ising machines against classical neural networks</article-title><source>Quantum Sci. Technol.</source><year>2017</year><volume>2</volume><fpage>044002</fpage></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Böhm</surname><given-names>F</given-names></name><etal/></person-group><article-title>Understanding dynamics of coherent Ising machines through simulation of large-scale 2D Ising models</article-title><source>Nat. Commun.</source><year>2018</year><volume>9</volume><fpage>5020</fpage><pub-id pub-id-type=\"pmid\">30479329</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Turitsyn</surname><given-names>SK</given-names></name><name><surname>Bednyakova</surname><given-names>AE</given-names></name><name><surname>Fedoruk</surname><given-names>MP</given-names></name><name><surname>Papernyi</surname><given-names>SB</given-names></name><name><surname>Clements</surname><given-names>WRL</given-names></name></person-group><article-title>Inverse four-wave mixing and self-parametric amplification in optical fibre</article-title><source>Nat. Photonics</source><year>2015</year><volume>9</volume><fpage>608</fpage><pub-id pub-id-type=\"pmid\">26345290</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Okawachi</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Dual-pumped degenerate Kerr oscillator in a silicon nitride microresonator</article-title><source>Opt. Lett.</source><year>2015</year><volume>40</volume><fpage>5267</fpage><pub-id pub-id-type=\"pmid\">26565851</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Takesue</surname><given-names>H</given-names></name><name><surname>Inagaki</surname><given-names>T</given-names></name></person-group><article-title>10 GHz clock time-multiplexed degenerate optical parametric oscillators for a photonic Ising spin network</article-title><source>Opt. Lett.</source><year>2016</year><volume>41</volume><fpage>4273</fpage><pub-id pub-id-type=\"pmid\">27628375</pub-id></element-citation></ref><ref id=\"CR39\"><label>39.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Okawachi</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Quantum random number generator using a microresonator-based Kerr oscillator</article-title><source>Opt. Lett.</source><year>2016</year><volume>41</volume><fpage>4194</fpage><pub-id pub-id-type=\"pmid\">27628355</pub-id></element-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gaeta</surname><given-names>AL</given-names></name><name><surname>Lipson</surname><given-names>M</given-names></name><name><surname>Kippenberg</surname><given-names>TJ</given-names></name></person-group><article-title>Photonic-chip-based frequency combs</article-title><source>Nat. Photonics</source><year>2019</year><volume>13</volume><fpage>158</fpage></element-citation></ref><ref id=\"CR41\"><label>41.</label><mixed-citation publication-type=\"other\">Jang, J. K., Okawachi, Y. & Gaeta, A. L. Dynamics of coupled microresonator-based degenerate optical parametric oscillators. In <italic>Conference on Lasers and Electro-Optics</italic>, FTh1E.4 (Optical Society of America, 2018).</mixed-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luke</surname><given-names>K</given-names></name><name><surname>Dutt</surname><given-names>A</given-names></name><name><surname>Poitras</surname><given-names>CB</given-names></name><name><surname>Lipson</surname><given-names>M</given-names></name></person-group><article-title>Overcoming Si<sub>3</sub>N<sub>4</sub> film stress limitations for high quality factor ring resonators</article-title><source>Opt. Express</source><year>2013</year><volume>21</volume><fpage>22829</fpage><lpage>22833</lpage><pub-id pub-id-type=\"pmid\">24104169</pub-id></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Soldano</surname><given-names>LB</given-names></name><etal/></person-group><article-title>Planar monomode optical couplers based on multimode interference effects</article-title><source>J. Lightwave Technol.</source><year>1992</year><volume>10</volume><fpage>1843</fpage><lpage>1850</lpage></element-citation></ref><ref id=\"CR44\"><label>44.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cunningham</surname><given-names>JE</given-names></name><etal/></person-group><article-title>Highly-efficient thermally-tuned resonant optical filters</article-title><source>Opt. Express</source><year>2010</year><volume>18</volume><fpage>19055</fpage><pub-id pub-id-type=\"pmid\">20940799</pub-id></element-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Joshi</surname><given-names>C</given-names></name><etal/></person-group><article-title>Thermally controlled comb generation and soliton modelocking in microresonators</article-title><source>Opt. Lett.</source><year>2016</year><volume>41</volume><fpage>2565</fpage><pub-id pub-id-type=\"pmid\">27244415</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lugiato</surname><given-names>LA</given-names></name><name><surname>Lefever</surname><given-names>R</given-names></name></person-group><article-title>Spatial dissipative structures in passive optical systems</article-title><source>Phys. Rev. Lett.</source><year>1987</year><volume>58</volume><fpage>2209</fpage><pub-id pub-id-type=\"pmid\">10034681</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dudley</surname><given-names>JM</given-names></name><name><surname>Coen</surname><given-names>S</given-names></name></person-group><article-title>Coherence properties of supercontinuum spectra generated in photonic crystal and tapered optical fibers</article-title><source>Opt. Lett.</source><year>2002</year><volume>27</volume><fpage>1180</fpage><lpage>1182</lpage><pub-id pub-id-type=\"pmid\">18026400</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ji</surname><given-names>X</given-names></name><etal/></person-group><article-title>Ultra-low-loss on-chip resonators with sub-milliwatt parametric oscillation threshold</article-title><source>Optica</source><year>2017</year><volume>4</volume><fpage>619</fpage></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reck</surname><given-names>M</given-names></name><name><surname>Zeilinger</surname><given-names>A</given-names></name><name><surname>Bernstein</surname><given-names>HJ</given-names></name><name><surname>Bertani</surname><given-names>P</given-names></name></person-group><article-title>Experimental realization of any discrete unitary operator</article-title><source>Phys. Rev. Lett.</source><year>1994</year><volume>73</volume><fpage>58</fpage><lpage>61</lpage><pub-id pub-id-type=\"pmid\">10056719</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Clements</surname><given-names>WR</given-names></name><name><surname>Humphreys</surname><given-names>PC</given-names></name><name><surname>Metcalf</surname><given-names>BJ</given-names></name><name><surname>Kolthammer</surname><given-names>WS</given-names></name><name><surname>Walmsley</surname><given-names>IA</given-names></name></person-group><article-title>Optimal design for universal multiport interferometers</article-title><source>Optica</source><year>2016</year><volume>3</volume><fpage>1460</fpage><lpage>1465</lpage></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shen</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Deep learning with coherent nanophotonic circuits</article-title><source>Nat. Photonics</source><year>2017</year><volume>11</volume><fpage>441</fpage></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>MacFarlane</surname><given-names>N</given-names></name><name><surname>Kossey</surname><given-names>MR</given-names></name><name><surname>Stroud</surname><given-names>JR</given-names></name><name><surname>Foster</surname><given-names>MA</given-names></name><name><surname>Foster</surname><given-names>AC</given-names></name></person-group><article-title>A multi-layer platform for low-loss nonlinear silicon photonics</article-title><source>APL Photonics</source><year>2019</year><volume>4</volume><fpage>110809</fpage></element-citation></ref><ref id=\"CR53\"><label>53.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Watts</surname><given-names>MR</given-names></name><etal/></person-group><article-title>Adiabatic thermo-optic Mach–Zehnder switch</article-title><source>Opt. Lett.</source><year>2013</year><volume>38</volume><fpage>733</fpage><lpage>735</lpage><pub-id pub-id-type=\"pmid\">23455281</pub-id></element-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Joo</surname><given-names>J</given-names></name><name><surname>Park</surname><given-names>J</given-names></name><name><surname>Kim</surname><given-names>G</given-names></name></person-group><article-title>Cost-effective 2 × 2 silicon nitride Mach–Zehnder interferometric (MZI) thermo-optic switch</article-title><source>IEEE Photonics Technol. Lett.</source><year>2018</year><volume>30</volume><fpage>740</fpage><lpage>743</lpage></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807929</article-id><article-id pub-id-type=\"pmc\">PMC7431592</article-id><article-id pub-id-type=\"publisher-id\">70970</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70970-7</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>High-throughput microCT scanning of small specimens: preparation, packing, parameters and post-processing</article-title></title-group><contrib-group><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Hipsley</surname><given-names>Christy A.</given-names></name><address><email>christy.hipsley@unimelb.edu.au</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Aguilar</surname><given-names>Rocio</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Black</surname><given-names>Jay R.</given-names></name><xref ref-type=\"aff\" rid=\"Aff4\">4</xref></contrib><contrib contrib-type=\"author\"><name><surname>Hocknull</surname><given-names>Scott A.</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff5\">5</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.1008.9</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2179 088X</institution-id><institution>School of BioSciences, </institution><institution>University of Melbourne, </institution></institution-wrap>BioSciences 4, Building 147, Parkville, VIC 3010 Australia </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.436717.0</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0500 6540</institution-id><institution>Museums Victoria, </institution></institution-wrap>GPO Box 666, Melbourne, VIC 3001 Australia </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.1002.3</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 7857</institution-id><institution>School of Biological Sciences, </institution><institution>Monash University, </institution></institution-wrap>Clayton, VIC Australia </aff><aff id=\"Aff4\"><label>4</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.1008.9</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2179 088X</institution-id><institution>School of Earth Sciences, </institution><institution>University of Melbourne, </institution></institution-wrap>Melbourne, VIC Australia </aff><aff id=\"Aff5\"><label>5</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.452644.5</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2215 0059</institution-id><institution>Queensland Museum, Geosciences, </institution></institution-wrap>122 Gerler Rd., Hendra, QLD 4011 Australia </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13863</elocation-id><history><date date-type=\"received\"><day>25</day><month>2</month><year>2020</year></date><date date-type=\"accepted\"><day>4</day><month>8</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">High-resolution X-ray microcomputed tomography, or microCT (μCT), enables the digital imaging of whole objects in three dimensions. The power of μCT to visualize internal features without disarticulation makes it particularly valuable for the study of museum collections, which house millions of physical specimens documenting the spatio-temporal patterns of life. Despite the potential for comparative analyses, most μCT studies include limited numbers of museum specimens, due to the challenges of digitizing numerous individuals within a project scope. Here we describe a method for high-throughput μCT scanning of hundreds of small (< 2 cm) specimens in a single container, followed by individual labelling and archival storage. We also explore the effects of various packing materials and multiple specimens per capsule to minimize sample movement that can degrade image quality, and hence μCT investment. We demonstrate this protocol on vertebrate fossils from Queensland Museum, Australia, as part of an effort to track community responses to climate change over evolutionary time. This system can be easily modified for other types of wet and dry material amenable to X-ray attenuation, including geological, botanical and zoological samples, providing greater access to large-scale phenotypic data and adding value to global collections.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>3-D reconstruction</kwd><kwd>X-ray tomography</kwd><kwd>Palaeoecology</kwd><kwd>Imaging techniques</kwd></kwd-group><funding-group><award-group><funding-source><institution>Australian Research Council</institution></funding-source><award-id>DE180100629</award-id><principal-award-recipient><name><surname>Hipsley</surname><given-names>Christy A.</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">High-resolution X-ray microcomputed tomography, also known as HRXMT or microCT (μCT), is an increasingly powerful tool for the non-destructive investigation of whole objects. Functioning like a microscope with X-ray vision, μCT generates high fidelity 3D models of solid material from which the outer layers can be virtually dissected or removed, revealing the inner structures. Starting with radiographs of an object taken over multiple angles, a computer algorithm is used to digitally reconstruct a stack of 2D X-ray projections, or tomograms, into a 3D volume. Whereas in human medicine the X-ray source rotates around the patient (e.g., computerized axial tomography, or CAT scan), in μCT the object is typically fixed on a rotating stage while the X-ray tube remains stationary. The differential properties of the object’s matter, including thickness and atomic number, interact with the X-ray’s energy beam to determine the number of photons that pass through it to reach the detector on the other side. This decrease in electromagnetic radiation, termed X-ray attenuation, results in detector pixels with grayscale values proportional to the radiopacity of the material, meaning that dense regions such as bone or rock appear white or light gray (radiopaque), while muscle or skin appears dark (radiolucent). The improved resolution of μCT over standard imaging techniques can achieve a detail detectability down to 200 nm (0.2 µm)—less than the diameter of a single red blood cell.</p><p id=\"Par3\">Since publication of the first X-ray microtomographic figures nearly four decades ago<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>, μCT has had profound impacts across scientific disciplines. Studies in biomedicine, zoology, geology and paleontology now regularly incorporate μCT images<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>–<xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>, and open source software for the quantitative analysis of volumetric data is developing rapidly (e.g., Fiji<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>, Blob3D<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>, Dragonfly [Object Research Systems (ORS) Inc., Montreal, Canada]). Within the biological sciences, μCT has been particularly valuable for the study of museum collections, which contain millions of often small, delicate and unique specimens not amenable to traditional (destructive and/or irreversible) preparation. Worldwide, these collections span taxonomic, geographic and temporal distributions, providing a wealth of information for understanding the past, present and future of biodiversity<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup>. Creation of cybertypes, or virtual models of type material, is another emerging application of μCT technology<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>, allowing researchers around the world to interact with voucher specimens. When combined with downstream analyses like geometric morphometrics, these models can be used to test taxonomic hypotheses that could shorten the lagtime between species discovery and formal description<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref>,<xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>, thus expanding the taxonomic bottleneck.</p><p id=\"Par4\">Despite the increasing use of μCT in systematic biology<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup>, a major challenge remains in the practical imaging of high numbers of museum specimens within a project scope, for example in the context of large-scale analyses of phenotypic variation. This has proven difficult because of the need for samples to remain motionless during scan time (minutes to hours), and because each individual must be digitally labelled to match the physical specimen’s identity and hence retain important information, e.g., material type, locality, stratigraphic age. These issues have so far limited community-level μCT analyses, which could provide important insights into evolutionary responses to environmental change in small, rare, and/or fossilized taxa<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. While other mass digitization efforts of museum collections are already under way, they are typically limited to high resolution photographs of entire drawers to capture external morphology<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. However internal structures are among the fastest responders to environmental drivers, suggesting an untapped role for μCT data in climate change research<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. Especially in the Anthropocene, μCT offers a promising technique to identify the evolutionary consequences of human activities in natural history collections at both the internal organismal and ecosystem levels<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>.</p><p id=\"Par5\">Although some digital morphology studies have included hundreds of<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup> or over a thousand<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref>–<xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup> 3D models, their images are often aggregated using different sources (museums, repositories, laboratories), methods (surface scanning, μCT), and parameters (X-ray energy, voxel size, number of projections), making it difficult to integrate and compare findings. For smaller specimens in particular, variation in spatial resolution can result in significant measurement errors, for example in estimated volumes or relationships between traits<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref>,<xref ref-type=\"bibr\" rid=\"CR25\">25</xref></sup>. Limitations on time and money for μCT scanning also mean that specimen data are often collected haphazardly, constraining systematic analyses to one or few individuals per species<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>. This trade-off between data accessibility and data quantity (and quality<sup><xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup>) precludes the opportunity for more rigorous investigations of phenotypic diversity in time and space, as well as focused analyses of variation in a single taxon or locality. Common biodiversity metrics such as species identity, richness, and evenness are inferable from μCT data, e.g.<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref>,<xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup>, especially for cryptic taxa and species complexes with few observable differences<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref>–<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. Therefore, optimization of the scanning process for high resolution 3D images of multiple specimens would not only contribute to our understanding of how biodiversity responds to global change, but also to the value of museum collections and researchers’ abilities to access them digitally.</p><p id=\"Par6\">Here we outline steps for high-throughput μCT scanning of small (< 2 cm) fossils, meant to facilitate advanced exploration of museum collections and allow researchers with limited access to specialized facilities the opportunity to maximize their investments. In contrast to other high-throughput μCT methods that have focused on clinical evaluations<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref>,<xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup> or automated phenotyping of laboratory strains (e.g., mice<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup>, rice<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup>, zebrafish<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup>), we concentrate on the practical arrangement of accessioned museum specimens so that each individual can be identified and labelled in the 3D volume, followed by long-term storage and archiving. We illustrate this method using vertebrate (reptile, amphibian and mammal) fossils from the paleontological collections of Queensland Museum, Australia, as part of a larger effort to identify changes in morphology and species assemblages over geological time. We succeed in maximizing the quantity of fossils per container to over 200 specimens to generate high quality 3D models for comparative analyses, while minimizing associated time, handling, and error. These steps should be applicable to any small dry objects amendable to X-ray attenuation including geological material, plant tissues, and invertebrates, and wet specimens provided they can be mounted inside small capsules or other containers<sup><xref ref-type=\"bibr\" rid=\"CR37\">37</xref>,<xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>.</p></sec><sec id=\"Sec2\"><title>Methods</title><sec id=\"Sec3\"><title>Preparation</title><p id=\"Par7\">The first step is the only time the user is required to handle specimen material directly, we therefore recommend the use of featherweight spring or feather light forceps and secondary containment (see Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref> for an overview of packing material). Although specimens of varying size can be scanned together, it is preferable to organize fossils into batches of roughly similar material properties for X-ray optimization. Thin, unbuffered, neutral pH tissue is ideal for specimen packing and eventual long-term storage. Other required materials are small clear two-piece pharmaceutical capsules, paper and/or plastic drinking straws, archival paper and pen for labelling, a 50 ml plastic centrifuge tube with cap, and medium density polyethylene foam. Suggested suppliers and estimated costs of material per tube are listed in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Equipment for dense fossil packing prior to μCT scanning: (<bold>a</bold>) litter tray, (<bold>b</bold>) paper and/or plastic straws, (<bold>c</bold>) clear two-piece pharmaceutical capsules, (<bold>d</bold>) archival tissue, (<bold>e</bold>) dry fossil specimen, (<bold>f</bold>) supporting material (paper, foam), (<bold>g</bold>) centrifuge tube with cap, (<bold>h</bold>) archival paper for labels shown inside a size 4 capsule, (<bold>i</bold>) forceps, (<bold>j</bold>) archival pen. Estimated costs and material suppliers are listed in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>.</p></caption><graphic xlink:href=\"41598_2020_70970_Fig1_HTML\" id=\"MO1\"/></fig><table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Estimated costs in Australian dollars (AUD) for material to μCT scan a 50 ml cylinder fully packed with 84 capsules holding 1 small (< 1 cm long) specimen each.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Item</th><th align=\"left\">Total cost per scan (AUD)</th></tr></thead><tbody><tr><td align=\"left\">Centrifuge Falcon Tube 50 mL polypropylene (PP), conical bottom with screw cap<sup>a</sup></td><td char=\".\" align=\"char\">0.60</td></tr><tr><td align=\"left\">Hard 2-piece clear gelatine capsules, size 4 (volume 0.23 ml)<sup>b,</sup>*</td><td char=\".\" align=\"char\">2.10</td></tr><tr><td align=\"left\">Standard paper straw (6 × 135 mm)<sup>c</sup></td><td char=\".\" align=\"char\">0.27</td></tr><tr><td align=\"left\">Green’s Lens Tissue, un-buffered TIS-L Soft, acid-free, long fibred 9gsm repair/wrapping tissue (609 × 914 mm)<sup>d</sup></td><td char=\".\" align=\"char\">0.76</td></tr><tr><td align=\"left\">Feather Forcep Sharp E122S, 0.23 mm gauge thick<sup>e</sup></td><td char=\".\" align=\"char\">4.90</td></tr><tr><td align=\"left\">Ethafoam 220, medium density, non-cross linked Polyethylene (PE) closed cell foam (2,400 × 1,200 mm)<sup>f</sup></td><td char=\".\" align=\"char\">1.00</td></tr><tr><td align=\"left\">Litter tray<sup>g</sup></td><td char=\".\" align=\"char\">4.00</td></tr><tr><td align=\"left\">Label paper<sup>h</sup></td><td char=\".\" align=\"char\">0.10</td></tr><tr><td align=\"left\">Archival pen<sup>i</sup></td><td char=\".\" align=\"char\">6.54</td></tr><tr><td align=\"left\">TOTAL</td><td char=\".\" align=\"char\">20.30</td></tr></tbody></table><table-wrap-foot><p>Note that many items (e.g., Falcon tube, straws, forceps, foam, litter tray, pen) can be reused multiple times, thus decreasing the cost for subsequent scans</p><p>*Clear 2-piece vegetable capsules are also available, although we did not test their performance in μCT scans.</p><p><sup>a</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://corning.com.au\">https://corning.com.au</ext-link>.</p><p><sup>b</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://gelcapsules.com.au\">https://gelcapsules.com.au</ext-link>.</p><p><sup>c</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.greenpack.com.au\">https://www.greenpack.com.au</ext-link>.</p><p><sup>d</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.archivalsurvival.com.au\">https://www.archivalsurvival.com.au</ext-link>.</p><p><sup>e</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.entosupplies.com.au\">https://www.entosupplies.com.au</ext-link>.</p><p><sup>f</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.foamsales.com.au\">https://www.foamsales.com.au</ext-link>.</p><p><sup>g</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.kmart.com.au\">https://www.kmart.com.au</ext-link>.</p><p><sup>h</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.talasonline.com\">https://www.talasonline.com</ext-link>.</p><p><sup>i</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.newtownartsupplies.com.au/\">https://www.newtownartsupplies.com.au/</ext-link>.</p></table-wrap-foot></table-wrap></p></sec><sec id=\"Sec4\"><title>Packing procedure</title><p id=\"Par8\">Briefly, each specimen is wrapped in archival tissue and placed inside a capsule with its label, several of which are inserted lengthwise into a straw. Up to 14 standard paper straws cut to 9 cm in length fit inside a closed 50 ml Falcon tube, with each straw holding 6 size 4 capsules. For easier tracking of specimens in the 3D volume, we recommend leaving one capsule empty in different positions along the length of some straws (see post-processing section below). Following this method, over 80 specimens < 1 cm long can be scanned in a single tube. This quantity can be doubled or tripled when 2 or 3 fossils (separated by tissue) are packed inside the same capsule, allowing a maximum of 252 specimens. However, scans with more than 3 specimens per capsule showed a high (≥ 50%) rate of movement during rotation, compared to those with fewer (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>). We therefore limit packing to 1–2 specimens per capsule, provided they can be distinguished from one another in relative size and/or shape. Specimens up to 2 cm long can be similarly packed inside size 00 capsules. Larger diameter paper or smoothie straws sliced lengthwise hold 3 size 00 capsules in a 50 ml tube (Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>b), which can be scanned together with smaller straws as needed.<table-wrap id=\"Tab2\"><label>Table 2</label><caption><p>Estimated movement among 408 fossils μCT scanned using various packing materials to secure the specimen inside each capsule.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" rowspan=\"2\">Packing material</th><th align=\"left\" colspan=\"3\">Number of specimens per capsule (size 4) that moved during scanning</th><th align=\"left\" rowspan=\"2\">Archival?</th><th align=\"left\" rowspan=\"2\">Comments</th></tr><tr><th align=\"left\">1–2</th><th align=\"left\">3</th><th align=\"left\"> > 3</th></tr></thead><tbody><tr><td align=\"left\">Green's Lens<sup>a</sup></td><td align=\"left\">0/100 (0%)</td><td align=\"left\">2/39 (5.1%)</td><td align=\"left\">25/50 (50%)</td><td align=\"left\">Yes</td><td align=\"left\">Ideal consistency</td></tr><tr><td align=\"left\">Kim Tech wipes<sup>b</sup></td><td align=\"left\">0/100 (0%)</td><td align=\"left\">4/39 (10.3%)</td><td align=\"left\">27/50 (54%)</td><td align=\"left\">No</td><td align=\"left\">100% fibre, not acid free</td></tr><tr><td align=\"left\">Ethafoam 220<sup>c</sup></td><td align=\"left\">10/10 (100%)</td><td align=\"left\">10/10 (100%)</td><td align=\"left\">10/10 (100%)</td><td align=\"left\">No</td><td align=\"left\">Slippery and hard</td></tr><tr><td align=\"left\">Tyvek<sup>d</sup></td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">Yes</td><td align=\"left\">Not tested, too waxy</td></tr><tr><td align=\"left\">Renaissance<sup>e</sup></td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">–</td><td align=\"left\">Yes</td><td align=\"left\">Not tested, too rigid</td></tr></tbody></table><table-wrap-foot><p><sup>a</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.archivalsurvival.com.au\">https://www.archivalsurvival.com.au</ext-link>.</p><p><sup>b</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.kcprofessional.com.au\">https://www.kcprofessional.com.au</ext-link>.</p><p><sup>c</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.foamsales.com.au\">https://www.foamsales.com.au</ext-link>.</p><p><sup>d</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.dupont.com.au\">https://www.dupont.com.au</ext-link>.</p><p><sup>e</sup><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.lightimpressionsdirect.com\">https://www.lightimpressionsdirect.com</ext-link>.</p></table-wrap-foot></table-wrap></p><p id=\"Par9\">Labels for each specimen are written in Indian or archival ink on uncoated acid-free paper, providing a unique and durable identifier that will be matched in the 3D volume. Our labels followed a two-part system containing specimen ID (e.g., museum accession number) and a code for straw and capsule position. Using permanent marker, each straw is labelled with a letter (A, B, C…N for 14 straws) and each capsule is labelled with its straw’s letter and a number from 1 to 6, denoting its position in the straw from top to bottom (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>a). This system can be amended to suit user needs, for example by including abbreviations for taxon if known and/or material (e.g., ‘il’ for ilium, ‘max’ for maxilla). These codes can also be extracted from the digital labels and used as variables in downstream analyses, e.g., in the R statistical package geomorph<sup><xref ref-type=\"bibr\" rid=\"CR39\">39</xref></sup> or MorphoJ<sup><xref ref-type=\"bibr\" rid=\"CR40\">40</xref></sup>. Record labels with capsule and straw positions, including any empty capsules as a marker, since these will be checked off later during unpacking.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Specimen labelling and packing: (<bold>a</bold>) 50 ml Falcon tube with a labelled straw and capsules, (<bold>b</bold>) a single fossil wrapped in Green’s Lens tissue to minimise movement during image acquisition, (<bold>c</bold>) loading capsules into the paper straw using a thinner cocktail straw to gently push them down, (<bold>d</bold>) a packed 50 ml Falcon tube mounted on a glass rod inside the μCT machine; note that the cap-side is facing down (<bold>e</bold>) the packed tube from above, and (<bold>f</bold>) the diagram of straw arrangements in (<bold>e</bold>), noting the position of a larger blue straw and paper used as a divider.</p></caption><graphic xlink:href=\"41598_2020_70970_Fig2_HTML\" id=\"MO2\"/></fig></p><p id=\"Par10\">Once labelling is complete, the specimens, labels, capsules and straws are aligned for packing. Each specimen is wrapped in a small envelope of Green’s Lens tissue (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>b), and empty spaces in the capsule are filled with tissue so the specimen does not contact its walls. This creates more distance between adjacent samples, allowing easier separation (i.e. segmentation) in the 3D volume. The tissue also prevents movement during image acquisition (Table <xref rid=\"Tab2\" ref-type=\"table\">2</xref>), which can lead to motion artefacts in the reconstruction like shadows or streaking. Finally, the label is inserted into the capsule with the ID facing out for long-term storage, and the capsules are loaded into their respective straws in the correct order. A thinner cocktail straw can be used to gently push the capsules down without compressing ones below it (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>c).</p><p id=\"Par11\">To load the packed straws into the Falcon tube, first fill the conical tip with polyethylene or other firm material to create a flat surface. Insert the straws with position 1 towards this end, which will be facing up in the μCT machine. The flat surface of the cap on the other end can be fixed to a glass rod or dowel using a hot glue gun to elevate the tube from the machine’s stage (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>d). This avoids including the metal clamp in the scan which could affect X-ray optimization, and centres the tube vertically and horizontally relative to the detector. Draw a diagram of the straw arrangement with the list of specimen labels, noting where paper or empty straws are used to separate them for easier identification (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>e,f). This material will be visible in reconstructed cross-sections, allowing one to match the 3D volume to the diagram. Finally, medium density foam can be cut and placed as filler around the straws and under the cap of the tube before closing. This foam is recommended over tissue or other supporting material as it acts as an effective shock absorber during rotation. Secure the tube onto the stage, making sure it can rotate freely without touching the X-ray source or detector.</p></sec><sec id=\"Sec5\"><title>X-ray parameters</title><p id=\"Par12\">Scan settings vary depending on the equipment, material size and properties, and desired results. Here we report parameters as optimized on a Phoenix nanotom m (GE Sensing & Inspection Technologies GmbH, Wunstorf, Germany) equipped with a 180 kV, 20 W high-power nanofocus X-ray tube and DXR detector (3,072 × 2,400 pixels). As with all microtomographic imaging, initial calibration of the detector is largely software-driven to reduce potential noise due to defective pixels or incident light. We calibrated the detector by taking an offset image (dark field) and two sets of gain images (flat field) at the same energy of the scan and at half the current, where images were averaged over 100 snapshots of the detector with a skip of 10. For all scans our instrument was fitted with a tungsten target, although molybdenum may be preferable for amber<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. Fossils can be radiodense, in such cases reconstruction artefacts may occur when lower energy X-rays are attenuated at the surface, or when stronger X-rays hit the detector without passing through the object first (beam hardening)<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. A thin metal filter placed in front of the X-ray source can overcome these issues by removing lower energy photons, although other parameters like current, voltage, or exposure time should be modified to achieve appropriate contrast.</p><p id=\"Par13\">Ideally the number of images for reconstruction is equal to the maximum width of the scanned object in pixels × π/2<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref>,<xref ref-type=\"bibr\" rid=\"CR41\">41</xref></sup>. For a sample optimized to fill the full width of our detector, this would result in nearly 5,000 projections at a resolution of 10.6 μm. Such a data set would be massive in terms of scan time and storage (up to 70 GB before reconstruction), in addition to the computing power and time needed to reconstruct and render each of the 3D files individually. To improve the μCT process for smaller specimens without sacrificing image quality, we divided the Falcon tube into three segments by moving the position of the stage up or down between scans, while keeping all other settings identical. This produced separate volumes of equally high quality while avoiding the need to move the detector back and/or the tube forward to accommodate its length, thereby decreasing resolution. These volumes can be merged using Avizo (Thermo Fisher Scientific) or other dedicated software to recreate the entire tube in high resolution. However, for faster processing we aligned each segment to include two rows of size 4 capsules (or one row of size 00), so they could be separately visualized and labelled in the 3D volume, if preferred.</p><p id=\"Par14\">For all reconstructions we followed the standard protocol of the Phoenix datos|x software (GE Sensing & Inspection Technologies GmbH, Wunstorf, Germany), which tests for object movement by comparing the first and last 2D projections. At high resolution, even minute movements can cause visible artefacts in the reconstructed images<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref>, <xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>. Instead of correcting for movement of single specimens during reconstruction by shifting the axis of rotation (which would in turn generate artefacts in the rest of the tube), we identified and labelled those specimens in post-processing so they could be rescanned. Other settings applied during reconstruction were a 3 × 3 median filter and an ROI filter.</p></sec><sec id=\"Sec6\"><title>Computed tomography post-processing</title><p id=\"Par15\">Following reconstruction, digital labelling of the specimens is performed in 3D volume rendering software. Here we describe steps using VGStudioMax 3.1 (Volume Graphics, Heidelberg, Germany), although other options for free or commercial licenses are available (see Table 7 in Keklikoglou et al.<sup><xref ref-type=\"bibr\" rid=\"CR38\">38</xref></sup>). To match the tube orientation to the diagram (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>f), the volume can be rotated in the 3D window or by using the registration tool in 2D. Straws can then be labelled digitally with the indicator tool or by creating a region of interest (ROI) to name each one by its letter (A, B, C… N). Similar-sized capsules should be aligned across the straws horizontally, making it easy to identify capsule positions in the 2D and 3D windows. For example, starting from the top (tip) of the tube in straw A, specimens should be ordered as A1, A2…A6, with capsules in the neighbouring straw B following the same order. Verifying the positions of empty capsules is another safeguard to ensure correct orientation. Using ROIs, the specimens can be segmented and renamed in the rendering software to match the specimen labels (e.g., museum accession number plus straw/capsule position). False-colouring straws or capsule rows differently in the 3D volume also helps to track specimen order. Any specimens with obvious artefacts can be marked with their labels followed by the word ‘REDO’, to be set aside during unpacking so they can be scanned again under improved conditions, i.e., repositioned in the capsule or with better tissue support.</p></sec><sec id=\"Sec7\"><title>Unpacking and storage</title><p id=\"Par16\">Capsules are unpacked from the straws in the same manner as they were put in, by inserting a cocktail straw into one end and pushing it through (Fig. <xref rid=\"Fig2\" ref-type=\"fig\">2</xref>c). We recommend checking specimens off the list as they are being unpacked as a final test of correct labelling. These samples can be stored long-term inside the capsules since archival tissue and paper was used.</p><p id=\"Par17\">Specimens for rescanning can be included in the next batch and labelled digitally with their ID, new straw and capsule position, and the word ‘REDONE’ for transparency. This process can be repeated as many times as necessary to achieve optimal scan quality for all material (e.g., labelled as REDONE_2, REDONE_3, etc.). Likewise, all labelled straws and Falcon tubes can be reused in subsequent scans, which will further ensure consistency in the packing and labelling process.</p></sec></sec><sec id=\"Sec8\"><title>Results and discussion</title><p id=\"Par18\">Optimal settings for our fossils in terms of time, resolution and file size were three separate scans of the Falcon tube at 40 mm focus-to-object distance (FOD) and 225 mm focus-to-detector distance (FDD), using 40 kV, 300 µA, and 0.5 s exposure for 1,400 images, with a frame average of 3 and image skip of 1. This configuration allowed us to decrease the width of the detector to 2,000 pixels, meaning that each X-ray image was 9.15 MB. The Y-axis of the stage was shifted down by 29 mm between scans to allow some overlap between segments (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a). For each segment this resulted in a scan time of 47 min and 17.8 µm voxel size, with reconstructed volumes around 10 GB each (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>b).<fig id=\"Fig3\"><label>Figure 3</label><caption><p>High-throughput μCT results: (<bold>a</bold>) the first X-ray image from each scanned segment of the 50 ml tube from top (tip) to bottom (cap), containing 72 frog and lizard specimens. (<bold>b</bold>) 3D renderings of the individual segments in the same positions, at 17.8 µm voxel size, (<bold>c</bold>) the stitched 3D volume at the same resolution, with fossils colour-coded by straw, (<bold>d</bold>) reconstructed cross-section of the same tube in side and (<bold>e</bold>) top views, (<bold>f</bold>) cross-section of an agamid lizard jaw showing contrast between the matrix, bone, and teeth, (<bold>g</bold>) cross-section of a skink mandible with a shadow motion artefact, (<bold>h</bold>) an example of beam hardening caused by a dense surface.</p></caption><graphic xlink:href=\"41598_2020_70970_Fig3_HTML\" id=\"MO3\"/></fig></p><p id=\"Par19\">This process is in stark contrast to the effort needed to scan each specimen individually at the same resolution, requiring over 65 h for a fully packed tube (one specimen per capsule), excluding time to swap samples. Automated sample changers are available for some µCT machines (e.g., Bruker SkyScan, Bruker Biosciences Pty Ltd), although these are still limited to under 20 specimens. Reconstructing and rendering each µCT file separately would also require substantial computing time, making the task unfeasible for individual researchers wishing to work on large data sets. Instead, we stitched the three segments together into a volume of equally high resolution (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>c), allowing access to multiple specimens by opening a single 30 GB file.</p><p id=\"Par20\">The image quality achieved by our method is appropriate for numerous applications in biological systematics, including geometric morphometrics, functional analyses, and morphological descriptions. Cross-sectional µCT images revealed sharp contrast between the hydroxyapatite of the bone, dentine, and enamel against the clay and crystalline calcium carbonate-rich matrix, while offering precise details of specimen morphology as well as the dividing walls of the capsules, straws and paper (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>d–f). In few cases artefacts were observed in the reconstruction due to movement (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>g) or beam hardening (Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>h). These specimens were labelled as ‘REDO’ in the 3D volume and set aside during unpacking to be rescanned, either with additional tissue in the capsule or using a filter, respectively. In earlier scans we observed some capsules in straws that had been slightly crushed by the one above it. These straws were packed using a solid dowel to push down the capsules, hence why we recommend using a cocktail straw instead.</p><p id=\"Par21\">Other examples of material scanned using our high-throughput method are shown in Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>. Fossilized frog ilia and varanid osteoderms (bony deposits in the scales of some lizards) yielded excellent results following this protocol (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>a,b), with hundreds of 3D models generated for minimal time, money and effort. We also applied our system to larger mammalian specimens, for example fossilized rodent jaws from the Middle Pleistocene Mt Etna<sup><xref ref-type=\"bibr\" rid=\"CR42\">42</xref></sup>. This material is larger and denser than the herpetological samples, requiring slightly different parameters for µCT scanning. For larger/denser fossils, a 0.1 mm copper plate was secured under the collimator to reduce beam hardening and other artefacts, and X-ray voltage was increased to 50–80 kV. The resulting reconstructed images could be easily segmented using density-based approaches in VGStudioMax, like the region growing tool to separate teeth from bone (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>c).<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Scanned fossil material and its applications: (<bold>a</bold>) 3D rendering of 73 fossil frog ilia scanned in a single tube, (<bold>b</bold>) a subsample of varanid osteoderms in 3D, (<bold>c</bold>) mandible of the rainforest rodent <italic>Pogonomys</italic> from the Riversleigh World Heritage site with molars colour coded and jaw bone rendered semi-transparent, (<bold>d</bold>) surface mesh of the same specimen composed of 109,213 vertices (file size 10.5 MB), (<bold>e</bold>) photograph of children holding a 3D print of enlarged fossil skink jaws at Capricorn Caves Fossil Open Day, (<bold>f</bold>) point cloud comparison of fossil tree frog (Hylidae) ilia found in Capricorn Caves at 10–20 cm depth (left) and 40–50 cm depth (right), representing a time difference of 2–3,000 years. Warmer colours denote regions with greater shape differences, as estimated in CloudCompare v.2.10.2.</p></caption><graphic xlink:href=\"41598_2020_70970_Fig4_HTML\" id=\"MO4\"/></fig></p><p id=\"Par22\">Stewardship of µCT data is still a major challenge for natural history museums, which are now tasked with the curation of rapidly expanding digital datasets associated with the physical collections<sup><xref ref-type=\"bibr\" rid=\"CR43\">43</xref></sup>. There is currently no standard for 3D data storage, meaning that the fate of µCT images and their metadata is often individual or institution-driven. We retain at least two copies of the original X-rays and reconstructions in different locations (one cloud-based, one physical), while working on the prepared volume on a regularly backed-up server. Davies et al.<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup> provide a summary of file types that should be retained for 3D digital morphology analyses, with best practices recommended when storage space is not a problem. Online data repositories are another attractive option for long-term data storage, several of which are free and cater to 3D digital datasets generated for biological research<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>, e.g., Zenodo (zenodo.org), MorphoSource (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.morphosource.org\">www.morphosource.org</ext-link>).</p><p id=\"Par23\">For most downstream analyses, µCT data will be converted into polygonal surface models such as STL (sterolithography) files, which capture the geometric shape of a 3D object using triangles or vertices to describe its surface (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>d). These models, also known as surface meshes, are virtually interactive and can be manipulated, viewed, and measured using the open source software MeshLab (<ext-link ext-link-type=\"uri\" xlink:href=\"https://meshlab.sourceforge.net/\">https://meshlab.sourceforge.net/</ext-link>), among others. They are also small in terms of data storage, making them easy to distribute online and providing wider access to museum collections. Finally, surface meshes can be scaled up for displays and educational programs, for example using 3D printing. Following digital labelling and unpacking of our specimens, we extracted STL models of each fossil from its ROI in VGStudioMax, while retaining its original identity.</p><p id=\"Par24\">Many of the fossils included in this project were excavated from cave deposits at Capricorn Caves in Rockhampton, Queensland, Australia. As part of an annual science communication event called Capricorn Caves Fossil Open Day<sup><xref ref-type=\"bibr\" rid=\"CR44\">44</xref></sup>, we 3D printed select specimens to engage the public in local biodiversity. There, palaeontologists referred to the enlarged 3D models to assist in the interpretation of microfossils, allowing children to interact with replicas of local animals from thousands of years in the past (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>e). In addition to public outreach, our future work will focus on analyzing the generated µCT data using a growing toolkit of bioinformatic approaches, including deep learning (AI), 3D landmark-based geometric morphometrics, and high-density point cloud comparisons of fossil and living forms (Fig. <xref rid=\"Fig4\" ref-type=\"fig\">4</xref>f).</p></sec><sec id=\"Sec9\"><title>Conclusions</title><p id=\"Par25\">Current research on museum-based µCT data encompasses an extraordinary array of material and topics, including tetrapod origins<sup><xref ref-type=\"bibr\" rid=\"CR45\">45</xref></sup>, echolocation in bats<sup><xref ref-type=\"bibr\" rid=\"CR46\">46</xref></sup>and whales<sup><xref ref-type=\"bibr\" rid=\"CR47\">47</xref></sup>, limb reduction in lizards<sup><xref ref-type=\"bibr\" rid=\"CR48\">48</xref>,<xref ref-type=\"bibr\" rid=\"CR49\">49</xref></sup>, mammalian limb development<sup><xref ref-type=\"bibr\" rid=\"CR50\">50</xref>,<xref ref-type=\"bibr\" rid=\"CR51\">51</xref></sup>, primate neuroanatomy<sup><xref ref-type=\"bibr\" rid=\"CR52\">52</xref>,<xref ref-type=\"bibr\" rid=\"CR53\">53</xref></sup>, reproduction in insects<sup><xref ref-type=\"bibr\" rid=\"CR54\">54</xref></sup> and plants<sup><xref ref-type=\"bibr\" rid=\"CR55\">55</xref></sup>, paleoecology of Precambrian biota<sup><xref ref-type=\"bibr\" rid=\"CR56\">56</xref></sup>, seafloor biomass estimation<sup><xref ref-type=\"bibr\" rid=\"CR57\">57</xref></sup> and amber inclusions<sup><xref ref-type=\"bibr\" rid=\"CR58\">58</xref></sup>, to name a few. Typically these studies involve few specimens, due to the challenges of capturing high resolution 3D data for large sample sizes.</p><p id=\"Par26\">Here we demonstrate how simple, inexpensive equipment like drinking straws and pharmaceutical capsules can be modified for high-throughput µCT scanning of hundreds of fossils in a single container, providing large-scale morphological datasets for comparative analyses. To date we have scanned over 1,000 accessioned museum specimens following this technique, including frogs, lizards, snakes and mammals. These data will contribute to ongoing efforts to identify evolutionary responses to climate change in Australia’s fossil record, where reptiles and amphibians constitute half of the terrestrial vertebrate diversity, and where increasing aridity has transformed large parts of the continent. This method can also be adapted for other (non-biological) objects, by creating custom-made holders and stages, and seeking µCT scanners with differing capacities. Some users may wish to scan individual specimens or holotypes at the highest possible resolution, in which case our protocol offers an efficient pathway for determining which specimens should be elevated to type status. At the same time we note that fossils can be difficult to scan, being highly variable in shape, composition, and density. Thus while µCT is a powerful tool for most small specimens, in some cases other non-destructive techniques such as magnetic resonance imaging (MRI), synchrotron, and confocal microscopy may be more appropriate.</p></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><ack><title>Acknowledgements</title><p>This work was supported by an Australian Research Council DECRA (DE180100629) to C.A.H., with scans performed through the Melbourne TrACEES platform. Danielle Measday, Conservator of Natural Sciences at Museums Victoria, advised on and provided archival papers for testing. We thank Kristen Spring, Rochelle Lawrence, Joanne Wilkinson, and Jonathan Cramb, along with many volunteers at the Queensland Museum, for their assistance in generating the large quantity of specimens made available for this project.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>C.A.H. and S.H. conceived the project; S.H. selected specimens for µCT scanning; C.A.H., R.A., and J.R.B. designed the methodology; J.R.B. operated the µCT machine; C.A.H. and R.A. collected and analysed the data and led manuscript writing. All authors contributed to manuscript drafts and gave final approval for publication.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>All X-ray and µCT data shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>a–e are available on MorphoSource (<ext-link ext-link-type=\"uri\" xlink:href=\"http://morphosource.org/\">http://morphosource.org/</ext-link>).</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interests</title><p id=\"Par27\">The authors declare no competing interests.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sato</surname><given-names>T</given-names></name><name><surname>Ikeda</surname><given-names>O</given-names></name><name><surname>Yamakoshi</surname><given-names>Y</given-names></name><name><surname>Tsubouchi</surname><given-names>M</given-names></name></person-group><article-title>X-ray tomography for microstructural objects</article-title><source>Appl. Opt.</source><year>1981</year><volume>20</volume><fpage>3880</fpage><lpage>3883</lpage><pub-id pub-id-type=\"pmid\">20372286</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Elliott</surname><given-names>JC</given-names></name><name><surname>Dover</surname><given-names>SD</given-names></name></person-group><article-title>X-ray microtomography</article-title><source>J. Microsc.</source><year>1982</year><volume>126</volume><fpage>211</fpage><lpage>213</lpage><pub-id pub-id-type=\"pmid\">7086891</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Elliott</surname><given-names>JC</given-names></name><name><surname>Dover</surname><given-names>SD</given-names></name></person-group><article-title>X-ray microscopy using computerized axial tomography</article-title><source>J. Microsc.</source><year>1985</year><volume>138</volume><fpage>329</fpage><lpage>331</lpage><pub-id pub-id-type=\"pmid\">4032470</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Sutton</surname><given-names>M</given-names></name><name><surname>Rahman</surname><given-names>I</given-names></name><name><surname>Garwood</surname><given-names>R</given-names></name></person-group><source>Techniques for Virtual Palaeontology</source><year>2014</year><publisher-loc>London</publisher-loc><publisher-name>Wiley-Blackwell</publisher-name><fpage>208</fpage></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Davies</surname><given-names>TG</given-names></name><etal/></person-group><article-title>Open data and digital morphology</article-title><source>Proc. R. Soc. B</source><year>2017</year><volume>284</volume><fpage>20170194</fpage><pub-id pub-id-type=\"pmid\">28404779</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gutiérrez</surname><given-names>Y</given-names></name><name><surname>Ott</surname><given-names>D</given-names></name><name><surname>Töpperwien</surname><given-names>M</given-names></name><name><surname>Salditt</surname><given-names>T</given-names></name><name><surname>Scherber</surname><given-names>C</given-names></name></person-group><article-title>X-ray computed tomography and its potential in ecological research: a review of studies and optimization of specimen preparation</article-title><source>Ecol. Evol.</source><year>2018</year><volume>8</volume><fpage>7717</fpage><lpage>7732</lpage><pub-id pub-id-type=\"pmid\">30151184</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schindelin</surname><given-names>J</given-names></name><etal/></person-group><article-title>Fiji: an open-source platform for biological-image analysis</article-title><source>Nat. Methods</source><year>2012</year><volume>9</volume><fpage>676</fpage><lpage>682</lpage><pub-id pub-id-type=\"pmid\">22743772</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ketcham</surname><given-names>RA</given-names></name></person-group><article-title>Computational methods for quantitative analysis of three-dimensional features in geological specimens</article-title><source>Geosphere</source><year>2005</year><volume>1</volume><fpage>32</fpage><lpage>41</lpage></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Page</surname><given-names>LM</given-names></name><name><surname>MacFadden</surname><given-names>BJ</given-names></name><name><surname>Fortes</surname><given-names>JA</given-names></name><name><surname>Soltis</surname><given-names>PS</given-names></name><name><surname>Riccardi</surname><given-names>G</given-names></name></person-group><article-title>Digitization of biodiversity collections reveals biggest data on biodiversity</article-title><source>Bioscience</source><year>2015</year><volume>65</volume><fpage>841</fpage><lpage>842</lpage></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Faulwetter</surname><given-names>S</given-names></name><name><surname>Vasileiadou</surname><given-names>A</given-names></name><name><surname>Kouratoras</surname><given-names>M</given-names></name><name><surname>Dailianis</surname><given-names>T</given-names></name><name><surname>Arvanitidis</surname><given-names>C</given-names></name></person-group><article-title>Micro-computed tomography: introducing new dimensions to taxonomy</article-title><source>ZooKeys</source><year>2013</year><volume>263</volume><fpage>1</fpage><lpage>45</lpage></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Akkari</surname><given-names>N</given-names></name><etal/></person-group><article-title>New avatars for Myriapods: complete 3D morphology of type specimens transcends conventional species description (Myriapoda, Chilopoda)</article-title><source>PLoS ONE</source><year>2018</year><volume>13</volume><fpage>0200158</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0200158</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fontaine</surname><given-names>B</given-names></name><name><surname>Perrard</surname><given-names>A</given-names></name><name><surname>Bouchet</surname><given-names>P</given-names></name></person-group><article-title>21 years of shelf life between discovery and description of new species</article-title><source>Curr. Biol.</source><year>2012</year><volume>22</volume><fpage>R943</fpage><lpage>R944</lpage><pub-id pub-id-type=\"pmid\">23174292</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hipsley</surname><given-names>CA</given-names></name><name><surname>Sherratt</surname><given-names>E</given-names></name></person-group><article-title>Psychology, not technology, is our biggest challenge to open digital morphology data</article-title><source>Sci. Data.</source><year>2019</year><volume>6</volume><fpage>41</fpage><pub-id pub-id-type=\"pmid\">31028285</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Blagoderov</surname><given-names>V</given-names></name><name><surname>Kitching</surname><given-names>IJ</given-names></name><name><surname>Livermore</surname><given-names>L</given-names></name><name><surname>Simonsen</surname><given-names>TJ</given-names></name><name><surname>Smith</surname><given-names>VS</given-names></name></person-group><article-title>No specimen left behind: industrial scale digitization of natural history collections</article-title><source>Zookeys</source><year>2012</year><volume>209</volume><fpage>133</fpage><lpage>146</lpage></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rogers</surname><given-names>N</given-names></name></person-group><article-title>Museum drawers go digital</article-title><source>Science</source><year>2016</year><volume>352</volume><fpage>762</fpage><lpage>765</lpage><pub-id pub-id-type=\"pmid\">27174969</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Meineke</surname><given-names>EK</given-names></name><name><surname>Davies</surname><given-names>TJ</given-names></name><name><surname>Daru</surname><given-names>BH</given-names></name><name><surname>Davis</surname><given-names>CC</given-names></name></person-group><article-title>Biological collections for understanding biodiversity in the Anthropocene</article-title><source>Philos. Trans. R. Soc. B</source><year>2018</year><volume>374</volume><fpage>20170386</fpage></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schmitt</surname><given-names>CJ</given-names></name><name><surname>Cook</surname><given-names>JA</given-names></name><name><surname>Zamudio</surname><given-names>KR</given-names></name><name><surname>Edwards</surname><given-names>SV</given-names></name></person-group><article-title>Museum specimens of terrestrial vertebrates are sensitive indicators of environmental change in the Anthropocene</article-title><source>Philos. Trans. R. Soc. B</source><year>2018</year><volume>374</volume><fpage>20170387</fpage></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sherratt</surname><given-names>E</given-names></name><name><surname>Gower</surname><given-names>DJ</given-names></name><name><surname>Klingenberg</surname><given-names>CP</given-names></name><name><surname>Wilkinson</surname><given-names>M</given-names></name></person-group><article-title>Evolution of cranial shape in caecilians (Amphibia: Gymnophiona)</article-title><source>Evol. Biol.</source><year>2014</year><volume>41</volume><fpage>528</fpage><lpage>545</lpage></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Watanabe</surname><given-names>A</given-names></name><etal/></person-group><article-title>Ecomorphological diversification in squamates from conserved pattern of cranial integration</article-title><source>Proc. Natl. Acad. Sci.</source><year>2019</year><volume>116</volume><fpage>14688</fpage><lpage>14697</lpage><pub-id pub-id-type=\"pmid\">31262818</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Simon</surname><given-names>MN</given-names></name><name><surname>Machado</surname><given-names>FA</given-names></name><name><surname>Marroig</surname><given-names>G</given-names></name></person-group><article-title>High evolutionary constraints limited adaptive responses to past climate changes in toad skulls</article-title><source>Proc. R. Soc. B-Biol. Sci.</source><year>2016</year><volume>283</volume><fpage>20161783</fpage></element-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sherratt</surname><given-names>E</given-names></name><name><surname>Serb</surname><given-names>JM</given-names></name><name><surname>Adams</surname><given-names>DC</given-names></name></person-group><article-title>Rates of morphological evolution, asymmetry and morphological integration of shell shape in scallops</article-title><source>BMC Evol. Biol.</source><year>2017</year><volume>17</volume><fpage>248</fpage><pub-id pub-id-type=\"pmid\">29216839</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chira</surname><given-names>AM</given-names></name><etal/></person-group><article-title>Correlates of rate heterogeneity in avian ecomorphological traits</article-title><source>Ecol. Lett.</source><year>2018</year><volume>21</volume><fpage>1505</fpage><lpage>1514</lpage><pub-id pub-id-type=\"pmid\">30133084</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Percival</surname><given-names>CJ</given-names></name><etal/></person-group><article-title>The effect of automated landmark identification on morphometric analyses</article-title><source>J. Anat.</source><year>2019</year><volume>234</volume><fpage>917</fpage><lpage>935</lpage><pub-id pub-id-type=\"pmid\">30901082</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bouxsein</surname><given-names>ML</given-names></name><etal/></person-group><article-title>Guidelines for assessment of bone microstructure in rodents using micro –computed tomography</article-title><source>J. Bone Miner. Res.</source><year>2010</year><volume>25</volume><fpage>1468</fpage><lpage>1486</lpage><pub-id pub-id-type=\"pmid\">20533309</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Broeckhoven</surname><given-names>C</given-names></name><name><surname>du Plessis</surname><given-names>A</given-names></name></person-group><article-title>X-ray microtomography in herpetological research: a review</article-title><source>Amphibia-Reptilia</source><year>2018</year><volume>39</volume><fpage>377</fpage><lpage>401</lpage></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Marcy</surname><given-names>AE</given-names></name><name><surname>Fruciano</surname><given-names>C</given-names></name><name><surname>Phillips</surname><given-names>MJ</given-names></name><name><surname>Mardon</surname><given-names>K</given-names></name><name><surname>Weisbecker</surname><given-names>V</given-names></name></person-group><article-title>Low resolution scans can provide a sufficiently accurate, cost- and time-effective alternative to high resolution scans for 3D shape analyses</article-title><source>PeerJ</source><year>2018</year><volume>6</volume><fpage>5032</fpage><pub-id pub-id-type=\"doi\">10.7717/peerj.5032</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gray</surname><given-names>JA</given-names></name><name><surname>McDowell</surname><given-names>MC</given-names></name><name><surname>Hutchinson</surname><given-names>MN</given-names></name><name><surname>Jones</surname><given-names>ME</given-names></name></person-group><article-title>Geometric morphometrics provides an alternative approach for interpreting the affinity of fossil lizard jaws</article-title><source>J. Herpetol.</source><year>2017</year><volume>51</volume><fpage>375</fpage><lpage>382</lpage></element-citation></ref><ref id=\"CR28\"><label>28.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thorn</surname><given-names>KM</given-names></name><name><surname>Hutchinson</surname><given-names>MN</given-names></name><name><surname>Archer</surname><given-names>M</given-names></name><name><surname>Lee</surname><given-names>MSY</given-names></name></person-group><article-title>A new scincid lizard from the Miocene of northern Australia, and the evolutionary history of social skinks (Scincidae: Egerniinae)</article-title><source>J. Vertebr. Paleontol.</source><year>2019</year><volume>39</volume><fpage>1</fpage></element-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chaplin</surname><given-names>K</given-names></name><name><surname>Sumner</surname><given-names>J</given-names></name><name><surname>Hipsley</surname><given-names>CA</given-names></name><name><surname>Melville</surname><given-names>J</given-names></name></person-group><article-title>An integrative approach using phylogenomics and high-resolution X-ray computed tomography for species delimitation in cryptic taxa</article-title><source>Syst. Biol.</source><year>2019</year><volume>69</volume><fpage>syz048</fpage><pub-id pub-id-type=\"doi\">10.1093/sysbio/syz048</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Melville</surname><given-names>J</given-names></name><etal/></person-group><article-title>Integrating phylogeography and high-resolution X-ray CT reveals five new cryptic species and multiple hybrid zones among Australian earless dragons</article-title><source>R. Soc. Open Sci.</source><year>2019</year><volume>6</volume><fpage>191166</fpage><pub-id pub-id-type=\"doi\">10.1098/rsos.191166</pub-id><pub-id pub-id-type=\"pmid\">31903207</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Caro</surname><given-names>A</given-names></name><name><surname>Gómez-Moliner</surname><given-names>BJ</given-names></name><name><surname>Madeira</surname><given-names>MJ</given-names></name></person-group><article-title>Integrating multilocus DNA data and 3D geometric morphometrics to elucidate species boundaries in the case of <italic>Pyrenaearia</italic> (Pulmonata: Hygromiidae)</article-title><source>Mol. Phylogenet. Evol.</source><year>2019</year><volume>132</volume><fpage>194</fpage><lpage>206</lpage><pub-id pub-id-type=\"pmid\">30550961</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Winkelmann</surname><given-names>CT</given-names></name><name><surname>Wise</surname><given-names>LD</given-names></name></person-group><article-title>High-throughput micro-computed tomography imaging as a method to evaluate rat and rabbit fetal skeletal abnormalities for developmental toxicity studies</article-title><source>J. Pharmacol. Tox. Met.</source><year>2009</year><volume>59</volume><fpage>156</fpage><lpage>165</lpage></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sevilla</surname><given-names>RS</given-names></name><etal/></person-group><article-title>Development and optimization of a high-throughput micro-computed tomography imaging method incorporating a novel analysis technique to evaluate bone mineral density of arthritic joints in a rodent model of collagen induced arthritis</article-title><source>Bone</source><year>2015</year><volume>73</volume><fpage>32</fpage><lpage>41</lpage><pub-id pub-id-type=\"pmid\">25482211</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wong</surname><given-names>MD</given-names></name><name><surname>Maezawa</surname><given-names>Y</given-names></name><name><surname>Lerch</surname><given-names>JP</given-names></name><name><surname>Henkelman</surname><given-names>RM</given-names></name></person-group><article-title>Automated pipeline for anatomical phenotyping of mouse embryos using micro-CT</article-title><source>Development</source><year>2014</year><volume>141</volume><fpage>2533</fpage><lpage>2541</lpage><pub-id pub-id-type=\"pmid\">24850858</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>D</given-names></name><etal/></person-group><article-title>Combining high-throughput micro-CT-RGB phenotyping and genome-wide association study to dissect the genetic architecture of tiller growth in rice</article-title><source>J. Exp. Bot.</source><year>2019</year><volume>70</volume><fpage>545</fpage><lpage>561</lpage><pub-id pub-id-type=\"pmid\">30380099</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ding</surname><given-names>Y</given-names></name><etal/></person-group><article-title>Computational 3D histological phenotyping of whole zebrafish by X-ray histotomography</article-title><source>Elife</source><year>2019</year><volume>8</volume><fpage>44898</fpage><pub-id pub-id-type=\"doi\">10.7554/eLife.44898.001</pub-id></element-citation></ref><ref id=\"CR37\"><label>37.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Staedtler</surname><given-names>YM</given-names></name><name><surname>Masson</surname><given-names>D</given-names></name><name><surname>Schönenberger</surname><given-names>J</given-names></name></person-group><article-title>Plant tissues in 3D via X-ray tomography: simple contrasting methods allow high resolution imaging</article-title><source>PLoS ONE</source><year>2013</year><volume>8</volume><fpage>75295</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0075295</pub-id></element-citation></ref><ref id=\"CR38\"><label>38.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Keklikoglou</surname><given-names>K</given-names></name><etal/></person-group><article-title>Micro-computed tomography for natural history specimens: a handbook of best practice protocols</article-title><source>Eur. J. Taxon.</source><year>2019</year><volume>522</volume><fpage>1</fpage><lpage>55</lpage></element-citation></ref><ref id=\"CR39\"><label>39.</label><mixed-citation publication-type=\"other\">Adams, D., Collyer, M. & Kaliontzopoulou, A. Geomorph: Software for geometric morphometric analyses. R package version 3.1.0. <ext-link ext-link-type=\"uri\" xlink:href=\"https://cran.r-project.org/package=geomorph\">https://cran.r-project.org/package=geomorph</ext-link> (2019).</mixed-citation></ref><ref id=\"CR40\"><label>40.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Klingenberg</surname><given-names>CP</given-names></name></person-group><article-title>MorphoJ: an integrated software package for geometric morphometrics</article-title><source>Mol. Ecol. Resour.</source><year>2011</year><volume>11</volume><fpage>353</fpage><lpage>357</lpage><pub-id pub-id-type=\"pmid\">21429143</pub-id></element-citation></ref><ref id=\"CR41\"><label>41.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>du Plessis</surname><given-names>A</given-names></name><name><surname>Broeckhoven</surname><given-names>C</given-names></name><name><surname>Guelpa</surname><given-names>A</given-names></name><name><surname>le Roux</surname><given-names>SG</given-names></name></person-group><article-title>Laboratory X-ray micro-computed tomography: a user guideline for biological samples</article-title><source>Gigascience</source><year>2017</year><volume>6</volume><fpage>1</fpage><lpage>11</lpage><pub-id pub-id-type=\"pmid\">28419369</pub-id></element-citation></ref><ref id=\"CR42\"><label>42.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hocknull</surname><given-names>SA</given-names></name><name><surname>Zhao</surname><given-names>JX</given-names></name><name><surname>Feng</surname><given-names>YX</given-names></name><name><surname>Webb</surname><given-names>GE</given-names></name></person-group><article-title>Responses of middle Pleistocene rainforest vertebrates to climate change in Australia</article-title><source>Earth Planet. Sci. Lett.</source><year>2007</year><volume>264</volume><fpage>317</fpage><lpage>331</lpage></element-citation></ref><ref id=\"CR43\"><label>43.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hedrick</surname><given-names>BP</given-names></name><etal/></person-group><article-title>Digitization and the future of natural history collections</article-title><source>Bioscience</source><year>2020</year><volume>70</volume><fpage>243</fpage><lpage>251</lpage></element-citation></ref><ref id=\"CR44\"><label>44.</label><mixed-citation publication-type=\"other\">Lawrence, R. A. & Hocknull, S. Engaging the public with small vertebrate fossils and utilizing citizen science to maximise scientific discovery at Capricorn Caves, Central Eastern Queensland, Australia. <italic>J. Vertebr. Paleontol. Program Abstr.</italic><bold>139</bold> (2019).</mixed-citation></ref><ref id=\"CR45\"><label>45.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Long</surname><given-names>JA</given-names></name><name><surname>Young</surname><given-names>GC</given-names></name><name><surname>Holland</surname><given-names>T</given-names></name><name><surname>Senden</surname><given-names>TJ</given-names></name><name><surname>Fitzgerald</surname><given-names>EM</given-names></name></person-group><article-title>An exceptional Devonian fish from Australia sheds light on tetrapod origins</article-title><source>Nature</source><year>2006</year><volume>444</volume><fpage>199</fpage><lpage>202</lpage><pub-id pub-id-type=\"pmid\">17051154</pub-id></element-citation></ref><ref id=\"CR46\"><label>46.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arbour</surname><given-names>JH</given-names></name><name><surname>Curtis</surname><given-names>AA</given-names></name><name><surname>Santana</surname><given-names>SE</given-names></name></person-group><article-title>Signatures of echolocation and dietary ecology in the adaptive evolution of skull shape in bats</article-title><source>Nat. Commun.</source><year>2019</year><volume>10</volume><fpage>2036</fpage><pub-id pub-id-type=\"pmid\">31048713</pub-id></element-citation></ref><ref id=\"CR47\"><label>47.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Park</surname><given-names>T</given-names></name><name><surname>Fitzgerald</surname><given-names>EM</given-names></name><name><surname>Evans</surname><given-names>AR</given-names></name></person-group><article-title>Ultrasonic hearing and echolocation in the earliest toothed whales</article-title><source>Biol. Lett.</source><year>2016</year><volume>12</volume><fpage>20160060</fpage><pub-id pub-id-type=\"doi\">10.1098/rsbl.2016.0060</pub-id><pub-id pub-id-type=\"pmid\">27072406</pub-id></element-citation></ref><ref id=\"CR48\"><label>48.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Müller</surname><given-names>J</given-names></name><etal/></person-group><article-title>Eocene lizard from Germany reveals amphisbaenian origins</article-title><source>Nature</source><year>2011</year><volume>473</volume><fpage>364</fpage><lpage>367</lpage><pub-id pub-id-type=\"pmid\">21593869</pub-id></element-citation></ref><ref id=\"CR49\"><label>49.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Miralles</surname><given-names>A</given-names></name><etal/></person-group><article-title>Distinct patterns of desynchronized limb regression in Malagasy scincine lizards (Squamata, Scincidae)</article-title><source>PLoS ONE</source><year>2015</year><volume>10</volume><fpage>0126074</fpage><pub-id pub-id-type=\"doi\">10.1371/journal.pone.0126074</pub-id></element-citation></ref><ref id=\"CR50\"><label>50.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weisbecker</surname><given-names>V</given-names></name></person-group><article-title>Monotreme ossification sequences and the riddle of mammalian skeletal development</article-title><source>Evolution</source><year>2011</year><volume>65</volume><fpage>1323</fpage><lpage>1335</lpage><pub-id pub-id-type=\"pmid\">21521190</pub-id></element-citation></ref><ref id=\"CR51\"><label>51.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Newton</surname><given-names>AH</given-names></name><etal/></person-group><article-title>Letting the ‘cat’ out of the bag: pouch young development of the extinct Tasmanian tiger revealed by X-ray computed tomography</article-title><source>R. Soc. Open Sci.</source><year>2018</year><volume>5</volume><fpage>171914</fpage><pub-id pub-id-type=\"doi\">10.1098/rsos.171914</pub-id><pub-id pub-id-type=\"pmid\">29515893</pub-id></element-citation></ref><ref id=\"CR52\"><label>52.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hublin</surname><given-names>JJ</given-names></name><etal/></person-group><article-title>New fossils from Jebel Irhoud, Morocco and the pan-African origin of Homo sapiens</article-title><source>Nature</source><year>2017</year><volume>546</volume><fpage>289</fpage><lpage>292</lpage><pub-id pub-id-type=\"pmid\">28593953</pub-id></element-citation></ref><ref id=\"CR53\"><label>53.</label><mixed-citation publication-type=\"other\">Beaudet, A. & Gilissen, E. Fossil primate endocasts: perspectives from advanced imaging techniques In <italic>Digital Endocasts: from Skulls to Brains</italic> (eds. Bruner, E., Ogihara, N. & Tanabe, H.) 47–58 (Springer, Berlin, 2018).</mixed-citation></ref><ref id=\"CR54\"><label>54.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wulff</surname><given-names>NC</given-names></name><name><surname>Lehmann</surname><given-names>AW</given-names></name><name><surname>Hipsley</surname><given-names>CA</given-names></name><name><surname>Lehmann</surname><given-names>GUC</given-names></name></person-group><article-title>Copulatory courtship by bushcricket genital titillators revealed by functional morphology, μCT scanning for 3D reconstruction and female sense structures</article-title><source>Arthropod Struct. Dev.</source><year>2015</year><volume>44</volume><fpage>388</fpage><lpage>397</lpage><pub-id pub-id-type=\"pmid\">26014975</pub-id></element-citation></ref><ref id=\"CR55\"><label>55.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gee</surname><given-names>CT</given-names></name></person-group><article-title>Applying microCT and 3D visualization to Jurassic silicified conifer seed cones: a virtual advantage over thin-sectioning</article-title><source>Appl. Plant Sci.</source><year>2013</year><volume>1</volume><fpage>1300039</fpage><pub-id pub-id-type=\"doi\">10.3732/apps.1300039</pub-id></element-citation></ref><ref id=\"CR56\"><label>56.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Meyer</surname><given-names>M</given-names></name><etal/></person-group><article-title>Three-dimensional microCT analysis of the Ediacara fossil <italic>Pteridinium</italic> simplex sheds new light on its ecology and phylogenetic affinity</article-title><source>Precambrian Res.</source><year>2014</year><volume>249</volume><fpage>79</fpage><lpage>87</lpage></element-citation></ref><ref id=\"CR57\"><label>57.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gooday</surname><given-names>AJ</given-names></name><name><surname>Sykes</surname><given-names>D</given-names></name><name><surname>Goral</surname><given-names>T</given-names></name><name><surname>Zubkov</surname><given-names>MV</given-names></name><name><surname>Glover</surname><given-names>AG</given-names></name></person-group><article-title>Micro-CT 3D imaging reveals the internal structure of three abyssal xenophyophore species (Protista, Foraminifera) from the eastern equatorial Pacific Ocean</article-title><source>Sci. Rep.</source><year>2018</year><volume>8</volume><fpage>12103</fpage><pub-id pub-id-type=\"doi\">10.1038/s41598-018-30186-2</pub-id><pub-id pub-id-type=\"pmid\">30108286</pub-id></element-citation></ref><ref id=\"CR58\"><label>58.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dunlop</surname><given-names>JA</given-names></name><etal/></person-group><article-title>Microtomography of the Baltic amber tick <italic>Ixodes succineus</italic> reveals affinities with the modern Asian disease vector <italic>Ixodes ovatus</italic></article-title><source>BMC Evol. Biol.</source><year>2016</year><volume>16</volume><fpage>203</fpage><pub-id pub-id-type=\"pmid\">27724841</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" xmlns:xlink=\"http://www.w3.org/1999/xlink\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Sci Rep</journal-id><journal-id journal-id-type=\"iso-abbrev\">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type=\"epub\">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32807896</article-id><article-id pub-id-type=\"pmc\">PMC7431593</article-id><article-id pub-id-type=\"publisher-id\">70734</article-id><article-id pub-id-type=\"doi\">10.1038/s41598-020-70734-3</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Micro-CT scan with virtual dissection of left ventricle is a non-destructive, reproducible alternative to dissection and weighing for left ventricular size</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Doost</surname><given-names>Ata</given-names></name><xref ref-type=\"aff\" rid=\"Aff1\">1</xref></contrib><contrib contrib-type=\"author\"><name><surname>Rangel</surname><given-names>Alejandra</given-names></name><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><contrib contrib-type=\"author\"><name><surname>Nguyen</surname><given-names>Quang</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\"><name><surname>Morahan</surname><given-names>Grant</given-names></name><xref ref-type=\"aff\" rid=\"Aff3\">3</xref></contrib><contrib contrib-type=\"author\" corresp=\"yes\"><name><surname>Arnolda</surname><given-names>Leonard</given-names></name><address><email>larnolda@uow.edu.au</email></address><xref ref-type=\"aff\" rid=\"Aff1\">1</xref><xref ref-type=\"aff\" rid=\"Aff2\">2</xref></contrib><aff id=\"Aff1\"><label>1</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.1001.0</institution-id><institution-id institution-id-type=\"ISNI\">0000 0001 2180 7477</institution-id><institution>Australian National University Medical School, </institution></institution-wrap>Canberra, ACT Australia </aff><aff id=\"Aff2\"><label>2</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.1007.6</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 0486 528X</institution-id><institution>Illawarra Health and Medical Research Institute (IHMRI), </institution><institution>University of Wollongong, </institution></institution-wrap>Building 32, Wollongong, NSW 2522 Australia </aff><aff id=\"Aff3\"><label>3</label><institution-wrap><institution-id institution-id-type=\"GRID\">grid.1012.2</institution-id><institution-id institution-id-type=\"ISNI\">0000 0004 1936 7910</institution-id><institution>Centre for Diabetes Research, Harry Perkins Institute of Medical Research, </institution><institution>University of Western Australia, </institution></institution-wrap>Perth, Australia </aff></contrib-group><pub-date pub-type=\"epub\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"pmc-release\"><day>17</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>13853</elocation-id><history><date date-type=\"received\"><day>29</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>13</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type=\"OpenAccess\"><license-p><bold>Open Access</bold>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type=\"uri\" xlink:href=\"http://creativecommons.org/licenses/by/4.0/\">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id=\"Abs1\"><p id=\"Par1\">Micro-CT scan images enhanced by iodine staining provide high-resolution visualisation of soft tissues in laboratory mice. We have compared Micro-CT scan-derived left ventricular (LV) mass with dissection and weighing. Ex-vivo micro-CT scan images of the mouse hearts were obtained following staining by iodine. The LV was segmented and its volume was assessed using a semi-automated method by Drishti software. The left ventricle was then dissected in the laboratory and its actual weight was measured and compared against the estimated results. LV mass was calculated multiplying its estimated volume and myocardial specific gravity. Thirty-five iodine-stained post-natal mouse hearts were studied. Mice were of either sex and 68 to 352 days old (median age 202 days with interquartile range 103 to 245 days) at the time of sacrifice. Samples were from 20 genetically diverse strains. Median mouse body weight was 29 g with interquartile range 24 to 34 g. Left Ventricular weights ranged from 40.0 to 116.7 mg. The segmented LV mass estimated from micro-CT scan and directly measured dissected LV mass were strongly correlated (R<sup>2</sup> = 0. 97). Segmented LV mass derived from Micro-CT images was very similar to the physically dissected LV mass (mean difference = 0.09 mg; 95% confidence interval − 3.29 mg to 3.1 mg). Micro-CT scanning provides a non-destructive, efficient and accurate visualisation tool for anatomical analysis of animal heart models of human cardiovascular conditions. Iodine-stained soft tissue imaging empowers researchers to perform qualitative and quantitative assessment of the cardiac structures with preservation of the samples for future histological analysis.</p></abstract><kwd-group kwd-group-type=\"npg-subject\"><title>Subject terms</title><kwd>Cardiovascular diseases</kwd><kwd>Cardiovascular biology</kwd><kwd>Biological techniques</kwd><kwd>Anatomy</kwd><kwd>Cardiology</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id=\"Sec1\"><title>Introduction</title><p id=\"Par2\">Left ventricular (LV) mass has been shown to be prognostic of cardiovascular morbidity and mortality irrespective of traditional risk factors<sup><xref ref-type=\"bibr\" rid=\"CR1\">1</xref>–<xref ref-type=\"bibr\" rid=\"CR3\">3</xref></sup>. Estimation of LV mass can be performed by several imaging modalities including echocardiography, cardiac computerised tomography scan (CT scan) and cardiac magnetic resonance (CMR)<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. Echocardiography has been traditionally established as the most widely used diagnostic tool despite inherent limitations<sup><xref ref-type=\"bibr\" rid=\"CR4\">4</xref></sup>. CMR is accurate and without risks of radiation but its availability, cost and patient tolerance make it an unattractive tool for clinical use<sup><xref ref-type=\"bibr\" rid=\"CR5\">5</xref></sup>. Despite radiation and use of contrast agents, cardiac CT scan is another viable option for assessment of LV mass with high spatial resolution<sup><xref ref-type=\"bibr\" rid=\"CR6\">6</xref></sup>.\n</p><p id=\"Par3\">Cardiac imaging in mice as opposed to humans poses problems that arise chiefly from their small size and fast heart rates<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup>. Variable results have been reported with CMR in mice<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref>–<xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup> where fast heart rates pose a challenge and cost limits availability. Radiation exposure is not a significant impediment to studying mouse hearts but fast heart rates remain a challenge<sup><xref ref-type=\"bibr\" rid=\"CR11\">11</xref></sup>. <italic>Ex-vivo</italic> measurements of mouse tissue are more easily implemented and have been evaluated here.</p><p id=\"Par4\">Our aim was to develop a non-destructive method of estimating <italic>ex-vivo</italic> left ventricular mass/tissue-volume in mice as part of a project aimed at identifying genes that contribute to left ventricular hypertrophy in the Collaborative Cross (CC)<sup><xref ref-type=\"bibr\" rid=\"CR12\">12</xref></sup>. Various CC strains have been housed in different locations and have been moved through their history. The initial breeding sites<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup> were in Oak Ridge (USA), Perth (Australia) and Nairobi (Kenya) and these populations have moved to Chapel Hill<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup> (USA), Beijing (China) and Tel Aviv<sup><xref ref-type=\"bibr\" rid=\"CR13\">13</xref></sup> (Israel) respectively. This has created some difficulty with access to the strains for phenotyping. The ability to study the CC strains using fixed tissue allows access to more strains at a greatly reduced cost with fewer regulatory impediments compared to shipping live animals. Additionally, the use of a non-destructive method allows subsequent analysis by other methods including histological examination. Therefore, we evaluated the reliability of staining enhanced micro-CT scan for measuring the LV tissue–volume/mass in the mouse.</p><p id=\"Par5\">We applied staining enhanced micro-CT scan for high resolution imaging of cardiovascular structure in laboratory mice to reconstruct the LV and measure the LV tissue–volume/mass. Micro-CT is one of a number of new tomographic techniques that have revolutionised the study of embryogenesis by permitting the viewing and analysis of complex 3-dimensional structures had previously been studied by analysis of serial 2-dimensional histological sections<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>–<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. The applications of micro-CT have included quantitative volumetric analysis at very small scale<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup> and the detailed geometric assessment of fine cardiac structures<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. Virtual dissection of micro-CT 3-dimensional images provides non-destructive measurement of left-ventricular tissue volume allowing both secondary uses and repeated measurements neither of which are possible with physical dissection. Estimated LV mass was compared with the directly measured LV mass after dissection as this has been the common comparator in many imaging studies although, for small volumes, histomorphometry has occasionally been used<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. Our results support the use of micro-CT to estimate LV tissue volume, particularly in circumstances where it is desirable to leave the preserved specimen intact for other subsequent use.</p></sec><sec id=\"Sec2\"><title>Results</title><p id=\"Par6\">Thirty-five mouse hearts were included in this study. Mice were of either sex and 68 to 352 days old (median age 202 days with interquartile range 103 to 245 days) at the time of sacrifice. Samples were from 20 genetically diverse strains including 18 strains of The Collaborative Cross<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup>. Median mouse body weight was 29 g with interquartile range 24 to 34 g.</p><sec id=\"Sec3\"><title>Differences between LV mass estimated from micro-CT (virtual dissection) and directly measured, dissected LV mass (physical dissection)</title><p id=\"Par7\">A scatter plot of estimated LV mass estimated from micro-CT and the measured LV mass is shown in Fig. <xref rid=\"Fig1\" ref-type=\"fig\">1</xref>. The two measures are closely correlated (R<sup>2</sup> = 0.96). Individual paired measurements lie close to and on either side of the line of identity with a slope that is close to 1.<fig id=\"Fig1\"><label>Figure 1</label><caption><p>Scatter plot of left ventricular (LV) mass estimated from virtual dissection of micro CT versus directly-measured, dissected LV mass (median of 3 measurements). The solid line is the line of equality. The dashed line is a plot of the linear regression equation.</p></caption><graphic xlink:href=\"41598_2020_70734_Fig1_HTML\" id=\"MO1\"/></fig></p><p id=\"Par8\">Figure <xref rid=\"Fig2\" ref-type=\"fig\">2</xref> shows the absolute and percentage differences between LV mass estimated from virtual dissection of CT images of whole heart plotted against the mean of the two measurements (virtual and physical dissection of LV). There was no significant bias. The 95% confidence interval for difference between virtual and physical dissection were − 3.3 mg to 3.1 mg for absolute weight and from − 4.2 to plus 4% of LV mass.<fig id=\"Fig2\"><label>Figure 2</label><caption><p>Difference between CT-estimated (virtual dissection) and directly-measured LV mass (physical dissection) versus mean left ventricular mass from the two methods. The upper panel shows the difference in grams and the lower panel the difference as a percentage of LV mass. <italic>CI</italic> confidence interval.</p></caption><graphic xlink:href=\"41598_2020_70734_Fig2_HTML\" id=\"MO2\"/></fig></p></sec><sec id=\"Sec4\"><title>Repeatability of virtual dissection</title><sec id=\"Sec5\"><title>Quantitative analysis</title><p id=\"Par9\">A scatter plot of measurements of LV tissue volume by independent virtual dissections is shown in Fig. <xref rid=\"Fig3\" ref-type=\"fig\">3</xref>. Paired measurements are closely correlated. Individual measures lie close to and on either side of the line of identity with a slope that is close to 1.<fig id=\"Fig3\"><label>Figure 3</label><caption><p>Scatter plot of independent virtual dissections of cardiac CT scans to yield paired repeated measurements of left ventricular tissue volume (μL). The solid line is the line of equality and the dashed line is the plotted linear regression equation.</p></caption><graphic xlink:href=\"41598_2020_70734_Fig3_HTML\" id=\"MO3\"/></fig></p><p id=\"Par10\">Figure <xref rid=\"Fig4\" ref-type=\"fig\">4</xref> shows the difference in volume between independent measurements of LV tissue volume from virtual dissection of CT images of whole heart plotted against the mean of the two measurements. There was no significant bias. The 95% confidence interval for difference between repeated virtual dissection was − 3.8 µL to 3.5 µL.<fig id=\"Fig4\"><label>Figure 4</label><caption><p>Difference between independent measurements of left ventricular tissue volume from virtual dissections of cardiac CT scans versus mean left ventricular tissue volume. <italic>CI</italic> confidence interval.</p></caption><graphic xlink:href=\"41598_2020_70734_Fig4_HTML\" id=\"MO4\"/></fig></p></sec><sec id=\"Sec6\"><title>Qualitative analysis</title><p id=\"Par11\">There were some differences in the approaches taken by Observers 1 & 2. An obvious example demonstrating these differences is shown in Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>. Observer 1 tended to take the shortest path from the margin of the RV septal surface to the epicardial surface whereas Observer 2 tended to follow the contour of the endocardial surface of the RV. In the example shown in Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref> the area of LV selected by Observer 2 is about 89% of the area selected by Observer 1. Figure <xref rid=\"Fig5\" ref-type=\"fig\">5</xref> shows differences between Virtual Dissection by Observer 1 (5a) & 2 (5b) over the RV surface. It is likely that a detailed protocol would attenuate these differences. Clot in the LV cavity (a3i) was excluded by Observer 1 but not by Observer 2.<fig id=\"Fig5\"><label>Figure 5</label><caption><p>Images from one heart showing differences in Virtual dissection by observer 1 (<bold>a</bold>) and Observer 2 (<bold>b)</bold> and physical dissection (<bold>c)</bold>. Images were chosen to illustrate points made in the text rather than to represent average differences. Selections made by Observer 1 are labelled <bold>a</bold>1, <bold>a</bold>2 etc<italic>.</italic> Selections made by Observer 2 are labelled <bold>b</bold>1, <bold>b</bold>2 etc<italic>.</italic> Photographs of the physically dissected heart are shown in the right panel labelled <bold>c</bold>. Panel <bold>a1</bold> shows the region selected as being the left ventricle (LV) by Observer 1 (blue shaded area) and Panel <bold>b1</bold> shows the region selected as being the LV by Observer 2 (green shaded area). The area selected as being LV by Observer 1 (panel <bold>a</bold>2) was greater than the area selected as LV by Observer 2 (panel <bold>b</bold>2). Panels <bold>a</bold>3 and <bold>b</bold>3 show the three-dimensional structure of the heart with the unselected surfaces in brown and the selected areas in blue (Observer1, <bold>a</bold>3) or green (Observer 2, <bold>b</bold>3). Observer 1 and 2 show close agreement in removal of supra ventricular structures. The surface attributed to right ventricular free wall by Observer 1 is less than the surface attributed to right ventricular free wall by Observer 2. <bold>a3i</bold> shows LV clot segmented and removed by Observer 1. Photographs of cut surface of LV and base of heart <bold>(c</bold>1), cut surface of LV and apex of heart <bold>(c</bold>2) and the LV with cut surfaces apposed and the right ventricle removed exposing the septal surface of the RV (<bold>c</bold>3). Imprecision in physical dissection is apparent in the ragged edges where the RV free wall was removed.</p></caption><graphic xlink:href=\"41598_2020_70734_Fig5_HTML\" id=\"MO5\"/></fig></p></sec></sec><sec id=\"Sec7\"><title>Repeatability of measurement of mass in physically dissected LV</title><p id=\"Par12\">Repeated measurements were closely aligned to the line of identity with a narrow 95% confidence interval from − 1.6 to 2.2 mg (Figs. <xref rid=\"Fig6\" ref-type=\"fig\">6</xref>, <xref rid=\"Fig7\" ref-type=\"fig\">7</xref>). This measurement error was approximately 60% of the difference between Virtual Dissection (mass estimated from micro-CT) and Physical Dissection with weighing. The errors that arise from inaccuracy in Physical Dissection per se (Fig. <xref rid=\"Fig5\" ref-type=\"fig\">5</xref>c) cannot be easily measured since physical dissection is a destructive technique that cannot be repeated on the same sample.<fig id=\"Fig6\"><label>Figure 6</label><caption><p>Scatter plot comparing repeated measurements of mass of a dissected left ventricle on an analytical balance. The line of equality (solid line) and the plotted regression equation (dashed line) are virtually superimposed.</p></caption><graphic xlink:href=\"41598_2020_70734_Fig6_HTML\" id=\"MO6\"/></fig><fig id=\"Fig7\"><label>Figure 7</label><caption><p>Difference between independent measurements on an analytical balance of LV tissue mass from a single physical dissection of the LV. Note this analytical error is likely a small part of the error associated with dissection of the heart for direct measurement of LV mass. <italic>CI</italic> confidence interval.</p></caption><graphic xlink:href=\"41598_2020_70734_Fig7_HTML\" id=\"MO7\"/></fig></p></sec></sec><sec id=\"Sec8\"><title>Discussion</title><p id=\"Par13\">We applied staining enhanced micro-CT scan for high resolution imaging of cardiovascular structure in laboratory mice to reconstruct the LV and measure the LV tissue–volume/mass. We have demonstrated that Virtual Dissection of X-ray micro-CT scans of iodine stained mouse hearts to determine LV size can replace physical dissection and weighing.</p><p id=\"Par14\">Micro-CT is one of a number of new tomographic techniques that have revolutionised the study of embryogenesis by permitting the viewing and analysis of complex 3-dimensional structures that previously been studied by analysis of serial 2-dimensional histological sections<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>–<xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup>. The applications of micro-CT have included quantitative volumetric analysis at very small scale<sup><xref ref-type=\"bibr\" rid=\"CR16\">16</xref></sup> and the detailed geometric assessment of fine cardiac structures<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup>. Virtual dissection of micro-CT, 3-dimensional images provides non-destructive measurement of left-ventricular tissue volume allowing both secondary uses and repeated measurements neither of which are possible with physical dissection. Estimated LV mass was compared with the directly measured LV mass after dissection as this has been the common comparator in many imaging studies although, for small volumes, histomorphometry has occasionally been used<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>. Our results support the use of micro-CT to estimate LV tissue volume, particularly in circumstances where it is desirable to leave the preserved specimen intact for other subsequent use.</p><p id=\"Par15\">This non-destructive method lends itself to the assessment of repeatability that is not possible with physical dissection, provides a permanent record of the three-dimensional relationship of cardiac structures that others have used to identify cardiac defects<sup><xref ref-type=\"bibr\" rid=\"CR14\">14</xref>,<xref ref-type=\"bibr\" rid=\"CR15\">15</xref></sup> and permits the use of cardiac tissue for other purposes. The micro-CT scanning protocol with iodine staining used here offers a simple, quick and inexpensive method to phenotype cardiovascular and other soft tissue structures in laboratory mice. Micro-CT has previously been widely used to study birth defects<sup><xref ref-type=\"bibr\" rid=\"CR2\">2</xref>,<xref ref-type=\"bibr\" rid=\"CR16\">16</xref>,<xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup> where its ability to provide a three-dimensional representation at very high resolution is unrivalled. Micro-CT can resolve structures that are much too small to be checked by physical dissection and weighing<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup>. Thus, it would be inappropriate to regard dissection and weighing as providing a “gold standard” for assessing the accuracy of micro-CT determination of LV size.</p><p id=\"Par16\">Measuring LV mass is the traditional method of assessing left ventricular hypertrophy in large animals but it poses some problems when applied to very small mouse hearts. Removal of great vessels, atria and free wall of the right ventricle is time-consuming and difficult to complete accurately because of the small size of the organs. Thus, Ghanem et al.<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup> note, ‘For the assessment of left-ventricular mass (LVM) in mice histomorphometry used to be the “golden standard,” (sic) as necropsy and assessment of wet heart weight has been found to be inaccurate particularly in smaller mice’ and Franco et al.<sup><xref ref-type=\"bibr\" rid=\"CR7\">7</xref></sup> did not attempt to remove the free wall of the RV because “dissection of the free wall was prone to error”. Quantification of these errors is difficult as the method is destructive and cannot be repeated on the same heart. The limits of determination of volume by micro-CT is readily quantified<sup><xref ref-type=\"bibr\" rid=\"CR20\">20</xref></sup> and the repeatability in practical applications can be measured.</p><p id=\"Par17\">Micro-CT scanning compares very well with existing methods for determining LV mass. The level of agreement we noted between the micro-CT and measuring mass after dissection (95% CI − 3.3 mg to 3.1 mg or − 4.2% to plus 4%) is closer than most previously reported echocardiographic or MRI measurement of LV mass in live animals, shown in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref>. Only a study by Dawson et al.<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup>, using “<italic>in house</italic>” equipment and software, reported results that were close to ours in the level of accuracy for 3D echo (− 5.3 mg to 5.5 mg or − 8% to 8.3%) and MRI (− 5.6 mg to 4 mg or − 8.5 to 6%). The span of the 95% CI in other studies in Table <xref rid=\"Tab1\" ref-type=\"table\">1</xref> ranged from 30 to 106 mg.<table-wrap id=\"Tab1\"><label>Table 1</label><caption><p>Mean with upper and lower 95% confidence limits for agreement between measured LV mass and mass in mg estimated by various imaging modalities.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\">Method</th><th align=\"left\">Author</th><th align=\"left\">Upper 95% CI</th><th align=\"left\">Mean</th><th align=\"left\">Lower 95% CI</th></tr></thead><tbody><tr><td align=\"left\" rowspan=\"4\">M-mode echocardiography</td><td align=\"left\">Gardin<sup><xref ref-type=\"bibr\" rid=\"CR33\">33</xref></sup></td><td align=\"left\"> − 11.0</td><td align=\"left\"> − 47.7</td><td align=\"left\"> − 84.5</td></tr><tr><td align=\"left\">Collins<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup></td><td align=\"left\">91.6</td><td align=\"left\">38.4</td><td align=\"left\"> − 14.8</td></tr><tr><td align=\"left\">Kiatchoosakun<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup></td><td align=\"left\">38.9</td><td align=\"left\">13.1</td><td align=\"left\"> − 12.7</td></tr><tr><td align=\"left\">Donner<sup><xref ref-type=\"bibr\" rid=\"CR36\">36</xref></sup></td><td align=\"left\">18.1</td><td align=\"left\">4.5</td><td align=\"left\"> − 9.1</td></tr><tr><td align=\"left\" rowspan=\"3\">2D echocardiography</td><td align=\"left\">Collins<sup><xref ref-type=\"bibr\" rid=\"CR34\">34</xref></sup></td><td align=\"left\">29</td><td align=\"left\">1</td><td align=\"left\"> − 27</td></tr><tr><td align=\"left\">Kiatchoosakun<sup><xref ref-type=\"bibr\" rid=\"CR35\">35</xref></sup></td><td align=\"left\">35.3</td><td align=\"left\">10.2</td><td align=\"left\"> − 14.9</td></tr><tr><td align=\"left\">Dawson<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup></td><td align=\"left\">6</td><td align=\"left\"> − 10</td><td align=\"left\"> − 36</td></tr><tr><td align=\"left\">3D echocardiography</td><td align=\"left\">Dawson<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup></td><td align=\"left\">5.5</td><td align=\"left\">0.1</td><td align=\"left\"> − 5.3</td></tr><tr><td align=\"left\" rowspan=\"3\">Magnetic resonance imaging</td><td align=\"left\">Franco<sup><xref ref-type=\"bibr\" rid=\"CR9\">9</xref></sup></td><td align=\"left\">2.41</td><td align=\"left\"> − 15.4</td><td align=\"left\">32.3</td></tr><tr><td align=\"left\">Ruff<sup><xref ref-type=\"bibr\" rid=\"CR10\">10</xref></sup></td><td align=\"left\">17.0</td><td align=\"left\">0.093</td><td align=\"left\"> − 18.3</td></tr><tr><td align=\"left\">Dawson<sup><xref ref-type=\"bibr\" rid=\"CR8\">8</xref></sup></td><td align=\"left\">4.00</td><td align=\"left\"> − 0.80</td><td align=\"left\"> − 5.6</td></tr><tr><td align=\"left\">Micro-CT scan</td><td align=\"left\">This study</td><td align=\"left\">3.10</td><td align=\"left\"> − 0.09</td><td align=\"left\"> − 3.29</td></tr></tbody></table><table-wrap-foot><p>Values for Gardin are calculated from tabulated data in their paper. Values for Franco and Ruff are measured from the figures provided in their papers.</p></table-wrap-foot></table-wrap></p><p id=\"Par18\">The percentage error compared to weighing (− 4% to 4%) was also very good in comparison with studies in humans where MRI is considered to provide a “gold standard”. Farber et al.<sup><xref ref-type=\"bibr\" rid=\"CR21\">21</xref></sup> compared MRI to explanted hearts obtaining levels of agreement from − 26% to 15% which is a much broader range than obtained in our study.</p><p id=\"Par19\">It is likely that the level of agreement we observed between micro-CT and dissection is close to the notional “repeatability” of dissection and weighing. The “error” in physical dissection is likely to be substantially larger than the error in determining mass on an analytical balance. The first error could only be assessed by simulation on a phantom whereas the latter error estimated by 95% CI for repeated determination of mass on the same piece of tissue was − 1.6 mg to 2.3 mg. This amounts to approximately 60% of the CI for the difference between micro-CT and dissection.</p><p id=\"Par20\">In our study repeatability was − 5% to 4% of LV mass. Moreover, our qualitative analysis suggests reproducibility could be improved by developing a more detailed protocol. Better repeatability and accuracy might also be achieved by using fully automated methods, for example by the implementation of Deep Learning for LV segmentation<sup><xref ref-type=\"bibr\" rid=\"CR22\">22</xref></sup>. In the best of the in vivo animal studies Dawson et al. reported 95% CI for intra-observer repeatability from − 4 to 12% of LV mass determined by 3-D echo. Another study of repeatability of MRI<sup><xref ref-type=\"bibr\" rid=\"CR23\">23</xref></sup> reported 95%CI from − 8 mg to 11 mg (− 10.5% to 14%). Interstudy repeatability with MRI in humans was − 8% to 7% in a study by Grothues et al.<sup><xref ref-type=\"bibr\" rid=\"CR24\">24</xref></sup>.</p><p id=\"Par21\">As an alternative to weighing, Ghanem et al.<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup> compared echocardiographic measurements of LV mass with serial sectioning at 500 μM intervals and 3D reconstruction of the LV. Micro CT scanning is conceptually similar to this approach of serial sections with computerised 3D reconstruction.</p><p id=\"Par22\">Micro-CT scan has several advantages over histomorphometry, considered the reference standard for analysis of cardiac development<sup><xref ref-type=\"bibr\" rid=\"CR17\">17</xref></sup>, including being non-destructive to tissue, having short image acquisition time and high resolution. However, micro-CT scan has limitations such as radiation and need for use of a contrast agent to visualise soft tissues. Iodine enhances contrast by diffusion through tissue layers and binding to glycogen within muscle cells<sup><xref ref-type=\"bibr\" rid=\"CR25\">25</xref>,<xref ref-type=\"bibr\" rid=\"CR26\">26</xref></sup> and increasing X-ray absorption. High resolution CT can provide information content comparable to that of conventional histological sectioning and staining<sup><xref ref-type=\"bibr\" rid=\"CR19\">19</xref></sup> with the additional benefit of providing 3-dimensional information.</p></sec><sec id=\"Sec9\"><title>Conclusion</title><p id=\"Par23\">In conclusion, results obtained through the micro-CT scan and iodine staining provided high-quality morphological information at the micrometre scale. The ability to study fixed tissue that is more easily transported than live animals allows collaboration more easily with colleagues at distant locations to study unique collections of mice. The clear advantages of micro-CT scan images over histology include a less tedious sample preparation protocol, and less time-consuming without the need for sectioning samples which could impose risks of tissue destruction and artefacts. Moreover, data obtained by the micro-CT scan enables 3D visualisation of slices in multiple imaging planes. We have confirmed that analysis of micro-CT scan data can provide accurate measurements of cardiac chamber mass. This enables investigators to use an efficient and reliable method to assess cardiac structures of larger quantity in a shorter time period without destruction of tissues.</p></sec><sec id=\"Sec10\"><title>Methods</title><sec id=\"Sec11\"><title>Sample preparation</title><p id=\"Par24\">Mice were from the Geniad Collaborative Cross Colony<sup><xref ref-type=\"bibr\" rid=\"CR18\">18</xref></sup> or from colonies held at ANU bearing a spontaneous mutation in the ALMS1 gene<sup><xref ref-type=\"bibr\" rid=\"CR27\">27</xref></sup>. Hearts were harvested from mice studied under ANU-AEEC approved protocols A2013/30 (28 mice) and A2013/47 (7 mice). Tissue collection was performed after euthanasia under isoflurane anaesthesia. Heart and great vessels were excised and then fixed in a 4% formalin solution until imaging preparation.</p><p id=\"Par25\">Formalin-fixed hearts were then immersed in ethanol in order to remove formalin from heart tissue. To prevent a further and abrupt shrinkage of the fixed sample, each heart was subjected to a graded series of ethanol solutions beginning with 20% ethanol for 24 h, then 50%, 70% and 90% ethanol for 1 day each. Following this stage, hearts were submerged in the 1.5% iodine potassium iodide (I<sub>2</sub>kI) in 90% ethanol solution. Stained hearts then were imaged at 72 h. Samples were washed in absolute ethanol and then placed on the CT scanner.</p></sec><sec id=\"Sec12\"><title>Image acquisition</title><p id=\"Par26\">A commercial Caliper Quantum FX micro-CT scanner system (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.perkinelmer.com\">www.perkinelmer.com</ext-link>) was used to scan the mice hearts. The tissue specimen is placed on a stationary loading dock of the scanner, positioning it between the rotating system of X-ray source and detector. The scanning time was pre-set at 3 min with the field of view (FOV) 10 mm in diameter, and the chosen mode of fine quality. Other setup parameters of Caliper Quantum FX micro-CT scanner include Voltage 90 kV, Capture Small, CT 200µA, Live 80uA. The maximum resolution achievable by this setting is 10 µm/voxel. The resultant images were stored as DICOM series in coronal, axial and sagittal views. Post processing of the raw DICOM data, reconstruction and visualisation were performed using the Drishti software package<sup><xref ref-type=\"bibr\" rid=\"CR28\">28</xref></sup> (Australian National University).</p></sec><sec id=\"Sec13\"><title>Left ventricular volume assessment (virtual dissection)</title><p id=\"Par27\">The semi-automated edge-based segmentation technique<sup><xref ref-type=\"bibr\" rid=\"CR29\">29</xref></sup> was used in Drishti Paint, a function of Drishti, in order to separate the LV from the rest of the heart. Using Drishti Paint, left ventricular boundaries were manually delineated in 10–15 CT scan slices. Afterwards, Drishti Paint would collate the tagged slices and in-between slices to create a segregated left ventricle from the whole heart. The result was manually adjusted to ensure accurate definition of the cardiac contours. Segmented LV myocardial volume is calculated by the Drishti software in µL.</p><p id=\"Par28\">To standardise LV volume measurement between samples, multiple parameters were followed to minimise inter-sample measurement errors. The left ventricle was defined from the aortic ring at the base to the left ventricular apex. Interventricular septum and mitral valve papillary muscles were also included as part of the LV volume. The right ventricular (RV) free wall was excluded. Virtual Dissection was performed independently by two observers. Differences were examined quantitatively (see “Analyses” below) and qualitatively.</p></sec><sec id=\"Sec14\"><title>Left ventricular mass calculation</title><p id=\"Par29\">LV mass was calculated from the product of ventricular muscle volume calculated by Drishti and specific gravity of heart muscle, estimated from the average of the directly measured weights and the average of the estimated volumes of all left ventricles.</p></sec><sec id=\"Sec15\"><title>Physical left ventricular dissection</title><p id=\"Par30\">The LV was dissected from the rest of the mouse cardiac structure under magnification and the mass of the dissected LV was measured using a high-performance analytical balance, (Shimadzu AUW220D) paying careful attention to the operating environment as described in the manual. Three sets of measurements were made on different days and the median value was used for comparison. Conceptually, there are two sources of error in Physical Left Ventricular Dissection. The larger error is likely to be the mechanical dissection of a small organ manually under a dissecting microscope. Unfortunately, the destructive nature of this approach renders impossible the assessment of repeatability. The smaller error is likely to be the measurement error which was assessed by comparing the first two of three determinations of mass.</p></sec><sec id=\"Sec16\"><title>Analyses</title><p id=\"Par31\">The principal methods employed were those described by Bland and Altman to quantify agreement between two methods of measurement<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref>,<xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>. Prior exploratory analysis included graphing the data in scatter plots and regression analysis<sup><xref ref-type=\"bibr\" rid=\"CR32\">32</xref></sup>.</p><sec id=\"Sec17\"><title>Left ventricular mass estimated by segmentation of whole heart CT images and weight of dissected left ventricle</title><p id=\"Par32\">Differences between paired values were plotted against their mean. The mean difference indicates the presence (or absence) of bias between the 2 measurements. We also plotted the 95% confidence interval (mean ± 1.96 standard deviations of the difference) to indicate the span within which 95% of differences between paired measurements would be expected to lie. When it appeared possible that either the mean difference or variability of the differences increased as the magnitude of the measurement increased we elected to plot the percentage difference<sup><xref ref-type=\"bibr\" rid=\"CR30\">30</xref></sup> against the mean of the 2 measurements rather than log transforming the values, as initially recommended<sup><xref ref-type=\"bibr\" rid=\"CR31\">31</xref></sup>, because we believed that percentages were more likely to be intuitively comprehended by the reader than logarithms and their antilogarithms. Moreover, expressing the level of agreement as a percentage permits comparison with assessments made in larger hearts, for example, human hearts.\n</p></sec></sec><sec id=\"Sec18\"><title>Compliance with ethical practice</title><p id=\"Par33\">All tissues and animals used in this study were handled in compliance with The Australian Code for the Responsible Conduct of Research, 2007 and Australian National University Animal Experimentation Ethics Committee (ANU-AEEC).\n</p></sec></sec></body><back><fn-group><fn><p><bold>Publisher's note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><ack><title>Acknowledgements</title><p>All imaging was performed at the imaging and cytometry facility at John Curtin School of Medical Research (JCSMR), Australian National University (ANU). We thank Ms Cathy Gillespie, manager, and all other staff at JCSMR. We thank Geniad Pty Ltd for providing CC mice. This manuscript was not funded.</p></ack><notes notes-type=\"author-contribution\"><title>Author contributions</title><p>Each named author has substantially contributed to the design of the work, analysis drafting and revising this manuscript. A.D., implemented the methods including sample preparation, staining and imaging. A.D., and L.A., performed analysis and were major contributors in writing the manuscript. All authors have read and approved the final manuscript.</p></notes><notes notes-type=\"data-availability\"><title>Data availability</title><p>Data and material used for this manuscript are stored in our research office and are available on request.</p></notes><notes id=\"FPar1\" notes-type=\"COI-statement\"><title>Competing interest</title><p id=\"Par34\">The authors declare no competing interest.</p></notes><ref-list id=\"Bib1\"><title>References</title><ref id=\"CR1\"><label>1.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hoang</surname><given-names>K</given-names></name><etal/></person-group><article-title>LV mass as a predictor of CVD events in older adults with and without metabolic syndrome and diabetes</article-title><source>JACC. Cardiovasc. Imaging</source><year>2015</year><volume>8</volume><fpage>1007</fpage><lpage>1015</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jcmg.2015.04.019</pub-id><pub-id pub-id-type=\"pmid\">26319502</pub-id></element-citation></ref><ref id=\"CR2\"><label>2.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lieb</surname><given-names>W</given-names></name><etal/></person-group><article-title>The natural history of left ventricular geometry in the community: clinical correlates and prognostic significance of change in LV geometric pattern</article-title><source>JACC. Cardiovasc. Imaging</source><year>2014</year><volume>7</volume><fpage>870</fpage><lpage>878</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jcmg.2014.05.008</pub-id><pub-id pub-id-type=\"pmid\">25129518</pub-id></element-citation></ref><ref id=\"CR3\"><label>3.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tsao</surname><given-names>CW</given-names></name><etal/></person-group><article-title>Left ventricular structure and risk of cardiovascular events: a framingham heart study cardiac magnetic resonance study</article-title><source>J. Am. Heart Assoc.</source><year>2015</year><volume>4</volume><fpage>e002188</fpage><pub-id pub-id-type=\"pmid\">26374295</pub-id></element-citation></ref><ref id=\"CR4\"><label>4.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Celebi</surname><given-names>AS</given-names></name><name><surname>Yalcin</surname><given-names>H</given-names></name><name><surname>Yalcin</surname><given-names>F</given-names></name></person-group><article-title>Current cardiac imaging techniques for detection of left ventricular mass</article-title><source>Cardiovasc. Ultrasound</source><year>2010</year><volume>8</volume><fpage>19</fpage><pub-id pub-id-type=\"doi\">10.1186/1476-7120-8-19</pub-id><pub-id pub-id-type=\"pmid\">20515461</pub-id></element-citation></ref><ref id=\"CR5\"><label>5.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Katz</surname><given-names>J</given-names></name><etal/></person-group><article-title>Estimation of human myocardial mass with MR imaging</article-title><source>Radiology</source><year>1988</year><volume>169</volume><fpage>495</fpage><lpage>498</lpage><pub-id pub-id-type=\"doi\">10.1148/radiology.169.2.2971985</pub-id><pub-id pub-id-type=\"pmid\">2971985</pub-id></element-citation></ref><ref id=\"CR6\"><label>6.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Klein</surname><given-names>R</given-names></name><name><surname>Ametepe</surname><given-names>ES</given-names></name><name><surname>Yam</surname><given-names>Y</given-names></name><name><surname>Dwivedi</surname><given-names>G</given-names></name><name><surname>Chow</surname><given-names>BJ</given-names></name></person-group><article-title>Cardiac CT assessment of left ventricular mass in mid-diastasis and its prognostic value</article-title><source>Eur. Heart J. Cardiovasc. Imaging</source><year>2017</year><volume>18</volume><fpage>95</fpage><lpage>102</lpage><pub-id pub-id-type=\"doi\">10.1093/ehjci/jev357</pub-id><pub-id pub-id-type=\"pmid\">26850628</pub-id></element-citation></ref><ref id=\"CR7\"><label>7.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Badea</surname><given-names>CT</given-names></name><name><surname>Bucholz</surname><given-names>E</given-names></name><name><surname>Hedlund</surname><given-names>LW</given-names></name><name><surname>Rockman</surname><given-names>HA</given-names></name><name><surname>Johnson</surname><given-names>GA</given-names></name></person-group><article-title>Imaging methods for morphological and functional phenotyping of the rodent heart</article-title><source>Toxicol. Pathol.</source><year>2006</year><volume>34</volume><fpage>111</fpage><lpage>117</lpage><pub-id pub-id-type=\"doi\">10.1080/01926230500404126</pub-id><pub-id pub-id-type=\"pmid\">16507552</pub-id></element-citation></ref><ref id=\"CR8\"><label>8.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dawson</surname><given-names>D</given-names></name><etal/></person-group><article-title>Quantitative 3-dimensional echocardiography for accurate and rapid cardiac phenotype characterization in mice</article-title><source>Circulation</source><year>2004</year><volume>110</volume><fpage>1632</fpage><lpage>1637</lpage><pub-id pub-id-type=\"doi\">10.1161/01.CIR.0000142049.14227.AD</pub-id><pub-id pub-id-type=\"pmid\">15364813</pub-id></element-citation></ref><ref id=\"CR9\"><label>9.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Franco</surname><given-names>F</given-names></name><name><surname>Dubois</surname><given-names>SK</given-names></name><name><surname>Peshock</surname><given-names>RM</given-names></name><name><surname>Shohet</surname><given-names>RV</given-names></name></person-group><article-title>Magnetic resonance imaging accurately estimates LV mass in a transgenic mouse model of cardiac hypertrophy</article-title><source>Am. J. Physiol.</source><year>1998</year><volume>274</volume><fpage>H679</fpage><lpage>H683</lpage><pub-id pub-id-type=\"pmid\">9486274</pub-id></element-citation></ref><ref id=\"CR10\"><label>10.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ruff</surname><given-names>J</given-names></name><etal/></person-group><article-title>Magnetic resonance microimaging for noninvasive quantification of myocardial function and mass in the mouse</article-title><source>Magn. Reson. Med.</source><year>1998</year><volume>40</volume><fpage>43</fpage><lpage>48</lpage><pub-id pub-id-type=\"doi\">10.1002/mrm.1910400106</pub-id><pub-id pub-id-type=\"pmid\">9660551</pub-id></element-citation></ref><ref id=\"CR11\"><label>11.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Badea</surname><given-names>CT</given-names></name><name><surname>Hedlund</surname><given-names>LW</given-names></name><name><surname>Cook</surname><given-names>J</given-names></name><name><surname>Berridge</surname><given-names>BR</given-names></name><name><surname>Johnson</surname><given-names>GA</given-names></name></person-group><article-title>Micro-CT imaging assessment of dobutamine-induced cardiac stress in rats</article-title><source>J. Pharmacol. Toxicol. Methods</source><year>2011</year><volume>63</volume><fpage>24</fpage><lpage>29</lpage><pub-id pub-id-type=\"doi\">10.1016/j.vascn.2010.04.002</pub-id><pub-id pub-id-type=\"pmid\">20399875</pub-id></element-citation></ref><ref id=\"CR12\"><label>12.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Churchill</surname><given-names>GA</given-names></name><etal/></person-group><article-title>The collaborative cross, a community resource for the genetic analysis of complex traits</article-title><source>Nat. Genet.</source><year>2004</year><volume>36</volume><fpage>1133</fpage><lpage>1137</lpage><pub-id pub-id-type=\"doi\">10.1038/ng1104-1133</pub-id><pub-id pub-id-type=\"pmid\">15514660</pub-id></element-citation></ref><ref id=\"CR13\"><label>13.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Threadgill</surname><given-names>DW</given-names></name><name><surname>Churchill</surname><given-names>GA</given-names></name></person-group><article-title>Ten years of the collaborative cross</article-title><source>Genetics</source><year>2012</year><volume>190</volume><fpage>291</fpage><lpage>294</lpage><pub-id pub-id-type=\"doi\">10.1534/genetics.111.138032</pub-id><pub-id pub-id-type=\"pmid\">22345604</pub-id></element-citation></ref><ref id=\"CR14\"><label>14.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Degenhardt</surname><given-names>K</given-names></name><name><surname>Wright</surname><given-names>A</given-names></name><name><surname>Horng</surname><given-names>D</given-names></name><name><surname>Padmanabhan</surname><given-names>A</given-names></name><name><surname>Epstein</surname><given-names>J</given-names></name></person-group><article-title>Rapid 3D phenotyping of cardiovascular development in mouse embryos by micro-CT with iodine staining</article-title><source>Circ. Cardiovasc. Imaging</source><year>2010</year><volume>3</volume><fpage>314</fpage><lpage>322</lpage><pub-id pub-id-type=\"doi\">10.1161/CIRCIMAGING.109.918482</pub-id><pub-id pub-id-type=\"pmid\">20190279</pub-id></element-citation></ref><ref id=\"CR15\"><label>15.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>AJ</given-names></name><etal/></person-group><article-title>Microcomputed tomography provides high accuracy congenital heart disease diagnosis in neonatal and fetal mice</article-title><source>Circ. Cardiovasc. Imaging</source><year>2013</year><volume>6</volume><fpage>551</fpage><lpage>559</lpage><pub-id pub-id-type=\"doi\">10.1161/CIRCIMAGING.113.000279</pub-id><pub-id pub-id-type=\"pmid\">23759365</pub-id></element-citation></ref><ref id=\"CR16\"><label>16.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>JS</given-names></name><name><surname>Min</surname><given-names>J</given-names></name><name><surname>Recknagel</surname><given-names>AK</given-names></name><name><surname>Riccio</surname><given-names>M</given-names></name><name><surname>Butcher</surname><given-names>JT</given-names></name></person-group><article-title>Quantitative three-dimensional analysis of embryonic chick morphogenesis via microcomputed tomography</article-title><source>Anat. Rec. (Hoboken)</source><year>2011</year><volume>294</volume><fpage>1</fpage><lpage>10</lpage><pub-id pub-id-type=\"doi\">10.1002/ar.21276</pub-id><pub-id pub-id-type=\"pmid\">21207522</pub-id></element-citation></ref><ref id=\"CR17\"><label>17.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ghanem</surname><given-names>A</given-names></name><etal/></person-group><article-title>Echocardiographic assessment of left ventricular mass in neonatal and adult mice: accuracy of different echocardiographic methods</article-title><source>Echocardiography</source><year>2006</year><volume>23</volume><fpage>900</fpage><lpage>907</lpage><pub-id pub-id-type=\"doi\">10.1111/j.1540-8175.2006.00323.x</pub-id><pub-id pub-id-type=\"pmid\">17069614</pub-id></element-citation></ref><ref id=\"CR18\"><label>18.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morahan</surname><given-names>G</given-names></name><name><surname>Balmer</surname><given-names>L</given-names></name><name><surname>Monley</surname><given-names>D</given-names></name></person-group><article-title>Establishment of “the gene mine”: a resource for rapid identification of complex trait genes</article-title><source>Mamm. Genome</source><year>2008</year><volume>19</volume><fpage>390</fpage><lpage>393</lpage><pub-id pub-id-type=\"doi\">10.1007/s00335-008-9134-9</pub-id><pub-id pub-id-type=\"pmid\">18716834</pub-id></element-citation></ref><ref id=\"CR19\"><label>19.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zanette</surname><given-names>I</given-names></name><name><surname>Weitkamp</surname><given-names>T</given-names></name><name><surname>Le Duc</surname><given-names>G</given-names></name><name><surname>Pfeiffer</surname><given-names>F</given-names></name></person-group><article-title>X-ray grating-based phase tomography for 3D histology</article-title><source>RSC Adv.</source><year>2013</year><volume>3</volume><fpage>19816</fpage><lpage>19819</lpage><pub-id pub-id-type=\"doi\">10.1039/c3ra41372a</pub-id></element-citation></ref><ref id=\"CR20\"><label>20.</label><mixed-citation publication-type=\"other\">Behrooz, A., Tseng, J.-C. & Meganack, J. Image resolution in MicroCT: principles and characterization of the quantum FX and quantum GX systems. <italic>Preclin. Vivo Imaging</italic> 1–5 (2016).</mixed-citation></ref><ref id=\"CR21\"><label>21.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Farber</surname><given-names>NJ</given-names></name><etal/></person-group><article-title>Ex vivo cardiovascular magnetic resonance measurements of right and left ventricular mass compared with direct mass measurement in excised hearts after transplantation: a first human SSFP comparison</article-title><source>J. Cardiovasc. Magn. Reson.</source><year>2014</year><volume>16</volume><fpage>74</fpage><pub-id pub-id-type=\"doi\">10.1186/s12968-014-0074-0</pub-id><pub-id pub-id-type=\"pmid\">25315015</pub-id></element-citation></ref><ref id=\"CR22\"><label>22.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>C</given-names></name><etal/></person-group><article-title>Deep learning for cardiac image segmentation: a review</article-title><source>Front. Cardiovasc. Med.</source><year>2020</year><volume>7</volume><fpage>25</fpage><pub-id pub-id-type=\"doi\">10.3389/fcvm.2020.00025</pub-id><pub-id pub-id-type=\"pmid\">32195270</pub-id></element-citation></ref><ref id=\"CR23\"><label>23.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zuo</surname><given-names>Z</given-names></name><etal/></person-group><article-title>Assessment of longitudinal reproducibility of mice LV function parameters at 11.7 T derived from self-gated CINE MRI</article-title><source>Biomed Res. Int.</source><year>2017</year><volume>2017</volume><fpage>8392952</fpage><pub-id pub-id-type=\"pmid\">28321415</pub-id></element-citation></ref><ref id=\"CR24\"><label>24.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grothues</surname><given-names>F</given-names></name><etal/></person-group><article-title>Comparison of interstudy reproducibility of cardiovascular magnetic resonance with two-dimensional echocardiography in normal subjects and in patients with heart failure or left ventricular hypertrophy</article-title><source>Am. J. Cardiol.</source><year>2002</year><volume>90</volume><fpage>29</fpage><lpage>34</lpage><pub-id pub-id-type=\"doi\">10.1016/S0002-9149(02)02381-0</pub-id><pub-id pub-id-type=\"pmid\">12088775</pub-id></element-citation></ref><ref id=\"CR25\"><label>25.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jeffery</surname><given-names>NS</given-names></name><name><surname>Stephenson</surname><given-names>RS</given-names></name><name><surname>Gallagher</surname><given-names>JA</given-names></name><name><surname>Jarvis</surname><given-names>JC</given-names></name><name><surname>Cox</surname><given-names>PG</given-names></name></person-group><article-title>Micro-computed tomography with iodine staining resolves the arrangement of muscle fibres</article-title><source>J. Biomech.</source><year>2011</year><volume>44</volume><fpage>189</fpage><lpage>192</lpage><pub-id pub-id-type=\"doi\">10.1016/j.jbiomech.2010.08.027</pub-id><pub-id pub-id-type=\"pmid\">20846653</pub-id></element-citation></ref><ref id=\"CR26\"><label>26.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lecker</surname><given-names>DN</given-names></name><name><surname>Kumari</surname><given-names>S</given-names></name><name><surname>Khan</surname><given-names>A</given-names></name></person-group><article-title>Iodine binding capacity and iodine binding energy of glycogen</article-title><source>J. Polym. Sci. Part A Polym. Chem.</source><year>1997</year><volume>35</volume><fpage>1409</fpage><lpage>1412</lpage><pub-id pub-id-type=\"doi\">10.1002/(SICI)1099-0518(199706)35:8<1409::AID-POLA9>3.0.CO;2-U</pub-id></element-citation></ref><ref id=\"CR27\"><label>27.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arsov</surname><given-names>T</given-names></name><etal/></person-group><article-title>Fat aussie—a new Alstrom syndrome mouse showing a critical role for ALMS1 in obesity, diabetes, and spermatogenesis</article-title><source>Mol. Endocrinol.</source><year>2006</year><volume>20</volume><fpage>1610</fpage><lpage>1622</lpage><pub-id pub-id-type=\"doi\">10.1210/me.2005-0494</pub-id><pub-id pub-id-type=\"pmid\">16513793</pub-id></element-citation></ref><ref id=\"CR28\"><label>28.</label><mixed-citation publication-type=\"other\">Limaye, A. Drishti: a volume exploration and presentation tool. In <italic>Proceedings of SPIE</italic><bold>8506</bold>, (2012).</mixed-citation></ref><ref id=\"CR29\"><label>29.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sharma</surname><given-names>N</given-names></name><name><surname>Aggarwal</surname><given-names>LM</given-names></name></person-group><article-title>Automated medical image segmentation techniques</article-title><source>J. Med. Phys.</source><year>2010</year><volume>35</volume><fpage>3</fpage><lpage>14</lpage><pub-id pub-id-type=\"doi\">10.4103/0971-6203.58777</pub-id><pub-id pub-id-type=\"pmid\">20177565</pub-id></element-citation></ref><ref id=\"CR30\"><label>30.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Altman</surname><given-names>DG</given-names></name><name><surname>Bland</surname><given-names>JM</given-names></name></person-group><article-title>Commentary on quantifying agreement between two methods of measurement</article-title><source>Clin. Chem.</source><year>2002</year><volume>48</volume><fpage>801</fpage><lpage>802</lpage><pub-id pub-id-type=\"doi\">10.1093/clinchem/48.5.801</pub-id></element-citation></ref><ref id=\"CR31\"><label>31.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bland</surname><given-names>JM</given-names></name><name><surname>Altman</surname><given-names>DG</given-names></name></person-group><article-title>Measuring agreement in method comparison studies</article-title><source>Stat. Methods Med. Res.</source><year>1999</year><volume>8</volume><fpage>135</fpage><lpage>160</lpage><pub-id pub-id-type=\"doi\">10.1177/096228029900800204</pub-id><pub-id pub-id-type=\"pmid\">10501650</pub-id></element-citation></ref><ref id=\"CR32\"><label>32.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Anscombe</surname><given-names>FJ</given-names></name></person-group><article-title>Graphs in statistical analysis</article-title><source>Am. Stat.</source><year>1973</year><volume>27</volume><fpage>17</fpage><lpage>21</lpage></element-citation></ref><ref id=\"CR33\"><label>33.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gardin</surname><given-names>JM</given-names></name><name><surname>Siri</surname><given-names>FM</given-names></name><name><surname>Kitsis</surname><given-names>RN</given-names></name><name><surname>Edwards</surname><given-names>JG</given-names></name><name><surname>Leinwand</surname><given-names>LA</given-names></name></person-group><article-title>Echocardiographic assessment of left ventricular mass and systolic function in mice</article-title><source>Circ. Res.</source><year>1995</year><volume>76</volume><fpage>907</fpage><lpage>914</lpage><pub-id pub-id-type=\"doi\">10.1161/01.RES.76.5.907</pub-id><pub-id pub-id-type=\"pmid\">7729009</pub-id></element-citation></ref><ref id=\"CR34\"><label>34.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Collins</surname><given-names>KA</given-names></name><etal/></person-group><article-title>Accuracy of echocardiographic estimates of left ventricular mass in mice</article-title><source>Am. J. Physiol. Circ. Physiol.</source><year>2001</year><volume>280</volume><fpage>H1954</fpage><lpage>H1962</lpage><pub-id pub-id-type=\"doi\">10.1152/ajpheart.2001.280.5.H1954</pub-id></element-citation></ref><ref id=\"CR35\"><label>35.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kiatchoosakun</surname><given-names>S</given-names></name><name><surname>Restivo</surname><given-names>J</given-names></name><name><surname>Kirkpatrick</surname><given-names>D</given-names></name><name><surname>Hoit</surname><given-names>BD</given-names></name></person-group><article-title>Assessment of left ventricular mass in mice: comparison between two-dimensional and m-mode echocardiography</article-title><source>Echocardiography</source><year>2002</year><volume>19</volume><fpage>199</fpage><lpage>205</lpage><pub-id pub-id-type=\"doi\">10.1046/j.1540-8175.2002.00199.x</pub-id><pub-id pub-id-type=\"pmid\">12022927</pub-id></element-citation></ref><ref id=\"CR36\"><label>36.</label><element-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Donner</surname><given-names>DG</given-names></name><name><surname>Kiriazis</surname><given-names>H</given-names></name><name><surname>Du</surname><given-names>X-J</given-names></name><name><surname>Marwick</surname><given-names>TH</given-names></name><name><surname>McMullen</surname><given-names>JR</given-names></name></person-group><article-title>Improving the quality of preclinical research echocardiography: observations, training, and guidelines for measurement</article-title><source>Am. J. Physiol. Heart Circ. Physiol.</source><year>2018</year><volume>315</volume><fpage>H58</fpage><lpage>H70</lpage><pub-id pub-id-type=\"doi\">10.1152/ajpheart.00157.2018</pub-id><pub-id pub-id-type=\"pmid\">29677464</pub-id></element-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Endocrinol.</journal-id><journal-title-group><journal-title>Frontiers in Endocrinology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2392</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849311</article-id><article-id pub-id-type=\"pmc\">PMC7431597</article-id><article-id pub-id-type=\"doi\">10.3389/fendo.2020.00540</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology</subject><subj-group><subject>Mini Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Immune System Remodelling by Prenatal Betamethasone: Effects on β-Cells and Type 1 Diabetes</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Perna-Barrull</surname><given-names>David</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/480435/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Gieras</surname><given-names>Anna</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/470125/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Rodriguez-Fernandez</surname><given-names>Silvia</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/506890/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Tolosa</surname><given-names>Eva</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/108996/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Vives-Pi</surname><given-names>Marta</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/442741/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Immunology Section, Germans Trias i Pujol Research Institute, Autonomous University of Barcelona</institution>, <addr-line>Badalona</addr-line>, <country>Spain</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Immunology, University Medical Center Hamburg-Eppendorf</institution>, <addr-line>Hamburg</addr-line>, <country>Germany</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Teresa Rodriguez-Calvo, Helmholtz Zentrum München, Germany</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Guoqiang Gu, Vanderbilt University, United States; Barak Blum, University of Wisconsin-Madison, United States</p></fn><corresp id=\"c001\">*Correspondence: Marta Vives-Pi <email>mvives@igtp.cat</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Diabetes: Molecular Mechanisms, a section of the journal Frontiers in Endocrinology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>540</elocation-id><history><date date-type=\"received\"><day>25</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>03</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Perna-Barrull, Gieras, Rodriguez-Fernandez, Tolosa and Vives-Pi.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Perna-Barrull, Gieras, Rodriguez-Fernandez, Tolosa and Vives-Pi</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Type 1 diabetes (T1D) is a multifactorial disease of unknown aetiology. Studies focusing on environment-related prenatal changes, which might have an influence on the development of T1D, are still missing. Drugs, such as betamethasone, are used during this critical period without exploring possible effects later in life. Betamethasone can interact with the development and function of the two main players in T1D, the immune system and the pancreatic β-cells. Short-term or persistent changes in any of these two players may influence the initiation of the autoimmune reaction against β-cells. In this review, we focus on the ability of betamethasone to induce alterations in the immune system, impairing the recognition of autoantigens. At the same time, betamethasone affects β-cell gene expression and apoptosis rate, reducing the danger signals that will attract unwanted attention from the immune system. These effects may synergise to hinder the autoimmune attack. In this review, we compile scattered evidence to provide a better understanding of the basic relationship between betamethasone and T1D, laying the foundation for future studies on human cohorts that will help to fully grasp the role of betamethasone in the development of T1D.</p></abstract><kwd-group><kwd>prenatal betamethasone</kwd><kwd>Type 1 diabetes</kwd><kwd>immune system</kwd><kwd>β cell</kwd><kwd>glucocorticoid</kwd></kwd-group><counts><fig-count count=\"1\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"88\"/><page-count count=\"9\"/><word-count count=\"6624\"/></counts></article-meta></front><body><sec id=\"s1\"><title>Betamethasone as an Emerging Environmental Factor in T1D</title><p>Type 1 diabetes (T1D) is an autoimmune disease caused by the selective destruction of insulin-producing β-cells. The trigger, however, remains unknown. Postnatal environmental determinants have been thoroughly studied as risk factors (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B2\" ref-type=\"bibr\">2</xref>) but a crucial phase for the immune system development, the late prenatal stage, has been poorly investigated. Specifically, the interaction of drugs commonly used during late pregnancy with T1D and the pancreatic β-cells remains unexplored. Nonetheless, some studies reveal the importance of the prenatal stage and the prematurity of the newborn in the development of T1D (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>–<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). An indirect demonstration of how critical the <italic>in utero</italic> environment is in T1D development arises from the studies in twins: heterozygotic twins have an increased concordance of T1D when compared to non-twin siblings (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>), underlining the potential relevance of prenatal factors and their influence in the development of autoimmunity.</p><p>Synthetic glucocorticoids, most often betamethasone, are routinely given to mothers at risk of preterm birth between 24 and 34 weeks of gestation. A single course of prenatal betamethasone reduces the occurrence and severity of respiratory distress syndrome and improves the survival chances in premature infants (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>, <xref rid=\"B9\" ref-type=\"bibr\">9</xref>). Another glucocorticoid used for lung maturation is dexamethasone and produces similar results on the newborn survivability (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). These synthetic glucocorticoids cross the placenta and accelerate foetal lung maturation, achieving maximum benefit between 24 h and 7 days after administration (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Betamethasone is a poor substrate for the glucocorticoid inactivating enzyme 11beta-hydroxysteroid-dehydrogenase 2 (11βHSD2), therefore, its bioactivity in the foetus lasts for several days (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>) and it is known to exert long-lasting effects on the hypothalamic-pituitary-adrenal (HPA) axis and cognition in children (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>).</p><p>Glucocorticoids exert their effects by binding nuclear receptors that are ligand-dependent transcription factors. They can regulate gene transcription, either by direct binding to DNA or by interacting with other transcription factors (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). Glucocorticoid receptors (GR) are ubiquitously expressed; however, due to the variation in the genomic location of GR binding, the transcriptional responses to glucocorticoids are cell type-specific (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Moreover, polymorphisms of the GR result in alterations in their responsiveness to glucocorticoids and in gene expression (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>). In addition, human GR receptor can be a target of endoncrine disruptors such as pesticides (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>) that, in combination with antenatal glucocorticoids, could increase developmental neurotoxicity (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>).</p><p>The general effects of glucocorticoids administered during pregnancy have been thoroughly reviewed (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). Considering the overwhelming use of betamethasone as the treatment of choice for respiratory distress syndrome in premature infants and the cell-specific response to glucocorticoids, in this review we will dissect the specific effects of betamethasone on the main cellular players in the context of T1D, namely immune cells and their targets, the β-cells of the pancreas.</p></sec><sec id=\"s2\"><title>Direct Effects of Betamethasone on the Immune System</title><p>Several cell types of the immune system are involved in the development of T1D, and disturbances in the activity of these cells, such as enhanced proinflammatory activity, can increase the risk to develop T1D (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). Below, the effect of betamethasone on different cell types of the immune system is detailed.</p><sec><title>Innate Immune Cells</title><p>Prenatal administration of betamethasone can induce an anti-inflammatory status in the newborn during the first days after delivery (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>), and this fact could be due to the immunomodulatory effects of betamethasone on innate immune cells.</p></sec><sec><title>Neutrophils</title><p>Neutrophils have gained interest in T1D aetiology due to their participation in the initial steps of autoimmunity against β-cells (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). Moreover, neutrophils are part of the islet leukocytic infiltrates of patients with T1D, and are accordingly reduced in peripheral blood at disease onset (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B26\" ref-type=\"bibr\">26</xref>).</p><p>A described effect of betamethasone is the increase in leukocyte counts in peripheral blood after treatment (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>), similarly to the effects of natural glucocorticoids during stress (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). Accordingly, neutrophil number and percentage were increased in human blood after betamethasone treatment (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>), correlating with the described neutrophil demargination into the blood vessels (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>–<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). Moreover, in humans, betamethasone reduces neutrophil motility and chemotaxis (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>), and can affect metabolism and cytokine production, i.e., reducing interleukin (IL)-8 and macrophage inflammatory protein alpha (MIP-1α) release (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). The inflammatory capacity of neutrophils is therefore reduced, as demonstrated in a lamb model of lung inflammation after betamethasone treatment, where gene expression of <italic>IL-1, IL-6, IL-8</italic>, and <italic>CCL2</italic> was suppressed (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>).</p></sec><sec><title>Monocytes</title><p>Monocytes are circulating innate immune cells that can become antigen-presenting cells (APCs), either macrophages, or dendritic cells (DCs). Thus, reprogramming monocytes may lead to changes in both differentiated cells. Betamethasone has an acute effect on the metabolism of monocytes, transiently reducing the production, and the secretion of IL-6 and reactive oxygen species. By contrast, the phagocytic activity of monocyte-derived APCs was not altered by betamethasone (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). In newborn children with low weight at birth, prenatal betamethasone administration induced a transient immunomodulatory effect in monocytes, causing diminished IL-6 and IL-10 release and downregulation of human leukocyte antigen DR (HLA-DR) expression (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>). Moreover, the total number of monocytes was reduced by betamethasone (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>). This effect was also assessed <italic>in vitro</italic>, demonstrating that glucocorticoids induce apoptosis in human monocytes (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>). Nevertheless, these results are controversial, and other authors reported that betamethasone does not affect monocytes' IL-6 production (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>).</p></sec><sec><title>Macrophages</title><p>Macrophages are crucial in the initial damage to β-cells in T1D. These tissue-resident APCs contribute to initiate specific immune responses (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>). In macrophages, betamethasone diminishes cytokine secretion (IL-8 and TNFα) (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>) and impairs their ability for antigen presentation to T cells (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>). These effects point to the induction of a regulatory profile in macrophages, similar as described in M2 macrophages (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>). Indeed, dexamethasone induces the polarization of the M2 phenotype (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>). Moreover, it was recently reported that dexamethasone increases the migration of macrophages by CD26 overexpression, a membrane glycoprotein with enzymatic capabilities involved in inflammation, and this could contribute to the egress of macrophages from inflamed tissue (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>).</p></sec><sec><title>Dendritic Cells</title><p>DCs are professional APCs with the ability to stimulate naïve T cells. In T1D, DCs are responsible for the presentation of β-cell autoantigens to T lymphocytes, initiating the adaptive autoimmune response against the insulin-producing cells. Similarly to the observed effect of dexamethasone on this cell type, DCs differentiated <italic>in vitro</italic> in the presence of betamethasone failed to achieve a fully mature status, showing a reduced capacity to stimulate the production of IL-17, a cytokine involved in autoimmune responses, by T lymphocytes (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). Furthermore, the release of proinflammatory cytokines was reduced by this drug in DCs. Betamethasone has been reported to induce tolerogenic Langerhans DCs (LDCs) in the skin of patients with psoriasis (<xref rid=\"B47\" ref-type=\"bibr\">47</xref>) and atopic dermatitis, which in turn arrest T helper (Th) 1 and Th2 responses (<xref rid=\"B48\" ref-type=\"bibr\">48</xref>). Similarly to the effects found in monocytes, human DCs differentiated with betamethasone showed a reduction of membrane expression of costimulatory molecules, such as CD40 and CD86, accompanied by a decrease in IL-12 secretion, an important cytokine for Th1 responses. These effects resulted in tolerogenic function in DCs and impaired ability to induce T lymphocyte proliferation (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>).</p></sec><sec><title>Natural Killer Cells</title><p>Natural Killer cells (NKc) are effector lymphocytes of the innate immune system. Their role in T1D is not completely understood, but abnormalities in this cell type may contribute to trigger autoimmune reactions against β-cells (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>). Little is known about the effects of betamethasone on NKc. Betamethasone tends to increase NKc activity in very preterm newborn babies (<32 weeks of gestation), supporting the maturation of this cell type (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>). However, other studies have reported that betamethasone reduces the number of NKc in newborn infants (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>). In adults, NKc showed a reduced cytolytic activity after topical betamethasone administration (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>).</p></sec><sec><title>Adaptative Immune Cells</title><p>Innate immune cells are crucial in the first phases of autoimmune diseases, but the final effector cells are the adaptative immune system cells, T and B lymphocytes. Modulation of these cells can dampen or exacerbate an autoimmune reaction.</p></sec><sec><title>T Lymphocytes</title><p>Betamethasone treatment results in T cell precursor apoptosis and, to a lesser extent, of mature T cells. Transient reduction in thymus weight and thymocyte numbers have been described after prenatal betamethasone administration in mice (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B52\" ref-type=\"bibr\">52</xref>). In humans, the thymus of the foetus of mothers that were prenatally treated with steroids showed delayed growth (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>). Moreover, a reduction of 20–30% of peripheral lymphocyte counts was observed in pregnant women after treatment with betamethasone, although this effect only lasted for 3 days (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>, <xref rid=\"B54\" ref-type=\"bibr\">54</xref>). Other glucocorticoids, like dexamethasone, have comparable effects on lymphocytes after prenatal treatment (<xref rid=\"B55\" ref-type=\"bibr\">55</xref>). In newborn children, a similar effect on lymphocyte counts has been described, mainly affecting CD4+ T lymphocytes (<xref rid=\"B56\" ref-type=\"bibr\">56</xref>). Data on lymphocyte counts are still controversial, since a different study reported an increase in CD3+ T lymphocytes in very preterm newborn babies (<32 gestational weeks) after betamethasone treatment (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>). Prenatal betamethasone administered to the experimental model of T1D, the non-obese diabetic (NOD) mouse, resulted in long-lasting changes in the T Cell Receptor (TCR) Vβ repertoire that persisted into adulthood (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). Importantly, the TCR Vβ families that diminished in frequency after prenatal steroid treatment included pathogenic Vβ domains (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>, <xref rid=\"B58\" ref-type=\"bibr\">58</xref>), so it is reasonable to speculate that betamethasone will protect against T1D. In humans, betamethasone reduces T lymphocyte proliferation capacity (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>), thus reducing clonal expansion. Overall, T cells will have an impaired capacity of interacting with β-cell antigens, thus contributing to prevent the autoimmune response.</p></sec><sec><title>B Lymphocytes</title><p>The role of B lymphocytes in the development of T1D is not completely understood. B cells produce autoantibodies to islet antigens that, even if extremely useful as predictive biomarkers for disease, do not appear pathogenic. Also, B cells are critical as APCs during the first stages of autoimmunity in T1D (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>). Betamethasone has a deleterious effect on mature B cells of NOD mouse (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>) and reduces their ability to produce antibodies, specifically IgE and IgG (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>, <xref rid=\"B61\" ref-type=\"bibr\">61</xref>). Other glucocorticoids, such as dexamethasone, show a similar deleterious effect (<xref rid=\"B62\" ref-type=\"bibr\">62</xref>), affecting early precursor B cells, whereas mature B cells –IgD positive– are resistant to glucocorticoid-induced apoptosis (<xref rid=\"B63\" ref-type=\"bibr\">63</xref>). The reduction observed in antibody production could be the result of impaired B cell receptor and Toll-like receptor 7 signalling, since without these signals B cells cannot switch their Ig isotype, thus reducing their functionality. At the same time, B cells have increased transcriptional activity of <italic>IL-10</italic>, amplifying the immunomodulatory capacity of these cells induced by glucocorticoids (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>).</p></sec></sec><sec id=\"s3\"><title>Effects of Betamethasone on the Target Cells of Autoimmune Diabetes, the Islet β-Cells</title><p>β-cells are the insulin-producing cells of the islets of Langerhans. The autoimmune destruction of these cells is the ultimate cause of T1D. β-cells also have an active role in their own destruction, facilitating the interaction with the immune system, and contributing to their own demise (<xref rid=\"B64\" ref-type=\"bibr\">64</xref>). Thus, the identification of changes induced by betamethasone may help to understand the possible outcome of this drug in the context of T1D. Studies performed in subjects with long term glucocorticoid treatment indicate that glucocorticoids can induce dysglycaemia, leading to diabetes (<xref rid=\"B65\" ref-type=\"bibr\">65</xref>, <xref rid=\"B66\" ref-type=\"bibr\">66</xref>). Glucocorticoids increase insulin resistance (<xref rid=\"B67\" ref-type=\"bibr\">67</xref>) without affecting β-cell mass (<xref rid=\"B68\" ref-type=\"bibr\">68</xref>). On the other hand, physiological endogenous levels of glucocorticoids are necessary for maintaining the regulation of insulin secretion by β-cells (<xref rid=\"B69\" ref-type=\"bibr\">69</xref>). Moreover, prenatal glucocorticoids support the maturation of β-cells by enhancing their glucose sensitivity due to increased expression of <italic>Glut2</italic> and <italic>Gck</italic> genes and by reducing apoptosis, similarly as the overexpression of surfactant proteins induced by glucocorticoids helps with the maturation of the foetal lungs (<xref rid=\"B70\" ref-type=\"bibr\">70</xref>). In a similar way, prenatal glucocorticoids enhance insulin secretion in rats due to the overexpression of <italic>Gck, Slca2</italic>, and <italic>Ins2</italic> genes in β-cells, despite β-cell mass is smaller than in non-treated animals (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>). Experimental data demonstrate that prenatal betamethasone reduces the risk of developing T1D in the NOD mice (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>), correlating to altered expression of genes related to metabolism and autoimmunity in β-cells. In a previous study we reported a reduction of <italic>Ccl2</italic> gene expression in β-cells, which may lead to a reduced recruitment of macrophages and monocytes. Moreover, an increase in <italic>Gad1</italic> gene expression, could promote tolerance to β-cells in NOD mice (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). Recent studies in other T1D experimental models indicate that short-term treatment with betamethasone during late pregnancy does not affect β-cell metabolism in later life (<xref rid=\"B72\" ref-type=\"bibr\">72</xref>, <xref rid=\"B73\" ref-type=\"bibr\">73</xref>). Glucocorticoid signalling can also cause epigenetic modifications in these cells. In fact, glucocorticoids impair the methylation of the DNA by altering the enzymes responsible for this process. Moreover, the prenatal period is a very sensitive phase during which the epigenome shows heightened plasticity to methylation modifications and these changes can be accumulated throughout life (<xref rid=\"B74\" ref-type=\"bibr\">74</xref>). Important β-cell functions, such as insulin secretion and islet cell mass homeostasis, are controlled by epigenetic mechanisms (<xref rid=\"B75\" ref-type=\"bibr\">75</xref>), and glucocorticoids can modify the epigenome of β-cells inducing changes that can affect their function in adults (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>), altering the efficiency of glucose metabolism (<xref rid=\"B77\" ref-type=\"bibr\">77</xref>, <xref rid=\"B78\" ref-type=\"bibr\">78</xref>).</p></sec><sec id=\"s4\"><title>How can Betamethasone Affect the Interplay Between the Immune System and β-Cells?</title><p>T1D is a multifactorial disease with complex interactions between the immune system and the pancreatic β-cells. Glucocorticoids are potent immune suppressors and are commonly used in patients with autoimmune diseases such as psoriasis or rheumatoid arthritis (<xref rid=\"B79\" ref-type=\"bibr\">79</xref>, <xref rid=\"B80\" ref-type=\"bibr\">80</xref>). Betamethasone, like other synthetic glucocorticoids, can reduce cytokine production and release, thereby inhibiting specific immune responses and blocking the initiation of an autoimmune attack to β-cells (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). Dampening the autoimmune reaction can be the most efficient form of preventing T1D, and it might be a consequence of the impaired functionality of innate and adaptative immune cells (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). On the one side, betamethasone diminishes the proinflammatory action of innate immune cells (neutrophils, macrophages, and NK cells). On the other side, this drug induces a tolerogenic antigen presentation in macrophages and DCs, limiting the possibilities to activate autoreactive T lymphocytes in the lymph nodes. In turn, lymphocytes are also affected by betamethasone, as aforementioned. T lymphocytes reduce their proliferation capacity, decreasing the number of cells that can kill the β-cells. At the same time, prenatal treatment critically reduces the number of developing thymocytes and induces a skewed TCR repertoire towards T cells with less affinity to β-cell autoantigens (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>). This fact will impair autoreactivity when APCs expose β-cell autoantigens in Major Histocompatibility Complex (MHC) molecules. Moreover, in the presence of betamethasone, T lymphocytes tend to differentiate to Th2 rather than autoimmunity-prone Th1 or Th17 cells (<xref rid=\"B81\" ref-type=\"bibr\">81</xref>), and it has been demonstrated that glucocorticoids exposure during foetal development can alter the HPA axis, impairing CD8+ T lymphocytes function later in life, making them less responsive against viral antigens (<xref rid=\"B82\" ref-type=\"bibr\">82</xref>), or blunting cortisol response against rhinovirus (<xref rid=\"B83\" ref-type=\"bibr\">83</xref>). In this sense, concerns have been raised about multiple doses of prenatal betamethasone, including an increased susceptibility to infections in children (<xref rid=\"B84\" ref-type=\"bibr\">84</xref>, <xref rid=\"B85\" ref-type=\"bibr\">85</xref>). B cell precursors are also affected by betamethasone, showing reduced antibody production (<xref rid=\"B63\" ref-type=\"bibr\">63</xref>). However, how these effects altogether might influence the development of T1D is not yet known. Taken together, these alterations suggest a rather positive effect leading to T1D protection, but this effect could depend, among other factors, on the concentration, and duration of the prenatal treatment. In addition, β-cell changes induced by betamethasone may enhance this protective effect. Especially, β-cell maturation and the acquisition of an apoptosis-resistant phenotype may be key factors in thwarting undesired autoimmune reactions (<xref rid=\"B70\" ref-type=\"bibr\">70</xref>). Moreover, alterations found in the expression of genes related to interactions between the immune system and β-cell reduce β-cell immunogenicity, hindering their direct interaction with immune system cells (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). This could help to avoid the activation of stray cytotoxic T cells with affinity to β-cell autoantigens. How long this effect is maintained is unknown, but it is reasonable to speculate that prenatal betamethasone could reprogramme some aspects of β-cell function until adult life, without affecting their intrinsic capacities as described for insulin secretion (<xref rid=\"B86\" ref-type=\"bibr\">86</xref>). Furthermore, glucocorticoid stimulation also induces epigenetic changes in the precursor β-cells (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>), and this could be the main actor behind the long-term effects observed after prenatal administration of betamethasone. We have summarised those studies that used betamethasone in their research (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>), focusing on their findings in the context of the immune system and β-cells, and the expected effect these changes would have on T1D. Further studies are needed to reveal the long-term effects of prenatal betamethasone treatment in the immune system and T1D (<xref rid=\"B87\" ref-type=\"bibr\">87</xref>).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Effects of prental betamethasone on β-cells and the immune system of the newborn. Created with BioRender.com.</p></caption><graphic xlink:href=\"fendo-11-00540-g0001\"/></fig><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Studies on the effect of betamethasone in the immune system and impact on T1D.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Species/substrates</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Main findings</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Expected effect on T1D</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>References</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ NKc cytolytic capacity</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protective</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B49\" ref-type=\"bibr\">49</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ IgE synthesis by B cells</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neutral</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B59\" ref-type=\"bibr\">59</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑ Insulin resistance (long-term)<break/> No effect in T1D prevalence (long-term)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neutral</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B80\" ref-type=\"bibr\">80</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑ T1D Hazard ratio after glucocorticoid treatment</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑ Risk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B81\" ref-type=\"bibr\">81</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑ Neutrophils<break/> ↓ Basophils, CD3+CD4+, and CD3+CD8+ T cells</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protective</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B27\" ref-type=\"bibr\">27</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human (adult monocytes)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ DCs costimulatory molecules<break/> ↓ IL-12p70 ↓ Th1 activation</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protective</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B37\" ref-type=\"bibr\">37</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human (adult skin cells)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ LDCs costimulatory molecules and HLA-DR<break/> ↓ Proinflammatory cytokines<break/> No effects in IL-10 secretion or ILT3 expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protective</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B45\" ref-type=\"bibr\">45</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human (cord blood of preterm babies)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑ NKc activity (<32 weeks gestation)<break/> ↓ Lymphocyte proliferation<break/> No effect in IL-6 secretion</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protective</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B38\" ref-type=\"bibr\">38</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human (cord blood)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ IL-6, IL-8 and TNFα secretion by macrophages</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protective</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B40\" ref-type=\"bibr\">40</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human (newborn and adult)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ migration and motility of newborn's neutrophils<break/> No effects in adult's neutrophils</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neutral</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B31\" ref-type=\"bibr\">31</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human (newborn)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ IL-8 and CCL3 secretion from neutrophils</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protective</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B33\" ref-type=\"bibr\">33</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human (newborn)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ HLA-DR expression on monocytes<break/> ↓ IL-6 and IL-10 in plasma</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protective</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B35\" ref-type=\"bibr\">35</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human (newborn)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑ CD3+ T cells and monocytes ↓ NKc</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑ Risk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B48\" ref-type=\"bibr\">48</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human (newborn)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ CD4<sup>+</sup> and CD25<sup>+</sup> T lymphocytes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protective</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B54\" ref-type=\"bibr\">54</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human (pregnant women)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑ Leukocytes and granulocytes<break/> ↓ Lymphocytes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neutral</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B25\" ref-type=\"bibr\">25</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human (pregnant women)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑ Leukocytes<break/> ↓ Lymphocytes and monocytes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neutral</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B36\" ref-type=\"bibr\">36</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human (pregnant women)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑ Neutrophils<break/> ↓ Lymphocytes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neutral</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B52\" ref-type=\"bibr\">52</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mouse (NOD)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ Immunogenicity ↑ Tolerance</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protective</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B30\" ref-type=\"bibr\">30</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mouse (NOD)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ T1D incidence ↓ Diabetogenic Vβ TCR</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protective</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B55\" ref-type=\"bibr\">55</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mouse</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ Impaired antigen presentation by macrophages</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protective</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B41\" ref-type=\"bibr\">41</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mouse</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ Th1 and Th2 induction by LDCs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protective</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B46\" ref-type=\"bibr\">46</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Mouse</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑ Apoptosis of thymocytes ↓ Thymus weight</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑ Risk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B50\" ref-type=\"bibr\">50</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sheep</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ IL-1, IL-6, IL-8, CCL2, and TLR4 expression</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protective</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B33\" ref-type=\"bibr\">33</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sheep</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ IL-6 and ROS from monocytes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protective</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B34\" ref-type=\"bibr\">34</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sheep</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">No long-term effects<break/> Improves preterm delivery adverse effects</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neutral</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B69\" ref-type=\"bibr\">69</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sheep</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">No impairment of insulin sensitivity ↑ Insulin signalling pathway</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neutral</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B71\" ref-type=\"bibr\">71</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rabbit</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ B cells IgG+</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neutral</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B58\" ref-type=\"bibr\">58</xref>)</td></tr></tbody></table><table-wrap-foot><p><italic>CCL, C-C motif ligand; HLA, human leukocyte antigen; ILT3, immunoglobulin-like transcript 3; IL, interleukin; IgE, immunoglobulin E; IgG, immunoglobulin G; LDCs, Langerhans dendritic cells of the skin; NKc, Natural killer cells; ROS, reactive oxygen species; T1D; type 1 diabetes; TCR, T cell receptor; Th, T helper; TLR, toll like receptor; TNF, tumour necrosis factor</italic>.</p></table-wrap-foot></table-wrap></sec><sec id=\"s5\"><title>Future Perspectives</title><p>The effectiveness of glucocorticoids has been demonstrated for a wide range of immunologically related diseases. However, their effects during the prenatal period, both in the immune system and the target tissue of T1D, are not fully characterised. Expanding the understanding of how they can affect self-tolerance and T1D could contribute to reduce the increasing incidence of this and other autoimmune diseases. Another key point is to determine whether the effects of glucocorticoid treatment found in immune system cells could result from changes induced in hematopoietic stem cells, thus explaining the alterations found in many immune cell types. Further studies are required to dissect the exact mechanism and the magnitude of these changes in the immune system and β-cells, because other factors, such as maternal nutrition or stress during pregnancy, could also veil betamethasone effects (<xref rid=\"B88\" ref-type=\"bibr\">88</xref>). Finally, epidemiological studies are needed to explore the effect of prenatal betamethasone on T1D. Finding pieces of evidence of the precise effects of betamethasone in T1D development could lead to improved neonatal care, with special focus on children with higher genetic risk to suffer from T1D.</p></sec><sec sec-type=\"conclusions\" id=\"s6\"><title>Conclusion</title><p>Knowledge about betamethasone action on the immune system is currently increasing, but it is very limited in the prenatal stage as well as in its consequences in the adulthood phase. Glucocorticoids have shown a plethora of effects, which depend on the duration of the treatment, the route of administration, the target tissue, etc. The key message of this review is that prenatal betamethasone affects the immune system cells, and these alterations may have long-term consequences. Simultaneously, betamethasone also alters β-cells towards a less immunogenic phenotype and could induce epigenetic modifications in immune cells and β-cell precursors. Considering these effects, it is tempting to speculate that betamethasone may act as a protective agent against human T1D when administered shortly before birth or in the perinatal period. Future immunological, metabolic, and epidemiological studies, together with the extrapolation of data from other glucocorticoids like dexamethasone, will shed light on the unanswered questions related to this prenatal treatment.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>DP-B and MV-P wrote the manuscript. AG, SR-F, and ET participated in the literature review, and edited the manuscript while adding additional insights. All authors read and approved the final version of the manuscript.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>MV-P holds a patent that relate to immunotherapy for T1D and is co-founder of Ahead Therapeutics S.L. SR-F is part-time employed at Ahead Therapeutics S.L. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.</p><p>The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>The authors thank Dr. Marta Murillo, Dr. Joan Bel, Dr. Federico Vázquez, and Prof. Manel Puig-Domingo, from University Hospital Germans Trias i Pujol, for fruitful discussions. Special thanks to DiabetesCero Foundation for their support.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work has been funded by the German Research Council (KFO296). DP-B was granted by DiabetesCero Foundation.</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Esposito</surname><given-names>S</given-names></name><name><surname>Toni</surname><given-names>G</given-names></name><name><surname>Tascini</surname><given-names>G</given-names></name><name><surname>Santi</surname><given-names>E</given-names></name><name><surname>Berioli</surname><given-names>MG</given-names></name><name><surname>Principi</surname><given-names>N</given-names></name></person-group>. <article-title>Environmental factors associated with type 1 diabetes</article-title>. <source>Front Endocrinol.</source> (<year>2019</year>) <volume>10</volume>:<fpage>1</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.3389/fendo.2019.00592</pub-id><pub-id pub-id-type=\"pmid\">31555211</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>DiMeglio</surname><given-names>LA</given-names></name><name><surname>Evans-Molina</surname><given-names>C</given-names></name><name><surname>Oram</surname><given-names>RA</given-names></name></person-group>. <article-title>Type 1 diabetes</article-title>. <source>Lancet.</source> (<year>2018</year>) <volume>391</volume>:<fpage>2449</fpage>–<lpage>62</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0140-6736(18)31320-5</pub-id><pub-id pub-id-type=\"pmid\">29916386</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>S</given-names></name><name><surname>Zhang</surname><given-names>M</given-names></name><name><surname>Tian</surname><given-names>H</given-names></name><name><surname>Liu</surname><given-names>Z</given-names></name><name><surname>Yin</surname><given-names>X</given-names></name><name><surname>Xi</surname><given-names>B</given-names></name></person-group>. <article-title>Preterm birth and risk of type 1 and type 2 diabetes: systematic review and meta-analysis</article-title>. <source>Obes Rev.</source> (<year>2014</year>) <volume>15</volume>:<fpage>804</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1111/obr.12214</pub-id><pub-id pub-id-type=\"pmid\">25073871</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Størdal</surname><given-names>K</given-names></name><name><surname>McArdle</surname><given-names>HJ</given-names></name><name><surname>Hayes</surname><given-names>H</given-names></name><name><surname>Tapia</surname><given-names>G</given-names></name><name><surname>Viken</surname><given-names>MK</given-names></name><name><surname>Lund-Blix</surname><given-names>NA</given-names></name><etal/></person-group>. <article-title>Prenatal iron exposure and childhood type 1 diabetes</article-title>. <source>Sci Rep.</source> (<year>2018</year>) <volume>8</volume>:<fpage>1</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-018-27391-4</pub-id><pub-id pub-id-type=\"pmid\">29311619</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Samuelsson</surname><given-names>U</given-names></name><name><surname>Oikarinen</surname><given-names>S</given-names></name><name><surname>Hyöty</surname><given-names>H</given-names></name><name><surname>Ludvigsson</surname><given-names>J</given-names></name></person-group>. <article-title>Low zinc in drinking water is associated with the risk of type 1 diabetes in children</article-title>. <source>Pediatr Diabetes.</source> (<year>2011</year>) <volume>12</volume>:<fpage>156</fpage>–<lpage>64</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1399-5448.2010.00678.x</pub-id><pub-id pub-id-type=\"pmid\">20920146</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nisticò</surname><given-names>L</given-names></name><name><surname>Iafusco</surname><given-names>D</given-names></name><name><surname>Galderisi</surname><given-names>A</given-names></name><name><surname>Fagnani</surname><given-names>C</given-names></name><name><surname>Cotichini</surname><given-names>R</given-names></name><name><surname>Toccaceli</surname><given-names>V</given-names></name><etal/></person-group>. <article-title>Emerging effects of early environmental factors over genetic background for type 1 diabetes susceptibility: evidence from a nationwide Italian twin study</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2012</year>) <volume>97</volume>:<fpage>E1483</fpage>–<lpage>91</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2011-3457</pub-id><pub-id pub-id-type=\"pmid\">22569240</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kumar</surname><given-names>D</given-names></name><name><surname>Gemayel</surname><given-names>NS</given-names></name><name><surname>Deapen</surname><given-names>D</given-names></name><name><surname>Kapadia</surname><given-names>D</given-names></name><name><surname>Yamashita</surname><given-names>PH</given-names></name><name><surname>Lee</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>North-American twins with IDDM: genetic, etiological, and clinical significance of disease concordance according to age, zygosity, and the interval after diagnosis in first twin</article-title>. <source>Diabetes.</source> (<year>1993</year>) <volume>42</volume>:<fpage>1351</fpage>–<lpage>63</lpage>. <pub-id pub-id-type=\"doi\">10.2337/diabetes.42.9.1351</pub-id><pub-id pub-id-type=\"pmid\">8349046</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liggins</surname><given-names>GC</given-names></name><name><surname>Howie</surname><given-names>RN</given-names></name></person-group>\n<article-title>A controlled trial of antepartum glucoccorticoid treatment for prevention of the respiratory distress syndrome in premature infants</article-title>. <source>Pediatrics.</source> (<year>1972</year>) <volume>50</volume>:<fpage>515</fpage>–<lpage>25</lpage>.<pub-id pub-id-type=\"pmid\">4561295</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Roberts</surname><given-names>D</given-names></name><name><surname>Brown</surname><given-names>J</given-names></name><name><surname>Medley</surname><given-names>N</given-names></name><name><surname>Dalziel</surname><given-names>SR</given-names></name></person-group>. <article-title>Antenatal corticosteroids for accelerating fetal lung maturation for women at risk of preterm birth</article-title>. <source>Cochrane Database Syst Rev.</source> (<year>2017</year>) <volume>3</volume>:<fpage>CD004454</fpage>. <pub-id pub-id-type=\"doi\">10.1002/14651858.CD004454.pub3</pub-id><pub-id pub-id-type=\"pmid\">28321847</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schmidt</surname><given-names>AF</given-names></name><name><surname>Kemp</surname><given-names>MW</given-names></name><name><surname>Kannan</surname><given-names>PS</given-names></name><name><surname>Kramer</surname><given-names>BW</given-names></name><name><surname>Newnham</surname><given-names>JP</given-names></name><name><surname>Kallapur</surname><given-names>SG</given-names></name><etal/></person-group>. <article-title>Antenatal dexamethasone vs. betamethasone dosing for lung maturation in fetal sheep</article-title>. <source>Pediatr Res.</source> (<year>2017</year>) <volume>81</volume>:<fpage>496</fpage>–<lpage>503</lpage>. <pub-id pub-id-type=\"doi\">10.1038/pr.2016.249</pub-id><pub-id pub-id-type=\"pmid\">27861467</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vidaeff</surname><given-names>AC</given-names></name><name><surname>Belfort</surname><given-names>MA</given-names></name><name><surname>Steer</surname><given-names>PJ</given-names></name></person-group>\n<article-title>Antenatal corticosteroids: a time for more careful scrutiny of the indications?</article-title>\n<source>BJOG.</source> (<year>2016</year>) <volume>123</volume>:<fpage>1067</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1111/1471-0528.13853</pub-id><pub-id pub-id-type=\"pmid\">26776668</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kajantie</surname><given-names>E</given-names></name><name><surname>Raivio</surname><given-names>T</given-names></name><name><surname>Jänne</surname><given-names>OA</given-names></name><name><surname>Hovi</surname><given-names>P</given-names></name><name><surname>Dunkel</surname><given-names>L</given-names></name><name><surname>Andersson</surname><given-names>S</given-names></name></person-group>. <article-title>Circulating glucocorticoid bioactivity in the preterm newborn after antenatal betamethasone treatment</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2004</year>) <volume>89</volume>:<fpage>3999</fpage>–<lpage>4003</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2004-0013</pub-id><pub-id pub-id-type=\"pmid\">15292340</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alexander</surname><given-names>N</given-names></name><name><surname>Rosenlöcher</surname><given-names>F</given-names></name><name><surname>Stalder</surname><given-names>T</given-names></name><name><surname>Linke</surname><given-names>J</given-names></name><name><surname>Distler</surname><given-names>W</given-names></name><name><surname>Morgner</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Impact of antenatal synthetic glucocorticoid exposure on endocrine stress reactivity in term-born children</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2012</year>) <volume>97</volume>:<fpage>3538</fpage>–<lpage>44</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2012-1970</pub-id><pub-id pub-id-type=\"pmid\">22869608</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alexander</surname><given-names>N</given-names></name><name><surname>Rosenlöcher</surname><given-names>F</given-names></name><name><surname>Dettenborn</surname><given-names>L</given-names></name><name><surname>Stalder</surname><given-names>T</given-names></name><name><surname>Linke</surname><given-names>J</given-names></name><name><surname>DIstler</surname><given-names>W</given-names></name><etal/></person-group>. <article-title>Impact of antenatal glucocorticoid therapy and risk of preterm delivery on intelligence in term-born children</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2016</year>) <volume>101</volume>:<fpage>581</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2015-2453</pub-id><pub-id pub-id-type=\"pmid\">26649618</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cain</surname><given-names>DW</given-names></name><name><surname>Cidlowski</surname><given-names>JA</given-names></name></person-group>. <article-title>Immune regulation by glucocorticoids</article-title>. <source>Nat Rev Immunol.</source> (<year>2017</year>) <volume>17</volume>:<fpage>233</fpage>–<lpage>47</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nri.2017.1</pub-id><pub-id pub-id-type=\"pmid\">28192415</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kardava</surname><given-names>L</given-names></name><name><surname>Germain</surname><given-names>RN</given-names></name><name><surname>Kumar</surname><given-names>P</given-names></name><name><surname>Tsang</surname><given-names>JS</given-names></name><name><surname>Gadkari</surname><given-names>M</given-names></name><name><surname>Franco</surname><given-names>LM</given-names></name><etal/></person-group>. <article-title>Immune regulation by glucocorticoids can be linked to cell type–dependent transcriptional responses</article-title>. <source>J Exp Med</source>. (<year>2019</year>) <volume>216</volume>:<fpage>384</fpage>–<lpage>406</lpage>. <pub-id pub-id-type=\"doi\">10.1084/jem.20180595</pub-id><pub-id pub-id-type=\"pmid\">30674564</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jewell</surname><given-names>CM</given-names></name><name><surname>Cidlowski</surname><given-names>JA</given-names></name></person-group>. <article-title>Molecular evidence for a link between the N363S glucocorticoid receptor polymorphism and altered gene expression</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2007</year>) <volume>92</volume>:<fpage>3268</fpage>–<lpage>77</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2007-0642</pub-id><pub-id pub-id-type=\"pmid\">17535992</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moraitis</surname><given-names>AG</given-names></name><name><surname>Block</surname><given-names>T</given-names></name><name><surname>Nguyen</surname><given-names>D</given-names></name><name><surname>Belanoff</surname><given-names>JK</given-names></name></person-group>. <article-title>The role of glucocorticoid receptors in metabolic syndrome and psychiatric illness</article-title>. <source>J Steroid Biochem Mol Biol.</source> (<year>2017</year>) <volume>165</volume>:<fpage>114</fpage>–<lpage>20</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jsbmb.2016.03.023</pub-id><pub-id pub-id-type=\"pmid\">27002803</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Liu</surname><given-names>R</given-names></name><name><surname>Gan</surname><given-names>J</given-names></name><name><surname>Liu</surname><given-names>J</given-names></name><name><surname>Liu</surname><given-names>W</given-names></name></person-group>. <article-title>Endocrine-disrupting effects of pesticides through interference with human glucocorticoid receptor</article-title>. <source>Environ Sci Technol.</source> (<year>2016</year>) <volume>50</volume>:<fpage>435</fpage>–<lpage>43</lpage>. <pub-id pub-id-type=\"doi\">10.1021/acs.est.5b03731</pub-id><pub-id pub-id-type=\"pmid\">26647222</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Slotkin</surname><given-names>TA</given-names></name><name><surname>Card</surname><given-names>J</given-names></name><name><surname>Seidler</surname><given-names>FJ</given-names></name></person-group>. <article-title>Chlorpyrifos developmental neurotoxicity: interaction with glucocorticoids in PC12 cells</article-title>. <source>Neurotoxicol Teratol.</source> (<year>2012</year>) <volume>34</volume>:<fpage>505</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ntt.2012.07.002</pub-id><pub-id pub-id-type=\"pmid\">22796634</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Solano</surname><given-names>ME</given-names></name><name><surname>Holmes</surname><given-names>MC</given-names></name><name><surname>Mittelstadt</surname><given-names>PR</given-names></name><name><surname>Chapman</surname><given-names>KE</given-names></name><name><surname>Tolosa</surname><given-names>E</given-names></name></person-group>. <article-title>Antenatal endogenous and exogenous glucocorticoids and their impact on immune ontogeny and long-term immunity</article-title>. <source>Semin Immunopathol.</source> (<year>2016</year>) <volume>38</volume>:<fpage>739</fpage>–<lpage>63</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00281-016-0575-z</pub-id><pub-id pub-id-type=\"pmid\">27465226</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Drexhage</surname><given-names>HA</given-names></name><name><surname>Dik</surname><given-names>WA</given-names></name><name><surname>Leenen</surname><given-names>PJM</given-names></name><name><surname>Versnel</surname><given-names>MA</given-names></name></person-group>. <article-title>The immune pathogenesis of type 1 diabetes: not only thinking outside the cell but also outside the islet and out of the box</article-title>. <source>Diabetes.</source> (<year>2016</year>) <volume>65</volume>:<fpage>2130</fpage>–<lpage>3</lpage>. <pub-id pub-id-type=\"doi\">10.2337/dbi16-0030</pub-id><pub-id pub-id-type=\"pmid\">27456621</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Faden</surname><given-names>M</given-names></name><name><surname>Holm</surname><given-names>M</given-names></name><name><surname>Allred</surname><given-names>E</given-names></name><name><surname>Fichorova</surname><given-names>R</given-names></name><name><surname>Dammann</surname><given-names>O</given-names></name><name><surname>Leviton</surname><given-names>A</given-names></name></person-group>. <article-title>Antenatal glucocorticoids and neonatal inflammation-associated proteins</article-title>. <source>Cytokine.</source> (<year>2016</year>) <volume>88</volume>:<fpage>199</fpage>–<lpage>208</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cyto.2016.09.015</pub-id><pub-id pub-id-type=\"pmid\">27668972</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Diana</surname><given-names>J</given-names></name><name><surname>Simoni</surname><given-names>Y</given-names></name><name><surname>Furio</surname><given-names>L</given-names></name><name><surname>Beaudoin</surname><given-names>L</given-names></name><name><surname>Agerberth</surname><given-names>B</given-names></name><name><surname>Barrat</surname><given-names>F</given-names></name><etal/></person-group>. <article-title>Crosstalk between neutrophils, B-1a cells and plasmacytoid dendritic cells initiates autoimmune diabetes</article-title>. <source>Nat Med.</source> (<year>2013</year>) <volume>19</volume>:<fpage>65</fpage>–<lpage>73</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nm.3042</pub-id><pub-id pub-id-type=\"pmid\">23242473</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Valle</surname><given-names>A</given-names></name><name><surname>Giamporcaro</surname><given-names>GM</given-names></name><name><surname>Scavini</surname><given-names>M</given-names></name><name><surname>Stabilini</surname><given-names>A</given-names></name><name><surname>Grogan</surname><given-names>P</given-names></name><name><surname>Bianconi</surname><given-names>E</given-names></name><etal/></person-group>. <article-title>Reduction of circulating neutrophils precedes and accompanies type 1 diabetes</article-title>. <source>Diabetes.</source> (<year>2013</year>) <volume>62</volume>:<fpage>2072</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.2337/db12-1345</pub-id><pub-id pub-id-type=\"pmid\">23349491</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Battaglia</surname><given-names>M</given-names></name><name><surname>Petrelli</surname><given-names>A</given-names></name><name><surname>Vecchio</surname><given-names>F</given-names></name></person-group>. <article-title>Neutrophils and type 1 diabetes</article-title>. <source>Curr Opin Endocrinol Diabetes Obes.</source> (<year>2019</year>) <volume>26</volume>:<fpage>201</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1097/MED.0000000000000485</pub-id><pub-id pub-id-type=\"pmid\">31157631</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kadanali</surname><given-names>S</given-names></name><name><surname>Ingeç</surname><given-names>M</given-names></name><name><surname>Küçüközkan</surname><given-names>T</given-names></name><name><surname>Börekçi</surname><given-names>B</given-names></name><name><surname>Kumtepe</surname><given-names>Y</given-names></name></person-group>. <article-title>Changes in leukocyte, granulocyte and lymphocyte counts following antenatal betamethasone administration to pregnant women</article-title>. <source>Int J Gynecol Obstet.</source> (<year>1997</year>) <volume>58</volume>:<fpage>269</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0020-7292(97)00085-4</pub-id><pub-id pub-id-type=\"pmid\">9286859</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cavalcanti</surname><given-names>DMH</given-names></name><name><surname>Lotufo</surname><given-names>CMC</given-names></name><name><surname>Borelli</surname><given-names>P</given-names></name><name><surname>Ferreira</surname><given-names>ZS</given-names></name><name><surname>Markus</surname><given-names>RP</given-names></name><name><surname>Farsky</surname><given-names>SHP</given-names></name></person-group>. <article-title>Endogenous glucocorticoids control neutrophil mobilization from bone marrow to blood and tissues in non-inflammatory conditions</article-title>. <source>Br J Pharmacol.</source> (<year>2007</year>) <volume>152</volume>:<fpage>1291</fpage>–<lpage>300</lpage>. <pub-id pub-id-type=\"doi\">10.1038/sj.bjp.0707512</pub-id><pub-id pub-id-type=\"pmid\">17982481</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jobe</surname><given-names>AH</given-names></name><name><surname>Milad</surname><given-names>MA</given-names></name><name><surname>Peppard</surname><given-names>T</given-names></name><name><surname>Jusko</surname><given-names>WJ</given-names></name></person-group>. <article-title>Pharmacokinetics and pharmacodynamics of intramuscular and oral betamethasone and dexamethasone in reproductive age women in india</article-title>. <source>Clin Transl Sci</source>. (<year>2019</year>) <volume>13</volume>:<fpage>391</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1111/cts.12724</pub-id><pub-id pub-id-type=\"pmid\">31808984</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fay</surname><given-names>ME</given-names></name><name><surname>Myers</surname><given-names>DR</given-names></name><name><surname>Kumar</surname><given-names>A</given-names></name><name><surname>Turbyfield</surname><given-names>CT</given-names></name><name><surname>Byler</surname><given-names>R</given-names></name><name><surname>Crawford</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Cellular softening mediates leukocyte demargination and trafficking, thereby increasing clinical blood counts</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2016</year>) <volume>113</volume>:<fpage>1987</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1508920113</pub-id><pub-id pub-id-type=\"pmid\">26858400</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weber</surname><given-names>PSD</given-names></name></person-group>. <article-title>Mechanisms of glucocorticoid-induced down-regulation of neutrophil L-selectin in cattle: evidence for effects at the gene-expression level and primarily on blood neutrophils</article-title>. <source>J Leukoc Biol.</source> (<year>2003</year>) <volume>75</volume>:<fpage>815</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.1189/jlb.1003505</pub-id><pub-id pub-id-type=\"pmid\">14761937</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Perna-Barrull</surname><given-names>D</given-names></name><name><surname>Rodriguez-Fernandez</surname><given-names>S</given-names></name><name><surname>Pujol-Autonell</surname><given-names>I</given-names></name><name><surname>Gieras</surname><given-names>A</given-names></name><name><surname>Ampudia-Carrasco</surname><given-names>RM</given-names></name><name><surname>Villalba</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Prenatal betamethasone interferes with immune system development and alters target cells in autoimmune diabetes</article-title>. <source>Sci Rep.</source> (<year>2019</year>) <volume>9</volume>:<fpage>1235</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-018-37878-9</pub-id><pub-id pub-id-type=\"pmid\">30718757</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fuenfer</surname><given-names>MM</given-names></name><name><surname>Herson</surname><given-names>VC</given-names></name><name><surname>Raye</surname><given-names>JR</given-names></name><name><surname>Woronick</surname><given-names>CL</given-names></name><name><surname>Eisenfeld</surname><given-names>L</given-names></name><name><surname>Ingardia</surname><given-names>CJ</given-names></name><etal/></person-group>. <article-title>The effect of betamethasone on neonatal neutrophil chemotaxis</article-title>. <source>Pediatr Res.</source> (<year>1987</year>) <volume>22</volume>:<fpage>150</fpage>–<lpage>2</lpage>. <pub-id pub-id-type=\"doi\">10.1203/00006450-198708000-00009</pub-id><pub-id pub-id-type=\"pmid\">3658539</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Irakam</surname><given-names>A</given-names></name><name><surname>Miskolci</surname><given-names>V</given-names></name><name><surname>Vancurova</surname><given-names>I</given-names></name><name><surname>Davidson</surname><given-names>D</given-names></name></person-group>. <article-title>Dose-related inhibition of proinflammatory cytokine release from neutrophils of the newborn by dexamethasone, betamethasone, and hydrocortisone</article-title>. <source>Neonatology.</source> (<year>2002</year>) <volume>82</volume>:<fpage>89</fpage>–<lpage>95</lpage>. <pub-id pub-id-type=\"doi\">10.1159/000063094</pub-id><pub-id pub-id-type=\"pmid\">12169830</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuypers</surname><given-names>E</given-names></name><name><surname>Collins</surname><given-names>JJP</given-names></name><name><surname>Kramer</surname><given-names>BW</given-names></name><name><surname>Ofman</surname><given-names>G</given-names></name><name><surname>Nitsos</surname><given-names>I</given-names></name><name><surname>Jane Pillow</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Intra-amniotic LPS and antenatal betamethasone: inflammation and maturation in preterm lamb lungs</article-title>. <source>Am J Physiol Lung Cell Mol Physiol.</source> (<year>2012</year>) <volume>302</volume>:<fpage>L380</fpage>. <pub-id pub-id-type=\"doi\">10.1152/ajplung.00338.2011</pub-id><pub-id pub-id-type=\"pmid\">22160306</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kramer</surname><given-names>BW</given-names></name><name><surname>Ikegami</surname><given-names>M</given-names></name><name><surname>Moss</surname><given-names>TJM</given-names></name><name><surname>Nitsos</surname><given-names>I</given-names></name><name><surname>Newnham</surname><given-names>JP</given-names></name><name><surname>Jobe</surname><given-names>AH</given-names></name></person-group>. <article-title>Antenatal betamethasone changes cord blood monocyte responses to endotoxin in preterm lambs</article-title>. <source>Pediatr Res.</source> (<year>2004</year>) <volume>55</volume>:<fpage>764</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1203/01.PDR.0000120678.72485.19</pub-id><pub-id pub-id-type=\"pmid\">14973182</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Palojärvi</surname><given-names>A</given-names></name><name><surname>Andersson</surname><given-names>S</given-names></name><name><surname>Turpeinen</surname><given-names>U</given-names></name><name><surname>Janér</surname><given-names>C</given-names></name><name><surname>Petäjä</surname><given-names>J</given-names></name></person-group>. <article-title>Antenatal betamethasone associates with transient immunodepression in very low birth weight infants</article-title>. <source>Neonatology.</source> (<year>2013</year>) <volume>104</volume>:<fpage>275</fpage>–<lpage>82</lpage>. <pub-id pub-id-type=\"doi\">10.1159/000353964</pub-id><pub-id pub-id-type=\"pmid\">24107413</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Diebel</surname><given-names>ND</given-names></name><name><surname>Parsons</surname><given-names>MT</given-names></name><name><surname>Spellacy</surname><given-names>WN</given-names></name></person-group>. <article-title>The effects of betamethasone on white blood cells during pregnancy with PPROM</article-title>. <source>J Perinat Med.</source> (<year>1998</year>) <volume>26</volume>:<fpage>204</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1515/jpme.1998.26.3.204</pub-id><pub-id pub-id-type=\"pmid\">9773380</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kalthoff</surname><given-names>FS</given-names></name><name><surname>Chung</surname><given-names>J</given-names></name><name><surname>Musser</surname><given-names>P</given-names></name><name><surname>Stuetz</surname><given-names>A</given-names></name></person-group>\n<article-title>Pimecrolimus does not affect the differentiation, maturation and function of human monocyte-derived dendritic cells, in contrast to corticosteroids</article-title>. <source>Clin Exp Immunol.</source> (<year>2003</year>) <volume>133</volume>:<fpage>350</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1046/j.1365-2249.2003.02225.x</pub-id><pub-id pub-id-type=\"pmid\">12930360</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kavelaars</surname><given-names>A</given-names></name><name><surname>Van Der Pompe</surname><given-names>G</given-names></name><name><surname>Bakker</surname><given-names>JM</given-names></name><name><surname>Van Hasselt</surname><given-names>PM</given-names></name><name><surname>Cats</surname><given-names>B</given-names></name><name><surname>Visser</surname><given-names>GHA</given-names></name><etal/></person-group>. <article-title>Altered immune function in human newborns after prenatal administration of betamethasone: enhanced natural killer cell activity and decreased T cell proliferation in cord blood</article-title>. <source>Pediatr Res.</source> (<year>1999</year>) <volume>45</volume>:<fpage>306</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.1203/00006450-199903000-00003</pub-id><pub-id pub-id-type=\"pmid\">10088646</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carrero</surname><given-names>JA</given-names></name><name><surname>McCarthy</surname><given-names>DP</given-names></name><name><surname>Ferris</surname><given-names>ST</given-names></name><name><surname>Wan</surname><given-names>X</given-names></name><name><surname>Hu</surname><given-names>H</given-names></name><name><surname>Zinselmeyer</surname><given-names>BH</given-names></name><etal/></person-group>. <article-title>Resident macrophages of pancreatic islets have a seminal role in the initiation of autoimmune diabetes of NOD mice</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2017</year>) <volume>114</volume>:<fpage>E10418</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1713543114</pub-id><pub-id pub-id-type=\"pmid\">29133420</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kasat</surname><given-names>K</given-names></name><name><surname>Patel</surname><given-names>H</given-names></name><name><surname>Predtechenska</surname><given-names>O</given-names></name><name><surname>Vancurova</surname><given-names>I</given-names></name><name><surname>Davidson</surname><given-names>D</given-names></name></person-group>. <article-title>Anti-inflammatory actions of endogenous and exogenous interleukin-10 versus glucocorticoids on macrophage functions of the newly born</article-title>. <source>J Perinatol.</source> (<year>2014</year>) <volume>34</volume>:<fpage>380</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1038/jp.2014.16</pub-id><pub-id pub-id-type=\"pmid\">24526008</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ponzin</surname><given-names>D</given-names></name><name><surname>Bellini</surname><given-names>F</given-names></name><name><surname>Chizzolini</surname><given-names>C</given-names></name></person-group>. <article-title>Antigen presentation is inhibited in vivo by betamethasone</article-title>. <source>Life Sci.</source> (<year>1995</year>) <volume>56</volume>:<fpage>1595</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1016/0024-3205(95)00126-Q</pub-id><pub-id pub-id-type=\"pmid\">7723588</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mantovani</surname><given-names>A</given-names></name><name><surname>Sica</surname><given-names>A</given-names></name><name><surname>Sozzani</surname><given-names>S</given-names></name><name><surname>Allavena</surname><given-names>P</given-names></name><name><surname>Vecchi</surname><given-names>A</given-names></name><name><surname>Locati</surname><given-names>M</given-names></name></person-group>. <article-title>The chemokine system in diverse forms of macrophage activation and polarization</article-title>. <source>Trends Immunol.</source> (<year>2004</year>) <volume>25</volume>:<fpage>677</fpage>–<lpage>86</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.it.2004.09.015</pub-id><pub-id pub-id-type=\"pmid\">15530839</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Teixeira</surname><given-names>CJ</given-names></name><name><surname>Santos-Silva</surname><given-names>JC</given-names></name><name><surname>de Souza</surname><given-names>DN</given-names></name><name><surname>Rafacho</surname><given-names>A</given-names></name><name><surname>Anhe</surname><given-names>GF</given-names></name><name><surname>Bordin</surname><given-names>S</given-names></name></person-group>. <article-title>Dexamethasone during pregnancy impairs maternal pancreatic β-cell renewal during lactation</article-title>. <source>Endocr Connect.</source> (<year>2019</year>) <volume>8</volume>:<fpage>120</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.1530/EC-18-0505</pub-id><pub-id pub-id-type=\"pmid\">30768422</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Diaz-Jimenez</surname><given-names>D</given-names></name><name><surname>Petrillo</surname><given-names>MG</given-names></name><name><surname>Busada</surname><given-names>JT</given-names></name><name><surname>Hermoso</surname><given-names>MA</given-names></name><name><surname>Cidlowski</surname><given-names>JA</given-names></name></person-group>. <article-title>Glucocorticoids mobilize macrophages by transcriptionally up-regulating the exopeptidase DPP4</article-title>. <source>J Biol Chem.</source> (<year>2020</year>) <volume>295</volume>:<fpage>3213</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.RA119.010894</pub-id><pub-id pub-id-type=\"pmid\">31988243</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alhadj Ali</surname><given-names>M</given-names></name><name><surname>Thrower</surname><given-names>SL</given-names></name><name><surname>Hanna</surname><given-names>SJ</given-names></name><name><surname>Coulman</surname><given-names>SA</given-names></name><name><surname>Birchall</surname><given-names>JC</given-names></name><name><surname>Wong</surname><given-names>FS</given-names></name><etal/></person-group>. <article-title>Topical steroid therapy induces pro-tolerogenic changes in langerhans cells in human skin</article-title>. <source>Immunology.</source> (<year>2015</year>) <volume>146</volume>:<fpage>411</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.1111/imm.12518</pub-id><pub-id pub-id-type=\"pmid\">26293297</pub-id></mixed-citation></ref><ref id=\"B48\"><label>48.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Matsui</surname><given-names>K</given-names></name><name><surname>Tamai</surname><given-names>S</given-names></name><name><surname>Ikeda</surname><given-names>R</given-names></name></person-group>\n<article-title>Betamethasone, but not tacrolimus, suppresses the development of Th2 cells mediated by langerhans cell-like dendritic cells</article-title>. <source>Biol Pharm Bull.</source> (<year>1980</year>) <volume>39</volume>:<fpage>1220</fpage>–<lpage>3</lpage>. <pub-id pub-id-type=\"doi\">10.1248/bpb.b16-00075</pub-id></mixed-citation></ref><ref id=\"B49\"><label>49.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>La Marca</surname><given-names>V</given-names></name><name><surname>Gianchecchi</surname><given-names>E</given-names></name><name><surname>Fierabracci</surname><given-names>A</given-names></name></person-group>. <article-title>Type 1 diabetes and its multi-factorial pathogenesis: the putative role of NK cells</article-title>. <source>Int J Mol Sci.</source> (<year>2018</year>) <volume>19</volume>:<fpage>794</fpage>. <pub-id pub-id-type=\"doi\">10.3390/ijms19030794</pub-id><pub-id pub-id-type=\"pmid\">29534427</pub-id></mixed-citation></ref><ref id=\"B50\"><label>50.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kotiranta-Ainamo</surname><given-names>A</given-names></name><name><surname>Apajasalo</surname><given-names>M</given-names></name><name><surname>Pohjavuori</surname><given-names>M</given-names></name><name><surname>Rautonen</surname><given-names>N</given-names></name><name><surname>Rautonen</surname><given-names>J</given-names></name></person-group>. <article-title>Mononuclear cell subpopulations in preterm and full-term neonates: independent effects of gestational age, neonatal infection, maternal pre- eclampsia, maternal betamethason therapy, and mode of delivery</article-title>. <source>Clin Exp Immunol.</source> (<year>1999</year>) <volume>115</volume>:<fpage>309</fpage>–<lpage>14</lpage>. <pub-id pub-id-type=\"doi\">10.1046/j.1365-2249.1999.00795.x</pub-id><pub-id pub-id-type=\"pmid\">9933458</pub-id></mixed-citation></ref><ref id=\"B51\"><label>51.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lesko</surname><given-names>MJ</given-names></name><name><surname>Lever</surname><given-names>RS</given-names></name><name><surname>Mackie</surname><given-names>RM</given-names></name><name><surname>Parrott</surname><given-names>DMV</given-names></name></person-group>. <article-title>The effect of topical steroid application on natural killer cell activity</article-title>. <source>Clin Exp Allergy.</source> (<year>1989</year>) <volume>19</volume>:<fpage>633</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-2222.1989.tb02759.x</pub-id><pub-id pub-id-type=\"pmid\">2598103</pub-id></mixed-citation></ref><ref id=\"B52\"><label>52.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Diepenbruck</surname><given-names>I</given-names></name><name><surname>Much</surname><given-names>CC</given-names></name><name><surname>Krumbholz</surname><given-names>A</given-names></name><name><surname>Kolster</surname><given-names>M</given-names></name><name><surname>Thieme</surname><given-names>R</given-names></name><name><surname>Thieme</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Effect of prenatal steroid treatment on the developing immune system</article-title>. <source>J Mol Med.</source> (<year>2013</year>) <volume>91</volume>:<fpage>1293</fpage>–<lpage>302</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00109-013-1069-2</pub-id><pub-id pub-id-type=\"pmid\">23851605</pub-id></mixed-citation></ref><ref id=\"B53\"><label>53.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jones</surname><given-names>CA</given-names></name><name><surname>Nisenbaum</surname><given-names>R</given-names></name><name><surname>De Souza</surname><given-names>LR</given-names></name><name><surname>Berger</surname><given-names>H</given-names></name></person-group>. <article-title>Antenatal corticosteroid administration is associated with decreased growth of the fetal thymus: a prospective cohort study</article-title>. <source>J Perinatol.</source> (<year>2020</year>) <volume>40</volume>:<fpage>30</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41372-019-0554-z</pub-id><pub-id pub-id-type=\"pmid\">31748655</pub-id></mixed-citation></ref><ref id=\"B54\"><label>54.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wallace</surname><given-names>EM</given-names></name><name><surname>Ekkel</surname><given-names>K</given-names></name><name><surname>Cotter</surname><given-names>T</given-names></name><name><surname>Tippetr</surname><given-names>C</given-names></name><name><surname>Catalano</surname><given-names>J</given-names></name></person-group>. <article-title>Haematological effects of betamethasone treatment in late pregnancy</article-title>. <source>Aust N Zeal J Obstet Gynaecol.</source> (<year>1998</year>) <volume>38</volume>:<fpage>396</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1479-828X.1998.tb03095.x</pub-id><pub-id pub-id-type=\"pmid\">9890217</pub-id></mixed-citation></ref><ref id=\"B55\"><label>55.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Denison</surname><given-names>FC</given-names></name><name><surname>Elliott</surname><given-names>CL</given-names></name><name><surname>Wallace</surname><given-names>EM</given-names></name></person-group>. <article-title>Dexamethasone-induced leucocytosis in pregnancy</article-title>. <source>Br J Obstet Gynaecol.</source> (<year>1997</year>) <volume>104</volume>:<fpage>851</fpage>–<lpage>3</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1471-0528.1997.tb12035.x</pub-id><pub-id pub-id-type=\"pmid\">9236656</pub-id></mixed-citation></ref><ref id=\"B56\"><label>56.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chabra</surname><given-names>S</given-names></name><name><surname>Cottrill</surname><given-names>C</given-names></name><name><surname>Rayens</surname><given-names>MK</given-names></name><name><surname>Cross</surname><given-names>R</given-names></name><name><surname>Lipke</surname><given-names>D</given-names></name><name><surname>Bruce</surname><given-names>M</given-names></name></person-group>. <article-title>Lymphocyte subsets in cord blood of preterm infants: effect of antenatal steroids</article-title>. <source>Biol Neonate.</source> (<year>1998</year>) <volume>74</volume>:<fpage>200</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1159/000014025</pub-id><pub-id pub-id-type=\"pmid\">9691160</pub-id></mixed-citation></ref><ref id=\"B57\"><label>57.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gieras</surname><given-names>A</given-names></name><name><surname>Gehbauer</surname><given-names>C</given-names></name><name><surname>Perna-Barrull</surname><given-names>D</given-names></name><name><surname>Engler</surname><given-names>JB</given-names></name><name><surname>Diepenbruck</surname><given-names>I</given-names></name><name><surname>Glau</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Prenatal administration of betamethasone causes changes in the T cell receptor repertoire influencing development of autoimmunity</article-title>. <source>Front Immunol.</source> (<year>2017</year>) <volume>8</volume>:<fpage>1505</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2017.01505</pub-id><pub-id pub-id-type=\"pmid\">29181000</pub-id></mixed-citation></ref><ref id=\"B58\"><label>58.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quinn</surname><given-names>A</given-names></name><name><surname>McInerney</surname><given-names>M</given-names></name><name><surname>Huffman</surname><given-names>D</given-names></name><name><surname>McInerney</surname><given-names>B</given-names></name><name><surname>Mayo</surname><given-names>S</given-names></name><name><surname>Haskins</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>T cells to a dominant epitope of GAD65 express a public CDR3 motif</article-title>. <source>Int Immunol.</source> (<year>2006</year>) <volume>18</volume>:<fpage>967</fpage>–<lpage>79</lpage>. <pub-id pub-id-type=\"doi\">10.1093/intimm/dxl033</pub-id><pub-id pub-id-type=\"pmid\">16641112</pub-id></mixed-citation></ref><ref id=\"B59\"><label>59.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wong</surname><given-names>FS</given-names></name><name><surname>Wen</surname><given-names>L</given-names></name><name><surname>Tang</surname><given-names>M</given-names></name><name><surname>Ramanathan</surname><given-names>M</given-names></name><name><surname>Visintin</surname><given-names>I</given-names></name><name><surname>Daugherty</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Investigation of the role of B-cells in type 1 diabetes in the NOD mouse</article-title>. <source>Diabetes.</source> (<year>2004</year>) <volume>53</volume>:<fpage>2581</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.2337/diabetes.53.10.2581</pub-id><pub-id pub-id-type=\"pmid\">15448087</pub-id></mixed-citation></ref><ref id=\"B60\"><label>60.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oehling</surname><given-names>A</given-names></name><name><surname>Crisci</surname><given-names>CD</given-names></name><name><surname>Sanz</surname><given-names>ML</given-names></name><name><surname>Subirá</surname><given-names>ML</given-names></name></person-group>. <article-title>Immunosuppressive effect of corticosteroids on rabbit's humoral and cellular response</article-title>. <source>Allergol Immunopathol.</source> (<year>1976</year>) <volume>4</volume>:<fpage>255</fpage>–<lpage>68</lpage>. <pub-id pub-id-type=\"pmid\">1086585</pub-id></mixed-citation></ref><ref id=\"B61\"><label>61.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Franco Del Prete</surname><given-names>G</given-names></name><name><surname>Vercelli</surname><given-names>D</given-names></name><name><surname>Tiri</surname><given-names>A</given-names></name><name><surname>Maggi</surname><given-names>E</given-names></name><name><surname>Rossi</surname><given-names>O</given-names></name><name><surname>Romagnani</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Effect of <italic>in vitro</italic> irradiation and cell cycle-inhibitory drugs on the spontaneous human IgE synthesis in vitro</article-title>. <source>J Allergy Clin Immunol.</source> (<year>1987</year>) <volume>79</volume>:<fpage>69</fpage>–<lpage>77</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0091-6749(87)80019-2</pub-id><pub-id pub-id-type=\"pmid\">3492525</pub-id></mixed-citation></ref><ref id=\"B62\"><label>62.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gruver-Yates</surname><given-names>AL</given-names></name><name><surname>Quinn</surname><given-names>MA</given-names></name><name><surname>Cidlowski</surname><given-names>JA</given-names></name></person-group>. <article-title>Analysis of glucocorticoid receptors and their apoptotic response to dexamethasone in male murine B cells during development</article-title>. <source>Endocrinology.</source> (<year>2014</year>) <volume>155</volume>:<fpage>463</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1210/en.2013-1473</pub-id><pub-id pub-id-type=\"pmid\">24196358</pub-id></mixed-citation></ref><ref id=\"B63\"><label>63.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lill-Elghanian</surname><given-names>D</given-names></name><name><surname>Schwartz</surname><given-names>K</given-names></name><name><surname>King</surname><given-names>L</given-names></name><name><surname>Fraker</surname><given-names>P</given-names></name></person-group>. <article-title>Glucocorticoid-induced apoptosis in early B cells from human bone marrow</article-title>. <source>Exp Biol Med.</source> (<year>2002</year>) <volume>227</volume>:<fpage>763</fpage>–<lpage>70</lpage>. <pub-id pub-id-type=\"doi\">10.1177/153537020222700907</pub-id><pub-id pub-id-type=\"pmid\">12324655</pub-id></mixed-citation></ref><ref id=\"B64\"><label>64.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Atkinson</surname><given-names>MA</given-names></name><name><surname>Bluestone</surname><given-names>JA</given-names></name><name><surname>Eisenbarth</surname><given-names>GS</given-names></name><name><surname>Hebrok</surname><given-names>M</given-names></name><name><surname>Herold</surname><given-names>KC</given-names></name><name><surname>Accili</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>How does type 1 diabetes develop?: the notion of homicide or β-cell suicide revisited</article-title>. <source>Diabetes.</source> (<year>2011</year>) <volume>60</volume>:<fpage>1370</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.2337/db10-1797</pub-id><pub-id pub-id-type=\"pmid\">21525508</pub-id></mixed-citation></ref><ref id=\"B65\"><label>65.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fichna</surname><given-names>M</given-names></name><name><surname>Fichna</surname><given-names>P</given-names></name></person-group>. <article-title>Glucocorticoids and beta-cell function</article-title>. <source>Endokrynol Pol.</source> (<year>2017</year>) <volume>68</volume>:<fpage>568</fpage>–<lpage>78</lpage>. <pub-id pub-id-type=\"doi\">10.5603/EP.2017.0060</pub-id><pub-id pub-id-type=\"pmid\">29168546</pub-id></mixed-citation></ref><ref id=\"B66\"><label>66.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rose</surname><given-names>AJ</given-names></name><name><surname>Herzig</surname><given-names>S</given-names></name></person-group>. <article-title>Metabolic control through glucocorticoid hormones: an update</article-title>. <source>Mol Cell Endocrinol.</source> (<year>2013</year>) <volume>380</volume>:<fpage>65</fpage>–<lpage>78</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.mce.2013.03.007</pub-id><pub-id pub-id-type=\"pmid\">23523966</pub-id></mixed-citation></ref><ref id=\"B67\"><label>67.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sheen</surname><given-names>JM</given-names></name><name><surname>Hsieh</surname><given-names>CS</given-names></name><name><surname>Tain</surname><given-names>YL</given-names></name><name><surname>Li</surname><given-names>SW</given-names></name><name><surname>Yu</surname><given-names>HR</given-names></name><name><surname>Chen</surname><given-names>CC</given-names></name><etal/></person-group>. <article-title>Programming effects of prenatal glucocorticoid exposure with a postnatal high-fat diet in diabetes mellitus</article-title>. <source>Int J Mol Sci.</source> (<year>2016</year>) <volume>17</volume>:<fpage>533</fpage>. <pub-id pub-id-type=\"doi\">10.3390/ijms17040533</pub-id><pub-id pub-id-type=\"pmid\">27070590</pub-id></mixed-citation></ref><ref id=\"B68\"><label>68.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sato</surname><given-names>S</given-names></name><name><surname>Saisho</surname><given-names>Y</given-names></name><name><surname>Inaishi</surname><given-names>J</given-names></name><name><surname>Kou</surname><given-names>K</given-names></name><name><surname>Murakami</surname><given-names>R</given-names></name><name><surname>Yamada</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>Effects of glucocorticoid treatment on β- and α-cell mass in Japanese adults with and without diabetes</article-title>. <source>Diabetes.</source> (<year>2015</year>) <volume>64</volume>:<fpage>2915</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.2337/db15-0151</pub-id><pub-id pub-id-type=\"pmid\">25883114</pub-id></mixed-citation></ref><ref id=\"B69\"><label>69.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fine</surname><given-names>NHF</given-names></name><name><surname>Doig</surname><given-names>CL</given-names></name><name><surname>Elhassan</surname><given-names>YS</given-names></name><name><surname>Vierra</surname><given-names>NC</given-names></name><name><surname>Marchetti</surname><given-names>P</given-names></name><name><surname>Bugliani</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Glucocorticoids reprogram b-cell signaling to preserve insulin secretion</article-title>. <source>Diabetes</source>. (<year>2018</year>) <volume>67</volume>:<fpage>278</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.2337/db16-1356</pub-id><pub-id pub-id-type=\"pmid\">29203512</pub-id></mixed-citation></ref><ref id=\"B70\"><label>70.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ghazalli</surname><given-names>N</given-names></name><name><surname>Wu</surname><given-names>X</given-names></name><name><surname>Walker</surname><given-names>S</given-names></name><name><surname>Trieu</surname><given-names>N</given-names></name><name><surname>Hsin</surname><given-names>LY</given-names></name><name><surname>Choe</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Glucocorticoid signaling enhances expression of glucose-sensing molecules in immature pancreatic beta-like cells derived from murine embryonic stem cells in vitro</article-title>. <source>Stem Cells Dev.</source> (<year>2018</year>) <volume>27</volume>:<fpage>898</fpage>–<lpage>909</lpage>. <pub-id pub-id-type=\"doi\">10.1089/scd.2017.0160</pub-id><pub-id pub-id-type=\"pmid\">29717618</pub-id></mixed-citation></ref><ref id=\"B71\"><label>71.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Santos-Silva</surname><given-names>JC</given-names></name><name><surname>da Silva</surname><given-names>PMR</given-names></name><name><surname>de Souza</surname><given-names>DN</given-names></name><name><surname>Teixeira</surname><given-names>CJ</given-names></name><name><surname>Bordin</surname><given-names>S</given-names></name><name><surname>Anhê</surname><given-names>GF</given-names></name></person-group>. <article-title>In utero exposure to dexamethasone programs the development of the pancreatic β- and α-cells during early postnatal life</article-title>. <source>Life Sci.</source> (<year>2020</year>) <volume>255</volume>:<fpage>117810</fpage>. <pub-id pub-id-type=\"doi\">10.1016/j.lfs.2020.117810</pub-id><pub-id pub-id-type=\"pmid\">32473248</pub-id></mixed-citation></ref><ref id=\"B72\"><label>72.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bansal</surname><given-names>A</given-names></name><name><surname>Alsweiler</surname><given-names>JM</given-names></name><name><surname>Oliver</surname><given-names>MH</given-names></name><name><surname>Jaquiery</surname><given-names>A</given-names></name><name><surname>Phua</surname><given-names>HH</given-names></name><name><surname>Dragunow</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Effects of preterm birth induced with or without exogenous glucocorticoids on the ovine glucose-insulin axis</article-title>. <source>Dev Orig Health Dis.</source> (<year>2020</year>) <fpage>1</fpage>–<lpage>13</lpage>. <pub-id pub-id-type=\"doi\">10.1017/S2040174419000916</pub-id><pub-id pub-id-type=\"pmid\">31937391</pub-id></mixed-citation></ref><ref id=\"B73\"><label>73.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Matteo</surname><given-names>R</given-names></name><name><surname>Hodgson</surname><given-names>DJ</given-names></name><name><surname>Bianco-Miotto</surname><given-names>T</given-names></name><name><surname>Nguyen</surname><given-names>V</given-names></name><name><surname>Owens</surname><given-names>JA</given-names></name><name><surname>Harding</surname><given-names>R</given-names></name><etal/></person-group>. <article-title>Betamethasone-exposed preterm birth does not impair insulin action in adult sheep</article-title>. <source>J Endocrinol.</source> (<year>2017</year>) <volume>232</volume>:<fpage>175</fpage>–<lpage>87</lpage>. <pub-id pub-id-type=\"doi\">10.1530/JOE-16-0300</pub-id><pub-id pub-id-type=\"pmid\">27821470</pub-id></mixed-citation></ref><ref id=\"B74\"><label>74.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zannas</surname><given-names>AS</given-names></name><name><surname>Chrousos</surname><given-names>GP</given-names></name></person-group>. <article-title>Epigenetic programming by stress and glucocorticoids along the human lifespan</article-title>. <source>Mol Psychiatry.</source> (<year>2017</year>) <volume>22</volume>:<fpage>640</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1038/mp.2017.35</pub-id><pub-id pub-id-type=\"pmid\">28289275</pub-id></mixed-citation></ref><ref id=\"B75\"><label>75.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gilbert</surname><given-names>ER</given-names></name><name><surname>Liu</surname><given-names>D</given-names></name></person-group>. <article-title>Epigenetics the missing link to understanding β-cell dysfunction in the pathogenesis of type 2 diabetes</article-title>. <source>Epigenetics.</source> (<year>2012</year>) <volume>7</volume>:<fpage>841</fpage>–<lpage>52</lpage>. <pub-id pub-id-type=\"doi\">10.4161/epi.21238</pub-id><pub-id pub-id-type=\"pmid\">22810088</pub-id></mixed-citation></ref><ref id=\"B76\"><label>76.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Seckl</surname><given-names>JR</given-names></name></person-group>. <article-title>Prenatal glucocorticoids and long-term programming</article-title>. <source>Eur J Endocrinol</source>. (<year>2004</year>) <volume>151</volume>(<issue>Suppl. 3</issue>):<fpage>U49</fpage>–<lpage>62</lpage>. <pub-id pub-id-type=\"doi\">10.1530/eje.0.151u049</pub-id><pub-id pub-id-type=\"pmid\">15554887</pub-id></mixed-citation></ref><ref id=\"B77\"><label>77.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kelly</surname><given-names>BA</given-names></name><name><surname>Lewandowski</surname><given-names>AJ</given-names></name><name><surname>Worton</surname><given-names>SA</given-names></name><name><surname>Davis</surname><given-names>EF</given-names></name><name><surname>Lazdam</surname><given-names>M</given-names></name><name><surname>Francis</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Antenatal glucocorticoid exposure and long-term alterations in aortic function and glucose metabolism</article-title>. <source>Pediatrics.</source> (<year>2012</year>) <volume>129</volume>:<fpage>e1282</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1542/peds.2011-3175</pub-id><pub-id pub-id-type=\"pmid\">22508917</pub-id></mixed-citation></ref><ref id=\"B78\"><label>78.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Norberg</surname><given-names>H</given-names></name><name><surname>Stålnacke</surname><given-names>J</given-names></name><name><surname>Nordenström</surname><given-names>A</given-names></name><name><surname>Norman</surname><given-names>M</given-names></name></person-group>. <article-title>Repeat antenatal steroid exposure and later blood pressure, arterial stiffness, and metabolic profile</article-title>. <source>J Pediatr.</source> (<year>2013</year>) <volume>163</volume>:<fpage>711</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jpeds.2013.03.074</pub-id><pub-id pub-id-type=\"pmid\">23651768</pub-id></mixed-citation></ref><ref id=\"B79\"><label>79.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Federman</surname><given-names>DG</given-names></name><name><surname>Froelich</surname><given-names>CW</given-names></name><name><surname>Kirsner</surname><given-names>RS</given-names></name></person-group>. <article-title>Topical psoriasis therapy</article-title>. <source>Am Fam Physician.</source> (<year>1999</year>) <volume>59</volume>:<fpage>957</fpage>–<lpage>62</lpage>. <pub-id pub-id-type=\"pmid\">10068717</pub-id></mixed-citation></ref><ref id=\"B80\"><label>80.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hench</surname><given-names>PS</given-names></name><name><surname>Kendall</surname><given-names>EC</given-names></name></person-group>. <article-title>The effect of a hormone of the adrenal cortex (17-hydroxy-11-dehydrocorticosterone; compound E) and of pituitary adrenocorticotropic hormone on rheumatoid arthritis</article-title>. <source>Proc Staff Meet Mayo Clin.</source> (<year>1949</year>) <volume>24</volume>:<fpage>181</fpage>–<lpage>97</lpage>. <pub-id pub-id-type=\"pmid\">18118071</pub-id></mixed-citation></ref><ref id=\"B81\"><label>81.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Walker</surname><given-names>LSK</given-names></name><name><surname>von Herrath</surname><given-names>M</given-names></name></person-group>. <article-title>CD4 T cell differentiation in type 1 diabetes</article-title>. <source>Clin Exp Immunol.</source> (<year>2016</year>) <volume>183</volume>:<fpage>16</fpage>–<lpage>29</lpage>. <pub-id pub-id-type=\"doi\">10.1111/cei.12672</pub-id><pub-id pub-id-type=\"pmid\">26102289</pub-id></mixed-citation></ref><ref id=\"B82\"><label>82.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hong</surname><given-names>JY</given-names></name><name><surname>Lim</surname><given-names>J</given-names></name><name><surname>Carvalho</surname><given-names>F</given-names></name><name><surname>Cho</surname><given-names>JY</given-names></name><name><surname>Vaidyanathan</surname><given-names>B</given-names></name><name><surname>Yu</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Long-term programming of CD8 T cell immunity by perinatal exposure to glucocorticoids</article-title>. <source>Cell.</source> (<year>2020</year>) <volume>180</volume>:<fpage>847</fpage>–<lpage>61.e15</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cell.2020.02.018</pub-id><pub-id pub-id-type=\"pmid\">32142678</pub-id></mixed-citation></ref><ref id=\"B83\"><label>83.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Korhonen</surname><given-names>LS</given-names></name><name><surname>Kortesluoma</surname><given-names>S</given-names></name><name><surname>Lukkarinen</surname><given-names>M</given-names></name><name><surname>Peltola</surname><given-names>V</given-names></name><name><surname>Pesonen</surname><given-names>H</given-names></name><name><surname>Pelto</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Prenatal maternal distress associates with a blunted cortisol response in rhinovirus-positive infants</article-title>. <source>Psychoneuroendocrinology.</source> (<year>2019</year>) <volume>107</volume>:<fpage>187</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.psyneuen.2019.05.023</pub-id><pub-id pub-id-type=\"pmid\">31146139</pub-id></mixed-citation></ref><ref id=\"B84\"><label>84.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vermillion</surname><given-names>ST</given-names></name><name><surname>Soper</surname><given-names>DE</given-names></name><name><surname>Newman</surname><given-names>RB</given-names></name></person-group>\n<article-title>Neonatal sepsis and death after multiple courses of antenatal betamethasone therapy</article-title>. <source>Am J Obstetr Gynecol.</source> (<year>2000</year>) <volume>183</volume>:<fpage>810</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1067/mob.2000.108838</pub-id></mixed-citation></ref><ref id=\"B85\"><label>85.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>YC</given-names></name><name><surname>Tseng</surname><given-names>HI</given-names></name><name><surname>Yang</surname><given-names>SN</given-names></name><name><surname>Lu</surname><given-names>CC</given-names></name><name><surname>Wu</surname><given-names>JR</given-names></name><name><surname>Dai</surname><given-names>ZK</given-names></name><etal/></person-group>. <article-title>Effects of antenatal corticosteroids on neonatal outcomes in very-low-birth-weight preterm newborns: a 10-year retrospective study in a medical center</article-title>. <source>Pediatr Neonatol.</source> (<year>2012</year>) <volume>53</volume>:<fpage>178</fpage>–<lpage>83</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.pedneo.2012.04.004</pub-id><pub-id pub-id-type=\"pmid\">22770106</pub-id></mixed-citation></ref><ref id=\"B86\"><label>86.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dalziel</surname><given-names>SR</given-names></name><name><surname>Walker</surname><given-names>NK</given-names></name><name><surname>Parag</surname><given-names>V</given-names></name><name><surname>Mantell</surname><given-names>C</given-names></name><name><surname>Rea</surname><given-names>HH</given-names></name><name><surname>Rodgers</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Cardiovascular risk factors after antenatal exposure to betamethasone: 30-Year follow-up of a randomised controlled trial</article-title>. <source>Lancet.</source> (<year>2005</year>) <volume>365</volume>:<fpage>1856</fpage>–<lpage>62</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0140-6736(05)66617-2</pub-id><pub-id pub-id-type=\"pmid\">15924982</pub-id></mixed-citation></ref><ref id=\"B87\"><label>87.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Greene</surname><given-names>NH</given-names></name><name><surname>Pedersen</surname><given-names>LH</given-names></name><name><surname>Liu</surname><given-names>S</given-names></name><name><surname>Olsen</surname><given-names>J</given-names></name></person-group>\n<article-title>Cohort profile: prenatal prescription corticosteroids and offspring diabetes: a national cohort study</article-title>. <source>Int J Epidemiol.</source> (<year>2013</year>) <volume>42</volume>:<fpage>186</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1093/ije/dys228</pub-id><pub-id pub-id-type=\"pmid\">23382365</pub-id></mixed-citation></ref><ref id=\"B88\"><label>88.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bloomfield</surname><given-names>FH</given-names></name></person-group>. <article-title>Impact of prematurity for pancreatic islet and beta-cell development</article-title>. <source>J Endocrinol.</source> (<year>2018</year>) <volume>238</volume>:<fpage>R161</fpage>–<lpage>71</lpage>. <pub-id pub-id-type=\"doi\">10.1530/JOE-18-0021</pub-id><pub-id pub-id-type=\"pmid\">29895718</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"brief-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Endocrinol.</journal-id><journal-title-group><journal-title>Frontiers in Endocrinology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2392</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849274</article-id><article-id pub-id-type=\"pmc\">PMC7431598</article-id><article-id pub-id-type=\"doi\">10.3389/fendo.2020.00481</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology</subject><subj-group><subject>Perspective</subject></subj-group></subj-group></article-categories><title-group><article-title>Collaboration Around Rare Bone Diseases Leads to the Unique Organizational Incentive of the Amsterdam Bone Center</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Eekhoff</surname><given-names>Elisabeth M. W.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/660498/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Micha</surname><given-names>Dimitra</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Forouzanfar</surname><given-names>Tymour</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>de Vries</surname><given-names>Teun J.</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/466360/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Netelenbos</surname><given-names>J. Coen</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Klein-Nulend</surname><given-names>Jenneke</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/791720/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>van Loon</surname><given-names>Jack J. W. A.</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/265078/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Lubbers</surname><given-names>Wouter D.</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Schwarte</surname><given-names>Lothar</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Schober</surname><given-names>Patrick</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/963502/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Raijmakers</surname><given-names>Pieter G. H. M.</given-names></name><xref ref-type=\"aff\" rid=\"aff7\"><sup>7</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Teunissen</surname><given-names>Bernd P.</given-names></name><xref ref-type=\"aff\" rid=\"aff7\"><sup>7</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1034058/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>de Graaf</surname><given-names>Pim</given-names></name><xref ref-type=\"aff\" rid=\"aff7\"><sup>7</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Lammertsma</surname><given-names>Adriaan A.</given-names></name><xref ref-type=\"aff\" rid=\"aff7\"><sup>7</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/67717/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Yaqub</surname><given-names>Maqsood M.</given-names></name><xref ref-type=\"aff\" rid=\"aff7\"><sup>7</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Botman</surname><given-names>Esmée</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/842605/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Treurniet</surname><given-names>Sanne</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/898965/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Smilde</surname><given-names>Bernard J.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/916754/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Bökenkamp</surname><given-names>Arend</given-names></name><xref ref-type=\"aff\" rid=\"aff8\"><sup>8</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Boonstra</surname><given-names>Anco</given-names></name><xref ref-type=\"aff\" rid=\"aff9\"><sup>9</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Kamp</surname><given-names>Otto</given-names></name><xref ref-type=\"aff\" rid=\"aff10\"><sup>10</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Nieuwenhuijzen</surname><given-names>Jakko A.</given-names></name><xref ref-type=\"aff\" rid=\"aff11\"><sup>11</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1031958/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Visser</surname><given-names>Marieke C.</given-names></name><xref ref-type=\"aff\" rid=\"aff12\"><sup>12</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Baayen</surname><given-names>Hans J. C.</given-names></name><xref ref-type=\"aff\" rid=\"aff13\"><sup>13</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Dahele</surname><given-names>Max</given-names></name><xref ref-type=\"aff\" rid=\"aff14\"><sup>14</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Eeckhout</surname><given-names>Guus A. M.</given-names></name><xref ref-type=\"aff\" rid=\"aff15\"><sup>15</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Goderie</surname><given-names>Thadé P. M.</given-names></name><xref ref-type=\"aff\" rid=\"aff16\"><sup>16</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1032843/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Smits</surname><given-names>Cas</given-names></name><xref ref-type=\"aff\" rid=\"aff17\"><sup>17</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Gilijamse</surname><given-names>Marjolijn</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1032582/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Karagozoglu</surname><given-names>K. Hakki</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>van de Valk</surname><given-names>Paul</given-names></name><xref ref-type=\"aff\" rid=\"aff18\"><sup>18</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Dickhoff</surname><given-names>Chris</given-names></name><xref ref-type=\"aff\" rid=\"aff19\"><sup>19</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1033140/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Moll</surname><given-names>Annette C.</given-names></name><xref ref-type=\"aff\" rid=\"aff20\"><sup>20</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Verbraak</surname><given-names>Frank F. D.</given-names></name><xref ref-type=\"aff\" rid=\"aff20\"><sup>20</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Curro-Tafili</surname><given-names>Katie K. R.</given-names></name><xref ref-type=\"aff\" rid=\"aff20\"><sup>20</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ghyczy</surname><given-names>Ebba A. E.</given-names></name><xref ref-type=\"aff\" rid=\"aff20\"><sup>20</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Rustemeyer</surname><given-names>Thomas</given-names></name><xref ref-type=\"aff\" rid=\"aff21\"><sup>21</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Saeed</surname><given-names>Peeroz</given-names></name><xref ref-type=\"aff\" rid=\"aff22\"><sup>22</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Maugeri</surname><given-names>Alessandra</given-names></name><xref ref-type=\"aff\" rid=\"aff23\"><sup>23</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Pals</surname><given-names>Gerard</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ridwan-Pramana</surname><given-names>Angela</given-names></name><xref ref-type=\"aff\" rid=\"aff24\"><sup>24</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Pekel</surname><given-names>Esther</given-names></name><xref ref-type=\"aff\" rid=\"aff25\"><sup>25</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Schoenmaker</surname><given-names>Ton</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/563024/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Lems</surname><given-names>Willem</given-names></name><xref ref-type=\"aff\" rid=\"aff26\"><sup>26</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Winters</surname><given-names>Henri A. H.</given-names></name><xref ref-type=\"aff\" rid=\"aff27\"><sup>27</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Botman</surname><given-names>Matthijs</given-names></name><xref ref-type=\"aff\" rid=\"aff27\"><sup>27</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Giannakópoulos</surname><given-names>Georgios F.</given-names></name><xref ref-type=\"aff\" rid=\"aff28\"><sup>28</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Koolwijk</surname><given-names>Peter</given-names></name><xref ref-type=\"aff\" rid=\"aff29\"><sup>29</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Janssen</surname><given-names>Jeroen J. W. M.</given-names></name><xref ref-type=\"aff\" rid=\"aff30\"><sup>30</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Kloen</surname><given-names>Peter</given-names></name><xref ref-type=\"aff\" rid=\"aff31\"><sup>31</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/990214/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Bravenboer</surname><given-names>Nathalie</given-names></name><xref ref-type=\"aff\" rid=\"aff32\"><sup>32</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/637849/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Smit</surname><given-names>Jan Maerten</given-names></name><xref ref-type=\"aff\" rid=\"aff27\"><sup>27</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Helder</surname><given-names>Marco N.</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/813425/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Amsterdam UMC, Department of Internal Medicine Section Endocrinology, Amsterdam Bone Center, Amsterdam Movement Sciences</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Amsterdam UMC, Department of Clinical Genetics, Amsterdam Bone Center, Amsterdam Movement Sciences</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Amsterdam UMC, Department of Oral and MaxilloFacial Surgery/Oral Pathology, Amsterdam Bone Center, Amsterdam Movement Sciences</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff6\"><sup>6</sup><institution>Amsterdam UMC, Department of Anaesthesiology</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff7\"><sup>7</sup><institution>Amsterdam UMC, Department of Radiology and Nuclear Medicine</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff8\"><sup>8</sup><institution>Amsterdam UMC, Emma Children's Hospital, Vrije Universiteit Amsterdam, Department of Pediatric Nephrology</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff9\"><sup>9</sup><institution>Amsterdam UMC, Department of Pulmonology</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff10\"><sup>10</sup><institution>Amsterdam UMC, Department of Cardiology</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff11\"><sup>11</sup><institution>Amsterdam UMC, Department of Urology</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff12\"><sup>12</sup><institution>Amsterdam UMC, Department of Neurology</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff13\"><sup>13</sup><institution>Amsterdam UMC, Department of Neurosurgery</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff14\"><sup>14</sup><institution>Amsterdam UMC, Department of Radiation Oncology</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff15\"><sup>15</sup><institution>Amsterdam UMC, Department Psychiatry</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff16\"><sup>16</sup><institution>Amsterdam UMC, Department of Otolaryngology—Head and Neck Surgery, Ear and Hearing</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff17\"><sup>17</sup><institution>Amsterdam UMC, Department of Otolaryngology—Head and Neck Surgery, Ear and Hearing, Amsterdam Public Health Research Institute</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff18\"><sup>18</sup><institution>Amsterdam UMC, Department of Pathology</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff19\"><sup>19</sup><institution>Amsterdam UMC, Thoracic and Endocrine Surgery, Department of Surgery and Cardiothoracic Surgery, Cancer Center Amsterdam</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff20\"><sup>20</sup><institution>Amsterdam UMC, AMC, Department of Ophtalmology</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff21\"><sup>21</sup><institution>Amsterdam UMC, Department of Dermatology</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff22\"><sup>22</sup><institution>Amsterdam UMC, Department of Ophtalmology</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff23\"><sup>23</sup><institution>Amsterdam UMC, Department of Clinical Genetics, Amsterdam Bone Center</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff24\"><sup>24</sup><institution>Amsterdam UMC, Dentistry and Prosthodontics Department of Oral and MaxilloFacial Surgery/Oral Pathology, Special Dentistry Foundation</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff25\"><sup>25</sup><institution>Amsterdam UMC, Department of Dietetics</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff26\"><sup>26</sup><institution>Amsterdam UMC, Department of Reumatology</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff27\"><sup>27</sup><institution>Amsterdam UMC, Department of Plastic, Reconstructive and Hand Surgery, Amsterdam Bone Center</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff28\"><sup>28</sup><institution>Amsterdam UMC, Department of Trauma Surgery</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff29\"><sup>29</sup><institution>Amsterdam UMC, Department of Physiology, Amsterdam Cardiovascular Science</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff30\"><sup>30</sup><institution>Amsterdam UMC, Department of Hematology</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff31\"><sup>31</sup><institution>Amsterdam UMC, Department of Orthopaedic Surgery</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff32\"><sup>32</sup><institution>Amsterdam UMC, Department of Clinical Chemistry, Amsterdam Bone Center, Amsterdam Movement Sciences</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Elaine Dennison, MRC Lifecourse Epidemiology Unit (MRC), United Kingdom</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: David M. Findlay, University of Adelaide, Australia; Monica De Mattei, University of Ferrara, Italy</p></fn><corresp id=\"c001\">*Correspondence: Elisabeth M. W. Eekhoff <email>emw.eekhoff@amsterdamumc.nl</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Bone Research, a section of the journal Frontiers in Endocrinology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>481</elocation-id><history><date date-type=\"received\"><day>23</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>17</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Eekhoff, Micha, Forouzanfar, de Vries, Netelenbos, Klein-Nulend, van Loon, Lubbers, Schwarte, Schober, Raijmakers, Teunissen, de Graaf, Lammertsma, Yaqub, Botman, Treurniet, Smilde, Bökenkamp, Boonstra, Kamp, Nieuwenhuijzen, Visser, Baayen, Dahele, Eeckhout, Goderie, Smits, Gilijamse, Karagozoglu, van de Valk, Dickhoff, Moll, Verbraak, Curro-Tafili, Ghyczy, Rustemeyer, Saeed, Maugeri, Pals, Ridwan-Pramana, Pekel, Schoenmaker, Lems, Winters, Botman, Giannakópoulos, Koolwijk, Janssen, Kloen, Bravenboer, Smit and Helder.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Eekhoff, Micha, Forouzanfar, de Vries, Netelenbos, Klein-Nulend, van Loon, Lubbers, Schwarte, Schober, Raijmakers, Teunissen, de Graaf, Lammertsma, Yaqub, Botman, Treurniet, Smilde, Bökenkamp, Boonstra, Kamp, Nieuwenhuijzen, Visser, Baayen, Dahele, Eeckhout, Goderie, Smits, Gilijamse, Karagozoglu, van de Valk, Dickhoff, Moll, Verbraak, Curro-Tafili, Ghyczy, Rustemeyer, Saeed, Maugeri, Pals, Ridwan-Pramana, Pekel, Schoenmaker, Lems, Winters, Botman, Giannakópoulos, Koolwijk, Janssen, Kloen, Bravenboer, Smit and Helder</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>In the field of rare bone diseases in particular, a broad care team of specialists embedded in multidisciplinary clinical and research environment is essential to generate new therapeutic solutions and approaches to care. Collaboration among clinical and research departments within a University Medical Center is often difficult to establish, and may be hindered by competition and non-equivalent cooperation inherent in a hierarchical structure. Here we describe the “collaborative organizational model” of the Amsterdam Bone Center (ABC), which emerged from and benefited the rare bone disease team. This team is often confronted with pathologically complex and under-investigated diseases. We describe the benefits of this model that still guarantees the autonomy of each team member, but combines and focuses our collective expertise on a clear shared goal, enabling us to capture synergistic and innovative opportunities for the patient, while avoiding self-interest and possible harmful competition.</p></abstract><kwd-group><kwd>rare bone diseases</kwd><kwd>amsterdam bone center (ABC)</kwd><kwd>collaborative organization</kwd><kwd>non-hierarchical</kwd><kwd>research</kwd><kwd>clinical</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Vrije Universiteit Amsterdam<named-content content-type=\"fundref-id\">10.13039/501100001833</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"3\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"24\"/><page-count count=\"7\"/><word-count count=\"4396\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Rare bone diseases (RBD) have, until recently, been a largely neglected area in healthcare. Their rarity and heterogeneity have unfortunately hindered their exploration at both clinical and scientific levels, even though more than 500 of the ~7,000 rare diseases are bone disorders (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B2\" ref-type=\"bibr\">2</xref>). The estimated incidence of RBD can vary, from around 15.7/100000 births for skeletal dysplasias (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>) which are the most common, to ultra-rare disorders of which only a few patients exist in the world, such as spondylo-ocular syndrome (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). However, in the last decade, the urgency to study and treat RBD has been boosted by the greater appreciation of the socioeconomic consequences associated with their chronic nature and severity, and by the wider availability of genetic diagnostics, patient advocacy, and the development of new pharmaceutical treatment options.</p><p>The focus of the Amsterdam UMC initially included the rare bone diseases (RBD) fibrodysplasia ossificans progressiva, osteogenesis imperfecta, fibrous dysplasia and hereditary osteoporosis, but encountered several obstacles. RBD are often extremely challenging to treat; clinical decisions are hindered by their complexity and lack of knowledge about their underlying pathology. Because standard treatment protocols do not exist for RBD, and off-label medications are typically required, a broad team of medical specialists is needed to design the right treatment approach for the individual patients. Ideally, because these diseases are so rare, such a team would be embedded in a multidisciplinary academic setting to facilitate urgently needed clinical and preclinical research. This provides access to research-oriented colleagues who have knowledge and affinity with relevant RBD and increases the likelihood of new insights and scientific breakthroughs to ultimate benefit the patients. Critical to maximizing progress is full collaboration between many different disciplines in a structure, where not only clinicians, but also clinical and basic researchers can efficiently interact across specialities and facilities. Such team structures and broad collaborative networks can be challenging to set up in academic centers due to other interests, competition, or non-equivalent cooperation (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>).</p><sec><title>Collaborative Organizational Model</title><p>Different opinions exist about the ideal organizational structure to facilitate successful cooperation of professionals from a wide variety of disciplines. Nonetheless, in most medical and research organizations, the traditional hierarchical pyramid still dominates. Such rigidly structured organizations that are managed “top-down” often fail to provide an optimum environment for self-motivation, creativity, engagement, and empathy, all important requirements for effective collaboration amongst colleagues (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>–<xref rid=\"B10\" ref-type=\"bibr\">10</xref>).</p><p>An alternative approach supports a less rigid hierarchy and the promotion of organic development of collaboration between colleagues in a culture of equality (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>–<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Fundamental to this is the recognition of the specific and complementary skills of each individual team member. There is increasing support of the idea that teams containing like-minded people with mutual and aligned interests can provide the basis for transparent, fair, and fruitful collaboration. Organizational models like this can achieve shared goals by stimulating an engaged, unforced and valued workforce mentality, in which individuality and freedom to show initiative is safeguarded (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>–<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). In such a model the aim is not the integration of all departments but an efficient collaboration between relevant partners driven by their balanced skills that are required to solve specific clinical or research questions. The overall goal is to improve patient care and to stimulate innovative research. The process is further enhanced by the critical input of patients in care and research. This kind of model is referred to in the literature as “collaborative organization,” and is considered an effective means of advancing both efficiency and innovation (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>–<xref rid=\"B10\" ref-type=\"bibr\">10</xref>).</p></sec></sec><sec id=\"s2\"><title>Amsterdam Bone Center</title><p>The Amsterdam Bone Center (ABC) was formed in late 2016, as a successful example of such “collaborative organizational model.” The ABC was an initiative of various clinical disciplines and researchers who wanted to pool their specialized skills, knowledge and experience across boundaries and their day to day scope, with the common goal of achieving new approaches to the diagnosis, care, and effective treatment of patients with RBD. Most of RBD treatment is still based on generic medical protocols which provide symptomatic relief, but effective future therapies that result in the recovery of the affected tissue will need detailed understanding of the underlying disease pathology, which is a challenging task. As a consequence, the ABC was initially focussed on RBD. Although the number of patients affected by some diseases was very limited, the level of required adapted complex care was very high. This resulted in extensive networking with many clinical departments such as plastic surgery, maxillofacial surgery, orthopedic surgery, thoracic surgery, traumatology, anaesthesiology, rehabilitation, urology, ear nose and throat surgery, audiology, ophthalmology, clinical genetics, rehabilitation, psychiatry, physiotherapy, social work, dietetics, gypsum master, cardiology, lung disease, nuclear medicine, radiology, neurology, neurosurgery, dermatology, radiotherapy, gastroenterology, endocrinology, pediatrics, rheumatology, and dentistry. In addition, the patient organizations have been actively involved. The multidisciplinary collaboration has been based on equality.</p><p>The ABC subsequently developed as a flat organization, where mutual interest, exchange of knowledge, and innovation have led to a vivid open collaboration between clinicians and researchers.</p><p>The ABC provides a bridge between clinicians and research laboratories whose partners are embedded in the Amsterdam Movement Sciences research institute, the latter of which embraces the targeted laboratories specializing in multifaceted aspects of research on bone tissue, dentition and the surrounding tissues. In this way, it connects expert groups focussed on osteocytes (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>), osteoblasts (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>), osteoclasts (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>), bone matrix formation (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>), and angiogenesis (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>), facilitating the study of bone differentiation and regeneration. With the aid of appropriate cell collection from RBD and control tissues, complex processes can be studied and interpreted in the physiological and pathological context. “Meet the expert” RBD sessions and annual RBD meetings help to keep the patients informed about the current research and progress. ABC education activities also extend to academic training at the bachelor, master and doctorate level by which enthusiasm for rare bone diseases is promoted in talented young professionals.</p></sec><sec id=\"s3\"><title>Management of the ABC</title><p>In place of the more typical hierarchical model in which all control is centralized to a Director, the ABC operates with a facilitating steering team, with one member in rotation functioning as the ABC chairman. The chairman conveys the consensus goals, ambitions, and decisions of the team. The different cultures and perspectives of the various collaborating departments are reflected in a steering team of four coordinators from the task force group, consisting of 2 preclinical theme leaders (from the Laboratory for Bone Metabolism of the Department of Clinical Chemistry and Cell Technology Laboratory of the Department of Oral and Maxillofacial Surgery) and 2 clinical theme leaders (from the Department of Internal Medicine section Endocrinology and the Department of Plastic Surgery). Steering team members are elected to their role for 2 years, based on their proven commitment to the ABC and their activities in promoting its interests.</p><p>In addition to the leading steering team, there is a task force which includes representatives from clinical and pre-clinical groups. These representatives are responsible for promoting their key themes [e.g., key themes are presently RBD, inflammatory bone diseases and bone oncology, complex fractures, and complex surrounding tissue injuries (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>)]. The task force comes together in brainstorm sessions to translate critical clinical questions into structured preclinical research lines, and move preclinical findings into the clinical environment. In this organizational model, the coordinating task force is not focused on safeguarding its own structure, but on leveraging its diverse expertise to drive adoption of new ideas across the ABC members, identify scientific gaps, support the finding of solutions, enhance ABC connectivity and crosstalk between themes where possible, give direction to future common goals, support optimal clinical care for patients, and provide high quality education and research.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>The organizational structure of the ABC with the main themes and partnerships. ABC, Amsterdam Bone Center; Max Fac, maxillo facial; Ortho-trauma, orthopedic surgery, traumatology; Int-endo, internal medicine, endocrinology; Radiol, radiology.</p></caption><graphic xlink:href=\"fendo-11-00481-g0001\"/></fig><p>An annual symposium will ensure that all groups working in bone research and clinics in the ABC can benefit and easily collaborate in an ideal setup for research and care. Yearly goals are suggested and proposed to the ABC community in these symposia, and subsequently set and evaluated by the task force based on extensive feedback. The ultimate goal is to become further embedded in a larger international network of centers for bone research in general, and RBD in particular, in order to meaningfully help patients and offer innovative diagnostics, to develop treatment options and recovery solutions for RBD and related bone diseases. The task force also monitors whether the activities of the leading steering team align with the goals of the wider ABC community. The obvious advantage of this lateral (“flat”) organization is that groups retain the freedom to pursue their own research choices, but they are encouraged to reach the best joint benefit.</p></sec><sec id=\"s4\"><title>Focus of RBD Within the ABC</title><p>The focus of the RBD theme of ABC was initially placed on four RBD, including fibrodysplasia ossificans progressiva (FOP) (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>–<xref rid=\"B19\" ref-type=\"bibr\">19</xref>), osteogenesis imperfecta (OI) (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>, <xref rid=\"B21\" ref-type=\"bibr\">21</xref>), fibrous dysplasia (FD) with an emphasis on skull (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>), and hereditary osteoporosis (her. OP) (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). This, repertoire was strategically composed based on the various clinical and research expertise available and on the possibility to match underlying etiology and clinical questions. A schematic overview of the differences and common ground of these RBD is given in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>. Based on this, it is clear that these diseases can serve as a paradigm for other RBD sharing a similar pathology, but also provide insight into general bone pathology.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Four genetic rare bone diseases that all dysregulate osteogenesis but in a different phase and on a different process in the cell. The arrow indicates the direction of the stem cell/fibroblast toward the development of osteocytes. FOP, fibrodysplasia ossificans progressiva; FD, fibrous dysplasia; OI, osteogenesis imperfecta; Her. OP, hereditary osteoporosis; ACVR1, activin A receptor 1; c-AMP, Cyclic adenosine monophosphate; COL, collagen; PLS, plastin; Ca, calcium.</p></caption><graphic xlink:href=\"fendo-11-00481-g0002\"/></fig></sec><sec id=\"s5\"><title>Achievements on RBD Within the ABC</title><p>A standardized approach to patient care of the four RBD is developed with the relevant clinical disciplines and patient organizations to create a patient-centered design. This has led to the implementation of a standardized route for care; its integration in numerous specialities is designed to thoroughly address all pathological aspects of each RBD (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). As a spin-off of the ABC structure, the RBD team has become an international referral center for FOP and it coordinates international studies on FOP, OI and hereditary OP, and FD.</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Patient Care Rare Bone Disease ABC, Amsterdam UMC. FOP, fibrodysplasia ossificans progressiva; FD, fibrous dysplasia; OI, osteogenesis imperfecta; Her-OP, hereditary osteoporosis; ABC, Amsterdam bone Center; DXA, Dual X-ray absorptiometry; [18F] NaF PET/CT, <sup>18</sup>F-Sodium Fluoride positron emission tomograph/-computed tomography; MRI, Magnetic resonance imaging; MDO, multidisciplinary consultation.</p></caption><graphic xlink:href=\"fendo-11-00481-g0003\"/></fig><p>Several preclinical research models have been developed to study the various RBD, including the culture of subdermal (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>) and periodontal ligament fibroblasts (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>) which can be converted to cartilage and bone-forming cells, or can be drivers for osteoclast formation (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B24\" ref-type=\"bibr\">24</xref>); this provides unique insight in rare bone diseases that primarily focuses on additional bone formation rather than affected bone degradation. Many signaling pathways for cartilage and osteogenic differentiation are reflected in these models, which facilitates their study in easily obtainable patient tissue. This collaboration has yielded the discovery of newly discovered genes for these RBD (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>, <xref rid=\"B23\" ref-type=\"bibr\">23</xref>); the investigation of their mechanism can help to shed light in possible therapeutic implications. The collaborative efforts have also led to innovative diagnostics, one example of which is a new modality for imaging of active heterotopic bone lesions in FOP patients with <sup>18</sup>F PET/CT (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>–<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Other advances include the development of new clinical trials with existing and new medications, translational projects on pharmacological therapy for RBD, and the development of new technology to quantify osteoclast activity <italic>ex vivo</italic>. The development of the RBD theme within the ABC structure has led to an increasing number of pre- and clinical scholarships, awarded from Amsterdam UMC AMS as well as other international universities, patient associations, and national and European funding organizations, in collaboration with pharmaceutical/industrial companies. This supports a rapidly developing academic trajectory resulting in many Ph.D. projects and dissertations.</p></sec><sec id=\"s6\"><title>Future Plans of the ABC</title><p>Regarding the future treatment of RBD, Regenerative Medicine (RM) is one of the main research priorities of the ABC. This specific focus within ABC aims meaningful repair/regeneration by exploiting the plasticity of the body's own cells. This requires extensive knowledge of the pathological mechanism of the disease extending from molecular interactions at the cellular level, to the influence of and inter-relationship with the surrounding tissues and other systemic factors. The aforementioned preclinical RBD models and findings can potentially be integrated with RM strategies in order to achieve synergy in disease control and tissue regeneration. This specific expertise within ABC extends to more prevalent bone disorders which are genetically less well-defined but which nonetheless may also benefit from therapeutic developments on RBD; these may include multifactorial osteoporosis, and immune-related bone diseases. The ultimate goal is to establish a regeneration center based on the development of new pathophysiological models for the realization of individualized treatment and prevention. In addition, ongoing future plans include the development of orthoplastic centers and the expansion of our network to more national, European and international collaborators outside the Amsterdam UMC.</p><p>In conclusion, this article we have outlined the establishment and development of the Amsterdam Bone Center, where “collaborative organization” encourages the cooperation of all relevant clinic and research teams. Specifically, we have successfully established a patient-centered, multidisciplinary focus on RBD including the development of targeted innovative diagnostics, clinical and research protocols and studies. Recognition of the different cultures and perspectives of the departments represented in the ABC, shared collaborative leadership, and a diverse and well-functioning task force is critical to maintaining a balanced and successful collaboration that advances science and innovation, and improves patient care.</p><p>Knowledge of this model may be useful to other organizations aiming to establish or enhance the growth of clinical-academic collaboration.</p></sec><sec sec-type=\"data-availability\" id=\"s7\"><title>Data Availability Statement</title><p>All datasets presented in this study are included in the article/supplementary material.</p></sec><sec id=\"s8\"><title>Author Contributions</title><p>This article on a unique collaborative, interdisciplinary concept in medical research arising from the rare bone diseases pillar of the Amsterdam Bone Centre was initiated by EE with contributions to all crude versions from DM, TF, TV, JCN, JK-N, JL, PKl, NB, JS, and MH. The subfinal version was prepared and sanctioned by this core group of authors. All remaining authors WDL, LS, PS, PR, BT, PG, AL, MY, EB, ST, BS, ABö, ABoo, OK, JAN, MV, HB, MD, GE, TG, CS, MG, KK, PV, CD, ACM, FV, KC-T, EG, TR, PS, AM, GP, AR-P, EP, TS, WL, HW, MB, GG, PKo, and JJ are active ABC members and have contributed for many years on rare bone diseases and to the success of this unique collaborative initiative. All these authors have had the opportunity to further improve the manuscript, these comments were incorporated by EE and DM in the submitted version.</p></sec><sec id=\"s9\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with one of the authors WL.</p></sec></body><back><ack><p>We are grateful for the good collaboration with the department of rehabilitation (Louise Sabelis, M.D. Ph.D.), the department of gastroenterology (M.A.J.M. Jacobs M.D., Ph.D.), in addition to all other collaborative partners of the Amsterdam UMC, AMS, ACTA as national and international partners, who are large in number and hopefully can participate in a next article. Our special gratefulness goes to the patients and patient organizations of all RBDs for their collaboration and important critical participation.</p></ack><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haendel</surname><given-names>M</given-names></name><name><surname>Vasilevsky</surname><given-names>N</given-names></name><name><surname>Unni</surname><given-names>D</given-names></name><name><surname>Bologa</surname><given-names>C</given-names></name><name><surname>Harris</surname><given-names>N</given-names></name><name><surname>Rehm</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>How many rare diseases are there?</article-title>\n<source>Nat Rev Drug Discov.</source> (<year>2020</year>) <volume>19</volume>:<fpage>77</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1038/d41573-019-00180-y</pub-id><pub-id pub-id-type=\"pmid\">32020066</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McCarthy</surname><given-names>EF</given-names></name></person-group>. <article-title>Genetic diseases of bones and joints</article-title>. <source>Semin Diagn Pathol</source>. (<year>2011</year>) <volume>28</volume>:<fpage>26</fpage>–<lpage>36</lpage>. <pub-id pub-id-type=\"doi\">10.1053/j.semdp.2011.01.004</pub-id><pub-id pub-id-type=\"pmid\">21675375</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bonafe</surname><given-names>L</given-names></name><name><surname>Cormier-Daire</surname><given-names>V</given-names></name><name><surname>Hall</surname><given-names>C</given-names></name><name><surname>Lachman</surname><given-names>R</given-names></name><name><surname>Mortier</surname><given-names>G</given-names></name><name><surname>Mundlos</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Nosology and classification on genetic skeletal disorders</article-title>. <source>Am J Med Genet A.</source> (<year>2015</year>) <volume>167A</volume>:<fpage>2869</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.1002/ajmg.a.37365</pub-id><pub-id pub-id-type=\"pmid\">26394607</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Munns</surname><given-names>CF</given-names></name><name><surname>Fahiminiya</surname><given-names>S</given-names></name><name><surname>Poudel</surname><given-names>N</given-names></name><name><surname>Munteanu</surname><given-names>MC</given-names></name><name><surname>Majewski</surname><given-names>J</given-names></name><name><surname>Sillence</surname><given-names>DO</given-names></name><etal/></person-group>. <article-title>Homozygosity for frameshift mutations in XYLT2 result in a spondylo-ocular syndrome with bone fragility, cataracts, and hearing defects</article-title>. <source>Am J Hum Genet.</source> (<year>2015</year>) <volume>96</volume>:<fpage>971</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ajhg.2015.04.017</pub-id><pub-id pub-id-type=\"pmid\">26027496</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>EJ</surname><given-names>Salkeld</given-names></name></person-group>\n<article-title>The Value of collaborative research in rare disease research</article-title>. <source>Exp Opin Orphan Drugs.</source> (<year>2016</year>) <volume>4</volume>:<fpage>687</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1080/21678707.2016.1183480</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Robertson</surname><given-names>P</given-names></name></person-group>\n<article-title>Collaborative organizing: An “ideal type” for a new paradigm</article-title>. In: <source>Research in Organizational Change and Development (Research in Organizational Change and Development)</source>, <publisher-name>Emerald Group Publishing Limited</publisher-name>\n<publisher-loc>Bingley</publisher-loc> (<year>2002</year>). p. <fpage>205</fpage>–<lpage>67</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0897-3016(99)12008-1</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cross</surname><given-names>P</given-names></name><name><surname>Gray</surname><given-names>P</given-names></name><name><surname>Cunningham</surname><given-names>S</given-names></name><name><surname>Showers</surname><given-names>S</given-names></name><name><surname>Thomas</surname><given-names>RJ</given-names></name></person-group>\n<article-title>The collaborative organization: how to make employee networks really work</article-title>. <source>MIT Sloan Management Rev.</source> (<year>2010</year>) <volume>52</volume>:<fpage>83</fpage>–<lpage>90</lpage>.</mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Swensen</surname><given-names>S</given-names></name><name><surname>Kabcenell</surname><given-names>A</given-names></name><name><surname>Shanafelt</surname><given-names>T</given-names></name></person-group>. <article-title>Physician-organization collaboration reduces physician burnout and promotes engagement: the mayo clinic experience</article-title>. <source>J Healthcare Manage.</source> (<year>2016</year>) <volume>61</volume>:<fpage>105</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.1097/00115514-201603000-00008</pub-id><pub-id pub-id-type=\"pmid\">27111930</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hardin</surname><given-names>L</given-names></name><name><surname>Kilian</surname><given-names>A</given-names></name><name><surname>Spykerman</surname><given-names>K</given-names></name></person-group>\n<article-title>Competing health care systems and complex patients: an inter-professional collaboration to improve outcomes and reduce health care costs</article-title>. <source>J Int Educ Pract.</source> (<year>2017</year>) <volume>7</volume>:<fpage>5</fpage>–<lpage>10</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.xjep.2017.01.002</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shanafelt</surname><given-names>TD</given-names></name><name><surname>Noseworthy</surname><given-names>JH</given-names></name></person-group>. <article-title>Executive leadership and physician well-being: nine organizational strategies to promote engagement and reduce burnout</article-title>. <source>Mayo Clin Proc</source>. (<year>2017</year>) <volume>92</volume>:<fpage>129</fpage>–<lpage>46</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.mayocp.2016.10.004</pub-id><pub-id pub-id-type=\"pmid\">27871627</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>C</given-names></name><name><surname>Bakker</surname><given-names>AD</given-names></name><name><surname>Klein-Nulend</surname><given-names>J</given-names></name><name><surname>Bravenboer</surname><given-names>N</given-names></name></person-group>. <article-title>Studies on osteocytes in their 3D native matrix versus 2D <italic>in vitro</italic> models</article-title>. <source>Curr Osteoporos Rep.</source> (<year>2019</year>) <volume>17</volume>:<fpage>207</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s11914-019-00521-1</pub-id><pub-id pub-id-type=\"pmid\">31240566</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Micha</surname><given-names>D</given-names></name><name><surname>Voermans</surname><given-names>E</given-names></name><name><surname>Eekhoff</surname><given-names>MEW</given-names></name><name><surname>van Essen</surname><given-names>HW</given-names></name><name><surname>Zandieh-Doulabi</surname><given-names>B</given-names></name><name><surname>Netelenbos</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>Inhibition of TGFβ signaling decreases osteogenic differentiation of fibrodysplasia ossificans progressiva fibroblasts in a novel <italic>in vitro</italic> model of the disease</article-title>. <source>Bone.</source> (<year>2016</year>) <volume>84</volume>:<fpage>169</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bone.2016.01.004</pub-id><pub-id pub-id-type=\"pmid\">26769004</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schoenmaker</surname><given-names>T</given-names></name><name><surname>Wouters</surname><given-names>F</given-names></name><name><surname>Micha</surname><given-names>D</given-names></name><name><surname>Forouzanfar</surname><given-names>T</given-names></name><name><surname>Netelenbos</surname><given-names>C</given-names></name><name><surname>Eekhoff</surname><given-names>EMW</given-names></name><etal/></person-group>. <article-title>The effect of Activin-A on periodontal ligament fibroblasts-mediated osteoclast formation in healthy donors and in patients with fibrodysplasia ossificans progressiva</article-title>. <source>J Cell Physiol.</source> (<year>2019</year>) <volume>234</volume>:<fpage>10238</fpage>–<lpage>47</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jcp.27693</pub-id><pub-id pub-id-type=\"pmid\">30417373</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Javaheri</surname><given-names>B</given-names></name><name><surname>Bravenboer</surname><given-names>N</given-names></name><name><surname>Bakker</surname><given-names>AD</given-names></name><name><surname>van der Veen</surname><given-names>A</given-names></name><name><surname>de Souza</surname><given-names>RL</given-names></name><name><surname>Saxon</surname><given-names>L</given-names></name><etal/></person-group>. <article-title><italic>In vivo</italic> models of mechanical loading</article-title>. <source>Methods Mol Biol.</source> (<year>2019</year>) <volume>1914</volume>:<fpage>369</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1007/978-1-4939-8997-3_22</pub-id><pub-id pub-id-type=\"pmid\">30729478</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>V</given-names></name><name><surname>Helder</surname><given-names>MN</given-names></name><name><surname>Bravenboer</surname><given-names>N</given-names></name><name><surname>Ten Bruggenkate</surname><given-names>CM</given-names></name><name><surname>Jin</surname><given-names>J</given-names></name><name><surname>Klein-Nulend</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Bone tissue regeneration in the oral and maxillofacial region: a review on the application of stem cells and new strategies to improve vascularization</article-title>. <source>Stem. Cells Int.</source> (<year>2019</year>) <volume>2019</volume>:<fpage>6279721</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2019/6279721</pub-id><pub-id pub-id-type=\"pmid\">32082383</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eekhoff</surname><given-names>EMW</given-names></name><name><surname>Botman</surname><given-names>E</given-names></name><name><surname>Netelenbos</surname><given-names>JC</given-names></name><name><surname>de Graaf</surname><given-names>P</given-names></name><name><surname>Bravenboer</surname><given-names>N</given-names></name><name><surname>Micha</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>18F NaF PET/CT scan as an early marker of heterotopic ossification in fibrodysplasia ossificans progressiva</article-title>. <source>Bone.</source> (<year>2018</year>) <volume>109</volume>:<fpage>143</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bone.2017.08.012</pub-id><pub-id pub-id-type=\"pmid\">28826841</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eekhoff</surname><given-names>EMW</given-names></name><name><surname>Netelenbos</surname><given-names>JC</given-names></name><name><surname>de Graaf</surname><given-names>P</given-names></name><name><surname>Hoebink</surname><given-names>M</given-names></name><name><surname>Bravenboer</surname><given-names>N</given-names></name><name><surname>Micha</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Flare-up after maxillofacial surgery in a patient with fibrodysplasia ossificans progressiva: an [<sup>18</sup>F]-NaF PET/CT study and a systematic review</article-title>. <source>JBMR Plus.</source> (<year>2017</year>) <volume>2</volume>:<fpage>55</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jbm4.10008</pub-id><pub-id pub-id-type=\"pmid\">30283890</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Botman</surname><given-names>E</given-names></name><name><surname>Raijmakers</surname><given-names>PGHM</given-names></name><name><surname>Yaqub</surname><given-names>M</given-names></name><name><surname>Teunissen</surname><given-names>B</given-names></name><name><surname>Netelenbos</surname><given-names>C</given-names></name><name><surname>Lubbers</surname><given-names>W</given-names></name><etal/></person-group>. <article-title><italic>Evolution</italic> of heterotopic bone in fibrodysplasia ossificans progressiva: an [<sup>18</sup>F]NaF PET/CT study</article-title>. <source>Bone.</source> (<year>2019</year>) <volume>124</volume>:<fpage>1</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bone.2019.03.009</pub-id><pub-id pub-id-type=\"pmid\">30858149</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hsiao</surname><given-names>EC</given-names></name><name><surname>Di Rocco</surname><given-names>M</given-names></name><name><surname>Cali</surname><given-names>A</given-names></name><name><surname>Zasloff</surname><given-names>M</given-names></name><name><surname>Al Mukaddam</surname><given-names>M</given-names></name><name><surname>Pignolo</surname><given-names>RJ</given-names></name><etal/></person-group>. <article-title>Special considerations for clinical trials in fibrodysplasia ossificans progressiva (FOP)</article-title>\n<source>Br J Clin Pharmacol</source>. (<year>2019</year>) <volume>85</volume>:<fpage>1199</fpage>–<lpage>207</lpage>. <pub-id pub-id-type=\"doi\">10.1111/bcp.13777</pub-id><pub-id pub-id-type=\"pmid\">30281842</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cayami</surname><given-names>FK</given-names></name><name><surname>Maugeri</surname><given-names>A</given-names></name><name><surname>Treurniet</surname><given-names>S</given-names></name><name><surname>Setijowati</surname><given-names>ED</given-names></name><name><surname>Teunissen</surname><given-names>BP</given-names></name><name><surname>Eekhoff</surname><given-names>EMW</given-names></name><etal/></person-group>. <article-title>The first family with adult osteogenesis imperfecta caused by a novel homozygous mutation in CREB3L1</article-title>. <source>Mol Genet Genomic Med.</source> (<year>2019</year>) <volume>7</volume>:<fpage>e823</fpage>. <pub-id pub-id-type=\"doi\">10.1002/mgg3.823</pub-id><pub-id pub-id-type=\"pmid\">31207160</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kloen</surname><given-names>P</given-names></name><name><surname>Donders</surname><given-names>JCE</given-names></name><name><surname>Eekhoff</surname><given-names>EMW</given-names></name><name><surname>Hamdy</surname><given-names>RC</given-names></name></person-group>. <article-title>Pauwels osteotomy for femoral neck nonunion in two adult siblings with osteogenesis imperfecta</article-title>. <source>Hip Pelvis</source>. (<year>2018</year>) <volume>30</volume>:<fpage>53</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.5371/hp.2018.30.1.53</pub-id><pub-id pub-id-type=\"pmid\">29564298</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vierhout</surname><given-names>L</given-names></name><name><surname>Eekhoff</surname><given-names>EM</given-names></name><name><surname>van der Waal</surname><given-names>I</given-names></name></person-group>. <article-title>An adolescent boy with fibrous dysplasia of the maxillary bone</article-title>. <source>NTVG.</source> (<year>2012</year>) <volume>119</volume>:<fpage>541</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"pmid\">23236738</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Van Dijk</surname><given-names>FS</given-names></name><name><surname>Micha</surname><given-names>D</given-names></name><name><surname>Zillikens</surname><given-names>C</given-names></name><name><surname>Micha</surname><given-names>D</given-names></name><name><surname>Riessland</surname><given-names>M</given-names></name><name><surname>Marcelis</surname><given-names>CLM</given-names></name><etal/></person-group>. <article-title>PLS3 mutations in X-linked osteoporosis with fractures</article-title>. <source>N Engl J Med.</source> (<year>2013</year>) <volume>369</volume>:<fpage>1529</fpage>–<lpage>36</lpage>. <pub-id pub-id-type=\"doi\">10.1056/NEJMoa1308223</pub-id><pub-id pub-id-type=\"pmid\">24088043</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>de Vries</surname><given-names>TJ</given-names></name><name><surname>Schoenmaker</surname><given-names>T</given-names></name><name><surname>Micha</surname><given-names>D</given-names></name><name><surname>Hogervorst</surname><given-names>J</given-names></name><name><surname>Bouskla</surname><given-names>S</given-names></name><name><surname>Forouzanfar</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>Periodontal ligament fibroblasts as a cell model to study osteogenesis and osteoclastogenesis in fibrodysplasia ossificans progressiva</article-title>. <source>Bone</source>. (<year>2018</year>) <volume>109</volume>:<fpage>168</fpage>–<lpage>77</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bone.2017.07.007</pub-id><pub-id pub-id-type=\"pmid\">28705683</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Physiol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Physiol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Physiol.</journal-id><journal-title-group><journal-title>Frontiers in Physiology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-042X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32848881</article-id><article-id pub-id-type=\"pmc\">PMC7431600</article-id><article-id pub-id-type=\"doi\">10.3389/fphys.2020.00961</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Physiology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Lack of Connexins 40 and 45 Reduces Local and Conducted Vasoconstrictor Responses in the Murine Afferent Arterioles</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Møller</surname><given-names>Sophie</given-names></name><xref rid=\"c001\" ref-type=\"corresp\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/909176/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Jacobsen</surname><given-names>Jens Christian Brings</given-names></name><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/26528/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Holstein-Rathlou</surname><given-names>Niels-Henrik</given-names></name><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/28869/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Sorensen</surname><given-names>Charlotte M.</given-names></name><uri xlink:type=\"simple\" xlink:href=\"https://loop.frontiersin.org/people/858821/overview\"/></contrib></contrib-group><aff>\n<institution>Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen</institution>, <addr-line>Copenhagen</addr-line>, <country>Denmark</country>\n</aff><author-notes><fn id=\"fn1\" fn-type=\"edited-by\"><p>Edited by: Mauricio P. Boric, Pontificia Universidad Católica de Chile, Chile</p></fn><fn id=\"fn2\" fn-type=\"edited-by\"><p>Reviewed by: William F. Jackson, Michigan State University, United States; Armin Kurtz,University of Regensburg, Germany</p></fn><corresp id=\"c001\">*Correspondence: Sophie Møller, <email>sophie.moller@gmail.com</email><email>sopm@nexs.ku.dk</email></corresp><fn id=\"fn3\" fn-type=\"other\"><p>This article was submitted to Vascular Physiology, a section of the journal Frontiers in Physiology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>07</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>961</elocation-id><history><date date-type=\"received\"><day>14</day><month>2</month><year>2020</year></date><date date-type=\"accepted\"><day>15</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Møller, Jacobsen, Holstein-Rathlou and Sorensen.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Møller, Jacobsen, Holstein-Rathlou and Sorensen</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>The juxtaglomerular apparatus (JGA) is an essential structure in the regulation of renal function. The JGA embodies two major functions: tubuloglomerular feedback (TGF) and renin secretion. TGF is one of the mechanisms mediating renal autoregulation. It is initiated by an increase in tubular NaCl concentration at the macula densa cells. This induces a local afferent arteriolar vasoconstriction and a conducted response that can be measured several 100 μm upstream from the juxtaglomerular segment. This spread of the vasomotor response into the surrounding vasculature likely plays a key role in renal autoregulation, and it requires the presence of gap junctions, intercellular pores based on connexin (Cx) proteins. Several Cx isoforms are expressed in the JGA and in the arteriolar wall. Disruption of this communication pathway is associated with reduced TGF, dysregulation of renin secretion, and hypertension. We examine if the absence of Cx40 or Cx45, expressed in the endothelial and vascular smooth muscle cells respectively, attenuates afferent arteriolar local and conducted vasoconstriction. Afferent arterioles from wildtype and Cx-deficient mice (Cx40 and Cx45) were studied using the isolated perfused juxtamedullary nephron preparation. Vasoconstriction was induced <italic>via</italic> electrical pulse stimulation at the glomerular entrance. Inner afferent arteriolar diameter was measured locally and upstream to evaluate conducted vasoconstriction. Electrical stimulation induced local vasoconstriction in all groups. The local vasoconstriction was significantly smaller when Cx40 was absent. The vasoconstriction decreased in magnitude with increasing distance from the stimulation site. In both Cx40 and Cx45 deficient mice, the vasoconstriction conducted a shorter distance along the vessel compared to wild-type mice. In Cx40 deficient arterioles, this may be caused by a smaller local vasoconstriction. Collectively, these findings imply that Cx40 and Cx45 are central for normal vascular reactivity and, therefore, likely play a key role in TGF-induced regulation of afferent arteriolar resistance.</p></abstract><kwd-group><kwd>gap junction</kwd><kwd>vascular conducted response</kwd><kwd>electrical pulse stimulation</kwd><kwd>renal</kwd><kwd>intercellular communication</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn1\">Dynamical Systems Interdisciplinary Network, University of Copenhagen</funding-source></award-group><award-group><funding-source id=\"cn2\">Edith Waagens and Frode Waagens Foundation</funding-source></award-group></funding-group><counts><fig-count count=\"4\"/><table-count count=\"2\"/><equation-count count=\"1\"/><ref-count count=\"57\"/><page-count count=\"10\"/><word-count count=\"7624\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"sec1\"><title>Introduction</title><p>The juxtaglomerular apparatus (JGA) is a specialized structure, formed by the afferent and efferent arteriole, thick limb of the ascending loop of Henle, and mesangial cells. The JGA embodies two major renal functions: tubuloglomerular feedback (TGF), which is part of the mechanism underlying renal autoregulation, and renin secretion. The JGA consists of different cell types with specialized functions: the macula densa (MD) cells, juxtaglomerular cells, mesangial cells, vascular smooth muscle cells (VSMCs), and endothelial cells (ECs). The MD cell of the thick limb of the ascending loop of Henle functions as a NaCl sensor. An increase in NaCl concentration in the tubular fluid eventually causes the VSMC in the afferent arteriole to contract to reduce glomerular filtration rate (GFR) and the juxtaglomerular cells of the afferent arteriole to reduce renin secretion. Mesangial cells are located between the MD cells (the sensor) and the afferent arteriole (the effector). They are believed to play an important role in the signal transduction from the MD to the afferent arteriole (<xref rid=\"ref12\" ref-type=\"bibr\">Goligorsky et al., 1997</xref>).</p><p>The TGF signal originates in the MD cells and elicits local vasoconstriction in the juxtaglomerular part of the afferent arteriole. The response, however, encompasses the entire afferent arteriole, the distal part of the interlobular artery, and travels into the afferent arterioles of neighboring nephrons (<xref rid=\"ref38\" ref-type=\"bibr\">Peti-Peterdi, 2006</xref>; <xref rid=\"ref32\" ref-type=\"bibr\">Marsh et al., 2009</xref>). This indicates that the vasoconstrictor signal is conducted within the vascular wall most likely through gap junctions (GJs) coupling ECs to ECs, VSMCs to VSMCs, and ECs to VSMCs.</p><p>GJs are fluid-filled pores that allow the movement of small molecules and current between neighboring cells. GJs are made up of two hemichannels, connexons, built from connexins (Cxs) (<xref rid=\"ref36\" ref-type=\"bibr\">Nielsen et al., 2012</xref>). Six Cxs of the same or different isoforms can assemble to form a connexon, and the two connexons from neighboring cells can dock to form a functional GJ channel (<xref rid=\"ref35\" ref-type=\"bibr\">Moreno, 2004</xref>). Several Cx isoforms are expressed in the JGA (<xref rid=\"ref17\" ref-type=\"bibr\">Hanner et al., 2010</xref>). Similarly, in preglomerular arterioles, expression of Cx37, Cx40, Cx43, and Cx45 (<xref rid=\"ref46\" ref-type=\"bibr\">Wagner, 2008</xref>; <xref rid=\"ref25\" ref-type=\"bibr\">Just et al., 2009</xref>; <xref rid=\"ref42\" ref-type=\"bibr\">Schweda et al., 2009</xref>) is found, enabling the cells of the vessel wall, both smooth muscle and endothelial, to act as a syncytium (<xref rid=\"ref12\" ref-type=\"bibr\">Goligorsky et al., 1997</xref>; <xref rid=\"ref8\" ref-type=\"bibr\">Dora et al., 2003</xref>; <xref rid=\"ref15\" ref-type=\"bibr\">Haefliger et al., 2004</xref>; <xref rid=\"ref38\" ref-type=\"bibr\">Peti-Peterdi, 2006</xref>; <xref rid=\"ref50\" ref-type=\"bibr\">Yao et al., 2009</xref>).</p><p>In the vasculature, Cx40 is the most widely expressed isoform and is predominantly found in the ECs (<xref rid=\"ref7\" ref-type=\"bibr\">de Wit et al., 2003</xref>; <xref rid=\"ref42\" ref-type=\"bibr\">Schweda et al., 2009</xref>), as well as in renin-producing cells (<xref rid=\"ref14\" ref-type=\"bibr\">Haefliger et al., 2001</xref>). In mice, deletion of Cx40 leads to the development of renin-dependent hypertension (<xref rid=\"ref6\" ref-type=\"bibr\">de Wit et al., 2000</xref>, <xref rid=\"ref7\" ref-type=\"bibr\">2003</xref>; <xref rid=\"ref10\" ref-type=\"bibr\">Figueroa et al., 2003</xref>; <xref rid=\"ref47\" ref-type=\"bibr\">Wagner et al., 2007</xref>; <xref rid=\"ref25\" ref-type=\"bibr\">Just et al., 2009</xref>; <xref rid=\"ref44\" ref-type=\"bibr\">Sorensen et al., 2012</xref>) and reduced conduction of vasodilation (<xref rid=\"ref6\" ref-type=\"bibr\">de Wit et al., 2000</xref>). Cx45 is expressed in the VSMCs (<xref rid=\"ref46\" ref-type=\"bibr\">Wagner, 2008</xref>) and mesangial cells (<xref rid=\"ref28\" ref-type=\"bibr\">Kurtz et al., 2009</xref>; <xref rid=\"ref17\" ref-type=\"bibr\">Hanner et al., 2010</xref>), and lack of Cx45 expression causes lethal malformations in the vasculature (<xref rid=\"ref27\" ref-type=\"bibr\">Krüger et al., 2000</xref>; <xref rid=\"ref18\" ref-type=\"bibr\">Hanner et al., 2008</xref>; <xref rid=\"ref46\" ref-type=\"bibr\">Wagner, 2008</xref>). Therefore, only a conditional deletion of Cx45 is possible in mice. Previous studies in these mice show that Cx45 plays a role in the propagation of VSMC calcium waves and in TGF (<xref rid=\"ref18\" ref-type=\"bibr\">Hanner et al., 2008</xref>; <xref rid=\"ref34\" ref-type=\"bibr\">Møller et al., 2020</xref>). This calcium signaling may be one mechanism by which Cx45 affects vascular conducted responses.</p><p>Once the signal from MD has reached the afferent arteriole, the vascular conduction can spread dilation or constriction along the vessel wall, depending on the blood flow needs (<xref rid=\"ref46\" ref-type=\"bibr\">Wagner, 2008</xref>). Conducted vasoconstriction occurs when depolarizing current propagates upstream <italic>via</italic> GJs (<xref rid=\"ref13\" ref-type=\"bibr\">Gustafsson and Holstein-Rathlou, 1999</xref>), from the afferent arterioles to the larger arteries, and changes the vascular resistance, thus reducing renal blood flow (<xref rid=\"ref48\" ref-type=\"bibr\">Wagner et al., 1997</xref>).</p><p>In this study, we aim to test if a lack of two specific Cxs affects vascular conduction in the afferent arteriole. Specifically, we explore the roles of Cx40 and Cx45 in the conduction of vasoconstrictor responses to electrical pulse stimulation.</p></sec><sec sec-type=\"materials|methods\" id=\"sec2\"><title>Materials and Methods</title><sec id=\"sec3\"><title>Animal Preparation</title><p>Procedures were approved by the Danish National Animal Experiments Inspectorate. All animals were kept in the animal facility at University of Copenhagen and received tap water and standard chow <italic>ad libitum</italic>. Genotyping was done on DNA from ear clippings using the DirectPCR kit (Viagen Biotech; Los Angeles, CA).</p><p>Cx40 wildtype (Cx40 WT) and Cx40 knockout (Cx40 KO) mice were on a mixed C57Bl/6 × 129S4/SvJae genetic background. Mice were purchased for breeding from the European Mouse Mutant Archive (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.infrafrontier.eu\">infrafrontier.eu</ext-link>, Munich, Germany). Kidneys from a total of 15 adult mice were used for the studies (Cx40 WT <italic>n</italic> = 8, five females and three males and Cx40 KO <italic>n</italic> = 7, four females and three males). The age of the mice ranged from 4 to 14 months.</p><p>Homozygous female mice with floxed Cx45 gene (Cx45<sup>fl/fl</sup>, >87% C57Bl/6 background) were mated with homozygous Cx45<sup>fl/fl:Nestin-Cre</sup> males, >87% C57Bl/6 background. Breeding pairs were a generous gift from Dr. Klaus Willecke, University of Bonn, Germany; the mice have been described previously (<xref rid=\"ref33\" ref-type=\"bibr\">Maxeiner et al., 2005</xref>). In their offspring, the Cx45 coding DNA is replaced with an enhanced green fluorescence protein (eGFP) in cells expressing the intermediate filament Nestin during development. The Cx45<sup>fl/fl</sup> mice are phenotypically wildtype and are used as such (WT). The Cx45<sup>fl/fl:Nestin-Cre</sup> mice are used as our knockout (KO). Kidneys from 20 adult mice were used for the studies (Cx45 WT <italic>n</italic> = 9, three females and six males and Cx45 KO <italic>n</italic> = 11, nine females and two males), ranging from 2 to 6 months of age.</p></sec><sec id=\"sec4\"><title>Imaging System and Software</title><p>The experimental setup is shown in <xref rid=\"fig1\" ref-type=\"fig\">Figure 1</xref>. The vasculature was viewed and recorded using an Olympus BX50WI microscope with a digital 12-bit CCD camera (Pixelfly, PCO, Kelheim, Germany) mounted on the microscope. Images were recorded using the CamWare software (PCO, Kelheim, Germany). Renal perfusion pressure (RPP) readings were obtained using the PowerLab/8SP data acquisition system (ADInstruments, Colorado Springs, CO) and viewed using LabChart 7 software (ADInstruments, Colorado Springs, CO). Post-experimental evaluations of afferent arteriolar diameter were performed offline using ImageJ (National Institute of Health, Bethesda, MD).</p><fig id=\"fig1\" position=\"float\"><label>Figure 1</label><caption><p>Schematic of the imaging system and experimental setup used. Schematic showing the setup used to measure the afferent arteriolar diameter in response to electrical stimulation.</p></caption><graphic xlink:href=\"fphys-11-00961-g001\"/></fig></sec><sec id=\"sec5\"><title>Surgical Procedure</title><p>Experiments were performed using the <italic>in vitro</italic> perfused juxtamedullary nephron preparation, developed by <xref rid=\"ref4\" ref-type=\"bibr\">Casellas and Navar (1984)</xref>, and adapted to mice (<xref rid=\"ref19\" ref-type=\"bibr\">Harrison-Bernard et al., 2003</xref>; <xref rid=\"ref44\" ref-type=\"bibr\">Sorensen et al., 2012</xref>). Mice were anesthetized with pentobarbital (50 g/kg <italic>i.p.</italic>, Mebumal, SAD, Denmark). The abdominal aorta was catheterized and immediately perfused with Tyrode’s solution (in mmol/L: 136.9 NaCl, 0.42 NaH<sub>2</sub>PO<sub>4</sub>, 11.9 NaHCO<sub>3</sub>, 2.7 KCl, 2.2 MgCl<sub>2</sub>, 5.6 d-glucose, and 1.8 CaCl<sub>2</sub>) containing 5% BSA (ICPbio International, Auckland, New Zealand) and an amino acid mixture (in mmol/L: 2 alanine, 2 glycine, 5 glutamine, 1 serine, and supplemented with MEM amino acid solution, all from Sigma-Aldrich, Copenhagen, Denmark) at pH 7.4. Kidneys were excised, and the cannula was advanced into the renal artery of the left kidney. The kidney was decapsulated and sectioned longitudinally exposing the intact papilla, which was reflected revealing the inner cortical surface. Venous tissue on the cortical surface was cut, allowing access to the arterial vasculature. Perfusion was isolated to the inner cortical vessels by ligating larger arterial vessels (Dafilon 10/0 sutures, B. Braun Vet Care GmbH, Tuttlingen, Germany).</p><p>The cannula system comprised of a 27-gauge blunt needle and a polyethylene (PE-10) line, connected to a servo-nulling pressure system (Instrumentation for Physiology and Medicine, San Diego, CA), allowing for continuous RPP monitoring. RPP was maintained at 95 mmHg throughout the experiment by adjusting the regulator, controlling the flow of 95% O<sub>2</sub>-5% CO<sub>2</sub> gas mixture to the perfusion solution reservoir.</p></sec><sec id=\"sec6\"><title>Experimental Procedure: Application of Electrical Pulse Stimulation</title><p>Borosilicate glass micropipettes (CMA Microdialysis, Kista, Sweden) were pulled on a horizontal Flaming/Brown micropipette puller (Model P-87, Sutter Instrument Co., Novato, CA) and back-filled with a 2 mol/L NaCl solution containing Lissamine green (Sigma-Aldrich, Copenhagen, Denmark). The microelectrode was connected to a stimulus isolator (ISO-flex, A.M.P.I., Jerusalem, Israel) connected to a stimulator (Model S44, Grass Instrument Co., Quincy, MA) and an oscilloscope (1200 A, Hewlett-Packard, Palo Alto, CA). The microelectrode (0.5–0.8 MΩ resistance) was placed at the glomerular entrance of the afferent arteriole, where local afferent vasoconstriction was induced by electrical pulse stimulation (2.5 Hz frequency, 300 ms pulse duration, and 90 V amplitude; <xref rid=\"ref39\" ref-type=\"bibr\">Salomonsson et al., 2002</xref>; <xref rid=\"ref37\" ref-type=\"bibr\">Palanker et al., 2008</xref>), as shown in <xref rid=\"fig2\" ref-type=\"fig\">Figure 2</xref>. A wire was placed in the tissue chamber to serve as the grounding electrode. After a recovery period, an experimental protocol consisting of 30 s recordings of baseline, electrical pulse stimulation, and recovery periods was initiated. The vessels were stimulated for 30 s, a period which was sufficient for it to reach a new steady-state diameter (<xref rid=\"ref45\" ref-type=\"bibr\">Steinhausen et al., 1997</xref>).</p><fig id=\"fig2\" position=\"float\"><label>Figure 2</label><caption><p>Illustration of the microelectrode positioning for electrical pulse stimulation. <bold>(A)</bold> Stimulation microelectrode was positioned at the glomerular entrance of the afferent arteriole. The diameter was measured at sites labeled (>), corresponding to the stimulation site and sites (50 μm apart) upstream from the microelectrode (maximum distance reached only in some experiments were 450 μm). Schematic representation inspired by <xref rid=\"ref48\" ref-type=\"bibr\">Wagner et al. (1997)</xref>. <bold>(B)</bold> Image of the experimental setup showing the stimulation electrode placed immediately upstream from glomerulus (G) just above the afferent arteriole (AA).</p></caption><graphic xlink:href=\"fphys-11-00961-g002\"/></fig></sec><sec id=\"sec7\"><title>Diameter Measurements</title><p>The afferent arteriolar diameter was measured by tracking the vessel edges and the inner diameter was measured every 10 s throughout the experiment. The diameter was measured at the stimulation point at the glomerular entrance (local response) as well as every 50 μm upstream from the glomerulus (conducted response; <xref rid=\"fig2\" ref-type=\"fig\">Figures 2</xref>, <xref rid=\"fig3\" ref-type=\"fig\">3</xref>) as far as it was possible to track the vessel (<xref rid=\"tab1\" ref-type=\"table\">Table 1</xref>). Each data point represents the average of three consecutive measurements (after 10, 20, and 30 s of stimulation) in the same position.</p><table-wrap id=\"tab1\" position=\"float\"><label>Table 1</label><caption><p>Mean values of body weight prior to kidney removal, age, pre-stimulated vessel diameter and traceable vessel length starting at the stimulation point on the afferent arteriole.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th colspan=\"2\" rowspan=\"1\">\n</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">N</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Body wt. (g)</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Age (weeks)</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">AA diameter (μm)</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Measured vessel length (μm)</th></tr></thead><tbody><tr><td align=\"left\" valign=\"middle\" rowspan=\"3\" colspan=\"1\">Cx40</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">WT</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">8</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">33.1 ± 1.3</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">31.8 ± 5.6</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">28.7 ± 5.3</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">259.4 ± 58.2</td></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">KO</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">7</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">27.9 ± 1.7</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">41.9 ± 4.1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">15.5 ± 3.4</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">178.6 ± 26.4</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<italic>p</italic> = 0.04</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">NS</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<italic>p</italic> = 0.04</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">NS</td></tr><tr><td align=\"left\" valign=\"middle\" rowspan=\"3\" colspan=\"1\">Cx45</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">WT</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">9</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">27.4 ± 1.3<xref rid=\"tfn1\" ref-type=\"table-fn\">\n<sup>*</sup>\n</xref>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">16.4 ± 1.1<xref rid=\"tfn2\" ref-type=\"table-fn\">\n<sup>***</sup>\n</xref>\n</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">30.6 ± 2.7</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">361.1 ± 28.6</td></tr><tr><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">KO</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">11</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">22.5 ± 1.6<xref rid=\"tfn3\" ref-type=\"table-fn\">\n<sup>#</sup>\n</xref>\n</td><td align=\"left\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">15.9 ± 1.2<xref rid=\"tfn4\" ref-type=\"table-fn\">\n<sup>###</sup>\n</xref>\n</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">23.6 ± 3.1</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">295.5 ± 41.8</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">\n<italic>p</italic> = 0.007</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">NS</td><td align=\"center\" valign=\"middle\" rowspan=\"1\" colspan=\"1\">NS</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">NS</td></tr></tbody></table><table-wrap-foot><p>Cx40, connexin 40; Cx45, connexin 45; WT, wildtype; KO, knockout; AA, afferent arteriole; NS, not significant.</p><fn id=\"tfn1\"><label>*</label><p>\n<italic>p</italic> < 0.05;</p></fn><fn id=\"tfn2\"><label>***</label><p>\n<italic>p</italic> < 0.001 vs. Cx40 WT;</p></fn><fn id=\"tfn3\"><label>#</label><p>\n<italic>p</italic> < 0.05;</p></fn><fn id=\"tfn4\"><label>###</label><p>\n<italic>p</italic> < 0.001 vs. Cx40 KO.</p></fn></table-wrap-foot></table-wrap><p>Experiments were included if they fulfilled the following: (1) electrical stimulation induced a minimum of 5% constriction in the vessel diameter (local response), (2) post-stimulation, the vessel recovered to at least 50% of its baseline diameter, and (3) diameters were measurable at a minimum of three positions along the vessel (including stimulation site).</p></sec><sec id=\"sec8\"><title>Data Handling and Statistical Analyses</title><p>Graphical presentation and statistical analyses were performed using SigmaPlot (Systat Software Inc. San Jose, CA, USA), MatLab (MathWorks, Natick, MA, USA), and Rstudio (version 1.3.959; Boston, MA). Vessel diameter during stimulation was normalized to the respective mean resting diameter at the given distance from the stimulation site. Normalized and pooled data for contraction at the stimulation site (0 μm) from each experimental series were evaluated using a one-way ANOVA to test for differences between WT and KO. A one-sample <italic>t</italic>-test testing against 0 was used to evaluate significant constriction at each measurement point.</p><p>Length constants of the conducted vasoconstriction were calculated in MATLAB® (The MathWorks Inc., R2017b) for each type of animal by a non-linear least squares fit of the data to the equation <italic>y</italic> = <italic>ae<sup>bx</sup></italic>, where <italic>y</italic> is the change in diameter at upstream distance <italic>x</italic> from the stimulation site, <italic>a</italic> is the <italic>y</italic>-intercept, and <italic>b</italic> is the decay rate of the response along the vessel. When conducted vasoconstriction reached 0 or a negative value, constriction was set to 0 for the remaining measurements. The maximum distance from the stimulation point at which the individual vessel could be visualized varied from 100 to 500 μm. Consequently, there were more measurements at the shorter distances. All measurements at a given distance were pooled for each group. Therefore, in each fit, the shorter distances with more measurements weighs in more heavily than longer distances with fewer measurements (<xref rid=\"fig4\" ref-type=\"fig\">Figure 4</xref>; constants shown in <xref rid=\"tab2\" ref-type=\"table\">Table 2</xref>). Each group gives rise to a single fit. To enable statistical comparison of the decay rates between groups, a boot strapping procedure was applied (source code available in <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Data</xref>). In brief, for each group of animals, a set of data points were obtained by drawing randomly, with replacement, from the original data set. The single exponential decay model was then fitted to this new data set to obtain estimates of <italic>a</italic> and <italic>b</italic>. The procedure was repeated 1,000 times, giving new sets of <italic>y</italic>-interceptions and decay rates. Differences in the latter between WT and KO groups were analyzed using a two-tailed <italic>t</italic>-test.</p><table-wrap id=\"tab2\" position=\"float\"><label>Table 2</label><caption><p>Summary of conducted response characteristics for WT and KO animals of both strains.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Strain</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">a, 95% CI (μm)</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">b (×10<sup>−3</sup>), 95% CI (×10<sup>−3</sup>) (1/μm)</th><th align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Length constant (−1/b, μm)</th></tr></thead><tbody><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cx40 WT</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.26, (0.20, 0.33)<xref rid=\"tfn5\" ref-type=\"table-fn\">\n<sup>*</sup>\n</xref>\n</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">−3.8, (−6.0, −1.7)</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">363</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cx40 KO</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.15, (0.092, 0.20)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">−5.6, (−10.7, −0.47)</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">179</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cx45 WT</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.23, (0.17, 0.29)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">−2.2, (−3.8, −0.53)<xref rid=\"tfn6\" ref-type=\"table-fn\">\n<sup>**</sup>\n</xref>\n</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">455</td></tr><tr><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">Cx45 KO</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">0.17, (0.12, 0.21)</td><td align=\"left\" valign=\"top\" rowspan=\"1\" colspan=\"1\">−4.5, (−7.1, −2.0)</td><td align=\"center\" valign=\"top\" rowspan=\"1\" colspan=\"1\">222</td></tr></tbody></table><table-wrap-foot><p>Values are calculated by fitting data to a single exponential decaying function:<inline-formula><mml:math id=\"M1\"><mml:mi mathvariant=\"normal\">y</mml:mi><mml:mo>=</mml:mo><mml:mi mathvariant=\"normal\">a</mml:mi><mml:msup><mml:mi mathvariant=\"normal\">e</mml:mi><mml:mi mathvariant=\"normal\">bx</mml:mi></mml:msup></mml:math></inline-formula>\n<italic>y</italic>=aebx where <italic>y</italic> is the relative change in diameter at distance <italic>x</italic> upstream from the stimulation site, <italic>a</italic> is the <italic>y</italic>-intercept, and <italic>b</italic> is the decay rate of the response along the vessel. Significance levels were evaluated using bootstrapping procedure.</p><fn id=\"tfn5\"><label>*</label><p>\n<italic>p</italic> < 0.01 Cx40 WT vs. Cx40 KO;</p></fn><fn id=\"tfn6\"><label>**</label><p>\n<italic>p</italic> < 0.05 Cx45 WT vs. Cx45 KO.</p></fn></table-wrap-foot></table-wrap><p>Previous studies have shown no difference in the measured renal vascular responses between male and female mice, and data were therefore pooled for statistical comparison (<xref rid=\"ref1\" ref-type=\"bibr\">Boesen et al., 2012</xref>; <xref rid=\"ref2\" ref-type=\"bibr\">Brasen et al., 2018</xref>; <xref rid=\"ref11\" ref-type=\"bibr\">Gohar et al., 2019</xref>; <xref rid=\"ref34\" ref-type=\"bibr\">Møller et al., 2020</xref>). <italic>p</italic> ≤ 0.05 was considered statistically significant. All values are reported as means ± SEM unless otherwise stated.</p></sec></sec><sec sec-type=\"results\" id=\"sec9\"><title>Results</title><sec id=\"sec10\"><title>Animals</title><p>Mean values of body weight, age, pre-stimulated vessel diameter (measured at 95 mmHg), and traceable vessel length are summarized in <xref rid=\"tab1\" ref-type=\"table\">Table 1</xref>. Body weights were significantly higher in Cx40 WT mice (33.1 ± 1.3 g) compared to Cx40 KO mice (27.9 ± 1.7 g, <italic>p</italic> = 0.04) and in Cx45 WT mice (27.4 ± 1.3 g) compared to Cx45 KO mice (22.5 ± 1.6 g, <italic>p</italic> = 0.007). Mice from the Cx40 strain were significantly heavier than the Cx45 strain. Age was not different within the groups; however, mice from the Cx40 strain were older than the mice from the Cx45 strain. Resting vessel diameter measured at the glomerular entrance was significantly different between Cx40 WT and Cx40 KO (<italic>p</italic> = 0.04), but not between Cx45 WT and Cx45 KO, or between groups. Maximal traceable vessel length (afferent arteriole and in some cases interlobular arteries) was not significantly different between or within groups.</p></sec><sec id=\"sec11\"><title>Afferent Arteriolar Diameter</title><p>\n<xref rid=\"fig2\" ref-type=\"fig\">Figure 2</xref> shows an afferent arteriole branching from an interlobular artery and the micropipette used to deliver the electrical pulse stimulation.</p><p>A typical recording of the afferent arteriolar diameter from a Cx40 WT mouse is shown in <xref rid=\"fig3\" ref-type=\"fig\">Figure 3A</xref> and the normalized diameter is shown in <xref rid=\"fig3\" ref-type=\"fig\">Figure 3B</xref>. The electrical stimulation (shown by the arrow at 30 s) produces strong vasoconstriction locally (0 μm). Vessel diameter is reduced by approximately 40% of its baseline diameter within the first 20 s of stimulation. Termination of the electrical stimulation (shown by the T-bar at 60 s) results in a quick return to near control diameters. Locally, diameter returned to approximately 85% of its baseline diameter within the first 10 s and by the end of the 30-s recovery, the diameter has returned to near baseline value. The magnitude of the vasoconstriction response is seen to decrease with increasing distances upstream from the stimulation site. At 400 μm, electrical stimulation induced no detectable vasoconstriction in the example shown.</p><fig id=\"fig3\" position=\"float\"><label>Figure 3</label><caption><p>Representative trace of a typical experimental recording from Cx40 WT. <bold>(A)</bold> Absolute changes in afferent arteriolar diameter as a function of time at the stimulation site (0) and at increasing distances from the stimulation site (100–400). <bold>(B)</bold> Changes in normalized afferent arteriolar diameter as a function of time (diameter was normalized by the corresponding local mean diameter prior to the electrical stimulation). The solid line (0) represents the diameter measurements at the stimulation site, while dotted/dashed lines represent upstream measurements. The arrow and T-bar indicate when electrical stimulation was initiated and stopped.</p></caption><graphic xlink:href=\"fphys-11-00961-g003\"/></fig><p>\n<xref rid=\"fig4\" ref-type=\"fig\">Figure 4</xref> shows the relative reduction in vessel diameter as a function of distance from the stimulation site in Cx40 WT and KO (<xref rid=\"fig4\" ref-type=\"fig\">Figures 4A</xref>,<xref rid=\"fig4\" ref-type=\"fig\">B</xref>). <xref rid=\"fig4\" ref-type=\"fig\">Figures 4C</xref>,<xref rid=\"fig4\" ref-type=\"fig\">D</xref> show data from Cx45 WT and KO, respectively. A data point (♦) in any of the figures represents the average of all the measurements made at that given distance ± SEM. The exponential curve fit is weighted according to the number of observations at a given distance (source code available in <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Data</xref>). Thus, data points close to the stimulation site with many observations weigh in more heavily than data points further away with fewer observations. Data for the fitted curves are shown in <xref rid=\"tab2\" ref-type=\"table\">Table 2</xref>.</p><fig id=\"fig4\" position=\"float\"><label>Figure 4</label><caption><p>Effect of electrical stimulation on afferent arteriolar diameter in WT and KO of Cx40 and Cx45 mice. Changes in afferent arteriolar diameter as a function of distance from the stimulation point. Data are constriction relative to the local diameter measured at rest. Data are presented as mean ± SEM. The lines are fits to the data points weighted according to the number of observations at each distance and fitted to an exponential model: y = ae<sup>bx</sup> (please see Methods and Materials section). Data points without error bars: <italic>n</italic> = 1. <bold>(A)</bold> Cx40 WT (<italic>n</italic> = 8) and <bold>(B)</bold> Cx40 KO (<italic>n</italic> = 7). <bold>(C)</bold> Cx45 WT (<italic>n</italic> = 9) and <bold>(D)</bold> Cx45 KO (<italic>n</italic> = 11). <sup>*</sup>\n<italic>p</italic> < 0.05 vs. 0.</p></caption><graphic xlink:href=\"fphys-11-00961-g004\"/></fig><p>Comparing measurements at the stimulation site directly between groups (one-way ANOVA) showed that relative vasoconstriction between Cx40 WT and KO (0.23 ± 0.03 vs. 0.13 ± 0.03; <italic>p</italic> < 0.05) was significantly different. Albeit numerically different, no significant difference was found between Cx45 WT and KO (0.22 ± 0.05 vs. 0.16 ± 0.03; <italic>p</italic> = 0.32). This is in line with the fitted data (<xref rid=\"tab2\" ref-type=\"table\">Table 2</xref>), where the <italic>y</italic>-intersection (<italic>a</italic>) is equivalent to the local vasoconstriction.</p><p>The decay rate (<italic>b</italic>) of the conducted vasoconstriction evaluated using bootstrapping was significantly smaller in Cx45KO compared to Cx45 WT (<italic>p</italic> < 0.05, <xref rid=\"tab2\" ref-type=\"table\">Table 2</xref>) corresponding to a larger length constant in the Cx45WT. There was no significant difference in decay rate between Cx40WT and Cx40KO although it appeared smaller in Cx40 KO.</p><p>In all groups, vasoconstriction decreases with increasing distance. Comparing the relative afferent arteriolar constriction in each group against 0, the data showed that in Cx40 WT a significant constriction was measurable up to 200 μm (<xref rid=\"fig4\" ref-type=\"fig\">Figure 4A</xref>), in Cx40 KO the distance was 50 μm (<xref rid=\"fig4\" ref-type=\"fig\">Figure 4B</xref>). In the Cx45 group, a significant constriction could be measured at 300 μm in the WT arterioles (<xref rid=\"fig4\" ref-type=\"fig\">Figure 4C</xref>) and at 250 μm in the KO (<xref rid=\"fig4\" ref-type=\"fig\">Figure 4D</xref>).</p></sec></sec><sec sec-type=\"discussions\" id=\"sec12\"><title>Discussion</title><p>The present study investigated the importance of intercellular communication <italic>via</italic> GJs, through Cx40 and Cx45, in the conduction of afferent arteriolar vasoconstriction in response to local electrical stimulation. We have previously shown that lack of Cx40 or Cx45 significantly reduces TGF (<xref rid=\"ref44\" ref-type=\"bibr\">Sorensen et al., 2012</xref>; <xref rid=\"ref34\" ref-type=\"bibr\">Møller et al., 2020</xref>). Here, we examine if this reduction is caused by a decrease in the vascular conduction of vasoconstriction. The local and conducted vasoconstrictor responses were measured during stimulation and compared to the diameter measured before stimulation. Decay of constriction was computed for the stimulation period for data pooled from each mouse strain.</p><p>VSMCs and ECs work together to coordinate and propagate constriction and dilation along the vessel wall (<xref rid=\"ref102\" ref-type=\"bibr\">Li et al., 2015</xref>). The conducted vasomotor response is important in the regulation of blood flow and resistance in vascular networks (<xref rid=\"ref105\" ref-type=\"bibr\">Gustafsson et al., 2001</xref>) and depends on GJs (<xref rid=\"ref120\" ref-type=\"bibr\">Segal et al., 1989</xref>; <xref rid=\"ref110\" ref-type=\"bibr\">Hakim et al., 2008</xref>; <xref rid=\"ref101\" ref-type=\"bibr\">Braunstein et al., 2009</xref>). In afferent arterioles, ECs are primarily coupled through Cx40 (<xref rid=\"ref100\" ref-type=\"bibr\">Arensbak et al., 2001</xref>) and VSMCs through Cx45 (<xref rid=\"ref34\" ref-type=\"bibr\">Møller et al., 2020</xref>). GJs also play a role in the signaling between ECs and VSMCs (<xref rid=\"ref104\" ref-type=\"bibr\">Emerson and Segal, 2000</xref>) through myoendothelial GJs (MEGJs; <xref rid=\"ref15\" ref-type=\"bibr\">Haefliger et al., 2004</xref>). It is unknown which Cxs are part of MEGJs in the renal vasculature (<xref rid=\"ref46\" ref-type=\"bibr\">Wagner, 2008</xref>) but Cx45 or Cx40 are likely candidates. In Cx40 KO mice, renal endothelium derived hyperpolarization was significantly reduced compared to WT, suggesting that Cx40 is part of the renal MEGJ (<xref rid=\"ref2\" ref-type=\"bibr\">Brasen et al., 2018</xref>). Also, Cx40 has been found in junctions between EC and renin secreting cells (transformed VSMC; <xref rid=\"ref14\" ref-type=\"bibr\">Haefliger et al., 2001</xref>) supporting that Cx40 could be part of the MEGJ.</p><p>Both mouse strains used in the present study have been characterized previously. The Cx40 KO was hypertensive (<xref rid=\"ref6\" ref-type=\"bibr\">de Wit et al., 2000</xref>; <xref rid=\"ref46\" ref-type=\"bibr\">Wagner, 2008</xref>; <xref rid=\"ref25\" ref-type=\"bibr\">Just et al., 2009</xref>), but RBF was similar in Cx40 WT and KO (<xref rid=\"ref25\" ref-type=\"bibr\">Just et al., 2009</xref>). Segmental arterial and afferent arteriolar diameters were not significantly different (<xref rid=\"ref44\" ref-type=\"bibr\">Sorensen et al., 2012</xref>), but heart weight and afferent wall trans-sectional area were increased in Cx40 KO mice (<xref rid=\"ref44\" ref-type=\"bibr\">Sorensen et al., 2012</xref>; <xref rid=\"ref24\" ref-type=\"bibr\">Jacobsen and Sorensen, 2015</xref>), possibly due to the hypertension. The Cx45 KO was reported to be slightly hypertensive (<xref rid=\"ref18\" ref-type=\"bibr\">Hanner et al., 2008</xref>) or normotensive (<xref rid=\"ref40\" ref-type=\"bibr\">Schmidt et al., 2012</xref>). Segmental arterial and afferent arteriolar diameters were not significantly different between Cx45 WT and KO, but wall trans-sectional area was decreased in afferent arterioles from Cx45 KO (<xref rid=\"ref34\" ref-type=\"bibr\">Møller et al., 2020</xref>). In both strains, TGF was found to be reduced in the KO (<xref rid=\"ref44\" ref-type=\"bibr\">Sorensen et al., 2012</xref>; <xref rid=\"ref34\" ref-type=\"bibr\">Møller et al., 2020</xref>), whereas the myogenic response was unaffected by the reduction in intercellular communication (<xref rid=\"ref24\" ref-type=\"bibr\">Jacobsen and Sorensen, 2015</xref>; <xref rid=\"ref34\" ref-type=\"bibr\">Møller et al., 2020</xref>).</p><p>Electrical pulse stimulation induced a local and a conducted vasoconstriction response in afferent arterioles from Cx40 and Cx45 WT and KO mice. The local response measured at the stimulation site was significantly smaller in Cx40 KO compared to Cx40 WT. However, in cremaster arterioles from Cx40 KO mice, local vasoconstriction elicited with electrical stimulation or KCl was similar to that observed in WT mice (<xref rid=\"ref10\" ref-type=\"bibr\">Figueroa et al., 2003</xref>; <xref rid=\"ref49\" ref-type=\"bibr\">Wölfle et al., 2007</xref>). As shown before, the afferent arteriolar diameter measured in the juxtaglomerular segment in Cx40 KO was smaller than in WT and smaller than the diameter measured 100 μm upstream (<xref rid=\"ref44\" ref-type=\"bibr\">Sorensen et al., 2012</xref>). This could suggest an increased baseline afferent arteriolar constriction at the site closest to the glomerulus when Cx40 is not present. Eliminating TGF did not normalize afferent arteriolar diameter, suggesting that TGF is not causing the constriction. Importantly, afferent arteriolar diameter measured upstream from the juxtaglomerular site was not significantly smaller in Cx40 KO (<xref rid=\"ref44\" ref-type=\"bibr\">Sorensen et al., 2012</xref>). No significant difference in the local vasoconstriction was found between Cx45 WT and KO even though it appeared smaller in Cx45 KO. However, this could be due to the relatively small effects being measured resulting in low statistical power. In mice lacking Cx45 in VSMC, KCl-induced local vasoconstriction was similar to the constriction elicited in WT mice (<xref rid=\"ref40\" ref-type=\"bibr\">Schmidt et al., 2012</xref>). In isolated segmental arteries from both strains, no difference was found between WT and KO when assessing the vasoconstrictor response to NE (<xref rid=\"ref2\" ref-type=\"bibr\">Brasen et al., 2018</xref>; <xref rid=\"ref34\" ref-type=\"bibr\">Møller et al., 2020</xref>). Collectively, these data suggest that the renal vascular reactivity in Cx40 and Cx45 KO is not reduced. Possibly, the decreased juxtaglomerular afferent arteriolar diameter in Cx40 KO affects the local vasoconstriction elicited at this site.</p><p>Although the decay rate of vasoconstriction seemed slightly increased in Cx40 KO, no significant difference was found between Cx40 WT and KO. This is in accordance with results obtained in skeletal muscle arterioles, where KCl-induced conducted vasoconstriction was unaffected in Cx40 KO mice (<xref rid=\"ref6\" ref-type=\"bibr\">de Wit et al., 2000</xref>). It is generally believed that electrical signals travel predominantly in the endothelial layer (<xref rid=\"ref26\" ref-type=\"bibr\">Kapela et al., 2018</xref>), which is coupled mainly by Cx40. In isolated preglomerular arteries from Cx40 KO, electrical stimulation did not elicit an increase in VSMC intracellular Ca<sup>2+</sup> measured 500 μm from the stimulation site (<xref rid=\"ref44\" ref-type=\"bibr\">Sorensen et al., 2012</xref>). However, no measurements were made at shorter distances. In addition, an electrical signal may travel further than what can be measured when using increases in intracellular Ca<sup>2+</sup> or vasoconstriction as parameters. Comparing the constriction at a given distance against 0 revealed that in Cx40 KO the constriction was only significant at 50 μm. This could be due to the smaller local constriction.</p><p>In contract, in Cx45 KO, the decay rate was significantly increased compared to WT. Possibly, Cx45 is part of the MEGJ in the afferent arteriolar wall, and its removal by conditional knockout could reduce communication between the VSMC layer and the endothelium, hence reducing the mechanical response from the VSMC layer. Myoendothelial junctions are unevenly distributed along the wall. The spread of depolarization from VSMCs that are coupled to the endothelium, to VSMCs that are not, could be central for a distant mechanical response to electrical stimulation. In addition, although the endothelium is normally considered the main path for a longitudinal spread of current in the microcirculation, the presence of VSMC-VSMC coupling by Cx45-containing GJs may also contribute. A possible proposed mechanism is through Ca<sup>2+</sup> signaling as Cx45 is known to be involved in the propagation of Ca<sup>2+</sup> waves in VSMC isolated from afferent arterioles (<xref rid=\"ref18\" ref-type=\"bibr\">Hanner et al., 2008</xref>). Comparing the constriction against 0 showed that in Cx45 KO the constriction was only significant up to 250 μm compared to 300 μm in the WT.</p><p>The structure of the vascular wall may affect both the local vasoconstriction and conduction properties. The vascular wall in renal vessels with a diameter <40 μm is thicker compared to vessels from skeletal muscles (<xref rid=\"ref22\" ref-type=\"bibr\">Hosoyamada et al., 2015</xref>), which would reduce conduction in afferent arterioles. In both KO strains, changes in wall trans-sectional area (WTA) and stress sensitivity in the afferent arteriole have been found (<xref rid=\"ref24\" ref-type=\"bibr\">Jacobsen and Sorensen, 2015</xref>; <xref rid=\"ref34\" ref-type=\"bibr\">Møller et al., 2020</xref>). However, WTA and stress sensitivity seemed to be increased in Cx40 KO, whereas it was decreased in Cx45 KO. Whether this can account for the increase in decay rate observed in Cx45 KO remains to be established. Another possibility is that deletion of Cx40 or Cx45 changes the expression of other GJs or ion channels responsible for eliciting the vasoconstriction. Deletion of Cx40 reduced expression of Cx37 in the vasculature (<xref rid=\"ref43\" ref-type=\"bibr\">Simon and McWhorter, 2003</xref>; <xref rid=\"ref5\" ref-type=\"bibr\">de Wit, 2010</xref>), which could affect the vascular response. Whether this could also be the case for vascular ion channels is currently not known.</p><p>Differences in myogenic activity at the chosen RPP of 95 mmHg may also influence the obtained results. Resting membrane potential of VSMCs in the afferent arteriole in isolated perfused rat kidneys was approximately −40 mV at 80 mmHg RPP (<xref rid=\"ref31\" ref-type=\"bibr\">Loutzenhiser et al., 1997</xref>). We assume that a comparable resting membrane potential is present in mouse afferent arterioles at an RPP of 95 mmHg. Under these conditions, the afferent arteriole has substantial myogenic tone and addition of nifedipine, an inhibitor of L-type Ca<sup>2+</sup> channels, significantly increased afferent arteriolar diameter (<xref rid=\"ref44\" ref-type=\"bibr\">Sorensen et al., 2012</xref>). In Cx40 KO mice, afferent arteriolar diameter remained constant even though RPP was increased from 75 to 95 mmHg (<xref rid=\"ref44\" ref-type=\"bibr\">Sorensen et al., 2012</xref>) and in Cx45 KO mice the same RPP increase induced a significant reduction in afferent arteriolar diameter (<xref rid=\"ref34\" ref-type=\"bibr\">Møller et al., 2020</xref>), suggesting that both strains have myogenic tone in the present experimental setup. As no differences have been found in the myogenic response between KO and WT from both strains (<xref rid=\"ref24\" ref-type=\"bibr\">Jacobsen and Sorensen, 2015</xref>; <xref rid=\"ref34\" ref-type=\"bibr\">Møller et al., 2020</xref>) it does not seem likely that differences in myogenic tone are affecting the electrically induced vascular responses observed in the present experiments.</p><p>A number of limitations are present in our experiments. Due to the experimental setup, it is not possible to position the stimulation pipette next to the vascular wall. A varying layer of tubular tissue will separate the pipette tip and the vascular wall. This limitation has been mitigated by only including arterioles with a minimum local constriction of 5%. In addition, the preparation is not fixed and the arterioles have some vasomotion that could affect the measurements. Consequently, we used an average of the diameter measured over 30 s. Vasoconstriction is not the most sensitive measure to quantify vascular conduction. Changes in membrane potential can be measured using sharp electrodes or patch-clamp (<xref rid=\"ref29\" ref-type=\"bibr\">Li et al., 2004</xref>). This method is not applicable in our preparation, where the cells of the vascular wall are not freely accessible. Another factor that should be taken into consideration is the age span of the mice (2–14 months). Laboratory mice reach sexual maturity at 8–12 weeks of age (<xref rid=\"ref9\" ref-type=\"bibr\">Dutta and Sengupta, 2016</xref>); past the age of 18 months, these mice are considered old (<xref rid=\"ref16\" ref-type=\"bibr\">Hagan, 2017</xref>). We know that age does not affect the myogenic response (<xref rid=\"ref30\" ref-type=\"bibr\">Lott et al., 2004</xref>), unless subjects are sedentary (<xref rid=\"ref21\" ref-type=\"bibr\">Hong and Lee, 2017</xref>). In WKY and SHR rats, both cortical and juxtamedullary autoregulatory activity were similar between ages 10 and 70 weeks (<xref rid=\"ref23\" ref-type=\"bibr\">Iversen et al., 1998</xref>) even though MAP increased significantly in SHR. These findings indicate that vascular function is intact at 14 months of age, and we are not aware of any study that has directly addressed the effect of aging on vascular conduction in the mouse afferent arteriole. Lastly, we have used mixed sexes. Renal autoregulation in the normal physiological state is identical in males and females (<xref rid=\"ref20\" ref-type=\"bibr\">Hilliard et al., 2011</xref>; <xref rid=\"ref3\" ref-type=\"bibr\">Brown et al., 2012</xref>) as is renal vascular responses to angiotensin II and PE (<xref rid=\"ref41\" ref-type=\"bibr\">Schneider et al., 2010</xref>). Thus, the use of mixed sexes would likely not affect the results.</p><p>In conclusion, we have shown that knockout of Cx40 reduces local vasoconstriction but does not significantly change the conduction of vasoconstriction. Vascular deletion of Cx45 significantly reduces the vascular conduction induced by electrical stimulation in afferent arterioles. Consequently, vasoconstriction in response to a comparable stimulus is conveyed shorter when intercellular communication between cells in the vascular wall is reduced in the renal microcirculation. This may in turn affect the ability to regulate and autoregulate renal perfusion.</p></sec><sec sec-type=\"data-availability\" id=\"sec13\"><title>Data Availability Statement</title><p>All datasets generated and source code for this study are included in the article/<xref ref-type=\"sec\" rid=\"sec21\">Supplementary Material</xref>.</p></sec><sec id=\"sec14\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by the Danish National Animal Experiments Inspectorate, Ministry of Environment and Food of Denmark, and Danish Veterinary and Food Administration.</p></sec><sec id=\"sec15\"><title>Author Contributions</title><p>SM and CS designed the animal experiments. SM performed animal experiments and drafted manuscript. SM, CS, and JJ analyzed data and prepared figures and tables. SM, JJ, N-HH-R, and CS interpreted results, edited and revised manuscript and approved final version of manuscript.</p><sec id=\"sec16\" sec-type=\"coi\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></sec></body><back><ack><p>This work is part of the Dynamical Systems Interdisciplinary Network, University of Copenhagen. We thank Max Salomonsson, M.D. Ph.D. for consultation on the electrical stimulation protocol. We also thank Kristoffer Racz for genotyping our connexin 45 mice.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> The study was supported by the Dynamical Systems Interdisciplinary Network, University of Copenhagen and Edith Waagens and Frode Waagens Foundation.</p></fn></fn-group><sec id=\"sec21\" sec-type=\"supplementary-material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fphys.2020.00961/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fphys.2020.00961/full#supplementary-material</ext-link>.</p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Data_Sheet_1.ZIP\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"ref100\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arensbak</surname><given-names>B.</given-names></name><name><surname>Mikkelsen</surname><given-names>H. B.</given-names></name><name><surname>Gustafsson</surname><given-names>F.</given-names></name><name><surname>Christensen</surname><given-names>T.</given-names></name><name><surname>Holstein-Rathlou</surname><given-names>N. -H.</given-names></name></person-group> (<year>2001</year>). <article-title>Expression of connexin 37, 40, and 43 mRNA and protein in renal preglomerular arterioles</article-title>. <source>Histochem. Cell Biol.</source>\n<volume>115</volume>, <fpage>479</fpage>–<lpage>487</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s004180100275</pub-id><pub-id pub-id-type=\"pmid\">11455448</pub-id></mixed-citation></ref><ref id=\"ref1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boesen</surname><given-names>E. I.</given-names></name><name><surname>Crislip</surname><given-names>G. R.</given-names></name><name><surname>Sullivan</surname><given-names>J. C.</given-names></name></person-group> (<year>2012</year>). <article-title>Use of ultrasound to assess renal reperfusion and P-selectin expression following unilateral renal ischemia</article-title>. <source>Am. J. Physiol. Ren. Physiol.</source>\n<volume>303</volume>, <fpage>F1333</fpage>–<lpage>F1340</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajprenal.00406.2012</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">22933301</pub-id></mixed-citation></ref><ref id=\"ref2\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brasen</surname><given-names>J. C.</given-names></name><name><surname>de Wit</surname><given-names>C.</given-names></name><name><surname>Sorensen</surname><given-names>C. M.</given-names></name></person-group> (<year>2018</year>). <article-title>Myoendothelial coupling through Cx40 contributes to EDH-induced vasodilation in murine renal arteries: evidence from experiments and modelling</article-title>. <source>Acta Physiol.</source>\n<volume>222</volume>, <fpage>1</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.1111/apha.12906</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">28613412</pub-id></mixed-citation></ref><ref id=\"ref101\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Braunstein</surname><given-names>T. H.</given-names></name><name><surname>Sorensen</surname><given-names>C. M.</given-names></name><name><surname>Holstein-Rathlou</surname><given-names>N. -H.</given-names></name></person-group> (<year>2009</year>). <article-title>Connexin abundance in resistance vessels from the renal microcirculation in normo- and hypertensive rats</article-title>. <source>APMIS</source>. <volume>117</volume>, <fpage>268</fpage>–<lpage>276</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1600-0463.2009.02432.x</pub-id><pub-id pub-id-type=\"pmid\">19338515</pub-id></mixed-citation></ref><ref id=\"ref3\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brown</surname><given-names>R. D.</given-names></name><name><surname>Hilliard</surname><given-names>L. M.</given-names></name><name><surname>Head</surname><given-names>G. A.</given-names></name><name><surname>Jones</surname><given-names>E. S.</given-names></name><name><surname>Widdop</surname><given-names>R. E.</given-names></name><name><surname>Denton</surname><given-names>K. M.</given-names></name></person-group> (<year>2012</year>). <article-title>Sex differences in the pressor and tubuloglomerular feedback response to angiotensin II</article-title>. <source>Hypertension</source>\n<volume>59</volume>, <fpage>129</fpage>–<lpage>135</lpage>. <pub-id pub-id-type=\"doi\">10.1161/HYPERTENSIONAHA.111.178715</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">22124434</pub-id></mixed-citation></ref><ref id=\"ref4\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Casellas</surname><given-names>D.</given-names></name><name><surname>Navar</surname><given-names>L. G.</given-names></name></person-group> (<year>1984</year>). <article-title>In vitro perfusion of juxtamedullary nephrons in rats</article-title>. <source>Am. J. Physiol. Ren. Physiol.</source>\n<volume>246</volume>, <fpage>F349</fpage>–<lpage>F358</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajprenal.1984.246.3.F349</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">6703068</pub-id></mixed-citation></ref><ref id=\"ref5\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>de Wit</surname><given-names>C.</given-names></name></person-group> (<year>2010</year>). <article-title>Different pathways with distinct properties conduct dilations in the microcirculation in vivo</article-title>. <source>Cardiovasc. Res.</source>\n<volume>85</volume>, <fpage>604</fpage>–<lpage>613</lpage>. <pub-id pub-id-type=\"doi\">10.1093/cvr/cvp340</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">19820254</pub-id></mixed-citation></ref><ref id=\"ref6\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>de Wit</surname><given-names>C.</given-names></name><name><surname>Bolz</surname><given-names>S. -S.</given-names></name><name><surname>Kirchhoff</surname><given-names>S.</given-names></name><name><surname>Krüger</surname><given-names>O.</given-names></name><name><surname>Willecke</surname><given-names>K.</given-names></name><name><surname>Pohl</surname><given-names>U.</given-names></name></person-group> (<year>2000</year>). <article-title>Impaired conduction of vasodilation along arterioles in connexin40- deficient mice</article-title>. <source>Circ. Res.</source>\n<volume>86</volume>, <fpage>649</fpage>–<lpage>655</lpage>. <pub-id pub-id-type=\"doi\">10.1161/01.RES.86.6.649</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">10747000</pub-id></mixed-citation></ref><ref id=\"ref7\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>de Wit</surname><given-names>C.</given-names></name><name><surname>Roos</surname><given-names>F.</given-names></name><name><surname>Bolz</surname><given-names>S.-S.</given-names></name><name><surname>Pohl</surname><given-names>U.</given-names></name></person-group> (<year>2003</year>). <article-title>Lack of vascular connexin 40 is associated with hypertension and irregular arteriolar vasomotion</article-title>. <source>Physiol. Genomics</source>\n<volume>13</volume>, <fpage>169</fpage>–<lpage>177</lpage>. <pub-id pub-id-type=\"doi\">10.1152/physiolgenomics.00169.2002</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">12700362</pub-id></mixed-citation></ref><ref id=\"ref8\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dora</surname><given-names>K. A.</given-names></name><name><surname>Xia</surname><given-names>J.</given-names></name><name><surname>Duling</surname><given-names>B. R.</given-names></name></person-group> (<year>2003</year>). <article-title>Endothelial cell signaling during conducted vasomotor responses</article-title>. <source>Am. J. Physiol. Heart Circ. Physiol.</source>\n<volume>285</volume>, <fpage>119</fpage>–<lpage>126</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpheart.00643.2002</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">12793976</pub-id></mixed-citation></ref><ref id=\"ref9\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dutta</surname><given-names>S.</given-names></name><name><surname>Sengupta</surname><given-names>P.</given-names></name></person-group> (<year>2016</year>). <article-title>Men and mice: relating their ages</article-title>. <source>Life Sci.</source>\n<volume>152</volume>, <fpage>244</fpage>–<lpage>248</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.lfs.2015.10.025</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">26596563</pub-id></mixed-citation></ref><ref id=\"ref104\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Emerson</surname><given-names>G. G.</given-names></name><name><surname>Segal</surname><given-names>S. S.</given-names></name></person-group> (<year>2000</year>). <article-title>Electrical coupling between endothelial cells and smooth muscle cells in hamster feed arteries</article-title>. <source>Circ. Res.</source>\n<volume>87</volume>, <fpage>474</fpage>–<lpage>479</lpage>. <pub-id pub-id-type=\"doi\">10.1161/01.RES.87.6.474</pub-id><pub-id pub-id-type=\"pmid\">10988239</pub-id></mixed-citation></ref><ref id=\"ref10\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Figueroa</surname><given-names>X. F.</given-names></name><name><surname>Paul</surname><given-names>D. L.</given-names></name><name><surname>Simon</surname><given-names>A. M.</given-names></name><name><surname>Goodenough</surname><given-names>D. A.</given-names></name><name><surname>Day</surname><given-names>K. H.</given-names></name><name><surname>Damon</surname><given-names>D. N.</given-names></name><etal/></person-group>. (<year>2003</year>). <article-title>Central role of connexin40 in the propagation of electrically activated vasodilation in mouse cremasteric arterioles in vivo</article-title>. <source>Circ. Res.</source>\n<volume>92</volume>, <fpage>793</fpage>–<lpage>800</lpage>. <pub-id pub-id-type=\"doi\">10.1161/01.RES.0000065918.90271.9A</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">12637364</pub-id></mixed-citation></ref><ref id=\"ref11\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gohar</surname><given-names>E. Y.</given-names></name><name><surname>Cook</surname><given-names>A. K.</given-names></name><name><surname>Pollock</surname><given-names>D. M.</given-names></name><name><surname>Inscho</surname><given-names>E. W.</given-names></name></person-group> (<year>2019</year>). <article-title>Afferent arteriole responsiveness to endothelin receptor activation: does sex matter?</article-title>\n<source>Biol. Sex Differ.</source>\n<volume>10</volume>:<fpage>1</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s13293-018-0218-2</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">30606254</pub-id></mixed-citation></ref><ref id=\"ref12\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Goligorsky</surname><given-names>M. S.</given-names></name><name><surname>Iijima</surname><given-names>K.</given-names></name><name><surname>Krivenko</surname><given-names>Y.</given-names></name><name><surname>Tsukahara</surname><given-names>H.</given-names></name><name><surname>Hu</surname><given-names>Y.</given-names></name><name><surname>Moore</surname><given-names>L. C.</given-names></name></person-group> (<year>1997</year>). <article-title>Role of mesangial cells in macula densa to afferent arteriole information transfer</article-title>. <source>Clin. Exp. Pharmacol. Physiol.</source>\n<volume>24</volume>, <fpage>527</fpage>–<lpage>531</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1440-1681.1997.tb01240.x</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">9248672</pub-id></mixed-citation></ref><ref id=\"ref105\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gustafsson</surname><given-names>F.</given-names></name><name><surname>Andreasen</surname><given-names>D.</given-names></name><name><surname>Salomonsson</surname><given-names>M.</given-names></name><name><surname>Jensen</surname><given-names>B. L.</given-names></name><name><surname>Holstein-Rathlou</surname><given-names>N. -H.</given-names></name></person-group> (<year>2001</year>). <article-title>Conducted vasoconstriction in rat mesenteric arterioles: role for dihydropyridine-insensitive Ca<sup>2+</sup> channels</article-title>. <source>Am. J. Physiol. Heart Circ. Physiol.</source>\n<volume>280</volume>, <fpage>H582</fpage>–<lpage>H590</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpheart.2001.280.2.H582</pub-id><pub-id pub-id-type=\"pmid\">11158955</pub-id></mixed-citation></ref><ref id=\"ref13\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gustafsson</surname><given-names>F.</given-names></name><name><surname>Holstein-Rathlou</surname><given-names>N. -H.</given-names></name></person-group> (<year>1999</year>). <article-title>Angiotensin II modulates conducted vasoconstriction to norepinephrine and local electrical stimulation in rat mesenteric arterioles</article-title>. <source>Cardiovasc. Res.</source>\n<volume>44</volume>, <fpage>176</fpage>–<lpage>184</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0008-6363(99)00174-1</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">10615401</pub-id></mixed-citation></ref><ref id=\"ref14\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haefliger</surname><given-names>J. -A.</given-names></name><name><surname>Demotz</surname><given-names>S.</given-names></name><name><surname>Braissant</surname><given-names>O.</given-names></name><name><surname>Suter</surname><given-names>E.</given-names></name><name><surname>Waeber</surname><given-names>B.</given-names></name><name><surname>Nicod</surname><given-names>P.</given-names></name><etal/></person-group>. (<year>2001</year>). <article-title>Connexins 40 and 43 are differentially regulated within the kidneys of rats with renovascular hypertension</article-title>. <source>Kidney Int.</source>\n<volume>60</volume>, <fpage>190</fpage>–<lpage>201</lpage>. <pub-id pub-id-type=\"doi\">10.1046/j.1523-1755.2001.00786.x</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">11422751</pub-id></mixed-citation></ref><ref id=\"ref15\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haefliger</surname><given-names>J. -A.</given-names></name><name><surname>Nicod</surname><given-names>P.</given-names></name><name><surname>Meda</surname><given-names>P.</given-names></name></person-group> (<year>2004</year>). <article-title>Contribution of connexins to the function of the vascular wall</article-title>. <source>Cardiovasc. Res.</source>\n<volume>62</volume>, <fpage>345</fpage>–<lpage>356</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cardiores.2003.11.015</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">15094354</pub-id></mixed-citation></ref><ref id=\"ref16\"><mixed-citation publication-type=\"other\"><person-group person-group-type=\"author\"><name><surname>Hagan</surname><given-names>C</given-names></name></person-group> (<year>2017</year>). When are mice considered old? Available at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.jax.org/news-and-insights/jax-blog/2017/november/when-are-mice-considered-old\">https://www.jax.org/news-and-insights/jax-blog/2017/november/when-are-mice-considered-old</ext-link> (Accessed April 9, 2019).</mixed-citation></ref><ref id=\"ref110\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hakim</surname><given-names>C. H.</given-names></name><name><surname>Jackson</surname><given-names>W. F.</given-names></name><name><surname>Segal</surname><given-names>S. S.</given-names></name></person-group> (<year>2008</year>). <article-title>Connexin isoform expression in smooth muscle cells and endothelial cells of hamster cheek pouch arterioles and retractor feed arteries</article-title>. <source>Microcirculation</source>\n<volume>15</volume>, <fpage>503</fpage>–<lpage>514</lpage>. <pub-id pub-id-type=\"doi\">10.1080/10739680801982808</pub-id><pub-id pub-id-type=\"pmid\">19086260</pub-id></mixed-citation></ref><ref id=\"ref17\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hanner</surname><given-names>F.</given-names></name><name><surname>Sorensen</surname><given-names>C. M.</given-names></name><name><surname>Holstein-Rathlou</surname><given-names>N. -H.</given-names></name><name><surname>Peti-Peterdi</surname><given-names>J.</given-names></name></person-group> (<year>2010</year>). <article-title>Connexins and the kidney</article-title>. <source>Am. J. Phys. Regul. Integr. Comp. Phys.</source>\n<volume>298</volume>, <fpage>R1143</fpage>–<lpage>R1155</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpregu.00808.2009</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">20164205</pub-id></mixed-citation></ref><ref id=\"ref18\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hanner</surname><given-names>F.</given-names></name><name><surname>Maltzahn</surname><given-names>J.</given-names><prefix>von</prefix></name><name><surname>Maxeiner</surname><given-names>S.</given-names></name><name><surname>Toma</surname><given-names>I.</given-names></name><name><surname>Sipos</surname><given-names>A.</given-names></name><name><surname>Krüger</surname><given-names>O.</given-names></name><etal/></person-group>. (<year>2008</year>). <article-title>Connexin45 is expressed in the juxtaglomerular apparatus and is involved in the regulation of renin secretion and blood pressure</article-title>. <source>Am. J. Phys. Regul. Integr. Comp. Phys.</source>\n<volume>295</volume>, <fpage>R371</fpage>–<lpage>R380</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpregu.00468.2007</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">18579650</pub-id></mixed-citation></ref><ref id=\"ref19\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Harrison-Bernard</surname><given-names>L. M.</given-names></name><name><surname>Cook</surname><given-names>A. K.</given-names></name><name><surname>Oliverio</surname><given-names>M. I.</given-names></name><name><surname>Coffman</surname><given-names>T. M.</given-names></name></person-group> (<year>2003</year>). <article-title>Renal segmental microvascular responses to ANG II in AT1A receptor null mice</article-title>. <source>Am. J. Physiol. Ren. Physiol.</source>\n<volume>284</volume>, <fpage>F538</fpage>–<lpage>F545</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajprenal.00340.2002</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">12429556</pub-id></mixed-citation></ref><ref id=\"ref20\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hilliard</surname><given-names>L. M.</given-names></name><name><surname>Nematbakhsh</surname><given-names>M.</given-names></name><name><surname>Kett</surname><given-names>M. M.</given-names></name><name><surname>Teichman</surname><given-names>E.</given-names></name><name><surname>Sampson</surname><given-names>A. K.</given-names></name><name><surname>Widdop</surname><given-names>R. E.</given-names></name><etal/></person-group>. (<year>2011</year>). <article-title>Gender differences in pressure-natriuresis and renal autoregulation: role of the angiotensin type 2 receptor</article-title>. <source>Hypertension</source>\n<volume>57</volume>, <fpage>275</fpage>–<lpage>282</lpage>. <pub-id pub-id-type=\"doi\">10.1161/HYPERTENSIONAHA.110.166827</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">21189402</pub-id></mixed-citation></ref><ref id=\"ref21\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hong</surname><given-names>K. -S.</given-names></name><name><surname>Lee</surname><given-names>M. -G.</given-names></name></person-group> (<year>2017</year>). <article-title>Rescue effect of exercise on impaired arteriolar myogenic response with advancing age</article-title>. <source>Exerc. Sci.</source>\n<volume>26</volume>, <fpage>8</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.15857/ksep.2017.26.1.8</pub-id></mixed-citation></ref><ref id=\"ref22\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hosoyamada</surname><given-names>Y.</given-names></name><name><surname>Ichimura</surname><given-names>K.</given-names></name><name><surname>Sakai</surname><given-names>T.</given-names></name></person-group> (<year>2015</year>). <article-title>Organ specificity and functional relevance of the arterial structure: a comparative study in the kidney and the skeletal muscle with electron microscopy</article-title>. <source>J. Vasc. Res.</source>\n<volume>52</volume>, <fpage>265</fpage>–<lpage>272</lpage>. <pub-id pub-id-type=\"doi\">10.1159/000442978</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">26824773</pub-id></mixed-citation></ref><ref id=\"ref23\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Iversen</surname><given-names>B. M.</given-names></name><name><surname>Amann</surname><given-names>K.</given-names></name><name><surname>Kvam</surname><given-names>F. I.</given-names></name><name><surname>Wang</surname><given-names>X.</given-names></name><name><surname>Ofstad</surname><given-names>J.</given-names></name></person-group> (<year>1998</year>). <article-title>Increased glomerular capillary pressure and size mediate glomerulosclerosis in SHR juxtamedullary cortex</article-title>. <source>Am. J. Phys.</source>\n<volume>274</volume>, <fpage>F365</fpage>–<lpage>F373</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajprenal.1998.274.2.F365</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">9486232</pub-id></mixed-citation></ref><ref id=\"ref24\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jacobsen</surname><given-names>J. C. B.</given-names></name><name><surname>Sorensen</surname><given-names>C. M.</given-names></name></person-group> (<year>2015</year>). <article-title>Influence of connexin40 on the renal myogenic response in murine afferent arterioles</article-title>. <source>Phys. Rep.</source>\n<volume>3</volume>, <fpage>1</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.14814/phy2.12416</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">26009638</pub-id></mixed-citation></ref><ref id=\"ref25\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Just</surname><given-names>A.</given-names></name><name><surname>Kurtz</surname><given-names>L.</given-names></name><name><surname>de Wit</surname><given-names>C.</given-names></name><name><surname>Wagner</surname><given-names>C.</given-names></name><name><surname>Kurtz</surname><given-names>A.</given-names></name><name><surname>Arendshorst</surname><given-names>W. J.</given-names></name></person-group> (<year>2009</year>). <article-title>Connexin 40 mediates the tubuloglomerular feedback contribution to renal blood flow autoregulation</article-title>. <source>J. Am. Soc. Nephrol.</source>\n<volume>20</volume>, <fpage>1577</fpage>–<lpage>1585</lpage>. <pub-id pub-id-type=\"doi\">10.1681/ASN.2008090943</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">19443640</pub-id></mixed-citation></ref><ref id=\"ref26\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kapela</surname><given-names>A.</given-names></name><name><surname>Behringer</surname><given-names>E. J.</given-names></name><name><surname>Segal</surname><given-names>S. S.</given-names></name><name><surname>Tsoukias</surname><given-names>N. M.</given-names></name></person-group> (<year>2018</year>). <article-title>Biophysical properties of microvascular endothelium: requirements for initiating and conducting electrical signals</article-title>. <source>Microcirculation</source>\n<volume>25</volume>, <fpage>1</fpage>–<lpage>15</lpage>. <pub-id pub-id-type=\"doi\">10.1111/micc.12429</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">29117630</pub-id></mixed-citation></ref><ref id=\"ref27\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krüger</surname><given-names>O.</given-names></name><name><surname>Plum</surname><given-names>A.</given-names></name><name><surname>Kim</surname><given-names>J. -S.</given-names></name><name><surname>Winterhager</surname><given-names>E.</given-names></name><name><surname>Maxeiner</surname><given-names>S.</given-names></name><name><surname>Hallas</surname><given-names>G.</given-names></name><etal/></person-group>. (<year>2000</year>). <article-title>Defective vascular development in connexin 45-deficient mice</article-title>. <source>Development</source>\n<volume>127</volume>, <fpage>4179</fpage>–<lpage>4193</lpage>., PMID: <pub-id pub-id-type=\"pmid\">10976050</pub-id></mixed-citation></ref><ref id=\"ref28\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kurtz</surname><given-names>L.</given-names></name><name><surname>Janssen-Bienhold</surname><given-names>U.</given-names></name><name><surname>Kurtz</surname><given-names>A.</given-names></name><name><surname>Wagner</surname><given-names>C.</given-names></name></person-group> (<year>2009</year>). <article-title>Connexin expression in renin-producing cells</article-title>. <source>J. Am. Soc. Nephrol.</source>\n<volume>20</volume>, <fpage>506</fpage>–<lpage>512</lpage>. <pub-id pub-id-type=\"doi\">10.1681/ASN.2008030252</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">19073828</pub-id></mixed-citation></ref><ref id=\"ref29\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>W. -C.</given-names></name><name><surname>Soffe</surname><given-names>S. R.</given-names></name><name><surname>Roberts</surname><given-names>A.</given-names></name></person-group> (<year>2004</year>). <article-title>A direct comparison of whole cell patch and sharp electrodes by simultaneous recording from single spinal neurons in frog tadpoles</article-title>. <source>J. Neurophysiol.</source>\n<volume>92</volume>, <fpage>380</fpage>–<lpage>386</lpage>. <pub-id pub-id-type=\"doi\">10.1152/jn.01238.2003</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">14999043</pub-id></mixed-citation></ref><ref id=\"ref102\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>L.</given-names></name><name><surname>Zhang</surname><given-names>W.</given-names></name><name><surname>Shi</surname><given-names>W. Y.</given-names></name><name><surname>Ma</surname><given-names>K. -T.</given-names></name><name><surname>Zhao</surname><given-names>L.</given-names></name><name><surname>Wang</surname><given-names>Y.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>The enhancement of Cx45 expression and function in renal interlobar artery of spontaneously hypertensive rats at different age</article-title>. <source>Kidney Blood Press. Res.</source>\n<volume>40</volume>, <fpage>52</fpage>–<lpage>65</lpage>. <pub-id pub-id-type=\"doi\">10.1159/000368482</pub-id><pub-id pub-id-type=\"pmid\">25791497</pub-id></mixed-citation></ref><ref id=\"ref30\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lott</surname><given-names>M. E. J.</given-names></name><name><surname>Herr</surname><given-names>M. D.</given-names></name><name><surname>Sinoway</surname><given-names>L. I.</given-names></name></person-group> (<year>2004</year>). <article-title>Effects of transmural pressure on brachial artery mean blood velocity dynamics in humans</article-title>. <source>J. Appl. Physiol.</source>\n<volume>93</volume>, <fpage>2137</fpage>–<lpage>2146</lpage>. <pub-id pub-id-type=\"doi\">10.1152/japplphysiol.00443.2002</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">12391123</pub-id></mixed-citation></ref><ref id=\"ref31\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Loutzenhiser</surname><given-names>R.</given-names></name><name><surname>Chilton</surname><given-names>L.</given-names></name><name><surname>Trottier</surname><given-names>G.</given-names></name></person-group> (<year>1997</year>). <article-title>Membrane potential measurements in renal afferent and efferent arterioles: actions of angiotensin II</article-title>. <source>Am. J. Physiol.</source>\n<volume>273</volume>, <fpage>F307</fpage>–<lpage>F314</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajprenal.1997.273.2.F307</pub-id><pub-id pub-id-type=\"pmid\">9277592</pub-id></mixed-citation></ref><ref id=\"ref32\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Marsh</surname><given-names>D. J.</given-names></name><name><surname>Toma</surname><given-names>I.</given-names></name><name><surname>Sosnovtseva</surname><given-names>O. V.</given-names></name><name><surname>Peti-Peterdi</surname><given-names>J.</given-names></name><name><surname>Holstein-Rathlou</surname><given-names>N. -H.</given-names></name></person-group> (<year>2009</year>). <article-title>Electrotonic vascular signal conduction and nephron synchronization</article-title>. <source>Am. J. Physiol. Ren. Physiol.</source>\n<volume>296</volume>, <fpage>F751</fpage>–<lpage>F761</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajprenal.90669.2008</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">19116241</pub-id></mixed-citation></ref><ref id=\"ref33\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maxeiner</surname><given-names>S.</given-names></name><name><surname>Dedek</surname><given-names>K.</given-names></name><name><surname>Janssen-Bienhold</surname><given-names>U.</given-names></name><name><surname>Ammermüller</surname><given-names>J.</given-names></name><name><surname>Brune</surname><given-names>H.</given-names></name><name><surname>Kirsch</surname><given-names>T.</given-names></name><etal/></person-group>. (<year>2005</year>). <article-title>Deletion of connexin45 in mouse retinal neurons disrupts the rod/cone signaling pathways between AII amacrine and ON cone bipolar cells and leads to impaired visual transmission</article-title>. <source>J. Neurosci.</source>\n<volume>25</volume>, <fpage>566</fpage>–<lpage>576</lpage>. <pub-id pub-id-type=\"doi\">10.1523/JNEUROSCI.3232-04.2005</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">15659592</pub-id></mixed-citation></ref><ref id=\"ref34\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Møller</surname><given-names>S.</given-names></name><name><surname>Jacobsen</surname><given-names>J. C. B.</given-names></name><name><surname>Braunstein</surname><given-names>T. H.</given-names></name><name><surname>Holstein-Rathlou</surname><given-names>N. -H.</given-names></name><name><surname>Sorensen</surname><given-names>C. M.</given-names></name></person-group> (<year>2020</year>). <article-title>Influence of connexin45 on renal autoregulation</article-title>. <source>Am. J. Physiol. Ren. Physiol.</source>\n<volume>318</volume>, <fpage>F732</fpage>–<lpage>F740</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajprenal.00185.2019</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">31984796</pub-id></mixed-citation></ref><ref id=\"ref35\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moreno</surname><given-names>A. P.</given-names></name></person-group> (<year>2004</year>). <article-title>Biophysical properties of homomeric and heteromultimeric channels formed by cardiac connexins</article-title>. <source>Cardiovasc. Res.</source>\n<volume>62</volume>, <fpage>276</fpage>–<lpage>286</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cardiores.2004.03.003</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">15094348</pub-id></mixed-citation></ref><ref id=\"ref36\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nielsen</surname><given-names>M. S.</given-names></name><name><surname>Axelsen</surname><given-names>L. N.</given-names></name><name><surname>Sorgen</surname><given-names>P. L.</given-names></name><name><surname>Verma</surname><given-names>V.</given-names></name><name><surname>Delmar</surname><given-names>M.</given-names></name><name><surname>Holstein-Rathlou</surname><given-names>N. -H.</given-names></name></person-group> (<year>2012</year>). <article-title>Gap junctions</article-title>. <source>Compr. Physiol.</source>\n<volume>2</volume>, <fpage>1981</fpage>–<lpage>2035</lpage>. <pub-id pub-id-type=\"doi\">10.1002/cphy.c110051</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">23723031</pub-id></mixed-citation></ref><ref id=\"ref37\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Palanker</surname><given-names>D.</given-names></name><name><surname>Vankov</surname><given-names>A.</given-names></name><name><surname>Freyvert</surname><given-names>Y.</given-names></name><name><surname>Huie</surname><given-names>P.</given-names></name></person-group> (<year>2008</year>). <article-title>Pulsed electrical stimulation for control of vasculature: temporary vasoconstriction and permanent thrombosis</article-title>. <source>Bioelectromagnetics</source>\n<volume>29</volume>, <fpage>100</fpage>–<lpage>107</lpage>. <pub-id pub-id-type=\"doi\">10.1002/bem.20368</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">17918191</pub-id></mixed-citation></ref><ref id=\"ref38\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Peti-Peterdi</surname><given-names>J.</given-names></name></person-group> (<year>2006</year>). <article-title>Calcium wave of tubuloglomerular feedback</article-title>. <source>Am. J. Physiol. Ren. Physiol.</source>\n<volume>291</volume>, <fpage>F473</fpage>–<lpage>F480</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajprenal.00425.2005</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">16495210</pub-id></mixed-citation></ref><ref id=\"ref39\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Salomonsson</surname><given-names>M.</given-names></name><name><surname>Gustafsson</surname><given-names>F.</given-names></name><name><surname>Andreasen</surname><given-names>D.</given-names></name><name><surname>Jensen</surname><given-names>B. L.</given-names></name><name><surname>Holstein-Rathlou</surname><given-names>N. -H.</given-names></name></person-group> (<year>2002</year>). <article-title>Local electric stimulation causes conducted calcium response in rat interlobular arteries</article-title>. <source>Am. J. Physiol. Ren. Physiol.</source>\n<volume>283</volume>, <fpage>F473</fpage>–<lpage>F480</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajprenal.00247.2001</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">12167598</pub-id></mixed-citation></ref><ref id=\"ref40\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schmidt</surname><given-names>V. J.</given-names></name><name><surname>Jobs</surname><given-names>A.</given-names></name><name><surname>Maltzahn</surname><given-names>J.</given-names><prefix>von</prefix></name><name><surname>Wörsdörfer</surname><given-names>P.</given-names></name><name><surname>Willecke</surname><given-names>K.</given-names></name><name><surname>de Wit</surname><given-names>C.</given-names></name></person-group> (<year>2012</year>). <article-title>Connexin45 is expressed in vascular smooth muscle but its function remains elusive</article-title>. <source>PLoS One</source>\n<volume>7</volume>:<fpage>e42287</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0042287</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">22848755</pub-id></mixed-citation></ref><ref id=\"ref41\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schneider</surname><given-names>M. P.</given-names></name><name><surname>Wach</surname><given-names>P. F.</given-names></name><name><surname>Durley</surname><given-names>M. K.</given-names></name><name><surname>Pollock</surname><given-names>J. S.</given-names></name><name><surname>Pollock</surname><given-names>D. M.</given-names></name></person-group> (<year>2010</year>). <article-title>Sex differences in acute ANG II-mediated hemodynamic responses in mice</article-title>. <source>Am. J. Phys. Regul. Integr. Comp. Phys.</source>\n<volume>299</volume>, <fpage>R899</fpage>–<lpage>R906</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpregu.00638.2009</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">20573987</pub-id></mixed-citation></ref><ref id=\"ref42\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schweda</surname><given-names>F.</given-names></name><name><surname>Kurtz</surname><given-names>L.</given-names></name><name><surname>de Wit</surname><given-names>C.</given-names></name><name><surname>Janssen-Bienhold</surname><given-names>U.</given-names></name><name><surname>Kurtz</surname><given-names>A.</given-names></name><name><surname>Wagner</surname><given-names>C.</given-names></name></person-group> (<year>2009</year>). <article-title>Substitution of connexin40 with connexin45 prevents hyperreninemia and attenuates hypertension</article-title>. <source>Kidney Int.</source>\n<volume>75</volume>, <fpage>482</fpage>–<lpage>489</lpage>. <pub-id pub-id-type=\"doi\">10.1038/ki.2008.637</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">19109587</pub-id></mixed-citation></ref><ref id=\"ref120\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Segal</surname><given-names>S. S.</given-names></name><name><surname>Damon</surname><given-names>D. N.</given-names></name><name><surname>Duling</surname><given-names>B. R.</given-names></name></person-group> (<year>1989</year>). <article-title>Propagation of vasomotor responses coordinates arteriolar resistances</article-title>. <source>Am. J. Physiol. Heart Circ. Physiol.</source>\n<volume>256</volume>, <fpage>H832</fpage>–<lpage>H837</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpheart.1989.256.3.h832</pub-id></mixed-citation></ref><ref id=\"ref43\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Simon</surname><given-names>A. M.</given-names></name><name><surname>McWhorter</surname><given-names>A. R.</given-names></name></person-group> (<year>2003</year>). <article-title>Decreased intercellular dye-transfer and downregulation of non-ablated connexins in aortic endothelium deficient in connexin37 and connexin40</article-title>. <source>J. Cell Sci.</source>\n<volume>116</volume>, <fpage>2223</fpage>–<lpage>2236</lpage>. <pub-id pub-id-type=\"doi\">10.1242/jcs.00429</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">12697838</pub-id></mixed-citation></ref><ref id=\"ref44\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sorensen</surname><given-names>C. M.</given-names></name><name><surname>Giese</surname><given-names>I.</given-names></name><name><surname>Braunstein</surname><given-names>T. H.</given-names></name><name><surname>Brasen</surname><given-names>J. C.</given-names></name><name><surname>Salomonsson</surname><given-names>M.</given-names></name><name><surname>Holstein-Rathlou</surname><given-names>N. -H.</given-names></name></person-group> (<year>2012</year>). <article-title>Role of connexin40 in the autoregulatory response of the afferent arteriole</article-title>. <source>Am. J. Physiol. Ren. Physiol.</source>\n<volume>303</volume>, <fpage>F855</fpage>–<lpage>F863</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajprenal.00026.2012</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">22811484</pub-id></mixed-citation></ref><ref id=\"ref45\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Steinhausen</surname><given-names>M.</given-names></name><name><surname>Endlich</surname><given-names>K.</given-names></name><name><surname>Nobiling</surname><given-names>R.</given-names></name><name><surname>Parekh</surname><given-names>N.</given-names></name><name><surname>Schütt</surname><given-names>F.</given-names></name></person-group> (<year>1997</year>). <article-title>Electrically induced vasomotor responses and their propagation in rat renal vessels in vivo</article-title>. <source>J. Physiol.</source>\n<volume>505</volume>, <fpage>493</fpage>–<lpage>501</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1469-7793.1997.493bb.x</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">9423188</pub-id></mixed-citation></ref><ref id=\"ref46\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wagner</surname><given-names>C.</given-names></name></person-group> (<year>2008</year>). <article-title>Function of connexins in the renal circulation</article-title>. <source>Kidney Int.</source>\n<volume>73</volume>, <fpage>547</fpage>–<lpage>555</lpage>. <pub-id pub-id-type=\"doi\">10.1038/sj.ki.5002720</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">18075497</pub-id></mixed-citation></ref><ref id=\"ref47\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wagner</surname><given-names>C.</given-names></name><name><surname>de Wit</surname><given-names>C.</given-names></name><name><surname>Kurtz</surname><given-names>L.</given-names></name><name><surname>Grünberger</surname><given-names>C.</given-names></name><name><surname>Kurtz</surname><given-names>A.</given-names></name><name><surname>Schweda</surname><given-names>F.</given-names></name></person-group> (<year>2007</year>). <article-title>Connexin40 is essential for the pressure control of renin synthesis and secretion</article-title>. <source>Circ. Res.</source>\n<volume>100</volume>, <fpage>556</fpage>–<lpage>563</lpage>. <pub-id pub-id-type=\"doi\">10.1161/01.RES.0000258856.19922.45</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">17255527</pub-id></mixed-citation></ref><ref id=\"ref48\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wagner</surname><given-names>A. J.</given-names></name><name><surname>Holstein-Rathlou</surname><given-names>N. -H.</given-names></name><name><surname>Marsh</surname><given-names>D. J.</given-names></name></person-group> (<year>1997</year>). <article-title>Internephron coupling by conducted vasomotor responses in normotensive and spontaneously hypertensive rats</article-title>. <source>Am. J. Physiol.</source>\n<volume>272</volume>, <fpage>F372</fpage>–<lpage>F379</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajprenal.1997.272.3.F372</pub-id><pub-id pub-id-type=\"pmid\">9087681</pub-id></mixed-citation></ref><ref id=\"ref49\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wölfle</surname><given-names>S. E.</given-names></name><name><surname>Schmidt</surname><given-names>V. J.</given-names></name><name><surname>Hoepfl</surname><given-names>B.</given-names></name><name><surname>Gebert</surname><given-names>A.</given-names></name><name><surname>Alcoléa</surname><given-names>S.</given-names></name><name><surname>Gros</surname><given-names>D.</given-names></name><etal/></person-group>. (<year>2007</year>). <article-title>Connexin45 cannot replace the function of Connexin40 in along arterioles</article-title>. <source>Circ. Res.</source>\n<volume>101</volume>, <fpage>1292</fpage>–<lpage>1299</lpage>. <pub-id pub-id-type=\"doi\">10.1161/CIRCRESAHA.107.163279</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">17932322</pub-id></mixed-citation></ref><ref id=\"ref50\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yao</surname><given-names>J.</given-names></name><name><surname>Oite</surname><given-names>T.</given-names></name><name><surname>Kitamura</surname><given-names>M.</given-names></name></person-group> (<year>2009</year>). <article-title>Gap junctional intercellular communication in the juxtaglomerular apparatus</article-title>. <source>Am. J. Physiol. Ren. Physiol.</source>\n<volume>296</volume>, <fpage>F939</fpage>–<lpage>F946</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajprenal.90612.2008</pub-id>, PMID: <pub-id pub-id-type=\"pmid\">19073638</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Neurosci</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Neurosci</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Neurosci.</journal-id><journal-title-group><journal-title>Frontiers in Neuroscience</journal-title></journal-title-group><issn pub-type=\"ppub\">1662-4548</issn><issn pub-type=\"epub\">1662-453X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32848566</article-id><article-id pub-id-type=\"pmc\">PMC7431601</article-id><article-id pub-id-type=\"doi\">10.3389/fnins.2020.00795</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Neuroscience</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Tracing Pilots’ Situation Assessment by Neuroadaptive Cognitive Modeling</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Klaproth</surname><given-names>Oliver W.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/931591/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Vernaleken</surname><given-names>Christoph</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Krol</surname><given-names>Laurens R.</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/139572/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Halbruegge</surname><given-names>Marc</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/218703/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zander</surname><given-names>Thorsten O.</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/6112/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Russwinkel</surname><given-names>Nele</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/218677/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Airbus Central R&T</institution>, <addr-line>Hamburg</addr-line>, <country>Germany</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Chair of Cognitive Modelling in Dynamic Systems, Department of Psychology and Ergonomics, Technische Universität Berlin</institution>, <addr-line>Berlin</addr-line>, <country>Germany</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Airbus Defence and Space</institution>, <addr-line>Manching</addr-line>, <country>Germany</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Zander Laboratories B.V.</institution>, <addr-line>Amsterdam</addr-line>, <country>Netherlands</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Chair of Neuroadaptive Human-Computer Interaction, Brandenburg University of Technology</institution>, <addr-line>Cottbus-Senftenberg</addr-line>, <country>Germany</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Davide Valeriani, Massachusetts Eye and Ear Infirmary and Harvard Medical School, United States</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Frederic Dehais, Institut Supérieur de l’Aéronautique et de l’Espace (ISAE-SUPAERO), France; Pietro Aricò, Sapienza University of Rome, Italy; Gianluca Di Flumeri, Sapienza University of Rome, Italy</p></fn><corresp id=\"c001\">*Correspondence: Oliver W. Klaproth, <email>oliver.klaproth@airbus.com</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Neural Technology, a section of the journal Frontiers in Neuroscience</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>14</volume><elocation-id>795</elocation-id><history><date date-type=\"received\"><day>08</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>07</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Klaproth, Vernaleken, Krol, Halbruegge, Zander and Russwinkel.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Klaproth, Vernaleken, Krol, Halbruegge, Zander and Russwinkel</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>This study presents the integration of a passive brain-computer interface (pBCI) and cognitive modeling as a method to trace pilots’ perception and processing of auditory alerts and messages during operations. Missing alerts on the flight deck can result in out-of-the-loop problems that can lead to accidents. By tracing pilots’ perception and responses to alerts, cognitive assistance can be provided based on individual needs to ensure they maintain adequate situation awareness. Data from 24 participating aircrew in a simulated flight study that included multiple alerts and air traffic control messages in single pilot setup are presented. A classifier was trained to identify pilots’ neurophysiological reactions to alerts and messages from participants’ electroencephalogram (EEG). A neuroadaptive ACT-R model using EEG data was compared to a conventional normative model regarding accuracy in representing individual pilots. Results show that passive BCI can distinguish between alerts that are processed by the pilot as task-relevant or irrelevant in the cockpit based on the recorded EEG. The neuroadaptive model’s integration of this data resulted in significantly higher performance of 87% overall accuracy in representing individual pilots’ responses to alerts and messages compared to 72% accuracy of a normative model that did not consider EEG data. We conclude that neuroadaptive technology allows for implicit measurement and tracing of pilots’ perception and processing of alerts on the flight deck. Careful handling of uncertainties inherent to passive BCI and cognitive modeling shows how the representation of pilot cognitive states can be improved iteratively for providing assistance.</p></abstract><kwd-group><kwd>situation awareness</kwd><kwd>aviation</kwd><kwd>brain-computer-interfaces</kwd><kwd>ACT-R</kwd><kwd>human-automation interaction</kwd></kwd-group><counts><fig-count count=\"5\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"70\"/><page-count count=\"13\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Irrespective of ubiquitous automation, current-generation commercial and business aircraft still rely on pilots to resolve critical situations caused, among others, by system malfunctions. Pilots need to maintain situational awareness (SA) so they can assume manual control or intervene when necessary. It is essential for flight safety that pilots understand the criticality of flight deck alerts, and do not accidentally miss alerts, e.g., due to high workload and cognitive tunneling (<xref rid=\"B18\" ref-type=\"bibr\">Dehais et al., 2014</xref>). Human-machine interfaces on the flight deck therefore need to ensure messages are processed correctly to reduce the risk of out-of-the-loop problems (<xref rid=\"B27\" ref-type=\"bibr\">Endsley and Kiris, 1995</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Berberian et al., 2017</xref>). Failed, delayed or otherwise inadequate response to flight deck alerts has been associated with several fatal accidents in the past (<xref rid=\"B1\" ref-type=\"bibr\">Air Accident Investigation and Aviation Safety Board, 2006</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Aviation Safety Council, 2016</xref>).</p><p>Automation has transformed pilots’ role from hands-on flying to monitoring system displays which is ill-matched to human cognitive capabilities (<xref rid=\"B9\" ref-type=\"bibr\">Bainbridge, 1983</xref>) and facilitates more superficial processing of information (<xref rid=\"B25\" ref-type=\"bibr\">Endsley, 2017</xref>). Furthermore, reduced-crew (e.g., single-pilot) operations can increase demands on pilots in commercial aircraft through elevated workload of remaining crew (<xref rid=\"B34\" ref-type=\"bibr\">Harris et al., 2015</xref>) and higher complexity imposed by additional automation (<xref rid=\"B8\" ref-type=\"bibr\">Bailey et al., 2017</xref>). More complex automation can impede the detection of divergence in the situation assessment by human operator and automated system, neither of which may adequately reflect reality (<xref rid=\"B57\" ref-type=\"bibr\">Rußwinkel et al., 2020</xref>). We believe that neurotechnologies can be used for cognitive enhancement and support of pilots in face of increased demands (<xref rid=\"B59\" ref-type=\"bibr\">Scerbo, 2006</xref>; <xref rid=\"B16\" ref-type=\"bibr\">Cinel et al., 2019</xref>). One way to achieve this is by monitoring the pilots’ cognitive states and performance during flight deck operations in order to detect the onset of such divergence e.g., cognitive phenomena that may lead to out-of-the-loop situations. Being able to detect such cognitive states, corrective measures may be initiated to prevent or reduce risk of out-of-the-loop situations and to maintain the high level of safety in aviation.</p><sec id=\"S1.SS1\"><title>OOTL and Situation Awareness</title><p>Out-of-the-loop problems arise when pilots lack SA (<xref rid=\"B26\" ref-type=\"bibr\">Endsley and Jones, 2011</xref>). SA is progressively developed through the levels of perception (1), comprehension (2), and projection (3) of a situation’s elements. Missing critical alerts impairs situation perception and inhibits the development of higher SA levels. In a study on pilot errors, the vast majority of errors could be accounted to incorrect perception (70.3%) and comprehension (20.3%) of situations (<xref rid=\"B35\" ref-type=\"bibr\">Jones and Endsley, 1996</xref>).</p><p>Situational awareness is commonly measured by sampling with the help of probing questions. Probes can give insights into pilots’ deeper understanding of a situation as well as whether or not a probed piece of information can be retrieved from memory. However, probing methods either require flight scenarios to be frozen (e.g., <xref rid=\"B24\" ref-type=\"bibr\">Endsley, 2000</xref>) or incur extra workload (<xref rid=\"B51\" ref-type=\"bibr\">Pierce, 2012</xref>) when assessing pilots’ SA. Physiological (e.g., <xref rid=\"B12\" ref-type=\"bibr\">Berka et al., 2006</xref>; <xref rid=\"B65\" ref-type=\"bibr\">van Dijk et al., 2011</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Di Flumeri et al., 2019</xref>) and performance-based metrics (e.g., <xref rid=\"B66\" ref-type=\"bibr\">Vidulich and McMillan, 2000</xref>) are less direct measures of memory contents, but they can be used unobtrusively in operations (see <xref rid=\"B26\" ref-type=\"bibr\">Endsley and Jones, 2011</xref>, for a summary of measures). As an example, <xref rid=\"B65\" ref-type=\"bibr\">van Dijk et al. (2011)</xref> showed how eye tracking can serve as an indicator of pilots’ perceptual and attentional processes. The abundance of visual information in the cockpit, however, makes tracing visual attention very challenging and susceptible to selective ignoring and inattentional blindness (<xref rid=\"B32\" ref-type=\"bibr\">Haines, 1991</xref>; <xref rid=\"B49\" ref-type=\"bibr\">Most et al., 2005</xref>).</p><p>Alerts in the cockpit are presented both visually and acoustically, while acoustic stimuli have shown to be more effective in attracting attention (<xref rid=\"B63\" ref-type=\"bibr\">Spence and Driver, 1997</xref>). Physiological responses to alert stimuli may reveal whether or not alerts have been perceived and processed. For example, event-related potentials (ERPs) in operators’ electroencephalogram (EEG) were proposed as indicators of attended and unattended stimuli in the assessment of SA (<xref rid=\"B23\" ref-type=\"bibr\">Endsley, 1995</xref>). <xref rid=\"B19\" ref-type=\"bibr\">Dehais et al. (2016)</xref> demonstrated that ERP components indeed allow to differentiate between missed and processed auditory stimuli in the cockpit, even in single trials (<xref rid=\"B20\" ref-type=\"bibr\">Dehais et al., 2019</xref>). They noted that these differences are primarily reflected in early perceptual and late attentional stages of auditory processing. According to <xref rid=\"B20\" ref-type=\"bibr\">Dehais et al. (2019)</xref>, failure to adequately perceive or process an alert is likely due to excessive demand to cognitive resources in terms of attention and memory at a central executive level. In addition, deterministic modeling individual processed or missed alerts requires lots of data about the situation and the pilot’s state and neurophysiological measures can help reduce uncertainty.</p><p>Thus, by monitoring what stimuli are provided when and checking for ERPs at stimulus onset, perception of a situation could be tracked in real-time (<xref rid=\"B67\" ref-type=\"bibr\">Wilson, 2000</xref>). After that, performance metrics in terms of comparing pilots’ actual behavior to normative procedures can provide information on later SA stages. In contrast to product-focused measures, this process-based approach of situation assessment (<xref rid=\"B58\" ref-type=\"bibr\">Sarter and Sarter, 2003</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Durso and Sethumadhavan, 2008</xref>) allows to also capture implicit components of SA (<xref rid=\"B24\" ref-type=\"bibr\">Endsley, 2000</xref>) that might be overlooked in SA probing.</p></sec><sec id=\"S1.SS2\"><title>Requirements for Cognitive State Assessment</title><p>As cognitive states underlying situation assessment are not directly observable, their detection and prediction in this study is approached from different angles by neurophysiological measures and cognitive modeling. Consistent monitoring of a pilot’s situation assessment in flight requires tracing what elements of a situation are perceived and processed. Tracing perceptual and cognitive processing can best be done implicitly by interpreting psycho-physiological measures so as not to increase the pilots’ load or otherwise interfere with operations. As we are interested in event-related cognitive processing, i.e., the processing of specific visual or auditory alerts, one requirement is that the onset of these alerts is captured accurately (<xref rid=\"B46\" ref-type=\"bibr\">Luck, 2014</xref>). This allows the timing of each alert to be synchronized with a measurement of the pilots’ neuroelectric activity, which is sensitive to even slight temporal misalignments. This activity can then be analyzed relative to each alert’s exact onset, allowing alert-specific cognitive states to be decoded. Such automated, non-intrusive detection of cognitive processing can be done using a passive brain-computer interface (pBCI), based on a continuous measurement of brain activity (<xref rid=\"B69\" ref-type=\"bibr\">Zander and Kothe, 2011</xref>; <xref rid=\"B40\" ref-type=\"bibr\">Krol et al., 2018</xref>).</p><p>If unprocessed alerts are detected, cognitive assistance can be offered depending on the alert’s significance for the course of the operation. In order to assess the significance of a missed alert, its impact on SA and the operation can be simulated. This way, critical drops in pilot performance can be anticipated and assistance can be provided to prevent the pilot from getting out of the loop. This simulation can be performed using cognitive models that capture the characteristics of the human cognitive system such as resource limitations.</p></sec><sec id=\"S1.SS3\"><title>Cognitive Pilot Models</title><p>ACT-R<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup> (<xref rid=\"B3\" ref-type=\"bibr\">Anderson et al., 2004</xref>) is the most comprehensive and widely used architecture to build models that can simulate, predict, and keep track of cognitive dynamics. It is based on accumulated research about the human brain’s modular architecture, where each module maps onto a different functional area of the brain. In its current 7.14 version the ACT-R architecture comprises separate modules for declarative and procedural memory, temporal, and intentional (i.e., “goal”) processing and visual, aural, motor, speech modules for limited perceptual-motor capabilities. While highly interconnected within themselves, exchange of symbolic information between modules is constrained by a small number of interfaces that are modeled as buffers (<xref rid=\"B2\" ref-type=\"bibr\">Anderson, 2007</xref>)<sup><xref ref-type=\"fn\" rid=\"footnote2\">2</xref></sup>. These intermodular connections meet in the procedural memory module (representing the caudate of the basal ganglia; <xref rid=\"B4\" ref-type=\"bibr\">Anderson et al., 2008</xref>), where condition-action statements (i.e., “productions”) are triggered depending on buffer contents. Actions can be defined for example in terms of memory retrieval, directing attention or manipulating the outside world through speech or motor actions. Based on sub-symbolic mechanisms such as utility learning, spreading activation, memory decay, and random noise, ACT-R models can adapt to dynamic environments and represent average human behavior in non-deterministic fashion.</p><p>ACT-R has frequently been used for modeling pilots’ cognitive dynamics (e.g., <xref rid=\"B15\" ref-type=\"bibr\">Byrne and Kirlik, 2005</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Gluck, 2010</xref>; <xref rid=\"B62\" ref-type=\"bibr\">Somers and West, 2013</xref>). It allows for the creation of cognitive models according to specific task descriptions, e.g., a goal-directed hierarchical task analysis (HTA; <xref rid=\"B23\" ref-type=\"bibr\">Endsley, 1995</xref>; <xref rid=\"B64\" ref-type=\"bibr\">Stanton, 2006</xref>). When this task description focuses on maintaining good SA, a normative cognitive model can be developed that acts in order to optimize SA. Normative models can be compared to individual pilot behavior to detect deviations and to make inferences about individual pilots’ SA. Tracing individual behavior (model-tracing; <xref rid=\"B30\" ref-type=\"bibr\">Fu et al., 2006</xref>) can suffer from epistemic uncertainty (<xref rid=\"B36\" ref-type=\"bibr\">Kiureghian and Ditlevsen, 2009</xref>), for example, when it is unknown why a pilot did not react to an alert. This uncertainty can be reduced by using physiological data alongside system inputs to build richer models of individual performance (<xref rid=\"B50\" ref-type=\"bibr\">Olofsen et al., 2010</xref>; <xref rid=\"B53\" ref-type=\"bibr\">Putze et al., 2015</xref>; <xref rid=\"B54\" ref-type=\"bibr\">Reifman et al., 2018</xref>). However, sensor data inaccuracies can introduce a different, aleatory kind of uncertainty that is hard to assign to individual observations and needs to be considered in design of adaptive models (<xref rid=\"B36\" ref-type=\"bibr\">Kiureghian and Ditlevsen, 2009</xref>).</p><p>ACT-R has gained popularity in modeling human autonomy interaction. The work of <xref rid=\"B53\" ref-type=\"bibr\">Putze et al. (2015)</xref> showed how an ACT-R model allows to modulate interface complexity according to operator workload measured in EEG. <xref rid=\"B10\" ref-type=\"bibr\">Ball et al. (2010)</xref> have developed a synthetic teammate able to pilot unmanned aerial vehicles and communicate with human teammates based on an extensive model of SA (see also <xref rid=\"B56\" ref-type=\"bibr\">Rodgers et al., 2013</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Freiman et al., 2018</xref>). Both these models demonstrate how selected human capabilities such as piloting and communicating (<xref rid=\"B47\" ref-type=\"bibr\">McNeese et al., 2018</xref>) or being empathic to operators’ cognitive state (<xref rid=\"B53\" ref-type=\"bibr\">Putze et al., 2015</xref>) can be allocated to an ACT-R model in human autonomy teaming.</p></sec><sec id=\"S1.SS4\"><title>Neuroadaptive Technology</title><p>Neuroadaptive technology refers to technology that uses cognitive state assessments as implicit input in order to enable intelligent forms of adaptation (<xref rid=\"B70\" ref-type=\"bibr\">Zander et al., 2016</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Krol and Zander, 2017</xref>). One way to achieve this, is to maintain a model that is continuously updated using measures of situational parameters as well as the corresponding cognitive states of the user (e.g., <xref rid=\"B41\" ref-type=\"bibr\">Krol et al., 2020</xref>). Adaptive actions can then be initiated based on the information provided by the model. Cognitive states can be assessed in different ways. Generally, certain cognitive states result, on average, in specific patterns of brain activity, and can be inferred from brain activity if the corresponding pattern distributions are known. As patterns differ to some extent between individuals and even between sessions, it is usually necessary to record multiple samples of related brain activity in order to describe the pattern distribution of cognitive responses in an individual. Given a sufficient amount of samples of a sufficiently distinct pattern, a so-called classifier can be calibrated which is capable of detecting these patterns in real time, with typical single-trial accuracies between 65 and 95% (<xref rid=\"B45\" ref-type=\"bibr\">Lotte et al., 2007</xref>).</p><p>Importantly, since these cognitive states occur as a natural consequence of the ongoing interaction, no additional effort is required, nor task load induced, for them to be made detectable. It is thus possible to use a measure of a user’s cognitive state as implicit input, referring to input that was acquired without this being deliberately communicated by the operator (<xref rid=\"B60\" ref-type=\"bibr\">Schmidt, 2000</xref>; <xref rid=\"B68\" ref-type=\"bibr\">Zander et al., 2014</xref>). Among other things, this has already been used for adaptive automation. For example, without the pilots explicitly communicating anything, a measure of their brain activity revealed indices of e.g., engagement or workload, allowing the automation to be increased or decreased accordingly (e.g., <xref rid=\"B52\" ref-type=\"bibr\">Pope et al., 1995</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Bailey et al., 2003</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Aricò et al., 2016</xref>).</p><p>In the cockpit, each alert can be expected to elicit specific cortical activity, e.g., an ERP. If this activity can be decoded to reveal whether or not the alert has been perceived, and potentially whether and how it was processed, it can be used as implicit input. Since such input can be obtained from an ongoing measurement of the pilots’ brain activity, no additional demands are placed on the pilots. By interpreting this information alongside historic pilot responses and further operational parameters, an informed decision can be made about the current cognitive state of the pilots and recommended adaptive steps.</p></sec><sec id=\"S1.SS5\"><title>Current Study</title><p>The remainder of this article describes the implementation and application of a concept for tracing individual pilots’ perception and processing of aural alerts based on neuroadaptive cognitive modeling. In contrast to conventional measures of SA, this method is designed for application in operations that require unobtrusive tracing of cognitive states. The method is applied to explore how to anticipate pilot behavior and when to offer assistance according to their cognitive state. To this end, we test (1) the feasibility of distinguishing between processed and missed alerts based on pilots’ brain activity, (2) whether individual pilot behavior can be anticipated using cognitive models, and (3) how the methods of pBCI and cognitive modeling can be integrated. Results are discussed regarding their implications for cognitive assistance on the flight deck and potential benefits for single pilot operations. Limitations are addressed to explore what else is needed in cognitive assistance for the anticipation and prevention of out-of-the-loop situations.</p></sec></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><p>This research complied with the American Psychological Association Code of Ethics and was approved by the Institutional Review Board at TU Berlin. Informed consent was obtained from each participant.</p><sec id=\"S2.SS1\"><title>Participants</title><p>Twenty-four aircrew (one female) with a mean age of 49.08 years (SD = 6.08) participated in the flight simulator study. Participants were predominantly military pilots with an average experience of 3230 h of flight (SD = 2330.71), of which on average 51.21 h (SD = 90.76) were performed in the previous year. All participating aircrew had normal or corrected to normal vision, all but two were right-handed.</p></sec><sec id=\"S2.SS2\"><title>Procedure</title><p>Participating aircrew were asked for information on their flight experience and physical health relevant for physiological data assessment in the simulator. After application of EEG sensors, participants performed a desktop-based auditory oddball training paradigm (<xref rid=\"B17\" ref-type=\"bibr\">Debener et al., 2005</xref>). Participants performed 10 blocks during each of which a sequence of 60 auditory tones was presented. Each tone could be either a standard tone of 350 Hz occurring 70–80% of the time, a target deviant tone of 650 Hz (10–15%), or non-target deviant (2000 Hz, 10–15%). There was a variable interval between stimulus onsets of 1.5 ± 0.2 s, and a self-paced break after each block. Each tone lasted 339 ms. Participants were instructed to count the target tones in each block with eyes open, and to verbally report their count after each block to ensure they stayed attentive during the task. Thus, the standard tones represent frequent but task-irrelevant events, target tones represent rare task-relevant events, and the deviants were rare but task-irrelevant.</p><p>Following this, participants were seated in the simulator and briefed on the flying task. For the flight scenario, participants were instructed to avoid communicating with the experimenter during the scenario but were allowed to think aloud and to perform readbacks of air traffic control (ATC) messages just as they would during a normal flight. After the scenario, a debriefing session was conducted in order to collect feedback from participants.</p></sec><sec id=\"S2.SS3\"><title>Simulator and Scenario</title><p>Participants flew a mission in the fixed-base cockpit simulator of a mission aircraft similar to current-generation business jets certified according to EASA CS-23, which may be operated by a single pilot. The mission was implemented and simulated using the open source flight simulation software “FlightGear 3.4”<sup><xref ref-type=\"fn\" rid=\"footnote3\">3</xref></sup>. Participants’ task was to perform a fictitious routine VIP passenger transport from Ingolstadt-Manching (ETSI) to Kassel (EDVK) airport. To keep workload levels associated with basic flying low, the scenario started with the aircraft already airborne at cruise flight level (FL 250) with autopilot (altitude and NAV<sup><xref ref-type=\"fn\" rid=\"footnote4\">4</xref></sup> mode) engaged. According to the flight management system (FMS) flight plan presented, the remaining flight time was approximately 40 min in fair weather conditions. To maintain speed, thrust had to be adjusted manually, since the aircraft was – like most business jets today – not equipped with auto-thrust. To simulate interactions with ATC and to ensure a consistent flow of the scenario for all participants, pilots were presented with pre-recorded routine ATC instructions relating to flight level and heading changes at fixed time intervals after the start of the scenario.</p><p>Also, at pre-defined times, pilots would encounter a series of flight deck alerts of varying, but generally increasing severity. First, 4 min into the scenario, the main fuel pump in the right wing tank failed, resulting in a caution level flight deck alert and, subsequently, the display of a simple recovery procedure, which was automatically presented as electronic checklist. After 6 min, a small fuel leak appeared in the right fuel tank, which had initially no salient flight deck effects and would therefore go mostly unnoticed. Contributing to this was a TCAS traffic advisory (caution level alert) after approximately 7 min, which would coincide with an ATC instruction to descend due to traffic (e.g., “F-UO<sup><xref ref-type=\"fn\" rid=\"footnote5\">5</xref></sup>, due to traffic, descend and maintain FL 280” or “F-UO, direct TUSOS and descend FL 200”). Moreover, to simulate the effects of an intermittent spurious alert, and to divert pilot attention from the FUEL format to decrease the chance of the pilot noticing the leak, an identical caution-level alert of an electrical bus system failure was triggered four times throughout the scenario. This alert would automatically be removed after 5 s without any pilot action, and before pilots were able to access the associated recovery procedure. When the fuel leak had caused a fuel imbalance exceeding a certain threshold, a caution-level alert relating to the imbalance would be raised. The associated procedure would then guide pilots through several steps intended to find the root cause of the fuel imbalance. The scenario ended once an in-flight fire of the left engine initiated after 16:40 min, resulting in a warning level alert, had successfully been extinguished by the pilot. To make sure that all participants encountered all events of the scenario, speed warnings were issued dynamically by the simulated ATC whenever airspeed did not remain within a predefined range.</p><p><xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref> gives an overview of events’ position on the flight path while <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref> shows the vertical profile including timing of events during the flight task. Normative responses to these events would result in the following respective parameter changes:</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Lateral profile of simulator task including events, waypoints, and geographic information.</p></caption><graphic xlink:href=\"fnins-14-00795-g001\"/></fig><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Vertical profile of simulator task including events, altitude in meters, and timing in seconds.</p></caption><graphic xlink:href=\"fnins-14-00795-g002\"/></fig><list list-type=\"simple\"><list-item><label>•</label><p>ATC 1: Altitude-Select 280 and Speed-Select 220.</p></list-item><list-item><label>•</label><p>ATC 2: Altitude-Select 300.</p></list-item><list-item><label>•</label><p>Fuel Pump Failure: Right-Main-Pump Off.</p></list-item><list-item><label>•</label><p>Electrical Systems Alert 1: No parameter change<sup><xref ref-type=\"fn\" rid=\"footnote6\">6</xref></sup>.</p></list-item><list-item><label>•</label><p>ATC 3: Altitude-Select 280.</p></list-item><list-item><label>•</label><p>TCAS TA-Alert: No parameter change.</p></list-item><list-item><label>•</label><p>ATC 4: Altitude-Select 300.</p></list-item><list-item><label>•</label><p>Electrical Systems Alert 2: No parameter change.</p></list-item><list-item><label>•</label><p>ATC 5: Altitude-Select 320.</p></list-item><list-item><label>•</label><p>ATC 6: Heading-Select 325.</p></list-item><list-item><label>•</label><p>Electrical Systems Alert 3: No parameter change.</p></list-item><list-item><label>•</label><p>Fuel Imbalance: Fuel-X-Feed True (not included in data analysis).</p></list-item><list-item><label>•</label><p>ATC 7: Heading-Select 350.</p></list-item><list-item><label>•</label><p>Electrical Systems Alert 4: No parameter change.</p></list-item></list></sec><sec id=\"S2.SS4\"><title>EEG</title><p>Electroencephalogram was recorded continuously at 500 Hz using a mobile, wireless LiveAmp amplifier (Brain Products, Gilching, Germany) using 32 active Ag/AgCl electrodes arranged on actiCAP caps according to the international 10–20 system and referenced to FCz. EEG was synchronized with both the desktop stimuli and the flight events using the Lab Streaming Layer (<xref rid=\"B38\" ref-type=\"bibr\">Kothe, 2014</xref>) software framework to ensure that EEG data could be related to the respective simulator events with adequate temporal resolution. In particular, FlightGear was configured to log the status of each of the alarms and send it at 100 Hz to a UDP port, where a custom Python script listened for incoming data and immediately forwarded each packet through LSL. A change in alert status could then be interpreted as the on- or offset of the alert.</p></sec><sec id=\"S2.SS5\"><title>ERP Classification</title><p>A windowed-means classifier (<xref rid=\"B13\" ref-type=\"bibr\">Blankertz et al., 2011</xref>) was calibrated on the EEG data recorded for each individual participant during the oddball paradigm to distinguish between their neurophysiological response to two different categories of tones. Features were the mean amplitudes of eight consecutive non-overlapping time windows of 50 ms each starting at 150 ms following onset of the auditory tone, after band-pass filtering the signal between 0.3 and 20 Hz. Shrinkage-regularized linear discriminant analysis was used to separate the classes. A fivefold cross-validation with margins of five was used to obtain estimates of the classifier’s parameters and accuracy. We focused on distinguishing between standard versus target tones, i.e., task-irrelevant versus task-relevant events. The classification algorithm was implemented using BCILAB (<xref rid=\"B39\" ref-type=\"bibr\">Kothe and Makeig, 2013</xref>).</p><p>The trained classifier was optimally capable of distinguishing between the two categories of tones based solely on the participant’s brain activity following each tone’s onset. Having trained the classifier on detecting differences between these events in an abstract oddball task, we then applied the classifier to the data recorded during that same participant’s flight. This thus allowed us to investigate to what extent flight deck alerts could be reliably identified as the comparable equivalent of “standard” (task-irrelevant, unimportant) or “target” (etc.) tones, based solely on the pilots’ EEG data less than 1 second after onset of each event. For each simulated flight event, the classifier returned a number between 1 and 2, signifying that the neurophysiological response was closest to the activity following standard (1) or target (2) tones in the oddball paradigm, respectively.</p></sec><sec id=\"S2.SS6\"><title>Cognitive Model</title><p>A normative and a neuroadaptive cognitive model were created following a HTA performed with a subject matter expert for the flight scenario using ACT-R. For the HTA and the cognitive model, good SA level 1 was defined as perceiving and paying attention to all auditory stimuli provided in the scenario. While adequacy of responses depended on the type of alert or contents of ATC messages, the time limit for initiating a first reaction to an alert was set to 25 s for all events. As the spurious electrical bus alerts disappeared before pilots were able to react, they are not included in the analysis of this article. The interface between the models and the simulator/Flight Gear was implemented as an extended version of ACT-CV (<xref rid=\"B33\" ref-type=\"bibr\">Halbrügge, 2013</xref>), where log files of cockpit system states recorded with a sampling rate of 20 Hz served as ACT-R task environment.</p><p>Both normative and neuroadaptive model were based on a routine loop consisting of monitoring flight parameters and managing thrust accordingly in order to have comparable workload as participants in the simulator; however, cognitively plausible modeling of workload and accuracy in thrust management was beyond the focus of this study and therefore not evaluated. The routine loop was temporarily exited when an aural alert was perceived. The normative model shifted its attention to read the warning message and initiate the corresponding procedure.</p><p>In order to illustrate the model’s flow of information from one module to another with respect to ACT-R’s neuroanatomical assumptions, associated brain areas as described by <xref rid=\"B4\" ref-type=\"bibr\">Anderson et al. (2008)</xref> and <xref rid=\"B14\" ref-type=\"bibr\">Borst et al. (2013)</xref> will be given in parentheses behind each module. The validation of activity predicted by the model with brain imaging data was beyond the scope of this article. For example of the fuel pump failure alert the model would go through the following steps: (1) a chunk representing a sound activates the aural module (mapped to the superior temporal gyrus) by being put in the model’s aural-location buffer. (2) Next, this information allows the procedural module (basal ganglia) to fire a production that starts counting seconds passed since the alert with the temporal module and that decodes the sound as an alert sound using the aural buffer. This latter information would trigger productions that (3) make the model shift its visual attention to the warning display by calling on the visual module’s (fusiform gyrus) visual-location buffer and (4) read the written fuel pump failure message using the visual buffer. (5) The following production would result in calling up the corresponding pump failure checklist, memorizing its first item (i.e., pressing the right main fuel pump pushbutton) in the imaginal buffer (intraparietal sulcus, representing the model’s short-term memory problem state). (6) Then, using its motor module (precentral gyrus), the model acts as if pressing the pump pushbutton (without changing any of the flight parameters) before (7) reading and carrying out the remaining checklist items in the same fashion while it keeps counting. (8) Finally when the count in the temporal module has reached 25 s, the module checks the flight parameters for the state of the right main fuel pump’s pushbutton to verify whether the pilot has carried out the action required by the first checklist item as memorized in the model’s imaginal buffer.</p><p>As the normative model assumed that pilots will correctly process each alert, adequate responses were scored as correct and inadequate (i.e., commission errors) as well as lacking and too late responses (i.e., omission errors) as incorrect classification of behavior. Adequacy and timeliness of responses were scored according to criteria assessed in the HTA with subject matter experts. For example, if an ATC message requested a flight level change to 300, entering an altitude-select of 300 in the flight control unit within a time window of 25 s was scored as good performance; all other responses such as entering an altitude-select of 280 or entering the correct altitude-select after 25 s were classified as missed ATC message. The fraction of incorrect classifications was treated as epistemic uncertainty (μ<sub>Epistemic</sub>) as the model had no information about why the pilot did not respond as expected.</p><p>The neuroadaptive model considered individual brain activity when classifying behavior to reduce this uncertainty. pBCI data were provided to the model along with the cockpit systems data. After each acoustic alert and message was decoded, the neuroadaptive model checked if the sound was processed as task-relevant by the participant according to pBCI data before shifting its visual attention to read the alert’s or message’s actual content. To build and improve on the normative model’s accuracy, the neuroadaptive model assumed that alerts will be processed correctly. If pBCI data showed that a message was processed as irrelevant (classifier output <1.5), the model scored lacking or inadequate responses as correct behavior classification. If the message was processed as relevant but no adequate response can be found, the model scored its classification as incorrect and treats these cases as epistemic uncertainty.</p><p>Responses were assessed for 10 events for each of the 21 pilots whereof eight ATC messages, one amber, and one red alert. Model accuracies were computed across participants as the fraction of correct classifications in all events. Normative and neuroadaptive model were compared by a paired samples <italic>t</italic>-test. Effect size is reported as Cohen’s <italic>d</italic><sub>av</sub> (<xref rid=\"B43\" ref-type=\"bibr\">Lakens, 2013</xref>). Aleatory uncertainty (μ<sub>Aleatory</sub>) was defined as one minus EEG classifier accuracy. Though aleatory uncertainty affects correct and incorrect classifications, an accuracy corrected for aleatory uncertainty was computed for the neuroadaptive model. The distribution of lacking and inadequate responses was tested for a relationship with EEG classifications by a Chi-square test. A detailed description of the cognitive model including the overall approach and modeling decisions made can be found in <xref rid=\"B37\" ref-type=\"bibr\">Klaproth et al. (2020)</xref>.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>ERP Classification</title><p><xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref> shows the grand-average ERPs on channel Pz for the standard and target tones during the oddball experiment on three electrode sites. Note that there is a delay. We had previously estimated our stimulus presentation pipeline to contain a lag of approximately 150 ms. This would coincide with the common interpretation that the initial negative peak visible in these plots is the N100.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>The grand-average ERP of 21 participants showing responses to target and standard tones on channel Cz. Shaded area indicates the standard error of the mean.</p></caption><graphic xlink:href=\"fnins-14-00795-g003\"/></fig><p>The classifier was trained to detect the differences between single-trial ERPs using all 32 channels and had a cross-validated averaged accuracy of 86%. Given the class imbalance between the standard deviant tones, chance level was not at 50% for this binary classifier. Instead, significant classification accuracy (<italic>p</italic> < 0.05) is reached at 78%. The classes could be separated with significant accuracy for all but three participants. This was in part due to technical issues with the EEG recording. These three participants were excluded from further analysis.</p><p>The classifier trained on data from the oddball paradigm was subsequently applied to data following four flight events: ATC messages, the spurious electrical bus system failure alert, the fuel imbalance alert, and the fire alert. These classification results provided information to be used in the neuroadaptive cognitive model.</p></sec><sec id=\"S3.SS2\"><title>Cognitive Model</title><p>The normative model correctly described participants’ behavior for 162 of the total 210 observed events (<italic>M</italic><sub>Normative</sub> = 0.72, SD = 0.09), indicating that participants missed to respond to 48 events. The neuroadaptive model was able to simulate 182 of participant’s responses correctly (<italic>M</italic><sub>Neuroadaptive</sub> = 0.87, SD = 0.13, see <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>), resulting in a significant added value of including pBCI data compared to the normative model [<italic>t</italic>(20) = 5.62, <italic>p</italic> < 0.01, <italic>d</italic><sub>av</sub> = 1.3]. <xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref> shows the respective models’ accuracies for each of the 21 pilots.</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Mean model accuracies, error bars indicate standard deviations across participants.</p></caption><graphic xlink:href=\"fnins-14-00795-g004\"/></fig><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Mean model accuracies per model and participant.</p></caption><graphic xlink:href=\"fnins-14-00795-g005\"/></fig><p>Epistemic uncertainties for the models are μ<sub>Epistemic</sub> = 0.28 for the normative and μ<sub>Epistemic</sub> = 0.13 for the neuroadaptive model. The added value of the neuroadaptive over the normative model is 0.15, so the neuroadaptive model’s accuracy corrected for EEG-classifier accuracy of 0.88 is 0.85 with μ<sub>Epistemic</sub> = 0.15 and μ<sub>Aleatory</sub> = 0.02.</p><p>Of the 58 events left unexplained by the normative model, 22 events did not show a response to the respective alert or message and 36 showed an incorrect response by the participant. Chi-square tests yielded no significant relationship between EEG classifier output (standard/target) and the event having missing or incorrect responses [χ<sup>2</sup>(1, <italic>N</italic> = 58) = 1.04, <italic>p</italic> = 0.31), i.e., pBCI-data do not predict whether a participant will respond incorrectly or not at all to missed alerts.</p></sec></sec><sec id=\"S4\"><title>Discussion</title><p>The use of increasingly complex and less traceable automation can result in out-of-the loop situations thanks to different assessment of situations by pilot and automated system. Results of this study have demonstrated the feasibility of implicitly detecting and handling of emerging divergence in situation assessment with the help of a neuroadaptive cognitive model.</p><p>Using a pBCI for real-time assessment of cognitive responses evoked by events in the cockpit provides insight into subjective situational interpretations. Such information is highly dependent on the context sensitive, individual state of the operator and can hardly, if at all, be inferred by purely behavioral or environmental measures. In general, we conclude that the combination of pBCI approaches with advanced methods of cognitive modeling, leads to an increase in the reliability and capability of the resulting cognitive model – introducing the idea of neuroadaptive cognitive modeling – as shown in this study.</p><p>Specifically, the ERP produced by the oddball paradigm shows clear differences between the different categories of tones. In particular, a P300 at Pz clearly distinguishes between target (task-relevant) and standard (task-irrelevant) tones. Based on these differences in single-trial event-related activity, the classifier was capable of distinguishing between target and standard tones with single-trial accuracies significantly higher than chance in the training session.</p><p>The improvement in the cognitive model that resulted from including the pBCI output indicates that it is possible to obtain informative cognitive state information based on a pilot’s brain activity immediately following an auditory event. The fact that the classifier decoding this information was trained in a desktop setting demonstrates that no elaborate training sessions are required.</p><p>Normative model results suggest that individual pilot behavior can be traced and anticipated by a cognitive model. By comparing individual pilots’ actions to the normative model behavior, deviations could be detected and inferences about SA could be made without intruding the task (<xref rid=\"B66\" ref-type=\"bibr\">Vidulich and McMillan, 2000</xref>). Twenty-eight percent of epistemic uncertainty, with lacking and incorrect responses evenly distributed, indicate that additional diagnostic information is required for meaningful analysis and support in cases of deviating behavior.</p><p>The improvement in accuracy for the neuroadaptive model demonstrate how individual behavior models can benefit from the integration of physiological data. Not only can top-down modeling of human cognition in a task be complemented by bottom-up integration of (neuro-) behavioral data for example to account for behavioral moderators (e.g., <xref rid=\"B55\" ref-type=\"bibr\">Ritter et al., 2004</xref>), it can also provide contextual information required for situation-dependent interpretation of EEG data. The different types of uncertainties inherent to model tracing and pBCI determined the model’s systematic design: pBCI data could only be used to reduce the fraction of the normative model’s unexplained behavior to deal with aleatory uncertainty.</p><p>The method’s limitations are quantified in terms of uncertainty. Later SA stages need to be monitored to increase accuracy in pilot modeling. Measures of additional physiological indicators might be connected in line to further reduce both epistemic uncertainty with new types of information, and aleatory with joint probability distributions. For example, gaze data such as visual search behavior in response to alerts could be indicative of comprehension problems and reinforce or challenge pBCI classifications of alerts being perceived or not. Other indicators, for example the error-related negativity component of the ERP, could help to identify situations where operators have low comprehension or are out of the loop (<xref rid=\"B11\" ref-type=\"bibr\">Berberian et al., 2017</xref>).</p><p>Any cockpit application of passive BCI technology requires a thorough consideration regarding the intrusiveness of the measurement, the intended function(s) enabled by the BCI, as well as the safety and airworthiness implications associated with this function. The intrusiveness perceived by pilots will mainly depend on how well the (dry) EEG electrodes can be integrated for example into the interior lining of a pilot helmet or the headband of a headset. The intended cockpit (assistance) function, in turn, will mainly determine the airworthiness certification and associated validation effort required.</p><p>If the system described in this article is merely be used to enhance the efficiency of the already certified flight deck alerting system of an aircraft, the design assurance level required from an airworthiness and safety perspective could be lower compared to a solution where a passive BCI-based cockpit function is an integral part of the aircraft’s safety net. In the latter case, the airworthiness effort will be substantial irrespective of whether AI and/or machine learning are used or not. Although evaluated offline after data collection, the methods presented in this paper are well-suited to be applied online without substantial modifications. While the abstract oddball task can replace more realistic alternatives to gather training data, and thus substantially shorten the amount of time required to do so, it may still be necessary to gather new training data before each flight due to the natural non-stationarity present in EEG activity. For a truly walk-up-and-use neuroadaptive solution, a subject-independent classifier would be required (e.g., <xref rid=\"B28\" ref-type=\"bibr\">Fazli et al., 2009</xref>). Monitoring pilots’ ERPs in response to alerts gives diagnostic value. Detection of inattentional deafness in early, perceptual ERP components could trigger communication of the alert in alternative modalities (e.g., tactile or visual; <xref rid=\"B44\" ref-type=\"bibr\">Liu et al., 2016</xref>). For unattended alerts detected in later ERP components, cockpit automation could prioritize and choose to postpone reminders in case of minor criticality. Withholding information that is not alert-related can be effective in forcing pilots’ attention onto the alert, but it may be accompanied by decrease in pilots’ authority and associated risks, for example to resilience in unexpected situations and technology acceptance.</p><p>The simulator setting likely introduced biases in task engagement and density of events in the scenario. Measuring system input from pilots while they monitor instruments in real flight conditions may not provide enough data to make inferences about cognitive states. This emphasizes the need for additional behavioral measures (e.g., neurophysiological activity, speech, or gaze) to provide individual assistance.</p><p>Pilots are capable of anticipating complex system behavior but reports of automation surprises and out-of-the-loop situations stress the importance of a shared understanding of situations by pilot and cockpit automation. Increasing complexity of automation should therefore go together with a paradigm shift toward human-autonomy teaming based on a shared understanding of the situation. This includes bi-directional communication whenever a significant divergence in the understanding of a situation occurs to provide information missing for shared awareness of the human autonomy team (<xref rid=\"B61\" ref-type=\"bibr\">Shively et al., 2017</xref>). Anticipation of divergences and understanding human information needs to ensure shared awareness remains a challenge for human autonomy teaming (<xref rid=\"B47\" ref-type=\"bibr\">McNeese et al., 2018</xref>). By addressing divergences in human and autonomy situation assessment, critical situations might be prevented or at least resolved before they result in incidents or accidents. Tracing pilots’ perception of cockpit events represents a first step toward this goal.</p></sec><sec id=\"S5\"><title>Conclusion</title><p>A pBCI allows to implicitly monitor whether pilots have correctly processed alerts or messages without intruding the mission using a classifier trained in a desktop setting. The integration of pBCI data in cognitive pilot models significantly improves the accuracy in following up with pilots’ situation assessment. Tracing pilots’ situation assessment through neuroadaptive cognitive modeling may facilitate the early detection of divergences in situation assessment in human autonomy teams. While sensor obtrusiveness and computational limitations may obstruct application, neuroadaptive cognitive modeling could help to tracing of pilots’ situation awareness and enable adaptive alerting.</p></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>The datasets presented in this article were mainly collected using aircrew employed by Airbus. For privacy and confidentiality reasons, they are not readily available; requests to address the datasets should be directed to oliver.klaproth@airbus.com.</p></sec><sec id=\"S7\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by the Ethik-Kommission Fakultät V – Verkehrs – und Maschinensysteme Institut für Psychologie und Arbeitswissenschaft TU Berlin. The patients/participants provided their written informed consent to participate in this study.</p></sec><sec id=\"S8\"><title>Author Contributions</title><p>OK, CV, LK, TZ, and NR designed the experiment. CV designed the flight task. LK and TZ designed the EEG trainings. OK and NR created the cognitive model. MH created the interface between flight simulator data and ACT-R. OK, CV, and LK performed the experiments and drafted the manuscript. OK and LK analyzed the data. OK, CV, LK, MH, TZ, and NR edited, revised, and approved the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>OK was employed by Airbus Central R&T. CV was employed by Airbus Defence and Space. LK and TZ were employed by Zander Laboratories B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>The authors thank Brain Products GmbH (Gilching, Germany) for supporting this research by providing an additional LiveAmp system including electrodes. Furthermore, the authors would like to thank Dr. Daniel Dreyer, Christophe Chavagnac, and all participating air crew for their fantastic support.</p></ack><fn-group><fn id=\"footnote1\"><label>1</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://act-r.psy.cmu.edu/\">http://act-r.psy.cmu.edu/</ext-link></p></fn><fn id=\"footnote2\"><label>2</label><p>The “two streams hypothesis” (<xref rid=\"B48\" ref-type=\"bibr\">Milner and Goodale, 2008</xref>) is implemented for the visual and aural module, resulting in a visual- and aural-location buffer for the where-pathway and visual and aural buffers for the what-pathway in the respective modules.</p></fn><fn id=\"footnote3\"><label>3</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://home.flightgear.org/\">http://home.flightgear.org/</ext-link></p></fn><fn id=\"footnote4\"><label>4</label><p>NAV mode is a managed lateral navigation mode of the autoflight system in which the aircraft follows the flight plan programmed in the FMS.</p></fn><fn id=\"footnote5\"><label>5</label><p>Abbreviated callsign, spoken FOXTROT UNIFORM OSCAR.</p></fn><fn id=\"footnote6\"><label>6</label><p>The spurious electrical alert would vanish by itself irrespective of any flight crew action; the normative response to a TCAS TA alert is to visually acquire the intruding aircraft and to prepare for a subsequent evasive maneuver, should a so-called “Resolution Advisory (RA)” alert follow.</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><collab>Air Accident Investigation and Aviation Safety Board</collab> (<year>2006</year>). <source><italic>Aircraft Accident Report Helios Airways Flight HCY522 Boeing 737-31S at Grammitko, Hellas on 14 August 2005.</italic></source> Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.aaiu.ie/sites/default/files/Hellenic%20Republic%20Accident%20Helios%20Airways%20B737-31S%20HCY522%20Grammatiko%20Hellas%202005-085-14.pdf\">http://www.aaiu.ie/sites/default/files/Hellenic%20Republic%20Accident%20Helios%20Airways%20B737-31S%20HCY522%20Grammatiko%20Hellas%202005-085-14.pdf</ext-link>\n<comment>(accessed December 19, 2019)</comment>.</mixed-citation></ref><ref id=\"B2\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Anderson</surname><given-names>J. R.</given-names></name></person-group> (<year>2007</year>). <source><italic>Oxford Series On Cognitive Models And Architectures. How Can The Human Mind Occur In The Physical Universe?.</italic></source>\n<publisher-loc>Oxford</publisher-loc>: <publisher-name>Oxford University Press</publisher-name>.</mixed-citation></ref><ref id=\"B3\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Anderson</surname><given-names>J. R.</given-names></name><name><surname>Bothell</surname><given-names>D.</given-names></name><name><surname>Byrne</surname><given-names>M. D.</given-names></name><name><surname>Douglass</surname><given-names>S.</given-names></name><name><surname>Lebiere</surname><given-names>C.</given-names></name><name><surname>Qin</surname><given-names>Y.</given-names></name></person-group> (<year>2004</year>). <article-title>An integrated theory of the mind.</article-title>\n<source><italic>Psychol. Rev.</italic></source>\n<volume>111</volume>\n<fpage>1036</fpage>–<lpage>1060</lpage>. <pub-id pub-id-type=\"doi\">10.1037/0033-295X.111.4.1036</pub-id>\n<pub-id pub-id-type=\"pmid\">15482072</pub-id></mixed-citation></ref><ref id=\"B4\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Anderson</surname><given-names>J. R.</given-names></name><name><surname>Fincham</surname><given-names>J. M.</given-names></name><name><surname>Qin</surname><given-names>Y.</given-names></name><name><surname>Stocco</surname><given-names>A.</given-names></name></person-group> (<year>2008</year>). <article-title>A central circuit of the mind.</article-title>\n<source><italic>Trends Cogn. Sci.</italic></source>\n<volume>12</volume>\n<fpage>136</fpage>–<lpage>143</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.tics.2008.01.006</pub-id>\n<pub-id pub-id-type=\"pmid\">18329948</pub-id></mixed-citation></ref><ref id=\"B5\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aricò</surname><given-names>P.</given-names></name><name><surname>Borghini</surname><given-names>G.</given-names></name><name><surname>Di Flumeri</surname><given-names>G.</given-names></name><name><surname>Colosimo</surname><given-names>A.</given-names></name><name><surname>Bonelli</surname><given-names>S.</given-names></name><name><surname>Golfetti</surname><given-names>A.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>Adaptive automation triggered by EEG-based mental workload index: a passive brain-computer interface application in realistic air traffic control environment.</article-title>\n<source><italic>Front. Hum. Neurosci.</italic></source>\n<volume>10</volume>:<issue>539</issue>. <pub-id pub-id-type=\"doi\">10.3389/fnhum.2016.00539</pub-id>\n<pub-id pub-id-type=\"pmid\">27833542</pub-id></mixed-citation></ref><ref id=\"B6\"><mixed-citation publication-type=\"journal\"><collab>Aviation Safety Council</collab> (<year>2016</year>). <source><italic>Aviation Occurrence Report: 4 February, 2015, TransAsia Airways Flight GE235 ATR72-212A: Loss of Control and Crashed into Keelung River Three Nautical Miles East of Songshan Airport. Aviation Occurrence Report ASC-AOR-16-06-001.</italic></source> Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.asc.gov.tw/upload/acd_att/ASC-AOR-16-06-001%20EN.pdf\">https://www.asc.gov.tw/upload/acd_att/ASC-AOR-16-06-001%20EN.pdf</ext-link>\n<comment>(accessed December 19, 2019)</comment>.</mixed-citation></ref><ref id=\"B7\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Bailey</surname><given-names>N. R.</given-names></name><name><surname>Scerbo</surname><given-names>M. W.</given-names></name><name><surname>Freeman</surname><given-names>F. G.</given-names></name><name><surname>Mikulka</surname><given-names>P. J.</given-names></name><name><surname>Scott</surname><given-names>L. A.</given-names></name></person-group> (<year>2003</year>). “<article-title>A brain-based adaptive automation system and situation awareness: The role of complacency potential</article-title>,” in <source><italic>Proceedings of the Human Factors and Ergonomics Society 47th Annual Meeting</italic></source>, <publisher-loc>Santa Monica, CA</publisher-loc>.</mixed-citation></ref><ref id=\"B8\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Bailey</surname><given-names>R. E.</given-names></name><name><surname>Kramer</surname><given-names>L. J.</given-names></name><name><surname>Kennedy</surname><given-names>K. D.</given-names></name><name><surname>Stephens</surname><given-names>C. L.</given-names></name><name><surname>Etherington</surname><given-names>T. J.</given-names></name></person-group> (<year>2017</year>). “<article-title>An assessment of reduced crew and single pilot operations in commercial transport aircraft operations</article-title>,” in <source><italic>Proceedings of the IEEE/AIAA 36th Digital Avionics Systems Conference (DASC)</italic></source>, <publisher-loc>St. Petersburg, FL</publisher-loc>.</mixed-citation></ref><ref id=\"B9\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Bainbridge</surname><given-names>L.</given-names></name></person-group> (<year>1983</year>). “<article-title>Ironies of automation</article-title>,” in <source><italic>Automatica: 19.1983,6. Analysis, Design and Evaluation Of Man-Machine Systems: Control Frontiers in Knowledge Based And Man-Machine Systems</italic></source>, <role>ed.</role>\n<person-group person-group-type=\"editor\"><name><surname>Johannsen</surname><given-names>G.</given-names></name></person-group> (<publisher-loc>Oxford</publisher-loc>: <publisher-name>Pergamon Press</publisher-name>), <fpage>129</fpage>–<lpage>135</lpage>. <pub-id pub-id-type=\"doi\">10.1016/B978-0-08-029348-6.50026-9</pub-id></mixed-citation></ref><ref id=\"B10\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ball</surname><given-names>J.</given-names></name><name><surname>Myers</surname><given-names>C.</given-names></name><name><surname>Heiberg</surname><given-names>A.</given-names></name><name><surname>Cooke</surname><given-names>N. J.</given-names></name><name><surname>Matessa</surname><given-names>M.</given-names></name><name><surname>Freiman</surname><given-names>M.</given-names></name><etal/></person-group> (<year>2010</year>). <article-title>The synthetic teammate project.</article-title>\n<source><italic>Comput. Math. Organ. Theor.</italic></source>\n<volume>16</volume>\n<fpage>271</fpage>–<lpage>299</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s10588-010-9065-3</pub-id></mixed-citation></ref><ref id=\"B11\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Berberian</surname><given-names>B.</given-names></name><name><surname>Somon</surname><given-names>B.</given-names></name><name><surname>Sahaï</surname><given-names>A.</given-names></name><name><surname>Gouraud</surname><given-names>J.</given-names></name></person-group> (<year>2017</year>). <article-title>The out-of-the-loop brain: a neuroergonomic approach of the human automation interaction.</article-title>\n<source><italic>Ann. Rev. Control</italic></source>\n<volume>44</volume>\n<fpage>303</fpage>–<lpage>315</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.arcontrol.2017.09.010</pub-id></mixed-citation></ref><ref id=\"B12\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Berka</surname><given-names>C.</given-names></name><name><surname>Levendowski</surname><given-names>D. J.</given-names></name><name><surname>Davis</surname><given-names>G.</given-names></name><name><surname>Whitmoyer</surname><given-names>M.</given-names></name><name><surname>Hale</surname><given-names>K.</given-names></name><name><surname>Fuchs</surname><given-names>K.</given-names></name></person-group> (<year>2006</year>). “<article-title>Objective measures of situational awareness using neurophysiology technology</article-title>,” in <source><italic>Augmented Cognition: Past, Present and Future</italic></source>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>Schmorrow</surname><given-names>D.</given-names></name><name><surname>Stanney</surname><given-names>K.</given-names></name><name><surname>Reeves</surname><given-names>L.</given-names></name></person-group> (<publisher-loc>Arlington, VA</publisher-loc>: <publisher-name>Strategic Analysis Inc</publisher-name>), <fpage>145</fpage>–<lpage>154</lpage>.</mixed-citation></ref><ref id=\"B13\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Blankertz</surname><given-names>B.</given-names></name><name><surname>Lemm</surname><given-names>S.</given-names></name><name><surname>Treder</surname><given-names>M.</given-names></name><name><surname>Haufe</surname><given-names>S.</given-names></name><name><surname>Müller</surname><given-names>K.-R.</given-names></name></person-group> (<year>2011</year>). <article-title>Single-trial analysis and classification of ERP components–a tutorial.</article-title>\n<source><italic>Neuroimage</italic></source>\n<volume>56</volume>\n<fpage>814</fpage>–<lpage>825</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuroimage.2010.06.048</pub-id>\n<pub-id pub-id-type=\"pmid\">20600976</pub-id></mixed-citation></ref><ref id=\"B14\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Borst</surname><given-names>J.</given-names></name><name><surname>Nijboer</surname><given-names>M.</given-names></name><name><surname>Taatgen</surname><given-names>N.</given-names></name><name><surname>Anderson</surname><given-names>J. R.</given-names></name></person-group> (<year>2013</year>). “<article-title>A data-driven mapping of five ACT-R modules on the brain</article-title>,” in <source><italic>Proceedings of the 12th International Conference on Cognitive Modeling</italic></source>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>West</surname><given-names>R. L.</given-names></name><name><surname>Stewart</surname><given-names>T. C.</given-names></name></person-group> (<publisher-loc>Ottawa</publisher-loc>: <publisher-name>Carleton University</publisher-name>).</mixed-citation></ref><ref id=\"B15\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Byrne</surname><given-names>M. D.</given-names></name><name><surname>Kirlik</surname><given-names>A.</given-names></name></person-group> (<year>2005</year>). <article-title>Using computational cognitive modeling to diagnose possible sources of aviation error.</article-title>\n<source><italic>Intern. J. Aviat. Psychol.</italic></source>\n<volume>15</volume>\n<fpage>135</fpage>–<lpage>155</lpage>. <pub-id pub-id-type=\"doi\">10.1207/s15327108ijap1502_2</pub-id></mixed-citation></ref><ref id=\"B16\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cinel</surname><given-names>C.</given-names></name><name><surname>Valeriani</surname><given-names>D.</given-names></name><name><surname>Poli</surname><given-names>R.</given-names></name></person-group> (<year>2019</year>). <article-title>Neurotechnologies for human cognitive augmentation: current state of the art and future prospects.</article-title>\n<source><italic>Front. Hum. Neurosci.</italic></source>\n<volume>13</volume>:<issue>13</issue>. <pub-id pub-id-type=\"doi\">10.3389/fnhum.2019.00013</pub-id>\n<pub-id pub-id-type=\"pmid\">30766483</pub-id></mixed-citation></ref><ref id=\"B17\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Debener</surname><given-names>S.</given-names></name><name><surname>Makeig</surname><given-names>S.</given-names></name><name><surname>Delorme</surname><given-names>A.</given-names></name><name><surname>Engel</surname><given-names>A. K.</given-names></name></person-group> (<year>2005</year>). <article-title>What is novel in the novelty oddball paradigm? Functional significance of the novelty P3 event-related potential as revealed by independent component analysis.</article-title>\n<source><italic>Cogn. Brain Res.</italic></source>\n<volume>22</volume>\n<fpage>309</fpage>–<lpage>321</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cogbrainres.2004.09.006</pub-id>\n<pub-id pub-id-type=\"pmid\">15722203</pub-id></mixed-citation></ref><ref id=\"B18\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dehais</surname><given-names>F.</given-names></name><name><surname>Causse</surname><given-names>M.</given-names></name><name><surname>Vachon</surname><given-names>F.</given-names></name><name><surname>Régis</surname><given-names>N.</given-names></name><name><surname>Menant</surname><given-names>E.</given-names></name><name><surname>Tremblay</surname><given-names>S.</given-names></name></person-group> (<year>2014</year>). <article-title>Failure to detect critical auditory alerts in the cockpit: evidence for inattentional deafness.</article-title>\n<source><italic>Hum. Fact.</italic></source>\n<volume>56</volume>\n<fpage>631</fpage>–<lpage>644</lpage>. <pub-id pub-id-type=\"doi\">10.1177/0018720813510735</pub-id>\n<pub-id pub-id-type=\"pmid\">25029890</pub-id></mixed-citation></ref><ref id=\"B19\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Dehais</surname><given-names>F.</given-names></name><name><surname>Roy</surname><given-names>R. N.</given-names></name><name><surname>Gateau</surname><given-names>T.</given-names></name><name><surname>Scanella</surname><given-names>S.</given-names></name></person-group> (<year>2016</year>). “<article-title>Auditory alarm misperceptions in the cockpit: An EEG-study of inattentional deafness</article-title>,” in <source><italic>Proceedings of the 10th Foundations of Augmented Cognition: Neuroergonomics and Operational Neuroscience Lecture Notes In Computer Science Lecture Notes in Artificial Intelligence</italic></source>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>Schmorrow</surname><given-names>D. D.</given-names></name><name><surname>Fidopiastis</surname><given-names>C. M.</given-names></name></person-group> (<publisher-loc>Cham</publisher-loc>: <publisher-name>Springer</publisher-name>), <fpage>177</fpage>–<lpage>187</lpage>. <pub-id pub-id-type=\"doi\">10.1007/978-3-319-39955-3_17</pub-id></mixed-citation></ref><ref id=\"B20\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dehais</surname><given-names>F.</given-names></name><name><surname>Roy</surname><given-names>R. N.</given-names></name><name><surname>Scannella</surname><given-names>S.</given-names></name></person-group> (<year>2019</year>). <article-title>Inattentional deafness to auditory alarms: Inter-individual differences, electrophysiological signature and single trial classification.</article-title>\n<source><italic>Behav. Brain Res.</italic></source>\n<volume>360</volume>\n<fpage>51</fpage>–<lpage>59</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbr.2018.11.045</pub-id>\n<pub-id pub-id-type=\"pmid\">30508609</pub-id></mixed-citation></ref><ref id=\"B21\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Di Flumeri</surname><given-names>G.</given-names></name><name><surname>De Crescenzio</surname><given-names>F.</given-names></name><name><surname>Berberian</surname><given-names>B.</given-names></name><name><surname>Ohneiser</surname><given-names>O.</given-names></name><name><surname>Kramer</surname><given-names>J.</given-names></name><name><surname>Aricò</surname><given-names>P.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Brain-computer interface-based adaptive automation to prevent out-of-the-loop phenomenon in air traffic controllers dealing with highly automated systems.</article-title>\n<source><italic>Front. Hum. Neurosci.</italic></source>\n<volume>13</volume>:<issue>296</issue>. <pub-id pub-id-type=\"doi\">10.3389/fnhum.2019.00296</pub-id>\n<pub-id pub-id-type=\"pmid\">31555113</pub-id></mixed-citation></ref><ref id=\"B22\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Durso</surname><given-names>F. T.</given-names></name><name><surname>Sethumadhavan</surname><given-names>A.</given-names></name></person-group> (<year>2008</year>). <article-title>Situation awareness: Understanding dynamic environments.</article-title>\n<source><italic>Hum. Fact.</italic></source>\n<volume>50</volume>\n<fpage>442</fpage>–<lpage>448</lpage>. <pub-id pub-id-type=\"doi\">10.1518/001872008X288448</pub-id>\n<pub-id pub-id-type=\"pmid\">18689051</pub-id></mixed-citation></ref><ref id=\"B23\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Endsley</surname><given-names>M. R.</given-names></name></person-group> (<year>1995</year>). <article-title>Measurement of situation awareness in dynamic systems.</article-title>\n<source><italic>Hum. Fact.</italic></source>\n<volume>37</volume>\n<fpage>65</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.1518/001872095779049499</pub-id>\n<pub-id pub-id-type=\"pmid\">27006588</pub-id></mixed-citation></ref><ref id=\"B24\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Endsley</surname><given-names>M. R.</given-names></name></person-group> (<year>2000</year>). “<article-title>Direct measurement of situation awareness: validity and use of SAGAT</article-title>,” in <source><italic>Situation Awareness Analysis And Measurement</italic></source>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>Endsley</surname><given-names>M. R.</given-names></name><name><surname>Garland</surname><given-names>D. J.</given-names></name></person-group> (<publisher-loc>Mahwah, NJ</publisher-loc>: <publisher-name>Lawrence Erlbaum Associates</publisher-name>), <fpage>147</fpage>–<lpage>173</lpage>.</mixed-citation></ref><ref id=\"B25\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Endsley</surname><given-names>M. R.</given-names></name></person-group> (<year>2017</year>). <article-title>From here to autonomy.</article-title>\n<source><italic>Hum. Fact.</italic></source>\n<volume>59</volume>\n<fpage>5</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.1177/0018720816681350</pub-id>\n<pub-id pub-id-type=\"pmid\">28146676</pub-id></mixed-citation></ref><ref id=\"B26\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Endsley</surname><given-names>M. R.</given-names></name><name><surname>Jones</surname><given-names>D. G.</given-names></name></person-group> (<year>2011</year>). <source><italic>Designing for Situation Awareness: An Approach To User-Centered Design.</italic></source>\n<publisher-loc>Boca Raton, FL</publisher-loc>: <publisher-name>CRC Press</publisher-name>.</mixed-citation></ref><ref id=\"B27\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Endsley</surname><given-names>M. R.</given-names></name><name><surname>Kiris</surname><given-names>E. O.</given-names></name></person-group> (<year>1995</year>). <article-title>The out-of-the-loop performance problem and level of control in automation.</article-title>\n<source><italic>Hum. Fact.</italic></source>\n<volume>37</volume>\n<fpage>381</fpage>–<lpage>394</lpage>. <pub-id pub-id-type=\"doi\">10.1518/001872095779064555</pub-id>\n<pub-id pub-id-type=\"pmid\">27006588</pub-id></mixed-citation></ref><ref id=\"B28\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fazli</surname><given-names>S.</given-names></name><name><surname>Popescu</surname><given-names>F.</given-names></name><name><surname>Danóczy</surname><given-names>M.</given-names></name><name><surname>Blankertz</surname><given-names>B.</given-names></name><name><surname>Müller</surname><given-names>K. R.</given-names></name><name><surname>Grozea</surname><given-names>C.</given-names></name></person-group> (<year>2009</year>). <article-title>Subject-independent mental state classification in single trials.</article-title>\n<source><italic>Neural Netw.</italic></source>\n<volume>22</volume>, <fpage>1305</fpage>–<lpage>1312</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neunet.2009.06.003</pub-id>\n<pub-id pub-id-type=\"pmid\">19560898</pub-id></mixed-citation></ref><ref id=\"B29\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Freiman</surname><given-names>M.</given-names></name><name><surname>Caisse</surname><given-names>M.</given-names></name><name><surname>Ball</surname><given-names>J.</given-names></name><name><surname>Halverson</surname><given-names>T.</given-names></name><name><surname>Myers</surname><given-names>C.</given-names></name></person-group> (<year>2018</year>). “<article-title>Empirically identified gaps in a situation awareness model for human-machine coordination</article-title>,” in <source><italic>Proceedings of the 2018 IEEE International Conference on Cognitive and Computational Aspects of Situation Management (CogSIMA)</italic></source>, <publisher-loc>Piscataway, NJ</publisher-loc>.</mixed-citation></ref><ref id=\"B30\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fu</surname><given-names>W.-T.</given-names></name><name><surname>Bothell</surname><given-names>D.</given-names></name><name><surname>Douglass</surname><given-names>S.</given-names></name><name><surname>Haimson</surname><given-names>C.</given-names></name><name><surname>Sohn</surname><given-names>M.-H.</given-names></name><name><surname>Anderson</surname><given-names>J.</given-names></name></person-group> (<year>2006</year>). <article-title>Toward a real-time model-based training system.</article-title>\n<source><italic>Interact. Comput.</italic></source>\n<volume>18</volume>\n<fpage>1215</fpage>–<lpage>1241</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.intcom.2006.07.011</pub-id></mixed-citation></ref><ref id=\"B31\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Gluck</surname><given-names>K. A.</given-names></name></person-group> (<year>2010</year>). “<article-title>Cognitive architectures for human factors in aviation</article-title>,” in <source><italic>Human Factors in Aviation</italic></source>, <edition>2nd Edn</edition>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>Salas</surname><given-names>E.</given-names></name><name><surname>Maurino</surname><given-names>D.</given-names></name></person-group> (<publisher-loc>New York, NY</publisher-loc>: <publisher-name>Elsevier</publisher-name>), <fpage>375</fpage>–<lpage>400</lpage>. <pub-id pub-id-type=\"doi\">10.1016/B978-0-12-374518-7.00012-2</pub-id></mixed-citation></ref><ref id=\"B32\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Haines</surname><given-names>R. F.</given-names></name></person-group> (<year>1991</year>). “<article-title>A breakdown in simultaneous information processing</article-title>,” in <source><italic>Presbyopia Research: From Molecular Biology To Visual Adaptation</italic></source>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>Obrecht</surname><given-names>G.</given-names></name><name><surname>Stark</surname><given-names>L. W.</given-names></name></person-group> (<publisher-loc>New York, NY</publisher-loc>: <publisher-name>Springer</publisher-name>), <fpage>171</fpage>–<lpage>175</lpage>. <pub-id pub-id-type=\"doi\">10.1007/978-1-4757-2131-7_17</pub-id></mixed-citation></ref><ref id=\"B33\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Halbrügge</surname><given-names>M.</given-names></name></person-group> (<year>2013</year>). “<article-title>ACT-CV - Bridging the gap between cognitive models and the outer world</article-title>,” in <source><italic>Grundlagen und Anwendungen der Mensch-Maschine-Interaktion: 10. Berliner Werkstatt Mensch-Maschine-Systeme = Foundations and Applications Of Human Machine Interaction</italic></source>, <role>ed.</role>\n<person-group person-group-type=\"editor\"><name><surname>Brandenburg</surname><given-names>E.</given-names></name></person-group> (<publisher-loc>Berlin</publisher-loc>: <publisher-name>Springer</publisher-name>), <fpage>205</fpage>–<lpage>210</lpage>.</mixed-citation></ref><ref id=\"B34\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Harris</surname><given-names>D.</given-names></name><name><surname>Stanton</surname><given-names>N. A.</given-names></name><name><surname>Starr</surname><given-names>A.</given-names></name></person-group> (<year>2015</year>). <article-title>Spot the difference: operational event sequence diagrams as a formal method for work allocation in the development of single-pilot operations for commercial aircraft.</article-title>\n<source><italic>Ergonomics</italic></source>\n<volume>58</volume>\n<fpage>1773</fpage>–<lpage>1791</lpage>. <pub-id pub-id-type=\"doi\">10.1080/00140139.2015.1044574</pub-id>\n<pub-id pub-id-type=\"pmid\">25909684</pub-id></mixed-citation></ref><ref id=\"B35\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jones</surname><given-names>D. G.</given-names></name><name><surname>Endsley</surname><given-names>M. R.</given-names></name></person-group> (<year>1996</year>). <article-title>Sources of situation awareness errors in aviation.</article-title>\n<source><italic>Aviat. Space Environ. Med.</italic></source>\n<volume>67</volume>\n<fpage>507</fpage>–<lpage>512</lpage>.<pub-id pub-id-type=\"pmid\">8827130</pub-id></mixed-citation></ref><ref id=\"B36\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kiureghian</surname><given-names>A. D.</given-names></name><name><surname>Ditlevsen</surname><given-names>O.</given-names></name></person-group> (<year>2009</year>). <article-title>Aleatory or epistemic? Does it matter?.</article-title>\n<source><italic>Struct. Saf.</italic></source>\n<volume>31</volume>\n<fpage>105</fpage>–<lpage>112</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.strusafe.2008.06.020</pub-id></mixed-citation></ref><ref id=\"B37\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Klaproth</surname><given-names>O. W.</given-names></name><name><surname>Halbruegge</surname><given-names>M.</given-names></name><name><surname>Krol</surname><given-names>L. R.</given-names></name><name><surname>Vernaleken</surname><given-names>C.</given-names></name><name><surname>Zander</surname><given-names>T. O.</given-names></name><name><surname>Rußwinkel</surname><given-names>N.</given-names></name></person-group> (<year>2020</year>). <article-title>A neuroadaptive cognitive model for dealing with uncertainty in tracing pilots’ cognitive state.</article-title>\n<source><italic>Top. Cogn. Sci.</italic></source>\n<pub-id pub-id-type=\"doi\">10.1111/tops.12515</pub-id>\n<pub-id pub-id-type=\"pmid\">32666616</pub-id></mixed-citation></ref><ref id=\"B38\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kothe</surname><given-names>C. A.</given-names></name></person-group> (<year>2014</year>). <source><italic>Lab Streaming Layer.</italic></source> Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/sccn/labstreaminglayer\">https://github.com/sccn/labstreaminglayer</ext-link>\n<comment>(accessed December 19, 2019)</comment>.</mixed-citation></ref><ref id=\"B39\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kothe</surname><given-names>C. A.</given-names></name><name><surname>Makeig</surname><given-names>S.</given-names></name></person-group> (<year>2013</year>). <article-title>BCILAB: a platform for brain-computer interface development.</article-title>\n<source><italic>Neural Eng.</italic></source>\n<volume>10</volume>:<issue>56014</issue>\n<pub-id pub-id-type=\"doi\">10.1088/1741-2560/10/5/056014</pub-id></mixed-citation></ref><ref id=\"B40\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Krol</surname><given-names>L. R.</given-names></name><name><surname>Andreessen</surname><given-names>L.</given-names></name><name><surname>Zander</surname><given-names>T. O.</given-names></name></person-group> (<year>2018</year>). “<article-title>Passive brain-computer interfaces: A perspective on increased interactivity</article-title>,” in <source><italic>Brain-Computer Interfaces Handbook: Technological and Theoretical Advances</italic></source>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>Nam</surname><given-names>C. S.</given-names></name><name><surname>Nijholt</surname><given-names>A.</given-names></name><name><surname>Lotte</surname><given-names>F.</given-names></name></person-group> (<publisher-loc>Boca Raton, FL</publisher-loc>: <publisher-name>CRC Press</publisher-name>), <fpage>69</fpage>–<lpage>86</lpage>. <pub-id pub-id-type=\"doi\">10.1201/9781351231954-3</pub-id></mixed-citation></ref><ref id=\"B41\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krol</surname><given-names>L. R.</given-names></name><name><surname>Haselager</surname><given-names>P.</given-names></name><name><surname>Zander</surname><given-names>T. O.</given-names></name></person-group> (<year>2020</year>). <article-title>Cognitive and affective probing: a tutorial and review of active learning for neuroadaptive technology.</article-title>\n<source><italic>J. Neural Eng.</italic></source>\n<volume>17</volume>:<issue>012001</issue>. <pub-id pub-id-type=\"doi\">10.1088/1741-2552/ab5bb5</pub-id>\n<pub-id pub-id-type=\"pmid\">31770724</pub-id></mixed-citation></ref><ref id=\"B42\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Krol</surname><given-names>L. R.</given-names></name><name><surname>Zander</surname><given-names>T. O.</given-names></name></person-group> (<year>2017</year>). “<article-title>Passive BCI-based neuroadaptive systems</article-title>,” in <source><italic>Proceedings of the 7th Graz Brain Computer Interface Conference</italic></source>, <publisher-loc>Graz</publisher-loc>.</mixed-citation></ref><ref id=\"B43\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lakens</surname><given-names>D.</given-names></name></person-group> (<year>2013</year>). <article-title>Calculating and reporting effect sizes to facilitate cumulative science: a practical primer for t-tests and ANOVAs.</article-title>\n<source><italic>Front. Psychol.</italic></source>\n<volume>4</volume>:<issue>863</issue>. <pub-id pub-id-type=\"doi\">10.3389/fpsyg.2013.00863</pub-id>\n<pub-id pub-id-type=\"pmid\">24324449</pub-id></mixed-citation></ref><ref id=\"B44\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>J.</given-names></name><name><surname>Gardi</surname><given-names>A.</given-names></name><name><surname>Ramasamy</surname><given-names>S.</given-names></name><name><surname>Lim</surname><given-names>Y.</given-names></name><name><surname>Sabatini</surname><given-names>R.</given-names></name></person-group> (<year>2016</year>). <article-title>Cognitive pilot-aircraft interface for single-pilot operations.</article-title>\n<source><italic>Knowl. Based Syst.</italic></source>\n<volume>112</volume>\n<fpage>37</fpage>–<lpage>53</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.knosys.2016.08.031</pub-id></mixed-citation></ref><ref id=\"B45\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lotte</surname><given-names>F.</given-names></name><name><surname>Congedo</surname><given-names>M.</given-names></name><name><surname>Lécuyer</surname><given-names>A.</given-names></name><name><surname>Lamarche</surname><given-names>F.</given-names></name><name><surname>Arnaldi</surname><given-names>B.</given-names></name></person-group> (<year>2007</year>). <article-title>A review of classification algorithms for EEG-based brain-computer interfaces.</article-title>\n<source><italic>Neural Eng.</italic></source>\n<volume>4</volume>\n<fpage>R1</fpage>–<lpage>R13</lpage>. <pub-id pub-id-type=\"doi\">10.1088/1741-2560/4/2/R01</pub-id></mixed-citation></ref><ref id=\"B46\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Luck</surname><given-names>S. J.</given-names></name></person-group> (<year>2014</year>). <source><italic>An Introduction to the Event-Related Potential Technique</italic></source>. <publisher-loc>Cambridge</publisher-loc>: <publisher-name>MIT Press.</publisher-name></mixed-citation></ref><ref id=\"B47\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McNeese</surname><given-names>N. J.</given-names></name><name><surname>Demir</surname><given-names>M.</given-names></name><name><surname>Cooke</surname><given-names>N. J.</given-names></name><name><surname>Myers</surname><given-names>C.</given-names></name></person-group> (<year>2018</year>). <article-title>Teaming with a synthetic teammate: Insights into human-autonomy teaming.</article-title>\n<source><italic>Hum. Fact.</italic></source>\n<volume>60</volume>\n<fpage>262</fpage>–<lpage>273</lpage>. <pub-id pub-id-type=\"doi\">10.1177/0018720817743223</pub-id>\n<pub-id pub-id-type=\"pmid\">29185818</pub-id></mixed-citation></ref><ref id=\"B48\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Milner</surname><given-names>A. D.</given-names></name><name><surname>Goodale</surname><given-names>M. A.</given-names></name></person-group> (<year>2008</year>). <article-title>Two visual systems re-viewed.</article-title>\n<source><italic>Neuropsychologia</italic></source>\n<volume>46</volume>\n<fpage>774</fpage>–<lpage>785</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuropsychologia.2007.10.005</pub-id>\n<pub-id pub-id-type=\"pmid\">18037456</pub-id></mixed-citation></ref><ref id=\"B49\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Most</surname><given-names>S. B.</given-names></name><name><surname>Scholl</surname><given-names>B. J.</given-names></name><name><surname>Clifford</surname><given-names>E. R.</given-names></name><name><surname>Simons</surname><given-names>D. J.</given-names></name></person-group> (<year>2005</year>). <article-title>What you see is what you set: Sustained inattentional blindness and the capture of awareness.</article-title>\n<source><italic>Psychol. Rev.</italic></source>\n<volume>112</volume>\n<fpage>217</fpage>–<lpage>242</lpage>. <pub-id pub-id-type=\"doi\">10.1037/0033-295X.112.1.217</pub-id>\n<pub-id pub-id-type=\"pmid\">15631594</pub-id></mixed-citation></ref><ref id=\"B50\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Olofsen</surname><given-names>E.</given-names></name><name><surname>van Dongen</surname><given-names>H. P. A.</given-names></name><name><surname>Mott</surname><given-names>C. G.</given-names></name><name><surname>Balkin</surname><given-names>T. J.</given-names></name><name><surname>Terman</surname><given-names>D.</given-names></name></person-group> (<year>2010</year>). <article-title>Current approaches and challenges to development of an individualized sleep and performance prediction model.</article-title>\n<source><italic>Open Sleep J.</italic></source>\n<volume>3</volume>\n<fpage>24</fpage>–<lpage>43</lpage>. <pub-id pub-id-type=\"doi\">10.2174/1874620901003010024</pub-id></mixed-citation></ref><ref id=\"B51\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pierce</surname><given-names>R. S.</given-names></name></person-group> (<year>2012</year>). <article-title>The effect of SPAM administration during a dynamic simulation.</article-title>\n<source><italic>Hum. Factors</italic></source>\n<volume>54</volume>, <fpage>838</fpage>–<lpage>848</lpage>. <pub-id pub-id-type=\"doi\">10.1177/0018720812439206</pub-id>\n<pub-id pub-id-type=\"pmid\">23156627</pub-id></mixed-citation></ref><ref id=\"B52\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pope</surname><given-names>A. T.</given-names></name><name><surname>Bogart</surname><given-names>E. H.</given-names></name><name><surname>Bartolome</surname><given-names>D. S.</given-names></name></person-group> (<year>1995</year>). <article-title>Biocybernetic system evaluates indices of operator engagement in automated task.</article-title>\n<source><italic>Biol. Psychol.</italic></source>\n<volume>40</volume>\n<fpage>187</fpage>–<lpage>195</lpage>. <pub-id pub-id-type=\"doi\">10.1016/0301-0511(95)05116-3</pub-id><pub-id pub-id-type=\"pmid\">7647180</pub-id></mixed-citation></ref><ref id=\"B53\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Putze</surname><given-names>F.</given-names></name><name><surname>Schultz</surname><given-names>T.</given-names></name><name><surname>Propper</surname><given-names>R.</given-names></name></person-group> (<year>2015</year>). “<article-title>Dummy model based workload modeling</article-title>,” in <source><italic>Proceedings of the 2015 IEEE International Conference on Systems, Man, and Cybernetics</italic></source>, <publisher-loc>Hong Kong</publisher-loc>.</mixed-citation></ref><ref id=\"B54\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reifman</surname><given-names>J.</given-names></name><name><surname>Ramakrishnan</surname><given-names>S.</given-names></name><name><surname>Liu</surname><given-names>J.</given-names></name><name><surname>Kapela</surname><given-names>A.</given-names></name><name><surname>Doty</surname><given-names>T. J.</given-names></name><name><surname>Balkin</surname><given-names>T. J.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>2B-Alert App: a mobile application for real-time individualized prediction of alertness.</article-title>\n<source><italic>Sleep Res.</italic></source>\n<volume>2018</volume>:<issue>e12725</issue>. <pub-id pub-id-type=\"doi\">10.1111/jsr.12725</pub-id>\n<pub-id pub-id-type=\"pmid\">30033688</pub-id></mixed-citation></ref><ref id=\"B55\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Ritter</surname><given-names>F. E.</given-names></name><name><surname>Reifers</surname><given-names>A.</given-names></name><name><surname>Klein</surname><given-names>L. C.</given-names></name><name><surname>Quigley</surname><given-names>K.</given-names></name><name><surname>Schoelles</surname><given-names>M.</given-names></name></person-group> (<year>2004</year>). “<article-title>Using cognitive modeling to study behavior moderators: pre-task appraisal and anxiety</article-title>,” in <source><italic>Proceedings of the Human Factors and Ergonomics Society</italic></source>, <publisher-loc>Santa Monica, CA</publisher-loc>.</mixed-citation></ref><ref id=\"B56\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rodgers</surname><given-names>S. M.</given-names></name><name><surname>Myers</surname><given-names>C. W.</given-names></name><name><surname>Ball</surname><given-names>J.</given-names></name><name><surname>Freiman</surname><given-names>M. D.</given-names></name></person-group> (<year>2013</year>). <article-title>Toward a situation model in a cognitive architecture.</article-title>\n<source><italic>Comput. Math. Organ. Theor.</italic></source>\n<volume>19</volume>\n<fpage>313</fpage>–<lpage>345</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s10588-012-9134-x</pub-id></mixed-citation></ref><ref id=\"B57\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Rußwinkel</surname><given-names>N.</given-names></name><name><surname>Vernaleken</surname><given-names>C.</given-names></name><name><surname>Klaproth</surname><given-names>O. W.</given-names></name></person-group> (<year>2020</year>). “<article-title>Towards cognitive assistance and teaming in aviation by inferring pilot’s mental State</article-title>,” in <source><italic>Proceedings of the 3rd International Conference on Intelligent Human Systems Integration</italic></source>, <publisher-loc>Modena</publisher-loc>.</mixed-citation></ref><ref id=\"B58\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sarter</surname><given-names>N.</given-names></name><name><surname>Sarter</surname><given-names>M.</given-names></name></person-group> (<year>2003</year>). <article-title>Neuroergonomics: Opportunities and challenges of merging cognitive neuroscience with cognitive ergonomics.</article-title>\n<source><italic>Theor. Issues Ergon. Sci.</italic></source>\n<volume>4</volume>\n<fpage>142</fpage>–<lpage>150</lpage>. <pub-id pub-id-type=\"doi\">10.1080/1463922021000020882</pub-id></mixed-citation></ref><ref id=\"B59\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Scerbo</surname><given-names>M. W.</given-names></name></person-group> (<year>2006</year>). “<article-title>Adaptive automation</article-title>,” in <source><italic>Neuroergonomics: The Brain At Work</italic></source>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>Parasuraman</surname><given-names>R.</given-names></name><name><surname>Rizzo</surname><given-names>M.</given-names></name></person-group> (<publisher-loc>New York, NY</publisher-loc>: <publisher-name>Oxford Universtity Press</publisher-name>), <fpage>239</fpage>–<lpage>252</lpage>. <pub-id pub-id-type=\"doi\">10.1093/acprof:oso/9780195177619.003.0016</pub-id></mixed-citation></ref><ref id=\"B60\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schmidt</surname><given-names>A.</given-names></name></person-group> (<year>2000</year>). <article-title>Implicit human computer interaction through context.</article-title>\n<source><italic>Pers. Technol.</italic></source>\n<volume>4</volume>\n<fpage>191</fpage>–<lpage>199</lpage>. <pub-id pub-id-type=\"doi\">10.1007/BF01324126</pub-id></mixed-citation></ref><ref id=\"B61\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Shively</surname><given-names>R. J.</given-names></name><name><surname>Lachter</surname><given-names>J.</given-names></name><name><surname>Brandt</surname><given-names>S. L.</given-names></name><name><surname>Matessa</surname><given-names>M.</given-names></name><name><surname>Battiste</surname><given-names>V.</given-names></name><name><surname>Johnson</surname><given-names>W. W.</given-names></name></person-group> (<year>2017</year>). “<article-title>Why human-autonomy teaming?</article-title>,” in <source><italic>Advances in Neuroergonomics and Cognitive Engineering</italic></source>, <role>ed.</role>\n<person-group person-group-type=\"editor\"><name><surname>Baldwin</surname><given-names>C.</given-names></name></person-group> (<publisher-loc>Cham</publisher-loc>: <publisher-name>Springer</publisher-name>), <fpage>3</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1007/978-3-319-60642-2_1</pub-id></mixed-citation></ref><ref id=\"B62\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Somers</surname><given-names>S.</given-names></name><name><surname>West</surname><given-names>R.</given-names></name></person-group> (<year>2013</year>). “<article-title>Steering control in a flight Simulator using ACT-R</article-title>,” in <source><italic>Proceedings of the 12th International Conference on Cognitive Modeling</italic></source>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>West</surname><given-names>R. L.</given-names></name><name><surname>Stewart</surname><given-names>T. C.</given-names></name></person-group> (<publisher-loc>Ottawa</publisher-loc>: <publisher-name>Carleton University</publisher-name>).</mixed-citation></ref><ref id=\"B63\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Spence</surname><given-names>C.</given-names></name><name><surname>Driver</surname><given-names>J.</given-names></name></person-group> (<year>1997</year>). “<article-title>Audiovisual links in attention: implications for interface design: job design and product design</article-title>,” in <source><italic>Engineering Psychology And Cognitive Ergonomics</italic></source>, <role>ed.</role>\n<person-group person-group-type=\"editor\"><name><surname>Harris</surname><given-names>D.</given-names></name></person-group> (<publisher-loc>Hampshire</publisher-loc>: <publisher-name>Ashgate</publisher-name>), <fpage>185</fpage>–<lpage>192</lpage>. <pub-id pub-id-type=\"doi\">10.4324/9781315094489-24</pub-id></mixed-citation></ref><ref id=\"B64\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stanton</surname><given-names>N. A.</given-names></name></person-group> (<year>2006</year>). <article-title>Hierarchical task analysis: Developments, applications, and extensions.</article-title>\n<source><italic>Appl. Ergon.</italic></source>\n<volume>37</volume>\n<fpage>55</fpage>–<lpage>79</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.apergo.2005.06.003</pub-id>\n<pub-id pub-id-type=\"pmid\">16139236</pub-id></mixed-citation></ref><ref id=\"B65\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van Dijk</surname><given-names>H.</given-names></name><name><surname>van de Merwe</surname><given-names>K.</given-names></name><name><surname>Zon</surname><given-names>R.</given-names></name></person-group> (<year>2011</year>). <article-title>A coherent impression of the pilots’ situation awareness: studying relevant human factors tools.</article-title>\n<source><italic>Aviat. Psychol.</italic></source>\n<volume>21</volume>\n<fpage>343</fpage>–<lpage>356</lpage>. <pub-id pub-id-type=\"doi\">10.1080/10508414.2011.606747</pub-id></mixed-citation></ref><ref id=\"B66\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Vidulich</surname><given-names>M.</given-names></name><name><surname>McMillan</surname><given-names>G.</given-names></name></person-group> (<year>2000</year>). “<article-title>The global implicit measure: evaluation of metrics for cockpit adaptation</article-title>,” in <source><italic>Contemporary Ergonomics 2000</italic></source>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>McCabe</surname><given-names>P. T.</given-names></name><name><surname>Hanson</surname><given-names>M. A.</given-names></name><name><surname>Robertson</surname><given-names>S. A.</given-names></name></person-group> (<publisher-loc>London</publisher-loc>: <publisher-name>Taylor and Francis</publisher-name>), <fpage>75</fpage>–<lpage>80</lpage>.</mixed-citation></ref><ref id=\"B67\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Wilson</surname><given-names>G. F.</given-names></name></person-group> (<year>2000</year>). “<article-title>Strategies for psychophysiological assessment of situation awareness</article-title>,” in <source><italic>Situation Awareness Analysis And Measurement</italic></source>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>Endsley</surname><given-names>M. R.</given-names></name><name><surname>Garland</surname><given-names>D. J.</given-names></name></person-group> (<publisher-loc>Mahwah, NJ</publisher-loc>: <publisher-name>Lawrence Erlbaum Associates</publisher-name>), <fpage>175</fpage>–<lpage>188</lpage>.</mixed-citation></ref><ref id=\"B68\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Zander</surname><given-names>T. O.</given-names></name><name><surname>Brönstrup</surname><given-names>J.</given-names></name><name><surname>Lorenz</surname><given-names>R.</given-names></name><name><surname>Krol</surname><given-names>L. R.</given-names></name></person-group> (<year>2014</year>). “<article-title>Towards BCI-based implicit control in human-computer interaction</article-title>,” in <source><italic>Human-Computer Interaction Series. Advances in Physiological Computing</italic></source>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>Fairclough</surname><given-names>S. H.</given-names></name><name><surname>Gilleade</surname><given-names>K.</given-names></name></person-group> (<publisher-loc>London</publisher-loc>: <publisher-name>Springer</publisher-name>), <fpage>67</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1007/978-1-4471-6392-3_4</pub-id></mixed-citation></ref><ref id=\"B69\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zander</surname><given-names>T. O.</given-names></name><name><surname>Kothe</surname><given-names>C.</given-names></name></person-group> (<year>2011</year>). <article-title>Towards passive brain-computer interfaces: applying brain-computer interface technology to human-machine systems in general.</article-title>\n<source><italic>Neural Eng.</italic></source>\n<volume>8</volume>:<issue>25005</issue>\n<pub-id pub-id-type=\"doi\">10.1088/1741-2560/8/2/025005</pub-id></mixed-citation></ref><ref id=\"B70\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zander</surname><given-names>T. O.</given-names></name><name><surname>Krol</surname><given-names>L. R.</given-names></name><name><surname>Birbaumer</surname><given-names>N. P.</given-names></name><name><surname>Gramann</surname><given-names>K.</given-names></name></person-group> (<year>2016</year>). <article-title>Neuroadaptive technology enables implicit cursor control based on medial prefrontal cortex activity.</article-title>\n<source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source>\n<volume>113</volume>\n<fpage>14898</fpage>–<lpage>14903</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1605155114</pub-id>\n<pub-id pub-id-type=\"pmid\">27956633</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Endocrinol.</journal-id><journal-title-group><journal-title>Frontiers in Endocrinology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2392</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849268</article-id><article-id pub-id-type=\"pmc\">PMC7431602</article-id><article-id pub-id-type=\"doi\">10.3389/fendo.2020.00465</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Osteoimmunology: The Regulatory Roles of T Lymphocytes in Osteoporosis</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Wenjuan</given-names></name><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/613024/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Dang</surname><given-names>Kai</given-names></name><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/895546/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Huai</surname><given-names>Ying</given-names></name><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/762023/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Qian</surname><given-names>Airong</given-names></name><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/538119/overview\"/></contrib></contrib-group><aff><institution>Lab for Bone Metabolism, Xi'an Key Laboratory of Special Medicine and Health Engineering, Key Lab for Space Biosciences and Biotechnology, Research Center for Special Medicine and Health Systems Engineering, NPU-UAB Joint Laboratory for Bone Metabolism, School of Life Sciences, Northwestern Polytechnical University</institution>, <addr-line>Xi'an</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Lilian Irene Plotkin, Indiana University Bloomington, United States</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Rajeev Aurora, Saint Louis University, United States; Anna Teti, University of L'Aquila, Italy</p></fn><corresp id=\"c001\">*Correspondence: Airong Qian <email>qianair@nwpu.edu.cn</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Bone Research, a section of the journal Frontiers in Endocrinology</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†These authors have contributed equally to this work and share first authorship</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>465</elocation-id><history><date date-type=\"received\"><day>31</day><month>12</month><year>2019</year></date><date date-type=\"accepted\"><day>15</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Zhang, Dang, Huai and Qian.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Zhang, Dang, Huai and Qian</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Immune imbalance caused bone loss. Osteoimmunology is emerging as a new interdisciplinary field to explore the shared molecules and interactions between the skeletal and immune systems. In particular, T lymphocytes (T cells) play pivotal roles in the regulation of bone health. However, the roles and mechanisms of T cells in the treatment of osteoporosis are not fully understood. The present review aims to summarize the essential regulatory roles of T cells in the pathophysiology of various cases of osteoporosis and the development of T cell therapy for osteoporosis from osteoimmunology perspective. As T cell-mediated immunomodulation inhibition reduced bone loss, there is an increasing interest in T cell therapy in an attempt to treat osteoporosis. In summary, the T cell therapy may be further pursued as an immunomodulatory strategy for the treatment of osteoporosis, which can provide a novel perspective for drug development in the future.</p></abstract><kwd-group><kwd>osteoimmunology</kwd><kwd>T lymphocytes</kwd><kwd>osteoporosis</kwd><kwd>bone formation</kwd><kwd>bone resorption</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Natural Science Foundation of China<named-content content-type=\"fundref-id\">10.13039/501100001809</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"0\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"112\"/><page-count count=\"8\"/><word-count count=\"6943\"/></counts></article-meta></front><body><sec id=\"s1\"><title>Introductions</title><p>Osteoporosis is a prevailing metabolic bone disease in both men > 50 years and postmenopausal women, which increases bone fragility and may further result in bone fractures, thus significantly leading to serious health problems for patients (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Worldwide, nearly 200 million people are diagnosed with osteoporosis annually, even leading to almost 9 million osteoporotic fractures (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). In the US, it was approximately 53.6 million of the adult population of years > 50 who suffered from osteoporosis and low bone mass (54% of the population) (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>). In fact, osteoporosis patients not only suffer from the enormous pain and disability but also bring a huge economic burden for patients and their families. In the US, it has been estimated that the financial costs associated with bone fractures will reach $25.3 billion by the end of 2025 (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>).</p><p>In traditional view, osteoporosis was considered as the imbalance of bone remodeling between osteoclasts and osteoblasts (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Recently, the immune system was reported to regulate the bone system, which promoted the emergence of interdisciplinary field of osteoimmunology (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>–<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). The immune and bone systems share the same microenvironment. The immune system regulates osteocytes by the secretion of inflammatory factors and related ligand, which further affects bone formation and bone resorption (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>, <xref rid=\"B10\" ref-type=\"bibr\">10</xref>). T cells, B cells, and cytokines are important regulatory factors in the bone resorption. Among them, T cells play pivotal roles in the regulation of bone remodeling (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B12\" ref-type=\"bibr\">12</xref>). The osteoclast differentiation was enhanced, and the bone mineral density was decreased in the nude mice (T cell deficient), which was due to the immune imbalance of T cells promoting osteoclast differentiation and bone resorption (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>). In pathophysiological condition, activated T cells secreted multiple inflammatory factors and related ligands such as TNF-α, IL-1, IL-6, IL-17, and CD40L, which enhanced bone resorption and disrupted bone balance, resulting in bone loss (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Th17 cells are mainly involved in inducing bone resorption (osteoclastogenesis) (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>), while Treg cells are major suppressors of bone loss (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>) by inhibiting differentiation of monocytes into osteoclasts (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B20\" ref-type=\"bibr\">20</xref>, <xref rid=\"B21\" ref-type=\"bibr\">21</xref>). These reports indicated that immune imbalance promoted osteoclast differentiation, further leading to bone loss. However, the roles of T cells in osteoporosis and the underlying mechanism of T cells in the regulation of bone system are still unclear.</p><p>Recently, there is an increasing interest in immune therapies especially T cell therapies for the treatment of osteoporosis (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). For example, antiretroviral therapy worsens HIV-induced bone loss (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>), which may be an important future approach to treat osteoporosis in human. That is because T cell reconstitution induces RANKL and TNFα production by B-cells and/or T-cells, which further enhancing bone resorption and bone loss. T cell therapy became the effective strategy for the treatment of osteoporosis. For example, RANKL/RANK inhibition may be an attractive approach for the treatment of postmenopausal osteoporosis (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). Sclareol is a natural product (initially isolated from the leaves and flowers of <italic>Salvia sclarea</italic>) with immune regulation and anti-inflammatory effects, and it prevents ovariectomy-induced bone loss <italic>in vivo</italic> and inhibits osteoclastogenesis <italic>in vitro</italic> via suppressing NF-κB and MAPK/ERK signaling pathways (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Thus, it will be essential to develop T cell therapy that may be a huge potential for the treatment of osteoporosis in future clinical applications.</p><p>Herein, we briefly highlight the roles of T cells in various types of osteoporosis and uncover novel mechanisms of osteoimmunology, which provides new insight for clinical implications in the treatment of osteoporosis. Nonetheless, the underlying mechanisms of bone-immune interactions need to be further dissected, and an accumulative evidence continues to be made in favor of regulation roles of immune cells in osteoporosis. Most importantly, the T cell therapy may represent a suitable and potential approach to reinstate aberrant bone remodeling in the bone metabolism diseases.</p></sec><sec id=\"s2\"><title>Osteoimmunology and The Regulation of T Cell Cytokines in Osteoporosis</title><p>Osteoimmunology is the intricate interaction between the immune system and the bone system (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>–<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). The RANKL/RANK/OPG pathway is essential for the differentiation of bone-resorbing osteoclasts and immune regulation (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>, <xref rid=\"B27\" ref-type=\"bibr\">27</xref>). Activated T cells directly produce RANKL, which further stimulates osteoclast formation (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>, <xref rid=\"B29\" ref-type=\"bibr\">29</xref>). RANKL and RANK were identified as key factors in the mediation of bone remodeling, especially in the osteoclast formation (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>, <xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Furthermore, the activated RANK facilitated the expression of tumor necrosis factor (TNF) receptor-associated factors (TRAFs), such as TRAF6, which leads to osteoclast differentiation (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>, <xref rid=\"B32\" ref-type=\"bibr\">32</xref>). In OVX mice, the low-dose RANKL of CD8<sup>+</sup> Treg cells decreased the expression of inflammatory and osteoclastogenic cytokines, thus suppressing bone resorption (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>). Multiple cytokines produced by T cell including interleukin (IL)-12, IL-17, IL-18, and TNF-α were involved in RANK signaling, and thus play essential roles in regulating osteoclastogenesis and osteoclast differentiation (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). In addition, activated T cells suppress osteoclast differentiation by the antiviral cytokine IFN-γ (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). Various inflammatory cytokines were necessary and sufficient for bone metabolism (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). IL-17A also upregulates the expression of RANK, thus promoting the osteoclastogenic activity of RANKL (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). All these studies indicated that T cell cytokines play essential roles in osteoporosis, which may be the potential targets for the treatment of osteoporosis. Various T cell cytokines are listed in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Roles of various T cell cytokines in the regulation of osteoclastogenesis.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Cytokine</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Source</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Modulation of immunology</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Osteoclastogenic function</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>References</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RANKL</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Th17 cells</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Osteoclast differentiation dendritic cells (DCs) maturation</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Osteoclast activation via RANK</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B37\" ref-type=\"bibr\">37</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RANK</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Osteoclasts, DCs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DCs activation</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Osteoclast differentiation and activation</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B38\" ref-type=\"bibr\">38</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">OPG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Osteoclasts</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Decoy receptor for RANKL</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits osteoclastogenesis</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B39\" ref-type=\"bibr\">39</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TNFα</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Th17, macrophage DCs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pro-inflammatory cytokine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Indirect osteoclastic activation through RANKL</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B37\" ref-type=\"bibr\">37</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">M-CSF</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Th1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pro-inflammatory</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits osteoclastogenesis</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B38\" ref-type=\"bibr\">38</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Th2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Humoral immunity</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits osteoclastogenesis</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B40\" ref-type=\"bibr\">40</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Macrophage, DCs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pro-inflammation, Th17 induction</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Activation of osteoclastogenesis</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B41\" ref-type=\"bibr\">41</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-7</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">T cells</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pro-inflammatory cytokine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits osteoclast formation</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B42\" ref-type=\"bibr\">42</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-8</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-10</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Regulatory T (Treg)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Anti-inflammatory</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Suppress bone resorption</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B43\" ref-type=\"bibr\">43</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-17</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">T cells</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pro-inflammatory cytokine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RANKL expression and vigorous pro-inflammatory potency</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B44\" ref-type=\"bibr\">44</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-27</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Macrophage and DCs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Th1and Treg Th17 induction</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits osteoclast formation, blocking receptor activator of NF-κB (RANK)-dependent osteoclastogenesis</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B45\" ref-type=\"bibr\">45</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-12</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Antigen-presenting cells</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pro-inflammatory cytokine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits RANKL-stimulated Osteoclastogenesis</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B46\" ref-type=\"bibr\">46</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-15</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">NK cells</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Pro-inflammatory cytokine</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Enhances RANK ligand (RANKL) and macrophage colony-stimulating factor expression</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B47\" ref-type=\"bibr\">47</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-23</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Macrophage and DCs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Th17 induction</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Indirect osteoclast activation</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B48\" ref-type=\"bibr\">48</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IFN-γ</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Th1, NK cells</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cellular immunity</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inhibits osteoclastogenesis</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B41\" ref-type=\"bibr\">41</xref>)</td></tr></tbody></table></table-wrap></sec><sec id=\"s3\"><title>The T Cells in The Regulation of Various Osteoporosis</title><p>T cells perform a dual role in the regulation of bone remodeling: resting T cells protect osteoclasts from bone resorption, and activated T cells actively regulate the osteoclasts generation. This review aims to summarize the regulatory roles of T cells in various types of osteoporosis such as chronic inflammation-induced osteoporosis, senile osteoporosis, estrogen deficiency-induced osteoporosis, parathyroid hormone (PTH)-induced osteoporosis, and glucocorticoid-induced osteoporosis (GIO).</p><sec><title>The Regulatory Roles of T Cells in Chronic Inflammation-Induced Osteoporosis</title><p>Osteoporosis commonly occurred in various chronic inflammatory diseases, such as rheumatic arthritis (RA), gout, psoriatic disease, osteoarthritis, and axial spondylarthritis and even leads to functional disability and increased mortality (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>–<xref rid=\"B52\" ref-type=\"bibr\">52</xref>). It is interesting to note that Tregs play pivotal roles in inflammation-induced bone loss by inhibiting the functions of Th17 cells (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B53\" ref-type=\"bibr\">53</xref>). In particular, Foxp3+ Treg cells play an indispensable role in bone and hematopoietic homeostasis acting on osteoclast development and function (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>). In addition, in inflammation condition, the expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), as well as by inflammatory cytokines such as TNFα, IL-1β, and IL-6 was induced and produced to promote osteoclast differentiation mediated by the RANKL-RANK and calcium signaling (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). INFγ, the main Th1 cytokine, can strongly inhibit osteoclast differentiation <italic>in vitro</italic> through the proteasomal degradation of TRAF6, indicating that T cells regulate osteoclastogenesis (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). The T cell subset, Tregs, also suppresses osteoclast formation and bone resorbing <italic>in vitro</italic> (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>). CTLA-4 is the most essential regulator in the Treg-mediated inhibition of osteoclast differentiation, whereas the major cytokines of Tregs-TGFβ and IL-10 do not possess any essential roles (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>). All these studies suggest that T cells and their related cytokine play pivotal roles in the regulation of osteoporosis, and they may be the potential therapeutic targets for bone loss.</p><p>Generally, chronic inflammatory diseases are associated with bone resorption. HIV-infected men had low CD4 T cells, which is inversely associated with bone loss (<xref rid=\"B55\" ref-type=\"bibr\">55</xref>). Some studies suggest that T cells are not associated with bone mineral density in HIV-infected patients treated with combination antiretroviral therapy (cART) (<xref rid=\"B56\" ref-type=\"bibr\">56</xref>). However, cART seems to influence bone mineral density (BMD) with the protective effect. Therefore, the regulatory roles for activated T cells in the pathogenesis of osteoporosis warrant further investigation. In RA patients, the enhanced osteoclast differentiation and activation lead to bone erosion and systematic osteoporosis (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>). Indeed, inflammatory cytokines including RANKL, TNFα, IL-6, and IL-1 were elevated in RA patients, which promoted the osteoclast differentiation (<xref rid=\"B58\" ref-type=\"bibr\">58</xref>). Taken together, these studies suggest that the T cells may determine the osteoclast differentiation in the chronic inflammatory diseases, and the T cell regulatory therapy could potentially have significant impact on the drug development for osteoporosis. However, whether the T cell therapy is efficient for osteoporosis in clinical studies needs further investigation.</p></sec><sec><title>The Regulation Roles of T Cells in Senile Osteoporosis</title><p>Aging is always accompanied with the imbalance between bone formation and resorption, causing skeletal microarchitecture damage and bone loss (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>). The production of naïve T cells is severely impaired due to a decreased output of lymphoid cells from the bone marrow and the deterioration of the thymus (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>). Incidence and severity of osteoporosis are increased in the older population (<xref rid=\"B61\" ref-type=\"bibr\">61</xref>). The prevalence of low BMD is associated with immune activation and senescence induced by HIV infection (<xref rid=\"B62\" ref-type=\"bibr\">62</xref>). Total T cells were increased in the bone marrow (BM) with age, especially the highly differentiated CD8<sup>+</sup> T cells without the expression of the co-stimulatory molecule CD28, while natural killer T (NKT) cells, monocytes, and naïve CD8<sup>+</sup> T cells were decreased in the BM with age (<xref rid=\"B63\" ref-type=\"bibr\">63</xref>). It seems that the immune system abnormality plays important roles in the regulation of senile osteoporosis.</p><p>Recent discoveries suggest that T cell dysfunction induced the accumulation of cytokines, immunological mediators, and transcription factors, which affect osteoclast and osteoblast in the elderly (<xref rid=\"B64\" ref-type=\"bibr\">64</xref>). Cytokines such as IL-6, TNF-α, and IL-1 increased with age (<xref rid=\"B65\" ref-type=\"bibr\">65</xref>, <xref rid=\"B66\" ref-type=\"bibr\">66</xref>). IL-1 and TNF-α activate the inducible NOS (iNOS) pathway, which inhibited osteoblast differentiation and enhanced osteoblast apoptosis <italic>in vitro</italic> (<xref rid=\"B67\" ref-type=\"bibr\">67</xref>). IL-12 derived from T cells, alone or combined with IL-18, was identified to inhibit osteoclast formation <italic>in vitro</italic> (<xref rid=\"B68\" ref-type=\"bibr\">68</xref>). IL-4 regulated osteoclast differentiation through the antagonism between STAT6 and NF-kB signaling (<xref rid=\"B69\" ref-type=\"bibr\">69</xref>). In addition, T cell mediated the bone balance by the inhibition of osteoclastogenesis through the crucial immunoregulatory control, mainly OPG expression and simultaneous production of cytokines (<xref rid=\"B64\" ref-type=\"bibr\">64</xref>). IFN-g, IL-12, and IL-18 inhibited the RANKL-induced maturation and activation of osteoclasts (<xref rid=\"B64\" ref-type=\"bibr\">64</xref>). Furthermore, senescent T cells impaired the production of IFN-γ, OPG, and osteoclast-inhibiting cytokines, which increased the incidence of aged osteoporosis. In addition, cytokines such as TGFβ and RANKL secreted by activated T cells can activate p38 MAPKs and further regulate bone development and remodeling. P38α MAPK mediates osteoclast proliferation and bone remodeling in an aging-dependent manner (<xref rid=\"B70\" ref-type=\"bibr\">70</xref>). Overall, T cells and their cytokines play important roles in the regulation of aged osteoporosis, which may be the novel targets for the treatment of osteoporosis, suggesting that T cell therapy could be used as immunotherapy and may be beneficial in counteracting immunosenescence in old population. Meanwhile, in females, osteoporosis occurrence is generally attributed to the decrease in estrogen, thus leading to estrogen deficiency-induced osteoporosis. The underlying mechanisms of T cells involved in the mediation of the postmenopausal osteoporosis were dissected in the next section, The Regulatory Roles of T Cells in Estrogen Deficiency-Induced Osteoporosis.</p></sec><sec><title>The Regulatory Roles of T Cells in Estrogen Deficiency-Induced Osteoporosis</title><p>The loss of estrogen initiates the inflammatory changes of bone-microenvironment state, inducing a rapid phase of bone loss leading to osteoporosis in half of postmenopausal women. In postmenopausal women, estrogen deficiency stimulates CD4<sup>+</sup> T cell dysregulation and induces elevated circulating levels of inflammatory cytokines, especially TNFα, IFN-γ, IL-17, RANKL, and CD40L (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>–<xref rid=\"B74\" ref-type=\"bibr\">74</xref>). These cytokines exert impressive regulatory effects on bone resorption. For example, TNF-α was overexpressed in the BM in postmenopausal osteoporosis, which promotes RANKL-induced osteoclast formation through the activation of NF-κB and PI3K/Akt signaling (<xref rid=\"B74\" ref-type=\"bibr\">74</xref>). Besides, TNF-α was identified to induce both autophagy and apoptosis in osteoblasts to enhance bone loss in postmenopausal women (<xref rid=\"B75\" ref-type=\"bibr\">75</xref>). Besides, estrogen deficiency increased the number of the costimulatory factors, CD40L, expressed on activated T cells, inducing the expressions of M-CSF and RANKL on stromal cells and downregulating the production of OPG, ultimately resulting in a remarkable increase in osteoclast numbers (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>, <xref rid=\"B77\" ref-type=\"bibr\">77</xref>). The pro-osteoclastic cytokines, such as IL-6, TNF-α, and IL-1, were increased significantly in estrogen deficiency-induced osteoporosis (<xref rid=\"B78\" ref-type=\"bibr\">78</xref>). All these studies indicated that the inflammatory cytokines and costimulatory factors of T cells changed significantly in estrogen deficiency-induced osteoporosis, which may provide the novel perspective for the treatment of bone loss in postmenopausal women.</p><p>Moreover, estrogen deficiency stimulates the IL-17 differentiation of Th17 cells (<xref rid=\"B79\" ref-type=\"bibr\">79</xref>) and augments the expression levels of pro-osteoclastogenic cytokines, such as TNF-a, IL-6, and RANKL, ultimately leading to bone loss. Nevertheless, IL-17 receptor deficiency induced more serious bone loss in OVX mice than that in control groups, implying that IL-17 may possess the bone protective effects (<xref rid=\"B80\" ref-type=\"bibr\">80</xref>). The pro-osteoclastogenic cytokine changes were reversed with the supplementary oral estrogen, indicating that estrogen may suppress Th17 differentiation and IL-17 production to protect bone health (<xref rid=\"B81\" ref-type=\"bibr\">81</xref>). In summary, in postmenopausal women, both aging and hormonal deficiency stimulate the deregulation of T cells contributing to the inflammatory, which increased bone resorption, resulting in a bone loss or osteoporosis. We believe that focusing on the potential biological mechanisms of T cells is of paramount importance for developing novel therapy strategies for the treatment of postmenopausal osteoporosis. However, further confirmation in phase I/II trials is needed to validate these strategies in a broader clinical evaluation.</p></sec><sec><title>The Regulatory Roles of T Cells in PTH-Induced Osteoporosis</title><p>PTH is a key calciotropic hormone and a critical regulator for postnatal skeletal development (<xref rid=\"B82\" ref-type=\"bibr\">82</xref>). The secretion of inflammatory or osteoclastogenic cytokines of T cells and bone cells was facilitated under long-term PTH administration, such as RANKL, TNF-α, and IL-17, which promoted the bone resorption (<xref rid=\"B83\" ref-type=\"bibr\">83</xref>). PTH induced bone loss via the expansion of intestinal TNF<sup>+</sup> T and Th17 cells, and the increase in their S1P-receptor-1 mediated egress from the intestine and recruitment to the BM (<xref rid=\"B84\" ref-type=\"bibr\">84</xref>). So targeting the gut microbiota or T cell migration may represent novel therapeutic strategies for PTH-induced osteoporosis. In addition, PTH exploited CD4<sup>+</sup> T cells to induce TNFα production that enhances the formation of IL-17A secreting Th17 T cells. Both TNFα and IL-17 further facilitated the development of an increased RANKL/OPG ratio favorable to osteoclastic bone resorption (<xref rid=\"B85\" ref-type=\"bibr\">85</xref>). Moreover, PTH boosted the production of TNF-α and RANKL in CD4<sup>+</sup> T cells, which triggered osteoclastogenic generation and bone resorption activity (<xref rid=\"B86\" ref-type=\"bibr\">86</xref>). Clinical studies also showed that PTH treatment increased Th17 cell numbers and the IL-17 production in humans with primary hyperparathyroidism (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). IL-17 intensified PTH-induced bone loss through the stimulation of the RANKL production in osteoblast-lineage cells, which is parallel to the roles of IL-17 in estrogen deficiency-induced osteoporosis.</p><p>Notably, T cells also secreted PTH receptors involved in the regulation of trabecular bone development (<xref rid=\"B87\" ref-type=\"bibr\">87</xref>). For example, T cells promoted the signals of BMSC proliferation through the combination of CD40L on T cells and its receptor on BMSC, weakening the bone catabolic activity of cPTH, leading to a reduction of the RANKL to OPG ratio and osteoclastogenic activity (<xref rid=\"B88\" ref-type=\"bibr\">88</xref>). Several studies found that the intermittent PTH administration at low dosage increased bone formation and bone mass, thus attenuating bone loss (<xref rid=\"B89\" ref-type=\"bibr\">89</xref>, <xref rid=\"B90\" ref-type=\"bibr\">90</xref>). The deletion of PTH receptor in BM mesenchymal progenitors results in a rapid increase in BM adipocyte accompanied with the reduction of bone mass. Given the essential regulatory roles of T cells for the PTH-induced bone loss, particular attention will be paid toward the combinations of intermittent PTH (iPTH) and T cell therapy for PTH-induced osteoporosis.</p></sec><sec><title>The Regulatory Roles of T Cells in GIO</title><p>Glucocorticoids (GCs) are extensively used for the treatment of immune and inflammatory disorders due to their powerful immunosuppressive and anti-inflammatory actions (<xref rid=\"B91\" ref-type=\"bibr\">91</xref>, <xref rid=\"B92\" ref-type=\"bibr\">92</xref>). However, long-term exogenous GC therapy might cause rapid and pronounced bone loss and subsequently osteoporosis (<xref rid=\"B93\" ref-type=\"bibr\">93</xref>, <xref rid=\"B94\" ref-type=\"bibr\">94</xref>). The pathogenesis of GIO was predominantly attributed to the fact that GCs impaired bone formation by reduction of osteoblast differentiation and activity via the expression of the osteoblast-specific transcription factor runt-related Runx2 (<xref rid=\"B95\" ref-type=\"bibr\">95</xref>–<xref rid=\"B97\" ref-type=\"bibr\">97</xref>). In addition, the long-term GC administration affects bone remodeling by whittling the insulin-like growth factor (IGF) in ossification (<xref rid=\"B98\" ref-type=\"bibr\">98</xref>). GCs enhanced the expression levels of RANKL in both osteoblasts and stromal cells, which triggered osteoclastogenesis and activated osteoclastic bone resorption by binding to the RANKL receptor RANK (<xref rid=\"B99\" ref-type=\"bibr\">99</xref>), thus resulting in the primary phase of rapid bone loss. On the other hand, GCs contributed to the apoptosis of certain T cell subsets, further augmented the secretion of RANKL, and directly induced osteoclast differentiation (<xref rid=\"B100\" ref-type=\"bibr\">100</xref>). Interestingly, different T cell subsets exhibit distinct sensitivity to GC-induced apoptosis. For example, Th17 cells, as an osteoclastogenesis-promoting factor, are resistant to GC-induced apoptosis and cytokine suppression mostly through the high production of IL-17 and RANKL (<xref rid=\"B79\" ref-type=\"bibr\">79</xref>). Therefore, GC therapy fails to inhibit the Th17 cell activation and the IL-17 and RANKL production. Excessive GCs could reduce the production of OPG, further promoting osteoclast differentiation and resulting in bone resorption. Given above, we assert that T cell therapy may be effective for the GC-induced osteoporosis.</p></sec></sec><sec id=\"s4\"><title>T Cell Therapy for Osteoporosis</title><p>T cells and their secreted cytokines are responsible for bone resorption in various osteoporosis. T cell therapy may be a potentially therapeutic approach to osteoporosis. For example, anti-inflammatory therapies have shown good potential in an animal model, although they have not been widely used clinically to treat osteoporosis (<xref rid=\"B101\" ref-type=\"bibr\">101</xref>). Immune modulation therapy such as probiotics was considered as a novel strategy for bone loss (<xref rid=\"B102\" ref-type=\"bibr\">102</xref>–<xref rid=\"B104\" ref-type=\"bibr\">104</xref>). RANKL was considered as an activator of dendritic cell (DC) expression in T cells. Anti-RANKL therapeutic antibody drug, denosumab, has been successfully applied in the treatment of osteoporosis in clinics (<xref rid=\"B105\" ref-type=\"bibr\">105</xref>–<xref rid=\"B107\" ref-type=\"bibr\">107</xref>). In addition, a novel vaccine targeting RANKL by introducing a p-nitrophenylalanine at a single site in mRANKL immunization could prevent OVX-induced bone loss in mice (<xref rid=\"B108\" ref-type=\"bibr\">108</xref>). Notably, anti-RANKL antibody inhibited osteoporosis and bone destruction, but possesses no therapeutic effect on RA disease. Therefore, it is necessary to rethink about the underlying mechanisms of bone-related diseases.</p><p>Recently, extracts and natural products derived from traditional Chinese medicine (TCM) have great potential as well as advantages in the prevention and treatment of osteoporosis in terms of good therapeutic effect, low toxicity, and side effects (<xref rid=\"B109\" ref-type=\"bibr\">109</xref>, <xref rid=\"B110\" ref-type=\"bibr\">110</xref>), and they have gained increasing attention from the medical community. For example, polysaccharides derived from persimmon leaves down-regulated RANKL-induced activation of mitogen-activated protein kinases (MAPKs) to suppress the nuclear factor of NFATc1 expression, thus possessing anti-osteoporotic effects in OVX-induced bone loss. The natural product cyperenoic acid is a terpenoid isolated from the medicinal plant <italic>Croton crassifolius</italic>, and it suppressed osteoclast differentiation by inhibiting the NF-κB pathway and suppressed RANKL expression (<xref rid=\"B111\" ref-type=\"bibr\">111</xref>). Baohuoside I is an active component of Herba Epimedii with the immune regulation functions of T cells and antioxidant activity, which serves as a candidate for treating postmenopausal osteoporosis (<xref rid=\"B112\" ref-type=\"bibr\">112</xref>). All these results indicated that drugs from TCM possess anti-osteoporosis effects by the regulation of T cells, and they may show great potential as therapeutic agents for osteoporosis. However, further experimental and clinical research remains to be specifically conducted to explore the cellular and molecular mechanisms of the drugs from TCM.</p></sec><sec id=\"s5\"><title>Conclusion and Perspective</title><p>The pathogen clearance of various types of osteoporosis would be impaired or would delay bone resorption due to the dysfunction of the T cells. Therefore, understanding the roles of T cells in the pathogenesis of osteoporosis and the mechanisms underlying these pathologies between the immune system and the bone system may lead to the development of new treatments for osteoporosis. However, further studies, especially clinical studies, are required to explore the safety of T cell therapy for bone loss.</p></sec><sec id=\"s6\"><title>Author Contributions</title><p>WZ designed, wrote and revised the whole manuscript. YH wrote the manuscript. AQ and KD helped to revise the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s7\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was funded by the National Natural Science Foundation of China (No. 81901917), China's Postdoctoral Science Fund (No. 2017M623249), and the Key Research and Development Project of Shaanxi Province (No. 2018SF-363).</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dai</surname><given-names>Z</given-names></name><name><surname>Zhang</surname><given-names>Y</given-names></name><name><surname>Lu</surname><given-names>N</given-names></name><name><surname>Felson</surname><given-names>DT</given-names></name><name><surname>Kiel</surname><given-names>DP</given-names></name><name><surname>Sahni</surname><given-names>S</given-names></name></person-group>. <article-title>Association between dietary fiber intake and bone loss in the Framingham Offspring Study</article-title>. <source>J Bone Miner Res.</source> (<year>2018</year>) <volume>33</volume>:<fpage>241</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jbmr.3308</pub-id><pub-id pub-id-type=\"pmid\">29024045</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yaacobi</surname><given-names>E</given-names></name><name><surname>Sanchez</surname><given-names>D</given-names></name><name><surname>Maniar</surname><given-names>H</given-names></name><name><surname>Horwitz</surname><given-names>DS</given-names></name></person-group>. <article-title>Surgical treatment of osteoporotic fractures: an update on the principles of management</article-title>. <source>Injury.</source> (<year>2017</year>) <volume>48</volume>:<fpage>S34</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.injury.2017.08.036</pub-id><pub-id pub-id-type=\"pmid\">28882375</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wright</surname><given-names>NC</given-names></name><name><surname>Looker</surname><given-names>AC</given-names></name><name><surname>Saag</surname><given-names>KG</given-names></name><name><surname>Curtis</surname><given-names>JR</given-names></name><name><surname>Delzell</surname><given-names>ES</given-names></name><name><surname>Randall</surname><given-names>S</given-names></name><etal/></person-group>\n<article-title>The recent prevalence of osteoporosis and low bone mass in the United States based on bone mineral density at the femoral neck or lumbar spine</article-title>. <source>J Bone Miner Res.</source> (<year>2016</year>) <volume>29</volume>:<fpage>2520</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jbmr.2269</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>B</given-names></name><name><surname>Wang</surname><given-names>C-Y</given-names></name></person-group>. <article-title>Osteoporosis: the result of an ‘aged’ bone microenvironment</article-title>. <source>Trends Mol Med.</source> (<year>2016</year>) <volume>22</volume>:<fpage>641</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.molmed.2016.06.002</pub-id><pub-id pub-id-type=\"pmid\">27354328</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Raisz</surname><given-names>LG</given-names></name><name><surname>Seeman</surname><given-names>E</given-names></name></person-group>. <article-title>Causes of age-related bone loss and bone fragility: an alternative view</article-title>. <source>Eur J Bone Miner. Res.</source> (<year>2002</year>) <volume>16</volume>:<fpage>1948</fpage>–<lpage>52</lpage>. <pub-id pub-id-type=\"doi\">10.1359/jbmr.2001.16.11.1948</pub-id><pub-id pub-id-type=\"pmid\">11697790</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Okamoto</surname><given-names>K</given-names></name><name><surname>Nakashima</surname><given-names>T</given-names></name><name><surname>Shinohara</surname><given-names>M</given-names></name><name><surname>Negishi-Koga</surname><given-names>T</given-names></name><name><surname>Komatsu</surname><given-names>N</given-names></name><name><surname>Terashima</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Osteoimmunology: the conceptual framework unifying the immune and skeletal systems</article-title>. <source>Physiol Rev.</source> (<year>2017</year>) <volume>97</volume>:<fpage>1295</fpage>–<lpage>349</lpage>. <pub-id pub-id-type=\"doi\">10.1152/physrev.00036.2016</pub-id><pub-id pub-id-type=\"pmid\">28814613</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Srivastava</surname><given-names>RK</given-names></name></person-group>. <article-title>Osteoimmunology: the nexus between bone and immune system</article-title>. <source>Front Biosci.</source> (<year>2018</year>) <volume>23</volume>:<fpage>464</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.2741/4600</pub-id><pub-id pub-id-type=\"pmid\">28930556</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Takayanagi</surname><given-names>H</given-names></name></person-group>\n<article-title>Osteoimmunology and the effects of the immune system on bone</article-title>. <source>Nat Rev Rheumatol.</source> (<year>2009</year>) <volume>5</volume>:<fpage>667</fpage>–<lpage>76</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrrheum.2009.217</pub-id><pub-id pub-id-type=\"pmid\">19884898</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Takayanagi</surname><given-names>H</given-names></name></person-group>. <article-title>Osteoimmunology in 2014: two-faced immunology-from osteogenesis to bone resorption</article-title>. <source>Nat Rev Rheumatol.</source> (<year>2015</year>) <volume>11</volume>:<fpage>74</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrrheum.2014.219</pub-id><pub-id pub-id-type=\"pmid\">25561367</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Okamoto</surname><given-names>K</given-names></name><name><surname>Takayanagi</surname><given-names>H</given-names></name></person-group>\n<article-title>Osteoimmunology</article-title>. <source>Cold Spring Harb Perspect Med.</source> (<year>2018</year>) <volume>9</volume>:<fpage>a031245</fpage>\n<pub-id pub-id-type=\"doi\">10.1101/cshperspect.a031245</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Srivastava</surname><given-names>RK</given-names></name><name><surname>Dar</surname><given-names>HY</given-names></name><name><surname>Mishra</surname><given-names>PK</given-names></name></person-group>. <article-title>Immunoporosis: immunology of osteoporosis-role of t cells</article-title>. <source>Front. Immunol.</source> (<year>2018</year>) <volume>9</volume>:<fpage>657</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2018.00657</pub-id><pub-id pub-id-type=\"pmid\">29675022</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kalyan</surname><given-names>S</given-names></name></person-group>. <article-title>It may seem inflammatory, but some T cells are innately healing to the bone</article-title>. <source>J Bone Miner Res.</source> (<year>2016</year>) <volume>31</volume>:<fpage>1997</fpage>–<lpage>2000</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jbmr.2875</pub-id><pub-id pub-id-type=\"pmid\">27207251</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Harmer</surname><given-names>D</given-names></name><name><surname>Falank</surname><given-names>C</given-names></name><name><surname>Reagan</surname><given-names>MR</given-names></name></person-group>. <article-title>Interleukin-6 interweaves the bone marrow microenvironment, bone loss, and multiple myeloma</article-title>. <source>Front Endocrinol.</source> (<year>2019</year>) <volume>9</volume>:<fpage>788</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fendo.2018.00788</pub-id><pub-id pub-id-type=\"pmid\">30671025</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Toraldo</surname><given-names>G</given-names></name><name><surname>Li</surname><given-names>A</given-names></name><name><surname>Yang</surname><given-names>X</given-names></name><name><surname>Weitzmann</surname><given-names>MN</given-names></name></person-group>. <article-title>B cells and T cells are critical for the preservation of bone homeostasis and attainment of peak bone mass <italic>in vivo</italic></article-title>. <source>Blood.</source> (<year>2007</year>) <volume>109</volume>:<fpage>3839</fpage>–<lpage>48</lpage>. <pub-id pub-id-type=\"doi\">10.1182/blood-2006-07-037994</pub-id><pub-id pub-id-type=\"pmid\">17202317</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weitzmann</surname><given-names>MN</given-names></name><name><surname>Ofotokun</surname><given-names>I</given-names></name></person-group>. <article-title>Physiological and pathophysiological bone turnover-role of the immune system</article-title>. <source>Nat Rev Endocrinol.</source> (<year>2016</year>) <volume>12</volume>:<fpage>518</fpage>–<lpage>32</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrendo.2016.91</pub-id><pub-id pub-id-type=\"pmid\">27312863</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fuller</surname><given-names>K</given-names></name><name><surname>Murphy</surname><given-names>C</given-names></name><name><surname>Kirstein</surname><given-names>B</given-names></name><name><surname>Fox</surname><given-names>SW</given-names></name><name><surname>Chambers</surname><given-names>TJ</given-names></name></person-group>. <article-title>TNFalpha potently activates osteoclasts, through a direct action independent of and strongly synergistic with RANKL</article-title>. <source>Endocrinol.</source> (<year>2002</year>) <volume>143</volume>:<fpage>1108</fpage>–<lpage>18</lpage>. <pub-id pub-id-type=\"doi\">10.1210/endo.143.3.8701</pub-id><pub-id pub-id-type=\"pmid\">11861538</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dar</surname><given-names>HY</given-names></name><name><surname>Shukla</surname><given-names>P</given-names></name><name><surname>Mishra</surname><given-names>PK</given-names></name><name><surname>Anupam</surname><given-names>R</given-names></name><name><surname>Mondal</surname><given-names>RK</given-names></name><name><surname>Tomar</surname><given-names>GB</given-names></name><etal/></person-group>. <article-title><italic>Lactobacillus acidophilus</italic> inhibits bone loss and increases bone heterogeneity in osteoporotic mice via modulating Treg-Th17 cell balance</article-title>. <source>Bone Rep.</source> (<year>2018</year>) <volume>8</volume>:<fpage>46</fpage>–<lpage>56</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bonr.2018.02.001</pub-id><pub-id pub-id-type=\"pmid\">29955622</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Buchwald</surname><given-names>ZS</given-names></name><name><surname>Kiesel</surname><given-names>JR</given-names></name><name><surname>Yang</surname><given-names>C</given-names></name><name><surname>DiPaolo</surname><given-names>R</given-names></name><name><surname>Novack</surname><given-names>DV</given-names></name><name><surname>Aurora</surname><given-names>R</given-names></name></person-group>. <article-title>Osteoclast-induced Foxp3+ CD8 T-cells limit bone loss in mice</article-title>. <source>Bone.</source> (<year>2013</year>) <volume>56</volume>:<fpage>163</fpage>–<lpage>73</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bone.2013.05.024</pub-id><pub-id pub-id-type=\"pmid\">23756229</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Glowacki</surname><given-names>AJ</given-names></name><name><surname>Yoshizawa</surname><given-names>S</given-names></name><name><surname>Jhunjhunwala</surname><given-names>S</given-names></name><name><surname>Vieira</surname><given-names>AE</given-names></name><name><surname>Garlet</surname><given-names>GP</given-names></name><name><surname>Sfeir</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>Prevention of inflammation-mediated bone loss in murine and canine periodontal disease via recruitment of regulatory lymphocytes</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2013</year>) <volume>110</volume>:<fpage>18525</fpage>–<lpage>30</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1302829110</pub-id><pub-id pub-id-type=\"pmid\">24167272</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luo</surname><given-names>C</given-names></name><name><surname>Wang</surname><given-names>L</given-names></name><name><surname>Sun</surname><given-names>C</given-names></name><name><surname>Li</surname><given-names>D</given-names></name></person-group>. <article-title>Estrogen enhances the functions of CD4+CD25+Foxp3+ regulatory T cells that suppress osteoclast differentiation and bone resorption <italic>in vitro</italic></article-title>. <source>Cell Mol Immunol.</source> (<year>2011</year>) <volume>8</volume>:<fpage>50</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1038/cmi.2010.54</pub-id><pub-id pub-id-type=\"pmid\">21200384</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yuan</surname><given-names>FL</given-names></name><name><surname>Li</surname><given-names>X</given-names></name><name><surname>Lu</surname><given-names>W-G</given-names></name><name><surname>Xu</surname><given-names>R-S</given-names></name><name><surname>Zhao</surname><given-names>Y-Q</given-names></name><name><surname>Li</surname><given-names>C-W</given-names></name><etal/></person-group>. <article-title>Regulatory T cells as a potent target for controlling bone loss</article-title>. <source>Biochem Biophys Res Commun.</source> (<year>2010</year>) <volume>402</volume>:<fpage>173</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbrc.2010.09.120</pub-id><pub-id pub-id-type=\"pmid\">20920469</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tanaka</surname><given-names>Y</given-names></name></person-group>. <article-title>Clinical immunity in bone and joints</article-title>. <source>J Bone Miner Metab.</source> (<year>2018</year>) <volume>37</volume>:<fpage>2</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00774-018-0965-5</pub-id><pub-id pub-id-type=\"pmid\">30324535</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ofotokun</surname><given-names>I</given-names></name><name><surname>Titanji</surname><given-names>K</given-names></name><name><surname>Vikulina</surname><given-names>T</given-names></name><name><surname>Roser-Page</surname><given-names>S</given-names></name><name><surname>Yamaguchi</surname><given-names>M</given-names></name><name><surname>Zayzafoon</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Role of T-cell reconstitution in HIV-1 antiretroviral therapy-induced bone loss</article-title>. <source>Nat Commun.</source> (<year>2016</year>) <volume>6</volume>:<fpage>8282</fpage>. <pub-id pub-id-type=\"doi\">10.1038/ncomms9282</pub-id><pub-id pub-id-type=\"pmid\">26392000</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Santana-Davila</surname><given-names>R</given-names></name><name><surname>Show</surname><given-names>LQ</given-names></name></person-group>. <article-title>The use of combination immunotherapies as frontline therapy for non-small cell lung cancer</article-title>. <source>Future Oncol</source>. (<year>2018</year>) <volume>14</volume>:<fpage>191</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.2217/fon-2017-0124</pub-id><pub-id pub-id-type=\"pmid\">29334785</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jin</surname><given-names>HM</given-names></name><name><surname>Shao</surname><given-names>ZX</given-names></name><name><surname>Wang</surname><given-names>QQ</given-names></name><name><surname>Miao</surname><given-names>JS</given-names></name><name><surname>Bai</surname><given-names>XQ</given-names></name><name><surname>Liu</surname><given-names>Q</given-names></name><etal/></person-group>. <article-title>Sclareol prevents ovariectomy-induced bone loss <italic>in vivo</italic> and inhibits osteoclastogenesis <italic>in vitro</italic> via suppressing NF-κB and MAPK/ERK signaling pathways</article-title>. <source>Food Funct.</source> (<year>2019</year>) <volume>10</volume>:<fpage>6556</fpage>. <pub-id pub-id-type=\"doi\">10.1039/C9FO00206E</pub-id><pub-id pub-id-type=\"pmid\">31549110</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bozec</surname><given-names>A</given-names></name><name><surname>Zaiss</surname><given-names>MM</given-names></name></person-group>. <article-title>T regulatory cells in bone remodelling</article-title>. <source>Curr Osteoporos Rep.</source> (<year>2017</year>) <volume>15</volume>:<fpage>121</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s11914-017-0356-1</pub-id><pub-id pub-id-type=\"pmid\">28432597</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Walsh</surname><given-names>MC</given-names></name><name><surname>Choi</surname><given-names>Y</given-names></name></person-group>. <article-title>Biology of the RANKL-RANK-OPG system in immunity, bone, and beyond</article-title>. <source>Front Immunol.</source> (<year>2014</year>) <volume>5</volume>:<fpage>511</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2014.00511</pub-id><pub-id pub-id-type=\"pmid\">25368616</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Takayanagi</surname><given-names>H</given-names></name><name><surname>Ogasawara</surname><given-names>K</given-names></name><name><surname>Hida</surname><given-names>S</given-names></name><name><surname>Chiba</surname><given-names>T</given-names></name><name><surname>Taniguchi</surname><given-names>T</given-names></name></person-group>. <article-title>T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-gamma</article-title>. <source>Nature.</source> (<year>2000</year>) <volume>408</volume>:<fpage>600</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1038/35046102</pub-id><pub-id pub-id-type=\"pmid\">11117749</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Theill</surname><given-names>LE</given-names></name><name><surname>Boyle</surname><given-names>WJ</given-names></name><name><surname>Penninger</surname><given-names>JM</given-names></name></person-group>. <article-title>RANK-L and RANK: T cells, bone loss, and mammalian evolution</article-title>. <source>Annu Rev Immunol.</source> (<year>2002</year>) <volume>20</volume>:<fpage>795</fpage>–<lpage>823</lpage>. <pub-id pub-id-type=\"doi\">10.1146/annurev.immunol.20.100301.064753</pub-id><pub-id pub-id-type=\"pmid\">11861618</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ginaldi</surname><given-names>L</given-names></name><name><surname>Benedetto</surname><given-names>MCD</given-names></name><name><surname>Martinis</surname><given-names>MD</given-names></name></person-group>. <article-title>Osteoporosis, inflammation and ageing</article-title>. <source>Immun Ageing.</source> (<year>2005</year>) <volume>2</volume>:<fpage>1</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1186/1742-4933-2-14</pub-id><pub-id pub-id-type=\"pmid\">15679897</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schett</surname><given-names>G</given-names></name><name><surname>David</surname><given-names>JP</given-names></name></person-group>. <article-title>The multiple faces of autoimmune-mediated bone loss</article-title>. <source>Nat Rev Endocrinol.</source> (<year>2010</year>) <volume>6</volume>:<fpage>698</fpage>–<lpage>706</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrendo.2010.190</pub-id><pub-id pub-id-type=\"pmid\">21045788</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nagy</surname><given-names>V</given-names></name><name><surname>Penninger</surname><given-names>JM</given-names></name></person-group>. <article-title>The RANKL-RANK story</article-title>. <source>Gerontol.</source> (<year>2015</year>) <volume>61</volume>:<fpage>534</fpage>–<lpage>42</lpage>. <pub-id pub-id-type=\"doi\">10.1159/000371845</pub-id><pub-id pub-id-type=\"pmid\">25720990</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pacifici</surname><given-names>R</given-names></name></person-group>. <article-title>T cells, osteoblasts, and osteocytes: interacting lineages key for the bone anabolic and catabolic activities of parathyroid hormone</article-title>. <source>Ann N Y Acad Sci.</source> (<year>2016</year>) <volume>1364</volume>:<fpage>11</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1111/nyas.12969</pub-id><pub-id pub-id-type=\"pmid\">26662934</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>JY</given-names></name><name><surname>D'Amelio</surname><given-names>P</given-names></name><name><surname>Robinson</surname><given-names>J</given-names></name><name><surname>Walker</surname><given-names>LD</given-names></name><name><surname>Vaccaro</surname><given-names>C</given-names></name><name><surname>Luo</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>IL-17A is increased in humans with primary hyperparathyroidism and mediates PTH-induced bone loss in mice</article-title>. <source>Cell Metab.</source> (<year>2015</year>) <volume>22</volume>:<fpage>799</fpage>–<lpage>810</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cmet.2015.09.012</pub-id><pub-id pub-id-type=\"pmid\">26456334</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kotake</surname><given-names>S</given-names></name><name><surname>Nanke</surname><given-names>Y</given-names></name><name><surname>Mogi</surname><given-names>M</given-names></name><name><surname>Kawamoto</surname><given-names>M</given-names></name><name><surname>Furuya</surname><given-names>T</given-names></name><name><surname>Yago</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>IFN-γ-producing human T cells directly induce osteoclastogenesis from human monocytes via the expression of RANKL</article-title>. <source>Eur J Immunol.</source> (<year>2005</year>) <volume>35</volume>:<fpage>3353</fpage>–<lpage>63</lpage>. <pub-id pub-id-type=\"doi\">10.1002/eji.200526141</pub-id><pub-id pub-id-type=\"pmid\">16220542</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Adamopoulos</surname><given-names>IE</given-names></name><name><surname>Chao</surname><given-names>CC</given-names></name><name><surname>Geissler</surname><given-names>R</given-names></name><name><surname>Laface</surname><given-names>D</given-names></name><name><surname>Bowman</surname><given-names>EP</given-names></name></person-group>. <article-title>Interleukin17A upregulates receptor activator of NF-κB on osteoclast precursors</article-title>. <source>Arthritis Res Ther.</source> (<year>2010</year>) <volume>12</volume>:<fpage>1</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1186/ar2936</pub-id><pub-id pub-id-type=\"pmid\">20167120</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boyce</surname><given-names>BF</given-names></name><name><surname>Xing</surname><given-names>L</given-names></name></person-group>. <article-title>Functions of RANKL/RANK/OPG in bone modeling and remodeling</article-title>. <source>Arch Biochem Biophys.</source> (<year>2008</year>) <volume>473</volume>:<fpage>139</fpage>–<lpage>46</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.abb.2008.03.018</pub-id><pub-id pub-id-type=\"pmid\">18395508</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Adamopoulos</surname><given-names>IE</given-names></name><name><surname>Bowman</surname><given-names>EP</given-names></name></person-group>. <article-title>Immune regulation of bone loss by Th17 cells in oestrogen-deficient osteoporosis</article-title>. <source>Eur J Clin Invest.</source> (<year>2013</year>) <volume>43</volume>:<fpage>1195</fpage>–<lpage>203</lpage>. <pub-id pub-id-type=\"doi\">10.1111/eci.12158</pub-id><pub-id pub-id-type=\"pmid\">24033116</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Harrington</surname><given-names>L</given-names></name></person-group>. <article-title>Expanding the effector CD4 T-cell repertoire: the Th17 lineage</article-title>. <source>Curr Opin Immunol.</source> (<year>2006</year>) <volume>18</volume>:<fpage>349</fpage>–<lpage>56</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.coi.2006.03.017</pub-id><pub-id pub-id-type=\"pmid\">16616472</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mangashetti</surname><given-names>LS</given-names></name><name><surname>Khapli</surname><given-names>SM</given-names></name><name><surname>Wani</surname><given-names>MR</given-names></name></person-group>. <article-title>IL-4 inhibits bone-resorbing activity of mature osteoclasts by affecting NF-KB and Ca<sup>2+</sup> Signaling</article-title>. <source>J Immunol.</source> (<year>2005</year>) <volume>175</volume>:<fpage>917</fpage>–<lpage>25</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.175.2.917</pub-id><pub-id pub-id-type=\"pmid\">16002690</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yun</surname><given-names>TJ</given-names></name><name><surname>Chaudhary</surname><given-names>PM</given-names></name><name><surname>Shu</surname><given-names>GL</given-names></name><name><surname>Frazer</surname><given-names>JK</given-names></name><name><surname>Ewings</surname><given-names>MK</given-names></name><name><surname>Schwartz</surname><given-names>SM</given-names></name><etal/></person-group>. <article-title>OPG/FDCR-1, a TNF receptor family member, is expressed in lymphoid cells and is up-regulated by ligating CD40</article-title>. <source>J Immunol.</source> (<year>1998</year>) <volume>161</volume>:<fpage>6113</fpage>–<lpage>21</lpage>. <pub-id pub-id-type=\"pmid\">9834095</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sun-Kyeong</surname><given-names>L</given-names></name><name><surname>Kalinowski</surname><given-names>JF</given-names></name><name><surname>Jastrzebski</surname><given-names>SL</given-names></name><name><surname>Lynn</surname><given-names>P</given-names></name><name><surname>Lorenzo</surname><given-names>JA</given-names></name></person-group>\n<article-title>Interleukin-7 is a direct inhibitor of <italic>in vitro</italic> osteoclastogenesis</article-title>. <source>Endocrinology</source>. (<year>2003</year>) <volume>144</volume>:<fpage>3524</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.1210/en.2002-221057</pub-id><pub-id pub-id-type=\"pmid\">12865334</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wing</surname><given-names>K</given-names></name><name><surname>Yamaguchi</surname><given-names>T</given-names></name><name><surname>Sakaguchi</surname><given-names>S</given-names></name></person-group>. <article-title>Cell-autonomous and -non-autonomous roles of CTLA-4 in immune regulation</article-title>. <source>Trends Immunol.</source> (<year>2011</year>) <volume>32</volume>:<fpage>428</fpage>–<lpage>33</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.it.2011.06.002</pub-id><pub-id pub-id-type=\"pmid\">21723783</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arboleya</surname><given-names>L</given-names></name><name><surname>Castañeda</surname><given-names>S</given-names></name></person-group>\n<article-title>Osteoimmunology: the study of the relationship between the immune system and bone tissue</article-title>. <source>Reumatol Clin</source>. (<year>2013</year>) <volume>9</volume>:<fpage>303</fpage>–<lpage>15</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.reumae.2013.02.004</pub-id><pub-id pub-id-type=\"pmid\">23727459</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Woodward</surname><given-names>J</given-names></name></person-group>. <article-title>Regulation of haematopoietic progenitor cell proliferation and survival The involvement of the osteoblast</article-title>. <source>Cell Adh Migr.</source> (<year>2010</year>) <volume>4</volume>:<fpage>4</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.4161/cam.4.1.10106</pub-id><pub-id pub-id-type=\"pmid\">19949313</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nagata</surname><given-names>N</given-names></name></person-group>. <article-title>Inhibition of RANKL-induced osteoclast formation in mouse bone marrow cells IL-12 : involvement of IFN-γ possibly induced from non-T cell population</article-title>. <source>Bone.</source> (<year>2003</year>) <volume>33</volume>:<fpage>721</fpage>–<lpage>32</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S8756-3282(03)00213-8</pub-id><pub-id pub-id-type=\"pmid\">14555278</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hayday</surname><given-names>AC</given-names></name></person-group>. <article-title>γ<italic>δ</italic> T Cells and the lymphoid stress-surveillance response</article-title>. <source>Immunity.</source> (<year>2009</year>) <volume>31</volume>:<fpage>184</fpage>–<lpage>96</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.immuni.2009.08.006</pub-id><pub-id pub-id-type=\"pmid\">19699170</pub-id></mixed-citation></ref><ref id=\"B48\"><label>48.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sato</surname><given-names>K</given-names></name><name><surname>Suematsu</surname><given-names>A</given-names></name><name><surname>Okamoto</surname><given-names>K</given-names></name><name><surname>Yamaguchi</surname><given-names>A</given-names></name><name><surname>Morishita</surname><given-names>Y</given-names></name><name><surname>Kadono</surname><given-names>Y</given-names></name><etal/></person-group>. <article-title>Th17 functions as an osteoclastogenic helper T cell subset that links T cell activation and bone destruction</article-title>. <source>J Exp Med.</source> (<year>2006</year>) <volume>203</volume>:<fpage>2673</fpage>–<lpage>82</lpage>. <pub-id pub-id-type=\"doi\">10.1084/jem.20061775</pub-id><pub-id pub-id-type=\"pmid\">17088434</pub-id></mixed-citation></ref><ref id=\"B49\"><label>49.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Clayton</surname><given-names>ES</given-names></name><name><surname>Hochberg</surname><given-names>MC</given-names></name></person-group>. <article-title>Osteoporosis and osteoarthritis, rheumatoid arthritis and spondylarthropathies</article-title>. <source>Curr Osteoporos Rep.</source> (<year>2013</year>) <volume>11</volume>:<fpage>257</fpage>–<lpage>62</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s11914-013-0172-1</pub-id><pub-id pub-id-type=\"pmid\">24085651</pub-id></mixed-citation></ref><ref id=\"B50\"><label>50.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gulati</surname><given-names>AM</given-names></name><name><surname>Michelsen</surname><given-names>B</given-names></name><name><surname>Diamantopoulos</surname><given-names>A</given-names></name><name><surname>Grandaunet</surname><given-names>B</given-names></name><name><surname>Salvesen</surname><given-names>Ø</given-names></name><name><surname>Kavanaugh</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Osteoporosis in psoriatic arthritis: a cross-sectional study of an outpatient clinic population</article-title>. <source>RMD Open.</source> (<year>2018</year>) <volume>4</volume>:<fpage>e000631</fpage>. <pub-id pub-id-type=\"doi\">10.1136/rmdopen-2017-000631</pub-id><pub-id pub-id-type=\"pmid\">30018797</pub-id></mixed-citation></ref><ref id=\"B51\"><label>51.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schett</surname><given-names>G</given-names></name><name><surname>Gravallese</surname><given-names>E</given-names></name></person-group>. <article-title>Bone erosion in rheumatoid arthritis: mechanisms, diagnosis and treatment</article-title>. <source>Nat Rev Rheumatol.</source> (<year>2012</year>) <volume>8</volume>:<fpage>656</fpage>–<lpage>64</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrrheum.2012.153</pub-id><pub-id pub-id-type=\"pmid\">23007741</pub-id></mixed-citation></ref><ref id=\"B52\"><label>52.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ødegård</surname><given-names>S</given-names></name><name><surname>Landewé</surname><given-names>R</given-names></name><name><surname>van der Heijde</surname><given-names>D</given-names></name><name><surname>Kvien</surname><given-names>TK</given-names></name><name><surname>Mowinckel</surname><given-names>P</given-names></name><name><surname>Uhlig</surname><given-names>T</given-names></name></person-group>. <article-title>Association of early radiographic damage with impaired physical function in rheumatoid arthritis: a ten-year, longitudinal observational study in 238 patients</article-title>. <source>Arthritis Rheum.</source> (<year>2006</year>) <volume>54</volume>:<fpage>68</fpage>–<lpage>75</lpage>. <pub-id pub-id-type=\"doi\">10.1002/art.21548</pub-id><pub-id pub-id-type=\"pmid\">16385497</pub-id></mixed-citation></ref><ref id=\"B53\"><label>53.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zaiss</surname><given-names>MM</given-names></name><name><surname>Axmann</surname><given-names>R</given-names></name><name><surname>Zwerina</surname><given-names>J</given-names></name><name><surname>Polzer</surname><given-names>K</given-names></name><name><surname>Gückel</surname><given-names>E</given-names></name><name><surname>Skapenko</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Treg cells suppress osteoclast formation: a new link between the immune system and bone</article-title>. <source>Arthritis Rheum.</source> (<year>2007</year>) <volume>56</volume>:<fpage>4104</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.1002/art.23138</pub-id><pub-id pub-id-type=\"pmid\">18050211</pub-id></mixed-citation></ref><ref id=\"B54\"><label>54.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fischer</surname><given-names>L</given-names></name><name><surname>Herkner</surname><given-names>C</given-names></name><name><surname>Kitte</surname><given-names>R</given-names></name><name><surname>Dohnke</surname><given-names>S</given-names></name><name><surname>Riewaldt</surname><given-names>J</given-names></name><name><surname>Kretschmer</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Foxp3<sup>+</sup> regulatory T cells in bone and hematopoietic homeostasis</article-title>. <source>Front Endocrinol.</source> (<year>2019</year>) <volume>10</volume>:<fpage>578</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fendo.2019.00578</pub-id><pub-id pub-id-type=\"pmid\">31551927</pub-id></mixed-citation></ref><ref id=\"B55\"><label>55.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kwak</surname><given-names>MK</given-names></name><name><surname>Lee</surname><given-names>EJ</given-names></name><name><surname>Park</surname><given-names>JW</given-names></name><name><surname>Park</surname><given-names>SY</given-names></name><name><surname>Kim</surname><given-names>BJ</given-names></name><name><surname>Kim</surname><given-names>TH</given-names></name><etal/></person-group>. <article-title>CD4 cell count is inversly associated with lumbar spine bone mass in HIV-infected men under the age of 50 years</article-title>. <source>Oateoporosis Int.</source> (<year>2019</year>) <volume>30</volume>:<fpage>1501</fpage>–<lpage>30</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00198-019-05115-2</pub-id><pub-id pub-id-type=\"pmid\">30915506</pub-id></mixed-citation></ref><ref id=\"B56\"><label>56.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krikke</surname><given-names>M</given-names></name><name><surname>Klomberg</surname><given-names>RCW</given-names></name><name><surname>Veer</surname><given-names>EVD</given-names></name><name><surname>Tesselaar</surname><given-names>K</given-names></name><name><surname>Verhaar</surname><given-names>HJJ</given-names></name><name><surname>Hoepelman</surname><given-names>AIM</given-names></name><etal/></person-group>. <article-title>Osteoporosis and osteopenia are not associated with T-cell activation in older cART-treated HIV-infected patients</article-title>. <source>Neth J Med.</source> (<year>2017</year>) <volume>75</volume>:<fpage>138</fpage>–<lpage>44</lpage>. <pub-id pub-id-type=\"pmid\">28522769</pub-id></mixed-citation></ref><ref id=\"B57\"><label>57.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jung</surname><given-names>YK</given-names></name><name><surname>Kang</surname><given-names>YM</given-names></name><name><surname>Han</surname><given-names>S</given-names></name></person-group>. <article-title>Osteoclasts in the inflammatory arthritis: implications for pathologic osteolysis</article-title>. <source>Immune Netw.</source> (<year>2019</year>) <volume>19</volume>:<fpage>1</fpage>–<lpage>13</lpage>. <pub-id pub-id-type=\"doi\">10.4110/in.2019.19.e2</pub-id><pub-id pub-id-type=\"pmid\">30838157</pub-id></mixed-citation></ref><ref id=\"B58\"><label>58.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Braun</surname><given-names>T</given-names></name><name><surname>Zwerina</surname><given-names>J</given-names></name></person-group>. <article-title>Positive regulators of osteoclastogenesis and bone resorption in rheumatoid arthritis</article-title>. <source>Arthritis Res Ther.</source> (<year>2011</year>) <volume>13</volume>:<fpage>1</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1186/ar3380</pub-id><pub-id pub-id-type=\"pmid\">21861862</pub-id></mixed-citation></ref><ref id=\"B59\"><label>59.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fei</surname><given-names>D</given-names></name><name><surname>Zhang</surname><given-names>Y</given-names></name><name><surname>Wu</surname><given-names>J</given-names></name><name><surname>Zhang</surname><given-names>H</given-names></name><name><surname>Liu</surname><given-names>A</given-names></name><name><surname>He</surname><given-names>X</given-names></name><etal/></person-group>. <article-title>Cav 1.2 regulates osteogenesis of bone marrow-derived mesenchymal stem cells via canonical Wnt pathway in age-related osteoporosis</article-title>. <source>Aging Cell.</source> (<year>2019</year>) <volume>18</volume>:<fpage>e12967</fpage>. <pub-id pub-id-type=\"doi\">10.1111/acel.12967</pub-id><pub-id pub-id-type=\"pmid\">31120193</pub-id></mixed-citation></ref><ref id=\"B60\"><label>60.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pangrazzi</surname><given-names>L</given-names></name><name><surname>Weinberger</surname><given-names>B</given-names></name></person-group>. <article-title>T cells, aging and senescence</article-title>. <source>Exp Gerontol</source>. (<year>2020</year>) <volume>22</volume>:<fpage>110887</fpage>. <pub-id pub-id-type=\"doi\">10.1016/j.exger.2020.110887</pub-id><pub-id pub-id-type=\"pmid\">32092501</pub-id></mixed-citation></ref><ref id=\"B61\"><label>61.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Borrelli</surname><given-names>J</given-names></name><name><surname>Anglen</surname><given-names>JO</given-names></name></person-group> (editors). <article-title>Arthroplasty for the treatment of fractures in the older patient (indications and current techniques) || The relationship of peak bone mass, aging, and bone loss to osteoporosis and fragility fractures</article-title>. In: <source>Arthroplasty for the Treatment of Fractures in the Older Patient Chapter</source>. <publisher-loc>New York, NY</publisher-loc>: <publisher-name>Springer</publisher-name> (<year>2018</year>). p. <fpage>3</fpage>–<lpage>17</lpage>.</mixed-citation></ref><ref id=\"B62\"><label>62.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jiménez</surname><given-names>B</given-names></name><name><surname>Sainz</surname><given-names>T</given-names></name><name><surname>Díaz</surname><given-names>L</given-names></name><name><surname>Mellado</surname><given-names>MJ</given-names></name><name><surname>Navarro</surname><given-names>ML</given-names></name><name><surname>Rojo</surname><given-names>P</given-names></name></person-group>. <article-title>Low bone mineral density in vertically HIV-infected children and adolescents: risk factors and the role of T-cell activation and senescence</article-title>. <source>Pediatr Infect Dis J.</source> (<year>2017</year>) <volume>36</volume>:<fpage>578</fpage>–<lpage>83</lpage>. <pub-id pub-id-type=\"doi\">10.1097/INF.0000000000001506</pub-id><pub-id pub-id-type=\"pmid\">28005690</pub-id></mixed-citation></ref><ref id=\"B63\"><label>63.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Naismith</surname><given-names>E</given-names></name><name><surname>Pangrazzi</surname><given-names>L</given-names></name><name><surname>Grasse</surname><given-names>M</given-names></name><name><surname>Keller</surname><given-names>M</given-names></name><name><surname>Miggitsch</surname><given-names>C</given-names></name><name><surname>Weinberger</surname><given-names>B</given-names></name><etal/></person-group>. <article-title>Peripheral antibody concentrations are associated with highly differentiated T cells and inflammatory processes in the human bone marrow</article-title>. <source>Immun Ageing.</source> (<year>2019</year>) <volume>16</volume>:<fpage>21</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s12979-019-0161-z</pub-id><pub-id pub-id-type=\"pmid\">31462901</pub-id></mixed-citation></ref><ref id=\"B64\"><label>64.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martinis</surname><given-names>MD</given-names></name><name><surname>Benedetto</surname><given-names>MCD</given-names></name><name><surname>Mengoli</surname><given-names>LP</given-names></name><name><surname>Ginaldi</surname><given-names>L</given-names></name></person-group>. <article-title>Senile osteoporosis: is it an immune-mediated disease?</article-title>\n<source>Inflamm Res.</source> (<year>2006</year>) <volume>55</volume>:<fpage>399</fpage>–<lpage>404</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00011-006-6034-x</pub-id><pub-id pub-id-type=\"pmid\">17109066</pub-id></mixed-citation></ref><ref id=\"B65\"><label>65.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mundy</surname><given-names>GR</given-names></name></person-group>. <article-title>Osteoporosis and inflammation</article-title>. <source>Nutr Rev.</source> (<year>2007</year>) <volume>65</volume>:<fpage>S147</fpage>–<lpage>51</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1753-4887.2007.tb00353.x</pub-id><pub-id pub-id-type=\"pmid\">18240539</pub-id></mixed-citation></ref><ref id=\"B66\"><label>66.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Deng</surname><given-names>L</given-names></name><name><surname>Hu</surname><given-names>G</given-names></name><name><surname>Jin</surname><given-names>L</given-names></name><name><surname>Wang</surname><given-names>C</given-names></name><name><surname>Niu</surname><given-names>H</given-names></name></person-group>. <article-title>Involvement of microRNA-23b in TNF-α-reduced BMSC osteogenic differentiation via targeting runx2</article-title>. <source>J Bone Miner Metab.</source> (<year>2018</year>) <volume>36</volume>:<fpage>648</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00774-017-0886-8</pub-id><pub-id pub-id-type=\"pmid\">29234953</pub-id></mixed-citation></ref><ref id=\"B67\"><label>67.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wei</surname><given-names>S</given-names></name><name><surname>Kitaura</surname><given-names>H</given-names></name><name><surname>Zhou</surname><given-names>P</given-names></name><name><surname>Ross</surname><given-names>FP</given-names></name><name><surname>Teitelbaum</surname><given-names>SL</given-names></name></person-group>. <article-title>IL-1 mediates TNF-induced osteoclastogenesis</article-title>. <source>J Clin Invest.</source> (<year>2005</year>) <volume>115</volume>:<fpage>282</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1172/JCI200523394</pub-id><pub-id pub-id-type=\"pmid\">15668736</pub-id></mixed-citation></ref><ref id=\"B68\"><label>68.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Horwood</surname><given-names>NJ</given-names></name><name><surname>Elliott</surname><given-names>J</given-names></name><name><surname>Martin</surname><given-names>TJ</given-names></name><name><surname>Gillespie</surname><given-names>MT</given-names></name></person-group>. <article-title>IL-12 alone and in synergy with IL-18 inhibits osteoclast formation <italic>in vitro</italic></article-title>. <source>J Immunol.</source> (<year>2001</year>) <volume>166</volume>:<fpage>4915</fpage>–<lpage>21</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.166.8.4915</pub-id><pub-id pub-id-type=\"pmid\">11290769</pub-id></mixed-citation></ref><ref id=\"B69\"><label>69.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abu-Amer</surname><given-names>Y</given-names></name></person-group>. <article-title>IL-4 abrogates osteoclastogenesis through STAT6-dependent inhibition of NF-κB</article-title>. <source>J Clin Invest.</source> (<year>2001</year>) <volume>107</volume>:<fpage>1375</fpage>–<lpage>85</lpage>. <pub-id pub-id-type=\"doi\">10.1172/JCI10530</pub-id><pub-id pub-id-type=\"pmid\">11390419</pub-id></mixed-citation></ref><ref id=\"B70\"><label>70.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cong</surname><given-names>Q</given-names></name><name><surname>Jia</surname><given-names>H</given-names></name><name><surname>Li</surname><given-names>P</given-names></name><name><surname>Qiu</surname><given-names>S</given-names></name><name><surname>Yeh</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>Y</given-names></name><etal/></person-group>. <article-title>p38α MAPK regulates proliferation and differentiation of osteoclast progenitors and bone remodeling in an aging-dependent manner</article-title>. <source>Sci Rep.</source> (<year>2017</year>) <volume>7</volume>:<fpage>45964</fpage>. <pub-id pub-id-type=\"doi\">10.1038/srep45964</pub-id><pub-id pub-id-type=\"pmid\">28382965</pub-id></mixed-citation></ref><ref id=\"B71\"><label>71.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Fu</surname><given-names>Q</given-names></name><name><surname>Ren</surname><given-names>Z</given-names></name><name><surname>Wang</surname><given-names>Y</given-names></name><name><surname>Wang</surname><given-names>C</given-names></name><name><surname>Shen</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>Changes of serum cytokines-related Th1/Th2/Th17 concentration in patients with postmenopausal osteoporosis</article-title>. <source>Gynecol Endocrinol.</source> (<year>2015</year>) <volume>31</volume>:<fpage>183</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.3109/09513590.2014.975683</pub-id><pub-id pub-id-type=\"pmid\">25384921</pub-id></mixed-citation></ref><ref id=\"B72\"><label>72.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Giorgio</surname><given-names>M</given-names></name><name><surname>Patrizia</surname><given-names>DA</given-names></name><name><surname>Roberta</surname><given-names>F</given-names></name><name><surname>Giacomina</surname><given-names>B</given-names></name></person-group>. <article-title>Bone-immune cell crosstalk: bone diseases</article-title>. <source>J Immunol Res.</source> (<year>2015</year>) <volume>2015</volume>:<fpage>1</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1155/2015/108451</pub-id><pub-id pub-id-type=\"pmid\">26000310</pub-id></mixed-citation></ref><ref id=\"B73\"><label>73.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sang</surname><given-names>C</given-names></name><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Zhang</surname><given-names>Y</given-names></name><name><surname>Chen</surname><given-names>F</given-names></name><name><surname>Cao</surname><given-names>X</given-names></name><name><surname>Guo</surname><given-names>L</given-names></name></person-group>. <article-title>TNF-α promotes osteoclastogenesis through JNK signaling-dependent induction of Semaphorin3D expression in estrogen-deficiency induced osteoporosis</article-title>. <source>J Cell Physiol.</source> (<year>2017</year>) <volume>232</volume>:<fpage>3396</fpage>–<lpage>408</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jcp.25784</pub-id><pub-id pub-id-type=\"pmid\">28059444</pub-id></mixed-citation></ref><ref id=\"B74\"><label>74.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zha</surname><given-names>L</given-names></name><name><surname>He</surname><given-names>L</given-names></name><name><surname>Liang</surname><given-names>Y</given-names></name><name><surname>Qin</surname><given-names>H</given-names></name><name><surname>Yu</surname><given-names>B</given-names></name><name><surname>Chang</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>TNF-α contributes to postmenopausal osteoporosis by synergistically promoting RANKL-induced osteoclast formation</article-title>. <source>Biomed Pharmacother.</source> (<year>2018</year>) <volume>102</volume>:<fpage>369</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.biopha.2018.03.080</pub-id><pub-id pub-id-type=\"pmid\">29571022</pub-id></mixed-citation></ref><ref id=\"B75\"><label>75.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Du</surname><given-names>D</given-names></name><name><surname>Zhou</surname><given-names>Z</given-names></name><name><surname>Zhu</surname><given-names>L</given-names></name><name><surname>Hu</surname><given-names>X</given-names></name><name><surname>Lu</surname><given-names>J</given-names></name><name><surname>Shi</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>TNF-α suppresses osteogenic differentiation of MSCs by accelerating P2Y2 receptor in estrogen-deficiency induced osteoporosis</article-title>. <source>Bone.</source> (<year>2018</year>) <volume>117</volume>:<fpage>161</fpage>–<lpage>70</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bone.2018.09.012</pub-id><pub-id pub-id-type=\"pmid\">30236554</pub-id></mixed-citation></ref><ref id=\"B76\"><label>76.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Douin-Echinard</surname><given-names>V</given-names></name><name><surname>Laffont</surname><given-names>S</given-names></name><name><surname>Seillet</surname><given-names>C</given-names></name><name><surname>Delpy</surname><given-names>L</given-names></name><name><surname>Krust</surname><given-names>A</given-names></name><name><surname>Chambon</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Estrogen receptor alpha, but not beta, is required for optimal dendritic cell differentiation and CD40-induced cytokine production</article-title>. <source>J Immunol.</source> (<year>2008</year>) <volume>180</volume>:<fpage>3661</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.180.6.3661</pub-id><pub-id pub-id-type=\"pmid\">18322171</pub-id></mixed-citation></ref><ref id=\"B77\"><label>77.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zheng</surname><given-names>L</given-names></name><name><surname>Wang</surname><given-names>W</given-names></name><name><surname>Ni</surname><given-names>J</given-names></name><name><surname>Mao</surname><given-names>X</given-names></name><name><surname>Song</surname><given-names>D</given-names></name><name><surname>Liu</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>Role of autophagy in tumor necrosis factor-α-induced apoptosis of osteoblast cells</article-title>. <source>J Investig Med.</source> (<year>2017</year>) <volume>65</volume>:<fpage>1014</fpage>–<lpage>20</lpage>. <pub-id pub-id-type=\"doi\">10.1136/jim-2017-000426</pub-id><pub-id pub-id-type=\"pmid\">28634253</pub-id></mixed-citation></ref><ref id=\"B78\"><label>78.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moffett</surname><given-names>SP</given-names></name><name><surname>Zmuda</surname><given-names>JM</given-names></name><name><surname>Oakley</surname><given-names>JI</given-names></name><name><surname>Beck</surname><given-names>TJ</given-names></name><name><surname>Cauley</surname><given-names>JA</given-names></name><name><surname>Stone</surname><given-names>KL</given-names></name><etal/></person-group>. <article-title>Tumor necrosis factor-α polymorphism, bone strength phenotypes, and the risk of fracture in older women</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2005</year>) <volume>90</volume>:<fpage>3491</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2004-2235</pub-id><pub-id pub-id-type=\"pmid\">15797957</pub-id></mixed-citation></ref><ref id=\"B79\"><label>79.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tyagi</surname><given-names>AM</given-names></name><name><surname>Srivastava</surname><given-names>K</given-names></name><name><surname>Mansoori</surname><given-names>MN</given-names></name><name><surname>Trivedi</surname><given-names>R</given-names></name><name><surname>Chattopadhyay</surname><given-names>N</given-names></name><name><surname>Singh</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Estrogen deficiency induces the differentiation of IL-17 secreting Th17 cells: a new candidate in the pathogenesis of osteoporosis</article-title>. <source>PLoS ONE.</source> (<year>2012</year>) <volume>7</volume>:<fpage>e44552</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0044552</pub-id><pub-id pub-id-type=\"pmid\">22970248</pub-id></mixed-citation></ref><ref id=\"B80\"><label>80.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xu</surname><given-names>F</given-names></name><name><surname>Dong</surname><given-names>Y</given-names></name><name><surname>Huang</surname><given-names>X</given-names></name><name><surname>Chen</surname><given-names>P</given-names></name><name><surname>Huang</surname><given-names>S</given-names></name></person-group>. <article-title>Pioglitazone affects the OPG/RANKL/RANK system and increase osteoclastogenesis</article-title>. <source>Mol Med Rep.</source> (<year>2016</year>) <volume>14</volume>:<fpage>2289</fpage>–<lpage>96</lpage>. <pub-id pub-id-type=\"doi\">10.3892/mmr.2016.5515</pub-id><pub-id pub-id-type=\"pmid\">27430581</pub-id></mixed-citation></ref><ref id=\"B81\"><label>81.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pacifici</surname><given-names>R</given-names></name></person-group>. (<year>2007</year>). <article-title>T cells and post menopausal osteoporosis in murine models</article-title>. <source>Arthritis Res Ther</source>. <volume>9</volume>:<fpage>1</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1186/ar2126</pub-id><pub-id pub-id-type=\"pmid\">17349065</pub-id></mixed-citation></ref><ref id=\"B82\"><label>82.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wein</surname><given-names>MN</given-names></name><name><surname>Kronenberg</surname><given-names>HM</given-names></name></person-group>. <article-title>Regulation of bone remodeling by parathyroid hormone</article-title>. <source>Cold Spring Harb Perspect Med.</source> (<year>2018</year>) <volume>8</volume>:<fpage>a031237</fpage>. <pub-id pub-id-type=\"doi\">10.1101/cshperspect.a031237</pub-id><pub-id pub-id-type=\"pmid\">29358318</pub-id></mixed-citation></ref><ref id=\"B83\"><label>83.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Roberto</surname><given-names>P</given-names></name></person-group>. <article-title>The Role of IL-17 and TH17 cells in the bone catabolic activity of PTH</article-title>. <source>Front Immunol.</source> (<year>2016</year>) <volume>7</volume>:<fpage>57</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2016.00057</pub-id><pub-id pub-id-type=\"pmid\">26925062</pub-id></mixed-citation></ref><ref id=\"B84\"><label>84.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>M</given-names></name><name><surname>Tyagi</surname><given-names>AM</given-names></name><name><surname>Li</surname><given-names>JY</given-names></name><name><surname>Adams</surname><given-names>J</given-names></name><name><surname>Denning</surname><given-names>TL</given-names></name><name><surname>Weitzmann</surname><given-names>MN</given-names></name><etal/></person-group>. <article-title>PTH induces bone loss via microbial-dependent expansion of intestinal TNF + T Cells and Th17 cells</article-title>. <source>Nat Commun</source>. (<year>2020</year>) <volume>11</volume>:<fpage>468</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41467-019-14148-4</pub-id><pub-id pub-id-type=\"pmid\">31980603</pub-id></mixed-citation></ref><ref id=\"B85\"><label>85.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Neale Weitzmann</surname><given-names>M</given-names></name><name><surname>Pacifici</surname><given-names>R</given-names></name></person-group>. <article-title>Parathyroid diseases and T cells</article-title>. <source>Curr Osteoporosis Rep.</source> (<year>2017</year>) <volume>15</volume>:<fpage>135</fpage>–<lpage>41</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s11914-017-0359-y</pub-id><pub-id pub-id-type=\"pmid\">28421466</pub-id></mixed-citation></ref><ref id=\"B86\"><label>86.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pacifici</surname><given-names>R</given-names></name></person-group>. <article-title>T cells: critical bone regulators in health and disease</article-title>. <source>Bone</source>. (<year>2010</year>) <volume>47</volume>:<fpage>461</fpage>–<lpage>71</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bone.2010.04.611</pub-id><pub-id pub-id-type=\"pmid\">20452473</pub-id></mixed-citation></ref><ref id=\"B87\"><label>87.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>JY</given-names></name><name><surname>Walker</surname><given-names>LD</given-names></name><name><surname>Tyagi</surname><given-names>AM</given-names></name><name><surname>Adams</surname><given-names>J</given-names></name><name><surname>Weitzmann</surname><given-names>MN</given-names></name><name><surname>Pacifici</surname><given-names>R</given-names></name></person-group>. <article-title>The sclerostin-independent bone anabolic activity of intermittent PTH treatment is mediated by T-Cell–produced Wnt10b</article-title>. <source>J Bone Miner Res.</source> (<year>2014</year>) <volume>29</volume>:<fpage>43</fpage>–<lpage>54</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jbmr.2044</pub-id><pub-id pub-id-type=\"pmid\">24357520</pub-id></mixed-citation></ref><ref id=\"B88\"><label>88.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hesham</surname><given-names>T</given-names></name><name><surname>Brahmchetna</surname><given-names>B</given-names></name><name><surname>Jau-Yi</surname><given-names>L</given-names></name><name><surname>Jonathan</surname><given-names>A</given-names></name><name><surname>Tatsuya</surname><given-names>K</given-names></name><name><surname>Neale</surname><given-names>WM</given-names></name><etal/></person-group>. <article-title>Disruption of PTH receptor 1 in t cells protects against pth-induced bone loss</article-title>. <source>PLoS ONE.</source> (<year>2010</year>) <volume>5</volume>:<fpage>e12290</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0012290</pub-id><pub-id pub-id-type=\"pmid\">20808842</pub-id></mixed-citation></ref><ref id=\"B89\"><label>89.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fan</surname><given-names>Y</given-names></name><name><surname>Hanai</surname><given-names>J-I</given-names></name><name><surname>Le</surname><given-names>PT</given-names></name><name><surname>Bi</surname><given-names>R</given-names></name><name><surname>Maridas</surname><given-names>D</given-names></name><name><surname>DeMambro</surname><given-names>V</given-names></name><etal/></person-group>. <article-title>Parathyroid hormone directs bone marrow mesenchymal cell fate</article-title>. <source>Cell Metab.</source> (<year>2017</year>) <volume>25</volume>:<fpage>661</fpage>–<lpage>72</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cmet.2017.01.001</pub-id><pub-id pub-id-type=\"pmid\">28162969</pub-id></mixed-citation></ref><ref id=\"B90\"><label>90.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sang</surname><given-names>WK</given-names></name><name><surname>Pajevic</surname><given-names>PD</given-names></name><name><surname>Selig</surname><given-names>M</given-names></name><name><surname>Barry</surname><given-names>KJ</given-names></name><name><surname>Yang</surname><given-names>JY</given-names></name><name><surname>Chan</surname><given-names>SS</given-names></name><etal/></person-group>\n<article-title>Intermittent parathyroid hormone administration converts quiescent lining cells to active osteoblasts</article-title>. <source>J Bone Miner Res.</source> (<year>2012</year>) <volume>27</volume>:<fpage>2075</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jbmr.1665</pub-id><pub-id pub-id-type=\"pmid\">22623172</pub-id></mixed-citation></ref><ref id=\"B91\"><label>91.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Geurtzen</surname><given-names>K</given-names></name><name><surname>Vernet</surname><given-names>A</given-names></name><name><surname>Freidin</surname><given-names>A</given-names></name><name><surname>Rauner</surname><given-names>M</given-names></name><name><surname>Hofbauer</surname><given-names>LC</given-names></name><name><surname>Schneider</surname><given-names>JE</given-names></name><etal/></person-group>. <article-title>Immune suppressive and bone inhibitory effects of prednisolone in growing and regenerating zebrafish tissues</article-title>. <source>J Bone Miner Res.</source> (<year>2017</year>) <volume>32</volume>:<fpage>2476</fpage>–<lpage>88</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jbmr.3231</pub-id><pub-id pub-id-type=\"pmid\">28771888</pub-id></mixed-citation></ref><ref id=\"B92\"><label>92.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sato</surname><given-names>AY</given-names></name><name><surname>Peacock</surname><given-names>M</given-names></name><name><surname>Bellido</surname><given-names>T</given-names></name></person-group>. <article-title>Glucocorticoid excess in bone and muscle</article-title>. <source>Clin Rev Bone Miner Metab.</source> (<year>2018</year>) <volume>16</volume>:<fpage>1</fpage>–<lpage>15</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s12018-018-9242-3</pub-id><pub-id pub-id-type=\"pmid\">29962904</pub-id></mixed-citation></ref><ref id=\"B93\"><label>93.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Compston</surname><given-names>J</given-names></name></person-group>. <article-title>Glucocorticoid-induced osteoporosis: an update</article-title>. <source>Endocrine.</source> (<year>2018</year>) <volume>61</volume>:<fpage>7</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s12020-018-1588-2</pub-id><pub-id pub-id-type=\"pmid\">29691807</pub-id></mixed-citation></ref><ref id=\"B94\"><label>94.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>N</given-names></name><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Yang</surname><given-names>JX</given-names></name></person-group>. <article-title>Growth factor progranulin blocks tumor necrosis factor-α-mediated inhibition of osteoblast differentiation</article-title>. <source>Genet Mol Res.</source> (<year>2016</year>) <fpage>15</fpage>. <pub-id pub-id-type=\"doi\">10.4238/gmr.15038126</pub-id><pub-id pub-id-type=\"pmid\">27706709</pub-id></mixed-citation></ref><ref id=\"B95\"><label>95.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rauch</surname><given-names>A</given-names></name><name><surname>Seitz</surname><given-names>S</given-names></name><name><surname>Baschant</surname><given-names>U</given-names></name><name><surname>Schilling</surname><given-names>AF</given-names></name><name><surname>Illing</surname><given-names>A</given-names></name><name><surname>Stride</surname><given-names>B</given-names></name><etal/></person-group>. <article-title>Glucocorticoids suppress bone formation by attenuating osteoblast differentiation via the monomeric glucocorticoid receptor</article-title>. <source>Cell Metab.</source> (<year>2010</year>) <volume>11</volume>:<fpage>517</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cmet.2010.05.005</pub-id><pub-id pub-id-type=\"pmid\">20519123</pub-id></mixed-citation></ref><ref id=\"B96\"><label>96.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reid</surname><given-names>IR</given-names></name></person-group>\n<article-title>Glucocorticoid-induced osteoporosis</article-title>. <source>New Engl J Med.</source> (<year>2010</year>) <volume>14</volume>:<fpage>279</fpage>–<lpage>98</lpage>. <pub-id pub-id-type=\"doi\">10.1053/beem.2000.0074</pub-id></mixed-citation></ref><ref id=\"B97\"><label>97.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weinstein</surname><given-names>RS</given-names></name></person-group>. <article-title>Glucocorticoid-induced bone disease</article-title>. <source>N Engl J Med.</source> (<year>2011</year>) <volume>365</volume>:<fpage>62</fpage>–<lpage>70</lpage>. <pub-id pub-id-type=\"doi\">10.1056/NEJMcp1012926</pub-id><pub-id pub-id-type=\"pmid\">21732837</pub-id></mixed-citation></ref><ref id=\"B98\"><label>98.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Beattie</surname><given-names>J</given-names></name><name><surname>Al-Khafaji</surname><given-names>H</given-names></name><name><surname>Noer</surname><given-names>PR</given-names></name><name><surname>Alkharobi</surname><given-names>HE</given-names></name><name><surname>Alhodhodi</surname><given-names>A</given-names></name><name><surname>Meade</surname><given-names>J</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Insulin-like growth factor-binding protein action in bone tissue: a key role for pregnancy- associated plasma protein-A</article-title>. <source>Front Endocrinol.</source>\n<volume>9</volume>:<fpage>31</fpage>\n<pub-id pub-id-type=\"doi\">10.3389/fendo.2018.00510</pub-id></mixed-citation></ref><ref id=\"B99\"><label>99.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wehmeyer</surname><given-names>C</given-names></name><name><surname>Pap</surname><given-names>T</given-names></name><name><surname>Buckley</surname><given-names>CD</given-names></name><name><surname>Naylor</surname><given-names>AJ</given-names></name></person-group>. <article-title>The role of stromal cells in inflammatory bone loss</article-title>. <source>Clin Exp Immunol.</source> (<year>2017</year>) <volume>189</volume>:<fpage>1</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1111/cei.12979</pub-id><pub-id pub-id-type=\"pmid\">28419440</pub-id></mixed-citation></ref><ref id=\"B100\"><label>100.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Banuelos</surname><given-names>J</given-names></name><name><surname>Lu</surname><given-names>NZ</given-names></name></person-group>. <article-title>A gradient of glucocorticoid sensitivity among helper T cell cytokines</article-title>. <source>Cytokine Growth Factor Rev.</source> (<year>2016</year>) <volume>31</volume>:<fpage>27</fpage>–<lpage>35</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cytogfr.2016.05.002</pub-id><pub-id pub-id-type=\"pmid\">27235091</pub-id></mixed-citation></ref><ref id=\"B101\"><label>101.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xie</surname><given-names>Y</given-names></name><name><surname>Zhang</surname><given-names>LC</given-names></name><name><surname>Xiong</surname><given-names>Q</given-names></name><name><surname>Gao</surname><given-names>YP</given-names></name><name><surname>Ge</surname><given-names>W</given-names></name><name><surname>Tang</surname><given-names>PF</given-names></name></person-group>. <article-title>Bench-to-bedside strategies for osteoporotic fracture: from osteoimmunology to mechanosensation</article-title>. <source>Bone Res</source>. (<year>2019</year>) <volume>7</volume>:<fpage>259</fpage>–<lpage>71</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41413-019-0066-7</pub-id><pub-id pub-id-type=\"pmid\">31646015</pub-id></mixed-citation></ref><ref id=\"B102\"><label>102.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dar</surname><given-names>HY</given-names></name><name><surname>Singh</surname><given-names>A</given-names></name><name><surname>Shukla</surname><given-names>P</given-names></name><name><surname>Anupam</surname><given-names>R</given-names></name><name><surname>Mondal</surname><given-names>RK</given-names></name><name><surname>Mishra</surname><given-names>PK</given-names></name><etal/></person-group>. <article-title>High dietary salt intake correlates with modulated Th17-Treg cell balance resulting in enhanced bone loss and impaired bone-microarchitecture in male mice</article-title>. <source>Sci Rep</source>. (<year>2018</year>) <volume>8</volume>:<fpage>2503</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-018-20896-y</pub-id><pub-id pub-id-type=\"pmid\">29410520</pub-id></mixed-citation></ref><ref id=\"B103\"><label>103.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kennel</surname><given-names>KA</given-names></name><name><surname>Drake</surname><given-names>MT</given-names></name></person-group>. <article-title>Adverse effects of bisphosphonates: implications for osteoporosis management</article-title>. <source>Mayo Clin Proc.</source> (<year>2009</year>) <volume>84</volume>:<fpage>632</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0025-6196(11)60752-0</pub-id><pub-id pub-id-type=\"pmid\">19567717</pub-id></mixed-citation></ref><ref id=\"B104\"><label>104.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sharma</surname><given-names>D</given-names></name><name><surname>Singh</surname><given-names>J</given-names></name></person-group>\n<article-title>Synthesis and characterization of fatty acid grafted chitosan polymer and their nanomicelles for non-viral gene delivery applications</article-title>. <source>Bioconjug Chem.</source> (<year>2017</year>) <volume>28</volume>:<fpage>2772</fpage>–<lpage>83</lpage>. <pub-id pub-id-type=\"doi\">10.1021/acs.bioconjchem.7b00505</pub-id><pub-id pub-id-type=\"pmid\">29040803</pub-id></mixed-citation></ref><ref id=\"B105\"><label>105.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bone</surname><given-names>H</given-names></name><name><surname>Brandi</surname><given-names>M</given-names></name><name><surname>Brown</surname><given-names>J</given-names></name><name><surname>Chapurlat</surname><given-names>R</given-names></name><name><surname>Cummings</surname><given-names>S</given-names></name><name><surname>Czerwinski</surname><given-names>E</given-names></name><etal/></person-group>. <article-title>10 years of denosumab treatment in postmenopausal women with osteoporosis: results from the phase 3 randomised FREEDOM trial and open-label extension</article-title>. <source>Lancet Diabetes Endocrinol.</source> (<year>2017</year>) <volume>5</volume>:<fpage>513</fpage>–<lpage>23</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S2213-8587(17)30138-9</pub-id><pub-id pub-id-type=\"pmid\">28546097</pub-id></mixed-citation></ref><ref id=\"B106\"><label>106.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Juji</surname><given-names>T</given-names></name><name><surname>Hertz</surname><given-names>M</given-names></name><name><surname>Aoki</surname><given-names>K</given-names></name><name><surname>Horie</surname><given-names>D</given-names></name><name><surname>Ohya</surname><given-names>K</given-names></name><name><surname>Gautam</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>A novel therapeutic vaccine approach, targeting RANKL, prevents bone destruction in bone related disorders</article-title>. <source>J Bone Miner Metabol.</source> (<year>2002</year>) <volume>20</volume>:<fpage>266</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s007740200038</pub-id><pub-id pub-id-type=\"pmid\">12203031</pub-id></mixed-citation></ref><ref id=\"B107\"><label>107.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zaheer</surname><given-names>S</given-names></name><name><surname>LeBoff</surname><given-names>M</given-names></name><name><surname>Lewiecki</surname><given-names>EM</given-names></name></person-group>. <article-title>Denosumab for the treatment of osteoporosis</article-title>. <source>Expert Opin Drug Metab Toxicol.</source> (<year>2015</year>) <volume>11</volume>:<fpage>461</fpage>–<lpage>70</lpage>. <pub-id pub-id-type=\"doi\">10.1517/17425255.2015.1000860</pub-id><pub-id pub-id-type=\"pmid\">25614274</pub-id></mixed-citation></ref><ref id=\"B108\"><label>108.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>F</given-names></name><name><surname>Li</surname><given-names>H</given-names></name><name><surname>Zhai</surname><given-names>Q</given-names></name><name><surname>Li</surname><given-names>FY</given-names></name><name><surname>Wu</surname><given-names>TL</given-names></name><name><surname>Sha</surname><given-names>X</given-names></name><etal/></person-group>. <article-title>A new vaccine targeting RANKL, prepared by incorporation of an unnatural amino acid into RANKL, prevents OVX-induced bone loss in mice</article-title>. <source>Biochem Biophys Res Commun.</source> (<year>2018</year>) <volume>499</volume>:<fpage>648</fpage>–<lpage>54</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbrc.2018.03.205</pub-id><pub-id pub-id-type=\"pmid\">29604276</pub-id></mixed-citation></ref><ref id=\"B109\"><label>109.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Curate</surname><given-names>F</given-names></name></person-group>. <article-title>Osteoporosis and paleopathology: a review</article-title>. <source>J Anthropol Sci.</source> (<year>2014</year>) <volume>92</volume>:<fpage>119</fpage>–<lpage>46</lpage>. <pub-id pub-id-type=\"pmid\">24607995</pub-id></mixed-citation></ref><ref id=\"B110\"><label>110.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>YH</given-names></name><name><surname>Li</surname><given-names>B</given-names></name><name><surname>Zheng</surname><given-names>XF</given-names></name><name><surname>Chen</surname><given-names>JW</given-names></name><name><surname>Chen</surname><given-names>K</given-names></name><name><surname>Jiang</surname><given-names>SD</given-names></name><etal/></person-group>. <article-title>Oxidative damage to osteoblasts can be alleviated by early autophagy through the endoplasmic reticulum stress pathway-implications for the treatment of osteoporosis</article-title>. <source>Free Radic Biol Med.</source> (<year>2014</year>) <volume>77</volume>:<fpage>10</fpage>–<lpage>20</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.freeradbiomed.2014.08.028</pub-id><pub-id pub-id-type=\"pmid\">25224042</pub-id></mixed-citation></ref><ref id=\"B111\"><label>111.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chawalitpong</surname><given-names>S</given-names></name><name><surname>Chokchaisiri</surname><given-names>R</given-names></name><name><surname>Suksamrarn</surname><given-names>A</given-names></name><name><surname>Katayama</surname><given-names>S</given-names></name><name><surname>Mitani</surname><given-names>T</given-names></name><name><surname>Nakamura</surname><given-names>T</given-names></name></person-group>. <article-title>Cyperenoic acid suppresses osteoclast differentiation and delays bone loss in a senile osteoporosis mouse model by inhibiting non-canonical NF-κB pathway</article-title>. <source>Sci Rep.</source> (<year>2018</year>) <volume>8</volume>:<fpage>5625</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-018-23912-3</pub-id><pub-id pub-id-type=\"pmid\">29618833</pub-id></mixed-citation></ref><ref id=\"B112\"><label>112.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xi</surname><given-names>YH</given-names></name><name><surname>Jiang</surname><given-names>TW</given-names></name><name><surname>Yu</surname><given-names>JM</given-names></name><name><surname>Xue</surname><given-names>MT</given-names></name><name><surname>Xu</surname><given-names>N</given-names></name><name><surname>Wen</surname><given-names>JK</given-names></name><etal/></person-group>. <article-title>Preliminary studies on the anti-osteoporosis activity of Baohuoside I</article-title>. <source>Biomed Pharmacother.</source> (<year>2019</year>) <volume>115</volume>:<fpage>108850</fpage>. <pub-id pub-id-type=\"doi\">10.1016/j.biopha.2019.108850</pub-id><pub-id pub-id-type=\"pmid\">31004988</pub-id></mixed-citation></ref></ref-list><glossary><def-list><title>Abbreviations</title><def-item><term>BMMs</term><def><p>Bone marrow macrophages</p></def></def-item><def-item><term>BMSCs</term><def><p>bone marrow stromal cells</p></def></def-item><def-item><term>Cbfa1</term><def><p>core-binding factor subunit alpha-1</p></def></def-item><def-item><term>DC</term><def><p>dendritic cell</p></def></def-item><def-item><term>GCs</term><def><p>glucocorticoids</p></def></def-item><def-item><term>GIO</term><def><p>glucocorticoid-induced osteoporosis</p></def></def-item><def-item><term>GSK-3β</term><def><p>glycogen synthase kinase 3β</p></def></def-item><def-item><term>IGF</term><def><p>insulin-like growth factor</p></def></def-item><def-item><term>IFN</term><def><p>interferon</p></def></def-item><def-item><term>M-CSF</term><def><p>macrophage-colony stimulating factor</p></def></def-item><def-item><term>MSCs</term><def><p>mesenchymal stem cells</p></def></def-item><def-item><term>NFATc1</term><def><p>nuclear factor of activated T cells cytoplasmic 1</p></def></def-item><def-item><term>NKT</term><def><p>natural killer T cells</p></def></def-item><def-item><term>iNOS</term><def><p>inducible NOS</p></def></def-item><def-item><term>RANKL</term><def><p>nuclear factor-kappa-B ligand</p></def></def-item><def-item><term>OVX</term><def><p>ovariectomized</p></def></def-item><def-item><term>OPG</term><def><p>osteoprotegerin</p></def></def-item><def-item><term>PTH</term><def><p>parathyroid hormone</p></def></def-item><def-item><term>T cells</term><def><p>T lymphocytes</p></def></def-item><def-item><term>TRAF6</term><def><p>TNF receptor associated factor 6</p></def></def-item><def-item><term>Runx2</term><def><p>Transcription Factor 2</p></def></def-item><def-item><term>RANK</term><def><p>receptor activator of NF-kB ligand</p></def></def-item><def-item><term>RA</term><def><p>rheumatoid arthritis.</p></def></def-item></def-list></glossary></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Immunol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Immunol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Immunol.</journal-id><journal-title-group><journal-title>Frontiers in Immunology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-3224</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849596</article-id><article-id pub-id-type=\"pmc\">PMC7431603</article-id><article-id pub-id-type=\"doi\">10.3389/fimmu.2020.01699</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Immunology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>IL-27 Regulated CD4<sup>+</sup>IL-10<sup>+</sup> T Cells in Experimental Sjögren Syndrome</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Qi</surname><given-names>Jingjing</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Zhuoya</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Tang</surname><given-names>Xiaojun</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Li</surname><given-names>Wenchao</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Weiwei</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Yao</surname><given-names>Genhong</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/942759/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Rheumatology and Immunology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School</institution>, <addr-line>Nanjing</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Immunology, College of Basic Medical Science, Dalian Medical University, Dalian</institution>, <addr-line>Liaoning</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Huji Xu, Tsinghua University, China</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Howard A. Young, National Cancer Institute at Frederick, United States; Umesh S. Deshmukh, Oklahoma Medical Research Foundation, United States</p></fn><corresp id=\"c001\">*Correspondence: Genhong Yao <email>yaogenhong@nju.edu.cn</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†These authors have contributed equally to this work</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1699</elocation-id><history><date date-type=\"received\"><day>07</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>25</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Qi, Zhang, Tang, Li, Chen and Yao.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Qi, Zhang, Tang, Li, Chen and Yao</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Interleukin 27 (IL-27) plays diverse immune regulatory roles in autoimmune disorders and promotes the generation of IL-10–producing CD4<sup>+</sup> T cells characterized by producing the immunosuppressive cytokine IL-10. However, whether IL-27 participates in pathological progress of Sjögren syndrome (SS) through regulating CD4<sup>+</sup>IL-10<sup>+</sup> T cells remains unknown. Here we aimed to explore the potential role of IL-27 and CD4<sup>+</sup>IL-10<sup>+</sup> T cells in the pathogenesis of SS. The IL-27 gene knockout non-obese diabetic (<italic>Il-27</italic><sup>−/−</sup>NOD) mice were generated and injected with exogenous IL-27. Exogenous injection of IL-27 and neutralization of IL-27 with anti–IL-27 antibody in NOD mice were performed. The histopathologic changes in submandibular glands, lacrimal glands and lung, salivary flow rate, and percentages of CD4<sup>+</sup>IL-10<sup>+</sup> T cells were determined. And, ovalbumin-immunized C57L/B6 mice were injected with IL-27 to detect the percentage of CD4<sup>+</sup>IL-10<sup>+</sup> T cells. <italic>In vitro</italic>, splenic naive T cells from C57L/B6 mice were cultured with IL-27 for 4 days to induce the differentiation of CD4<sup>+</sup>IL-10<sup>+</sup> T cells. In addition, IL-27, IL-10, and CD4<sup>+</sup>IL-10<sup>+</sup> T cells were determined in health control and SS patients. The results showed that <italic>Il-27</italic><sup>−/−</sup>NOD mice had more severe disease and lower level of CD4<sup>+</sup>IL-10<sup>+</sup> T cells than control mice. And IL-27 promoted the generation and differentiation of CD4<sup>+</sup>IL-10<sup>+</sup> T cells <italic>in vivo</italic> and <italic>in vitro</italic> significantly. In agreement with the findings in the SS-like mice, patients with SS showed lower levels of IL-27, IL-10, and CD4<sup>+</sup>IL-10<sup>+</sup> T cells. Our findings indicated that IL-27 deficiency aggravated SS by regulating CD4<sup>+</sup>IL-10<sup>+</sup> T cells. Targeting IL-27 and CD4<sup>+</sup>IL-10<sup>+</sup> T cells may be a novel therapy for patients with SS.</p></abstract><kwd-group><kwd>Sjögren syndrome</kwd><kwd>CD4<sup>+</sup>IL-10<sup>+</sup> T cells</kwd><kwd>interleukin-27</kwd><kwd>interleukin-10</kwd><kwd>immunosuppression</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Natural Science Foundation of China<named-content content-type=\"fundref-id\">10.13039/501100001809</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"5\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"51\"/><page-count count=\"10\"/><word-count count=\"5577\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Sjögren syndrome (SS) is a chronic autoimmune disease that typically presents with dry eyes and mouth as a result of lymphocytic infiltration in salivary (SGs) and lacrimal glands (LGs). Sjögren syndrome is characterized by the presence of autoreactive T and B cells in exocrine glands and circulating antibodies against several autologous antigens, such as the autoantibodies against SS antigen A (SSA)/Ro and SS antigen B (SSB)/La and the Fc fragment of immunoglobulin G (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B2\" ref-type=\"bibr\">2</xref>). The current treatments of SS are limited to be symptomatic, as a result of the elusive pathogenesis and diverse syndrome. According to reports, the dysregulated cytokine network contributes to the occurrence and development of SS. Thus, targeting to cytokine as the potential therapies in SS should be explored (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>).</p><p>Interleukin 27 (IL-27) is a heterodimeric immunological factor of the IL-12 cytokine family and consists of p28 and Epstein-Barr virus–induced gene 3 (EBi3) subunits. Interleukin 27 mainly produced by antigen-presenting cells (APCs) upon stimulation of innate immune receptors (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>–<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). The IL-27 receptor is composed of the unique chain IL-27Rα (also called TCCR or WSX-1) and gp130 (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). Interleukin 27Rα is widely expressed in the immune cells (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). The ligation of IL-27 and its receptor induce intracellular signaling via heterogeneous Jak/STAT pathways, with predominant activation of STAT1 and STAT3 (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>–<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Interleukin 27 was preliminarily characterized as a pro-inflammatory cytokine with T<sub>H</sub>1 induction. However, studies with infectious and autoimmune inflammatory models had reported that IL-27Rα deficiency mice developed excessive, pathological inflammation responding to a variety of challenges (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). And the anti-inflammatory role of IL-27 signaling has been illustrated in many recent studies (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>–<xref rid=\"B17\" ref-type=\"bibr\">17</xref>).</p><p>Hunter and colleagues showed the molecular mechanisms underlying suppressive characters of IL-27, which indicated that IL-27 could reduce IL-2 production during T<sub>H</sub>1 differentiation and promote the development of regulatory T (Treg) cells specialized to control T<sub>H</sub>1-mediated immunity at local sites of inflammation (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Interleukin 27 signaling in DCs played a key role in antigen-induced peripheral tolerance, which relied on the ability of IL-27 to induce T cell–derived IL-10 and interferon γ (IFN-γ) (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). And the study found that IL-27 and IL-6 induced T<sub>H</sub>1 and T<sub>H</sub>2 cells, as well as T<sub>H</sub>17 cells to secrete IL-10 (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). Interleukin 27 promoted the expression of inhibitory receptors on T cells <italic>in vivo</italic> and <italic>in vitro</italic> (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). Furthermore, studies reported that IL-27 drove the generation (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>) and differentiation of IL-10–producing murine type 1 regulatory T (Tr1) cells by inducing three key factors: the transcription factor c-Maf, the cytokine IL-21, and the costimulatory receptor ICOS (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>, <xref rid=\"B25\" ref-type=\"bibr\">25</xref>).</p><p>Tr1 cells are a subset of T cells that have strong immunosuppressive properties and predominantly produce IL-10 with variable amounts of IFN-γ, IL-2, and transforming growth factor β (TGF-β), but do not express transcription factor Fork head box 3 (Foxp3) (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>, <xref rid=\"B26\" ref-type=\"bibr\">26</xref>, <xref rid=\"B27\" ref-type=\"bibr\">27</xref>). There is a lot of research focused on Tr1 cells to suppress innate and adaptive immunity to alleviate inflammatory pathologies, in particular autoimmune diseases (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>–<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). Interleukin 27 could limit autoimmune diseases by stimulating IL-10–secreting T cells (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B32\" ref-type=\"bibr\">32</xref>). Nevertheless, the role of IL-27 and CD4<sup>+</sup>IL-10<sup>+</sup> T cells in SS remained unknown.</p><p>Here, our results showed that CD4<sup>+</sup>IL-10<sup>+</sup> T cells related to SS pathogenesis and reduced generation of CD4<sup>+</sup>IL-10<sup>+</sup> T cells was ascribed to decreased IL-27 in SS. Our findings indicated that targeting to IL-27 and CD4<sup>+</sup>IL-10<sup>+</sup> T cells is a new direction for the SS treatment.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Study Population</title><p>A total of 31 SS patients and 34 health controls (HCs) from the Department of Rheumatology and Immunology, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing University, were enrolled. Written informed consent was obtained from all subjects. Whole-blood samples were obtained. Plasma was isolated and frozen at −80°C until use. The study was approved by the ethics committee of our institute.</p></sec><sec><title>Mice</title><p>Seven-week-old female NOD and IL-27 gene knockout female NOD (<italic>Il-27</italic><sup>−/−</sup>NOD) and 5-week-old female C57L/B6 mice were purchased from the Model Animal Research Center of Nanjing University and maintained under specific-pathogen–free conditions in the animal center of the Affiliated Drum Tower Hospital of Nanjing University Medical School.</p></sec><sec><title>Salivary Flow Rate</title><p>After anesthetization, the mice were stimulated with pilocarpine 0.1 mg pilocarpine/kg body weight injected intraperitoneally (i.p.). The whole saliva was obtained from the oral cavity for 15 min. Saliva volume was determined gravimetrically.</p></sec><sec><title>Histological Analysis</title><p>For histological analysis, mice were euthanized. Submandibular glands, LGs, and lung tissues were collected and embedded in paraffin for hematoxylin and eosin staining.</p></sec><sec><title>Preparation of Single Cell Suspensions</title><p>Spleen was isolated, and the red blood cells were lysed with lysing buffer for single cell suspension preparation. Peripheral blood mononuclear cells in blood from SS patients and healthy volunteers were isolated with Ficoll-Hypaque by density gradient centrifugation.</p></sec><sec><title>Flow Cytometry</title><p>Antibodies (eBioscience) were diluted at optimal concentration for cell immunostaining. To avoid non-specific binding to Fc receptors, isotype-matched antibodies were used as controls. For analysis of intracellular IL-10, cells were stimulated with 20 ng/mL PMA plus 1 μg/mL ionomycin with 5 μg/mL of brefeldin A (Enzo LifeScience, East Farmingdale, NY, USA) at 37°C for 4 h before harvest. First, surface CD4 with anti–mouse/human CD4–fluorescein isothiocyanate was stained. After that, cells were fixed with Cytofix/Cytoperm solution (BD Pharmingen), incubated with anti–mouse IL-10–APC or anti–human IL-10–APC and analyzed on a FACS Calibur flow cytometer (BD Biosciences, Mountain View, CA, USA).</p></sec><sec><title>Immunization</title><p>Seven-week-old female C57L/B6 mice were sensitized with 10 μg ovalbumin (OVA) in complete Freund's adjuvant intradermally as the described methods (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>).</p></sec><sec><title><italic>In vivo</italic> Treatment With IL-27 and Anti–IL-27</title><p>In the IL-27 treatment experiments, 11-week-old female NOD mice or OVA-immunized 8-week-old female C57L/B6 mice were injected with 200 ng/mouse IL-27 (BioLegend) once a day for a total of seven times. The control mice were injected with same volume of phosphate-buffered saline (PBS). In the anti-IL-27 treatment experiments, female 11-week-old NOD mice were injected with 0.5 mg/mouse anti-IL-27 (BioLegend) or with commensurable IgG2a isotype once i.p. Mice were sacrificed on the seventh day.</p></sec><sec><title><italic>In vitro</italic> Tr1 Cell Differentiation</title><p>Splenic CD4<sup>+</sup>CD62L<sup>+</sup> naive T cells were purified with Micro Beads (Miltenyi Biotec) from 6- to 8-week-old female C57L/B6 mice, with purity of more than 90%. Then naive T cells are cultured in a 96-well plate bound with 4 μg/mL anti-CD3e antibodies (eBioscience) at a density of 1 × 10<sup>6</sup>/mL in RPMI 1640 supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin, in the presence of 2 μg/mL anti-CD28 antibodies with 20 ng/mL mouse recombinant IL-27 (rIL-27) (BioLegend) or not for 4 days.</p></sec><sec><title>Cytokine Quantification</title><p>The plasma IL-10 and IL-27 were measured by standard sandwich enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) according to the manufacturer's instructions.</p></sec><sec><title>Statistical Analysis</title><p>Statistical analysis was performed with Prism software version 5.0 (GraphPad Software). Differences in means ± SEM were evaluated with Student <italic>t</italic> test or one-way analysis of variance followed by Dunnett test. <italic>P</italic> < 0.05 was considered statistically significant.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title><italic>Il-27</italic><sup>−/−</sup>NOD Mice Displayed Severe SS</title><p>Interleukin 27 has been shown to attenuate multiple autoimmune disorders by regulating the response of T cells and decreasing the production of inflammatory cytokines (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>–<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). To investigate the potential role of IL-27 in SS, we compared the severity of SS-like symptoms in <italic>Il-27</italic><sup>−/−</sup>NOD and wild-type NOD mice. We found that <italic>Il-27</italic><sup>−/−</sup>NOD mice developed rash (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>) and had swollen SGs compared to NOD mice (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Figures S1A,B</xref>). Histopathologic analysis results showed more severe inflammation in SG (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Figure S1C</xref>), LGs (<xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Figure S1D</xref>), and lung (<xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Figure S1E</xref>) of <italic>Il-27</italic><sup>−/−</sup>NOD mice. The level of salivary flow rate decreased significantly in <italic>Il-27</italic><sup>−/−</sup>NOD mice (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>). These data indicated that the IL-27 deficiency aggravated SS in NOD mice.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Interleukin 27 gene deficiency aggravated SS in NOD mice. <bold>(A)</bold> The physical appearance manifestation of rash in 12-week-old female NOD and <italic>Il-27</italic><sup>−/−</sup>NOD mice. <bold>(B–D)</bold> Histological analysis, SGs, LGs, and lung from representative NOD and <italic>Il-27</italic><sup>−/−</sup>NOD mice stained with hematoxylin and eosin to assess inflammation (top, magnification ×100; bottom, magnification ×400), <italic>n</italic> = 5.</p></caption><graphic xlink:href=\"fimmu-11-01699-g0001\"/></fig><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Lower levels of salivary flow rate and CD4<sup>+</sup>IL-10<sup>+</sup> T cells in <italic>Il-27</italic><sup>−/−</sup>NOD mice. <bold>(A)</bold> Histological analysis, SG from representative 12-week-old female NOD and <italic>Il-27</italic><sup>−/−</sup>NOD and IL-27–treated <italic>Il-27</italic><sup>−/−</sup>NOD mice stained with hematoxylin and eosin to assess inflammation (top, magnification ×100; bottom, magnification ×400). <bold>(B)</bold> The lymphocyte infiltration in SG of mice were evaluated for histological scores. <bold>(C)</bold> Salivary flow rate, <bold>(D)</bold> representative flow cytometry results, and <bold>(E)</bold> the percentage and <bold>(F)</bold> absolute number of splenic CD4<sup>+</sup>IL-10<sup>+</sup> T cells in NOD and <italic>Il-27</italic><sup>−/−</sup>NOD and IL-27–treated <italic>Il-27</italic><sup>−/−</sup>NOD mice. Error bars indicate SEM. *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, ***<italic>p</italic> < 0.001, <italic>n</italic> = 5.</p></caption><graphic xlink:href=\"fimmu-11-01699-g0002\"/></fig></sec><sec><title>CD4<sup>+</sup>IL-10<sup>+</sup> T Cells Decreased in <italic>Il-27<sup>−/−</sup></italic>NOD Mice</title><p>To explore the effects of IL-27 gene deficiency on inflammation in NOD mice, the mice were divided into three groups: NOD, <italic>Il-27</italic><sup>−/−</sup>NOD, and <italic>Il-27</italic><sup>−/−</sup>NOD mice with IL-27 treatment. More infiltrating lymphocytes and larger infiltrating area were seen in SG of <italic>Il-27</italic><sup>−/−</sup> NOD mice compared to those of NOD and <italic>Il-27</italic><sup>−/−</sup> NOD mice treated with IL-27 (<xref ref-type=\"fig\" rid=\"F2\">Figures 2A,B</xref>). We collected the whole saliva from the oral cavity and found the saliva volume of <italic>Il-27</italic><sup>−/−</sup> NOD mice reduced significantly, while exogenous IL-27 treatment could restore the salivary flow rate of <italic>Il-27</italic><sup>−/−</sup>NOD mice (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>). The proportion and absolute number of splenic CD4<sup>+</sup>IL-10<sup>+</sup> T cells of <italic>Il-27</italic><sup>−/−</sup>NOD mice were lower than those of wild-type NOD mice. Exogenous IL-27 treatment restored the proportion (<xref ref-type=\"fig\" rid=\"F2\">Figures 2D,E</xref>) and absolute number of splenic CD4<sup>+</sup>IL-10<sup>+</sup> T cells (<xref ref-type=\"fig\" rid=\"F2\">Figure 2F</xref>) significantly in <italic>Il-27</italic><sup>−/−</sup>NOD mice. These findings suggested that IL-27 gene deficiency aggravated SS-like symptoms in NOD mice, and exogenous IL-27 treatment could reverse the decreasing salivary flow rate, splenic CD4<sup>+</sup>IL-10<sup>+</sup> T cells, and serious inflammation.</p></sec><sec><title>Exogenous IL-27 Up-Regulated CD4<sup>+</sup>IL-10<sup>+</sup> T Cells in NOD Mice</title><p>Given the aggravated SS-like symptoms and the decreased CD4<sup>+</sup>IL-10<sup>+</sup> T cells in <italic>Il-27</italic><sup>−/−</sup>NOD mice, we sought to investigate whether exogenous IL-27 treatment could up-regulate CD4<sup>+</sup>IL-10<sup>+</sup> T cells and suppress inflammation in NOD mice. NOD mice were injected i.p. with recombinant IL-27, and the infiltration in SG, salivary flow rate, and splenic CD4<sup>+</sup>IL-10<sup>+</sup> T cells were examined. We found NOD mice treated with IL-27 showed fewer lymphocytes in SG (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>) and LG (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Figure S2A</xref>) and higher level of salivary flow rate (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>). Meanwhile, we used flow cytometry as the gating strategy (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Figure S2C</xref>) to characterize the proportions and numbers of splenic CD4<sup>+</sup>IL-10<sup>+</sup> T cells. We found significantly higher percentage, but not more numbers (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Figure S2D</xref>), of splenic CD4<sup>+</sup>IL-10<sup>+</sup> T cells in IL-27–treated NOD mice (<xref ref-type=\"fig\" rid=\"F3\">Figures 3E,F</xref>). To further determine the significance of IL-27–induced of CD4<sup>+</sup>IL-10<sup>+</sup> T cells in SS, anti-IL-27 and homologous isotype IgG2a were injected i.p. to NOD mice. As expected, we found the infiltration in SG (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>) and LG (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Figure S2B</xref>) aggravated, the salivary flow rate reduced significantly (<xref ref-type=\"fig\" rid=\"F3\">Figure 3D</xref>), and splenic CD4<sup>+</sup>IL-10<sup>+</sup> T-cell proportion, but not the numbers (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Figure S2E</xref>), decreased significantly (<xref ref-type=\"fig\" rid=\"F3\">Figures 3G,H</xref>) in anti-IL-27–injected NOD mice. These results consist with our findings in <italic>Il-27</italic><sup>−/−</sup>NOD mice and support the notion that IL-27 could inhibit SS-like symptoms in NOD by promoting CD4<sup>+</sup>IL-10<sup>+</sup> T cells.</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Interleukin 27 suppressed the inflammation in NOD mice. <bold>(A,C)</bold> Histological analysis, SG from representative mice stained with hematoxylin and eosin to assess inflammation (magnification ×100). Arrows indicate lymphocytes infiltrating focus. <bold>(B,D)</bold> Salivary flow rate of 12-week-old female NOD mice. <bold>(E,G)</bold> Representative flow cytometry results and <bold>(F,H)</bold> the percentage of splenic CD4<sup>+</sup>IL-10<sup>+</sup> T cells in NOD mice. Error bars indicate SEM. *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, ***<italic>p</italic> < 0.001, <italic>n</italic> = 5.</p></caption><graphic xlink:href=\"fimmu-11-01699-g0003\"/></fig></sec><sec><title>IL-27 Promoted the Development of CD4<sup>+</sup>IL-10<sup>+</sup> T Cells</title><p>Next, the C57BL/6 mice were immunized with OVA and treated with PBS or IL-27 as schedule (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>). Results showed that IL-27 remarkably promoted the generation of CD4<sup>+</sup>IL-10<sup>+</sup> T cells <italic>in vivo</italic> (<xref ref-type=\"fig\" rid=\"F4\">Figures 4B,C</xref>). In addition, we cultured splenic naive T cells from C57BL/6 mice with rIL-27 <italic>in vitro</italic> to investigate the effects of IL-27 on the differentiation of CD4<sup>+</sup>IL-10<sup>+</sup> T cells <italic>in vitro</italic>. And we found that IL-27 up-regulated the differentiation of CD4<sup>+</sup>IL-10<sup>+</sup> T cells significantly (<xref ref-type=\"fig\" rid=\"F4\">Figures 4D,E</xref>). Collectively, these results suggested that IL-27 significantly promoted generation and differentiation of CD4<sup>+</sup>IL-10<sup>+</sup> T cells <italic>in vivo</italic> and <italic>in vitro</italic>.</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>CD4<sup>+</sup>IL-10<sup>+</sup> T cells generation and differentiation were promoted by IL-27. <bold>(A)</bold> IL-27 treatment schedule. <bold>(B)</bold> Representative flow cytometry results and <bold>(C)</bold> the percentage of splenic CD4<sup>+</sup>IL-10<sup>+</sup> T cells in IL-27–treated or control OVA-immunized 9-week-old female C57BL/6 mice. <bold>(D)</bold> Representative flow cytometry results, and <bold>(E)</bold> the percentage of differentiated CD4<sup>+</sup>IL-10<sup>+</sup> T cells. Error bars indicate SEM. *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, <italic>n</italic> = 5.</p></caption><graphic xlink:href=\"fimmu-11-01699-g0004\"/></fig></sec><sec><title>IL-27 Correlated With CD4<sup>+</sup>IL-10<sup>+</sup> T Cells in SS Patients</title><p>To examine the role and relationship of IL-27 and CD4<sup>+</sup>IL-10<sup>+</sup> T cells in SS pathogenesis and whether they are correlated with known markers of SS disease, such as anti-SSA/Ro antibodies, anti-SSB/La antibodies, and antinuclear antibodies (ANAs). We detected the levels of plasma IL-27, IL-10, and peripheral CD4<sup>+</sup>IL-10<sup>+</sup> T cells in HCs and SS patients. In addition, we analyzed the correlation between CD4<sup>+</sup>IL-10<sup>+</sup> T cells and anti-SSA antibodies, anti-SSB antibodies, and ANAs in SS patients (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Table S1</xref>). Results showed that the levels of plasma IL-27 (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>) and IL-10 (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>) and percentage of peripheral CD4<sup>+</sup>IL-10<sup>+</sup> T cells (<xref ref-type=\"fig\" rid=\"F5\">Figures 5C,D</xref>) in SS patients were lower than those in HCs. CD4<sup>+</sup>IL-10<sup>+</sup> T cells were negatively correlated with anti-SSA antibodies (<xref ref-type=\"fig\" rid=\"F5\">Figure 5E</xref>), but not anti-SSB antibodies (<xref ref-type=\"fig\" rid=\"F5\">Figure 5F</xref>) and ANAs (<xref ref-type=\"fig\" rid=\"F5\">Figure 5G</xref>). These data support the view that CD4<sup>+</sup>IL-10<sup>+</sup> T cells regulated by IL-27 participated in SS pathogenesis.</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>Interleukin 27 was related to decreased CD4<sup>+</sup>IL-10<sup>+</sup> T cells in SS patients. <bold>(A,B)</bold> Plasma levels of IL-27 and IL-10 in HCs (<italic>n</italic> = 14) and SS patients (<italic>n</italic> = 14) were detected by ELISA. <bold>(C)</bold> Representative flow cytometry results and <bold>(D)</bold> the percentage of CD4<sup>+</sup>IL-10<sup>+</sup> T cells in HCs (<italic>n</italic> = 20) and SS patients (<italic>n</italic> = 17). Sjögren syndrome patients were divided into antibodies-positive (+) and antibodies-negative (–) two groups. The correlations of CD4<sup>+</sup>IL-10<sup>+</sup> T cells with <bold>(E)</bold> anti-SSA antibodies, <bold>(F)</bold> anti-SSB antibodies, and <bold>(G)</bold> ANAs were analyzed in SS patients. Error bars indicate SEM. *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01.</p></caption><graphic xlink:href=\"fimmu-11-01699-g0005\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Our study here has shown that IL-27–regulated CD4<sup>+</sup>IL-10<sup>+</sup> T cells participated in SS pathogenesis. <italic>Il-27</italic><sup>−/−</sup>NOD mice displayed severe SS disease. Exogenous IL-27 improved the symptoms of SS by promoting the generation of Tr1 cells in <italic>Il-27</italic><sup>−/−</sup>NOD and NOD mice. And, antibody neutralization of IL-27 not only exacerbated inflammation but also reduced splenic CD4<sup>+</sup>IL-10<sup>+</sup> T cells significantly in NOD mice. Moreover, SS patients have lower levels of IL-27, IL-10, and CD4<sup>+</sup>IL-10<sup>+</sup> T cells. And, CD4<sup>+</sup>IL-10<sup>+</sup> T cells were negatively correlated with anti-SSA antibodies. Together, these results have demonstrated a critical role of IL-27 in the pathogenesis of SS.</p><p>The anti-inflammatory properties of IL-27 have been reported in several autoimmune diseases, and IL-27 has been proposed as a therapy to modify inflammatory conditions by regulating T-cell responses (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). However, the contradictory effects of IL-27 have been reported in type 1 diabetes (T1D). Recent investigation reported that IL-27 not only showed immunomodulatory function, but also was a compensatory effort of dendritic cells against the ongoing inflammation in T1D patients (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>). On the contrary, Ciecko et al. (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>) reported that IL-27 contributes to the pathogenesis of T1D in NOD mice by altering the balance of Treg and T<sub>H</sub>1 cells and enhancing the effector function of CD8 T cells, although it was reported that serum IL-27 was strongly elevated in patients with SS (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>), which is inconsistent with our results. This may be because SS patients were particularly associated with interstitial lung disease complications in their study. Another study reported that the levels of IL-27 and IL-23 increased significantly in the serum and urine of systemic lupus erythematosus patients with and without lupus nephritis compared with healthy control (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>), which may account for the pleiotopic roles of IL-27 in several cases of autoimmunity. Up to date, many studies have reported that IL-27 showed a protective role in a variety of diseases, and exogenous IL-27 could suppress multiple autoimmune diseases by promoting Tr1 cells or other mechanisms (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B35\" ref-type=\"bibr\">35</xref>, <xref rid=\"B36\" ref-type=\"bibr\">36</xref>, <xref rid=\"B41\" ref-type=\"bibr\">41</xref>, <xref rid=\"B42\" ref-type=\"bibr\">42</xref>). Lee et al. (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>) reported that exogenous IL-27 could induce a suppressive effect on SS development by regulating T<sub>H</sub>17 pro-inflammatory activity. Our previous study showed that IL-27 decreased significantly in SS patients and SS-like NOD mice, and mesenchymal stem cells (MSCs) alleviated SS by elevating the level of IL-27 (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>). In this study, we found that IL-27 alleviated SS by inducing IL-10–producing CD4<sup>+</sup> T cells. The different properties of IL-27 in the pathogenic conditions may account for the complex effects of IL-27 on different lymphocyte populations, which play a pleiotropic role in the development and progression of disease in NOD mice. Thus, the specific effects of IL-27 in SS need further investigation. In our studies, we compared the SS-like symptoms in NOD and <italic>Il-27</italic><sup>−/−</sup>NOD mice and found the protective function of IL-27 by regulating Tr1 cells in SS.</p><p>Tr1 cells, a T-cell population with distinct suppressive function, are characterized by secreting high amounts of IL-10 and variable amounts of IFN-γ, IL-2, and TGF-β, depending on the microenvironment and the disease context (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>), while none of the biomarkers are yet the ideal candidates for Tr1 cells. Studies suggested that IL-10–producing CD4<sup>+</sup> T cells may represent a heterogeneous cell population reflecting different cell origins, maturation stage, or different functions of Tr1 cells (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>). Tr1 cells suppressed immune responses mainly by producing IL-10 (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). A number of investigators have also documented that Tr1 cells could prevent T cell–mediated diseases via a TGF-β-dependent mechanism in addition to IL-10 (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>, <xref rid=\"B47\" ref-type=\"bibr\">47</xref>). Interleukin 27 was the dominant factor for IL-10–producing T cells and worked together with TGF-β to further enhance Tr1 differentiation (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). And IL-27 limited autoimmune disorders by promoting Tr1 cells. In this study, exogenous IL-27 ameliorated SS-like syndromes in NOD mice by promoting the generation of CD4<sup>+</sup>IL-10<sup>+</sup> T cells in NOD and <italic>Il-27</italic><sup>−/−</sup>NOD mice, whereas IFN-γ and TGF-β as the important factors involved in the induction and function of Tr1 cells should be considered and need to be identified.</p><p>Although the deficiency of IL-27 signal resulted in more serious SS-like symptoms and fewer splenic CD4<sup>+</sup>IL-10<sup>+</sup> T cells in NOD mice, the percentage of CD4<sup>+</sup>IL-10<sup>+</sup> T cells was very low and showed no significant difference in SG of NOD and <italic>Il-27</italic><sup>−/−</sup>NOD mice (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Figure S3</xref>). The improved SS-like syndromes in IL-27–treated NOD mice might be due to that splenic CD4<sup>+</sup>IL-10<sup>+</sup> T cells could affect already established inflammation in SG and saliva flow indirectly via IL-10 to regulate other immune responses in the immune system.</p><p>Many different cytokines can induce JAKs and STATs activation to regulate fundamental biological processes (<xref rid=\"B48\" ref-type=\"bibr\">48</xref>). It has been reported that IL-27 induced IL-10–producing Tr1 cells generation by activating STAT1 and STAT3. While other cytokines that signal, such as IFN-α or IL-6, alone or in combination, could promote the generation of Tr1 cells via STAT3 (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>, <xref rid=\"B50\" ref-type=\"bibr\">50</xref>). Brockmann et al. reported that IL-10 signaling controlled the differentiation and regulatory activity of Tr1 cells via P38MAPK and not by STAT3 (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>). These studies suggested that STAT1 and/or STAT3 were activated in the Tr1 cells depending on different cytokine signals.</p><p>In summary, we have demonstrated that IL-27 could promote the development and differentiation of CD4<sup>+</sup>IL-10<sup>+</sup> T cells <italic>in vitro</italic> and <italic>in vivo</italic>. Interleukin 27 gene deficiency resulted in decreased CD4<sup>+</sup>IL-10<sup>+</sup> T cells and aggravated the severity of SS-like symptoms of NOD mice. Exogenous IL-27 ameliorated <italic>Il-27</italic><sup>−/−</sup>NOD and NOD mice by regulating CD4<sup>+</sup>IL-10<sup>+</sup> T cells. Notably, CD4<sup>+</sup>IL-10<sup>+</sup> T cells decreased in SS patients, which may be correlated to IL-27. These findings suggested that IL-27 played a crucial role in the pathogenesis of SS by regulating CD4<sup>+</sup>IL-10<sup>+</sup> T cells. We propose that targeting IL-27 and CD4<sup>+</sup>IL-10<sup>+</sup> T cells may be a new direction for the SS treatment.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The studies were reviewed and approved by Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School. Written informed consent was obtained from all subjects.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>JQ and ZZ participated in study design, data collection, data analysis, data interpretation, and drafting the paper. XT, WL, and WC participated in patient recruitment, animal experiments, and data collection. GY supervised the whole research, designed the study, interpreted the data, and wrote the paper. All authors read and approved the manuscript.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by the National Natural Science Foundation of China (NSFC) (grant nos. 81770061, 81970062, and 81571583 to GY) and Key Project supported by Medical Science and technology development Foundation, Nanjing Department of Health (grant no. ZKX18024 to GY).</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s9\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fimmu.2020.01699/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fimmu.2020.01699/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Data_Sheet_1.PDF\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mavragani</surname><given-names>CP</given-names></name></person-group>. <article-title>Mechanisms and new strategies for primary Sjogren's syndrome</article-title>. <source>Annu Rev Med.</source> (<year>2017</year>) <volume>68</volume>:<fpage>331</fpage>–<lpage>43</lpage>. <pub-id pub-id-type=\"doi\">10.1146/annurev-med-043015-123313</pub-id><pub-id pub-id-type=\"pmid\">28099084</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mavragani</surname><given-names>CP</given-names></name><name><surname>Moutsopoulos</surname><given-names>HM</given-names></name></person-group>. <article-title>Sjogren's syndrome</article-title>. <source>Annu Rev Pathol.</source> (<year>2014</year>) <volume>9</volume>:<fpage>273</fpage>–<lpage>85</lpage>. <pub-id pub-id-type=\"doi\">10.1146/annurev-pathol-012513-104728</pub-id><pub-id pub-id-type=\"pmid\">24050623</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Roescher</surname><given-names>N</given-names></name><name><surname>Tak</surname><given-names>PP</given-names></name><name><surname>Illei</surname><given-names>GG</given-names></name></person-group>. <article-title>Cytokines in Sjogren's syndrome</article-title>. <source>Oral Dis.</source> (<year>2009</year>) <volume>15</volume>:<fpage>519</fpage>–<lpage>26</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1601-0825.2009.01582.x</pub-id><pub-id pub-id-type=\"pmid\">19519622</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kimura</surname><given-names>D</given-names></name><name><surname>Miyakoda</surname><given-names>M</given-names></name><name><surname>Kimura</surname><given-names>K</given-names></name><name><surname>Honma</surname><given-names>K</given-names></name><name><surname>Hara</surname><given-names>H</given-names></name><name><surname>Yoshida</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Interleukin-27-producing CD4<sup>+</sup> T cells regulate protective immunity during malaria parasite infection</article-title>. <source>Immunity.</source> (<year>2016</year>) <volume>44</volume>:<fpage>672</fpage>–<lpage>82</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.immuni.2016.02.011</pub-id><pub-id pub-id-type=\"pmid\">26968425</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yoshida</surname><given-names>H</given-names></name><name><surname>Hunter</surname><given-names>CA</given-names></name></person-group>. <article-title>The immunobiology of interleukin-27</article-title>. <source>Annu Rev Immunol.</source> (<year>2015</year>) <volume>33</volume>:<fpage>417</fpage>–<lpage>43</lpage>. <pub-id pub-id-type=\"doi\">10.1146/annurev-immunol-032414-112134</pub-id><pub-id pub-id-type=\"pmid\">25861977</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Batten</surname><given-names>M</given-names></name><name><surname>Li</surname><given-names>J</given-names></name><name><surname>Yi</surname><given-names>S</given-names></name><name><surname>Kljavin</surname><given-names>NM</given-names></name><name><surname>Danilenko</surname><given-names>DM</given-names></name><name><surname>Lucas</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Interleukin 27 limits autoimmune encephalomyelitis by suppressing the development of interleukin 17-producing T cells</article-title>. <source>Nat Immunol.</source> (<year>2006</year>) <volume>7</volume>:<fpage>929</fpage>–<lpage>36</lpage>. <pub-id pub-id-type=\"doi\">10.1038/ni1375</pub-id><pub-id pub-id-type=\"pmid\">16906167</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pflanz</surname><given-names>S</given-names></name><name><surname>Timans</surname><given-names>JC</given-names></name><name><surname>Cheung</surname><given-names>J</given-names></name><name><surname>Rosales</surname><given-names>R</given-names></name><name><surname>Kanzler</surname><given-names>H</given-names></name><name><surname>Gilbert</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4+ T cells</article-title>. <source>Immunity.</source> (<year>2002</year>) <volume>16</volume>:<fpage>779</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S1074-7613(02)00324-2</pub-id><pub-id pub-id-type=\"pmid\">12121660</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pflanz</surname><given-names>S</given-names></name><name><surname>Hibbert</surname><given-names>L</given-names></name><name><surname>Mattson</surname><given-names>J</given-names></name><name><surname>Rosales</surname><given-names>R</given-names></name><name><surname>Vaisberg</surname><given-names>E</given-names></name><name><surname>Bazan</surname><given-names>JF</given-names></name><etal/></person-group>. <article-title>WSX-1 and glycoprotein 130 constitute a signal-transducing receptor for IL-27</article-title>. <source>J Immunol.</source> (<year>2004</year>) <volume>172</volume>:<fpage>2225</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.172.4.2225</pub-id><pub-id pub-id-type=\"pmid\">14764690</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Villarino</surname><given-names>AV</given-names></name><name><surname>Larkin</surname><given-names>J</given-names><suffix>III</suffix></name><name><surname>Saris</surname><given-names>CJ</given-names></name><name><surname>Caton</surname><given-names>AJ</given-names></name><name><surname>Lucas</surname><given-names>S</given-names></name><name><surname>Wong</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>Positive and negative regulation of the IL-27 receptor during lymphoid cell activation</article-title>. <source>J Immunol.</source> (<year>2005</year>) <volume>174</volume>:<fpage>7684</fpage>–<lpage>91</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.174.12.7684</pub-id><pub-id pub-id-type=\"pmid\">15944269</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Artis</surname><given-names>D</given-names></name><name><surname>Villarino</surname><given-names>A</given-names></name><name><surname>Silverman</surname><given-names>M</given-names></name><name><surname>He</surname><given-names>W</given-names></name><name><surname>Thornton</surname><given-names>EM</given-names></name><name><surname>Mu</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>The IL-27 receptor (WSX-1) is an inhibitor of innate and adaptive elements of type 2 immunity</article-title>. <source>J Immunol.</source> (<year>2004</year>) <volume>173</volume>:<fpage>5626</fpage>–<lpage>34</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.173.9.5626</pub-id><pub-id pub-id-type=\"pmid\">15494513</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Takeda</surname><given-names>A</given-names></name><name><surname>Hamano</surname><given-names>S</given-names></name><name><surname>Yamanaka</surname><given-names>A</given-names></name><name><surname>Hanada</surname><given-names>T</given-names></name><name><surname>Ishibashi</surname><given-names>T</given-names></name><name><surname>Mak</surname><given-names>TW</given-names></name><etal/></person-group>. <article-title>Cutting edge: role of IL-27/WSX-1 signaling for induction of T-bet through activation of STAT1 during initial Th1 commitment</article-title>. <source>J Immunol.</source> (<year>2003</year>) <volume>170</volume>:<fpage>4886</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.170.10.4886</pub-id><pub-id pub-id-type=\"pmid\">12734330</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hibbert</surname><given-names>L</given-names></name><name><surname>Pflanz</surname><given-names>S</given-names></name><name><surname>De Waal Malefyt</surname><given-names>R</given-names></name><name><surname>Kastelein</surname><given-names>RA</given-names></name></person-group>. <article-title>IL-27 and IFN-alpha signal via Stat1 and Stat3 and induce T-Bet and IL-12Rbeta2 in naive T cells</article-title>. <source>J Interferon Cytokine Res.</source> (<year>2003</year>) <volume>23</volume>:<fpage>513</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.1089/10799900360708632</pub-id><pub-id pub-id-type=\"pmid\">14565860</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yoshida</surname><given-names>H</given-names></name><name><surname>Hamano</surname><given-names>S</given-names></name><name><surname>Senaldi</surname><given-names>G</given-names></name><name><surname>Covey</surname><given-names>T</given-names></name><name><surname>Faggioni</surname><given-names>R</given-names></name><name><surname>Mu</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>WSX-1 is required for the initiation of Th1 responses and resistance to L. major infection</article-title>. <source>Immunity.</source> (<year>2001</year>) <volume>15</volume>:<fpage>569</fpage>–<lpage>78</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S1074-7613(01)00206-0</pub-id><pub-id pub-id-type=\"pmid\">11672539</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>X</given-names></name><name><surname>Deng</surname><given-names>R</given-names></name><name><surname>Chi</surname><given-names>W</given-names></name><name><surname>Hua</surname><given-names>X</given-names></name><name><surname>Lu</surname><given-names>F</given-names></name><name><surname>Bian</surname><given-names>F</given-names></name><etal/></person-group>. <article-title>IL-27 signaling deficiency develops Th17-enhanced Th2-dominant inflammation in murine allergic conjunctivitis model</article-title>. <source>Allergy.</source> (<year>2019</year>) <volume>74</volume>:<fpage>910</fpage>–<lpage>21</lpage>. <pub-id pub-id-type=\"doi\">10.1111/all.13691</pub-id><pub-id pub-id-type=\"pmid\">30515838</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>L</given-names></name><name><surname>Cao</surname><given-names>J</given-names></name><name><surname>Li</surname><given-names>C</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name></person-group>. <article-title>IL-27/IL-27 receptor signaling provides protection in <italic>Clostridium difficile</italic>-induced colitis</article-title>. <source>J Infect Dis.</source> (<year>2018</year>) <volume>217</volume>:<fpage>198</fpage>–<lpage>207</lpage>. <pub-id pub-id-type=\"doi\">10.1093/infdis/jix581</pub-id><pub-id pub-id-type=\"pmid\">29140433</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fitzgerald</surname><given-names>DC</given-names></name><name><surname>Zhang</surname><given-names>GX</given-names></name><name><surname>El-Behi</surname><given-names>M</given-names></name><name><surname>Fonseca-Kelly</surname><given-names>Z</given-names></name><name><surname>Li</surname><given-names>H</given-names></name><name><surname>Yu</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Suppression of autoimmune inflammation of the central nervous system by interleukin 10 secreted by interleukin 27-stimulated T cells</article-title>. <source>Nat Immunol.</source> (<year>2007</year>) <volume>8</volume>:<fpage>1372</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1038/ni1540</pub-id><pub-id pub-id-type=\"pmid\">17994023</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Do</surname><given-names>J</given-names></name><name><surname>Kim</surname><given-names>D</given-names></name><name><surname>Kim</surname><given-names>S</given-names></name><name><surname>Valentin-Torres</surname><given-names>A</given-names></name><name><surname>Dvorina</surname><given-names>N</given-names></name><name><surname>Jang</surname><given-names>E</given-names></name><etal/></person-group>. <article-title>Treg-specific IL-27Ralpha deletion uncovers a key role for IL-27 in Treg function to control autoimmunity</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2017</year>) <volume>114</volume>:<fpage>10190</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1703100114</pub-id><pub-id pub-id-type=\"pmid\">28874534</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hall</surname><given-names>AO</given-names></name><name><surname>Beiting</surname><given-names>DP</given-names></name><name><surname>Tato</surname><given-names>C</given-names></name><name><surname>John</surname><given-names>B</given-names></name><name><surname>Oldenhove</surname><given-names>G</given-names></name><name><surname>Lombana</surname><given-names>CG</given-names></name><etal/></person-group>. <article-title>The cytokines interleukin 27 and interferon-gamma promote distinct Treg cell populations required to limit infection-induced pathology</article-title>. <source>Immunity.</source> (<year>2012</year>) <volume>37</volume>:<fpage>511</fpage>–<lpage>23</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.immuni.2012.06.014</pub-id><pub-id pub-id-type=\"pmid\">22981537</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Villarino</surname><given-names>AV</given-names></name><name><surname>Stumhofer</surname><given-names>JS</given-names></name><name><surname>Saris</surname><given-names>CJ</given-names></name><name><surname>Kastelein</surname><given-names>RA</given-names></name><name><surname>de Sauvage</surname><given-names>FJ</given-names></name><name><surname>Hunter</surname><given-names>CA</given-names></name></person-group>. <article-title>IL-27 limits IL-2 production during Th1 differentiation</article-title>. <source>J Immunol.</source> (<year>2006</year>) <volume>176</volume>:<fpage>237</fpage>–<lpage>47</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.176.1.237</pub-id><pub-id pub-id-type=\"pmid\">16365415</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thome</surname><given-names>R</given-names></name><name><surname>Moore</surname><given-names>JN</given-names></name><name><surname>Mari</surname><given-names>ER</given-names></name><name><surname>Rasouli</surname><given-names>J</given-names></name><name><surname>Hwang</surname><given-names>D</given-names></name><name><surname>Yoshimura</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Induction of peripheral tolerance in ongoing autoimmune inflammation requires interleukin 27 signaling in dendritic cells</article-title>. <source>Front Immunol.</source> (<year>2017</year>) <volume>8</volume>:<fpage>1392</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2017.01392</pub-id><pub-id pub-id-type=\"pmid\">29163476</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stumhofer</surname><given-names>JS</given-names></name><name><surname>Silver</surname><given-names>JS</given-names></name><name><surname>Laurence</surname><given-names>A</given-names></name><name><surname>Porrett</surname><given-names>PM</given-names></name><name><surname>Harris</surname><given-names>TH</given-names></name><name><surname>Turka</surname><given-names>LA</given-names></name><etal/></person-group>. <article-title>Interleukins 27 and 6 induce STAT3-mediated T cell production of interleukin 10</article-title>. <source>Nat Immunol.</source> (<year>2007</year>) <volume>8</volume>:<fpage>1363</fpage>–<lpage>71</lpage>. <pub-id pub-id-type=\"doi\">10.1038/ni1537</pub-id><pub-id pub-id-type=\"pmid\">17994025</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>DeLong</surname><given-names>JH</given-names></name><name><surname>O'Hara Hall</surname><given-names>A</given-names></name><name><surname>Rausch</surname><given-names>M</given-names></name><name><surname>Moodley</surname><given-names>D</given-names></name><name><surname>Perry</surname><given-names>J</given-names></name><name><surname>Park</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>IL-27 and TCR stimulation promote T cell expression of multiple inhibitory receptors</article-title>. <source>Immunohorizons.</source> (<year>2019</year>) <volume>3</volume>:<fpage>13</fpage>–<lpage>25</lpage>. <pub-id pub-id-type=\"doi\">10.4049/immunohorizons.1800083</pub-id><pub-id pub-id-type=\"pmid\">31356173</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Awasthi</surname><given-names>A</given-names></name><name><surname>Carrier</surname><given-names>Y</given-names></name><name><surname>Peron</surname><given-names>JP</given-names></name><name><surname>Bettelli</surname><given-names>E</given-names></name><name><surname>Kamanaka</surname><given-names>M</given-names></name><name><surname>Flavell</surname><given-names>RA</given-names></name><etal/></person-group>. <article-title>A dominant function for interleukin 27 in generating interleukin 10-producing anti-inflammatory T cells</article-title>. <source>Nat Immunol.</source> (<year>2007</year>) <volume>8</volume>:<fpage>1380</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1038/ni1541</pub-id><pub-id pub-id-type=\"pmid\">17994022</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pot</surname><given-names>C</given-names></name><name><surname>Jin</surname><given-names>H</given-names></name><name><surname>Awasthi</surname><given-names>A</given-names></name><name><surname>Liu</surname><given-names>SM</given-names></name><name><surname>Lai</surname><given-names>CY</given-names></name><name><surname>Madan</surname><given-names>R</given-names></name><etal/></person-group>. <article-title>Cutting edge: IL-27 induces the transcription factor c-Maf, cytokine IL-21, and the costimulatory receptor ICOS that coordinately act together to promote differentiation of IL-10-producing Tr1 cells</article-title>. <source>J Immunol.</source> (<year>2009</year>) <volume>183</volume>:<fpage>797</fpage>–<lpage>801</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.0901233</pub-id><pub-id pub-id-type=\"pmid\">19570826</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Murugaiyan</surname><given-names>G</given-names></name><name><surname>Mittal</surname><given-names>A</given-names></name><name><surname>Lopez-Diego</surname><given-names>R</given-names></name><name><surname>Maier</surname><given-names>LM</given-names></name><name><surname>Anderson</surname><given-names>DE</given-names></name><name><surname>Weiner</surname><given-names>HL</given-names></name></person-group>. <article-title>IL-27 is a key regulator of IL-10 and IL-17 production by human CD4+ T cells</article-title>. <source>J Immunol.</source> (<year>2009</year>) <volume>183</volume>:<fpage>2435</fpage>–<lpage>43</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.0900568</pub-id><pub-id pub-id-type=\"pmid\">19625647</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vieira</surname><given-names>PL</given-names></name><name><surname>Christensen</surname><given-names>JR</given-names></name><name><surname>Minaee</surname><given-names>S</given-names></name><name><surname>O'Neill</surname><given-names>EJ</given-names></name><name><surname>Barrat</surname><given-names>FJ</given-names></name><name><surname>Boonstra</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>IL-10-secreting regulatory T cells do not express Foxp3 but have comparable regulatory function to naturally occurring CD4+CD25+ regulatory T cells</article-title>. <source>J Immunol.</source> (<year>2004</year>) <volume>172</volume>:<fpage>5986</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.172.10.5986</pub-id><pub-id pub-id-type=\"pmid\">15128781</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mbongue</surname><given-names>JC</given-names></name><name><surname>Rawson</surname><given-names>J</given-names></name><name><surname>Garcia</surname><given-names>PA</given-names></name><name><surname>Gonzalez</surname><given-names>N</given-names></name><name><surname>Cobb</surname><given-names>J</given-names></name><name><surname>Kandeel</surname><given-names>F</given-names></name><etal/></person-group>. <article-title>Reversal of new onset type 1 diabetes by oral salmonella-based combination therapy and mediated by regulatory T-cells in NOD mice</article-title>. <source>Front Immunol.</source> (<year>2019</year>) <volume>10</volume>:<fpage>320</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2019.00320</pub-id><pub-id pub-id-type=\"pmid\">30863412</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cook</surname><given-names>L</given-names></name><name><surname>Stahl</surname><given-names>M</given-names></name><name><surname>Han</surname><given-names>X</given-names></name><name><surname>Nazli</surname><given-names>A</given-names></name><name><surname>MacDonald</surname><given-names>KN</given-names></name><name><surname>Wong</surname><given-names>MQ</given-names></name><etal/></person-group>. <article-title>Suppressive and gut reparative functions of human type 1 T-regulatory cells</article-title>. <source>Gastroenterology.</source> (<year>2019</year>) <volume>157</volume>:<fpage>1584</fpage>–<lpage>98</lpage>. <pub-id pub-id-type=\"doi\">10.1053/j.gastro.2019.09.002</pub-id><pub-id pub-id-type=\"pmid\">31513797</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>H</given-names></name><name><surname>Gagliani</surname><given-names>N</given-names></name><name><surname>Ishigame</surname><given-names>H</given-names></name><name><surname>Huber</surname><given-names>S</given-names></name><name><surname>Zhu</surname><given-names>S</given-names></name><name><surname>Esplugues</surname><given-names>E</given-names></name><etal/></person-group>. <article-title>Intestinal type 1 regulatory T cells migrate to periphery to suppress diabetogenic T cells and prevent diabetes development</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2017</year>) <volume>114</volume>:<fpage>10443</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1705599114</pub-id><pub-id pub-id-type=\"pmid\">28894001</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mfarrej</surname><given-names>B</given-names></name><name><surname>Tresoldi</surname><given-names>E</given-names></name><name><surname>Stabilini</surname><given-names>A</given-names></name><name><surname>Paganelli</surname><given-names>A</given-names></name><name><surname>Caldara</surname><given-names>R</given-names></name><name><surname>Secchi</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Generation of donor-specific Tr1 cells to be used after kidney transplantation and definition of the timing of their <italic>in vivo</italic> infusion in the presence of immunosuppression</article-title>. <source>J Transl Med.</source> (<year>2017</year>) <volume>15</volume>:<fpage>40</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s12967-017-1133-8</pub-id><pub-id pub-id-type=\"pmid\">28222739</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gagliani</surname><given-names>N</given-names></name><name><surname>Jofra</surname><given-names>T</given-names></name><name><surname>Stabilini</surname><given-names>A</given-names></name><name><surname>Valle</surname><given-names>A</given-names></name><name><surname>Atkinson</surname><given-names>M</given-names></name><name><surname>Roncarolo</surname><given-names>MG</given-names></name><etal/></person-group>. <article-title>Antigen-specific dependence of Tr1-cell therapy in preclinical models of islet transplant</article-title>. <source>Diabetes.</source> (<year>2010</year>) <volume>59</volume>:<fpage>433</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.2337/db09-1168</pub-id><pub-id pub-id-type=\"pmid\">19934002</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tait Wojno</surname><given-names>ED</given-names></name><name><surname>Hunter</surname><given-names>CA</given-names></name><name><surname>Stumhofer</surname><given-names>JS</given-names></name></person-group>. <article-title>The immunobiology of the interleukin-12 family: room for discovery</article-title>. <source>Immunity.</source> (<year>2019</year>) <volume>50</volume>:<fpage>851</fpage>–<lpage>70</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.immuni.2019.03.011</pub-id><pub-id pub-id-type=\"pmid\">30995503</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Suzuki</surname><given-names>M</given-names></name><name><surname>Yokota</surname><given-names>M</given-names></name><name><surname>Ozaki</surname><given-names>S</given-names></name><name><surname>Matsumoto</surname><given-names>T</given-names></name></person-group>. <article-title>Intranasal administration of IL-27 ameliorates nasal allergic responses and symptoms</article-title>. <source>Int Arch Allergy Immunol.</source> (<year>2019</year>) <volume>178</volume>:<fpage>101</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1159/000493398</pub-id><pub-id pub-id-type=\"pmid\">30428479</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhu</surname><given-names>X</given-names></name><name><surname>Liu</surname><given-names>Z</given-names></name><name><surname>Liu</surname><given-names>JQ</given-names></name><name><surname>Zhu</surname><given-names>J</given-names></name><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Davis</surname><given-names>JP</given-names></name><etal/></person-group>. <article-title>Systemic delivery of IL-27 by an adeno-associated viral vector inhibits T cell-mediated colitis and induces multiple inhibitory pathways in T cells</article-title>. <source>J Leukocyte Biol.</source> (<year>2016</year>) <volume>100</volume>:<fpage>403</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1189/jlb.3A1215-540R</pub-id><pub-id pub-id-type=\"pmid\">27106672</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moon</surname><given-names>SJ</given-names></name><name><surname>Park</surname><given-names>JS</given-names></name><name><surname>Heo</surname><given-names>YJ</given-names></name><name><surname>Kang</surname><given-names>CM</given-names></name><name><surname>Kim</surname><given-names>EK</given-names></name><name><surname>Lim</surname><given-names>MA</given-names></name><etal/></person-group>. <article-title><italic>In vivo</italic> action of IL-27: reciprocal regulation of Th17 and Treg cells in collagen-induced arthritis</article-title>. <source>Exp Mol Med.</source> (<year>2013</year>) <volume>45</volume>:<fpage>e46</fpage>. <pub-id pub-id-type=\"doi\">10.1038/emm.2013.89</pub-id><pub-id pub-id-type=\"pmid\">24091748</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Niedbala</surname><given-names>W</given-names></name><name><surname>Cai</surname><given-names>B</given-names></name><name><surname>Wei</surname><given-names>X</given-names></name><name><surname>Patakas</surname><given-names>A</given-names></name><name><surname>Leung</surname><given-names>BP</given-names></name><name><surname>McInnes</surname><given-names>IB</given-names></name><etal/></person-group>. <article-title>Interleukin 27 attenuates collagen-induced arthritis</article-title>. <source>Ann Rheum Dis.</source> (<year>2008</year>) <volume>67</volume>:<fpage>1474</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1136/ard.2007.083360</pub-id><pub-id pub-id-type=\"pmid\">18199596</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parackova</surname><given-names>Z</given-names></name><name><surname>Vrabcova</surname><given-names>P</given-names></name><name><surname>Zentsova</surname><given-names>I</given-names></name><name><surname>Kayserova</surname><given-names>J</given-names></name><name><surname>Richtrova</surname><given-names>I</given-names></name><name><surname>Sojka</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Enhanced STAT3 phosphorylation and PD-L1 expression in myeloid dendritic cells indicate impaired IL-27Ralpha signaling in type 1 diabetes</article-title>. <source>Sci Rep.</source> (<year>2020</year>) <volume>10</volume>:<fpage>493</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-020-57507-8</pub-id><pub-id pub-id-type=\"pmid\">31949260</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ciecko</surname><given-names>AE</given-names></name><name><surname>Foda</surname><given-names>B</given-names></name><name><surname>Barr</surname><given-names>JY</given-names></name><name><surname>Ramanathan</surname><given-names>S</given-names></name><name><surname>Atkinson</surname><given-names>MA</given-names></name><name><surname>Serreze</surname><given-names>DV</given-names></name><etal/></person-group>. <article-title>Interleukin-27 is essential for type 1 diabetes development and Sjogren syndrome-like inflammation</article-title>. <source>Cell Rep.</source> (<year>2019</year>) <volume>29</volume>:<fpage>3073</fpage>–<lpage>86.e5</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.celrep.2019.11.010</pub-id><pub-id pub-id-type=\"pmid\">31801074</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xia</surname><given-names>L</given-names></name><name><surname>Shen</surname><given-names>H</given-names></name><name><surname>Zhao</surname><given-names>L</given-names></name><name><surname>Lu</surname><given-names>J</given-names></name></person-group>. <article-title>Elevated levels of interleukin-27 in patients with Sjogren's syndrome</article-title>. <source>Scand J Rheumatol.</source> (<year>2012</year>) <volume>41</volume>:<fpage>73</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.3109/03009742.2011.620574</pub-id><pub-id pub-id-type=\"pmid\">22118194</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xia</surname><given-names>LP</given-names></name><name><surname>Li</surname><given-names>BF</given-names></name><name><surname>Shen</surname><given-names>H</given-names></name><name><surname>Lu</surname><given-names>J</given-names></name></person-group>. <article-title>Interleukin-27 and interleukin-23 in patients with systemic lupus erythematosus: possible role in lupus nephritis</article-title>. <source>Scand J Rheumatol.</source> (<year>2015</year>) <volume>44</volume>:<fpage>200</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.3109/03009742.2014.962080</pub-id><pub-id pub-id-type=\"pmid\">25562331</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sugiyama</surname><given-names>N</given-names></name><name><surname>Nakashima</surname><given-names>H</given-names></name><name><surname>Yoshimura</surname><given-names>T</given-names></name><name><surname>Sadanaga</surname><given-names>A</given-names></name><name><surname>Shimizu</surname><given-names>S</given-names></name><name><surname>Masutani</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Amelioration of human lupus-like phenotypes in MRL/lpr mice by overexpression of interleukin 27 receptor alpha (WSX-1)</article-title>. <source>Ann Rheum Dis.</source> (<year>2008</year>) <volume>67</volume>:<fpage>1461</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1136/ard.2007.077537</pub-id><pub-id pub-id-type=\"pmid\">18094002</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fitzgerald</surname><given-names>DC</given-names></name><name><surname>Ciric</surname><given-names>B</given-names></name><name><surname>Touil</surname><given-names>T</given-names></name><name><surname>Harle</surname><given-names>H</given-names></name><name><surname>Grammatikopolou</surname><given-names>J</given-names></name><name><surname>Das Sarma</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Suppressive effect of IL-27 on encephalitogenic Th17 cells and the effector phase of experimental autoimmune encephalomyelitis</article-title>. <source>J Immunol.</source> (<year>2007</year>) <volume>179</volume>:<fpage>3268</fpage>–<lpage>75</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.179.5.3268</pub-id><pub-id pub-id-type=\"pmid\">17709543</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>BH</given-names></name><name><surname>Carcamo</surname><given-names>WC</given-names></name><name><surname>Chiorini</surname><given-names>JA</given-names></name><name><surname>Peck</surname><given-names>AB</given-names></name><name><surname>Nguyen</surname><given-names>CQ</given-names></name></person-group>. <article-title>Gene therapy using IL-27 ameliorates Sjogren's syndrome-like autoimmune exocrinopathy</article-title>. <source>Arthritis Res Ther.</source> (<year>2012</year>) <volume>14</volume>:<fpage>R172</fpage>. <pub-id pub-id-type=\"doi\">10.1186/ar3925</pub-id><pub-id pub-id-type=\"pmid\">22827855</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yao</surname><given-names>G</given-names></name><name><surname>Qi</surname><given-names>J</given-names></name><name><surname>Liang</surname><given-names>J</given-names></name><name><surname>Shi</surname><given-names>B</given-names></name><name><surname>Chen</surname><given-names>W</given-names></name><name><surname>Li</surname><given-names>W</given-names></name><etal/></person-group>. <article-title>Mesenchymal stem cell transplantation alleviates experimental Sjogren's syndrome through IFN-beta/IL-27 signaling axis</article-title>. <source>Theranostics.</source> (<year>2019</year>) <volume>9</volume>:<fpage>8253</fpage>–<lpage>65</lpage>. <pub-id pub-id-type=\"doi\">10.7150/thno.37351</pub-id><pub-id pub-id-type=\"pmid\">31754394</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sakaguchi</surname><given-names>S</given-names></name><name><surname>Mikami</surname><given-names>N</given-names></name><name><surname>Wing</surname><given-names>JB</given-names></name><name><surname>Tanaka</surname><given-names>A</given-names></name><name><surname>Ichiyama</surname><given-names>K</given-names></name><name><surname>Ohkura</surname><given-names>N</given-names></name></person-group>. <article-title>Regulatory T cells and human disease</article-title>. <source>Annu Rev Immunol.</source> (<year>2020</year>) <volume>38</volume>:<fpage>541</fpage>–<lpage>66</lpage>. <pub-id pub-id-type=\"doi\">10.1146/annurev-immunol-042718-041717</pub-id><pub-id pub-id-type=\"pmid\">32017635</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Levings</surname><given-names>MK</given-names></name><name><surname>Sangregorio</surname><given-names>R</given-names></name><name><surname>Galbiati</surname><given-names>F</given-names></name><name><surname>Squadrone</surname><given-names>S</given-names></name><name><surname>de Waal Malefyt</surname><given-names>R</given-names></name><name><surname>Roncarolo</surname><given-names>MG</given-names></name></person-group>. <article-title>IFN-alpha and IL-10 induce the differentiation of human type 1 T regulatory cells</article-title>. <source>J Immunol.</source> (<year>2001</year>) <volume>166</volume>:<fpage>5530</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.166.9.5530</pub-id><pub-id pub-id-type=\"pmid\">11313392</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Singh</surname><given-names>B</given-names></name><name><surname>Krawetz</surname><given-names>MD</given-names></name><name><surname>De Lima</surname><given-names>RM</given-names></name><name><surname>Mukherjee</surname><given-names>R</given-names></name><name><surname>Chaturvedi</surname><given-names>P</given-names></name><name><surname>Lee-Chan</surname><given-names>E</given-names></name><etal/></person-group>. <article-title>Role of TGF-beta in self-peptide regulation of autoimmunity</article-title>. <source>Arch Immunol Ther Exp.</source> (<year>2018</year>) <volume>66</volume>:<fpage>11</fpage>–<lpage>19</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00005-017-0482-6</pub-id><pub-id pub-id-type=\"pmid\">28733878</pub-id></mixed-citation></ref><ref id=\"B48\"><label>48.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Salas</surname><given-names>A</given-names></name><name><surname>Hernandez-Rocha</surname><given-names>C</given-names></name><name><surname>Duijvestein</surname><given-names>M</given-names></name><name><surname>Faubion</surname><given-names>W</given-names></name><name><surname>McGovern</surname><given-names>D</given-names></name><name><surname>Vermeire</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>JAK-STAT pathway targeting for the treatment of inflammatory bowel disease</article-title>. <source>Nat Rev Gastroenterol Hepatol.</source> (<year>2020</year>) <volume>17</volume>:<fpage>323</fpage>–<lpage>37</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41575-020-0273-0</pub-id><pub-id pub-id-type=\"pmid\">32203403</pub-id></mixed-citation></ref><ref id=\"B49\"><label>49.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jin</surname><given-names>JO</given-names></name><name><surname>Han</surname><given-names>X</given-names></name><name><surname>Yu</surname><given-names>Q</given-names></name></person-group>. <article-title>Interleukin-6 induces the generation of IL-10-producing Tr1 cells and suppresses autoimmune tissue inflammation</article-title>. <source>J Autoimmun.</source> (<year>2013</year>) <volume>40</volume>:<fpage>28</fpage>–<lpage>44</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jaut.2012.07.009</pub-id><pub-id pub-id-type=\"pmid\">22921334</pub-id></mixed-citation></ref><ref id=\"B50\"><label>50.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Corre</surname><given-names>B</given-names></name><name><surname>Perrier</surname><given-names>J</given-names></name><name><surname>El Khouri</surname><given-names>M</given-names></name><name><surname>Cerboni</surname><given-names>S</given-names></name><name><surname>Pellegrini</surname><given-names>S</given-names></name><name><surname>Michel</surname><given-names>F</given-names></name></person-group>. <article-title>Type I interferon potentiates T-cell receptor mediated induction of IL-10-producing CD4<sup>+</sup> T cells</article-title>. <source>Eur J Immunol.</source> (<year>2013</year>) <volume>43</volume>:<fpage>2730</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1002/eji.201242977</pub-id><pub-id pub-id-type=\"pmid\">23839924</pub-id></mixed-citation></ref><ref id=\"B51\"><label>51.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brockmann</surname><given-names>L</given-names></name><name><surname>Gagliani</surname><given-names>N</given-names></name><name><surname>Steglich</surname><given-names>B</given-names></name><name><surname>Giannou</surname><given-names>AD</given-names></name><name><surname>Kempski</surname><given-names>J</given-names></name><name><surname>Pelczar</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>IL-10 receptor signaling is essential for TR1 cell function <italic>in vivo</italic></article-title>. <source>J Immunol.</source> (<year>2017</year>) <volume>198</volume>:<fpage>1130</fpage>–<lpage>41</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.1601045</pub-id><pub-id pub-id-type=\"pmid\">28003377</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Oncol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Oncol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Oncol.</journal-id><journal-title-group><journal-title>Frontiers in Oncology</journal-title></journal-title-group><issn pub-type=\"epub\">2234-943X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850451</article-id><article-id pub-id-type=\"pmc\">PMC7431604</article-id><article-id pub-id-type=\"doi\">10.3389/fonc.2020.01398</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Oncology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Development and Validation of a Radiomics Nomogram Model for Predicting Postoperative Recurrence in Patients With Esophageal Squamous Cell Cancer Who Achieved pCR After Neoadjuvant Chemoradiotherapy Followed by Surgery</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Qiu</surname><given-names>Qingtao</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/667044/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Duan</surname><given-names>Jinghao</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/859238/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Deng</surname><given-names>Hongbin</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Han</surname><given-names>Zhujun</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Gu</surname><given-names>Jiabing</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/598493/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Yue</surname><given-names>Ning J.</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/171874/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Yin</surname><given-names>Yong</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/730363/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences</institution>, <addr-line>Jinan</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Medical Imaging Ultrasonography, Second Affiliated Hospital of Nanjing Medical University</institution>, <addr-line>Nanjing</addr-line>, <country>China</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Radiation Oncology, Yantai Yuhuangding Hospital</institution>, <addr-line>Yantai</addr-line>, <country>China</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Radiation Oncology, The Cancer Institute of New Jersey</institution>, <addr-line>New Brunswick, NJ</addr-line>, <country>United States</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Abhishek Ashok Solanki, Loyola University Chicago, United States</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Alexander F. I. Osman, Al-Neelain University, Sudan; Zhenyu Liu, Institute of Automation (CAS), China</p></fn><corresp id=\"c001\">*Correspondence: Yong Yin <email>yinyongsd@126.com</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Radiation Oncology, a section of the journal Frontiers in Oncology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>1398</elocation-id><history><date date-type=\"received\"><day>13</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>02</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Qiu, Duan, Deng, Han, Gu, Yue and Yin.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Qiu, Duan, Deng, Han, Gu, Yue and Yin</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p><bold>Background and purpose:</bold> Although patients with esophageal squamous cell carcinoma (ESCC) can achieve a pathological complete response (pCR) after neoadjuvant chemoradiotherapy (nCRT) followed by surgery, one-third of these patients with a pCR may still experience recurrence. The aim of this study is to develop and validate a predictive model to estimate recurrence-free survival (RFS) in those patients who achieved pCR.</p><p><bold>Materials and methods:</bold> Two hundred six patients with ESCC were enrolled and divided into a training cohort (<italic>n</italic> = 146) and a validation cohort (<italic>n</italic> = 60). Radiomic features were extracted from contrast-enhanced computed tomography (CT) images of each patient. Feature reduction was then implemented in two steps, including a multiple segmentation test and least absolute shrinkage and selection operator (LASSO) Cox proportional hazards regression method. A radiomics signature was subsequently constructed and evaluated. For better prediction performance, a clinical nomogram based on clinical risk factors and a nomogram incorporating the radiomics signature and clinical risk factors was built. Finally, the prediction models were further validated by calibration and the clinical usefulness was examined in the validation cohort to determine the optimal prediction model.</p><p><bold>Results:</bold> The radiomics signature was constructed using eight radiomic features and displayed a significant correlation with RFS. The nomogram incorporating the radiomics signature with clinical risk factors achieved optimal performance compared with the radiomics signature (<italic>P</italic> < 0.001) and clinical nomogram (<italic>P</italic> < 0.001) in both the training cohort [C-index (95% confidence interval [CI]), 0.746 (0.680–0.812) vs. 0.685 (0.620–0.750) vs. 0.614 (0.538–0.690), respectively] and validation cohort [C-index (95% CI), 0.724 (0.696–0.752) vs. 0.671 (0.624–0.718) vs. 0.629 (0.597–0.661), respectively]. The calibration curve and decision curve analysis revealed that the radiomics nomogram outperformed the other two models.</p><p><bold>Conclusions:</bold> A radiomics nomogram model incorporating radiomics features and clinical factors has been developed and has the improved ability to predict the postoperative recurrence risk in patients with ESCC who achieved pCR after nCRT followed by surgery.</p></abstract><kwd-group><kwd>esophageal squamous cell carcinoma</kwd><kwd>neoadjuvant chemoradiotherapy</kwd><kwd>radiomics</kwd><kwd>pathological complete response</kwd><kwd>recurrence</kwd></kwd-group><counts><fig-count count=\"5\"/><table-count count=\"2\"/><equation-count count=\"1\"/><ref-count count=\"40\"/><page-count count=\"10\"/><word-count count=\"6130\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Esophageal cancer (EC) is the fourth most prevalent cancer in China, has a poor prognosis and is the sixth leading cause of death worldwide (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B2\" ref-type=\"bibr\">2</xref>). Esophageal squamous cell carcinoma (ESCC) is the most common subtype of esophageal malignancy and the cause of the highest morbidity in China compared to other developing countries (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B4\" ref-type=\"bibr\">4</xref>).</p><p>Despite multidisciplinary advances in EC treatment, surgery is still the curative strategy of choice. However, neoadjuvant chemotherapy (nCT) or neoadjuvant chemoradiotherapy (nCRT) followed by surgery has been shown to achieve a better outcome for patients with EC compared with surgery alone (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>, <xref rid=\"B6\" ref-type=\"bibr\">6</xref>), and is related to improvements in overall survival and disease-free survival. Notably, 15–30% of all patients with EC treated with nCRT followed by surgery achieve a pathological complete response (pCR) (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>–<xref rid=\"B9\" ref-type=\"bibr\">9</xref>) and ~45% of patients with ESCC achieve pCR (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B11\" ref-type=\"bibr\">11</xref>), where pCR is defined as no histological evidence of the tumor in the surgical specimen. However, approximately one-third of those patients achieving pCR still experience recurrence within 2 years after treatment (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Once recurrence occurs, the patient's prognosis is usually poor, with a reported survival time of 3–10 months (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Therefore, the ability to predict the likelihood of recurrence in patients with EC who have achieved pCR is important. It is a very essential way to ensure that an appropriately tailored treatment strategy is implemented early in the cohort of patient with a high risk of recurrence.</p><p>Many studies have been focused on predicting the pCR (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>–<xref rid=\"B16\" ref-type=\"bibr\">16</xref>), but few studies have investigated the prediction of recurrence in patients achieving pCR. Barbetta et al. developed a multivariate competing risk regression model based on clinical and pathological factors and determined that poor tumor differentiation is an independent risk factor predicting recurrence in patients with EC who achieved pCR after undergoing neoadjuvant therapy plus surgical resection (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). On the other hand, as a fundamental component of clinical oncology, medical imaging, including computed tomography (CT), plays a vital role in monitoring treatment outcomes (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>) and can provide a good description of EC tumors (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). Moreover, an image processing technology called radiomics converts medical images into mineable high-throughput data and may potentially improve the diagnostic, prognostic, and predictive accuracy (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Radiomics models have been shown to exhibit higher performance than conventional clinical models in predicting treatment outcomes (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>–<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). However, to the best of our knowledge, no previous radiomic studies have been focused on and conducted to predict recurrence in patients with ESCC who achieved pCR. Thus, a reasonable hypothesis is that radiomics may play an important role in predicting the recurrence risk in patients who achieved pCR. In the present study, we aimed to develop and validate a radiomics signature-based model using the pretreatment CT images to estimate recurrence-free survival (RFS) in patients with ESCC who achieved pCR after receiving nCRT followed by surgery.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Study Design</title><p>The overall research workflow is depicted in <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>, and a detailed description is provided in the <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Methods</xref>.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Workflow of this study.</p></caption><graphic xlink:href=\"fonc-10-01398-g0001\"/></fig></sec><sec><title>Patients</title><p>Based on the pathological data derived from surgical specimens, 303 consecutive patients who achieved pCR at the Shandong Cancer Hospital and Institute between April 2015 and October 2017 were selected for this study. Ethical approval of this study was obtained from the Institutional Review Board at Shandong Cancer Hospital and Institute, and the need for informed consent was waived because this study employed a retrospective design. The inclusion and exclusion criteria are described in <xref ref-type=\"supplementary-material\" rid=\"SM1\">Figure S1</xref>. The enrolled patients were divided into a training cohort and a validation cohort with the cutoff date of November 1, 2016.</p><p>Clinical factors, including gender, age, <italic>T</italic> and <italic>N</italic> stages, tumor location, pathological differentiation derived from medical records before nCRT, and the length of resected esophagus measured after surgery, were recorded.</p></sec><sec><title>CT Image Acquisition</title><p>CT images captured before nCRT were collected for all patients. All patients underwent standard chest contrast-enhanced CT scanning with a Philips CT scanner (Brilliance iCT 128, Philips Medical System, the Netherlands). The scanning protocol was a 120 kV tube voltage, 406 mA tube current, 5 mm slice thickness, 0.8984 × 0.8984 mm/pixel in-plane resolution, and helical scanning mode. In this study, enhanced CT images were used for tumor delineation and feature extraction because of the well-differentiated tumor borders.</p></sec><sec><title>Tumor Segmentation</title><p>Several radiation oncologists with over 5 years of professional experience manually delineated the gross tumor volumes (GTVs). The contoured GTVs were cross-checked slice-by-slice by different experienced radiation oncologists. Since manual delineation is prone to interobserver variability, an independent radiation oncologist delineated the GTVs for 50 randomly selected patients with ESCC to evaluate and confirm the reproducibility of the radiomic features and to reduce the effect of the uncertainty in the manual tumor delineation on feature extraction. All delineation tasks were performed in on a MIM Maestro Workstation (version 6.8.2, MIM Software Inc., USA).</p></sec><sec><title>Radiomic Feature Extraction and Selection</title><p>Radiomic features were automatically extracted using the SlicerRadomics extension in 3D Slicer (version 4.8.1, <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.slicer.org\">http://www.slicer.org</ext-link>, USA), an open source, easy-to-use medical image analysis software, from each contoured GTV (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Seven hundred eleven radiomic features were extracted, and the detailed descriptions are reported in the <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Methods</xref>.</p><p>Two steps were included in feature selection. First, the intraclass correlation coefficient (ICC) (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B26\" ref-type=\"bibr\">26</xref>) was calculated to quantify the reproducibility of features extracted from 50 randomly selected patients by two oncologists and to acquire robust radiomic features that were not affected by the variability in tumor segmentation. Second, the most useful predictive features based on the reproducible features identified in the previous step were selected using the least absolute shrinkage and selection operator (LASSO) Cox regression model (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B28\" ref-type=\"bibr\">28</xref>), which is used to reduce high-dimensional data. Ten-fold cross-validation was used in the parameter tuning phase of the LASSO algorithm to extract the effective and predictive features (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>).</p></sec><sec><title>Radiomics Signature and Nomogram Construction</title><p>After feature selection, a radiomics signature, also known as radiomics score or rad-score, was established from a linear combination of features and corresponding weights.</p><p>For visualization, a multivariate Cox proportional hazards model was utilized to build a clinical and a radiomics nomogram. The factors included in the clinical nomogram were gender, age, <italic>T</italic> stage, <italic>N</italic> stage, length of resection, tumor location, and pathological differentiation. A radiomics nomogram was constructed with the addition of the radiomics signature to the afore mentioned conventional clinical factors to ascertain the model with the optimal predictive performance.</p></sec><sec><title>Validation of the Radiomics Signature and Nomograms</title><p>The correlation between the radiomics signature and RFS was first evaluated using the Kaplan-Meier survival analysis in the training cohort and then validated in the validation cohort. A threshold or cutoff point was generated using X-tile software (version 3.6.1) (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Subsequently, the patients with radiomics signature values greater than the threshold were allocated into a high-risk group and patients with values less than the threshold were allocated into the low-risk group. The log-rank test was used to measure the difference in survival curves of the low-risk and high-risk groups. The discrimination power, which is defined as the agreement between the predicted and actual RFS probability, and the clinical usefulness that quantifies the net benefits at different threshold probabilities were used to evaluate the performance of radiomics signature and nomograms. In this study, the discrimination power was evaluated with Harrell's concordance index (C-index) in both the training and validation cohorts (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). In addition, we used a calibration curve to intuitively assess the predictive accuracy and the agreement between the actual RFS and the RFS predicted by the nomograms (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). The calibration curve shows the actual RFS (ordinate) and the predicted RFS (abscissa) in a two-dimensional coordinate system. Then, we used the Akaike information criterion (AIC) to assess the risk of overfitting. A smaller AIC value indicates a better fit of the model. Finally, a decision curve, which quantified the net benefits at a threshold ranging from 0 to 1 in the validation cohort, was plotted to determine the clinical usefulness of the nomogram (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). Higher clinical utility is observed the farther away the decision curve is from the two extreme curves (treat-all and treat-none).</p></sec><sec><title>Statistical Analysis</title><p>Statistical analyses were performed in R (version 3.6.1, <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.r-project.org\">http://www.r-project.org</ext-link>), an open source programming language and software environment for statistical computing and graphics. The packages in R used in this study are listed in <xref ref-type=\"supplementary-material\" rid=\"SM4\">Table S1</xref>. Comparisons of patient characteristics were performed using the Mann–Whitney <italic>U</italic>-test or two-sample <italic>t</italic>-test, as appropriate. An ICC value > 0.95 indicated strong reproducibility (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). The reported statistical significance levels were all two-sided. The statistical significance level was set to 0.05.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>Patients' Clinical Characteristics</title><p>The clinical characteristics of the patients in the two cohorts are summarized in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> and showed consistent demographic distributions. In the present study, 146 and 60 patients with ESCC were enrolled in the training and validation cohorts, respectively. No significant differences were observed in the two cohorts, with <italic>P</italic>-values ranging from 0.386 to 0.709. The RFS times and recurrence status of the enrolled patients were determined based on the follow-up information.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Demographic and clinical characteristics of patients with ESCC in the training cohort and validation cohort.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Characteristic</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Training cohort</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Validation cohort</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Gender</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Male</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">111 (76.0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">45 (75.0)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Female</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35 (24.0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15 (25.0)</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Age (years)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Mean</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60.83</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">59.98</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Range</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">43–78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">38–76</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><italic><bold>T</bold></italic>\n<bold>stage</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   T1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30 (20.5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12 (20.0)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   T2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23 (15.8)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8 (13.3)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   T3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">86 (58.9)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">37 (61.7)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   T4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7 (4.8)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3 (5.0)</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><italic><bold>N</bold></italic>\n<bold>stage</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   N0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">96 (65.8)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">36 (60.0)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   N1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">36 (24.7)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21 (35.0)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   N2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9 (6.2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (3.3)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   N3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5 (3.4)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (1.7)</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Length of Resection</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   ≤5 cm</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">102 (69.9)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40 (66.7)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   5–10 cm</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">42 (28.8)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19 (31.7)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   ≥10 cm</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (1.4)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (1.7)</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Location</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Lower</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35 (24.0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9 (15.0)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Middle</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">103 (70.5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">47 (78.3)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Upper</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8 (5.5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (6.7)</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Pathological Differentiation</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">41 (28.1)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12 (20)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Middle</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">68 (46.6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31 (51.7)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   High</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">37 (25.3)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17 (28.3)</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"3\" rowspan=\"1\"><bold>Follow-Up Time (Months)</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Median</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13.5</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Range</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1–38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2–35</td></tr></tbody></table><table-wrap-foot><p><italic>All the data except Age in above table are numbers of patients, with percentages in parentheses. No difference was found between training cohort and validation cohort in either the clinical characteristics or recurrence status (p = 0.386–0.709). ESCC, esophageal squamous cell carcinoma</italic>.</p></table-wrap-foot></table-wrap></sec><sec><title>Results of the Radiomic Feature Selection</title><p>In the first step of the reproducible feature selection, 478 of the 711 extracted radiomic features had an ICC value > 0.95, indicating the high reproducibility among multiple segmentations. The excluded and selected radiomic features are presented in <xref ref-type=\"supplementary-material\" rid=\"SM5\">Table S2</xref>.</p><p>In the second step of the predictive feature selection, eight radiomic features with non-zero coefficients were selected from the 478 reproducible features based on the LASSO Cox regression model for the survival analysis. The parameter tuning phase of the regression model and the feature space reduction are depicted in <xref ref-type=\"supplementary-material\" rid=\"SM2\">Figure S2</xref>. The selected features with corresponding coefficients and ICC values are listed in <xref ref-type=\"supplementary-material\" rid=\"SM6\">Table S3</xref>.</p></sec><sec><title>Radiomics Signature Construction and Validation Results</title><p>The radiomics signature was constructed using the following formula:</p><disp-formula id=\"E1\"><mml:math id=\"M1\"><mml:mtable columnalign=\"left\"><mml:mtr><mml:mtd><mml:mtext>Radiomics Signature</mml:mtext><mml:mo>=</mml:mo><mml:mo>−</mml:mo><mml:mtext>Original</mml:mtext><mml:mo>_</mml:mo><mml:mtext>GLSZM</mml:mtext><mml:mo>_</mml:mo><mml:mtext>SZNUN</mml:mtext><mml:mo>×</mml:mo><mml:mtext>1</mml:mtext><mml:mo>.</mml:mo><mml:mn>2200</mml:mn></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mtext>                             </mml:mtext><mml:mo>+</mml:mo><mml:mtext>Wavelet</mml:mtext><mml:mo>_</mml:mo><mml:mtext>LHL</mml:mtext><mml:mo>_</mml:mo><mml:mtext>FirOrd</mml:mtext><mml:mo>_</mml:mo><mml:mtext>Skewness</mml:mtext><mml:mo>×</mml:mo><mml:mn>0</mml:mn><mml:mo>.</mml:mo><mml:mn>0358</mml:mn></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mtext>                             </mml:mtext><mml:mo>+</mml:mo><mml:mtext>Wavelet</mml:mtext><mml:mo>_</mml:mo><mml:mtext>LHH</mml:mtext><mml:mo>_</mml:mo><mml:mtext>GLSZM</mml:mtext><mml:mo>_</mml:mo><mml:mtext>SAE</mml:mtext><mml:mo>×</mml:mo><mml:mn>0</mml:mn><mml:mo>.</mml:mo><mml:mn>9097</mml:mn></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mtext>                             </mml:mtext><mml:mo>+</mml:mo><mml:mtext>Wavelet</mml:mtext><mml:mo>_</mml:mo><mml:mtext>HHH</mml:mtext><mml:mo>_</mml:mo><mml:mtext>GLSZM</mml:mtext><mml:mo>_</mml:mo><mml:mtext>LGLZE</mml:mtext><mml:mo>×</mml:mo><mml:mn>0</mml:mn><mml:mo>.</mml:mo><mml:mn>6696</mml:mn></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mtext>                             </mml:mtext><mml:mo>+</mml:mo><mml:mtext>Wavelet</mml:mtext><mml:mo>_</mml:mo><mml:mtext>HHL</mml:mtext><mml:mo>_</mml:mo><mml:mtext>GLCM</mml:mtext><mml:mo>_</mml:mo><mml:mtext>MCC</mml:mtext><mml:mo>×</mml:mo><mml:mn>0</mml:mn><mml:mo>.</mml:mo><mml:mn>7100</mml:mn></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mtext>                             </mml:mtext><mml:mo>−</mml:mo><mml:mtext>Wavelet</mml:mtext><mml:mo>_</mml:mo><mml:mtext>HHL</mml:mtext><mml:mo>_</mml:mo><mml:mtext>GLRLM</mml:mtext><mml:mo>_</mml:mo><mml:mtext>SRLGLE</mml:mtext><mml:mo>×</mml:mo><mml:mn>0</mml:mn><mml:mo>.</mml:mo><mml:mn>0288</mml:mn></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mn>                             </mml:mn><mml:mo>−</mml:mo><mml:mtext>Wavelet</mml:mtext><mml:mo>_</mml:mo><mml:mtext>HHL</mml:mtext><mml:mo>_</mml:mo><mml:mtext>GLSZM</mml:mtext><mml:mo>_</mml:mo><mml:mtext>LALGLE</mml:mtext><mml:mo>×</mml:mo><mml:mn>0</mml:mn><mml:mo>.</mml:mo><mml:mn>5297</mml:mn></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mtext>                             </mml:mtext><mml:mo>+</mml:mo><mml:mtext>Wavelet</mml:mtext><mml:mo>_</mml:mo><mml:mtext>LLL</mml:mtext><mml:mo>_</mml:mo><mml:mtext>GLSZM</mml:mtext><mml:mo>_</mml:mo><mml:mtext>ZoneVar</mml:mtext><mml:mo>×</mml:mo><mml:mtext> </mml:mtext><mml:mn>0</mml:mn><mml:mo>.</mml:mo><mml:mn>2152</mml:mn></mml:mtd></mml:mtr></mml:mtable></mml:math></disp-formula><p>The optimal cutoff point of 0.52 was generated from the X-tile plot shown in <xref ref-type=\"supplementary-material\" rid=\"SM3\">Figure S3</xref> to identify the low-risk and high-risk subgroups. Therefore, patients with were divided into a low-risk group (radiomics signature ≤ 0.52) and high-risk group (radiomics signature > 0.52). The distributions of radiomics signature values calculated from the training and validation cohorts are presented in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>. The Kaplan-Meier plots of the low-risk and high-risk groups in both the training and validation cohorts are illustrated in <xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>. In the training cohort, the radiomics signature was significantly associated with RFS (<italic>P</italic> < 0.0001; hazard ratio [HR], 2.479; 95% confidence interval [CI], 1.458–4.251). Then, this finding was confirmed in the validation cohort (<italic>P</italic> < 0.0001; HR, 3.606; 95% CI, 1.742–7.464). The mean RFS times of the low-risk and high-risk groups were 21.24 and 13.74 months in the training cohort, and 21.13 and 12.27 months in the validation cohort, respectively.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Bar plot of the radiomics signature value for each patient in the training cohort <bold>(A)</bold> and the validation cohort <bold>(B)</bold>. Patients with radiomics signature values >0.52 were allocated into the high-risk group, while patients with values ≤0.52 were allocated into the low-risk group. Recurrent and censored patients were marked with a different color.</p></caption><graphic xlink:href=\"fonc-10-01398-g0002\"/></fig><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Kaplan-Meier survival analyses of high-risk and low-risk groups divided by radiomics signature in training cohort <bold>(A)</bold> and validation cohort <bold>(B)</bold>. Significant differences were observed in both training cohort (log-rank test <italic>P</italic> < 0.0001) and validation cohort (log-rank test <italic>P</italic> < 0.0001). It indicates that radiomics signature significantly associated with RFS. Dashed line in the two-sided CI of survival curves.</p></caption><graphic xlink:href=\"fonc-10-01398-g0003\"/></fig></sec><sec><title>Nomogram Construction and Validation Results</title><p>Combined with the conventional clinical factors, radiomics signature and Cox proportional hazards model, a clinical nomogram and radiomics nomogram were built, as presented in <xref ref-type=\"fig\" rid=\"F4\">Figures 4A,B</xref>. The corresponding calibration curve of these two nomograms for the probability of recurrence-free survival at 1 and 2 years after surgery are depicted in <xref ref-type=\"fig\" rid=\"F4\">Figures 4C,D</xref>, respectively. The calibration curve showed better agreement and goodness-of-fit between the RFS predicted by the radiomics nomogram and actual RFS probability for both 1 and 2-years RFS.</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Nomograms developed using the training cohort. <bold>(A)</bold> The nomogram incorporates clinical risk factors. <bold>(B)</bold> The nomogram incorporates the radiomics signature and clinical risk factors. Calibration curves of the clinical nomogram <bold>(C)</bold> and radiomics nomogram <bold>(D)</bold> were plotted to assess the agreement between RFS probability predicted by the nomogram and the observed RFS.</p></caption><graphic xlink:href=\"fonc-10-01398-g0004\"/></fig><p>The C-index with 95% CI and AIC estimates for the different models, including radiomics signature, clinical nomogram, and radiomics nomogram, were calculated and listed in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>. The radiomics nomogram yielded an optimal C-index value of 0.746 (95% CI, 0.680–0.812) in the training cohort and 0.724 (95% CI, 0.696–0.752) in the validation cohort. The discrimination performance of the radiomics signature increased significantly when the radiomics signature was integrated with clinical risk factors compared with each feature set alone (<italic>P</italic> < 0.0001 for each comparison). Additionally, among all the three prediction models, the radiomics nomogram yielded the lowest AIC value in both the training (582.843) and validation (603.927) cohorts. A decision curve was further plotted in <xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>, and it showed that the radiomics nomogram produced a greater net benefit than the clinical nomograms and the radiomics signature.</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Comparison of the discriminating performance of the radiomics signature, clinical nomogram, and radiomics nomogram.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Model</bold></th><th valign=\"top\" align=\"center\" colspan=\"4\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Training cohort</bold></th><th valign=\"top\" align=\"center\" colspan=\"4\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Validation cohort</bold></th></tr><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>C-index</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>95% CI</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>p</italic>-value</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>AIC</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>C-index</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>95% CI</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>p</italic>-value</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>AIC</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Radiomics signature</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.685</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.620–0.750</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>Ψ</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">596.615</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.671</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.624–0.718</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>Ψ</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">609.273</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Clinical nomogram</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.614</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.538–0.690</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>ζ</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">614.049</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.629</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.597–0.661</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>ζ</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">635.411</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Radiomics nomogram</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.746</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.680–0.812</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>§</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">582.043</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.724</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.696–0.752</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>§</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">603.927</td></tr></tbody></table><table-wrap-foot><fn id=\"TN1\"><label>Ψ</label><p><italic>The comparison of CI between radiomics signature and clinical nomogram</italic>.</p></fn><fn id=\"TN2\"><label>ζ</label><p><italic>The comparison of CI between clinical nomogram and Radiomics nomogra</italic>.</p></fn><fn id=\"TN3\"><label>§</label><p><italic>The comparison of CI between radiomics nomogram and radiomics signature</italic>.</p></fn></table-wrap-foot></table-wrap><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>Decision curves of the three models and two extreme curves were plotted based on the validation cohort. When considering the decision curve drawn for different thresholds of probability, patients will add more net benefits when the threshold probability is >0.10. Therefore, the use of the radiomics nomogram to predict recurrence in patients who achieved pCR has a greater benefit than the use of the radiomics signature and clinical nomogram.</p></caption><graphic xlink:href=\"fonc-10-01398-g0005\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>We developed three models for predicting the recurrence risk in patients with ESCC who had achieved pCR after treatment with nCRT followed by surgery. Among the three models, the radiomics nomogram had the best discrimination power and was further confirmed to exhibit superior calibration and clinical utility. Although the radiomics signature displayed a significant correlation with the RFS times, we assumed that it will achieve better performance when combined with other predictors; this hypothesis was verified. The radiomics nomogram model incorporating the radiomics signature and clinical risk factors exhibited a higher predictive power for predicting RFS at the 1 and 2-years time points. The radiomics nomogram was useful to clinical physicians for an early evaluation of long-term outcomes.</p><p>The constructed radiomics signature consisted of eight features, and nearly all of the selected predictive features were wavelet-based features, similar to the results of several other studies (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>–<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). The potential explanation is that the multifrequency decomposition of the original CT image provided useful information about tumor heterogeneity for evaluating treatment outcomes. In addition, the eight features and 146 patients are in the proper proportion and number for the construction of the radiomics signature, according to the description of overfitting of the prediction model in a previous study (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). Therefore, the constructed signature efficiently stratified those patients into low-risk and high-risk groups.</p><p>The radiomics signature model incorporated some individual radiomic features as predictors to explore the clinical utility of features that have been explored and investigated in many studies (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B28\" ref-type=\"bibr\">28</xref>, <xref rid=\"B32\" ref-type=\"bibr\">32</xref>–<xref rid=\"B34\" ref-type=\"bibr\">34</xref>); notably, radiomics signatures constructed from 24 to 14 features were used to preoperatively predict the lymph node metastasis in patients with rectal cancer (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>) and EC (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>), respectively. Similarly, in the current study, a high discrimination of the radiomics signature model was observed in both the training cohort (C-index, 0.685; 95% CI, 0.620–0.750) and validation cohort (C-index, 0.671; 95% CI, 0.624–0.718). Moreover, the distribution of radiomics signature in both the training and validation cohorts confirmed its stability. In a recent study, poor tumor differentiation (HR, 2.28; <italic>P</italic> = 0.022) and an advanced clinical stage (HR, 1.89; <italic>P</italic> = 0.042) were predictors of recurrence in the esophageal adenocarcinoma subgroup, and poor tumor differentiation was the only risk factor predicting recurrence (HR, 2.28, <italic>P</italic> = 0.009) in the total cohort of patients with EC achieving pCR after nCRT and surgery (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). In our study, the HR of the high radiomics signature value (training cohort: HR = 2.479, <italic>P</italic> < 0.0001; validation cohort: HR = 3.606, <italic>P</italic> < 0.0001) was sufficient to confirm that it is an independent predictor of RFS. Because tumor differentiation and clinical stages are potential predictors of recurrence, we must consider whether clinical factors achieve more accurate predictions of recurrence than the radiomics signature. Thus, we developed a clinical nomogram that incorporates clinical factors, including gender, age, <italic>T</italic> stage, <italic>N</italic> stage, tumor location, length of resection, and tumor differentiation. These clinical factors are generally readily available during treatment and the collection of this information does not increase the burden on patients as a result of the additional examinations. Compared with the radiomics signature model, tumor differentiation is not a dominant variable in the clinical nomogram, as shown in <xref ref-type=\"fig\" rid=\"F4\">Figures 4A,B</xref>. It may be caused by the nuances in the dataset or confounding by other factors in the process of model development (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). Although the clinical prediction model we established achieved acceptable discrimination in both the training cohort (C-index, 0.614, 95% CI, 0.538–0.690) and validation cohort (C-index, 0.629, 95% CI, 0.597–0.661), the C-index values of the model were still lower than the radiomics signature model, and the statistically significant differences were observed in both the training cohort (<italic>P</italic> < 0.001) and the validation cohort (<italic>P</italic> < 0.001). The possible interpretation is that the information dimension from the limited clinical factors was lower than the high-dimensional feature space mined from medical images for reflecting the spatial heterogeneity of tumors. If insufficient information is used to develop a prediction model, a low-discrimination model will very likely be the result.</p><p>Many studies have reported improvements in the predictive accuracy in models combining radiomic features or signatures with clinical risk factors (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>, <xref rid=\"B36\" ref-type=\"bibr\">36</xref>–<xref rid=\"B38\" ref-type=\"bibr\">38</xref>). For example, the nomogram models developed by combining radiomic signatures and clinical factors have shown outstanding performance in the prediction of cognitive impairment (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>) and in the differentiation of renal angiomyolipoma (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>). This strategy was adopted in the present study and a radiomics nomogram was developed by incorporating the radiomics signature into the clinical nomogram. The developed radiomics nomogram achieved remarkable discrimination power in both the training cohort (C-index, 0.746, 95% CI, 0.680–0.812) and validation cohort (C-index, 0.724, 95% CI, 0.696–0.752). The discrimination power of the radiomics nomogram outperformed both the radiomics signature and clinical nomogram as individual predictors (<italic>P</italic> < 0.001). Our finding supports the hypothesis that a more holistic model is obtained when non-radiomic features (such as clinical factors) are incorporated, which is described in criterion six of the radiomics quality score (RQS) proposed by Lambin (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>), the founder of the concept of radiomics (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>). Our results also confirmed that this hypothesis is feasible with the optimal discrimination model obtained from the nomogram combining the radiomics signature and clinical factors. The radiomics nomogram is an easy-to-use scoring model with the ability to assess the recurrence risk of individual patients. The generated calibration curves and AICs were used to address the important and final arguments for the utilization of the nomogram. The developed radiomics nomogram model is useful to predict the probability of recurrence for an individual patient and can be used in postoperative assessments of the individual recurrence risk in patients achieving pCR. Furthermore, a decision curve analysis was conducted to reduce the bias caused by the clinical consequences of a particular level of discrimination or degree of miscalibration in the discrimination and calibration curve (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). The decision curves generated from the validation cohort revealed that the radiomics nomogram is potentially advantageous in predicting the recurrence risk in patients achieving pCR compared with the other two models presented in this study.</p><p>Our study has a few limitations. First, other imaging modalities that are commonly available in clinical practice, such as positron emission tomography (PET) and magnetic resonance imaging (MRI), were not included in this study, and further studies are needed to determine whether the developed model is suitable for those imaging modalities. Second, a subgroup analysis based on disease stage was not conducted in this study due to the limited cohort size. In addition, the use of nCRT as new treatment strategy has achieved encouraging long-term outcomes. The survival analysis of patients treated with nCRT was not included in this study because it has only been adopted and implemented in our hospital for a few years and an inadequate number of patients was available for this type of study. Our future studies will focus on subgroup analyses of survival and the risk of recurrence, as well as the utilization of radiomics to predict clinical outcomes of nCRT in those subgroups.</p></sec><sec sec-type=\"conclusions\" id=\"s5\"><title>Conclusion</title><p>We have developed and validated a radiomics nomogram model that incorporates both radiomics signatures and clinical factors to predict the postoperative ESCC recurrence risk of patients who achieved pCR after nCRT followed by surgery. As a holistic predictive model, the radiomics nomogram may serve as a powerful tool in the evaluation of clinical outcomes in those patients with ESCC.</p></sec><sec sec-type=\"data-availability\" id=\"s6\"><title>Data Availability Statement</title><p>The original contributions presented in the study are included in the article and <xref ref-type=\"sec\" rid=\"s10\">Supplementary Material</xref>, further inquiries can be directed to the corresponding author.</p></sec><sec id=\"s7\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by the Institutional Review Board at Shandong Cancer Hospital and Institute. Written informed consent was not required, due to the retrospective nature of the study.</p></sec><sec id=\"s8\"><title>Author Contributions</title><p>QQ, JD, and YY: conceptualization. QQ and JD: methodology. QQ, JD, and JG: software, data curation, and visualization. QQ, HD, and ZH: investigation. YY: supervision and project administration. JD and YY: funding acquisition. QQ, JD, HD, ZH, JG, NY, and YY: writing-original draft, writing-review, and editing. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s9\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This study was supported by the Key Support Program of Natural Science Foundation of Shandong Province (Grant No. ZR2019LZL017), the Taishan Scholars Project of Shandong Province (Grant No. ts201712098), the National Key Research and Development Program of China (Grant No. 2017YFC0113202), the National Natural Science Foundation of China (Grant No. 81901743), and the Key Research and Development Plan of Shandong Province (Grant Nos. 2018GSF118006 and 2019GSF108134).</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s10\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fonc.2020.01398/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fonc.2020.01398/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><label>Figure S1</label><caption><p>Flowchart of enrolled ESCC patients in this study. CT, computed tomography. ESCC, esophageal squamous cell carcinoma. pCR, pathologic complete response. pCR patient, patients who achieved pCR after treatment with nCRT followed by surgery.</p></caption><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM2\"><label>Figure S2</label><caption><p>Feature selection using the least absolute shrinkage and selection operator (LASSO) with a Cox regression model.</p></caption><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM3\"><label>Figure S3</label><caption><p>X-tile plot of the radiomics signature in the training data set.</p></caption><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM4\"><label>Table S1</label><caption><p>Packages in R used in this study.</p></caption><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM5\"><label>Table S2</label><caption><p>Radiomic features reduction after multiple segmentation test and least absolute shrinkage and selection operator (LASSO) method in each category.</p></caption><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM6\"><label>Table S3</label><caption><p>Selected radiomics features and the corresponding coefficient and ICC values.</p></caption><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>W</given-names></name><name><surname>Zheng</surname><given-names>R</given-names></name><name><surname>Baade</surname><given-names>PD</given-names></name><name><surname>Zhang</surname><given-names>S</given-names></name><name><surname>Zeng</surname><given-names>H</given-names></name><name><surname>Bray</surname><given-names>F</given-names></name><etal/></person-group>\n<article-title>Cancer statistics in China, 2015</article-title>. <source>CA Cancer J Clin.</source> (<year>2016</year>) <volume>66</volume>:<fpage>115</fpage>–<lpage>32</lpage>. <pub-id pub-id-type=\"doi\">10.3322/caac.21338</pub-id><pub-id pub-id-type=\"pmid\">26808342</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Short</surname><given-names>MW</given-names></name><name><surname>Burgers</surname><given-names>KG</given-names></name><name><surname>Fry</surname><given-names>VT</given-names></name></person-group>. <article-title>Esophageal cancer</article-title>. <source>Am Family Physician.</source> (<year>2017</year>) <volume>95</volume>:<fpage>22</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"pmid\">28075104</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bray</surname><given-names>F</given-names></name><name><surname>Ferlay</surname><given-names>J</given-names></name><name><surname>Soerjomataram</surname><given-names>I</given-names></name><name><surname>Siegel</surname><given-names>RL</given-names></name><name><surname>Torre</surname><given-names>LA</given-names></name><name><surname>Jemal</surname><given-names>A</given-names></name></person-group>. <article-title>Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries</article-title>. <source>CA Cancer J Clin.</source> (<year>2018</year>) <volume>68</volume>:<fpage>394</fpage>–<lpage>424</lpage>. <pub-id pub-id-type=\"doi\">10.3322/caac.21492</pub-id><pub-id pub-id-type=\"pmid\">30207593</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Di Pardo</surname><given-names>BJ</given-names></name><name><surname>Bronson</surname><given-names>NW</given-names></name><name><surname>Diggs</surname><given-names>BS</given-names></name><name><surname>Thomas</surname><given-names>CR</given-names><suffix>Jr</suffix></name><name><surname>Hunter</surname><given-names>JG</given-names></name><name><surname>Dolan</surname><given-names>JP</given-names></name></person-group>. <article-title>The global burden of esophageal cancer: a disability-adjusted life-year approach</article-title>. <source>World J Surg.</source> (<year>2016</year>) <volume>40</volume>:<fpage>395</fpage>–<lpage>401</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00268-015-3356-2</pub-id><pub-id pub-id-type=\"pmid\">26630937</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ronellenfitsch</surname><given-names>U</given-names></name><name><surname>Schwarzbach</surname><given-names>M</given-names></name><name><surname>Hofheinz</surname><given-names>R</given-names></name><name><surname>Kienle</surname><given-names>P</given-names></name><name><surname>Kieser</surname><given-names>M</given-names></name><name><surname>Slanger</surname><given-names>TE</given-names></name><etal/></person-group>. <article-title>Preoperative chemo(radio)therapy versus primary surgery for gastroesophageal adenocarcinoma: systematic review with meta-analysis combining individual patient and aggregate data</article-title>. <source>Eur J Cancer</source>. (<year>2013</year>) <volume>49</volume>:<fpage>3149</fpage>–<lpage>58</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ejca.2013.05.029</pub-id><pub-id pub-id-type=\"pmid\">23800671</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sunde</surname><given-names>B</given-names></name><name><surname>Klevebro</surname><given-names>F</given-names></name><name><surname>Johar</surname><given-names>A</given-names></name><name><surname>Johnsen</surname><given-names>G</given-names></name><name><surname>Jacobsen</surname><given-names>AB</given-names></name><name><surname>Glenjen</surname><given-names>NI</given-names></name><etal/></person-group>. <article-title>Health-related quality of life in a randomized trial of neoadjuvant chemotherapy or chemoradiotherapy plus surgery in patients with oesophageal cancer (NeoRes trial)</article-title>. <source>Br J Surg.</source> (<year>2019</year>) <volume>106</volume>:<fpage>1452</fpage>–<lpage>63</lpage>. <pub-id pub-id-type=\"doi\">10.1002/bjs.11246</pub-id><pub-id pub-id-type=\"pmid\">31436322</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van Hagen</surname><given-names>P</given-names></name><name><surname>Hulshof</surname><given-names>MC</given-names></name><name><surname>van Lanschot</surname><given-names>JJ</given-names></name><name><surname>Steyerberg</surname><given-names>EW</given-names></name><name><surname>van Berge Henegouwen</surname><given-names>MI</given-names></name><name><surname>Wijnhoven</surname><given-names>BP</given-names></name><etal/></person-group>\n<article-title>Preoperative chemoradiotherapy for esophageal or junctional cancer</article-title>. <source>N Engl J Med.</source> (<year>2012</year>) <volume>366</volume>:<fpage>2074</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.1056/NEJMoa1112088</pub-id><pub-id pub-id-type=\"pmid\">22646630</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mariette</surname><given-names>C</given-names></name><name><surname>Dahan</surname><given-names>L</given-names></name><name><surname>Mornex</surname><given-names>F</given-names></name><name><surname>Maillard</surname><given-names>E</given-names></name><name><surname>Thomas</surname><given-names>PA</given-names></name><name><surname>Meunier</surname><given-names>B</given-names></name><etal/></person-group>\n<article-title>Surgery alone versus chemoradiotherapy followed by surgery for stage I and II esophageal cancer: final analysis of randomized controlled phase III trial FFCD 9901</article-title>. <source>J Clin Oncol.</source> (<year>2014</year>) <volume>32</volume>:<fpage>2416</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.1200/JCO.2013.53.6532</pub-id><pub-id pub-id-type=\"pmid\">24982463</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Donahue</surname><given-names>JM</given-names></name><name><surname>Nichols</surname><given-names>FC</given-names></name><name><surname>Li</surname><given-names>Z</given-names></name><name><surname>Schomas</surname><given-names>DA</given-names></name><name><surname>Allen</surname><given-names>MS</given-names></name><name><surname>Cassivi</surname><given-names>SD</given-names></name><etal/></person-group>. <article-title>Complete pathologic response after neoadjuvant chemoradiotherapy for esophageal cancer is associated with enhanced survival</article-title>. <source>Ann Thoracic Surg.</source> (<year>2009</year>) <volume>87</volume>:<fpage>392</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.athoracsur.2008.11.001</pub-id><pub-id pub-id-type=\"pmid\">19161745</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xi</surname><given-names>M</given-names></name><name><surname>Yang</surname><given-names>Y</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Yang</surname><given-names>H</given-names></name><name><surname>Merrell</surname><given-names>KW</given-names></name><name><surname>Hallemeier</surname><given-names>CL</given-names></name><etal/></person-group>. <article-title>Multi-institutional analysis of recurrence and survival after neoadjuvant chemoradiotherapy of esophageal cancer: impact of histology on recurrence patterns and outcomes</article-title>. <source>Ann Surg.</source> (<year>2019</year>) <volume>269</volume>:<fpage>663</fpage>–<lpage>70</lpage>. <pub-id pub-id-type=\"doi\">10.1097/SLA.0000000000002670</pub-id><pub-id pub-id-type=\"pmid\">29334555</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Barbetta</surname><given-names>A</given-names></name><name><surname>Sihag</surname><given-names>S</given-names></name><name><surname>Nobel</surname><given-names>T</given-names></name><name><surname>Hsu</surname><given-names>M</given-names></name><name><surname>Tan</surname><given-names>KS</given-names></name><name><surname>Bains</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Patterns and risk of recurrence in patients with esophageal cancer with a pathologic complete response after chemoradiotherapy followed by surgery</article-title>. <source>J Thoracic Cardiovasc Surg.</source> (<year>2018</year>) <volume>157</volume>:<fpage>1249</fpage>–<lpage>59</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jtcvs.2018.09.136</pub-id><pub-id pub-id-type=\"pmid\">30655068</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parry</surname><given-names>K</given-names></name><name><surname>Visser</surname><given-names>E</given-names></name><name><surname>van Rossum</surname><given-names>PS</given-names></name><name><surname>Mohammad</surname><given-names>NH</given-names></name><name><surname>Ruurda</surname><given-names>JP</given-names></name><name><surname>van Hillegersberg</surname><given-names>R</given-names></name></person-group>. <article-title>Prognosis and treatment after diagnosis of recurrent esophageal carcinoma following esophagectomy with curative intent</article-title>. <source>Ann Surg Oncol.</source> (<year>2015</year>) <volume>22</volume>(<issue>Suppl.3</issue>):<fpage>S1292</fpage>–<lpage>300</lpage>. <pub-id pub-id-type=\"doi\">10.1245/s10434-015-4840-5</pub-id><pub-id pub-id-type=\"pmid\">26334295</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>Z</given-names></name><name><surname>He</surname><given-names>B</given-names></name><name><surname>Zhuang</surname><given-names>X</given-names></name><name><surname>Gao</surname><given-names>X</given-names></name><name><surname>Wang</surname><given-names>D</given-names></name><name><surname>Li</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>CT-based radiomic signatures for prediction of pathologic complete response in esophageal squamous cell carcinoma after neoadjuvant chemoradiotherapy</article-title>. <source>J Radiat Res.</source> (<year>2019</year>) <volume>60</volume>:<fpage>538</fpage>–<lpage>45</lpage>. <pub-id pub-id-type=\"doi\">10.1093/jrr/rrz027</pub-id><pub-id pub-id-type=\"pmid\">31111948</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tan</surname><given-names>Z</given-names></name><name><surname>Yang</surname><given-names>H</given-names></name><name><surname>Wen</surname><given-names>J</given-names></name><name><surname>Luo</surname><given-names>K</given-names></name><name><surname>Liu</surname><given-names>Q</given-names></name><name><surname>Hu</surname><given-names>Y</given-names></name><etal/></person-group>. <article-title>Clinical predictors of pathologically response after neoadjuvant chemoradiotherapy for esophageal squamous cell carcinoma: long term outcomes of a phase II study</article-title>. <source>J Thoracic Dis.</source> (<year>2018</year>) <volume>10</volume>:<fpage>5254</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.21037/jtd.2018.08.88</pub-id><pub-id pub-id-type=\"pmid\">30416772</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hamai</surname><given-names>Y</given-names></name><name><surname>Hihara</surname><given-names>J</given-names></name><name><surname>Emi</surname><given-names>M</given-names></name><name><surname>Furukawa</surname><given-names>T</given-names></name><name><surname>Murakami</surname><given-names>Y</given-names></name><name><surname>Nishibuchi</surname><given-names>I</given-names></name><etal/></person-group>\n<article-title>Preoperative prediction of a pathologic complete response of esophageal squamous cell carcinoma to neoadjuvant chemoradiotherapy</article-title>. <source>Surgery.</source> (<year>2018</year>) <volume>164</volume>:<fpage>40</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.surg.2018.01.011</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>RW</given-names></name><name><surname>Chao</surname><given-names>YK</given-names></name><name><surname>Wen</surname><given-names>YW</given-names></name><name><surname>Chang</surname><given-names>HK</given-names></name><name><surname>Tseng</surname><given-names>CK</given-names></name><name><surname>Chan</surname><given-names>SC</given-names></name><etal/></person-group>\n<article-title>Predictors of pathological complete response to neoadjuvant chemoradiotherapy for esophageal squamous cell carcinoma</article-title>. <source>World J Surg Oncol.</source> (<year>2014</year>) <volume>12</volume>:<fpage>170</fpage>\n<pub-id pub-id-type=\"doi\">10.1186/1477-7819-12-170</pub-id><pub-id pub-id-type=\"pmid\">24885430</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Qiu</surname><given-names>Q</given-names></name><name><surname>Duan</surname><given-names>J</given-names></name><name><surname>Duan</surname><given-names>Z</given-names></name><name><surname>Meng</surname><given-names>X</given-names></name><name><surname>Ma</surname><given-names>C</given-names></name><name><surname>Zhu</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Reproducibility and non-redundancy of radiomic features extracted from arterial phase CT scans in hepatocellular carcinoma patients: impact of tumor segmentation variability</article-title>. <source>Quant Imag Med Surg.</source> (<year>2019</year>) <volume>9</volume>:<fpage>453</fpage>–<lpage>64</lpage>. <pub-id pub-id-type=\"doi\">10.21037/qims.2019.03.02</pub-id><pub-id pub-id-type=\"pmid\">31032192</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shen</surname><given-names>C</given-names></name><name><surname>Liu</surname><given-names>Z</given-names></name><name><surname>Wang</surname><given-names>Z</given-names></name><name><surname>Guo</surname><given-names>J</given-names></name><name><surname>Zhang</surname><given-names>H</given-names></name><name><surname>Wang</surname><given-names>Y</given-names></name><etal/></person-group>. <article-title>Building CT radiomics based nomogram for preoperative esophageal cancer patients lymph node metastasis prediction</article-title>. <source>Transl Oncol.</source> (<year>2018</year>) <volume>11</volume>:<fpage>815</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.tranon.2018.04.005</pub-id><pub-id pub-id-type=\"pmid\">29727831</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gillies</surname><given-names>RJ</given-names></name><name><surname>Kinahan</surname><given-names>PE</given-names></name><name><surname>Hricak</surname><given-names>H</given-names></name></person-group>. <article-title>Radiomics: images are more than pictures, they are data</article-title>. <source>Radiology</source>. (<year>2016</year>) <volume>278</volume>:<fpage>563</fpage>–<lpage>77</lpage>. <pub-id pub-id-type=\"doi\">10.1148/radiol.2015151169</pub-id><pub-id pub-id-type=\"pmid\">26579733</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mattonen</surname><given-names>SA</given-names></name><name><surname>Palma</surname><given-names>DA</given-names></name><name><surname>Johnson</surname><given-names>C</given-names></name><name><surname>Louie</surname><given-names>AV</given-names></name><name><surname>Landis</surname><given-names>M</given-names></name><name><surname>Rodrigues</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Detection of local cancer recurrence after stereotactic ablative radiation therapy for lung cancer: physician performance versus radiomic assessment</article-title>. <source>Int J Radiat Oncol Biol Phys.</source> (<year>2016</year>) <volume>94</volume>:<fpage>1121</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijrobp.2015.12.369</pub-id><pub-id pub-id-type=\"pmid\">26907916</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fave</surname><given-names>X</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Yang</surname><given-names>J</given-names></name><name><surname>Mackin</surname><given-names>D</given-names></name><name><surname>Balter</surname><given-names>P</given-names></name><name><surname>Gomez</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Delta-radiomics features for the prediction of patient outcomes in non-small cell lung cancer</article-title>. <source>Sci Rep.</source> (<year>2017</year>) <volume>7</volume>:<fpage>588</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-017-00665-z</pub-id><pub-id pub-id-type=\"pmid\">28373718</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fried</surname><given-names>DV</given-names></name><name><surname>Mawlawi</surname><given-names>O</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Fave</surname><given-names>X</given-names></name><name><surname>Zhou</surname><given-names>S</given-names></name><name><surname>Ibbott</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Stage III non-small cell lung cancer: prognostic value of FDG PET quantitative imaging features combined with clinical prognostic factors</article-title>. <source>Radiology</source>. (<year>2016</year>) <volume>278</volume>:<fpage>214</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.1148/radiol.2015142920</pub-id><pub-id pub-id-type=\"pmid\">26176655</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fried</surname><given-names>DV</given-names></name><name><surname>Tucker</surname><given-names>SL</given-names></name><name><surname>Zhou</surname><given-names>S</given-names></name><name><surname>Liao</surname><given-names>Z</given-names></name><name><surname>Mawlawi</surname><given-names>O</given-names></name><name><surname>Ibbott</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Prognostic value and reproducibility of pretreatment CT texture features in stage III non-small cell lung cancer</article-title>. <source>Int J Radiat Oncol Biol Phys.</source> (<year>2014</year>) <volume>90</volume>:<fpage>834</fpage>–<lpage>42</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijrobp.2014.07.020</pub-id><pub-id pub-id-type=\"pmid\">25220716</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>J</given-names></name><name><surname>Aguilera</surname><given-names>T</given-names></name><name><surname>Shultz</surname><given-names>D</given-names></name><name><surname>Gudur</surname><given-names>M</given-names></name><name><surname>Rubin</surname><given-names>DL</given-names></name><name><surname>Loo</surname><given-names>BW</given-names><suffix>Jr</suffix></name><etal/></person-group>. <article-title>Early-stage non-small cell lung cancer: quantitative imaging characteristics of (18)f fluorodeoxyglucose PET/CT allow prediction of distant metastasis</article-title>. <source>Radiology</source>. (<year>2016</year>) <volume>281</volume>:<fpage>270</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1148/radiol.2016151829</pub-id><pub-id pub-id-type=\"pmid\">27046074</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van Griethuysen</surname><given-names>JJM</given-names></name><name><surname>Fedorov</surname><given-names>A</given-names></name><name><surname>Parmar</surname><given-names>C</given-names></name><name><surname>Hosny</surname><given-names>A</given-names></name><name><surname>Aucoin</surname><given-names>N</given-names></name><name><surname>Narayan</surname><given-names>V</given-names></name><etal/></person-group>. <article-title>Computational radiomics system to decode the radiographic phenotype</article-title>. <source>Cancer Res.</source> (<year>2017</year>) <volume>77</volume>:<fpage>e104</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1158/0008-5472.CAN-17-0339</pub-id><pub-id pub-id-type=\"pmid\">29092951</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parmar</surname><given-names>C</given-names></name><name><surname>Rios Velazquez</surname><given-names>E</given-names></name><name><surname>Leijenaar</surname><given-names>R</given-names></name><name><surname>Jermoumi</surname><given-names>M</given-names></name><name><surname>Carvalho</surname><given-names>S</given-names></name><name><surname>Mak</surname><given-names>RH</given-names></name><etal/></person-group>. <article-title>Robust Radiomics feature quantification using semiautomatic volumetric segmentation</article-title>. <source>PLoS ONE.</source> (<year>2014</year>) <volume>9</volume>:<fpage>e102107</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0102107</pub-id><pub-id pub-id-type=\"pmid\">25025374</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>Y</given-names></name><name><surname>Liu</surname><given-names>Z</given-names></name><name><surname>He</surname><given-names>L</given-names></name><name><surname>Chen</surname><given-names>X</given-names></name><name><surname>Pan</surname><given-names>D</given-names></name><name><surname>Ma</surname><given-names>Z</given-names></name><etal/></person-group>. <article-title>Radiomics signature: a potential biomarker for the prediction of disease-free survival in early-stage (I or II) non-small cell lung cancer</article-title>. <source>Radiology.</source> (<year>2016</year>) <volume>281</volume>:<fpage>947</fpage>–<lpage>57</lpage>. <pub-id pub-id-type=\"doi\">10.1148/radiol.2016152234</pub-id><pub-id pub-id-type=\"pmid\">27347764</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>YQ</given-names></name><name><surname>Liang</surname><given-names>CH</given-names></name><name><surname>He</surname><given-names>L</given-names></name><name><surname>Tian</surname><given-names>J</given-names></name><name><surname>Liang</surname><given-names>CS</given-names></name><name><surname>Chen</surname><given-names>X</given-names></name><etal/></person-group>. <article-title>Development and validation of a radiomics nomogram for preoperative prediction of lymph node metastasis in colorectal cancer</article-title>. <source>J Clin Oncol.</source> (<year>2016</year>) <volume>34</volume>:<fpage>2157</fpage>–<lpage>64</lpage>. <pub-id pub-id-type=\"doi\">10.1200/JCO.2015.65.9128</pub-id><pub-id pub-id-type=\"pmid\">27138577</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xie</surname><given-names>C</given-names></name><name><surname>Yang</surname><given-names>P</given-names></name><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Xu</surname><given-names>L</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name><name><surname>Li</surname><given-names>X</given-names></name><etal/></person-group>. <article-title>Sub-region based radiomics analysis for survival prediction in oesophageal tumours treated by definitive concurrent chemoradiotherapy</article-title>. <source>EBioMedicine</source>. (<year>2019</year>) <volume>44</volume>:<fpage>289</fpage>–<lpage>97</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ebiom.2019.05.023</pub-id><pub-id pub-id-type=\"pmid\">31129097</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Camp</surname><given-names>RL</given-names></name><name><surname>Dolled-Filhart</surname><given-names>M</given-names></name><name><surname>Rimm</surname><given-names>DL</given-names></name></person-group>. <article-title>X-tile: a new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization</article-title>. <source>Clin Cancer Res.</source> (<year>2004</year>) <volume>10</volume>:<fpage>7252</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1158/1078-0432.CCR-04-0713</pub-id><pub-id pub-id-type=\"pmid\">15534099</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cui</surname><given-names>Y</given-names></name><name><surname>Tha</surname><given-names>KK</given-names></name><name><surname>Terasaka</surname><given-names>S</given-names></name><name><surname>Yamaguchi</surname><given-names>S</given-names></name><name><surname>Wang</surname><given-names>J</given-names></name><name><surname>Kudo</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Prognostic imaging biomarkers in glioblastoma: development and independent validation on the basis of multiregion and quantitative analysis of MR images</article-title>. <source>Radiology.</source> (<year>2016</year>) <volume>278</volume>:<fpage>546</fpage>–<lpage>53</lpage>. <pub-id pub-id-type=\"doi\">10.1148/radiol.2015150358</pub-id><pub-id pub-id-type=\"pmid\">26348233</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liang</surname><given-names>W</given-names></name><name><surname>Yang</surname><given-names>P</given-names></name><name><surname>Huang</surname><given-names>R</given-names></name><name><surname>Xu</surname><given-names>L</given-names></name><name><surname>Wang</surname><given-names>J</given-names></name><name><surname>Liu</surname><given-names>W</given-names></name><etal/></person-group>. <article-title>A combined nomogram model to preoperatively predict histologic grade in pancreatic neuroendocrine tumors</article-title>. <source>Clin Cancer Res.</source> (<year>2019</year>) <volume>25</volume>:<fpage>584</fpage>–<lpage>94</lpage>. <pub-id pub-id-type=\"doi\">10.1158/1078-0432.CCR-18-1305</pub-id><pub-id pub-id-type=\"pmid\">30397175</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>S</given-names></name><name><surname>Zheng</surname><given-names>J</given-names></name><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Wu</surname><given-names>Z</given-names></name><name><surname>Shi</surname><given-names>S</given-names></name><name><surname>Huang</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Development and validation of an MRI-based radiomics signature for the preoperative prediction of lymph node metastasis in bladder cancer</article-title>. <source>EBioMedicine.</source> (<year>2018</year>) <volume>34</volume>:<fpage>76</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ebiom.2018.07.029</pub-id><pub-id pub-id-type=\"pmid\">30078735</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>S</given-names></name><name><surname>Zheng</surname><given-names>J</given-names></name><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Yu</surname><given-names>H</given-names></name><name><surname>Shi</surname><given-names>S</given-names></name><name><surname>Xie</surname><given-names>W</given-names></name><etal/></person-group>. <article-title>A radiomics nomogram for the preoperative prediction of lymph node metastasis in bladder cancer</article-title>. <source>Clin Cancer Res.</source> (<year>2017</year>) <volume>23</volume>:<fpage>6904</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1158/1078-0432.CCR-17-1510</pub-id><pub-id pub-id-type=\"pmid\">28874414</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Frank</surname><given-names>E</given-names></name><name><surname>Harrell</surname><given-names>J</given-names></name></person-group>\n<source>Regression Modeling Strategies: With Applications to Linear Models, Logistic and Ordinal Regression, and Survival Analysis</source>. <publisher-loc>New York, NY</publisher-loc>: <publisher-name>Spinger</publisher-name> (<year>2015</year>). <pub-id pub-id-type=\"doi\">10.1007/978-3-319-19425-7</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Anthony</surname><given-names>GJ</given-names></name><name><surname>Cunliffe</surname><given-names>A</given-names></name><name><surname>Castillo</surname><given-names>R</given-names></name><name><surname>Pham</surname><given-names>N</given-names></name><name><surname>Guerrero</surname><given-names>T</given-names></name><name><surname>Armato</surname><given-names>SG</given-names><suffix>3rd</suffix></name><etal/></person-group>. <article-title>Incorporation of pre-therapy (18) F-FDG uptake data with CT texture features into a radiomics model for radiation pneumonitis diagnosis</article-title>. <source>Med Phys.</source> (<year>2017</year>) <volume>44</volume>:<fpage>3686</fpage>–<lpage>94</lpage>. <pub-id pub-id-type=\"doi\">10.1002/mp.12282</pub-id><pub-id pub-id-type=\"pmid\">28422299</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>K</given-names></name><name><surname>Lin</surname><given-names>Y</given-names></name><name><surname>Yang</surname><given-names>L</given-names></name><name><surname>Wang</surname><given-names>Y</given-names></name><name><surname>Cai</surname><given-names>S</given-names></name><name><surname>Pang</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>A multipredictor model to predict the conversion of mild cognitive impairment to Alzheimer's disease by using a predictive nomogram</article-title>. <source>Neuropsychopharmacology</source>. (<year>2019</year>) <volume>45</volume>:<fpage>358</fpage>–<lpage>66</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41386-019-0551-0</pub-id><pub-id pub-id-type=\"pmid\">31634898</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nie</surname><given-names>P</given-names></name><name><surname>Yang</surname><given-names>G</given-names></name><name><surname>Wang</surname><given-names>Z</given-names></name><name><surname>Yan</surname><given-names>L</given-names></name><name><surname>Miao</surname><given-names>W</given-names></name><name><surname>Hao</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>A CT-based radiomics nomogram for differentiation of renal angiomyolipoma without visible fat from homogeneous clear cell renal cell carcinoma</article-title>. <source>Eur Radiol.</source> (<year>2019</year>) <volume>30</volume>:<fpage>1274</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00330-019-06427-x</pub-id><pub-id pub-id-type=\"pmid\">31506816</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lambin</surname><given-names>P</given-names></name><name><surname>Leijenaar</surname><given-names>RTH</given-names></name><name><surname>Deist</surname><given-names>TM</given-names></name><name><surname>Peerlings</surname><given-names>J</given-names></name><name><surname>de Jong</surname><given-names>EEC</given-names></name><name><surname>van Timmeren</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Radiomics: the bridge between medical imaging and personalized medicine</article-title>. <source>Nat Rev Clin Oncol.</source> (<year>2017</year>) <volume>14</volume>:<fpage>749</fpage>–<lpage>62</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrclinonc.2017.141</pub-id><pub-id pub-id-type=\"pmid\">28975929</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lambin</surname><given-names>P</given-names></name><name><surname>Rios-Velazquez</surname><given-names>E</given-names></name><name><surname>Leijenaar</surname><given-names>R</given-names></name><name><surname>Carvalho</surname><given-names>S</given-names></name><name><surname>van Stiphout</surname><given-names>RG</given-names></name><name><surname>Granton</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Radiomics: extracting more information from medical images using advanced feature analysis</article-title>. <source>Eur J Cancer</source>. (<year>2012</year>) <volume>48</volume>:<fpage>441</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ejca.2011.11.036</pub-id><pub-id pub-id-type=\"pmid\">22257792</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Immunol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Immunol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Immunol.</journal-id><journal-title-group><journal-title>Frontiers in Immunology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-3224</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849510</article-id><article-id pub-id-type=\"pmc\">PMC7431608</article-id><article-id pub-id-type=\"doi\">10.3389/fimmu.2020.01496</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Immunology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>IL-34 Actions on FOXP3<sup>+</sup> Tregs and CD14<sup>+</sup> Monocytes Control Human Graft Rejection</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Bézie</surname><given-names>Séverine</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/512917/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Freuchet</surname><given-names>Antoine</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/969669/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Sérazin</surname><given-names>Céline</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/942476/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Salama</surname><given-names>Apolline</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/970742/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Vimond</surname><given-names>Nadège</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Anegon</surname><given-names>Ignacio</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"author-notes\" rid=\"fn003\"><sup>‡</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/41971/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Guillonneau</surname><given-names>Carole</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><xref ref-type=\"author-notes\" rid=\"fn003\"><sup>‡</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/197378/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Centre de Recherche en Transplantation et Immunologie UMR1064, INSERM, Université de Nantes</institution>, <addr-line>Nantes</addr-line>, <country>France</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes</institution>, <addr-line>Nantes</addr-line>, <country>France</country></aff><aff id=\"aff3\"><sup>3</sup><institution>LabEx IGO “Immunotherapy, Graft, Oncology”</institution>, <addr-line>Nantes</addr-line>, <country>France</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Markus Neurath, University Hospital Erlangen, Germany</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Depei Wu, Soochow University Medical College, China; Edoardo Fiorillo, National Research Council (CNR), Italy</p></fn><corresp id=\"c001\">*Correspondence: Carole Guillonneau <email>carole.guillonneau@univ-nantes.fr</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Cytokines and Soluble Mediators in Immunity, a section of the journal Frontiers in Immunology</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†These authors share first authorship</p></fn><fn fn-type=\"other\" id=\"fn003\"><p>‡These authors share last authorship</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1496</elocation-id><history><date date-type=\"received\"><day>31</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>08</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Bézie, Freuchet, Sérazin, Salama, Vimond, Anegon and Guillonneau.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Bézie, Freuchet, Sérazin, Salama, Vimond, Anegon and Guillonneau</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Cytokines are major players regulating immune responses toward inflammatory and tolerogenic results. In organ and bone marrow transplantation, new reagents are needed to inhibit tissue destructive mechanisms and eventually induce immune tolerance without overall immunosuppression. IL-34 is a cytokine with no significant homology with any other cytokine but that acts preferentially through CSF-1R, as CSF-1 does, and through PTPζ and CD138. Although IL-34 and CSF-1 share actions, a detailed analysis of their effects on immune cells needs further research. We previously showed that both CD4<sup>+</sup> and CD8<sup>+</sup> FOXP3<sup>+</sup> Tregs suppress effector T cells through the production of IL-34, but not CSF-1, and that this action was mediated through antigen-presenting cells. We showed here by single-cell RNAseq and cytofluorimetry that different subsets of human monocytes expressed different levels of CSF-1R, CD138, and PTPζ and that both CD4<sup>+</sup> and CD8<sup>+</sup> FOXP3<sup>+</sup> Tregs expressed higher levels of CSF-1R than conventional T cells. The effects of IL-34 differed in the survival of these different subpopulations of monocytes and RNAseq analysis showed several genes differentially expressed between IL-34, CSF-1, M0, M1, and also M2 macrophages. Acute graft-vs.-host disease (aGVHD) in immunodeficient NSG mice injected with human PBMCs was decreased when treated with IL-34 in combination with an anti-CD45RC mAb that depleted conventional T cells. When IL-34-differentiated monocytes were used to expand Tregs <italic>in vitro</italic>, both CD4<sup>+</sup> and CD8<sup>+</sup> FOXP3<sup>+</sup> Tregs were highly enriched and this effect was superior to the one obtained with CSF-1. Human CD8<sup>+</sup> Tregs expanded <italic>in vitro</italic> with IL-34-differentiated allogeneic monocytes suppressed human immune responses in an NSG mouse aGVHD model humanized with hPBMCs. Overall, we showed that IL-34 induced the differentiation of human monocytes with a particular transcriptional profile and these cells favored the development of potent suppressor FOXP3<sup>+</sup> Tregs.</p></abstract><kwd-group><kwd>IL-34</kwd><kwd>transplantation</kwd><kwd>tolerance</kwd><kwd>monocyte</kwd><kwd>Treg</kwd><kwd>cell therapy</kwd><kwd>GVHD</kwd><kwd>CSF-1R</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Agence Nationale de la Recherche<named-content content-type=\"fundref-id\">10.13039/501100001665</named-content></funding-source></award-group><award-group><funding-source id=\"cn002\">Agence de la Biomédecine<named-content content-type=\"fundref-id\">10.13039/501100006005</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"7\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"53\"/><page-count count=\"16\"/><word-count count=\"9098\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Organ and bone marrow transplantation is the only treatment for patients suffering from a number of diseases. In organ transplantation, the use of immunosuppressors has allowed remarkable success in the short and medium term graft survival, but unwanted side effects still lead to high morbidity and mortality, even when avoiding excessive immunosuppression (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). In bone marrow transplantation, acute and chronic GVHD are very frequent complications with high mortality and morbidity and thus with high unmet clinical needs (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B3\" ref-type=\"bibr\">3</xref>). In the long term, immunosuppressors can even be deleterious in the establishment of tolerance (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Therefore, new treatments are needed that will be more specific for allogeneic immune responses and/or induce fewer side effects and that would allow, at the least, to decrease the use of immunosuppressors. Cytokines and enzymes controlling metabolic pathways have been described as powerful tools for controlling immune responses and it is important to identify new mediators of immune tolerance. Interleukin-34 (IL-34) is a cytokine, described for the first time in 2008 (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Although IL-34 shares no homology with macrophage colony-stimulating factor (CSF-1 or M-CSF) in its amino acid sequence, they share a common receptor (CSF-1R or CD115) and IL-34 also has two distinct receptors, protein-tyrosine-phosphatase zeta (PTPζ) and CD138 (syndecan-1) (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>), suggesting additional roles for IL-34. In addition, the affinity of IL-34 for CSF-1R is higher than the one of CSF-1 and the binding mode to CSF-1R, as well as signaling of both cytokines, are different (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). Until now, studies have demonstrated that IL-34 is released by some cell types and is involved in the differentiation and survival of macrophages, monocytes, and dendritic cells (DCs) in response to inflammation, in the development of microglia and Langerhans cells (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>, <xref rid=\"B10\" ref-type=\"bibr\">10</xref>). More recent articles have described the immunoregulatory properties of IL-34 (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B12\" ref-type=\"bibr\">12</xref>). We have demonstrated that IL-34 is secreted by FOXP3<sup>+</sup> CD4<sup>+</sup> and CD8<sup>+</sup> regulatory T cells (Tregs) in human and CD8<sup>+</sup>CD45RC<sup>low/−</sup> Tregs in rat. We also demonstrated that blockade of IL-34 <italic>in vitro</italic> in human and rat co-culture suppression assays inhibited both CD4<sup>+</sup> and CD8<sup>+</sup> Tregs suppressive function. Most importantly, we also showed that IL-34 treatment <italic>in vivo</italic> in a rat model of cardiac allograft induced transplant tolerance through the differentiation of macrophages toward a regulatory profile and subsequent induction of CD4<sup>+</sup> and CD8<sup>+</sup> Tregs by these macrophages (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). This role had never been evidenced before and needed to be explored in humans. We therefore investigated the tolerogenic effect of IL-34 on monocytes/macrophages and the mechanisms by which CD4<sup>+</sup> and CD8<sup>+</sup> Tregs were generated. Since CD4<sup>+</sup> and CD8<sup>+</sup> Tregs produce IL-34, our hypothesis was that IL-34 acts in autocrine and paracrine fashions to reinforce immune tolerance. Thus, we analyzed the expression of IL-34 receptors (CSF-1R, CD138, and PTPζ) on human monocytes and T cells and assessed the effect of IL-34 on human monocytes by single cell and bulk RNAseq. We also analyzed the effects of IL-34 on human Treg cell generation and evaluated in immune humanized mice the suppressive function of CD8<sup>+</sup> Tregs differentiated using IL-34-treated human monocytes in a model of acute GVHD.</p><p>In the present manuscript we report that IL-34 can act on CD14<sup>++</sup>CSF-1R<sup>+</sup>PTPζ<sup>+</sup> monocytes and CD4<sup>+</sup> or CD8<sup>+</sup> FOXP3<sup>+</sup>CSF-1R<sup>+</sup> Tregs in an autocrine manner. We demonstrate that IL-34 action on monocytes results in differentiation toward a regulatory macrophage profile different from M2 macrophages, as shown by transcriptomic profiling. We demonstrate also that naive and effector precursor T cell depletion using anti-CD45RC mAbs results in synergistic enhanced IL-34 tolerogenic action <italic>in vivo</italic>. <italic>In vitro</italic>, we show that IL-34 is more efficient at inducing FOXP3<sup>+</sup> Tregs than CSF-1 and that these FOXP3<sup>+</sup> Tregs can efficiently control GVHD <italic>in vivo</italic> in a model of immune humanized immunodeficient mice.</p><p>Altogether, these data provide new informations on this new function of IL-34 on regulating Treg activity.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Healthy Volunteers' Blood Collection and PBMC Separation</title><p>Blood from healthy individuals was obtained at the Etablissement Français du Sang (Nantes, France). Written informed consent was provided according to institutional guidelines. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Paque density-gradient centrifugation (Eurobio, Courtaboeuf, France). Red cells and platelets were eliminated using a hypotonic solution and centrifugation.</p></sec><sec><title>Cell Isolation</title><p>CD14<sup>++</sup>CD16<sup>−</sup>, CD14<sup>++</sup>CD16<sup>+</sup>, and CD14<sup>dim</sup>CD16<sup>++</sup> subsets were FACS Aria sorted from PBMCs based on size morphology and CD14<sup>++/dim</sup>CD16<sup>++/−</sup> expression for differentiation with IL-34 (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 1E</xref>). Total CD14<sup>+</sup> monocytes were isolated using a negative selection kit (Miltenyi Biotec., Bergisch Gladbach, Germany) for phosphorylation analysis, or by magnetic depletion (Dynabeads, Invitrogen) of CD3<sup>+</sup>, CD16<sup>+</sup>, and CD19<sup>+</sup>, then FACS Aria sorting of CD14<sup>++</sup> cells for both RNA sequencing analysis and Treg expansion. CD8<sup>+</sup> Tregs were obtained by enrichment of PBMCs in T cells (to 80% T cells) by magnetic depletion of CD19<sup>+</sup>, CD14<sup>+</sup>, and CD16<sup>+</sup> and then sorting of CD3<sup>+</sup>CD4<sup>−</sup>CD45RC<sup>low/−</sup> cells (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 4A</xref>) using FACS ARIA II (BD Biosciences, Mountain View, CA, USA). Allogeneic APCs were isolated by magnetic depletion of CD3<sup>+</sup> cells from PBMCs.</p></sec><sec><title>Quantification of CSF-1R and PTPζ Signaling Pathway Activation</title><p>Freshly sorted CD14<sup>+</sup>CD16<sup>−</sup> monocytes were plated at 1 × 10<sup>6</sup> cells/ml in fetal bovine serum (FBS)-free RPMI 1640 medium (1% penicillin-streptomycin, 1 mM glutamine, 1% NEAA, 10 mM Hepes, 1 mM sodium pyruvate) in low attachment round-bottomed 96-well plates (Perkin-Elmer, Inc., Waltham, MA, USA), and left untouched for 2 h before adding IL-34 or CSF-1 at a final concentration of 100 ng/ml. Analysis of the phosphorylation of AKT and ERK1/2 after 1, 3, 5, 10, and 15 min was performed by flow cytometry following the BD Biosciences Phosflow protocol, using the BD Cytofix Fixation buffer and BD Phosflow Perm Buffer III (BD Biosciences), as well as phospho-AKT (Ser473) and phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) primary goat antibodies (Cell Signaling Technology, Leiden, The Netherlands), and goat anti-rabbit IgG(H+L)-AF647 (Life Technologies, ThermoFisher Scientific) secondary antibody.</p></sec><sec><title>Differentiation of Monocytes and Expansion of Tregs</title><p>Monocytes were seeded at 1 × 10<sup>6</sup> cells/mL in complete RPMI 1640 medium supplemented with 10% FBS and IL-34 (2 nM, eBiosciences, ThermoFisher Scientific, Waltham, MA, USA) or CSF-1 (2 nM, R&D Systems, Bio-techne, Minneapolis, MN, USA) and macrophages were harvested at day 6. M1 macrophages were obtained by supplementing the medium with granulocyte-macrophage colony-stimulating factor (GM-CSF, 10 ng/mL, Cellgenix, Freiburg, Germany) over 5 days and by addition of interferon-gamma (IFNγ, 1000 U/mL, Miltenyi Biotec) from day 5 until day 7 of culture. M2 macrophages were obtained by supplementing the medium with CSF-1 (25 ng/mL, R&D Systems Biotechne) for 5 days and by addition of IL-4 (20 ng/mL, Cellgenix) and IL-10 (20 ng/mL, R&D Systems Biotechne) from day 5 until day 7 of culture. Lipopolysaccharide (LPS, 100 ng/mL, Sigma Aldrich, Saint-Louis, MO, USA) was added in the culture for the last 24 h for cytokine dosage. Macrophages were harvested using Trypsin (TryPLE, Gibco, ThermoFisher Scientific) at day 7.</p><p>Allogeneic PBMCs were seeded at 1 × 10<sup>6</sup> in 24-well plate in Iscove's modified Dulbecco's medium (IMDM), supplemented with 2 mM glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 5% human AB serum with IL-34- or CSF-1- differentiated macrophages at a ratio of PBMCs:macrophages 5:1 and cultured for 14 days.</p><p>CD8<sup>+</sup>CD45RC<sup>low/−</sup> Tregs were seeded at 5 × 10<sup>5</sup> cells/cm<sup>2</sup>/500 μl in flat-bottom plates coated with anti-CD3 mAb (1 μg/mL, OKT3, hybridoma from the European Collection of Cell Culture), in complete RPMI 1640 medium supplemented with 10% FBS, IL-2 (1,000 U/mL, Proleukin, Novartis), IL-15 (10 ng/mL, Miltenyi Biotec) and soluble anti-CD28 mAb (1 μg/mL, CD28.2, hybridoma from the European Collection of Cell Culture) in the presence of IL-34-differentiated macrophages or allogeneic APCs irradiated (35 Gy) at 1:4 Treg:IL-34-macrophage or APC ratio. CD8<sup>+</sup> Tregs were stimulated again using anti-CD3 and anti-CD28 mAbs at day 7 of culture and IL-2 and IL-15 were freshly added at days 0, 2, 4, 7, 10 and 12.</p></sec><sec><title>Monoclonal Antibodies and Flow Cytometry</title><p>Antibodies used are listed in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Table 1</xref>. For analysis of intracellular cytokines, Tregs were incubated with PMA, ionomycin, and brefeldine A (10 μg/ml) for 4 h before staining. Fc receptors were blocked (BD Biosciences) before staining and cells were permeabilized with a Fix/Perm kit (Ebiosciences).</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>List of antibodies used.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Marker</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Clone</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Provider</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD14</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">M5E2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD16</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3G8</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD115</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">9-4D2-1E4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PTPζ</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Polyclonal</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bioss</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD138</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MI15</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SK7</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RPA-T4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD8</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RPA-T8</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD25</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">M-A251</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD45RC</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MT2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IQProduct</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD19</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HIB19</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD56</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">B159</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD335</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">9E2/Nkp46</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Biolegend</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD86</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2331</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD80</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">L307.4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD40</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5C3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD206</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">19.2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD169</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7-239</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD163</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GHI/61</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD209a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DCN46</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD36</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HIT2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD1a</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HI149</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-34</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">578416</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">R&D System</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TGFβ1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TW4-9E7</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FOXP3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">259D/C7</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IFNγ</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">B27</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tbet</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">O4-46</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GITR</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">REA841</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Miltenyi Biotec</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PD-1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EH12.1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD127</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">hIL-7R-M21</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD28</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD28.2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD27</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">M-T271</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD45RA</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HI100</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HLA-DR</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">L243</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD154</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TRAP1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TRAIL</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RIK-2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CD103</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ber-ACT8</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">hCD45</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HI30</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">mCD45</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">30-F11</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BD Biosciences</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phospho-Akt (Ser473)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D9E</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cell Signaling Technology</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D13.14.4E</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cell Signaling Technology</td></tr></tbody></table></table-wrap><p>Fluorescence was measured with LSR II or Canto II cytometers (BD Biosciences) and analyzed with FLOWJO software (Tree Star, Inc., Ashland, OR, USA).</p></sec><sec><title>ELISA</title><p>IL-10 and IL-12p40 were quantified in the supernatant of monocytes cultured for 6 days as well as control M1 macrophages, and both were stimulated for the last 24 h with LPS at 100 ng/ml using Human IL-10 ELISA Set and Human IL-12p40 ELISA Set performed according to manufacturer's instructions (BD Biosciences).</p></sec><sec><title>DGE-RNA Sequencing</title><p>CD14<sup>++</sup>CD16<sup>−</sup> monocytes were sorted by FACS Aria and lysed in RLT Buffer (Qiagen). RNeasy-Mini Kits (Qiagen) were used to isolate total RNA that was then processed for RNA sequencing. A protocol of 3′ Digital Gene Expression (DGE) RNA-sequencing was performed as previously described (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Library was run on an Illumina NextSeq 550 high-output (2 × 75 pb) (Genom'IC platform, Cochin Institute, Paris). Reads 1 encode for well-specific barcodes and unique molecular identifiers (UMIs) whereas Reads 2 encode for 3' mRNA sequences and were aligned to human genome reference (hg19). Count matrix was generated by counting sample-specific UMI associated with genes for each sample. Differentially expressed genes between conditions were calculated using R package Deseq2 (Bioconductor) by first applying a regularized log transformation (rlog). Genes with adjusted <italic>p</italic>-value inferior to 0.05 were considered as differentially expressed. Heatmaps were generated by scaling and center genes expression. Finally, a volcano plot was designed by plotting -Log10 of adjusted <italic>p</italic>-value in function of log2 Fold Change; highlighted genes correspond to differentially expressed genes. The accession number for DGE-RNA sequencing raw data and processed data is GEO: <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"GSE151194\">GSE151194</ext-link>.</p></sec><sec><title>Single Cell RNAseq Analysis</title><p>An online public dataset of 10X genomics (<ext-link ext-link-type=\"uri\" xlink:href=\"https://support.10xgenomics.com/single-cell-gene-expression/datasets/3.0.2/5k_pbmc_v3_nextgem\">https://support.10xgenomics.com/single-cell-gene-expression/datasets/3.0.2/5k_pbmc_v3_nextgem</ext-link>) was used to analyze gene expression of SDC1 (CD138), PTPRZ1 (PTPz) and CSF-1R in human PBMCs. Data were processed with “Seurat” package (version 3.1.3) in R software (RStudio, Inc., Boston). To eliminate unwanted cells (debris and doublets), cells with fewer than 200 genes or more than 4,000 genes were excluded. Then, cells with more than 10% of mitochondrial genes were excluded from the downstream analysis. Single cell transcriptomes were first normalized (log normalization) and then scaled. The most variable genes were found according to the variance stabilizing transformation (vst) method and were used to perform Principal Component Analysis (PCA). Clustering was performed on the first nine principal components, and hPBMC subsets were characterized according to expression of common membrane markers. Finally, a supervised analysis was performed to classify CD14<sup>++</sup>CD16<sup>−</sup>, CD14<sup>++</sup>CD16<sup>+</sup>, and CD14<sup>dim</sup>CD16<sup>++</sup> monocytes.</p></sec><sec><title>Immune Humanized Mouse aGVHD Model</title><p>This study was carried out according to permit numbers APAFIS 3168 from the Ministry of Research. Eight to twelve-week-old NOD/SCID/<italic>Il2r</italic>γ<sup>−/−</sup> (NSG) mice were bred in our own animal facilities in SPF conditions (accreditation number C44-278). 1.5 × 10<sup>7</sup> human PBMCs were intravenously injected in 1.5 Gy-irradiated NSG mice the day before, as previously described (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>, <xref rid=\"B15\" ref-type=\"bibr\">15</xref>). Human PBMCs were monitored in blood and GVHD development was evaluated by body weight loss (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>, <xref rid=\"B15\" ref-type=\"bibr\">15</xref>). Human recombinant IL-34 (0.4 or 0.8 mg/kg/2.5 d for 20 days; from eBiosciences) and/or anti-human CD45RC mAbs (0.8 mg/kg/2.5 d for 20 days, MT2 or ABIS-45RC clones) were injected intraperitoneally. PBMCs were i.v. injected alone or with Tregs in a range of PBMC:Treg ratio from 1:0.5 to 1:2.</p></sec><sec><title>Statistical Analysis</title><p>Two-way repeated measure ANOVA was used to analyze mouse weight loss over time and Log Rank (Mantel Cox) test was used to analyze mouse survival. Friedman test with Dunn's multiple comparison test were used to compare monocyte frequency in PBMCs. Two-way ANOVA and Bonferroni post-test were used to analyze the survival of monocytes during the culture, phenotype of monocyte subsets and expanded Tregs. Mann Whitney <italic>U</italic>-test was used to compare the IL-10/IL-12p40 ratio in the supernatants of cultured macrophages.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>CSF-1R and PTPζ Are Both Expressed on CD14<sup>++</sup> Monocytes and CSF-1R Is Also Expressed on FOXP3<sup>+</sup> CD4<sup>+</sup> and CD8<sup>+</sup> Tregs</title><p>We previously showed that IL-34 produced by FOXP3<sup>+</sup> Tregs acted at least on human monocytes <italic>in vitro</italic> (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). To get a better overview of IL-34 action on the immune system, we analyzed the expression of its reported receptors CSF-1R (also called CD115), CD138 (also called SDC1), and PTPζ (also called PTPRZ1) on whole PBMCs using a public single cell RNAseq dataset (<ext-link ext-link-type=\"uri\" xlink:href=\"https://support.10xgenomics.com/single-cell-gene-expression/datasets/3.0.2/5k_pbmc_v3_nextgem\">https://support.10xgenomics.com/single-cell-gene-expression/datasets/3.0.2/5k_pbmc_v3_nextgem</ext-link>). We observed that CSF-1R single cell mRNA expression was restricted to monocytes and not significantly expressed by resting T, B and NK cells (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). Analysis of markers of non-classical (CD14<sup>dim</sup>CD16<sup>++</sup>), intermediate (CD14<sup>++</sup>CD16<sup>+</sup>), or classical (CD14<sup>++</sup>CD16<sup>−</sup>) monocytes/macrophages (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B17\" ref-type=\"bibr\">17</xref>) showed that CSF-1R was expressed in all three populations of monocytes (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>) with a higher expression in non-classical and intermediate monocytes. In contrast, CD138 and PTPzeta mRNA expression was not detectable in resting PBMCs (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figures 1A,B</xref>). However, we were able to detect PTPζ protein expression in all monocyte subsets and we also confirmed that CSF-1R was expressed by all monocytes, and both with a higher expression level in non-classical monocytes (<xref ref-type=\"fig\" rid=\"F1\">Figures 1C,D</xref>). Nevertheless, since CSF-1R<sup>+</sup> and PTPζ<sup>+</sup> classical monocyte frequency in PBMCs is much higher than CSF-1R<sup>+</sup> and PTPζ<sup>+</sup> intermediate and non-classical monocytes (<xref ref-type=\"fig\" rid=\"F1\">Figure 1E</xref>), it suggests that IL-34 will mostly act on CD14<sup>++</sup>CD16<sup>−</sup> monocytes.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>CSF-1R and PTPζ expression is restricted to monocytes and FOXP3<sup>+</sup> Tregs. PBMCs were analyzed for CSF-1R expression at single cell transcriptional <bold>(A,B)</bold> and proteomic levels <bold>(C–F)</bold>. <bold>(A)</bold> Top: UMAP visualization of a public dataset of resting Human PBMC single cell RNA-seq from one healthy volunteer for which subsets of monocytes, T cells, B cells, and NK cells were identified by antibody staining. Bottom: CSF-1R expression in total PBMCs. One point represents one cell. Relative expression level is scaled from gray to dark blue. <bold>(B)</bold> Monocyte subsets were further subdivided based on RNA (RNAseq, bottom left) and protein expressions (CITEseq, bottom right) of CD14 (left) and FCGR3A (CD16) (right) summarized in the UMAP visualization (upper left), and subsets were analyzed for CSF-1R RNA expression (upper middle and right). One point represents one cell. Relative expression level is scaled from gray to dark blue (RNA expression) or from gray to dark green (protein expression). Upper Right: Violin plot representing the expression level of mRNA for CSF-1R in CD14<sup>++</sup>CD16<sup>−</sup> monocytes (red), in CD16<sup>++</sup>CD14<sup>dim</sup> monocytes (pink), and in CD14<sup>++</sup>CD16<sup>+</sup> monocytes (purple). <bold>(C)</bold> Representative gating strategy for FACS analysis of CSF-1R, PTPζ, and CD138 expression in living (DAPI<sup>−</sup>) non-NK cells (CD56<sup>−</sup>NKp46<sup>−</sup>) CD14<sup>++/dim</sup>CD16<sup>++/+/−</sup> cell subsets from PBMCs. Representative from three individuals. <bold>(D)</bold> Frequency (left) of CSF-1R, PTPζ, and CD138 expressing cells and expression level (MFI) of CSF-1R and PTPζ (right) in CD14<sup>++/dim</sup>CD16<sup>++/+/−</sup> cell subsets. <italic>n</italic> = 3 individuals. <bold>(E)</bold> Frequency of CSF-1R<sup>+</sup>, PTPζ<sup>+</sup>, and CD138<sup>+</sup> monocytes in total PBMCs. <italic>n</italic> = 3 individuals. <bold>(F)</bold> Frequency of CSF-1R expressing cells in stimulated (black) or not (white) FOXP3<sup>+/−</sup> CD4<sup>+</sup> or CD8<sup>+</sup> T cells. <italic>n</italic> = 5 individuals. Mann Whitney tests, *<italic>p</italic>< 0.05, **<italic>p</italic> < 0.01.</p></caption><graphic xlink:href=\"fimmu-11-01496-g0001\"/></fig><p>To better comprehend whether IL-34 could act directly on Tregs, we further analyzed CSF-1R and PTPζ expression in total CD4<sup>+</sup> or CD8<sup>+</sup> T cells compared to FOXP3<sup>+</sup> CD4<sup>+</sup> or CD8<sup>+</sup> Tregs (<xref ref-type=\"fig\" rid=\"F1\">Figure 1F</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figures 1C,D</xref>). We observed a significant expression of CSF-1R in non-stimulated FOXP3<sup>+</sup> CD4<sup>+</sup> and CD8<sup>+</sup> Tregs compared to total CD4<sup>+</sup> and CD8<sup>+</sup> T cells, respectively (<xref ref-type=\"fig\" rid=\"F1\">Figure 1F</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 1C</xref>). The expression was even higher following stimulation, although it remains lower than on monocytes. We did not observe expression of PTPζ on Treg cells (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 1D</xref>).</p><p>Altogether, these results suggest that IL-34 can act on CD14<sup>++</sup> monocytes, likely through CSF-1R and PTPζ and on FOXP3<sup>+</sup> Tregs through CSF-1R in PBMCs.</p></sec><sec><title>IL-34 Preferentially Acts Through CD14<sup>++</sup>CSF-1R<sup>+</sup>PTPζ<sup>+</sup> Monocytes to Induce Immunoregulation</title><p>We and others have shown that IL-34 induces differentiation of human CD14<sup>++</sup> monocytes into macrophages with regulatory properties (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>). However, we observed that CSF-1R and PTPζ expressions was higher on non-classical and intermediate than classical monocytes, thus we investigated in each of the three subpopulations the survival and maturation upon IL-34 treatment compared to M1- and M2-macrophages differentiated with GM-CSF+IFNγ or CSF-1+IL-4+IL-10, respectively, as controls (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>) (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref> and cell sorting in <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 1E</xref>). Classical monocytes were largely predominant over intermediate and non-classical monocytes among PBMCs (about 18.8 vs. 4.7 vs. 1.8%, respectively, <xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>), and together with intermediate monocytes had a lower survival rate after 6-days culture than non-classical monocytes (10.6 vs. 24.7 vs. 21.2% for CD14<sup>++</sup>CD16<sup>−</sup>, CD14<sup>dim</sup>CD16<sup>++</sup>, and CD14<sup>++</sup>CD16<sup>+</sup>, respectively, <xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>). Comparing the phenotype, classical monocytes differentiated with IL-34 expressed higher levels than non-classical monocytes of M2-type markers CD163, CD36, CD169, CD206, CD14, and TRAIL (<xref ref-type=\"fig\" rid=\"F2\">Figure 2D</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 1F</xref>), displayed an anti-inflammatory cytokine secretion profile (<xref ref-type=\"fig\" rid=\"F2\">Figure 2E</xref>), were isolated (vs. in clumps for non-classical differentiated monocytes) and displayed fewer dendrites under macroscopic observation (vs. intermediate and non-classical monocytes) (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 1G</xref>). Intermediate monocytes had an intermediate phenotype, closer to classical than non-classical monocytes (<xref ref-type=\"fig\" rid=\"F2\">Figures 2D–E</xref>). Interestingly, non-classical monocytes expressed high levels of the M2-associated marker CD209a after culture in the presence of IL-34 (<xref ref-type=\"fig\" rid=\"F2\">Figure 2D</xref>). Finally, CD11b was more expressed in classical and intermediate monocytes, in accordance with previous observations (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>, <xref rid=\"B20\" ref-type=\"bibr\">20</xref>).</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>CD14<sup>++</sup> monocytes are the main mediators of IL-34-induced immunoregulation. <bold>(A)</bold> Schematic depicting conditions and timing of supplementation in cytokines in monocyte cultures. LPS was added for the last 24 h for cytokine release analysis only. <bold>(B)</bold> Frequency of monocyte subsets in PBMCs of healthy individuals. <italic>n</italic> = 8 individuals. Mann Whitney tests, *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, ***<italic>p</italic> < 0.001. <bold>(C)</bold> Living cell count over 6-days culture normalized to day 0 (=100%). <italic>n</italic> = 3–14 individuals. M1 (dark gray dotted line) and M2 (light gray dashed line) macrophages mean survival of three individuals after 7-days culture is shown. Two-way ANOVA and Bonferroni post-test. *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, ***<italic>p</italic> < 0.001. <bold>(D)</bold> Monocyte subsets were cultured for 7 days in the presence of IL-34 and analyzed for surface marker expression. Top: Geometric mean of fluorescence +/– SEM out of three experiments is represented over time. M1 (dark gray dotted line) and M2 (light gray dashed line) macrophages mean of fluorescence of three individuals after 7-days culture is shown. Mann Whitney <italic>U</italic>-test, *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, and ***<italic>p</italic> < 0.001. Bottom: Representative histograms of FACS staining. CD14<sup>++</sup>CD16<sup>−</sup> (blue line), CD14<sup>dim</sup>CD16<sup>++</sup> (red line), and CD14<sup>++</sup>CD16<sup>+</sup> (green line). Isotypic control is shown in filled gray. <bold>(E)</bold> IL-10/IL-12p40 ratios secreted by LPS-activated macrophages were quantified in supernatants at day 6. <italic>n</italic> = 3–5 individuals. Mann Whitney <italic>U</italic>-test, *<italic>p</italic> < 0.05.</p></caption><graphic xlink:href=\"fimmu-11-01496-g0002\"/></fig><p>These results show that IL-34 is more efficient at inducing M2-like macrophages from classical and intermediate monocytes than non-classical monocytes and suggest that CD14<sup>++</sup>CSF-1R<sup>+</sup>PTPζ<sup>+</sup> monocytes are the cells through which IL-34 induces immunoregulation.</p></sec><sec><title>IL-34 Efficiently Induces Regulatory Macrophages From Classical Monocytes Expressing Different Genes Than CSF-1-Treated Macrophages</title><p>We further investigated the signal induced in CD14<sup>++</sup>CD16<sup>−</sup> classical monocytes by IL-34 after binding the CSF-1R and PTPζ receptors in comparison to the signal induced by CSF-1 binding CSF-1R only. We observed a significant increase in the levels of phosphorylated AKT (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>) and ERK1/2 (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>) at 3 and 5 min following the addition of both IL-34 and CSF-1, compared to medium alone. CSF-1 induced non-significant slighter and higher levels of AKT and ERK1/2 phosphorylation compared to IL-34. After 6 days of culture, we observed morphological differences in the presence of IL-34 compared to CSF-1, with fewer dendrites and a more rounded morphology for IL-34-differentiated macrophages (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>), suggesting a difference in the phenotype of the differentiated macrophages. To further understand the similarities and differences of the IL-34 vs. CSF-1 induced macrophages, we performed a 3' digital gene expression RNA-sequencing (DGEseq) and compared freshly isolated CD14<sup>++</sup> monocytes (M0), 6-days differentiated macrophages in the presence of GM-CSF+IFNγ (M1), CSF-1+IL-4+IL-10 (M2), IL-34 alone, or CSF-1 alone (<xref ref-type=\"fig\" rid=\"F3\">Figures 3D–F</xref>). Transcriptomic clustering (<xref ref-type=\"fig\" rid=\"F3\">Figure 3D</xref>), principal component (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 2A</xref>), and Pearson correlation (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 2B</xref>) analyses highlighted the transcriptional changes following differentiation and indicated clear divergence between CD14<sup>++</sup> monocytes (M0) and M1-macrophages vs. all other groups and a clear convergence between M2-macrophages, IL-34-macrophages, and CSF-1-macrophages (<xref ref-type=\"fig\" rid=\"F3\">Figure 3D</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figures 2A,B</xref>). Further analysis of significant genes differentially expressed between IL-34 and CSF-1-macrophages revealed differential expression of 61 genes, with an upregulation of the expression of some interesting genes. Among those genes, we identified <italic>PDK4</italic>, a metabolic checkpoint for macrophage differentiation, <italic>CHI3L1</italic>, a carbohydrate-binding lectin that may play a role in tissue remodeling and cell capacity to respond to the environment involved in regulating Th2 cell responses and M2 macrophages differentiation, <italic>FCER1A</italic>, a receptor expressed by DCs that can play pro- or anti-inflammatory roles, and <italic>CD300A</italic>, a cell membrane receptor that contains classical ITIM motifs and negatively regulates Toll-like receptor (TLR) signaling mediated by MYD88 through the activation of PTPN6 and of macrophages in animal models (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). In contrast, we observed a down-regulation of <italic>MARCO</italic>, a marker of pro-inflammatory macrophages in IL-34-differentiated macrophages compared to CSF-1-differentiated macrophages (<xref ref-type=\"fig\" rid=\"F3\">Figure 3E</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 2C</xref>). Interestingly, further analysis of typical markers of macrophages (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>) showed a preferential expression of some genes, such as <italic>arginase-1</italic> (<italic>ARG1</italic>) in IL-34 macrophages, compared to CSF-1, M1, and M2-differenciated macrophages, or <italic>IDO1</italic> that was found expressed only in M1 macrophages (<xref ref-type=\"fig\" rid=\"F3\">Figure 3F</xref>). Other genes, like <italic>IL-10</italic>, in contrast were expressed by M2, IL-34, and CSF-1 macrophages.</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>IL-34-induced macrophages display a transcriptome close, but not identical, to M2-type and CSF-1-induced macrophages. <bold>(A,B)</bold> CD14<sup>++</sup> monocytes were cultured with IL-34 or CSF-1 for 1, 3, 5, 10, and 15 min and analyzed for phosphorylation of AKT <bold>(A)</bold> and ERK1/2 <bold>(B)</bold> by flow cytometry. Results are represented as a percentage of baseline levels (T0). <italic>n</italic> = 4 individuals. Two-way ANOVA and Bonferroni post-test compared to medium alone. *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, ***<italic>p</italic> < 0.001. <bold>(C)</bold> Photos of CD14<sup>++</sup> monocytes after 6 days of culture in the presence of IL-34 or CSF-1. X20 magnification. <bold>(D–F)</bold> CD14<sup>++</sup> monocytes were cultured for 6 days with IL-34 or CSF-1 and analyzed by DGE-RNAseq for gene expression. <bold>(D)</bold> Expression levels of differentially expressed genes between each condition are presented as a heatmap. Each column represents one sample. Blue color represents low expressed genes and red color represents highly expressed genes. The color bar shows experimental conditions. M0 are freshly sorted monocytes. <bold>(E)</bold> Volcano plot highlighting overexpressed genes (on the right, red dots) and under-expressed genes (on the left, blue dots) in IL-34-differentiated macrophages as compared with CSF-1-differentiated macrophages. The <italic>p</italic>-value adjusted cut-off is 0.05. <bold>(F)</bold> Heatmap representing expression of M1 and M2 macrophage genes in samples. Gene expression was normalized with regularized log transformations (rlog) algorithm (Deseq2), center and scaled. Blue color represents low expressed genes and red color represents highly expressed genes. Supervised clustering was performed to order samples. The color bar corresponds to experimental conditions.</p></caption><graphic xlink:href=\"fimmu-11-01496-g0003\"/></fig><p>Thus, IL-34 induced a high activation of monocytes through CSF-1R, subsequently inducing macrophages with a specific signature conferring regulatory/anti-inflammatory functions.</p></sec><sec><title>IL-34 Prolongs Survival in a Model of Humanized Acute GVHD Through Treg Expansion Rather Than Generation of Induced Treg From Naive T Cells</title><p>We highlighted previously that IL-34 treatment in a model of cardiac allo-transplantation resulted in the induction of highly suppressive Tregs through M2-like macrophages <italic>in vivo</italic> in rat and <italic>ex vivo</italic> in human (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). However, whether IL-34-induced Tregs resulted from the expansion of natural pre-existing Tregs or from newly converted Tregs from naive/effector T cells was not clear. Thus, we used an anti-CD45RC antibody (mAb) that specifically eliminates naive and precursor effector T cells (Teff) (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>) and depleted <italic>in vivo</italic> CD45RC<sup>high</sup> Teff cells using a short-term course of anti-CD45RC mAb (as we previously described) in immunodeficient NOD/SCID/IL2rγ<sup>null</sup> (NSG) mice injected with human PBMCs with or without IL-34 administration (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figures 3A,B</xref>). We observed that low-dose anti-CD45RC mAb treatment significantly delayed GVHD occurrence from 13.25 ± 0.9 days (mean survival) to 22.67 ± 2.7 days (<xref ref-type=\"fig\" rid=\"F4\">Figures 4B,C</xref>). Although, low dose IL-34 treatment every 2.5 days at 0.8 mg/kg over 20 days was not sufficient to delay GVHD; IL-34 recombinant protein in combination with anti-CD45RC mAb therapy synergized and inhibited GVHD mortality in 66% of mice (<xref ref-type=\"fig\" rid=\"F4\">Figures 4B,C</xref>). Analysis of mouse blood showed an efficient depletion of CD45RC<sup>high</sup> cells during the anti-CD45RC mAb treatment with no impact on the engraftment of other human PBMC subsets (<xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 3</xref>).</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>IL-34 in combination with depletion of naive cells prolongs survival in an acute GVHD humanized model. <bold>(A)</bold> Schematic depicting the GVHD model in humanized mice. NSG mice were injected with human PBMCs, treated or not with IL-34 protein and/or anti-CD45RC mAbs for 20 days, and followed for body weight loss. <bold>(B)</bold> Evolution of mouse body weight over time, normalized to the weight before the injection of PBMCs (D0), after no treatment (black line), IL-34 treatment (blue line), anti-CD45RC mAb treatment (green line), isotype Ig control treatment (gray line), and dual IL-34 + anti-CD45RC mAb treatment (red line). <italic>n</italic> = 3–16. Mean ± SEM is represented. Two Way repeated measure ANOVA, *<italic>p</italic> < 0.05, ***<italic>p</italic> < 0.001. <bold>(C)</bold> Percentage of mouse survival over time. <italic>n</italic> = 3–16. Log Rank (Mantel-Cox) test, *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, ***<italic>p</italic> < 0.001.</p></caption><graphic xlink:href=\"fimmu-11-01496-g0004\"/></fig><p>These results suggest that Teff cell depletion in combination with IL-34 administration can more efficiently control immune responses.</p></sec><sec><title>IL-34 Induces, More Efficiently Than CSF-1, FOXP3<sup>+</sup> Tregs Which Delay Xenogeneic GVHD</title><p>We have previously shown that long-term tolerance in an allogeneic transplant model in rats treated with IL-34 was due to CD4<sup>+</sup> and CD8<sup>+</sup> Tregs that can control transplant rejection upon adoptive cell transfer (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). We also showed that human Tregs expanded from total PBMCs in the presence of IL-34-differentiated allogeneic macrophages suppressed immune response <italic>in vitro</italic> more potently than Tregs generated with monocytes in the absence of IL-34 (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). However, we did not assess whether this effect was comparable between IL-34 and CSF-1 or how these Tregs generated with IL-34 <italic>in vitro</italic> behaved <italic>in vivo</italic>. To do so, CD14<sup>++</sup> monocytes from healthy volunteers were cell-sorted and differentiated in the presence of IL-34 or CSF-1 for 6 days and then added to allogeneic PBMCs for 14 days in the presence of IL-2 and IL-15 and a polyclonal stimulation. We thus observed that in both CD4<sup>+</sup> and CD8<sup>+</sup> T cells, IL-34 increased more efficiently the frequency of CD25<sup>+</sup>FOXP3<sup>+</sup> Tregs than CSF-1 (<xref ref-type=\"fig\" rid=\"F5\">Figures 5A,B</xref>), and this increase was even more significant for FOXP3<sup>+</sup>CD8<sup>+</sup> Tregs for which CSF-1 had little effect (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>). In addition, analysis of the number of CD4<sup>+</sup> and CD8<sup>+</sup> Tregs following a 14-day expansion in the presence of IL-34-differentiated macrophages demonstrated a higher number of total Tregs (both CD4<sup>+</sup> and CD8<sup>+</sup>) compared to expansion in the presence of CSF-1-differentiated macrophages (<xref ref-type=\"fig\" rid=\"F5\">Figure 5C</xref>).</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>IL-34 potentiates differentiation of CD4<sup>+</sup> and CD8<sup>+</sup> FOXP3<sup>+</sup>\n<italic>in vitro</italic> more effectively than CSF-1. <bold>(A)</bold> Representative FACS staining of CD25 and FOXP3 expression in CD4<sup>+</sup> (left) and CD8<sup>+</sup> (right) T cells after 14-day culture of PBMCs with either IL-34-differentiated macrophages (middle) or CSF-1-differentiated (bottom) macrophages compared to fresh cells (upper). <bold>(B)</bold> Frequency of FOXP3 positive cells in CD4<sup>+</sup> (dotted lines) and CD8<sup>+</sup> (solid line) T cells before and after expansion with IL-34- (red lines) or CSF-1- (blue lines) differentiated macrophages. Two-way ANOVA, *<italic>p</italic> < 0.05, ***<italic>p</italic> < 0.001. <bold>(C)</bold> CD4<sup>+</sup> and CD8<sup>+</sup> Tregs count harvested after 14 days of culture with IL-34- (red bars) or CSF-1- (blue bars) differentiated macrophages in fold expansion. Two-way ANOVA, *<italic>p</italic> < 0.05. <bold>(D)</bold> Schematic depicting cell culture. CD14<sup>++</sup> monocytes were sorted from a healthy volunteer (HV#1), cultured for 6 days in the presence of IL-34, then added to CD8<sup>+</sup>CD45RC<sup>low/−</sup> Tregs harvested from another healthy volunteer (HV#2) and 14-day cultured in the presence of a polyclonal stimulation once per week and IL-2 + IL-15 supplementation three times per week. <bold>(E)</bold> Treg cell count harvested after 14 days of culture with IL-34-differentiated macrophages or freshly isolated APCs normalized to Treg cell count seeded at day 0. <bold>(F)</bold> IL-34-Tregs (red bars) were analyzed by flow cytometry for Treg-associated marker expression as compared to before expansion (fresh cells, black bars). <italic>n</italic> = 3 individuals. Two-way ANOVA and Bonferroni post-test, *<italic>p</italic> < 0.05, ***<italic>p</italic> < 0.001.</p></caption><graphic xlink:href=\"fimmu-11-01496-g0005\"/></fig><p>We previously reported that polyclonal or chimeric antigen receptor (CAR)-modified CD8<sup>+</sup> Tregs can be efficiently expanded <italic>in vitro</italic> and control xenogeneic GVHD <italic>in vivo</italic> (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>, <xref rid=\"B15\" ref-type=\"bibr\">15</xref>). Given the efficacy of IL-34 to preferentially expand FOXP3<sup>+</sup> Tregs, we then assessed the therapeutic benefit of using IL-34 in the CD8<sup>+</sup> Treg expansion process for cell therapy. For this, we cultured naive CD8<sup>+</sup>CD45RC<sup>low/−</sup> Tregs from PBMCs for 14 days in the presence of macrophages differentiated from CD14<sup>++</sup> monocytes by IL-34 compared to freshly isolated APCs, IL-2, and IL-15 cytokines, and a low polyclonal anti-CD3/anti-CD28 mAbs stimulation (<xref ref-type=\"fig\" rid=\"F5\">Figure 5D</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 4A</xref>). We obtained more than an 100-fold expansion of CD8<sup>+</sup> Tregs with either IL-34-differentiated macrophages (named IL-34-Tregs) or untreated macrophages (named Tregs) (<xref ref-type=\"fig\" rid=\"F5\">Figure 5E</xref>). After expansion, IL-34-Tregs were highly enriched in FOXP3<sup>+</sup> cells, expressed higher levels of surface markers commonly related to CD4<sup>+</sup> and CD8<sup>+</sup> Tregs, such as GITR and PD-1, and cytokines such as TGFβ, IFNγ, and IL-34 that we have demonstrated as being mediators of CD8<sup>+</sup> Treg-suppressive activity (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>, <xref rid=\"B24\" ref-type=\"bibr\">24</xref>) (<xref ref-type=\"fig\" rid=\"F5\">Figure 5F</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figure 4B</xref>).</p><p>Finally, we assessed the suppressive function of IL-34-Tregs <italic>in vivo</italic> in a xenogeneic model of acute GVHD (<xref ref-type=\"fig\" rid=\"F6\">Figures 6A–C</xref>). NSG mice were first injected with human PBMCs to induce a xenogeneic acute GVHD and were either treated or not with IL-34-Tregs in a range of PBMC:Treg ratios (<xref ref-type=\"fig\" rid=\"F6\">Figures 6A–C</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM2\">Supplementary Figures 4C,D</xref>). We observed that IL-34-Tregs significantly delayed body weight loss (<xref ref-type=\"fig\" rid=\"F6\">Figure 6B</xref>) and mouse survival (<xref ref-type=\"fig\" rid=\"F6\">Figure 6C</xref>) in a dose-dependent manner compared to the control group.</p><fig id=\"F6\" position=\"float\"><label>Figure 6</label><caption><p>Cell therapy with IL-34-expanded CD8<sup>+</sup> Tregs delays aGVHD. <bold>(A)</bold> Schematic depicting treatment of mice in the model of xenogeneic GVHD. PBMCs injected are syngeneic to the expanded Tregs co-injected. <bold>(B)</bold> Mouse body weight follow-up and <bold>(C)</bold> mouse survival after PBMC injection (D0) with or without Tregs expanded in the presence of IL-34-differentiated macrophages in a range of PBMC:Tregs ratio. <italic>n</italic> = 3–8. <bold>(B)</bold> Two-way RM ANOVA, **<italic>p</italic> < 0.01. <bold>(C)</bold> Log Rank (Mantel Cox) test. *<italic>p</italic> < 0.05.</p></caption><graphic xlink:href=\"fimmu-11-01496-g0006\"/></fig><p>Altogether, these results demonstrate that IL-34 is beneficial for FOXP3<sup>+</sup> Treg expansion <italic>ex vivo</italic> and that CD8<sup>+</sup> Tregs expanded with IL-34 can control graft rejection in a dose-dependent manner.</p></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Altogether, we have demonstrated that IL-34-treated CD14<sup>++</sup>CSF-1R<sup>+</sup>PTPζ<sup>+</sup> monocytes were differentiated into pro-tolerogenic macrophages with a specific signature able to efficiently expand and potentiate FOXP3<sup>+</sup> Tregs <italic>in vitro</italic> and <italic>in vivo</italic> to control anti-donor immune responses (<xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref>).</p><fig id=\"F7\" position=\"float\"><label>Figure 7</label><caption><p>Integrated scheme of the regulatory actions of IL-34-differentiated macrophages and their ability to potentiate FOXP3<sup>+</sup> Tregs. (1) IL-34 exogenously administered or from endogenous sources, such as from Treg, acts through CSF-1R to preferentially differentiate classical and intermediate monocytes into regulatory macrophages (2). (3) IL-34-differentiated macrophages expand and enhance the suppressive phenotype of both CD4<sup>+</sup>CD25<sup>+</sup>CD127<sup>low</sup> and CD8<sup>+</sup>CD45RC<sup>low/−</sup> Tregs. IL-34 secretion by Tregs maintains and increases the regulatory loop and can act in an autocrine fashion on Tregs. (4) In GVHD in NSG mouse, expanded CD8<sup>+</sup> Tregs efficiently delay GVHD incidence. Dashed arrow, induction; solid arrow, binding.</p></caption><graphic xlink:href=\"fimmu-11-01496-g0007\"/></fig><p>We found the expression of CSF-1R and PTPζ mostly on CD14<sup>++</sup> classical and intermediate monocytes, although we found a more significant expression of both receptors on non-classical CD16<sup>++</sup> monocytes (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B26\" ref-type=\"bibr\">26</xref>). As for CSF-1, IL-34 could polarize all three subtypes of monocytes into type 2 (M2) macrophages depending on the environment (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). Non-classical macrophages in particular play an important role in the control of immune responses and have also been associated with wound-healing and resolution of inflammation in damaged tissues (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). PTPζ expression was mostly reported in the brain and, more recently, in the kidney (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B29\" ref-type=\"bibr\">29</xref>), while its expression on monocytes has only been suggested by western blotting (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>); thus, our study confirms that both CSF-1R and PTPζ are expressed at the protein level by monocytes, suggesting that IL-34 action on monocytes through both PTPζ and CSF-1R could explain the differential effect compared to CSF-1. The intracellular signaling through PTPζ in monocytes still needs to be analyzed. The differential effects of IL-34 and CSF-1 can also be explained by the different binding characteristics and signaling through the CSF-1R that are discussed below.</p><p>We did not observe CSF-1R and PTPζ expression on resting total T cells, including Tregs, in single cell RNAseq data analysis of total PBMCs, probably because of the low frequency of Tregs and the low frequency of CSF-1R in Tregs compared to monocytes. However, using antibody staining, we were able to find a low expression of the protein CSF-1R on resting CD4<sup>+</sup> and CD8<sup>+</sup> FOXP3<sup>+</sup> Tregs and upon stimulation this expression was significantly increased on activated CD4<sup>+</sup> and CD8<sup>+</sup> FOXP3<sup>+</sup> Tregs. Thus it is possible that IL-34 acts directly on Treg polarization as TGFβ and IL-2, or on Treg function, in addition to acting through monocytes (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>), and this will need to be further investigated.</p><p>Surprisingly, we did not observe any expression of CSF-1R in expanded FOXP3<sup>+</sup> Tregs (data not shown), suggesting a transient expression of CSF-1R in Tregs upon activation and a narrow window for IL-34 to act directly on those cells. This further suggests a synergistic effect of IL-34 on monocytes and recently activated Tregs that supports the therapeutic strategy based on a short course treatment with IL-34 to induce tolerogenic monocytes and Tregs right after an immune challenge.</p><p>Although IL-34 and CSF-1 bind to the same receptor, CSF-1R, on the same cells, IL-34 can also act through PTPζ binding on monocytes, resulting in a different potential to induce FOXP3<sup>+</sup> Tregs <italic>in vitro</italic>. They are several hypotheses to explain this important difference in their respective capacity to induce FOXP3<sup>+</sup> Tregs (both CD4<sup>+</sup> and CD8<sup>+</sup>). IL-34 and CSF-1 have very different sequences and structures, as well as a different affinity for CSF-1R (IL-34 has an affinity 34-fold superior to the one of CSF-1 for CSF-1R) (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B32\" ref-type=\"bibr\">32</xref>), and although they establish structurally similar binding to CSF-1R, it is possible that the subsequent signaling and the signaling and transcriptional pathways involved in the differentiation of the monocytes to macrophages and the phenotype of the differentiated macrophages are different (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>, <xref rid=\"B34\" ref-type=\"bibr\">34</xref>). The higher affinity of IL-34 to CSF-1R would suggest a more important signal transduction for IL-34 compared to CSF-1. In addition, the expression of PTPζ probably impacts on CSF-1R-signaling in monocytes. Whether PTPζ reinforces, weakens, fastens, or slows down the signal induced through CSF-1R needs further investigation. We observed that IL-34 and CSF-1 induced in a similar manner the phosphorylation of AKT and ERK1/2, two molecules involved in the signaling of both CSF-1R and PTPζ molecules. In addition, although we did not observe striking differences in the global transcriptomic profile of 6-days differentiated macrophages with either IL-34 or CSF-1, we did observe several functionally important genes differentially regulated. <italic>Arginase-1</italic> mRNA was highly and specifically increased in IL-34-differentiated macrophages. Arginase-1 degrades arginine, deprives NO synthase of its substrate, down-regulates nitric oxide production, and is one of the key factors by which regulatory macrophages or myeloid-derived suppressor cells suppress T cell responses (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>, <xref rid=\"B36\" ref-type=\"bibr\">36</xref>). Arginase-1<sup>+</sup> macrophages also promote wound-healing and decrease T cell activation and induce it when tolerance is sought or when targeting Arginase-1 in cancer is the focus of current efforts (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>). We also observed significant upregulation of other genes, such as <italic>PDK4</italic>, a metabolic checkpoint for macrophage differentiation (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>), <italic>CHI3L1</italic>, a marker of M2 macrophages (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>), <italic>FCER1A</italic>, a receptor expressed by DCs and a few monocytes that can play pro- or anti-inflammatory roles (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>, <xref rid=\"B42\" ref-type=\"bibr\">42</xref>), or <italic>CD300A</italic>, a negative regulator of TLR signaling in IL-34-differentiated macrophages compared to CSF-1-differentiated macrophages, emphasizing the differences between IL-34 vs. CSF-1. Interestingly, we found several genes involved in macrophage phagocytosis downregulated [i.e., <italic>MARCO</italic> (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>–<xref rid=\"B45\" ref-type=\"bibr\">45</xref>), <italic>A2M</italic> (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>, <xref rid=\"B47\" ref-type=\"bibr\">47</xref>), <italic>VSIG4</italic> (<xref rid=\"B48\" ref-type=\"bibr\">48</xref>), or <italic>COLEC12</italic> (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>, <xref rid=\"B50\" ref-type=\"bibr\">50</xref>)] or inhibitors of phagocytosis upregulated such as <italic>CD300A</italic> (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>) in IL-34-differentiated macrophages compared to CSF-1-differentiated macrophages, suggesting a decreased capacity to phagocytes compared to CSF-1 (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>), but this will need further investigation. Although we found a low number of genes differentially regulated between CSF-1- and IL-34-differentiated macrophages, these markers emphasized the difference of activity on CSF-1R and/or the impact of the exclusive binding of IL-34 on PTPζ. The role of these different genes on the observed promoting effect of IL-34 on Treg induction will also need further investigation.</p><p>The capacity of IL-34 to induce both CD4<sup>+</sup> and CD8<sup>+</sup> Tregs is interesting as it would suggest that both CD4<sup>+</sup> and CD8<sup>+</sup> FOXP3<sup>+</sup> cells could be expanded together without cell sorting from total PBMCs and then the final product, enriched in both Treg subsets, could be administered subsequently <italic>in vivo</italic>. Maybe elimination of Teff and naive cells using anti-CD45RC mAbs, for example, as we showed <italic>in vivo</italic> that it was beneficial for IL-34-therapeutic potential, would also be beneficial <italic>in vitro</italic> in the expansion protocol (i.e., depletion of CD45RC<sup>+</sup> cells by cell sorting). These results obtained with the anti-CD45RC mAb suggest that naive/effector T cells were not involved in IL-34 establishment of a control of immune responses and that Tregs were rather expanded cells than newly-generated cells. Although we cannot conclude on a direct effect of IL-34 on Tregs in this experiment, since human IL-34 does not cross-react on murine cells and can only act on human cells and since in this model of humanized mice, GVHD is mediated mostly by T cells, this suggests a direct effect of IL-34 on Tregs and will need to be the subject of further investigations. The synergy between IL-34 and anti-CD45RC mAb also suggests that <italic>in vivo</italic> IL-34 efficacy may be limited by Teff cells. Although the synergistic capacity of CD4<sup>+</sup> and CD8<sup>+</sup> Tregs is not yet clear, both subsets could show complementary effects and it could be beneficial to administer them together to patients (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). IL-34 could also be used <italic>in vivo</italic> together with Treg cell therapy to promote the persistence and the function of the induced Tregs, as is done with low-dose IL-2 or rapamycin (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>, <xref rid=\"B53\" ref-type=\"bibr\">53</xref>), by enrichment of the environment with tolerogenic macrophages and by direct action on Tregs. We have tested <italic>in vivo</italic> the FOXP3<sup>+</sup>CD8<sup>+</sup> Tregs induced in the 14-day <italic>ex vivo</italic> expansion in a model of xenogeneic GVHD in immune-humanized mice, and we have observed a similar protective potential of the Tregs compared to what we have previously demonstrated using polyclonally expanded CD8<sup>+</sup> Tregs (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>). Thus, it suggests that efficient Tregs were expanded, even from total PBMCs as a starting material, which shows similar protection compared to Tregs expanded without IL-34. Thus, an important advantage of using IL-34 would be the co-expansion of CD4<sup>+</sup> and CD8<sup>+</sup> FOXP3<sup>+</sup> Tregs from total PBMCs. Also, this suggests that upon improvement of this protocol, with for example selective effector T cell depletion before expansion, it could result in improved protection.</p><p>Altogether, our results highlight the potential of IL-34 to favor the development of FOXP3<sup>+</sup> Tregs and suggest that this cytokine should be further considered for <italic>in vitro</italic> use or <italic>in vivo</italic> therapy.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/<xref ref-type=\"sec\" rid=\"s9\">Supplementary Material</xref>.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by Ministry of Research. Blood from healthy individuals was obtained at the Etablissement Français du Sang (Nantes, France). Written informed consent was provided according to institutional guidelines.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>CG and IA contributed conception and design of the study. CG, SB, and IA wrote sections of the manuscript. SB, AF, CS, AS, and NV performed experiments and analyzed data. All authors contributed to manuscript revision, read, and approved the submitted version.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>CG, IA, and SB have patents on IL-34 that are pending and are entitled to a share in net income generated from the licensing of these patent rights for commercial development. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>We thank Dimitri Meistermann and Eric Charpentier for their advice on biostatistical analysis. We thank P. Guerif, I. Guihard, and A. Fleury from the CHU of Nantes for their help. We thank the humanized rodents platform from the Labex IGO. We thank the Fondation Progreffe for financial support and Crédit Agricole for the donation of the FACSAria.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was realized in the context and partially funded by the Labex IGO program, supported by the National Research Agency via the investment of the future program ANR-11-LABX-0016-01. This work was also realized in the context of the IHU-Cesti project which received French Government financial support managed by the National Research Agency via the investment of the future program ANR-10-IBHU-005. The IHU-Cesti project is also supported by Nantes Metropole and the Pays de la Loire Region. This work was funded by the Agence Nationale de la Recherche ANR-17-CE18-0008, the Fondation du rein Don de Soi – Don de Vie 2017 FdR-Trans-Forme/FRM and the Agence de la Biomedecine.</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s9\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fimmu.2020.01496/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fimmu.2020.01496/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Table_1.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"SM2\"><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Marcen</surname><given-names>R</given-names></name></person-group>. <article-title>Immunosuppressive drugs in kidney transplantation: impact on patient survival, and incidence of cardiovascular disease, malignancy and infection</article-title>. <source>Drugs.</source> (<year>2009</year>) <volume>69</volume>:<fpage>2227</fpage>–<lpage>43</lpage>. <pub-id pub-id-type=\"doi\">10.2165/11319260-000000000-00000</pub-id><pub-id pub-id-type=\"pmid\">19852526</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Horwitz</surname><given-names>ME</given-names></name><name><surname>Sullivan</surname><given-names>KM</given-names></name></person-group>. <article-title>Chronic graft-versus-host disease</article-title>. <source>Blood Rev.</source> (<year>2006</year>) <volume>20</volume>:<fpage>15</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.blre.2005.01.007</pub-id><pub-id pub-id-type=\"pmid\">16426941</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wingard</surname><given-names>JR</given-names></name><name><surname>Majhail</surname><given-names>NS</given-names></name><name><surname>Brazauskas</surname><given-names>R</given-names></name><name><surname>Wang</surname><given-names>Z</given-names></name><name><surname>Sobocinski</surname><given-names>KA</given-names></name><name><surname>Jacobsohn</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Long-term survival and late deaths after allogeneic hematopoietic cell transplantation</article-title>. <source>J Clin Oncol.</source> (<year>2011</year>) <volume>29</volume>:<fpage>2230</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1200/JCO.2010.33.7212</pub-id><pub-id pub-id-type=\"pmid\">21464398</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Meier-Kriesche</surname><given-names>HU</given-names></name><name><surname>Li</surname><given-names>S</given-names></name><name><surname>Gruessner</surname><given-names>RW</given-names></name><name><surname>Fung</surname><given-names>JJ</given-names></name><name><surname>Bustami</surname><given-names>RT</given-names></name><name><surname>Barr</surname><given-names>ML</given-names></name><etal/></person-group>. <article-title>Immunosuppression: evolution in practice and trends, 1994-2004</article-title>. <source>Am J Transplant.</source> (<year>2006</year>) <volume>6</volume>:<fpage>1111</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1600-6143.2006.01270.x</pub-id><pub-id pub-id-type=\"pmid\">16613591</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lin</surname><given-names>H</given-names></name><name><surname>Lee</surname><given-names>E</given-names></name><name><surname>Hestir</surname><given-names>K</given-names></name><name><surname>Leo</surname><given-names>C</given-names></name><name><surname>Huang</surname><given-names>M</given-names></name><name><surname>Bosch</surname><given-names>E</given-names></name><etal/></person-group>. <article-title>Discovery of a cytokine and its receptor by functional screening of the extracellular proteome</article-title>. <source>Science.</source> (<year>2008</year>) <volume>320</volume>:<fpage>807</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1126/science.1154370</pub-id><pub-id pub-id-type=\"pmid\">18467591</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nandi</surname><given-names>S</given-names></name><name><surname>Cioce</surname><given-names>M</given-names></name><name><surname>Yeung</surname><given-names>YG</given-names></name><name><surname>Nieves</surname><given-names>E</given-names></name><name><surname>Tesfa</surname><given-names>L</given-names></name><name><surname>Lin</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Receptor-type protein-tyrosine phosphatase zeta is a functional receptor for interleukin-34</article-title>. <source>J Biol Chem.</source> (<year>2013</year>) <volume>288</volume>:<fpage>21972</fpage>–<lpage>86</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M112.442731</pub-id><pub-id pub-id-type=\"pmid\">23744080</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Segaliny</surname><given-names>AI</given-names></name><name><surname>Brion</surname><given-names>R</given-names></name><name><surname>Mortier</surname><given-names>E</given-names></name><name><surname>Maillasson</surname><given-names>M</given-names></name><name><surname>Cherel</surname><given-names>M</given-names></name><name><surname>Jacques</surname><given-names>Y</given-names></name><etal/></person-group>. <article-title>Syndecan-1 regulates the biological activities of interleukin-34</article-title>. <source>Biochim Biophys Acta.</source> (<year>2015</year>) <volume>1853</volume>:<fpage>1010</fpage>–<lpage>21</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbamcr.2015.01.023</pub-id><pub-id pub-id-type=\"pmid\">25662098</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>H</given-names></name><name><surname>Leo</surname><given-names>C</given-names></name><name><surname>Chen</surname><given-names>X</given-names></name><name><surname>Wong</surname><given-names>BR</given-names></name><name><surname>Williams</surname><given-names>LT</given-names></name><name><surname>Lin</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>The mechanism of shared but distinct CSF-1R signaling by the non-homologous cytokines IL-34 and CSF-1</article-title>. <source>Biochim Biophys Acta.</source> (<year>2012</year>) <volume>1824</volume>:<fpage>938</fpage>–<lpage>45</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbapap.2012.04.012</pub-id><pub-id pub-id-type=\"pmid\">22579672</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>Y</given-names></name><name><surname>Szretter</surname><given-names>KJ</given-names></name><name><surname>Vermi</surname><given-names>W</given-names></name><name><surname>Gilfillan</surname><given-names>S</given-names></name><name><surname>Rossini</surname><given-names>C</given-names></name><name><surname>Cella</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia</article-title>. <source>Nat Immunol.</source> (<year>2012</year>) <volume>13</volume>:<fpage>753</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.1038/ni.2360</pub-id><pub-id pub-id-type=\"pmid\">22729249</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Greter</surname><given-names>M</given-names></name><name><surname>Lelios</surname><given-names>I</given-names></name><name><surname>Pelczar</surname><given-names>P</given-names></name><name><surname>Hoeffel</surname><given-names>G</given-names></name><name><surname>Price</surname><given-names>J</given-names></name><name><surname>Leboeuf</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Stroma-derived interleukin-34 controls the development and maintenance of langerhans cells and the maintenance of microglia</article-title>. <source>Immunity.</source> (<year>2012</year>) <volume>37</volume>:<fpage>1050</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.immuni.2012.11.001</pub-id><pub-id pub-id-type=\"pmid\">23177320</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Guillonneau</surname><given-names>C</given-names></name><name><surname>Bezie</surname><given-names>S</given-names></name><name><surname>Anegon</surname><given-names>I</given-names></name></person-group>. <article-title>Immunoregulatory properties of the cytokine IL-34</article-title>. <source>Cell Mol Life Sci.</source> (<year>2017</year>) <volume>74</volume>:<fpage>2569</fpage>–<lpage>86</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00018-017-2482-4</pub-id><pub-id pub-id-type=\"pmid\">28258292</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bezie</surname><given-names>S</given-names></name><name><surname>Picarda</surname><given-names>E</given-names></name><name><surname>Ossart</surname><given-names>J</given-names></name><name><surname>Tesson</surname><given-names>L</given-names></name><name><surname>Usal</surname><given-names>C</given-names></name><name><surname>Renaudin</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>IL-34 is a Treg-specific cytokine and mediates transplant tolerance</article-title>. <source>J Clin Invest.</source> (<year>2015</year>) <volume>125</volume>:<fpage>3952</fpage>–<lpage>64</lpage>. <pub-id pub-id-type=\"doi\">10.1172/JCI81227</pub-id><pub-id pub-id-type=\"pmid\">26389674</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Picarda</surname><given-names>E</given-names></name><name><surname>Bezie</surname><given-names>S</given-names></name><name><surname>Boucault</surname><given-names>L</given-names></name><name><surname>Autrusseau</surname><given-names>E</given-names></name><name><surname>Kilens</surname><given-names>S</given-names></name><name><surname>Meistermann</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Transient antibody targeting of CD45RC induces transplant tolerance and potent antigen-specific regulatory T cells</article-title>. <source>JCI insight.</source> (<year>2017</year>) <volume>2</volume>:<fpage>e90088</fpage>. <pub-id pub-id-type=\"doi\">10.1172/jci.insight.90088</pub-id><pub-id pub-id-type=\"pmid\">28194440</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bezie</surname><given-names>S</given-names></name><name><surname>Meistermann</surname><given-names>D</given-names></name><name><surname>Boucault</surname><given-names>L</given-names></name><name><surname>Kilens</surname><given-names>S</given-names></name><name><surname>Zoppi</surname><given-names>J</given-names></name><name><surname>Autrusseau</surname><given-names>E</given-names></name><etal/></person-group>. <article-title><italic>Ex vivo</italic> expanded human non-cytotoxic CD8(+)CD45RC(low/-) Tregs efficiently delay skin graft rejection and GVHD in humanized mice</article-title>. <source>Front Immunol.</source> (<year>2017</year>) <volume>8</volume>:<fpage>2014</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2017.02014</pub-id><pub-id pub-id-type=\"pmid\">29445370</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bezie</surname><given-names>S</given-names></name><name><surname>Charreau</surname><given-names>B</given-names></name><name><surname>Vimond</surname><given-names>N</given-names></name><name><surname>Lasselin</surname><given-names>J</given-names></name><name><surname>Gerard</surname><given-names>N</given-names></name><name><surname>Nerriere-Daguin</surname><given-names>V</given-names></name><etal/></person-group>. <article-title>Human CD8+ Tregs expressing a MHC-specific CAR display enhanced suppression of human skin rejection and GVHD in NSG mice</article-title>. <source>Blood Adv.</source> (<year>2019</year>) <volume>3</volume>:<fpage>3522</fpage>–<lpage>38</lpage>. <pub-id pub-id-type=\"doi\">10.1182/bloodadvances.2019000411</pub-id><pub-id pub-id-type=\"pmid\">31730699</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ziegler-Heitbrock</surname><given-names>L</given-names></name><name><surname>Ancuta</surname><given-names>P</given-names></name><name><surname>Crowe</surname><given-names>S</given-names></name><name><surname>Dalod</surname><given-names>M</given-names></name><name><surname>Grau</surname><given-names>V</given-names></name><name><surname>Hart</surname><given-names>DN</given-names></name><etal/></person-group>. <article-title>Nomenclature of monocytes and dendritic cells in blood</article-title>. <source>Blood.</source> (<year>2010</year>) <volume>116</volume>:<fpage>e74</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.1182/blood-2010-02-258558</pub-id><pub-id pub-id-type=\"pmid\">20628149</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reynolds</surname><given-names>G</given-names></name><name><surname>Haniffa</surname><given-names>M</given-names></name></person-group>. <article-title>Human and mouse mononuclear phagocyte networks: a tale of two species?</article-title>\n<source>Front Immunol.</source> (<year>2015</year>) <volume>6</volume>:<fpage>330</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2015.00330</pub-id><pub-id pub-id-type=\"pmid\">26124761</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Foucher</surname><given-names>ED</given-names></name><name><surname>Blanchard</surname><given-names>S</given-names></name><name><surname>Preisser</surname><given-names>L</given-names></name><name><surname>Garo</surname><given-names>E</given-names></name><name><surname>Ifrah</surname><given-names>N</given-names></name><name><surname>Guardiola</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>IL-34 induces the differentiation of human monocytes into immunosuppressive macrophages antagonistic effects of GM-CSF and IFNgamma</article-title>. <source>PLoS ONE.</source> (<year>2013</year>) <volume>8</volume>:<fpage>e56045</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0056045</pub-id><pub-id pub-id-type=\"pmid\">23409120</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Murray</surname><given-names>PJ</given-names></name></person-group>. <article-title>Macrophage polarization</article-title>. <source>Annu Rev Physiol.</source> (<year>2017</year>) <volume>79</volume>:<fpage>541</fpage>–<lpage>66</lpage>. <pub-id pub-id-type=\"doi\">10.1146/annurev-physiol-022516-034339</pub-id><pub-id pub-id-type=\"pmid\">27813830</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>Z</given-names></name><name><surname>Buki</surname><given-names>K</given-names></name><name><surname>Vaaraniemi</surname><given-names>J</given-names></name><name><surname>Gu</surname><given-names>G</given-names></name><name><surname>Vaananen</surname><given-names>HK</given-names></name></person-group>. <article-title>The critical role of IL-34 in osteoclastogenesis</article-title>. <source>PLoS ONE.</source> (<year>2011</year>) <volume>6</volume>:<fpage>e18689</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0018689</pub-id><pub-id pub-id-type=\"pmid\">21494622</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Valiate</surname><given-names>BVS</given-names></name><name><surname>Alvarez</surname><given-names>RU</given-names></name><name><surname>Karra</surname><given-names>L</given-names></name><name><surname>Queiroz-Junior</surname><given-names>CM</given-names></name><name><surname>Amaral</surname><given-names>FA</given-names></name><name><surname>Levi-Schaffer</surname><given-names>F</given-names></name><etal/></person-group>. <article-title>The immunoreceptor CD300a controls the intensity of inflammation and dysfunction in a model of Ag-induced arthritis in mice</article-title>. <source>J Leukoc Biol.</source> (<year>2019</year>) <volume>106</volume>:<fpage>957</fpage>–<lpage>66</lpage>. <pub-id pub-id-type=\"doi\">10.1002/JLB.3A1018-389R</pub-id><pub-id pub-id-type=\"pmid\">31107994</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Viola</surname><given-names>A</given-names></name><name><surname>Munari</surname><given-names>F</given-names></name><name><surname>Sanchez-Rodriguez</surname><given-names>R</given-names></name><name><surname>Scolaro</surname><given-names>T</given-names></name><name><surname>Castegna</surname><given-names>A</given-names></name></person-group>. <article-title>The metabolic signature of macrophage responses</article-title>. <source>Front Immunol.</source> (<year>2019</year>) <volume>10</volume>:<fpage>1462</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2019.01462</pub-id><pub-id pub-id-type=\"pmid\">31333642</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bezie</surname><given-names>S</given-names></name><name><surname>Anegon</surname><given-names>I</given-names></name><name><surname>Guillonneau</surname><given-names>C</given-names></name></person-group>. <article-title>Advances on CD8+ Treg cells and their potential in transplantation</article-title>. <source>Transplantation.</source> (<year>2018</year>) <volume>102</volume>:<fpage>1467</fpage>–<lpage>78</lpage>. <pub-id pub-id-type=\"doi\">10.1097/TP.0000000000002258</pub-id><pub-id pub-id-type=\"pmid\">29688996</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Flippe</surname><given-names>L</given-names></name><name><surname>Bezie</surname><given-names>S</given-names></name><name><surname>Anegon</surname><given-names>I</given-names></name><name><surname>Guillonneau</surname><given-names>C</given-names></name></person-group>. <article-title>Future prospects for CD8(+) regulatory T cells in immune tolerance</article-title>. <source>Immunol Rev.</source> (<year>2019</year>) <volume>292</volume>:<fpage>209</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1111/imr.12812</pub-id><pub-id pub-id-type=\"pmid\">31593314</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kapellos</surname><given-names>TS</given-names></name><name><surname>Bonaguro</surname><given-names>L</given-names></name><name><surname>Gemund</surname><given-names>I</given-names></name><name><surname>Reusch</surname><given-names>N</given-names></name><name><surname>Saglam</surname><given-names>A</given-names></name><name><surname>Hinkley</surname><given-names>ER</given-names></name><etal/></person-group>. <article-title>Human monocyte subsets and phenotypes in major chronic inflammatory diseases</article-title>. <source>Front Immunol.</source> (<year>2019</year>) <volume>10</volume>:<fpage>2035</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2019.02035</pub-id><pub-id pub-id-type=\"pmid\">31543877</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ziegler-Heitbrock</surname><given-names>L</given-names></name></person-group>. <article-title>The CD14+ CD16+ blood monocytes: their role in infection and inflammation</article-title>. <source>J Leukoc Biol.</source> (<year>2007</year>) <volume>81</volume>:<fpage>584</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.1189/jlb.0806510</pub-id><pub-id pub-id-type=\"pmid\">17135573</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Murray</surname><given-names>PJ</given-names></name><name><surname>Allen</surname><given-names>JE</given-names></name><name><surname>Biswas</surname><given-names>SK</given-names></name><name><surname>Fisher</surname><given-names>EA</given-names></name><name><surname>Gilroy</surname><given-names>DW</given-names></name><name><surname>Goerdt</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Macrophage activation and polarization: nomenclature and experimental guidelines</article-title>. <source>Immunity.</source> (<year>2014</year>) <volume>41</volume>:<fpage>14</fpage>–<lpage>20</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.immuni.2014.06.008</pub-id><pub-id pub-id-type=\"pmid\">25035950</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thomas</surname><given-names>G</given-names></name><name><surname>Tacke</surname><given-names>R</given-names></name><name><surname>Hedrick</surname><given-names>CC</given-names></name><name><surname>Hanna</surname><given-names>RN</given-names></name></person-group>. <article-title>Nonclassical patrolling monocyte function in the vasculature</article-title>. <source>Arteriosc Thromb Vasc Biol.</source> (<year>2015</year>) <volume>35</volume>:<fpage>1306</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.1161/ATVBAHA.114.304650</pub-id><pub-id pub-id-type=\"pmid\">25838429</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sanchez-Nino</surname><given-names>MD</given-names></name><name><surname>Sanz</surname><given-names>AB</given-names></name><name><surname>Ortiz</surname><given-names>A</given-names></name></person-group>. <article-title>Chronicity following ischaemia-reperfusion injury depends on tubular-macrophage crosstalk involving two tubular cell-derived CSF-1R activators: CSF-1 and IL-34</article-title>. <source>Nephrol Dialysis Transplant.</source> (<year>2016</year>) <volume>31</volume>:<fpage>1409</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.1093/ndt/gfw026</pub-id><pub-id pub-id-type=\"pmid\">27190368</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zwicker</surname><given-names>S</given-names></name><name><surname>Bureik</surname><given-names>D</given-names></name><name><surname>Bosma</surname><given-names>M</given-names></name><name><surname>Martinez</surname><given-names>GL</given-names></name><name><surname>Almer</surname><given-names>S</given-names></name><name><surname>Bostrom</surname><given-names>EA</given-names></name></person-group>. <article-title>Receptor-Type protein-tyrosine phosphatase zeta and colony stimulating factor-1 receptor in the intestine: cellular expression and cytokine- and chemokine responses by interleukin-34 and colony stimulating factor-1</article-title>. <source>PLoS ONE.</source> (<year>2016</year>) <volume>11</volume>:<fpage>e0167324</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0167324</pub-id><pub-id pub-id-type=\"pmid\">27898738</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scheinecker</surname><given-names>C</given-names></name><name><surname>Goschl</surname><given-names>L</given-names></name><name><surname>Bonelli</surname><given-names>M</given-names></name></person-group>. <article-title>Treg cells in health and autoimmune diseases: new insights from single cell analysis</article-title>. <source>J Autoimmun.</source> (<year>2019</year>) <volume>110</volume>:<fpage>102376</fpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jaut.2019.102376</pub-id><pub-id pub-id-type=\"pmid\">31862128</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Felix</surname><given-names>J</given-names></name><name><surname>Elegheert</surname><given-names>J</given-names></name><name><surname>Gutsche</surname><given-names>I</given-names></name><name><surname>Shkumatov</surname><given-names>AV</given-names></name><name><surname>Wen</surname><given-names>Y</given-names></name><name><surname>Bracke</surname><given-names>N</given-names></name><etal/></person-group>. <article-title>Human IL-34 and CSF-1 establish structurally similar extracellular assemblies with their common hematopoietic receptor</article-title>. <source>Structure.</source> (<year>2013</year>) <volume>21</volume>:<fpage>528</fpage>–<lpage>39</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.str.2013.01.018</pub-id><pub-id pub-id-type=\"pmid\">23478061</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chihara</surname><given-names>T</given-names></name><name><surname>Suzu</surname><given-names>S</given-names></name><name><surname>Hassan</surname><given-names>R</given-names></name><name><surname>Chutiwitoonchai</surname><given-names>N</given-names></name><name><surname>Hiyoshi</surname><given-names>M</given-names></name><name><surname>Motoyoshi</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>IL-34 and M-CSF share the receptor Fms but are not identical in biological activity and signal activation</article-title>. <source>Cell Death Differ.</source> (<year>2010</year>) <volume>17</volume>:<fpage>1917</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.1038/cdd.2010.60</pub-id><pub-id pub-id-type=\"pmid\">20489731</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boulakirba</surname><given-names>S</given-names></name><name><surname>Pfeifer</surname><given-names>A</given-names></name><name><surname>Mhaidly</surname><given-names>R</given-names></name><name><surname>Obba</surname><given-names>S</given-names></name><name><surname>Goulard</surname><given-names>M</given-names></name><name><surname>Schmitt</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>IL-34 and CSF-1 display an equivalent macrophage differentiation ability but a different polarization potential</article-title>. <source>Sci Rep.</source> (<year>2018</year>) <volume>8</volume>:<fpage>256</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-017-18433-4</pub-id><pub-id pub-id-type=\"pmid\">29321503</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scalea</surname><given-names>JR</given-names></name><name><surname>Lee</surname><given-names>YS</given-names></name><name><surname>Davila</surname><given-names>E</given-names></name><name><surname>Bromberg</surname><given-names>JS</given-names></name></person-group>. <article-title>Myeloid-derived suppressor cells and their potential application in transplantation</article-title>. <source>Transplantation.</source> (<year>2018</year>) <volume>102</volume>:<fpage>359</fpage>–<lpage>67</lpage>. <pub-id pub-id-type=\"doi\">10.1097/TP.0000000000002022</pub-id><pub-id pub-id-type=\"pmid\">29189485</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arlauckas</surname><given-names>SP</given-names></name><name><surname>Garren</surname><given-names>SB</given-names></name><name><surname>Garris</surname><given-names>CS</given-names></name><name><surname>Kohler</surname><given-names>RH</given-names></name><name><surname>Oh</surname><given-names>J</given-names></name><name><surname>Pittet</surname><given-names>MJ</given-names></name><etal/></person-group>. <article-title>Arg1 expression defines immunosuppressive subsets of tumor-associated macrophages</article-title>. <source>Theranostics.</source> (<year>2018</year>) <volume>8</volume>:<fpage>5842</fpage>–<lpage>54</lpage>. <pub-id pub-id-type=\"doi\">10.7150/thno.26888</pub-id><pub-id pub-id-type=\"pmid\">30613266</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bronte</surname><given-names>V</given-names></name><name><surname>Murray</surname><given-names>PJ</given-names></name></person-group>. <article-title>Understanding local macrophage phenotypes in disease: modulating macrophage function to treat cancer</article-title>. <source>Nat Med.</source> (<year>2015</year>) <volume>21</volume>:<fpage>117</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nm.3794</pub-id><pub-id pub-id-type=\"pmid\">25654601</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Steggerda</surname><given-names>SM</given-names></name><name><surname>Bennett</surname><given-names>MK</given-names></name><name><surname>Chen</surname><given-names>J</given-names></name><name><surname>Emberley</surname><given-names>E</given-names></name><name><surname>Huang</surname><given-names>T</given-names></name><name><surname>Janes</surname><given-names>JR</given-names></name><etal/></person-group>. <article-title>Inhibition of arginase by CB-1158 blocks myeloid cell-mediated immune suppression in the tumor microenvironment</article-title>. <source>J Immunother Cancer.</source> (<year>2017</year>) <volume>5</volume>:<fpage>101</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s40425-017-0308-4</pub-id><pub-id pub-id-type=\"pmid\">29254508</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Min</surname><given-names>BK</given-names></name><name><surname>Park</surname><given-names>S</given-names></name><name><surname>Kang</surname><given-names>HJ</given-names></name><name><surname>Kim</surname><given-names>DW</given-names></name><name><surname>Ham</surname><given-names>HJ</given-names></name><name><surname>Ha</surname><given-names>CM</given-names></name><etal/></person-group>. <article-title>Pyruvate dehydrogenase kinase is a metabolic checkpoint for polarization of macrophages to the M1 phenotype</article-title>. <source>Front Immunol.</source> (<year>2019</year>) <volume>10</volume>:<fpage>944</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2019.00944</pub-id><pub-id pub-id-type=\"pmid\">31134063</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rehli</surname><given-names>M</given-names></name><name><surname>Niller</surname><given-names>HH</given-names></name><name><surname>Ammon</surname><given-names>C</given-names></name><name><surname>Langmann</surname><given-names>S</given-names></name><name><surname>Schwarzfischer</surname><given-names>L</given-names></name><name><surname>Andreesen</surname><given-names>R</given-names></name><etal/></person-group>. <article-title>Transcriptional regulation of CHI3L1, a marker gene for late stages of macrophage differentiation</article-title>. <source>J Biol Chem.</source> (<year>2003</year>) <volume>278</volume>:<fpage>44058</fpage>–<lpage>67</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M306792200</pub-id><pub-id pub-id-type=\"pmid\">12933821</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tang-Huau</surname><given-names>TL</given-names></name><name><surname>Gueguen</surname><given-names>P</given-names></name><name><surname>Goudot</surname><given-names>C</given-names></name><name><surname>Durand</surname><given-names>M</given-names></name><name><surname>Bohec</surname><given-names>M</given-names></name><name><surname>Baulande</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Human <italic>in vivo</italic>-generated monocyte-derived dendritic cells and macrophages cross-present antigens through a vacuolar pathway</article-title>. <source>Nat Commun.</source> (<year>2018</year>) <volume>9</volume>:<fpage>2570</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41467-018-04985-0</pub-id><pub-id pub-id-type=\"pmid\">29967419</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shin</surname><given-names>JS</given-names></name><name><surname>Greer</surname><given-names>AM</given-names></name></person-group>. <article-title>The role of FcepsilonRI expressed in dendritic cells and monocytes</article-title>. <source>Cell Mol Life Sci.</source> (<year>2015</year>) <volume>72</volume>:<fpage>2349</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00018-015-1870-x</pub-id><pub-id pub-id-type=\"pmid\">25715742</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Georgoudaki</surname><given-names>AM</given-names></name><name><surname>Prokopec</surname><given-names>KE</given-names></name><name><surname>Boura</surname><given-names>VF</given-names></name><name><surname>Hellqvist</surname><given-names>E</given-names></name><name><surname>Sohn</surname><given-names>S</given-names></name><name><surname>Ostling</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Reprogramming tumor-associated macrophages by antibody targeting inhibits cancer progression and metastasis</article-title>. <source>Cell Rep.</source> (<year>2016</year>) <volume>15</volume>:<fpage>2000</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.celrep.2016.04.084</pub-id><pub-id pub-id-type=\"pmid\">27210762</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kang</surname><given-names>MJ</given-names></name><name><surname>Jang</surname><given-names>AR</given-names></name><name><surname>Park</surname><given-names>JY</given-names></name><name><surname>Ahn</surname><given-names>JH</given-names></name><name><surname>Lee</surname><given-names>TS</given-names></name><name><surname>Kim</surname><given-names>DY</given-names></name><etal/></person-group>. <article-title>IL-10 protects mice from the lung infection of Acinetobacter baumannii and contributes to bacterial clearance by regulating STAT3-mediated MARCO expression in macrophages</article-title>. <source>Front Immunol.</source> (<year>2020</year>) <volume>11</volume>:<fpage>270</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2020.00270</pub-id><pub-id pub-id-type=\"pmid\">32153580</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thuong</surname><given-names>NT</given-names></name><name><surname>Tram</surname><given-names>TT</given-names></name><name><surname>Dinh</surname><given-names>TD</given-names></name><name><surname>Thai</surname><given-names>PV</given-names></name><name><surname>Heemskerk</surname><given-names>D</given-names></name><name><surname>Bang</surname><given-names>ND</given-names></name><etal/></person-group>. <article-title>MARCO variants are associated with phagocytosis, pulmonary tuberculosis susceptibility and Beijing lineage</article-title>. <source>Genes Immun.</source> (<year>2016</year>) <volume>17</volume>:<fpage>419</fpage>–<lpage>25</lpage>. <pub-id pub-id-type=\"doi\">10.1038/gene.2016.43</pub-id><pub-id pub-id-type=\"pmid\">27853145</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Valentin-Weigand</surname><given-names>P</given-names></name><name><surname>Traore</surname><given-names>MY</given-names></name><name><surname>Blobel</surname><given-names>H</given-names></name><name><surname>Chhatwal</surname><given-names>GS</given-names></name></person-group>. <article-title>Role of α<sub>2</sub>-macroglobulin in phagocytosis of group A and C streptococci</article-title>. <source>FEMS Microbiol Lett.</source> (<year>1990</year>) <volume>58</volume>:<fpage>321</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1574-6968.1990.tb13997.x</pub-id><pub-id pub-id-type=\"pmid\">1699839</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Federici Canova</surname><given-names>D</given-names></name><name><surname>Pavlov</surname><given-names>AM</given-names></name><name><surname>Norling</surname><given-names>LV</given-names></name><name><surname>Gobbetti</surname><given-names>T</given-names></name><name><surname>Brunelleschi</surname><given-names>S</given-names></name><name><surname>Le Fauder</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Alpha-2-macroglobulin loaded microcapsules enhance human leukocyte functions and innate immune response</article-title>. <source>J Control Release.</source> (<year>2015</year>) <volume>217</volume>:<fpage>284</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jconrel.2015.09.021</pub-id><pub-id pub-id-type=\"pmid\">26385167</pub-id></mixed-citation></ref><ref id=\"B48\"><label>48.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Helmy</surname><given-names>KY</given-names></name><name><surname>Katschke</surname><given-names>KJ</given-names><suffix>Jr</suffix></name><name><surname>Gorgani</surname><given-names>NN</given-names></name><name><surname>Kljavin</surname><given-names>NM</given-names></name><name><surname>Elliott</surname><given-names>JM</given-names></name><name><surname>Diehl</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>CRIg: a macrophage complement receptor required for phagocytosis of circulating pathogens</article-title>. <source>Cell.</source> (<year>2006</year>) <volume>124</volume>:<fpage>915</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cell.2005.12.039</pub-id><pub-id pub-id-type=\"pmid\">16530040</pub-id></mixed-citation></ref><ref id=\"B49\"><label>49.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nakamura</surname><given-names>K</given-names></name><name><surname>Funakoshi</surname><given-names>H</given-names></name><name><surname>Miyamoto</surname><given-names>K</given-names></name><name><surname>Tokunaga</surname><given-names>F</given-names></name><name><surname>Nakamura</surname><given-names>T</given-names></name></person-group>. <article-title>Molecular cloning and functional characterization of a human scavenger receptor with C-type lectin (SRCL), a novel member of a scavenger receptor family</article-title>. <source>Biochem Biophys Res Commun.</source> (<year>2001</year>) <volume>280</volume>:<fpage>1028</fpage>–<lpage>35</lpage>. <pub-id pub-id-type=\"doi\">10.1006/bbrc.2000.4210</pub-id><pub-id pub-id-type=\"pmid\">11162630</pub-id></mixed-citation></ref><ref id=\"B50\"><label>50.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coombs</surname><given-names>PJ</given-names></name><name><surname>Graham</surname><given-names>SA</given-names></name><name><surname>Drickamer</surname><given-names>K</given-names></name><name><surname>Taylor</surname><given-names>ME</given-names></name></person-group>. <article-title>Selective binding of the scavenger receptor C-type lectin to Lewisx trisaccharide and related glycan ligands</article-title>. <source>J Biol Chem.</source> (<year>2005</year>) <volume>280</volume>:<fpage>22993</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M504197200</pub-id><pub-id pub-id-type=\"pmid\">15845541</pub-id></mixed-citation></ref><ref id=\"B51\"><label>51.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Simhadri</surname><given-names>VR</given-names></name><name><surname>Andersen</surname><given-names>JF</given-names></name><name><surname>Calvo</surname><given-names>E</given-names></name><name><surname>Choi</surname><given-names>SC</given-names></name><name><surname>Coligan</surname><given-names>JE</given-names></name><name><surname>Borrego</surname><given-names>F</given-names></name></person-group>. <article-title>Human CD300a binds to phosphatidylethanolamine and phosphatidylserine, and modulates the phagocytosis of dead cells</article-title>. <source>Blood.</source> (<year>2012</year>) <volume>119</volume>:<fpage>2799</fpage>–<lpage>809</lpage>. <pub-id pub-id-type=\"doi\">10.1182/blood-2011-08-372425</pub-id><pub-id pub-id-type=\"pmid\">22302738</pub-id></mixed-citation></ref><ref id=\"B52\"><label>52.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tahvildari</surname><given-names>M</given-names></name><name><surname>Dana</surname><given-names>R</given-names></name></person-group>. <article-title>Low-dose IL-2 therapy in transplantation, autoimmunity, inflammatory diseases</article-title>. <source>J Immunol.</source> (<year>2019</year>) <volume>203</volume>:<fpage>2749</fpage>–<lpage>55</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.1900733</pub-id><pub-id pub-id-type=\"pmid\">31740549</pub-id></mixed-citation></ref><ref id=\"B53\"><label>53.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hester</surname><given-names>J</given-names></name><name><surname>Schiopu</surname><given-names>A</given-names></name><name><surname>Nadig</surname><given-names>SN</given-names></name><name><surname>Wood</surname><given-names>KJ</given-names></name></person-group>. <article-title>Low-dose rapamycin treatment increases the ability of human regulatory T cells to inhibit transplant arteriosclerosis <italic>in vivo</italic></article-title>. <source>Am J Transplant.</source> (<year>2012</year>) <volume>12</volume>:<fpage>2008</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1600-6143.2012.04065.x</pub-id><pub-id pub-id-type=\"pmid\">22500984</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Microbiol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Microbiol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Microbiol.</journal-id><journal-title-group><journal-title>Frontiers in Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-302X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849429</article-id><article-id pub-id-type=\"pmc\">PMC7431609</article-id><article-id pub-id-type=\"doi\">10.3389/fmicb.2020.01827</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Microbiology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Evaluation of Enzymatic Cleaning on Food Processing Installations and Food Products Bacterial Microflora</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Delhalle</surname><given-names>Laurent</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/955303/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Taminiau</surname><given-names>Bernard</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Fastrez</surname><given-names>Sebastien</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Fall</surname><given-names>Abdoulaye</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ballesteros</surname><given-names>Marina</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Burteau</surname><given-names>Sophie</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Daube</surname><given-names>Georges</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Fundamental and Applied Research for Animals and Health, Department of Food Science, University of Liège</institution>, <addr-line>Liège</addr-line>, <country>Belgium</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Realco SA</institution>, <addr-line>Ottignies-Louvain-la-Neuve</addr-line>, <country>Belgium</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Genalyse Partner SA</institution>, <addr-line>Herstal</addr-line>, <country>Belgium</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Lin Lin, Jiangsu University, China</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Moshe Shemesh, Agricultural Research Organization, Israel; Lucilla Iacumin, University of Udine, Italy</p></fn><corresp id=\"c001\">*Correspondence: Laurent Delhalle, <email>l.delhalle@uliege.be</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1827</elocation-id><history><date date-type=\"received\"><day>16</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>12</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Delhalle, Taminiau, Fastrez, Fall, Ballesteros, Burteau and Daube.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Delhalle, Taminiau, Fastrez, Fall, Ballesteros, Burteau and Daube</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Biofilms are a permanent source of contamination in food industries and could harbor various types of microorganisms, such as spoiling bacteria. New strategies, such as enzymatic cleaning, have been proposed to eradicate them. The purpose of this study was to evaluate the impact of enzymatic cleaning on the microbial flora of installations in a processing food industry and of the final food product throughout its shelf life. A total of 189 samples were analyzed by classical microbiology and 16S rDNA metagenetics, including surface samples, cleaning-in-place (CIP) systems, and food products (at D<sub>0</sub>, D<sub>end of the shelf life</sub>, and D<sub>end of the shelf life +7 days</sub>). Some surfaces were highly contaminated with spoiling bacteria during conventional cleaning while the concentration of the total flora decreased during enzymatic cleaning. Although the closed circuits were cleaned with conventional cleaning before enzymatic cleaning, there was a significant release of microorganisms from some parts of the installations during enzymatic treatment. A significant difference in the total flora in the food products at the beginning of the shelf life was observed during enzymatic cleaning compared to the conventional cleaning, with a reduction of up to 2 log CFU/g. Metagenetic analysis of the food samples at the end of their shelf life showed significant differences in bacterial flora between conventional and enzymatic cleaning, with a decrease of spoiling bacteria (<italic>Leuconostoc</italic> sp.). Enzymatic cleaning has improved the hygiene of the food processing instillations and the microbial quality of the food throughout the shelf life. Although enzymatic cleaning is not yet commonly used in the food industry, it should be considered in combination with conventional sanitizing methods to improve plant hygiene.</p></abstract><kwd-group><kwd>enzyme</kwd><kwd>cleaning</kwd><kwd>food</kwd><kwd>microflora</kwd><kwd>spoilage</kwd><kwd>contamination</kwd><kwd>metagenetics</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Service Public de Wallonie<named-content content-type=\"fundref-id\">10.13039/501100009595</named-content></funding-source><award-id rid=\"cn001\">Grant no. 7320</award-id></award-group></funding-group><counts><fig-count count=\"6\"/><table-count count=\"5\"/><equation-count count=\"2\"/><ref-count count=\"103\"/><page-count count=\"17\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Biofilms are multicellular communities held together by a self-produced, extra polymeric substance (EPS). The mechanisms that different bacteria employ to form biofilms vary, depending on environmental conditions and specific strain attributes (<xref rid=\"B60\" ref-type=\"bibr\">López et al., 2010</xref>). Several studies have demonstrated the presence of biofilms in various food industries, such as breweries, dairies, fresh vegetables industries, poultry and meat cutting plant (<xref rid=\"B64\" ref-type=\"bibr\">Marchand et al., 2012</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Giaouris et al., 2014</xref>; <xref rid=\"B51\" ref-type=\"bibr\">Kim et al., 2017</xref>; <xref rid=\"B2\" ref-type=\"bibr\">Adator et al., 2018</xref>; <xref rid=\"B72\" ref-type=\"bibr\">Parijs and Steenackers, 2018</xref>). Biofilms are a source of microbial contamination leading to food spoilage and shelf life reduction and a potential way of pathogen transmission (<xref rid=\"B101\" ref-type=\"bibr\">Wirtanen and Salo, 2016</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Giaouris and Simões, 2018</xref>). In particular, approximately 60% of food-borne infections results from microbial transfer from equipment surfaces to processed foods (<xref rid=\"B8\" ref-type=\"bibr\">Bridier et al., 2015</xref>). Product-contact surfaces in the food process may contaminate the product directly, i.e., the product touching over the surface will potentially lead to microbial contamination (<xref rid=\"B39\" ref-type=\"bibr\">Gibson et al., 1999</xref>).</p><p>Bacteria embedded in a biofilm are 100–1000 times more resistant to cleaning and sanitizing chemicals than the corresponding planktonic cells (<xref rid=\"B40\" ref-type=\"bibr\">Gilbert et al., 2002</xref>). Nevertheless, CIP procedures still leave residual microorganisms on equipment surfaces, thus resulting in biofilm formation (<xref rid=\"B7\" ref-type=\"bibr\">Bremer et al., 2006</xref>; <xref rid=\"B53\" ref-type=\"bibr\">Kumari and Sarkar, 2016</xref>; <xref rid=\"B72\" ref-type=\"bibr\">Parijs and Steenackers, 2018</xref>). The time it takes for biofilm to form depends on the frequency of cleaning and disinfection regimes: if there is a long period between cleaning/disinfection treatments, then there is more time for biofilm to form on surfaces (<xref rid=\"B39\" ref-type=\"bibr\">Gibson et al., 1999</xref>). Increased biofilm resistance to conventional treatment enhances the need to develop new control strategies (<xref rid=\"B95\" ref-type=\"bibr\">Simões et al., 2010</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Coughlan et al., 2016</xref>).</p><p>New strategies has been proposed to eliminate biofilms, i.e., by using enzymes, phages, and bioregulation (<xref rid=\"B14\" ref-type=\"bibr\">Coughlan et al., 2016</xref>). The use of enzyme-based detergents as biocleaners, also known as “green chemicals,” can be a viable option to overcome biofilms in the food industry (<xref rid=\"B58\" ref-type=\"bibr\">Lequette et al., 2010</xref>; <xref rid=\"B96\" ref-type=\"bibr\">Stiefel et al., 2016</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Fleming and Rumbaugh, 2017</xref>). Formulations containing several different enzymes are a successful biofilm control strategy (<xref rid=\"B14\" ref-type=\"bibr\">Coughlan et al., 2016</xref>). In industrial environments, numerous microbial species coexist within the same biofilm, thus increasing the biochemical heterogeneity of the matrix. Efficient formulations may therefore be composed of mixtures of enzymes with different substrates to destabilize the EPS, such as proteases, cellulases, polysaccharide depolymerases, alginate lyases, dispersin B, and DNAses (<xref rid=\"B8\" ref-type=\"bibr\">Bridier et al., 2015</xref>).</p><p>However, studies evaluating the efficacy of enzyme-based detergents have been conducted in labs or pilot plant scale models (<xref rid=\"B71\" ref-type=\"bibr\">Oulahal et al., 2007</xref>; <xref rid=\"B58\" ref-type=\"bibr\">Lequette et al., 2010</xref>; <xref rid=\"B55\" ref-type=\"bibr\">Lefebvre et al., 2016</xref>; <xref rid=\"B96\" ref-type=\"bibr\">Stiefel et al., 2016</xref>; <xref rid=\"B68\" ref-type=\"bibr\">Nagaraj et al., 2017</xref>). Lab models have their own advantages and their own limitations, but they could never mimic the real conditions that can be encountered in industries. This study evaluates the impact of enzymatic cleaning protocols on the microbial flora of installations in the processed food industry and of the final food product throughout its shelf life. The objective is to assess whether enzymatic cleaning could be considered as an alternative to conventional cleaning in food industries to improve the microbial ecology of food processing surfaces and equipment’s.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Food Process</title><p>The study was carried out from August to December 2016 in a Belgian food company that produces ready-to-eat lasagne. The production is fully automatic via the successive addition of layers of Bolognese sauce, bechamel sauce, and lasagne sheets. The sauces are pre-cooked and placed successively in a tray via a dosing system. Several filling machines containing sauces are present along the production chain. Lasagne sheets were handled by robots equipped with suction cups. Finally, a layer of grated cheese is placed on top. The food products are packed under a modified atmosphere containing 50% N<sub>2</sub> and 50% CO<sub>2</sub>. The duration of the production cycle is 48 h continuously with three production cycles per week in a room at 20°C. The production chain is described in <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Food production chain of lasagne.</p></caption><graphic xlink:href=\"fmicb-11-01827-g001\"/></fig></sec><sec id=\"S2.SS2\"><title>Cleaning Methods</title><p>The basic sequence of the cleaning and disinfection operations (herein, referred together as sanitation) for the open surface and closed circuits is as follows: (1) a pre-rinse with cold water to remove largest residues; (2) cleaning with detergent to remove remaining residues; (3) an intermediate water rinse to remove detergents; (4) disinfection with a chemical agent; and (5) a cold water rinse to remove the disinfectant (<xref rid=\"B95\" ref-type=\"bibr\">Simões et al., 2010</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Forsythe and Hayes, 2012</xref>). In this company, the installations are cleaned and disinfected three times a week after 48 h of production. For the open surfaces, the installations are cleaned by an alkaline chlorinated solution (EnduroPlus VE6, conc. 3%, Diversey, United States) and by an acid agent (EnduroEco VE9, conc. 3%, Diversey, United States) at 40°C for 15 min per each step. The installations are disinfected by quaternary ammonium (Divosan Extra VT55L, conc. 1%, Diversey, United States) and (peracetic acid Divosan Actif VT5, conc. 1%, Diversey, United States) at room temperature for 15 min each disinfectant. For closed circuits, the pipes are cleaned three times a week with caustic soda (Cipton HD VC151, conc. 3%, Diversey, United States) at 85°C for 90 min by the CIP system. An additional cleaning process is carried out once a week with an acid (Pascal VA5, conc. 1%, Diversey, United States) at 85°C for 15 min, followed by disinfection with peracetic acid (Divosan Actif VT5, conc. 1%, Diversey, United States) at 20°C for 30 min. This cleaning and disinfection protocol of the facilities is referred as “conventional cleaning” in this article.</p><p>The enzymatic cleaners were developed by the French National Institute for Research in Agronomy (INRA; Villeneuve d’Ascq, France) and were formulated by a commercial company (Realco, Louvain La Neuve, Belgium). The formulation consists of several enzymes targeting the components of EPS, surfactants, and dispersing and chelating agents (<xref rid=\"B58\" ref-type=\"bibr\">Lequette et al., 2010</xref>). Two types of enzymatic cleaning protocols were applied for open surfaces: “reinforced” and “routine” enzymatic cleaning. The reinforced enzymatic cleaning is distinguished from routine enzymatic cleaning by longer treatment duration and higher number and concentration of enzymes. For open surfaces, the installations are cleaned by an alkaline chlorinated solution (EnduroPlus VE6, conc. 3%, Diversey, United States) at 40°C for 15 min, followed by foaming enzymatic solutions at 40°C for 30 min for reinforced enzymatic cleaning (Enzyfoam SG, conc. 3%, Realco, Belgium) and for 15 min for the routine enzymatic cleaning (Bioremfoam, conc. 3%, Realco, Belgium).</p><p>The closed circuits are cleaned with enzymatic detergents only once when enzymatic treatment of the facilities is initiated (Biorem A1 + Biorem 10, conc. 0,5% and 0,1%, respectively, Realco, Belgium). The conventional cleaning protocol was applied before using enzymatic detergent at 45°C and pH 7 for 60 min and at pH 9 for 60 min.</p><p>For this study, different cleaning treatments were tested over time: conventional cleaning, reinforced enzymatic cleaning, routine enzymatic cleaning 3X/week, 2X/week, and 1X/week, then conventional cleaning again and reinforced enzymatic cleaning. The solutions were applied according to the manufacturer’s recommendations. All factors and the food product remained unchanged, except for the cleaning and disinfection protocol. <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> describes the different cleaning and disinfection protocols applied in this study.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>Cleaning and disinfection protocols for open surfaces and closed circuits.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ZONE</td><td valign=\"top\" align=\"center\" colspan=\"7\" rowspan=\"1\">Open Plant Cleaning (OPC) of the surfaces</td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Cleaning In Place (CIP) of closed circuits and tanks</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Period (wk)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1–3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4–5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">6–7</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8–9</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">10–11</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">12–13</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14–15</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">1–15</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4</td></tr><tr><td valign=\"top\" align=\"center\" colspan=\"11\" rowspan=\"1\"><hr/></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Protocol</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Conventional cleaning</td><td valign=\"top\" align=\"center\" colspan=\"4\" rowspan=\"1\">Enzymatic cleaning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Conventional cleaning 2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Enzymatic cleaning</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"4\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\"><hr/></td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reinforced enzymatic cleaning</td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">Routine enzymatic cleaning</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reinforced enzymatic cleaning 2</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Conventional treatment</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reinforced enzymatic cleaning</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Frequency</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3x/wk (every 48 h)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3x/wk (every 48 h)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3x/wk (every 48 h)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2x/wk with enzymatic cleaning + 1x/wk with conventional cleaning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1x/wk with enzymatic cleaning + 2x/wk with conventional cleaning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3x/wk (every 48 h)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3x/wk (every 48 h)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3x/wk (every 48 h)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1x/wk (before the production week)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">In addition to the conventional treatment</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cleaning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Alkaline chlorinated15 min, 40°C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Alkaline chlorinated15 min, 40°C</td><td valign=\"top\" align=\"justify\" colspan=\"3\" rowspan=\"1\">Alkaline chlorinated15 min, 40°C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Alkaline chlorinated15 min, 40°C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Alkaline chlorinated15 min, 40°C”</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Caustic soda90 min, 85°C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Acid agent15 min, 85°C</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"justify\" colspan=\"3\" rowspan=\"1\">Rinsing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Acid agent15 min,40°C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Foaming enzymatic detergent 3% (protease, lipase, amylase, oxydo reductase) 30 min, 40°C</td><td valign=\"top\" align=\"justify\" colspan=\"3\" rowspan=\"1\">Foaming enzymatic detergent 3% (protease, lipase, amylase)15 min, 40°C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Acid agent15 min, 40°C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Foaming enzymatic detergent 3% (protease, lipase, amylase, oxydo reductase) 30 min, 40°C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Alkaline chlorinated30 min, 85°C</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Enzymatic detergent (protease, lipase, amylase, oxydo reductase)pH 7, 60 min, 45°CpH 9, 60 min, 45°C</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"justify\" colspan=\"3\" rowspan=\"1\">Rinsing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Disinfection</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Quaternary ammonium15 min, 40°C</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Quaternary ammonium15 min, 40°C</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Peracetic acid15 min, 20°C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Peracetic acid15 min, 20°C</td><td valign=\"top\" align=\"justify\" colspan=\"3\" rowspan=\"1\">Peracetic acid15 min, 20°C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Peracetic acid15 min, 20°C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Peracetic acid15 min, 20°C</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Peracetic acid30 min, 20°C</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Peracetic acid30 min, 20°C</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"justify\" colspan=\"3\" rowspan=\"1\">Rinsing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing</td></tr></tbody></table></table-wrap></sec><sec id=\"S2.SS3\"><title>Sampling Collection</title><p>The surface samples were the pasta conveyor belt and the nozzles of the dosing machines for Bolognese and bechamel sauces. Surface sampling was conducted several times per week in accordance with the requirements of ISO 18593 regarding surface sampling techniques (<xref rid=\"B47\" ref-type=\"bibr\">ISO, 2016</xref>) with sterile wipes (KW-P8030, Conformat, France). The wipes were placed in a sterile plastic bag (IUL 2456, IUL instruments, Spain) with neutralizing buffer (DifcoTM Neutralizing Buffer, Becton, Dickinson and Company, United States). One liter of cleaning water from the closed circuits was collected during enzymatic cleaning into autoclaved glass bottles (GLS 80, DURAN, Germany). The lasagnes were collected at the end of the food chain production line after sealing the tray under modified atmosphere. The food product was the same throughout the study with the same recipe and same packaging. All the samples were placed in a refrigerated box (4°C) and transferred on the same day to the laboratory for analysis. The food samples were stored in a fridge at 8°C to be analyzed at D<sub>0</sub>, D<sub>end the shelf life</sub>, and D<sub>end of the shelf life + 7 days</sub>. <xref rid=\"T2\" ref-type=\"table\">Table 2</xref> describes the samples collected during the study in relation to the cleaning protocol.</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Samples analyzed by classical microbiology and metagenetic analyses.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cleaning method</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Samples</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">n</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Conventional cleaning</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ingredients (pasta, bolognese sauce, béchamel sauce, and cheese)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surfaces</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Food products at D<sub>0</sub>, D<sub>end of the shelf life</sub>, and D<sub>end of the shelf+ 7 days</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">39</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Enzymatic cleaning</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surfaces</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reinforced enzymatic cleaning (OPC + CIP)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Food products at D<sub>0</sub>, D<sub>end of the shelf life</sub>, and D<sub>end of the shelf + 7 days</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cleaning water during enzymatic cleaning</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Routine enzymatic cleaning 3x/wk</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surfaces</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Food products at D<sub>0</sub>, D<sub>end of the shelf life</sub>, and D<sub>end of the shelf + 7 days</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Routine enzymatic cleaning 2x/wk</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surfaces</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Food products at D<sub>0</sub>, D<sub>end of the shelf life</sub>, and D<sub>end of the shelf + 7 days</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Routine enzymatic cleaning 1x/wk</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surfaces</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Food products at D<sub>0</sub>, D<sub>end of the shelf life</sub>, and D<sub>end of the shelf + 7 days</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Conventional cleaning 2</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surfaces</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Food products at D<sub>0</sub>, D<sub>end of the shelf life</sub>, and D<sub>end of the shelf + 7 days</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Enzymatic cleaning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reinforced enzymatic cleaning 2 (OPC)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surfaces</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Food products at D<sub>0</sub>, D<sub>end of the shelf life</sub>, and D<sub>end of the shelf + 7 days</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">189</td></tr></tbody></table><table-wrap-foot><attrib><italic>OPC, open plant cleaning; CIP, cleaning in place; D<sub>0</sub>, food product analyzed at the beginning of the shelf life; D<sub><italic>end of the shelf life</italic></sub>, food product analyzed at the end of the shelf life; D<sub><italic>end of the shelf +</italic> 7 <italic>days</italic></sub>, food product analyzed seven days after the end of the shelf life.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S2.SS4\"><title>Microbiological Analyses</title><p>One laboratory licensed by the Belgian Ministry of Public Health and accredited in accordance with the requirements of the ISO 17,025 standard performed all the microbiological analyses. All samples were stored chilled and were examined within 24 h.</p><p>Twenty-five grams (25 g) of food or wipes from surfaces were put in a Stomacher bag with a mesh screen liner (80-μm pore size; ref 80015, BioMérieux, France) under aseptic conditions. Physiological water was automatically added to each bag at 1:10 dilution (Dilumat, BioMérieux, France), and the samples were homogenized for 2 min in a Stomacher (Bagmixer, Interscience, France). From this primary suspension, decimal dilutions in physiological water (8.5 g/L sodium chloride) were prepared for microbiological analysis, and 0.1 mL aliquots of the appropriate dilutions were plated onto media for each analysis in triplicate (Spiral plater, DW Scientific, United Kingdom). For the enzymatic cleaning water from closed circuits, one liter (1 L) water from CIP was filtered through 0.45-μm sterile filters (HABG047S6, Merck, Germany).</p><p>The following microbiological analyses were performed:</p><list list-type=\"simple\"><list-item><label>1.</label><p>Aerobic colony counts, following the requirements of the modified ISO 4833 standard using PCA (Plate Count Agar, #3544475, Bio-Rad, Marnes-la-Coquette, France) at 22°C and incubation for 72 h;</p></list-item><list-item><label>2.</label><p>Anaerobic colony counts, following the requirements of the ISO 6222 standard using the Reinforced Clostridial agar (BO0251M, Thermo Fischer Scientific, Waltham, United States) at 22°C and incubated for 24 h under anaerobic conditions.</p></list-item></list><p>Aerobic colony count were also assessed for surface samples and for cleaning water from closed circuits.</p><p>Aerobic colony count is evaluated using the ISO 4833:2003 standard for which incubation temperature of the plates is performed at 30°C. However several studies used a lower incubation temperature than that indicated in the ISO 4833:2003 standard for the detection of psychrophilic bacteria in foodstuffs (<xref rid=\"B24\" ref-type=\"bibr\">Ercolini et al., 2009</xref>; <xref rid=\"B83\" ref-type=\"bibr\">Pothakos et al., 2012</xref>, <xref rid=\"B82\" ref-type=\"bibr\">2015</xref>; <xref rid=\"B88\" ref-type=\"bibr\">Ribeiro Júnior et al., 2018</xref>; <xref rid=\"B91\" ref-type=\"bibr\">Samapundo et al., 2019</xref>). <xref rid=\"B83\" ref-type=\"bibr\">Pothakos et al. (2012)</xref> have shown a consistent underestimation of the microbial flora with the total viable counts on plates incubated at 30°C (representing the mesophiles) compared on plates incubated at 22°C (indicating the psychrotrophs) for 86 food samples covering a wide range of foods products.</p></sec><sec id=\"S2.SS5\"><title>ATPmetry</title><p>Adenosine triphosphate tests are one of the most commonly used hygiene monitoring indicator to check cleaning effectiveness as they are simple and easy to use and provide immediate results (<xref rid=\"B46\" ref-type=\"bibr\">ICMSF, 2012</xref>). The surface samples and cleaning water from closed circuits were tested to measure ATP concentration with a commercial kit (QGA-100C, LuminUltra Technologies SAS, Canada), and the results were expressed as pg cATP/ml.</p></sec><sec id=\"S2.SS6\"><title>16S rDNA Extraction and High-Throughput Sequencing</title><p>Bacterial DNA was extracted from each primary suspension, previously stored at −80°C, using the DNEasy Blood and Tissue kit (QIAGEN, Belgium), following the manufacturer’s recommendations.</p><p>The resulting DNA extracts were eluted in DNase/RNase-free water, and their concentration and purity were evaluated using optical density with the NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scientific, United States) by measuring the ratio of the absorbance at 260 nm and 280 nm (A260/280) and at 260 and 230 nm (A260/230). If the DNA concentration exceeds 200 ng/μl, the DNA is diluted 5-fold to avoid PCR inhibition. DNA samples were stored at – 20°C until 16S rRNA amplicon sequencing. PCR-amplification of the V1-V3 region of the 16S rDNA library preparation was performed with the following primers (with Illumina overhand adapters): forward (5′-GAGAGTTTGATYMTGGCTCAG-3′) and reverse (5′-ACCGCGGCTGCTGGCAC-3′). Each PCR product was purified with the Agencourt AMPure XP beads kit (Beckman Coulter; Pasadena, United States) and submitted to a second PCR round for indexing using the Nextera XT index primers 1 and 2. Thermocycling conditions consisted of a denaturation step of 4 min at 94°C, followed by 25 cycles of denaturation (15 s at 94°C), annealing (45 s at 56°C), and extension (60 s at 72°C), with a final elongation step (8 min at 72°C). These amplifications were performed on an EP Mastercycler Gradient System device (Eppendorf, Hamburg, Germany). The PCR products of approximately 650 nucleotides were run on 1% agarose gel electrophoresis, and the DNA fragments were plugged out and purified using a Wizard SV PCR purification kit (Promega Benelux, Netherlands). After purification, PCR products were quantified using the Quanti-IT PicoGreen (Thermo Fisher Scientific, United States) and were diluted to 10 ng/μL. A final quantification by quantitative (q)PCR of each sample in the library was performed using the KAPA SYBR<sup>®</sup> FAST qPCR Kit (KapaBiosystems, United States) before normalization, pooling, and sequencing on a MiSeq sequencer using V3 reagents (Illumina, United States).</p></sec><sec id=\"S2.SS7\"><title>Bioinformatics Analysis</title><p>The 16S rRNA gene sequence reads were processed with MOTHUR (<xref rid=\"B92\" ref-type=\"bibr\">Schloss et al., 2009</xref>). The quality of all sequence reads was denoised using the Pyronoise algorithm implemented in MOTHUR. The sequences were checked for the presence of chimeric amplification using ChimeraSlayer (developed by the Broad Institute<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup>). The obtained read sets were compared to a reference data set of aligned sequences of the corresponding region derived from the SILVA database of full-length rRNA gene sequences<sup><xref ref-type=\"fn\" rid=\"footnote2\">2</xref></sup> implemented in MOTHUR (<xref rid=\"B84\" ref-type=\"bibr\">Pruesse et al., 2007</xref>; <xref rid=\"B85\" ref-type=\"bibr\">Quast et al., 2012</xref>). The final reads were clustered into OTUs using the nearest neighbor algorithm of MOTHUR with a 0.03 distance unit cut-off. A taxonomic identity was attributed to each OTU by comparison to the SILVA database using an 80% homogeneity cut-off. As MOTHUR is not dedicated to the taxonomic assignment beyond the genus level, all unique sequences for each OTU were compared to the SILVA dataset 111 using a BLASTN algorithm. For each OTU, a consensus-detailed taxonomic identification was given based on the identity (<1% mismatch with the aligned sequence) and the metadata associated with the best hit (validated bacterial species or not) (<xref rid=\"B19\" ref-type=\"bibr\">Delcenserie et al., 2014</xref>).</p></sec><sec id=\"S2.SS8\"><title>16S rDNA Data Analysis</title><p>A correcting factor for 16S rDNA gene copy numbers was applied for any taxon <italic>i</italic> (equation 1) (<xref rid=\"B50\" ref-type=\"bibr\">Kembel et al., 2012</xref>; <xref rid=\"B61\" ref-type=\"bibr\">Louca et al., 2018</xref>).</p><disp-formula id=\"S2.E1\"><label>(1)</label><mml:math id=\"M1\"><mml:mrow><mml:msub><mml:mi>A</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mrow><mml:msub><mml:mi>N</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mo>×</mml:mo><mml:msub><mml:mi>C</mml:mi><mml:mi>i</mml:mi></mml:msub></mml:mrow></mml:mrow></mml:math></disp-formula><p>where A<sub>i</sub> is the real abundance of 16S genes from that taxon; N<sub>i</sub> is the number of reads for the taxon in the sample; and C<sub>i</sub> is the genomic 16S copy number of that taxon. To obtain each gene copy number, Ribosomal RNA Database (rrnDB) (<xref rid=\"B97\" ref-type=\"bibr\">Stoddard et al., 2015</xref>) and EzBioCloud database (<xref rid=\"B102\" ref-type=\"bibr\">Yoon et al., 2017</xref>) were used.</p><p>Then, to compare the relative abundance of OTUs, the number of reads was normalized as described by <xref rid=\"B13\" ref-type=\"bibr\">Chaillou et al. (2015)</xref>. The reads counts of each sample were divided by a sample-specific scaling factor (<xref rid=\"B30\" ref-type=\"bibr\">Fougy et al., 2016</xref>; <xref rid=\"B90\" ref-type=\"bibr\">Rouger et al., 2017</xref>):</p><disp-formula id=\"S2.E2\"><label>(2)</label><mml:math id=\"M2\"><mml:mrow><mml:msub><mml:mi>S</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mo>=</mml:mo><mml:mrow><mml:msub><mml:mi>N</mml:mi><mml:mi>i</mml:mi></mml:msub><mml:mo>/</mml:mo><mml:msub><mml:mi>m</mml:mi><mml:mi>e</mml:mi></mml:msub></mml:mrow></mml:mrow></mml:math></disp-formula><p>where S<sub>i</sub> is the normalization factor associated with sample; N<sub>i</sub> is the number of total reads in the sample I; m<sub>e</sub> is the median value of total reads for all the samples of the dataset. Reads counts of all samples were then transformed into a percentage of each OTUs.</p></sec><sec id=\"S2.SS9\"><title>Statistical Analysis</title><p>Mann–Withney test was used to compare the classical microbiology and ATPmetry results in relation with the cleaning treatments using the R software (<xref rid=\"B87\" ref-type=\"bibr\">R Core Team, 2008</xref>). All tests were considered significant when <italic>p</italic> ≤ 0.05. When non-colony was detected in the classical microbiology results, a value of half-limit of detection was used for the calculations (<xref rid=\"B44\" ref-type=\"bibr\">Hutchison et al., 2005</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Ghafir et al., 2008</xref>).</p><p>The richness estimation (Chao1 estimator) and microbial biodiversity (inverse of the Simpson index, coverage index) were evaluated using MOTHUR (version 1.40.5)<sup><xref ref-type=\"fn\" rid=\"footnote3\">3</xref></sup> (<xref rid=\"B89\" ref-type=\"bibr\">Riquelme et al., 2015</xref>). The coverage index measures how well an environment was sampled and indicates the percentage of individuals sampled in a microbial community. The analysis is included with a coverage index of at least 0.9 (<xref rid=\"B57\" ref-type=\"bibr\">Lemos et al., 2011</xref>). Chao1 index estimates diversity from the abundance data (importance of rare OTUs). The inverse Simpson index reflects the effective number of species: the higher its value, the greater the diversity (with 1 as the lowest possible number).</p><p>Using the STAMP (v2+) software<sup><xref ref-type=\"fn\" rid=\"footnote4\">4</xref></sup>, a two-sided Welch’s <italic>t</italic>-test was performed on metagenetic results, and confidence intervals were calculated according to the Newcombe–Wilson method. The differences were considered significant when <italic>p</italic> ≤ 0.05 (<xref rid=\"B73\" ref-type=\"bibr\">Parks et al., 2014</xref>).</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Ingredients</title><p>The ingredients were analyzed once to evaluate the potential source of contamination in the food process. The results of ACC are in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>. As expected, only a few microorganisms are present in the Bolognese and bechamel sauces due the cooking process. The dough, bechamel sauce, and cheese have low bacterial concentrations. The concentration of anaerobic colony was under the Dl in the ingredients.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Aerobic Colony Counts (ACC) (log CFU/g) and relative abundances of the dominant bacterial groups (%) in ingredients.</p></caption><graphic xlink:href=\"fmicb-11-01827-g002\"/></fig><p><xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref> illustrates also the results of the metagenetic analysis. To facilitate the interpretation of the graph, three groups of bacteria, namely “Spoilers,” “Lactic acid bacteria,” and “Others” were created based on the predominant groups identified. “Spoilers” include bacteria belonging to the <italic>Enterobacteriaceae</italic> family, <italic>Pseudomonas</italic> sp., <italic>Leuconostoc</italic> sp., and <italic>Brochotrix thermosphacta</italic>, as well as spore-forming bacteria which include <italic>Bacillus</italic> sp., <italic>Geobacillus</italic> sp., <italic>Paenibacillus</italic> sp., and <italic>Anoxybacillus</italic> sp. “Lactic acid bacteria” includes <italic>Lactococcus</italic> sp., <italic>Lactobacillus</italic> sp., and <italic>Streptococcus</italic> sp., whereas “Others” includes <italic>Stenotrophomonas maltophilia</italic> and all microorganisms that are present less than 1% in the analyzed sample and do not belong to the bacterial groups listed above.</p><p>Lactic acid bacteria are the most dominant in the cheese (<italic>Lactococcus lactis</italic>, 80.3%) and in the bechamel sauce (<italic>Streptococcus</italic> sp., 70.7%). A high number of taxa classified as “Others” are identified in the dough and Bolognese sauce (35.9 and 63.7%, respectively). Potential spoilers are present in the dough (55.4%), Bolognese sauce (35.9%), and bechamel sauce (24.2%).</p><p><xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Material</xref> summarizes the ACC, metagenetic results, microbial richness, and diversity indicates of all samples collected in this study. All the ingredient samples have a coverage index above 0.9. The bechamel sauce has the highest value for the Chao1 index but the lowest value for the inverse Simpson index, indicating that many OTUs are related to the same bacterial species. Meanwhile, the Bolognese sauce has the lowest value for the Chao1 index but the highest value for the inverse Simpson, indicating that there are many bacterial species with few OTUs.</p></sec><sec id=\"S3.SS2\"><title>Surfaces</title><p><xref rid=\"T3\" ref-type=\"table\">Table 3</xref> present the results of the ACC and ATPmetry for surface samples. The results indicate that some surfaces were highly contaminated during conventional cleaning, and significant decreases between conventional and enzymatic cleaning were observed for ACC (<italic>p</italic> = 0.042) and ATPmetry (<italic>p</italic> = 0.002).</p><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>ACC (log CFU/cm<sup>2</sup>) and ATPmetry (pg cATP/ml) for processing surfaces in relation with cleaning treatment.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cleaning treatment</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">ACC (log CFU/g)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">ATPmetry (pg cATP/ml)</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Group</td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Mann–Whitney Test - Conventional cleaning (Group A), Enzymatic cleaning (Group B)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\"><hr/></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">P50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">P90</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Pmax</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">n</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">P50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">P90</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Pmax</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ACC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ATPmetry</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Conventional cleaning</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">39,7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">929,2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic> = 0.042</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic> = 0.002</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Enzymatic cleaning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reinforced enzymatic cleaning</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3,2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Routine enzymatic cleaning 3x/wk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Routine enzymatic cleaning 2x/wk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">DL</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Routine enzymatic cleaning 1x/wk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><Dl</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Conventional cleaning</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><Dl</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Enzymatic cleaning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reinforced enzymatic cleaning 2 (OPC)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic>ACC, aerobic colony counts; Dl, detection limit; P50, percentile 50; P90, percentile 90; Pmax, percentile 100.</italic></attrib></table-wrap-foot></table-wrap><p>Metagenetic analyses indicate that the proportion of spoilers remained relatively stable on the surface during the different cleaning treatments (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref> and Supplementary Material). However, the composition of microorganisms in this group changed with the cleaning method. <italic>Pseudomonas</italic> sp. (16.6 ± 32.5%) and <italic>Enterobacteriaceae</italic> (46.3 ± 60.8%) were the most abundant bacteria during the first conventional cleaning. Spore-forming bacteria were the most predominant group during curative enzymatic cleaning (49.1 ± 61.7%), routine enzymatic cleaning 3x/week (56.6 ± 75.7%), 2X/week (39.4 ± 48.5%), and 1x week (49.7 ± 49.5%). During the second conventional cleaning period, microbiological diversity increased, having a high proportion of “others” (78.12 ± 51.7%). Finally, when the second curative cleaning was reinstated, spore-forming bacteria reappeared at a high proportion (38.7 ± 61.7%). Figure F presents the results of the statistical analysis on the metagenetic data (Welsh t-test). Significant differences between conventional cleaning and enzymatic cleaning were noted on <italic>Streptococcus</italic> sp. (<italic>p</italic> < 0.029) and spore-forming bacteria (<italic>p</italic> < 0.046). These were more present during enzymatic cleaning. The implementation of the enzymatic cleaning on surfaces led to a decrease in bacterial concentration and a shift in bacterial composition. The coverage index for surface sampling was 0.986–0.993. Chao1 index values were relatively constant during the cleaning treatments. An increase in microbial diversity was observed after the implementation of the enzymatic cleaning with an increasing inverse Simpson index.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Percentiles 50, 90 and maximum of Aerobic Colony Counts (ACC) (log CFU/cm<sup>2</sup>) and mean abundances of the dominant bacterial groups (%) on processing surfaces in different cleaning treatments.</p></caption><graphic xlink:href=\"fmicb-11-01827-g003\"/></fig></sec><sec id=\"S3.SS3\"><title>Closed Circuits</title><p><xref rid=\"T4\" ref-type=\"table\">Table 4</xref> shows the results of classical microbiology analysis after the enzymatic treatment of closed circuits (closing pipes and tanks) during the CIP treatment. This treatment was carried once during the first reinforced enzymatic cleaning due to its practicality. No microorganisms and low ATP concentration were detected in the rinsing water before enzymatic cleaning. However, they both increased during enzymatic treatment in some part of the process, indicating a release of microorganisms and organic compounds during treatment.</p><table-wrap id=\"T4\" position=\"float\"><label>TABLE 4</label><caption><p>ACC (log CFU/ml) and ATPmetry (pg cATP/ml) for the closed circuits during enzymatic cleaning treatment.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Closed circuits</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ACC</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">ATPmetry</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(log CFU/ml)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">(pg cATP/ml)</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Rinsing water</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><Dl</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.6</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Line 1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10748.5</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Line 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1230.5</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dough machine 1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">735.9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dough machine 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1598.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bolognese cooking tank</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><Dl</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bechamel cooking tank</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><Dl</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.5</td></tr></tbody></table><table-wrap-foot><attrib><italic>ACC, aerobic colony counts; Dl, Detection Limit.</italic></attrib></table-wrap-foot></table-wrap><p><xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref> and <xref rid=\"T3\" ref-type=\"table\">Table 3</xref> indicate that a high proportion of spoilers was released in line 1 (98.1%), which were mainly <italic>Pseudomonas</italic> and spore forming bacteria, in dough machine 1 (46.6%), mainly <italic>Enterobacteriaceae</italic>, and in dough machine 2 (68.1%), mainly <italic>Enterobacteriaceae</italic> and <italic>Pseudomonas</italic>. “Others” was predominant in line 2 (99.9%). Although no bacteria were detected by classical microbiology in the bechamel and Bolognese tanks, spoiling bacteria were detected in high proportions (98.0% and 70.5%), respectively. <italic>Pseudomonas and Enterobacteriaceae</italic> were mainly present in the Bolognese tank, whereas spore-forming bacteria were mainly detected in the bechamel tank. The coverage index values ranged from 0.941 to 0.995. The minimum values for Chao1 and inverse Simpson index were from line 2 and bechamel tank, having the presence of one dominant bacterial species, <italic>Stenotrophomonas maltophilia</italic> and <italic>Brochotrix thermosphacta</italic>, respectively. The highest values for Chao1 and inverse Simpson index were from the dough machine 2, which was also the sample with the highest concentration of total flora.</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Aerobic Colony Counts (ACC) (log CFU/ml) and abundances of the dominant bacterial groups (%) in the closed circuits during the reinforced enzymatic treatments.</p></caption><graphic xlink:href=\"fmicb-11-01827-g004\"/></fig></sec><sec id=\"S3.SS4\"><title>Food Products</title><p><xref rid=\"T5\" ref-type=\"table\">Table 5</xref> lists the ACC and ANCC values in the final product at D<sub>0</sub>, D<sub>end of the shelf life</sub>, and D<sub>end of the shelf life +7 days</sub> in relation to the cleaning method. As expected, the total flora increased during the shelf life. The P90 of ACC and ANCC decreased in food products analyzed at D<sub>0</sub> by about 2 log UFC/g after the implementation of enzymatic cleaning, increased when conventional cleaning is reinstated, and decreased again after enzymatic cleaning. Significant differences for ACC were found between conventional and enzymatic cleaning for the finished products at D<sub>0</sub> (<italic>p</italic> = 0.011), D<sub>end of the shelf life</sub> (<italic>p</italic> = 0.029) and D<sub>end of the shelf life +7 days</sub> (<italic>p</italic> = 0.027), and for ANCC at D<sub>0</sub> (<italic>p</italic> = 0.010) and D<sub>end of the shelf life +7 days</sub> (<italic>p</italic> = 0.004).</p><table-wrap id=\"T5\" position=\"float\"><label>TABLE 5</label><caption><p>ACC and ANCC of the food product at D<sub>0</sub>, D<sub>end of the shelf life</sub>, and D<sub>end of the shelf life +7 days</sub> for different cleaning treatments.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><tbody><tr><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><bold>Cleaning treatment</bold></td><td valign=\"top\" align=\"center\" colspan=\"9\" rowspan=\"1\"><bold>ACC</bold></td><td valign=\"top\" align=\"center\" colspan=\"9\" rowspan=\"1\"><bold>ANCC</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Group</bold></td><td valign=\"top\" align=\"center\" colspan=\"6\" rowspan=\"1\"><bold>Mann–Whitney Test − Conventional cleaning (Group A.) Enzymatic cleaning (Group B)</bold></td></tr><tr><td valign=\"top\" colspan=\"3\" rowspan=\"1\"/><td valign=\"top\" colspan=\"9\" rowspan=\"1\"/><td valign=\"top\" colspan=\"9\" rowspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>ACC</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>ANCC</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>D<sub>0</sub></bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>D<sub>end of the shelf life</sub></bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>D<sub>end of the shelf life + 7 days</sub></bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>D<sub>0</sub></bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>D<sub>end of the shelf life</sub></bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>D<sub>end of the shelf life + 7 days</sub></bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>D<sub>0</sub></bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>D<sub>end of the shelf life</sub></bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>D<sub>end of the shelf life + 7 days</sub></bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>D<sub>0</sub></bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>D<sub>end of the shelf life</sub></bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>D<sub>end of the shelf life + 7 days</sub></bold></td></tr><tr><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><hr/></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><hr/></td></tr><tr><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>n</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>P50</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>P90</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Pmax</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>P50</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>P90</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Pmax</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>P50</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>P90</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Pmax</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>P50</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>P90</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Pmax</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>P50</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>P90</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Pmax</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>P50</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>P90</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Pmax</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Conventional cleaning</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,68</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,76</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,53</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,62</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,99</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,46</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9,41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic> = 0.011</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic> = 0.029</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic> = 0.027</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic> = 0.010</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic> = 0.04</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Enzymatic cleaning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reinforced enzymatic cleaning (OPC + CIP)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9,1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9,2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><Dl</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,56</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,62</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9,11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9,18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,93</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,07</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Routine enzymatic cleaning 3x/wk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6,6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6,66</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,79</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,36</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Routine enzymatic cleaning 2x/wk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><Dl</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,65</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,81</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,79</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Routine enzymatic cleaning 1x/wk</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0,7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,04</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,62</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,66</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Conventional cleaning 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6,8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9,3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9,5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,63</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5,59</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,43</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,81</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,53</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Enzymatic cleaning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reinforced enzymatic cleaning 2 (OPC)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5,5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6,0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6,1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5,3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6,0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6,1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,82</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5,51</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9,16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9,26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,54</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,63</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">B</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic>NS, not significant; ACC, aerobic colony counts; ANCC, anaerobic colony counts; D<sub>0</sub>, food product analyzed at the beginning of the shelf life; D<sub><italic>end of the shelf life</italic></sub>, food product analyzed at the end of the shelf life; D<sub><italic>end of the shelf +</italic> 7 <italic>days</italic></sub>, food product analyzed 7 days after the end of the shelf life; Dl, detection limit; P50, percentile 50; P90, percentile 90; Pmax, percentile 100.</italic></attrib></table-wrap-foot></table-wrap><p>From <xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref> and <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>, the main bacteria present at the start of shelf life were lactic acid bacteria (<italic>Lactococcu</italic>s sp.), regardless of the cleaning treatment. The proportion of spoilers decreased in the finished products at D<sub>end of the shelf life +7 days</sub> after enzymatic cleaning, except when the enzymatic cleaning frequency was reduced to once a week. <xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref> shows significant differences between conventional and enzymatic cleaning for the bacteria in the “Others” group in the product analyzed at D<sub>0</sub>, for <italic>Leuconostoc</italic> sp. and <italic>Lactobacillus</italic> sp. at D<sub>end of the shelf life</sub>, and <italic>Leuconostoc</italic> sp. at D<sub>end of the shelf life +7 days</sub>. The decrease in spoiling bacteria was compensated by the higher proportion of lactic acid bacteria. The coverage index ranged from 0.988–0.996 for the food products. Chao1 and the inverse Simpson index were relatively stable throughout the shelf life of the food products. The number of OTUs and the microbial diversity were constant and relatively lower compared to with the other samples.</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Percentiles 50, 90 and maximum of Aerobic Colony Counts (ACC) (log CFU/g) and mean abundances of the dominant bacterial groups (%) in the food products throughout their shelf life after different cleaning treatments.</p></caption><graphic xlink:href=\"fmicb-11-01827-g005\"/></fig><fig id=\"F6\" position=\"float\"><label>FIGURE 6</label><caption><p>Bacterial species with a statistical difference between conventional and enzymatic cleanings of processing surfaces and in the lasagne throughout its shelf life.</p></caption><graphic xlink:href=\"fmicb-11-01827-g006\"/></fig></sec></sec><sec id=\"S4\"><title>Discussion</title><p>Cleaning and disinfection are the major day-to-day controls for hard-surface vectors in food product contamination. If effective, they can reduce hazards within the processing environment (<xref rid=\"B43\" ref-type=\"bibr\">Holah, 2013</xref>). The cleaning process removes food residues, soils, and organic matters that accumulate on processing equipment and surfaces during production (<xref rid=\"B32\" ref-type=\"bibr\">Gabric et al., 2016</xref>). Microorganisms adhering on the food product’s contact surfaces could be an important source of potential contamination, thus leading to serious hygienic problems and economic losses due to food spoilage (<xref rid=\"B43\" ref-type=\"bibr\">Holah, 2013</xref>). Biofilms are the dominant lifestyle of bacteria and are also likely found within food industry premises. Bacteria that reside, accumulate, and persist in biofilms on surfaces with risks of subsequent transfer to food products are threats to food quality and safety (<xref rid=\"B33\" ref-type=\"bibr\">Galié et al., 2018</xref>). In the food industry, aggressive chemicals, such as sodium hydroxide and sodium hypochlorite, together with clean-in-place techniques are often used to mitigate undesirable biofilm effects. However, such approaches are not always effective for biofilm control (<xref rid=\"B66\" ref-type=\"bibr\">Meireles et al., 2016</xref>). Correct cleaning and disinfection strategies for biofilm eradication and prevention with documented effects under relevant conditions are necessary to overcome biofilms in food process industries (<xref rid=\"B95\" ref-type=\"bibr\">Simões et al., 2010</xref>).</p><p>Classical culture methods, such as agar plating, are not effective to detect biofilms due to the difficulty in culturing many biofilm bacteria, as well as the “viable but non-culturable” (VBNC) form with low metabolic activity. VBNC cells can be detected by using a culture-independent technique (<xref rid=\"B36\" ref-type=\"bibr\">Gião and Keevil, 2014</xref>). Developed in the last decades, next-generation sequencing methods have contributed immensely to the exploration of food microbiota (<xref rid=\"B34\" ref-type=\"bibr\">Galimberti et al., 2015</xref>) in beverages (<xref rid=\"B23\" ref-type=\"bibr\">Elizaquível et al., 2015</xref>; <xref rid=\"B77\" ref-type=\"bibr\">Pérez-Cataluña et al., 2018</xref>), vegetables (<xref rid=\"B54\" ref-type=\"bibr\">Lee et al., 2017</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Gu et al., 2018</xref>), dairy (<xref rid=\"B69\" ref-type=\"bibr\">Nalbantoglu et al., 2014</xref>; <xref rid=\"B12\" ref-type=\"bibr\">Ceugniez et al., 2017</xref>; <xref rid=\"B81\" ref-type=\"bibr\">Porcellato et al., 2018</xref>), seafood (<xref rid=\"B74\" ref-type=\"bibr\">Parlapani et al., 2018</xref>; <xref rid=\"B94\" ref-type=\"bibr\">Silbande et al., 2018</xref>), and meat products (<xref rid=\"B18\" ref-type=\"bibr\">De Filippis et al., 2013</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Benson et al., 2014</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Greppi et al., 2015</xref>; <xref rid=\"B80\" ref-type=\"bibr\">Połka et al., 2015</xref>; <xref rid=\"B98\" ref-type=\"bibr\">Stoops et al., 2015</xref>; <xref rid=\"B103\" ref-type=\"bibr\">Zhao et al., 2015</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Delhalle et al., 2016</xref>; <xref rid=\"B63\" ref-type=\"bibr\">Mann et al., 2016</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Carrizosa et al., 2017</xref>; <xref rid=\"B10\" ref-type=\"bibr\">Cauchie et al., 2017</xref>; <xref rid=\"B49\" ref-type=\"bibr\">Kaur et al., 2017</xref>; <xref rid=\"B78\" ref-type=\"bibr\">Peruzy et al., 2019</xref>). However, only a few studies have investigated the microbial flora of food processing surfaces and equipment using next-generation sequencing methodologies (<xref rid=\"B5\" ref-type=\"bibr\">Bokulich and Mills, 2013</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Bokulich et al., 2013</xref>; <xref rid=\"B65\" ref-type=\"bibr\">Mayo et al., 2014</xref>). The objective of this study was to evaluate the impact of enzymatic cleaning on the microbial flora of installations in a processed food industry, as well as in the final food product, using classical microbiology and metagenetic analysis. A food product with a very low initial bacterial flora concentration was selected to assess the change in the microbial flora from facilities to the final food product throughout its shelf life.</p><p>The interpretation of metagenetic analysis can sometimes be difficult when the number of identified microorganisms is high. To facilitate this, we summarized the results in the graphs by forming three groups of bacteria. “Spoilers” group contains microorganisms described as spoiling bacteria of various meat-based foodstuff (<xref rid=\"B48\" ref-type=\"bibr\">Iulietto et al., 2015</xref>; <xref rid=\"B3\" ref-type=\"bibr\">André et al., 2017</xref>). Lactic acid bacteria’s role is more ambiguous as they could be either be spoiling or protective, depending on the food product, environment, and species (<xref rid=\"B82\" ref-type=\"bibr\">Pothakos et al., 2015</xref>). Lastly, “Others” contained the bacterial taxa present at very low proportions (<1%) and were considered to have a minor impact on the microbial quality of the food product throughout the its shelf life (<xref rid=\"B10\" ref-type=\"bibr\">Cauchie et al., 2017</xref>).</p><p>Aerobic colony count values of the Bolognese and bechamel sauces were low due to the thermal process involved in the preparation of the ingredients. Lactic acid bacteria were dominant in the cheese and bechamel sauce. <italic>Lactococcus lactis</italic> is commonly used as a starter bacteria for cheese production (<xref rid=\"B31\" ref-type=\"bibr\">Fox and McSweeney, 2017</xref>). The milk used for the preparation of bechamel sauce contains an initial bacterial flora, which is eliminated in the cooking step. <italic>Streptococcus sp.</italic> is commonly found in raw milk (<xref rid=\"B86\" ref-type=\"bibr\">Quigley et al., 2013</xref>) and is heat-resistant; therefore, it could contaminate post-pasteurization (<xref rid=\"B28\" ref-type=\"bibr\">Flint et al., 2002</xref>). <italic>Enterobacteriaceae</italic> was identified in the Bolognese sauce and dough at a high proportion and is commonly present in red meat products (<xref rid=\"B22\" ref-type=\"bibr\">Doulgeraki et al., 2012</xref>) and wheat dough (<xref rid=\"B21\" ref-type=\"bibr\">Dinardo et al., 2019</xref>). <italic>Pseudomonas</italic> sp. was also present in the dough at a high proportion and has been identified in wheat dough previously (<xref rid=\"B11\" ref-type=\"bibr\">Celano et al., 2016</xref>; <xref rid=\"B17\" ref-type=\"bibr\">De Angelis et al., 2019</xref>; <xref rid=\"B67\" ref-type=\"bibr\">Menezes et al., 2020</xref>). Although the concentration of microorganisms in the ingredients was very low, they could be the starting point for contamination and lead to the spoilage of food products (<xref rid=\"B45\" ref-type=\"bibr\">ICMFS, 2006</xref>).</p><p>Some surfaces were highly contaminated during the first part of the study with conventional cleaning. These results are in accordance with other studies which evaluated the microbial concentration on food contact surfaces (<xref rid=\"B1\" ref-type=\"bibr\">Abdallah et al., 2014</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Cunault et al., 2019</xref>). Because some equipment are difficult to clean, some organic residues could still be present even after cleaning and disinfection (<xref rid=\"B16\" ref-type=\"bibr\">da Costa Luciano et al., 2016</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Gabric et al., 2016</xref>). The presence of organic residues promotes biofilm formation, which could be a permanent source of contamination (<xref rid=\"B101\" ref-type=\"bibr\">Wirtanen and Salo, 2016</xref>). The average concentration of AAC and ATP on surface decreased after the enzymatic cleaning. The implementation of enzymatic cleaning on surfaces resulted to a decrease in bacterial concentration and a shift in bacterial composition. <italic>Pseudomonas</italic> sp. and <italic>Enterobacteriaceae</italic> were mainly present during conventional cleaning, and they are described as biofilm formers and potential spoilers (<xref rid=\"B60\" ref-type=\"bibr\">López et al., 2010</xref>). The increasing proportion of spore-forming bacteria during enzymatic cleaning could be attributed to their higher resistance against the stress brought by the sanitizing process (<xref rid=\"B62\" ref-type=\"bibr\">Maillard, 2016</xref>). <italic>Brochotrix thermosphacta</italic> and <italic>Enterobacteriaceae</italic> have disappeared from surfaces when enzymatic cleaning was done three times per week.</p><p>Although the closed circuits were treated with conventional cleaning prior to enzymatic cleaning, there is a significant release in microorganisms and ATP in some parts of the installations during the treatment, especially for lines 1 and 2 and dough machines 1 and 2. No bacteria was detected by classical microbiology in the Bolognese and bechamel tanks due to the thermal inactivation of microorganisms during the cooking process (<xref rid=\"B45\" ref-type=\"bibr\">ICMFS, 2006</xref>). Bacteria identified in the dough machine and Bolognese and bechamel tanks are related to the ingredients used in these equipment’s. Several studies have described that conventional sanitizing process does not completely eliminate the microbial flora in closing pipes (<xref rid=\"B56\" ref-type=\"bibr\">Lelièvre et al., 2002</xref>; <xref rid=\"B7\" ref-type=\"bibr\">Bremer et al., 2006</xref>; <xref rid=\"B52\" ref-type=\"bibr\">Kumari and Sarkar, 2014</xref>, <xref rid=\"B53\" ref-type=\"bibr\">2016</xref>; <xref rid=\"B59\" ref-type=\"bibr\">Liu et al., 2014</xref>). For example, Parijs and Steeenackers. have shown that microbial contamination after the CIP process in several breweries was reduced by less than 75% in 52% of the samples and was even increased in 24% of the samples, indicating that CIP is insufficient, and improving antimicrobial treatments is essential (<xref rid=\"B72\" ref-type=\"bibr\">Parijs and Steenackers, 2018</xref>). A high proportion of <italic>Pseudomonas</italic> sp. was found for the line 1 (67,9%) and to a lesser extent in other equipment as the dough machine 2 (36, 8%) and the Bolognese tank (37,6%). <italic>Pseudomonas</italic> spp. is among the bacteria most frequently isolated from surfaces in the food industry and it produces multispecies biofilm on the wall of tanks and pipelines before heat processing (<xref rid=\"B93\" ref-type=\"bibr\">Shirtliff et al., 2002</xref>; <xref rid=\"B64\" ref-type=\"bibr\">Marchand et al., 2012</xref>). Spore-forming bacteria were detected in the bechamel tank at a high proportion (97,9%). Several previous studies assessed CIP procedures to eliminate spore-forming bacteria and demonstrated that the efficacy is related to the several parameters such as the surface chemistry, shear forces and the detergent applied during the cleaning (<xref rid=\"B56\" ref-type=\"bibr\">Lelièvre et al., 2002</xref>; <xref rid=\"B99\" ref-type=\"bibr\">Sundberg et al., 2011</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Faille et al., 2013</xref>). Sporulation could occur in biofilms, suggesting that biofilms would be a significant source of food contamination with spores (<xref rid=\"B100\" ref-type=\"bibr\">Wijman et al., 2007</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Faille et al., 2014</xref>).</p><p>Expectedly, ACC results in the food products showed an increasing concentration of the bacterial flora during shelf life. After the implementation of enzymatic cleaning, the bacterial flora at the beginning of the shelf life decreased to 2 log CFU/g. The hygiene of food installations has a measurable effect on the food product, especially when the initial concentration of the food products is very low (<xref rid=\"B45\" ref-type=\"bibr\">ICMFS, 2006</xref>). The products at D<sub>0</sub> were mainly composed of <italic>Lactococcus</italic> sp., which was mainly present in the cheese used as topping of the lasagne. Spoiling bacteria, such as <italic>Enterobacteriaceae sp.</italic> and <italic>Leuconostoc</italic> sp., were predominant in the food product along its shelf life during conventional cleaning. After the implementation of enzymatic cleaning, the proportion of spoiling bacteria decreased in favor of lactic acid bacteria, such as <italic>Lactobacillus</italic> sp. and <italic>Lactococcus</italic> sp., during the shelf life of the products. However, when the frequency of enzymatic cleaning was reduced to once a week, the proportion of spoiling bacteria increased again. Therefore, a minimum frequency of enzymatic cleaning is necessary to maintain a low proportion of spoiling bacteria in food products. Although there was a significant decrease in <italic>Leuconostoc</italic> sp. in favor of <italic>Lactobacillus sp.</italic>, it was not possible to affirm that the food product was not spoiled. Complementary tests, such as sensory analyses, are necessary to confirm that the product maintains all its microbial and technical qualities throughout the shelf life.</p><p>Several studies in meat microbiology have established that spoilage is caused by only a fraction of the initial microbial flora that dominates the food product throughout its shelf life (<xref rid=\"B70\" ref-type=\"bibr\">Nychas et al., 2008</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Doulgeraki et al., 2012</xref>). These spoilage microorganisms have been designated as E(S)SOs due to their ability to eventually become dominant in the spoilage flora (<xref rid=\"B70\" ref-type=\"bibr\">Nychas et al., 2008</xref>; <xref rid=\"B76\" ref-type=\"bibr\">Pennacchia et al., 2011</xref>). <italic>Leuconostoc</italic> sp. was described as a spoilage bacteria in several food products, such as ready-to-eat food products (<xref rid=\"B79\" ref-type=\"bibr\">Petruzzi et al., 2017</xref>; <xref rid=\"B75\" ref-type=\"bibr\">Pellissery et al., 2020</xref>). This bacterium can grow very quickly and dominate the bacterial flora of food products packaged in modified atmosphere, even if its initial concentration is very low (<xref rid=\"B22\" ref-type=\"bibr\">Doulgeraki et al., 2012</xref>). During conventional cleaning, it was present in the food product at a very low proportion at the beginning of the shelf life and became the most predominant bacteria at the end of the shelf life. After enzymatic cleaning, its proportion was reduced in the food products along the shelf life. It was also detected on surfaces at a very low proportion, regardless of the cleaning treatment. However, ACC showed that bacterial concentration on surfaces was reduced during enzymatic treatment, including that of <italic>Leuconostoc</italic> sp. It is therefore likely that the decrease in the concentration of <italic>Leuconostoc</italic> sp. in the food installations leads to its reduction in the final food product.</p><p>Few studies are available comparing different cleaning methods on the microbial ecology in agro-industrial facilities and no studies have used high-throughput sequencing methods to describe the microbial population of industrial facilities and finished products according to different types of cleaning. High throughput sequencing coupled with classical microbiology methods provides useful information on the dynamics of bacterial populations according to environmental conditions, such as the change of cleaning methods. This study was carried out in a processing industry which produces the same product along the time and where the bacterial concentration in the food product is low at the beginning of its shelf life. In those conditions, it is possible to observe changes of the microbial flora in the food products following the change of cleaning procedures of the installations, which would probably be more difficult to observe in foodstuffs with more variable and higher bacterial flora concentration such as perishable foods. Future studies could also be conducted to evaluate cleaning methods on the microbial flora of instillations and the impact on finished products in other food sectors such as dairies, meat or fish industries.</p><p>A decrease in the microbial flora concentration and the proportion of spoilage bacteria on installations surfaces was observed after enzymatic cleaning compared to conventional cleaning. At the same time, a reduction of the initial concentration at the beginning of the shelf life and a reduction of the spoiling bacteria throughout the shelf life of the food product was also observed. Enzymic cleaning has led to an improvement in the hygiene of the facilities and the microbial quality of the food throughout the shelf life. Although its effectiveness in removing organic residues and biofilms is increasingly studied, this type of cleaning is not yet commonly used in the food industry. Depending on the installations, production method, and the food product itself with its specific risks, enzymatic cleaning should be considered in combination with conventional sanitizing methods to improve plant hygiene.</p></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/\">https://www.ncbi.nlm.nih.gov/</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"PRJNA615390\">PRJNA615390</ext-link>.</p></sec><sec id=\"S6\"><title>Author Contributions</title><p>LD contributed to design and followed up the study. BT performed the statistical analysis on metagenetic results. SF and MB contributed to technical support for enzymatic cleaners. AF carried out the bioinformatics on metagenetic analysis. SB carried out the metagenetic analysis. GD supervised the study. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>MB and SF were employed by the Realco. SB and AF were employed by the Genalyse Partner. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by the Directorate General for Economy, Employment, and Research of the Walloon Region (DGO6) of Belgium (Grant No. 7320).</p></fn></fn-group><ack><p>The authors thank the employees of the Genalyse Partner and Realco for their technical support.</p></ack><fn-group><fn id=\"footnote1\"><label>1</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://microbiomeutil.sourceforge.net/#A_CS\">http://microbiomeutil.sourceforge.net/#A_CS</ext-link></p></fn><fn id=\"footnote2\"><label>2</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.arb-silva.de/\">http://www.arb-silva.de/</ext-link>, version v1.2.11.</p></fn><fn id=\"footnote3\"><label>3</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.mothur.org\">http://www.mothur.org</ext-link></p></fn><fn id=\"footnote4\"><label>4</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"https://beikolab.cs.dal.ca/software/STAMP\">https://beikolab.cs.dal.ca/software/STAMP</ext-link></p></fn></fn-group><sec id=\"S9\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fmicb.2020.01827/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fmicb.2020.01827/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"TS1\"><media xlink:href=\"Table_1.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abdallah</surname><given-names>M.</given-names></name><name><surname>Benoliel</surname><given-names>C.</given-names></name><name><surname>Drider</surname><given-names>D.</given-names></name><name><surname>Dhulster</surname><given-names>P.</given-names></name><name><surname>Chihib</surname><given-names>N.-E.</given-names></name></person-group> (<year>2014</year>). <article-title>Biofilm formation and persistence on abiotic surfaces in the context of food and medical environments.</article-title>\n<source><italic>Arch. Microbiol.</italic></source>\n<volume>196</volume>\n<fpage>453</fpage>–<lpage>472</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00203-014-0983-1</pub-id>\n<pub-id pub-id-type=\"pmid\">24744186</pub-id></mixed-citation></ref><ref id=\"B2\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Adator</surname><given-names>E. H.</given-names></name><name><surname>Cheng</surname><given-names>M.</given-names></name><name><surname>Holley</surname><given-names>R.</given-names></name><name><surname>McAllister</surname><given-names>T.</given-names></name><name><surname>Narvaez-Bravo</surname><given-names>C.</given-names></name></person-group> (<year>2018</year>). <article-title>Ability of Shiga toxigenic <italic>Escherichia coli</italic> to survive within dry-surface biofilms and transfer to fresh lettuce.</article-title>\n<source><italic>Int. J. Food Microbiol.</italic></source>\n<volume>269</volume>\n<fpage>52</fpage>–<lpage>59</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijfoodmicro.2018.01.014</pub-id>\n<pub-id pub-id-type=\"pmid\">29421358</pub-id></mixed-citation></ref><ref id=\"B3\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>André</surname><given-names>S.</given-names></name><name><surname>Vallaeys</surname><given-names>T.</given-names></name><name><surname>Planchon</surname><given-names>S.</given-names></name></person-group> (<year>2017</year>). <article-title>Spore-forming bacteria responsible for food spoilage.</article-title>\n<source><italic>Res. Microbiol.</italic></source>\n<volume>168</volume>\n<fpage>379</fpage>–<lpage>387</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.resmic.2016.10.003</pub-id>\n<pub-id pub-id-type=\"pmid\">27989764</pub-id></mixed-citation></ref><ref id=\"B4\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Benson</surname><given-names>A. K.</given-names></name><name><surname>David</surname><given-names>J. R. D.</given-names></name><name><surname>Gilbreth</surname><given-names>S. E.</given-names></name><name><surname>Smith</surname><given-names>G.</given-names></name><name><surname>Nietfeldt</surname><given-names>J.</given-names></name><name><surname>Legge</surname><given-names>R.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Microbial successions are associated with changes in chemical profiles of a model refrigerated fresh pork sausage during an 80-day shelf life study.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>80</volume>\n<fpage>5178</fpage>–<lpage>5194</lpage>. <pub-id pub-id-type=\"doi\">10.1128/AEM.00774-14</pub-id>\n<pub-id pub-id-type=\"pmid\">24928886</pub-id></mixed-citation></ref><ref id=\"B5\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bokulich</surname><given-names>N. A.</given-names></name><name><surname>Mills</surname><given-names>D. A.</given-names></name></person-group> (<year>2013</year>). <article-title>Facility-specific “House” microbiome drives microbial landscapes of artisan cheesemaking plants.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>79</volume>\n<fpage>5214</fpage>–<lpage>5223</lpage>. <pub-id pub-id-type=\"doi\">10.1128/AEM.00934-13</pub-id>\n<pub-id pub-id-type=\"pmid\">23793641</pub-id></mixed-citation></ref><ref id=\"B6\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bokulich</surname><given-names>N. A.</given-names></name><name><surname>Ohta</surname><given-names>M.</given-names></name><name><surname>Richardson</surname><given-names>P. M.</given-names></name><name><surname>Mills</surname><given-names>D. A.</given-names></name></person-group> (<year>2013</year>). <article-title>Monitoring seasonal changes in winery-resident microbiota.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>8</volume>:<issue>e66437</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0066437</pub-id>\n<pub-id pub-id-type=\"pmid\">23840468</pub-id></mixed-citation></ref><ref id=\"B7\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bremer</surname><given-names>P. J.</given-names></name><name><surname>Fillery</surname><given-names>S.</given-names></name><name><surname>McQuillan</surname><given-names>A. J.</given-names></name></person-group> (<year>2006</year>). <article-title>Laboratory scale Clean-In-Place (CIP) studies on the effectiveness of different caustic and acid wash steps on the removal of dairy biofilms.</article-title>\n<source><italic>Int. J. Food Microbiol.</italic></source>\n<volume>106</volume>\n<fpage>254</fpage>–<lpage>262</lpage>. <pub-id pub-id-type=\"doi\">10.1016/J.IJFOODMICRO.2005.07.004</pub-id>\n<pub-id pub-id-type=\"pmid\">16216371</pub-id></mixed-citation></ref><ref id=\"B8\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bridier</surname><given-names>A.</given-names></name><name><surname>Sanchez-Vizuete</surname><given-names>P.</given-names></name><name><surname>Guilbaud</surname><given-names>M.</given-names></name><name><surname>Piard</surname><given-names>J.-C.</given-names></name><name><surname>Naïtali</surname><given-names>M.</given-names></name><name><surname>Briandet</surname><given-names>R.</given-names></name></person-group> (<year>2015</year>). <article-title>Biofilm-associated persistence of food-borne pathogens.</article-title>\n<source><italic>Food Microbiol.</italic></source>\n<volume>45</volume>\n<fpage>167</fpage>–<lpage>178</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fm.2014.04.015</pub-id>\n<pub-id pub-id-type=\"pmid\">25500382</pub-id></mixed-citation></ref><ref id=\"B9\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carrizosa</surname><given-names>E.</given-names></name><name><surname>Benito</surname><given-names>M. J.</given-names></name><name><surname>Ruiz-Moyano</surname><given-names>S.</given-names></name><name><surname>Hernández</surname><given-names>A.</given-names></name><name><surname>Villalobos</surname><given-names>M. D. C.</given-names></name><name><surname>Martín</surname><given-names>A.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Bacterial communities of fresh goat meat packaged in modified atmosphere.</article-title>\n<source><italic>Food Microbiol.</italic></source>\n<volume>65</volume>\n<fpage>57</fpage>–<lpage>63</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fm.2017.01.023</pub-id>\n<pub-id pub-id-type=\"pmid\">28400020</pub-id></mixed-citation></ref><ref id=\"B10\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cauchie</surname><given-names>E.</given-names></name><name><surname>Gand</surname><given-names>M.</given-names></name><name><surname>Kergourlay</surname><given-names>G.</given-names></name><name><surname>Taminiau</surname><given-names>B.</given-names></name><name><surname>Delhalle</surname><given-names>L.</given-names></name><name><surname>Korsak</surname><given-names>N.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>The use of 16S rRNA gene metagenetic monitoring of refrigerated food products for understanding the kinetics of microbial subpopulations at different storage temperatures: the example of white pudding.</article-title>\n<source><italic>Int. J. Food Microbiol.</italic></source>\n<volume>247</volume>\n<fpage>70</fpage>–<lpage>78</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijfoodmicro.2016.10.012</pub-id>\n<pub-id pub-id-type=\"pmid\">27751567</pub-id></mixed-citation></ref><ref id=\"B11\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Celano</surname><given-names>G.</given-names></name><name><surname>De Angelis</surname><given-names>M.</given-names></name><name><surname>Minervini</surname><given-names>F.</given-names></name><name><surname>Gobbetti</surname><given-names>M.</given-names></name></person-group> (<year>2016</year>). <article-title>Different flour microbial communities drive to sourdoughs characterized by diverse bacterial strains and free amino acid profiles.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>7</volume>:<issue>1770</issue>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2016.01770</pub-id>\n<pub-id pub-id-type=\"pmid\">27877165</pub-id></mixed-citation></ref><ref id=\"B12\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ceugniez</surname><given-names>A.</given-names></name><name><surname>Taminiau</surname><given-names>B.</given-names></name><name><surname>Coucheney</surname><given-names>F.</given-names></name><name><surname>Jacques</surname><given-names>P.</given-names></name><name><surname>Delcenserie</surname><given-names>V.</given-names></name><name><surname>Daube</surname><given-names>G.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Use of a metagenetic approach to monitor the bacterial microbiota of & quot; Tomme d’Orchies & quot; cheese during the ripening process.</article-title>\n<source><italic>Int. J. Food Microbiol.</italic></source>\n<volume>247</volume>\n<fpage>65</fpage>–<lpage>69</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijfoodmicro.2016.10.034</pub-id>\n<pub-id pub-id-type=\"pmid\">27817942</pub-id></mixed-citation></ref><ref id=\"B13\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chaillou</surname><given-names>S.</given-names></name><name><surname>Chaulot-Talmon</surname><given-names>A.</given-names></name><name><surname>Caekebeke</surname><given-names>H.</given-names></name><name><surname>Cardinal</surname><given-names>M.</given-names></name><name><surname>Christieans</surname><given-names>S.</given-names></name><name><surname>Denis</surname><given-names>C.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage.</article-title>\n<source><italic>ISME J.</italic></source>\n<volume>9</volume>\n<fpage>1105</fpage>–<lpage>1118</lpage>. <pub-id pub-id-type=\"doi\">10.1038/ismej.2014.202</pub-id>\n<pub-id pub-id-type=\"pmid\">25333463</pub-id></mixed-citation></ref><ref id=\"B14\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coughlan</surname><given-names>L. M.</given-names></name><name><surname>Cotter</surname><given-names>P. D.</given-names></name><name><surname>Hill</surname><given-names>C.</given-names></name><name><surname>Alvarez-Ordóñez</surname><given-names>A.</given-names></name></person-group> (<year>2016</year>). <article-title>New weapons to fight old enemies: novel strategies for the (Bio)control of bacterial biofilms in the food industry.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>7</volume>:<issue>1641</issue>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2016.01641</pub-id>\n<pub-id pub-id-type=\"pmid\">27803696</pub-id></mixed-citation></ref><ref id=\"B15\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cunault</surname><given-names>C.</given-names></name><name><surname>Faille</surname><given-names>C.</given-names></name><name><surname>Calabozo-Delgado</surname><given-names>A.</given-names></name><name><surname>Benezech</surname><given-names>T.</given-names></name></person-group> (<year>2019</year>). <article-title>Structure and resistance to mechanical stress and enzymatic cleaning of <italic>Pseudomonas fluorescens</italic> biofilms formed in fresh-cut ready to eat washing tanks.</article-title>\n<source><italic>J. Food Eng.</italic></source>\n<volume>262</volume>\n<fpage>154</fpage>–<lpage>161</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jfoodeng.2019.06.006</pub-id></mixed-citation></ref><ref id=\"B16\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>da Costa Luciano</surname><given-names>C.</given-names></name><name><surname>Olson</surname><given-names>N.</given-names></name><name><surname>Tipple</surname><given-names>A. F. V.</given-names></name><name><surname>Alfa</surname><given-names>M.</given-names></name></person-group> (<year>2016</year>). <article-title>Evaluation of the ability of different detergents and disinfectants to remove and kill organisms in traditional biofilm.</article-title>\n<source><italic>Am. J. Infect. Control</italic></source>\n<volume>44</volume>\n<fpage>e243</fpage>–<lpage>e249</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ajic.2016.03.040</pub-id>\n<pub-id pub-id-type=\"pmid\">27234012</pub-id></mixed-citation></ref><ref id=\"B17\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Angelis</surname><given-names>M.</given-names></name><name><surname>Minervini</surname><given-names>F.</given-names></name><name><surname>Siragusa</surname><given-names>S.</given-names></name><name><surname>Rizzello</surname><given-names>C. G.</given-names></name><name><surname>Gobbetti</surname><given-names>M.</given-names></name></person-group> (<year>2019</year>). <article-title>Wholemeal wheat flours drive the microbiome and functional features of wheat sourdoughs.</article-title>\n<source><italic>Int. J. Food Microbiol.</italic></source>\n<volume>302</volume>\n<fpage>35</fpage>–<lpage>46</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijfoodmicro.2018.08.009</pub-id>\n<pub-id pub-id-type=\"pmid\">30177230</pub-id></mixed-citation></ref><ref id=\"B18\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Filippis</surname><given-names>F.</given-names></name><name><surname>La Storia</surname><given-names>A.</given-names></name><name><surname>Villani</surname><given-names>F.</given-names></name><name><surname>Ercolini</surname><given-names>D.</given-names></name></person-group> (<year>2013</year>). <article-title>Exploring the sources of bacterial spoilers in beefsteaks by culture-independent high-throughput sequencing.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>8</volume>:<issue>e70222</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0070222</pub-id>\n<pub-id pub-id-type=\"pmid\">23936168</pub-id></mixed-citation></ref><ref id=\"B19\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Delcenserie</surname><given-names>V.</given-names></name><name><surname>Taminiau</surname><given-names>B.</given-names></name><name><surname>Delhalle</surname><given-names>L.</given-names></name><name><surname>Nezer</surname><given-names>C.</given-names></name><name><surname>Doyen</surname><given-names>P.</given-names></name><name><surname>Crevecoeur</surname><given-names>S.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Microbiota characterization of a belgian protected designation of origin cheese. Herve cheese, using metagenomic analysis.</article-title>\n<source><italic>J. Dairy Sci.</italic></source>\n<volume>97</volume>\n<fpage>6046</fpage>–<lpage>6056</lpage>. <pub-id pub-id-type=\"doi\">10.3168/jds.2014-8225</pub-id>\n<pub-id pub-id-type=\"pmid\">25064656</pub-id></mixed-citation></ref><ref id=\"B20\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Delhalle</surname><given-names>L.</given-names></name><name><surname>Korsak</surname><given-names>N.</given-names></name><name><surname>Taminiau</surname><given-names>B.</given-names></name><name><surname>Nezer</surname><given-names>C.</given-names></name><name><surname>Burteau</surname><given-names>S.</given-names></name><name><surname>Delcenserie</surname><given-names>V.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>Exploring the bacterial diversity of belgian steak tartare using metagenetics and quantitative real-time PCR analysis.</article-title>\n<source><italic>J. Food Prot.</italic></source>\n<volume>79</volume>\n<fpage>220</fpage>–<lpage>229</lpage>. <pub-id pub-id-type=\"doi\">10.4315/0362-028X.JFP-15-185</pub-id>\n<pub-id pub-id-type=\"pmid\">26818982</pub-id></mixed-citation></ref><ref id=\"B21\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dinardo</surname><given-names>F. R.</given-names></name><name><surname>Minervini</surname><given-names>F.</given-names></name><name><surname>De Angelis</surname><given-names>M.</given-names></name><name><surname>Gobbetti</surname><given-names>M.</given-names></name><name><surname>Gänzle</surname><given-names>M. G.</given-names></name></person-group> (<year>2019</year>). <article-title>Dynamics of <italic>Enterobacteriaceae</italic> and lactobacilli in model sourdoughs are driven by pH and concentrations of sucrose and ferulic acid.</article-title>\n<source><italic>LWT</italic></source>\n<volume>114</volume>:<issue>108394</issue>\n<pub-id pub-id-type=\"doi\">10.1016/j.lwt.2019.108394</pub-id></mixed-citation></ref><ref id=\"B22\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Doulgeraki</surname><given-names>A. I.</given-names></name><name><surname>Ercolini</surname><given-names>D.</given-names></name><name><surname>Villani</surname><given-names>F.</given-names></name><name><surname>Nychas</surname><given-names>G. J. E.</given-names></name></person-group> (<year>2012</year>). <article-title>Spoilage microbiota associated to the storage of raw meat in different conditions.</article-title>\n<source><italic>Int. J. Food Microbiol.</italic></source>\n<volume>157</volume>\n<fpage>130</fpage>–<lpage>141</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijfoodmicro.2012.05.020</pub-id>\n<pub-id pub-id-type=\"pmid\">22682877</pub-id></mixed-citation></ref><ref id=\"B23\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Elizaquível</surname><given-names>P.</given-names></name><name><surname>Pérez-Cataluña</surname><given-names>A.</given-names></name><name><surname>Yépez</surname><given-names>A.</given-names></name><name><surname>Aristimuño</surname><given-names>C.</given-names></name><name><surname>Jiménez</surname><given-names>E.</given-names></name><name><surname>Cocconcelli</surname><given-names>P. S.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>Pyrosequencing vs. culture-dependent approaches to analyze lactic acid bacteria associated to chicha, a traditional maize-based fermented beverage from Northwestern Argentina.</article-title>\n<source><italic>Int. J. Food Microbiol.</italic></source>\n<volume>198</volume>\n<fpage>9</fpage>–<lpage>18</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijfoodmicro.2014.12.027</pub-id>\n<pub-id pub-id-type=\"pmid\">25584777</pub-id></mixed-citation></ref><ref id=\"B24\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ercolini</surname><given-names>D.</given-names></name><name><surname>Russo</surname><given-names>F.</given-names></name><name><surname>Nasi</surname><given-names>A.</given-names></name><name><surname>Ferranti</surname><given-names>P.</given-names></name><name><surname>Villani</surname><given-names>F.</given-names></name></person-group> (<year>2009</year>). <article-title>Mesophilic and psychrotrophic bacteria from meat and their spoilage potential in vitro and in beef.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>75</volume>\n<fpage>1990</fpage>–<lpage>2001</lpage>. <pub-id pub-id-type=\"doi\">10.1128/AEM.02762-08</pub-id>\n<pub-id pub-id-type=\"pmid\">19201980</pub-id></mixed-citation></ref><ref id=\"B25\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Faille</surname><given-names>C.</given-names></name><name><surname>Bénézech</surname><given-names>T.</given-names></name><name><surname>Blel</surname><given-names>W.</given-names></name><name><surname>Ronse</surname><given-names>A.</given-names></name><name><surname>Ronse</surname><given-names>G.</given-names></name><name><surname>Clarisse</surname><given-names>M.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>Role of mechanical vs. chemical action in the removal of adherent <italic>Bacillus</italic> spores during CIP procedures.</article-title>\n<source><italic>Food Microbiol.</italic></source>\n<volume>33</volume>\n<fpage>149</fpage>–<lpage>157</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fm.2012.09.010</pub-id>\n<pub-id pub-id-type=\"pmid\">23200646</pub-id></mixed-citation></ref><ref id=\"B26\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Faille</surname><given-names>C.</given-names></name><name><surname>Bénézech</surname><given-names>T.</given-names></name><name><surname>Midelet-Bourdin</surname><given-names>G.</given-names></name><name><surname>Lequette</surname><given-names>Y.</given-names></name><name><surname>Clarisse</surname><given-names>M.</given-names></name><name><surname>Ronse</surname><given-names>G.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Sporulation of <italic>Bacillus</italic> spp. within biofilms: a potential source of contamination in food processing environments.</article-title>\n<source><italic>Food Microbiol.</italic></source>\n<volume>40</volume>\n<fpage>64</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fm.2013.12.004</pub-id>\n<pub-id pub-id-type=\"pmid\">24549199</pub-id></mixed-citation></ref><ref id=\"B27\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fleming</surname><given-names>D.</given-names></name><name><surname>Rumbaugh</surname><given-names>K.</given-names></name></person-group> (<year>2017</year>). <article-title>Approaches to dispersing medical biofilms.</article-title>\n<source><italic>Microorganisms</italic></source>\n<volume>5</volume>:<issue>15</issue>. <pub-id pub-id-type=\"doi\">10.3390/microorganisms5020015</pub-id>\n<pub-id pub-id-type=\"pmid\">28368320</pub-id></mixed-citation></ref><ref id=\"B28\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Flint</surname><given-names>S.</given-names></name><name><surname>Brooks</surname><given-names>J.</given-names></name><name><surname>Bremer</surname><given-names>P.</given-names></name><name><surname>Walker</surname><given-names>K.</given-names></name><name><surname>Hausman</surname><given-names>E.</given-names></name></person-group> (<year>2002</year>). <article-title>The resistance to heat of thermo-resistant streptococci attached to stainless steel in the presence of milk.</article-title>\n<source><italic>J. Ind. Microbiol. Biotechnol.</italic></source>\n<volume>28</volume>\n<fpage>134</fpage>–<lpage>136</lpage>. <pub-id pub-id-type=\"doi\">10.1038/sj.jim.7000229</pub-id>\n<pub-id pub-id-type=\"pmid\">12074086</pub-id></mixed-citation></ref><ref id=\"B29\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Forsythe</surname><given-names>S.</given-names></name><name><surname>Hayes</surname><given-names>P. R.</given-names></name></person-group> (<year>2012</year>). <source><italic>Food hygiene, Microbiology and HACCP</italic></source>, <role>ed.</role>\n<person-group person-group-type=\"editor\"><name><surname>Forsythe</surname><given-names>S.</given-names></name></person-group> (<publisher-loc>Betlin</publisher-loc>: <publisher-name>Springer</publisher-name>).</mixed-citation></ref><ref id=\"B30\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fougy</surname><given-names>L.</given-names></name><name><surname>Desmonts</surname><given-names>M.-H.</given-names></name><name><surname>Coeuret</surname><given-names>G.</given-names></name><name><surname>Fassel</surname><given-names>C.</given-names></name><name><surname>Hamon</surname><given-names>E.</given-names></name><name><surname>Hézard</surname><given-names>B.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>Reducing salt in raw pork sausages increases spoilage and correlates with reduced bacterial diversity.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>82</volume>\n<fpage>3928</fpage>–<lpage>3939</lpage>. <pub-id pub-id-type=\"doi\">10.1128/AEM.00323-16</pub-id>\n<pub-id pub-id-type=\"pmid\">27107120</pub-id></mixed-citation></ref><ref id=\"B31\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Fox</surname><given-names>P. F.</given-names></name><name><surname>McSweeney</surname><given-names>P. L. H.</given-names></name></person-group> (<year>2017</year>). “<article-title>Cheese: an overview</article-title>,” in <source><italic>Cheese: Chemistry, Physics and Microbiology</italic></source>, <edition>4th Edn</edition> (<publisher-loc>Amsterdam</publisher-loc>: <publisher-name>Academic Press</publisher-name>), <fpage>5</fpage>–<lpage>21</lpage>.</mixed-citation></ref><ref id=\"B32\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Gabric</surname><given-names>D.</given-names></name><name><surname>Galic</surname><given-names>K.</given-names></name><name><surname>Timmerman</surname><given-names>H.</given-names></name></person-group> (<year>2016</year>). “<article-title>Cleaning of surfaces</article-title>,” in <source><italic>Handbook of Hygiene Control in the Food Industry</italic></source>, <edition>2nd Edn</edition>, (<publisher-loc>Amsterdam</publisher-loc>: <publisher-name>Woodhead Publishing Ltd</publisher-name>), <fpage>447</fpage>–<lpage>463</lpage>.</mixed-citation></ref><ref id=\"B33\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Galié</surname><given-names>S.</given-names></name><name><surname>García-Gutiérrez</surname><given-names>C.</given-names></name><name><surname>Miguélez</surname><given-names>E. M.</given-names></name><name><surname>Villar</surname><given-names>C. J.</given-names></name><name><surname>Lombó</surname><given-names>F.</given-names></name></person-group> (<year>2018</year>). <article-title>Biofilms in the food industry: health aspects and control methods.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>9</volume>:<issue>898</issue>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2018.00898</pub-id>\n<pub-id pub-id-type=\"pmid\">29867809</pub-id></mixed-citation></ref><ref id=\"B34\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Galimberti</surname><given-names>A.</given-names></name><name><surname>Bruno</surname><given-names>A.</given-names></name><name><surname>Mezzasalma</surname><given-names>V.</given-names></name><name><surname>De Mattia</surname><given-names>F.</given-names></name><name><surname>Bruni</surname><given-names>I.</given-names></name><name><surname>Labra</surname><given-names>M.</given-names></name></person-group> (<year>2015</year>). <article-title>Emerging DNA-based technologies to characterize food ecosystems.</article-title>\n<source><italic>Food Res. Int.</italic></source>\n<volume>69</volume>\n<fpage>424</fpage>–<lpage>433</lpage>. <pub-id pub-id-type=\"doi\">10.1016/J.FOODRES.2015.01.017</pub-id></mixed-citation></ref><ref id=\"B35\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ghafir</surname><given-names>Y.</given-names></name><name><surname>China</surname><given-names>B.</given-names></name><name><surname>Dierick</surname><given-names>K.</given-names></name><name><surname>De Zutter</surname><given-names>L.</given-names></name><name><surname>Daube</surname><given-names>G.</given-names></name></person-group> (<year>2008</year>). <article-title>Hygiene indicator microorganisms for selected pathogens on beef, pork, and poultry meats in Belgium.</article-title>\n<source><italic>J. Food Prot.</italic></source>\n<volume>71</volume>\n<fpage>35</fpage>–<lpage>45</lpage>. <pub-id pub-id-type=\"doi\">10.4315/0362-028x-71.1.35</pub-id>\n<pub-id pub-id-type=\"pmid\">18236660</pub-id></mixed-citation></ref><ref id=\"B36\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gião</surname><given-names>M. S.</given-names></name><name><surname>Keevil</surname><given-names>C. W.</given-names></name></person-group> (<year>2014</year>). <article-title>Listeria monocytogenes can form biofilms in tap water and enter into the viable but non-cultivable state.</article-title>\n<source><italic>Microb. Ecol.</italic></source>\n<volume>67</volume>\n<fpage>603</fpage>–<lpage>611</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00248-013-0364-3</pub-id>\n<pub-id pub-id-type=\"pmid\">24452996</pub-id></mixed-citation></ref><ref id=\"B37\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Giaouris</surname><given-names>E.</given-names></name><name><surname>Heir</surname><given-names>E.</given-names></name><name><surname>Hébraud</surname><given-names>M.</given-names></name><name><surname>Chorianopoulos</surname><given-names>N.</given-names></name><name><surname>Langsrud</surname><given-names>S.</given-names></name><name><surname>Møretrø</surname><given-names>T.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Attachment and biofilm formation by foodborne bacteria in meat processing environments: causes, implications, role of bacterial interactions and control by alternative novel methods.</article-title>\n<source><italic>Meat Sci.</italic></source>\n<volume>97</volume>\n<fpage>298</fpage>–<lpage>309</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.meatsci.2013.05.023</pub-id>\n<pub-id pub-id-type=\"pmid\">23747091</pub-id></mixed-citation></ref><ref id=\"B38\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Giaouris</surname><given-names>E. E.</given-names></name><name><surname>Simões</surname><given-names>M. V.</given-names></name></person-group> (<year>2018</year>). <article-title>Pathogenic biofilm formation in the food industry and alternative control strategies.</article-title>\n<source><italic>Foodborne Dis.</italic></source>\n<volume>2018</volume>\n<fpage>309</fpage>–<lpage>377</lpage>. <pub-id pub-id-type=\"doi\">10.1016/B978-0-12-811444-5.00011-7</pub-id></mixed-citation></ref><ref id=\"B39\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gibson</surname><given-names>H.</given-names></name><name><surname>Taylor</surname><given-names>J. H.</given-names></name><name><surname>Hall</surname><given-names>K. E.</given-names></name><name><surname>Holah</surname><given-names>J. T.</given-names></name></person-group> (<year>1999</year>). <article-title>Effectiveness of cleaning techniques used in the food industry in terms of the removal of bacterial biofilms.</article-title>\n<source><italic>J. Appl. Microbiol.</italic></source>\n<volume>87</volume>\n<fpage>41</fpage>–<lpage>48</lpage>. <pub-id pub-id-type=\"doi\">10.1046/j.1365-2672.1999.00790.x</pub-id>\n<pub-id pub-id-type=\"pmid\">10432586</pub-id></mixed-citation></ref><ref id=\"B40\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gilbert</surname><given-names>P.</given-names></name><name><surname>Allison</surname><given-names>D. G.</given-names></name><name><surname>McBain</surname><given-names>A. J.</given-names></name></person-group> (<year>2002</year>). <article-title>Biofilms in vitro and in vivo: do singular mechanisms imply cross-resistance?</article-title>\n<source><italic>J. Appl. Microbiol.</italic></source>\n<volume>92</volume>\n<fpage>98S</fpage>–<lpage>110S</lpage>. <pub-id pub-id-type=\"doi\">10.1046/j.1365-2672.92.5s1.5.x</pub-id><pub-id pub-id-type=\"pmid\">12000619</pub-id></mixed-citation></ref><ref id=\"B41\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Greppi</surname><given-names>A.</given-names></name><name><surname>Ferrocino</surname><given-names>I.</given-names></name><name><surname>La Storia</surname><given-names>A.</given-names></name><name><surname>Rantsiou</surname><given-names>K.</given-names></name><name><surname>Ercolini</surname><given-names>D.</given-names></name><name><surname>Cocolin</surname><given-names>L.</given-names></name></person-group> (<year>2015</year>). <article-title>Monitoring of the microbiota of fermented sausages by culture independent rRNA-based approaches.</article-title>\n<source><italic>Int. J. Food Microbiol.</italic></source>\n<volume>212</volume>\n<fpage>67</fpage>–<lpage>75</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijfoodmicro.2015.01.016</pub-id>\n<pub-id pub-id-type=\"pmid\">25724303</pub-id></mixed-citation></ref><ref id=\"B42\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gu</surname><given-names>G.</given-names></name><name><surname>Ottesen</surname><given-names>A.</given-names></name><name><surname>Bolten</surname><given-names>S.</given-names></name><name><surname>Ramachandran</surname><given-names>P.</given-names></name><name><surname>Reed</surname><given-names>E.</given-names></name><name><surname>Rideout</surname><given-names>S.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Shifts in spinach microbial communities after chlorine washing and storage at compliant and abusive temperatures.</article-title>\n<source><italic>Food Microbiol.</italic></source>\n<volume>73</volume>\n<fpage>73</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fm.2018.01.002</pub-id>\n<pub-id pub-id-type=\"pmid\">29526229</pub-id></mixed-citation></ref><ref id=\"B43\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Holah</surname><given-names>J. T.</given-names></name></person-group> (<year>2013</year>). “<article-title>Cleaning and disinfection practices in food processing</article-title>,” in <source><italic>Hygiene in Food Processing: Principles and Practice</italic></source>, <edition>2nd Edn</edition>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>Holah</surname><given-names>J.</given-names></name><name><surname>White</surname><given-names>B.</given-names></name><name><surname>Lelieveld</surname><given-names>H.</given-names></name></person-group> (<publisher-loc>Amsterdam</publisher-loc>: <publisher-name>Elsevier Inc</publisher-name>), <fpage>259</fpage>–<lpage>304</lpage>. <pub-id pub-id-type=\"doi\">10.1533/9780857098634.3.259</pub-id></mixed-citation></ref><ref id=\"B44\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hutchison</surname><given-names>M. L.</given-names></name><name><surname>Walters</surname><given-names>L. D.</given-names></name><name><surname>Avery</surname><given-names>S. M.</given-names></name><name><surname>Reid</surname><given-names>C.-A.</given-names></name><name><surname>Wilson</surname><given-names>D.</given-names></name><name><surname>Howell</surname><given-names>M.</given-names></name><etal/></person-group> (<year>2005</year>). <article-title>A comparison of wet-dry swabbing and excision sampling methods for microbiological testing of bovine, porcine, and ovine carcasses at red meat slaughterhouses.</article-title>\n<source><italic>J. Food Prot.</italic></source>\n<volume>68</volume>\n<fpage>2155</fpage>–<lpage>2162</lpage>. <pub-id pub-id-type=\"doi\">10.4315/0362-028x-68.10.2155</pub-id>\n<pub-id pub-id-type=\"pmid\">16245723</pub-id></mixed-citation></ref><ref id=\"B45\"><mixed-citation publication-type=\"book\"><collab>ICMFS</collab> (<year>2006</year>). <source><italic>Microorganisms in Foods 6: Microbial Ecology of Food Commodities</italic></source>, <edition>2nd Edn</edition>\n<publisher-loc>New York, NY</publisher-loc>: <publisher-name>Kuwer Academic/plenum Publishers</publisher-name>.</mixed-citation></ref><ref id=\"B46\"><mixed-citation publication-type=\"book\"><collab>ICMSF</collab> (<year>2012</year>). “<article-title>Microorganisms in foods 7: microbiological testing in food safety management</article-title>,” in <source><italic>International Commission for The Microbiological Specifications of Foods</italic></source>, (<publisher-loc>New York, NY</publisher-loc>: <publisher-name>Springer-Verlag</publisher-name>).</mixed-citation></ref><ref id=\"B47\"><mixed-citation publication-type=\"book\"><collab>ISO</collab> (<year>2016</year>). <source><italic>ISO 18593 - Microbiology of the Food Chain - Horizontal Methods for Surface Sampling.</italic></source>\n<publisher-loc>Geneva</publisher-loc>: <publisher-name>International Organization for Standardization</publisher-name>.</mixed-citation></ref><ref id=\"B48\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Iulietto</surname><given-names>M. F.</given-names></name><name><surname>Sechi</surname><given-names>P.</given-names></name><name><surname>Borgogni</surname><given-names>E.</given-names></name><name><surname>Cenci-Goga</surname><given-names>B. T.</given-names></name></person-group> (<year>2015</year>). <article-title>Meat spoilage: a critical review of a neglected alteration due to ropy slime producing bacteria.</article-title>\n<source><italic>Ital. J. Anim. Sci.</italic></source>\n<volume>14</volume>:<issue>4011</issue>\n<pub-id pub-id-type=\"doi\">10.4081/ijas.2015.4011</pub-id></mixed-citation></ref><ref id=\"B49\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kaur</surname><given-names>J.</given-names></name><name><surname>Lee</surname><given-names>S.</given-names></name><name><surname>Sharma</surname><given-names>A.</given-names></name><name><surname>Park</surname><given-names>Y.-S.</given-names></name></person-group> (<year>2017</year>). <article-title>DNA profiling of Leuconostoc mesenteroides strains isolated from fermented foods and farm produce in Korea by repetitive-element PCR.</article-title>\n<source><italic>Food Sci. Biotechnol.</italic></source>\n<volume>26</volume>\n<fpage>1667</fpage>–<lpage>1673</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s10068-017-0189-9</pub-id>\n<pub-id pub-id-type=\"pmid\">30263704</pub-id></mixed-citation></ref><ref id=\"B50\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kembel</surname><given-names>S. W.</given-names></name><name><surname>Wu</surname><given-names>M.</given-names></name><name><surname>Eisen</surname><given-names>J. A.</given-names></name><name><surname>Green</surname><given-names>J. L.</given-names></name></person-group> (<year>2012</year>). <article-title>Incorporating 16S gene copy number information improves estimates of microbial diversity and abundance.</article-title>\n<source><italic>PLoS Comput. Biol.</italic></source>\n<volume>8</volume>:<issue>e1002743</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pcbi.1002743</pub-id>\n<pub-id pub-id-type=\"pmid\">23133348</pub-id></mixed-citation></ref><ref id=\"B51\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>S.-H.</given-names></name><name><surname>Park</surname><given-names>C.</given-names></name><name><surname>Lee</surname><given-names>E.-J.</given-names></name><name><surname>Bang</surname><given-names>W.-S.</given-names></name><name><surname>Kim</surname><given-names>Y.-J.</given-names></name><name><surname>Kim</surname><given-names>J.-S.</given-names></name></person-group> (<year>2017</year>). <article-title>Biofilm formation of campylobacter strains isolated from raw chickens and its reduction with DNase I treatment.</article-title>\n<source><italic>Food Control</italic></source>\n<volume>71</volume>\n<fpage>94</fpage>–<lpage>100</lpage>. <pub-id pub-id-type=\"doi\">10.1016/J.FOODCONT.2016.06.038</pub-id></mixed-citation></ref><ref id=\"B52\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kumari</surname><given-names>S.</given-names></name><name><surname>Sarkar</surname><given-names>P. K.</given-names></name></person-group> (<year>2014</year>). <article-title>In vitro model study for biofilm formation by <italic>Bacillus cereus</italic> in dairy chilling tanks and optimization of clean-in-place (CIP) regimes using response surface methodology.</article-title>\n<source><italic>Food Control</italic></source>\n<volume>36</volume>\n<fpage>153</fpage>–<lpage>158</lpage>. <pub-id pub-id-type=\"doi\">10.1016/J.FOODCONT.2013.08.014</pub-id></mixed-citation></ref><ref id=\"B53\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kumari</surname><given-names>S.</given-names></name><name><surname>Sarkar</surname><given-names>P. K.</given-names></name></person-group> (<year>2016</year>). <article-title><italic>Bacillus cereus</italic> hazard and control in industrial dairy processing environment.</article-title>\n<source><italic>Food Control</italic></source>\n<volume>69</volume>\n<fpage>20</fpage>–<lpage>29</lpage>. <pub-id pub-id-type=\"doi\">10.1016/J.FOODCONT.2016.04.012</pub-id></mixed-citation></ref><ref id=\"B54\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>M.</given-names></name><name><surname>Song</surname><given-names>J. H.</given-names></name><name><surname>Jung</surname><given-names>M. Y.</given-names></name><name><surname>Lee</surname><given-names>S. H.</given-names></name><name><surname>Chang</surname><given-names>J. Y.</given-names></name></person-group> (<year>2017</year>). <article-title>Large-scale targeted metagenomics analysis of bacterial ecological changes in 88 kimchi samples during fermentation.</article-title>\n<source><italic>Food Microbiol.</italic></source>\n<volume>66</volume>\n<fpage>173</fpage>–<lpage>183</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fm.2017.05.002</pub-id>\n<pub-id pub-id-type=\"pmid\">28576366</pub-id></mixed-citation></ref><ref id=\"B55\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lefebvre</surname><given-names>E.</given-names></name><name><surname>Vighetto</surname><given-names>C.</given-names></name><name><surname>Di Martino</surname><given-names>P.</given-names></name><name><surname>Larreta Garde</surname><given-names>V.</given-names></name><name><surname>Seyer</surname><given-names>D.</given-names></name></person-group> (<year>2016</year>). <article-title>Synergistic antibiofilm efficacy of various commercial antiseptics, enzymes and EDTA: a study of <italic>Pseudomonas aeruginosa</italic> and Staphylococcus aureus biofilms.</article-title>\n<source><italic>Int. J. Antimicrob. Agents</italic></source>\n<volume>48</volume>\n<fpage>181</fpage>–<lpage>188</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijantimicag.2016.05.008</pub-id>\n<pub-id pub-id-type=\"pmid\">27424598</pub-id></mixed-citation></ref><ref id=\"B56\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lelièvre</surname><given-names>C.</given-names></name><name><surname>Legentilhomme</surname><given-names>P.</given-names></name><name><surname>Gaucher</surname><given-names>C.</given-names></name><name><surname>Legrand</surname><given-names>J.</given-names></name><name><surname>Faille</surname><given-names>C.</given-names></name><name><surname>Bénézech</surname><given-names>T.</given-names></name></person-group> (<year>2002</year>). <article-title>Cleaning in place: effect of local wall shear stress variation on bacterial removal from stainless steel equipment.</article-title>\n<source><italic>Chem. Eng. Sci.</italic></source>\n<volume>57</volume>\n<fpage>1287</fpage>–<lpage>1297</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0009-2509(02)00019-2</pub-id></mixed-citation></ref><ref id=\"B57\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lemos</surname><given-names>L. N.</given-names></name><name><surname>Fulthorpe</surname><given-names>R. R.</given-names></name><name><surname>Triplett</surname><given-names>E. W.</given-names></name><name><surname>Roesch</surname><given-names>L. F. W.</given-names></name></person-group> (<year>2011</year>). <article-title>Rethinking microbial diversity analysis in the high throughput sequencing era.</article-title>\n<source><italic>J. Microbiol. Methods</italic></source>\n<volume>86</volume>\n<fpage>42</fpage>–<lpage>51</lpage>. <pub-id pub-id-type=\"doi\">10.1016/J.MIMET.2011.03.014</pub-id>\n<pub-id pub-id-type=\"pmid\">21457733</pub-id></mixed-citation></ref><ref id=\"B58\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lequette</surname><given-names>Y.</given-names></name><name><surname>Boels</surname><given-names>G.</given-names></name><name><surname>Clarisse</surname><given-names>M.</given-names></name><name><surname>Faille</surname><given-names>C.</given-names></name></person-group> (<year>2010</year>). <article-title>Using enzymes to remove biofilms of bacterial isolates sampled in the food-industry.</article-title>\n<source><italic>Biofouling</italic></source>\n<volume>26</volume>\n<fpage>421</fpage>–<lpage>431</lpage>. <pub-id pub-id-type=\"doi\">10.1080/08927011003699535</pub-id>\n<pub-id pub-id-type=\"pmid\">20198521</pub-id></mixed-citation></ref><ref id=\"B59\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>X.</given-names></name><name><surname>Tang</surname><given-names>B.</given-names></name><name><surname>Gu</surname><given-names>Q.</given-names></name><name><surname>Yu</surname><given-names>X.</given-names></name></person-group> (<year>2014</year>). <article-title>Elimination of the formation of biofilm in industrial pipes using enzyme cleaning technique.</article-title>\n<source><italic>MethodsX</italic></source>\n<volume>1</volume>\n<fpage>130</fpage>–<lpage>136</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.mex.2014.08.008</pub-id>\n<pub-id pub-id-type=\"pmid\">26150944</pub-id></mixed-citation></ref><ref id=\"B60\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>López</surname><given-names>D.</given-names></name><name><surname>Vlamakis</surname><given-names>H.</given-names></name><name><surname>Kolter</surname><given-names>R.</given-names></name></person-group> (<year>2010</year>). <article-title>Biofilms.</article-title>\n<source><italic>Cold Spring Harb. Perspect. Biol.</italic></source>\n<volume>2</volume>:<issue>a000398</issue>. <pub-id pub-id-type=\"doi\">10.1101/cshperspect.a000398</pub-id>\n<pub-id pub-id-type=\"pmid\">20519345</pub-id></mixed-citation></ref><ref id=\"B61\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Louca</surname><given-names>S.</given-names></name><name><surname>Doebeli</surname><given-names>M.</given-names></name><name><surname>Parfrey</surname><given-names>L. W.</given-names></name></person-group> (<year>2018</year>). <article-title>Correcting for 16S rRNA gene copy numbers in microbiome surveys remains an unsolved problem.</article-title>\n<source><italic>Microbiome</italic></source>\n<volume>6</volume>:<issue>41</issue>. <pub-id pub-id-type=\"doi\">10.1186/s40168-018-0420-9</pub-id>\n<pub-id pub-id-type=\"pmid\">29482646</pub-id></mixed-citation></ref><ref id=\"B62\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Maillard</surname><given-names>J. Y.</given-names></name></person-group> (<year>2016</year>). “<article-title>Testing the effectiveness of disinfectants and sanitizers</article-title>,” in <source><italic>Handbook of Hygiene Control in the Food Industry</italic></source>, <edition>2nd Edn</edition>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>Holah</surname><given-names>J.</given-names></name><name><surname>Mostert</surname><given-names>M. A.</given-names></name><name><surname>Lelieveld</surname><given-names>H.</given-names></name></person-group> (<publisher-loc>Amsterdam</publisher-loc>: <publisher-name>Elsevier Inc</publisher-name>), <fpage>569</fpage>–<lpage>586</lpage>. <pub-id pub-id-type=\"doi\">10.1016/b978-0-08-100155-4.00037-6</pub-id></mixed-citation></ref><ref id=\"B63\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mann</surname><given-names>E.</given-names></name><name><surname>Wetzels</surname><given-names>S. U.</given-names></name><name><surname>Pinior</surname><given-names>B.</given-names></name><name><surname>Metzler-Zebeli</surname><given-names>B. U.</given-names></name><name><surname>Wagner</surname><given-names>M.</given-names></name><name><surname>Schmitz-Esser</surname><given-names>S.</given-names></name></person-group> (<year>2016</year>). <article-title>Psychrophile spoilers dominate the bacterial microbiome in musculature samples of slaughter pigs.</article-title>\n<source><italic>Meat Sci.</italic></source>\n<volume>117</volume>\n<fpage>36</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.meatsci.2016.02.034</pub-id>\n<pub-id pub-id-type=\"pmid\">26943946</pub-id></mixed-citation></ref><ref id=\"B64\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Marchand</surname><given-names>S.</given-names></name><name><surname>De Block</surname><given-names>J.</given-names></name><name><surname>De Jonghe</surname><given-names>V.</given-names></name><name><surname>Coorevits</surname><given-names>A.</given-names></name><name><surname>Heyndrickx</surname><given-names>M.</given-names></name><name><surname>Herman</surname><given-names>L.</given-names></name></person-group> (<year>2012</year>). <article-title>Biofilm formation in milk production and processing environments; influence on milk quality and safety.</article-title>\n<source><italic>Compr. Rev. Food Sci. Food Saf.</italic></source>\n<volume>11</volume>\n<fpage>133</fpage>–<lpage>147</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1541-4337.2011.00183.x</pub-id></mixed-citation></ref><ref id=\"B65\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mayo</surname><given-names>B.</given-names></name><name><surname>Rachid</surname><given-names>C. T. C. C.</given-names></name><name><surname>Alegría</surname><given-names>A.</given-names></name><name><surname>Leite</surname><given-names>A. M. O.</given-names></name><name><surname>Peixoto</surname><given-names>R. S.</given-names></name><name><surname>Delgado</surname><given-names>S.</given-names></name></person-group> (<year>2014</year>). <article-title>Impact of next generation sequencing techniques in food microbiology.</article-title>\n<source><italic>Curr. Genomics</italic></source>\n<volume>15</volume>\n<fpage>293</fpage>–<lpage>309</lpage>. <pub-id pub-id-type=\"doi\">10.2174/1389202915666140616233211</pub-id>\n<pub-id pub-id-type=\"pmid\">25132799</pub-id></mixed-citation></ref><ref id=\"B66\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Meireles</surname><given-names>A.</given-names></name><name><surname>Borges</surname><given-names>A.</given-names></name><name><surname>Giaouris</surname><given-names>E.</given-names></name><name><surname>Simões</surname><given-names>M.</given-names></name></person-group> (<year>2016</year>). <article-title>The current knowledge on the application of anti-biofilm enzymes in the food industry.</article-title>\n<source><italic>Food Res. Int.</italic></source>\n<volume>86</volume>\n<fpage>140</fpage>–<lpage>146</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.foodres.2016.06.006</pub-id></mixed-citation></ref><ref id=\"B67\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Menezes</surname><given-names>L. A. A.</given-names></name><name><surname>Sardaro</surname><given-names>M. L. S.</given-names></name><name><surname>Duarte</surname><given-names>R. T. D.</given-names></name><name><surname>Mazzon</surname><given-names>R. R.</given-names></name><name><surname>Neviani</surname><given-names>E.</given-names></name><name><surname>Gatti</surname><given-names>M.</given-names></name><etal/></person-group> (<year>2020</year>). <article-title>Sourdough bacterial dynamics revealed by metagenomic analysis in Brazil.</article-title>\n<source><italic>Food Microbiol.</italic></source>\n<volume>85</volume>:<issue>103302</issue>. <pub-id pub-id-type=\"doi\">10.1016/j.fm.2019.103302</pub-id>\n<pub-id pub-id-type=\"pmid\">31500708</pub-id></mixed-citation></ref><ref id=\"B68\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nagaraj</surname><given-names>V.</given-names></name><name><surname>Skillman</surname><given-names>L.</given-names></name><name><surname>Li</surname><given-names>D.</given-names></name><name><surname>Xie</surname><given-names>Z.</given-names></name><name><surname>Ho</surname><given-names>G.</given-names></name></person-group> (<year>2017</year>). <article-title>Control of biofouling by xanthine oxidase on seawater reverse osmosis membranes from a desalination plant: enzyme production and screening of bacterial isolates from the full-scale plant.</article-title>\n<source><italic>Lett. Appl. Microbiol.</italic></source>\n<volume>65</volume>\n<fpage>73</fpage>–<lpage>81</lpage>. <pub-id pub-id-type=\"doi\">10.1111/lam.12747</pub-id>\n<pub-id pub-id-type=\"pmid\">28418590</pub-id></mixed-citation></ref><ref id=\"B69\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nalbantoglu</surname><given-names>U.</given-names></name><name><surname>Cakar</surname><given-names>A.</given-names></name><name><surname>Dogan</surname><given-names>H.</given-names></name><name><surname>Abaci</surname><given-names>N.</given-names></name><name><surname>Ustek</surname><given-names>D.</given-names></name><name><surname>Sayood</surname><given-names>K.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Metagenomic analysis of the microbial community in kefir grains.</article-title>\n<source><italic>Food Microbiol.</italic></source>\n<volume>41</volume>\n<fpage>42</fpage>–<lpage>51</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fm.2014.01.014</pub-id>\n<pub-id pub-id-type=\"pmid\">24750812</pub-id></mixed-citation></ref><ref id=\"B70\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nychas</surname><given-names>G. J. E.</given-names></name><name><surname>Skandamis</surname><given-names>P. N.</given-names></name><name><surname>Tassou</surname><given-names>C. C.</given-names></name><name><surname>Koutsoumanis</surname><given-names>K. P.</given-names></name></person-group> (<year>2008</year>). <article-title>Meat spoilage during distribution.</article-title>\n<source><italic>Meat Sci.</italic></source>\n<volume>78</volume>\n<fpage>77</fpage>–<lpage>89</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.meatsci.2007.06.020</pub-id>\n<pub-id pub-id-type=\"pmid\">22062098</pub-id></mixed-citation></ref><ref id=\"B71\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oulahal</surname><given-names>N.</given-names></name><name><surname>Martial-Gros</surname><given-names>A.</given-names></name><name><surname>Bonneau</surname><given-names>M.</given-names></name><name><surname>Blum</surname><given-names>L. J.</given-names></name></person-group> (<year>2007</year>). <article-title>Removal of meat biofilms from surfaces by ultrasounds combined with enzymes and/or a chelating agent.</article-title>\n<source><italic>Innov. Food Sci. Emerg. Technol.</italic></source>\n<volume>8</volume>\n<fpage>192</fpage>–<lpage>196</lpage>. <pub-id pub-id-type=\"doi\">10.1016/J.IFSET.2006.10.001</pub-id></mixed-citation></ref><ref id=\"B72\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parijs</surname><given-names>I.</given-names></name><name><surname>Steenackers</surname><given-names>H. P.</given-names></name></person-group> (<year>2018</year>). <article-title>Competitive inter-species interactions underlie the increased antimicrobial tolerance in multispecies brewery biofilms.</article-title>\n<source><italic>ISME J.</italic></source>\n<volume>12</volume>\n<fpage>2061</fpage>–<lpage>2075</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41396-018-0146-5</pub-id>\n<pub-id pub-id-type=\"pmid\">29858577</pub-id></mixed-citation></ref><ref id=\"B73\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parks</surname><given-names>D. H.</given-names></name><name><surname>Tyson</surname><given-names>G. W.</given-names></name><name><surname>Hugenholtz</surname><given-names>P.</given-names></name><name><surname>Beiko</surname><given-names>R. G.</given-names></name></person-group> (<year>2014</year>). <article-title>STAMP: statistical analysis of taxonomic and functional profiles.</article-title>\n<source><italic>Bioinformatics</italic></source>\n<volume>30</volume>\n<fpage>3123</fpage>–<lpage>3124</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bioinformatics/btu494</pub-id>\n<pub-id pub-id-type=\"pmid\">25061070</pub-id></mixed-citation></ref><ref id=\"B74\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parlapani</surname><given-names>F. F.</given-names></name><name><surname>Michailidou</surname><given-names>S.</given-names></name><name><surname>Anagnostopoulos</surname><given-names>D. A.</given-names></name><name><surname>Sakellariou</surname><given-names>A. K.</given-names></name><name><surname>Pasentsis</surname><given-names>K.</given-names></name><name><surname>Psomopoulos</surname><given-names>F.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Microbial spoilage investigation of thawed common cuttlefish (<italic>Sepia officinalis</italic>) stored at 2°C using next generation sequencing and volatilome analysis.</article-title>\n<source><italic>Food Microbiol.</italic></source>\n<volume>76</volume>\n<fpage>518</fpage>–<lpage>525</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fm.2018.08.004</pub-id>\n<pub-id pub-id-type=\"pmid\">30166182</pub-id></mixed-citation></ref><ref id=\"B75\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Pellissery</surname><given-names>A. J.</given-names></name><name><surname>Vinayamohan</surname><given-names>P. G.</given-names></name><name><surname>Amalaradjou</surname><given-names>M. A. R.</given-names></name><name><surname>Venkitanarayanan</surname><given-names>K.</given-names></name></person-group> (<year>2020</year>). “<article-title>Spoilage bacteria and meat quality</article-title>,” in <source><italic>Meat Quality Analysis</italic></source>, (<publisher-loc>Amsterdam</publisher-loc>: <publisher-name>Academic Press</publisher-name>), <fpage>307</fpage>–<lpage>334</lpage>. <pub-id pub-id-type=\"doi\">10.1016/b978-0-12-819233-7.00017-3</pub-id></mixed-citation></ref><ref id=\"B76\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pennacchia</surname><given-names>C.</given-names></name><name><surname>Ercolini</surname><given-names>D.</given-names></name><name><surname>Villani</surname><given-names>F.</given-names></name></person-group> (<year>2011</year>). <article-title>Spoilage-related microbiota associated with chilled beef stored in air or vacuum pack.</article-title>\n<source><italic>Food Microbiol.</italic></source>\n<volume>28</volume>\n<fpage>84</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fm.2010.08.010</pub-id>\n<pub-id pub-id-type=\"pmid\">21056779</pub-id></mixed-citation></ref><ref id=\"B77\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pérez-Cataluña</surname><given-names>A.</given-names></name><name><surname>Elizaquível</surname><given-names>P.</given-names></name><name><surname>Carrasco</surname><given-names>P.</given-names></name><name><surname>Espinosa</surname><given-names>J.</given-names></name><name><surname>Reyes</surname><given-names>D.</given-names></name><name><surname>Wacher</surname><given-names>C.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Diversity and dynamics of lactic acid bacteria in Atole agrio, a traditional maize-based fermented beverage from South-Eastern Mexico, analysed by high throughput sequencing and culturing.</article-title>\n<source><italic>Antonie Van Leeuwenhoek</italic></source>\n<volume>111</volume>\n<fpage>385</fpage>–<lpage>399</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s10482-017-0960-1</pub-id>\n<pub-id pub-id-type=\"pmid\">29058140</pub-id></mixed-citation></ref><ref id=\"B78\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Peruzy</surname><given-names>M. F.</given-names></name><name><surname>Murru</surname><given-names>N.</given-names></name><name><surname>Yu</surname><given-names>Z.</given-names></name><name><surname>Cnockaert</surname><given-names>M.</given-names></name><name><surname>Joossens</surname><given-names>M.</given-names></name><name><surname>Proroga</surname><given-names>Y. T. R.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Determination of the microbiological contamination in minced pork by culture dependent and 16S amplicon sequencing analysis.</article-title>\n<source><italic>Int. J. Food Microbiol.</italic></source>\n<volume>290</volume>\n<fpage>27</fpage>–<lpage>35</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijfoodmicro.2018.09.025</pub-id>\n<pub-id pub-id-type=\"pmid\">30292676</pub-id></mixed-citation></ref><ref id=\"B79\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Petruzzi</surname><given-names>L.</given-names></name><name><surname>Corbo</surname><given-names>M. R.</given-names></name><name><surname>Sinigaglia</surname><given-names>M.</given-names></name><name><surname>Bevilacqua</surname><given-names>A.</given-names></name></person-group> (<year>2017</year>). “<article-title>Microbial spoilage of foods: fundamentals</article-title>,” in <source><italic>The Microbiological Quality of Food: Foodborne Spoilers</italic></source>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>Bevilacqua</surname><given-names>A.</given-names></name><name><surname>Corbo</surname><given-names>M. R.</given-names></name><name><surname>Sinigaglia</surname><given-names>M.</given-names></name></person-group> (<publisher-loc>Amsterdam</publisher-loc>: <publisher-name>Elsevier Inc</publisher-name>), <fpage>1</fpage>–<lpage>21</lpage>.</mixed-citation></ref><ref id=\"B80\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Połka</surname><given-names>J.</given-names></name><name><surname>Rebecchi</surname><given-names>A.</given-names></name><name><surname>Pisacane</surname><given-names>V.</given-names></name><name><surname>Morelli</surname><given-names>L.</given-names></name><name><surname>Puglisi</surname><given-names>E.</given-names></name></person-group> (<year>2015</year>). <article-title>Bacterial diversity in typical Italian salami at different ripening stages as revealed by high-throughput sequencing of 16S rRNA amplicons.</article-title>\n<source><italic>Food Microbiol.</italic></source>\n<volume>46</volume>\n<fpage>342</fpage>–<lpage>356</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fm.2014.08.023</pub-id>\n<pub-id pub-id-type=\"pmid\">25475305</pub-id></mixed-citation></ref><ref id=\"B81\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Porcellato</surname><given-names>D.</given-names></name><name><surname>Aspholm</surname><given-names>M.</given-names></name><name><surname>Skeie</surname><given-names>S. B.</given-names></name><name><surname>Monshaugen</surname><given-names>M.</given-names></name><name><surname>Brendehaug</surname><given-names>J.</given-names></name><name><surname>Mellegård</surname><given-names>H.</given-names></name></person-group> (<year>2018</year>). <article-title>Microbial diversity of consumption milk during processing and storage.</article-title>\n<source><italic>Int. J. Food Microbiol.</italic></source>\n<volume>266</volume>\n<fpage>21</fpage>–<lpage>30</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijfoodmicro.2017.11.004</pub-id>\n<pub-id pub-id-type=\"pmid\">29161642</pub-id></mixed-citation></ref><ref id=\"B82\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pothakos</surname><given-names>V.</given-names></name><name><surname>Devlieghere</surname><given-names>F.</given-names></name><name><surname>Villani</surname><given-names>F.</given-names></name><name><surname>Björkroth</surname><given-names>J.</given-names></name><name><surname>Ercolini</surname><given-names>D.</given-names></name></person-group> (<year>2015</year>). <article-title>Lactic acid bacteria and their controversial role in fresh meat spoilage.</article-title>\n<source><italic>Meat Sci.</italic></source>\n<volume>109</volume>\n<fpage>66</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.meatsci.2015.04.014</pub-id>\n<pub-id pub-id-type=\"pmid\">25972087</pub-id></mixed-citation></ref><ref id=\"B83\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pothakos</surname><given-names>V.</given-names></name><name><surname>Samapundo</surname><given-names>S.</given-names></name><name><surname>Devlieghere</surname><given-names>F.</given-names></name></person-group> (<year>2012</year>). <article-title>Total mesophilic counts underestimate in many cases the contamination levels of psychrotrophic lactic acid bacteria (LAB) in chilled-stored food products at the end of their shelf-life.</article-title>\n<source><italic>Food Microbiol.</italic></source>\n<volume>32</volume>\n<fpage>437</fpage>–<lpage>443</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fm.2012.07.011</pub-id>\n<pub-id pub-id-type=\"pmid\">22986212</pub-id></mixed-citation></ref><ref id=\"B84\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pruesse</surname><given-names>E.</given-names></name><name><surname>Quast</surname><given-names>C.</given-names></name><name><surname>Knittel</surname><given-names>K.</given-names></name><name><surname>Fuchs</surname><given-names>B. M.</given-names></name><name><surname>Ludwig</surname><given-names>W.</given-names></name><name><surname>Peplies</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2007</year>). <article-title>SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB.</article-title>\n<source><italic>Nucleic Acids Res.</italic></source>\n<volume>35</volume>\n<fpage>7188</fpage>–<lpage>7196</lpage>. <pub-id pub-id-type=\"doi\">10.1093/nar/gkm864</pub-id>\n<pub-id pub-id-type=\"pmid\">17947321</pub-id></mixed-citation></ref><ref id=\"B85\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quast</surname><given-names>C.</given-names></name><name><surname>Pruesse</surname><given-names>E.</given-names></name><name><surname>Yilmaz</surname><given-names>P.</given-names></name><name><surname>Gerken</surname><given-names>J.</given-names></name><name><surname>Schweer</surname><given-names>T.</given-names></name><name><surname>Yarza</surname><given-names>P.</given-names></name><etal/></person-group> (<year>2012</year>). <article-title>The SILVA ribosomal RNA gene database project: improved data processing and web-based tools.</article-title>\n<source><italic>Nucleic Acids Res.</italic></source>\n<volume>41</volume>\n<fpage>D590</fpage>–<lpage>D596</lpage>. <pub-id pub-id-type=\"doi\">10.1093/nar/gks1219</pub-id>\n<pub-id pub-id-type=\"pmid\">23193283</pub-id></mixed-citation></ref><ref id=\"B86\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quigley</surname><given-names>L.</given-names></name><name><surname>O’Sullivan</surname><given-names>O.</given-names></name><name><surname>Stanton</surname><given-names>C.</given-names></name><name><surname>Beresford</surname><given-names>T. P.</given-names></name><name><surname>Ross</surname><given-names>R. P.</given-names></name><name><surname>Fitzgerald</surname><given-names>G. F.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>The complex microbiota of raw milk.</article-title>\n<source><italic>FEMS Microbiol. Rev.</italic></source>\n<volume>37</volume>\n<fpage>664</fpage>–<lpage>698</lpage>. <pub-id pub-id-type=\"doi\">10.1111/1574-6976.12030</pub-id>\n<pub-id pub-id-type=\"pmid\">23808865</pub-id></mixed-citation></ref><ref id=\"B87\"><mixed-citation publication-type=\"book\"><collab>R Core Team</collab> (<year>2008</year>). <source><italic>R: A Language and Environment for Statistical Computing.</italic></source>\n<publisher-loc>Vienna</publisher-loc>: <publisher-name>R Foundation for Statistical Computing</publisher-name>.</mixed-citation></ref><ref id=\"B88\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ribeiro Júnior</surname><given-names>J. C.</given-names></name><name><surname>de Oliveira</surname><given-names>A. M.</given-names></name><name><surname>Silva</surname><given-names>F.</given-names></name><name><surname>de</surname><given-names>G.</given-names></name><name><surname>Tamanini</surname><given-names>R.</given-names></name><name><surname>de Oliveira</surname><given-names>A. L. M.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>The main spoilage-related psychrotrophic bacteria in refrigerated raw milk.</article-title>\n<source><italic>J. Dairy Sci.</italic></source>\n<volume>101</volume>\n<fpage>75</fpage>–<lpage>83</lpage>. <pub-id pub-id-type=\"doi\">10.3168/jds.2017-13069</pub-id>\n<pub-id pub-id-type=\"pmid\">29102138</pub-id></mixed-citation></ref><ref id=\"B89\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Riquelme</surname><given-names>C.</given-names></name><name><surname>Câmara</surname><given-names>S.</given-names></name><name><surname>de Enes Dapkevicius</surname><given-names>M. L. N.</given-names></name><name><surname>Vinuesa</surname><given-names>P.</given-names></name><name><surname>da Silva</surname><given-names>C. C. G.</given-names></name><name><surname>Malcata</surname><given-names>F. X.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>Characterization of the bacterial biodiversity in Pico cheese (an artisanal Azorean food).</article-title>\n<source><italic>Int. J. Food Microbiol.</italic></source>\n<volume>192</volume>\n<fpage>86</fpage>–<lpage>94</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijfoodmicro.2014.09.031</pub-id>\n<pub-id pub-id-type=\"pmid\">25440551</pub-id></mixed-citation></ref><ref id=\"B90\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rouger</surname><given-names>A.</given-names></name><name><surname>Remenant</surname><given-names>B.</given-names></name><name><surname>Prévost</surname><given-names>H.</given-names></name><name><surname>Zagorec</surname><given-names>M.</given-names></name></person-group> (<year>2017</year>). <article-title>A method to isolate bacterial communities and characterize ecosystems from food products: validation and utilization in as a reproducible chicken meat model.</article-title>\n<source><italic>Int. J. Food Microbiol.</italic></source>\n<volume>247</volume>\n<fpage>38</fpage>–<lpage>47</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijfoodmicro.2016.04.028</pub-id>\n<pub-id pub-id-type=\"pmid\">27184973</pub-id></mixed-citation></ref><ref id=\"B91\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Samapundo</surname><given-names>S.</given-names></name><name><surname>de Baenst</surname><given-names>I.</given-names></name><name><surname>Aerts</surname><given-names>M.</given-names></name><name><surname>Cnockaert</surname><given-names>M.</given-names></name><name><surname>Devlieghere</surname><given-names>F.</given-names></name><name><surname>Van Damme</surname><given-names>P.</given-names></name></person-group> (<year>2019</year>). <article-title>Tracking the sources of psychrotrophic bacteria contaminating chicken cuts during processing.</article-title>\n<source><italic>Food Microbiol.</italic></source>\n<volume>81</volume>\n<fpage>40</fpage>–<lpage>50</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fm.2018.06.003</pub-id>\n<pub-id pub-id-type=\"pmid\">30910087</pub-id></mixed-citation></ref><ref id=\"B92\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schloss</surname><given-names>P. D.</given-names></name><name><surname>Westcott</surname><given-names>S. L.</given-names></name><name><surname>Ryabin</surname><given-names>T.</given-names></name><name><surname>Hall</surname><given-names>J. R.</given-names></name><name><surname>Hartmann</surname><given-names>M.</given-names></name><name><surname>Hollister</surname><given-names>E. B.</given-names></name><etal/></person-group> (<year>2009</year>). <article-title>Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>75</volume>\n<fpage>7537</fpage>–<lpage>7541</lpage>. <pub-id pub-id-type=\"doi\">10.1128/AEM.01541-09</pub-id>\n<pub-id pub-id-type=\"pmid\">19801464</pub-id></mixed-citation></ref><ref id=\"B93\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shirtliff</surname><given-names>M. E.</given-names></name><name><surname>Mader</surname><given-names>J. T.</given-names></name><name><surname>Camper</surname><given-names>A. K.</given-names></name></person-group> (<year>2002</year>). <article-title>Molecular interactions in biofilms.</article-title>\n<source><italic>Chem. Biol.</italic></source>\n<volume>9</volume>\n<fpage>859</fpage>–<lpage>871</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S1074-5521(02)00198-9</pub-id><pub-id pub-id-type=\"pmid\">12204685</pub-id></mixed-citation></ref><ref id=\"B94\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Silbande</surname><given-names>A.</given-names></name><name><surname>Cornet</surname><given-names>J.</given-names></name><name><surname>Cardinal</surname><given-names>M.</given-names></name><name><surname>Chevalier</surname><given-names>F.</given-names></name><name><surname>Rochefort</surname><given-names>K.</given-names></name><name><surname>Smith-Ravin</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Characterization of the spoilage potential of pure and mixed cultures of bacterial species isolated from tropical yellowfin tuna (<italic>Thunnus albacares</italic>).</article-title>\n<source><italic>J. Appl. Microbiol.</italic></source>\n<volume>124</volume>\n<fpage>559</fpage>–<lpage>571</lpage>. <pub-id pub-id-type=\"doi\">10.1111/jam.13663</pub-id>\n<pub-id pub-id-type=\"pmid\">29222941</pub-id></mixed-citation></ref><ref id=\"B95\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Simões</surname><given-names>M.</given-names></name><name><surname>Simões</surname><given-names>L. C.</given-names></name><name><surname>Vieira</surname><given-names>M. J.</given-names></name></person-group> (<year>2010</year>). <article-title>A review of current and emergent biofilm control strategies.</article-title>\n<source><italic>LWT Food Sci. Technol.</italic></source>\n<volume>43</volume>\n<fpage>573</fpage>–<lpage>583</lpage>. <pub-id pub-id-type=\"doi\">10.1016/J.LWT.2009.12.008</pub-id></mixed-citation></ref><ref id=\"B96\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stiefel</surname><given-names>P.</given-names></name><name><surname>Mauerhofer</surname><given-names>S.</given-names></name><name><surname>Schneider</surname><given-names>J.</given-names></name><name><surname>Maniura-Weber</surname><given-names>K.</given-names></name><name><surname>Rosenberg</surname><given-names>U.</given-names></name><name><surname>Ren</surname><given-names>Q.</given-names></name></person-group> (<year>2016</year>). <article-title>Enzymes enhance biofilm removal efficiency of cleaners.</article-title>\n<source><italic>Antimicrob. Agents Chemother.</italic></source>\n<volume>60</volume>\n<fpage>3647</fpage>–<lpage>3652</lpage>. <pub-id pub-id-type=\"doi\">10.1128/AAC.00400-16</pub-id>\n<pub-id pub-id-type=\"pmid\">27044552</pub-id></mixed-citation></ref><ref id=\"B97\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stoddard</surname><given-names>S. F.</given-names></name><name><surname>Smith</surname><given-names>B. J.</given-names></name><name><surname>Hein</surname><given-names>R.</given-names></name><name><surname>Roller</surname><given-names>B. R. K.</given-names></name><name><surname>Schmidt</surname><given-names>T. M.</given-names></name></person-group> (<year>2015</year>). <article-title>rrnDB: improved tools for interpreting rRNA gene abundance in bacteria and archaea and a new foundation for future development.</article-title>\n<source><italic>Nucleic Acids Res.</italic></source>\n<volume>43</volume>\n<fpage>D593</fpage>–<lpage>D598</lpage>. <pub-id pub-id-type=\"doi\">10.1093/nar/gku1201</pub-id>\n<pub-id pub-id-type=\"pmid\">25414355</pub-id></mixed-citation></ref><ref id=\"B98\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stoops</surname><given-names>J.</given-names></name><name><surname>Ruyters</surname><given-names>S.</given-names></name><name><surname>Busschaert</surname><given-names>P.</given-names></name><name><surname>Spaepen</surname><given-names>R.</given-names></name><name><surname>Verreth</surname><given-names>C.</given-names></name><name><surname>Claes</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>Bacterial community dynamics during cold storage of minced meat packaged under modified atmosphere and supplemented with different preservatives.</article-title>\n<source><italic>Food Microbiol.</italic></source>\n<volume>48</volume>\n<fpage>192</fpage>–<lpage>199</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fm.2014.12.012</pub-id>\n<pub-id pub-id-type=\"pmid\">25791008</pub-id></mixed-citation></ref><ref id=\"B99\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sundberg</surname><given-names>M.</given-names></name><name><surname>Christiansson</surname><given-names>A.</given-names></name><name><surname>Lindahl</surname><given-names>C.</given-names></name><name><surname>Wahlund</surname><given-names>L.</given-names></name><name><surname>Birgersson</surname><given-names>C.</given-names></name></person-group> (<year>2011</year>). <article-title>Cleaning effectiveness of chlorine-free detergents for use on dairy farms.</article-title>\n<source><italic>J. Dairy Res.</italic></source>\n<volume>78</volume>\n<fpage>105</fpage>–<lpage>110</lpage>. <pub-id pub-id-type=\"doi\">10.1017/S0022029910000762</pub-id>\n<pub-id pub-id-type=\"pmid\">21134310</pub-id></mixed-citation></ref><ref id=\"B100\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wijman</surname><given-names>J. G. E.</given-names></name><name><surname>De Leeuw</surname><given-names>P. P. L. A.</given-names></name><name><surname>Moezelaar</surname><given-names>R.</given-names></name><name><surname>Zwietering</surname><given-names>M. H.</given-names></name><name><surname>Abee</surname><given-names>T.</given-names></name></person-group> (<year>2007</year>). <article-title>Air-liquid interface biofilms of <italic>Bacillus cereus</italic>: formation, sporulation, and dispersion.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>73</volume>\n<fpage>1481</fpage>–<lpage>1488</lpage>. <pub-id pub-id-type=\"doi\">10.1128/AEM.01781-06</pub-id>\n<pub-id pub-id-type=\"pmid\">17209076</pub-id></mixed-citation></ref><ref id=\"B101\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Wirtanen</surname><given-names>G.</given-names></name><name><surname>Salo</surname><given-names>S.</given-names></name></person-group> (<year>2016</year>). “<article-title>Biofilm risks</article-title>,” in <source><italic>Handbook of Hygiene Control in the Food Industry</italic></source> (<publisher-loc>Cambridge</publisher-loc>: <publisher-name>Woodhead Publishing</publisher-name>), <fpage>55</fpage>–<lpage>79</lpage>. <pub-id pub-id-type=\"doi\">10.1016/B978-0-08-100155-4.00005-4</pub-id></mixed-citation></ref><ref id=\"B102\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yoon</surname><given-names>S.-H.</given-names></name><name><surname>Ha</surname><given-names>S.-M.</given-names></name><name><surname>Kwon</surname><given-names>S.</given-names></name><name><surname>Lim</surname><given-names>J.</given-names></name><name><surname>Kim</surname><given-names>Y.</given-names></name><name><surname>Seo</surname><given-names>H.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies.</article-title>\n<source><italic>Int. J. Syst. Evol. Microbiol.</italic></source>\n<volume>67</volume>\n<fpage>1613</fpage>–<lpage>1617</lpage>. <pub-id pub-id-type=\"doi\">10.1099/ijsem.0.001755</pub-id>\n<pub-id pub-id-type=\"pmid\">28005526</pub-id></mixed-citation></ref><ref id=\"B103\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhao</surname><given-names>F.</given-names></name><name><surname>Zhou</surname><given-names>G.</given-names></name><name><surname>Ye</surname><given-names>K.</given-names></name><name><surname>Wang</surname><given-names>S.</given-names></name><name><surname>Xu</surname><given-names>X.</given-names></name><name><surname>Li</surname><given-names>C.</given-names></name></person-group> (<year>2015</year>). <article-title>Microbial changes in vacuum-packed chilled pork during storage.</article-title>\n<source><italic>Meat. Sci.</italic></source>\n<volume>100</volume>\n<fpage>145</fpage>–<lpage>149</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.meatsci.2014.10.004</pub-id>\n<pub-id pub-id-type=\"pmid\">25460118</pub-id></mixed-citation></ref></ref-list><glossary><title>Abbreviations</title><def-list id=\"DL1\"><def-item><term>ACC</term><def><p>aerobic colony count</p></def></def-item><def-item><term>ANCC</term><def><p>anaerobic colony counts</p></def></def-item><def-item><term>ATP</term><def><p>adenosine triphosphate</p></def></def-item><def-item><term>CIP</term><def><p>cleaning-in-place</p></def></def-item><def-item><term>Dl</term><def><p>detection limit</p></def></def-item><def-item><term>ESSOs</term><def><p>ephemeral/specific spoilage organisms</p></def></def-item><def-item><term>OPC</term><def><p>open plant cleaning</p></def></def-item><def-item><term>OTU</term><def><p>operational taxonomic unit</p></def></def-item><def-item><term>P50</term><def><p>percentile 50</p></def></def-item><def-item><term>P90</term><def><p>percentile 90</p></def></def-item><def-item><term>Pmax</term><def><p>percentile 100</p></def></def-item><def-item><term>qPCR</term><def><p>quantitative polymerase chain reaction.</p></def></def-item></def-list></glossary></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Endocrinol.</journal-id><journal-title-group><journal-title>Frontiers in Endocrinology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2392</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849277</article-id><article-id pub-id-type=\"pmc\">PMC7431610</article-id><article-id pub-id-type=\"doi\">10.3389/fendo.2020.00484</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>New Surgical Model for Bone–Muscle Injury Reveals Age and Gender-Related Healing Patterns in the 5 Lipoxygenase (5LO) Knockout Mouse</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Biguetti</surname><given-names>Claudia Cristina</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/593383/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Couto</surname><given-names>Maira Cristina Rondina</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Silva</surname><given-names>Ana Claudia Rodrigues</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Shindo</surname><given-names>João Vitor Tadashi Cosin</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Rosa</surname><given-names>Vinicius Mateus</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1047031/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Shinohara</surname><given-names>André Luis</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Andreo</surname><given-names>Jesus Carlos</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1046084/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Duarte</surname><given-names>Marco Antonio Hungaro</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Zhiying</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Brotto</surname><given-names>Marco</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/704081/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Matsumoto</surname><given-names>Mariza Akemi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Basic Sciences, School of Dentistry, São Paulo State University (UNESP)</institution>, <addr-line>Araçatuba</addr-line>, <country>Brazil</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Bone-Muscle Research Center, College of Nursing and Health Innovation, University of Texas at Arlington</institution>, <addr-line>Arlington, TX</addr-line>, <country>United States</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Health Sciences, Universidade Do Sagrado Coração</institution>, <addr-line>Bauru</addr-line>, <country>Brazil</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Bauru School of Dentistry, University of São Paulo, FOB-USP</institution>, <addr-line>São Paulo</addr-line>, <country>Brazil</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Giacomina Brunetti, University of Bari Aldo Moro, Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Michaël R. Laurent, University Hospitals Leuven, Belgium; Michaela Tencerova, Institute of Physiology (ASCR), Czechia</p></fn><corresp id=\"c001\">*Correspondence: Claudia Cristina Biguetti <email>claudia.biguetti@uta.edu</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Bone Research, a section of the journal Frontiers in Endocrinology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>484</elocation-id><history><date date-type=\"received\"><day>19</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>22</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Biguetti, Couto, Silva, Shindo, Rosa, Shinohara, Andreo, Duarte, Wang, Brotto and Matsumoto.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Biguetti, Couto, Silva, Shindo, Rosa, Shinohara, Andreo, Duarte, Wang, Brotto and Matsumoto</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Signaling lipid mediators released from 5 lipoxygenase (5LO) pathways influence both bone and muscle cells, interfering in their proliferation and differentiation capacities. A major limitation to studying inflammatory signaling pathways in bone and muscle healing is the inadequacy of available animal models. We developed a surgical injury model in the <italic>vastus lateralis</italic> (VL) muscle and femur in 129/SvEv littermates mice to study simultaneous musculoskeletal (MSK) healing in male and female, young (3 months) and aged (18 months) WT mice compared to mice lacking 5LO (5LOKO). MSK defects were surgically created using a 1-mm punch device in the VA muscle followed by a 0.5-mm round defect in the femur. After days 7 and 14 post-surgery, the specimens were removed for microtomography (microCT), histopathology, and immunohistochemistry analyses. In addition, non-injured control skeletal muscles along with femur and L5 vertebrae were analyzed. Bones were microCT phenotyped, revealing that aged female WT mice presented reduced BV/TV and trabecular parameters compared to aged males and aged female 5LOKO mice. Skeletal muscles underwent a customized targeted lipidomics investigation for profiling and quantification of lipid signaling mediators (LMs), evidencing age, and gender related-differences in aged female 5LOKO mice compared to matched WT. Histological analysis revealed a suitable bone-healing process with osteoid deposition at day 7 post-surgery, followed by woven bone at day 14 post-surgery, observed in all young mice. Aged WT females displayed increased inflammatory response at day 7 post-surgery, delayed bone matrix maturation, and increased TRAP immunolabeling at day 14 post-surgery compared to 5LOKO females. Skeletal muscles of aged animals showed higher levels of inflammation in comparison to young controls at day 14 post-surgery; however, inflammatory process was attenuated in aged 5LOKO mice compared to aged WT. In conclusion, this new model shows that MSK healing is influenced by age, gender, and the 5LO pathway, which might serve as a potential target to investigate therapeutic interventions and age-related MSK diseases. Our new model is suitable for bone–muscle crosstalk studies.</p></abstract><kwd-group><kwd>5 lipoxygenase</kwd><kwd>bone</kwd><kwd>lipid mediators</kwd><kwd>aging</kwd><kwd>muscle</kwd><kwd>tissue healing</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Fundação de Amparo à Pesquisa do Estado de São Paulo<named-content content-type=\"fundref-id\">10.13039/501100001807</named-content></funding-source><award-id rid=\"cn001\">#13/04714-8</award-id><award-id rid=\"cn001\">2018/08913</award-id><award-id rid=\"cn001\">2018/19406-0</award-id></award-group><award-group><funding-source id=\"cn002\">National Institutes of Health<named-content content-type=\"fundref-id\">10.13039/100000002</named-content></funding-source><award-id rid=\"cn002\">PO1 AG039355</award-id><award-id rid=\"cn002\">R01AG056504</award-id><award-id rid=\"cn002\">R01AG060341</award-id></award-group></funding-group><counts><fig-count count=\"8\"/><table-count count=\"3\"/><equation-count count=\"0\"/><ref-count count=\"87\"/><page-count count=\"20\"/><word-count count=\"14149\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Investigation of bone and muscle homeostasis has in recent years expanded to biochemical crosstalk via the secretion of different molecules, such as signaling lipid mediators (LMs) derived from essential polyunsaturated fatty acids (PUFA), with potential implications on aging, inflammation, and tissue healing (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>–<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). In this context, although skeletal muscle possesses great plasticity in response to physiologic stimuli (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>, <xref rid=\"B6\" ref-type=\"bibr\">6</xref>), its capacity for adaptation/regeneration is dependent on many different factors, such as the nature and extent of the stimulus (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>), viability of satellite cells, aging, and inflammation (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>–<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). Indeed, higher inflammatory marker levels in elderly populations are directly associated with loss of muscle mass and strength (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>), as well with the reduced capacity for bone healing, decreasing in the viability of osteoprogenitor cells (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). As observed in mice, aging also causes changes in skeletal muscle in a variable range of LM (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>), such as AA-derived eicosanoids, eicosapentaenoic acid (EPA), as well as Omega 3 (ω-3) PUFA derivatives [e.g., docosahexaenoic acid [DHA]], in an age- and gender-dependent manner. In the face of these multiple factors (inflammation, aging, and gender), healing outcomes on the MSK system can vary from complete tissue regeneration to fibrosis, affecting the functional recovery of these tissues with important clinical implications (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). However, investigations are still ongoing on the specific cellular and molecular factors involved in the interconnected and interdependent healing of bone and muscle.</p><p>Due to the close anatomical relationship of bone and muscle, traumatic bone injuries (open bone fractures or surgical trauma) involve damage to the adjacent muscles, requiring simultaneous tissue healing of both compartments (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Skeletal muscle assists bone healing, not only when it is used as a flap (or a second periosteum) for improving bone defect vascularization (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>–<xref rid=\"B16\" ref-type=\"bibr\">16</xref>), but also the osteogenic potential of satellite cells (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). <italic>In vitro</italic> studies have suggested that osteocytes can enhance <italic>in vitro</italic> myogenesis and <italic>ex vivo</italic> muscle contractility by different mechanisms, such as through the Wnt/β-Catenin pathway (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>) and the secretion of prostaglandin E<sub>2</sub> (PGE<sub>2</sub>) key LM, which has a positive impact in the myogenic differentiation of primary mouse myoblasts/myotubes (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>).</p><p>Skeletal muscle is mainly composed by muscle fibers surrounded by myogenic progenitors (satellite cells) with the capacity to proliferate and induce new fiber formation to restore injured tissue, followed by the upregulation and expression of Myoblast Determination Protein 1 (MyoD), as demonstrated in murine models of muscle regeneration (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B17\" ref-type=\"bibr\">17</xref>). MyoD is a quintessential protein in mammals that belongs to the Muscle Regulatory Family of proteins and is necessary for myoblast differentiation into myotubes (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). While new myotubes are formed, other injured muscle fibers undergo to degeneration/atrophy due to injuries followed by the upregulation and expression of Muscle Ring Finger-1 (MuRF1) (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>). MuRF1 belongs to the family of ubiquitin ligases, is proposed to trigger muscle protein degradation via ubiquitination, serving as a marker for fiber degeneration/atrophy (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). MyoD and MuRF1 are both suitable markers for models of muscle regeneration/remodeling in skeletal muscle post-surgery. At the same time, bone has mesenchymal cell-derived osteoblasts, which can transfer from a quiescent stage into rapid proliferation and differentiation after certain stimuli and expression of Runt-related Transcription Factor-2 (Runx-2) (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). After new bone deposition, hematopoietic-derived osteoclasts regulated by osteoblasts, and inflammatory mediators contribute to bone maturation by a coordinated resorption of new bone matrix, followed by expression of different markers, such as Tartrate Resistant Acid Phosphatase (TRAP) (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>).</p><p>Apart from the specificity of each cell type involved in skeletal muscle and/or bone regeneration, general tissue healing post-injury requires an initial and transient inflammatory phase, with release of several cytokines and inflammatory mediators, such as AA-derived signaling lipid mediators released from ciclooxigenase-2 (COX2) and 5-Lipoxygenase (5LO) pathways (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>–<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). These processes inherent to the inflammatory response are essential in directing vascular events and leukocytes migration, through the release of PGs and LTs (LTB4 and/or CysLTs), respectively (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B26\" ref-type=\"bibr\">26</xref>). In addition, evidence from other studies have also supported a crucial modulatory role of COXs and 5LO pathways and their products, PGs and LTs, on bone and muscle cells in different models of tissue repair/regeneration (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B27\" ref-type=\"bibr\">27</xref>–<xref rid=\"B31\" ref-type=\"bibr\">31</xref>).</p><p>While COX2 seems to affect bone healing in a positive manner by inducing new bone formation and angiogenesis (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B33\" ref-type=\"bibr\">33</xref>), the 5LO pathway and its final products are supposed to contribute to osteoclastic differentiation and consequent bone resorption (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>, <xref rid=\"B34\" ref-type=\"bibr\">34</xref>–<xref rid=\"B36\" ref-type=\"bibr\">36</xref>) and inhibited bone formation <italic>in vitro</italic> (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>). Male mice genetically deficient (knockout) for COX2 expression (COX2 KO mice), presented delayed bone formation in femur fractures, but with significant rescue after periosteal injection of the prostaglandin E2 receptor 4 (EP4) agonist (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>). On the other hand, male young mice lacking 5LO expression presented increased cortical thickness (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>) and accelerated fracture healing in endochondral bones (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). Previous studies comparing 129 SvEv WT with homozygous KO for 5LO have also shown a relevant role of 5LO activity in periodontal inflammation and bone resorption (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>). Furthermore, the pharmacological inhibition of 5LO (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>) or antagonism of CysLT1-receptor also enhanced endochondral bone healing in rats (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>). In skeletal muscle, LTB4 contributes to muscle regeneration by enhancing the proliferation and differentiation of satellite cells (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>). <italic>In vitro</italic> studies revealed that the 5LO pathway is one of the major sources of extracellular ROS release in skeletal muscle (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>). Significant upregulation of LTB4 pathway has been particularly found in muscle tissue in chronic inflammatory conditions (polymyositis or dermatomyositis) and seems to be associated with muscle weakness (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>). However, the role of 5LO on muscle healing remains elusive, and no previous studies have addressed the effects of 5LO inhibition or its complete deletion in animal models.</p><p>Finally, it is important to mention that 5LO activity measured by the amount of LTs are significantly higher in females than in males, in humans (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>, <xref rid=\"B45\" ref-type=\"bibr\">45</xref>), and rodents (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>–<xref rid=\"B48\" ref-type=\"bibr\">48</xref>). These differences in gender generally associate with a higher incidence and severity of inflammatory chronic conditions and autoimmune diseases in females than males, such as rheumatoid arthritis and asthma, and positively associate with increased levels of LTs (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>). Thus, it is reasonable to hypothesize that gender is an important factor that can modulate the action of lipid mediators on the MSK system-healing capacity.</p><p>Therefore, efforts on development/characterization of animal models of simultaneous bone–muscle healing might support a comprehensive understanding on the role of lipid mediators in this process to provide insights that could lead to the identification of potential targets for the treatment and/or new interventions (e.g., specific diets/exercises) of MSK injuries. Our primary objective was to develop a new surgical mouse model that better represents the pathophysiological conditions for the simultaneous injury of bone and muscle without major catastrophic failure. As a secondary objective, we then utilized this model to investigate healing patterns during aging. We further studied the influence of gender and 5LO using the 5LOKO mouse. Our key findings revealed that aging delayed both muscle and bone healing in mice, with bones recovering faster than muscles, maybe indicating a role of muscles, to act as bone sentinels. We observed the major negative effects in aged WT females. Importantly, despite the aging negative effects, 5LOKO aged females presented an improved skeletal phenotype and healing outcomes compared to the young WT mice.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Animals</title><p>In this study, we utilized 129/SvEv wild type (WT) and 5LO (homozygous knockout for 5LO<sup>tm1Fun</sup>, designated here as 5LOKO). Littermate controls 129/SvEv and 5LOKO mice were used as previously described by other studies using this strain of 5LOKO mice (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>) and from the same source (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>). Mice were obtained from Central Animal Facility for Special Mice of the School of Medicine of Ribeirão Preto—University of São Paulo, Brazil (CCCE-FMRP-USP) and were housed in the Central Animal Facility of Universidade Sagrado Coração (USC), Bauru, São Paulo, Brazil, under the approval of IACUC protocol #9589271017 (from Institutional Ethic Committee on Animal Use) and following the normative regulations provided by the National Council for the Control of Animal Experimentation in Brazil (CONCEA). Our design included four experimental groups, each containing 20 mice: 3-month old (young) males and females, and 18-month old (aged) males and females. The mice received sterile water and sterile standard solid mice chow (Nuvital, Curitiba, PR, Brazil) <italic>ad libitum</italic> and were housed at temperature-controlled rooms (22–25°C). We conducted estrus cycle analysis (Toluine blue O vaginal smears) to determine post-menopausal stage in all aged female mice (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>, <xref rid=\"B51\" ref-type=\"bibr\">51</xref>). All animal procedures were cared for in accordance with the CONCEA and following the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Institute of Laboratory Animal Resources (U.S.), as well as the ARRIVE guidelines recommendations (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>). Experimental groups for bone–muscle surgical injury model were comprised of five animals per group/time point (7 and 14 days) and used for microtomography (microCT), histological, and immunohistochemical analysis. Prior and during the experimental protocol, animals from each group were allocated in groups of five mice per cage and the cages were codified by the researcher supervisor and technician in order to minimize bias. Naïve bones (left femur and L5 vertebrae) were used for skeletal phenotyping by microCT analysis. One 129SvEv WT aged and one young female were additionally used for pilot studies for the surgical model. Naïve gastrocnemius muscles were collected for lipidomics analyses at the Bone–Muscle Research Center, <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.uta.edu/conhi/research/bmrc/index.php\">https://www.uta.edu/conhi/research/bmrc/index.php</ext-link>, University of Texas–Arlington.</p></sec><sec><title>Experimental Protocol for Surgical Muscle and Bone Injury</title><p>We developed a new surgical model for the simultaneous injury of bone and muscle to simulate conditions of trauma and/or surgeries in a controlled and reproducible manner, where both tissues are damaged, but without catastrophic damage, such as an injury that occurs in automobile accidents or with military personnel. Mice were anesthetized with intraperitoneal injection of 80 mg/kg ketamine chloride (Dopalen, Agribrands Brasil, Paulínia, SP, Brazil) and 160 mg/kg xylazine chloride (Anasedan, Agribrands Brasil, Paulínia, SP, Brazil). Additional local anesthesia was provided by using 5 μL of intramuscular injection of Mepivacaine Hydrochloride 2% with Epinephrine 1:100,000 (Mepiadre®, DFL, Rio de Janeiro, Brazil) vasoconstrictor, to provide hemostasis and additional comfort in the first hours after surgery. All animals were measured in relation to body weight (BW) and rostrocaudal (RC) length before the experimental procedures. Then, mice were positioned in lateral decubitus under a stereomicroscope providing 5× magnification (DF Vasconcellos, São Paulo, SP, Brazil). The right lower limb was gently shaved, disinfected with polyvinylpyrrolidone prior to a 10-mm vertical incision to expose the Vastus Lateralis (VL) underlying muscle. A 1-mm micro-punch device for rodents (Härte Instrumentos Cirúrgicos, Ribeirao Preto, Brazil) was used to create a muscle defect to a depth of 1.5 mm until it reached the femoral bone. To produce a subjacent monocortical bone defect in the femur, midshaft femoral bone was drilled using a 0.50-mm pilot drill (NTI-Kahla GmbH Rotary Dental Instruments, Kahla, Thüringen, Germany) to a maximum depth of 1 mm at 600 rpm, using a surgical motor (NSK-Nakanishi International, Kanuma, Tochigi, Japan). Bone defects were created under cold saline solution irrigation to avoid thermal necrosis in bone and surrounding tissues. Only subcutaneous tissue (not muscle) was sutured with 5–0 silk suture (Ethicon, Johnson & Johnson, São Paulo, Brazil). All groups of animals were submitted to surgery, and the actual surgical procedure (creation of defects) was performed by a single trained, expert surgeon (CCB), who was blinded at this point of the experiment. No antibiotics and anti-inflammatory drugs were administered to the animals after the MSK-injury so as not to interfere with experimental design. At the first 72 h after surgery, the feed was supplied from the bottom compartment of the cage to prevent, but not impede, the recovering mice from getting up in the cage, which could result in additional unintended damage. The suture naturally fell off 3–4 days post-surgery when the skin was clinically healed. Mice were provided sterile water <italic>ad libitum</italic> and were fed with sterile standard solid mice chow (Nuvital, Curitiba, PR, Brazil) throughout all experimental periods of this study.</p></sec><sec><title>Sample Collection</title><p>At the end of experimental time points, mice were euthanized for sample collection. The VL muscle was dissected from the quadriceps femoris muscles, embedded in optimal cutting temperature compound (OCT) (Tissue-Tek, Sakura Finetek, Torrance, CA, USA) and immediately frozen in liquid nitrogen for cryostat sectioning. Gastrocnemius muscle samples were collected and immediately frozen in liquid nitrogen for targeted quantification of LMs via lipidomics, as previously described (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Injured femurs, as well as femur controls and L5 vertebrae, were fixed in phosphate buffered saline (PBS) buffered formalin (10%) solution (pH 7.2) for 48 h at room temperature. Bone specimens were washed overnight in running water and maintained in 70% hydrous ethanol for microCT scanning. After microCT scanning, bone specimens were decalcified in 4.13% EDTA (pH 7.2) following histological processing protocols, as previously described (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>).</p></sec><sec><title>Micro CT</title><p>Bone specimens (injured, control femurs, and L5 vertebrae) were scanned for qualitative and quantitative analyses by microCT. Controls used for bone phenotyping (10 biological replicates) from each group were pooled (from 7 to 14 days), since they represented controls. Five biological replicates of injured femurs were used from each group. First, specimens were rehydrated in saline solution for 10 min before scanning. Sample scanning was performed by using a Skyscan 1174 System (Skyscan, Kontich, Belgium) at 50 kV, 800 μA, with a 0.5-mm aluminum filter, 180 degrees of rotation and exposure range of 1 degree and a 14-μm-pixel-size resolution, as previously described (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). Briefly, projections were first reconstructed using the NRecon software (Bruker microCT, Kontich, Belgium), realigned in Data Viewer for 2D images. Tridimensional images were obtained by using CTVox (Bruker microCT, Kontich, Belgium). Quantitative parameters were assessed using CTAn (Bruker microCT, Kontich, Belgium), based on previous guidelines and recommendations (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>), as well as similar regions of interest (ROI) for L5 vertebrae and femur analysis (<xref rid=\"B55\" ref-type=\"bibr\">55</xref>–<xref rid=\"B57\" ref-type=\"bibr\">57</xref>). For skeletal phenotype, L5- vertebral body was evaluated considering a ROI, 1.5 mm in diameter and 3 mm in length. Femur was analyzed considering two regions: a 2-mm length of the cortical of mid-diaphysis and a 1.5-mm length of the cancellous compartment of distal metaphysis. Evaluated parameters for skeletal phenotype comprised bone volume (BV, mm<sup>3</sup>), fraction of bone volume [Bone Volume/Tissue Volume (BV/TV), %], trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). Cross-sectional volume (mm<sup>3</sup>) was evaluated only for the cortical mid-diaphysis of femur. Bone defects were evaluated for BV/TV (%) by using a cylindrical ROI, 0.5 mm in diameter and 0.5 mm in depth, considering the size of monortical defect.</p></sec><sec><title>Histological Processing for Injured Bone</title><p>After microCT scanning, femur samples containing the region of defect were washed and immersed in buffered 4.13% EDTA (pH 7.2) for decalcification for 3 weeks. Then, specimens were processed for histological embedding in paraffin blocks. Semi-serial 5-μm histological transversal slices were obtained from the central area of the bone defect and were used for Hematoxylin and Eosin (H&E), modified Goldner's Trichrome/Alcian Blue (<xref rid=\"B58\" ref-type=\"bibr\">58</xref>), and Picrosirius red staining for birefringence and immunohistochemistry for 5LO, Runx2, and TRAP.</p></sec><sec><title>Histological Processing for Injured Muscles</title><p>Muscle samples were processed as previously described (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>, <xref rid=\"B60\" ref-type=\"bibr\">60</xref>). VL muscles were embedded in OCT and sectioned at temperature of −20°C (Leica, CM 1850, Nussloch, Germany). Eight semi-serial 8-μm transversal sections were obtained from each specimen separated by at least 40 μm, comprising the area of defect and adjacent areas. Histological sections from days 7- to 14 post-surgery were used for H&E staining. Histological sections from 7 days post-surgery were used for immunohistochemistry for MuRF1 and MyoD.</p></sec><sec><title>Histopathological Analysis of Bone and Muscle Healing</title><p>Bone and muscle samples stained with H&E were used for histopathological analysis by two blinded examiners (CCB and MAM). Bone healing was evaluated considering the presence of inflammatory infiltration, connective tissue, cartilage, newly formed bone (containing new osteoblasts and osteocytes differentiation), osteoclasts, and blood vessels in remodeling areas. Modified Goldner's Trichrome/Alcian Blue was also used as a complementary approach to identify newly formed bone, cartilage, and connective tissue. Five biological replicates from muscle samples were also used for histomorphometric analysis. The histological region for healing comprised 1 mm<sup>2</sup> in the central area of muscle defect, as well the adjacent region of central damage, for evaluation of cross-sectional area (CSA) of fibers. CSA measurement was performed with two technical replicates from each animal, from which six photomicrographs at 100× magnification (technical replicates) were captured and evaluated using the software SigmaScan Pro 5.0 (Systat Software Inc., Chicago, USA). Histomorphometric parameters included inflammatory infiltrate in the area of injury and fibers with centralized myonuclei surrounding the region of the defect. We used histological sections (three technical replicates) from five animals (biological replicates) per group. Quantification of histological parameters was performed using four random histological fields from each histological section. Histological fields were captured at 100× using an oil immersion objective (Carl Zeiss Jena GmbH, Jena, Germany). A grid image was superimposed on each histological field containing a total of 100 points in a quadrangular area by using the ImageJ software (Version 1.51, National Institutes of Health, Bethesda, Maryland, USA), as previously described (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>). Only the points coincident (considered as the intersection point of the vertical and horizontal lines) with the histological parameters were considered, and the total number of points was obtained to calculate the area density for each healing component in each section. Results were presented as means ± SDs of the area density for each bone and muscle healing parameter.</p></sec><sec><title>Birefringence Analysis for Collagenous Content in Bone Defects</title><p>Quality and quantity of bone matrix deposition was performed using the Picrosirius-polarization method and birefringence analysis. Two histological fields (two biological replicates) of each femur defects stained with Picrosirius Red were captured with a 10× objective under polarizing lens coupled to a binocular inverted microscope (Leica DM IRB/E) and analyzed as previously described (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>, <xref rid=\"B61\" ref-type=\"bibr\">61</xref>). Green birefringence color indicates thin fibers; yellow and red colors at birefringence analysis indicate thick and organized collagen fibers (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>, <xref rid=\"B58\" ref-type=\"bibr\">58</xref>). Briefly, spectra of green, yellow, and red colors were defined by RGB values, and the quantity of pixels-squared was calculated for each field by using the AxioVision 4.8 software (CarlZeiss). After calculations of each spectrum fiber, total area was also calculated by the sum of each color spectrum. Means and standard deviation (SD) considering the two technical replicates (histological fields) and five biological replicates (number of animals per group) were calculated for each group, considering strain, gender and age.</p></sec><sec><title>Immunohistochemistry</title><p>Histological sections from femur defects were used for individual immune detection of TRAP (sc30832), Runx2 (sc8566), and 5LO (sc136195). Muscle samples were used for individual immune detection of MuRF-1 (sc398608) and MyoD (sc377460). All primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Paraffin-embedded histological sections were rehydrated, and antigen retrieval was performed by boiling the slides in 10 mM sodium citrate buffer (pH 6) for 30 min at 100°C. Sections were pre-incubated with 3% Hydrogeroxidase Block (Spring Bioscience Corporation, CA, USA) and then incubated with 7% Non-fat Dry Milk to block serum proteins. Frozen muscles samples were incubated in ice-cold acetone prior to incubation with horse serum for protein blocking. Anti-TRAP, anti-Runx2, Anti-MuRF1, and Anti-MyoD primary antibodies were diluted at 1:100, while 5LO was diluted in 1:200 in diluent solution and then incubated for 1 h at room temperature. Goat-On-Rodent HRP-Polymer PromARM (cat # GHP516G, Biocare Medical, Pacheco, CA) was used as a detection method for TRAP and Runx-2, and sections were incubated for 40 min at room temperature following manufacturer instructions. Anti-Mouse HRP-Polymer made in goat (ImmPRESS, Vector Laboratories, San Diego, CA) was used as a detection method for 5LO, MuRF-1, and MyoD. The identification of antigen–antibody reaction was performed using 3-3′-diaminobenzidine (DAB). Bone samples labeled for TRAP, Runx-2, and 5LO were counterstained with Mayer's hematoxylin. Muscle samples were left without counterstaining to be used for optical density quantification. Negative controls were performed by using only diluents solution instead of primary antibody.</p></sec><sec><title>Quantification of Immunohistochemical Markers</title><p>Quantification of all markers was performed by using four histological fields from each histological section captured, using a 100× oil immersion objective (Carl Zeiss Jena GmbH, Jena, Germany). Quantification of TRAP was performed at the 14-day time point; most TRAP+ cells were found in areas of bone remodeling. Runx2, MuRF-1, and MyoD labeling were analyzed at the 7-day time point. Pixels quantification was performed with Aperio Image Scope v. 12.3.3 (Leica Biosystems, Buffalo Grove, USA), used for immunohistochemical analysis in pathology (<xref rid=\"B62\" ref-type=\"bibr\">62</xref>). Images were analyzed by choosing the algorithm Positive Pixel Count in Aperio Image Scope (Leica Biosystems, Buffalo Grove, USA). Subsequently, all positive pixels were calculated, and the results were expressed as Positivity (number of positive pixels/number total). Negative controls tissues were used for confirmation of negative labeling and detection. Results were presented as the means and SD for each marker.</p></sec><sec><title>Targeted Lipidomics</title><p>Targeted lipidomics was performed following our previously published quantification method (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>) with some modifications. Briefly, aliquoted frozen muscle tissue (50–100 mg) was applied for LC-MS/MS-based lipidomic analysis. All components of LC-MS/MS system are from Shimadzu Scientific Instruments, Inc. (Columbia, MD). The LC system was equipped with four pumps (Pump A/B: LC-30AD, Pump C/D: LC-20AD XR), a SIL-30AC autosampler (AS), and a CTO-30A column oven containing a two-channel, six-port switching valve. The LC separation was conducted on a C8 column (Ultra C8, 150 × 2.1 mm, 3 μm, RESTEK, Manchaca, TX) along with a Halo guard column (Optimize Technologies, Oregon City, OR). The MS/MS analysis was performed on Shimadzu LCMS-8050 triple quadrupole mass spectrometer. The instrument was operated and optimized under both positive and negative electrospray and multiple reaction monitoring modes (± ESI MRM). Standard lipid mediators and corresponding isotope-labeled lipid mediator internal standards (IS) were purchased from Cayman Chemical Co. (Ann Arbor, MI). All analyses and data processing were completed on Shimadzu LabSolutions V5.91 software (Columbia, MD). Four total bioactive lipids [EPA, DHA, AA, 11,12-epoxyeicosatrienoic acid [11,12-EET], and PGE<sub>2</sub>] were quantified from muscle samples in this study. Results were presented quantity of each lipid in muscle (pg/mg muscle).</p></sec><sec><title>Statistical Analysis</title><p>Quantitative data were first analyzed for distribution of normality using Shapiro-Wilk normality test. Possible outliers were identified using the ROUT method, using the maximum Q of 1% for False Discovery Rate (FDR) (<xref rid=\"B63\" ref-type=\"bibr\">63</xref>). The effect of age and/or gender and/or genotype for quantitative parameters were analyzed by two-way ANOVA followed by Bonferroni correction. Values of <italic>p</italic> < 0.05 were considered statistically significant.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>Differences in Gross Anatomy and Skeletal Phenotype of 5LOKO Aged Mice</title><p>First, we investigated the impact of aging on body weight, RC length, and microtomographic parameters for skeletal phenotype in male and female 129/SvEv WT and 5LOKO mice (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>, <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Statistically significant differences for the body weight (grams) and RC length (cm) were found between genders in WT groups: higher values for young and aged male compared to female mice. Also, young 5LOKO male presented increased body weight compared to females, and aged 5LOKO male mice presented increased RC length compared to females. Considering differences between genotypes, aged 5LOKO males showed higher RC length compared to aged controls, while no differences were found between aged or young WT vs. 5LOKO females considering these parameters.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Macroscopic anatomy and skeletal phenotype of young and aged WT and 5LOKO mice: <bold>(A)</bold> Male and female, young (3 months old) and aged (18 months old) 129/SvEv WT and 5LOKO mice. <bold>(B)</bold> Axial and Coronal sections of L5 vertebrae, femur distal metaphysis, and mid-diaphysis. Images were obtained after scanning in Skyscan 1174 System (Skyscan, Kontich, Belgium). Projections were first reconstructed using the NRecon software and realigned in Data Viewer (Bruker, Kontich, Belgium) for 2D images.</p></caption><graphic xlink:href=\"fendo-11-00484-g0001\"/></fig><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Macroscopic features and skeletal phenotyping in WT vs. 5LOKO mice.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Parameters</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Sites/Groups</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Y WT ♂</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Y WT ♀</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>A WT ♂</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>A WT ♀</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Y KO ♂</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Y KO ♀</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>A KO ♂</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>A KO ♀</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BW (grams)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">27.97 ± 3.22<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>&</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">22.54 ± 2.36<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>&</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">29.06 ± 3.22<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>&</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">23.96 ± 3.58<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>&</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">25.90 ± 3.07<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>&</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">20.61 ± 2.00<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>&</sup></xref><xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">29.04 ± 2.59</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">26.07 ± 2.49<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RC (cm)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">–</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">9.07 ± 0.27<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>&</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8.43 ± 0.30<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>&</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8.66 ± 0.30<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>#</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8.51 ± 0.47</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">9.00 ± 0.23</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8.50 ± 0.16</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">9.37 ± 0.49<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>&</sup></xref><xref ref-type=\"table-fn\" rid=\"TN2\"><sup>#</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8.79 ± 0.42<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>&</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BV (mm<sup>3</sup>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Femur diaphysis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.70 ± 0.09</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.90 ± 0.80</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2.22 ± 0.31</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.62 ± 0.32</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.98 ± 0.22</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.59 ± 0.41</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.70 ± 0.26</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.74 ± 0.64</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BV/TV (%)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Femur metaphysis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">16.98 ± 5.12</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">23.99 ± 5.81<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">19.08 ± 2.87</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">12.30 ± 2.20<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref><xref ref-type=\"table-fn\" rid=\"TN2\"><sup>#</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">28.42 ± 7.00</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">27.91 ± 54.98</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">27.30 ± 3.92</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">21.00 ± 4.19<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>#</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">L5 vertebrae</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">35.88 ± 4.87</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">44.16 ± 4.35<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">33.68 ± 7.45</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">25.62 ± 6.65<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref><xref ref-type=\"table-fn\" rid=\"TN2\"><sup>#</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">40.08 ± 3.28</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">34.15 ± 5.25</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">42.63 ± 4.41</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">42.27 ± 7.06<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>#</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tb.Th (mm)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Femur metaphysis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.18 ± 0.01</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.19 ± 0.02</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.17 ± 0.02</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.16 ± 0.03</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.19 ± 0.03</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.19 ± 0.04</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.21 ± 0.06</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.21 ± 0.02</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">L5 vertebrae</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.09 ± 0.01</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.10 ± 0.01</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.10 ± 0.02</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.10 ± 0.03</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.10 ± 0.01</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.13 ± 0.02</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.12 ± 0.01</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.17 ± 0.03</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tb.Sp (mm)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Femur metaphysis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.46 ± 0.18</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.33 ± 0.07<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.53 ± 0.12</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.86 ± 0.39<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.33 ± 0.09</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.30 ± 0.06<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.31 ± 0.109<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>&</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.70 ± 0.07<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref><xref ref-type=\"table-fn\" rid=\"TN3\"><sup>&</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">L5 vertebrae</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.29 ± 0.03</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.24 ± 0.02</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.34 ± 0.06<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>#</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.32 ± 0.07<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>#</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.20 ± 0.03</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.21 ± 0.04</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.21 ± 0.01<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>#</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.17 ± 0.03<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>#</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Tb.N (1/mm)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Femur metaphysis</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.05 ± 0.16</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.18 ± 0.30</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.99 ± 0.15</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.61 ± 0.11</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.45 ± 0.12</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.67 ± 0.43<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.53 ± 0.32</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">0.85 ± 0.26<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">L5 vertebrae</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3.22 ± 0.25</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3.51 ± 0.34<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2.60 ± 0.66<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>&</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.28 ± 0.36<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref><sup><italic>#&</italic></sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3.96 ± 0.39</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3.15 ± 0.18</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3.07 ± 0.26</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2.18 ± 0.28<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>#</sup></xref></td></tr></tbody></table><table-wrap-foot><p><italic>Results are presented as the means (± SD) for each parameter</italic>.</p><p>In the comparison between columns, symbol</p><fn id=\"TN1\"><label>*</label><p>indicates comparison between columns: the effect of age (Y vs. A) in groups of same genotype and gender (e.g., Y ♀ WT vs. A ♀ WT); symbol</p></fn><fn id=\"TN2\"><label>#</label><p>indicates the effect of different genotypes (WT vs. KO) in groups of same age and gender (e.g., Y ♀ WT vs. Y ♀ KO); and symbol</p></fn><fn id=\"TN3\"><label>&</label><p><italic>indicates the effect of gender (♂ vs. ♀) in the same genotype and age (e.g., Y ♀ WT vs. Y ♂ WT). Statistically significant differences are indicated between groups with equal symbols (p < 0.05). Y = 3 months; A = 18 months. BW, Body weight; RC, rostrocaudal length</italic>.</p></fn></table-wrap-foot></table-wrap><p>We evaluated the cortical diaphysis of femur (BV) and morphological parameters in cancellous compartments of femur (distal metaphysis) and the L5-vertebral body. No statistical differences were found in the cortical diaphysis of femur, considering age, gender, or genotype (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>); although qualitative differences were observed in the transversal sections of femur diaphysis of aged WT females compared to aged WT males and aged 5LOKO females. Aged WT and aged 5LOKO females displayed increased trabecular spacing (Tb.Sp) in femur metaphysis compared to their young controls (<italic>p</italic> < 0.05), but presented no differences comparing both genotypes. The effect of age led to a reduced BV/TV in femur metaphysis of aged WT females compared to young controls (<italic>p</italic> < 0.05). In a comparison of genotypes, aged female WT mice presented a significantly reduced BV/TV in femur metaphysis in comparison to aged 5LOKO female mice. Major differences were also found in trabecular parameters of L5. The effect of age led to a reduced BV/TV and Tb.N in aged WT female compared to young controls. In comparison of genotypes, aged female WT mice presented a significantly reduced BV/TV and Tb.N in comparison to aged 5LOKO female mice. Both aged male and female WT mice presented an increased Tb.Sp compared to 5LOKO controls. In general, WT mice were most influenced by aging compared to 5LOKO (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p></sec><sec><title>Targeted Lipidomics of Skeletal Muscle</title><p>In order to analyze the endogenous quantities of eicosanoid lipid mediators relevant in the inflammatory process of naïve skeletal muscle, we determined the concentration (pg/mg) of PGE<sub>2</sub>, 11,12-EET, AA, EPA, and DHA (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). Considering the effect of age, WT mice presented a decrease in levels of DHA compared to their young controls, while aged 5LOKO mice presented an increase in this LM when compared with their young controls. Aged WT female mice presented an increase in levels of 11,12-EET compared to young WT female mice and compared to aged WT male mice. Considering the effect of genotype, the amounts of AA, DHA, and EPA were significantly decreased in aged female WT mice compared to aged female KO mice. Levels of 11,12-EET were significantly increased in aged 5LOKO male mice compared to aged WT male mice and also compared to aged female 5LOKO mice. Aged male WT mice also had a decrease in the levels of DHA and EPA compared to aged KO mice. Also, young and aged female WT mice had a decrease in the levels of PGE<sub>2</sub> comparing with 5LOKO matched controls. Considering the effect of gender, levels of PGE<sub>2</sub> were significantly increased in aged female KO mice, with significant differences compared to aged male KO mice.</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Targeted lipidomics of skeletal muscle in WT vs. 5LOKO mice.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Groups Lipids (pg/mg)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Y WT ♂</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Y WT ♀</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>A WT ♂</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>A WT ♀</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Y KO ♂</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Y KO ♀</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>A KO ♂</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>A KO ♀</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,136 ± 163</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,340 ± 177</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3,221 ± 193</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3,125 ± 370<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,652 ± 81</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,473 ± 633</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,481 ± 859</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5,288 ± 613<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>#</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PGE<sub>2</sub></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.8 ± 3.65</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.9 ± 2.26<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.77 ± 2.59</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.9 ± 3.06<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.23 ± 0.64<xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20.73 ± 15.22<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.9 ± 2.78<xref ref-type=\"table-fn\" rid=\"TN6\"><sup>&</sup></xref><xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">45.87 ± 29<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>#</sup></xref><xref ref-type=\"table-fn\" rid=\"TN4\"><sup>&</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">11,12-EET</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6 ± 1.37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8 ± 3.36</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 ± 0.70<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>#</sup></xref><xref ref-type=\"table-fn\" rid=\"TN6\"><sup>&</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10 ± 3.28<xref ref-type=\"table-fn\" rid=\"TN6\"><sup>&</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.90 ± 3.36<xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.26 ± 2.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23.8 ± 8.03<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>#</sup></xref><xref ref-type=\"table-fn\" rid=\"TN6\"><sup>&</sup></xref><xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.8 ± 1.66<xref ref-type=\"table-fn\" rid=\"TN6\"><sup>&</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DHA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,386 ± 847<xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,899 ± 769<xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6,078 ± 586<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>#</sup></xref><xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,333 ± 1,571<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>#</sup></xref><xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9,679 ± 643<xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11,580 ± 2133<xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17,006 ± 1408<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>#</sup></xref><xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15,659 ± 2128<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>#</sup></xref><xref ref-type=\"table-fn\" rid=\"TN4\"><sup>*</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">EPA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">197 ± 21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">211 ± 24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">189 ± 26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">172 ± 29<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">292 ± 23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">325 ± 65<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">391 ± 108</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">401 ± 106<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>#</sup></xref></td></tr></tbody></table><table-wrap-foot><p><italic>Results are presented as the means (±SD) for each lipid mediator</italic>.</p><p>In the comparison between columns, symbol</p><fn id=\"TN4\"><label>*</label><p>indicates comparison between columns: the effect of age (Y vs. A) in groups of same genotype and gender (e.g., Y ♀ WT vs. A ♀ WT); symbol</p></fn><fn id=\"TN5\"><label>#</label><p>indicates the effect of different genotypes (WT vs. KO) in groups of same age and gender (e.g., Y ♀ WT vs. Y ♀ KO); and symbol</p></fn><fn id=\"TN6\"><label>&</label><p><italic>indicates the effect of gender (♂ vs. ♀) in the same genotype and age (e.g., Y ♀ WT vs. Y ♂ WT). Statistically significant differences are indicated between groups with equal symbols (p < 0.05). Y = 3 months; A = 18 months</italic>.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Development of the Surgical Protocol</title><p>For developing the surgical MSK injury, we created a defect in the VA muscle (1-mm in diameter) of the right lower limb, followed by a subjacent monocortical defect (0.5 mm diameter) in the midshaft of femoral bone (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>). After animal anesthesia and preparation under the stereomicroscope, the time of surgical procedure and suture was no longer than 10 min. No fractures or other additional complications were observed following the surgeries. There was no evidence of weight loss, infection, and persistent inflammation in surgical sites. The subcutaneous sutures fell off 3–4 days post-surgery when the skin was clinically healed by day 7 post-surgery. The day after the surgery, animals were able to ambulate properly and presented no major signs of distress. Importantly, of the 80 animals used in this study, two aged 5LOKO female mice died immediately after the anesthesia by accidental excess of local anesthesia combined with sedation and were replaced by two other animals. No animals were lost due to the surgical procedure and/or during the recovery phase or up to the 14-day post-surgery period.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Establishment of the novel surgical bone-muscle model of simultaneous injury: <bold>(A)</bold> Images of the surgical model: I to IV—(I) <italic>vastus lateralis</italic> and femur anatomic localization in mouse and the schematic representation of muscle and bone surgical injury; (II) clinical aspect of muscle and bone injury; (III) 1-mm micro punch created a muscle defect to a depth of 1.5-mm until reaching the femoral bone; (IV) view of the subsequent defect induced by bone drilling using a 0.5-mm pilot drill at 600 rpm under constant irrigation. <bold>(B)</bold> Representative images for the immunolabeling of 5LO (arrows) in bone and muscle of young WT 7 days post-surgery (b), muscle (m), and periosteum (p) surrounding the bone defect. <bold>(C–F)</bold> MicroCT analysis: Femur containing bone defects were scanned with the microCT System (Skyscan 1174; Skyscan, Kontich, Belgium). <bold>(C)</bold> MicroCT 3D qualitative images were obtained from 7 to 14 days post-surgery in young and aged, male and female, and WT and 5LOKO mice. <bold>(D)</bold> ROI determined in the site of femur injury. <bold>(E,F)</bold> Bone Volume Fraction (BV/TV, %) was quantified at days 7 and 14 post-surgery, and results are presented as mean ± SD. Different letters indicate significant differences between young and aged mice for each sex and strain (<italic>p</italic> < 0.05).</p></caption><graphic xlink:href=\"fendo-11-00484-g0002\"/></fig></sec><sec><title>5LO Is Detected in WT Mice in Bone- and Muscle-Injured Tissues</title><p>Histological slides from young male WT mice at 7 days post-surgery were used for the immunolabeling of 5LO. 5LO-positive cells were found on the cytoplasm of inflammatory cells in the granulation tissue of bone healing sites. They were also found in cells of the connective tissue of the periosteum and in the endomysium of VA muscle (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). Results for negative immunolabeling of 5LO in 5LOKO mouse tissue are found in <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure 1</xref>.</p></sec><sec><title>Micro CT and Birefringence Analysis for Bone Healing</title><p>The proportion of mineralized bone was measured in sites of bone defect by Micro CT (<xref ref-type=\"fig\" rid=\"F2\">Figures 2C–F</xref>). The effect of age was observed in bone healing of all groups at 7 and 14 days post-surgery, with reduced BV/TV (%) in the site of bone defects (<xref ref-type=\"fig\" rid=\"F2\">Figures 2D,E</xref>). No significant differences were found for genders and genotypes. Considering the quality and quantity of the new collagen fibers in sites of bone healing, aged female 5LOKO mice presented a significant increase in red spectra fibers 7 days post-surgery compared to young controls (<xref ref-type=\"fig\" rid=\"F3\">Figures 3A,B</xref>). After 14 days post-injury, no significant differences were detected in the comparison among both young groups and aged groups at the same time point (<xref ref-type=\"fig\" rid=\"F3\">Figures 3A,B</xref>). Despite the difference pointed out at day 7 post-surgery in relation to the red fibers, total collagenous content did not show any significant differences among the groups in both time points (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>).</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Birefringence analysis of collagen fibers during bone healing in young and aged WT and 5LOKO mice. <bold>(A)</bold> Representative images of the birefringence analysis are seen under polarized light. In the last panel, 14 days post-surgery of aged animals, green, yellow and red fibers bundles are exemplified by arrows in each respective color. Green birefringence color indicates thin fibers, while yellow and red colors indicate thicker collagen fibers, as indicated by arrows and in the <bold>(B)</bold>. <bold>(B)</bold> Proportion of different thickness of collagen fibers (color spectrum) considering the total of collagen in each time point post-surgery (7 and 14 days post-surgery). Slides stained with Picrosirius red and captured at 10× were analyzed in Image-analysis software (AxioVision, v. 4.8, CarlZeiss). <bold>(C)</bold> Total area of collagen fibers (pixel<sup>2</sup>) considering the sum of each spectrum of birefringence. Results are presented as mean and SD of pixels<sup>2</sup> for each color in the birefringence analysis. Symbol * indicates statistically significant difference between young female KO and aged female KO (<italic>p</italic> < 0.05).</p></caption><graphic xlink:href=\"fendo-11-00484-g0003\"/></fig></sec><sec><title>Histopathological Analysis for Bone Healing</title><p>Our histopathological analyses showed that the bone injury site had been filled with woven bone in young female WT mice at day 7 post-surgery, characterized by a maturing process at day 14 post-surgery with osteoclastic resorption of the primary trabeculae and osteoblastic deposition of lamellar bone. In contrast, the aged female WT showed an intense leukocyte infiltration of granulation tissue in the bone defect area at day 7 post-surgery and the presence of primary bone at day 14 post-surgery, predominantly located at the defect walls. The effect of age was also observed in male WT mice. Young male WT mice presented with a similar healing process compared to the young female mice considering the same time points, while in the aged male WT mice, the defect was almost closed by maturing bone at day 14 post-surgery. In 5LOKO animals, female young mice presented a similar healing pattern to their male counterparts 7 days post-surgery, when the defect was filled with primary trabecular bone, and at day 14 post-surgery, when an advanced maturing process was observed by the presence of mature lamellar bone, similarly to the WT genotype. We noted that aged female 5LOKO mice healed the defect with a thin layer of maturing bone filling the bone defect at day 14 post-surgery, while in the aged 5LOKO male mice, it was mostly filled with mature trabecular bone. Comparing the genotypes, we observed major protective effects in aged female 5LOKO compared to aged WT mice, since aged 5LOKO presented an improved bone healing compared to aged WT females, with less inflammation at day 7 post-surgery a more mature bone at day 14 post-surgery in 5LOKO females (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>).</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Histopathological characterization of bone healing 7 and 14 days post-surgery: Male and female, young (3 months old), and aged (18 months old) 129/SvEv WT and 5LOKO mice underwent our surgical injury model, and bone specimens were evaluated at 7 and 14 days post-surgery. <bold>(A)</bold> Representative images from young and aged animals (WT and 5LOKO) stained with H&E and Goldner's Trichrome/Alcian Blue. Images were captured at 10× magnification. <bold>(B)</bold> H&E representative images from aged animals at 14 days were captured at 100× magnification. Newly formed bone (NB), Osteoclast (blue arrows), Osteoblast (arrowhead), Blood Vessel (BV).</p></caption><graphic xlink:href=\"fendo-11-00484-g0004\"/></fig></sec><sec><title>Histopathological and Histomorphometric Analysis for Skeletal Muscle Healing</title><p>The effects of age were most detrimental regarding the histopathological description of aged WT mice, compared to the controls in both genders. At day 7 post-surgery, a key observation in the site of muscle injury of young WT male mice was the presence of loose connective tissue with eventual mononuclear leukocytes, while aged male WT mice showed a mixture of connective and adipose tissue infiltrated by neutrophils. The same pattern was observed in the group of WT female animals, except that at day 7 post-surgery, the young female mice presented dense connective tissue at the site of muscle injury. In the comparison of different genotypes, 5LOKO young animals, both male and female, showed clear differences as compared to WT mice especially at day 7 post-surgery, when muscle cells with centralized myonuclei could be seen surrounded by connective tissue. However, both male and female aged 5LOKO mice exhibited an injury site filled by connective tissue. Only at day 14 post-surgery, some muscle cells were observed in the injury site in young male and female WT mice, while in the aged mice the site of injury showed a disorganized connective tissue with adipose cells and focal mononuclear and neutrophil leukocytes infiltrate. Nonetheless, at the site of muscle injury in 5LOKO young males, numerous muscle fibers with centralized myonuclei could be seen at day 14 post-surgery. The key difference we found in the site of injury in the aged male 5LOKO animals was that a higher amount of connective tissue could be observed surrounding the muscle fibers. At this same period, female 5LOKO young mice also showed some muscle fibers with centralized myonuclei, but connective tissue was predominant. Muscle fibers with centralized myonuclei were also seen in the defect of 5LOKO aged female mice, but a loose connective tissue highly infiltrated by neutrophils was noted surrounding the muscle fibers (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>). Non-inflammed connective tissue was also observed among muscle fibers in adjacent areas of the injured sites of both WT and 5LOKO female and male aged mice (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>).</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>Histopathological analysis during muscle healing in young and aged WT and 5LOKO mice: Male and female, young (3 months old), and aged (18 months old) 129/SvEv WT and 5LOKO mice underwent simultaneous surgical muscle and bone injury, and bone specimens were evaluated at days 7 and 14 post-surgery. <bold>(A)</bold> Representative images from the central area of muscle injury. Blue arrowheads indicate centralized myonuclei in the region of damage. <bold>(B)</bold> Representative images from the adjacent area of injury. Histological transversal sections were stained with H&E. Images were captured at 100× magnification. Green arrows indicate connective tissue among muscle fibers.</p></caption><graphic xlink:href=\"fendo-11-00484-g0005\"/></fig><p>From the histomorphometric analysis, the percentage of muscle inflammation did not present significant differences at day 7 post-surgery, but was significantly increased in all aged animals at day 14 post-surgery, evidencing the detrimental effect of aging in muscle healing of both genotypes and genders. When comparing the 5LOKO male and female groups with their matched WT strains, it was detected that muscle inflammation decreased in both male and female aged 5LOKO at day 14 post-surgery. We also determined that aged female WT mice was the only group that showed a significant decrease in muscle fiber CSA when compared to their young WT group at day 14 post-surgery. We also identified an increase in the number of centralized myonuclei in aged 5KOLO female mice between day 7 and 14 post-surgery (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>).</p><table-wrap id=\"T3\" position=\"float\"><label>Table 3</label><caption><p>Histomorphometry of injured muscles in WT vs. 5LOKO mice.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Parameters</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Groups/Periods</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Y WT ♂</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Y WT ♀</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>A WT ♂</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>A WT ♀</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Y KO ♂</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Y KO ♀</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>A KO ♂</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>A KO ♀</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Muscle Inflam (%)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7 days</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16 ± 5<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14 ± 2<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14 ± 5<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15 ± 10<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11 ± 3<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13 ± 4<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16 ± 4<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14 ± 7<sup>a</sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14 days</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14 ± 6<sup>a</sup><xref ref-type=\"table-fn\" rid=\"TN7\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18 ± 9<sup>a</sup><xref ref-type=\"table-fn\" rid=\"TN7\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">43 ± 8<sup>b</sup><xref ref-type=\"table-fn\" rid=\"TN7\"><sup>*</sup></xref><xref ref-type=\"table-fn\" rid=\"TN8\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">38 ± 7<sup>b</sup><xref ref-type=\"table-fn\" rid=\"TN7\"><sup>*</sup></xref><xref ref-type=\"table-fn\" rid=\"TN8\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10 ± 4<sup>a</sup><xref ref-type=\"table-fn\" rid=\"TN7\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11 ± 5<sup>a</sup><xref ref-type=\"table-fn\" rid=\"TN7\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25 ± 7<sup>b</sup><xref ref-type=\"table-fn\" rid=\"TN7\"><sup>*</sup></xref><xref ref-type=\"table-fn\" rid=\"TN8\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22 ± 4<sup>b</sup><xref ref-type=\"table-fn\" rid=\"TN7\"><sup>*</sup></xref><xref ref-type=\"table-fn\" rid=\"TN8\"><sup>#</sup></xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CSA (μm<sup>2</sup>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7 days</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,088 ± 661<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">977 ± 474<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">894 ± 515<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">955 ± 424<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,341 ± 715<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1021 ± 516<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">994 ± 199<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">751 ± 290<sup>a</sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14 days</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,298 ± 724<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,127 ± 446<sup>a</sup><xref ref-type=\"table-fn\" rid=\"TN7\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">739 ± 94<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">505 ± 101<sup>a</sup><xref ref-type=\"table-fn\" rid=\"TN7\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,451 ± 432<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,361 ± 530<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">869 ± 383<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">741 ± 319<sup>a</sup></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Centralized Myonuclei (%)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7 days</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24 ± 18<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26 ± 20<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20 ± 11<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19 ± 10<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">37 ± 13<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">34 ± 8<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26 ± 4<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24 ± 10<sup>a</sup></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14 days</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23 ± 16<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28 ± 17<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21 ± 18<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25 ± 11<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">39 ± 17<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">37 ± 14<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29 ± 15<sup>a</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">37 ± 12<sup>b</sup></td></tr></tbody></table><table-wrap-foot><p><italic>Results are presented as the means (±SD) for each parameter</italic>.</p><p>In the comparison between lines, different letters (a vs. b) indicate significant difference for time points (7 vs. 14 days post-surgery) of the same group in each evaluated parameter (p < 0.05). In the comparison between columns, symbol</p><fn id=\"TN7\"><label>*</label><p>indicates comparison between columns: the effect of age (Y vs. A) in groups of same genotype and gender (e.g., Y ♀ WT vs. A ♀ WT); symbol</p></fn><fn id=\"TN8\"><label>#</label><p><italic>indicates the effect of different genotypes (WT vs. KO) in groups of same age and gender (e.g., Y ♀ WT vs. Y ♀ KO). Statistically significant differences are indicated between groups with equal symbols (p < 0.05). Y = 3 months; A = 18 months. No statistical significant differences were found between genders (♂ vs. ♀) in the comparison of the same genotype and age (e.g., Y ♀ WT vs. Y ♂ WT)</italic>.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Immunolabeling and Histomorphometric Analysis of Muscle and Bone Markers of Remodeling</title><p>Immunolabeling for both MuRF1 and MyoD was performed at day 7 post-surgery and positive pixel quantification was performed. MuRF1 was predominant in muscle nuclei (<xref ref-type=\"fig\" rid=\"F6\">Figure 6AA'</xref>). The effect of age in MuRF1 immunolabeling (e.g., young male WT vs. aged male WT) was observed in both studied strains and genders, with a significant decrease in these markers for all aged animals (<xref ref-type=\"fig\" rid=\"F6\">Figure 6B</xref>). No significant differences were obtained for MuRF1 among the genotypes and genders (<xref ref-type=\"fig\" rid=\"F6\">Figure 6B</xref>). For MyoD, aged male and female WT mice showed a decreased immunolabeling for MyoD when compared to the matched gender young mice, but no effects of age were observed in 5LOKO mice (<xref ref-type=\"fig\" rid=\"F6\">Figure 6C</xref>). No significant differences were found considering the effect of gender and genotype.</p><fig id=\"F6\" position=\"float\"><label>Figure 6</label><caption><p>Histomorphometric characterization and Immunolabeling of MuRF1 and MyoD in WT and 5LOKO mice: <bold>(A)</bold> Representative images for MuRF1 immunostaining and MyoD immunostaining (arrows) in male young SvEv WT mice at 7 days post-surgery. DAB was used for the antigen–antibody reaction. Muscle samples were left without counterstaining to be used for optical density quantification. <bold>(A–A</bold><bold>′</bold><bold>)</bold> Positive Pixel quantification was performed by the software Aperio Image Scope v/ 12.3.3 (Leica Biosystems, Buffalo Grove, USA). Images were analyzed with algorithm Positive Pixel Count version 9 and are expressed as Positivity (number of positive pixels/number total). Negative controls (NC) were used for calibration and confirmation. <bold>(B,C)</bold> Results from positive pixel quantification for MuRF1 and MyoD were presented as means ± SD for each marker. Different letters indicate significant differences between young (3 months old) and aged (18 months old) mice for each sex and strain (<italic>p</italic> < 0.05).</p></caption><graphic xlink:href=\"fendo-11-00484-g0006\"/></fig><p>Dynamics of bone modeling/remodeling in bone-injured sites were evaluated with anti-Runx-2 and anti-TRAP antibodies (<xref ref-type=\"fig\" rid=\"F7\">Figure 7A</xref>). Considering the effect of age for Runx-2, a significant decreased in the area density of positive cells were found in aged 5LOKO male mice compared to young controls. Considering the effect of genotype, both male and female 5LOKO young mice demonstrated an increase in the area density of Runx-2+ cells compared to their matched WT controls at day 7 post-surgery (<xref ref-type=\"fig\" rid=\"F7\">Figure 7B</xref>). TRAP immunolabeling was significantly increased in the bone damage sites of female aged WT animals when compared to 5LOKO mice at day 14 post-surgery (<xref ref-type=\"fig\" rid=\"F7\">Figures 7A,C</xref>).</p><fig id=\"F7\" position=\"float\"><label>Figure 7</label><caption><p>Histomorphometric characterization and Immunolabeling of Runx-2 and TRAP in WT and 5LOKO mice: <bold>(A)</bold> Representative images for Runx2 immunostaining and TRAP immunostaining (arrows) of male and female, young (3 months old), and aged (18 months old) 129/SvEv WT and 5LOKO mice. Identification of antigen–antibody reaction was performed using DAB, counterstained with Mayer's hematoxylin. <bold>(B,C)</bold> Pixels quantification of Runx2 <bold>(B)</bold> and TRAP <bold>(C)</bold> was performed by the software Aperio Image Scope v/ 12.3.3 (Leica Biosystems, Buffalo Grove, USA). Images were analyzed with algorithm Positive Pixel Count version 9 and are expressed as positivity (umber of positive pixels/number total). Negative controls were used for calibration and confirmation. Results were presented as the mean and SD for each marker. Different letters indicate significant differences in between young (3 months old) and aged (18 months old) mice for each sex and strain (<italic>p</italic> < 0.05); symbol * indicates significant differences between WT vs. 5LOKO at the same age.</p></caption><graphic xlink:href=\"fendo-11-00484-g0007\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Aging and inflammation can negatively affect the MSK system of male and females in a different manner and proportion (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>, <xref rid=\"B64\" ref-type=\"bibr\">64</xref>, <xref rid=\"B65\" ref-type=\"bibr\">65</xref>). In our results for bone phenotyping, we found significant differences in macroscopic features comparing genders, with higher values (BW and/or RC length) for males compared to females in both genotypes. We observed major detrimental aging effects in female WT mice, with decreased in trabecular bone parameters (BV/TV and Tb.N) in L5 vertebral body compared to aged WT males.</p><p>Considering the genotype, aged 5LOKO mice also displayed a higher size compared to aged WT, but additional studies are necessary to observe the proportion of adipose tissue and muscles in mice lacking 5LOKO. A previous study has associated the 5LO gene (also called Alox5) as a candidate gene for obesity and low bone mass when 5LOKO (in the C57Bl/6 background) is subjected under long-term treatment with high fat diet (containing 45% fat by kcal and 3.0g of AA). Briefly, in this previous study, male and female 5LOKO mice under this treatment presented decrease in Tb.N compared to WT (at final age of 16 weeks, or ~ 4 months) (<xref rid=\"B66\" ref-type=\"bibr\">66</xref>). In our comparisons by microCT, we observed an increased bone quality in young and aged 5LOKO mice, compared to WT controls. It is also important to emphasize the differences in this particular methodology: 129Sv background, different ages (4 months for young and 18 months for aged mice) and a standard diet. In agreement, a previous study using radiology and bone histomorphometry has shown that both male and female 5LOKO mice, at 7 weeks age, from either 129Sv or mixed C57Bl/6x129sv background, have increased cortical bone compared with WT controls (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). Our study suggested that 5LO knockout protects against age-related bone loss in females, since the effect of aging on 5LOKO females was attenuated and they presented an increased BV/TV and Tb.N in femur and L5 compared to aged WT controls. It is also important to emphasize the differences in this particular methodology: different ages (4 months for young and 18 months for aged mice) and a standard diet.</p><p>It has been demonstrated that females have higher 5LO activity and/or higher LT production compared to males which could impact other pathways of the eicosanoid system (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>). Using a unique customized approach of targeted lipidomics in gastrocnemius muscles of young and aged C57Bl/6 mice, a previous study has demonstrated lipid signaling is age- and gender-dependent (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Our results from targeted lipidomics in gastrocnemius muscles demonstrate that the deletion of 5LO in mice significantly impact the profiling and quantities of lipid mediators in skeletal muscles of 5LOKO mice. In accordance, the endogenous levels of AA, EPA, and DHA were significantly decreased in aged female WT mice compared to aged female 5LOKO. Levels of 11,12-EET were significantly increased in aged 5LOKO male mice compared to aged WT mice. Aged male WT mice also presented a decrease in the levels of DHA and EPA compared to aged KO mice. Also, levels of PGE<sub>2</sub> were significantly increased in skeletal muscle of aged female KO mice compared to aged male KO mice. Considering the effect of gender, levels of PGE<sub>2</sub> were significantly increased while 11,12-EET was decreased in aged female KO mice, with significant differences compared to aged male KO mice. It is tempting to postulate that such combination of an elevation of PGE<sub>2</sub>, which was demonstrated at nanomolar levels to enhance myogenic differentiation of C2C12 mouse muscle cells and increase <italic>ex vivo</italic> contractile force (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B4\" ref-type=\"bibr\">4</xref>), and the decreased levels in 11,12-EET could combine in the female 5LOKO to promote improved MSK healing. Although AA-derived lipid mediators are known to play a major pro-inflammatory role, PGE<sub>2</sub> can have a dual role, modulating both pro- and anti-inflammatory responses (<xref rid=\"B67\" ref-type=\"bibr\">67</xref>). Furthermore, PGE<sub>2</sub> can accelerate myogenesis differentiation (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>) and induce osteogenesis (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B30\" ref-type=\"bibr\">30</xref>, <xref rid=\"B32\" ref-type=\"bibr\">32</xref>). EPA and DHA are involved in the lipid-mediator profile switching during resolution of inflammation. In this context, EPA-derived mediators have a low pro-inflammatory potential, whereas DHA is a major source for the final anti-inflammatory products D-series resolvins and protectins required for inflammation resolution without fibrosis (<xref rid=\"B68\" ref-type=\"bibr\">68</xref>–<xref rid=\"B70\" ref-type=\"bibr\">70</xref>). EET acids, such as 11,12-EET, are generated by the activity of cytochrome p450 (CYP) enzymes on AA (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>). 11,12-EET can play a modulatory role on COX-2 activity and attenuate the synthesis of PGE<sub>2</sub> during LPS-induced inflammatory response in rat monocytes (<xref rid=\"B72\" ref-type=\"bibr\">72</xref>). Other previous studies have shown that 11,12-EET can play anti-inflammatory activities by inhibition TNFα-induced VCAM-1, E-selectin, and ICAM-1 expression in endothelial cells (<xref rid=\"B73\" ref-type=\"bibr\">73</xref>). In our results, aged 5LOKO females presented an increased amount of PGE<sub>2</sub> compared to aged WT females, and conversely, aged 5LOKO females presented a decreased amount of 11,12-EET. An important consideration about the effects of LMs, myokines, osteokines, and secreted factors from bone-muscle and tissues in general is that we should apply accepted knowledge from endocrinology to this growing and fertile field of investigation.</p><p>To investigate the role of 5LO in the process of simultaneous bone and muscle healing, we developed a new surgical model of MSK injury in 5LOKO animals. Despite the importance of <italic>in vivo</italic> investigation for the comprehension of bone healing, previous rodent models of bone and muscle damage focus their attention exclusively on bone formation (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>, <xref rid=\"B74\" ref-type=\"bibr\">74</xref>), while the majority of studies of the on MSK healing addresses skeletal muscle (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>, <xref rid=\"B75\" ref-type=\"bibr\">75</xref>) or bone, but separately (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>, <xref rid=\"B39\" ref-type=\"bibr\">39</xref>). In our study, we performed a moderated simultaneous bone and muscle surgical damage, with a 1-mm diameter defect in muscle and a 0.5-mm diameter defect in the subjacent bone (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>), in order to avoid further surgical complications, such as femur fracture in aged animals. Our primary goal was to mirror human conditions of simultaneous tissue damage where recovery is feasible and to avoid any type of major catastrophic failure. Our results revealed a different pattern and timing in bone and muscle healing in WT mice. Interestingly, the bone defects of young male and female mice were completely filled with woven bone by day 7 post-surgery, and maturing by day 14 post-surgery, as previously demonstrated for bone healing in the tibia of mice, but in a non-simultaneous muscle defect model (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>). On the contrary, muscle healing is clearly delayed in the same animals. In the site of muscle injury of young male and female WT mice there was still connective tissue with eventual mononuclear leukocytes at 7 days post-surgery, with no significant changes in area density of inflammatory infiltrate at 14 days post-surgery. It is important to mention that bone has significant capacity for remodeling/regeneration regarding bone lining cells and/or osteoprogenitors lineages as stable cells, which are found in quiescent stage in physiologic conditions, but can undergo rapid proliferation in response to an injury (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). On the other hand, muscle fibers rapidly undergo degeneration after extensive damage (<xref rid=\"B77\" ref-type=\"bibr\">77</xref>, <xref rid=\"B78\" ref-type=\"bibr\">78</xref>), and damaged muscle cells can still be found after 6 days in mouse models of surgical injury (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). Simultaneously and in response to the mediators released to the site of injury, satellite cells can proliferate and differentiate in new myotubes followed by upregulation of MyoD (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). This early process was observed during the first 7 days in mouse models of muscle injury (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B78\" ref-type=\"bibr\">78</xref>). MuRF1 was used in this study as a marker for fiber degeneration as a consequence of the surgical injury, as previously described (<xref rid=\"B79\" ref-type=\"bibr\">79</xref>), while MyoD was used as a marker of new fibers. MyoD induces the expression of muscle-specific genes in myoblasts and rapid cell proliferation in models of crush-induced injury, but it may be undetectable in newly formed myotubes (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>). We detected both MuRF1 and MyoD 7 days post-injury. An intriguing possibility is that both the degradation of muscle cells and the burst in satellite cell activity and myoblast proliferation could lead to a surge of myokines near the bone damage, which in turn helps bone to accelerate regeneration, and it occurs with muscle flaps, the skeletal muscle functions to assist in the faster recovery of bone tissue.</p><p>Other previous models of combined bone and muscle injury have been done using models of volumetric muscle loss injury, and they are more common rats. Usually combined bone and muscle injury models in rodents involve volumetric muscle-loss injuries, followed by endogenously healing osteotomy (<xref rid=\"B80\" ref-type=\"bibr\">80</xref>) or severe open fractures (<xref rid=\"B81\" ref-type=\"bibr\">81</xref>–<xref rid=\"B83\" ref-type=\"bibr\">83</xref>) and non-endogenously healing femur defects (<xref rid=\"B84\" ref-type=\"bibr\">84</xref>). Due to the nature of trauma and embryologic origin of femur, bone fractures generally heal throughout a cartilaginous callus formation, previous to woven bone deposition (<xref rid=\"B80\" ref-type=\"bibr\">80</xref>), while in our mouse model the bone healing was noted to be predominantly driven by intramembranous healing. Together, these previous studies have also evidenced impaired bone healing related with concomitant muscle trauma and increased levels of inflammatory markers in sites of injury combined, compared to isolated injuries (<xref rid=\"B80\" ref-type=\"bibr\">80</xref>). When muscles are severely traumatized, the sustained inflammation diminishes mechanical bone strength and decreases mineralized matrix in the site of injury (<xref rid=\"B81\" ref-type=\"bibr\">81</xref>). Interestingly, these previous studies evaluated bone and muscle defects with or without combination, to permit additional comparisons and better determine the role of muscle in bone healing and <italic>vice versa</italic>. Comparatively, in our study we only produced combined injuries for primary study due the limited number of animals for studying 3 variables (two ages, genders, and genotypes). Additionally, our model does not aim to challenge bone and muscle healing with critical injuries, and it is performed in a very controlled manner, in order to avoid complications related to the surgical procedure. In this way, it is possible to investigate the MKS healing in compromised and fragile animals, such as aged mice.</p><p>Considering the advantages of our animal model, we decided by developing this combined defects in mice, considering a number of advantages and the cost–benefit related to mice when comparing with other rodents, such as the possibility to explore genetic approaches (e.g., 5LOKO mouse model utilized in this study); the reduced size compared to rats, which consequently reduces quantities of experimental drugs; and reduced experimental periods (<xref rid=\"B85\" ref-type=\"bibr\">85</xref>). In the context of bone–muscle crosstalk field, mouse is a suitable animal model for pharmacological and genetic interventions. Also, while timing of muscle and bone healing ranges from 4 to 12 weeks in rats, this type of MSK injury in mice was evaluated along 14 days. Even including longer time points, it might not exceed 4 weeks due the accelerated mice metabolism compared to other species. Comparing our model with preclinical orthopedic mice models (e.g., closed and open fractures), our study provides a surgical model that can be consistently reproduced with a rapid and simple surgery, minimal timing consuming, improving the animal recovery and avoid the risk of infections (<xref rid=\"B86\" ref-type=\"bibr\">86</xref>). On the other hand, since it is a non-critical muscle and bone defect, the present model does not challenge beyond physiological bone-muscle healing mechanisms in non-compromised animals (e.g., young adult and/or health). This is a major advantage in the context of normal aging.</p><p>During aging, the MSK system shows a decreased capacity for healing, associated with increased levels of inflammation (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B87\" ref-type=\"bibr\">87</xref>). Our results revealed a significant impairment on both bone and muscle healing in aged mice when compared to the young controls. The fraction of newly formed (BV/TV, %) bone analyzed by microCT was significantly decreased in all aged mice, considering both genotypes and genders (<xref ref-type=\"fig\" rid=\"F2\">Figures 2C–F</xref>). Despite increased amount of thicker collagen fibers in young 5LOKO females compared to aged controls, no major differences were found in the collagen content of bone matrix (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>). In skeletal muscles, a significant increase in inflammatory infiltrated in the VL muscles was noted at day 7 and 14 post-surgery (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). CSA was also significantly decreased in aged WT females compared to the young controls, while aged 5LOKO females maintained the CSA compared to young 5LOKO females (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). Additionally, while MuRF1 immunolabeling was increased in male and female aged mice (both genotypes), decreased immunolabeling for MyoD was observed to the matched young groups (for WT genotype), suggesting a reduced capacity for remodeling and regeneration along aging (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>). These observations confirm a detrimental effect of aging in all groups, but with the attenuated effect of the aging effect in 5LOKO mice, especially in histopathological comparisons between aged WT vs. aged 5LOKO females.</p><p>After 7 days of surgical trauma, injured muscle, periosteum, and bone of WT mice were assessed for 5LO expression (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). In support of 5LO roles on inflammation (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>), 5LO positive cells were predominantly found in the leucocytes infiltrating bone and muscle healing sites, but also were found in the constitutive cells of connective tissue from the periosteum. In agreement with our findings, a mouse model of femur closed-fracture healing has demonstrated strong positive immunolabeling of 5LO in bone marrow leukocytes, periosteum, osteoclast, muscle interstitial cells, and chondrocytes until 7 days post-injury (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). Given the limitations of immunohistochemistry techniques, future molecular investigation is necessary to explore 5LO in injured and non-injured bone and muscle cells by other approaches.</p><p>No previous studies have addressed the effects of 5LO or its metabolites in combined MSK injuries. We next evaluated the influence of 5LO on bone and muscle healing in different ages and gender, by comparing WT vs. 5LOKO. Isolated evaluation of bone injury using bone fracture healing models have previously demonstrated that genetic deletion of 5LO in mice (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>) or its pharmacological inhibition (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>, <xref rid=\"B40\" ref-type=\"bibr\">40</xref>) accelerate fracture callus formation in young male rodents. An important finding in our studies was that young female and male 5LOKO mice presented significantly increased Runx-2 expression in comparison with matched WT animals at day 7 post-injury, but with no significant differences in BV/TV of newly formed bone. Additionally, genetic deletion of 5LO in females led to attenuated inflammatory response after the simultaneous injury. While an intense inflammatory response in the bone-injury site of aged WT females 7 days post-injury with a delay in healing (14 days) was clear, aged 5LOKO presented less inflammatory infiltration and earlier mature bone (7 days) (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). TRAP-positive osteoclasts were also increased in aged females WT compared to 5LOKO at day 14 post-surgery (<xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref>), in agreement with the role of LTs on osteoclast formation in bone resorption and inflamed environments (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>, <xref rid=\"B35\" ref-type=\"bibr\">35</xref>). These results suggest that the 5LO signaling pathway may play a negative role on bone healing. Furthermore, young 5LOKO mice displayed advanced healing in VL muscle defects compared to WT mice, with centralized myonuclei surrounded by connective tissue while highly inflamed muscles could be seen in WT animals. Both male and female aged 5LOKO mice exhibited an injury site filled by connective tissue, while aged controls presented connective and adipose tissue (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). Interestingly, the number of positive MyoD cells was already decreased in young 5LOKO mice 7 days post-injury compared to WT (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>), but the 5LOKO presented a higher amount of centralized myonuclei indicative of muscle regeneration by day 14 post-surgery (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). Furthermore, MyoD positive cells were decreased in aged WT, compared to the young WT mice, but not in aged 5LOKO mice compared to young 5LOKO mice (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>). Future studies could conduct longitudinal studies to help further clarify the roles of 5LO in modulating the levels of muscle regulatory proteins.</p><p>In summary, our results demonstrate that 5LOKO mice present important differences in bone phenotyping and lipid profiling and quantification in skeletal muscles. Notably, the deletion of 5LO plays a protective role in bone of aged female 5LOKO mice and significantly impacts the capacity of MSK healing during aging. It is important to emphasize that this study presents some limitations, including detailed functional approaches and molecular quantification of 5LO and its products in both age and genders. However, our detailed lipidomic analyses, along with the bone phenotyping, allowed us to we hypothesize that changes found in skeletal muscle levels of LMs (particularly the intriguing aforementioned combination of elevation of PGE<sub>2</sub> and reduction of 11,12-EET) in 5LOKO may contribute to explain the improved MSK healing in these mice. Reinforcing this hypothesis, aged 5LOKO females mice presented increased amount of anti-inflammatory lipids (EPA and DHA) in comparison to aged WT females. We propose that future studies could investigate the levels of LMs in injured sites as well as expand the investigation on the role of 5LO in the MSK system, by using pharmacological and molecular-genetic approaches. The new model of simultaneous bone-muscle injury is pathophysiologically relevant for future studies in bone–muscle crosstalk, as well as suitable to advance the understanding of MSK healing during aging (<xref ref-type=\"fig\" rid=\"F8\">Figure 8</xref>). We finally suggest the 5LO signaling pathway as a potential target for interventions against post-menopausal osteoporosis.</p><fig id=\"F8\" position=\"float\"><label>Figure 8</label><caption><p>Graphical abstract of the new surgical protocol for MSK combined injury and methodologies performed to evaluate bone-muscle injury in mice. Other possibilities are suggested to investigate signaling molecules involved in bone and muscle crosstalk during tissue regeneration. CM, centralized myonuclei; V, blood vessels.</p></caption><graphic xlink:href=\"fendo-11-00484-g0008\"/></fig></sec><sec sec-type=\"conclusions\" id=\"s5\"><title>Conclusion</title><p>This study described a suitable mice model of simultaneous bone–muscle healing in order to contribute to the investigation of future bone–muscle crosstalk studies (<xref ref-type=\"fig\" rid=\"F8\">Figure 8</xref>), revealing that simultaneous bone–muscle healing was influenced by aging, gender, and the 5LO pathway. In general, 5LOKO aged female mice presented improved healing capacity when compared to their matched WT groups. Our lipidomic analyses elicited intriguing possibilities for the roles of lipid signaling mediators in skeletal aging and MSK healing. Our studies suggest that 5LO plays a crucial role in both modulation and resolution of inflammation during aging.</p></sec><sec sec-type=\"data-availability\" id=\"s6\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p></sec><sec id=\"s7\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by Ethic Committee on Animal Use of the Sagrado Coração University (CEUA/USC).</p></sec><sec id=\"s8\"><title>Author Contributions</title><p>CB and MM contributed to the conception and design, the acquisition, analysis, and interpretation, drafted the manuscript, critically revised the manuscript, gave final approval, and agreed to be accountable for all aspects of work. MB contributed to the analysis and interpretation, drafted the manuscript, critically revised the manuscript, gave final approval, and agreed to be accountable for all aspects of work. MC, ACS, JS, VR, ALS, and ZW contributed to the acquisition, analysis, and interpretation of the manuscript, critically revised the manuscript, gave final approval, and agreed to be accountable for all aspects of work. JA contributed to the conception and design, the interpretation and critically revised the manuscript, gave final approval, and agreed to be accountable for all aspects of work. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s9\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>The authors would like to thank Dr. Lynda Bonewald for her contributions in discussing with the authors important concepts and ideas that helped improve the quality of the manuscript, Rafael Ortiz for his excellent technical assistance with Image Scope Software, and Matthew Fiedler for English revision.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by grants #2018/08913-9 (CB and MM) from São Paulo Research Foundation (FAPESP). ACS was supported by FAPESP #2018/19406-0. MM was partially supported from FAPESP #13/04714-8. We are grateful for instrumentation support with microCT System from School Bauru of Dentistry, University of São Paulo and additional funding support from USP and UNESP. This work was supported in part by NIH-National Institutes of Aging (NIA) PO1 AG039355 (MB). CB and MB were partially supported by NIA R01AG056504 and R01AG060341 (MB), and the George W. and Hazel M. Jay and Evanston Research Endowments (MB). We are grateful for instrumentation support from Shimadzu Scientific Instruments, Inc.</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s10\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fendo.2020.00484/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fendo.2020.00484/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Data_Sheet_1.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brotto</surname><given-names>M</given-names></name><name><surname>Bonewald</surname><given-names>L</given-names></name></person-group>. <article-title>Bone and muscle: interactions beyond mechanical</article-title>. <source>Bone.</source> (<year>2015</year>) <volume>80</volume>:<fpage>109</fpage>–<lpage>14</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bone.2015.02.010</pub-id><pub-id pub-id-type=\"pmid\">26453500</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brotto</surname><given-names>M</given-names></name><name><surname>Johnson</surname><given-names>ML</given-names></name></person-group>. <article-title>Endocrine crosstalk between muscle and bone</article-title>. <source>Curr Osteoporos Rep.</source> (<year>2014</year>) <volume>12</volume>:<fpage>135</fpage>–<lpage>41</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s11914-014-0209-0</pub-id><pub-id pub-id-type=\"pmid\">24667990</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>J</given-names></name><name><surname>Romero-Suarez</surname><given-names>S</given-names></name><name><surname>Lara</surname><given-names>N</given-names></name><name><surname>Mo</surname><given-names>C</given-names></name><name><surname>Kaja</surname><given-names>S</given-names></name><name><surname>Brotto</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Crosstalk between MLO-Y4 osteocytes and C2C12 muscle cells is mediated by the Wnt/beta-catenin pathway</article-title>. <source>JBMR Plus.</source> (<year>2017</year>) <volume>1</volume>:<fpage>86</fpage>–<lpage>100</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jbm4.10015</pub-id><pub-id pub-id-type=\"pmid\">29104955</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mo</surname><given-names>C</given-names></name><name><surname>Zhao</surname><given-names>R</given-names></name><name><surname>Vallejo</surname><given-names>J</given-names></name><name><surname>Igwe</surname><given-names>O</given-names></name><name><surname>Bonewald</surname><given-names>L</given-names></name><name><surname>Wetmore</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Prostaglandin E2 promotes proliferation of skeletal muscle myoblasts via EP4 receptor activation</article-title>. <source>Cell Cycle.</source> (<year>2015</year>) <volume>14</volume>:<fpage>1507</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.1080/15384101.2015.1026520</pub-id><pub-id pub-id-type=\"pmid\">25785867</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Frontera</surname><given-names>WR</given-names></name><name><surname>Ochala</surname><given-names>J</given-names></name></person-group>. <article-title>Skeletal muscle: a brief review of structure and function</article-title>. <source>Calcif Tissue Int.</source> (<year>2015</year>) <volume>96</volume>:<fpage>183</fpage>–<lpage>95</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00223-014-9915-y</pub-id><pub-id pub-id-type=\"pmid\">25294644</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Matsakas</surname><given-names>A</given-names></name><name><surname>Patel</surname><given-names>K</given-names></name></person-group>. <article-title>Skeletal muscle fibre plasticity in response to selected environmental and physiological stimuli</article-title>. <source>Histol Histopathol.</source> (<year>2009</year>) <volume>24</volume>:<fpage>611</fpage>–<lpage>29</lpage>. <pub-id pub-id-type=\"doi\">10.14670/HH-24.611</pub-id><pub-id pub-id-type=\"pmid\">19283669</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Snijders</surname><given-names>T</given-names></name><name><surname>Nederveen</surname><given-names>JP</given-names></name><name><surname>McKay</surname><given-names>BR</given-names></name><name><surname>Joanisse</surname><given-names>S</given-names></name><name><surname>Verdijk</surname><given-names>LB</given-names></name><name><surname>van Loon</surname><given-names>LJC</given-names></name></person-group>. <article-title>Satellite cells in human skeletal muscle plasticity</article-title>. <source>Front Physiol.</source> (<year>2015</year>) <volume>6</volume>:<fpage>283</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fphys.2015.00283</pub-id><pub-id pub-id-type=\"pmid\">26557092</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Charge</surname><given-names>SB</given-names></name><name><surname>Rudnicki</surname><given-names>MA</given-names></name></person-group>. <article-title>Cellular and molecular regulation of muscle regeneration</article-title>. <source>Physiol Rev.</source> (<year>2004</year>) <volume>84</volume>:<fpage>209</fpage>–<lpage>38</lpage>. <pub-id pub-id-type=\"doi\">10.1152/physrev.00019.2003</pub-id><pub-id pub-id-type=\"pmid\">14715915</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Howard</surname><given-names>EE</given-names></name><name><surname>Pasiakos</surname><given-names>SM</given-names></name><name><surname>Blesso</surname><given-names>CN</given-names></name><name><surname>Fussell</surname><given-names>MA</given-names></name><name><surname>Rodriguez</surname><given-names>NR</given-names></name></person-group>. <article-title>Divergent roles of inflammation in skeletal muscle recovery from injury</article-title>. <source>Front Physiol.</source> (<year>2020</year>) <volume>11</volume>:<fpage>87</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fphys.2020.00087</pub-id><pub-id pub-id-type=\"pmid\">32116792</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schaap</surname><given-names>LA</given-names></name><name><surname>Pluijm</surname><given-names>SM</given-names></name><name><surname>Deeg</surname><given-names>DJ</given-names></name><name><surname>Harris</surname><given-names>TB</given-names></name><name><surname>Kritchevsky</surname><given-names>SB</given-names></name><name><surname>Newman</surname><given-names>AB</given-names></name><etal/></person-group>. <article-title>Higher inflammatory marker levels in older persons: associations with 5-year change in muscle mass and muscle strength</article-title>. <source>J Gerontol A Biol Sci Med Sci.</source> (<year>2009</year>) <volume>64</volume>:<fpage>1183</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1093/gerona/glp097</pub-id><pub-id pub-id-type=\"pmid\">19622801</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gibon</surname><given-names>E</given-names></name><name><surname>Lu</surname><given-names>L</given-names></name><name><surname>Goodman</surname><given-names>SB</given-names></name></person-group>. <article-title>Aging, inflammation, stem cells, and bone healing</article-title>. <source>Stem Cell Res Ther.</source> (<year>2016</year>) <volume>7</volume>:<fpage>44</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s13287-016-0300-9</pub-id><pub-id pub-id-type=\"pmid\">27006071</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>Z</given-names></name><name><surname>Bian</surname><given-names>L</given-names></name><name><surname>Mo</surname><given-names>C</given-names></name><name><surname>Kukula</surname><given-names>M</given-names></name><name><surname>Schug</surname><given-names>KA</given-names></name><name><surname>Brotto</surname><given-names>M</given-names></name></person-group>. <article-title>Targeted quantification of lipid mediators in skeletal muscles using restricted access media-based trap-and-elute liquid chromatography-mass spectrometry</article-title>. <source>Anal Chim Acta.</source> (<year>2017</year>) <volume>984</volume>:<fpage>151</fpage>–<lpage>61</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.aca.2017.07.024</pub-id><pub-id pub-id-type=\"pmid\">28843558</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schindeler</surname><given-names>A</given-names></name><name><surname>Liu</surname><given-names>R</given-names></name><name><surname>Little</surname><given-names>DG</given-names></name></person-group>. <article-title>The contribution of different cell lineages to bone repair: exploring a role for muscle stem cells</article-title>. <source>Differentiation.</source> (<year>2009</year>) <volume>77</volume>:<fpage>12</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.diff.2008.09.007</pub-id><pub-id pub-id-type=\"pmid\">19281760</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Utvag</surname><given-names>SE</given-names></name><name><surname>Iversen</surname><given-names>KB</given-names></name><name><surname>Grundnes</surname><given-names>O</given-names></name><name><surname>Reikeras</surname><given-names>O</given-names></name></person-group>. <article-title>Poor muscle coverage delays fracture healing in rats</article-title>. <source>Acta Orthop Scand.</source> (<year>2002</year>) <volume>73</volume>:<fpage>471</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1080/00016470216315</pub-id><pub-id pub-id-type=\"pmid\">12358124</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>R</given-names></name><name><surname>Schindeler</surname><given-names>A</given-names></name><name><surname>Little</surname><given-names>DG</given-names></name></person-group>\n<article-title>The potential role of muscle in bone repair</article-title>. <source>J Musculoskelet Neuronal Interact.</source> (<year>2010</year>) <volume>10</volume>:<fpage>71</fpage>–<lpage>6</lpage>.<pub-id pub-id-type=\"pmid\">20190382</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chan</surname><given-names>JK</given-names></name><name><surname>Harry</surname><given-names>L</given-names></name><name><surname>Williams</surname><given-names>G</given-names></name><name><surname>Nanchahal</surname><given-names>J</given-names></name></person-group>. <article-title>Soft-tissue reconstruction of open fractures of the lower limb: muscle versus fasciocutaneous flaps</article-title>. <source>Plast Reconstr Surg.</source> (<year>2012</year>) <volume>130</volume>:<fpage>284e</fpage>−<lpage>95</lpage>. <pub-id pub-id-type=\"doi\">10.1097/PRS.0b013e3182589e63</pub-id><pub-id pub-id-type=\"pmid\">22842425</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fuchtbauer</surname><given-names>EM</given-names></name><name><surname>Westphal</surname><given-names>H</given-names></name></person-group>. <article-title>MyoD and myogenin are coexpressed in regenerating skeletal muscle of the mouse</article-title>. <source>Dev Dyn.</source> (<year>1992</year>) <volume>193</volume>:<fpage>34</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1002/aja.1001930106</pub-id><pub-id pub-id-type=\"pmid\">1311614</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weintraub</surname><given-names>H</given-names></name><name><surname>Davis</surname><given-names>R</given-names></name><name><surname>Tapscott</surname><given-names>S</given-names></name><name><surname>Thayer</surname><given-names>M</given-names></name><name><surname>Krause</surname><given-names>M</given-names></name><name><surname>Benezra</surname><given-names>R</given-names></name><etal/></person-group>. <article-title>The myoD gene family: nodal point during specification of the muscle cell lineage</article-title>. <source>Science.</source> (<year>1991</year>) <volume>251</volume>:<fpage>761</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1126/science.1846704</pub-id><pub-id pub-id-type=\"pmid\">1846704</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Koyama</surname><given-names>S</given-names></name><name><surname>Hata</surname><given-names>S</given-names></name><name><surname>Witt</surname><given-names>CC</given-names></name><name><surname>Ono</surname><given-names>Y</given-names></name><name><surname>Lerche</surname><given-names>S</given-names></name><name><surname>Ojima</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Muscle RING-finger protein-1 (MuRF1) as a connector of muscle energy metabolism and protein synthesis</article-title>. <source>J Mol Biol.</source> (<year>2008</year>) <volume>376</volume>:<fpage>1224</fpage>–<lpage>36</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jmb.2007.11.049</pub-id><pub-id pub-id-type=\"pmid\">18222470</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Florencio-Silva</surname><given-names>R</given-names></name><name><surname>Sasso</surname><given-names>GR</given-names></name><name><surname>Sasso-Cerri</surname><given-names>E</given-names></name><name><surname>Simoes</surname><given-names>MJ</given-names></name><name><surname>Cerri</surname><given-names>PS</given-names></name></person-group>. <article-title>Biology of bone tissue: structure, function, and factors that influence bone cells</article-title>. <source>Biomed Res Int.</source> (<year>2015</year>) <volume>2015</volume>:<fpage>421746</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2015/421746</pub-id><pub-id pub-id-type=\"pmid\">26247020</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Biguetti</surname><given-names>CC</given-names></name><name><surname>Vieira</surname><given-names>AE</given-names></name><name><surname>Cavalla</surname><given-names>F</given-names></name><name><surname>Fonseca</surname><given-names>AC</given-names></name><name><surname>Colavite</surname><given-names>PM</given-names></name><name><surname>Silva</surname><given-names>RM</given-names></name><etal/></person-group>. <article-title>CCR2 contributes to F4/80+ cells migration along intramembranous bone healing in maxilla, but its deficiency does not critically affect the healing outcome</article-title>. <source>Front Immunol.</source> (<year>2018</year>) <volume>9</volume>:<fpage>1804</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2018.01804</pub-id><pub-id pub-id-type=\"pmid\">30147688</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gharaibeh</surname><given-names>B</given-names></name><name><surname>Chun-Lansinger</surname><given-names>Y</given-names></name><name><surname>Hagen</surname><given-names>T</given-names></name><name><surname>Ingham</surname><given-names>SJ</given-names></name><name><surname>Wright</surname><given-names>V</given-names></name><name><surname>Fu</surname><given-names>F</given-names></name><etal/></person-group>. <article-title>Biological approaches to improve skeletal muscle healing after injury and disease</article-title>. <source>Birth Defects Res C Embryo Today.</source> (<year>2012</year>) <volume>96</volume>:<fpage>82</fpage>–<lpage>94</lpage>. <pub-id pub-id-type=\"doi\">10.1002/bdrc.21005</pub-id><pub-id pub-id-type=\"pmid\">22457179</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>JT</given-names></name><name><surname>Kasukonis</surname><given-names>B</given-names></name><name><surname>Dunlap</surname><given-names>G</given-names></name><name><surname>Perry</surname><given-names>R</given-names></name><name><surname>Washington</surname><given-names>T</given-names></name><name><surname>Wolchok</surname><given-names>JC</given-names></name></person-group>. <article-title>Regenerative repair of volumetric muscle loss injury is sensitive to age</article-title>. <source>Tissue Eng A.</source> (<year>2020</year>) <volume>26</volume>:<fpage>3</fpage>–<lpage>14</lpage>. <pub-id pub-id-type=\"doi\">10.1089/ten.tea.2019.0034</pub-id><pub-id pub-id-type=\"pmid\">31064280</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Loi</surname><given-names>F</given-names></name><name><surname>Cordova</surname><given-names>LA</given-names></name><name><surname>Pajarinen</surname><given-names>J</given-names></name><name><surname>Lin</surname><given-names>TH</given-names></name><name><surname>Yao</surname><given-names>Z</given-names></name><name><surname>Goodman</surname><given-names>SB</given-names></name></person-group>. <article-title>Inflammation, fracture and bone repair</article-title>. <source>Bone.</source> (<year>2016</year>) <volume>86</volume>:<fpage>19</fpage>–<lpage>30</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bone.2016.02.020</pub-id><pub-id pub-id-type=\"pmid\">26946132</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bennett</surname><given-names>M</given-names></name><name><surname>Gilroy</surname><given-names>DW</given-names></name></person-group>\n<article-title>Lipid mediators in inflammation</article-title>. <source>Microbiol Spectr.</source> (<year>2016</year>) <volume>4</volume>:<fpage>343</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1128/microbiolspec.MCHD-0035-2016</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Radmark</surname><given-names>O</given-names></name><name><surname>Werz</surname><given-names>O</given-names></name><name><surname>Steinhilber</surname><given-names>D</given-names></name><name><surname>Samuelsson</surname><given-names>B</given-names></name></person-group>. <article-title>5-Lipoxygenase, a key enzyme for leukotriene biosynthesis in health and disease</article-title>. <source>Biochim Biophys Acta.</source> (<year>2015</year>) <volume>1851</volume>:<fpage>331</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbalip.2014.08.012</pub-id><pub-id pub-id-type=\"pmid\">25152163</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lin</surname><given-names>HN</given-names></name><name><surname>O'Connor</surname><given-names>JP</given-names></name></person-group>. <article-title>Immunohistochemical localization of key arachidonic acid metabolism enzymes during fracture healing in mice</article-title>. <source>PLoS ONE.</source> (<year>2014</year>) <volume>9</volume>:<fpage>e88423</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0088423</pub-id><pub-id pub-id-type=\"pmid\">24516658</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manigrasso</surname><given-names>MB</given-names></name><name><surname>O'Connor</surname><given-names>JP</given-names></name></person-group>. <article-title>Accelerated fracture healing in mice lacking the 5-lipoxygenase gene</article-title>. <source>Acta Orthop.</source> (<year>2010</year>) <volume>81</volume>:<fpage>748</fpage>–<lpage>55</lpage>. <pub-id pub-id-type=\"doi\">10.3109/17453674.2010.533931</pub-id><pub-id pub-id-type=\"pmid\">21067431</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martel-Pelletier</surname><given-names>J</given-names></name><name><surname>Mineau</surname><given-names>F</given-names></name><name><surname>Fahmi</surname><given-names>H</given-names></name><name><surname>Laufer</surname><given-names>S</given-names></name><name><surname>Reboul</surname><given-names>P</given-names></name><name><surname>Boileau</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>Regulation of the expression of 5-lipoxygenase-activating protein/5-lipoxygenase and the synthesis of leukotriene B(4) in osteoarthritic chondrocytes: role of transforming growth factor beta and eicosanoids</article-title>. <source>Arthritis Rheum.</source> (<year>2004</year>) <volume>50</volume>:<fpage>3925</fpage>–<lpage>33</lpage>. <pub-id pub-id-type=\"doi\">10.1002/art.20632</pub-id><pub-id pub-id-type=\"pmid\">15593193</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>O'Connor</surname><given-names>JP</given-names></name><name><surname>Manigrasso</surname><given-names>MB</given-names></name><name><surname>Kim</surname><given-names>BD</given-names></name><name><surname>Subramanian</surname><given-names>S</given-names></name></person-group>. <article-title>Fracture healing and lipid mediators</article-title>. <source>Bonekey Rep.</source> (<year>2014</year>) <volume>3</volume>:<fpage>517</fpage>. <pub-id pub-id-type=\"doi\">10.1038/bonekey.2014.12</pub-id><pub-id pub-id-type=\"pmid\">24795811</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tagliaferri</surname><given-names>C</given-names></name><name><surname>Wittrant</surname><given-names>Y</given-names></name><name><surname>Davicco</surname><given-names>MJ</given-names></name><name><surname>Walrand</surname><given-names>S</given-names></name><name><surname>Coxam</surname><given-names>V</given-names></name></person-group>. <article-title>Muscle and bone, two interconnected tissues</article-title>. <source>Ageing Res Rev.</source> (<year>2015</year>) <volume>21</volume>:<fpage>55</fpage>–<lpage>70</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.arr.2015.03.002</pub-id><pub-id pub-id-type=\"pmid\">25804855</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Simon</surname><given-names>AM</given-names></name><name><surname>O'Connor</surname><given-names>JP</given-names></name></person-group>. <article-title>Dose and time-dependent effects of cyclooxygenase-2 inhibition on fracture-healing</article-title>. <source>J Bone Joint Surg Am.</source> (<year>2007</year>) <volume>89</volume>:<fpage>500</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.2106/JBJS.F.00127</pub-id><pub-id pub-id-type=\"pmid\">17332098</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xie</surname><given-names>C</given-names></name><name><surname>Liang</surname><given-names>B</given-names></name><name><surname>Xue</surname><given-names>M</given-names></name><name><surname>Lin</surname><given-names>AS</given-names></name><name><surname>Loiselle</surname><given-names>A</given-names></name><name><surname>Schwarz</surname><given-names>EM</given-names></name><etal/></person-group>. <article-title>Rescue of impaired fracture healing in COX-2-/- mice via activation of prostaglandin E2 receptor subtype 4</article-title>. <source>Am J Pathol.</source> (<year>2009</year>) <volume>175</volume>:<fpage>772</fpage>–<lpage>85</lpage>. <pub-id pub-id-type=\"doi\">10.2353/ajpath.2009.081099</pub-id><pub-id pub-id-type=\"pmid\">19628768</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Garcia</surname><given-names>C</given-names></name><name><surname>Boyce</surname><given-names>BF</given-names></name><name><surname>Gilles</surname><given-names>J</given-names></name><name><surname>Dallas</surname><given-names>M</given-names></name><name><surname>Qiao</surname><given-names>M</given-names></name><name><surname>Mundy</surname><given-names>GR</given-names></name><etal/></person-group>. <article-title>Leukotriene B4 stimulates osteoclastic bone resorption both <italic>in vitro</italic> and <italic>in vivo</italic></article-title>. <source>J Bone Miner Res.</source> (<year>1996</year>) <volume>11</volume>:<fpage>1619</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jbmr.5650111105</pub-id><pub-id pub-id-type=\"pmid\">8915769</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moura</surname><given-names>AP</given-names></name><name><surname>Taddei</surname><given-names>SR</given-names></name><name><surname>Queiroz-Junior</surname><given-names>CM</given-names></name><name><surname>Madeira</surname><given-names>MF</given-names></name><name><surname>Rodrigues</surname><given-names>LF</given-names></name><name><surname>Garlet</surname><given-names>GP</given-names></name><etal/></person-group>. <article-title>The relevance of leukotrienes for bone resorption induced by mechanical loading</article-title>. <source>Bone.</source> (<year>2014</year>) <volume>69</volume>:<fpage>133</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bone.2014.09.019</pub-id><pub-id pub-id-type=\"pmid\">25270168</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bonewald</surname><given-names>LF</given-names></name><name><surname>Flynn</surname><given-names>M</given-names></name><name><surname>Qiao</surname><given-names>M</given-names></name><name><surname>Dallas</surname><given-names>MR</given-names></name><name><surname>Mundy</surname><given-names>GR</given-names></name><name><surname>Boyce</surname><given-names>BF</given-names></name></person-group>. <article-title>Mice lacking 5-lipoxygenase have increased cortical bone thickness</article-title>. <source>Adv Exp Med Biol.</source> (<year>1997</year>) <volume>433</volume>:<fpage>299</fpage>–<lpage>302</lpage>. <pub-id pub-id-type=\"doi\">10.1007/978-1-4899-1810-9_63</pub-id><pub-id pub-id-type=\"pmid\">9561156</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Traianedes</surname><given-names>K</given-names></name><name><surname>Dallas</surname><given-names>MR</given-names></name><name><surname>Garrett</surname><given-names>IR</given-names></name><name><surname>Mundy</surname><given-names>GR</given-names></name><name><surname>Bonewald</surname><given-names>L</given-names></name></person-group>. <article-title>5-Lipoxygenase metabolites inhibit bone formation <italic>in vitro</italic></article-title>. <source>Endocrinology</source>. (<year>1998</year>) <volume>139</volume>:<fpage>3178</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.1210/endo.139.7.6115</pub-id><pub-id pub-id-type=\"pmid\">9645691</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lopes</surname><given-names>DEM</given-names></name><name><surname>Jabr</surname><given-names>CL</given-names></name><name><surname>Dejani</surname><given-names>NN</given-names></name><name><surname>Saraiva</surname><given-names>AC</given-names></name><name><surname>Aquino</surname><given-names>SG</given-names></name><name><surname>Medeiros</surname><given-names>A</given-names></name><etal/></person-group>\n<article-title>Inhibition of 5-Lipoxygenase (5-Lo) attenuates inflammation and bone resorption in Lipopolysaccharide (Lps)-induced periodontal disease</article-title>. <source>J Periodontol.</source> (<year>2017</year>) <volume>91</volume>:<fpage>1</fpage>–<lpage>18</lpage>. <pub-id pub-id-type=\"doi\">10.1902/jop.2017.170210</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cottrell</surname><given-names>JA</given-names></name><name><surname>O'Connor</surname><given-names>JP</given-names></name></person-group>. <article-title>Pharmacological inhibition of 5-lipoxygenase accelerates and enhances fracture-healing</article-title>. <source>J Bone Joint Surg Am.</source> (<year>2009</year>) <volume>91</volume>:<fpage>2653</fpage>–<lpage>65</lpage>. <pub-id pub-id-type=\"doi\">10.2106/JBJS.H.01844</pub-id><pub-id pub-id-type=\"pmid\">19884440</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wixted</surname><given-names>JJ</given-names></name><name><surname>Fanning</surname><given-names>PJ</given-names></name><name><surname>Gaur</surname><given-names>T</given-names></name><name><surname>O'Connell</surname><given-names>SL</given-names></name><name><surname>Silva</surname><given-names>J</given-names></name><name><surname>Mason-Savas</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Enhanced fracture repair by leukotriene antagonism is characterized by increased chondrocyte proliferation and early bone formation: a novel role of the cysteinyl LT-1 receptor</article-title>. <source>J Cell Physiol.</source> (<year>2009</year>) <volume>221</volume>:<fpage>31</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jcp.21809</pub-id><pub-id pub-id-type=\"pmid\">19544365</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sun</surname><given-names>R</given-names></name><name><surname>Ba</surname><given-names>X</given-names></name><name><surname>Cui</surname><given-names>L</given-names></name><name><surname>Xue</surname><given-names>Y</given-names></name><name><surname>Zeng</surname><given-names>X</given-names></name></person-group>. <article-title>Leukotriene B4 regulates proliferation and differentiation of cultured rat myoblasts via the BLT1 pathway</article-title>. <source>Mol Cells.</source> (<year>2009</year>) <volume>27</volume>:<fpage>403</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s10059-009-0053-8</pub-id><pub-id pub-id-type=\"pmid\">19390820</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zuo</surname><given-names>L</given-names></name><name><surname>Christofi</surname><given-names>FL</given-names></name><name><surname>Wright</surname><given-names>VP</given-names></name><name><surname>Bao</surname><given-names>S</given-names></name><name><surname>Clanton</surname><given-names>TL</given-names></name></person-group>. <article-title>Lipoxygenase-dependent superoxide release in skeletal muscle</article-title>. <source>J Appl Physiol.</source> (<year>1985</year>) <volume>97</volume>:<fpage>661</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1152/japplphysiol.00096.2004</pub-id><pub-id pub-id-type=\"pmid\">15107407</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Loell</surname><given-names>I</given-names></name><name><surname>Alemo Munters</surname><given-names>L</given-names></name><name><surname>Pandya</surname><given-names>J</given-names></name><name><surname>Zong</surname><given-names>M</given-names></name><name><surname>Alexanderson</surname><given-names>H</given-names></name><name><surname>Fasth</surname><given-names>AE</given-names></name><etal/></person-group>\n<article-title>Activated LTB4 pathway in muscle tissue of patients with polymyositis or dermatomyositis</article-title>. <source>Ann Rheum Dis.</source> (<year>2013</year>) <volume>72</volume>:<fpage>293</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1136/annrheumdis-2012-201294</pub-id><pub-id pub-id-type=\"pmid\">22736094</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pergola</surname><given-names>C</given-names></name><name><surname>Dodt</surname><given-names>G</given-names></name><name><surname>Rossi</surname><given-names>A</given-names></name><name><surname>Neunhoeffer</surname><given-names>E</given-names></name><name><surname>Lawrenz</surname><given-names>B</given-names></name><name><surname>Northoff</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>ERK-mediated regulation of leukotriene biosynthesis by androgens: a molecular basis for gender differences in inflammation and asthma</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2008</year>) <volume>105</volume>:<fpage>19881</fpage>–<lpage>86</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.0809120105</pub-id><pub-id pub-id-type=\"pmid\">19064924</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pergola</surname><given-names>C</given-names></name><name><surname>Rogge</surname><given-names>A</given-names></name><name><surname>Dodt</surname><given-names>G</given-names></name><name><surname>Northoff</surname><given-names>H</given-names></name><name><surname>Weinigel</surname><given-names>C</given-names></name><name><surname>Barz</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Testosterone suppresses phospholipase D, causing sex differences in leukotriene biosynthesis in human monocytes</article-title>. <source>FASEB J.</source> (<year>2011</year>) <volume>25</volume>:<fpage>3377</fpage>–<lpage>87</lpage>. <pub-id pub-id-type=\"doi\">10.1096/fj.11-182758</pub-id><pub-id pub-id-type=\"pmid\">21693622</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pace</surname><given-names>S</given-names></name><name><surname>Pergola</surname><given-names>C</given-names></name><name><surname>Dehm</surname><given-names>F</given-names></name><name><surname>Rossi</surname><given-names>A</given-names></name><name><surname>Gerstmeier</surname><given-names>J</given-names></name><name><surname>Troisi</surname><given-names>F</given-names></name><etal/></person-group>. <article-title>Androgen-mediated sex bias impairs efficiency of leukotriene biosynthesis inhibitors in males</article-title>. <source>J Clin Invest.</source> (<year>2017</year>) <volume>127</volume>:<fpage>3167</fpage>–<lpage>76</lpage>. <pub-id pub-id-type=\"doi\">10.1172/JCI92885</pub-id><pub-id pub-id-type=\"pmid\">28737505</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weinblatt</surname><given-names>ME</given-names></name><name><surname>Kremer</surname><given-names>JM</given-names></name><name><surname>Coblyn</surname><given-names>JS</given-names></name><name><surname>Helfgott</surname><given-names>S</given-names></name><name><surname>Maier</surname><given-names>AL</given-names></name><name><surname>Petrillo</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Zileuton, a 5-lipoxygenase inhibitor in rheumatoid arthritis</article-title>. <source>J Rheumatol.</source> (<year>1992</year>) <volume>19</volume>:<fpage>1537</fpage>–<lpage>41</lpage>. <pub-id pub-id-type=\"pmid\">1334515</pub-id></mixed-citation></ref><ref id=\"B48\"><label>48.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Savari</surname><given-names>S</given-names></name><name><surname>Chandrashekar</surname><given-names>NK</given-names></name><name><surname>Osman</surname><given-names>J</given-names></name><name><surname>Douglas</surname><given-names>D</given-names></name><name><surname>Bellamkonda</surname><given-names>K</given-names></name><name><surname>Jonsson</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Cysteinyl leukotriene 1 receptor influences intestinal polyp incidence in a gender-specific manner in the ApcMin/+ mouse model</article-title>. <source>Carcinogenesis.</source> (<year>2016</year>) <volume>37</volume>:<fpage>491</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1093/carcin/bgw031</pub-id><pub-id pub-id-type=\"pmid\">26979937</pub-id></mixed-citation></ref><ref id=\"B49\"><label>49.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pace</surname><given-names>S</given-names></name><name><surname>Sautebin</surname><given-names>L</given-names></name><name><surname>Werz</surname><given-names>O</given-names></name></person-group>. <article-title>Sex-biased eicosanoid biology: impact for sex differences in inflammation and consequences for pharmacotherapy</article-title>. <source>Biochem Pharmacol.</source> (<year>2017</year>) <volume>145</volume>:<fpage>1</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bcp.2017.06.128</pub-id><pub-id pub-id-type=\"pmid\">28647490</pub-id></mixed-citation></ref><ref id=\"B50\"><label>50.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Byers</surname><given-names>SL</given-names></name><name><surname>Wiles</surname><given-names>MV</given-names></name><name><surname>Dunn</surname><given-names>SL</given-names></name><name><surname>Taft</surname><given-names>RA</given-names></name></person-group>. <article-title>Mouse estrous cycle identification tool and images</article-title>. <source>PLoS ONE.</source> (<year>2012</year>) <volume>7</volume>:<fpage>e35538</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0035538</pub-id><pub-id pub-id-type=\"pmid\">22514749</pub-id></mixed-citation></ref><ref id=\"B51\"><label>51.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cora</surname><given-names>MC</given-names></name><name><surname>Kooistra</surname><given-names>L</given-names></name><name><surname>Travlos</surname><given-names>G</given-names></name></person-group>. <article-title>Vaginal cytology of the laboratory rat and mouse: review and criteria for the staging of the estrous cycle using stained vaginal smears</article-title>. <source>Toxicol Pathol.</source> (<year>2015</year>) <volume>43</volume>:<fpage>776</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1177/0192623315570339</pub-id><pub-id pub-id-type=\"pmid\">25739587</pub-id></mixed-citation></ref><ref id=\"B52\"><label>52.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kilkenny</surname><given-names>C</given-names></name><name><surname>Browne</surname><given-names>WJ</given-names></name><name><surname>Cuthi</surname><given-names>I</given-names></name><name><surname>Emerson</surname><given-names>M</given-names></name><name><surname>Altman</surname><given-names>DG</given-names></name></person-group>. <article-title>Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research</article-title>. <source>Vet Clin Pathol.</source> (<year>2012</year>) <volume>41</volume>:<fpage>27</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1939-165X.2012.00418.x</pub-id><pub-id pub-id-type=\"pmid\">22390425</pub-id></mixed-citation></ref><ref id=\"B53\"><label>53.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Biguetti</surname><given-names>CC</given-names></name><name><surname>De Oliva</surname><given-names>AH</given-names></name><name><surname>Healy</surname><given-names>K</given-names></name><name><surname>Mahmoud</surname><given-names>RH</given-names></name><name><surname>Custodio</surname><given-names>IDC</given-names></name><name><surname>Constantino</surname><given-names>DH</given-names></name><etal/></person-group>. <article-title>Medication-related osteonecrosis of the jaws after tooth extraction in senescent female mice treated with zoledronic acid: microtomographic, histological and immunohistochemical characterization</article-title>. <source>PLoS ONE.</source> (<year>2019</year>) <volume>14</volume>:<fpage>e0214173</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0214173</pub-id><pub-id pub-id-type=\"pmid\">31199812</pub-id></mixed-citation></ref><ref id=\"B54\"><label>54.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bouxsein</surname><given-names>ML</given-names></name><name><surname>Boyd</surname><given-names>SK</given-names></name><name><surname>Christiansen</surname><given-names>BA</given-names></name><name><surname>Guldberg</surname><given-names>RE</given-names></name><name><surname>Jepsen</surname><given-names>KJ</given-names></name><name><surname>Muller</surname><given-names>R</given-names></name></person-group>. <article-title>Guidelines for assessment of bone microstructure in rodents using micro-computed tomography</article-title>. <source>J Bone Miner Res.</source> (<year>2010</year>) <volume>25</volume>:<fpage>1468</fpage>–<lpage>86</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jbmr.141</pub-id><pub-id pub-id-type=\"pmid\">20533309</pub-id></mixed-citation></ref><ref id=\"B55\"><label>55.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gao</surname><given-names>C</given-names></name><name><surname>Chen</surname><given-names>BP</given-names></name><name><surname>Sullivan</surname><given-names>MB</given-names></name><name><surname>Hui</surname><given-names>J</given-names></name><name><surname>Ouellet</surname><given-names>JA</given-names></name><name><surname>Henderson</surname><given-names>JE</given-names></name><etal/></person-group>. <article-title>Micro CT analysis of spine architecture in a mouse model of scoliosis</article-title>. <source>Front Endocrinol.</source> (<year>2015</year>) <volume>6</volume>:<fpage>38</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fendo.2015.00038</pub-id><pub-id pub-id-type=\"pmid\">25852647</pub-id></mixed-citation></ref><ref id=\"B56\"><label>56.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gingery</surname><given-names>A</given-names></name><name><surname>Subramaniam</surname><given-names>M</given-names></name><name><surname>Pitel</surname><given-names>KS</given-names></name><name><surname>Reese</surname><given-names>JM</given-names></name><name><surname>Cicek</surname><given-names>M</given-names></name><name><surname>Lindenmaier</surname><given-names>LB</given-names></name><etal/></person-group>. <article-title>The effects of a novel hormonal breast cancer therapy, endoxifen, on the mouse skeleton</article-title>. <source>PLoS ONE.</source> (<year>2014</year>) <volume>9</volume>:<fpage>e98219</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0098219</pub-id><pub-id pub-id-type=\"pmid\">24853369</pub-id></mixed-citation></ref><ref id=\"B57\"><label>57.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sheng</surname><given-names>ZF</given-names></name><name><surname>Xu</surname><given-names>K</given-names></name><name><surname>Ma</surname><given-names>YL</given-names></name><name><surname>Liu</surname><given-names>JH</given-names></name><name><surname>Dai</surname><given-names>RC</given-names></name><name><surname>Zhang</surname><given-names>YH</given-names></name><etal/></person-group>. <article-title>Zoledronate reverses mandibular bone loss in osteoprotegerin-deficient mice</article-title>. <source>Osteoporos Int.</source> (<year>2009</year>) <volume>20</volume>:<fpage>151</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00198-008-0640-0</pub-id><pub-id pub-id-type=\"pmid\">18496637</pub-id></mixed-citation></ref><ref id=\"B58\"><label>58.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thompson</surname><given-names>J</given-names></name><name><surname>Mendoza</surname><given-names>F</given-names></name><name><surname>Tan</surname><given-names>E</given-names></name><name><surname>Bertol</surname><given-names>JW</given-names></name><name><surname>Gaggar</surname><given-names>AS</given-names></name><name><surname>Jun</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>A cleft lip and palate gene, Irf6, is involved in osteoblast differentiation of craniofacial bone</article-title>. <source>Dev Dyn</source>. (<year>2019</year>) <volume>248</volume>:<fpage>221</fpage>–<lpage>32</lpage>. <pub-id pub-id-type=\"doi\">10.1002/dvdy.13</pub-id><pub-id pub-id-type=\"pmid\">30684382</pub-id></mixed-citation></ref><ref id=\"B59\"><label>59.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dare</surname><given-names>LR</given-names></name><name><surname>Dias</surname><given-names>DV</given-names></name><name><surname>Rosa Junior</surname><given-names>GM</given-names></name><name><surname>Bueno</surname><given-names>CR</given-names></name><name><surname>Buchaim</surname><given-names>RL</given-names></name><name><surname>Rodrigues Ade</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>Effect of beta-hydroxy-beta-methylbutyrate in masticatory muscles of rats</article-title>. <source>J Anat.</source> (<year>2015</year>) <volume>226</volume>:<fpage>40</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1111/joa.12256</pub-id><pub-id pub-id-type=\"pmid\">25400135</pub-id></mixed-citation></ref><ref id=\"B60\"><label>60.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krag</surname><given-names>TO</given-names></name><name><surname>Pinos</surname><given-names>T</given-names></name><name><surname>Nielsen</surname><given-names>TL</given-names></name><name><surname>Brull</surname><given-names>A</given-names></name><name><surname>Andreu</surname><given-names>AL</given-names></name><name><surname>Vissing</surname><given-names>J</given-names></name></person-group>. <article-title>Differential muscle involvement in mice and humans affected by McArdle disease</article-title>. <source>J Neuropathol Exp Neurol.</source> (<year>2016</year>) <volume>75</volume>:<fpage>441</fpage>–<lpage>54</lpage>. <pub-id pub-id-type=\"doi\">10.1093/jnen/nlw018</pub-id><pub-id pub-id-type=\"pmid\">27030740</pub-id></mixed-citation></ref><ref id=\"B61\"><label>61.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Biguetti</surname><given-names>CC</given-names></name><name><surname>Cavalla</surname><given-names>F</given-names></name><name><surname>Silveira</surname><given-names>EV</given-names></name><name><surname>Tabanez</surname><given-names>AP</given-names></name><name><surname>Francisconi</surname><given-names>CF</given-names></name><name><surname>Taga</surname><given-names>R</given-names></name><etal/></person-group>. <article-title>HGMB1 and RAGE as essential components of Ti osseointegration process in Mice</article-title>. <source>Front Immunol.</source> (<year>2019</year>) <volume>10</volume>:<fpage>709</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2019.00709</pub-id><pub-id pub-id-type=\"pmid\">31024546</pub-id></mixed-citation></ref><ref id=\"B62\"><label>62.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fonseca</surname><given-names>FP</given-names></name><name><surname>de Andrade</surname><given-names>BA</given-names></name><name><surname>Rangel</surname><given-names>AL</given-names></name><name><surname>Della Coletta</surname><given-names>R</given-names></name><name><surname>Lopes</surname><given-names>MA</given-names></name><name><surname>de Almeida</surname><given-names>OP</given-names></name><etal/></person-group>. <article-title>Tissue microarray is a reliable method for immunohistochemical analysis of pleomorphic adenoma</article-title>. <source>Oral Surg Oral Med Oral Pathol Oral Radiol.</source> (<year>2014</year>) <volume>117</volume>:<fpage>81</fpage>–<lpage>8</lpage>\n<pub-id pub-id-type=\"doi\">10.1016/j.oooo.2013.08.029</pub-id><pub-id pub-id-type=\"pmid\">24332331</pub-id></mixed-citation></ref><ref id=\"B63\"><label>63.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Motulsky</surname><given-names>HJ</given-names></name><name><surname>Brown</surname><given-names>RE</given-names></name></person-group>. <article-title>Detecting outliers when fitting data with nonlinear regression – a new method based on robust nonlinear regression and the false discovery rate</article-title>. <source>BMC Bioinformatics.</source> (<year>2006</year>) <volume>7</volume>:<fpage>123</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1471-2105-7-123</pub-id><pub-id pub-id-type=\"pmid\">16526949</pub-id></mixed-citation></ref><ref id=\"B64\"><label>64.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Elis</surname><given-names>S</given-names></name><name><surname>Courtland</surname><given-names>HW</given-names></name><name><surname>Wu</surname><given-names>Y</given-names></name><name><surname>Rosen</surname><given-names>CJ</given-names></name><name><surname>Sun</surname><given-names>H</given-names></name><name><surname>Jepsen</surname><given-names>KJ</given-names></name><etal/></person-group>. <article-title>Elevated serum levels of IGF-1 are sufficient to establish normal body size and skeletal properties even in the absence of tissue IGF-1</article-title>. <source>J Bone Miner Res.</source> (<year>2010</year>) <volume>25</volume>:<fpage>1257</fpage>–<lpage>66</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jbmr.20</pub-id><pub-id pub-id-type=\"pmid\">20200935</pub-id></mixed-citation></ref><ref id=\"B65\"><label>65.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Elis</surname><given-names>S</given-names></name><name><surname>Wu</surname><given-names>Y</given-names></name><name><surname>Courtland</surname><given-names>HW</given-names></name><name><surname>Sun</surname><given-names>H</given-names></name><name><surname>Rosen</surname><given-names>CJ</given-names></name><name><surname>Adamo</surname><given-names>ML</given-names></name><etal/></person-group>. <article-title>Increased serum IGF-1 levels protect the musculoskeletal system but are associated with elevated oxidative stress markers and increased mortality independent of tissue igf1 gene expression</article-title>. <source>Aging Cell.</source> (<year>2011</year>) <volume>10</volume>:<fpage>547</fpage>–<lpage>50</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1474-9726.2011.00683.x</pub-id><pub-id pub-id-type=\"pmid\">21418509</pub-id></mixed-citation></ref><ref id=\"B66\"><label>66.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Le</surname><given-names>P</given-names></name><name><surname>Kawai</surname><given-names>M</given-names></name><name><surname>Bornstein</surname><given-names>S</given-names></name><name><surname>DeMambro</surname><given-names>VE</given-names></name><name><surname>Horowitz</surname><given-names>MC</given-names></name><name><surname>Rosen</surname><given-names>CJ</given-names></name></person-group>. <article-title>A high-fat diet induces bone loss in mice lacking the Alox5 gene</article-title>. <source>Endocrinology.</source> (<year>2012</year>) <volume>153</volume>:<fpage>6</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.1210/en.2011-0082</pub-id><pub-id pub-id-type=\"pmid\">22128029</pub-id></mixed-citation></ref><ref id=\"B67\"><label>67.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vachier</surname><given-names>I</given-names></name><name><surname>Chanez</surname><given-names>P</given-names></name><name><surname>Bonnans</surname><given-names>C</given-names></name><name><surname>Godard</surname><given-names>P</given-names></name><name><surname>Bousquet</surname><given-names>J</given-names></name><name><surname>Chavis</surname><given-names>C</given-names></name></person-group>. <article-title>Endogenous anti-inflammatory mediators from arachidonate in human neutrophils</article-title>. <source>Biochem Biophys Res Commun.</source> (<year>2002</year>) <volume>290</volume>:<fpage>219</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1006/bbrc.2001.6155</pub-id><pub-id pub-id-type=\"pmid\">11779156</pub-id></mixed-citation></ref><ref id=\"B68\"><label>68.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Calder</surname><given-names>PC</given-names></name></person-group>. <article-title>Omega-3 fatty acids and inflammatory processes</article-title>. <source>Nutrients.</source> (<year>2010</year>) <volume>2</volume>:<fpage>355</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.3390/nu2030355</pub-id><pub-id pub-id-type=\"pmid\">22254027</pub-id></mixed-citation></ref><ref id=\"B69\"><label>69.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Levy</surname><given-names>BD</given-names></name><name><surname>Clish</surname><given-names>CB</given-names></name><name><surname>Schmidt</surname><given-names>B</given-names></name><name><surname>Gronert</surname><given-names>K</given-names></name><name><surname>Serhan</surname><given-names>CN</given-names></name></person-group>. <article-title>Lipid mediator class switching during acute inflammation: signals in resolution</article-title>. <source>Nat Immunol.</source> (<year>2001</year>) <volume>2</volume>:<fpage>612</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1038/89759</pub-id><pub-id pub-id-type=\"pmid\">11429545</pub-id></mixed-citation></ref><ref id=\"B70\"><label>70.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Serhan</surname><given-names>CN</given-names></name></person-group>. <article-title>Pro-resolving lipid mediators are leads for resolution physiology</article-title>. <source>Nature.</source> (<year>2014</year>) <volume>510</volume>:<fpage>92</fpage>–<lpage>101</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature13479</pub-id><pub-id pub-id-type=\"pmid\">24899309</pub-id></mixed-citation></ref><ref id=\"B71\"><label>71.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thomson</surname><given-names>SJ</given-names></name><name><surname>Askari</surname><given-names>A</given-names></name><name><surname>Bishop-Bailey</surname><given-names>D</given-names></name></person-group>. <article-title>Anti-inflammatory effects of epoxyeicosatrienoic acids</article-title>. <source>Int J Vasc Med.</source> (<year>2012</year>) <volume>2012</volume>:<fpage>605101</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2012/605101</pub-id><pub-id pub-id-type=\"pmid\">22848834</pub-id></mixed-citation></ref><ref id=\"B72\"><label>72.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kozak</surname><given-names>W</given-names></name><name><surname>Aronoff</surname><given-names>DM</given-names></name><name><surname>Boutaud</surname><given-names>O</given-names></name><name><surname>Kozak</surname><given-names>A</given-names></name></person-group>. <article-title>11,12-epoxyeicosatrienoic acid attenuates synthesis of prostaglandin E2 in rat monocytes stimulated with lipopolysaccharide</article-title>. <source>Exp Biol Med (Maywood)</source>. (<year>2003</year>) <volume>228</volume>:<fpage>786</fpage>–<lpage>94</lpage>. <pub-id pub-id-type=\"doi\">10.1177/15353702-0322807-03</pub-id><pub-id pub-id-type=\"pmid\">12876297</pub-id></mixed-citation></ref><ref id=\"B73\"><label>73.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Node</surname><given-names>K</given-names></name><name><surname>Huo</surname><given-names>Y</given-names></name><name><surname>Ruan</surname><given-names>X</given-names></name><name><surname>Yang</surname><given-names>B</given-names></name><name><surname>Spiecker</surname><given-names>M</given-names></name><name><surname>Ley</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Anti-inflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids</article-title>. <source>Science.</source> (<year>1999</year>) <volume>285</volume>:<fpage>1276</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1126/science.285.5431.1276</pub-id><pub-id pub-id-type=\"pmid\">10455056</pub-id></mixed-citation></ref><ref id=\"B74\"><label>74.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Takikawa</surname><given-names>S</given-names></name><name><surname>Matsui</surname><given-names>N</given-names></name><name><surname>Kokubu</surname><given-names>T</given-names></name><name><surname>Tsunoda</surname><given-names>M</given-names></name><name><surname>Fujioka</surname><given-names>H</given-names></name><name><surname>Mizuno</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Low-intensity pulsed ultrasound initiates bone healing in rat nonunion fracture model</article-title>. <source>J Ultrasound Med.</source> (<year>2001</year>) <volume>20</volume>:<fpage>197</fpage>–<lpage>205</lpage>. <pub-id pub-id-type=\"doi\">10.7863/jum.2001.20.3.197</pub-id><pub-id pub-id-type=\"pmid\">11270523</pub-id></mixed-citation></ref><ref id=\"B75\"><label>75.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>JT</given-names></name><name><surname>Kasukonis</surname><given-names>BM</given-names></name><name><surname>Brown</surname><given-names>LA</given-names></name><name><surname>Washington</surname><given-names>TA</given-names></name><name><surname>Wolchok</surname><given-names>JC</given-names></name></person-group>. <article-title>Recovery from volumetric muscle loss injury: a comparison between young and aged rats</article-title>. <source>Exp Gerontol.</source> (<year>2016</year>) <volume>83</volume>:<fpage>37</fpage>–<lpage>46</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.exger.2016.07.008</pub-id><pub-id pub-id-type=\"pmid\">27435497</pub-id></mixed-citation></ref><ref id=\"B76\"><label>76.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mouraret</surname><given-names>S</given-names></name><name><surname>Hunter</surname><given-names>DJ</given-names></name><name><surname>Bardet</surname><given-names>C</given-names></name><name><surname>Brunski</surname><given-names>JB</given-names></name><name><surname>Bouchard</surname><given-names>P</given-names></name><name><surname>Helms</surname><given-names>JA</given-names></name></person-group>. <article-title>A pre-clinical murine model of oral implant osseointegration</article-title>. <source>Bone.</source> (<year>2014</year>) <volume>58</volume>:<fpage>177</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bone.2013.07.021</pub-id><pub-id pub-id-type=\"pmid\">23886841</pub-id></mixed-citation></ref><ref id=\"B77\"><label>77.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhao</surname><given-names>W</given-names></name><name><surname>Lu</surname><given-names>H</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name><name><surname>Ransohoff</surname><given-names>RM</given-names></name><name><surname>Zhou</surname><given-names>L</given-names></name></person-group>. <article-title>CX3CR1 deficiency delays acute skeletal muscle injury repair by impairing macrophage functions</article-title>. <source>FASEB J.</source> (<year>2016</year>) <volume>30</volume>:<fpage>380</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1096/fj.14-270090</pub-id><pub-id pub-id-type=\"pmid\">26443824</pub-id></mixed-citation></ref><ref id=\"B78\"><label>78.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>X</given-names></name><name><surname>Zhao</surname><given-names>W</given-names></name><name><surname>Ransohoff</surname><given-names>RM</given-names></name><name><surname>Zhou</surname><given-names>L</given-names></name></person-group>. <article-title>Infiltrating macrophages are broadly activated at the early stage to support acute skeletal muscle injury repair</article-title>. <source>J Neuroimmunol.</source> (<year>2018</year>) <volume>317</volume>:<fpage>55</fpage>–<lpage>66</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jneuroim.2018.01.004</pub-id><pub-id pub-id-type=\"pmid\">29325905</pub-id></mixed-citation></ref><ref id=\"B79\"><label>79.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moriscot</surname><given-names>AS</given-names></name><name><surname>Baptista</surname><given-names>IL</given-names></name><name><surname>Bogomolovas</surname><given-names>J</given-names></name><name><surname>Witt</surname><given-names>C</given-names></name><name><surname>Hirner</surname><given-names>S</given-names></name><name><surname>Granzier</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>MuRF1 is a muscle fiber-type II associated factor and together with MuRF2 regulates type-II fiber trophicity and maintenance</article-title>. <source>J Struct Biol.</source> (<year>2010</year>) <volume>170</volume>:<fpage>344</fpage>–<lpage>53</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jsb.2010.02.001</pub-id><pub-id pub-id-type=\"pmid\">20149877</pub-id></mixed-citation></ref><ref id=\"B80\"><label>80.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hurtgen</surname><given-names>BJ</given-names></name><name><surname>Henderson</surname><given-names>BEP</given-names></name><name><surname>Ward</surname><given-names>CL</given-names></name><name><surname>Goldman</surname><given-names>SM</given-names></name><name><surname>Garg</surname><given-names>K</given-names></name><name><surname>McKinley</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>Impairment of early fracture healing by skeletal muscle trauma is restored by FK506</article-title>. <source>BMC Musculosk Disord.</source> (<year>2017</year>) <volume>18</volume>:<fpage>253</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s12891-017-1617-y</pub-id><pub-id pub-id-type=\"pmid\">28606129</pub-id></mixed-citation></ref><ref id=\"B81\"><label>81.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hurtgen</surname><given-names>BJ</given-names></name><name><surname>Ward</surname><given-names>Cl</given-names></name><name><surname>Garg</surname><given-names>K</given-names></name><name><surname>Pollot</surname><given-names>BE</given-names></name><name><surname>Goldman</surname><given-names>SM</given-names></name><name><surname>McKinley</surname><given-names>TO</given-names></name><etal/></person-group>. <article-title>Severe muscle trauma triggers heightened and prolonged local musculoskeletal inflammation and impairs adjacent tibia fracture healing</article-title>. <source>J Musculoskelet Neuronal Interact.</source> (<year>2016</year>) <volume>16</volume>:<fpage>122</fpage>–<lpage>34</lpage>. <pub-id pub-id-type=\"pmid\">27282456</pub-id></mixed-citation></ref><ref id=\"B82\"><label>82.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pollot</surname><given-names>BE</given-names></name><name><surname>Goldman</surname><given-names>SM</given-names></name><name><surname>Wenke</surname><given-names>JC</given-names></name><name><surname>Corona</surname><given-names>BT</given-names></name></person-group>. <article-title>Decellularized extracellular matrix repair of volumetric muscle loss injury impairs adjacent bone healing in a rat model of complex musculoskeletal trauma</article-title>. <source>J Trauma Acute Care Surg.</source> (<year>2016</year>) <volume>81</volume>:<fpage>S184</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1097/TA.0000000000001212</pub-id><pub-id pub-id-type=\"pmid\">27533905</pub-id></mixed-citation></ref><ref id=\"B83\"><label>83.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Utvåg</surname><given-names>SE</given-names></name><name><surname>Grundnes</surname><given-names>O</given-names></name><name><surname>Rindal</surname><given-names>DB</given-names></name><name><surname>Reikerås</surname><given-names>O</given-names></name></person-group>. <article-title>Influence of extensive muscle injury on fracture healing in rat tibia</article-title>. <source>J Orthop Trauma.</source> (<year>2003</year>) <volume>17</volume>:<fpage>430</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1097/00005131-200307000-00007</pub-id><pub-id pub-id-type=\"pmid\">12843728</pub-id></mixed-citation></ref><ref id=\"B84\"><label>84.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Willett</surname><given-names>NJ</given-names></name><name><surname>Li</surname><given-names>MA</given-names></name><name><surname>Uhrig</surname><given-names>BA</given-names></name><name><surname>Boerckel</surname><given-names>JD</given-names></name><name><surname>Huebsch</surname><given-names>N</given-names></name><name><surname>Lundgren</surname><given-names>TL</given-names></name><etal/></person-group>. <article-title>Attenuated human bone morphogenetic protein-2-mediated bone regeneration in a rat model of composite bone and muscle injury</article-title>. <source>Tissue Eng Part C Methods.</source> (<year>2013</year>) <volume>19</volume>:<fpage>316</fpage>–<lpage>25</lpage>. <pub-id pub-id-type=\"doi\">10.1089/ten.tec.2012.0290</pub-id><pub-id pub-id-type=\"pmid\">22992043</pub-id></mixed-citation></ref><ref id=\"B85\"><label>85.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vandamme</surname><given-names>TF</given-names></name></person-group>\n<article-title>Use of rodents as models of human diseases</article-title>. <source>J Pharm Bioallied Sci.</source> (<year>2014</year>) <volume>6</volume>:<fpage>2</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.4103/0975-7406.124301</pub-id><pub-id pub-id-type=\"pmid\">24459397</pub-id></mixed-citation></ref><ref id=\"B86\"><label>86.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schindeler</surname><given-names>A</given-names></name><name><surname>Mills</surname><given-names>RJ</given-names></name><name><surname>Bobyn</surname><given-names>JD</given-names></name></person-group>. <article-title>Little DG preclinical models for orthopedic research and bone tissue engineering</article-title>. <source>J Orthop Res.</source> (<year>2018</year>) <volume>36</volume>:<fpage>832</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jor.23824</pub-id><pub-id pub-id-type=\"pmid\">29205478</pub-id></mixed-citation></ref><ref id=\"B87\"><label>87.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cholok</surname><given-names>D</given-names></name><name><surname>Lee</surname><given-names>E</given-names></name><name><surname>Lisiecki</surname><given-names>J</given-names></name><name><surname>Agarwal</surname><given-names>S</given-names></name><name><surname>Loder</surname><given-names>S</given-names></name><name><surname>Ranganathan</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Traumatic muscle fibrosis: from pathway to prevention</article-title>. <source>J Trauma Acute Care Surg.</source> (<year>2017</year>) <volume>82</volume>:<fpage>174</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.1097/TA.0000000000001290</pub-id><pub-id pub-id-type=\"pmid\">27787441</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Microbiol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Microbiol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Microbiol.</journal-id><journal-title-group><journal-title>Frontiers in Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-302X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849469</article-id><article-id pub-id-type=\"pmc\">PMC7431611</article-id><article-id pub-id-type=\"doi\">10.3389/fmicb.2020.01890</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Microbiology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Comparative Microbiomics of Tephritid Frugivorous Pests (Diptera: Tephritidae) From the Field: A Tale of High Variability Across and Within Species</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>De Cock</surname><given-names>Maarten</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/703107/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Virgilio</surname><given-names>Massimiliano</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/119094/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Vandamme</surname><given-names>Peter</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/336509/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Bourtzis</surname><given-names>Kostas</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/43395/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>De Meyer</surname><given-names>Marc</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Willems</surname><given-names>Anne</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/282149/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Royal Museum for Central Africa</institution>, <addr-line>Tervuren</addr-line>, <country>Belgium</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent University</institution>, <addr-line>Ghent</addr-line>, <country>Belgium</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Insect Pest Control Laboratory, Joint Food and Agriculture Organization of the UnitedNations/International Atomic Energy Agency (FAO/IAEA) Division of Nuclear Techniques in Food and Agriculture</institution>, <addr-line>Vienna</addr-line>, <country>Austria</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Brian Weiss, Yale University, United States</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Beatriz Sabater-Munoz, Polytechnic University of Valencia, Spain; Elisabeth Margaretha Bik, uBiome, United States</p></fn><corresp id=\"c001\">*Correspondence: Maarten De Cock, <email>maarten.decock@ugent.be</email>; <email>maarten_de_cock@hotmail.com</email></corresp><corresp id=\"c002\">Massimiliano Virgilio, <email>massimiliano.virgilio@africamuseum.be</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Systems Microbiology, a section of the journal Frontiers in Microbiology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1890</elocation-id><history><date date-type=\"received\"><day>02</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>20</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 De Cock, Virgilio, Vandamme, Bourtzis, De Meyer and Willems.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>De Cock, Virgilio, Vandamme, Bourtzis, De Meyer and Willems</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>The family Tephritidae includes some of the most notorious insect pests of agricultural and horticultural crops in tropical and sub-tropical regions. Despite the interest in the study of their gut microbiome, our present knowledge is largely based on the analysis of laboratory strains. In this study, we present a first comparative analysis of the gut microbiome profiles of field populations of ten African and Mediterranean tephritid pests. For each species, third instar larvae were sampled from different locations and host fruits and compared using 16S rRNA amplicon sequencing and a multi-factorial sampling design. We observed considerable variation in gut microbiome diversity and composition both between and within fruit fly species. A “core” microbiome, shared across all targeted species, could only be identified at most at family level (Enterobacteriaceae). At genus level only a few bacterial genera (<italic>Klebsiella</italic>, <italic>Enterobacter</italic>, and <italic>Bacillus</italic>) were present in most, but not all, samples, with high variability in their relative abundance. Higher relative abundances were found for seven bacterial genera in five of the fruit fly species considered. These were <italic>Erwinia</italic> in <italic>Bactrocera oleae</italic>, <italic>Lactococcus</italic> in <italic>B. zonata</italic>, <italic>Providencia</italic> in <italic>Ceratitis flexuosa</italic>, <italic>Klebsiella</italic>, and <italic>Rahnella</italic> in <italic>C. podocarpi</italic> and <italic>Acetobacter</italic> and <italic>Serratia</italic> in <italic>C. rosa</italic>. With the possible exception of <italic>C. capitata</italic> and <italic>B. dorsalis</italic> (the two most polyphagous species considered) we could not detect obvious relationships between fruit fly dietary breadth and microbiome diversity or abundance patterns. Similarly, our results did not suggest straightforward differences between the microbiome profiles of species belonging to C<italic>eratitis</italic> and the closely related <italic>Bactrocera/Zeugodacus</italic>. These results provide a first comparative analysis of the gut microbiomes of field populations of multiple economically relevant tephritids and provide base line information for future studies that will further investigate the possible functional role of the observed associations.</p></abstract><kwd-group><kwd>gut microbiomics</kwd><kwd>insects</kwd><kwd><italic>Bactrocera</italic></kwd><kwd><italic>Zeugodacus</italic></kwd><kwd><italic>Ceratitis</italic></kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Belgian Federal Science Policy Office<named-content content-type=\"fundref-id\">10.13039/501100002749</named-content></funding-source><award-id rid=\"cn001\">BR/154/PI/SYMDIV</award-id></award-group></funding-group><counts><fig-count count=\"3\"/><table-count count=\"4\"/><equation-count count=\"0\"/><ref-count count=\"100\"/><page-count count=\"13\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Plants are able to produce a wide variety of allelochemicals that act as deterrents against phytophagy. The capability of phytophagous insects to overcome these toxic compounds is strictly associated with the insect feeding preferences and host plant range. This is thought to represent an important evolutionary process promoting insect speciation and, ultimately insect-plant co-evolution (<xref rid=\"B4\" ref-type=\"bibr\">Aluja and Norrbom, 1999</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Després et al., 2007</xref>; <xref rid=\"B98\" ref-type=\"bibr\">Winkler and Mitter, 2016</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Chen et al., 2017</xref>). The family of the Tephritidae (Diptera), commonly referred to as fruit flies, consists worldwide of more than 4500 species distributed over 500 genera (<xref rid=\"B96\" ref-type=\"bibr\">White and Elson-Harris, 1992</xref>; <xref rid=\"B85\" ref-type=\"bibr\">Uchôa, 2012</xref>). Multiple species are found on all continents, excluding Antarctica, but they mainly thrive in tropical and sub-tropical environments. Although the majority infests the seed-bearing organs of plants, about half of the 4500 fruit fly species use the actual fruits for their own reproduction. Eggs are laid in ripening fruits and the different stages of larval development take place within the fruit. Larvae leave the fruit before pupation, pupate in the soil in order to emerge and become adult fruit flies (<xref rid=\"B32\" ref-type=\"bibr\">Christenson and Foote, 1960</xref>; <xref rid=\"B4\" ref-type=\"bibr\">Aluja and Norrbom, 1999</xref>). This larval development causes damage to the fruit, both directly by damaging the fruit tissue, and indirectly by accelerating the rotting process and increasing infestation by other insects, fungi and bacteria (<xref rid=\"B76\" ref-type=\"bibr\">Pierre, 2007</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Badii et al., 2015</xref>; <xref rid=\"B78\" ref-type=\"bibr\">Qin et al., 2015</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Alvarez et al., 2016</xref>). Fruit flies are found in both wild and commercial fruits and because of this, infestations by fruit flies can have huge economic impacts on the agricultural sector.</p><p>As many other phytophagous insects, tephritids can differ widely in their degree of host plant specialization and attack only one host plant species (monophagous flies), only one genus of host plant species (stenophagous), different genera within the same family (oligophagous) or a wide range of hosts belonging to several unrelated plant families (polyphagous). However, the functional classification based on feeding preferences is sometimes ambiguous as flies are also sporadically recorded not only on their “natural” host plants (<italic>sensu</italic>\n<xref rid=\"B3\" ref-type=\"bibr\">Aluja and Mangan, 2008</xref>) but also, and sporadically, on “unconventional” hosts (<xref rid=\"B38\" ref-type=\"bibr\">De Meyer et al., 2015</xref>; <xref rid=\"B49\" ref-type=\"bibr\">Hafsi et al., 2016</xref>). Previous phylogenetic research suggested that the evolutionary relationships observed in fruit flies might be related to their feeding preferences and host plant specialization (<xref rid=\"B89\" ref-type=\"bibr\">Virgilio et al., 2009</xref>). In particular, strong specialization on host plant species (i.e., monophagy and stenophagy) seems to be associated with the capacity to metabolize toxic secondary compounds of the host plant enabling fruit flies to exploit hosts inaccessible to polyphagous flies (<xref rid=\"B45\" ref-type=\"bibr\">Erbout et al., 2011</xref>; <xref rid=\"B73\" ref-type=\"bibr\">Pavlidi et al., 2013</xref>, <xref rid=\"B74\" ref-type=\"bibr\">2017</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Ben-Yosef et al., 2015</xref>). Because of the overall importance of microbial symbionts, it has been hypothesized that microbes might play a crucial role in shaping the dietary range and host plant specialization of herbivorous insects (microbial facilitation hypothesis (<xref rid=\"B55\" ref-type=\"bibr\">Janson et al., 2008</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Brucker and Bordenstein, 2012</xref>; <xref rid=\"B44\" ref-type=\"bibr\">Douglas, 2013</xref>; <xref rid=\"B52\" ref-type=\"bibr\">Hansen and Moran, 2014</xref>; <xref rid=\"B51\" ref-type=\"bibr\">Hammer and Bowers, 2015</xref>). However, it is not entirely clear how important the relative contribution of microbial symbionts is in facilitating host plant shifts and host plant specialization compared to other processes, including the capacity of insects to produce plastic metabolic responses when changing host plant (<xref rid=\"B75\" ref-type=\"bibr\">Pfennig et al., 2010</xref>).</p><p>In recent years, an increasing number of studies have focused on the gut microbiome of tephritid fruit flies (<xref rid=\"B63\" ref-type=\"bibr\">Lauzon et al., 2000</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Bourtzis and Miller, 2003</xref>; <xref rid=\"B87\" ref-type=\"bibr\">van den Bosch and Welte, 2016</xref>; <xref rid=\"B31\" ref-type=\"bibr\">Cheng et al., 2017</xref>; <xref rid=\"B79\" ref-type=\"bibr\">Ras et al., 2017</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Cáceres et al., 2019</xref>). Largely thanks to the emergence of high throughput sequencing (HTS) techniques which facilitated the analysis of complex assemblages generally including thousands of Amplicon Sequence Variants (ASVs) (<xref rid=\"B91\" ref-type=\"bibr\">Wang A. et al., 2014</xref>; <xref rid=\"B92\" ref-type=\"bibr\">Wang H. et al., 2014</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Andongma et al., 2015</xref>). As observed in other insects, evidence has emerged that bacteria help to overcome pesticides (<xref rid=\"B31\" ref-type=\"bibr\">Cheng et al., 2017</xref>) and boost host defenses (<xref rid=\"B21\" ref-type=\"bibr\">Ben-Yosef et al., 2015</xref>) or generally increase longevity of fruit flies (<xref rid=\"B70\" ref-type=\"bibr\">Niyazi et al., 2004</xref>; <xref rid=\"B15\" ref-type=\"bibr\">Behar et al., 2008a</xref>; <xref rid=\"B50\" ref-type=\"bibr\">Hamden et al., 2013</xref>; <xref rid=\"B80\" ref-type=\"bibr\">Sacchetti et al., 2013</xref>). Complex relationships may exist between the feeding strategy and the gut microbiome with the general expectation that monophagous flies might harbor a more specialized gut microbiome, while polyphagous species should harbor a more diverse and less specialized gut microbiome. Precedence for this kind of relationship was found in <italic>Bactrocera oleae</italic>, a strict monophagous species. Studies have unveiled a close evolutionary relationship between <italic>B. oleae</italic> and the bacterial species <italic>”Candidatus</italic> Erwinia dacicola<italic>”</italic> (<xref rid=\"B29\" ref-type=\"bibr\">Capuzzo et al., 2005</xref>; <xref rid=\"B46\" ref-type=\"bibr\">Estes et al., 2009</xref>). It has been shown that this bacterial species has an important role in facilitating the digestion of olives, and that its absence may strongly reduce survival rate of <italic>B. oleae</italic> in the field (<xref rid=\"B19\" ref-type=\"bibr\">Ben-Yosef et al., 2008</xref>, <xref rid=\"B21\" ref-type=\"bibr\">2015</xref>).</p><p>Most of the currently available research on tephritid gut microbiomics focuses on fruit fly laboratory populations (i.e., fed with artificial diets) and often aims at investigating the optimal rearing conditions for species of interest for the sterile insect technique (SIT) (<xref rid=\"B12\" ref-type=\"bibr\">Augustinos et al., 2015</xref>, <xref rid=\"B13\" ref-type=\"bibr\">2019</xref>; <xref rid=\"B60\" ref-type=\"bibr\">Kyritsis et al., 2017</xref>, <xref rid=\"B61\" ref-type=\"bibr\">2019</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Asimakis et al., 2019</xref>) while the composition and levels of variability of microbiome profiles of wild tephritid flies are far less known.</p><p>A number of studies have targeted one (<xref rid=\"B92\" ref-type=\"bibr\">Wang H. et al., 2014</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Deutscher et al., 2018</xref>; <xref rid=\"B66\" ref-type=\"bibr\">Malacrinò et al., 2018</xref>; <xref rid=\"B37\" ref-type=\"bibr\">De Cock et al., 2019</xref>) or a few (<xref rid=\"B69\" ref-type=\"bibr\">Morrow et al., 2015</xref>) fruit fly species and compared the microbiomes of wild and laboratory populations. Other studies investigated relationships between the microbiome composition of a single fruit fly species and the host plant attacked (<xref rid=\"B99\" ref-type=\"bibr\">Zaada et al., 2019</xref>) or the geographic origin of larvae (<xref rid=\"B48\" ref-type=\"bibr\">Hadapad et al., 2015</xref>; <xref rid=\"B58\" ref-type=\"bibr\">Koskinioti et al., 2019</xref>). Regardless of that, there is still the need for a better understanding of patterns of variability of microbiome profiles in wild flies, and studies providing wide inter- and intra-specific comparisons in field conditions are, to our knowledge, currently missing.</p><p>The present study aimed at providing a first wide-range comparative analysis of the microbiome profiles of tephritid flies as observed under field conditions (i.e., from larvae sampled while feeding on their natural host plants). In this respect, we characterized the microbiome profiles of representative monophagous, stenophagous, oligophagous and polyphagous species from three economically important genera in the Mediterranean region and Sub-Saharan Africa.</p><p>Due to the relatively high heterogeneity previously observed for the microbiome of both laboratory and field populations of <italic>Ceratitis capitata</italic> (<xref rid=\"B37\" ref-type=\"bibr\">De Cock et al., 2019</xref>) we decided to characterize the intra-specific variability of microbiome assemblages by considering field populations from replicated sampling sites and host plants. This approach aimed at verifying the presence of particular groups of gut symbionts consistently associated to the targeted fruit fly species while disentangling the effects of geographic variability and host plant choice.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Sample Collection and Experimental Setup</title><p>We targeted three tephritid genera of economic relevance (<italic>Bactrocera, Zeugodacus</italic>, and <italic>Ceratitis</italic>) including ten representative fruit fly species [<italic>B. dorsalis</italic> (Hendel), <italic>B. oleae</italic> (Rossi), <italic>B. zonata</italic> (Saunders), <italic>Z. cucurbitae</italic> (Coquillett), <italic>C. capitata</italic> (Wiedemann), <italic>C. cosyra</italic> (Walker), <italic>C. flexuosa</italic> (Walker), <italic>C. podocarpi</italic> (Bezzi), <italic>C. quilicii</italic>, De Meyer, Mwatawala & Virgilio, and <italic>C. rosa</italic>, Karsch]. The species selection covered a range of feeding strategies including monophagy (<italic>B. oleae, C. flexuosa</italic>), stenophagy (<italic>C. podocarpi</italic>), oligophagy (<italic>Z. cucurbitae</italic>), and polyphagy (at increasing levels of polyphagy: <italic>C. cosyra, B. zonata, C. quilicii, C. rosa, C. capitata</italic>, and <italic>B. dorsalis</italic>).</p><p>A first part of this study was based on a balanced sampling design (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table S1</xref>), as required for ANOVA/PERMANOVA (see below). Here we considered five fruit fly species (<italic>B. dorsalis</italic>, <italic>Z. cucurbitae, B. oleae</italic>, <italic>C. capitata</italic>, and <italic>C. quilicii</italic>) and, for each of them, the geographic variability of microbiome assemblages was estimated by collecting samples from two arbitrarily chosen locations in two different African or European countries. Similarly, intraspecific variability associated to host-plant choice was estimated by collecting three replicate samples in fruits from two randomly chosen host plant species at each location (see <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Tables S1</xref>, <xref ref-type=\"supplementary-material\" rid=\"TS2\">S2</xref>). As in <xref rid=\"B37\" ref-type=\"bibr\">De Cock et al. (2019)</xref>, we tried to reduce inter-individual variability by pooling, for each sample, the dissected guts of five third instar larvae. This first balanced experiment (dataset A) included a total of 60 samples as obtained from 300 dissected guts.</p><p>This dataset was then expanded with 33 additional samples from the ten fruit fly species listed above, collected from additional host plants and sampling locations (see details in <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Tables S1</xref>, <xref ref-type=\"supplementary-material\" rid=\"TS2\">S2</xref>). This allowed considering a larger dataset (dataset B) including a total of 93 samples obtained from the dissection of 465 larval guts which was used for a wider range of statistics (see below).</p></sec><sec id=\"S2.SS2\"><title>Laboratory Procedures</title><p>After collection in the field or in the rearing facilities of partner Institutions (see Acknowledgments), larvae were immediately stored in 70% ethanol before being transferred to the Royal Museum for Central Africa (Tervuren, Belgium). There, individual larvae were rinsed again in 70% ethanol for 30 s and washed in sterile phosphate buffed saline (PBS) water. The complete gut was dissected under sterile conditions as detailed in <xref rid=\"B37\" ref-type=\"bibr\">De Cock et al. (2019)</xref>. Although we acknowledge that the use of diluted rather than absolute ethanol as a killing and preserving agent is suboptimal and might have affected the gut microbial community, contributing to the variability of microbiome profiles across experimental replicates (see <xref rid=\"B37\" ref-type=\"bibr\">De Cock et al., 2019</xref>). We eventually considered this was the only suitable methodological approach to keep the larval tissues soft and allow dissections. From each gut, DNA was extracted using the Qiagen DNAeasy kit, as per manufacturer’s instructions. After DNA extraction the identity of each larva was confirmed via DNA barcoding as described in <xref rid=\"B90\" ref-type=\"bibr\">Virgilio et al. (2012)</xref> and DNA concentrations were quantified using a Qubit fluorometer (Thermo Fisher Scientific). We only selected DNA extracts from larvae having a correct identification and DNA concentrations higher than 1 ng/μl. For each sample, three replicates were prepared, each consisting of the pooled DNA extracts from five individual larvae (normalized DNA concentrations). This way about 951 larvae were processed from which 358 DNA extracts needed to be rejected (199 wrong identification, 82 failed identification and 77 DNA concentration to low). From the remaining 593 DNA extract, 465 extracts were selected to create our pooled samples. A mock community was composed consisting of the DNAs of 18 pure bacterial strains (see <xref ref-type=\"supplementary-material\" rid=\"TS3\">Supplementary Table S3</xref>) obtained from the BCCM/LMG Bacteria Collection<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup>. The species were selected based on literature reports of their occurrence in fruit fly guts. Bacterial strains were individually grown following the BCCM/LMG catalog instructions. DNA was extracted using the Qiagen DNAeasy kit, and mixed in equal concentrations (DNA concentration: 10 ng/μl). This mock sample and a blank sample were also included in sample preparation and sequencing protocol as, positive and negative control.</p><p>Genomic library preparation for 16S rDNA metagenomics relied on the Nextera XT kit (<xref rid=\"B54\" ref-type=\"bibr\">Illumina, 2016</xref>). In a first step, the primers 341F and 806R (insert size 465 bp), targeting the V3–V4 region of the 16S ribosomal RNA (<xref rid=\"B83\" ref-type=\"bibr\">Takahashi et al., 2014</xref>), were used to amplify the targeted region of the bacterial 16S rRNA, simultaneously two Illumina sequencing adapters were attached to the target DNA fragment. In a second step, dual-index barcodes were attached to the Illumina sequencing adapters. If needed, this second step was repeated to increase DNA yield. A final check of quality and fragment size was performed via an Agilent 2100 Bioanalyzer. Libraries were sequenced on an Illumina MiqSeq platform (300 bp paired end sequencing) by Macrogen (Amsterdam).</p></sec><sec id=\"S2.SS3\"><title>Data Analysis</title><p>Read quality was evaluated using FastQC (<xref rid=\"B10\" ref-type=\"bibr\">Andrews, 2014</xref>). The pipeline DADA2 (<xref rid=\"B28\" ref-type=\"bibr\">Callahan et al., 2016</xref>), implemented in R, was used for data filtering. This pipeline is based in a self-learning algorithm, which sets up a parametric error model that fits the raw data. This model is then used to infer sequencing error. In DADA2, raw reads were trimmed, demultiplexed, filtered and paired (<xref rid=\"B28\" ref-type=\"bibr\">Callahan et al., 2016</xref>). Processed reads were assigned to Amplicon Sequence Variants (ASVs) according to the Bayesian classifier method implemented by DADA2 (<xref rid=\"B94\" ref-type=\"bibr\">Wang et al., 2007</xref>) (percentage of identity = 97% similarity, <italic>p</italic>-min-consensus = 0.51). Taxonomic assignment of ASV relied on the Silva v132 (26) database. The robustness of the assignment was double-checked against the RDP (<xref rid=\"B34\" ref-type=\"bibr\">Cole et al., 2014</xref>) and Greengenes databases (<xref rid=\"B40\" ref-type=\"bibr\">DeSantis et al., 2006</xref>, data not shown). The full analytical pipeline is detailed in <xref ref-type=\"supplementary-material\" rid=\"TS4\">Supplementary Table S4</xref>. As in <xref rid=\"B37\" ref-type=\"bibr\">De Cock et al. (2019)</xref>, before analyses, single- and doubletons reads were filtered out to reduce possible biases due to sequencing error. For comparative analysis, normalized data, based on the median sample number of reads, was used (<xref rid=\"B36\" ref-type=\"bibr\">de Cárcer et al., 2011</xref>).</p><p>The data were processed in both univariate and multivariate frameworks. The effects of Fruit Fly Species (FFSp), Location (Lo), and Host plant (Ho) on univariate patterns of alpha diversity, as estimated by the Simpson index D (<xref rid=\"B81\" ref-type=\"bibr\">Sagar and Sharma, 2012</xref>), were tested via Analysis of Variance (ANOVA) (<xref rid=\"B86\" ref-type=\"bibr\">Underwood, 1997</xref>). Comparisons of multivariate patterns were done by using Permutational Multivariate Analysis of Variance (PERMANOVA, <xref rid=\"B8\" ref-type=\"bibr\">Anderson, 2017</xref>) and Permutational Multivariate Analysis of Dispersion (PERMDISP, <xref rid=\"B6\" ref-type=\"bibr\">Anderson, 2001</xref>). We used PERMANOVA to test differences in the relative abundance of ASVs (2749 in total, see section “Results”), while, as the PERMDISP routine of <xref rid=\"B6\" ref-type=\"bibr\">Anderson (2001)</xref> can only be implemented on a maximum of 500 variables, this analysis was implemented on the relative abundance of genera (401 in total, see section “Results”). In order to reduce differences in scale among variables while preserving information about taxa proportions, we transformed the multivariate data following <xref rid=\"B33\" ref-type=\"bibr\">Clarke (1993)</xref>. This approach allowed reducing the importance of dominant, compared to the less abundant, taxa and to better identify more subtle changes in the abundance of non-dominant species. We compared the possible impact of data transformation by implementing both (1) presence-absence transformation (as an example of extreme transformation severely affecting abundance proportions) and (2) fourth-root transformation (as an example of less aggressive transformation, of common use in community ecology. For both ANOVA and PERMANOVA a three-way factorial setup was adopted with fruit fly Species (FFSp) as a fixed, orthogonal factor and Location [Lo(FFSp)] and Host Plant [Ho(FFSpxLo)] as random, nested factors. For PERMDISP, that only allows two-way designs (<xref rid=\"B7\" ref-type=\"bibr\">Anderson, 2006</xref>), we tested the effects of FFSp, and either [Lo(FFSp)] or [Ho(FFSp)]. <italic>A posteriori</italic> pairwise comparisons of significant factors were implemented via Tukey’s Honestly Significant Difference (HSD) test (<xref rid=\"B1\" ref-type=\"bibr\">Abdi and Williams, 2010</xref>) for ANOVA and permutational t-statistics for PERMANOVA and PERMDISP (<xref rid=\"B6\" ref-type=\"bibr\">Anderson, 2001</xref>, <xref rid=\"B8\" ref-type=\"bibr\">2017</xref>). Probability values of repeated <italic>a posteriori</italic> tests were corrected for Type I errors using the False Discovery Rate procedure (<xref rid=\"B17\" ref-type=\"bibr\">Benjamini and Hochberg, 1995</xref>) with experiment-wise probability <italic>p</italic> = 0.05. In order to increase the power of the multivariate <italic>a posteriori</italic> test (<xref rid=\"B86\" ref-type=\"bibr\">Underwood, 1997</xref>), we increased the number of permutable units (<xref rid=\"B8\" ref-type=\"bibr\">Anderson, 2017</xref>) by pooling together the replicates of non-significant terms. Following <xref rid=\"B36\" ref-type=\"bibr\">de Cárcer et al. (2011)</xref>, we repeated multivariate tests on both data fourth-root transformed to the median and presence/absence data. The analysis of presence/absence data allowed stressing the possible effects of less abundant taxa.</p><p>Further investigation of the gut microbiome composition was done using the packages Phyloseq (<xref rid=\"B67\" ref-type=\"bibr\">McMurdie and Holmes, 2013</xref>), Vegan (<xref rid=\"B72\" ref-type=\"bibr\">Oksanen et al., 2019</xref>), and ggplot2 (<xref rid=\"B97\" ref-type=\"bibr\">Wickham, 2009</xref>), as implemented in R version 3.1.0. Principal Coordinates Analyses (PCoAs) based on Bray-Curtis distance (<xref rid=\"B25\" ref-type=\"bibr\">Bray and Curtis, 1957</xref>) were calculated for both fourth-root transformed data and presence/absence data. PCoAs for separated species were not incorporated due to the relatively small proportion of variance represented in PCoAs and to the relatively small sample size of samples available for each host and location. ASVs were pooled based on the bacterial genera and the percentage contribution of each of these genera to the average Bray-Curtis dissimilarity between fruit fly species was calculated using SIMPER (<xref rid=\"B33\" ref-type=\"bibr\">Clarke, 1993</xref>) on standardized, untransformed data. A permutational test based on 10,000 iterations was used to identify bacterial genera significantly differing between fruit fly species. Repeated permutational tests were corrected using FDR (<xref rid=\"B17\" ref-type=\"bibr\">Benjamini and Hochberg, 1995</xref>) at an experiment-wise <italic>p</italic> < 0.01. The results of SIMPER pairwise tests were summarized by considering only those bacterial genera (a) significantly differing between fruit fly species and (b) with an average contribution to dissimilarity higher than 5%.</p></sec></sec><sec id=\"S3\"><title>Results</title><p>The MiSeq Illumina run produced more than 19 × 10<sup>6</sup> paired-end (PE) reads (average per sample = 213185.07; <italic>SD</italic> = 72270.58). Following quality assessment in FastQC (<xref rid=\"B10\" ref-type=\"bibr\">Andrews, 2014</xref>), the forward and reverse reads were trimmed at respectively 240 and 210 bp. Based on read quality, a strict error rate (max N’s = 0, max error rate = 1, see <xref ref-type=\"supplementary-material\" rid=\"TS4\">Supplementary Table S4</xref>) was applied in DADA2. After filtering, demultiplexing and merging about 5.4 × 10<sup>6</sup> reads, 2749 unique ASVs were identified. The analysis of reads from the positive control did not suggest relevant biases while reads corresponding to 11 ASVs detected in the negative control (see <xref ref-type=\"supplementary-material\" rid=\"TS5\">Supplementary Table S5</xref>) were eliminated from the datasets to avoid possible biases.</p><p>The 2749 ASVs were assigned to 401 genera belonging to 142 different families and 22 phyla (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1</xref>). Of these phyla, Proteobacteria was by far the most dominant, representing 89.25% of all reads, followed by Firmicutes (8.43%), Bacteroidetes (0.95%), Actinobacteria (0.83%), Epsilonbacteraeota (0.22%), and Tenericutes (0.18%). The remaining phyla represented only about 0.01% of total reads. The phylum Proteobacteria consisted of 62 bacterial families, mainly represented by Enterobacteriaceae (65.60% of all reads), Acetobacteraceae (16.72%), Rhizobiaceae (3.37%), and Burkholderiaceae (0.69%) (<xref ref-type=\"supplementary-material\" rid=\"FS2\">Supplementary Figure S2</xref>). The phylum Firmicutes consisted of 27 bacterial families, mainly represented by Leuconostocaceae (4.16%), Streptococcaceae (2.60%), and Lactobacillaceae (0.52%) (<xref ref-type=\"supplementary-material\" rid=\"FS2\">Supplementary Figure S2</xref>). The phylum Bacteroidetes consisted of 23 bacterial families, mainly represented by Weeksellaceae (0.57%), Dysgonomonadaceae (0.14%), and Flavobacteriaceae (0.10%) (<xref ref-type=\"supplementary-material\" rid=\"FS2\">Supplementary Figure S2</xref>). The phylum Actinobacteria consisted of 33 bacterial families, mainly represented by Microbacteriaceae (0.29%) and Corynebacteriaceae (0.27%) (<xref ref-type=\"supplementary-material\" rid=\"FS2\">Supplementary Figure S2</xref>). The remaining phyla are all represented by only one or a few bacterial families. Of the above-mentioned phyla, only Proteobacteria was present in every sample. The phylum Firmicutes was present in almost all samples (> 90%) but had a very low abundance in some samples. The phyla Bacteroidetes and Actinobacteria were present in most samples, respectively, 64 and 73%. All remaining phyla were present in less than 25% of the samples. At bacterial family level, only the family of Enterobacteriaceae was present in all samples. Of the remaining bacterial families only Moraxellaceae, Burkholderiaceae, Streptococcaceae, Acetobacteraceae, Bacillaceae, Corynebacteriaceae, Leuconostocaceae and Staphylococcaceae were present in more than half of the samples. At bacterial genus level there were no genera present in every sample and only a few genera were present in the majority of the samples, including <italic>Klebsiella</italic> (96.43% of samples), <italic>Bacillus</italic> (96.43%), <italic>Enterobacter</italic> (92.86%), and <italic>Acinetobacter</italic> (89.29%). However, high variability between samples, and replicates could be observed with no bacterial genera dominant across all samples. A detailed overview of the most abundant bacterial genera for each fruit fly species can be found in <xref ref-type=\"supplementary-material\" rid=\"TS11\">Supplementary Table S11</xref>. PERMANOVA on fourth-root transformed data (Dataset A, <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, and <xref ref-type=\"supplementary-material\" rid=\"TS6\">Supplementary Table S6</xref>) showed that the gut microbiome composition significantly differs between fruit fly species (<italic>p</italic> < 0.01) and host plants (<italic>p</italic> < 0.001). PERMANOVA on presence/absence data (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref> and <xref ref-type=\"supplementary-material\" rid=\"TS7\">Supplementary Table S7</xref>) could also detect a significant effect of location, suggesting that the gut microbiome of conspecific samples from different locations differs with respect to the less abundant ASVs. The <italic>post hoc</italic> tests on fourth-root transformed data (pooled for location) showed significant differences in all pairwise comparisons with <italic>B. oleae</italic> as well as between <italic>B. dorsalis</italic> and all other species but <italic>C. capitata</italic>, between <italic>Z. cucurbitae</italic> and all other species but <italic>C. capitata</italic>, and between <italic>C. quilicii</italic> and all other species but <italic>C. capitata</italic> (<xref ref-type=\"supplementary-material\" rid=\"TS4\">Supplementary Table S4</xref>). The <italic>post hoc</italic> comparison also provided indications on variability of the gut microbiome composition of the same species when feeding on different host plants. While the microbiome profiles of <italic>Z. cucurbitae</italic> and <italic>C. quilicii</italic> did not show significant variation across host plants, in both <italic>B. dorsalis</italic> and <italic>C. capitata</italic>, we found differences in most (all but one) pairwise comparisons (<xref ref-type=\"supplementary-material\" rid=\"TS6\">Supplementary Table S6</xref>). <italic>Post hoc</italic> comparison on presence/absence data did not reveal any significant effect (<xref ref-type=\"supplementary-material\" rid=\"TS7\">Supplementary Table S7</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>PERMANOVA (fourth-root transformed and presence/absence data; dataset A) testing differences in the microbiome profiles (2,749 ASVs considered) of five fruit fly species (FFSp, <italic>B. dorsalis, Z. cucurbitae, B. oleae, C. capitata, C. quilicii)</italic> sampled in two locations [Lo(FFSp)] from two host plants within each location [Ho(FFSpxLo)].</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>df</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>MS</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic>-value</td><td rowspan=\"1\" colspan=\"1\"/></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Fourth-root transformed data</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FSp</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23502.690</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.521</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">**</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lo(FFSp)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6675.461</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.220</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.189</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">n.s.</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ho(FFSpxLo)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5472.781</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.548</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.000</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">***</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Residual</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2148.190</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">59</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Presence/Absence data</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FFSp</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18961.396</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.557</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.000</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">***</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lo(FFSp)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7414.919</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.345</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.041</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ho(FFSpxLo)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5514.679</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.733</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.000</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">***</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Residual</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2018.200</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">59</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic>***p < 0.001, **p < 0.01, *p < 0.05, n.s.p > 0.05.</italic></attrib></table-wrap-foot></table-wrap><p>Pooling the taxonomically assigned ASVs for the balanced experiment (Dataset A), by genus resulted in a dataset of 401 distinct bacterial genera. On both fourth-root transformed and presence/absence data, PERMDISP revealed significant effects of fruit fly species (<italic>p</italic> < 0.01) and host (<italic>p</italic> < 0.001) on multivariate dispersion (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref> and <xref ref-type=\"supplementary-material\" rid=\"TS8\">Supplementary Tables S8</xref>, <xref ref-type=\"supplementary-material\" rid=\"TS9\">S9</xref>). Although the average dissimilarity between replicates in <italic>B. oleae</italic> (as calculated from fourth-root transformed data) was lower than in all other species, we did not observe significant differences in the <italic>post hoc</italic> comparisons between species (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>, <xref ref-type=\"supplementary-material\" rid=\"TS8\">Supplementary Tables S8</xref>, <xref ref-type=\"supplementary-material\" rid=\"TS9\">S9</xref>).</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>PERMDISP (fourth-root transformed and presence/absence data; dataset A) testing differences in the microbiome profiles (401 bacterial genera considered) of five fruit fly species (<italic>B. dorsalis</italic>, <italic>Z. cucurbitae</italic>, <italic>B. oleae</italic>, <italic>C. capitata</italic>, <italic>C. quilicii</italic>) sampled from four different host plants.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>df</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>MS</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic></td><td rowspan=\"1\" colspan=\"1\"/></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Fourth-root transformed data</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FFSp</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1555.209</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.668</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.004</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">**</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ho(FFSp)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">233.240</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.179</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.000</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">***</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Residual</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">37.745</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">59</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Presence/Absence-data</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FFSp</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">749.530</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.637</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.003</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">**</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ho(FFSp)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">98.143</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.822</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.045</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">**</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Residual</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53.860</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">59</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><hr/></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Average within-group dissimilarities</bold></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\"><bold>Fourth-root transformed data</bold></td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\"><bold>Presence/Absence-data</bold></td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><hr/></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>B. dorsalis</italic></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">63.213</td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">65.326</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Z. cucurbitae</italic></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">79.797</td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">51.782</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>B. oleae</italic></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">14.186</td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">66.527</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>C. capitata</italic></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">83.920</td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">65.283</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>C. quilicii</italic></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">82.305</td><td valign=\"top\" align=\"center\" colspan=\"3\" rowspan=\"1\">69.623</td></tr></tbody></table><table-wrap-foot><attrib><italic>***p < 0.001, **p < 0.01, n.s. p > 0.05.</italic></attrib></table-wrap-foot></table-wrap><p>The PCOAs of the five species included in the balanced experiments (Dataset A, <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>) only accounted for a relatively limited amount of variation, explaining in total 27.9 and 23.7% of variability (PC1 + PC2, 4th root transformed data and presence-absence, respectively). The 95% confidence ellipses allowed resolving <italic>B. oleae</italic> from all other species. Adding the additional species to the PCOA (data not shown), allowed accounting for 25.0 and 18.8% of variability (PC1 + PC2, 4th root transformed data and presence-absence, respectively). Again, inspection of the 95% confidence ellipses showed that only <italic>B. oleae</italic> clustered separately from all other species. Even when removing <italic>B. oleae</italic> from the PCOA an extensive overlap between the different species was still observed. The preliminary analysis of separate PCoAs for each of the fruit fly species targeted in this study did not provide additional suggestions on possible patterns related to location or host-plant (data not shown).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Multivariate ordination (PCOA) of gut microbial assemblages in five target fruit fly species (<italic>B. dorsalis, Z. cucurbitae, B. oleae, C. capitata, C. quilicii;</italic> dataset A); Shape = Fruit fly genus, Color = Fruit fly species; Left: Abundance data; Right: Presence-absence data.</p></caption><graphic xlink:href=\"fmicb-11-01890-g001\"/></fig><p>The mean alpha diversity, as estimated by the Simpson index, across all samples (Dataset A) was <italic>D</italic> = 0.62 (median: 0.72, SD: 0.28). ANOVA revealed a significant effect of both fruit fly species (<italic>p</italic> < 0.05) and host plant (<italic>p</italic> < 0.01) on gut microbiome diversity (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>, <xref ref-type=\"supplementary-material\" rid=\"TS10\">Supplementary Table S10</xref>, and <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). <italic>Post hoc</italic> tests revealed significantly lower alpha diversity in <italic>B. oleae</italic> (<italic>D</italic> = 0.234; SD: 0.27) (<xref ref-type=\"supplementary-material\" rid=\"TS10\">Supplementary Table S10</xref>) compared to all other species except <italic>C. capitata</italic>. Effects of host plants on gut microbiome diversity were found for <italic>B. oleae</italic> between two varieties of <italic>Olea europaea</italic> and for <italic>C. quilicii</italic> between <italic>Harpephyllum caffrum</italic> and <italic>Eriobotrya japonica</italic> (<xref ref-type=\"supplementary-material\" rid=\"TS10\">Supplementary Table S10</xref>).</p><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>ANOVA testing differences in alpha diversity (as estimated by the Simpson index, D; dataset A) of microbiome profiles of five fruit fly species (FFSp, <italic>B. dorsalis, Z. cucurbitae, B. oleae, C. capitata, C. quilicii)</italic> sampled in two locations [Lo(FFSp)] from two host plants within each location [Ho(FFSpxLo)].</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>df</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Mean Sq</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F</italic>-value</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>p</italic></td><td rowspan=\"1\" colspan=\"1\"/></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"6\" rowspan=\"1\"><bold>Abundance-data</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FFSp</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.602</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.651</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.043</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lo(FFSp)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.107</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.008</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.461</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">n.s.</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ho(FFSpxLo)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.106</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.072</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">***</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Residual</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.026</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic>***p < 0.001, *p < 0.05, n.s.p > 0.05.</italic></attrib></table-wrap-foot></table-wrap><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Alpha diversity (Simpson index, D) of gut microbial assemblages in five target fruit fly species (<italic>B. dorsalis, Z. cucurbitae, B. oleae, C. capitata, C. quilicii</italic>; dataset B) sampled in two locations, from two host plants at each location.</p></caption><graphic xlink:href=\"fmicb-11-01890-g002\"/></fig><p>The permutational similarity percentage (SIMPER) analysis (<xref rid=\"B33\" ref-type=\"bibr\">Clarke, 1993</xref>) (Dataset B, <xref rid=\"T4\" ref-type=\"table\">Table 4</xref>, <xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>, and <xref ref-type=\"supplementary-material\" rid=\"TS12\">Supplementary Table S12</xref>) suggested that five of the 10 investigated fruit fly species had characteristic associations with one or more bacterial genera. These putative associations were observed in all (9 out of 9) pairwise comparisons involving (a) <italic>C. flexuosa</italic>, which showed comparably higher abundances of reads from the genus <italic>Providencia</italic> (average abundance = 31.73%, <italic>SD</italic> = 24.45%) (b) <italic>C. podocarpi</italic>, with higher abundances of <italic>Klebsiella</italic> (average abundance = 52.83%, <italic>SD</italic> = 62.55%) and <italic>Rahnella</italic> (average abundance = 17.70%, <italic>SD</italic> = 25.03%)<italic>;</italic> (c) <italic>C. rosa</italic> with higher abundances of <italic>Acetobacter</italic> (average abundance = 55.30%, <italic>SD</italic> = 11.45%) and <italic>Serratia</italic> (average abundance = 0.06%, <italic>SD</italic> = 0.10%); (d) <italic>B. oleae</italic>, with higher abundances of <italic>Erwinia</italic> (average abundance = 93.28%, <italic>SD</italic> = 19.98%) and (e) <italic>B. zonata</italic>, with significantly higher abundances of <italic>Lactococcus</italic> (average abundance = 22.63%, <italic>SD</italic> = 38.71%). Other bacterial genera significantly contributed to the dissimilarity in most of the pairwise comparisons, such as <italic>Morganella</italic> and <italic>Pantoea</italic> in <italic>C. capitata</italic>, <italic>Enterobacter</italic> in <italic>B. dorsalis</italic> and <italic>Gluconobacter</italic> in <italic>C. quilicii.</italic> In <italic>C. cosyra</italic> none of the bacterial genera significantly contributed to more of 5% to the dissimilarity in at least five out of nine pairwise tests.</p><table-wrap id=\"T4\" position=\"float\"><label>TABLE 4</label><caption><p>Pairwise SIMPER permutational tests (10,000 iterations) between fruit fly species (<italic>C. capitata, C. flexuosa, C. podocarpi, C. quilicii, C. rosa, C. cosyra, B. dorsalis, B. oleae, B. zonata, Z. cucurbitae</italic>; dataset B).</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bacterial genera significantly contributing to > 5% dissimilarity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>C. capitata</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>C. flexuosa</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>C. podocarpi</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>C. quilicii</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>C. rosa</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>C. cosyra</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>B. dorsalis</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>B. oleae</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>B. zonata</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Z. cucurbitae</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Proportion of significant pairwise tests (FDR <italic>p</italic> < 0.05)</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>C. capitata</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Morganella</italic></td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6/9</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Pantoea</italic></td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5/9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>C. flexuosa</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Providencia</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9/9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>C. podocarpi</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Klebsiella</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9/9</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Rahnella</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9/9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>C. quilicii</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Gluconobacter</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8/9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>C. rosa</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Acetobacter</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9/9</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Serratia</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9/9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>C. cosyra</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">−</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>B. dorsalis</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Enterobacter</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7/9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>B. oleae</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Erwinia</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9/9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>B. zonata</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Lactococcus</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9/9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Z. cucurbitae</italic></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Lactococcus</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5/9</td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Ochrobactrum</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">*</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5/9</td></tr></tbody></table><table-wrap-foot><attrib><italic>Results are reported for bacterial genera producing significant differences in at least 5 pairwise tests out of 9 (*: FDR-corrected p < 0.01) and contributing to > 5% of dissimilarity between groups. The complete results are available in <xref ref-type=\"supplementary-material\" rid=\"TS12\">Supplementary Table 13</xref>.</italic></attrib></table-wrap-foot></table-wrap><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Abundances (as estimated in number of reads; dataset B) of most representative bacterial genera in ten targeted fruit fly species. Results are reported for bacterial genera producing significant differences in at least 8 pairwise tests out of 9 (FDR-corrected <italic>p</italic> < 0.01) and contributing to > 5% of dissimilarity between groups. For each species, significance letters for pairwise tests are indicated (see also <xref rid=\"T4\" ref-type=\"table\">Table 4</xref> and <xref ref-type=\"supplementary-material\" rid=\"TS12\">Supplementary Table 12</xref>).</p></caption><graphic xlink:href=\"fmicb-11-01890-g003\"/></fig></sec><sec id=\"S4\"><title>Discussion</title><p>One of the main difficulties in the analysis of relationships between gut microbiome profiles and life history traits, including host plant choice, is represented by the high intra- and -interspecific variability of gut microbiomes that include thousands of ASVs (e.g., see <xref rid=\"B37\" ref-type=\"bibr\">De Cock et al., 2019</xref>). Differences between microbiome profiles can be related to life history traits and environmental factors including life stage (<xref rid=\"B64\" ref-type=\"bibr\">Lauzon et al., 2009</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Andongma et al., 2015</xref>), diet (<xref rid=\"B82\" ref-type=\"bibr\">Santo Domingo et al., 1998</xref>; <xref rid=\"B39\" ref-type=\"bibr\">De Vries et al., 2004</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Colman et al., 2012</xref>; <xref rid=\"B69\" ref-type=\"bibr\">Morrow et al., 2015</xref>), or technical artifacts (<xref rid=\"B37\" ref-type=\"bibr\">De Cock et al., 2019</xref>). Consistent with what has been reported in other studies on frugivorous tephritids (<xref rid=\"B84\" ref-type=\"bibr\">Thaochan et al., 2010</xref>; <xref rid=\"B93\" ref-type=\"bibr\">Wang et al., 2011</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Andongma et al., 2015</xref>; <xref rid=\"B69\" ref-type=\"bibr\">Morrow et al., 2015</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Augustinos et al., 2019</xref>), the gut microbiome profiles of third instar larvae of the ten fruit fly species targeted by the present study were mainly composed of Proteobacteria and Firmicutes which together represented more than 98.49% of reads in all tephritid species targeted. <xref rid=\"B9\" ref-type=\"bibr\">Andongma et al. (2015)</xref> suggested that Proteobacteria might be the most abundant phylum in earlier developmental stages of <italic>Bactrocera</italic>, while Firmicutes the most abundant in adult stages, possibly as a result of changes in habitat and diet. The dominance of Proteobacteria in larval stages is consistent with what is observed in the present work not only for <italic>Bactrocera</italic> and <italic>Zeugodacus</italic> but also for <italic>Ceratitis</italic> and it further confirms variation in microbiome profiles across developmental stages, as also described in <italic>C. capitata</italic> (<xref rid=\"B37\" ref-type=\"bibr\">De Cock et al., 2019</xref>).</p><p>Previous studies reported contrasting results on the most abundant gut bacterial families in tephritid fruit flies. While most of these studies report Enterobacteriaceae (Proteobacteria) as a major, dominant component of fruit fly gut microbiomes (<xref rid=\"B59\" ref-type=\"bibr\">Kuzina et al., 2001</xref>; <xref rid=\"B16\" ref-type=\"bibr\">Behar et al., 2008b</xref>; <xref rid=\"B93\" ref-type=\"bibr\">Wang et al., 2011</xref>; <xref rid=\"B92\" ref-type=\"bibr\">Wang H. et al., 2014</xref>) there are also notable exceptions such in <xref rid=\"B9\" ref-type=\"bibr\">Andongma et al. (2015)</xref> where Comamonadaceae are shown to represent a dominant taxon in immature stages of <italic>B. dorsalis</italic>. The dominance of Enterobacteriaceae, as the major component of the gut microbiome of most of the targeted species was confirmed by the results of the present study, with the notable exception of <italic>C. quilicii</italic> and <italic>C. rosa</italic> for which Acetobacteraceae (Proteobacteria) was the bacterial family with the highest abundance. At genus level and ASV level, we observed a high variability both between fruit fly species and within species. Only a few bacterial genera (<italic>Klebsiella</italic>, <italic>Enterobacter</italic>, and <italic>Bacillus</italic>) were present in a large proportion of samples, albeit with high variability in their relative abundance. Patterns observed for <italic>C. capitata</italic> were generally in line with what previously observed in laboratory populations (<xref rid=\"B37\" ref-type=\"bibr\">De Cock et al., 2019</xref>), with Proteobacteria and Firmicutes as the most abundant phyla and Enterobacteriaceae representing the most abundant family.</p><p>Regardless the relatively high number of individual guts used for this screening, the microbiome profiles of larvae collected in the field from their natural host plants showed highly variable patterns both between and within species, with intraspecific variation often, but not always, showing significant changes according to the host plant attacked. Interspecific variation of microbiome profiles was significantly affected by larval diet only in the two most polyphagous fruit fly species, <italic>B. dorsalis</italic> and <italic>C. capitata</italic>. Similarly, both the polyphagous <italic>C. quilicii</italic> and the monophagous <italic>B. oleae</italic> seemed also affected by host plant, even if to a lesser extent (i.e., they showed differences in univariate patterns of diversity but not in their multivariate patterns) while the oligophagous <italic>Z. cucurbitae</italic> did not seem significantly affected by host-plant choice. Regardless of that, the geographic variability of microbiome profiles from fruit fly populations thousands of km distant was relatively limited (relatively, as significant effects could only be observed from the analysis of presence/absence data). This suggests that the variable patterns observed across fruit fly species and host plants (particularly for <italic>B. dorsalis</italic> and <italic>C. capitata</italic>), are geographically consistent, even at large spatial scales (i.e., across different countries in the same continent). More focused experimental designs (i.e., based on a larger number of replicated samples and hosts) are now needed for a more detailed characterization of changes in the microbiome profiles of <italic>B. dorsalis</italic> and <italic>C. capitata</italic> across different host fruits. It would also be of interest to include the microbiome of the fruit that the larvae are sampled from in these studies.</p><p>Similarly, we did not find indications of obvious relationships either between microbiome profile diversity and fruit fly dietary breadth or between the microbiome profiles of the three different genera targeted in this study (<italic>Ceratitis</italic> on one hand and the closely related <italic>Bactrocera/Zeugodacus</italic> on the other).</p><p>From our observation, a core microbiome for the targeted fruit fly species could be defined only at family level, where the family Enterobacteriaceae was the single recurrent element in all samples. At genus or ASV level, however, we could not identify universal core microbiome elements shared by all fruit fly species tested. For individual fruit fly species, however, we could identify a set of key bacterial genera whose abundance was significantly higher in particular fruit fly species, irrespective of the host plant or sampling location considered. Major differences (i.e., significantly higher in all pairwise comparisons implemented) were found in the abundance patterns of seven bacterial genera in five fruit fly species considered. These were <italic>Erwinia</italic> in <italic>B. oleae</italic>, <italic>Lactococcus</italic> in <italic>B. zonata</italic>, <italic>Providencia</italic> in <italic>C. flexuosa</italic>, <italic>Klebsiella</italic>, and <italic>Rahnella</italic> in <italic>C. podocarpi</italic> and <italic>Acetobacter</italic> and <italic>Serratia</italic> in <italic>C. rosa</italic>. Other but less pronounced differences (as significant in a large proportion of pairwise comparison but not in all) were found for genera such as <italic>Ochrobactrum</italic> in <italic>Z. cucurbitae</italic>, <italic>Gluconobacter</italic> in <italic>C. quilicii</italic> and <italic>Enterobacter</italic> in <italic>B. dorsalis</italic>. Further experimental validation is now needed to verify the generality of these patterns and to test the occurrence of stable associations between larval dietary niche and the presence of the above-mentioned gut symbionts.</p><p>In herbivorous insects, gut microbes can aid with the breakdown of complex polysaccharides that make up the plant cell wall, or supplement the nutritionally poor plant diet with nitrogen, vitamins and sterols (<xref rid=\"B43\" ref-type=\"bibr\">Douglas, 2009</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Ben-Yosef et al., 2010</xref>, <xref rid=\"B20\" ref-type=\"bibr\">2014</xref>). There is also evidence that they take part in the detoxification of plant allelochemicals (<xref rid=\"B51\" ref-type=\"bibr\">Hammer and Bowers, 2015</xref>). The relationship between the genus <italic>Erwinia</italic> and <italic>B. oleae</italic> has been studied extensively as it is a prime example of the coevolution between an insect and its gut microbiome (<xref rid=\"B29\" ref-type=\"bibr\">Capuzzo et al., 2005</xref>; <xref rid=\"B20\" ref-type=\"bibr\">Ben-Yosef et al., 2014</xref>, <xref rid=\"B21\" ref-type=\"bibr\">2015</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Estes et al., 2014</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Blow et al., 2016</xref>; <xref rid=\"B74\" ref-type=\"bibr\">Pavlidi et al., 2017</xref>). It is hypothesized that the close relationship with this bacterium allows <italic>B. oleae</italic> to exploit olives as a food source by detoxifying plant defense compounds (<xref rid=\"B21\" ref-type=\"bibr\">Ben-Yosef et al., 2015</xref>) and providing additional nutrition (<xref rid=\"B20\" ref-type=\"bibr\">Ben-Yosef et al., 2014</xref>). “<italic>Candidatus</italic> Erwinia dacicola” allows larvae of <italic>B. oleae</italic> to develop in unripe olives, which contain high concentrations of the toxin oleuropein (<xref rid=\"B21\" ref-type=\"bibr\">Ben-Yosef et al., 2015</xref>; <xref rid=\"B74\" ref-type=\"bibr\">Pavlidi et al., 2017</xref>). As such, host-associated microbial communities seem to play an important role in the evolution and possibly speciation of the host (<xref rid=\"B100\" ref-type=\"bibr\">Zilber-Rosenberg and Rosenberg, 2008</xref>), in particular in fruit flies (<xref rid=\"B15\" ref-type=\"bibr\">Behar et al., 2008a</xref>; <xref rid=\"B18\" ref-type=\"bibr\">Ben-Yosef et al., 2010</xref>, <xref rid=\"B20\" ref-type=\"bibr\">2014</xref>, <xref rid=\"B21\" ref-type=\"bibr\">2015</xref>).</p><p>The bacterial genus <italic>Ochrobactrum</italic>, which in our study showed higher abundance in <italic>Z. cucurbitae</italic> in a number of interspecific pairwise comparisons, has often been reported as a plant endophyte of Cucurbitaceae (<xref rid=\"B95\" ref-type=\"bibr\">Weller et al., 2006</xref>; <xref rid=\"B2\" ref-type=\"bibr\">Akbaba and Ozaktan, 2018</xref>) and described in a number of cucurbit feeder fruit flies including <italic>Z. cucurbitae</italic> (<xref rid=\"B68\" ref-type=\"bibr\">Mishra and Sharma, 2018</xref>), <italic>Z. tau</italic> (pumpkin fly) (<xref rid=\"B57\" ref-type=\"bibr\">Khan et al., 2014</xref>; <xref rid=\"B77\" ref-type=\"bibr\">Prabhakar et al., 2013</xref>; <xref rid=\"B65\" ref-type=\"bibr\">Luo et al., 2018</xref>), and in the polyphagous <italic>B. tryoni</italic> (<xref rid=\"B56\" ref-type=\"bibr\">Jessup and Mccarthy, 1993</xref>). Similarly, the bacterial genus <italic>Rahnella</italic> which we consistently found in higher abundances in the gut microbiome of <italic>C. podocarpi</italic> has been reported in different species of bark beetles (<xref rid=\"B62\" ref-type=\"bibr\">Lacey et al., 2007</xref>; <xref rid=\"B88\" ref-type=\"bibr\">Vasanthakumar et al., 2009</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Brady et al., 2014</xref>; <xref rid=\"B53\" ref-type=\"bibr\">Hernández-García et al., 2017</xref>), many of which feed on bark of coniferous trees. While these beetles and <italic>C. podocarpi</italic> do not share a taxonomic link, they do share a similar host: <italic>C. podocarpi</italic> exclusively targets members of the family Podocarpaceae, which also belong to the group of conifer trees. Even more so, the fruits of <italic>Afrocarpus falcatus</italic> (syn. <italic>Podocarpus falcatus</italic>) are known to be edible, but very resinous (source ICRAF Agroforestree Database; <xref rid=\"B71\" ref-type=\"bibr\">Oduol et al., 1988</xref>). The presence of <italic>Rahnella</italic> in the gut of these insects could be linked to the presence of this resin, which is also found plentiful in other coniferous trees.</p></sec><sec id=\"S5\"><title>Conclusion</title><p>Consistent with literature, we found that the gut microbiome of all fruit fly species included in the present study, was composed mainly of members of the bacterial phyla Proteobacteria and Firmicutes. At family level, we found that the family of Enterobacteriaceae was the dominant component in most species, except in <italic>C. quilicii</italic> and <italic>C. rosa</italic> where Acetobacteraceae was the dominant bacterial family. Despite heterogeneous abundances, we consistently observed Enterobacteriaceae across all samples, making it the single bacterial family that could be considered a part of the “core” gut microbiome. At genus level and ASV level, we observed a high variability both between fruit fly species (regardless of fruit fly genus) and within species. As such, we could not identify “core” gut microbiome members at genus or ASV levels that were shared across the targeted fruit flies. The few bacterial genera (<italic>Klebsiella</italic>, <italic>Enterobacter</italic> and <italic>Bacillus</italic>) that were present in most samples, showed a high variability in their relative abundance. Interestingly, we observed that interspecific variation of microbiome profiles was significantly affected by larval diet only in a part of the targeted fruit fly species (i.e., the most polyphagous ones, <italic>B. dorsalis</italic> and <italic>C. capitata</italic>), and that the observed patterns were geographically consistent. Finally, we could identify a number of bacterial genera (such as <italic>Erwinia</italic>, <italic>Ochrobactrum</italic> and <italic>Rahnella</italic>) that were consistently associated with particular fruit fly species (respectively <italic>B. oleae</italic>, <italic>Z. cucurbitae</italic>, and <italic>C. podocarpi</italic>). With these results, the present study provides a first comparative analysis of the gut microbiome of major fruit fly pests as well as, new base line information for future studies that will further investigate the functional role of the described associations.</p></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/\">https://www.ncbi.nlm.nih.gov/</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SRR8741994\">SRR8741994</ext-link> to <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SRR8742034\">SRR8742034</ext-link>.</p></sec><sec id=\"S7\"><title>Author Contributions</title><p>MDM, AW, MV, KB, and PV designed the research and secured the funding. MDC and MV designed and performed the experiments. MDC and MV analyzed the data with input from all other authors. MDC drafted the manuscript. All authors proofread, edited, and approved the manuscript.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This study was funded by the Belgian Federal Science Policy administration (BelSPO) BRAIN-be program (grant BR/154/PI/SYMDIV) and co-funded by the Coordinated Research Project (CRP) “SIT and Related Technologies to Manage Major Insect Plant Pests” (D40045) of the International Atomic Energy Agency.</p></fn></fn-group><ack><p>We would like to thank the following research institutions for their contribution to the collection of fruit fly samples: University of Thessaly, Greece; Sokoine University of Agriculture, Tanzania; Wilberforce Okeka and International Center for Insect Physiology and Ecology, Kenya; E. Mondlane University, Mozambique; CIRAD, La Réunion France; University of Pretoria, South Africa; and Citrus Research International (Nelspruit), South Africa.</p></ack><fn-group><fn id=\"footnote1\"><label>1</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://bccm.belspo.be/\">http://bccm.belspo.be/</ext-link></p></fn></fn-group><sec id=\"S10\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fmicb.2020.01890/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fmicb.2020.01890/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"FS1\"><label>FIGURE S1</label><caption><p>Bubble plot representing the bacterial phylum composition per sample. Bubble color: number of bacterial genera per phylum; Bubble size: Log of reads per phylum; X-axis: Sample labels build with a consistent structure: XX_YY_ZZ in which XX is fruit fly species, YY is sample location and ZZ is host plant.</p></caption><media xlink:href=\"Image_1.JPEG\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS2\"><label>FIGURE S2</label><caption><p>Relative abundances (%, as estimated from number of reads) of the dominant bacterial families per fruit fly species. Error bars (SD) as calculated from averaged three replicates per species are indicated.</p></caption><media xlink:href=\"Image_2.JPEG\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS1\"><label>TABLE S1</label><caption><p>Overview of sample design implemented in this study. Blue: balanced experiment including five species (<italic>B. dorsalis, Z. cucurbitae, B. oleae, C. capitata, C. quilicii</italic>) sampled in two locations from two host plants within each location (three replicate samples for each host plant species); Gray: additional samples complementing the balanced experiment and including additional fruit fly species (and including <italic>B. zonata, C. cosyra, C. rosa, C. flexuosa, C. podocarpi</italic>), locations and host plants. Blue = dataset A; Blue + Gray = dataset B.</p></caption><media xlink:href=\"Table_1.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS2\"><label>TABLE S2</label><caption><p>Collection data of samples considered in this study.</p></caption><media xlink:href=\"Table_2.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS3\"><label>TABLE S3</label><caption><p>Detailed list ofthe strain composition of the MOCK community.</p></caption><media xlink:href=\"Table_3.XLSX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS4\"><label>TABLE S4</label><caption><p>Detailed analytical pipeline implemented for genomic data filtering and taxon assignment.</p></caption><media xlink:href=\"Table_4.docx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS5\"><label>TABLE S5</label><caption><p>Identification and number of reads for ASVs that were detected in the negative control.</p></caption><media xlink:href=\"Table_5.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS6\"><label>TABLE S6</label><caption><p><italic>A posteriori</italic> pairwise comparisons (permutational t-statistics) for the significant effects detected by the PERMANOVA test reported in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> (fourth root transformed data; dataset A). ‘***’ = <italic>p</italic> < 0.001, ‘**’ = <italic>p</italic> < 0.01, ‘**’ = <italic>p</italic> < 0.05, ‘n.s.’ = <italic>p</italic> > 0.05.</p></caption><media xlink:href=\"Table_6.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS7\"><label>TABLE S7</label><caption><p><italic>A posteriori</italic> pairwise comparisons (permutational t-statistics) for the significant effects detected by the PERMANOVA test reported in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> (presence-absence data; dataset A). ‘***’ = <italic>p</italic> < 0.001, ‘**’ = <italic>p</italic> < 0.01 ‘*’ = <italic>p</italic> < 0.05, ‘n.s.’ = <italic>p</italic> > 0.05.</p></caption><media xlink:href=\"Table_7.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS8\"><label>TABLE S8</label><caption><p><italic>A posteriori</italic> pairwise comparisons (permutational t-statistics) for the significant effects detected by the PERMDISP test reported in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref> (fourth root transformed data; dataset A). ‘***’ = <italic>p</italic> < 0.001, ‘**’ = <italic>p</italic> < 0.01, ‘*’ = <italic>p</italic> < 0.05, ‘n.s.’ = <italic>p</italic> > 0.05.</p></caption><media xlink:href=\"Table_8.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS9\"><label>TABLE S9</label><caption><p><italic>A posteriori</italic> pairwise comparisons (permutational t-statistics) for the significant effects detected by the PERMDISP test reported in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref> (presence-absence data; dataset A). ‘***’ = <italic>p</italic> < 0.001, ‘**’ = <italic>p</italic> < 0.01, ‘*’ = <italic>p</italic> < 0.05, ‘n.s.’ = <italic>p</italic> > 0.05.</p></caption><media xlink:href=\"Table_9.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS10\"><label>TABLE S10</label><caption><p><italic>A posteriori</italic> pairwise comparisons (Student-Newman-Keuls test) for the significant effects detected by the ANOVA test reported in <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>. ‘***’ = <italic>p</italic> < 0.001, ‘**’ = <italic>p</italic> < 0.01, ‘*’ = <italic>p</italic> < 0.05, ‘n.s.’ = <italic>p</italic> > 0.05 (Dataset A).</p></caption><media xlink:href=\"Table_10.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS11\"><label>TABLE S11</label><caption><p>Most abundant bacterial genera observed in each fruit fly species (% as estimated from number of reads).</p></caption><media xlink:href=\"Table_11.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS12\"><label>TABLE S12</label><caption><p>Permutational SIMPER analysis (10,000 iterations; dataset B) of the microbiome assemblages of 10 fruit fly species (<italic>C. capitata, C. flexuosa, C. podocarpi, C. quilicii, C. rosa, C.cosyra, B. dorsalis, B. oleae, B. zonata, Z. cucurbitae</italic>). sd: standard deviation of contribution to dissimilarity between groups; ratio: average to sd ratio; FDR p: probability value after False Discovery Rate correction (<xref rid=\"B17\" ref-type=\"bibr\">Benjamini and Hochberg, 1995</xref>); *: FDR p < 0.05, **: FDR p < 0.01. Results are reported for bacterial genera contributing up to 95% of dissimilarity between fruit fly species. Bacterial genera with contribution to average group dissimilarity >5% and significant FDR p are highlighted in blue.</p></caption><media xlink:href=\"Table_12.XLSX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Abdi</surname><given-names>H.</given-names></name><name><surname>Williams</surname><given-names>L.</given-names></name></person-group> (<year>2010</year>). “<article-title>Tukey’s honestly signiflcant difierence (HSD) test</article-title>,” in <source><italic>Encyclopedia of Research Design</italic></source>, <role>ed.</role>\n<person-group person-group-type=\"editor\"><name><surname>Salkind</surname><given-names>N. J.</given-names></name></person-group> (<publisher-loc>Thousand Oaks, CA</publisher-loc>: <publisher-name>SAGA Plubications, Inc</publisher-name>), <fpage>1</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.4135/9781412961288.n181</pub-id></mixed-citation></ref><ref id=\"B2\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Akbaba</surname><given-names>M.</given-names></name><name><surname>Ozaktan</surname><given-names>H.</given-names></name></person-group> (<year>2018</year>). <article-title>Biocontrol of angular leaf spot disease and colonization of cucumber (<italic>Cucumis sativus</italic> L.) by endophytic bacteria.</article-title>\n<source><italic>Egypt. J. Biol. Pest Control</italic></source>\n<volume>28</volume>\n<fpage>1</fpage>–<lpage>10</lpage>. <pub-id pub-id-type=\"doi\">10.1186/s41938-017-0020-1</pub-id></mixed-citation></ref><ref id=\"B3\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aluja</surname><given-names>M.</given-names></name><name><surname>Mangan</surname><given-names>R. L</given-names></name></person-group> (<year>2008</year>). <article-title>Fruit fly (<italic>Diptera</italic>: <italic>Tephritidae</italic>) host stats determination: critical conceptual, methodological, and regulatory considerations.</article-title>\n<source><italic>Annu. Rev. Entomol.</italic></source>\n<volume>53</volume>\n<fpage>473</fpage>–<lpage>502</lpage>. <pub-id pub-id-type=\"doi\">10.1146/annurev.ento.53.103106.093350</pub-id>\n<pub-id pub-id-type=\"pmid\">17877455</pub-id></mixed-citation></ref><ref id=\"B4\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Aluja</surname><given-names>M.</given-names></name><name><surname>Norrbom</surname><given-names>A.</given-names></name></person-group> (<year>1999</year>). <source><italic>Fruit Flies (Tephritidae): Phylogeny and Evolution of Behavior.</italic></source>\n<publisher-loc>Boca Raton</publisher-loc>: <publisher-name>Taylor & Francis</publisher-name>, <pub-id pub-id-type=\"doi\">10.1201/9781420074468</pub-id></mixed-citation></ref><ref id=\"B5\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Alvarez</surname><given-names>S.</given-names></name><name><surname>Evans</surname><given-names>E. A.</given-names></name><name><surname>Hodges</surname><given-names>A. W.</given-names></name></person-group> (<year>2016</year>). <source><italic>Estimated Costs and Regional Economic Impacts of the Oriental Fruit Fly Outbreak in Miami-Dade County, Florida.</italic></source>\n<publisher-loc>Edis, FL</publisher-loc>: <publisher-name>UF/IFAS Extension</publisher-name>.</mixed-citation></ref><ref id=\"B6\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Anderson</surname><given-names>M. J.</given-names></name></person-group> (<year>2001</year>). <article-title>A new method for non-parametric multivariate analysis of variance.</article-title>\n<source><italic>Austral Ecol.</italic></source>\n<volume>26</volume>\n<fpage>32</fpage>–<lpage>46</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1442-9993.2001.01070.pp.x</pub-id></mixed-citation></ref><ref id=\"B7\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Anderson</surname><given-names>M. J.</given-names></name></person-group> (<year>2006</year>). <article-title>Distance-based tests for homogeneity of multivariate dispersions.</article-title>\n<source><italic>Biometrics</italic></source>\n<volume>62</volume>\n<fpage>245</fpage>–<lpage>253</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1541-0420.2005.00440.x</pub-id>\n<pub-id pub-id-type=\"pmid\">16542252</pub-id></mixed-citation></ref><ref id=\"B8\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Anderson</surname><given-names>M. J.</given-names></name></person-group> (<year>2017</year>). <article-title>Permutational multivariate analysis of variance (PERMANOVA).</article-title>\n<source><italic>Wiley StatsRef Stat. Ref. Online</italic></source>\n<fpage>1</fpage>–<lpage>15</lpage>. <pub-id pub-id-type=\"doi\">10.1002/9781118445112.stat07841</pub-id></mixed-citation></ref><ref id=\"B9\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Andongma</surname><given-names>A. A.</given-names></name><name><surname>Wan</surname><given-names>L.</given-names></name><name><surname>Dong</surname><given-names>Y.-C.</given-names></name><name><surname>Li</surname><given-names>P.</given-names></name><name><surname>Desneux</surname><given-names>N.</given-names></name><name><surname>White</surname><given-names>J. A.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>Pyrosequencing reveals a shift in symbiotic bacteria populations across life stages of <italic>Bactrocera dorsalis</italic>.</article-title>\n<source><italic>Sci. Rep.</italic></source>\n<volume>5</volume>:<issue>9470</issue>. <pub-id pub-id-type=\"doi\">10.1038/srep09470</pub-id>\n<pub-id pub-id-type=\"pmid\">25822599</pub-id></mixed-citation></ref><ref id=\"B10\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Andrews</surname><given-names>S.</given-names></name></person-group> (<year>2014</year>). <source><italic>FastQC a Quality Control tool for High Throughput Sequence Data.</italic></source>\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.bioinformatics.babraham.ac.uk/projects/fastqc/\">http://www.bioinformatics.babraham.ac.uk/projects/fastqc/</ext-link></mixed-citation></ref><ref id=\"B11\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Asimakis</surname><given-names>E. D.</given-names></name><name><surname>Khan</surname><given-names>M.</given-names></name><name><surname>Stathopoulou</surname><given-names>P.</given-names></name><name><surname>Caceres</surname><given-names>C.</given-names></name><name><surname>Bourtzis</surname><given-names>K.</given-names></name><name><surname>Tsiamis</surname><given-names>G.</given-names></name></person-group> (<year>2019</year>). <article-title>The effect of diet and radiation on the bacterial symbiome of the melon fly, <italic>Zeugodacus cucurbitae</italic> (Coquillett).</article-title>\n<source><italic>BMC Biotechnol.</italic></source>\n<volume>19</volume>:<issue>88</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12896-019-0578-7</pub-id>\n<pub-id pub-id-type=\"pmid\">31847902</pub-id></mixed-citation></ref><ref id=\"B12\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Augustinos</surname><given-names>A. A.</given-names></name><name><surname>Kyritsis</surname><given-names>G. A.</given-names></name><name><surname>Papadopoulos</surname><given-names>N. T.</given-names></name><name><surname>Abd-Alla</surname><given-names>A. M. M.</given-names></name><name><surname>Cáceres</surname><given-names>C.</given-names></name><name><surname>Bourtzis</surname><given-names>K.</given-names></name></person-group> (<year>2015</year>). <article-title>Exploitation of the medfly gut microbiota for the enhancement of sterile insect technique: use of <italic>Enterobacter</italic> sp. in larval diet-based probiotic applications.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>10</volume>:<issue>e0136459</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0136459</pub-id>\n<pub-id pub-id-type=\"pmid\">26325068</pub-id></mixed-citation></ref><ref id=\"B13\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Augustinos</surname><given-names>A. A.</given-names></name><name><surname>Tsiamis</surname><given-names>G.</given-names></name><name><surname>Cáceres</surname><given-names>C.</given-names></name><name><surname>Abd-Alla</surname><given-names>A. M. M.</given-names></name><name><surname>Bourtzis</surname><given-names>K.</given-names></name></person-group> (<year>2019</year>). <article-title>Taxonomy, diet, and developmental stage contribute to the structuring of gut-associated bacterial communities in tephritid pest species.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>10</volume>:<issue>2004</issue>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2019.02004</pub-id>\n<pub-id pub-id-type=\"pmid\">31555239</pub-id></mixed-citation></ref><ref id=\"B14\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Badii</surname><given-names>K. B.</given-names></name><name><surname>Billah</surname><given-names>M. K.</given-names></name><name><surname>Afreh Nuamah</surname><given-names>K.</given-names></name><name><surname>Obeng Ofori</surname><given-names>D.</given-names></name><name><surname>Nyarko</surname><given-names>G.</given-names></name></person-group> (<year>2015</year>). <article-title>Review of the pest status, economic impact and management of fruit-infesting flies (<italic>Diptera</italic>: <italic>Tephritidae</italic>) in Africa.</article-title>\n<source><italic>African J. Agric. Res.</italic></source>\n<volume>10</volume>\n<fpage>1488</fpage>–<lpage>1498</lpage>. <pub-id pub-id-type=\"doi\">10.5897/AJAR2014.9278</pub-id></mixed-citation></ref><ref id=\"B15\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Behar</surname><given-names>A.</given-names></name><name><surname>Jurkevitch</surname><given-names>E.</given-names></name><name><surname>Yuval</surname><given-names>B.</given-names></name></person-group> (<year>2008a</year>). <article-title>Bringing back the fruit into fruit fly-bacteria interactions.</article-title>\n<source><italic>Mol. Ecol.</italic></source>\n<volume>17</volume>\n<fpage>1375</fpage>–<lpage>1386</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-294X.2008.03674.x</pub-id>\n<pub-id pub-id-type=\"pmid\">18302695</pub-id></mixed-citation></ref><ref id=\"B16\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Behar</surname><given-names>A.</given-names></name><name><surname>Yuval</surname><given-names>B.</given-names></name><name><surname>Jurkevitch</surname><given-names>E.</given-names></name></person-group> (<year>2008b</year>). <article-title>Gut bacterial communities in the Mediterranean fruit fly (<italic>Ceratitis capitata</italic>) and their impact on host longevity.</article-title>\n<source><italic>J. Insect Physiol.</italic></source>\n<volume>54</volume>\n<fpage>1377</fpage>–<lpage>1383</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jinsphys.2008.07.011</pub-id>\n<pub-id pub-id-type=\"pmid\">18706909</pub-id></mixed-citation></ref><ref id=\"B17\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Benjamini</surname><given-names>Y.</given-names></name><name><surname>Hochberg</surname><given-names>Y.</given-names></name></person-group> (<year>1995</year>). <article-title>Controlling the false discovery rate?: a practical and powerful approach to multiple testing.</article-title>\n<source><italic>J. R. Stat. Soc. Ser. B</italic></source>\n<volume>57</volume>\n<fpage>289</fpage>–<lpage>300</lpage>. <pub-id pub-id-type=\"doi\">10.2307/2346101</pub-id></mixed-citation></ref><ref id=\"B18\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ben-Yosef</surname><given-names>M.</given-names></name><name><surname>Aharon</surname><given-names>Y.</given-names></name><name><surname>Jurkevitch</surname><given-names>E.</given-names></name><name><surname>Yuval</surname><given-names>B.</given-names></name></person-group> (<year>2010</year>). <article-title>Give us the tools and we will do the job: symbiotic bacteria affect olive fly fitness in a diet-dependent fashion.</article-title>\n<source><italic>Proc. R. Soc. Biol. Sci.</italic></source>\n<volume>277</volume>\n<fpage>1545</fpage>–<lpage>1552</lpage>. <pub-id pub-id-type=\"doi\">10.1098/rspb.2009.2102</pub-id>\n<pub-id pub-id-type=\"pmid\">20071385</pub-id></mixed-citation></ref><ref id=\"B19\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ben-Yosef</surname><given-names>M.</given-names></name><name><surname>Jurkevitch</surname><given-names>E.</given-names></name><name><surname>Yuval</surname><given-names>B.</given-names></name></person-group> (<year>2008</year>). <article-title>Effect of bacteria on nutritional status and reproductive success of the Mediterranean fruit fly <italic>Ceratitis capitata</italic>.</article-title>\n<source><italic>Physiol. Entomol.</italic></source>\n<volume>33</volume>\n<fpage>145</fpage>–<lpage>154</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-3032.2008.00617.x</pub-id></mixed-citation></ref><ref id=\"B20\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ben-Yosef</surname><given-names>M.</given-names></name><name><surname>Pasternak</surname><given-names>Z.</given-names></name><name><surname>Jurkevitch</surname><given-names>E.</given-names></name><name><surname>Yuval</surname><given-names>B.</given-names></name></person-group> (<year>2014</year>). <article-title>Symbiotic bacteria enable olive flies (<italic>Bactrocera oleae</italic>) to exploit intractable sources of nitrogen.</article-title>\n<source><italic>J. Evol. Biol.</italic></source>\n<volume>27</volume>\n<fpage>2695</fpage>–<lpage>2705</lpage>. <pub-id pub-id-type=\"doi\">10.1111/jeb.12527</pub-id>\n<pub-id pub-id-type=\"pmid\">25403559</pub-id></mixed-citation></ref><ref id=\"B21\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ben-Yosef</surname><given-names>M.</given-names></name><name><surname>Pasternak</surname><given-names>Z.</given-names></name><name><surname>Jurkevitch</surname><given-names>E.</given-names></name><name><surname>Yuval</surname><given-names>B.</given-names></name></person-group> (<year>2015</year>). <article-title>Symbiotic bacteria enable olive fly larvae to overcome host defences.</article-title>\n<source><italic>R. Soc. open Sci.</italic></source>\n<volume>2</volume>:<issue>150170</issue>. <pub-id pub-id-type=\"doi\">10.1098/rsos.150170</pub-id>\n<pub-id pub-id-type=\"pmid\">26587275</pub-id></mixed-citation></ref><ref id=\"B22\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Blow</surname><given-names>F.</given-names></name><name><surname>Gioti</surname><given-names>A.</given-names></name><name><surname>Starns</surname><given-names>D.</given-names></name><name><surname>Ben-Yosef</surname><given-names>M.</given-names></name><name><surname>Pasternak</surname><given-names>Z.</given-names></name><name><surname>Jurkevitch</surname><given-names>E.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>Draft genome sequence of the <italic>Bactrocera oleae</italic> symbiont “candidatus erwinia dacicola”.</article-title>\n<source><italic>Genome Announc.</italic></source>\n<volume>4</volume>:<issue>e896-16</issue>. <pub-id pub-id-type=\"doi\">10.1128/genomeA.00896-16</pub-id>\n<pub-id pub-id-type=\"pmid\">27634990</pub-id></mixed-citation></ref><ref id=\"B23\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Bourtzis</surname><given-names>K.</given-names></name><name><surname>Miller</surname><given-names>T.</given-names></name></person-group> (<year>2003</year>). <source><italic>Insect Symbiosis.</italic></source>\n<publisher-loc>Boca Raton</publisher-loc>: <publisher-name>CRC Press</publisher-name>.</mixed-citation></ref><ref id=\"B24\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brady</surname><given-names>C.</given-names></name><name><surname>Hunter</surname><given-names>G.</given-names></name><name><surname>Kirk</surname><given-names>S.</given-names></name><name><surname>Arnold</surname><given-names>D.</given-names></name><name><surname>Denman</surname><given-names>S.</given-names></name></person-group> (<year>2014</year>). <article-title><italic>Rahnella</italic> victoriana sp. nov., <italic>Rahnella</italic> bruchi sp. nov., <italic>Rahnella</italic> woolbedingensis sp. nov., classification of <italic>Rahnella</italic> genomospecies 2 and 3 as <italic>Rahnella</italic> variigena sp. nov. and <italic>Rahnella</italic> inusitata sp. nov., respectively and emended description of the genus.</article-title>\n<source><italic>Syst. Appl. Microbiol.</italic></source>\n<volume>37</volume>\n<fpage>545</fpage>–<lpage>552</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.syapm.2014.09.001</pub-id>\n<pub-id pub-id-type=\"pmid\">25264035</pub-id></mixed-citation></ref><ref id=\"B25\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bray</surname><given-names>J. R.</given-names></name><name><surname>Curtis</surname><given-names>J. T.</given-names></name></person-group> (<year>1957</year>). <article-title>An ordination of the upland forest communities of southern Wisconsin.</article-title>\n<source><italic>Ecol. Monogr.</italic></source>\n<volume>27</volume>\n<fpage>325</fpage>–<lpage>349</lpage>. <pub-id pub-id-type=\"doi\">10.2307/1942268</pub-id></mixed-citation></ref><ref id=\"B26\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brucker</surname><given-names>R. M.</given-names></name><name><surname>Bordenstein</surname><given-names>S. R.</given-names></name></person-group> (<year>2012</year>). <article-title>The roles of host evolutionary relationships (Genus: <italic>Nasonia</italic>) and development in structuring microbial communities.</article-title>\n<source><italic>Evolution</italic></source>\n<volume>66</volume>\n<fpage>349</fpage>–<lpage>362</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1558-5646.2011.01454.x</pub-id>\n<pub-id pub-id-type=\"pmid\">22276533</pub-id></mixed-citation></ref><ref id=\"B27\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cáceres</surname><given-names>C.</given-names></name><name><surname>Tsiamis</surname><given-names>G.</given-names></name><name><surname>Yuval</surname><given-names>B.</given-names></name><name><surname>Jurkevitch</surname><given-names>E.</given-names></name><name><surname>Bourtzis</surname><given-names>K.</given-names></name></person-group> (<year>2019</year>). <article-title>Joint FAO/IAEA Coordinated Research Project on ‘Use of symbiotic bacteria to reduce mass-rearing costs and increase mating success in selected fruit pests in support of SIT application.</article-title>\n<source><italic>BMC Microbiol.</italic></source>\n<volume>19</volume>:<issue>284</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12866-019-1644-y</pub-id>\n<pub-id pub-id-type=\"pmid\">31870284</pub-id></mixed-citation></ref><ref id=\"B28\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Callahan</surname><given-names>B. J.</given-names></name><name><surname>McMurdie</surname><given-names>P. J.</given-names></name><name><surname>Rosen</surname><given-names>M. J.</given-names></name><name><surname>Han</surname><given-names>A. W.</given-names></name><name><surname>Johnson</surname><given-names>A. J. A.</given-names></name><name><surname>Holmes</surname><given-names>S. P.</given-names></name></person-group> (<year>2016</year>). <article-title>DADA2: high-resolution sample inference from Illumina amplicon data.</article-title>\n<source><italic>Nat. Methods</italic></source>\n<volume>13</volume>\n<fpage>581</fpage>–<lpage>583</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nmeth.3869</pub-id>\n<pub-id pub-id-type=\"pmid\">27214047</pub-id></mixed-citation></ref><ref id=\"B29\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Capuzzo</surname><given-names>C.</given-names></name><name><surname>Firrao</surname><given-names>G.</given-names></name><name><surname>Mazzon</surname><given-names>L.</given-names></name><name><surname>Squartini</surname><given-names>A.</given-names></name><name><surname>Girolami</surname><given-names>V.</given-names></name></person-group> (<year>2005</year>). <article-title>“<italic>Candidatus</italic> Erwinia dacicola”, a coevolved symbiotic bacterium of the olive fly <italic>Bactrocera oleae</italic> (Gmelin).</article-title>\n<source><italic>Int. J. Syst. Evol. Microbiol.</italic></source>\n<volume>55</volume>\n<fpage>1641</fpage>–<lpage>1647</lpage>. <pub-id pub-id-type=\"doi\">10.1099/ijs.0.63653-0</pub-id>\n<pub-id pub-id-type=\"pmid\">16014495</pub-id></mixed-citation></ref><ref id=\"B30\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>R.</given-names></name><name><surname>Wang</surname><given-names>Z.</given-names></name><name><surname>Chen</surname><given-names>J.</given-names></name><name><surname>Jiang</surname><given-names>L.-Y.</given-names></name><name><surname>Qiao</surname><given-names>G.-X.</given-names></name></person-group> (<year>2017</year>). <article-title>Insect-bacteria parallel evolution in multiple-co-obligate-aphid association: a case in <italic>Lachninae</italic> (<italic>Hemiptera</italic>: <italic>Aphididae</italic>).</article-title>\n<source><italic>Sci. Rep.</italic></source>\n<volume>7</volume>:<issue>10204</issue>. <pub-id pub-id-type=\"doi\">10.1038/s41598-017-10761-9</pub-id>\n<pub-id pub-id-type=\"pmid\">28860659</pub-id></mixed-citation></ref><ref id=\"B31\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cheng</surname><given-names>D.</given-names></name><name><surname>Guo</surname><given-names>Z.</given-names></name><name><surname>Riegler</surname><given-names>M.</given-names></name><name><surname>Xi</surname><given-names>Z.</given-names></name><name><surname>Liang</surname><given-names>G.</given-names></name><name><surname>Xu</surname><given-names>Y.</given-names></name></person-group> (<year>2017</year>). <article-title>Gut symbiont enhances insecticide resistance in a significant pest, the oriental fruit fly <italic>Bactrocera dorsalis</italic> (Hendel).</article-title>\n<source><italic>Microbiome</italic></source>\n<volume>5</volume>:<issue>13</issue>. <pub-id pub-id-type=\"doi\">10.1186/s40168-017-0236-z</pub-id>\n<pub-id pub-id-type=\"pmid\">28143582</pub-id></mixed-citation></ref><ref id=\"B32\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Christenson</surname><given-names>L. D.</given-names></name><name><surname>Foote</surname><given-names>R. H.</given-names></name></person-group> (<year>1960</year>). <article-title>Biology of fruit flies.</article-title>\n<source><italic>Annu. Rev. Entomol.</italic></source>\n<volume>5</volume>\n<fpage>171</fpage>–<lpage>192</lpage>. <pub-id pub-id-type=\"doi\">10.1146/annurev.en.05.010160.001131</pub-id></mixed-citation></ref><ref id=\"B33\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Clarke</surname><given-names>K. R.</given-names></name></person-group> (<year>1993</year>). <article-title>Non-parametric multivariate analyses of changes in community structure.</article-title>\n<source><italic>Aust. J. Ecol.</italic></source>\n<volume>18</volume>\n<fpage>117</fpage>–<lpage>143</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1442-9993.1993.tb00438.x</pub-id></mixed-citation></ref><ref id=\"B34\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cole</surname><given-names>J. R.</given-names></name><name><surname>Wang</surname><given-names>Q.</given-names></name><name><surname>Fish</surname><given-names>J. A.</given-names></name><name><surname>Chai</surname><given-names>B.</given-names></name><name><surname>McGarrell</surname><given-names>D. M.</given-names></name><name><surname>Sun</surname><given-names>Y.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Ribosomal database project: data and tools for high throughput rRNA analysis.</article-title>\n<source><italic>Nucleic Acids Res.</italic></source>\n<volume>42</volume>\n<fpage>633</fpage>–<lpage>642</lpage>. <pub-id pub-id-type=\"doi\">10.1093/nar/gkt1244</pub-id>\n<pub-id pub-id-type=\"pmid\">24288368</pub-id></mixed-citation></ref><ref id=\"B35\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Colman</surname><given-names>D. R.</given-names></name><name><surname>Toolson</surname><given-names>E. C.</given-names></name><name><surname>Takacs-Vesbach</surname><given-names>C. D.</given-names></name></person-group> (<year>2012</year>). <article-title>Do diet and taxonomy influence insect gut bacterial communities?</article-title>\n<source><italic>Mol. Ecol.</italic></source>\n<volume>21</volume>\n<fpage>5124</fpage>–<lpage>5137</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-294X.2012.05752.x</pub-id>\n<pub-id pub-id-type=\"pmid\">22978555</pub-id></mixed-citation></ref><ref id=\"B36\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>de Cárcer</surname><given-names>D. A.</given-names></name><name><surname>Denman</surname><given-names>S. E.</given-names></name><name><surname>McSweeney</surname><given-names>C.</given-names></name><name><surname>Morrison</surname><given-names>M.</given-names></name></person-group> (<year>2011</year>). <article-title>Evaluation of subsampling-based normalization strategies for tagged high-throughput sequencing data sets from gut microbiomes.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>77</volume>\n<fpage>8795</fpage>–<lpage>8798</lpage>. <pub-id pub-id-type=\"doi\">10.1128/AEM.05491-11</pub-id>\n<pub-id pub-id-type=\"pmid\">21984239</pub-id></mixed-citation></ref><ref id=\"B37\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Cock</surname><given-names>M.</given-names></name><name><surname>Virgilio</surname><given-names>M.</given-names></name><name><surname>Vandamme</surname><given-names>P.</given-names></name><name><surname>Augustinos</surname><given-names>A. A.</given-names></name><name><surname>Bourtzis</surname><given-names>K.</given-names></name><name><surname>Willems</surname><given-names>A.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Impact of sample preservation and manipulation on insect gut microbiome profiling. A test case with fruit flies (<italic>Diptera</italic>, <italic>Tephritidae</italic>).</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>10</volume>:<issue>2833</issue>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2019.02833</pub-id>\n<pub-id pub-id-type=\"pmid\">31921020</pub-id></mixed-citation></ref><ref id=\"B38\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Meyer</surname><given-names>M.</given-names></name><name><surname>Delatte</surname><given-names>H.</given-names></name><name><surname>Mwatawala</surname><given-names>M.</given-names></name><name><surname>Quilici</surname><given-names>S.</given-names></name><name><surname>Vayssières</surname><given-names>J.-F.</given-names></name><name><surname>Virgilio</surname><given-names>M.</given-names></name></person-group> (<year>2015</year>). <article-title>A review of the current knowledge on <italic>Zeugodacus cucurbitae</italic> (Coquillett) (<italic>Diptera</italic>, <italic>Tephritidae</italic>) in Africa, with a list of species included in <italic>Zeugodacus</italic>.</article-title>\n<source><italic>Zookeys</italic></source>\n<volume>26</volume>\n<fpage>539</fpage>–<lpage>557</lpage>. <pub-id pub-id-type=\"doi\">10.3897/zookeys.540.9672</pub-id>\n<pub-id pub-id-type=\"pmid\">26798277</pub-id></mixed-citation></ref><ref id=\"B39\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Vries</surname><given-names>E. J.</given-names></name><name><surname>Jacobs</surname><given-names>G.</given-names></name><name><surname>Sabelis</surname><given-names>M. W.</given-names></name><name><surname>Menken</surname><given-names>S. B. J.</given-names></name><name><surname>Breeuwer</surname><given-names>J. A. J.</given-names></name></person-group> (<year>2004</year>). <article-title>Diet-dependent effects of gut bacteria on their insect host: the symbiosis of <italic>Erwinia</italic> sp. and western flower thrips.</article-title>\n<source><italic>R. Soc.</italic></source>\n<volume>271</volume>\n<fpage>2171</fpage>–<lpage>2178</lpage>. <pub-id pub-id-type=\"doi\">10.1098/rspb.2004.2817</pub-id>\n<pub-id pub-id-type=\"pmid\">15475338</pub-id></mixed-citation></ref><ref id=\"B40\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>DeSantis</surname><given-names>T. Z.</given-names></name><name><surname>Hugenholtz</surname><given-names>P.</given-names></name><name><surname>Larsen</surname><given-names>N.</given-names></name><name><surname>Rojas</surname><given-names>M.</given-names></name><name><surname>Brodie</surname><given-names>E. L.</given-names></name><name><surname>Keller</surname><given-names>K.</given-names></name><etal/></person-group> (<year>2006</year>). <article-title>Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>72</volume>\n<fpage>5069</fpage>–<lpage>5072</lpage>. <pub-id pub-id-type=\"doi\">10.1128/AEM.03006-05</pub-id>\n<pub-id pub-id-type=\"pmid\">16820507</pub-id></mixed-citation></ref><ref id=\"B41\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Després</surname><given-names>L.</given-names></name><name><surname>David</surname><given-names>J.-P.</given-names></name><name><surname>Gallet</surname><given-names>C.</given-names></name></person-group> (<year>2007</year>). <article-title>The evolutionary ecology of insect resistance to plant chemicals.</article-title>\n<source><italic>Trends Ecol. Evol.</italic></source>\n<volume>22</volume>\n<fpage>298</fpage>–<lpage>307</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.tree.2007.02.010</pub-id>\n<pub-id pub-id-type=\"pmid\">17324485</pub-id></mixed-citation></ref><ref id=\"B42\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Deutscher</surname><given-names>A. T.</given-names></name><name><surname>Burke</surname><given-names>C. M.</given-names></name><name><surname>Darling</surname><given-names>A. E.</given-names></name><name><surname>Riegler</surname><given-names>M.</given-names></name><name><surname>Reynolds</surname><given-names>O. L.</given-names></name><name><surname>Chapman</surname><given-names>T. A.</given-names></name></person-group> (<year>2018</year>). <article-title>Near full-length 16S rRNA gene next-generation sequencing revealed <italic>Asaia</italic> as a common midgut bacterium of wild and domesticated Queensland fruit fly larvae.</article-title>\n<source><italic>Microbiome</italic></source>\n<volume>6</volume>:<issue>85</issue>. <pub-id pub-id-type=\"doi\">10.1186/s40168-018-0463-y</pub-id>\n<pub-id pub-id-type=\"pmid\">29729663</pub-id></mixed-citation></ref><ref id=\"B43\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Douglas</surname><given-names>A. E.</given-names></name></person-group> (<year>2009</year>). <article-title>The microbial dimension in insect nutritional ecology.</article-title>\n<source><italic>Funct. Ecol.</italic></source>\n<volume>23</volume>\n<fpage>38</fpage>–<lpage>47</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-2435.2008.01442.x</pub-id></mixed-citation></ref><ref id=\"B44\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Douglas</surname><given-names>A. E.</given-names></name></person-group> (<year>2013</year>). <article-title>Microbial brokers of insect-plant interactions revisited.</article-title>\n<source><italic>J. Chem. Ecol.</italic></source>\n<volume>39</volume>\n<fpage>952</fpage>–<lpage>961</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s10886-013-0308-x</pub-id>\n<pub-id pub-id-type=\"pmid\">23793897</pub-id></mixed-citation></ref><ref id=\"B45\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Erbout</surname><given-names>N.</given-names></name><name><surname>Virgilio</surname><given-names>M.</given-names></name><name><surname>Lens</surname><given-names>L.</given-names></name><name><surname>Barr</surname><given-names>N.</given-names></name><name><surname>De Meyer</surname><given-names>M.</given-names></name></person-group> (<year>2011</year>). <article-title>Discrepancies between subgeneric classification and molecular phylogeny of Ceratitis (<italic>Diptera</italic>: <italic>Tephritidae</italic>), can the evolution of host use provide some clues?</article-title>\n<source><italic>Mol. Phylogenet. Evol.</italic></source>\n<volume>60</volume>\n<fpage>259</fpage>–<lpage>264</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ympev.2011.04.004</pub-id>\n<pub-id pub-id-type=\"pmid\">21545840</pub-id></mixed-citation></ref><ref id=\"B46\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Estes</surname><given-names>A. M.</given-names></name><name><surname>Hearn</surname><given-names>D. J.</given-names></name><name><surname>Bronstein</surname><given-names>J. L.</given-names></name><name><surname>Pierson</surname><given-names>E. A.</given-names></name></person-group> (<year>2009</year>). <article-title>The olive fly endosymbiont, “Candidatus Erwinia dacicola”, switches from an intracellular existence to an extracellular existence during host insect development.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>75</volume>\n<fpage>7097</fpage>–<lpage>7106</lpage>. <pub-id pub-id-type=\"doi\">10.1128/AEM.00778-09</pub-id>\n<pub-id pub-id-type=\"pmid\">19767463</pub-id></mixed-citation></ref><ref id=\"B47\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Estes</surname><given-names>A. M.</given-names></name><name><surname>Segura</surname><given-names>D. F.</given-names></name><name><surname>Jessup</surname><given-names>A.</given-names></name><name><surname>Wornoayporn</surname><given-names>V.</given-names></name><name><surname>Pierson</surname><given-names>E. A.</given-names></name></person-group> (<year>2014</year>). <article-title>Effect of the symbiont <italic>Candidatus</italic> Erwinia dacicola on mating success of the olive fly <italic>Bactrocera oleae</italic> (<italic>Diptera</italic>: <italic>Tephritidae</italic>).</article-title>\n<source><italic>Int. J. Trop. Insect Sci.</italic></source>\n<volume>34</volume>\n<fpage>S123</fpage>–<lpage>S131</lpage>. <pub-id pub-id-type=\"doi\">10.1017/S1742758414000174</pub-id></mixed-citation></ref><ref id=\"B48\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hadapad</surname><given-names>A. B.</given-names></name><name><surname>Prabhakar</surname><given-names>C. S.</given-names></name><name><surname>Chandekar</surname><given-names>S. C.</given-names></name><name><surname>Tripathi</surname><given-names>J.</given-names></name><name><surname>Hire</surname><given-names>R. S.</given-names></name></person-group> (<year>2015</year>). <article-title>Diversity of bacterial communities in the midgut of <italic>Bactrocera cucurbitae</italic> (<italic>Diptera</italic>: <italic>Tephritidae</italic>) populations and their potential use as attractants.</article-title>\n<source><italic>Pest Manag. Sci.</italic></source>\n<volume>72</volume>\n<fpage>1222</fpage>–<lpage>1230</lpage>. <pub-id pub-id-type=\"doi\">10.1002/ps.4102</pub-id>\n<pub-id pub-id-type=\"pmid\">26299539</pub-id></mixed-citation></ref><ref id=\"B49\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hafsi</surname><given-names>A.</given-names></name><name><surname>Facon</surname><given-names>B.</given-names></name><name><surname>Ravigné</surname><given-names>V.</given-names></name><name><surname>Chiroleu</surname><given-names>F.</given-names></name><name><surname>Quilici</surname><given-names>S.</given-names></name><name><surname>Chermiti</surname><given-names>B.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>Host plant range of a fruit fly community (<italic>Diptera</italic>: <italic>Tephritidae</italic>): does fruit composition influence larval performance?</article-title>\n<source><italic>BMC Ecol.</italic></source>\n<volume>16</volume>:<issue>40</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12898-016-0094-8</pub-id>\n<pub-id pub-id-type=\"pmid\">27650549</pub-id></mixed-citation></ref><ref id=\"B50\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hamden</surname><given-names>H.</given-names></name><name><surname>Guerfali</surname><given-names>M. M.</given-names></name><name><surname>Fadhl</surname><given-names>S.</given-names></name><name><surname>Saidi</surname><given-names>M.</given-names></name><name><surname>Chevrier</surname><given-names>C.</given-names></name></person-group> (<year>2013</year>). <article-title>Fitness improvement of mass-reared sterile males of <italic>Ceratitis capitata</italic> (Vienna 8 strain) (<italic>Diptera</italic>: <italic>Tephritidae</italic>) after gut enrichment with probiotics.</article-title>\n<source><italic>J. Econ. Entomol.</italic></source>\n<volume>106</volume>\n<fpage>641</fpage>–<lpage>647</lpage>. <pub-id pub-id-type=\"doi\">10.1603/ec12362</pub-id>\n<pub-id pub-id-type=\"pmid\">23786049</pub-id></mixed-citation></ref><ref id=\"B51\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hammer</surname><given-names>T. J.</given-names></name><name><surname>Bowers</surname><given-names>M. D.</given-names></name></person-group> (<year>2015</year>). <article-title>Gut microbes may facilitate insect herbivory of chemically defended plants.</article-title>\n<source><italic>Oecologia</italic></source>\n<volume>179</volume>\n<fpage>1</fpage>–<lpage>14</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00442-015-3327-1</pub-id>\n<pub-id pub-id-type=\"pmid\">25936531</pub-id></mixed-citation></ref><ref id=\"B52\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hansen</surname><given-names>A. K.</given-names></name><name><surname>Moran</surname><given-names>N. A.</given-names></name></person-group> (<year>2014</year>). <article-title>The impact of microbial symbionts on host plant utilization by herbivorous insects.</article-title>\n<source><italic>Mol. Ecol.</italic></source>\n<volume>23</volume>\n<fpage>1473</fpage>–<lpage>1496</lpage>. <pub-id pub-id-type=\"doi\">10.1111/mec.12421</pub-id>\n<pub-id pub-id-type=\"pmid\">23952067</pub-id></mixed-citation></ref><ref id=\"B53\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hernández-García</surname><given-names>J. A.</given-names></name><name><surname>Briones-Roblero</surname><given-names>C. I.</given-names></name><name><surname>Rivera-Orduña</surname><given-names>F. N.</given-names></name><name><surname>Zúñiga</surname><given-names>G.</given-names></name></person-group> (<year>2017</year>). <article-title>Revealing the gut bacteriome of <italic>Dendroctonus</italic> bark beetles (<italic>Curculionidae</italic>: <italic>Scolytinae</italic>): diversity, core members and co-evolutionary patterns.</article-title>\n<source><italic>Sci. Rep.</italic></source>\n<volume>7</volume>\n<fpage>1</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-017-14031-6</pub-id>\n<pub-id pub-id-type=\"pmid\">28127051</pub-id></mixed-citation></ref><ref id=\"B54\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Illumina</surname><given-names>I.</given-names></name></person-group> (<year>2016</year>). <source><italic>Nextera DNA Library Prep Protocol Guide.</italic></source> Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nexteradna/nextera-dna-library-prep-reference-guide-15027987-01.pdf\">https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/samplepreps_nextera/nexteradna/nextera-dna-library-prep-reference-guide-15027987-01.pdf</ext-link>\n<comment>(accessed December 15, 2019)</comment>.</mixed-citation></ref><ref id=\"B55\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Janson</surname><given-names>E. M.</given-names></name><name><surname>Stireman</surname><given-names>J. O.</given-names></name><name><surname>Singer</surname><given-names>M. S.</given-names></name><name><surname>Abbot</surname><given-names>P.</given-names></name></person-group> (<year>2008</year>). <article-title>Phytophagous insect-microbe mutualisms and adaptive evolutionary diversification.</article-title>\n<source><italic>Evolution</italic></source>\n<volume>62</volume>\n<fpage>997</fpage>–<lpage>1012</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1558-5646.2008.00348.x</pub-id>\n<pub-id pub-id-type=\"pmid\">18298649</pub-id></mixed-citation></ref><ref id=\"B56\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jessup</surname><given-names>A. J.</given-names></name><name><surname>Mccarthy</surname><given-names>D.</given-names></name></person-group> (<year>1993</year>). <article-title>Host status of some australian-grown cucurbits to <italic>Bactrocera tryoni</italic> (Froggatt) (<italic>Diptera</italic>, <italic>Tephritidae</italic>) under laboratory conditions.</article-title>\n<source><italic>J. Aust. Entomol. Soc.</italic></source>\n<volume>32</volume>\n<fpage>97</fpage>–<lpage>98</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1440-6055.1993.tb00552.x</pub-id></mixed-citation></ref><ref id=\"B57\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Khan</surname><given-names>M.</given-names></name><name><surname>Mahin</surname><given-names>A. A.</given-names></name><name><surname>Pramanik</surname><given-names>M. K.</given-names></name><name><surname>Akter</surname><given-names>H.</given-names></name></person-group> (<year>2014</year>). <article-title>Identification of gut bacterial community and their effect on the fecundity of pumpkin fly, Bactrocera tau (Walker).</article-title>\n<source><italic>J. Entomol.</italic></source>\n<volume>11</volume>\n<fpage>68</fpage>–<lpage>77</lpage>. <pub-id pub-id-type=\"doi\">10.3923/je.2014.68.77</pub-id></mixed-citation></ref><ref id=\"B58\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Koskinioti</surname><given-names>P.</given-names></name><name><surname>Ras</surname><given-names>E.</given-names></name><name><surname>Augustinos</surname><given-names>A. A.</given-names></name><name><surname>Tsiamis</surname><given-names>G.</given-names></name><name><surname>Beukeboom</surname><given-names>L. W.</given-names></name><name><surname>Caceres</surname><given-names>C.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>The effects of geographic origin and antibiotic treatment on the gut symbiotic communities of <italic>Bactrocera oleae</italic> populations.</article-title>\n<source><italic>Entomol. Exp. Appl.</italic></source>\n<volume>167</volume>\n<fpage>197</fpage>–<lpage>208</lpage>. <pub-id pub-id-type=\"doi\">10.1111/eea.12764</pub-id></mixed-citation></ref><ref id=\"B59\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuzina</surname><given-names>L. V.</given-names></name><name><surname>Peloquin</surname><given-names>J. J.</given-names></name><name><surname>Vacek</surname><given-names>D. C.</given-names></name><name><surname>Miller</surname><given-names>T. A.</given-names></name></person-group> (<year>2001</year>). <article-title>Isolation and identification of bacteria associated with adult laboratory Mexican fruit flies, <italic>Anastrepha ludens</italic> (<italic>Diptera</italic>: <italic>Tephritidae</italic>).</article-title>\n<source><italic>Curr. Microbiol.</italic></source>\n<volume>42</volume>\n<fpage>290</fpage>–<lpage>294</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s002840110219</pub-id>\n<pub-id pub-id-type=\"pmid\">11178731</pub-id></mixed-citation></ref><ref id=\"B60\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kyritsis</surname><given-names>G. A.</given-names></name><name><surname>Augustinos</surname><given-names>A. A.</given-names></name><name><surname>Cáceres</surname><given-names>C.</given-names></name><name><surname>Bourtzis</surname><given-names>K.</given-names></name></person-group> (<year>2017</year>). <article-title>Medfly gut microbiota and enhancement of the sterile insect technique: similarities and differences of <italic>Klebsiella oxytoca</italic> and <italic>Enterobacter</italic> sp. AA26 probiotics during the larval and adult stages of the VIENNA 8D53+ genetic sexing strain.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>8</volume>:<issue>2064</issue>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2017.02064</pub-id>\n<pub-id pub-id-type=\"pmid\">29163379</pub-id></mixed-citation></ref><ref id=\"B61\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kyritsis</surname><given-names>G. A.</given-names></name><name><surname>Augustinos</surname><given-names>A. A.</given-names></name><name><surname>Ntougias</surname><given-names>S.</given-names></name><name><surname>Papadopoulos</surname><given-names>N. T.</given-names></name><name><surname>Bourtzis</surname><given-names>K.</given-names></name><name><surname>Cáceres</surname><given-names>C.</given-names></name></person-group> (<year>2019</year>). <article-title><italic>Enterobacter</italic> sp. AA26 gut symbiont as a protein source for Mediterranean fruit fly mass-rearing and sterile insect technique applications.</article-title>\n<source><italic>BMC Microbiol.</italic></source>\n<volume>19</volume>:<issue>288</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12866-019-1651-z</pub-id>\n<pub-id pub-id-type=\"pmid\">31870292</pub-id></mixed-citation></ref><ref id=\"B62\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lacey</surname><given-names>L. A.</given-names></name><name><surname>Unruh</surname><given-names>T. R.</given-names></name><name><surname>Simkins</surname><given-names>H.</given-names></name><name><surname>Thomsen-Archer</surname><given-names>K.</given-names></name></person-group> (<year>2007</year>). <article-title>Gut bacteria associated with the Pacific Coast wireworm, <italic>Limonius canus</italic>, inferred from 16s rDNA sequences and their implications for control.</article-title>\n<source><italic>Phytoparasitica</italic></source>\n<volume>35</volume>\n<fpage>479</fpage>–<lpage>489</lpage>. <pub-id pub-id-type=\"doi\">10.1007/BF03020607</pub-id></mixed-citation></ref><ref id=\"B63\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lauzon</surname><given-names>C.</given-names></name><name><surname>Sjogren</surname><given-names>R.</given-names></name><name><surname>Prokopy</surname><given-names>R.</given-names></name></person-group> (<year>2000</year>). <article-title>Enzymatic capabilities of bacteria associated with apple maggot flies: a postulated role in attraction.</article-title>\n<source><italic>J. Chem. Ecol.</italic></source>\n<volume>26</volume>\n<fpage>953</fpage>–<lpage>967</lpage>. <pub-id pub-id-type=\"doi\">10.1023/A:1005460225664</pub-id></mixed-citation></ref><ref id=\"B64\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lauzon</surname><given-names>C. R.</given-names></name><name><surname>McCombs</surname><given-names>S. D.</given-names></name><name><surname>Potter</surname><given-names>S. E.</given-names></name><name><surname>Peabody</surname><given-names>N. C.</given-names></name></person-group> (<year>2009</year>). <article-title>Establishment and vertical passage of <italic>Enterobacter</italic> (<italic>Pantoea</italic>) <italic>agglomerans</italic> and <italic>Klebsiella pneumoniae</italic> through all life Stages of the Mediterranean fruit fly (<italic>Diptera</italic>: <italic>Tephritidae</italic>).</article-title>\n<source><italic>Ann. Entomol. Soc. Am.</italic></source>\n<volume>102</volume>\n<fpage>85</fpage>–<lpage>95</lpage>. <pub-id pub-id-type=\"doi\">10.1603/008.102.0109</pub-id></mixed-citation></ref><ref id=\"B65\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Luo</surname><given-names>M.</given-names></name><name><surname>Zhang</surname><given-names>H.</given-names></name><name><surname>Du</surname><given-names>Y.</given-names></name><name><surname>Idrees</surname><given-names>A.</given-names></name><name><surname>He</surname><given-names>L.</given-names></name><name><surname>Chen</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Molecular identification of cultivable bacteria in the gut of adult Bactrocera tau (Walker) and their trapping effect.</article-title>\n<source><italic>Pest Manag. Sci.</italic></source>\n<volume>74</volume>\n<fpage>2842</fpage>–<lpage>2850</lpage>. <pub-id pub-id-type=\"doi\">10.1002/ps.5074</pub-id>\n<pub-id pub-id-type=\"pmid\">29749026</pub-id></mixed-citation></ref><ref id=\"B66\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Malacrinò</surname><given-names>A.</given-names></name><name><surname>Campolo</surname><given-names>O.</given-names></name><name><surname>Medina</surname><given-names>R. F.</given-names></name><name><surname>Palmeri</surname><given-names>V.</given-names></name></person-group> (<year>2018</year>). <article-title>Instar- and host-associated differentiation of bacterial communities in the Mediterranean fruit fly <italic>Ceratitis capitata</italic>.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>13</volume>:<issue>e0194131</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0194131</pub-id>\n<pub-id pub-id-type=\"pmid\">29518170</pub-id></mixed-citation></ref><ref id=\"B67\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McMurdie</surname><given-names>P. J.</given-names></name><name><surname>Holmes</surname><given-names>S.</given-names></name></person-group> (<year>2013</year>). <article-title>Phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>8</volume>:<issue>e61217</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0061217</pub-id>\n<pub-id pub-id-type=\"pmid\">23630581</pub-id></mixed-citation></ref><ref id=\"B68\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mishra</surname><given-names>M.</given-names></name><name><surname>Sharma</surname><given-names>K.</given-names></name></person-group> (<year>2018</year>). <article-title>Characterization of culturable gut bacterial isolates from wild population of melon fruit fly (<italic>Bactrocera cucurbitae</italic>) and assessing their attractancy potential for sustainable pest management.</article-title>\n<source><italic>Phytoparasitica</italic></source>\n<volume>46</volume>\n<fpage>583</fpage>–<lpage>594</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s12600-018-0694-2</pub-id></mixed-citation></ref><ref id=\"B69\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morrow</surname><given-names>J. L.</given-names></name><name><surname>Frommer</surname><given-names>M.</given-names></name><name><surname>Shearman</surname><given-names>D. C. A.</given-names></name><name><surname>Riegler</surname><given-names>M.</given-names></name></person-group> (<year>2015</year>). <article-title>The microbiome of field-caught and laboratory-adapted Australian tephritid fruit fly species with different host plant use and specialisation.</article-title>\n<source><italic>Microb. Ecol.</italic></source>\n<volume>70</volume>\n<fpage>498</fpage>–<lpage>508</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00248-015-0571-1</pub-id>\n<pub-id pub-id-type=\"pmid\">25666536</pub-id></mixed-citation></ref><ref id=\"B70\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Niyazi</surname><given-names>N.</given-names></name><name><surname>Lauzon</surname><given-names>C. R.</given-names></name><name><surname>Shelly</surname><given-names>T. E.</given-names></name></person-group> (<year>2004</year>). <article-title>Effect of probiotic adult diets on fitness components of sterile male Mediterranean fruit flies (<italic>Diptera</italic>: <italic>Tephritidae</italic>) under laboratory and field cage conditions.</article-title>\n<source><italic>J. Econ. Entomol.</italic></source>\n<volume>97</volume>\n<fpage>1570</fpage>–<lpage>1580</lpage>. <pub-id pub-id-type=\"doi\">10.1603/0022-0493-97.5.1570</pub-id>\n<pub-id pub-id-type=\"pmid\">15568345</pub-id></mixed-citation></ref><ref id=\"B71\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oduol</surname><given-names>P. A.</given-names></name><name><surname>Muraya</surname><given-names>P.</given-names></name><name><surname>Fernandes</surname><given-names>E. C. M.</given-names></name><name><surname>Nair</surname><given-names>P. K. R.</given-names></name><name><surname>Oktingati</surname><given-names>A.</given-names></name><name><surname>Maghembe</surname><given-names>J.</given-names></name></person-group> (<year>1988</year>). <article-title>The agroforestry systems database at ICRAF.</article-title>\n<source><italic>Agrofor. Syst.</italic></source>\n<volume>6</volume>\n<fpage>253</fpage>–<lpage>270</lpage>. <pub-id pub-id-type=\"doi\">10.1007/BF02344762</pub-id></mixed-citation></ref><ref id=\"B72\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oksanen</surname><given-names>J.</given-names></name><name><surname>Blanchet</surname><given-names>F. G.</given-names></name><name><surname>Friendly</surname><given-names>M.</given-names></name><name><surname>Kindt</surname><given-names>R.</given-names></name><name><surname>Legendre</surname><given-names>P.</given-names></name><name><surname>Mcglinn</surname><given-names>D.</given-names></name><etal/></person-group> (<year>2019</year>). <source><italic>Package ‘ Vegan.</italic></source> Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://cran.r-project.org/web/packages/vegan/vegan.pdf\">https://cran.r-project.org/web/packages/vegan/vegan.pdf</ext-link>\n<comment>(accessed January 21, 2020)</comment>.</mixed-citation></ref><ref id=\"B73\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pavlidi</surname><given-names>N.</given-names></name><name><surname>Dermauw</surname><given-names>W.</given-names></name><name><surname>Rombauts</surname><given-names>S.</given-names></name><name><surname>Chrysargyris</surname><given-names>A.</given-names></name><name><surname>Thomas</surname><given-names>V. L.</given-names></name><name><surname>Vontas</surname><given-names>J.</given-names></name></person-group> (<year>2013</year>). <article-title>Analysis of the olive fruit fly <italic>Bactrocera oleae</italic> transcriptome and phylogenetic classification of the major detoxification gene families.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>8</volume>:<issue>e66533</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0066533</pub-id>\n<pub-id pub-id-type=\"pmid\">23824998</pub-id></mixed-citation></ref><ref id=\"B74\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pavlidi</surname><given-names>N.</given-names></name><name><surname>Gioti</surname><given-names>A.</given-names></name><name><surname>Wybouw</surname><given-names>N.</given-names></name><name><surname>Dermauw</surname><given-names>W.</given-names></name><name><surname>Ben-Yosef</surname><given-names>M.</given-names></name><name><surname>Yuval</surname><given-names>B.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Transcriptomic responses of the olive fruit fly <italic>Bactrocera oleae</italic> and its symbiont Candidatus <italic>Erwinia</italic> dacicola to olive feeding.</article-title>\n<source><italic>Sci. Rep.</italic></source>\n<volume>7</volume>:<issue>42633</issue>. <pub-id pub-id-type=\"doi\">10.1038/srep42633</pub-id>\n<pub-id pub-id-type=\"pmid\">28225009</pub-id></mixed-citation></ref><ref id=\"B75\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pfennig</surname><given-names>D. W.</given-names></name><name><surname>Wund</surname><given-names>M. A.</given-names></name><name><surname>Snell-Rood</surname><given-names>E. C.</given-names></name><name><surname>Cruickshank</surname><given-names>T.</given-names></name><name><surname>Schlichting</surname><given-names>C. D.</given-names></name><name><surname>Moczek</surname><given-names>A. P.</given-names></name></person-group> (<year>2010</year>). <article-title>Phenotypic plasticity’s impacts on diversification and speciation.</article-title>\n<source><italic>Trends Ecol. Evol.</italic></source>\n<volume>25</volume>\n<fpage>459</fpage>–<lpage>467</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.tree.2010.05.006</pub-id>\n<pub-id pub-id-type=\"pmid\">20557976</pub-id></mixed-citation></ref><ref id=\"B76\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Pierre</surname><given-names>R. Y.</given-names></name></person-group> (<year>2007</year>). <source><italic>Economic impact of a Mediterranean fruit fly Outbreak in Florida</italic></source>, PhD Thesis, <publisher-name>University of Florida</publisher-name>, <publisher-loc>Florida</publisher-loc>.</mixed-citation></ref><ref id=\"B77\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Prabhakar</surname><given-names>C. S.</given-names></name><name><surname>Sood</surname><given-names>P.</given-names></name><name><surname>Kanwar</surname><given-names>S. S.</given-names></name><name><surname>Sharma</surname><given-names>P. N.</given-names></name><name><surname>Kumar</surname><given-names>A.</given-names></name><name><surname>Mehta</surname><given-names>P. K.</given-names></name></person-group> (<year>2013</year>). <article-title>Isolation and characterization of gut bacteria of fruit fly, Bactrocera tau (Walker).</article-title>\n<source><italic>Phytoparasitica</italic></source>\n<volume>41</volume>\n<fpage>193</fpage>–<lpage>201</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s12600-012-0278-5</pub-id></mixed-citation></ref><ref id=\"B78\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Qin</surname><given-names>Y.</given-names></name><name><surname>Paini</surname><given-names>D. R.</given-names></name><name><surname>Wang</surname><given-names>C.</given-names></name><name><surname>Fang</surname><given-names>Y.</given-names></name><name><surname>Li</surname><given-names>Z.</given-names></name></person-group> (<year>2015</year>). <article-title>Global establishment risk of economically important fruit fly species (<italic>Tephritidae</italic>).</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>10</volume>:<fpage>6</fpage>–<lpage>13</lpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0116424</pub-id>\n<pub-id pub-id-type=\"pmid\">25588025</pub-id></mixed-citation></ref><ref id=\"B79\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ras</surname><given-names>E.</given-names></name><name><surname>Beukeboom</surname><given-names>L. W.</given-names></name><name><surname>Cáceres</surname><given-names>C.</given-names></name><name><surname>Bourtzis</surname><given-names>K.</given-names></name></person-group> (<year>2017</year>). <article-title>Review of the role of gut microbiota in mass rearing of the olive fruit fly, <italic>Bactrocera oleae</italic>, and its parasitoids.</article-title>\n<source><italic>Entomol. Exp. Appl.</italic></source>\n<volume>164</volume>\n<fpage>237</fpage>–<lpage>256</lpage>. <pub-id pub-id-type=\"doi\">10.1111/eea.12609</pub-id></mixed-citation></ref><ref id=\"B80\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sacchetti</surname><given-names>P.</given-names></name><name><surname>Ghiardi</surname><given-names>B.</given-names></name><name><surname>Granchietti</surname><given-names>A.</given-names></name><name><surname>Stefanini</surname><given-names>F. M.</given-names></name><name><surname>Belcari</surname><given-names>A.</given-names></name></person-group> (<year>2013</year>). <article-title>Development of probiotic diets for the olive fly: evaluation of their effects on fly longevity and fecundity.</article-title>\n<source><italic>Ann. Appl. Biol.</italic></source>\n<volume>164</volume>\n<fpage>138</fpage>–<lpage>150</lpage>. <pub-id pub-id-type=\"doi\">10.1111/aab.12088</pub-id></mixed-citation></ref><ref id=\"B81\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sagar</surname><given-names>R.</given-names></name><name><surname>Sharma</surname><given-names>G.</given-names></name></person-group> (<year>2012</year>). <article-title>Measurement of alpha diversity using Simpson index (1/λ): the jeopardy.</article-title>\n<source><italic>Environ. Skept. Critics</italic></source>\n<volume>1</volume>\n<fpage>23</fpage>–<lpage>24</lpage>.</mixed-citation></ref><ref id=\"B82\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Santo Domingo</surname><given-names>J. W.</given-names></name><name><surname>Kaufman</surname><given-names>M. G.</given-names></name><name><surname>Klug</surname><given-names>M. J.</given-names></name><name><surname>Holben</surname><given-names>W. E.</given-names></name><name><surname>Harris</surname><given-names>D.</given-names></name><name><surname>Tiedje</surname><given-names>J. M.</given-names></name></person-group> (<year>1998</year>). <article-title>Influence of diet on the structure and function of the bacterial hindgut community of crickets.</article-title>\n<source><italic>Mol. Ecol.</italic></source>\n<volume>7</volume>\n<fpage>761</fpage>–<lpage>767</lpage>. <pub-id pub-id-type=\"doi\">10.1046/j.1365-294x.1998.00390.x</pub-id></mixed-citation></ref><ref id=\"B83\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Takahashi</surname><given-names>S.</given-names></name><name><surname>Tomita</surname><given-names>J.</given-names></name><name><surname>Nishioka</surname><given-names>K.</given-names></name><name><surname>Hisada</surname><given-names>T.</given-names></name><name><surname>Nishijima</surname><given-names>M.</given-names></name></person-group> (<year>2014</year>). <article-title>Development of a prokaryotic universal primer for simultaneous analysis of <italic>Bacteria</italic> and <italic>Archaea</italic> using next-generation sequencing.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>9</volume>:<issue>e105592</issue>\n<pub-id pub-id-type=\"doi\">10.1371/0105592</pub-id></mixed-citation></ref><ref id=\"B84\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Thaochan</surname><given-names>N.</given-names></name><name><surname>Drew</surname><given-names>R. A. I.</given-names></name><name><surname>Hughes</surname><given-names>J. M.</given-names></name><name><surname>Vijaysegaran</surname><given-names>S.</given-names></name><name><surname>Chinajariyawong</surname><given-names>A.</given-names></name></person-group> (<year>2010</year>). <article-title>Alimentary tract bacteria isolated and identified with API-20E and molecular cloning techniques from Australian tropical fruit flies, <italic>Bactrocera cacuminata</italic> and <italic>B. tryoni</italic>.</article-title>\n<source><italic>J. Insect Sci.</italic></source>\n<volume>10</volume>:131. <pub-id pub-id-type=\"doi\">10.1673/031.010.13101</pub-id>\n<pub-id pub-id-type=\"pmid\">20883132</pub-id></mixed-citation></ref><ref id=\"B85\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Uchôa</surname><given-names>M. A.</given-names></name></person-group> (<year>2012</year>). “<article-title>Fruit flies (<italic>Diptera</italic>: <italic>Tephritoidea</italic>): biology, host plants, natural enemies, and the implications to their natural control</article-title>,” in <source><italic>Integrated Pest Management and Pest Control Current and Future Tactics</italic></source>, <role>eds</role>\n<person-group person-group-type=\"editor\"><name><surname>Larramendy</surname><given-names>M. L.</given-names></name><name><surname>Soloneski</surname><given-names>S.</given-names></name></person-group> (<publisher-loc>Rijeka</publisher-loc>: <publisher-name>InTechOpen</publisher-name>), <fpage>217</fpage>–<lpage>300</lpage>. <pub-id pub-id-type=\"doi\">10.5772/1383</pub-id></mixed-citation></ref><ref id=\"B86\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Underwood</surname><given-names>A. J.</given-names></name></person-group> (<year>1997</year>). <source><italic>Experiments in Ecology: their Logical Design and Interpretation using Analysis of Variance.</italic></source>\n<publisher-loc>Cambridge</publisher-loc>: <publisher-name>Cambridge University Press</publisher-name>.</mixed-citation></ref><ref id=\"B87\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van den Bosch</surname><given-names>T. J. M.</given-names></name><name><surname>Welte</surname><given-names>C. U.</given-names></name></person-group> (<year>2016</year>). <article-title>Detoxifying symbionts in agriculturally important pest insects.</article-title>\n<source><italic>Microb. Biotechnol.</italic></source>\n<volume>10</volume>\n<fpage>531</fpage>–<lpage>540</lpage>. <pub-id pub-id-type=\"doi\">10.1111/1751-7915.12483</pub-id>\n<pub-id pub-id-type=\"pmid\">27943632</pub-id></mixed-citation></ref><ref id=\"B88\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vasanthakumar</surname><given-names>A.</given-names></name><name><surname>Delalibera</surname><given-names>I.</given-names></name><name><surname>Handelsman</surname><given-names>J.</given-names></name><name><surname>Klepzig</surname><given-names>K. D.</given-names></name><name><surname>Schloss</surname><given-names>P. D.</given-names></name><name><surname>Raffa</surname><given-names>K. F.</given-names></name></person-group> (<year>2009</year>). <article-title>Characterization of gut-associated bacteria in larvae and adults of the southern pine beetle.</article-title>\n<source><italic>Environ. Entomol.</italic></source>\n<volume>35</volume>\n<fpage>1710</fpage>–<lpage>1717</lpage>.</mixed-citation></ref><ref id=\"B89\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Virgilio</surname><given-names>M.</given-names></name><name><surname>De Meyer</surname><given-names>M.</given-names></name><name><surname>White</surname><given-names>I. M.</given-names></name><name><surname>Backeljau</surname><given-names>T.</given-names></name></person-group> (<year>2009</year>). <article-title>African <italic>Dacus</italic> (<italic>Diptera</italic>: <italic>Tephritidae</italic>): molecular data and host plant associations do not corroborate morphology based classifications.</article-title>\n<source><italic>Mol. Phylogenet. Evol.</italic></source>\n<volume>51</volume>\n<fpage>531</fpage>–<lpage>539</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ympev.2009.01.003</pub-id>\n<pub-id pub-id-type=\"pmid\">19444961</pub-id></mixed-citation></ref><ref id=\"B90\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Virgilio</surname><given-names>M.</given-names></name><name><surname>Jordaens</surname><given-names>K.</given-names></name><name><surname>Breman</surname><given-names>F. C.</given-names></name><name><surname>Backeljau</surname><given-names>T.</given-names></name><name><surname>de Meyer</surname><given-names>M.</given-names></name></person-group> (<year>2012</year>). <article-title>Identifying insects with incomplete DNA barcode libraries, African fruit flies (<italic>Diptera</italic>: <italic>Tephritidae</italic>) as a test case.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>7</volume>:<issue>e31581</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0031581</pub-id>\n<pub-id pub-id-type=\"pmid\">22359600</pub-id></mixed-citation></ref><ref id=\"B91\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>A.</given-names></name><name><surname>Zhichao</surname><given-names>Y.</given-names></name><name><surname>Weiwei</surname><given-names>Z.</given-names></name><name><surname>Hongyu</surname><given-names>Z.</given-names></name></person-group> (<year>2014</year>). <article-title>Bacterial communities in the gut and reproductive organs of <italic>Bactrocera minax</italic> (<italic>Diptera</italic>: <italic>Tephritidae</italic>) based on 454 pyrosequencing.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>9</volume>:<issue>e106988</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0106988</pub-id>\n<pub-id pub-id-type=\"pmid\">25215866</pub-id></mixed-citation></ref><ref id=\"B92\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>H.</given-names></name><name><surname>Jin</surname><given-names>L.</given-names></name><name><surname>Peng</surname><given-names>T.</given-names></name><name><surname>Zhang</surname><given-names>H.</given-names></name><name><surname>Chen</surname><given-names>Q.</given-names></name><name><surname>Hua</surname><given-names>Y.</given-names></name></person-group> (<year>2014</year>). <article-title>Identification of cultivable bacteria in the intestinal tract of <italic>Bactrocera dorsalis</italic> from three different populations and determination of their attractive potential.</article-title>\n<source><italic>Pest Manag. Sci.</italic></source>\n<volume>70</volume>\n<fpage>80</fpage>–<lpage>87</lpage>. <pub-id pub-id-type=\"doi\">10.1002/ps.3528</pub-id>\n<pub-id pub-id-type=\"pmid\">23696217</pub-id></mixed-citation></ref><ref id=\"B93\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>H.</given-names></name><name><surname>Jin</surname><given-names>L.</given-names></name><name><surname>Zhang</surname><given-names>H.</given-names></name></person-group> (<year>2011</year>). <article-title>Comparison of the diversity of the bacterial communities in the intestinal tract of adult <italic>Bactrocera dorsalis</italic> from three different populations.</article-title>\n<source><italic>J. Appl. Microbiol.</italic></source>\n<volume>110</volume>\n<fpage>1390</fpage>–<lpage>1401</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-2672.2011.05001.x</pub-id>\n<pub-id pub-id-type=\"pmid\">21395953</pub-id></mixed-citation></ref><ref id=\"B94\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>Q.</given-names></name><name><surname>Garrity</surname><given-names>G. M.</given-names></name><name><surname>Tiedje</surname><given-names>J. M.</given-names></name><name><surname>Cole</surname><given-names>J. R.</given-names></name></person-group> (<year>2007</year>). <article-title>Naîve bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>73</volume>\n<fpage>5261</fpage>–<lpage>5267</lpage>. <pub-id pub-id-type=\"doi\">10.1128/AEM.00062-07</pub-id>\n<pub-id pub-id-type=\"pmid\">17586664</pub-id></mixed-citation></ref><ref id=\"B95\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weller</surname><given-names>S. A.</given-names></name><name><surname>Stead</surname><given-names>D. E.</given-names></name><name><surname>Young</surname><given-names>J. P. W.</given-names></name></person-group> (<year>2006</year>). <article-title>Recurrent outbreaks of rootmat in cucumber and tomato are associated with a monomorphic, cucumopine, Ri-plasmid harboured by various Alphaproteobacteria.</article-title>\n<source><italic>FEMS Microbiol. Lett.</italic></source>\n<volume>258</volume>\n<fpage>136</fpage>–<lpage>143</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1574-6968.2006.00214.x</pub-id>\n<pub-id pub-id-type=\"pmid\">16630268</pub-id></mixed-citation></ref><ref id=\"B96\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>White</surname><given-names>I. M.</given-names></name><name><surname>Elson-Harris</surname><given-names>M. M.</given-names></name></person-group> (<year>1992</year>). <source><italic>Fruit Flies of Economic Significance: their Identification and Bionomics.</italic></source>\n<publisher-loc>Wallingford</publisher-loc>: <publisher-name>CAB International</publisher-name>.</mixed-citation></ref><ref id=\"B97\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Wickham</surname><given-names>H.</given-names></name></person-group> (<year>2009</year>). <source><italic>ggplot2: Elegant graphics for Data Analysis.</italic></source>\n<publisher-loc>New York, NY</publisher-loc>: <publisher-name>Springer</publisher-name>, <pub-id pub-id-type=\"doi\">10.1007/978-0-387-98141-3</pub-id></mixed-citation></ref><ref id=\"B98\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Winkler</surname><given-names>I.</given-names></name><name><surname>Mitter</surname><given-names>C.</given-names></name></person-group> (<year>2016</year>). “<article-title>The phylogenetic dimension of insect/plant interactions: a review of recent evidence</article-title>,” in <source><italic>The Evolutionary Biology of Herbivorous Insects: Specialization, Speciation, and Radiation</italic></source>, <role>ed.</role>\n<person-group person-group-type=\"editor\"><name><surname>Abrahamson</surname><given-names>W. G.</given-names></name></person-group> (<publisher-loc>Oakland, CA</publisher-loc>: <publisher-name>University of California Press</publisher-name>), <fpage>240</fpage>–<lpage>263</lpage>. <pub-id pub-id-type=\"doi\">10.1525/california/9780520251328.003.0018</pub-id></mixed-citation></ref><ref id=\"B99\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zaada</surname><given-names>D. S. Y.</given-names></name><name><surname>Ben-Yosef</surname><given-names>M.</given-names></name><name><surname>Yuval</surname><given-names>B.</given-names></name><name><surname>Jurkevitch</surname><given-names>E.</given-names></name></person-group> (<year>2019</year>). <article-title>The host fruit amplifies mutualistic interaction between <italic>Ceratitis capitata</italic> larvae and associated bacteria.</article-title>\n<source><italic>BMC Biotechnol.</italic></source>\n<volume>19</volume>:<issue>92</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12896-019-0581-z</pub-id>\n<pub-id pub-id-type=\"pmid\">31847844</pub-id></mixed-citation></ref><ref id=\"B100\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zilber-Rosenberg</surname><given-names>I.</given-names></name><name><surname>Rosenberg</surname><given-names>E.</given-names></name></person-group> (<year>2008</year>). <article-title>Role of microorganisms in the evolution of animals and plants: the hologenome theory of evolution.</article-title>\n<source><italic>FEMS Microbiol. Rev.</italic></source>\n<volume>32</volume>\n<fpage>723</fpage>–<lpage>735</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1574-6976.2008.00123.x</pub-id>\n<pub-id pub-id-type=\"pmid\">18549407</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Microbiol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Microbiol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Microbiol.</journal-id><journal-title-group><journal-title>Frontiers in Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-302X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849435</article-id><article-id pub-id-type=\"pmc\">PMC7431612</article-id><article-id pub-id-type=\"doi\">10.3389/fmicb.2020.01835</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Microbiology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Effects of Gut Microbiome and Short-Chain Fatty Acids (SCFAs) on Finishing Weight of Meat Rabbits</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Fang</surname><given-names>Shaoming</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/776918/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Xuan</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/776938/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ye</surname><given-names>Xiaoxing</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1049236/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zhou</surname><given-names>Liwen</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/777169/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Xue</surname><given-names>Shuaishuai</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1049262/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Gan</surname><given-names>Qianfu</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/776934/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>College of Animal Science, Fujian Agriculture and Forestry University</institution>, <addr-line>Fuzhou</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>College of Life Science, Fujian Agriculture and Forestry University</institution>, <addr-line>Fuzhou</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: George Tsiamis, University of Patras, Greece</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Liang Xiao, Beijing Genomics Institute (BGI), China; María Velasco-Galilea, Institut de Recerca i Tecnologia Agroaliment ries (IRTA), Spain</p></fn><corresp id=\"c001\">*Correspondence: Qianfu Gan, <email>ganning707@163.com</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>These authors have contributed equally to this work</p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Systems Microbiology, a section of the journal Frontiers in Microbiology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1835</elocation-id><history><date date-type=\"received\"><day>17</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>13</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Fang, Chen, Ye, Zhou, Xue and Gan.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Fang, Chen, Ye, Zhou, Xue and Gan</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Understanding how the gut microbiome and short-chain fatty acids (SCFAs) affect finishing weight is beneficial to improve meat production in the meat rabbit industry. In this study, we identified 15 OTUs and 23 microbial species associated with finishing weight using 16S rRNA gene and metagenomic sequencing analysis, respectively. Among these, butyrate-producing bacteria of the family Ruminococcaceae were positively associated with finishing weight, whereas the microbial taxa related to intestinal damage and inflammation showed opposite effects. Furthermore, interactions of these microbial taxa were firstly found to be associated with finishing weight. Gut microbial functional capacity analysis revealed that CAZymes, such as galactosidase, xylanase, and glucosidase, could significantly affect finishing weight, given their roles in regulating nutrient digestibility. GOs related to the metabolism of several carbohydrates and amino acids also showed important effects on finishing weight. Additionally, both KOs and KEGG pathways related to the membrane transportation system and involved in aminoacyl-tRNA biosynthesis and butanoate metabolism could act as key factors in modulating finishing weight. Importantly, gut microbiome explained nearly 11% of the variation in finishing weight, and our findings revealed that a subset of metagenomic species could act as predictors of finishing weight. SCFAs levels, especially butyrate level, had critical impacts on finishing weight, and several finishing weight-associated species were potentially contributed to the shift in butyrate level. Thus, our results should give deep insights into how gut microbiome and SCFAs influence finishing weight of meat rabbits and provide essential knowledge for improving finishing weight by manipulating gut microbiome.</p></abstract><kwd-group><kwd>gut microbiome</kwd><kwd>SCFAs</kwd><kwd>metagenomic</kwd><kwd>16S rRNA gene</kwd><kwd>finishing weight</kwd><kwd>meat rabbits</kwd></kwd-group><counts><fig-count count=\"8\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"88\"/><page-count count=\"14\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>The gut microbiome comprises the collective genome of the trillions of microorganisms colonizing the intestinal tract, which is responsible for vital metabolic, immunological, and nutritional functions (<xref rid=\"B68\" ref-type=\"bibr\">Sidhu and van der Poorten, 2017</xref>). Short-chain fatty acids (SCFAs), mainly acetate, propionate, and butyrate, are recognized as important metabolites derived from the fermentation of indigestible dietary fibers by gut microbial species, which play fundamental roles in maintaining intestinal homeostasis and energy metabolism regulation (<xref rid=\"B61\" ref-type=\"bibr\">Priyadarshini et al., 2018</xref>).</p><p>Recently, the roles of gut microbiome and SCFAs in modulating growth performances of farm animals have been extensively studied. For instance, <xref rid=\"B70\" ref-type=\"bibr\">Wang J. et al. (2019)</xref> indicated that certain bacteria of the family Ruminococcaceae, the genus <italic>Faecalibacterium</italic>, and the genus <italic>Prevotella</italic> are involved in metabolizing dietary fibers which subsequently affect SCFA production, which exerted important roles in feed intake and body weight modulation in pigs. <xref rid=\"B17\" ref-type=\"bibr\">Du et al. (2018)</xref> demonstrated that both butyrate-producing bacteria <italic>Butyricimonas</italic> and the digestive enzyme family metabolic pathway could potentially affect the body weight of beef calves. Additionally, research on the gut microbiome of broiler chickens suggested that increased abundance of <italic>Lactobacillus</italic> and <italic>Bifidobacterium</italic>, and facilitated generations of lactate and SCFAs, had a positive influence on feed efficiency and body weight gain (<xref rid=\"B80\" ref-type=\"bibr\">Yu et al., 2019</xref>).</p><p>Finishing weight is regarded as a crucial growth trait of meat rabbits, which directly affects broiler rabbits’ meat production. However, only few studies have unraveled the relationship between the gut microbiome and finishing weight of rabbits. Zeng <italic>et al.</italic> reported that changes in the abundance of YS2, <italic>Bacteroides</italic>, <italic>Lactococcus</italic>, <italic>Lactobacillus</italic>, and <italic>Prevotella</italic> in the gut microbial communities of rabbits were associated with finishing weight (<xref rid=\"B82\" ref-type=\"bibr\">Zeng et al., 2015</xref>). <xref rid=\"B71\" ref-type=\"bibr\">Wang Q. et al. (2019)</xref> found that <italic>Coprococcus</italic> and <italic>Ruminococcaceae_UCG-004</italic> may hinder the production of pro-inflammatory factors that exert beneficial effects on the finishing weight of rabbits. Moreover, <xref rid=\"B56\" ref-type=\"bibr\">North et al. (2019)</xref> indicated that rabbits with greater abundances of Eubacteriaceae, Natranaerobiaceae, Peptococcaceae, and Syntrophomonadaceae in the gut microbial communities tended to gain more weight at finishing. Nevertheless, these investigations are based on 16S rRNA gene sequencing analysis, which cannot determine species and functional capacities that affect finishing weight. Additionally, the potential roles of SCFAs in modulating the finishing weight of meat rabbits remain unknown. Hence, the present study aimed to assess the effects of gut microbiome and SCFAs on finishing weight using 16S rRNA gene sequencing, metagenomic sequencing, and fecal SCFA level data. We not only identified a number of microbial taxa and metabolic functions associated with finishing weight but also unraveled the correlations between SCFA levels and finishing weight. In addition, we evaluated the role of the gut microbiome in the phenotypic variation of finishing weight. Such findings could improve our understanding of how the gut microbiome and SCFAs modulate finishing weight and thus provide important information for the meat rabbit industry.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Experimental Rabbits and Sample Collection</title><p>One hundred and five Ira rabbits (28 ± 2 days, 53 males and 52 females) with similar weaning weight were randomly distributed to separated cages (one rabbit per cage) and fed with the fatten diet (details are shown in <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table S1</xref>) until finishing (72 ± 2 days). Finishing body weight was measured, and hard fecal samples were collected. Finishing weights were sorted, and the top 5 rabbits with the highest body weight (high group) and the bottom 5 with the lowest body weight (low group) were selected for metagenomic sequencing. All rabbits were healthy and had not received antibiotics, anti-coccidial drugs, probiotics, or prebiotics during the experimental period. All fecal samples were snap frozen in liquid nitrogen for transportation and stored at −80°C until further processing.</p></sec><sec id=\"S2.SS2\"><title>16S rRNA Gene Sequencing</title><p>Microbial genomic DNA of fecal samples was extracted by the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions. The purity and integrity of total DNA was detected by using the NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, United States) and 1.5% agarose gel electrophoresis, respectively. Hypervariable regions V3–V4 of the 16S ribosomal RNA gene were then amplified by using the primer 341F:CCTACGGGNGGCWGCAG and 806R:GGACTACHVGGGTATCTAAT and sequenced by the HiSeq 2500 platform (Illumina, United States) according to the manufacturer’s manuals. QIIME (v.1.9.1) was used for the quality control process of sequencing data including filtering out of the primers, barcodes, and low-quality sequences (quality score < 20) (<xref rid=\"B7\" ref-type=\"bibr\">Caporaso et al., 2010</xref>). High-quality paired-end reads were merged into tags by using FLASH (v.1.2.11) (<xref rid=\"B51\" ref-type=\"bibr\">Magoc and Salzberg, 2011</xref>). We rarefied the library size of microbial sequences to 40,000 tags per sample to normalize the sequencing depth (<xref rid=\"B27\" ref-type=\"bibr\">Fu et al., 2015</xref>). USEARCH (v.10.0) was used to cluster tags into operational taxonomic units (OTUs) at 97% sequence similarity (<xref rid=\"B19\" ref-type=\"bibr\">Edgar, 2010</xref>). OTUs which had relative abundance < 0.1% and were presented in less than 1% of the experimental rabbits were filtered out before further analysis. OTU taxonomic category assignments were performed by using the SILVA database (v.132) (<xref rid=\"B63\" ref-type=\"bibr\">Quast et al., 2013</xref>).</p></sec><sec id=\"S2.SS3\"><title>Two-Part Model Analysis</title><p>After sex and cage rearing effect corrections, residuals of finishing weight phenotypic values were used for association analysis between finishing weight and relative abundances of OTUs by using a two-part model method as described before (<xref rid=\"B27\" ref-type=\"bibr\">Fu et al., 2015</xref>). In brief, the two-part model analysis was consisted of binary, quantitative, and meta models. The binary model describes a binomial analysis that assesses for the effect of the presence/absence of the gut microbe on finishing weight. The quantitative model tests for the association between finishing weight and the abundance of each OTU, but only the samples where that microbe is present were included in the analysis. The meta model was used to integrate the effect of both binary and quantitative analysis. The final association <italic>p</italic>-value was assigned from the minimum of <italic>p</italic>-values from the binary analysis, quantitative analysis, and meta-analysis. Skewness correction was performed by 1000 × permutation tests. False discovery rate (FDR)-adjusted <italic>p</italic> < 0.05 was set as the significance threshold.</p></sec><sec id=\"S2.SS4\"><title>Metagenomic Sequencing</title><p>A paired-end (PE) DNA library was constructed for each sample according to the manufacturer’s instruction (Illumina, United States), and sequencing was performed on an Illumina HiSeq 4000 platform. Quality control, adapter trimming, and low-quality read filtering of raw data were performed by using fastp (v.0.19.4) (<xref rid=\"B10\" ref-type=\"bibr\">Chen et al., 2018</xref>) and obtained an average of 64,042,801 high-quality reads for each sample. We assembled the high-quality reads into contigs using the MEGAHIT (v.1.1.3) (<xref rid=\"B46\" ref-type=\"bibr\">Li et al., 2015</xref>). The contigs with more than 200 bp in length were used for open reading frame (ORF) prediction with MetaGeneMark (v.2.10) (<xref rid=\"B88\" ref-type=\"bibr\">Zhu et al., 2010</xref>). A non-redundant gene catalog was established by removing the redundant genes from the predicted ORFs using Cd-hit (v.4.6.1) (<xref rid=\"B28\" ref-type=\"bibr\">Fu et al., 2012</xref>). The gene abundance profile was generated by mapping the high-quality reads against the non-redundant gene catalog using MOCAT (v2.0) (<xref rid=\"B43\" ref-type=\"bibr\">Kultima et al., 2016</xref>). DIAMOND (v.0.9.24) (<xref rid=\"B6\" ref-type=\"bibr\">Buchfink et al., 2015</xref>) was used for taxonomic category and GO term assignments of the predicted genes by aligning against the non-redundant (NR) database and Gene Ontology (GO) database, respectively. Carbohydrate-Active enZYmes (CAZymes) were annotated by hmmscan program in HMMER (v.3.1) (<xref rid=\"B18\" ref-type=\"bibr\">Eddy, 2011</xref>). GhostKOALA was used to extract KEGG Orthology (KO) and KEGG pathway annotation information from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (<xref rid=\"B39\" ref-type=\"bibr\">Kanehisa et al., 2016</xref>).</p></sec><sec id=\"S2.SS5\"><title>Co-abundance Group (CAG) Analysis</title><p>In order to construct CAG, we first calculated the correlation coefficients among the finishing weight-associated microbial taxa using the Sparse Correlations for Compositional data (SparCC) algorithm (<xref rid=\"B26\" ref-type=\"bibr\">Friedman and Alm, 2012</xref>). Then, CAGs were defined by Ward’s linkage hierarchical clustering method and PERMANOVA (999 permutations, <italic>p</italic> < 0.05) based on the SparCC correlation coefficient matrix through made4 and vegan R packages, respectively (<xref rid=\"B85\" ref-type=\"bibr\">Zhang et al., 2016</xref>). The CAG interaction networks were established based on FDR-adjusted <italic>p</italic> < 0.05 and | r| > 0.35 by CYTOSCAPE (v.3.7.0) (<xref rid=\"B66\" ref-type=\"bibr\">Shannon et al., 2003</xref>). Spearman’s correlation analysis with FDR correction was performed to compute the association between CAGs and finishing weight.</p></sec><sec id=\"S2.SS6\"><title>Estimating Phenotypic Variance Explained by the Gut Microbiome</title><p>To evaluate the contribution of the gut microbiome to the variation of finishing weight, we performed a 100× cross-validation (<xref rid=\"B27\" ref-type=\"bibr\">Fu et al., 2015</xref>). The data set was randomly divided into an 80% discovery data set and a 20% validation data set. In the discovery data set, we performed two-part model analysis to identify a number of (n) OTUs that were significantly associated with finishing weight at a certain <italic>p</italic>-value and assessed the effect sizes of binary and quantitative features (β<sub>1</sub> and β<sub>2</sub>) of each OTUs. In the validation data set, the effect of the gut microbiome on finishing weight (r<sub>m</sub>) was estimated by an additive model: r<sub>m</sub> = <inline-formula><mml:math id=\"INEQ2\"><mml:mrow><mml:munderover><mml:mo movablelimits=\"false\">∑</mml:mo><mml:mrow><mml:mi mathvariant=\"normal\">j</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mtext>n</mml:mtext></mml:mrow></mml:munderover><mml:mrow><mml:mo stretchy=\"false\">(</mml:mo><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">β</mml:mi><mml:mn>1</mml:mn></mml:msub><mml:mo>+</mml:mo><mml:msub><mml:mi>b</mml:mi><mml:mrow><mml:mtext>j</mml:mtext></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:mrow><mml:msub><mml:mi mathvariant=\"normal\">β</mml:mi><mml:mn>2</mml:mn></mml:msub><mml:mo>⁢</mml:mo><mml:msub><mml:mi>q</mml:mi><mml:mrow><mml:mtext>j</mml:mtext></mml:mrow></mml:msub></mml:mrow></mml:mrow><mml:mo stretchy=\"false\">)</mml:mo></mml:mrow></mml:mrow></mml:math></inline-formula>, b<sub>j</sub> and q<sub>j</sub> corresponding to the binary and quantitative feature of j OTU, respectively. We calculated the squared correlation coefficient (R<sup>2</sup>) between r<sub>m</sub> and the phenotypic value, which represents d or the phenotypic variance explained by the gut microbiome. To ensure validity and stability of the estimation, we repeated the cross-validation for 100 times and calculated the average value of the explained variations.</p></sec><sec id=\"S2.SS7\"><title>Determinations of SCFA in Fecal Samples</title><p>The concentrations of SCFAs were measured in the fecal samples using gas chromatography on an Agilent 7890A GC system with a flame ionization detector (Agilent Technologies, United States) according to the previously described method with some modifications (<xref rid=\"B65\" ref-type=\"bibr\">Reddivari et al., 2017</xref>). Hundred and fifty milligram fecal samples were homogenized in 1 ml of water –0.5% phosphoric acid by Vortex-Genie 2 (Scientific Industries, United States). The mixture was centrifuged at 15,000 rpm for 10 min at 4°C. The supernatant (600 μl) which was mixed with an equal volume of ethyl acetate was used for filtering by a 0.35 mm filter membrane and then transferred to a glass gas chromatography vial prior to injection into a GC instrument. The internal standard of 2-methyl butyrate at a final concentration of 10 mM was added to the organic phase to correct injection variability among the samples. 10–100 mM acetate, 1–10 mM propionate, and 1–10 mM butyrate were set as external standards, and five sample intervals were prepared to calculate retention times and create standard curves. The concentrations of SCFAs in fecal samples were determined by comparing to standard curves. The correlations between finishing weight-associated species and SCFA levels were analyzed by using the Spearman method.</p></sec><sec id=\"S2.SS8\"><title>Basic Statistical Analysis</title><p>The Wilcoxon rank-sum test with FDR correction was used to identify the metagenomic species and function capacities of the gut microbiome having significantly different enrichments between high and low finishing rabbits. To identify important species that could predict finishing weight variation, random forest analysis was performed by using species relative abundance data, and a predictor species was determined based on the mean decrease in accuracy of discrimination > 2 (<xref rid=\"B5\" ref-type=\"bibr\">Breiman, 2001</xref>). Two-sided unpaired Student’s <italic>t</italic>-test with FDR correction was performed to detect the differences in levels of SCFAs in fecal samples between high and low finishing weight rabbits. Except the differential metagenomic species which were visualized online by Interactive Tree Of Life (iTOL) (<xref rid=\"B45\" ref-type=\"bibr\">Letunic and Bork, 2019</xref>), the other plots were accomplished in R software.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Gut Microbiota Associated With Finishing Weight</title><p>The phylogenetic analysis of gut microbiota in experimental rabbits has been previously described (<xref rid=\"B21\" ref-type=\"bibr\">Fang et al., 2019</xref>, <xref rid=\"B20\" ref-type=\"bibr\">2020</xref>). Here, we focused on identifying the gut microbiota associated with finishing weight. The phenotypic distribution of finishing weight in experimental rabbits is shown in <xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1A</xref>. The phenotypic value of rabbits in the high finishing weight group was significantly different than that in the low finishing weight group (FDR adjusted <italic>P</italic> < 0.05, <xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1B</xref>).</p><p>To identify microbial taxa associated with finishing weight, we performed a two-part model analysis using the relative abundances of OTUs and phenotypic values in the experimental population. We identified 15 OTUs associated with finishing weight (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref> and <xref ref-type=\"supplementary-material\" rid=\"TS2\">Supplementary Table S2</xref>, FDR adjusted <italic>P</italic> < 0.05), of which 8 OTUs exhibited positive associations and the remaining showed negative associations. Among the OTUs positively associated with finishing weight, one was annotated to the order Mollicutes_RF9. Two were annotated to the family Ruminococcaceae, and one to the family Lachnospiraceae. Three OTUs were annotated to the genus <italic>Ruminococcaceae_UCG-014</italic>, and one to the genus <italic>Ruminococcus_1</italic>. As for the OTUs negatively associated with finishing weight, three OTUs were annotated to the family level (two belonging to the family Coriobacteriaceae and one to the family Alcaligenaceae) and four to the genus level (<italic>Ruminococcaceae_UCG-005</italic>, <italic>Tyzzerella_3</italic>, <italic>Ruminiclostridium</italic>, and <italic>Anaerotruncus</italic> one OTU each).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>The OTUs significantly associated with finishing weight (FDR adjusted <italic>P</italic> < 0.05) are shown as Z scores.</p></caption><graphic xlink:href=\"fmicb-11-01835-g001\"/></fig><p>Due to the great limitations of 16S rRNA gene sequencing in both resolution and accuracy at the species level, we performed metagenomic sequencing of five individuals with the highest and lowest phenotypic values, respectively. We used the Wilcoxon rank-sum test with metagenomic sequencing data to determine microbial species showing different enrichments between high and low finishing weight rabbits. A total of 23 species were identified (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref> and <xref ref-type=\"supplementary-material\" rid=\"TS3\">Supplementary Table S3</xref>, FDR adjusted <italic>P</italic> < 0.05), including four species from the family Ruminococcaceae <italic>Ruminococcus</italic> sp. <italic>CAG:353</italic>, <italic>Ruminococcus albus</italic>, <italic>Ruminococcus</italic> sp. <italic>HUN007</italic>, and <italic>Ruminococcus flavefaciens</italic>, which were abundant in rabbits with high finishing weight. In addition, <italic>Faecalibacterium prausnitzii</italic>, <italic>Bifidobacterium saeculare</italic>, <italic>Roseburia</italic> sp. <italic>CAG:303</italic>, <italic>Lactobacillus ruminis</italic>, <italic>butyrate-producing bacterium SS3/4</italic>, and <italic>Bacteroides pectinophilus CAG:437</italic> were also enriched in rabbits with high finishing weight. Conversely, we found that <italic>Ruminococcus gauvreauii</italic>, <italic>Ruminococcus bromii</italic>, and <italic>Ruminococcus obeum CAG:39</italic> (members of family Ruminococcaceae) were augmented in rabbits with low finishing weight. Additionally, <italic>Akkermansia muciniphila CAG:154</italic>, <italic>Butyrivibrio</italic> sp. <italic>XBB1001</italic>, <italic>Bacteroides fragilis</italic>, <italic>Clostridium</italic> sp. <italic>CAG:217</italic>, <italic>Coprococcus</italic> sp. <italic>ART55/1</italic>, <italic>Bacteroides thetaiotaomicron</italic>, <italic>Ruminiclostridium thermocellum</italic>, <italic>Tyzzerella nexilis</italic>, <italic>Coriobacteriaceae bacterium 68-1-3</italic>, and <italic>Anaerotruncus</italic> sp. <italic>CAG:390</italic> were also abundant in rabbits with low finishing weight.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>The phylogenetic relationships and abundances of metagenomic species enriched in high and low finishing weight rabbits. The phylogenetic tree of species is shown as the innermost layers. Labels with red and blue color represent high and low finishing weight groups, respectively. The different color strips in the third layer correspond to different families as indicated by the color code on the left. The outermost layers depict the relative abundances of differential species between high and low finishing weight individuals.</p></caption><graphic xlink:href=\"fmicb-11-01835-g002\"/></fig></sec><sec id=\"S3.SS2\"><title>Interactions of Microbial Taxa Correlated With Finishing Weight</title><p>Previous studies have demonstrated that the interactions of gut microbes play vital roles in host physiology, development, and growth (<xref rid=\"B64\" ref-type=\"bibr\">Ramayo-Caldas et al., 2016</xref>; <xref rid=\"B41\" ref-type=\"bibr\">Ke et al., 2019</xref>). To investigate the potential effect of interactions among the identified species on finishing weight, we constructed a co-abundance network based on SparCC correlation coefficients. The co-abundance network was consisted of two co-abundance groups (CAGs; <xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS2\">Supplementary Figure S2</xref>). The species that were positively associated with finishing weight were clustered into CAG 1 through intra-positive interactions, whereas CAG 2 was formed by intra-positive interactions among the species that were negatively associated with finishing weight. In addition, CAG 1 had positive associations with finishing weight, but CAG 2 was negatively correlated with finishing weight (<xref ref-type=\"fig\" rid=\"F3\">Figures 3B,C</xref>). Notably, when we analyzed the entire experimental population, the co-abundance network could also be established using finishing weight associated OTUs, and similar correlations were observed between the CAGs and finishing weight (<xref ref-type=\"supplementary-material\" rid=\"FS3\">Supplementary Figure S3</xref>).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Interactions among the species responding for finishing weight. <bold>(A)</bold> The interaction network of finishing weight-associated species. The red nodes represent positively associated species and the blue nodes represent negatively associated species. The node size indicates the average relative abundance of each species. Lines linked to nodes indicate significant correlations among the species (FDR adjusted <italic>P</italic> < 0.05, |r| > 0.45), with red and gray colors showing positive and negative correlations, respectively. The species are clustered into two co-abundance groups (CAGs) by PERMANOVA at P < 0.05. <bold>(B)</bold> The association of the abundance of CAG 1 with finishing weight. <bold>(C)</bold> The association of the abundance of CAG 2 with finishing weight.</p></caption><graphic xlink:href=\"fmicb-11-01835-g003\"/></fig></sec><sec id=\"S3.SS3\"><title>Functionalities of the Gut Microbiome Related to Finishing Weight</title><p>Functionalities of the gut microbiome related to finishing weight were detected by comparing the abundances of CAZymes, GOs, and KEGG items between rabbits with high and low finishing weights.</p><p>A total of 30 CAZymes with significantly different abundances in rabbits with high and low finishing weights were identified (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref> and <xref ref-type=\"supplementary-material\" rid=\"TS4\">Supplementary Table S4</xref>, FDR adjusted <italic>P</italic> < 0.05). Among these, 16 CAZymes especially galactosidase and xylanase (e.g., GH27, GH95, GH4, GH51, GH120, and GH11) were significantly enriched in the gut microbiome of rabbits with high finishing weight. 14 CAZymes, mainly glucosidase (e.g., GH30, GH1, and GH3) had significantly higher abundances in the gut microbiome of low finishing weight rabbits.</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>The differential CAzymes <bold>(A)</bold> and GOs <bold>(B)</bold> identified to associate with finishing weight. “**”, “***”, and “****” represents for FDR adjusted <italic>P</italic> < 0.01, <italic>P</italic> < 0.005, and <italic>P</italic> < 0.001, respectively.</p></caption><graphic xlink:href=\"fmicb-11-01835-g004\"/></fig><p>We also identified 39 GOs that showed different abundances between high and low finishing weight rabbits (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref> and <xref ref-type=\"supplementary-material\" rid=\"TS4\">Supplementary Table S4</xref>, FDR-adjusted <italic>P</italic> < 0.05). Twenty-two out of 39 GOs were more abundant in rabbits with high finishing weight, most of which were related to the metabolism of xylan, galactose, and arabinose (e.g., GO:0005997, GO:0006012, GO:0009045, and GO:0019572) and to the biosynthetic processes of asparagine, cysteine, and arginine (e.g., GO:0006421, GO:0004816, GO:0070981, GO:0004066, GO:0004817, GO:0019344, and GO:0006526). The remaining 17 GOs, predominant in rabbits with low finishing weight, were correlated with the metabolism of glucose (e.g., GO:0008865 and GO:0008878), tryptophan, tyrosine, and phenylalanine (e.g., GO:0004830, GO:0006571, GO:0006436, GO:0006437, GO:0009094, and GO:0006568).</p><p>Sixty KOs exhibited distinct abundances in rabbits with high and low finishing weight (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref> and <xref ref-type=\"supplementary-material\" rid=\"TS4\">Supplementary Table S4</xref>, FDR-adjusted <italic>P</italic> < 0.05). Twenty-six KOs were more abundant in high finishing weight rabbits, most of which were correlated with butanoate metabolism (e.g., K00171, K00169, and K03737), aminoacyl-tRNA biosynthesis (e.g., K01883, K01893, and K01876), and the cysteine and methionine metabolism (e.g., K00016, K00812, and K11358). The other 34 KOs related to tyrosine metabolism (e.g., K04072, K13954, K00274, and K18933), fructose and mannose metabolism (e.g., K16370, K01818, K02771, and K00879), ABC transporters (e.g., K10038, K05668, K19309, and K10554), and two-component system (e.g., K06596, K11614, K03412, and K01034) were plentiful in low finishing weight rabbits.</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>The KEGG function terms showing different enrichments between high and low finishing weight rabbits. <bold>(A)</bold> Heat map of KOs showing different enrichments between high and low finishing weight rabbits. The <italic>X</italic>-axis shows the sample IDs, e.g., H1 representing the individual 1 with high finishing weight. <bold>(B)</bold> Heat map of KEGG pathways showing different enrichments between high and low finishing weight rabbits.</p></caption><graphic xlink:href=\"fmicb-11-01835-g005\"/></fig><p>On the other hand, 25 differential enriched KEGG pathways were identified in the gut microbiome of rabbits with distinct finishing weights (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref> and <xref ref-type=\"supplementary-material\" rid=\"TS4\">Supplementary Table S4</xref>, FDR adjusted <italic>P</italic> < 0.05). Similarly, we found that aminoacyl-tRNA biosynthesis, glycolysis/gluconeogenesis, butanoate metabolism, and cysteine and methionine metabolism were more active in the gut microbial communities of high finishing weight rabbits. Meanwhile, in rabbits with low finishing weight, gut microbiota were more capable of operating ABC transporters and two-component system; additionally, the metabolism of tyrosine, fructose, and mannose was more active in these animals.</p></sec><sec id=\"S3.SS4\"><title>Gut Microbiome as a Predictor of Finishing Weight Variation</title><p>To assess whether the gut microbiome was able to predict finishing weight, we investigated how much degree of phenotypic variance of finishing weight was explained by the gut microbiome by performing 100 times cross-validation analyses at different <italic>p</italic>-value thresholds (ranging from 10<sup>–5</sup> to 0.1) using the OTU data. The OTUs identified at <italic>p</italic> = 1 × 10<sup>–5</sup> could explain 8.01% of the variations in finishing weight (<xref ref-type=\"fig\" rid=\"F6\">Figure 6A</xref>); at <italic>p</italic> = 0.1, the explained variation increased to 10.85%, given that more OTUs were included in the analysis as the threshold increased.</p><fig id=\"F6\" position=\"float\"><label>FIGURE 6</label><caption><p>The predictive role of the gut microbiome in finishing weight. <bold>(A)</bold> The variation of finishing weight explained by the gut microbiome at different <italic>P</italic>-values. <bold>(B)</bold> Metagenomic species were identified as being able to predict finishing weight.</p></caption><graphic xlink:href=\"fmicb-11-01835-g006\"/></fig><p>Then, to evaluate whether a subset of metagenomic species within the gut microbial community could predict the finishing weights of rabbits, we performed random forest analysis in high and low finishing weight rabbits. Thirty-one species were identified as being able to predict finishing weight and showed a substantial overlap with the species above identified as being associated with finishing weight (<xref ref-type=\"fig\" rid=\"F6\">Figure 6B</xref>).</p></sec><sec id=\"S3.SS5\"><title>Changes in Fecal SCFAs Linked to Finishing Weight</title><p>As SCFAs produced by gut microbiota exert important effects on host energy metabolism and intestinal health status regulation in animals (<xref rid=\"B14\" ref-type=\"bibr\">den Besten et al., 2013</xref>), we analyzed the levels of acetic acid, propionic acid, and butyric acid in the feces of rabbits with high and low finishing weights (<xref ref-type=\"fig\" rid=\"F7\">Figure 7A</xref>). Our results showed that the level of butyric acid was significantly greater in the feces of rabbits with high finishing weight in comparison to that in rabbits with low finishing weight (FDR adjusted <italic>P</italic> < 0.05), but the levels of propionic acid tended to decrease. No significant change in the levels of acetic acid was observed between high and low finishing weight rabbits. In addition, correlation analysis indicated that the level of butyric acid had a significantly positive association with finishing weight, while the levels of acetic acid and propionic acid showed the tendency of positive and negative association with finishing weight, respectively (<xref ref-type=\"fig\" rid=\"F7\">Figures 7B–D</xref>).</p><fig id=\"F7\" position=\"float\"><label>FIGURE 7</label><caption><p>Fecal short-chain fatty acid levels in high and low finishing weight rabbits and their correlations with finishing weight. <bold>(A)</bold> Comparisons of fecal short-chain fatty acid levels between high and low finishing weight rabbits. <bold>(B–D)</bold> Correlations between fecal short-chain fatty acid levels and finishing weight.</p></caption><graphic xlink:href=\"fmicb-11-01835-g007\"/></fig><p>We also analyzed the correlations between finishing weight-associated species and SCFAs levels. As shown in <xref ref-type=\"fig\" rid=\"F8\">Figure 8</xref>, nine out of ten species enriched in the high finishing weight individuals were positively associated with the level of butyric acid, and nine out of thirteen species augmented in the low finishing weight individuals were negatively correlated with the level of butyric acid. Both <italic>Anaerotruncus</italic> sp. <italic>CAG:390</italic> and <italic>Bacteroides thetaiotaomicron</italic> were more abundant in low finishing weight rabbits and had positive associations with the level of propionic acid. However, no significant correlations were observed between the level of acetic acid and finishing weight-associated species.</p><fig id=\"F8\" position=\"float\"><label>FIGURE 8</label><caption><p>Heat map showing correlations between finishing weight-associated species and SCFAs levels.</p></caption><graphic xlink:href=\"fmicb-11-01835-g008\"/></fig></sec></sec><sec id=\"S4\"><title>Discussion</title><p>Accumulating evidence indicates that the gut microbiome plays a pivotal role in nutrient metabolism, energy utilization, and health maintenance in domestic animals (<xref rid=\"B57\" ref-type=\"bibr\">O’Callaghan et al., 2016</xref>). Accordingly, the gut microbiome is considered to be an essential factor that affects growth of livestock animals. Thus, identifying gut microbiota as key for production performances could be a game changer in the livestock production industry (<xref rid=\"B52\" ref-type=\"bibr\">Maltecca et al., 2020</xref>). SCFAs are important metabolites generated by gut microbial fermentation of complex carbohydrates, which have emerged as key regulators in intestinal health and energy homeostasis regulation. However, the effects of gut microbiome and SCFAs on finishing weight of meat rabbits have received far less attention. Here, we systematically and comprehensively evaluated how the gut microbiome and SCFAs affect the finishing weight of meat rabbits.</p><p>We identified 15 OTUs that were significantly associated with finishing weight, of which eight OTUs were annotated to members of the family Ruminococcaceae (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Among these, <italic>Ruminococcaceae_UCG-014</italic> and <italic>Ruminococcus_1</italic> were positively associated with finishing weight, while <italic>Ruminococcaceae_UCG-005</italic> and <italic>Ruminiclostridium</italic> showed the opposite correlations. <italic>Ruminococcaceae_UCG-014</italic> belongs to butyrate-producing bacteria capable of degrading cellulose and hemicellulose in feeds and has a potential role in maintenance of intestinal health (<xref rid=\"B12\" ref-type=\"bibr\">Dai et al., 2018</xref>). <italic>Ruminococcus_1</italic> is another important player in butyrate production by fermenting complex non-digestible polysaccharides, which is considered to be related to intestinal anti-inflammatory responses (<xref rid=\"B76\" ref-type=\"bibr\">Xie et al., 2019</xref>). In contrast, <italic>Ruminococcaceae_UCG-005</italic> and <italic>Ruminiclostridium</italic> were positively correlated with diarrhea incidence and intestinal villi damage, respectively (<xref rid=\"B33\" ref-type=\"bibr\">Hung et al., 2019</xref>; <xref rid=\"B77\" ref-type=\"bibr\">Xing et al., 2019</xref>).</p><p>Family Ruminococcaceae encompasses many species. It is important to know which are associated with a good intestinal health status. Metagenomic sequencing analysis sheds light on this. Our results showed that different species from family Ruminococcaceae exert contrasting effects on finishing weight (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). For example, <italic>Ruminococcus albus</italic> and <italic>Ruminococcus flavefaciens</italic> were enriched in rabbits with high finishing weight, but <italic>Ruminococcus gauvreauii</italic> and <italic>Ruminococcus bromii</italic> were more abundant in low finishing weight individuals. Both <italic>R. albus</italic> and <italic>R. flavefaciens</italic> are well-known cellulolytic bacteria involved in the butyrate metabolic pathway and abundant in ruminants with high body weight gain (<xref rid=\"B8\" ref-type=\"bibr\">Carberry et al., 2012</xref>; <xref rid=\"B53\" ref-type=\"bibr\">Mao et al., 2017</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Izuddin et al., 2019</xref>). However, both <italic>R. gauvreauii</italic> and <italic>R. bromii</italic> are mucolytic bacteria which can induce chronic intestinal inflammation (<xref rid=\"B49\" ref-type=\"bibr\">Lyra et al., 2009</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Crost et al., 2018</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Fernandez et al., 2018</xref>). Moreover, <italic>R. bromii</italic> is negatively associated with body weight in pigs (<xref rid=\"B69\" ref-type=\"bibr\">Tran et al., 2018</xref>).</p><p>We identified several other species that could significantly affect finishing weight. For instance, <italic>Faecalibacterium prausnitzii</italic> is a major butyrate producer in the intestine, which has an important role in providing energy sources for enterocytes and fulfilling anti-inflammatory actions (<xref rid=\"B25\" ref-type=\"bibr\">Foditsch et al., 2014</xref>). <italic>Lactobacillus ruminis</italic> is a member of lactic acid bacteria that has multiple probiotic properties, including inhibition of intestinal pathogens, fortification of epithelial barrier functions, and modulation of immune responses (<xref rid=\"B81\" ref-type=\"bibr\">Yu et al., 2017</xref>). In agreement with previous studies (<xref rid=\"B58\" ref-type=\"bibr\">Oikonomou et al., 2013</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Gao et al., 2017</xref>), we found that these two species were more abundant in the gut microbiome of individuals with a higher body weight. In contrast, although both <italic>Akkermansia muciniphila</italic> and <italic>Bacteroides fragilis</italic> were considered to be promising candidates as probiotics (<xref rid=\"B30\" ref-type=\"bibr\">Gilbert et al., 2013</xref>; <xref rid=\"B87\" ref-type=\"bibr\">Zhang T. et al., 2019</xref>), the overgrowth of these two species in the gut of rabbits could result in the decreased butyrate yield and lead to the incidence of epizootic rabbit enteropathy (ERE, a severe gastrointestinal syndrome disease) (<xref rid=\"B38\" ref-type=\"bibr\">Jin et al., 2018</xref>). Hence, these two species augmenting in the gut microbiome of meat rabbits could be a potential trigger for reduction of finishing weight.</p><p>In order to make a microbial community-based inference, it is important not only to identify microbial taxa associated with finishing weight but also to understand the effects of the interactions among such taxa on finishing weight. Thus, co-abundance networks were established at both metagenomic species and OTU level. We found that co-occurrence and co-exclusion relationships exist among finishing weight-associated microbial taxa (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS3\">Supplementary Figure S3</xref>). Importantly, their interactions which could affect the finishing weight have intimate relations with their distinct roles in host metabolic and intestinal health regulation. Similarly, earlier studies have revealed that interactions of gut microbes affect the body weight in farm animals. Yang et al. demonstrated that the strong co-occurrence relationship between butyrate-producing bacteria (e.g., Ruminococcaceae) and lactic acid bacteria (e.g., <italic>Lactobacillus</italic>) may affect porcine body weight gain by regulating host appetite and feeding behavior (<xref rid=\"B78\" ref-type=\"bibr\">Yang et al., 2018</xref>). <xref rid=\"B84\" ref-type=\"bibr\">Zhang et al. (2018)</xref> suggested that the significant co-exclusion relationship between Enterobacteriaceae and Prevotellaceae was associated with the metabolism of SCFAs and energy, which provides an important direction for manipulating gut microbiota to improve the body weight in pigs. Additionally, <xref rid=\"B29\" ref-type=\"bibr\">Gao et al. (2017)</xref> and <xref rid=\"B50\" ref-type=\"bibr\">Ma et al. (2018)</xref> highlighted the important role of gut microbial interactions in improving growth performances of broiler chickens.</p><p>Distinct metagenomic functional capacities were also found to be associated with finishing weight. The CAZyme comparison analysis showed that galactosidase and xylanase were more abundant in rabbits with high finishing weight, while glucosidase was more abundant in low finishing weight rabbits (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>). Adding extra galactosidase and xylanase into feeds has been reported to enhance nutrient (e.g., fibers and amino acids) digestibility and improve the body weight of farm animals (<xref rid=\"B86\" ref-type=\"bibr\">Zhang et al., 2017</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Jasek et al., 2018</xref>). This could be used to explain why rabbits with a greater abundance of these two enzymes showed greater finishing weights. However, the effects of gut microbial glucosidase on body weight are conflicting. <xref rid=\"B13\" ref-type=\"bibr\">De Cesare et al. (2017)</xref> demonstrated that a higher abundance of glucosidase in the gut microbiome contributed to increased body weight gain of broiler chickens, whereas <xref rid=\"B67\" ref-type=\"bibr\">Shokryazdan et al. (2017)</xref> suggested that the reduced glucosidase enzyme activity in the intestine led to an increase in body weight in broiler chickens.</p><p>Our results also showed that GOs related to xylan, galactose, and arabinose metabolism were more abundant in rabbits with high finishing weight, while those associated with glucose metabolism showed higher abundances in low finishing weight rabbits (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>). Additionally, we found that the gut microbiota of rabbits with high finishing weight preferred to be involved in the biosynthesis of asparagine, cysteine, and arginine, but gut microbial communities in low finishing weight rabbits were more active in metabolizing tryptophan, tyrosine, and phenylalanine. Although asparagine is a non-essential amino acid, emerging evidence has demonstrated that it plays a key role in attenuating intestinal injury and improving the energy status of enterocytes, all of which are good for the health and growth of animals (<xref rid=\"B73\" ref-type=\"bibr\">Wang et al., 2015</xref>; <xref rid=\"B59\" ref-type=\"bibr\">Patra et al., 2019</xref>). Cysteine is able to regulate the antioxidant status and expression of anti-inflammatory genes in intestinal cells, which exert beneficial effects on body weight gain in pigs and chickens (<xref rid=\"B15\" ref-type=\"bibr\">Dilger and Baker, 2007</xref>; <xref rid=\"B79\" ref-type=\"bibr\">Yang and Liao, 2019</xref>). Arginine is a hub precursor for the synthesis of various important metabolic molecules, including NO and polyamines, exerting regulatory roles in the host’s metabolic processes (<xref rid=\"B24\" ref-type=\"bibr\">Flynn et al., 2002</xref>). Thus, it has been used to improve meat production in meat-producing animals (<xref rid=\"B32\" ref-type=\"bibr\">Hu et al., 2017</xref>; <xref rid=\"B48\" ref-type=\"bibr\">Liu et al., 2019</xref>). Conversely, gut microbiota is involved in aromatic amino acids (tryptophan, tyrosine, and phenylalanine) metabolism has been linked to chronic low-grade inflammation and metabolic disorders, which may hinder development and growth of animals (<xref rid=\"B72\" ref-type=\"bibr\">Wang et al., 2011</xref>; <xref rid=\"B1\" ref-type=\"bibr\">Agus et al., 2018</xref>).</p><p>On the other hand, the aminoacyl-tRNA biosynthesis pathway and the metabolic pathways of butanoate, cysteine, and methionine were more active in high finishing weight rabbits, whereas ABC transporters, and fructose, mannose, and tyrosine metabolic pathways were overrepresented in rabbits with low finishing weight (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>). The aminoacyl-tRNA biosynthesis pathway is an essential metabolic function of microorganisms and is intimately correlated with maturation of gastrointestinal microbiota (<xref rid=\"B83\" ref-type=\"bibr\">Zhang K. et al., 2019</xref>). More importantly, microbial mature status is fundamental for host development and growth (<xref rid=\"B74\" ref-type=\"bibr\">Wang X. et al., 2019</xref>). Several studies have demonstrated that the gut microbial butanoate metabolic pathway exerts beneficial effects on body weight of livestock due to its potential impact on increasing energy intake, improving intestinal histomorphometric characteristics (e.g., villi height and crypt depth), and modulating the immune system (<xref rid=\"B55\" ref-type=\"bibr\">Ndou et al., 2018</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Che et al., 2019</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Jacquier et al., 2019</xref>). Similar to the abovementioned cysteine, methionine is crucial in maintaining the functions of intestinal cells by regulating the redox status (<xref rid=\"B3\" ref-type=\"bibr\">Azad et al., 2019</xref>). Thus, enhanced methionine metabolism in the gut microbial communities contributed to bovine body weight gain (<xref rid=\"B37\" ref-type=\"bibr\">Jiao et al., 2017</xref>). On the other hand, ABC transporters involved in transporting a large variety of sterols, lipids, drugs, and primary and secondary metabolites (<xref rid=\"B31\" ref-type=\"bibr\">Hou et al., 2017</xref>) have recently been related to fat deposition, drug resistance, and colonic inflammation (<xref rid=\"B2\" ref-type=\"bibr\">Andersen et al., 2015</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Fang et al., 2017</xref>), which may do harm for growth performances in animals. Furthermore, enhanced fructose, mannose, and tyrosine metabolic pathways in the gut microbiome have been associated with host intestinal permeability and metabolic endotoxemia that implies their negative effects on growth of animals (<xref rid=\"B72\" ref-type=\"bibr\">Wang et al., 2011</xref>; <xref rid=\"B16\" ref-type=\"bibr\">Do et al., 2018</xref>).</p><p>To further emphasize the predictive role of the gut microbiome in finishing weight variation, we estimated that the gut microbiome could explain 7.23–10.85% of the variation in finishing weight (<xref ref-type=\"fig\" rid=\"F6\">Figure 6A</xref>), which is similar to host genetics on finishing weight (6.3–12.1%) (<xref rid=\"B60\" ref-type=\"bibr\">Piles et al., 2007</xref>). Moreover, we identified 31 species that could act as predictors of finishing weight, most of which are strongly associated with finishing weight (<xref ref-type=\"fig\" rid=\"F6\">Figure 6B</xref>). Consistent with prior studies, our findings suggested that the gut microbiome should be regarded as a key variable of body weight prediction model of farm animals (<xref rid=\"B74\" ref-type=\"bibr\">Wang X. et al., 2019</xref>; <xref rid=\"B75\" ref-type=\"bibr\">Wen et al., 2019</xref>).</p><p>Our results also revealed that the level of fecal butyric acid was significantly higher in rabbits with high finishing weight in comparison to rabbits with low finishing weight (<xref ref-type=\"fig\" rid=\"F7\">Figure 7A</xref>). Importantly, the level of butyric acid showed positive associations with finishing weight (<xref ref-type=\"fig\" rid=\"F7\">Figure 7B</xref>). Butyrate is not only recognized as an essential energy source but also acts as a signal transduction molecule of G-protein-coupled receptors (FFAR3, GPR109A) and as epigenetic regulators of gene expression by inhibiting histone deacetylase (HDAC) (<xref rid=\"B40\" ref-type=\"bibr\">Kasubuchi et al., 2015</xref>). Due to these important properties, butyrate exhibits wide energy and metabolic regulation abilities and strong anti-inflammatory effects that positively affect the body weight gain of animals (<xref rid=\"B4\" ref-type=\"bibr\">Bedford and Gong, 2018</xref>). Consistently, we found that several microbial species positively associated with finishing weight (such as, <italic>Faecalibacterium prausnitzii</italic>, <italic>Roseburia</italic> sp. <italic>CAG:303</italic>, and <italic>butyrate-producing bacterium SS3/4</italic>) were well-known butyrate producers (<xref rid=\"B62\" ref-type=\"bibr\">Qin et al., 2012</xref>) that had positive associations with butyric acid level (<xref ref-type=\"fig\" rid=\"F8\">Figure 8</xref>). Interestingly, we also observed that both <italic>Lactobacillus ruminis</italic> and <italic>Bifidobacterium saeculare</italic> were positively associated with butyric acid level. This is in accordance with previous findings which revealed that high levels of intestinal <italic>Lactobacillus</italic> sp. and <italic>Bifidobacterium</italic> sp. contributed to increased butyrate levels (<xref rid=\"B44\" ref-type=\"bibr\">Le Roy et al., 2015</xref>; <xref rid=\"B42\" ref-type=\"bibr\">Kim et al., 2020</xref>). In addition, our results showed the changes in the levels of acetic acid and propionic acid in the feces of rabbits with high and low finishing weight and their potential correlations with finishing weight (<xref ref-type=\"fig\" rid=\"F7\">Figures 7A,C,D</xref>). These findings were in agreement with previous studies suggested that both acetate and propionate were important players in modulating growth performances in animals (<xref rid=\"B47\" ref-type=\"bibr\">Liu et al., 2018</xref>; <xref rid=\"B54\" ref-type=\"bibr\">Min et al., 2019</xref>).</p><p>Our study, although limited by a small rabbit population, provides important information that supports the potential of gut microbiota manipulation in improving finishing weight of meat rabbits. However, from potential to action, the next steps are to validate the causative roles of some gut bacterial species in modulating finishing weight through specific pathogen-free (SPF) rabbits’ intervention experiment and to gain more mechanistic insights into the cross talk between gut microbiome and host and its effects on growth performances of meat rabbits by multi-omics studies.</p></sec><sec id=\"S5\"><title>Conclusion</title><p>In conclusion, the current study identified key microbial taxa associated with finishing weight. We also determined the gut microbial CAZymes, GOs, KOs, and KEGG pathways associated with finishing weight. We found that the gut microbiome could act as a key predictor of finishing weight variation. In addition, we emphasized the important effects of SCFAs on finishing weight, especially butyrate. Hence, our findings provide essential insights into how the gut microbiome and SCFAS affect finishing weight and imply that manipulating the gut microbial community could be an efficient strategy to improve finishing weight in the meat rabbit industry.</p></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ <xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Material</xref>.</p></sec><sec id=\"S7\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by the Animal Care and Use Committee (ACUC) in Fujian Agriculture and Forestry University.</p></sec><sec id=\"S8\"><title>Author Contributions</title><p>QG designed the experiments, analyzed the data, and wrote and revised the manuscript. SF and XC performed the experiments, analyzed the data, and wrote the manuscript. LZ, XY, and SX performed the experiments. All authors read and approved the final manuscript.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported in part by grants from the education and scientific research project for junior researchers of the Fujian Educational Bureau (JAT190124), the Fujian Agriculture and Forestry University International Science and Technology Cooperation and Exchange Program (KXb16003A), the Fujian Foreign Cooperation Project (2016I0001), and the Fujian Agriculture and Forestry University Science and Technology Innovation Special Fund (CXZX2017059).</p></fn></fn-group><ack><p>We are grateful to the manager Mr. Zhoulin Chen of the commercial Ira rabbit farm who provides the opportunity to collect samples and phenotype measurement and the workers who are making contributions to the management of the experimental rabbit population.</p></ack><sec id=\"S11\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fmicb.2020.01835/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fmicb.2020.01835/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"FS1\"><label>FIGURE S1</label><caption><p>Finishing weight phenotypic values of all rabbits <bold>(A)</bold> and high and low individuals <bold>(B)</bold>.</p></caption><media xlink:href=\"Image_1.TIFF\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS2\"><label>FIGURE S2</label><caption><p>The finishing weight associated species formed two clusters using Ward clustering algorithm.</p></caption><media xlink:href=\"Image_2.TIFF\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS3\"><label>FIGURE S3</label><caption><p><bold>(A,B)</bold> Constructions of CAGs and interactions network by using the finishing weight associated OTUs. <bold>(C,D)</bold> Associations between CAGs and finishing weight.</p></caption><media xlink:href=\"Image_3.TIFF\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS1\"><label>TABLE S1</label><caption><p>Composition of pellet diet for fattening rabbits.</p></caption><media xlink:href=\"Table_1.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS2\"><label>TABLE S2</label><caption><p>The OTUs showing significant associations with finishing weight.</p></caption><media xlink:href=\"Table_2.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS3\"><label>TABLE S3</label><caption><p>Differential microbial species between high and low finishing weight rabbits.</p></caption><media xlink:href=\"Table_3.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS4\"><label>TABLE S4</label><caption><p>Functionalities showing different enrichments between high and low finishing weight rabbits.</p></caption><media xlink:href=\"Table_4.xlsx\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Agus</surname><given-names>A.</given-names></name><name><surname>Planchais</surname><given-names>J.</given-names></name><name><surname>Sokol</surname><given-names>H.</given-names></name></person-group> (<year>2018</year>). <article-title>Gut microbiota regulation of tryptophan metabolism in health and disease.</article-title>\n<source><italic>Cell Host Microbe</italic></source>\n<volume>23</volume>\n<fpage>716</fpage>–<lpage>724</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.chom.2018.05.003</pub-id>\n<pub-id pub-id-type=\"pmid\">29902437</pub-id></mixed-citation></ref><ref id=\"B2\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Andersen</surname><given-names>V.</given-names></name><name><surname>Svenningsen</surname><given-names>K.</given-names></name><name><surname>Knudsen</surname><given-names>L. A.</given-names></name><name><surname>Hansen</surname><given-names>A. K.</given-names></name><name><surname>Holmskov</surname><given-names>U.</given-names></name><name><surname>Stensballe</surname><given-names>A.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>Novel understanding of ABC transporters ABCB1/MDR/P-glycoprotein, ABCC2/MRP2, and ABCG2/BCRP in colorectal pathophysiology.</article-title>\n<source><italic>World J. Gastroenterol.</italic></source>\n<volume>21</volume>\n<fpage>11862</fpage>–<lpage>11876</lpage>. <pub-id pub-id-type=\"doi\">10.3748/wjg.v21.i41.11862</pub-id>\n<pub-id pub-id-type=\"pmid\">26557010</pub-id></mixed-citation></ref><ref id=\"B3\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Azad</surname><given-names>M. A. K.</given-names></name><name><surname>Liu</surname><given-names>G.</given-names></name><name><surname>Bin</surname><given-names>P.</given-names></name><name><surname>Ding</surname><given-names>S.</given-names></name><name><surname>Kong</surname><given-names>X.</given-names></name><name><surname>Guan</surname><given-names>G.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Sulfur-containing amino acid supplementation to gilts from late pregnancy to lactation altered offspring’s intestinal microbiota and plasma metabolites.</article-title>\n<source><italic>Appl. Microbiol. Biotechnol.</italic></source>\n<volume>104</volume>\n<fpage>1227</fpage>–<lpage>1242</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00253-019-10302-6</pub-id>\n<pub-id pub-id-type=\"pmid\">31853564</pub-id></mixed-citation></ref><ref id=\"B4\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bedford</surname><given-names>A.</given-names></name><name><surname>Gong</surname><given-names>J.</given-names></name></person-group> (<year>2018</year>). <article-title>Implications of butyrate and its derivatives for gut health and animal production.</article-title>\n<source><italic>Anim. Nutr.</italic></source>\n<volume>4</volume>\n<fpage>151</fpage>–<lpage>159</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.aninu.2017.08.010</pub-id>\n<pub-id pub-id-type=\"pmid\">30140754</pub-id></mixed-citation></ref><ref id=\"B5\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Breiman</surname><given-names>L.</given-names></name></person-group> (<year>2001</year>). <article-title>Random forests.</article-title>\n<source><italic>Mach. Learn.</italic></source>\n<volume>45</volume>\n<fpage>5</fpage>–<lpage>32</lpage>. <pub-id pub-id-type=\"doi\">10.1023/A:1010933404324</pub-id></mixed-citation></ref><ref id=\"B6\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Buchfink</surname><given-names>B.</given-names></name><name><surname>Xie</surname><given-names>C.</given-names></name><name><surname>Huson</surname><given-names>D. H.</given-names></name></person-group> (<year>2015</year>). <article-title>Fast and sensitive protein alignment using DIAMOND.</article-title>\n<source><italic>Nat. Methods</italic></source>\n<volume>12</volume>\n<fpage>59</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nmeth.3176</pub-id>\n<pub-id pub-id-type=\"pmid\">25402007</pub-id></mixed-citation></ref><ref id=\"B7\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Caporaso</surname><given-names>J. G.</given-names></name><name><surname>Kuczynski</surname><given-names>J.</given-names></name><name><surname>Stombaugh</surname><given-names>J.</given-names></name><name><surname>Bittinger</surname><given-names>K.</given-names></name><name><surname>Bushman</surname><given-names>F. D.</given-names></name><name><surname>Costello</surname><given-names>E. K.</given-names></name><etal/></person-group> (<year>2010</year>). <article-title>QIIME allows analysis of high-throughput community sequencing data.</article-title>\n<source><italic>Nat. Methods</italic></source>\n<volume>7</volume>\n<fpage>335</fpage>–<lpage>336</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nmeth.f.303</pub-id>\n<pub-id pub-id-type=\"pmid\">20383131</pub-id></mixed-citation></ref><ref id=\"B8\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carberry</surname><given-names>C. A.</given-names></name><name><surname>Kenny</surname><given-names>D. A.</given-names></name><name><surname>Han</surname><given-names>S.</given-names></name><name><surname>McCabe</surname><given-names>M. S.</given-names></name><name><surname>Waters</surname><given-names>S. M.</given-names></name></person-group> (<year>2012</year>). <article-title>Effect of phenotypic residual feed intake and dietary forage content on the rumen microbial community of beef cattle.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>78</volume>\n<fpage>4949</fpage>–<lpage>4958</lpage>. <pub-id pub-id-type=\"doi\">10.1128/AEM.07759-11</pub-id>\n<pub-id pub-id-type=\"pmid\">22562991</pub-id></mixed-citation></ref><ref id=\"B9\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Che</surname><given-names>L.</given-names></name><name><surname>Hu</surname><given-names>Q.</given-names></name><name><surname>Wang</surname><given-names>R.</given-names></name><name><surname>Zhang</surname><given-names>D.</given-names></name><name><surname>Liu</surname><given-names>C.</given-names></name><name><surname>Zhang</surname><given-names>Y.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Inter-correlated gut microbiota and SCFAs changes upon antibiotics exposure links with rapid body-mass gain in weaned piglet model.</article-title>\n<source><italic>J. Nutr. Biochem.</italic></source>\n<volume>74</volume>:<issue>108246</issue>. <pub-id pub-id-type=\"doi\">10.1016/j.jnutbio.2019.108246</pub-id>\n<pub-id pub-id-type=\"pmid\">31671360</pub-id></mixed-citation></ref><ref id=\"B10\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>S.</given-names></name><name><surname>Zhou</surname><given-names>Y.</given-names></name><name><surname>Chen</surname><given-names>Y.</given-names></name><name><surname>Gu</surname><given-names>J.</given-names></name></person-group> (<year>2018</year>). <article-title>fastp: an ultra-fast all-in-one FASTQ preprocessor.</article-title>\n<source><italic>Bioinformatics</italic></source>\n<volume>34</volume>\n<fpage>i884</fpage>–<lpage>i890</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bioinformatics/bty560</pub-id>\n<pub-id pub-id-type=\"pmid\">30423086</pub-id></mixed-citation></ref><ref id=\"B11\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Crost</surname><given-names>E. H.</given-names></name><name><surname>Le Gall</surname><given-names>G.</given-names></name><name><surname>Laverde-Gomez</surname><given-names>J. A.</given-names></name><name><surname>Mukhopadhya</surname><given-names>I.</given-names></name><name><surname>Flint</surname><given-names>H. J.</given-names></name><name><surname>Juge</surname><given-names>N.</given-names></name></person-group> (<year>2018</year>). <article-title>Mechanistic insights into the cross-feeding of <italic>Ruminococcus gnavus</italic> and <italic>Ruminococcus bromii</italic> on host and dietary carbohydrates.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>9</volume>:<issue>2558</issue>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2018.02558</pub-id>\n<pub-id pub-id-type=\"pmid\">30455672</pub-id></mixed-citation></ref><ref id=\"B12\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dai</surname><given-names>S. J.</given-names></name><name><surname>Zhang</surname><given-names>K. Y.</given-names></name><name><surname>Ding</surname><given-names>X. M.</given-names></name><name><surname>Bai</surname><given-names>S. P.</given-names></name><name><surname>Luo</surname><given-names>Y. H.</given-names></name><name><surname>Wang</surname><given-names>J. P.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Effect of dietary non-phytate phosphorus levels on the diversity and structure of cecal microbiota in meat duck from 1 to 21 d of age.</article-title>\n<source><italic>Poult. Sci.</italic></source>\n<volume>97</volume>\n<fpage>2441</fpage>–<lpage>2450</lpage>. <pub-id pub-id-type=\"doi\">10.3382/ps/pey090</pub-id>\n<pub-id pub-id-type=\"pmid\">29617914</pub-id></mixed-citation></ref><ref id=\"B13\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Cesare</surname><given-names>A.</given-names></name><name><surname>Sirri</surname><given-names>F.</given-names></name><name><surname>Manfreda</surname><given-names>G.</given-names></name><name><surname>Moniaci</surname><given-names>P.</given-names></name><name><surname>Giardini</surname><given-names>A.</given-names></name><name><surname>Zampiga</surname><given-names>M.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Effect of dietary supplementation with <italic>Lactobacillus acidophilus</italic> D2/CSL (CECT 4529) on caecum microbioma and productive performance in broiler chickens.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>12</volume>:<issue>e0176309</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0176309</pub-id>\n<pub-id pub-id-type=\"pmid\">28472118</pub-id></mixed-citation></ref><ref id=\"B14\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>den Besten</surname><given-names>G.</given-names></name><name><surname>van Eunen</surname><given-names>K.</given-names></name><name><surname>Groen</surname><given-names>A. K.</given-names></name><name><surname>Venema</surname><given-names>K.</given-names></name><name><surname>Reijngoud</surname><given-names>D. J.</given-names></name><name><surname>Bakker</surname><given-names>B. M.</given-names></name></person-group> (<year>2013</year>). <article-title>The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism.</article-title>\n<source><italic>J. Lipid Res.</italic></source>\n<volume>54</volume>\n<fpage>2325</fpage>–<lpage>2340</lpage>. <pub-id pub-id-type=\"doi\">10.1194/jlr.R036012</pub-id>\n<pub-id pub-id-type=\"pmid\">23821742</pub-id></mixed-citation></ref><ref id=\"B15\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dilger</surname><given-names>R. N.</given-names></name><name><surname>Baker</surname><given-names>D. H.</given-names></name></person-group> (<year>2007</year>). <article-title>Oral N-acetyl-L-cysteine is a safe and effective precursor of cysteine.</article-title>\n<source><italic>J. Anim Sci.</italic></source>\n<volume>85</volume>\n<fpage>1712</fpage>–<lpage>1718</lpage>. <pub-id pub-id-type=\"doi\">10.2527/jas.2006-835</pub-id>\n<pub-id pub-id-type=\"pmid\">17371789</pub-id></mixed-citation></ref><ref id=\"B16\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Do</surname><given-names>M. H.</given-names></name><name><surname>Lee</surname><given-names>E.</given-names></name><name><surname>Oh</surname><given-names>M. J.</given-names></name><name><surname>Kim</surname><given-names>Y.</given-names></name><name><surname>Park</surname><given-names>H. Y.</given-names></name></person-group> (<year>2018</year>). <article-title>High-glucose or -fructose diet cause changes of the gut microbiota and metabolic disorders in mice without body weight change.</article-title>\n<source><italic>Nutrients</italic></source>\n<volume>10</volume>:<issue>639</issue>. <pub-id pub-id-type=\"doi\">10.3390/nu10060761</pub-id>\n<pub-id pub-id-type=\"pmid\">29899272</pub-id></mixed-citation></ref><ref id=\"B17\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Du</surname><given-names>R.</given-names></name><name><surname>Jiao</surname><given-names>S.</given-names></name><name><surname>Dai</surname><given-names>Y.</given-names></name><name><surname>An</surname><given-names>J.</given-names></name><name><surname>Lv</surname><given-names>J.</given-names></name><name><surname>Yan</surname><given-names>X.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Probiotic <italic>Bacillus amyloliquefaciens</italic> C-1 improves growth performance, stimulates GH/IGF-1, and regulates the gut microbiota of growth-retarded beef calves.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>9</volume>:<issue>2006</issue>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2018.02006</pub-id>\n<pub-id pub-id-type=\"pmid\">30210477</pub-id></mixed-citation></ref><ref id=\"B18\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eddy</surname><given-names>S. R.</given-names></name></person-group> (<year>2011</year>). <article-title>Accelerated profile HMM searches.</article-title>\n<source><italic>PLoS Comput. Biol.</italic></source>\n<volume>7</volume>:<issue>e1002195</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pcbi.1002195</pub-id>\n<pub-id pub-id-type=\"pmid\">22039361</pub-id></mixed-citation></ref><ref id=\"B19\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Edgar</surname><given-names>R. C.</given-names></name></person-group> (<year>2010</year>). <article-title>Search and clustering orders of magnitude faster than BLAST.</article-title>\n<source><italic>Bioinformatics</italic></source>\n<volume>26</volume>\n<fpage>2460</fpage>–<lpage>2461</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bioinformatics/btq461</pub-id>\n<pub-id pub-id-type=\"pmid\">20709691</pub-id></mixed-citation></ref><ref id=\"B20\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fang</surname><given-names>S.</given-names></name><name><surname>Chen</surname><given-names>X.</given-names></name><name><surname>Pan</surname><given-names>J.</given-names></name><name><surname>Chen</surname><given-names>Q.</given-names></name><name><surname>Zhou</surname><given-names>L.</given-names></name><name><surname>Wang</surname><given-names>C.</given-names></name><etal/></person-group> (<year>2020</year>). <article-title>Dynamic distribution of gut microbiota in meat rabbits at different growth stages and relationship with average daily gain (ADG).</article-title>\n<source><italic>BMC Microbiol.</italic></source>\n<volume>20</volume>:<issue>116</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12866-020-01797-5</pub-id>\n<pub-id pub-id-type=\"pmid\">32410629</pub-id></mixed-citation></ref><ref id=\"B21\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fang</surname><given-names>S.</given-names></name><name><surname>Chen</surname><given-names>X.</given-names></name><name><surname>Zhou</surname><given-names>L.</given-names></name><name><surname>Wang</surname><given-names>C.</given-names></name><name><surname>Chen</surname><given-names>Q.</given-names></name><name><surname>Lin</surname><given-names>R.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Faecal microbiota and functional capacity associated with weaning weight in meat rabbits.</article-title>\n<source><italic>Microb. Biotechnol.</italic></source>\n<volume>12</volume>\n<fpage>1441</fpage>–<lpage>1452</lpage>. <pub-id pub-id-type=\"doi\">10.1111/1751-7915.13485</pub-id>\n<pub-id pub-id-type=\"pmid\">31571427</pub-id></mixed-citation></ref><ref id=\"B22\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fang</surname><given-names>S.</given-names></name><name><surname>Xiong</surname><given-names>X.</given-names></name><name><surname>Su</surname><given-names>Y.</given-names></name><name><surname>Huang</surname><given-names>L.</given-names></name><name><surname>Chen</surname><given-names>C.</given-names></name></person-group> (<year>2017</year>). <article-title>16S rRNA gene-based association study identified microbial taxa associated with pork intramuscular fat content in feces and cecum lumen.</article-title>\n<source><italic>BMC Microbiol.</italic></source>\n<volume>17</volume>:<issue>162</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12866-017-1055-x</pub-id>\n<pub-id pub-id-type=\"pmid\">28724349</pub-id></mixed-citation></ref><ref id=\"B23\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fernandez</surname><given-names>J.</given-names></name><name><surname>Moreno</surname><given-names>F. J.</given-names></name><name><surname>Olano</surname><given-names>A.</given-names></name><name><surname>Clemente</surname><given-names>A.</given-names></name><name><surname>Villar</surname><given-names>C. J.</given-names></name><name><surname>Lombo</surname><given-names>F.</given-names></name></person-group> (<year>2018</year>). <article-title>A galacto-oligosaccharides preparation derived from lactulose protects against colorectal cancer development in an animal model.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>9</volume>:<issue>2004</issue>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2018.02004</pub-id>\n<pub-id pub-id-type=\"pmid\">30233512</pub-id></mixed-citation></ref><ref id=\"B24\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Flynn</surname><given-names>N. E.</given-names></name><name><surname>Meininger</surname><given-names>C. J.</given-names></name><name><surname>Haynes</surname><given-names>T. E.</given-names></name><name><surname>Wu</surname><given-names>G.</given-names></name></person-group> (<year>2002</year>). <article-title>The metabolic basis of arginine nutrition and pharmacotherapy.</article-title>\n<source><italic>Biomed. Pharmacother.</italic></source>\n<volume>56</volume>\n<fpage>427</fpage>–<lpage>438</lpage>. <pub-id pub-id-type=\"doi\">10.1016/s0753-3322(02)00273-1</pub-id><pub-id pub-id-type=\"pmid\">12481979</pub-id></mixed-citation></ref><ref id=\"B25\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Foditsch</surname><given-names>C.</given-names></name><name><surname>Santos</surname><given-names>T. M.</given-names></name><name><surname>Teixeira</surname><given-names>A. G.</given-names></name><name><surname>Pereira</surname><given-names>R. V.</given-names></name><name><surname>Dias</surname><given-names>J. M.</given-names></name><name><surname>Gaeta</surname><given-names>N.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Isolation and characterization of <italic>Faecalibacterium prausnitzii</italic> from calves and piglets.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>9</volume>:<issue>e116465</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0116465</pub-id>\n<pub-id pub-id-type=\"pmid\">25551453</pub-id></mixed-citation></ref><ref id=\"B26\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Friedman</surname><given-names>J.</given-names></name><name><surname>Alm</surname><given-names>E. J.</given-names></name></person-group> (<year>2012</year>). <article-title>Inferring correlation networks from genomic survey data.</article-title>\n<source><italic>PLoS Comput. Biol.</italic></source>\n<volume>8</volume>:<issue>e1002687</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pcbi.1002687</pub-id>\n<pub-id pub-id-type=\"pmid\">23028285</pub-id></mixed-citation></ref><ref id=\"B27\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fu</surname><given-names>J.</given-names></name><name><surname>Bonder</surname><given-names>M. J.</given-names></name><name><surname>Cenit</surname><given-names>M. C.</given-names></name><name><surname>Tigchelaar</surname><given-names>E. F.</given-names></name><name><surname>Maatman</surname><given-names>A.</given-names></name><name><surname>Dekens</surname><given-names>J. A.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>The gut microbiome contributes to a substantial proportion of the variation in blood lipids.</article-title>\n<source><italic>Circ. Res.</italic></source>\n<volume>117</volume>\n<fpage>817</fpage>–<lpage>824</lpage>. <pub-id pub-id-type=\"doi\">10.1161/CIRCRESAHA.115.306807</pub-id>\n<pub-id pub-id-type=\"pmid\">26358192</pub-id></mixed-citation></ref><ref id=\"B28\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fu</surname><given-names>L.</given-names></name><name><surname>Niu</surname><given-names>B.</given-names></name><name><surname>Zhu</surname><given-names>Z.</given-names></name><name><surname>Wu</surname><given-names>S.</given-names></name><name><surname>Li</surname><given-names>W.</given-names></name></person-group> (<year>2012</year>). <article-title>CD-HIT: accelerated for clustering the next-generation sequencing data.</article-title>\n<source><italic>Bioinformatics</italic></source>\n<volume>28</volume>\n<fpage>3150</fpage>–<lpage>3152</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bioinformatics/bts565</pub-id>\n<pub-id pub-id-type=\"pmid\">23060610</pub-id></mixed-citation></ref><ref id=\"B29\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gao</surname><given-names>P.</given-names></name><name><surname>Ma</surname><given-names>C.</given-names></name><name><surname>Sun</surname><given-names>Z.</given-names></name><name><surname>Wang</surname><given-names>L.</given-names></name><name><surname>Huang</surname><given-names>S.</given-names></name><name><surname>Su</surname><given-names>X.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Feed-additive probiotics accelerate yet antibiotics delay intestinal microbiota maturation in broiler chicken.</article-title>\n<source><italic>Microbiome</italic></source>\n<volume>5</volume>:<issue>91</issue>. <pub-id pub-id-type=\"doi\">10.1186/s40168-017-0315-1</pub-id>\n<pub-id pub-id-type=\"pmid\">28768551</pub-id></mixed-citation></ref><ref id=\"B30\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gilbert</surname><given-names>J. A.</given-names></name><name><surname>Krajmalnik-Brown</surname><given-names>R.</given-names></name><name><surname>Porazinska</surname><given-names>D. L.</given-names></name><name><surname>Weiss</surname><given-names>S. J.</given-names></name><name><surname>Knight</surname><given-names>R.</given-names></name></person-group> (<year>2013</year>). <article-title>Toward effective probiotics for autism and other neurodevelopmental disorders.</article-title>\n<source><italic>Cell</italic></source>\n<volume>155</volume>\n<fpage>1446</fpage>–<lpage>1448</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cell.2013.11.035</pub-id>\n<pub-id pub-id-type=\"pmid\">24360269</pub-id></mixed-citation></ref><ref id=\"B31\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hou</surname><given-names>Y. P.</given-names></name><name><surname>He</surname><given-names>Q. Q.</given-names></name><name><surname>Ouyang</surname><given-names>H. M.</given-names></name><name><surname>Peng</surname><given-names>H. S.</given-names></name><name><surname>Wang</surname><given-names>Q.</given-names></name><name><surname>Li</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Human gut microbiota associated with obesity in chinese children and adolescents.</article-title>\n<source><italic>Biomed. Res. Int.</italic></source>\n<volume>2017</volume>:<issue>7585989</issue>. <pub-id pub-id-type=\"doi\">10.1155/2017/7585989</pub-id>\n<pub-id pub-id-type=\"pmid\">29214176</pub-id></mixed-citation></ref><ref id=\"B32\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hu</surname><given-names>C. J.</given-names></name><name><surname>Jiang</surname><given-names>Q. Y.</given-names></name><name><surname>Zhang</surname><given-names>T.</given-names></name><name><surname>Yin</surname><given-names>Y. L.</given-names></name><name><surname>Li</surname><given-names>F. N.</given-names></name><name><surname>Deng</surname><given-names>J. P.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Dietary supplementation with arginine and glutamic acid modifies growth performance, carcass traits, and meat quality in growing-finishing pigs.</article-title>\n<source><italic>J. Anim. Sci.</italic></source>\n<volume>95</volume>\n<fpage>2680</fpage>–<lpage>2689</lpage>. <pub-id pub-id-type=\"doi\">10.2527/jas.2017.1388</pub-id>\n<pub-id pub-id-type=\"pmid\">28727042</pub-id></mixed-citation></ref><ref id=\"B33\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hung</surname><given-names>D. Y.</given-names></name><name><surname>Cheng</surname><given-names>Y. H.</given-names></name><name><surname>Chen</surname><given-names>W. J.</given-names></name><name><surname>Hua</surname><given-names>K. F.</given-names></name><name><surname>Pietruszka</surname><given-names>A.</given-names></name><name><surname>Dybus</surname><given-names>A.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title><italic>Bacillus licheniformis</italic>-fermented products reduce diarrhea incidence and alter the fecal microbiota community in weaning piglets.</article-title>\n<source><italic>Animals</italic></source>\n<volume>9</volume>:<issue>1145</issue>. <pub-id pub-id-type=\"doi\">10.3390/ani9121145</pub-id>\n<pub-id pub-id-type=\"pmid\">31847281</pub-id></mixed-citation></ref><ref id=\"B34\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Izuddin</surname><given-names>W. I.</given-names></name><name><surname>Loh</surname><given-names>T. C.</given-names></name><name><surname>Samsudin</surname><given-names>A. A.</given-names></name><name><surname>Foo</surname><given-names>H. L.</given-names></name><name><surname>Humam</surname><given-names>A. M.</given-names></name><name><surname>Shazali</surname><given-names>N.</given-names></name></person-group> (<year>2019</year>). <article-title>Effects of postbiotic supplementation on growth performance, ruminal fermentation and microbial profile, blood metabolite and GHR, IGF-1 and MCT-1 gene expression in post-weaning lambs.</article-title>\n<source><italic>BMC Vet. Res.</italic></source>\n<volume>15</volume>:<issue>315</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12917-019-2064-9</pub-id>\n<pub-id pub-id-type=\"pmid\">31477098</pub-id></mixed-citation></ref><ref id=\"B35\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jacquier</surname><given-names>V.</given-names></name><name><surname>Nelson</surname><given-names>A.</given-names></name><name><surname>Jlali</surname><given-names>M.</given-names></name><name><surname>Rhayat</surname><given-names>L.</given-names></name><name><surname>Brinch</surname><given-names>K. S.</given-names></name><name><surname>Devillard</surname><given-names>E.</given-names></name></person-group> (<year>2019</year>). <article-title>Bacillus subtilis 29784 induces a shift in broiler gut microbiome toward butyrate-producing bacteria and improves intestinal histomorphology and animal performance.</article-title>\n<source><italic>Poult. Sci.</italic></source>\n<volume>98</volume>\n<fpage>2548</fpage>–<lpage>2554</lpage>. <pub-id pub-id-type=\"doi\">10.3382/ps/pey602</pub-id>\n<pub-id pub-id-type=\"pmid\">30668816</pub-id></mixed-citation></ref><ref id=\"B36\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jasek</surname><given-names>A.</given-names></name><name><surname>Latham</surname><given-names>R. E.</given-names></name><name><surname>Manon</surname><given-names>A.</given-names></name><name><surname>Llamas-Moya</surname><given-names>S.</given-names></name><name><surname>Adhikari</surname><given-names>R.</given-names></name><name><surname>Poureslami</surname><given-names>R.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Impact of a multicarbohydrase containing alpha-galactosidase and xylanase on ileal digestible energy, crude protein digestibility, and ileal amino acid digestibility in broiler chickens.</article-title>\n<source><italic>Poult. Sci.</italic></source>\n<volume>97</volume>\n<fpage>3149</fpage>–<lpage>3155</lpage>. <pub-id pub-id-type=\"doi\">10.3382/ps/pey193</pub-id>\n<pub-id pub-id-type=\"pmid\">29897592</pub-id></mixed-citation></ref><ref id=\"B37\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jiao</surname><given-names>S.</given-names></name><name><surname>Cao</surname><given-names>H.</given-names></name><name><surname>Dai</surname><given-names>Y.</given-names></name><name><surname>Wu</surname><given-names>J.</given-names></name><name><surname>Lv</surname><given-names>J.</given-names></name><name><surname>Du</surname><given-names>R.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Effect of high-fat diet and growth stage on the diversity and composition of intestinal microbiota in healthy bovine livestock.</article-title>\n<source><italic>J. Sci. Food Agric.</italic></source>\n<volume>97</volume>\n<fpage>5004</fpage>–<lpage>5013</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jsfa.8380</pub-id>\n<pub-id pub-id-type=\"pmid\">28417460</pub-id></mixed-citation></ref><ref id=\"B38\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jin</surname><given-names>D. X.</given-names></name><name><surname>Zou</surname><given-names>H. W.</given-names></name><name><surname>Liu</surname><given-names>S. Q.</given-names></name><name><surname>Wang</surname><given-names>L. Z.</given-names></name><name><surname>Xue</surname><given-names>B.</given-names></name><name><surname>Wu</surname></name><etal/></person-group> (<year>2018</year>). <article-title>The underlying microbial mechanism of epizootic rabbit enteropathy triggered by a low fiber diet.</article-title>\n<source><italic>Sci. Rep.</italic></source>\n<volume>8</volume>\n<issue>12489</issue>. <pub-id pub-id-type=\"doi\">10.1038/s41598-018-30178-2</pub-id>\n<pub-id pub-id-type=\"pmid\">30131509</pub-id></mixed-citation></ref><ref id=\"B39\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kanehisa</surname><given-names>M.</given-names></name><name><surname>Sato</surname><given-names>Y.</given-names></name><name><surname>Kawashima</surname><given-names>M.</given-names></name><name><surname>Furumichi</surname><given-names>M.</given-names></name><name><surname>Tanabe</surname><given-names>M.</given-names></name></person-group> (<year>2016</year>). <article-title>KEGG as a reference resource for gene and protein annotation.</article-title>\n<source><italic>Nucleic Acids Res.</italic></source>\n<volume>44</volume>\n<fpage>D457</fpage>–<lpage>D462</lpage>. <pub-id pub-id-type=\"doi\">10.1093/nar/gkv1070</pub-id>\n<pub-id pub-id-type=\"pmid\">26476454</pub-id></mixed-citation></ref><ref id=\"B40\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kasubuchi</surname><given-names>M.</given-names></name><name><surname>Hasegawa</surname><given-names>S.</given-names></name><name><surname>Hiramatsu</surname><given-names>T.</given-names></name><name><surname>Ichimura</surname><given-names>A.</given-names></name><name><surname>Kimura</surname><given-names>I.</given-names></name></person-group> (<year>2015</year>). <article-title>Dietary gut microbial metabolites, short-chain fatty acids, and host metabolic regulation.</article-title>\n<source><italic>Nutrients</italic></source>\n<volume>7</volume>\n<fpage>2839</fpage>–<lpage>2849</lpage>. <pub-id pub-id-type=\"doi\">10.3390/nu7042839</pub-id>\n<pub-id pub-id-type=\"pmid\">25875123</pub-id></mixed-citation></ref><ref id=\"B41\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ke</surname><given-names>S.</given-names></name><name><surname>Fang</surname><given-names>S.</given-names></name><name><surname>He</surname><given-names>M.</given-names></name><name><surname>Huang</surname><given-names>X.</given-names></name><name><surname>Yang</surname><given-names>H.</given-names></name><name><surname>Yang</surname><given-names>B.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Age-based dynamic changes of phylogenetic composition and interaction networks of health pig gut microbiome feeding in a uniformed condition.</article-title>\n<source><italic>BMC Vet. Res.</italic></source>\n<volume>15</volume>:<issue>172</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12917-019-1918-5</pub-id>\n<pub-id pub-id-type=\"pmid\">31126262</pub-id></mixed-citation></ref><ref id=\"B42\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>H.</given-names></name><name><surname>Jeong</surname><given-names>Y.</given-names></name><name><surname>Kang</surname><given-names>S.</given-names></name><name><surname>You</surname><given-names>H. J.</given-names></name><name><surname>Ji</surname><given-names>G. E.</given-names></name></person-group> (<year>2020</year>). <article-title>Co-culture with <italic>Bifidobacterium catenulatum</italic> improves the growth, gut colonization, and butyrate production of <italic>Faecalibacterium prausnitzii</italic>: in vitro and in vivo studies.</article-title>\n<source><italic>Microorganisms</italic></source>\n<volume>8</volume>:<issue>788</issue>. <pub-id pub-id-type=\"doi\">10.3390/microorganisms8050788</pub-id>\n<pub-id pub-id-type=\"pmid\">32466189</pub-id></mixed-citation></ref><ref id=\"B43\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kultima</surname><given-names>J. R.</given-names></name><name><surname>Coelho</surname><given-names>L. P.</given-names></name><name><surname>Forslund</surname><given-names>K.</given-names></name><name><surname>Huerta-Cepas</surname><given-names>J.</given-names></name><name><surname>Li</surname><given-names>S. S.</given-names></name><name><surname>Driessen</surname><given-names>M.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>MOCAT2: a metagenomic assembly, annotation and profiling framework.</article-title>\n<source><italic>Bioinformatics</italic></source>\n<volume>32</volume>\n<fpage>2520</fpage>–<lpage>2523</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bioinformatics/btw183</pub-id>\n<pub-id pub-id-type=\"pmid\">27153620</pub-id></mixed-citation></ref><ref id=\"B44\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Le Roy</surname><given-names>C. I.</given-names></name><name><surname>Stsepetova</surname><given-names>J.</given-names></name><name><surname>Sepp</surname><given-names>E.</given-names></name><name><surname>Songisepp</surname><given-names>E.</given-names></name><name><surname>Claus</surname><given-names>S. P.</given-names></name><name><surname>Mikelsaar</surname><given-names>M.</given-names></name></person-group> (<year>2015</year>). <article-title>New insights into the impact of Lactobacillus population on host-bacteria metabolic interplay.</article-title>\n<source><italic>Oncotarget</italic></source>\n<volume>6</volume>\n<fpage>30545</fpage>–<lpage>30556</lpage>. <pub-id pub-id-type=\"doi\">10.18632/oncotarget.5906</pub-id>\n<pub-id pub-id-type=\"pmid\">26437083</pub-id></mixed-citation></ref><ref id=\"B45\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Letunic</surname><given-names>I.</given-names></name><name><surname>Bork</surname><given-names>P.</given-names></name></person-group> (<year>2019</year>). <article-title>Interactive Tree Of Life (iTOL) v4: recent updates and new developments.</article-title>\n<source><italic>Nucleic Acids Res.</italic></source>\n<volume>47</volume>\n<fpage>W256</fpage>–<lpage>W259</lpage>. <pub-id pub-id-type=\"doi\">10.1093/nar/gkz239</pub-id>\n<pub-id pub-id-type=\"pmid\">30931475</pub-id></mixed-citation></ref><ref id=\"B46\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>D.</given-names></name><name><surname>Liu</surname><given-names>C. M.</given-names></name><name><surname>Luo</surname><given-names>R.</given-names></name><name><surname>Sadakane</surname><given-names>K.</given-names></name><name><surname>Lam</surname><given-names>T. W.</given-names></name></person-group> (<year>2015</year>). <article-title>MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph.</article-title>\n<source><italic>Bioinformatics</italic></source>\n<volume>31</volume>\n<fpage>1674</fpage>–<lpage>1676</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bioinformatics/btv033</pub-id>\n<pub-id pub-id-type=\"pmid\">25609793</pub-id></mixed-citation></ref><ref id=\"B47\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>B.</given-names></name><name><surname>Wang</surname><given-names>W.</given-names></name><name><surname>Zhu</surname><given-names>X.</given-names></name><name><surname>Sun</surname><given-names>X.</given-names></name><name><surname>Xiao</surname><given-names>J.</given-names></name><name><surname>Li</surname><given-names>D.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Response of gut microbiota to dietary fiber and metabolic interaction with SCFAs in piglets.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>9</volume>:<issue>2344</issue>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2018.02344</pub-id>\n<pub-id pub-id-type=\"pmid\">30323803</pub-id></mixed-citation></ref><ref id=\"B48\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>S.</given-names></name><name><surname>Tan</surname><given-names>J.</given-names></name><name><surname>Hu</surname><given-names>Y.</given-names></name><name><surname>Jia</surname><given-names>X.</given-names></name><name><surname>Kogut</surname><given-names>M. H.</given-names></name><name><surname>Yuan</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Dietary l-arginine supplementation influences growth performance and B-cell secretion of immunoglobulin in broiler chickens.</article-title>\n<source><italic>J. Anim. Physiol. Anim. Nutr.</italic></source>\n<volume>103</volume>\n<fpage>1125</fpage>–<lpage>1134</lpage>.</mixed-citation></ref><ref id=\"B49\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lyra</surname><given-names>A.</given-names></name><name><surname>Rinttila</surname><given-names>T.</given-names></name><name><surname>Nikkila</surname><given-names>J.</given-names></name><name><surname>Krogius-Kurikka</surname><given-names>L.</given-names></name><name><surname>Kajander</surname><given-names>K.</given-names></name><name><surname>Malinen</surname><given-names>E.</given-names></name><etal/></person-group> (<year>2009</year>). <article-title>Diarrhoea-predominant irritable bowel syndrome distinguishable by 16S rRNA gene phylotype quantification.</article-title>\n<source><italic>World J. Gastroenterol.</italic></source>\n<volume>15</volume>\n<fpage>5936</fpage>–<lpage>5945</lpage>. <pub-id pub-id-type=\"doi\">10.3748/wjg.15.5936</pub-id>\n<pub-id pub-id-type=\"pmid\">20014457</pub-id></mixed-citation></ref><ref id=\"B50\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ma</surname><given-names>Y.</given-names></name><name><surname>Wang</surname><given-names>W.</given-names></name><name><surname>Zhang</surname><given-names>H.</given-names></name><name><surname>Wang</surname><given-names>J.</given-names></name><name><surname>Zhang</surname><given-names>W.</given-names></name><name><surname>Gao</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Supplemental <italic>Bacillus subtilis</italic> DSM 32315 manipulates intestinal structure and microbial composition in broiler chickens.</article-title>\n<source><italic>Sci. Rep.</italic></source>\n<volume>8</volume>:<issue>15358</issue>. <pub-id pub-id-type=\"doi\">10.1038/s41598-018-33762-8</pub-id>\n<pub-id pub-id-type=\"pmid\">30337568</pub-id></mixed-citation></ref><ref id=\"B51\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Magoc</surname><given-names>T.</given-names></name><name><surname>Salzberg</surname><given-names>S. L.</given-names></name></person-group> (<year>2011</year>). <article-title>FLASH: fast length adjustment of short reads to improve genome assemblies.</article-title>\n<source><italic>Bioinformatics</italic></source>\n<volume>27</volume>\n<fpage>2957</fpage>–<lpage>2963</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bioinformatics/btr507</pub-id>\n<pub-id pub-id-type=\"pmid\">21903629</pub-id></mixed-citation></ref><ref id=\"B52\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maltecca</surname><given-names>C.</given-names></name><name><surname>Bergamaschi</surname><given-names>M.</given-names></name><name><surname>Tiezzi</surname><given-names>F.</given-names></name></person-group> (<year>2020</year>). <article-title>The interaction between microbiome and pig efficiency: a review.</article-title>\n<source><italic>J. Anim. Breed Genet.</italic></source>\n<volume>137</volume>\n<fpage>4</fpage>–<lpage>13</lpage>. <pub-id pub-id-type=\"doi\">10.1111/jbg.12443</pub-id>\n<pub-id pub-id-type=\"pmid\">31576623</pub-id></mixed-citation></ref><ref id=\"B53\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mao</surname><given-names>H.</given-names></name><name><surname>Xia</surname><given-names>Y.</given-names></name><name><surname>Tu</surname><given-names>Y.</given-names></name><name><surname>Wang</surname><given-names>C.</given-names></name><name><surname>Diao</surname><given-names>Q.</given-names></name></person-group> (<year>2017</year>). <article-title>Effects of various weaning times on growth performance, rumen fermentation and microbial population of yellow cattle calves.</article-title>\n<source><italic>Asian Austral. J. Anim. Sci.</italic></source>\n<volume>30</volume>\n<fpage>1557</fpage>–<lpage>1562</lpage>. <pub-id pub-id-type=\"doi\">10.5713/ajas.16.0981</pub-id>\n<pub-id pub-id-type=\"pmid\">28423879</pub-id></mixed-citation></ref><ref id=\"B54\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Min</surname><given-names>B. R.</given-names></name><name><surname>Gurung</surname><given-names>N.</given-names></name><name><surname>Shange</surname><given-names>R.</given-names></name><name><surname>Solaiman</surname><given-names>S.</given-names></name></person-group> (<year>2019</year>). <article-title>Potential role of rumen microbiota in altering average daily gain and feed efficiency in meat goats fed simple and mixed pastures using bacterial tag-encoded FLX amplicon pyrosequencing1.</article-title>\n<source><italic>J. Anim. Sci.</italic></source>\n<volume>97</volume>\n<fpage>3523</fpage>–<lpage>3534</lpage>. <pub-id pub-id-type=\"doi\">10.1093/jas/skz193</pub-id>\n<pub-id pub-id-type=\"pmid\">31214714</pub-id></mixed-citation></ref><ref id=\"B55\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ndou</surname><given-names>S. P.</given-names></name><name><surname>Tun</surname><given-names>H. M.</given-names></name><name><surname>Kiarie</surname><given-names>E.</given-names></name><name><surname>Walsh</surname><given-names>M. C.</given-names></name><name><surname>Khafipour</surname><given-names>E.</given-names></name><name><surname>Nyachoti</surname><given-names>C. M.</given-names></name></person-group> (<year>2018</year>). <article-title>Dietary supplementation with flaxseed meal and oat hulls modulates intestinal histomorphometric characteristics, digesta- and mucosa-associated microbiota in pigs.</article-title>\n<source><italic>Sci. Rep.</italic></source>\n<volume>8</volume>:<issue>5880</issue>. <pub-id pub-id-type=\"doi\">10.1038/s41598-018-24043-5</pub-id>\n<pub-id pub-id-type=\"pmid\">29651010</pub-id></mixed-citation></ref><ref id=\"B56\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>North</surname><given-names>M. K.</given-names></name><name><surname>Zotte</surname><given-names>A. D.</given-names></name><name><surname>Hoffman</surname><given-names>L. C.</given-names></name></person-group> (<year>2019</year>). <article-title>Composition of rabbit caecal microbiota and the effects of dietary ouercetin supplementation and sex thereupon.</article-title>\n<source><italic>World Rabbit Sci.</italic></source>\n<volume>27</volume>\n<fpage>185</fpage>–<lpage>198</lpage>.</mixed-citation></ref><ref id=\"B57\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>O’Callaghan</surname><given-names>T. F.</given-names></name><name><surname>Ross</surname><given-names>R. P.</given-names></name><name><surname>Stanton</surname><given-names>C.</given-names></name><name><surname>Clarke</surname><given-names>G.</given-names></name></person-group> (<year>2016</year>). <article-title>The gut microbiome as a virtual endocrine organ with implications for farm and domestic animal endocrinology.</article-title>\n<source><italic>Domest Anim. Endocrinol.</italic></source>\n<volume>56</volume>(<issue>Suppl.</issue>), <fpage>S44</fpage>–<lpage>S55</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.domaniend.2016.05.003</pub-id>\n<pub-id pub-id-type=\"pmid\">27345323</pub-id></mixed-citation></ref><ref id=\"B58\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oikonomou</surname><given-names>G.</given-names></name><name><surname>Teixeira</surname><given-names>A. G.</given-names></name><name><surname>Foditsch</surname><given-names>C.</given-names></name><name><surname>Bicalho</surname><given-names>M. L.</given-names></name><name><surname>Machado</surname><given-names>V. S.</given-names></name><name><surname>Bicalho</surname><given-names>R. C.</given-names></name></person-group> (<year>2013</year>). <article-title>Fecal microbial diversity in pre-weaned dairy calves as described by pyrosequencing of metagenomic 16S rDNA. Associations of Faecalibacterium species with health and growth.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>8</volume>:<issue>e63157</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0063157</pub-id>\n<pub-id pub-id-type=\"pmid\">23646192</pub-id></mixed-citation></ref><ref id=\"B59\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Patra</surname><given-names>A. K.</given-names></name><name><surname>Geiger</surname><given-names>S.</given-names></name><name><surname>Schrapers</surname><given-names>K. T.</given-names></name><name><surname>Braun</surname><given-names>H. S.</given-names></name><name><surname>Gehlen</surname><given-names>H.</given-names></name><name><surname>Starke</surname><given-names>A.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Effects of dietary menthol-rich bioactive lipid compounds on zootechnical traits, blood variables and gastrointestinal function in growing sheep.</article-title>\n<source><italic>J. Anim. Sci. Biotechnol.</italic></source>\n<volume>10</volume>:<issue>86</issue>. <pub-id pub-id-type=\"doi\">10.1186/s40104-019-0398-6</pub-id>\n<pub-id pub-id-type=\"pmid\">31827785</pub-id></mixed-citation></ref><ref id=\"B60\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Piles</surname><given-names>M.</given-names></name><name><surname>Garcia-Tomas</surname><given-names>M.</given-names></name><name><surname>Rafel</surname><given-names>O.</given-names></name><name><surname>Ramon</surname><given-names>J.</given-names></name><name><surname>Ibanez-Escriche</surname><given-names>N.</given-names></name><name><surname>Varona</surname><given-names>L.</given-names></name></person-group> (<year>2007</year>). <article-title>Individual efficiency for the use of feed resources in rabbits.</article-title>\n<source><italic>J. Anim. Sci.</italic></source>\n<volume>85</volume>\n<fpage>2846</fpage>–<lpage>2853</lpage>. <pub-id pub-id-type=\"doi\">10.2527/jas.2006-218</pub-id>\n<pub-id pub-id-type=\"pmid\">17686894</pub-id></mixed-citation></ref><ref id=\"B61\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Priyadarshini</surname><given-names>M.</given-names></name><name><surname>Kotlo</surname><given-names>K. U.</given-names></name><name><surname>Dudeja</surname><given-names>P. K.</given-names></name><name><surname>Layden</surname><given-names>B. T.</given-names></name></person-group> (<year>2018</year>). <article-title>Role of short chain fatty acid receptors in intestinal physiology and pathophysiology.</article-title>\n<source><italic>Compr. Physiol.</italic></source>\n<volume>8</volume>\n<fpage>1091</fpage>–<lpage>1115</lpage>. <pub-id pub-id-type=\"doi\">10.1002/cphy.c170050</pub-id>\n<pub-id pub-id-type=\"pmid\">29978895</pub-id></mixed-citation></ref><ref id=\"B62\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Qin</surname><given-names>J.</given-names></name><name><surname>Li</surname><given-names>Y.</given-names></name><name><surname>Cai</surname><given-names>Z.</given-names></name><name><surname>Li</surname><given-names>S.</given-names></name><name><surname>Zhu</surname><given-names>J.</given-names></name><name><surname>Zhang</surname><given-names>F.</given-names></name><etal/></person-group> (<year>2012</year>). <article-title>A metagenome-wide association study of gut microbiota in type 2 diabetes.</article-title>\n<source><italic>Nature</italic></source>\n<volume>490</volume>\n<fpage>55</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature11450</pub-id>\n<pub-id pub-id-type=\"pmid\">23023125</pub-id></mixed-citation></ref><ref id=\"B63\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Quast</surname><given-names>C.</given-names></name><name><surname>Pruesse</surname><given-names>E.</given-names></name><name><surname>Yilmaz</surname><given-names>P.</given-names></name><name><surname>Gerken</surname><given-names>J.</given-names></name><name><surname>Schweer</surname><given-names>T.</given-names></name><name><surname>Yarza</surname><given-names>P.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>The SILVA ribosomal RNA gene database project: improved data processing and web-based tools.</article-title>\n<source><italic>Nucleic Acids Res.</italic></source>\n<volume>41</volume>\n<fpage>D590</fpage>–<lpage>D596</lpage>. <pub-id pub-id-type=\"doi\">10.1093/nar/gks1219</pub-id>\n<pub-id pub-id-type=\"pmid\">23193283</pub-id></mixed-citation></ref><ref id=\"B64\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ramayo-Caldas</surname><given-names>Y.</given-names></name><name><surname>Mach</surname><given-names>N.</given-names></name><name><surname>Lepage</surname><given-names>P.</given-names></name><name><surname>Levenez</surname><given-names>F.</given-names></name><name><surname>Denis</surname><given-names>C.</given-names></name><name><surname>Lemonnier</surname><given-names>G.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>Phylogenetic network analysis applied to pig gut microbiota identifies an ecosystem structure linked with growth traits.</article-title>\n<source><italic>ISME J.</italic></source>\n<volume>10</volume>\n<fpage>2973</fpage>–<lpage>2977</lpage>. <pub-id pub-id-type=\"doi\">10.1038/ismej.2016.77</pub-id>\n<pub-id pub-id-type=\"pmid\">27177190</pub-id></mixed-citation></ref><ref id=\"B65\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reddivari</surname><given-names>L.</given-names></name><name><surname>Veeramachaneni</surname><given-names>D. N. R.</given-names></name><name><surname>Walters</surname><given-names>W. A.</given-names></name><name><surname>Lozupone</surname><given-names>C.</given-names></name><name><surname>Palmer</surname><given-names>J.</given-names></name><name><surname>Hewage</surname><given-names>M. K. K.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Perinatal bisphenol a exposure induces chronic inflammation in rabbit offspring via modulation of gut bacteria and their metabolites.</article-title>\n<source><italic>mSystems</italic></source>\n<volume>2</volume>:<issue>e00093-17</issue>. <pub-id pub-id-type=\"doi\">10.1128/mSystems.00093-17</pub-id>\n<pub-id pub-id-type=\"pmid\">29034330</pub-id></mixed-citation></ref><ref id=\"B66\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shannon</surname><given-names>P.</given-names></name><name><surname>Markiel</surname><given-names>A.</given-names></name><name><surname>Ozier</surname><given-names>O.</given-names></name><name><surname>Baliga</surname><given-names>N. S.</given-names></name><name><surname>Wang</surname><given-names>J. T.</given-names></name><name><surname>Ramage</surname><given-names>D.</given-names></name><etal/></person-group> (<year>2003</year>). <article-title>Cytoscape: a software environment for integrated models of biomolecular interaction networks.</article-title>\n<source><italic>Genome Res.</italic></source>\n<volume>13</volume>\n<fpage>2498</fpage>–<lpage>2504</lpage>. <pub-id pub-id-type=\"doi\">10.1101/gr.1239303</pub-id>\n<pub-id pub-id-type=\"pmid\">14597658</pub-id></mixed-citation></ref><ref id=\"B67\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shokryazdan</surname><given-names>P.</given-names></name><name><surname>Faseleh Jahromi</surname><given-names>M.</given-names></name><name><surname>Liang</surname><given-names>J. B.</given-names></name><name><surname>Ramasamy</surname><given-names>K.</given-names></name><name><surname>Sieo</surname><given-names>C. C.</given-names></name><name><surname>Ho</surname><given-names>Y. W.</given-names></name></person-group> (<year>2017</year>). <article-title>Effects of a <italic>Lactobacillus salivarius</italic> mixture on performance, intestinal health and serum lipids of broiler chickens.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>12</volume>:<issue>e0175959</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0175959</pub-id>\n<pub-id pub-id-type=\"pmid\">28459856</pub-id></mixed-citation></ref><ref id=\"B68\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sidhu</surname><given-names>M.</given-names></name><name><surname>van der Poorten</surname><given-names>D.</given-names></name></person-group> (<year>2017</year>). <article-title>The gut microbiome.</article-title>\n<source><italic>Aust. Fam. Phys.</italic></source>\n<volume>46</volume>\n<fpage>206</fpage>–<lpage>211</lpage>.</mixed-citation></ref><ref id=\"B69\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tran</surname><given-names>H.</given-names></name><name><surname>Anderson</surname><given-names>C. L.</given-names></name><name><surname>Bundy</surname><given-names>J. W.</given-names></name><name><surname>Fernando</surname><given-names>S. C.</given-names></name><name><surname>Miller</surname><given-names>P. S.</given-names></name><name><surname>Burkey</surname><given-names>T. E.</given-names></name></person-group> (<year>2018</year>). <article-title>Effects of spray-dried porcine plasma on fecal microbiota in nursery pigs.</article-title>\n<source><italic>J. Anim. Sci.</italic></source>\n<volume>96</volume>\n<fpage>1017</fpage>–<lpage>1031</lpage>. <pub-id pub-id-type=\"doi\">10.1093/jas/skx034</pub-id>\n<pub-id pub-id-type=\"pmid\">29385463</pub-id></mixed-citation></ref><ref id=\"B70\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>J.</given-names></name><name><surname>Liu</surname><given-names>Y. J.</given-names></name><name><surname>Yang</surname><given-names>Y. Z.</given-names></name><name><surname>Bao</surname><given-names>C. L.</given-names></name><name><surname>Cao</surname><given-names>Y. H.</given-names></name></person-group> (<year>2019</year>). <article-title>High level expression of an acidic thermostable xylanase in <italic>Pichia pastoris</italic> and its application in weaned piglets.</article-title>\n<source><italic>J. Anim. Sci.</italic></source>\n<volume>98</volume>:<issue>skz364</issue>. <pub-id pub-id-type=\"doi\">10.1093/jas/skz364</pub-id>\n<pub-id pub-id-type=\"pmid\">31778535</pub-id></mixed-citation></ref><ref id=\"B71\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>Q.</given-names></name><name><surname>Fu</surname><given-names>W.</given-names></name><name><surname>Guo</surname><given-names>Y.</given-names></name><name><surname>Tang</surname><given-names>Y.</given-names></name><name><surname>Du</surname><given-names>H.</given-names></name><name><surname>Wang</surname><given-names>M.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Drinking warm water improves growth performance and optimizes the gut microbiota in early postweaning rabbits during winter.</article-title>\n<source><italic>Animals</italic></source>\n<volume>9</volume>:<issue>346</issue>. <pub-id pub-id-type=\"doi\">10.3390/ani9060346</pub-id>\n<pub-id pub-id-type=\"pmid\">31212853</pub-id></mixed-citation></ref><ref id=\"B72\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>T. J.</given-names></name><name><surname>Larson</surname><given-names>M. G.</given-names></name><name><surname>Vasan</surname><given-names>R. S.</given-names></name><name><surname>Cheng</surname><given-names>S.</given-names></name><name><surname>Rhee</surname><given-names>E. P.</given-names></name><name><surname>McCabe</surname><given-names>E.</given-names></name><etal/></person-group> (<year>2011</year>). <article-title>Metabolite profiles and the risk of developing diabetes.</article-title>\n<source><italic>Nat. Med.</italic></source>\n<volume>17</volume>\n<fpage>448</fpage>–<lpage>453</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nm.2307</pub-id>\n<pub-id pub-id-type=\"pmid\">21423183</pub-id></mixed-citation></ref><ref id=\"B73\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>X.</given-names></name><name><surname>Liu</surname><given-names>Y.</given-names></name><name><surname>Li</surname><given-names>S.</given-names></name><name><surname>Pi</surname><given-names>D.</given-names></name><name><surname>Zhu</surname><given-names>H.</given-names></name><name><surname>Hou</surname><given-names>Y.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>Asparagine attenuates intestinal injury, improves energy status and inhibits AMP-activated protein kinase signalling pathways in weaned piglets challenged with <italic>Escherichia coli</italic> lipopolysaccharide.</article-title>\n<source><italic>Br. J. Nutr.</italic></source>\n<volume>114</volume>\n<fpage>553</fpage>–<lpage>565</lpage>. <pub-id pub-id-type=\"doi\">10.1017/S0007114515001877</pub-id>\n<pub-id pub-id-type=\"pmid\">26277838</pub-id></mixed-citation></ref><ref id=\"B74\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>X.</given-names></name><name><surname>Tsai</surname><given-names>T.</given-names></name><name><surname>Deng</surname><given-names>F.</given-names></name><name><surname>Wei</surname><given-names>X.</given-names></name><name><surname>Chai</surname><given-names>J.</given-names></name><name><surname>Knapp</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Longitudinal investigation of the swine gut microbiome from birth to market reveals stage and growth performance associated bacteria.</article-title>\n<source><italic>Microbiome</italic></source>\n<volume>7</volume>:<issue>109</issue>. <pub-id pub-id-type=\"doi\">10.1186/s40168-019-0721-7</pub-id>\n<pub-id pub-id-type=\"pmid\">31362781</pub-id></mixed-citation></ref><ref id=\"B75\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wen</surname><given-names>C.</given-names></name><name><surname>Yan</surname><given-names>W.</given-names></name><name><surname>Sun</surname><given-names>C.</given-names></name><name><surname>Ji</surname><given-names>C.</given-names></name><name><surname>Zhou</surname><given-names>Q.</given-names></name><name><surname>Zhang</surname><given-names>D.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>The gut microbiota is largely independent of host genetics in regulating fat deposition in chickens.</article-title>\n<source><italic>ISME J.</italic></source>\n<volume>13</volume>\n<fpage>1422</fpage>–<lpage>1436</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41396-019-0367-2</pub-id>\n<pub-id pub-id-type=\"pmid\">30728470</pub-id></mixed-citation></ref><ref id=\"B76\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xie</surname><given-names>J.</given-names></name><name><surname>Liu</surname><given-names>Y.</given-names></name><name><surname>Chen</surname><given-names>B.</given-names></name><name><surname>Zhang</surname><given-names>G.</given-names></name><name><surname>Ou</surname><given-names>S.</given-names></name><name><surname>Luo</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Ganoderma lucidum polysaccharide improves rat DSS-induced colitis by altering cecal microbiota and gene expression of colonic epithelial cells.</article-title>\n<source><italic>Food Nutr. Res.</italic></source>\n<volume>63</volume>:<issue>1559</issue>. <pub-id pub-id-type=\"doi\">10.29219/fnr.v63.1559</pub-id>\n<pub-id pub-id-type=\"pmid\">30814921</pub-id></mixed-citation></ref><ref id=\"B77\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xing</surname><given-names>S. C.</given-names></name><name><surname>Huang</surname><given-names>C. B.</given-names></name><name><surname>Mi</surname><given-names>J. D.</given-names></name><name><surname>Wu</surname><given-names>Y. B.</given-names></name><name><surname>Liao</surname><given-names>X. D.</given-names></name></person-group> (<year>2019</year>). <article-title>Bacillus coagulans R11 maintained intestinal villus health and decreased intestinal injury in lead-exposed mice by regulating the intestinal microbiota and influenced the function of faecal microRNAs.</article-title>\n<source><italic>Environ. Pollut.</italic></source>\n<volume>255</volume>(<issue>Pt 2</issue>), <fpage>113139</fpage>. <pub-id pub-id-type=\"doi\">10.1016/j.envpol.2019.113139</pub-id>\n<pub-id pub-id-type=\"pmid\">31563774</pub-id></mixed-citation></ref><ref id=\"B78\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>H.</given-names></name><name><surname>Yang</surname><given-names>M.</given-names></name><name><surname>Fang</surname><given-names>S.</given-names></name><name><surname>Huang</surname><given-names>X.</given-names></name><name><surname>He</surname><given-names>M.</given-names></name><name><surname>Ke</surname><given-names>S.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Evaluating the profound effect of gut microbiome on host appetite in pigs.</article-title>\n<source><italic>BMC Microbiol.</italic></source>\n<volume>18</volume>:<issue>215</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12866-018-1364-8</pub-id>\n<pub-id pub-id-type=\"pmid\">30547751</pub-id></mixed-citation></ref><ref id=\"B79\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>Z.</given-names></name><name><surname>Liao</surname><given-names>S. F.</given-names></name></person-group> (<year>2019</year>). <article-title>Physiological effects of dietary amino acids on gut health and functions of swine.</article-title>\n<source><italic>Front. Vet. Sci.</italic></source>\n<volume>6</volume>:<issue>169</issue>. <pub-id pub-id-type=\"doi\">10.3389/fvets.2019.00169</pub-id>\n<pub-id pub-id-type=\"pmid\">31245390</pub-id></mixed-citation></ref><ref id=\"B80\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>M.</given-names></name><name><surname>Li</surname><given-names>Z.</given-names></name><name><surname>Chen</surname><given-names>W.</given-names></name><name><surname>Wang</surname><given-names>G.</given-names></name><name><surname>Cui</surname><given-names>Y.</given-names></name><name><surname>Ma</surname><given-names>X.</given-names></name></person-group> (<year>2019</year>). <article-title>Dietary supplementation with citrus extract altered the intestinal microbiota and microbial metabolite profiles and enhanced the mucosal immune homeostasis in yellow-feathered broilers.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>10</volume>:<issue>2662</issue>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2019.02662</pub-id>\n<pub-id pub-id-type=\"pmid\">31849855</pub-id></mixed-citation></ref><ref id=\"B81\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>X.</given-names></name><name><surname>Avall-Jaaskelainen</surname><given-names>S.</given-names></name><name><surname>Koort</surname><given-names>J.</given-names></name><name><surname>Lindholm</surname><given-names>A.</given-names></name><name><surname>Rintahaka</surname><given-names>J.</given-names></name><name><surname>von Ossowski</surname><given-names>I.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>A comparative characterization of different host-sourced <italic>Lactobacillus ruminis</italic> strains and their adhesive, inhibitory, and immunomodulating functions.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>8</volume>:<issue>657</issue>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2017.00657</pub-id>\n<pub-id pub-id-type=\"pmid\">28450859</pub-id></mixed-citation></ref><ref id=\"B82\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zeng</surname><given-names>B.</given-names></name><name><surname>Han</surname><given-names>S.</given-names></name><name><surname>Wang</surname><given-names>P.</given-names></name><name><surname>Wen</surname><given-names>B.</given-names></name><name><surname>Jian</surname><given-names>W.</given-names></name><name><surname>Guo</surname><given-names>W.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>The bacterial communities associated with fecal types and body weight of rex rabbits.</article-title>\n<source><italic>Sci. Rep.</italic></source>\n<volume>5</volume>:<issue>9342</issue>. <pub-id pub-id-type=\"doi\">10.1038/srep09342</pub-id>\n<pub-id pub-id-type=\"pmid\">25791609</pub-id></mixed-citation></ref><ref id=\"B83\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>K.</given-names></name><name><surname>Li</surname><given-names>B.</given-names></name><name><surname>Guo</surname><given-names>M.</given-names></name><name><surname>Liu</surname><given-names>G.</given-names></name><name><surname>Yang</surname><given-names>Y.</given-names></name><name><surname>Wang</surname><given-names>X.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Maturation of the goat rumen microbiota involves three stages of microbial colonization.</article-title>\n<source><italic>Animals</italic></source>\n<volume>9</volume>:<issue>1028</issue>. <pub-id pub-id-type=\"doi\">10.3390/ani9121028</pub-id>\n<pub-id pub-id-type=\"pmid\">31775375</pub-id></mixed-citation></ref><ref id=\"B84\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>L.</given-names></name><name><surname>Wu</surname><given-names>W.</given-names></name><name><surname>Lee</surname><given-names>Y. K.</given-names></name><name><surname>Xie</surname><given-names>J.</given-names></name><name><surname>Zhang</surname><given-names>H.</given-names></name></person-group> (<year>2018</year>). <article-title>Spatial heterogeneity and co-occurrence of mucosal and luminal microbiome across swine intestinal tract.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>9</volume>:<issue>48</issue>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2018.00048</pub-id>\n<pub-id pub-id-type=\"pmid\">29472900</pub-id></mixed-citation></ref><ref id=\"B85\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>Q.</given-names></name><name><surname>Wu</surname><given-names>Y.</given-names></name><name><surname>Wang</surname><given-names>J.</given-names></name><name><surname>Wu</surname><given-names>G.</given-names></name><name><surname>Long</surname><given-names>W.</given-names></name><name><surname>Xue</surname><given-names>Z.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>Accelerated dysbiosis of gut microbiota during aggravation of DSS-induced colitis by a butyrate-producing bacterium.</article-title>\n<source><italic>Sci. Rep.</italic></source>\n<volume>6</volume>:<issue>27572</issue>. <pub-id pub-id-type=\"doi\">10.1038/srep27572</pub-id>\n<pub-id pub-id-type=\"pmid\">27264309</pub-id></mixed-citation></ref><ref id=\"B86\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>S.</given-names></name><name><surname>Song</surname><given-names>J.</given-names></name><name><surname>Deng</surname><given-names>Z.</given-names></name><name><surname>Cheng</surname><given-names>L.</given-names></name><name><surname>Tian</surname><given-names>M.</given-names></name><name><surname>Guan</surname><given-names>W.</given-names></name></person-group> (<year>2017</year>). <article-title>Effects of combined alpha-galactosidase and xylanase supplementation on nutrient digestibility and growth performance in growing pigs.</article-title>\n<source><italic>Arch. Anim. Nutr.</italic></source>\n<volume>71</volume>\n<fpage>441</fpage>–<lpage>454</lpage>. <pub-id pub-id-type=\"doi\">10.1080/1745039X.2017.1389217</pub-id>\n<pub-id pub-id-type=\"pmid\">29110578</pub-id></mixed-citation></ref><ref id=\"B87\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>T.</given-names></name><name><surname>Li</surname><given-names>Q.</given-names></name><name><surname>Cheng</surname><given-names>L.</given-names></name><name><surname>Buch</surname><given-names>H.</given-names></name><name><surname>Zhang</surname><given-names>F.</given-names></name></person-group> (<year>2019</year>). <article-title>Akkermansia muciniphila is a promising probiotic.</article-title>\n<source><italic>Microb. Biotechnol.</italic></source>\n<volume>12</volume>\n<fpage>1109</fpage>–<lpage>1125</lpage>. <pub-id pub-id-type=\"doi\">10.1111/1751-7915.13410</pub-id>\n<pub-id pub-id-type=\"pmid\">31006995</pub-id></mixed-citation></ref><ref id=\"B88\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhu</surname><given-names>W.</given-names></name><name><surname>Lomsadze</surname><given-names>A.</given-names></name><name><surname>Borodovsky</surname><given-names>M.</given-names></name></person-group> (<year>2010</year>). <article-title>Ab initio gene identification in metagenomic sequences.</article-title>\n<source><italic>Nucleic Acids Res.</italic></source>\n<volume>38</volume>:<issue>e132</issue>. <pub-id pub-id-type=\"doi\">10.1093/nar/gkq275</pub-id>\n<pub-id pub-id-type=\"pmid\">20403810</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Microbiol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Microbiol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Microbiol.</journal-id><journal-title-group><journal-title>Frontiers in Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-302X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849479</article-id><article-id pub-id-type=\"pmc\">PMC7431613</article-id><article-id pub-id-type=\"doi\">10.3389/fmicb.2020.01928</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Microbiology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Exploring the Diversity Within the Genus <italic>Francisella</italic> – An Integrated Pan-Genome and Genome-Mining Approach</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Kumar</surname><given-names>Rajender</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/970601/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Bröms</surname><given-names>Jeanette E.</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/382030/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Sjöstedt</surname><given-names>Anders</given-names></name><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/17348/overview\"/></contrib></contrib-group><aff><institution>Department of Clinical Microbiology and Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University</institution>, <addr-line>Umeå</addr-line>, <country>Sweden</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Iain Sutcliffe, Northumbria University, United Kingdom</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Jean Challacombe, UC San Diego Health, United States; Jochen Blom, University of Giessen, Germany</p></fn><corresp id=\"c001\">*Correspondence: Anders Sjöstedt, <email>anders.sjostedt@umu.se</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Evolutionary and Genomic Microbiology, a section of the journal Frontiers in Microbiology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1928</elocation-id><history><date date-type=\"received\"><day>05</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>22</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Kumar, Bröms and Sjöstedt.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Kumar, Bröms and Sjöstedt</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Pan-genome analysis is a powerful method to explore genomic heterogeneity and diversity of bacterial species. Here we present a pan-genome analysis of the genus <italic>Francisella</italic>, comprising a dataset of 63 genomes and encompassing clinical as well as environmental isolates from distinct geographic locations. To determine the evolutionary relationship within the genus, we performed phylogenetic whole-genome studies utilizing the average nucleotide identity, average amino acid identity, core genes and non-recombinant loci markers. Based on the analyses, the phylogenetic trees obtained identified two distinct clades, A and B and a diverse cluster designated C. The sizes of the pan-, core-, cloud-, and shell-genomes of <italic>Francisella</italic> were estimated and compared to those of two other facultative intracellular pathogens, <italic>Legionella</italic> and <italic>Piscirickettsia</italic>. <italic>Francisella</italic> had the smallest core-genome, 692 genes, compared to 886 and 1,732 genes for <italic>Legionella</italic> and <italic>Piscirickettsia</italic> respectively, while the pan-genome of <italic>Legionella</italic> was more than twice the size of that of the other two genera. Also, the composition of the <italic>Francisella</italic> Type VI secretion system (T6SS) was analyzed. Distinct differences in the gene content of the T6SS were identified. <italic>In silico</italic> approaches performed to identify putative substrates of these systems revealed potential effectors targeting the cell wall, inner membrane, cellular nucleic acids as well as proteins, thus constituting attractive targets for site-directed mutagenesis. The comparative analysis performed here provides a comprehensive basis for the assessment of the phylogenomic relationship of members of the genus <italic>Francisella</italic> and for the identification of putative T6SS virulence traits.</p></abstract><kwd-group><kwd>whole-genome analysis</kwd><kwd>T6SS</kwd><kwd><italic>Francisella</italic></kwd><kwd>ANI</kwd><kwd>core-genome and pan-genome</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Medicinska Forskningsrådet<named-content content-type=\"fundref-id\">10.13039/501100006310</named-content></funding-source><award-id rid=\"cn001\">2013-4581</award-id></award-group><award-group><funding-source id=\"cn002\">Medicinska fakulteten, Umeå Universitet<named-content content-type=\"fundref-id\">10.13039/501100010794</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"7\"/><table-count count=\"3\"/><equation-count count=\"0\"/><ref-count count=\"98\"/><page-count count=\"17\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>The genus <italic>Francisella</italic> belongs to the γ-subclass of <italic>Proteobacteria</italic>, but shows no close relationship to other human pathogens (<xref rid=\"B79\" ref-type=\"bibr\">Sjöstedt, 2005</xref>). The genus is diverse with many species adapted to specific ecological niches and some of the pathogenic species to a very broad range of mammals, as well as fish (<xref rid=\"B80\" ref-type=\"bibr\">Sjöstedt, 2007</xref>; <xref rid=\"B5\" ref-type=\"bibr\">Birkbeck et al., 2011</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Colquhoun and Duodu, 2011</xref>; <xref rid=\"B77\" ref-type=\"bibr\">Sjödin et al., 2012</xref>; <xref rid=\"B60\" ref-type=\"bibr\">Pilo, 2018</xref>; <xref rid=\"B97\" ref-type=\"bibr\">Yon et al., 2019</xref>). A feature of the genus is an unusual fatty acid composition and a high lipid content of the cell wall (<xref rid=\"B79\" ref-type=\"bibr\">Sjöstedt, 2005</xref>). The important human pathogen, <italic>F. tularensis</italic>, has for 50 years been divided into several subspecies (<xref rid=\"B31\" ref-type=\"bibr\">Keim et al., 2007</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Kingry et al., 2013</xref>), the most important being subsp. <italic>holarctica</italic> and subsp. <italic>tularensis</italic>, both harboring isolates that cause human tularemia (<xref rid=\"B83\" ref-type=\"bibr\">Tärnvik and Berglund, 2003</xref>). This disease is rather common in many countries of the Northern hemisphere, however, isolates of subsp. <italic>tularensis</italic> are found in North America only (<xref rid=\"B32\" ref-type=\"bibr\">Kingry et al., 2013</xref>). Isolates of subsp. <italic>tularensis</italic>, in particular the lineage A1b, are the most virulent, both in humans but also in animal models (<xref rid=\"B37\" ref-type=\"bibr\">Kugeler et al., 2009</xref>). The designations of type A and type B are often used to designate subsp. <italic>tularensis</italic> and <italic>holarctica</italic>, but these have no formal approval. In addition, there is a third subspecies, subsp. <italic>mediasiatica</italic>, represented by strains from the Central Asian republics of former Soviet Union, but in contrast to the other subspecies, it has low virulence and has not been reported as a human pathogen (<xref rid=\"B58\" ref-type=\"bibr\">Olsufiev et al., 1959</xref>). The three subspecies demonstrate distinct genomic differences as demonstrated by multiple whole-genome sequences present in current databases. Some 30 years ago, <italic>F. novicida</italic> was recognized, a rare human pathogen with many isolates derived from environmental sources (<xref rid=\"B27\" ref-type=\"bibr\">Hollis et al., 1989</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Kingry et al., 2013</xref>). This is also true for a second species of the genus, <italic>F. philomiragia</italic>, which possesses distinct biochemical characteristics compared to <italic>F. tularensis</italic> (<xref rid=\"B27\" ref-type=\"bibr\">Hollis et al., 1989</xref>). As for <italic>F. novicida</italic>, the few cases of human <italic>F. philomiragia</italic>-infections that have been described are healthy individuals with a history of contact with natural water, e.g., near-drowning, or which are immunocompromised (<xref rid=\"B67\" ref-type=\"bibr\">Robles-Marhuenda et al., 2018</xref>).</p><p>In contrast to the aforementioned, since long recognized members of the genus <italic>Francisella</italic>, a large number of new species have been described during the last decade, often identified by genomic characterization of one or a few isolates. The rapidly expanding number of species demonstrate that the genus <italic>Francisella</italic> is very diverse, likely exists globally, and many species are adapted to highly specialized environmental niches (<xref rid=\"B27\" ref-type=\"bibr\">Hollis et al., 1989</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Clarridge et al., 1996</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Barns et al., 2005</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Kuske et al., 2006</xref>; <xref rid=\"B74\" ref-type=\"bibr\">Siddaramappa et al., 2011</xref>, <xref rid=\"B75\" ref-type=\"bibr\">2012</xref>; <xref rid=\"B62\" ref-type=\"bibr\">Qu et al., 2013</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Challacombe et al., 2017</xref>). The best-described example is <italic>F. noatunensis</italic>, an economically important pathogen that globally causes serious disease in farmed and wild fish in both salt and fresh water (<xref rid=\"B5\" ref-type=\"bibr\">Birkbeck et al., 2011</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Colquhoun and Duodu, 2011</xref>; <xref rid=\"B47\" ref-type=\"bibr\">McDermott and Palmeiro, 2013</xref>). Two subspecies have been recognized, subsp. <italic>noatunensis</italic> and subsp. <italic>orientalis.</italic> Recently, however, the latter was proposed to form a novel species; <italic>Francisella orientalis</italic> sp. nov., and an additional subspecies within the species <italic>F. noatunensis</italic> was suggested, i.e., subsp. <italic>chilensis</italic> subsp. nov. (<xref rid=\"B64\" ref-type=\"bibr\">Ramirez-Paredes et al., 2020</xref>). In addition, a multitude of potentially new <italic>Francisella</italic> species has been isolated globally from environmental sources, e.g., cooling water systems, from a wide variety of tick endosymbionts, as well as from human samples, e.g., skin lesions, or from immunocompromised patients near-drowning, with respiratory disease, or with cerebrospinal infection (<xref rid=\"B27\" ref-type=\"bibr\">Hollis et al., 1989</xref>; <xref rid=\"B93\" ref-type=\"bibr\">Wenger et al., 1989</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Clarridge et al., 1996</xref>; <xref rid=\"B94\" ref-type=\"bibr\">Whipp et al., 2003</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Barns et al., 2005</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Kuske et al., 2006</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Kugeler et al., 2008</xref>; <xref rid=\"B59\" ref-type=\"bibr\">Petersen et al., 2009</xref>; <xref rid=\"B28\" ref-type=\"bibr\">Huber et al., 2010</xref>; <xref rid=\"B74\" ref-type=\"bibr\">Siddaramappa et al., 2011</xref>, <xref rid=\"B75\" ref-type=\"bibr\">2012</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Kreizinger et al., 2013</xref>; <xref rid=\"B62\" ref-type=\"bibr\">Qu et al., 2013</xref>; <xref rid=\"B66\" ref-type=\"bibr\">Respicio-Kingry et al., 2013</xref>; <xref rid=\"B70\" ref-type=\"bibr\">Rydzewski et al., 2014</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Challacombe et al., 2017</xref>; <xref rid=\"B92\" ref-type=\"bibr\">Wang Y. et al., 2018</xref>; <xref rid=\"B88\" ref-type=\"bibr\">Vallesi et al., 2019</xref>).</p><p>In view of the rapidly evolving diversity within many bacterial genera and families, the need to obtain additional data to provide a robust platform for species delineation is essential. This is particularly true of the genus <italic>Francisella</italic>, since for many decades, there has been much ambiguity regarding the taxonomical relationships between many species and subspecies, further emphasized by the discoveries of previously unrecognized bacterial isolates with unclear taxonomic belonging. The rapidly evolving diversity within the genus <italic>Francisella</italic> many times challenges the traditional taxonomical classification, since several of the aforementioned isolates have only been identified by means of genetic characterization and may be unculturable, or are phenotypically ill-defined. To this end, recent work is attempting to define unambiguous criteria that can be generally applied to delineate bacterial species in <italic>Francisella</italic> as well as in other genera (<xref rid=\"B77\" ref-type=\"bibr\">Sjödin et al., 2012</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Challacombe et al., 2017</xref>). In this regard, the utility of the dramatically increasing amount of genomic data has to be incorporated in the species definition alongside other relevant, more traditional taxonomic data.</p><p>For the genus <italic>Francisella</italic>, a large number of completed and draft genome assemblies are available in biological sequence databases, such as the National Center for Biotechnology Information (NCBI) assembly database and the Joint Genome Institute (JGI) Genome Portal. These huge sequence datasets offer not only the possibility to understand the functional and evolutionary repertoire of bacterial genera, but also open up possibilities for developing therapies and engineering applications. The objective of this study was to elucidate the core- and pan-genome features of the <italic>Francisella</italic> genus to shed light on its diversity and characteristics, as well as to identify putative T6SS substrates <italic>in silico</italic>. Our analysis identifies conceptual and technical approaches that may be used for studies of pathogenicity, especially related to secretion systems.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Genomic Data Sets</title><p>All 62 publicly available (January 2018) whole genome sequences of <italic>Francisella</italic> bacteria were downloaded from the NCBI assembly database<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup> and used for analysis. <italic>Allofrancisella guangzhouensis</italic>, a species previously considered to be a member of the genus (<xref rid=\"B63\" ref-type=\"bibr\">Qu et al., 2016</xref>), was also included in the analysis, thus making the number of genomes analyzed 63. These complete genome assemblies cover almost the complete genus <italic>Francisella</italic>, comprising 14 species with various number of subspecies (a total number of 26; <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). As a starting point, any plasmid sequences present were removed from the assemblies. In the next step, whole genome comparisons were performed, and the average nucleotide identity (ANI) calculated in the <italic>pyani</italic> program (<xref rid=\"B61\" ref-type=\"bibr\">Pritchard et al., 2016</xref>), using the BlastN alignment tool with 1,020 nt long fragments as input sequences and other parameters used were default. For each pairwise genome comparison, an ANI matrix was generated along with a dendrogram. The same methodology was also applied to the genus <italic>Legionella</italic> (77 complete genomes) and <italic>Piscirickettsia</italic> (19 complete genomes), to allow comparisons to be made between the three genera. Only one representative of highly related species (ANI ≥ 99.5%) was used for further analysis of the pan-genome, phylogenomic analysis etc.</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>The 25 representative and complete <italic>Francisella</italic> genome assemblies, including their annotation, the bacterial niche as well as a description of their <italic>Francisella</italic> Pathogenicity Island (FPI).</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Strain No.</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Strain</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Strain abbreviation</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">No. of genes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">G + C content (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Genome size (bp)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Source</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">No. of FPI loci/category*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Accession number</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">References</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. halioticida</italic> DSM23729</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fha</italic> DSM23729</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,351</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2197430</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Giant abalone</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0/A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP022132\">NZ_CP022132</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B7\" ref-type=\"bibr\">Brevik et al., 2011</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. hispaniensis</italic> FSC454</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fhi</italic> FSC454</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,902</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1922599</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/C</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP018093\">NZ_CP018093</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B28\" ref-type=\"bibr\">Huber et al., 2010</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. noatunensis</italic> subsp. <italic>noatunensis</italic> FSC772</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fnn</italic> FSC772</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,891</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1933822</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Freshwater</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/P (A: <italic>pdpC/E</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP022207\">NZ_CP022207</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B50\" ref-type=\"bibr\">Mikalsen et al., 2007</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. noatunensis</italic> subsp. <italic>orientalis</italic> FNO12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fnor</italic> FNO12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,899</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1862215</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Fish</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/P (A: <italic>pdpC/E</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP011921\">NZ_CP011921</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B77\" ref-type=\"bibr\">Sjödin et al., 2012</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>5</bold></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. persica</italic> ATCC VR331</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fpe</italic> ATCCVR331</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,502</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1540768</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Tick</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/P (A: <italic>pdpD</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP013022\">NZ_CP013022</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B40\" ref-type=\"bibr\">Larson et al., 2016</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. philomiragia</italic> GA012794</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fph</italic> GA012794</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,082</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2148038</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/P (A: <italic>pdpC/E</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP009440\">NZ_CP009440</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B29\" ref-type=\"bibr\">Johnson et al., 2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. philomiragia</italic> GA012801</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fph</italic> GA012801</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,003</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2022507</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/P (A: <italic>pdpC/E</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP009444\">NZ_CP009444</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B29\" ref-type=\"bibr\">Johnson et al., 2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. philomiragia</italic> O319036</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fph</italic> O319036</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,859</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1919185</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Muskrat</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/P (A: <italic>pdpC/E</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP009442\">NZ_CP009442</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B29\" ref-type=\"bibr\">Johnson et al., 2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. philomiragia</italic> O319067</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fph</italic> O319067</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,992</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2045775</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Muskrat</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/P (A: <italic>pdpC/E</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP009436\">NZ_CP009436</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B29\" ref-type=\"bibr\">Johnson et al., 2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. philomiragia</italic> ATCC 25015</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fph</italic> ATCC25015</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,923</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2017400</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Muskrat</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/P (A: <italic>pdpC/E</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP010019\">NZ_CP010019</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B29\" ref-type=\"bibr\">Johnson et al., 2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. frigiditurris</italic> sp. nov. CA971460</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Ffr</italic> CA971460</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,846</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1855434</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Air-conditioning system</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/P (A: <italic>pdpC/E</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP009654\">NZ_CP009654</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B14\" ref-type=\"bibr\">Challacombe et al., 2017</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. endociliophora</italic> FSC1006</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fen</italic> FSC1006</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,972</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2015987</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Ciliate</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/P (A: <italic>iglI, pdpC/D/E, anmK</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP009574\">NZ_CP009574</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B78\" ref-type=\"bibr\">Sjödin et al., 2014</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. opportunistica</italic> sp. nov. MA067296</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fop</italic> MA067296</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,757</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1824527</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/P (A: <italic>pdpC/E</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP016930\">NZ_CP016930</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B38\" ref-type=\"bibr\">Kugeler et al., 2008</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. salina</italic> sp. nov. TX077308</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fsa</italic> TX077308</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,987</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2035931</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Seawater</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/P (A: <italic>pdpC/E</italic>; short, splitted <italic>iglG</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NC_015696\">NC_015696</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B74\" ref-type=\"bibr\">Siddaramappa et al., 2011</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. uliginis</italic> sp. nov. TX077310</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Ful</italic> TX077310</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2,073</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2237379</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Seawater</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/P (A: <italic>pdpC/E</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP016796\">NZ_CP016796</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B59\" ref-type=\"bibr\">Petersen et al., 2009</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. tularensis</italic> subsp. <italic>holarctica</italic> LVS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fth</italic> LVS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,961</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1892177</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Human/vaccine strain</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2/P (A: <italic>anmK</italic>; truncated <italic>pdpD</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP009694\">NZ_CP009694</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B68\" ref-type=\"bibr\">Rohmer et al., 2007</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. tularensis</italic> subsp. <italic>mediasiatica</italic> FSC147</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Ftm</italic> FSC147</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,930</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1893886</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Gerbil</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2/C</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NC_010677\">NC_010677</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B41\" ref-type=\"bibr\">Larsson et al., 2009</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. cf. novicida</italic> 3523</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fno</italic> 3523</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,879</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1945310</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/C</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NC_017449\">NC_017449</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B41\" ref-type=\"bibr\">Larsson et al., 2009</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. cf. novicida</italic> Fx1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fno</italic> Fx1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,834</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1913619</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/C</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NC_017450\">NC_017450</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B94\" ref-type=\"bibr\">Whipp et al., 2003</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. novicida</italic> AL972214</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fno</italic> AL972214</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,851</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1916455</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/P (A: <italic>pdpC/E</italic>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP009653\">NZ_CP009653</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B74\" ref-type=\"bibr\">Siddaramappa et al., 2011</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. novicida</italic> AZ067470</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fno</italic> AZ067470</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,872</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1890780</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/C</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP009682\">NZ_CP009682</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B4\" ref-type=\"bibr\">Birdsell et al., 2009</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">22</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. novicida</italic> D9876</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fno</italic> D9876</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,811</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1870206</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/C</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP009607\">NZ_CP009607</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B29\" ref-type=\"bibr\">Johnson et al., 2015</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. novicida</italic> PA107858</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fno</italic> PA107858</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,935</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1978958</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/C</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP016635\">NZ_CP016635</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B6\" ref-type=\"bibr\">Brett et al., 2012</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. novicida</italic> U112</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Fno</italic> U112</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,846</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1910592</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Water</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1/C</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP009633\">NZ_CP009633</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B68\" ref-type=\"bibr\">Rohmer et al., 2007</xref></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">25</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>F. tularensis</italic> subsp. <italic>tularensis</italic> SCHU S4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>Ftt</italic> SCHU S4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1,928</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1892789</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Human</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2/C (<italic>anmK</italic> is split into two ORFs)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP010290\">NZ_CP010290</ext-link></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><xref rid=\"B42\" ref-type=\"bibr\">Larsson et al., 2005</xref></td></tr></tbody></table><table-wrap-foot><attrib><italic>*A: absent, P: partial, C: complete (meaning that 18 FPI genes were identified).</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S2.SS2\"><title>Core- and Pan-Genome Analysis</title><p>The <italic>Francisella</italic> core- and pan-genome size was assessed in a manner similar to that previously reported, using iterative and combinatorial approaches (<xref rid=\"B84\" ref-type=\"bibr\">Tettelin et al., 2005</xref>; <xref rid=\"B49\" ref-type=\"bibr\">Meric et al., 2014</xref>; <xref rid=\"B51\" ref-type=\"bibr\">Mosquera-Rendon et al., 2016</xref>). To estimate the number of orthologous genes within the genus, we used the GET_HOMOLOGUES tool (<xref rid=\"B20\" ref-type=\"bibr\">Contreras-Moreira and Vinuesa, 2013</xref>) and the three clustering algorithms (i) bidirectional best-hit (BDBH) (<xref rid=\"B95\" ref-type=\"bibr\">Wolf and Koonin, 2012</xref>) COGtriangles (<xref rid=\"B36\" ref-type=\"bibr\">Kristensen et al., 2010</xref>) and (iii) OrthoMCL (Ortho Markov Cluster Algorithm) (<xref rid=\"B45\" ref-type=\"bibr\">Li et al., 2003</xref>). Orthologous genes were clustered using an E-value of >1e-05 and a query coverage of > 50%. Finally, the core-genome was defined as the set of genes shared by representative species/strains, while the pan-genome was defined as the sum of the core-genome and the set of auxiliary (i.e., available in more than 1 and less than 26 genomes) and exclusive (i.e., available in only one genome) genes. We validated the result of the pan-genome analysis by BPGA (Bacterial Pan Genome Analysis tool) that uses the USEARCH algorithm for fastest clustering (<xref rid=\"B16\" ref-type=\"bibr\">Chaudhari et al., 2016</xref>). The core- and pan-genomes, as well as their predicted sizes and trajectories, were obtained using the method proposed by Knight (<xref rid=\"B33\" ref-type=\"bibr\">Knight et al., 2017</xref>), the models/regression algorithms given by Tettelin (<xref rid=\"B84\" ref-type=\"bibr\">Tettelin et al., 2005</xref>, <xref rid=\"B85\" ref-type=\"bibr\">2008</xref>), and the binomial mixture model of Snipen (<xref rid=\"B81\" ref-type=\"bibr\">Snipen et al., 2009</xref>). For each method, the parameters used were default.</p><p>Curve fitting of the pan-genome was performed using a power-law regression based on Heaps’ law [y = A<sub>pan</sub>x<sup>Bpan</sup> + C<sub>pan</sub>], as previously described (<xref rid=\"B84\" ref-type=\"bibr\">Tettelin et al., 2005</xref>, <xref rid=\"B85\" ref-type=\"bibr\">2008</xref>; <xref rid=\"B65\" ref-type=\"bibr\">Rasko et al., 2008</xref>). The same protocol was also applied to estimate the core- and pan-genomes for the genera <italic>Legionella</italic> and <italic>Piscirickettsia</italic>. Further, the common core-genome shared by all three genera was estimated, based on individual core sets for each genus as input. In the next step, this “core of core” was functionally characterized using COG (Clusters of Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) annotations.</p></sec><sec id=\"S2.SS3\"><title>Phylogenomic Analysis</title><p>For whole-genome phylogenetic analysis of closely related <italic>F. tularensis</italic> strains, we used multiple approaches. First, we used the UBCG (Up-to-date bacterial core genes) approach, by utilizing its pipeline and default parameters<sup><xref ref-type=\"fn\" rid=\"footnote2\">2</xref></sup> (<xref rid=\"B52\" ref-type=\"bibr\">Na et al., 2018</xref>). First, all 26 genome assemblies were converted into <italic>bcg</italic> files using the UBCG.jar extract command. These files contain a label with full information about the strain/genome and strain details. Next, all markers, i.e., a set of 92 bacterial core genes, were identified from an up-to-date genome database using the hmmsearch program and default parameters.<sup><xref ref-type=\"fn\" rid=\"footnote3\">3</xref></sup> In the next step, multiple alignments were performed for each gene using the UBCG.jar align command with the MAFFT (Multiple Alignment Fast Fourier Transform) alignment program<sup><xref ref-type=\"fn\" rid=\"footnote4\">4</xref></sup> using default parameters. Each of the UBCG genes were aligned separately, before being concatenated into a single alignment. A highly resolved maximum likelihood tree was obtained using FastTree<sup><xref ref-type=\"fn\" rid=\"footnote5\">5</xref></sup> and visualized using the iTOL server.<sup><xref ref-type=\"fn\" rid=\"footnote6\">6</xref></sup> A bootstrap analysis was performed to determine the reliability of the branches obtained.</p><p>We also constructed a marker-based phylogenetic tree, by using the GET_PHYLOMARKERS software package in the default mode (<xref rid=\"B90\" ref-type=\"bibr\">Vinuesa et al., 2018</xref>), and with sets of single copy orthologous core-genomes as input. This analysis allows us to identify high-quality markers to estimate robust genome phylogenies from the UBCG, thereby resolving poor tree topologies. During the phylogenetic tree reconstruction, a set of sequential filters was applied to exclude recombinant alignments and horizontal gene transfer. A maximum likelihood (ML) phylogenetic tree was estimated from the concatenated set of top-ranking alignments at the DNA as well as at the protein levels, using the advanced general amino-acid replacement matrix model (LG) (<xref rid=\"B43\" ref-type=\"bibr\">Le and Gascuel, 2008</xref>) and MFP feature in the IQ-TREE (IQT) software (<xref rid=\"B56\" ref-type=\"bibr\">Nguyen et al., 2015</xref>). The remaining parameters were kept as default. The tree was visualized using the iTOL server. A bootstrap analysis was performed to determine the reliability of the branches obtained.</p></sec><sec id=\"S2.SS4\"><title>FPI Cluster Homology Searches</title><p>Comparative analyses of FPI/T6SS clusters were performed using the MultiGeneBlast program with default parameters<sup><xref ref-type=\"fn\" rid=\"footnote7\">7</xref></sup> (<xref rid=\"B48\" ref-type=\"bibr\">Medema et al., 2013</xref>). This program offers a BLAST-based tool to perform “architecture searches” with operons or gene clusters as basic units, instead of single genes. This allows for the identification of genomic loci containing homologs of specific user-specified gene combinations. As input query, we used sequences corresponding to the FPI cluster of the <italic>F. novicida</italic> strain U112 (accession number <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NZ_CP009633\">NZ_CP009633</ext-link>) to search a database containing all of the 26 representative <italic>Francisella</italic> species. To generate blast hits, we set the minimal sequence identify to 25% and the sequence coverage to 30%, while the rest of the parameters were kept as default. Using the same parameters, we also tested the FPI cluster of U112 against the bacterial domain in the NCBI gene bank database to look for the presence of FPI homologous genes in other bacterial genera. To estimate the G + C contents for the FPI cluster and for the whole genome, the following formula was used: (G + C)/(A + T + G + C) <sup>∗</sup> 100%. We also analyzed the amino acid composition of the FPI proteins (encoded by <italic>pdpA</italic> to <italic>anmK</italic>) and compared it with the amino acid composition of the rest of the genome. The first was calculated using the concatenated all FPI proteins only, while the second was calculated using the concatenated all protein sequences after excluding the FPI proteins. The genomes included in the analysis, in addition to <italic>F. novicida</italic> U112, were <italic>F. tularensis</italic> subsp. <italic>tularensis</italic> SCHU S4 (NZ_CP010290), <italic>F. cf. novicida</italic> Fx1 (NC_017450), and <italic>F. tularensis</italic> subsp. <italic>holarctica</italic> LVS (NZ_CP009694).</p></sec><sec id=\"S2.SS5\"><title>T6SS Effector Prediction</title><p>The Bastion6 program<sup><xref ref-type=\"fn\" rid=\"footnote8\">8</xref></sup> predicts T6SS effectors using a two-layer SVM-based ensemble model with optimized parameters (<xref rid=\"B91\" ref-type=\"bibr\">Wang J. W. et al., 2018</xref>). We employed this program to search for putative T6SS effectors encoded within the <italic>Francisella</italic> genomes, using the complete genome sequence of <italic>F. tularensis</italic> subsp. <italic>tularensis</italic> SCHU S4 as a reference genome. Predicted promiscuous effectors were selected based on an ensemble model result score of ≥0.5, and were functionally described and Gene Ontology (GO)-annotated with respect to their predicted biological process, molecular function or cellular component, using the PANNZER2 (Protein annotation with Z-scoRE) server (<xref rid=\"B87\" ref-type=\"bibr\">Toronen et al., 2018</xref>). We also used our hits to search the Pfam database<sup><xref ref-type=\"fn\" rid=\"footnote9\">9</xref></sup> for conserved domains of unknown functions, DUFs.</p><p>To specifically search for homologs of T6SS-dependent, ion-selective pore-forming effectors (<xref rid=\"B46\" ref-type=\"bibr\">Mariano et al., 2019</xref>) within the <italic>Francisella</italic> genus, we used the sequence for the effector Ssp6 (SMDB11_4673) of <italic>Serratia marcescens</italic> Db10 as query against the NR database (set as <italic>Francisella</italic> group) using the PSI-BLASTP program with default parameters. To specifically search for MIX effectors (<xref rid=\"B71\" ref-type=\"bibr\">Salomon et al., 2014</xref>) within the <italic>Francisella</italic> genome, we used the NR database from NCBI using the position specific iterative (PSI)-BLASTP with four iterations and other parameters kept as default. As queries, we used representative sequences for each of the following five classes of MIX effectors: MIX I - <italic>Vibrio parahaemolyticus</italic> VP1388 (accession: <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_797767\">NP_797767</ext-link>), MIX II - <italic>Proteus mirabilis</italic> IdsD (accession <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"SPY42138\">SPY42138</ext-link>), MIX III - <italic>Burkholderia thailandensis</italic> BTH_I2691 (accession <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"ABC38088\">ABC38088</ext-link>), MIX IV - <italic>Vibrio cholerae</italic> VCA0020 (accession <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_232421\">NP_232421</ext-link>) and MIX V – <italic>V. parahaemolyticus</italic> VPA1263 (accession <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"NP_800773\">NP_800773</ext-link>). Each query generated a set of identified hit protein sequences, which we used in a multiple sequence alignment analysis to identify the conserved sequence and predict putative signal peptides. Furthermore, by using the MultiGeneBlast program, the chromosomal location of the identified hits as well as the upstream and downstream ORFs were analyzed for the 26 representative complete genome sequence data set.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Whole Genome Comparisons</title><p>Whole-genome comparisons have the power to discriminate between strains and species with high resolution. For this purpose, all completely sequenced available <italic>Francisella</italic> genomes (a total of 63 when this study was initiated, see <xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S1</xref>) were selected for further analysis, out of which five were excluded since they were found to represent duplicated genomes. For the remaining 57 genomes, whole-genome sequence comparisons were performed in a pairwise fashion, by calculating and comparing the ANI (average nucleotide identity) (<xref rid=\"B97\" ref-type=\"bibr\">Yon et al., 2019</xref>), for each genome pair (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S2</xref>). ANI is a well-documented and robust method for comparing genomes and assessing species relationships (<xref rid=\"B34\" ref-type=\"bibr\">Konstantinidis et al., 2006</xref>). The pair-wise comparisons showed a minimum ANI of ∼74.2% for the most distant strains, while strains of the same subspecies showed an ANI of >97.0%. Only one representative of highly related species (ANI ≥ 99.5%) was used for further pan-genome analysis. This allowed us to down-select the genome set aimed to represent the entire genus <italic>Francisella</italic> to a total of 26 genomes (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). For pairwise ANI comparisons of the 26 genomes, see <xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S3</xref>. Noteworthy, we observed that <italic>Francisella philomiragia</italic> GA012794 and <italic>Francisella philomiragia</italic> GA012801, while named as belonging to the same species, show only about 93% ANI, according to the comparable algorithms ANIb (93.63%) and OrthoANI (93.9%), thus questioning their species belonging (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S3</xref>). The 26 genomes were found to represent two major groups; a large cluster which comprised all the human pathogens and for which the strains showed an ANI of 97.0 - 99.5%, and a second cluster that comprised strains that predominantly are environmental or water-related, and with ANI values of 74.2–90.4% (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Importantly, the minor variation (32.3 ± 0.4) in the G + C content of this genome dataset was indicative of a stable boundary delineation within the genus (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Average Nucleotide Identity (ANI) demonstrating nucleotide-level genomic similarity between the coding regions of indicated <italic>Francisella</italic> genomes. Pairwise comparisons for all 26 complete genomes were computed by BlastN using the Pyani Program. For strain abbreviations, see <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>.</p></caption><graphic xlink:href=\"fmicb-11-01928-g001\"/></fig><p>In the genus <italic>Legionella</italic>, a total of 77 complete genome assemblies were used for ANI analysis. Using the same down-selection process as for <italic>Francisella</italic>, 35 genomes were selected for further pan-genome analysis. The total genome size was larger than that of <italic>Francisella</italic>, and more diverse in sequence, since the minimum ANI was approximately 71%. The largest cluster within the genus belonged to species <italic>L. pneumophila</italic> and strains thereof, and showed an ANI of > 96%. All available genomes from the genus <italic>Piscirickettsia</italic> (19 in total) were derived from only two species, <italic>P. salmonis</italic> and <italic>P. litoralis</italic>, and showed ANI values ranging from 95.7–99.9% (data not shown).</p></sec><sec id=\"S3.SS2\"><title>Core-Genome and Pan-Genome Analyses of <italic>Francisella</italic></title><p>Bacterial genomes are dynamic entities that harbor essential genes and accessory elements, which may be unique to each community. The so called ‘core’ genomes constitute conserved genes present in all strains studied, while ‘dispensable’ genomes (also known as flexible or accessory genomes) are composed of genes absent from one or more of the strains (<xref rid=\"B84\" ref-type=\"bibr\">Tettelin et al., 2005</xref>). The latter usually pertains to supplementary biochemical pathways and functions that may confer a selective advantage to the microbe, such as ecological adaptation, antibiotic resistance, virulence mechanisms, or colonization of a new host. To estimate the pan- and core-genome sizes of <italic>Francisella</italic>, we used our down-selected 26 genomes, from 14 <italic>Francisella</italic> species, and the binomial mixture model of Snipen and collaborators (<xref rid=\"B81\" ref-type=\"bibr\">Snipen et al., 2009</xref>) and Tettelin and collaborators (<xref rid=\"B84\" ref-type=\"bibr\">Tettelin et al., 2005</xref>). We observed that the more genomes analyzed (i.e., increasing the data set), the bigger the estimated pan-genome size. At the same time, the rate of the increase was going down (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>). Thus, since the core/pan-genome ratio did not reach a distinct sharp plateau, we conclude that <italic>Francisella</italic> has an open pan-genome (<xref ref-type=\"fig\" rid=\"F2\">Figures 2A,B</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Pan-genome analysis of 26 <italic>Francisella</italic> genomes from 14 species. Estimates of pan-genome size <bold>(A)</bold> and <bold>(B)</bold> core-genome size, both with the Tettelin fit.</p></caption><graphic xlink:href=\"fmicb-11-01928-g002\"/></fig><p>Based on the 26 <italic>Francisella</italic> genomes (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>), and the use of three different algorithms (for details see section “Materials and Methods”) the pan-genome of the genus was predicted to comprise 4,053 genes. Amongst these, 692 genes (709 including paralogs) constituted the core genome, i.e., genes present in all genomes included in the analysis (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref> and <xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>). The core genome in turn, constituted approximately 36.1% of the mean number of CDS (692 vs. 1,915) (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). Together with the soft-core genomes, i.e., genes present in 95% of all genomes included in the analysis (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Figure S1</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Kaas et al., 2012</xref>), these 977 and highly conserved genes may provide information about the evolutionary history of the members of a genus. The remaining genes of the pan-genome were accessory genes, of which 2,179 constituted the cloud genome, i.e., strain-specific and rare genes present only in a few genomes (<xref rid=\"B89\" ref-type=\"bibr\">Vernikos et al., 2015</xref>), which might be rapidly gained or lost (<xref rid=\"B18\" ref-type=\"bibr\">Collins and Higgs, 2012</xref>). The remaining 897 genes constituted the shell genome, i.e., moderately conserved and dispensable genes, present in one or several genomes (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Figure S1</xref>). The cloud and shell genome subsets reflect both the evolutionary history of a lineage as well as adaptation of an organism to its particular environment (<xref rid=\"B55\" ref-type=\"bibr\">Nelson and Stegen, 2015</xref>).</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>Comparative core- and pan-genome analysis of the genera <italic>Francisella, Legionella</italic>, and <italic>Piscirickettsia</italic>.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Genus</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Complete genomes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Representative genome set*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Core-genome (no. genes)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Pan-genome (no. genes)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Core-genome/mean no. CDS (%)</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Francisella</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">63</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">692</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4053</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">36.1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Legionella</italic><sup>#</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">77</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">886</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8413</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Piscirickettsia</italic><sup>##</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1732</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3463</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">-</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Piscirickettsia</italic><sup>###</sup></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 + 1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1324</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4178</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">44.2</td></tr></tbody></table><table-wrap-foot><attrib><italic>*A representative genome set was used for the analysis (for details see section “Materials and Methods). <italic><sup>#</sup></italic>The genome of the <italic>Legionella</italic> endosymbiont of <italic>Polyplax serrata</italic> was excluded (Comment: Symbiotic bacterium from the lice of the genus <italic>Polyplax</italic>). <sup>##</sup>Pan-Genome analysis of <italic>Piscirickettsia salmonis</italic> by <xref rid=\"B57\" ref-type=\"bibr\">Nourdin-Galindo et al. (2017)</xref>. <sup>###</sup>For the genus <italic>Piscirickettsia</italic>, the sequenced genomes are derived from two species: <italic>Piscirickettsia salmonis</italic> (complete genomes exist for the different strains) and <italic>Piscirickettsia litoralis</italic> (only a scaffold genome exist for the single strain).</italic></attrib></table-wrap-foot></table-wrap><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Venn diagrams of core genomes from <italic>Francisella</italic>\n<bold>(A)</bold> and <italic>Legionella</italic>\n<bold>(B)</bold> generated by the BDBH, COG and OMCL strategies, using the GET_HOMOLOGUES tool. Singletons (genes present in only one copy in any genome) from 26 and 77 representative species sequences respectively were used as input.</p></caption><graphic xlink:href=\"fmicb-11-01928-g003\"/></fig><p>In <italic>Francisella</italic>, approximately 71% of the strain-specific genes were predicted to encode hypothetical proteins, while 29% encode functionally characterized proteins. The total number of coding genes and the genome size for each of the 26 representative <italic>Francisella</italic> genomes are provided in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>. From the core genome analyses, AAI (Average Amino-acid identity) was calculated using protein-coding sequences (CDSs) of the 26 selected genomes. A heat-map representing the degree of similarity of the genomes based on the average amino acid identities of their CDSs was constructed (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>), demonstrating the formation of two distinct groups. The observation also illustrates the microbial evolution and displays a functional relationship between different <italic>Francisella</italic> strains as well as species obtained from variable environments.</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>A heatmap representing the degree of similarity of genomes based on the average amino acid identities of their protein coding genes. The heatmap was derived from the high similarity (light yellow) and low similarity (dark orange) of CDSs derived from the 26 <italic>Francisella</italic> genomes. For strain abbreviations, see <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>.</p></caption><graphic xlink:href=\"fmicb-11-01928-g004\"/></fig></sec><sec id=\"S3.SS3\"><title>Functional Genome Analyses</title><p>By using the same approach as for <italic>Francisella</italic>, the core- and pan-genomes of the genus <italic>Legionella</italic> were estimated to be 886 and 8,413 genes, respectively, while the corresponding numbers for <italic>Piscirickettsia</italic> were 1,324 and 4,178 genes, respectively (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref> and <xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>). It should be noted that the core-genome size of <italic>Piscirickettsia</italic> may be affected by the lack of genomes of other species than <italic>P. salmonis</italic> and <italic>P. litoralis</italic>, and therefore appear to be larger than those of <italic>Francisella</italic> and <italic>Legionella</italic> (<xref rid=\"B57\" ref-type=\"bibr\">Nourdin-Galindo et al., 2017</xref>). We also compared the core-genome size to the mean number of CDS per genome. For <italic>Legionella</italic> this corresponded to 29.2% (886 <italic>vs.</italic> 3031) and for <italic>Piscirickettsia</italic> to 44.2% (1323 <italic>vs.</italic> 2995) (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). Furthermore, the “core of core” within all the three genera comprised 263 genes, while the corresponding numbers within <italic>Francisella</italic> and <italic>Legionella</italic> were 383 genes, within <italic>Piscirickettsia</italic> and <italic>Francisella</italic> 399 genes, and within <italic>Legionella</italic> and <italic>Piscirickettsia</italic> 472 genes (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). To assign biological functions to the genus orthologs (“core of core”), the corresponding amino acid sequences for all 263 shared genes were annotated using COG. This revealed that the majority (25.7%) of the proteins belonged to the COG category Translation, ribosomal structure and biogenesis, 7.5% to Energy production and conversion, 7.6% to Post-translational modification, protein turnover, and chaperones, and 3.3% were poorly categorized, or with unknown function (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Figure S2</xref>). We also mapped the protein cellular functions using KEGG. Genes were divided into five branches according to the biological pathways they are likely to participate in and the percentage of genes belonging to a particular category calculated to be as follows: (A), Cellular Processes (1.7%); (B), Environmental Information and Cellular Processing (6.8%); (C), Genetic Information Processing (26.9%); (D), Metabolism (63.1%); (E), Organismal Systems (0.39%), and (F), Human diseases (2.8%) (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Figure S3</xref>).</p><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>The size of the common core-genome shared between different genera.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Genera</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Common core-genome (no. genes)*</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Francisella</italic> vs. <italic>Legionella</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">383</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Piscirickettsia</italic> vs. <italic>Francisella</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">399</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Legionella</italic> vs. <italic>Piscirickettsia</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">472</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>Legionella</italic> vs. <italic>Piscirickettsia</italic> vs. <italic>Francisella</italic></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">263</td></tr></tbody></table><table-wrap-foot><attrib><italic>*The size was estimated using minimum sequence coverage and 50% sequence identity cut-off.</italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS4\"><title>Global Phylogeny of <italic>Francisella</italic></title><p>Phylogenetic relationship of bacteria is usually estimated by comparing sequences of homologous genes, typically the 16S rRNA gene. In the case of <italic>Francisella</italic>, however, the differences within the 16S rRNA sequence are very few (<xref rid=\"B14\" ref-type=\"bibr\">Challacombe et al., 2017</xref>), requiring the use of an alternative approach. While single gene-based phylogenetic trees have low inter-species discriminatory power, multi-gene approaches offer the possibility to create more robust phylogenetic trees (<xref rid=\"B13\" ref-type=\"bibr\">Castresana, 2007</xref>; <xref rid=\"B72\" ref-type=\"bibr\">Satoh et al., 2013</xref>). Thus, we explored the genetic diversity within the genus <italic>Francisella</italic> by inferring the phylogenomic relationship based on the genomic content. For this purpose, we used the up-to-date bacterial core gene set, UBCG, consisting of 92 core genes from 1,492 bacterial species covering 28 phyla. This robust phylogenomic method is universally applicable to any phyla of the domain <italic>Bacteria</italic> (<xref rid=\"B52\" ref-type=\"bibr\">Na et al., 2018</xref>). The obtained results clearly indicated two major and distinct clades, A and B, and an additional and diverse cluster designated C (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>). Most strains within clade A are pathogenic to mammals, e.g., members of the species <italic>F. tularensis</italic> and subspecies thereof, while clade B includes strains found in the marine environment, most of which are pathogenic to fish, but also some potentially pathogenic to humans, e.g., <italic>F. philomiragia</italic> and <italic>F. noatunensis</italic> and its subspecies. Clade B is more disparate than clade A. Clade C was found to comprise <italic>A. guangzhouensis</italic> 08HL01032T, the species <italic>F. frigiditurris</italic> sp. nov. CA971460, <italic>F. endociliophora</italic> FSC1006, <italic>F. uliginis</italic> sp. nov. TX077310, and <italic>F. halioticida</italic> DSM23729, most of which are associated with the marine environment (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Four species, <italic>F. hispaniensis</italic> FSC454, <italic>F. cf. novicida</italic> 3523, <italic>F. opportunistica</italic> sp. nov. MA067296, and <italic>F. persica</italic> ATCC VR331, differentiated into two groups and formed a small cluster phylogenetically rather close to clade A (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>). This global phylogenomic-based analysis also supported the ANI and AAI hierarchical cluster-based dendrograms (<xref ref-type=\"fig\" rid=\"F1\">Figures 1</xref>, <xref ref-type=\"fig\" rid=\"F4\">4</xref>). In addition to the aforementioned phylogenetic approaches, we also assessed the phylogeny based on the non-recombinant loci alignment, as a means to construct a phylogenetic tree of more accurate and precise topology. The 692 core genes (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>) were used for evaluating the phylogenies based on encoded proteins as well as DNA content. Top scoring phylogenetic markers were selected based on the criteria recommended by Vinuesa (<xref rid=\"B90\" ref-type=\"bibr\">Vinuesa et al., 2018</xref>), i.e., they should (i) be non-recombinant (<xref rid=\"B30\" ref-type=\"bibr\">Kaas et al., 2012</xref>), (ii) show a robust phylogenetic signal, and (iii) result in a coherent phylogenetic tree. In total, 43 proteins and 236 DNA-based markers were used for maximum likelihood (ML) phylogenetic estimation, generating two trees of almost identical topology (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref> and data not shown), confirming that our phylogeny is correct and optimal. Like the UBCG as well as ANI-based phylogenetic trees, the marker-based phylogenetic tree also formed three main clades (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>).</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Phylogenomics tree reconstruction by the UBCG software, using standard settings based on 92 up-to-date bacterial core genes, revealing three major clades <bold>(A–C)</bold>. Bootstrap values are presented at the branching points. For strain abbreviations, see <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>. The numbers 1 or 2 indicate that a given genome belongs to one of the two major groups identified with respect to FPI gene content; group 1 (complete FPI island with 18 genes) or group 2 (incomplete FPI, lacking the <italic>pdpC</italic> and <italic>pdpE</italic> genes). An asterisk indicates that additional FPI genes are missing for group 2 members. For genomes without numbers, see <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> for a description of their FPI gene content. Scale bar equals 0.1 substitutions per nucleotide position.</p></caption><graphic xlink:href=\"fmicb-11-01928-g005\"/></fig><fig id=\"F6\" position=\"float\"><label>FIGURE 6</label><caption><p>A maximum likelihood (ML) phylogenetic tree based on the non-recombinant loci concatenated set of top-ranking phylogenetic markers, revealing three major clades <bold>(A–C)</bold>. Bootstrap values are presented at the branching points. For strain abbreviations, see <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>. Scale bar equals 0.1 substitutions per nucleotide position.</p></caption><graphic xlink:href=\"fmicb-11-01928-g006\"/></fig><p><xref rid=\"B77\" ref-type=\"bibr\">Sjödin et al. (2012)</xref> previously reported a divergence of the <italic>Francisella</italic> genus into two distinct clades, with clade A comprising <italic>F. tularensis</italic>, <italic>F. novicida</italic>, <italic>F. hispaniensis</italic>, and <italic>F. persica</italic>, and clade B containing <italic>F. philomiragia</italic> and <italic>F. noatunensis</italic>. Our comprehensive phylogenetic analysis also confirmed this bifurcation of <italic>Francisella</italic> into two clades, with the addition of a third clade, clade C. Notably, in the UBCG analysis, the <italic>F. persica</italic> ATCC VR331 and <italic>F. opportunistica</italic> sp. nov. MA067296 showed a common ancestor and were closer to clade A, while in the selected marker-based trees, these species are more disparate (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>). The clade A of the marker-based phylogenetic tree comprised <italic>F. tularensis</italic> and subspecies thereof, with the addition of <italic>F. cf. novicida</italic> Fx1 (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>). Overall, our results based on selected markers therefore give additional support to the core-genome-based phylogenomic tree of the genus <italic>Francisella</italic>.</p></sec><sec id=\"S3.SS5\"><title>The <italic>Francisella</italic> FPI Cluster</title><p>The <italic>Francisella</italic> pathogenicity island (FPI) is a cluster of 16–19 genes, present in most of the <italic>Francisella</italic> genomes that have been sequenced to date. Although 16 FPI genes are highly conserved, 2–3 genes are absent or interrupted by stop codons in some strains (<xref rid=\"B53\" ref-type=\"bibr\">Nano and Schmerk, 2007</xref>). Intriguingly, the highly virulent <italic>Francisella</italic> strains contain two copies of the entire FPI, while the less virulent <italic>Francisella</italic> strains have a single copy (<xref rid=\"B82\" ref-type=\"bibr\">Spidlova and Stulik, 2017</xref>). We found that depending on species analyzed, the overall G + C content of the FPI was 3–5% lower than for the rest of the <italic>Francisella</italic> genome, ∼32% (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S4</xref>). Moreover, significant variations in G + C content within this region were also noted (data not shown; <xref rid=\"B54\" ref-type=\"bibr\">Nano et al., 2004</xref>). In support, a comparison of proteins encoded within the FPI and outside of the FPI demonstrated that the most over-represented amino acids within the FPI correspond to lysine, asparagine and serine, all of which are encoded by GC-poor codons (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S5</xref>). In contrast, the most under-represented amino acids within the FPI corresponded to alanine, glycine, valine, tryptophan, i.e., GC-rich codons, as well as methionine (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S5</xref>). Similar results were obtained for all of the four genomes investigated, i.e., <italic>F. tularensis</italic> subsp. <italic>holarctica</italic> LVS, <italic>F. noatunensis</italic> subsp. <italic>noatunensis</italic> FSC772, <italic>F. cf. novicida</italic> Fx1 and <italic>F. novicida</italic> U112 (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S5</xref>). To search for FPI genes within our 26 representative genomes, we used the MultiGeneBlast program and the FPI island of <italic>F. novicida</italic> U112 as query. Our results show that all of the 26 <italic>Francisella</italic> genomes had at least one copy of the FPI, except for <italic>A. guangzhouensis</italic> 08HL01032T (data not shown) and <italic>F. halioticida</italic> DSM23729, for which only the genes encoding IglA and IglB, i.e., the T6SS sheath proteins, were detected. The <italic>F. tularensis</italic> subsp. <italic>holarctica</italic> LVS, subsp. <italic>mediasiatica</italic> FSC147, and subsp. <italic>tularensis</italic> SCHU S4 all have two copies of the FPI as shown in <xref rid=\"T1\" ref-type=\"table\">Table 1</xref> and <xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Figure S4</xref>. Two out of the 26 genomes, those from <italic>F. endociliophora</italic> FSC1006 and <italic>F. salina</italic> sp. nov. TX077308, have a single FPI copy with three or more of the FPI genes missing or inactivated (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref> and <xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Figure S4</xref>). Interestingly, <italic>F. philomiragia</italic> GA012794 and <italic>F. endociliophora</italic> FSC1006 possess two additional T6SS clusters, both of which lack significant homology to the FPI cluster. Instead, our phylogenomic analysis suggested that they show most similarity to the T6SS of <italic>Escherichia coli</italic> (data not shown).</p><p>Based on FPI gene content and organization, two major groups could be distinguished within the <italic>Francisella</italic> genus. The first is characterized by the presence of an intact FPI cluster and includes, e.g., <italic>F. hispaniensis</italic> FSC454, <italic>F. tularensis</italic> subsp. <italic>mediasiatica</italic> FSC147 and <italic>F. tularensis</italic> subsp. <italic>tularensis</italic> SCHU S4, <italic>F. novicida</italic> U112, <italic>F. novicida</italic> PA107858, <italic>F. novicida</italic> D9876, <italic>F. novicida</italic> AZ067470, <italic>F. cf. novicida</italic> 3523, and <italic>F. cf. novicida</italic> Fx1. Most of the species belonging to this group clustered to clade A in the phylogenetic tree analysis. The second group is characterized by the presence of an FPI cluster, which lacks both the <italic>pdpC</italic> and <italic>pdpE</italic> genes. This group included, e.g., all strains of <italic>F. philomiragia</italic> and <italic>F. noatunensis</italic>, and <italic>F. noatunensis</italic> subsp. <italic>orientalis, F. frigiditurris</italic> sp. nov. CA971460, <italic>F. opportunistica</italic> sp. nov. MA067296, <italic>F. uliginis</italic> sp. nov. TX077310, <italic>F. salina</italic> sp. nov. TX077308, <italic>F. novicida</italic> AL972214, and <italic>F. endociliophora</italic> FSC1006, the latter being unique in that its FPI also lacks <italic>pdpD</italic>, <italic>anmK</italic>, and <italic>iglI</italic>, and exhibits gene rearrangements (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, <xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Figure S4</xref>, and <xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref>). With the exception of <italic>F. opportunistica</italic> sp. nov. MA067296 and <italic>F. novicida</italic> AL972214, all of this group belong to clade B or clade C according to our analysis. In addition, other variants of the FPI cluster were predicted from the analysis (<xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref>). For example, strain <italic>F. persica</italic> was found to lack the entire <italic>pdpD</italic> gene, while the same gene is truncated in both loci of the <italic>F. tularensis</italic> subsp. <italic>holarctica</italic> strain LVS. The <italic>anmK</italic> gene exists as two distinct truncated forms in <italic>F. tularensis</italic> subsp. <italic>tularensis</italic>, but is absent in subsp. <italic>holarctica</italic> (<xref ref-type=\"fig\" rid=\"F7\">Figure 7</xref> and <xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Figure S4</xref>). Recently, <xref rid=\"B8\" ref-type=\"bibr\">Brodmann et al., 2017</xref> reported that <italic>pdpC</italic>, <italic>pdpD</italic>, <italic>pdpE</italic> and <italic>anmK</italic> are dispensable for T6S.</p><fig id=\"F7\" position=\"float\"><label>FIGURE 7</label><caption><p>Comparative analysis of T6SS clusters in the genus <italic>Francisella</italic>. Shown are clusters from some representative species belonging to clades A and B that were identified in <xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>. For strain abbreviations, see <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>.</p></caption><graphic xlink:href=\"fmicb-11-01928-g007\"/></fig><p>We also searched the NCBI database for FPI homologs present outside of the genus <italic>Francisella</italic> using the BlastP program. As reported before, a few FPI proteins had homologs in T6SSs belonging to a wide range of species, e.g., IglA, IglB, and DotU, many of which have been demonstrated to be functionally conserved (<xref rid=\"B21\" ref-type=\"bibr\">De Bruin et al., 2007</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Bröms et al., 2010</xref>, <xref rid=\"B11\" ref-type=\"bibr\">2012</xref>). Interestingly, this category also included IglG, and to some extent IglI, both of which previously were reported to lack homologs in other bacteria (<xref rid=\"B10\" ref-type=\"bibr\">Bröms et al., 2011</xref>). Also homologs of AnmK were found in other bacterial species, as well as outside of the FPI cluster within <italic>Francisella</italic>. Based on homology, <italic>anmK</italic> is predicted to encode an anhydro-N-acetylmuramic acid kinase. In contrast, we could not find any homolog to PdpC outside of the genus <italic>Francisella</italic>. For the remaining FPI components, only one or a few homolog(s) outside of the genus exist(s), and then primarily in species closely related to <italic>Francisella</italic>, such as <italic>Piscirickettsia</italic> sp., <italic>Cysteiniphilum</italic> sp., <italic>Fangia hongkongensis</italic>, and <italic>Pseudofrancisella aestuarii.</italic> Taken together, our comparative analysis of the FPI gene cluster demonstrates that the FPI genes are highly similar within the genus, but share low similarities with T6SS genes of other bacterial species.</p></sec><sec id=\"S3.SS6\"><title>Putative T6SS Effectors</title><p>Effector protein identification is critical for the understanding of how the <italic>Francisella</italic> FPI promotes pathogenesis. So far, a few putative effectors encoded within the FPI have been identified by the use of different reporter assays (<xref rid=\"B2\" ref-type=\"bibr\">Barker et al., 2009</xref>; <xref rid=\"B11\" ref-type=\"bibr\">Bröms et al., 2012</xref>) and, more recently by a proteome-based approach combined with quantitative mass spectrometry (<xref rid=\"B24\" ref-type=\"bibr\">Eshraghi et al., 2016</xref>). Interestingly, the latter study also identified putative effector proteins encoded outside of the FPI for <italic>F. novicida</italic>, including OpiA. In a follow up study, this protein was shown to possess phosphatidylinositol 3-kinase-activity, alter phagosomal maturation, and, thereby, promote intracellular growth of <italic>F. novicida</italic> (<xref rid=\"B44\" ref-type=\"bibr\">Ledvina et al., 2018</xref>).</p><p>To search for putative T6SS effector proteins within the genome of <italic>F. tularensis</italic> subsp. <italic>tularensis</italic> SCHU S4, we used the Bastion6 machine learning predictor to identify putative T6SS effectors. A total of 144 promising candidates, all with a predicted ensemble score above 50%, were retrieved using Bastion6. All candidates, except for PdpB and PdpD, were encoded outside of the FPI. For further details about the hits, see supporting information in <xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S6</xref>. PANNZER2 in combination with gene ontology were used to functionally describe and annotate the putative effectors further. This analysis demonstrated that more than 1/3 of the putative effectors are predicted to act on cellular targets including the peptidoglycan cell wall (hydrolases), cellular nucleic acids and proteins (nucleases and proteolytic enzymes respectively), as well as the inner membrane (phospholipases) (see <xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S7</xref> for more details about the proteins putative function and localization). Among the top-ranked hits, three were predicted to possess hydrolase activity and, according to the Carbohydrate-Active Enzymes (CAZymes: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.cazy.org/\">http://www.cazy.org/</ext-link>) analysis, constitute members of the glycosyl hydrolases family 18. Four putative effectors had protein domains of no characterized function, i.e., DUF1338, DUF2147, DUF4124, and DUF4440 (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S7</xref>). Further investigation using the Pfam database suggested that these hits may be a putative metal hydrolase, a member of the lipocalin family, to possess an immunoglobulin-like (Ig-like) fold and to be a member of the nuclear transport factor 2 (NTF-2)-family, respectively. Among the putative effectors, we also identified an OmpA family protein (outer membrane lipoprotein; <xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S7</xref>). OmpA is a peptidoglycan-binding protein that is not physically part of the T6SS clusters, but has been suggested to share a functional relationship with some T6SS proteins (<xref rid=\"B73\" ref-type=\"bibr\">Shrivastava and Mande, 2008</xref>).</p><p>While we failed to identify any homologs to the ion-selective pore-forming T6SS effectors that were recently identified and suggested to be widespread within <italic>Enterobacteriaceae</italic> (<xref rid=\"B46\" ref-type=\"bibr\">Mariano et al., 2019</xref>), we also carried out an analysis searching for effectors with the previously identified N−terminal domains named MIX (Marker for type six effectors). Previously, T6SS effectors of various <italic>Proteobacteria</italic> were demonstrated to share this conserved motif and to group into five clans named MIX I-V (<xref rid=\"B71\" ref-type=\"bibr\">Salomon et al., 2014</xref>). We used known MIX sequences from representative clan members to search for MIX effectors in the genus <italic>Francisella</italic>. We failed to identify putative MIX effectors belonging to the MIX-II, III and IV clans, however, two <italic>Francisella</italic> proteins showed low sequence similarity to either MIX-I or MIX-V clan members. Both predicted effectors are mainly found in the marine and fish-pathogenic strains (Clade B), and in some mammalian pathogenic-species of <italic>Francisella</italic> (Clade A). The first putative effector (MIX-I) is a conserved hypothetical protein (locus tag: “FTT_1768c”), functionally predicted to be a Chitinase/glycoside hydrolase family 18 protein and also identified as a putative effector in the Bastion6 machine learning based predictor for T6SS effectors (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Table S7</xref>). The second (MIX-V) is an uncharacterized protein of the DUF3568 family (locus tag: “FTT_1416c”). Upon further comparative analysis of this protein, we found that members of this family are approximately 120–130 amino acids long and contain a highly conserved cysteine residue within the N-terminus. In agreement with a putative role as lipoproteins, the first 25 amino acids of the N-terminus were predicted to form a signal peptide, suggesting that prelipoproteins belonging to this family would be cleaved directly upstream of the conserved cysteine. Interestingly, some <italic>Francisella</italic> genomes were found to have duplicate or triplicate copies of the DUF3568-containing gene, including <italic>F. endociliophora</italic> strain FSC1006, <italic>F. halioticida</italic> strain DSM23729, and <italic>F. tularensis</italic> subsp. <italic>tularensis</italic> strain WY96. A DUF3568 neighborhood analysis did not provide any evidence for an association with the T6SS (data not shown). Remarkably, the DUF3568 domain-containing protein originally reported as <italic>F. tularensis</italic> Virulence Determinant protein (i.e., Flpp3) has been suggested to share structural homology to Bet v1 allergen proteins (<xref rid=\"B98\" ref-type=\"bibr\">Zook et al., 2015</xref>). Taken together, this analysis has revealed the presence of putative T6SS substrates encoded outside of the FPI within the <italic>Francisella</italic> genome. Functional characterization will be needed to determine whether they are indeed T6S substrates and if they contribute to bacterial virulence.</p></sec></sec><sec id=\"S4\"><title>Discussion</title><p>Bacterial taxonomy based on 16S rRNA sequencing has since long been the most important parameter to explore the phylogenetic relationships of bacteria and to assign genus- and species-belonging. A drawback, however, is that the resolution of the method is normally not sufficient to discriminate subspecies and that it is vulnerable to biases depending on primer sequence-matching in different species (<xref rid=\"B15\" ref-type=\"bibr\">Chan et al., 2012</xref>; <xref rid=\"B69\" ref-type=\"bibr\">Rosselli et al., 2016</xref>). Also, phenotypical and biochemical characteristics have been used as a basis for phylogenetic determination, however, these are traits that to some extent can be affected by choice of culture medium and other conditions (<xref rid=\"B86\" ref-type=\"bibr\">Tindall et al., 2010</xref>). Therefore, objective methods that show high resolution need to be implemented. One promising and rather often used method in this regard is based on determination of the relatedness by calculating the average nucleotide identity, as previously described (<xref rid=\"B26\" ref-type=\"bibr\">Han et al., 2016</xref>).</p><p>The present study constitutes a comprehensive comparative genomic characterization of the genus <italic>Francisella</italic>. The characterized divergences and similarities identified here represent an important contribution toward understanding the biology and evolution of <italic>Francisella</italic>. Importantly, the minor variation (32.3 ± 0.4) in the G + C content of this genome dataset was indicative of a stable boundary delineation within the genus. The distinctly lower G + C content of the FPI suggests that horizontal gene transfer has been a major factor driving the evolution of the FPI of <italic>Francisella</italic>. Indeed, Nano et al., suggested that the FPI originally had been acquired through horizontal gene transfer from an organism with a lower G + C content (<xref rid=\"B54\" ref-type=\"bibr\">Nano et al., 2004</xref>). Our findings support their conclusion and, additionally, we could demonstrate a distinct bias for GC-poor codons within the FPI. Thus, our findings are in agreement with findings in eubacterial and archaeal genomes demonstrating that a biased nucleotide-content causes a divergent amino acid composition of the encoded proteins (<xref rid=\"B76\" ref-type=\"bibr\">Singer and Hickey, 2000</xref>). In contrast, Larsson et al., postulated that the ancestor had been an organism with a higher G + C content, but our findings do not support the hypothesis (<xref rid=\"B41\" ref-type=\"bibr\">Larsson et al., 2009</xref>).</p><p>Our phylogenetic trees were based on analyses including the core genome, ANI, and non-recombinant loci alignment of 26 completely sequenced genomes. Since a multitude of analyses, including the established method UBCG that includes up-to-date core genes in the analysis, were performed and gave congruent results, the findings strongly corroborate previous phylogenetic analyses and further refine the relationships within the genus. This is the first time that UBCG has been implemented for the genus <italic>Francisella</italic>. Regardless of method used, the analysis provided unequivocal evidence for the existence of two genogroups, Clade A and Clade B, which has also been reported previously (<xref rid=\"B77\" ref-type=\"bibr\">Sjödin et al., 2012</xref>). Our phylogenetic trees closely resemble those previously reported by Sjödin et al. and Challacombe et al., but the variety of methods used in our study add much more robustness to the composition of the phylogenetic trees obtained. Clade A comprised mostly human pathogenic strains, predominantly belonging to <italic>F. tularensis</italic>, whereas clade B was more diverse and encompassed fish pathogens and strains rarely pathogenic to humans, such as <italic>F. noatunensis</italic> and <italic>F. philomiragia</italic>. The analysis also identified phylogenetic positions for recently characterized strains such as <italic>F. cf. novicida</italic> 3523, <italic>F. frigiditurris</italic> sp. nov. CA971460, <italic>F. opportunistica</italic> sp. nov. MA067296, <italic>F. uliginis</italic> sp. nov. TX077310, and <italic>F. salina</italic> sp. nov. TX077308.</p><p>The study by Challacombe et al. characterized four new species of the genus <italic>Francisella</italic> and demonstrated that the demarcation of new species in bacteria is quite challenging (<xref rid=\"B14\" ref-type=\"bibr\">Challacombe et al., 2017</xref>). This is in particular the case for isolates with similar genomic characteristics, but different physiological features, e.g., some being pathogenic, whereas others are opportunistic pathogens, or even non-pathogenic (<xref rid=\"B14\" ref-type=\"bibr\">Challacombe et al., 2017</xref>). The analyses by Challacombe, based on ANI, 16S rRNA, or a multilocus sequence typing scheme, gave congruent results and overall also agree with the taxonomic positions we identified. Collectively, the findings support the use of genomic analyses as a basis for species delineation and demonstrate a robustness in the phylogenetic trees of the genus. Thereby, the methods utilized herein are potent tools for a precise delineation of the taxonomical belonging of strains that will be identified in the future. In addition to the aforementioned study, Dietrich et al. reported the identification of three isolates of <italic>F. opportunistica</italic> sp. nov., from human blood and cerebrospinal fluid, which showed ANI inter-strain similarities of 99.9%, and 88.6% to the closest relative, the tick endosymbiont <italic>F. persica</italic> (<xref rid=\"B22\" ref-type=\"bibr\">Dietrich et al., 2019</xref>). In agreement, our ANI analysis of 26 complete genomes of <italic>Francisella</italic> demonstrated ANI values > 95% within species, and 74–95% between species. These values also concurred with the conclusions of the study by <xref rid=\"B1\" ref-type=\"bibr\">Appelt et al. (2019)</xref> in which <italic>F. tularensis</italic> isolates from Switzerland were analyzed. In this study, an ANI threshold of 99.5% was postulated to distinguish subspecies from each other.</p><p>In our analyses, we also included <italic>A. guangzhouensis</italic> strain 08HL01032T to determine its phylogenetic relationship with the genus <italic>Francisella</italic>. Prior to 2016, this strain was considered a member of the genus <italic>Francisella</italic>, however, based upon 16S RNA- and multilocus sequence typing-based analyses, it was reclassified as a separate genus (<xref rid=\"B63\" ref-type=\"bibr\">Qu et al., 2016</xref>). To date, this is the only complete genome available for this genus, but a scaffold assembly exists for <italic>A. inopinata</italic>. Interestingly, the phylogenetic tree obtained from the core genome comparative analysis clearly indicated that <italic>A. guangzhouensis</italic> 08HL01032T is an outlier, separate from the two main clusters of <italic>Francisella</italic> strains. However, it clustered with <italic>F. frigiditurris</italic> sp. nov. and the same relationship was also confirmed in the protein marker-based phylogenetic tree. Our further in-depth analysis concluded that these two strains of <italic>A. guangzhouensis</italic> and <italic>F. frigiditurris</italic> sp. nov. exhibited very similar ANI values vs. the SCHU S4 strain (74.5% vs ∼74%), the latter being the lowest value of all 26 <italic>Francisella</italic> genomes analyzed. Interestingly, in the recent study by Challacombe et al., <italic>F. frigiditurris</italic> sp. nov. was suggested to be a new member of the genus <italic>Francisella</italic> (<xref rid=\"B14\" ref-type=\"bibr\">Challacombe et al., 2017</xref>). Thus, <italic>A. guangzhouensis</italic> 08HL01032T may be closer to the genus <italic>Francisella</italic> than previously considered (<xref rid=\"B63\" ref-type=\"bibr\">Qu et al., 2016</xref>), and the classification of this strain as a member of a separate genus is therefore not clear-cut.</p><p>The nucleotide diversity was rather similar for <italic>Francisella</italic> and <italic>Legionella</italic>, 74% and 71%, respectively. The pan-genome of the latter was considerably larger, comprising 8,413 genes, whereas that of <italic>Francisella</italic> encompassed 4,053 genes. Of these, 692 genes, represented the core-genome, whereas the corresponding number for <italic>Legionella</italic> was 886 genes. The core genes are expected to play a role in the ability of these intracellular pathogens to survive within the specialized environment of phagocytic cells and protozoa, respectively. Still, as evidenced by the differences in the size of their pangenomes, both pathogens demonstrate a distinct genetic composition that likely contributes to unique features for the two genera. In this regard, a drawback in the genetic analysis of <italic>Francisella</italic> is the plethora of unannotated genes, however, a majority of these could still be assigned a function using COG or KEGG.</p><p>The FPI is essential for the virulence of <italic>Francisella</italic> and encodes a Type VI secretion system (T6SS) (<xref rid=\"B54\" ref-type=\"bibr\">Nano et al., 2004</xref>; <xref rid=\"B9\" ref-type=\"bibr\">Bröms et al., 2010</xref>). All of the 26 <italic>Francisella</italic> genomes possess at least one FPI copy, with the exception of <italic>F. halioticida</italic> DSM23729, for which only the genes encoding the T6SS sheath proteins, IglA and IglB, were detected. <italic>A. guangzhouensis</italic> 08HL01032T also lacked the island, as reported previously (<xref rid=\"B14\" ref-type=\"bibr\">Challacombe et al., 2017</xref>). Both, together with <italic>F. frigiditurris</italic> sp. nov. CA971460, exhibited among the lowest ANI values overall in our analysis. Since the latter strain possesses a typical FPI, low ANI values does not correlate with the absence of the FPI in the genome. Upon analyzing FPI gene content and organization, several groups could be distinguished, including those that (i) lacked the entire FPI, i.e., <italic>F. halioticida</italic> DSM23729, (ii) possessed one complete FPI copy or more, e.g., <italic>F. hispaniensis</italic> FSC454 (1 copy) and <italic>F. tularensis</italic> subsp. <italic>tularensis</italic> SCHU S4 (2 copies), (iii) lacked both of <italic>pdpC</italic> and <italic>pdpE</italic>, e.g., <italic>F. philomiragia</italic>, (iv) lacked a functional <italic>pdpD</italic> gene, i.e<italic>., F. persica</italic> ATCC VR331 and <italic>F. tularensis</italic> subsp. <italic>holarctica</italic> LVS, or (v), lacked all of <italic>pdpC, pdpD</italic> and <italic>pdpE</italic> genes (<italic>F. endociliophora</italic> FSC1006). Similar results were obtained in the previous study by Challacombe et al., which was based on 31 <italic>Francisella</italic> genomes in total (<xref rid=\"B14\" ref-type=\"bibr\">Challacombe et al., 2017</xref>). The advantage of using a larger genome data set is the possibility of finding unique FPI patterns not discovered before, however, all of the additional genomes that we included in our study could be sorted into the previously categorized FPI groups. We did, however, make one interesting observation, since we observed an additional FPI genogroup as represented by <italic>F. endociliophora</italic> FSC1006. The strain lacks <italic>pdpC</italic>, <italic>pdpD</italic>, <italic>pdpE</italic> as well as <italic>anmK</italic>, as previously reported (<xref rid=\"B14\" ref-type=\"bibr\">Challacombe et al., 2017</xref>), but, in addition, we identified a lack of the <italic>iglI</italic> gene. Thus, the repertoire of FPI variants is more diverse than previously reported. The additional genomes that have been sequenced upon completion of this study may add to this complexity. The lack of <italic>pdpC</italic> and <italic>pdpD</italic> in certain strains was reported previously (<xref rid=\"B24\" ref-type=\"bibr\">Eshraghi et al., 2016</xref>). The two genes have previously been suggested to encode effector proteins, in fact, <italic>pdpD</italic> was identified as an effector also in our computational screen. Thus, the acquisition of <italic>pdpC</italic> and <italic>pdpD</italic> genes may have been an important step toward pathogenesis in mammals, possibly facilitating host tropism. The role of <italic>pdpE</italic> is less clear, since studies indicate that mutant is as virulent as the parental strain (<xref rid=\"B10\" ref-type=\"bibr\">Bröms et al., 2011</xref>). Nevertheless, since loss of <italic>pdpE</italic> always is accompanied by loss of <italic>pdpC</italic>, our results suggest that these two proteins somehow may interact.</p><p>While the repertoire of effector proteins is quite abundant for some T6SS, e.g., <italic>V. cholerae</italic>, a modest number of substrates has been identified for the <italic>Francisella</italic> T6SS (<xref rid=\"B11\" ref-type=\"bibr\">Bröms et al., 2012</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Eshraghi et al., 2016</xref>). Naturally, this could simply be a consequence of low effector abundance, choice of strain and/or method to quantify secretion. Our findings of 144 promising candidates, most of them encoded outside of the FPI, therefore constitute interesting targets for site-directed mutagenesis. Among the top-ranked hits, we identified, e.g., glycosyl hydrolase active enzymes. One of the candidates was FTT_1768c, which shares some homology to MIX-I effector proteins, and was functionally predicted to be a Chitinase/glycoside hydrolase family 18 protein. In fact, the FTT_1768c protein was identified in a high-throughput yeast two-hybrid assay, revealing putative physical interactions to human proteins, including Vps35 (Vacuolar protein sorting-associated protein 35) (<xref rid=\"B23\" ref-type=\"bibr\">Dyer et al., 2010</xref>). The latter is a core component of the retromer complex, which controls vesicular transport within eukaryotic cells and consists of a membrane-associated sorting nexin dimer and a vacuolar protein sorting (Vps) trimer. Because of its essential role in vesicle trafficking, this transport pathway has emerged as an important target for intracellular bacterial pathogens to promote their survival and replication. For example, VPS35 and VPS26A, both components of the retromer, were recently shown to be required for the diversion of <italic>Brucella</italic>-containing vacuoles (BCVs) from the endolysosomal pathway and the establishment of the intracellular replicative niche (<xref rid=\"B12\" ref-type=\"bibr\">Casanova et al., 2019</xref>). Moreover, the Dot/Icm effector RidL of <italic>L. pneumophila</italic> inhibits retromer activity to promote intracellular replication by directly binding to the retromer subunit VPS29 (<xref rid=\"B25\" ref-type=\"bibr\">Finsel et al., 2013</xref>), thereby outcompeting essential retromer regulators (<xref rid=\"B96\" ref-type=\"bibr\">Yao et al., 2018</xref>). This raises the question of whether our identified hit, the Chitinase/glycoside hydrolase family 18 protein, plays a similar role in vesicle trafficking and intracellular survival of <italic>Francisella</italic>, and whether this involves a direct physical interaction with the retromer. To our knowledge, this has not been investigated. Interestingly, this putative effector is highly conserved among the different subspecies of <italic>F. tularensis</italic>, >99% identity, but is less conserved within the species <italic>F. philomiragia</italic> and <italic>F. noatunensis</italic>, 37–52%, that only rarely infect humans, possibly reflecting a difference in function. These candidate genes may therefore constitute interesting targets for designing novel strategies to prevent and control infections with species that belong to this highly diverse and environmentally adapted genus.</p><p>Collectively, the comparative genomic analysis performed provides a comprehensive basis for the assessment of the phylogenomic relationship of members of the genus <italic>Francisella</italic> and for the identification of putative T6SS virulence traits.</p></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>All datasets presented in this study are included in the article/<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary Material</xref>.</p></sec><sec id=\"S6\"><title>Author Contributions</title><p>RK, AS, and JB designed the study, analyzed the data, and wrote the manuscript. RK performed all experiments.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> RK acknowledges the MIMS and UCMR for funding at Umeå University.</p></fn></fn-group><ack><p>We would like to thank Igor Golovlev and Athar Alam for fruitful discussions. We acknowledge research funding for this work by grants 2013-4581 and 2013-8621 from the Swedish Research Council and a Biotechnology grant from the Medical Faculty, Umeå University, Umeå, Sweden (FS 2.1.6-2291-18), and the JC Kempe Memorial Foundation (JCK-1624).</p></ack><fn-group><fn id=\"footnote1\"><label>1</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/assembly\">https://www.ncbi.nlm.nih.gov/assembly</ext-link></p></fn><fn id=\"footnote2\"><label>2</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ezbiocloud.net/tools/ubcg\">https://www.ezbiocloud.net/tools/ubcg</ext-link></p></fn><fn id=\"footnote3\"><label>3</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://hmmer.org/\">http://hmmer.org/</ext-link></p></fn><fn id=\"footnote4\"><label>4</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ebi.ac.uk/Tools/msa/mafft/\">https://www.ebi.ac.uk/Tools/msa/mafft/</ext-link></p></fn><fn id=\"footnote5\"><label>5</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.microbesonline.org/fasttree/\">http://www.microbesonline.org/fasttree/</ext-link></p></fn><fn id=\"footnote6\"><label>6</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"https://itol.embl.de/\">https://itol.embl.de/</ext-link></p></fn><fn id=\"footnote7\"><label>7</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://multigeneblast.sourceforge.net/\">http://multigeneblast.sourceforge.net/</ext-link></p></fn><fn id=\"footnote8\"><label>8</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://bastion6.erc.monash.edu/\">http://bastion6.erc.monash.edu/</ext-link></p></fn><fn id=\"footnote9\"><label>9</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://pfam.xfam.org/\">http://pfam.xfam.org/</ext-link></p></fn></fn-group><sec id=\"S9\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fmicb.2020.01928/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fmicb.2020.01928/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"DS1\"><media xlink:href=\"Data_Sheet_1.PDF\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Appelt</surname><given-names>S.</given-names></name><name><surname>Koppen</surname><given-names>K.</given-names></name><name><surname>Radonic</surname><given-names>A.</given-names></name><name><surname>Drechsel</surname><given-names>O.</given-names></name><name><surname>Jacob</surname><given-names>D.</given-names></name><name><surname>Grunow</surname><given-names>R.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Genetic diversity and spatial segregation of <italic>Francisella tularensis</italic> subspecies <italic>holarctica</italic> in Germany.</article-title>\n<source><italic>Front. Cell. Infect. Microbiol.</italic></source>\n<volume>9</volume>:<issue>376</issue>. <pub-id pub-id-type=\"doi\">10.3389/Fcimb.2019.00376</pub-id>\n<pub-id pub-id-type=\"pmid\">31781515</pub-id></mixed-citation></ref><ref id=\"B2\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Barker</surname><given-names>J. R.</given-names></name><name><surname>Chong</surname><given-names>A.</given-names></name><name><surname>Wehrly</surname><given-names>T. D.</given-names></name><name><surname>Yu</surname><given-names>J. J.</given-names></name><name><surname>Rodriguez</surname><given-names>S. A.</given-names></name><name><surname>Liu</surname><given-names>J. R.</given-names></name><etal/></person-group> (<year>2009</year>). <article-title>The <italic>Francisella tularensis</italic> pathogenicity island encodes a secretion system that is required for phagosome escape and virulence.</article-title>\n<source><italic>Mol. Microbiol.</italic></source>\n<volume>74</volume>\n<fpage>1459</fpage>–<lpage>1470</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-2958.2009.06947.x</pub-id>\n<pub-id pub-id-type=\"pmid\">20054881</pub-id></mixed-citation></ref><ref id=\"B3\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Barns</surname><given-names>S. M.</given-names></name><name><surname>Grow</surname><given-names>C. C.</given-names></name><name><surname>Okinaka</surname><given-names>R. T.</given-names></name><name><surname>Keim</surname><given-names>P.</given-names></name><name><surname>Kuske</surname><given-names>C. R.</given-names></name></person-group> (<year>2005</year>). <article-title>Detection of diverse new <italic>Francisella</italic>-like bacteria in environmental samples.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>71</volume>\n<fpage>5494</fpage>–<lpage>5500</lpage>. <pub-id pub-id-type=\"doi\">10.1128/AEM.71.9.5494-5500.2005</pub-id>\n<pub-id pub-id-type=\"pmid\">16151142</pub-id></mixed-citation></ref><ref id=\"B4\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Birdsell</surname><given-names>D. N.</given-names></name><name><surname>Stewart</surname><given-names>T.</given-names></name><name><surname>Vogler</surname><given-names>A. J.</given-names></name><name><surname>Lawaczeck</surname><given-names>E.</given-names></name><name><surname>Diggs</surname><given-names>A.</given-names></name><name><surname>Sylvester</surname><given-names>T. L.</given-names></name><etal/></person-group> (<year>2009</year>). <article-title><italic>Francisella tularensis</italic> subsp. <italic>novicida</italic> isolated from a human in Arizona.</article-title>\n<source><italic>BMC Res. Notes</italic></source>\n<volume>2</volume>:<issue>223</issue>. <pub-id pub-id-type=\"doi\">10.1186/1756-0500-2-223</pub-id>\n<pub-id pub-id-type=\"pmid\">19895698</pub-id></mixed-citation></ref><ref id=\"B5\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Birkbeck</surname><given-names>T. H.</given-names></name><name><surname>Feist</surname><given-names>S. W.</given-names></name><name><surname>Verner-Jeffreys</surname><given-names>D. W.</given-names></name></person-group> (<year>2011</year>). <article-title><italic>Francisella</italic> infections in fish and shellfish.</article-title>\n<source><italic>J. Fish Dis.</italic></source>\n<volume>34</volume>\n<fpage>173</fpage>–<lpage>187</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-2761.2010.01226.x</pub-id>\n<pub-id pub-id-type=\"pmid\">21306585</pub-id></mixed-citation></ref><ref id=\"B6\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brett</surname><given-names>M.</given-names></name><name><surname>Doppalapudi</surname><given-names>A.</given-names></name><name><surname>Respicio-Kingry</surname><given-names>L. B.</given-names></name><name><surname>Myers</surname><given-names>D.</given-names></name><name><surname>Husband</surname><given-names>B.</given-names></name><name><surname>Pollard</surname><given-names>K.</given-names></name><etal/></person-group> (<year>2012</year>). <article-title><italic>Francisella novicida</italic> bacteremia after a near-drowning accident.</article-title>\n<source><italic>J. Clin. Microbiol.</italic></source>\n<volume>50</volume>\n<fpage>2826</fpage>–<lpage>2829</lpage>. <pub-id pub-id-type=\"doi\">10.1128/JCM.00995-12</pub-id>\n<pub-id pub-id-type=\"pmid\">22692740</pub-id></mixed-citation></ref><ref id=\"B7\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brevik</surname><given-names>Ø</given-names></name><name><surname>Ottem</surname><given-names>K.</given-names></name><name><surname>Kamaishi</surname><given-names>T.</given-names></name><name><surname>Watanabe</surname><given-names>K.</given-names></name><name><surname>Nylund</surname><given-names>A.</given-names></name></person-group> (<year>2011</year>). <article-title><italic>Francisella halioticida</italic> sp. nov., a pathogen of farmed giant abalone (<italic>Haliotis gigantea</italic>) in Japan.</article-title>\n<source><italic>J. Appl. Microbiol.</italic></source>\n<volume>111</volume>\n<fpage>1044</fpage>–<lpage>1056</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-2672.2011.05133.x</pub-id>\n<pub-id pub-id-type=\"pmid\">21883728</pub-id></mixed-citation></ref><ref id=\"B8\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brodmann</surname><given-names>M.</given-names></name><name><surname>Dreier</surname><given-names>R. F.</given-names></name><name><surname>Broz</surname><given-names>P.</given-names></name><name><surname>Basler</surname><given-names>M.</given-names></name></person-group> (<year>2017</year>). <article-title><italic>Francisella</italic> requires dynamic type VI secretion system and ClpB to deliver effectors for phagosomal escape.</article-title>\n<source><italic>Nat. Commun.</italic></source>\n<volume>8</volume>:<issue>15853</issue>. <pub-id pub-id-type=\"doi\">10.1038/Ncomms15853</pub-id>\n<pub-id pub-id-type=\"pmid\">28621333</pub-id></mixed-citation></ref><ref id=\"B9\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bröms</surname><given-names>J.</given-names></name><name><surname>Sjöstedt</surname><given-names>A.</given-names></name><name><surname>Lavander</surname><given-names>M.</given-names></name></person-group> (<year>2010</year>). <article-title>The role of the <italic>Francisella tularensis</italic> pathogenicity island in type VI secretion, intracellular survival, and modulation of host cell signaling.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>1</volume>:<issue>136</issue>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2010.00136</pub-id>\n<pub-id pub-id-type=\"pmid\">21687753</pub-id></mixed-citation></ref><ref id=\"B10\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bröms</surname><given-names>J. E.</given-names></name><name><surname>Lavander</surname><given-names>M.</given-names></name><name><surname>Meyer</surname><given-names>L.</given-names></name><name><surname>Sjöstedt</surname><given-names>A.</given-names></name></person-group> (<year>2011</year>). <article-title>IglG and IglI of the <italic>Francisella</italic> pathogenicity island are important virulence determinants of <italic>Francisella tularensis</italic> LVS.</article-title>\n<source><italic>Infect. Immunity</italic></source>\n<volume>79</volume>\n<fpage>3683</fpage>–<lpage>3696</lpage>. <pub-id pub-id-type=\"doi\">10.1128/iai.01344-10</pub-id>\n<pub-id pub-id-type=\"pmid\">21690239</pub-id></mixed-citation></ref><ref id=\"B11\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bröms</surname><given-names>J. E.</given-names></name><name><surname>Meyer</surname><given-names>L.</given-names></name><name><surname>Sun</surname><given-names>K.</given-names></name><name><surname>Lavander</surname><given-names>M.</given-names></name><name><surname>Söstedt</surname><given-names>A.</given-names></name></person-group> (<year>2012</year>). <article-title>Unique substrates secreted by the Type VI secretion system of <italic>Francisella tularensis</italic> during intramacrophage infection.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>7</volume>:<issue>e50473</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0050473</pub-id>\n<pub-id pub-id-type=\"pmid\">23185631</pub-id></mixed-citation></ref><ref id=\"B12\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Casanova</surname><given-names>A.</given-names></name><name><surname>Low</surname><given-names>S. H.</given-names></name><name><surname>Québatte</surname><given-names>M.</given-names></name><name><surname>Sedzicki</surname><given-names>J.</given-names></name><name><surname>Tschon</surname><given-names>T.</given-names></name><name><surname>Ketterer</surname><given-names>M.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>A role for the VPS retromer in <italic>Brucella</italic> intracellular replication revealed by genomewide siRNA screening.</article-title>\n<source><italic>mSphere</italic></source>\n<volume>4</volume>:<issue>e00380-19</issue>.</mixed-citation></ref><ref id=\"B13\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Castresana</surname><given-names>J.</given-names></name></person-group> (<year>2007</year>). <article-title>Topological variation in single-gene phylogenetic trees.</article-title>\n<source><italic>Genome Biol.</italic></source>\n<volume>8</volume>:<issue>216</issue>. <pub-id pub-id-type=\"doi\">10.1186/Gb-2007-8-6-216</pub-id>\n<pub-id pub-id-type=\"pmid\">17567929</pub-id></mixed-citation></ref><ref id=\"B14\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Challacombe</surname><given-names>J. F.</given-names></name><name><surname>Petersen</surname><given-names>J. M.</given-names></name><name><surname>Gallegos-Graves</surname><given-names>V.</given-names></name><name><surname>Hodge</surname><given-names>D.</given-names></name><name><surname>Pillai</surname><given-names>S.</given-names></name><name><surname>Kuske</surname><given-names>C. R.</given-names></name></person-group> (<year>2017</year>). <article-title>Whole-genome relationships among <italic>Francisella</italic> bacteria of diverse origins define new species and provide specific regions for detection.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>83</volume>:<issue>e02589-16</issue>. <pub-id pub-id-type=\"doi\">10.1128/AEM.02589-16</pub-id>\n<pub-id pub-id-type=\"pmid\">27881415</pub-id></mixed-citation></ref><ref id=\"B15\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chan</surname><given-names>J. Z. M.</given-names></name><name><surname>Halachev</surname><given-names>M. R.</given-names></name><name><surname>Loman</surname><given-names>N. J.</given-names></name><name><surname>Constantinidou</surname><given-names>C.</given-names></name><name><surname>Pallen</surname><given-names>M. J.</given-names></name></person-group> (<year>2012</year>). <article-title>Defining bacterial species in the genomic era: insights from the genus Acinetobacter.</article-title>\n<source><italic>BMC Microbiol.</italic></source>\n<volume>12</volume>:<issue>302</issue>. <pub-id pub-id-type=\"doi\">10.1186/1471-2180-12-302</pub-id>\n<pub-id pub-id-type=\"pmid\">23259572</pub-id></mixed-citation></ref><ref id=\"B16\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chaudhari</surname><given-names>N. M.</given-names></name><name><surname>Gupta</surname><given-names>V. K.</given-names></name><name><surname>Dutta</surname><given-names>C.</given-names></name></person-group> (<year>2016</year>). <article-title>BPGA- an ultra-fast pan-genome analysis pipeline.</article-title>\n<source><italic>Sci. Rep.</italic></source>\n<volume>6</volume>:<issue>24373</issue>. <pub-id pub-id-type=\"doi\">10.1038/Srep24373</pub-id>\n<pub-id pub-id-type=\"pmid\">27071527</pub-id></mixed-citation></ref><ref id=\"B17\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Clarridge</surname><given-names>J. E.</given-names><suffix>III</suffix></name><name><surname>Raich</surname><given-names>T. J.</given-names></name><name><surname>Sjöstedt</surname><given-names>A.</given-names></name><name><surname>Sandstrom</surname><given-names>G.</given-names></name><name><surname>Darouiche</surname><given-names>R. O.</given-names></name><name><surname>Shawar</surname><given-names>R. M.</given-names></name><etal/></person-group> (<year>1996</year>). <article-title>Characterization of two unusual clinically significant <italic>Francisella</italic> strains.</article-title>\n<source><italic>J. Clin. Microbiol.</italic></source>\n<volume>34</volume>\n<fpage>1995</fpage>–<lpage>2000</lpage>. <pub-id pub-id-type=\"doi\">10.1128/jcm.34.8.1995-2000.1996</pub-id>\n<pub-id pub-id-type=\"pmid\">8818897</pub-id></mixed-citation></ref><ref id=\"B18\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Collins</surname><given-names>R. E.</given-names></name><name><surname>Higgs</surname><given-names>P. G.</given-names></name></person-group> (<year>2012</year>). <article-title>Testing the infinitely many genes model for the evolution of the bacterial core genome and pangenome.</article-title>\n<source><italic>Mol. Biol. Evol.</italic></source>\n<volume>29</volume>\n<fpage>3413</fpage>–<lpage>3425</lpage>. <pub-id pub-id-type=\"doi\">10.1093/molbev/mss163</pub-id>\n<pub-id pub-id-type=\"pmid\">22752048</pub-id></mixed-citation></ref><ref id=\"B19\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Colquhoun</surname><given-names>D. J.</given-names></name><name><surname>Duodu</surname><given-names>S.</given-names></name></person-group> (<year>2011</year>). <article-title><italic>Francisella</italic> infections in farmed and wild aquatic organisms.</article-title>\n<source><italic>Vet. Res.</italic></source>\n<volume>42</volume>:<issue>47</issue>. <pub-id pub-id-type=\"doi\">10.1186/1297-9716-42-47</pub-id>\n<pub-id pub-id-type=\"pmid\">21385413</pub-id></mixed-citation></ref><ref id=\"B20\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Contreras-Moreira</surname><given-names>B.</given-names></name><name><surname>Vinuesa</surname><given-names>P.</given-names></name></person-group> (<year>2013</year>). <article-title>GET_HOMOLOGUES, a versatile software package for scalable and robust microbial pangenome analysis.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>79</volume>\n<fpage>7696</fpage>–<lpage>7701</lpage>. <pub-id pub-id-type=\"doi\">10.1128/Aem.02411-13</pub-id>\n<pub-id pub-id-type=\"pmid\">24096415</pub-id></mixed-citation></ref><ref id=\"B21\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Bruin</surname><given-names>O. M.</given-names></name><name><surname>Ludu</surname><given-names>J. S.</given-names></name><name><surname>Nano</surname><given-names>F. E.</given-names></name></person-group> (<year>2007</year>). <article-title>The <italic>Francisella</italic> pathogenicity island protein IglA localizes to the bacterial cytoplasm and is needed for intracellular growth.</article-title>\n<source><italic>BMC Microbiol.</italic></source>\n<volume>7</volume>:<issue>1</issue>. <pub-id pub-id-type=\"doi\">10.1186/1471-2180-7-1</pub-id>\n<pub-id pub-id-type=\"pmid\">17233889</pub-id></mixed-citation></ref><ref id=\"B22\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dietrich</surname><given-names>E. A.</given-names></name><name><surname>Kingry</surname><given-names>L. C.</given-names></name><name><surname>Kugeler</surname><given-names>K. J.</given-names></name><name><surname>Levy</surname><given-names>C.</given-names></name><name><surname>Yaglom</surname><given-names>H.</given-names></name><name><surname>Young</surname><given-names>J. W.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title><italic>Francisella opportunistica</italic> sp. nov., isolated from human blood and cerebrospinal fluid.</article-title>\n<source><italic>Int. J. Syst. Evol. Microbiol.</italic></source>\n<volume>70</volume>\n<fpage>1145</fpage>–<lpage>1151</lpage>. <pub-id pub-id-type=\"doi\">10.1099/ijsem.0.003891</pub-id>\n<pub-id pub-id-type=\"pmid\">31860434</pub-id></mixed-citation></ref><ref id=\"B23\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dyer</surname><given-names>M. D.</given-names></name><name><surname>Neff</surname><given-names>C.</given-names></name><name><surname>Dufford</surname><given-names>M.</given-names></name><name><surname>Rivera</surname><given-names>C. G.</given-names></name><name><surname>Shattuck</surname><given-names>D.</given-names></name><name><surname>Bassaganya-Riera</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2010</year>). <article-title>The human-bacterial pathogen protein interaction networks of <italic>Bacillus anthracis</italic>, <italic>Francisella tularensis</italic>, and <italic>Yersinia pestis</italic>.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>5</volume>:<issue>e12089</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0012089</pub-id>\n<pub-id pub-id-type=\"pmid\">20711500</pub-id></mixed-citation></ref><ref id=\"B24\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eshraghi</surname><given-names>A.</given-names></name><name><surname>Kim</surname><given-names>J.</given-names></name><name><surname>Walls</surname><given-names>A. C.</given-names></name><name><surname>Ledvina</surname><given-names>H. E.</given-names></name><name><surname>Miller</surname><given-names>C. N.</given-names></name><name><surname>Ramsey</surname><given-names>K. M.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>Secreted effectors encoded within and outside of the <italic>Francisella</italic> pathogenicity Island promote intramacrophage growth.</article-title>\n<source><italic>Cell Host Microbe</italic></source>\n<volume>20</volume>\n<fpage>573</fpage>–<lpage>583</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.chom.2016.10.008</pub-id>\n<pub-id pub-id-type=\"pmid\">27832588</pub-id></mixed-citation></ref><ref id=\"B25\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Finsel</surname><given-names>I.</given-names></name><name><surname>Ragaz</surname><given-names>C.</given-names></name><name><surname>Hoffmann</surname><given-names>C.</given-names></name><name><surname>Harrison</surname><given-names>C. F.</given-names></name><name><surname>Weber</surname><given-names>S.</given-names></name><name><surname>van Rahden</surname><given-names>V. A.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>The Legionella effector RidL inhibits retrograde trafficking to promote intracellular replication.</article-title>\n<source><italic>Cell Host Microbe</italic></source>\n<volume>14</volume>\n<fpage>38</fpage>–<lpage>50</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.chom.2013.06.001</pub-id>\n<pub-id pub-id-type=\"pmid\">23870312</pub-id></mixed-citation></ref><ref id=\"B26\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Han</surname><given-names>N.</given-names></name><name><surname>Qiang</surname><given-names>Y. J.</given-names></name><name><surname>Zhang</surname><given-names>W.</given-names></name></person-group> (<year>2016</year>). <article-title>ANItools web: a web tool for fast genome comparison within multiple bacterial strains.</article-title>\n<source><italic>Database</italic></source>\n<volume>2016</volume>:<issue>baw084</issue>. <pub-id pub-id-type=\"doi\">10.1093/database/baw084</pub-id>\n<pub-id pub-id-type=\"pmid\">27270714</pub-id></mixed-citation></ref><ref id=\"B27\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hollis</surname><given-names>D. G.</given-names></name><name><surname>Weaver</surname><given-names>R. E.</given-names></name><name><surname>Steigerwalt</surname><given-names>A. G.</given-names></name><name><surname>Wenger</surname><given-names>J. D.</given-names></name><name><surname>Moss</surname><given-names>C. W.</given-names></name><name><surname>Brenner</surname><given-names>D. J.</given-names></name></person-group> (<year>1989</year>). <article-title><italic>Francisella philomiragia</italic> comb. nov. (formerly <italic>Yersinia philomiragia</italic>) and <italic>Francisella tularensis</italic> biogroup <italic>novicida</italic> (formerly <italic>Francisella novicida</italic>) associated with human disease.</article-title>\n<source><italic>J. Clin. Microbiol.</italic></source>\n<volume>27</volume>\n<fpage>1601</fpage>–<lpage>1608</lpage>. <pub-id pub-id-type=\"doi\">10.1128/jcm.27.7.1601-1608.1989</pub-id>\n<pub-id pub-id-type=\"pmid\">2671019</pub-id></mixed-citation></ref><ref id=\"B28\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huber</surname><given-names>B.</given-names></name><name><surname>Escudero</surname><given-names>R.</given-names></name><name><surname>Busse</surname><given-names>H. J.</given-names></name><name><surname>Seibold</surname><given-names>E.</given-names></name><name><surname>Scholz</surname><given-names>H. C.</given-names></name><name><surname>Anda</surname><given-names>P.</given-names></name><etal/></person-group> (<year>2010</year>). <article-title>Description of <italic>Francisella hispaniensis</italic> sp. nov., isolated from human blood, reclassification of <italic>Francisella novicida</italic> (Larson et al. 1955) <xref rid=\"B58\" ref-type=\"bibr\">Olsufiev et al., 1959</xref> as <italic>Francisella tularensis</italic> subsp. <italic>novicida</italic> comb. nov. and emended description of the genus <italic>Francisella</italic>.</article-title>\n<source><italic>Int. J. Syst. Evol. Microbiol.</italic></source>\n<volume>60(Pt 8)</volume>\n<fpage>1887</fpage>–<lpage>1896</lpage>. <pub-id pub-id-type=\"doi\">10.1099/ijs.0.015941-0</pub-id>\n<pub-id pub-id-type=\"pmid\">19783615</pub-id></mixed-citation></ref><ref id=\"B29\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Johnson</surname><given-names>S. L.</given-names></name><name><surname>Daligault</surname><given-names>H. E.</given-names></name><name><surname>Davenport</surname><given-names>K. W.</given-names></name><name><surname>Coyne</surname><given-names>S. R.</given-names></name><name><surname>Frey</surname><given-names>K. G.</given-names></name><name><surname>Koroleva</surname><given-names>G. I.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>Genome sequencing of 18 <italic>Francisella</italic> strains to aid in assay development and testing.</article-title>\n<source><italic>Microbiol. Resour. Announc.</italic></source>\n<volume>3</volume>:<issue>e00147-15</issue>. <pub-id pub-id-type=\"doi\">10.1128/genomeA.00147-15</pub-id>\n<pub-id pub-id-type=\"pmid\">25931589</pub-id></mixed-citation></ref><ref id=\"B30\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kaas</surname><given-names>R. S.</given-names></name><name><surname>Friis</surname><given-names>C.</given-names></name><name><surname>Ussery</surname><given-names>D. W.</given-names></name><name><surname>Aarestrup</surname><given-names>F. M.</given-names></name></person-group> (<year>2012</year>). <article-title>Estimating variation within the genes and inferring the phylogeny of 186 sequenced diverse <italic>Escherichia coli</italic> genomes.</article-title>\n<source><italic>BMC Genomics</italic></source>\n<volume>13</volume>:<issue>577</issue>. <pub-id pub-id-type=\"doi\">10.1186/1471-2164-13-577</pub-id>\n<pub-id pub-id-type=\"pmid\">23114024</pub-id></mixed-citation></ref><ref id=\"B31\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Keim</surname><given-names>P.</given-names></name><name><surname>Johansson</surname><given-names>A.</given-names></name><name><surname>Wagner</surname><given-names>D. M.</given-names></name></person-group> (<year>2007</year>). <article-title>Molecular epidemiology, evolution, and ecology of <italic>Francisella</italic>.</article-title>\n<source><italic>Ann. N. Y. Acad. Sci.</italic></source>\n<volume>1105</volume>\n<fpage>30</fpage>–<lpage>66</lpage>. <pub-id pub-id-type=\"doi\">10.1196/annals.1409.011</pub-id>\n<pub-id pub-id-type=\"pmid\">17435120</pub-id></mixed-citation></ref><ref id=\"B32\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kingry</surname><given-names>L. C.</given-names></name><name><surname>Cummings</surname><given-names>J. E.</given-names></name><name><surname>Brookman</surname><given-names>K. W.</given-names></name><name><surname>Bommineni</surname><given-names>G. R.</given-names></name><name><surname>Tonge</surname><given-names>P. J.</given-names></name><name><surname>Slayden</surname><given-names>R. A.</given-names></name></person-group> (<year>2013</year>). <article-title>The <italic>Francisella tularensis</italic> FabI enoyl-acyl carrier protein reductase gene is essential to bacterial viability and is expressed during infection.</article-title>\n<source><italic>J. Bacteriol.</italic></source>\n<volume>195</volume>\n<fpage>351</fpage>–<lpage>358</lpage>. <pub-id pub-id-type=\"doi\">10.1128/jb.01957-12</pub-id>\n<pub-id pub-id-type=\"pmid\">23144254</pub-id></mixed-citation></ref><ref id=\"B33\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Knight</surname><given-names>D. R.</given-names></name><name><surname>Squire</surname><given-names>M. M.</given-names></name><name><surname>Collins</surname><given-names>D. A.</given-names></name><name><surname>Riley</surname><given-names>T. V.</given-names></name></person-group> (<year>2017</year>). <article-title>Genome analysis of <italic>Clostridium difficile</italic> PCR Ribotype 014 lineage in Australian Pigs and humans reveals a diverse genetic repertoire and signatures of long-Range interspecies transmission.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>7</volume>:<issue>2138</issue>. <pub-id pub-id-type=\"doi\">10.3389/Fmicb.2016.02138</pub-id>\n<pub-id pub-id-type=\"pmid\">28123380</pub-id></mixed-citation></ref><ref id=\"B34\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Konstantinidis</surname><given-names>K. T.</given-names></name><name><surname>Ramette</surname><given-names>A.</given-names></name><name><surname>Tiedje</surname><given-names>J. M.</given-names></name></person-group> (<year>2006</year>). <article-title>Toward a more robust assessment of intraspecies diversity, using fewer genetic markers.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>72</volume>\n<fpage>7286</fpage>–<lpage>7293</lpage>. <pub-id pub-id-type=\"doi\">10.1128/Aem.01398-06</pub-id>\n<pub-id pub-id-type=\"pmid\">16980418</pub-id></mixed-citation></ref><ref id=\"B35\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kreizinger</surname><given-names>Z.</given-names></name><name><surname>Hornok</surname><given-names>S.</given-names></name><name><surname>Dan</surname><given-names>A.</given-names></name><name><surname>Hresko</surname><given-names>S.</given-names></name><name><surname>Makrai</surname><given-names>L.</given-names></name><name><surname>Magyar</surname><given-names>T.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>Prevalence of <italic>Francisella tularensis</italic> and <italic>Francisella</italic>-like endosymbionts in the tick population of Hungary and the genetic variability of <italic>Francisella</italic>-like agents.</article-title>\n<source><italic>Vector Borne Zoon. Dis.</italic></source>\n<volume>13</volume>\n<fpage>160</fpage>–<lpage>163</lpage>. <pub-id pub-id-type=\"doi\">10.1089/vbz.2012.1065</pub-id>\n<pub-id pub-id-type=\"pmid\">23421891</pub-id></mixed-citation></ref><ref id=\"B36\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kristensen</surname><given-names>D. M.</given-names></name><name><surname>Kannan</surname><given-names>L.</given-names></name><name><surname>Coleman</surname><given-names>M. K.</given-names></name><name><surname>Wolf</surname><given-names>Y. I.</given-names></name><name><surname>Sorokin</surname><given-names>A.</given-names></name><name><surname>Koonin</surname><given-names>E. V.</given-names></name><etal/></person-group> (<year>2010</year>). <article-title>A low-polynomial algorithm for assembling clusters of orthologous groups from intergenomic symmetric best matches.</article-title>\n<source><italic>Bioinformatics</italic></source>\n<volume>26</volume>\n<fpage>1481</fpage>–<lpage>1487</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bioinformatics/btq229</pub-id>\n<pub-id pub-id-type=\"pmid\">20439257</pub-id></mixed-citation></ref><ref id=\"B37\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kugeler</surname><given-names>K. J.</given-names></name><name><surname>Mead</surname><given-names>P. S.</given-names></name><name><surname>Janusz</surname><given-names>A. M.</given-names></name><name><surname>Staples</surname><given-names>J. E.</given-names></name><name><surname>Kubota</surname><given-names>K. A.</given-names></name><name><surname>Chalcraft</surname><given-names>L. G.</given-names></name><etal/></person-group> (<year>2009</year>). <article-title>Molecular epidemiology of <italic>Francisella tularensis</italic> in the United States.</article-title>\n<source><italic>Clin. Infect. Dis.</italic></source>\n<volume>48</volume>\n<fpage>863</fpage>–<lpage>870</lpage>. <pub-id pub-id-type=\"doi\">10.1086/597261</pub-id>\n<pub-id pub-id-type=\"pmid\">19245342</pub-id></mixed-citation></ref><ref id=\"B38\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kugeler</surname><given-names>K. J.</given-names></name><name><surname>Mead</surname><given-names>P. S.</given-names></name><name><surname>McGowan</surname><given-names>K. L.</given-names></name><name><surname>Burnham</surname><given-names>J. M.</given-names></name><name><surname>Hogarty</surname><given-names>M. D.</given-names></name><name><surname>Ruchelli</surname><given-names>E.</given-names></name><etal/></person-group> (<year>2008</year>). <article-title>Isolation and characterization of a novel <italic>Francisella</italic> sp. from human cerebrospinal fluid and blood.</article-title>\n<source><italic>J. Clin. Microbiol.</italic></source>\n<volume>46</volume>\n<fpage>2428</fpage>–<lpage>2431</lpage>. <pub-id pub-id-type=\"doi\">10.1128/JCM.00698-08</pub-id>\n<pub-id pub-id-type=\"pmid\">18495864</pub-id></mixed-citation></ref><ref id=\"B39\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuske</surname><given-names>C. R.</given-names></name><name><surname>Barns</surname><given-names>S. M.</given-names></name><name><surname>Grow</surname><given-names>C. C.</given-names></name><name><surname>Merrill</surname><given-names>L.</given-names></name><name><surname>Dunbar</surname><given-names>J.</given-names></name></person-group> (<year>2006</year>). <article-title>Environmental survey for four pathogenic bacteria and closely related species using phylogenetic and functional genes.</article-title>\n<source><italic>J. Forensic. Sci.</italic></source>\n<volume>51</volume>\n<fpage>548</fpage>–<lpage>558</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1556-4029.2006.00131.x</pub-id>\n<pub-id pub-id-type=\"pmid\">16696701</pub-id></mixed-citation></ref><ref id=\"B40\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Larson</surname><given-names>M. A.</given-names></name><name><surname>Nalbantoglu</surname><given-names>U.</given-names></name><name><surname>Sayood</surname><given-names>K.</given-names></name><name><surname>Zentz</surname><given-names>E. B.</given-names></name><name><surname>Cer</surname><given-names>R. Z.</given-names></name><name><surname>Iwen</surname><given-names>P. C.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>Reclassification of <italic>Wolbachia persica</italic> as <italic>Francisella persica</italic> comb. nov and emended description of the family <italic>Francisella</italic>ceae.</article-title>\n<source><italic>Int. J. Syst. Evol. Microbiol.</italic></source>\n<volume>66</volume>\n<fpage>1200</fpage>–<lpage>1205</lpage>. <pub-id pub-id-type=\"doi\">10.1099/ijsem.0.000855</pub-id>\n<pub-id pub-id-type=\"pmid\">26747442</pub-id></mixed-citation></ref><ref id=\"B41\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Larsson</surname><given-names>P.</given-names></name><name><surname>Elfsmark</surname><given-names>D.</given-names></name><name><surname>Svensson</surname><given-names>K.</given-names></name><name><surname>Wikstrom</surname><given-names>P.</given-names></name><name><surname>Forsman</surname><given-names>M.</given-names></name><name><surname>Brettin</surname><given-names>T.</given-names></name><etal/></person-group> (<year>2009</year>). <article-title>Molecular evolutionary consequences of niche restriction in <italic>Francisella tularensis</italic>, a facultative intracellular pathogen.</article-title>\n<source><italic>PLoS Pathog.</italic></source>\n<volume>5</volume>:<issue>e1000472</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.ppat.1000472</pub-id>\n<pub-id pub-id-type=\"pmid\">19521508</pub-id></mixed-citation></ref><ref id=\"B42\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Larsson</surname><given-names>P.</given-names></name><name><surname>Oyston</surname><given-names>P. C.</given-names></name><name><surname>Chain</surname><given-names>P.</given-names></name><name><surname>Chu</surname><given-names>M. C.</given-names></name><name><surname>Duffield</surname><given-names>M.</given-names></name><name><surname>Fuxelius</surname><given-names>H. H.</given-names></name><etal/></person-group> (<year>2005</year>). <article-title>The complete genome sequence of <italic>Francisella tularensis</italic>, the causative agent of tularemia</article-title>. <source><italic>Nat. Genet.</italic></source>\n<volume>37</volume>, <fpage>153</fpage>–<lpage>159</lpage>. <pub-id pub-id-type=\"doi\">10.1038/ng1499</pub-id>\n<pub-id pub-id-type=\"pmid\">15640799</pub-id></mixed-citation></ref><ref id=\"B43\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Le</surname><given-names>S. Q.</given-names></name><name><surname>Gascuel</surname><given-names>O.</given-names></name></person-group> (<year>2008</year>). <article-title>An improved general amino acid replacement matrix.</article-title>\n<source><italic>Mol. Biol. Evol.</italic></source>\n<volume>25</volume>\n<fpage>1307</fpage>–<lpage>1320</lpage>. <pub-id pub-id-type=\"doi\">10.1093/molbev/msn067</pub-id>\n<pub-id pub-id-type=\"pmid\">18367465</pub-id></mixed-citation></ref><ref id=\"B44\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ledvina</surname><given-names>H. E.</given-names></name><name><surname>Kelly</surname><given-names>K. A.</given-names></name><name><surname>Eshraghi</surname><given-names>A.</given-names></name><name><surname>Plemel</surname><given-names>R. L.</given-names></name><name><surname>Peterson</surname><given-names>S. B.</given-names></name><name><surname>Lee</surname><given-names>B.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>A phosphatidylinositol 3-kinase effector alters phagosomal maturation to promote intracellular growth of <italic>Francisella</italic>.</article-title>\n<source><italic>Cell Host Microbe</italic></source>\n<volume>24</volume>:<issue>285</issue>. <pub-id pub-id-type=\"doi\">10.1016/j.chom.2018.07.003</pub-id>\n<pub-id pub-id-type=\"pmid\">30057173</pub-id></mixed-citation></ref><ref id=\"B45\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>L.</given-names></name><name><surname>Stoeckert</surname><given-names>C. J.</given-names></name><name><surname>Roos</surname><given-names>D. S.</given-names></name></person-group> (<year>2003</year>). <article-title>OrthoMCL: identification of ortholog groups for eukaryotic genomes.</article-title>\n<source><italic>Genome Res.</italic></source>\n<volume>13</volume>\n<fpage>2178</fpage>–<lpage>2189</lpage>. <pub-id pub-id-type=\"doi\">10.1101/gr.1224503</pub-id>\n<pub-id pub-id-type=\"pmid\">12952885</pub-id></mixed-citation></ref><ref id=\"B46\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mariano</surname><given-names>G.</given-names></name><name><surname>Trunk</surname><given-names>K.</given-names></name><name><surname>Williams</surname><given-names>D. J.</given-names></name><name><surname>Monlezun</surname><given-names>L.</given-names></name><name><surname>Strahl</surname><given-names>H.</given-names></name><name><surname>Pitt</surname><given-names>S. J.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>A family of Type VI secretion system effector proteins that form ion-selective pores.</article-title>\n<source><italic>Nat. Commun.</italic></source>\n<volume>10</volume>\n<fpage>1</fpage>–<lpage>15</lpage>.<pub-id pub-id-type=\"pmid\">30602773</pub-id></mixed-citation></ref><ref id=\"B47\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McDermott</surname><given-names>C.</given-names></name><name><surname>Palmeiro</surname><given-names>B.</given-names></name></person-group> (<year>2013</year>). <article-title>Selected emerging infectious diseases of ornamental fish.</article-title>\n<source><italic>Vet. Clin. North Am. Exot. Anim. Pract.</italic></source>\n<volume>16</volume>\n<fpage>261</fpage>–<lpage>282</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cvex.2013.01.006</pub-id>\n<pub-id pub-id-type=\"pmid\">23642862</pub-id></mixed-citation></ref><ref id=\"B48\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Medema</surname><given-names>M. H.</given-names></name><name><surname>Takano</surname><given-names>E.</given-names></name><name><surname>Breitling</surname><given-names>R.</given-names></name></person-group> (<year>2013</year>). <article-title>Detecting sequence homology at the gene cluster level with multigeneblast.</article-title>\n<source><italic>Mol. Biol. Evol.</italic></source>\n<volume>30</volume>\n<fpage>1218</fpage>–<lpage>1223</lpage>. <pub-id pub-id-type=\"doi\">10.1093/molbev/mst025</pub-id>\n<pub-id pub-id-type=\"pmid\">23412913</pub-id></mixed-citation></ref><ref id=\"B49\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Meric</surname><given-names>G.</given-names></name><name><surname>Yahara</surname><given-names>K.</given-names></name><name><surname>Mageiros</surname><given-names>L.</given-names></name><name><surname>Pascoe</surname><given-names>B.</given-names></name><name><surname>Maiden</surname><given-names>M. C. J.</given-names></name><name><surname>Jolley</surname><given-names>K. A.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>A reference pan-genome approach to comparative bacterial genomics: identification of novel epidemiological markers in pathogenic <italic>Campylobacter</italic>.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>9</volume>:<issue>e92798</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0092798</pub-id>\n<pub-id pub-id-type=\"pmid\">24676150</pub-id></mixed-citation></ref><ref id=\"B50\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mikalsen</surname><given-names>J.</given-names></name><name><surname>Olsen</surname><given-names>A. B.</given-names></name><name><surname>Tengs</surname><given-names>T.</given-names></name><name><surname>Colquhoun</surname><given-names>D. J.</given-names></name></person-group> (<year>2007</year>). <article-title><italic>Francisella philomiragia</italic> subsp. <italic>noatunensis</italic> subsp. nov., isolated from farmed Atlantic cod (<italic>Gadus morhua</italic> L.)</article-title>. <source><italic>Int. J. Syst. Evol. Microbiol.</italic></source>\n<volume>57</volume>, <fpage>1960</fpage>–<lpage>1965</lpage>. <pub-id pub-id-type=\"doi\">10.1099/ijs.0.64765-0</pub-id>\n<pub-id pub-id-type=\"pmid\">17766855</pub-id></mixed-citation></ref><ref id=\"B51\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mosquera-Rendon</surname><given-names>J.</given-names></name><name><surname>Rada-Bravo</surname><given-names>A. M.</given-names></name><name><surname>Cardenas-Brito</surname><given-names>S.</given-names></name><name><surname>Corredor</surname><given-names>M.</given-names></name><name><surname>Restrepo-Pineda</surname><given-names>E.</given-names></name><name><surname>Benitez-Paez</surname><given-names>A.</given-names></name></person-group> (<year>2016</year>). <article-title>Pangenome-wide and molecular evolution analyses of the <italic>Pseudomonas aeruginosa</italic> species.</article-title>\n<source><italic>BMC Genomics</italic></source>\n<volume>17</volume>:<issue>45</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12864-016-2364-4</pub-id>\n<pub-id pub-id-type=\"pmid\">26754847</pub-id></mixed-citation></ref><ref id=\"B52\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Na</surname><given-names>S. I.</given-names></name><name><surname>Kim</surname><given-names>Y. O.</given-names></name><name><surname>Yoon</surname><given-names>S. H.</given-names></name><name><surname>Ha</surname><given-names>S. M.</given-names></name><name><surname>Baek</surname><given-names>I.</given-names></name><name><surname>Chun</surname><given-names>J.</given-names></name></person-group> (<year>2018</year>). <article-title>UBCG: up-to-date bacterial core gene set and pipeline for phylogenomic tree reconstruction.</article-title>\n<source><italic>J. Microbiol.</italic></source>\n<volume>56</volume>\n<fpage>280</fpage>–<lpage>285</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s12275-018-8014-6</pub-id>\n<pub-id pub-id-type=\"pmid\">29492869</pub-id></mixed-citation></ref><ref id=\"B53\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nano</surname><given-names>F. E.</given-names></name><name><surname>Schmerk</surname><given-names>C.</given-names></name></person-group> (<year>2007</year>). <article-title>The <italic>Francisella</italic> pathogenicity island.</article-title>\n<source><italic>Ann. N. Y. Acad. Sci.</italic></source>\n<volume>1105</volume>\n<fpage>122</fpage>–<lpage>137</lpage>. <pub-id pub-id-type=\"doi\">10.1196/annals.1409.000</pub-id>\n<pub-id pub-id-type=\"pmid\">17395722</pub-id></mixed-citation></ref><ref id=\"B54\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nano</surname><given-names>F. E.</given-names></name><name><surname>Zhang</surname><given-names>N.</given-names></name><name><surname>Cowley</surname><given-names>S. C.</given-names></name><name><surname>Klose</surname><given-names>K. E.</given-names></name><name><surname>Cheung</surname><given-names>K. K.</given-names></name><name><surname>Roberts</surname><given-names>M. J.</given-names></name><etal/></person-group> (<year>2004</year>). <article-title>A <italic>Francisella tularensis</italic> pathogenicity island required for intramacrophage growth.</article-title>\n<source><italic>J. Bacteriol.</italic></source>\n<volume>186</volume>\n<fpage>6430</fpage>–<lpage>6436</lpage>. <pub-id pub-id-type=\"doi\">10.1128/jb.186.19.6430-6436.2004</pub-id>\n<pub-id pub-id-type=\"pmid\">15375123</pub-id></mixed-citation></ref><ref id=\"B55\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nelson</surname><given-names>W. C.</given-names></name><name><surname>Stegen</surname><given-names>J. C.</given-names></name></person-group> (<year>2015</year>). <article-title>The reduced genomes of Parcubacteria (OD1) contain signatures of a symbiotic lifestyle.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>6</volume>:<issue>713</issue>. <pub-id pub-id-type=\"doi\">10.3389/Fmicb.2015.00713</pub-id>\n<pub-id pub-id-type=\"pmid\">26257709</pub-id></mixed-citation></ref><ref id=\"B56\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nguyen</surname><given-names>L. T.</given-names></name><name><surname>Schmidt</surname><given-names>H. A.</given-names></name><name><surname>von Haeseler</surname><given-names>A.</given-names></name><name><surname>Minh</surname><given-names>B. Q.</given-names></name></person-group> (<year>2015</year>). <article-title>IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies.</article-title>\n<source><italic>Mol. Biol. Evol.</italic></source>\n<volume>32</volume>\n<fpage>268</fpage>–<lpage>274</lpage>. <pub-id pub-id-type=\"doi\">10.1093/molbev/msu300</pub-id>\n<pub-id pub-id-type=\"pmid\">25371430</pub-id></mixed-citation></ref><ref id=\"B57\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nourdin-Galindo</surname><given-names>G.</given-names></name><name><surname>Sanchez</surname><given-names>P.</given-names></name><name><surname>Molina</surname><given-names>C. F.</given-names></name><name><surname>Espinoza-Rojas</surname><given-names>D. A.</given-names></name><name><surname>Oliver</surname><given-names>C.</given-names></name><name><surname>Ruiz</surname><given-names>P.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Comparative pan-genome analysis of <italic>Piscirickettsia salmonis</italic> reveals genomic divergences within genogroups.</article-title>\n<source><italic>Front. Cell. Infect. Microbiol.</italic></source>\n<volume>7</volume>:<issue>459</issue>. <pub-id pub-id-type=\"doi\">10.3389/Fcimb.2017.00459</pub-id>\n<pub-id pub-id-type=\"pmid\">29164068</pub-id></mixed-citation></ref><ref id=\"B58\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Olsufiev</surname><given-names>N. G.</given-names></name><name><surname>Emelyanova</surname><given-names>O. S.</given-names></name><name><surname>Dunayeva</surname><given-names>T. N.</given-names></name></person-group> (<year>1959</year>). <article-title>Comparative study of strains of <italic>B. tularense</italic> in the old and new world and their taxonomy.</article-title>\n<source><italic>J. Hyg. Epidemiol. Microbiol. Immunol.</italic></source>\n<volume>3</volume>\n<fpage>138</fpage>–<lpage>149</lpage>.</mixed-citation></ref><ref id=\"B59\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Petersen</surname><given-names>J. M.</given-names></name><name><surname>Carlson</surname><given-names>J.</given-names></name><name><surname>Yockey</surname><given-names>B.</given-names></name><name><surname>Pillai</surname><given-names>S.</given-names></name><name><surname>Kuske</surname><given-names>C.</given-names></name><name><surname>Garbalena</surname><given-names>G.</given-names></name><etal/></person-group> (<year>2009</year>). <article-title>Direct isolation of <italic>Francisella</italic> spp. from environmental samples.</article-title>\n<source><italic>Lett. Appl. Microbiol.</italic></source>\n<volume>48</volume>\n<fpage>663</fpage>–<lpage>667</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1472-765X.2009.02589.x</pub-id>\n<pub-id pub-id-type=\"pmid\">19413814</pub-id></mixed-citation></ref><ref id=\"B60\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pilo</surname><given-names>P.</given-names></name></person-group> (<year>2018</year>). <article-title>Phylogenetic lineages of <italic>Francisella tularensis</italic> in animals.</article-title>\n<source><italic>Front. Cell. Infect. Microbiol.</italic></source>\n<volume>8</volume>:<issue>258</issue>. <pub-id pub-id-type=\"doi\">10.3389/fcimb.2018.00258</pub-id>\n<pub-id pub-id-type=\"pmid\">30109216</pub-id></mixed-citation></ref><ref id=\"B61\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pritchard</surname><given-names>L.</given-names></name><name><surname>Glover</surname><given-names>R. H.</given-names></name><name><surname>Humphris</surname><given-names>S.</given-names></name><name><surname>Elphinstone</surname><given-names>J. G.</given-names></name><name><surname>Toth</surname><given-names>I. K.</given-names></name></person-group> (<year>2016</year>). <article-title>Genomics and taxonomy in diagnostics for food security: soft-rotting enterobacterial plant pathogens.</article-title>\n<source><italic>Anal. Methods</italic></source>\n<volume>8</volume>\n<fpage>12</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1039/c5ay02550h</pub-id></mixed-citation></ref><ref id=\"B62\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Qu</surname><given-names>P. H.</given-names></name><name><surname>Chen</surname><given-names>S. Y.</given-names></name><name><surname>Scholz</surname><given-names>H. C.</given-names></name><name><surname>Busse</surname><given-names>H. J.</given-names></name><name><surname>Gu</surname><given-names>Q.</given-names></name><name><surname>Kampfer</surname><given-names>P.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title><italic>Francisella guangzhouensis</italic> sp. nov., isolated from air-conditioning systems.</article-title>\n<source><italic>Int. J. Syst. Evol. Microbiol.</italic></source>\n<volume>63(Pt 10)</volume>\n<fpage>3628</fpage>–<lpage>3635</lpage>. <pub-id pub-id-type=\"doi\">10.1099/ijs.0.049916-0</pub-id>\n<pub-id pub-id-type=\"pmid\">23606480</pub-id></mixed-citation></ref><ref id=\"B63\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Qu</surname><given-names>P. H.</given-names></name><name><surname>Li</surname><given-names>Y.</given-names></name><name><surname>Salam</surname><given-names>N.</given-names></name><name><surname>Chen</surname><given-names>S. Y.</given-names></name><name><surname>Liu</surname><given-names>L.</given-names></name><name><surname>Gu</surname><given-names>Q.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title><italic>Allfrancisella inopinata</italic> gen. nov., sp. nov. and <italic>Allofrancisella frigidaquae</italic> sp. nov., isolated from water-cooling systems, and transfer of <italic>Francisella guangzhouensis</italic>\n<xref rid=\"B62\" ref-type=\"bibr\">Qu et al., 2013</xref> to the new genus as <italic>Allofrancisella guangzhouensis</italic> comb. nov.</article-title>\n<source><italic>Int. J. Syst. Evol. Microbiol.</italic></source>\n<volume>66</volume>\n<fpage>4832</fpage>–<lpage>4838</lpage>. <pub-id pub-id-type=\"doi\">10.1099/ijsem.0.001437</pub-id>\n<pub-id pub-id-type=\"pmid\">27543089</pub-id></mixed-citation></ref><ref id=\"B64\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ramirez-Paredes</surname><given-names>J. G.</given-names></name><name><surname>Larsson</surname><given-names>P.</given-names></name><name><surname>Thompson</surname><given-names>K. D.</given-names></name><name><surname>Penman</surname><given-names>D. J.</given-names></name><name><surname>Busse</surname><given-names>H. J.</given-names></name><name><surname>Ohrman</surname><given-names>C.</given-names></name><etal/></person-group> (<year>2020</year>). <article-title>Reclassification of <italic>Francisella noatunensis</italic> subsp. <italic>orientalis</italic> Ottem et al. 2009 as <italic>Francisella orientalis</italic> sp. nov., <italic>Francisella noatunensis</italic> subsp. <italic>chilensis</italic> subsp. nov. and emended description of <italic>Francisella noatunensis</italic>.</article-title>\n<source><italic>Int. J. Syst. Evol. Microbiol.</italic></source>\n<volume>70</volume>\n<fpage>2034</fpage>–<lpage>2048</lpage>. <pub-id pub-id-type=\"doi\">10.1099/ijsem.0.004009</pub-id>\n<pub-id pub-id-type=\"pmid\">32160147</pub-id></mixed-citation></ref><ref id=\"B65\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rasko</surname><given-names>D. A.</given-names></name><name><surname>Rosovitz</surname><given-names>M. J.</given-names></name><name><surname>Myers</surname><given-names>G. S. A.</given-names></name><name><surname>Mongodin</surname><given-names>E. F.</given-names></name><name><surname>Fricke</surname><given-names>W. F.</given-names></name><name><surname>Gajer</surname><given-names>P.</given-names></name><etal/></person-group> (<year>2008</year>). <article-title>The pangenome structure of <italic>Escherichia coli</italic>: comparative genomic analysis of <italic>E. coli</italic> commensal and pathogenic isolates.</article-title>\n<source><italic>J. Bacteriol.</italic></source>\n<volume>190</volume>\n<fpage>6881</fpage>–<lpage>6893</lpage>. <pub-id pub-id-type=\"doi\">10.1128/Jb.00619-08</pub-id>\n<pub-id pub-id-type=\"pmid\">18676672</pub-id></mixed-citation></ref><ref id=\"B66\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Respicio-Kingry</surname><given-names>L. B.</given-names></name><name><surname>Byrd</surname><given-names>L.</given-names></name><name><surname>Allison</surname><given-names>A.</given-names></name><name><surname>Brett</surname><given-names>M.</given-names></name><name><surname>Scott-Waldron</surname><given-names>C.</given-names></name><name><surname>Galliher</surname><given-names>K.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>Cutaneous infection caused by a novel <italic>Francisella</italic> sp.</article-title>\n<source><italic>J. Clin. Microbiol.</italic></source>\n<volume>51</volume>\n<fpage>3456</fpage>–<lpage>3460</lpage>. <pub-id pub-id-type=\"doi\">10.1128/JCM.01105-13</pub-id>\n<pub-id pub-id-type=\"pmid\">23903547</pub-id></mixed-citation></ref><ref id=\"B67\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Robles-Marhuenda</surname><given-names>A.</given-names></name><name><surname>Vaca</surname><given-names>M.</given-names></name><name><surname>Romero</surname><given-names>P.</given-names></name><name><surname>Ferreira</surname><given-names>A.</given-names></name><name><surname>Lopez-Granados</surname><given-names>E.</given-names></name><name><surname>Arnalich</surname><given-names>F.</given-names></name></person-group> (<year>2018</year>). <article-title><italic>Francisella philomiragia</italic>: think of chronic granulomatous disease.</article-title>\n<source><italic>J. Clin. Immunol.</italic></source>\n<volume>38</volume>\n<fpage>257</fpage>–<lpage>259</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s10875-018-0498-7</pub-id>\n<pub-id pub-id-type=\"pmid\">29663155</pub-id></mixed-citation></ref><ref id=\"B68\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rohmer</surname><given-names>L.</given-names></name><name><surname>Fong</surname><given-names>C.</given-names></name><name><surname>Abmayr</surname><given-names>S.</given-names></name><name><surname>Wasnick</surname><given-names>M.</given-names></name><name><surname>Larson Freeman</surname><given-names>T. J.</given-names></name><name><surname>Radey</surname><given-names>M.</given-names></name><etal/></person-group> (<year>2007</year>). <article-title>Comparison of <italic>Francisella tularensis</italic> genomes reveals evolutionary events associated with the emergence of human pathogenic strains</article-title>. <source><italic>Genome Biol.</italic></source>\n<volume>8</volume>:<issue>R102</issue>. <pub-id pub-id-type=\"doi\">10.1186/gb-2007-8-6-r102</pub-id>\n<pub-id pub-id-type=\"pmid\">17550600</pub-id></mixed-citation></ref><ref id=\"B69\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rosselli</surname><given-names>R.</given-names></name><name><surname>Romoli</surname><given-names>O.</given-names></name><name><surname>Vitulo</surname><given-names>N.</given-names></name><name><surname>Vezzi</surname><given-names>A.</given-names></name><name><surname>Campanaro</surname><given-names>S.</given-names></name><name><surname>de Pascale</surname><given-names>F.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>Direct 16S rRNA-seq from bacterial communities: a PCR-independent approach to simultaneously assess microbial diversity and functional activity potential of each taxon.</article-title>\n<source><italic>Sci. Rep.</italic></source>\n<volume>6</volume>:<issue>32165</issue>. <pub-id pub-id-type=\"doi\">10.1038/Srep32165</pub-id>\n<pub-id pub-id-type=\"pmid\">27577787</pub-id></mixed-citation></ref><ref id=\"B70\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rydzewski</surname><given-names>K.</given-names></name><name><surname>Schulz</surname><given-names>T.</given-names></name><name><surname>Brzuszkiewicz</surname><given-names>E.</given-names></name><name><surname>Holland</surname><given-names>G.</given-names></name><name><surname>Luck</surname><given-names>C.</given-names></name><name><surname>Fleischer</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Genome sequence and phenotypic analysis of a first German <italic>Francisella</italic> sp. isolate (W12-1067) not belonging to the species <italic>Francisella tularensis</italic>.</article-title>\n<source><italic>BMC Microbiol.</italic></source>\n<volume>14</volume>:<issue>169</issue>. <pub-id pub-id-type=\"doi\">10.1186/1471-2180-14-169</pub-id>\n<pub-id pub-id-type=\"pmid\">24961323</pub-id></mixed-citation></ref><ref id=\"B71\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Salomon</surname><given-names>D.</given-names></name><name><surname>Kinch</surname><given-names>L. N.</given-names></name><name><surname>Trudgian</surname><given-names>D. C.</given-names></name><name><surname>Guo</surname><given-names>X. F.</given-names></name><name><surname>Klimko</surname><given-names>J. A.</given-names></name><name><surname>Grishin</surname><given-names>N. V.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Marker for type VI secretion system effectors.</article-title>\n<source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source>\n<volume>111</volume>\n<fpage>9271</fpage>–<lpage>9276</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1406110111</pub-id>\n<pub-id pub-id-type=\"pmid\">24927539</pub-id></mixed-citation></ref><ref id=\"B72\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Satoh</surname><given-names>S.</given-names></name><name><surname>Mimuro</surname><given-names>M.</given-names></name><name><surname>Tanaka</surname><given-names>A.</given-names></name></person-group> (<year>2013</year>). <article-title>Construction of a phylogenetic tree of photosynthetic prokaryotes based on average similarities of whole genome sequences.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>8</volume>:<issue>e70290</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0070290</pub-id>\n<pub-id pub-id-type=\"pmid\">23922968</pub-id></mixed-citation></ref><ref id=\"B73\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shrivastava</surname><given-names>S.</given-names></name><name><surname>Mande</surname><given-names>S. S.</given-names></name></person-group> (<year>2008</year>). <article-title>Identification and functional characterization of gene components of Type VI secretion system in bacterial genomes.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>3</volume>:<issue>e2955</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0002955</pub-id>\n<pub-id pub-id-type=\"pmid\">18698408</pub-id></mixed-citation></ref><ref id=\"B74\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Siddaramappa</surname><given-names>S.</given-names></name><name><surname>Challacombe</surname><given-names>J. F.</given-names></name><name><surname>Petersen</surname><given-names>J. M.</given-names></name><name><surname>Pillai</surname><given-names>S.</given-names></name><name><surname>Hogg</surname><given-names>G.</given-names></name><name><surname>Kuske</surname><given-names>C. R.</given-names></name></person-group> (<year>2011</year>). <article-title>Common ancestry and novel genetic traits of <italic>Francisella novicida</italic>-like isolates from North America and Australia as revealed by comparative genomic analyses.</article-title>\n<source><italic>Appl. Environ. Microbiol.</italic></source>\n<volume>77</volume>\n<fpage>5110</fpage>–<lpage>5122</lpage>. <pub-id pub-id-type=\"doi\">10.1128/AEM.00337-11</pub-id>\n<pub-id pub-id-type=\"pmid\">21666011</pub-id></mixed-citation></ref><ref id=\"B75\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Siddaramappa</surname><given-names>S.</given-names></name><name><surname>Challacombe</surname><given-names>J. F.</given-names></name><name><surname>Petersen</surname><given-names>J. M.</given-names></name><name><surname>Pillai</surname><given-names>S.</given-names></name><name><surname>Kuske</surname><given-names>C. R.</given-names></name></person-group> (<year>2012</year>). <article-title>Genetic diversity within the genus <italic>Francisella</italic> as revealed by comparative analyses of the genomes of two North American isolates from environmental sources.</article-title>\n<source><italic>BMC Genomics</italic></source>\n<volume>13</volume>:<issue>422</issue>. <pub-id pub-id-type=\"doi\">10.1186/1471-2164-13-422</pub-id>\n<pub-id pub-id-type=\"pmid\">22920915</pub-id></mixed-citation></ref><ref id=\"B76\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Singer</surname><given-names>G. A.</given-names></name><name><surname>Hickey</surname><given-names>D. A.</given-names></name></person-group> (<year>2000</year>). <article-title>Nucleotide bias causes a genomewide bias in the amino acid composition of proteins.</article-title>\n<source><italic>Mol. Biol. Ecol.</italic></source>\n<volume>17</volume>\n<fpage>1581</fpage>–<lpage>1588</lpage>. <pub-id pub-id-type=\"doi\">10.1093/oxfordjournals.molbev.a026257</pub-id>\n<pub-id pub-id-type=\"pmid\">11070046</pub-id></mixed-citation></ref><ref id=\"B77\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sjödin</surname><given-names>A.</given-names></name><name><surname>Svensson</surname><given-names>K.</given-names></name><name><surname>Öhrman</surname><given-names>C.</given-names></name><name><surname>Ahlinder</surname><given-names>J.</given-names></name><name><surname>Lindgren</surname><given-names>P.</given-names></name><name><surname>Duodu</surname><given-names>S.</given-names></name><etal/></person-group> (<year>2012</year>). <article-title>Genome characterisation of the genus <italic>Francisella</italic> reveals insight into similar evolutionary paths in pathogens of mammals and fish.</article-title>\n<source><italic>BMC Genomics</italic></source>\n<volume>13</volume>:<issue>268</issue>. <pub-id pub-id-type=\"doi\">10.1186/1471-2164-13-268</pub-id>\n<pub-id pub-id-type=\"pmid\">22727144</pub-id></mixed-citation></ref><ref id=\"B78\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sjödin</surname><given-names>A.</given-names></name><name><surname>Ohrman</surname><given-names>C.</given-names></name><name><surname>Backman</surname><given-names>S.</given-names></name><name><surname>Larkeryd</surname><given-names>A.</given-names></name><name><surname>Granberg</surname><given-names>M.</given-names></name><name><surname>Lundmark</surname><given-names>E.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Complete genome sequence of <italic>Francisella endociliophora</italic> strain FSC1006, isolated from a laboratory culture of the marine ciliate <italic>Euplotes raikovi</italic></article-title>. <source><italic>Genome Announc.</italic></source>\n<volume>26</volume>:<issue>e01227-14</issue>. <pub-id pub-id-type=\"doi\">10.1128/genomeA.01227-14</pub-id>\n<pub-id pub-id-type=\"pmid\">25428973</pub-id></mixed-citation></ref><ref id=\"B79\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Sjöstedt</surname><given-names>A.</given-names></name></person-group> (<year>2005</year>). “<article-title>Family XVII. <italic>Francisella</italic>ceae, genus I. <italic>Francisella</italic></article-title>,” in <source><italic>Bergey’s Manual of Systematic Bacteriology</italic></source>, <role>ed.</role>\n<person-group person-group-type=\"editor\"><name><surname>Brenner</surname><given-names>D. J.</given-names></name></person-group> (<publisher-loc>New York, NY</publisher-loc>: <publisher-name>Springer-Verlag</publisher-name>).</mixed-citation></ref><ref id=\"B80\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sjöstedt</surname><given-names>A.</given-names></name></person-group> (<year>2007</year>). <article-title>Tularemia: history, epidemiology, pathogen physiology, and clinical manifestations.</article-title>\n<source><italic>Ann. N. Y. Acad. Sci.</italic></source>\n<volume>1105</volume>\n<fpage>1</fpage>–<lpage>29</lpage>. <pub-id pub-id-type=\"doi\">10.1196/annals.1409.009</pub-id>\n<pub-id pub-id-type=\"pmid\">17395726</pub-id></mixed-citation></ref><ref id=\"B81\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Snipen</surname><given-names>L.</given-names></name><name><surname>Almoy</surname><given-names>T.</given-names></name><name><surname>Ussery</surname><given-names>D. W.</given-names></name></person-group> (<year>2009</year>). <article-title>Microbial comparative pan-genomics using binomial mixture models.</article-title>\n<source><italic>BMC Genomics</italic></source>\n<volume>10</volume>:<issue>385</issue>. <pub-id pub-id-type=\"doi\">10.1186/1471-2164-10-385</pub-id>\n<pub-id pub-id-type=\"pmid\">19691844</pub-id></mixed-citation></ref><ref id=\"B82\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Spidlova</surname><given-names>P.</given-names></name><name><surname>Stulik</surname><given-names>J.</given-names></name></person-group> (<year>2017</year>). <article-title><italic>Francisella tularensis</italic> type VI secretion system comes of age.</article-title>\n<source><italic>Virulence</italic></source>\n<volume>8</volume>\n<fpage>628</fpage>–<lpage>631</lpage>. <pub-id pub-id-type=\"doi\">10.1080/21505594.2016.1278336</pub-id>\n<pub-id pub-id-type=\"pmid\">28060573</pub-id></mixed-citation></ref><ref id=\"B83\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tärnvik</surname><given-names>A.</given-names></name><name><surname>Berglund</surname><given-names>L.</given-names></name></person-group> (<year>2003</year>). <article-title>Tularaemia.</article-title>\n<source><italic>Eur. Respir. J.</italic></source>\n<volume>21</volume>\n<fpage>361</fpage>–<lpage>373</lpage>.<pub-id pub-id-type=\"pmid\">12608453</pub-id></mixed-citation></ref><ref id=\"B84\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tettelin</surname><given-names>H.</given-names></name><name><surname>Masignani</surname><given-names>V.</given-names></name><name><surname>Cieslewicz</surname><given-names>M. J.</given-names></name><name><surname>Donati</surname><given-names>C.</given-names></name><name><surname>Medini</surname><given-names>D.</given-names></name><name><surname>Ward</surname><given-names>N. L.</given-names></name><etal/></person-group> (<year>2005</year>). <article-title>Genome analysis of multiple pathogenic isolates of <italic>Streptococcus agalactiae</italic>: implications for the microbial “pan-genome”.</article-title>\n<source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source>\n<volume>102</volume>\n<fpage>13950</fpage>–<lpage>13955</lpage>.<pub-id pub-id-type=\"pmid\">16172379</pub-id></mixed-citation></ref><ref id=\"B85\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tettelin</surname><given-names>H.</given-names></name><name><surname>Riley</surname><given-names>D.</given-names></name><name><surname>Cattuto</surname><given-names>C.</given-names></name><name><surname>Medini</surname><given-names>D.</given-names></name></person-group> (<year>2008</year>). <article-title>Comparative genomics: the bacterial pan-genome.</article-title>\n<source><italic>Curr. Opin. Microbiol.</italic></source>\n<volume>11</volume>\n<fpage>472</fpage>–<lpage>477</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.mib.2008.09.006</pub-id>\n<pub-id pub-id-type=\"pmid\">19086349</pub-id></mixed-citation></ref><ref id=\"B86\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tindall</surname><given-names>B. J.</given-names></name><name><surname>Rossello-Mora</surname><given-names>R.</given-names></name><name><surname>Busse</surname><given-names>H. J.</given-names></name><name><surname>Ludwig</surname><given-names>W.</given-names></name><name><surname>Kampfer</surname><given-names>P.</given-names></name></person-group> (<year>2010</year>). <article-title>Notes on the characterization of prokaryote strains for taxonomic purposes.</article-title>\n<source><italic>Int. J. Syst. Evol. Microbiol.</italic></source>\n<volume>60</volume>\n<fpage>249</fpage>–<lpage>266</lpage>. <pub-id pub-id-type=\"doi\">10.1099/ijs.0.016949-0</pub-id>\n<pub-id pub-id-type=\"pmid\">19700448</pub-id></mixed-citation></ref><ref id=\"B87\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Toronen</surname><given-names>P.</given-names></name><name><surname>Medlar</surname><given-names>A.</given-names></name><name><surname>Holm</surname><given-names>L.</given-names></name></person-group> (<year>2018</year>). <article-title>PANNZER2: a rapid functional annotation web server.</article-title>\n<source><italic>Nucleic Acids Res.</italic></source>\n<volume>46</volume>\n<fpage>W84</fpage>–<lpage>W88</lpage>. <pub-id pub-id-type=\"doi\">10.1093/nar/gky350</pub-id>\n<pub-id pub-id-type=\"pmid\">29741643</pub-id></mixed-citation></ref><ref id=\"B88\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vallesi</surname><given-names>A.</given-names></name><name><surname>Sjödin</surname><given-names>A.</given-names></name><name><surname>Petrelli</surname><given-names>D.</given-names></name><name><surname>Luporini</surname><given-names>P.</given-names></name><name><surname>Taddei</surname><given-names>A. R.</given-names></name><name><surname>Thelaus</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>A new species of the gamma-Proteobacterium <italic>Francisella</italic>, <italic>F. adeliensis</italic> Sp. Nov., Endocytobiont in an Antarctic marine ciliate and potential evolutionary forerunner of pathogenic species.</article-title>\n<source><italic>Microb. Ecol.</italic></source>\n<volume>77</volume>\n<fpage>587</fpage>–<lpage>596</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00248-018-1256-3</pub-id>\n<pub-id pub-id-type=\"pmid\">30187088</pub-id></mixed-citation></ref><ref id=\"B89\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vernikos</surname><given-names>G.</given-names></name><name><surname>Medini</surname><given-names>D.</given-names></name><name><surname>Riley</surname><given-names>D. R.</given-names></name><name><surname>Tettelin</surname><given-names>H.</given-names></name></person-group> (<year>2015</year>). <article-title>Ten years of pan-genome analyses.</article-title>\n<source><italic>Curr. Opin. Microbiol.</italic></source>\n<volume>23</volume>\n<fpage>148</fpage>–<lpage>154</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.mib.2014.11.016</pub-id>\n<pub-id pub-id-type=\"pmid\">25483351</pub-id></mixed-citation></ref><ref id=\"B90\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vinuesa</surname><given-names>P.</given-names></name><name><surname>Ochoa-Sanchez</surname><given-names>L. E.</given-names></name><name><surname>Contreras-Moreira</surname><given-names>B.</given-names></name></person-group> (<year>2018</year>). <article-title>GET_PHYLOMARKERS, a software package to select optimal orthologous clusters for phylogenomics and inferring pan-genome phylogenies, used for a critical geno-taxonomic revision of the Genus <italic>Stenotrophomonas</italic>.</article-title>\n<source><italic>Front. Microbiol.</italic></source>\n<volume>9</volume>:<issue>771</issue>. <pub-id pub-id-type=\"doi\">10.3389/Fmicb.2018.00771</pub-id>\n<pub-id pub-id-type=\"pmid\">29765358</pub-id></mixed-citation></ref><ref id=\"B91\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>J. W.</given-names></name><name><surname>Yang</surname><given-names>B. J.</given-names></name><name><surname>Leier</surname><given-names>A.</given-names></name><name><surname>Marquez-Lago</surname><given-names>T. T.</given-names></name><name><surname>Hayashida</surname><given-names>M.</given-names></name><name><surname>Rocker</surname><given-names>A.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Bastion6: a bioinformatics approach for accurate prediction of type VI secreted effectors.</article-title>\n<source><italic>Bioinformatics</italic></source>\n<volume>34</volume>\n<fpage>2546</fpage>–<lpage>2555</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bioinformatics/bty155</pub-id>\n<pub-id pub-id-type=\"pmid\">29547915</pub-id></mixed-citation></ref><ref id=\"B92\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>Y.</given-names></name><name><surname>Mao</surname><given-names>L.</given-names></name><name><surname>Sun</surname><given-names>Y.</given-names></name><name><surname>Wang</surname><given-names>Z.</given-names></name><name><surname>Zhang</surname><given-names>J.</given-names></name><name><surname>Zhang</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>A Novel <italic>Francisella</italic>-Like Endosymbiont in <italic>Haemaphysalis longicornis</italic> and <italic>Hyalomma asiaticum</italic>, China.</article-title>\n<source><italic>Vector Borne Zoonotic Dis.</italic></source>\n<volume>18</volume>\n<fpage>669</fpage>–<lpage>676</lpage>. <pub-id pub-id-type=\"doi\">10.1089/vbz.2017.2252</pub-id>\n<pub-id pub-id-type=\"pmid\">30106668</pub-id></mixed-citation></ref><ref id=\"B93\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wenger</surname><given-names>J. D.</given-names></name><name><surname>Hollis</surname><given-names>D. G.</given-names></name><name><surname>Weaver</surname><given-names>R. E.</given-names></name><name><surname>Baker</surname><given-names>C. N.</given-names></name><name><surname>Brown</surname><given-names>G. R.</given-names></name><name><surname>Brenner</surname><given-names>D. J.</given-names></name><etal/></person-group> (<year>1989</year>). <article-title>Infection caused by <italic>Francisella philomiragia</italic> (formerly <italic>Yersinia philomiragia</italic>). A newly recognized human pathogen.</article-title>\n<source><italic>Ann. Intern. Med.</italic></source>\n<volume>110</volume>\n<fpage>888</fpage>–<lpage>892</lpage>. <pub-id pub-id-type=\"doi\">10.7326/0003-4819-110-11-888</pub-id>\n<pub-id pub-id-type=\"pmid\">2541646</pub-id></mixed-citation></ref><ref id=\"B94\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Whipp</surname><given-names>M. J.</given-names></name><name><surname>Davis</surname><given-names>J. M.</given-names></name><name><surname>Lum</surname><given-names>G.</given-names></name><name><surname>de Boer</surname><given-names>J.</given-names></name><name><surname>Zhou</surname><given-names>Y.</given-names></name><name><surname>Bearden</surname><given-names>S. W.</given-names></name><etal/></person-group> (<year>2003</year>). <article-title>Characterization of a <italic>novicida</italic>-like subspecies of <italic>Francisella tularensis</italic> isolated in Australia.</article-title>\n<source><italic>J. Med. Microbiol.</italic></source>\n<volume>52(Pt 9)</volume>\n<fpage>839</fpage>–<lpage>842</lpage>. <pub-id pub-id-type=\"doi\">10.1099/jmm.0.05245-0</pub-id>\n<pub-id pub-id-type=\"pmid\">12909664</pub-id></mixed-citation></ref><ref id=\"B95\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wolf</surname><given-names>Y. I.</given-names></name><name><surname>Koonin</surname><given-names>E. V.</given-names></name></person-group> (<year>2012</year>). <article-title>A tight link between orthologs and bidirectional best hits in bacterial and archaeal genomes.</article-title>\n<source><italic>Genome Biol. Evol.</italic></source>\n<volume>4</volume>\n<fpage>1286</fpage>–<lpage>1294</lpage>. <pub-id pub-id-type=\"doi\">10.1093/gbe/evs100</pub-id>\n<pub-id pub-id-type=\"pmid\">23160176</pub-id></mixed-citation></ref><ref id=\"B96\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yao</surname><given-names>J.</given-names></name><name><surname>Yang</surname><given-names>F.</given-names></name><name><surname>Sun</surname><given-names>X.</given-names></name><name><surname>Wang</surname><given-names>S.</given-names></name><name><surname>Gan</surname><given-names>N.</given-names></name><name><surname>Liu</surname><given-names>Q.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Mechanism of inhibition of retromer transport by the bacterial effector RidL.</article-title>\n<source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source>\n<volume>115</volume>\n<fpage>E1446</fpage>–<lpage>E1454</lpage>.<pub-id pub-id-type=\"pmid\">29386389</pub-id></mixed-citation></ref><ref id=\"B97\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yon</surname><given-names>L.</given-names></name><name><surname>Duff</surname><given-names>J. P.</given-names></name><name><surname>Agren</surname><given-names>E. O.</given-names></name><name><surname>Erdelyi</surname><given-names>K.</given-names></name><name><surname>Ferroglio</surname><given-names>E.</given-names></name><name><surname>Godfroid</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Recent changes in infectious diseases in European wildlife.</article-title>\n<source><italic>J. Wildl. Dis.</italic></source>\n<volume>55</volume>\n<fpage>3</fpage>–<lpage>43</lpage>. <pub-id pub-id-type=\"doi\">10.7589/2017-07-172</pub-id>\n<pub-id pub-id-type=\"pmid\">30284963</pub-id></mixed-citation></ref><ref id=\"B98\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zook</surname><given-names>J.</given-names></name><name><surname>Mo</surname><given-names>G.</given-names></name><name><surname>Sisco</surname><given-names>N. J.</given-names></name><name><surname>Craciunescu</surname><given-names>F. M.</given-names></name><name><surname>Hansen</surname><given-names>D. T.</given-names></name><name><surname>Baravati</surname><given-names>B.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>NMR structure of <italic>Francisella tularensis</italic> virulence determinant reveals structural homology to Bet v1 allergen proteins.</article-title>\n<source><italic>Structure</italic></source>\n<volume>23</volume>\n<fpage>1116</fpage>–<lpage>1122</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.str.2015.03.025</pub-id>\n<pub-id pub-id-type=\"pmid\">26004443</pub-id></mixed-citation></ref></ref-list><glossary><title>Abbreviations</title><def-list id=\"DL1\"><def-item><term>AAI</term><def><p>Average Amino acid Identity</p></def></def-item><def-item><term>ANI</term><def><p>Average Nucleotide Identity</p></def></def-item><def-item><term>DUF</term><def><p>domain of unknown function</p></def></def-item><def-item><term><italic>F</italic></term><def><p><italic>Francisella</italic></p></def></def-item><def-item><term>FPI</term><def><p><italic>Francisella</italic> Pathogenicity Island</p></def></def-item><def-item><term>MIX</term><def><p>Marker for type six effectors</p></def></def-item><def-item><term>T6SS</term><def><p>Type VI secretion system.</p></def></def-item></def-list></glossary></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Endocrinol.</journal-id><journal-title-group><journal-title>Frontiers in Endocrinology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2392</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849295</article-id><article-id pub-id-type=\"pmc\">PMC7431614</article-id><article-id pub-id-type=\"doi\">10.3389/fendo.2020.00509</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Association Between Metabolic and Hormonal Derangements and Professional Exposure to Urban Pollution in a High Intensity Traffic Area</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Molfino</surname><given-names>Alessio</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/337797/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Amabile</surname><given-names>Maria Ida</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/449477/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Muscaritoli</surname><given-names>Maurizio</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/329728/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Germano</surname><given-names>Annunziata</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Alfano</surname><given-names>Rossella</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ramaccini</surname><given-names>Cesarina</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Spagnoli</surname><given-names>Alessandra</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/690810/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Cavaliere</surname><given-names>Liberato</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Marseglia</surname><given-names>Gianluca</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Nardone</surname><given-names>Antonio</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Muto</surname><given-names>Giuseppina</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Carbone</surname><given-names>Umberto</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Triassi</surname><given-names>Maria</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/491165/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Fiorito</surname><given-names>Silvana</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Translational and Precision Medicine, Sapienza University of Rome</institution>, <addr-line>Rome</addr-line>, <country>Italy</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Surgical Sciences, Sapienza University of Rome</institution>, <addr-line>Rome</addr-line>, <country>Italy</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Public Health, University Federico II</institution>, <addr-line>Naples</addr-line>, <country>Italy</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Public Health and Infectious Diseases, Sapienza University of Rome</institution>, <addr-line>Rome</addr-line>, <country>Italy</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Institute of Translational Pharmacology, CNR</institution>, <addr-line>Rome</addr-line>, <country>Italy</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Carla Lubrano, Sapienza University of Rome, Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Valeria Guglielmi, University of Rome Tor Vergata, Italy; Michela Zanetti, University of Trieste, Italy; Anastassia Amaro, University of Pennsylvania, United States</p></fn><corresp id=\"c001\">*Correspondence: Alessio Molfino <email>alessio.molfino@uniroma1.it</email></corresp><corresp id=\"c002\">Silvana Fiorito <email>silvana.fiorito@ift.cnr.it</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Obesity, a section of the journal Frontiers in Endocrinology</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†These authors have contributed equally to this work</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>509</elocation-id><history><date date-type=\"received\"><day>09</day><month>1</month><year>2020</year></date><date date-type=\"accepted\"><day>25</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Molfino, Amabile, Muscaritoli, Germano, Alfano, Ramaccini, Spagnoli, Cavaliere, Marseglia, Nardone, Muto, Carbone, Triassi and Fiorito.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Molfino, Amabile, Muscaritoli, Germano, Alfano, Ramaccini, Spagnoli, Cavaliere, Marseglia, Nardone, Muto, Carbone, Triassi and Fiorito</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p><bold>Rationale:</bold> Studies suggest a relation between exposure to air particulate matter (PM)<sub>2.5</sub> pollution and greater cardiovascular morbidity, as well as increased risk for obesity and diabetes. We aimed to identify association(s) between nutritional and metabolic status and exposure to environmental pollution in a cohort of policemen exposed to high levels of air pollution.</p><p><bold>Methods:</bold> We considered adult municipal policemen, working in an urban area at high-traffic density with documented high levels of air PM<sub>2.5</sub> (exposed group) compared to non-exposed policemen. Clinical characteristics, including the presence/absence of metabolic syndrome, were recorded, and serum biomarkers, including adiponectin, leptin, and ghrelin, were assessed.</p><p><bold>Results:</bold> One hundred ninety-nine participants were enrolled, 100 in the exposed group and 99 in the non-exposed group. Metabolic syndrome was documented in 32% of exposed group and in 52.5% of non-exposed group (<italic>P</italic> = 0.008). In the exposed group, we found a positive correlation between body mass index and serum leptin as well as in the non-exposed group (<italic>P</italic> < 0.0001). Within the exposed group, subjects with metabolic syndrome showed lower serum adiponectin (<italic>P</italic> < 0.0001) and higher leptin (<italic>P</italic> = 0.002) levels with respect to those without metabolic syndrome, whereas in the non-exposed group, subjects with metabolic syndrome showed only higher leptin levels when compared to those without metabolic syndrome (<italic>P</italic> = 0.01). Among the participants with metabolic syndrome, we found lower adiponectin levels in those of the exposed group with respect to the non-exposed ones (<italic>P</italic> = 0.007). When comparing the exposed and non-exposed groups, after stratifying participants for Homeostatic Model Assessment for Insulin Resistance >2.5, we found lower adiponectin levels in those of the exposed group with respect to the non-exposed ones (<italic>P</italic> = 0.038).</p><p><bold>Conclusions:</bold> Exposure to air PM pollution was associated with lower levels of adiponectin in adult males with metabolic syndrome.</p></abstract><kwd-group><kwd>air pollution</kwd><kwd>particulate matter</kwd><kwd>metabolic syndrome</kwd><kwd>insulin resistance</kwd><kwd>adiponectin</kwd><kwd>leptin</kwd></kwd-group><counts><fig-count count=\"3\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"49\"/><page-count count=\"8\"/><word-count count=\"5765\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>In industrialized countries, air pollution determines risks for human health (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Particulate and gaseous emissions from motor vehicles contribute to increase the air pollution (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). Diesel exhaust particles (DEPs), a category of particulate matter (PM) derived from diesel fossil fuels and combustible engines, are among the most abundant components of airborne PM with an aerodynamic diameter <2.5 μm (PM<sub>2.5</sub>) (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>) and are considered major contributors to traffic-related PM in urban areas (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B3\" ref-type=\"bibr\">3</xref>). The most part of DEP is composed by particles of nano-sized dimension (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>), which are small aggregates of carbonaceous particles (<100 nm), representing a severe problem for human health considering that they remain in the atmosphere for long periods and go beyond to the indoor air environment, and they can be breathed determining a more toxic effect deeply into the lungs with respect to the coarse particles (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>).</p><p>Particulate pollution is a major public health concern. Several epidemiological studies demonstrated that exposure to DEP is related to various cardiopulmonary, vascular, and oncologic diseases (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>–<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). Moreover, carbonaceous nanoparticles derived from diesel engine exhaust are classified as human carcinogens (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Recent data from large cohorts of subjects have shown strong associations between exposure to air PM<sub>2.5</sub> pollution and increased cardiovascular morbidity and mortality (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Long-term air pollution exposure to fine PM<sub>2.5</sub> above US Environmental Protection Agency–defined standards has been reported to be associated with higher risk of neurodegenerative diseases, including Alzheimer disease (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>).</p><p>Moreover, it has been reported that exposure to diesel exhaust PM may increase the risk for obesity and diabetes (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>–<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). Recently, it has been demonstrated that an early exposure to high levels of PM<sub>2.5</sub> during life represents a risk factor for development of adiposity and insulin resistance in the subsequent years, likely mediated at least in part by reactive oxygen species generation (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Exposure for a long period of time to ultrafine particles in areas near highways has been associated with stroke, ischemic heart disease, hypertension, and type 2 diabetes (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>). Robust data highlighted the association between long-term exposure to air pollution and type 2 diabetes and neurodegenerative disorders in adults, such as dementia and a general decline in cognition (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Another recent study identified the alteration of several metabolic pathways that mediate the development of asthma and cardiovascular diseases associated with ambient air pollutants (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). Moreover, a study conducted in non-obese children exposed to high concentrations of PM<sub>2.5</sub>, vs. low pollution controls, showed high circulating leptin and endothelin 1 levels, vitamin D deficiency, and food reward hormone dysregulation (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>).</p><p>At present, few data are available on the association between hormone dysregulation, metabolic derangement, and chronic exposure to environmental contaminants.</p><p>By the present study, we aimed at identifying association(s) between nutritional, metabolic, and hormonal derangements and exposure to environmental pollution in an Italian population of traffic policemen professionally exposed to high levels of air PM<sub>2.5</sub> compared to employees performing indoor administrative work in the same area.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><p>This is a cross-sectional study performed on adult male municipal policemen of the city of Naples, Italy. The male gender was chosen because of the high percentage of male subjects working in this field in this specific geographical area.</p><p>This study was carried out in accordance with the health surveillance program (D.L. n.81/08), approved to be conducted at the Department of Public Health, University of Naples “Federico II,” Italy. All the subjects gave written informed consent in accordance with the Declaration of Helsinki and authorized the use of the clinical data for research purposes. The privacy rights of human subjects were always observed. Exclusion criteria included the presence of highly catabolic diseases, such as cancer, chronic infections, and the absence of informed consent.</p><sec><title>Participant Demographic and Clinical Characteristics</title><p>We enrolled adult males professionally exposed to air pollution (or airborne nanoparticles) in an urban area at high traffic density (exposed group) and adult males non-exposed (at least 1 year indoor working) (non-exposed group) matched by body mass index (BMI).</p><p>Demographic characteristics, including age, weight, height, BMI, and comorbidities, such as hypertension, diabetes, cardiovascular disorders, and dyslipidemia, were recorded in all participants. The diagnostic criteria we used for metabolic syndrome were based on the National Cholesterol Education Program Adult Treatment Panel III clinical criteria for defining the metabolic syndrome.</p></sec><sec><title>Biomarkers</title><p>Blood samples were collected in all the participants on fasting condition and then centrifuged, and the serum was stored at −80°C at the Laboratory of the Department of Public Health, University of Naples “Federico II.” The samples were shipped to the Central Laboratory for analysis at the Department of Translational and Precision Medicine (formerly Department of Clinical Medicine), Sapienza University of Rome. The glycemic–insulinemic profile, including the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) index and the complete lipid profile (total cholesterol, high-density lipoprotein, low-density lipoprotein, triglycerides), has been assessed by enzymatic and/or colorimetric methods.</p><p>The serum levels of leptin and adiponectin, as adipokines, and ghrelin, as gastrointestinal peptide, have been assessed by enzyme-linked immunosorbent assays.</p><p>We additionally utilized leptin/BMI and adiponectin/BMI ratios in our analyses accounting for the well-known associations between leptin and adiponectin with adiposity, as previously shown (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>, <xref rid=\"B23\" ref-type=\"bibr\">23</xref>).</p></sec><sec><title>Statistical Analyses</title><p>Participants' characteristics were described using mean ± SD for continuous normally distributed variables, median and interquartile range for non–normally distributed variables, as appropriate, and categorical variables were presented as number of cases (percentages). Skewed variables were transformed to the natural logarithm (LN). A Shapiro–Wilk test was used to determine normality. Relations among variables were assessed through χ<sup>2</sup> tests, <italic>t</italic>-tests, analysis of variance, or Wilcoxon rank-sum tests, as appropriate. Spearman correlation index was used for non-parametric correlations.</p><p>A standard two-tailed <italic>P</italic> < 0.05 was considered statistically significant. All statistical analysis was performed in SPSS®.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>Characteristics of the Participants</title><p>A total of 200 participants were consecutively enrolled in the study during the second half of the year 2016: 100 adult males, who worked as traffic policemen exposed to airborne traffic–derived pollution in an urban area (Naples, Italy) at high traffic density (exposed group) (mean duration of the exposure was 20.2 years, min. 16, max. 29 years), and 100 adult males with administrative duties performed in the office in the same city (non-exposed group). One participant of the non-exposed group was excluded because he did not perform blood sampling and did not respond to the administered questionnaires. Therefore, 99 participants in the non-exposed group were studied.</p><p>According to the Air Quality Monitoring Report 2016 by the Regional Agency for Environmental Protection in Campania, Italy, during the 3 months before the enrollment, the traffic policemen were exposed to daily mean PM<sub>2.5</sub> values between 13.97 and 23.37 μg/m<sup>3</sup>. The same agency recommends maximum values of PM<sub>2.5</sub> of 25 μg/m<sup>3</sup> during the entire year (<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.arpacampania.it/home\">http://www.arpacampania.it/home</ext-link>). No differences were observed between exposed and non-exposed groups in terms of cigarette smoking. In particular, the exposed group included 30 smokers (30%), and 21 of them (70%) smoking more than 10 cigarettes/day. Non-exposed group included 31 smokers (31.3%), and 27 of them (87%) smoking more than 10 cigarettes/day.</p></sec><sec><title>Nutritional and Metabolic Profile of the Participants</title><p>No differences were observed between the two groups in terms of body weight, BMI, and lipid profile, whereas plasma glucose levels and HOMA-IR were higher in the non-exposed group compared to the exposed one (<italic>P</italic> = 0.009, <italic>P</italic> = 0.03, respectively) (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). In addition, BMI was ≥30 kg/m<sup>2</sup> in 22% of the exposed group and in 23.2% of the non-exposed group (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). The presence of comorbidities was as follows: diabetes was present in 7% in the exposed group and in 10.5% of the non-exposed group; hypertension was more frequent in the non-exposed group (<italic>P</italic> = 0.025); dyslipidemia was present in 45% of participants in the exposed group and 43.4% of the non-exposed ones (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Metabolic syndrome was documented in 32% of the exposed group and in the 52.5% of the non-exposed group (<italic>P</italic> = 0.008) (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Between the two groups, no differences were seen in terms of adiponectin, ghrelin, and leptin serum levels (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>), also when considering these values as ratio with BMI.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Participants' characteristics.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"left\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\"><bold>Exposed group</bold></th><th valign=\"top\" align=\"left\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\"><bold>Non-exposed group</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>P</italic></bold></th></tr><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Participants <italic>n</italic> = 100</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Participants <italic>n</italic> = 99</bold></th><th rowspan=\"1\" colspan=\"1\"/></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Age, years</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">58.0 (47.75, 61.0)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">61.0 (57.0, 63.0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.0001</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Body weight, kg</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">80.0 (75.0, 90.0)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">82.5 (75.0, 90.0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.52</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BMI, kg/m<sup>2</sup></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">26.2 (24.5, 28.7)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">27.3 (24.9, 29.4)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.28</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BMI ≥30 kg/m<sup>2</sup>, <italic>n</italic> (%)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">22 (22%)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">23 (23.2%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.84</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cholesterol, mg/dL</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">181.0 (160.8, 213.0)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">185.0 (158.5, 209.5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.81</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">LDL cholesterol, mg/dL<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">116.3 ± 37.1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">114.8 ± 35.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.78</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Triglycerides, mg/dL</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">116.5 (72.0, 151.8)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">107.0 (85.0, 156.0)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.93</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Fasting glucose levels, mg/dL</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">96.5 (89.0, 106.3)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">104.0 (92.0, 116.5)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.009</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insulinemia, μU/mL</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">10.5 (6.45, 17.15)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">12.5 (7.56, 18.4)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.10</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HOMA-IR index</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2.39 (1.46, 4.08)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3.16 (1.81, 5.05)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.03</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"4\" rowspan=\"1\"><bold>Comorbidities, Yes/No:</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hypertension</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">30/70</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">46/53</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.025</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Diabetes mellitus</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">7/93</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">10/89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.445</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Dyslipidemia</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">34/66</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">28/71</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.38</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Metabolic syndrome</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">32/68</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">52/47</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.008</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">LN adiponectin, ng/mL<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">11.02 ± 0.5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">11.09 ± 0.55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.39</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">LN ghrelin, ng/mL<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2.22 ± 0.23</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2.26 ± 0.20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.13</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Leptin, ng/mL</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.1 (0.63, 2.00)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1.14 (0.56, 1.99)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.81</td></tr></tbody></table><table-wrap-foot><p><italic>Median (interquartile range) is shown for non–normally distributed variables</italic>.</p><fn id=\"TN1\"><label>*</label><p><italic>Mean ± SD</italic>.</p></fn><p><italic>BMI, body mass index; LDL, low-density lipoprotein; HOMA-IR, Homeostatic Model Assessment for Insulin Resistance; LN, natural logarithm</italic>.</p></table-wrap-foot></table-wrap></sec><sec><title>Correlation Between BMI and Serum Adiponectin, Ghrelin, and Leptin in the Two Groups of Participants</title><p>In the exposed group, we found a negative correlation between BMI and serum adiponectin levels (ρ = −0.0205, <italic>P</italic> = 0.04) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>) and a positive correlation between BMI and serum leptin concentrations (ρ = 0.667, <italic>P</italic> < 0.0001) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). No significant correlation was documented between BMI and serum ghrelin levels (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p><bold>(A)</bold> Correlation between BMI and serum adiponectin, ghrelin, and leptin levels in the exposed group (<italic>n</italic> = 100). *<italic>P</italic> = 0.04, <sup>#</sup><italic>P</italic> < 0.0001. <bold>(B)</bold> Correlation between BMI and serum adiponectin, ghrelin, and leptin levels in the non-exposed group (<italic>n</italic> = 99). <sup>#</sup><italic>P</italic> < 0.0001.</p></caption><graphic xlink:href=\"fendo-11-00509-g0001\"/></fig><p>In the non-exposed group, we found a positive correlation between BMI and serum leptin (ρ = 0.546, <italic>P</italic> < 0.0001) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>), but no significant correlations were detected between BMI and serum adiponectin and ghrelin (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>).</p></sec><sec><title>Association Between Comorbidities, Metabolic Syndrome, and Serum Adiponectin, Ghrelin, and Leptin Levels in the Two Groups of Participants</title><p>In the exposed group, participants with diabetes showed lower serum adiponectin levels with respect to non-diabetic (<italic>P</italic> = 0.001). No association was observed between the other comorbidities, including obesity, hypertension, dyslipidemia, and serum adiponectin, ghrelin, and leptin levels. Subjects in this group with metabolic syndrome showed lower serum adiponectin levels and higher serum leptin levels with respect to those without metabolic syndrome (<italic>P</italic> < 0.0001, <italic>P</italic> = 0.002, respectively) (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>). This observation was confirmed when correcting per BMI both serum levels of adiponectin (adiponectin/BMI) (<italic>P</italic> < 0.0001) and leptin (leptin/BMI) (<italic>P</italic> = 0.003).</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p><bold>(A)</bold> Box-dot plot of serum adiponectin, ghrelin, and leptin levels in the exposed group in subjects with (<italic>n</italic> = 32) and without metabolic syndrome (<italic>n</italic> = 68). *<italic>P</italic> = 0.002, <sup>#</sup><italic>P</italic> < 0.0001. <bold>(B)</bold> Box-dot plot of serum adiponectin, ghrelin, and leptin levels in the non-exposed group in subjects with (<italic>n</italic> = 52) and without metabolic syndrome (<italic>n</italic> = 47). *<italic>P</italic> = 0.01.</p></caption><graphic xlink:href=\"fendo-11-00509-g0002\"/></fig><p>In the non-exposed group, no differences in serum adiponectin, ghrelin, and leptin levels were documented between diabetic and non-diabetic participants and when considering the presence of obesity, hypertension, and dyslipidemia. In the non-exposed subjects with metabolic syndrome, we found no significant difference in terms of serum adiponectin levels, whereas higher serum leptin levels were observed when compared to those without metabolic syndrome (<italic>P</italic> = 0.58, <italic>P</italic> = 0.01, respectively) (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). Also, in this group, this behavior was confirmed when correcting per BMI both serum levels of adiponectin (adiponectin/BMI) (<italic>P</italic> < 0.0001) and leptin (leptin/BMI) (<italic>P</italic> = 0.018).</p></sec><sec><title>Metabolic Syndrome and Metabolic Biomarkers Between the Two Groups of Participants</title><p>When comparing the two groups after stratifying participants for the presence of metabolic syndrome, we found lower serum adiponectin levels in the exposed group when compared to the non-exposed one (<italic>P</italic> = 0.007) (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). No differences were seen in terms of serum leptin, ghrelin, insulin, and fasting plasma glucose levels. Finally, when comparing the two groups, after stratifying participants for HOMA-IR > 2.5, as indicator of insulin resistance (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>), we found lower adiponectin serum levels in the exposed group with respect to the non-exposed (<italic>P</italic> = 0.038).</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Box-dot plot of serum adiponectin levels in subjects with metabolic syndrome of the non-exposed (<italic>n</italic> = 52) and of the exposed (<italic>n</italic> = 32) group (*<italic>P</italic> = 0.007).</p></caption><graphic xlink:href=\"fendo-11-00509-g0003\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>A large number of studies performed worldwide have shown that environmental pollution due to exhaust particles and gas from diesel engine vehicles heavily impacts human health (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>–<xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B9\" ref-type=\"bibr\">9</xref>, <xref rid=\"B10\" ref-type=\"bibr\">10</xref>). In the last 10 years, several scientific reports highlighted the dangerous effects for human health deriving from the exposure to DEP. The contact of micro (PM 10–2.5 μm) and/or nano (PM < 100 nm) sized particles with mucous membranes of the respiratory systems through breathing, the potential achievement of the brain tissue through nasal inhalation, and the direct absorption through the skin have been considered the main entrance doors of the particles into the human body. Several data both <italic>in vitro</italic> and <italic>in vivo</italic> have documented the deleterious effects of DEP. The impact on different cell systems has been assessed, as well as the underlying cellular mechanisms regulating the biological effects between pollution-derived particles (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>–<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). It has been widely demonstrated that the biological effects of urban traffic pollution are mediated by inflammatory mechanisms.</p><p>The results of our study, aimed at investigating the potential effects of environmental pollutants on metabolic and hormonal parameters in people exposed professionally to urban traffic, showed that, when comparing the two groups of participants (exposed vs. non-exposed), no differences were detectable in terms of serum adiponectin, ghrelin, and leptin levels. However, in the two groups we confirmed the presence of a positive correlation between BMI and leptin concentrations, as expected to be influenced by adiposity, while the negative correlation between BMI and adiponectin was present only in the exposed group, where also the presence of diabetes was associated with lower serum adiponectin levels. A similar behavior was shown regarding the association between metabolic syndrome and changes in terms of both adiponectin (lower) and leptin (higher) circulating levels only in the exposed group; in fact, in the non-exposed subjects, only leptin serum levels were higher in subjects with metabolic syndrome. More importantly, when comparing the two groups after stratifying participants for the presence of metabolic syndrome, as well as for HOMA-IR > 2.5 alone, we found significantly lower serum adiponectin levels in the exposed group with respect to the non-exposed. Among the metabolic and hormonal parameters (insulin, ghrelin, and leptin), our data suggest that the prolonged daily exposure to high levels of urban pollution, as documented by the report of the Regional Agency for Environmental Protection, significantly affected only the adiponectin serum levels.</p><p>Adiponectin is a 244-amino-acid protein, produced almost exclusively by white adipocyte cells, mostly by visceral depots (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>); it possesses a wide range of biological activities, mainly including an insulin-sensitizing and antiatherogenic function, and has been identified as a potent and pleiotropic regulator of inflammation (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>–<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). Experimental studies have provided strong evidences that major cellular responses to PM exposure include oxidative stress and inflammation (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>, <xref rid=\"B37\" ref-type=\"bibr\">37</xref>). The majority of the effects induced by exhaust emissions determine the triggering of inflammatory responses in exposed individuals and the gradual release of cytokines, reactive oxygen species, nitric oxide synthase, and other defense mechanisms, which ultimately expose the DNA to negative oxidative effects (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>). Moreover, several recent studies highlighted the fundamental role played by PM exposure in the pathogenesis of the metabolic syndrome and cardiovascular disorders (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>). Increased PM<sub>2.5</sub> exposure has been shown to promote elevations in blood pressure among healthy subjects and in particular among obese individuals who resulted more susceptible to the effects of ambient air pollution (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>). High levels of air PM with diameter ≤10 and 2.5 μm have been demonstrated to impair myocardial perfusion and increase myocardial oxygen demand in non-smoking patients with metabolic syndrome (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>). Air pollution has been implicated in the pathogenesis of metabolic syndrome associated with other metabolic disorders (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>, <xref rid=\"B43\" ref-type=\"bibr\">43</xref>).</p><p>A hormonal derangement, likely triggering the onset of the metabolic syndrome during prolonged exposure to PM<sub>10</sub>, has been reported (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>).</p><p>Our findings on reduced adiponectin serum levels in exposed subjects with metabolic syndrome are supported by a recent study reporting higher levels of cadherin 13 upon exposure to PM<sub>10</sub> that, resulting in reduced levels of free adiponectin, could affect insulin resistance, as adiponectin is crucial to its reduction. The authors suggested that long-term exposure to PM<sub>10</sub> may interfere with insulin metabolism, as adiponectin plays a central role in regulating insulin levels (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>, <xref rid=\"B46\" ref-type=\"bibr\">46</xref>).</p><p>A more recent study showed that exposure to 1-year average PM<sub>2.5</sub> is associated with an increased risk of metabolic syndrome and its components in adults without cardiovascular disease, thus indicating that PM<sub>2.5</sub> affects the onset of metabolic syndrome, which may lead to increase the risk of cardiovascular disease (<xref rid=\"B47\" ref-type=\"bibr\">47</xref>). A significant association of short- and long-term exposures to PM<sub>2.5</sub> with hypertension has been observed with a stronger relationship among studies of men from Asia, North America, and areas with higher air pollutant levels (<xref rid=\"B48\" ref-type=\"bibr\">48</xref>).</p><p>Regarding our study, we should consider that the city of Naples is on the seaside, exposed during almost all hours of the day to the sea breeze or wind. It is likely that these windy atmospheric conditions are able to disperse gaseous and particulate exhaust components decreasing considerably their inhaled portion, thus potentially reducing their dangerous effects. In this light, the metabolic derangements that we observed in a small percentage (32%) of our exposed group might be even worse in a more polluted metropolitan area.</p><p>Our study has limitations, including the absence of an objective assessment of physical activity/inactivity, in particular in the non-exposed group. However, we assume that the exposed group is more physically active that the non-exposed one at least during the working time.</p><p>We did not assess food intake, which might help in correlating metabolic and hormonal changes with energy and protein imbalance. To account for adiposity, we did not measure waist circumference, as well as body composition, although we corrected the levels of the biomarkers for body size (BMI).</p><p>We considered only male subjects, and more importantly, we are not able to ascertain the effect of a single subject's exposure to pollution and to cigarettes smoking, as potentially additive effects, and the change in hormonal and metabolic profile in a longitudinal fashion. Moreover, a higher percentage of hypertensive and metabolic syndrome individuals in the non-exposed cohort might have impacted on the correlations observed. However, considering that the exposed group is the one showing significantly higher metabolic dysregulations in terms of leptin and adiponectin levels, we believe that the data obtained within this group appear reliable, although a cause-effect investigation is needed to clarify these observations.</p><p>In conclusion, the significantly lower circulating adiponectin levels documented in the exposed group with metabolic syndrome appear clinically important, suggesting that the exposed group, despite protective factors such as non-sedentary daily activity, is subjected to undergo metabolic changes likely due to the observed reduced levels of the hormone adiponectin (which possesses a protective function against inflammation) potentially induced by environmental pollution. Based on these findings, it is possible to hypothesize that being constantly exposed to traffic-derived pollutants may induce metabolic derangements that could lead, if added to other risk factors, including genetic background and unbalanced dietary habits, to the development of chronic diseases including diabetes.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The datasets generated for this study are available on request to the corresponding author.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>AM designed research, analyzed data, and wrote the paper. MA collected the data and wrote the paper. MM and UC reviewed the paper. AG and RA conducted research and collected the data. CR performed laboratory dosage. AS performed statistical analyses. LC, GMa, AN, and GMu collected the data and reviewed the paper. MT conducted the research and reviewed the paper. SF designed research, wrote the paper, and had primary responsibility for final content. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a shared affiliation, though no other collaboration, with the authors AM, MA, MM, CR, and AS at the time of the review.</p></sec></body><back><ack><p>The results of the present study first appeared as conference abstract at the 12th International Conference on Cachexia, Sarcopenia & Muscle Wasting, Berlin 6–8 December 2019 (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>).</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> Institutional Funds of the Department of Translational and Precision Medicine, Sapienza University of Rome, and of the Department of Public Health, University Federico II, Naples, Italy for research activity.</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mannucci</surname><given-names>PM</given-names></name><name><surname>Franchini</surname><given-names>M</given-names></name></person-group>. <article-title>Health effects of ambient air pollution in developing countries</article-title>. <source>Int J Environ Res Public Health.</source> (<year>2017</year>) <volume>14</volume>:<fpage>E1048</fpage>. <pub-id pub-id-type=\"doi\">10.3390/ijerph14091048</pub-id><pub-id pub-id-type=\"pmid\">28895888</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Crinnion</surname><given-names>W</given-names></name></person-group>. <article-title>Particulate matter is a surprisingly common contributor to disease</article-title>. <source>Integr Med (Encinitas).</source> (<year>2017</year>) <volume>16</volume>:<fpage>8</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"pmid\">30881250</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Patel</surname><given-names>MM</given-names></name><name><surname>Chillrud</surname><given-names>SN</given-names></name><name><surname>Correa</surname><given-names>JC</given-names></name><name><surname>Hazi</surname><given-names>Y</given-names></name><name><surname>Feinberg</surname><given-names>M</given-names></name><name><surname>Kc</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Traffic-related particulate matter and acute respiratory symptoms among New York city area adolescents</article-title>. <source>Environ Health Perspect.</source> (<year>2010</year>) <volume>118</volume>:<fpage>1338</fpage>–<lpage>43</lpage>. <pub-id pub-id-type=\"doi\">10.1289/ehp.0901499</pub-id><pub-id pub-id-type=\"pmid\">20452882</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nemmar</surname><given-names>A</given-names></name><name><surname>Al-Maskari</surname><given-names>S</given-names></name><name><surname>Ali</surname><given-names>BH</given-names></name><name><surname>Al-Amri</surname><given-names>IS</given-names></name></person-group>. <article-title>Cardiovascular and lung inflammatory effects induced by systemically administered diesel exhaust particles in rats</article-title>. <source>Am J Physiol Lung Cell Mol Physiol.</source> (<year>2007</year>) <volume>292</volume>:<fpage>L664</fpage>–<lpage>70</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajplung.00240.2006</pub-id><pub-id pub-id-type=\"pmid\">17085524</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cassee</surname><given-names>FR</given-names></name><name><surname>Héroux</surname><given-names>ME</given-names></name><name><surname>Gerlofs-Nijland</surname><given-names>ME</given-names></name><name><surname>Kelly</surname><given-names>FJ</given-names></name></person-group>. <article-title>Particulate matter beyond mass: recent health evidence on the role of fractions, chemical constituents and sources of emission</article-title>. <source>Inhal Toxicol.</source> (<year>2013</year>) <volume>25</volume>:<fpage>802</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.3109/08958378.2013.850127</pub-id><pub-id pub-id-type=\"pmid\">24304307</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Donaldson</surname><given-names>K</given-names></name><name><surname>Poland</surname><given-names>CA</given-names></name></person-group>. <article-title>Inhaled nanoparticles and lung cancer - what we can learn from conventional particle toxicology</article-title>. <source>Swiss Med Wkly.</source> (<year>2012</year>) <volume>142</volume>:<fpage>w13547</fpage>. <pub-id pub-id-type=\"doi\">10.4414/smw.2012.13547</pub-id><pub-id pub-id-type=\"pmid\">22714122</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hamanaka</surname><given-names>RB</given-names></name><name><surname>Mutlu</surname><given-names>GM</given-names></name></person-group>. <article-title>Particulate matter air pollution: effects on the cardiovascular system</article-title>. <source>Front Endocrinol.</source> (<year>2018</year>) <volume>9</volume>:<fpage>680</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fendo.2018.00680</pub-id><pub-id pub-id-type=\"pmid\">30505291</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kelly</surname><given-names>FJ</given-names></name><name><surname>Fussell</surname><given-names>JC</given-names></name></person-group>. <article-title>Air pollution and airway disease</article-title>. <source>Clin Exp Allergy.</source> (<year>2011</year>) <volume>41</volume>:<fpage>1059</fpage>–<lpage>71</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-2222.2011.03776.x</pub-id><pub-id pub-id-type=\"pmid\">21623970</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shah</surname><given-names>AS</given-names></name><name><surname>Langrish</surname><given-names>JP</given-names></name><name><surname>Nair</surname><given-names>H</given-names></name><name><surname>McAllister</surname><given-names>DA</given-names></name><name><surname>Hunter</surname><given-names>AL</given-names></name><name><surname>Donaldson</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Global association of air pollution and heart failure: a systematic review and meta-analysis</article-title>. <source>Lancet.</source> (<year>2013</year>) <volume>382</volume>:<fpage>1039</fpage>–<lpage>48</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0140-6736(13)60898-3</pub-id><pub-id pub-id-type=\"pmid\">23849322</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Turner</surname><given-names>MC</given-names></name><name><surname>Krewski</surname><given-names>D</given-names></name><name><surname>Pope</surname><given-names>CA</given-names><suffix>3rd</suffix></name><name><surname>Chen</surname><given-names>Y</given-names></name><name><surname>Gapstur</surname><given-names>SM</given-names></name><name><surname>Thun</surname><given-names>MJ</given-names></name></person-group>. <article-title>Long-term ambient fine particulate matter air pollution and lung cancer in a large cohort of never-smokers</article-title>. <source>Am J Respir Crit Care Med.</source> (<year>2011</year>) <volume>184</volume>:<fpage>1374</fpage>–<lpage>81</lpage>. <pub-id pub-id-type=\"doi\">10.1164/rccm.201106-1011OC</pub-id><pub-id pub-id-type=\"pmid\">21980033</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><collab>IARC</collab></person-group> (<year>2012</year>). <source>IARC Monographs on Evaluation of Carcinogenic Risks to Humans. Volume 105: Diesel and Gasoline Engine Exhausts and Some Nitroarenes</source>. Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.iarc.fr/en/media-centre/iarcnews/2012/mono105-info.php\">http://www.iarc.fr/en/media-centre/iarcnews/2012/mono105-info.php</ext-link> (accessed June 30, 2019).</mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pope</surname><given-names>CA</given-names><suffix>3rd</suffix></name><name><surname>Burnett</surname><given-names>RT</given-names></name><name><surname>Thurston</surname><given-names>GD</given-names></name><name><surname>Thun</surname><given-names>MJ</given-names></name><name><surname>Calle</surname><given-names>EE</given-names></name><name><surname>Krewski</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Cardiovascular mortality and long-term exposure to particulate air pollution: epidemiological evidence of general pathophysiological pathways of disease</article-title>. <source>Circulation.</source> (<year>2004</year>) <volume>109</volume>:<fpage>71</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1161/01.CIR.0000108927.80044.7F</pub-id><pub-id pub-id-type=\"pmid\">14676145</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Miller</surname><given-names>KA</given-names></name><name><surname>Siscovick</surname><given-names>DS</given-names></name><name><surname>Sheppard</surname><given-names>L</given-names></name><name><surname>Shepherd</surname><given-names>K</given-names></name><name><surname>Sullivan</surname><given-names>JH</given-names></name><name><surname>Anderson</surname><given-names>GL</given-names></name><etal/></person-group>. <article-title>Long-term exposure to air pollution and incidence of cardiovascular events in women</article-title>. <source>N Engl J Med.</source> (<year>2007</year>) <volume>356</volume>:<fpage>447</fpage>–<lpage>58</lpage>. <pub-id pub-id-type=\"doi\">10.1056/NEJMoa054409</pub-id><pub-id pub-id-type=\"pmid\">17267905</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Calderón-Garcidueñas</surname><given-names>L</given-names></name><name><surname>Solt</surname><given-names>AC</given-names></name><name><surname>Henríquez-Roldán</surname><given-names>C</given-names></name><name><surname>Torres-Jardón</surname><given-names>R</given-names></name><name><surname>Nuse</surname><given-names>B</given-names></name><name><surname>Herritt</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Long-term air pollution exposure is associated with neuroinflammation, an altered innate immune response, disruption of the blood-brain barrier, ultrafine particulate deposition, and accumulation of amyloid beta-42 and alpha-synuclein in children and young adults</article-title>. <source>Toxicol Pathol.</source> (<year>2008</year>) <volume>36</volume>:<fpage>289</fpage>–<lpage>310</lpage>. <pub-id pub-id-type=\"doi\">10.1177/0192623307313011</pub-id><pub-id pub-id-type=\"pmid\">18349428</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Di Bonaventura</surname><given-names>M</given-names></name><name><surname>Nicolucci</surname><given-names>A</given-names></name><name><surname>Meincke</surname><given-names>H</given-names></name><name><surname>Le Lay</surname><given-names>A</given-names></name><name><surname>Fournier</surname><given-names>J</given-names></name></person-group>. <article-title>Obesity in Germany and Italy: prevalence, comorbidities, and associations with patient outcomes</article-title>. <source>Clinicoecon Outcomes Res.</source> (<year>2018</year>) <volume>10</volume>:<fpage>457</fpage>–<lpage>75</lpage>. <pub-id pub-id-type=\"doi\">10.2147/CEOR.S157673</pub-id><pub-id pub-id-type=\"pmid\">30197528</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xu</surname><given-names>X</given-names></name><name><surname>Yavar</surname><given-names>Z</given-names></name><name><surname>Verdin</surname><given-names>M</given-names></name><name><surname>Ying</surname><given-names>Z</given-names></name><name><surname>Mihai</surname><given-names>G</given-names></name><name><surname>Kampfrath</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>Effect of early particulate air pollution exposure on obesity in mice: role of p47phox</article-title>. <source>Arterioscler Thromb Vasc Biol.</source> (<year>2010</year>) <volume>30</volume>:<fpage>2518</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.1161/ATVBAHA.110.215350</pub-id><pub-id pub-id-type=\"pmid\">20864666</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Lane</surname><given-names>KJ</given-names></name><name><surname>Corlin</surname><given-names>L</given-names></name><name><surname>Patton</surname><given-names>AP</given-names></name><name><surname>Durant</surname><given-names>JL</given-names></name><name><surname>Thanikachalam</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Association of long-term near-highway exposure to ultrafine particles with cardiovascular diseases, diabetes and hypertension</article-title>. <source>Int J Environ Res Public Health.</source> (<year>2017</year>) <volume>14</volume>:<fpage>E461</fpage>. <pub-id pub-id-type=\"doi\">10.3390/ijerph14050461</pub-id><pub-id pub-id-type=\"pmid\">28445425</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Toledo-Corral</surname><given-names>CM</given-names></name><name><surname>Alderete</surname><given-names>TL</given-names></name><name><surname>Habre</surname><given-names>R</given-names></name><name><surname>Berhane</surname><given-names>K</given-names></name><name><surname>Lurmann</surname><given-names>FW</given-names></name><name><surname>Weigensberg</surname><given-names>MJ</given-names></name><etal/></person-group>. <article-title>Effects of air pollution exposure on glucose metabolism in Los Angeles minority children</article-title>. <source>Pediatr Obes.</source> (<year>2018</year>) <volume>13</volume>:<fpage>54</fpage>–<lpage>62</lpage>. <pub-id pub-id-type=\"doi\">10.1111/ijpo.12188</pub-id><pub-id pub-id-type=\"pmid\">27923100</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dimakakou</surname><given-names>E</given-names></name><name><surname>Johnston</surname><given-names>HJ</given-names></name><name><surname>Streftaris</surname><given-names>G</given-names></name><name><surname>Cherrie</surname><given-names>JW</given-names></name></person-group>. <article-title>Exposure to environmental and occupational particulate air pollution as a potential contributor to neurodegeneration and diabetes: a systematic review of epidemiological research</article-title>. <source>Int J Environ Res Public Health.</source> (<year>2018</year>) <volume>15</volume>:<fpage>E1704</fpage>. <pub-id pub-id-type=\"doi\">10.3390/ijerph15081704</pub-id><pub-id pub-id-type=\"pmid\">30096929</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jeong</surname><given-names>A</given-names></name><name><surname>Fiorito</surname><given-names>G</given-names></name><name><surname>Keski-Rahkonen</surname><given-names>P</given-names></name><name><surname>Imboden</surname><given-names>M</given-names></name><name><surname>Kiss</surname><given-names>A</given-names></name><name><surname>Robinot</surname><given-names>N</given-names></name><etal/></person-group>. <article-title>Perturbation of metabolic pathways mediates the association of air pollutants with asthma and cardiovascular diseases</article-title>. <source>Environ Int.</source> (<year>2018</year>) <volume>119</volume>:<fpage>334</fpage>–<lpage>45</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.envint.2018.06.025</pub-id><pub-id pub-id-type=\"pmid\">29990954</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Calderón-Garcidueñas</surname><given-names>L</given-names></name><name><surname>Franco-Lira</surname><given-names>M</given-names></name><name><surname>D'Angiulli</surname><given-names>A</given-names></name><name><surname>Rodríguez-Díaz</surname><given-names>J</given-names></name><name><surname>Blaurock-Busch</surname><given-names>E</given-names></name><name><surname>Busch</surname><given-names>Y</given-names></name><etal/></person-group>. <article-title>Mexico City normal weight children exposed to high concentrations of ambient PM2.5 show high blood leptin and endothelin-1, vitamin D deficiency, and food reward hormone dysregulation versus low pollution controls. Relevance for obesity and Alzheimer disease</article-title>. <source>Environ Res.</source> (<year>2015</year>) <volume>140</volume>:<fpage>579</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.envres.2015.05.012</pub-id><pub-id pub-id-type=\"pmid\">26037109</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>JH</given-names></name><name><surname>Reed</surname><given-names>DR</given-names></name><name><surname>Price</surname><given-names>RA</given-names></name></person-group>. <article-title>Leptin resistance is associated with extreme obesity and aggregates in families</article-title>. <source>Int J Obes Relat Metab Disord.</source> (<year>2001</year>) <volume>25</volume>:<fpage>1471</fpage>–<lpage>3</lpage>. <pub-id pub-id-type=\"doi\">10.1038/sj.ijo.0801736</pub-id><pub-id pub-id-type=\"pmid\">11673768</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Molfino</surname><given-names>A</given-names></name><name><surname>Kaysen</surname><given-names>GA</given-names></name><name><surname>Chertow</surname><given-names>GM</given-names></name><name><surname>Doyle</surname><given-names>J</given-names></name><name><surname>Delgado</surname><given-names>C</given-names></name><name><surname>Dwyer</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>Validating appetite assessment tools among patients receiving hemodialysis</article-title>. <source>J Ren Nutr.</source> (<year>2016</year>) <volume>26</volume>:<fpage>103</fpage>–<lpage>10</lpage>. <pub-id pub-id-type=\"doi\">10.1053/j.jrn.2015.09.002</pub-id><pub-id pub-id-type=\"pmid\">26522141</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Molfino</surname><given-names>A</given-names></name><name><surname>Rossi Fanelli</surname><given-names>F</given-names></name><name><surname>Laviano</surname><given-names>A</given-names></name></person-group>. <article-title>Sympathetic nervous system activity may link hyperphagia and fat deposition in human obesity</article-title>. <source>Am J Physiol Endocrinol Metab.</source> (<year>2007</year>) <volume>293</volume>:<fpage>E1129</fpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpendo.00500.2007</pub-id><pub-id pub-id-type=\"pmid\">17911351</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Su</surname><given-names>DS</given-names></name><name><surname>Serafino</surname><given-names>A</given-names></name><name><surname>Müller</surname><given-names>JO</given-names></name><name><surname>Jentoft</surname><given-names>RE</given-names></name><name><surname>Schlögl</surname><given-names>R</given-names></name><name><surname>Fiorito</surname><given-names>S</given-names></name></person-group>. <article-title>Cytotoxicity and inflammatory potential of soot particles of low-emission diesel engines</article-title>. <source>Environ Sci Technol.</source> (<year>2008</year>) <volume>42</volume>:<fpage>1761</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1021/es0716554</pub-id><pub-id pub-id-type=\"pmid\">18441832</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lawal</surname><given-names>AO</given-names></name></person-group>. <article-title>Diesel exhaust particles and the induction of macrophage activation and dysfunction</article-title>. <source>Inflammation.</source> (<year>2018</year>) <volume>41</volume>:<fpage>356</fpage>–<lpage>63</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s10753-017-0682-6</pub-id><pub-id pub-id-type=\"pmid\">29047037</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tseng</surname><given-names>CY</given-names></name><name><surname>Wang</surname><given-names>JS</given-names></name><name><surname>Chao</surname><given-names>MW</given-names></name></person-group>. <article-title>Causation by diesel exhaust particles of endothelial dysfunctions in cytotoxicity, pro-inflammation, permeability, and apoptosis induced by ROS generation</article-title>. <source>Cardiovasc Toxicol.</source> (<year>2017</year>) <volume>17</volume>:<fpage>384</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s12012-016-9364-0</pub-id><pub-id pub-id-type=\"pmid\">26965709</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fiorito</surname><given-names>S</given-names></name><name><surname>Mastrofrancesco</surname><given-names>A</given-names></name><name><surname>Cardinali</surname><given-names>G</given-names></name><name><surname>Rosato</surname><given-names>E</given-names></name><name><surname>Salsano</surname><given-names>F</given-names></name><name><surname>Su</surname><given-names>DS</given-names></name><etal/></person-group>\n<article-title>Effects of carbonaceous nanoparticles from low-emission and older diesel engines on human skin cells</article-title>. <source>Carbon.</source> (<year>2011</year>) <volume>49</volume>:<fpage>5038</fpage>–<lpage>48</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.carbon.2011.07.022</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mastrofrancesco</surname><given-names>A</given-names></name><name><surname>Alfè</surname><given-names>M</given-names></name><name><surname>Rosato</surname><given-names>E</given-names></name><name><surname>Gargiulo</surname><given-names>V</given-names></name><name><surname>Beatrice</surname><given-names>C</given-names></name><name><surname>Di Blasio</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Proinflammatory effects of diesel exhaust nanoparticles on scleroderma skin cells</article-title>. <source>J Immunol Res.</source> (<year>2014</year>) <volume>2014</volume>:<fpage>138751</fpage>\n<pub-id pub-id-type=\"doi\">10.1155/2014/138751</pub-id><pub-id pub-id-type=\"pmid\">24982919</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Steiner</surname><given-names>S</given-names></name><name><surname>Bisiq</surname><given-names>C</given-names></name><name><surname>Petri-Fink</surname><given-names>A</given-names></name><name><surname>Rothen-Rutishauer</surname><given-names>B</given-names></name></person-group>. <article-title>Diesel exhaust: current knowledge of adverse effects and underlying cellular mechanisms</article-title>. <source>Arch Toxicol.</source> (<year>2016</year>) <volume>90</volume>:<fpage>1541</fpage>–<lpage>53</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00204-016-1736-5</pub-id><pub-id pub-id-type=\"pmid\">27165416</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arita</surname><given-names>Y</given-names></name><name><surname>Kihara</surname><given-names>S</given-names></name><name><surname>Ouchi</surname><given-names>N</given-names></name><name><surname>Takahashi</surname><given-names>M</given-names></name><name><surname>Maeda</surname><given-names>K</given-names></name><name><surname>Miyagawa</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity</article-title>. <source>Biochem Biophys Res Commun.</source> (<year>1999</year>) <volume>257</volume>:<fpage>79</fpage>–<lpage>83</lpage>. <pub-id pub-id-type=\"doi\">10.1006/bbrc.1999.0255</pub-id><pub-id pub-id-type=\"pmid\">10092513</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dietz</surname><given-names>JJ</given-names></name><name><surname>Iglesias</surname><given-names>P</given-names></name></person-group>. <article-title>The role of the novel adipocyte derived hormone adiponectin in human disease</article-title>. <source>Eur J Endocrinol.</source> (<year>2003</year>) <volume>148</volume>:<fpage>293</fpage>–<lpage>300</lpage>. <pub-id pub-id-type=\"doi\">10.1530/eje.0.1480293</pub-id><pub-id pub-id-type=\"pmid\">12611609</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ohashi</surname><given-names>K</given-names></name><name><surname>Ouchi</surname><given-names>N</given-names></name><name><surname>Matsuzawa</surname><given-names>Y</given-names></name></person-group>. <article-title>Anti-inflammatory and anti-atherogenic properties of adiponectin</article-title>. <source>Biochimie.</source> (<year>2012</year>) <volume>94</volume>:<fpage>2137</fpage>–<lpage>42</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.biochi.2012.06.008</pub-id><pub-id pub-id-type=\"pmid\">22713764</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yamauchi</surname><given-names>T</given-names></name><name><surname>Kamon</surname><given-names>J</given-names></name><name><surname>Waki</surname><given-names>H</given-names></name><name><surname>Terauchi</surname><given-names>Y</given-names></name><name><surname>Kubota</surname><given-names>N</given-names></name><name><surname>Hara</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity</article-title>. <source>Nat Med.</source> (<year>2001</year>) <volume>7</volume>:<fpage>941</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1038/90984</pub-id><pub-id pub-id-type=\"pmid\">11479627</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yamamoto</surname><given-names>S</given-names></name><name><surname>Matsushita</surname><given-names>Y</given-names></name><name><surname>Nakagawa</surname><given-names>T</given-names></name><name><surname>Hayashi</surname><given-names>T</given-names></name><name><surname>Noda</surname><given-names>M</given-names></name><name><surname>Mizoue</surname><given-names>T</given-names></name></person-group>. <article-title>Circulating adiponectin levels and risk of type 2 diabetes in the Japanese</article-title>. <source>Nutr Diabetes.</source> (<year>2014</year>) <volume>4</volume>:<fpage>e130</fpage>. <pub-id pub-id-type=\"doi\">10.1038/nutd.2014.27</pub-id><pub-id pub-id-type=\"pmid\">25133442</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manzo</surname><given-names>ND</given-names></name><name><surname>LaGier</surname><given-names>AJ</given-names></name><name><surname>Slade</surname><given-names>R</given-names></name><name><surname>Ledbetter</surname><given-names>AD</given-names></name><name><surname>Richards</surname><given-names>JH</given-names></name><name><surname>Dye</surname><given-names>JA</given-names></name></person-group>. <article-title>Nitric oxide and superoxide mediate diesel particle effects in cytokine-treated mice and murine lung epithelial cells—implications for susceptibility to traffic- related air pollution</article-title>. <source>Part Fibre Toxicol.</source> (<year>2012</year>) <volume>9</volume>:<fpage>43</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1743-8977-9-43</pub-id><pub-id pub-id-type=\"pmid\">23151036</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Uski</surname><given-names>OJ</given-names></name><name><surname>Happo</surname><given-names>MS</given-names></name><name><surname>Jalava</surname><given-names>PI</given-names></name><name><surname>Brunner</surname><given-names>T</given-names></name><name><surname>Kelz</surname><given-names>J</given-names></name><name><surname>Obernberger</surname><given-names>I</given-names></name><etal/></person-group>. <article-title>Acute systemic and lung inflammation in C57Bl/6J mice after intratracheal aspiration of particulate matter from small-scale biomass combustion appliances based on old and modern technologies</article-title>. <source>Inhal Toxicol.</source> (<year>2012</year>) <volume>24</volume>:<fpage>952</fpage>–<lpage>65</lpage>. <pub-id pub-id-type=\"doi\">10.3109/08958378.2012.742172</pub-id><pub-id pub-id-type=\"pmid\">23216156</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manzetti</surname><given-names>S</given-names></name><name><surname>Andersen</surname><given-names>O</given-names></name></person-group>. <article-title>Biochemical and physiological effects from exhaust emissions. A review of the relevant literature</article-title>. <source>Pathophysiology.</source> (<year>2016</year>) <volume>23</volume>:<fpage>285</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.pathophys.2016.10.002</pub-id><pub-id pub-id-type=\"pmid\">27793419</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grundy</surname><given-names>SM</given-names></name><name><surname>Cleeman</surname><given-names>JI</given-names></name><name><surname>Daniels</surname><given-names>SR</given-names></name><name><surname>Donato</surname><given-names>KA</given-names></name><name><surname>Eckel</surname><given-names>RH</given-names></name><name><surname>Franklin</surname><given-names>BA</given-names></name><etal/></person-group>. <article-title>Diagnosis and management of the metabolic syndrome: an american heart association/national heart, lung, and blood institute scientific statement</article-title>. <source>Circulation.</source> (<year>2005</year>) <volume>112</volume>:<fpage>2735</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1161/CIRCULATIONAHA.105.169404</pub-id><pub-id pub-id-type=\"pmid\">16157765</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>W</given-names></name><name><surname>Wang</surname><given-names>L</given-names></name><name><surname>Li</surname><given-names>J</given-names></name><name><surname>Liu</surname><given-names>M</given-names></name><name><surname>Xu</surname><given-names>H</given-names></name><name><surname>Liu</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Short-term blood pressure responses to ambient fine particulate matter exposures at the extremes of global air pollution concentrations</article-title>. <source>Am J Hypertens.</source> (<year>2018</year>) <volume>31</volume>:<fpage>590</fpage>–<lpage>99</lpage>. <pub-id pub-id-type=\"doi\">10.1093/ajh/hpx216</pub-id><pub-id pub-id-type=\"pmid\">29409056</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>S</given-names></name><name><surname>Brook</surname><given-names>RD</given-names></name><name><surname>Huang</surname><given-names>W</given-names></name><name><surname>Fan</surname><given-names>Z</given-names></name><name><surname>Xu</surname><given-names>H</given-names></name><name><surname>Wu</surname><given-names>R</given-names></name><etal/></person-group>. <article-title>Extreme levels of ambient air pollution adversely impact cardiac and central aortic hemodynamics: the AIRCMD-China study</article-title>. <source>J Am Soc Hypertens.</source> (<year>2017</year>) <volume>11</volume>:<fpage>754</fpage>–<lpage>61</lpage>.e3. <pub-id pub-id-type=\"doi\">10.1016/j.jash.2017.09.009</pub-id><pub-id pub-id-type=\"pmid\">29031802</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dabass</surname><given-names>A</given-names></name><name><surname>Talbott</surname><given-names>EO</given-names></name><name><surname>Rager</surname><given-names>JR</given-names></name><name><surname>Marsh</surname><given-names>GM</given-names></name><name><surname>Venkat</surname><given-names>A</given-names></name><name><surname>Holguin</surname><given-names>F</given-names></name><etal/></person-group>\n<article-title>Systemic inflammatory markers associated with cardiovascular disease and acute and chronic exposure to fine particulate matter air pollution (PM2.5) among US NHANES adults with metabolic syndrome</article-title>. <source>Environ Res.</source> (<year>2018</year>) <volume>161</volume>:<fpage>485</fpage>–<lpage>91</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.envres.2017.11.042</pub-id><pub-id pub-id-type=\"pmid\">29223110</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wei</surname><given-names>Y</given-names></name><name><surname>Zhang</surname><given-names>JJ</given-names></name><name><surname>Li</surname><given-names>Z</given-names></name><name><surname>Gow</surname><given-names>A</given-names></name><name><surname>Chung</surname><given-names>KF</given-names></name><name><surname>Hu</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Chronic exposure to air pollution particles increases the risk of obesity and metabolic syndrome: findings from a natural experiment in Beijing</article-title>. <source>FASEB J.</source> (<year>2016</year>) <volume>30</volume>:<fpage>2115</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.1096/fj.201500142</pub-id><pub-id pub-id-type=\"pmid\">26891735</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eze</surname><given-names>IC</given-names></name><name><surname>Schaffner</surname><given-names>E</given-names></name><name><surname>Foraster</surname><given-names>M</given-names></name><name><surname>Imboden</surname><given-names>M</given-names></name><name><surname>von Eckardstein</surname><given-names>A</given-names></name><name><surname>Gerbase</surname><given-names>MW</given-names></name><etal/></person-group>. <article-title>Long-term exposure to ambient air pollution and metabolic syndrome in adults</article-title>. <source>PLoS ONE.</source> (<year>2015</year>) <volume>10</volume>:<fpage>e0130337</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0130337</pub-id><pub-id pub-id-type=\"pmid\">26103580</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hug</surname><given-names>C</given-names></name><name><surname>Wang</surname><given-names>J</given-names></name><name><surname>Ahmad</surname><given-names>NS</given-names></name><name><surname>Bogan</surname><given-names>JS</given-names></name><name><surname>Tsao</surname><given-names>TS</given-names></name><name><surname>Lodish</surname><given-names>HF</given-names></name></person-group>. <article-title>T-cadherin is a receptor for hexameric and high-molecular-weight forms of Acrp30/adiponectin</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2004</year>) <volume>101</volume>:<fpage>10308</fpage>–<lpage>13</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.0403382101</pub-id><pub-id pub-id-type=\"pmid\">15210937</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Imboden</surname><given-names>M</given-names></name><name><surname>Kumar</surname><given-names>A</given-names></name><name><surname>Curjuric</surname><given-names>I</given-names></name><name><surname>Adam</surname><given-names>M</given-names></name><name><surname>Thun</surname><given-names>GA</given-names></name><name><surname>Haun</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Modification of the association between PM10 and lung function decline by cadherin 13 polymorphisms in the SAPALDIA cohort: a genome-wide interaction analysis</article-title>. <source>Environ Health Perspect.</source> (<year>2015</year>) <volume>123</volume>:<fpage>72</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1289/ehp.1307398</pub-id><pub-id pub-id-type=\"pmid\">25127211</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>S</given-names></name><name><surname>Park</surname><given-names>H</given-names></name><name><surname>Kim</surname><given-names>S</given-names></name><name><surname>Lee</surname><given-names>EK</given-names></name><name><surname>Lee</surname><given-names>J</given-names></name><name><surname>Hong</surname><given-names>YS</given-names></name><etal/></person-group>. <article-title>Fine particulate matter and incidence of metabolic syndrome in non-CVD patients: a nationwide population-based cohort study</article-title>. <source>Int J Hyg Environ Health.</source> (<year>2019</year>) <volume>222</volume>:<fpage>533</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijheh.2019.01.010</pub-id><pub-id pub-id-type=\"pmid\">30797734</pub-id></mixed-citation></ref><ref id=\"B48\"><label>48.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>BY</given-names></name><name><surname>Qian</surname><given-names>Z</given-names></name><name><surname>Howard</surname><given-names>SW</given-names></name><name><surname>Vaughn</surname><given-names>MG</given-names></name><name><surname>Fan</surname><given-names>SJ</given-names></name><name><surname>Liu</surname><given-names>KK</given-names></name><etal/></person-group>. <article-title>Global association between ambient air pollution and blood pressure: a systematic review and meta-analysis</article-title>. <source>Environ Pollut.</source> (<year>2018</year>) <volume>235</volume>:<fpage>576</fpage>–<lpage>88</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.envpol.2018.01.001</pub-id><pub-id pub-id-type=\"pmid\">29331891</pub-id></mixed-citation></ref><ref id=\"B49\"><label>49.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Molfino</surname><given-names>A</given-names></name><name><surname>Muscaritoli</surname><given-names>M</given-names></name><name><surname>Germano</surname><given-names>A</given-names></name><name><surname>Ramaccini</surname><given-names>C</given-names></name><name><surname>Alfano</surname><given-names>R</given-names></name><name><surname>Spagnoli</surname><given-names>A</given-names></name><etal/></person-group>\n<article-title>Changes in metabolic, nutritional and hormonal profile among subjects professionally exposed to urban pollution in a high intensity traffic area</article-title>. <source>J Cachexia Sarcopenia Muscle.</source> (<year>2019</year>) <volume>10</volume>:<fpage>1378</fpage>–<lpage>435</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jcsm.12420</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"case-report\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Endocrinol.</journal-id><journal-title-group><journal-title>Frontiers in Endocrinology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2392</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849297</article-id><article-id pub-id-type=\"pmc\">PMC7431615</article-id><article-id pub-id-type=\"doi\">10.3389/fendo.2020.00512</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology</subject><subj-group><subject>Case Report</subject></subj-group></subj-group></article-categories><title-group><article-title>Contrast-Enhanced Ultrasound of Primary Squamous Cell Carcinoma of the Thyroid: A Case Report</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Sijie</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/957361/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Peng</surname><given-names>Qinghai</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/978450/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Qi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/977972/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Niu</surname><given-names>Chengcheng</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/852962/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Ultrasound Diagnosis, The Second Xiangya Hospital, Central South University</institution>, <addr-line>Changsha</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Research Center of Ultrasonography, The Second Xiangya Hospital, Central South University</institution>, <addr-line>Changsha</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Christoph Reiners, University Hospital Würzburg, Germany</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Pasqualino Malandrino, University of Catania, Italy; Daniela Pasquali, University of Campania Luigi Vanvitelli, Italy</p></fn><corresp id=\"c001\">*Correspondence: Chengcheng Niu <email>niuchengcheng@csu.edu.cn</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Cancer Endocrinology, a section of the journal Frontiers in Endocrinology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>512</elocation-id><history><date date-type=\"received\"><day>21</day><month>2</month><year>2020</year></date><date date-type=\"accepted\"><day>25</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Chen, Peng, Zhang and Niu.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Chen, Peng, Zhang and Niu</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p><bold>Introduction:</bold> Primary squamous cell carcinoma of the thyroid (ThyPSCC) is an extremely rare aggressive malignancy with a poor prognosis. However, almost no report thus far has investigated the microvasculature of ThyPSCC imaged using contrast-enhanced ultrasound.</p><p><bold>Case Report:</bold> A 59-year-old male patient presented to our hospital with progressively worsening hoarse voice symptoms for 20 days and was diagnosed with left unilateral vocal fold palsy. Ultrasonography revealed a solitary marked hypoechoic thyroid nodule with an unclear boundary in the inferior part of the left lobe. Color Doppler flow imaging showed a poor blood flow signal inside this nodule. Contrast-enhanced ultrasound images showed a persistent low peak enhancement of the nodule from its periphery to its center. The time-intensity curve displayed a wash-in time of 10 s, a time to peak of 37 s, a peak signal intensity of 24.5%, and a wash-out time of 70 s for the thyroid tumor. Finally, left hemithyroidectomy of the thyroid tumor was performed, and histopathologic and immunohistochemical evaluations confirmed the diagnosis of ThyPSCC. Postoperatively, the patient received a combination therapy of chemotherapy, radiotherapy, and targeted therapy, but the patient died 4 months after surgery.</p><p><bold>Conclusion:</bold> Primary squamous cell carcinoma of the thyroid is a rare but aggressive malignancy of the thyroid. Herein, we reported a case of ThyPSCC and its ultrasonography and pathologic findings.</p></abstract><kwd-group><kwd>thyroid cancer</kwd><kwd>thyroid nodules (TNs)</kwd><kwd>thyroid ultrasound (US)</kwd><kwd>primary squamous cell carcinoma</kwd><kwd>contrast enhanced ultrasound (CEUS)</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Natural Science Foundation of China<named-content content-type=\"fundref-id\">10.13039/501100001809</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"5\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"22\"/><page-count count=\"6\"/><word-count count=\"3011\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Primary squamous cell carcinoma of the thyroid (ThyPSCC) is a rare thyroid malignancy with high aggressiveness and poor prognosis, comprising ~0.1–1% of all primary thyroid carcinomas (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>–<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). Owing to the rapidly progressing and highly invasive nature of the malignancy, patients with ThyPSCC often present at an advanced stage and are difficult to diagnose in the early stage because of its rare incidence and lack of typical imaging findings (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B8\" ref-type=\"bibr\">8</xref>).</p><p>Thyroid ultrasonography and fine-needle aspiration biopsy (FNAB) are the diagnostic tools of choice for evaluating patients with suspected thyroid nodules (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). Contrast-enhanced ultrasound (CEUS), as a relatively novel US technique, is used to investigate the microvasculature of thyroid nodules and improve the diagnostic accuracy of thyroid nodules accompanied by the use of Thyroid Imaging Reporting and Data Systems for ultrasonographic features (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>–<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). However, very few published studies have reported the use of ultrasonography for ThyPSCC. To our knowledge, this is the first case describing the CEUS findings of ThyPSCC.</p></sec><sec id=\"s2\"><title>Case Report</title><p>A 59-year-old male patient presented to our hospital with progressively worsening hoarse voice symptoms for 20 days and was diagnosed with left unilateral vocal fold palsy. A high-resolution ultrasound instrument (Siemens Acuson S3000, Mountain View, CA, USA) equipped with a 4- to 9-MHz linear probe was used. Thyroid ultrasonography revealed a solitary 3.1 × 2.8 × 2.6-cm<sup>3</sup> marked hypoechoic thyroid nodule with an unclear boundary in the inferior part of the left lobe (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). This nodule exhibited many malignant ultrasound features, such as solid components, hypoechogenicity, and microlobulated margins. Color Doppler flow imaging (CDFI) showed poor blood flow signals in the nodule (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). Contrast-enhanced ultrasound was performed with a bolus intravenous injection of 3.0 mL of SonoVue (Bracco, Milan, Italy) followed by 5 mL of saline. Contrast pulse sequencing technology was used, and the time-intensity curves (TICs) of the nodule were calculated. The nodule began to be slowly enhanced from the periphery to the center at 10 s (wash-in time), and the enhancement reached its peak [time to peak (TTP)] at 37 s with a peak intensity of 24.5%. Then, the nodule slowly declined until all the microbubbles washed out at 70 s (<xref ref-type=\"fig\" rid=\"F1\">Figures 1C,D</xref>). Based on its malignant conventional ultrasound features and the poor microvasculature revealed by CEUS, we inferred that the nodule was a malignant tumor.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Ultrasonography images of primary squamous cell carcinoma of the thyroid. <bold>(A)</bold> Longitudinal gray-scale sonography revealed a solid marked hypoechoic thyroid nodule in the inferior part of the left lobe. <bold>(B)</bold> Color Doppler flow imaging showed a poor blood flow signal inside this nodule. <bold>(C)</bold> Contrast-enhanced ultrasound image showed a persistent low peak enhancement of the nodule at 37 s. <bold>(D)</bold> Time-intensity curves displayed the wash-in time of 10 s, TTP of 37 s, peak signal intensity of 24.5%, and wash-out time of 70 s for the thyroid tumor.</p></caption><graphic xlink:href=\"fendo-11-00512-g0001\"/></fig><p>After neck ultrasonography, the positron emission tomography–computed tomography was carried for evaluating the situation of distant metastases. Positron emission tomography–computed tomography showed a mass with increased glucose metabolism in the inferior part of the left thyroid lobe (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>), which indicated it as a malignant mass, whereas there was no evidence of lymph nodes metastasis and distant metastases. Then, ultrasonography-guided FNAB was performed for the left thyroid mass immediately. Cytologic examination by fine-needle aspiration (FNA) revealed sheets of tumor cells with giant deep-stained nuclei (Bethesda category V) (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). Finally, a left hemithyroidectomy of the thyroid tumor was undertaken. The lower edge of the tumor reached the upper mediastinum, and the depth of the tumor invaded the esophagus and trachea, which could not be completely removed. According to the eighth edition of the American Joint Committee on Cancer/Tumor Lymph Node Metastasis (TNM) staging system (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>), the patient was in TNM stage III (T4a N0 M0). Histopathological examination of hematoxylin and eosin staining showed that a carcinoma in the inferior part of the thyroid lobe (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>) had no obvious palisade arrangement, intercellular bridges, or keratinization with a cancer pearl (<xref ref-type=\"fig\" rid=\"F3\">Figures 3B–D</xref>). Immunohistochemically, tumor cells were positive for cytokeratin 19 (CK19, <xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>), cytokeratin 5 and 6 (CK5/6, <xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>), epithelial membrane antigen (EMA, <xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>), p40 (<xref ref-type=\"fig\" rid=\"F4\">Figure 4D</xref>), p63 (<xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>), and Ki-67 (30%+, <xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>) and negative for thyroglobulin (TG, <xref ref-type=\"fig\" rid=\"F5\">Figure 5C</xref>) and thyroid transcription factor 1 (TTF-1, <xref ref-type=\"fig\" rid=\"F5\">Figure 5D</xref>). In view of these findings, the tumor was diagnosed as poorly differentiated ThyPSCC. Postoperatively, the patient received two cycles of chemotherapy with docetaxel/cisplatin, intensity-modulated radiotherapy, and nimotuzumab-targeted therapy. However, the patient died 4 months after surgery.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p><bold>(A)</bold> A positron emission tomography–computed tomography scan showed increased <sup>18</sup>F-fluorodeoxyglucose metabolism in the left neck mass. <bold>(B)</bold> Preoperative fine-needle aspiration cytology of the mass demonstrated a few sheets of malignant-looking tumor cells with giant deep stained nuclei (hematoxylin and eosin, magnification × 400).</p></caption><graphic xlink:href=\"fendo-11-00512-g0002\"/></fig><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Hematoxylin and eosin staining of primary squamous cell carcinoma of the thyroid: <bold>(A)</bold> magnification × 8, <bold>(B)</bold> magnification × 20, <bold>(C)</bold> magnification × 100, <bold>(D)</bold> magnification × 400.</p></caption><graphic xlink:href=\"fendo-11-00512-g0003\"/></fig><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Immunohistochemical staining of primary squamous cell carcinoma of the thyroid (magnification × 200). Immunohistochemical staining for <bold>(A)</bold> CK19, <bold>(B)</bold> CK5/6, <bold>(C)</bold> EMA, <bold>(D)</bold> p40, all of which were deeply stained (positive).</p></caption><graphic xlink:href=\"fendo-11-00512-g0004\"/></fig><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>Immunohistochemical staining of primary squamous cell carcinoma of the thyroid (magnification × 200). Immunohistochemical staining for <bold>(A)</bold> p63, <bold>(B)</bold> Ki 67, <bold>(C)</bold> TG, <bold>(D)</bold> TTF-1, and p63 was deeply stain (positive); Ki67 proliferation index was 30%; TG and TTF-1 did not stain (negative).</p></caption><graphic xlink:href=\"fendo-11-00512-g0005\"/></fig></sec><sec sec-type=\"discussion\" id=\"s3\"><title>Discussion</title><p>Primary squamous cell carcinoma of the thyroid is a thyroid malignancy with extremely rare incidence, and the clinical diagnosis and treatment guidelines for this disease have no consensus (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). The biological behavior of ThyPSCC is aggressive, and the prognosis is poor, with a median overall survival of 4–24 months, which depends on the different tumor grades (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Yang et al. using the Surveillance, Epidemiology, and End Results Program database, reported that poorly differentiated tumor grade occupied the highest percentages of all graded tumors, and the median survival was 4 months, which is similar to the survival time in our case (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>).</p><p>High-frequency ultrasound, as the basic imaging modality in the diagnosis of thyroid nodules, has found gradually increasing differentiated thyroid cancers over recent years (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>). The ultrasonography imaging findings of ThyPSCC have seldom been published. Regarding the ultrasonography findings, Chen et al. (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>) reported that ThyPSCC presented as a thyroid mass with eggshell calcification, peripheral soft tissue with a blurred margin, and minimal vascular signals on CDFI sonography. In the case of Jang et al. (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>), ThyPSCC presented as a large, well-defined, lobulated, heterogeneously hypoechoic mass with diffuse microcalcifications on ultrasonography. Kondo et al. (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>) reported that a well-differentiated ThyPSCC showed a cystic hypoechoic mass with a smooth margin and rapidly grew with margin change blurring in 1 year. In our case, this poorly differentiated ThyPSCC presented as a solitary marked hypoechoic thyroid mass with an irregular margin and unclear boundary with a normal thyroid. The irregular margin and unclear boundary with normal thyroid corresponded to tumor invasion with adjacent tissue infiltration, which is consistent with the findings during the operation that tumor invasion with the esophagus cannot be completely removed. Poor blood flow signals on CDFI sonography and persistent hypoenhancement on CEUS of the mass are consistent with squamous cell carcinoma, which has no obvious vascularity on pathologic examination.</p><p>Many studies have investigated the application of CEUS to improve the diagnostic accuracy of thyroid nodules, despite its usage in ThyPSCC being scarce. Zhang et al. (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>) found that high/circular/equal enhancement indicated benign thyroid nodules, and low enhancement indicated malignant thyroid nodules. Ma et al. (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>) investigated whether incomplete, no ring or heterogeneous enhancement, later wash-in time, and low peak intensity on CEUS were independent risk factors in predicting malignant thyroid nodules. Deng et al. (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>) detected that papillary thyroid carcinomas (PTCs) exhibited low enhancement, a lower peak signal intensity, and a lower area under the curve (AUC) than peripheral thyroid parenchyma on CEUS (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B20\" ref-type=\"bibr\">20</xref>). In our study, the TICs of CEUS for ThyPSCC showed a wash-in time of 10 s, a TTP of 37 s, a peak signal intensity as low as 24.5%, and a wash-out time of 70 s. This is similar to the results of PTCs with a slow wash-in time, a lower peak signal intensity, and a lower AUC, as in previous reports (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). To our knowledge, no reports on CEUS imaging findings of ThyPSCC have appeared in the English-language literature. According to Jang et al. (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>), ThyPSCC showed a large heterogeneously enhancing thyroid mass with a large central non-enhancing portion on enhanced CT, which corresponded well with the squamous cell carcinoma portion with a necrotic portion in pathologic staining. Because of the rapid growth of squamous tumor cells, relatively few interstitial blood vessels in tumors were related to the low peak signal intensity and low AUC on CEUS.</p><p>With increasing malignancy in squamous cell carcinoma, the typical squamous cell carcinoma findings of intercellular bridges and keratinized cancer pearl can decrease or disappear. Immunohistochemical staining is useful in diagnosing primary thyroid cancer. In this case, positivity for CK5/6 and EMA and negativity for TTF-1 and TG expression predicted squamous cell carcinoma derivation and excluded the possibility of these common tumors (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B21\" ref-type=\"bibr\">21</xref>). Further positivity for p63 and Ki67 expression as poor prognostic markers was associated with its poorly differentiated tumor grade (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B22\" ref-type=\"bibr\">22</xref>).</p></sec><sec sec-type=\"conclusions\" id=\"s4\"><title>Conclusion</title><p>Primary squamous cell carcinoma of the thyroid is an extremely rare tumor, and very few studies describe its ultrasonographic imaging findings. It is difficult to establish a clinical guideline for diagnosis. Our case presents the CEUS features of ThyPSCC, indicating that the TICs of ThyPSCC are similar to the enhancing parameters of PTCs with a slow wash-in time, a lower peak signal intensity, and a lower AUC.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The datasets generated for this study are available on request to the corresponding author.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by the Ethics Committee of Second Xiangya Hospital, Central South University, China. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This project was funded by the National Natural Science Foundation of China (81974267), Hunan Provincial Natural Science Foundation of China (2018JJ2575), and Hunan Provincial Health Commission Research Foundation Project (B2019166).</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>S</given-names></name><name><surname>Li</surname><given-names>C</given-names></name><name><surname>Shi</surname><given-names>X</given-names></name><name><surname>Ma</surname><given-names>B</given-names></name><name><surname>Xu</surname><given-names>W</given-names></name><name><surname>Jiang</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Primary squamous cell carcinoma in the thyroid gland: a population-based analysis using the SEER database</article-title>. <source>World J Surg.</source> (<year>2019</year>) <volume>43</volume>:<fpage>1249</fpage>–<lpage>55</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00268-019-04906-2</pub-id><pub-id pub-id-type=\"pmid\">30719559</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Limberg</surname><given-names>J</given-names></name><name><surname>Ullmann</surname><given-names>TM</given-names></name><name><surname>Stefanova</surname><given-names>D</given-names></name><name><surname>Finnerty</surname><given-names>BM</given-names></name><name><surname>Beninato</surname><given-names>T</given-names></name><name><surname>Fahey</surname><given-names>TJ</given-names></name><etal/></person-group>. <article-title>Prognostic characteristics of primary squamous cell carcinoma of the thyroid: a national cancer database analysis</article-title>. <source>World J Surg.</source> (<year>2020</year>) <volume>44</volume>:<fpage>348</fpage>–<lpage>55</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00268-019-05098-5</pub-id><pub-id pub-id-type=\"pmid\">31399796</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Koyama</surname><given-names>S</given-names></name><name><surname>Fujiwara</surname><given-names>K</given-names></name><name><surname>Nosaka</surname><given-names>K</given-names></name><name><surname>Fukuhara</surname><given-names>T</given-names></name><name><surname>Morisaki</surname><given-names>T</given-names></name><name><surname>Miyake</surname><given-names>N</given-names></name><etal/></person-group>. <article-title>Immunohistochemical features of primary pure squamous cell carcinoma in the thyroid: an autopsy case</article-title>. <source>Case Rep Oncol.</source> (<year>2018</year>) <volume>11</volume>:<fpage>418</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1159/000490410</pub-id><pub-id pub-id-type=\"pmid\">30057536</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>SS</given-names></name><name><surname>Ye</surname><given-names>DX</given-names></name><name><surname>Wang</surname><given-names>B</given-names></name><name><surname>Xie</surname><given-names>C</given-names></name></person-group>. <article-title>The expressions of keratins and P63 in primary squamous cell carcinoma of the thyroid gland: an application of raman spectroscopy</article-title>. <source>Onco Targets Ther.</source> (<year>2020</year>) <volume>13</volume>:<fpage>585</fpage>–<lpage>91</lpage>. <pub-id pub-id-type=\"doi\">10.2147/OTT.S229436</pub-id><pub-id pub-id-type=\"pmid\">32021300</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yasumatsu</surname><given-names>R</given-names></name><name><surname>Sato</surname><given-names>M</given-names></name><name><surname>Uchi</surname><given-names>R</given-names></name><name><surname>Nakano</surname><given-names>T</given-names></name><name><surname>Hashimoto</surname><given-names>K</given-names></name><name><surname>Kogo</surname><given-names>R</given-names></name><etal/></person-group>. <article-title>The treatment and outcome analysis of primary squamous cell carcinoma of the thyroid</article-title>. <source>Auris Nasus Larynx.</source> (<year>2018</year>) <volume>45</volume>:<fpage>553</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.anl.2017.07.009</pub-id><pub-id pub-id-type=\"pmid\">28739190</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kao</surname><given-names>NH</given-names></name><name><surname>Tan</surname><given-names>CSH</given-names></name><name><surname>Koh</surname><given-names>AJ</given-names></name></person-group>. <article-title>The utility of immunohistochemistry in differentiating metastatic primary squamous cell carcinoma of the thyroid from a primary lung squamous cell carcinoma</article-title>. <source>Case Rep Endocrinol.</source> (<year>2019</year>) <volume>2019</volume>:<fpage>1</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1155/2019/8641267</pub-id><pub-id pub-id-type=\"pmid\">31772786</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jang</surname><given-names>JY</given-names></name><name><surname>Kwon</surname><given-names>KW</given-names></name><name><surname>Kim</surname><given-names>SW</given-names></name><name><surname>Youn</surname><given-names>I</given-names></name></person-group>. <article-title>Primary squamous cell carcinoma of thyroid gland with local recurrence: ultrasonographic and computed tomographic findings</article-title>. <source>Ultrasonography.</source> (<year>2014</year>) <volume>33</volume>:<fpage>143</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.14366/usg.13022</pub-id><pub-id pub-id-type=\"pmid\">24936508</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Raggio</surname><given-names>B</given-names></name><name><surname>Barr</surname><given-names>J</given-names></name><name><surname>Ghandour</surname><given-names>Z</given-names></name><name><surname>Friedlander</surname><given-names>P</given-names></name></person-group>. <article-title>Primary squamous cell carcinoma of the thyroid</article-title>. <source>Ochsner J.</source> (<year>2019</year>) <volume>19</volume>:<fpage>290</fpage>–<lpage>2</lpage>. <pub-id pub-id-type=\"doi\">10.31486/toj.18.0002</pub-id><pub-id pub-id-type=\"pmid\">31528145</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haugen</surname><given-names>BR</given-names></name><name><surname>Alexander</surname><given-names>EK</given-names></name><name><surname>Bible</surname><given-names>KC</given-names></name><name><surname>Doherty</surname><given-names>GM</given-names></name><name><surname>Mandel</surname><given-names>SJ</given-names></name><name><surname>Nikiforov</surname><given-names>YE</given-names></name><etal/></person-group>. <article-title>2015 American thyroid association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer: the American thyroid association guidelines task force on thyroid nodules and differentiated thyroid cancer</article-title>. <source>Thyroid.</source> (<year>2016</year>) <volume>26</volume>:<fpage>1</fpage>–<lpage>133</lpage>. <pub-id pub-id-type=\"doi\">10.1089/thy.2015.0020</pub-id><pub-id pub-id-type=\"pmid\">26462967</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tessler</surname><given-names>FN</given-names></name><name><surname>Middleton</surname><given-names>WD</given-names></name><name><surname>Grant</surname><given-names>EG</given-names></name><name><surname>Hoang</surname><given-names>JK</given-names></name><name><surname>Berland</surname><given-names>LL</given-names></name><name><surname>Teefey</surname><given-names>SA</given-names></name><etal/></person-group>. <article-title>ACR thyroid imaging, reporting and data system (TI-RADS): white paper of the ACR TI-RADS committee</article-title>. <source>J Am Coll Radiol.</source> (<year>2017</year>) <volume>14</volume>:<fpage>587</fpage>–<lpage>95</lpage>\n<pub-id pub-id-type=\"doi\">10.1016/j.jacr.2017.01.046</pub-id><pub-id pub-id-type=\"pmid\">28372962</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kwak</surname><given-names>JY</given-names></name><name><surname>Han</surname><given-names>KH</given-names></name><name><surname>Yoon</surname><given-names>JH</given-names></name><name><surname>Moon</surname><given-names>HJ</given-names></name><name><surname>Son</surname><given-names>EJ</given-names></name><name><surname>Park</surname><given-names>SH</given-names></name><etal/></person-group>. <article-title>Thyroid imaging reporting and data system for US features of nodules: a step in establishing better stratification of cancer risk</article-title>. <source>Radiology.</source> (<year>2011</year>) <volume>260</volume>:<fpage>892</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1148/radiol.11110206radiol.11110206</pub-id><pub-id pub-id-type=\"pmid\">21771959</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Peng</surname><given-names>Q</given-names></name><name><surname>Niu</surname><given-names>C</given-names></name><name><surname>Zhang</surname><given-names>Q</given-names></name><name><surname>Zhang</surname><given-names>M</given-names></name><name><surname>Chen</surname><given-names>S</given-names></name></person-group>. <article-title>Mummified thyroid nodules: conventional and contrast-enhanced ultrasound features</article-title>. <source>J Ultrasound Med.</source> (<year>2018</year>) <volume>38</volume>:<fpage>441</fpage>–<lpage>52</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jum.14712</pub-id><pub-id pub-id-type=\"pmid\">30099759</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Peng</surname><given-names>Q</given-names></name><name><surname>Niu</surname><given-names>C</given-names></name><name><surname>Zhang</surname><given-names>M</given-names></name><name><surname>Chen</surname><given-names>S</given-names></name></person-group>. <article-title>Sonographic characteristics of papillary thyroid carcinoma with coexistent hashimoto's thyroiditis: conventional ultrasound, acoustic radiation force impulse imaging and contrast-enhanced ultrasound</article-title>. <source>Ultrasound Med Biol.</source> (<year>2019</year>) <volume>45</volume>:<fpage>471</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.16/j.ultrasmedbio.2018.10.020</pub-id><pub-id pub-id-type=\"pmid\">30528690</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Casella</surname><given-names>C</given-names></name><name><surname>Ministrini</surname><given-names>S</given-names></name><name><surname>Galani</surname><given-names>A</given-names></name><name><surname>Mastriale</surname><given-names>F</given-names></name><name><surname>Cappelli</surname><given-names>C</given-names></name><name><surname>Portolani</surname><given-names>N</given-names></name></person-group>. <article-title>The new TNM staging system for thyroid cancer and the risk of disease downstaging</article-title>. <source>Front Endocrinol.</source> (<year>2018</year>) <volume>9</volume>:<fpage>541</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fendo.2018.00541</pub-id><pub-id pub-id-type=\"pmid\">30279679</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>Y</given-names></name><name><surname>Zhou</surname><given-names>P</given-names></name><name><surname>Tian</surname><given-names>SM</given-names></name><name><surname>Zhao</surname><given-names>YF</given-names></name><name><surname>Li</surname><given-names>JL</given-names></name><name><surname>Li</surname><given-names>L</given-names></name></person-group>. <article-title>Usefulness of combined use of contrast-enhanced ultrasound and TI-RADS classification for the differentiation of benign from malignant lesions of thyroid nodules</article-title>. <source>Eur Radiol.</source> (<year>2017</year>) <volume>27</volume>:<fpage>1527</fpage>–<lpage>36</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00330-016-4508-y</pub-id><pub-id pub-id-type=\"pmid\">27525973</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>YZ</given-names></name><name><surname>Xu</surname><given-names>T</given-names></name><name><surname>Gong</surname><given-names>HY</given-names></name><name><surname>Li</surname><given-names>CY</given-names></name><name><surname>Ye</surname><given-names>XH</given-names></name><name><surname>Lin</surname><given-names>HJ</given-names></name><etal/></person-group>. <article-title>Application of high-resolution ultrasound, real-time elastography, and contrast-enhanced ultrasound in differentiating solid thyroid nodules</article-title>. <source>Medicine.</source> (<year>2016</year>) <volume>95</volume>:<fpage>e5329</fpage>. <pub-id pub-id-type=\"doi\">10.1097/MD.000000000000532900005792-201611080-00016</pub-id><pub-id pub-id-type=\"pmid\">27828854</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>CY</given-names></name><name><surname>Tseng</surname><given-names>HS</given-names></name><name><surname>Lee</surname><given-names>CH</given-names></name><name><surname>Chan</surname><given-names>PW</given-names></name></person-group>. <article-title>Primary squamous cell carcinoma of the thyroid gland with eggshell calcification: sonographic and computed tomographic findings</article-title>. <source>J Ultrasound Med.</source> (<year>2010</year>) <volume>29</volume>:<fpage>1667</fpage>–<lpage>70</lpage>. <pub-id pub-id-type=\"doi\">10.7863/jum.2010.29.11.1667</pub-id><pub-id pub-id-type=\"pmid\">20966481</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kondo</surname><given-names>T</given-names></name><name><surname>Matsuyoshi</surname><given-names>A</given-names></name><name><surname>Matsuyoshi</surname><given-names>H</given-names></name><name><surname>Goto</surname><given-names>R</given-names></name><name><surname>Ono</surname><given-names>K</given-names></name><name><surname>Honda</surname><given-names>Y</given-names></name><etal/></person-group>. <article-title>A case of primary thyroid squamous cell cancer: transformation from benign tumour associated with chronic thyroiditis?</article-title>\n<source>BMJ Case Rep.</source> (<year>2009</year>) <volume>2009</volume>:<fpage>bcr10</fpage>.2008.1137. <pub-id pub-id-type=\"doi\">10.1136/bcr.10.2008.1137</pub-id><pub-id pub-id-type=\"pmid\">21686450</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ma</surname><given-names>JJ</given-names></name><name><surname>Ding</surname><given-names>H</given-names></name><name><surname>Xu</surname><given-names>BH</given-names></name><name><surname>Xu</surname><given-names>C</given-names></name><name><surname>Song</surname><given-names>LJ</given-names></name><name><surname>Huang</surname><given-names>BJ</given-names></name><etal/></person-group>. <article-title>Diagnostic performances of various gray-scale, color doppler, and contrast-enhanced ultrasonography findings in predicting malignant thyroid nodules</article-title>. <source>Thyroid.</source> (<year>2014</year>) <volume>24</volume>:<fpage>355</fpage>–<lpage>63</lpage>. <pub-id pub-id-type=\"doi\">10.1089/thy.2013.0150</pub-id><pub-id pub-id-type=\"pmid\">23978252</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Deng</surname><given-names>J</given-names></name><name><surname>Zhou</surname><given-names>P</given-names></name><name><surname>Tian</surname><given-names>SM</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Li</surname><given-names>JL</given-names></name><name><surname>Qian</surname><given-names>Y</given-names></name></person-group>. <article-title>Comparison of diagnostic efficacy of contrast-enhanced ultrasound, acoustic radiation force impulse imaging, and their combined use in differentiating focal solid thyroid nodules</article-title>. <source>PLoS ONE.</source> (<year>2014</year>) <volume>9</volume>:<fpage>e90674</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0090674PONE-D-13-30329</pub-id><pub-id pub-id-type=\"pmid\">24594879</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Struller</surname><given-names>F</given-names></name><name><surname>Senne</surname><given-names>M</given-names></name><name><surname>Falch</surname><given-names>C</given-names></name><name><surname>Kirschniak</surname><given-names>A</given-names></name><name><surname>Konigsrainer</surname><given-names>A</given-names></name><name><surname>Muller</surname><given-names>S</given-names></name></person-group>. <article-title>Primary squamous cell carcinoma of the thyroid: case report and systematic review of the literature</article-title>. <source>Int J Surg Case Rep.</source> (<year>2017</year>) <volume>37</volume>:<fpage>36</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijscr.2017.06.011</pub-id><pub-id pub-id-type=\"pmid\">28633125</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>W</given-names></name><name><surname>Ouyang</surname><given-names>Q</given-names></name><name><surname>Meng</surname><given-names>C</given-names></name><name><surname>Jing</surname><given-names>L</given-names></name><name><surname>Li</surname><given-names>X</given-names></name></person-group>. <article-title>Treatment optimization and prognostic considerations for primary squamous cell carcinoma of the thyroid</article-title>. <source>Gland Surg.</source> (<year>2019</year>) <volume>8</volume>:<fpage>683</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.21037/gs.2019.11.07</pub-id><pub-id pub-id-type=\"pmid\">32042676</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Immunol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Immunol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Immunol.</journal-id><journal-title-group><journal-title>Frontiers in Immunology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-3224</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849635</article-id><article-id pub-id-type=\"pmc\">PMC7431616</article-id><article-id pub-id-type=\"doi\">10.3389/fimmu.2020.01770</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Immunology</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>The Chimeric Antigen Receptor Detection Toolkit</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Hu</surname><given-names>Yifei</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/971296/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Huang</surname><given-names>Jun</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/644799/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Pritzker School of Molecular Engineering, University of Chicago</institution>, <addr-line>Chicago, IL</addr-line>, <country>United States</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Pritzker School of Medicine, University of Chicago</institution>, <addr-line>Chicago, IL</addr-line>, <country>United States</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Luca Gattinoni, Regensburg Center for Interventional Immunology (RCI), Germany</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Sanjivan Gautam, National Cancer Institute, National Institutes of Health (NIH), United States; Joseph Anthony Fraietta, University of Pennsylvania, United States</p></fn><corresp id=\"c001\">*Correspondence: Jun Huang <email>huangjun@uchicago.edu</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1770</elocation-id><history><date date-type=\"received\"><day>11</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>01</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Hu and Huang.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Hu and Huang</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Chimeric antigen receptor-T (CAR-T) cell therapy is a promising frontier of immunoengineering and cancer immunotherapy. Methods that detect, quantify, track, and visualize the CAR, have catalyzed the rapid advancement of CAR-T cell therapy from preclinical models to clinical adoption. For instance, CAR-staining/labeling agents have enabled flow cytometry analysis, imaging applications, cell sorting, and high-dimensional clinical profiling. Molecular assays, such as quantitative polymerase chain reaction, integration site analysis, and RNA-sequencing, have characterized CAR transduction, expression, and <italic>in vivo</italic> CAR-T cell expansion kinetics. <italic>In vitro</italic> visualization methods, including confocal and total internal reflection fluorescence microscopy, have captured the molecular details underlying CAR immunological synapse formation, signaling, and cytotoxicity. <italic>In vivo</italic> tracking methods, including two-photon microscopy, bioluminescence imaging, and positron emission tomography scanning, have monitored CAR-T cell biodistribution across blood, tissue, and tumor. Here, we review the plethora of CAR detection methods, which can operate at the genomic, transcriptomic, proteomic, and organismal levels. For each method, we discuss: (1) what it measures; (2) how it works; (3) its scientific and clinical importance; (4) relevant examples of its use; (5) specific protocols for CAR detection; and (6) its strengths and weaknesses. Finally, we consider current scientific and clinical needs in order to provide future perspectives for improved CAR detection.</p></abstract><kwd-group><kwd>chimeric antigen receptor (CAR)</kwd><kwd>T cell</kwd><kwd>detection</kwd><kwd>cancer immunotherapy</kwd><kwd>method</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Phi Beta Kappa Society<named-content content-type=\"fundref-id\">10.13039/100006288</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"4\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"88\"/><page-count count=\"16\"/><word-count count=\"11435\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Chimeric antigen receptor-T (CAR-T) cell therapy is a breakthrough application of adoptive cell therapy (ACT), a novel immunoengineering field where T cells are genetically modified <italic>ex vivo</italic> and infused for anti-tumor, anti-viral, or immunomodulatory effects <italic>in vivo</italic>. At the center of CAR-T cell therapy is the CAR, an engineered immunoreceptor consisting of an extracellular single-chain antibody fragment (scFv) and hinge, a transmembrane region, and intracellular signaling domains. The CAR directs T cells to recognize, activate, proliferate, and kill in response to scFv-driven recognition of tumor-associated antigens (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Since 2017, two formulations of anti-CD19 CAR-T cell therapy won FDA approval: Kymriah and Yescarta. Both formulations yielded unprecedented 40% complete response rates against relapsed/refractory B-cell leukemia and lymphoma (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B3\" ref-type=\"bibr\">3</xref>). These preliminary successes ignited interest in extending CAR-T cell therapies from hematological malignancies to solid tumors (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Currently, CARs that target human epidermal growth factor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII) against glioblastoma, GD2 disialoganglioside against neuroblastoma, and mesothelin (MSLN) against mesothelioma, are being evaluated in clinical trials (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>).</p><p>Although CAR-T cell therapy's preliminary clinical success in B cell cancers warrants optimism, there are several challenges in the CAR-T field that need to be addressed: (1) CAR-T cell therapy does not work on solid tumors; (2) clinical non-response/relapse mechanisms in B cell cancers need elucidation; (3) the <italic>in vivo</italic> biology of CAR-T cells in human subjects needs investigation; and (4) the molecular designs of the CAR immunoreceptor need optimization.</p><p>Accurate and reproducible CAR detection methods are required to address these challenges. Developing CAR-T cell therapy for solid tumors and elucidating clinical non-response/relapse mechanisms in B cell cancers require methods to stain and sort CAR-T cells from clinical samples for downstream applications, such as multiparameter flow cytometry and next-generation sequencing. Investigating <italic>in vivo</italic> CAR-T cell biology requires methods to track and assess <italic>in vivo</italic> CAR-T cell expansion kinetics, persistence, biodistribution, and effector functions in patients and animal models. Optimizing CAR molecular designs requires methods to characterize CAR signaling and visualize CAR immunological synapse formation at the molecular and cellular levels. Finally, development and application of any CAR detection methods for clinical trial laboratory operations should adhere to Good Clinical Laboratory Practice (GCLP) guidelines, which ensure high data quality, reduced false negative and false positive incidences, and replicability across independent GCLP-compliant laboratories (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>).</p><p>Here, we review current CAR detection methods. After describing the target and importance of each CAR detection method, we will discuss experimental protocols, examples of its application, as well as its strengths and weaknesses. Wherever possible, we will provide perspectives for method improvements. We will introduce CAR detection methods in the order of the level at which they operate: genomic, transcriptomic, proteomic, and organismal (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). By facilitating experimental design and planning, this review aims to catalyze basic, immunoengineering, and clinical research.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>CAR detection methods across multiple levels. CAR detection methods can operate at genomic, transcriptomic, proteomic, and organismal levels. At the genomic level, real-time quantitative PCR (qPCR) and digital PCR (dPCR) measure CAR vector copy number while integration site analysis determines sites of insertional mutagenesis. At the transcriptomic level, RNA-seq measures CAR mRNA abundance while RNAscope <italic>in situ</italic> hybridization (RNAscope ISH) determines the presence and subcellular localization of CAR mRNA molecules. At the proteomic level, staining agents facilitate flow cytometry and western blotting quantification of the CAR protein, while the Topanga reagent detects the CAR via luminescence. The CAR can also be fused with fluorescent proteins for fluorescence microscopy. At the organismal level, bioluminescence imaging (BLI) and positron emission tomography (PET) scanning determines the distribution of CAR-T cells between organs, while two-photon microscopy tracks single CAR-T cells in tissue.</p></caption><graphic xlink:href=\"fimmu-11-01770-g0001\"/></fig></sec><sec id=\"s2\"><title>CAR Detection at the Genomic Level</title><p>During the CAR manufacturing process, T cells are virally transduced with a CAR vector, which semi-randomly integrates into the T cell's genome. There are three main methods for detecting the integrated CAR vector: real-time quantitative polymerase chain reaction (qPCR, <xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>), digital polymerase chain reaction (dPCR, <xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>), and integration site analysis (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>). Using genomic DNA (gDNA), qPCR and dPCR determine the frequency while integration site analysis determines the genomic location of the CAR vector. Since gDNA is more stable than mRNA, proteins, or cryopreserved biospecimens, experiments involving these methods can be easier to coordinate. Importantly, these methods can help evaluate and optimize the safety profiles of alternative non-viral techniques for CAR vector delivery, including CRISPR/Cas9 and transposon-mediated insertion (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>).</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Genomic CAR detection. Real-time quantitative PCR (qPCR) and digital PCR (dPCR) measure CAR vector copy number (VCN) while integration site analysis determines sites of insertional mutagenesis. <bold>(A)</bold> With qPCR, target amplicons are amplified from genomic DNA (gDNA) with fluorescent probes. C<sub>q</sub> is calculated from fluorescence tracked over PCR cycles, which measures copy number. In the singleplex setup, vector and reference gene are amplified separately. In the multiplex setup vector and reference are amplified concurrently using two independent probes. <bold>(B)</bold> With dPCR, gDNA is partitioned into tiny droplets. Most droplets contain zero or one template copies. Target amplicons are amplified from each droplet separately, and the proportion of fluorescent droplets measures copy number. <bold>(C)</bold> With integration site analysis, gDNA is fragmented and ligated with adaptors in two steps with sonication or restriction enzymes, or in one step with tagmentation. Fragments containing either of the CAR vector flanks (left flank shown here) can be enriched and prepped for sequencing with multiple rounds of PCR. Mapping the reads to the genome determines sites of insertional mutagenesis.</p></caption><graphic xlink:href=\"fimmu-11-01770-g0002\"/></fig><sec><title>Real-Time Quantitative Polymerase Chain Reaction</title><p>qPCR measures the frequency of integrated CAR vector in the genome (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>). Using target-specific primers and fluorescent probes, qPCR amplifies and quantifies an amplicon over PCR cycles. The quantitation cycle (C<sub>q</sub>), when fluorescence exceeds background levels, measures an amplicon's relative (compared to another amplicon) or absolute (compared to a standard curve) copy number. With CAR-T cells' gDNA and CAR-specific primers and probes, qPCR can measure vector copy number (VCN)—the average vector copies per genome. VCN estimates CAR vector delivery efficiency and CAR-T cells' representation in a cell pool. Hence, VCN is an important quality metric for clinical-grade CAR-T cell infusions and a technical benchmark that non-viral forms of CAR vector delivery, such as transposon-mediated delivery, must improve upon (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). In both CAR-T cell research and clinical settings, qPCR helps monitor VCN from patient blood gDNA over the course of CAR-T treatment. These results have consistently shown strong correlations between CAR-T cell expansion, persistence, clinical response, and grade of side effects across multiple types of B cell cancers (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B8\" ref-type=\"bibr\">8</xref>).</p><p>Optimized CAR qPCR protocols have been developed to detect the anti-CD19 (clone FMC63) scFv. Wang et al. (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>) developed and validated TaqMan qPCR primers and probes for a ~130 bp amplicon from the FMC63 nucleotide sequence. In their assay, qPCR was performed side-by-side against FMC63 and <italic>GAPDH</italic> to measure CAR copy number and genome copies, respectively. These two measured values were used to calculate VCN. In addition to robustness across replicates, their qPCR assay achieved a minimum detection limit of 10 CAR copies per μL of blood and linear signal between 10 and 10<sup>7</sup> copies/μL. However, a singleplex design increases sample and reagent use, decreases throughput, and introduces pipetting noise. Multiplexed qPCR can address these issues. Kunz et al. developed a single copy gene-based duplex qPCR assay (SCG-DP-PCR) to measure VCN. In their assay, the FMC63 scFv and RNaseP (<italic>RPPH1</italic>) were simultaneously qPCR-amplified from the same gDNA sample using two independent fluorescent probes. Using <italic>RPPH1</italic> as an internal control, their duplex setup resulted in similar efficiency as the corresponding singleplex setup. As proof-of-principle, they used SCG-DP-PCR to measure longitudinal VCN from three sets of CAR-T patient blood gDNA samples (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>).</p><p>Overall, qPCR is a common and robust assay for monitoring CAR VCN. With well-designed primers and probes, qPCR is rapid, easily performed, and trustworthy for clinical use. qPCR can measure CAR vector delivery efficiency, on-treatment expansion kinetics, and persistence to predict clinical response (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B8\" ref-type=\"bibr\">8</xref>). As such, qPCR machines optimized and certified for CAR VCN measurements are now available commercially. However, qPCR has notable weaknesses. While robust at the population level, qPCR cannot differentiate subtle copy number differences. Hence, qPCR for VCN at the single-cell level is expected to be noisy and has never been implemented (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Other than VCN, qPCR cannot determine the CAR-T cells' phenotype or whether the CAR is actually expressed. CAR expression depends on the chosen viral promoter, local chromatin architecture, regulatory elements (i.e., promoters, enhancers, insulator sequences), DNA methylation, and biological noise. Hence, qPCR overestimates the number of functional CAR-T cells in a given population. Furthermore, the reliability of qPCR results depends on the target specificity of the primers and probes. In conclusion, qPCR is a clinically useful assay for monitoring VCN, but RNA-seq and flow cytometry may be more useful methods for determining CAR-T cell functionality in research settings.</p></sec><sec><title>Digital Polymerase Chain Reaction</title><p>Like qPCR, dPCR also measures VCN (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). However, dPCR measurements are more sensitive and precise. In brief, the gDNA template is partitioned into tiny droplets. Most droplets contain zero or one template molecule. PCR amplification of the target amplicon with a fluorescent probe occurs separately within each droplet. Subsequently, the droplets are flowed through an excitation source and detector, which measures each droplet's fluorescence. With Poisson statistics, the proportion of fluorescent droplets is used to calculate copy number (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). While qPCR relies on continuous intermediate fluorescence measurements from PCR cycles, dPCR relies only on end-point fluorescence. Therefore, dPCR is robust to amplification kinetics and suppresses noise. Like with qPCR, dPCR can be multiplexed to decrease sample and reagent use, increase throughput, and decrease pipetting noise.</p><p>Fehse et al. developed a duplex dPCR assay to concomitantly probe the anti-CD19 CAR and a reference gene. Their duplex dPCR assay achieved a minimum detection limit of 0.01% CAR-transduced cells from 100 ng of gDNA. As proof-of-principle, they applied their assay on five sets of Yescarta patient blood gDNA samples (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). The enhanced sensitivity from dPCR also enables single-cell VCN measurements. Santeramo et al. recently developed dPCR to measure VCN in single lentivirally transduced T cells. In their assay, target amplicons were first pre-amplified to generate sufficient template material, prior to dPCR for vector and reference amplicons. Their single-cell assay generated results that were consistent with bulk measurements (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>). However, their method has yet to be applied for CAR VCN measurements.</p><p>As a more sensitive assay for monitoring VCN, dPCR shares strengths and weaknesses with qPCR. Unlike qPCR, the increased sensitivity allows dPCR to measure VCN in single cells. Single-cell VCN measurements can capture cell-cell heterogeneity in transduction efficiency within a CAR-T cell infusion, which may impact clinical efficacy. However, dPCR-compatible machines are rarer, and dPCR reactions are more costly.</p></sec><sec><title>Integration Site Analysis</title><p>The presence and genomic location of the integrated CAR vector can be assayed via integration site analysis (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>). During CAR transduction, the CAR vector is randomly inserted into the genome (i.e., insertional mutagenesis), which can disrupt genes, trigger premalignant T cell proliferation, promote CAR-T cell efficacy, and influence clinical response (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>–<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). Integration site analysis maps sites of insertional mutagenesis using next-generation sequencing. Importantly, integration site analysis can characterize the integration loci biases of different CAR vector delivery techniques. In brief, integration site analysis involves fragmentation, PCR, and analysis steps. First, gDNA from the CAR-T cell sample is fragmented via restriction enzymes, transposases, or sonication. If necessary, adaptors are ligated onto the resulting DNA fragments. Then, fragments containing the CAR vector and flanking genome are enriched by PCR amplification, using a primer that anneals on the adaptor paired with a primer that anneals on the CAR vector. When this amplicon is sequenced, reads begin in the vector and extend into flanking human DNA, which can be aligned onto the human genome to reveal the integration loci.</p><p>Historically, integration site analysis employed restriction enzymes to fragment gDNA (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). However, restriction enzymes generated inconsistent results that depended on which restriction enzyme was used (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). The more up-to-date integration site pipeline for paired-end reads (INSPIIRED) eliminates restriction enzyme bias by using sonication for fragmentation. INSPIIRED includes well-documented steps for sonication, library preparation, Illumina paired-end sequencing, and bioinformatic site-calling (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>, <xref rid=\"B21\" ref-type=\"bibr\">21</xref>). INSPIIRED has been employed on anti-CD19 CAR-T cell therapy infusion samples, which showed that insertional mutagenesis near genes in cell-signaling and chromatin modification pathways predicted clinical response (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). The final and more elegant method for integration site mapping uses transposases to combine the fragmentation and ligation steps (i.e., tagmentation). In one step, the transposase agnostically fragments gDNA and inserts an adaptor for PCR amplification and Illumina sequencing. The tagmented gDNA can simultaneously be used for integration site analysis and chromatin accessibility profiling (via ATAC-seq) via the recently developed vector integration analysis with epigenomic assay (EpiVIA), which can be applied at both the bulk and single-cell level (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). In a clinical case study, Mu transposase-enabled integration site analysis characterized how lentiviral insertion of the CAR vector disrupted <italic>TET2</italic> and led to massive (94% of blood CD8<sup>+</sup> T cells) CAR-T cell expansion (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>). In addition, Tn5 transposase-enabled integration site analysis was employed to compare integration sites between γ-retrovirus, lentivirus, and <italic>piggyBac</italic> transposon-mediated gene transfer. Compared to viral transduction, the <italic>piggyBac</italic> transposon integrated less often near transcriptional start sites and more often into genomic safe harbors (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>).</p><p>Overall, integration site analysis is a clinically relevant CAR detection method for locating sites of insertional mutagenesis. Newer integration site analysis pipelines involving transposases significantly streamline benchwork and enable EpiVIA, which combines integration site analysis and ATAC-seq at the bulk and single-cell levels (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). Furthermore, the abundance of each CAR-T cell clone (each of which can be assumed to harbor unique integration sites) can be bioinformatically inferred from integration site sequencing data (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). However, capture efficiencies for most CAR integration site analysis methods are unavailable. Where available, capture efficiencies are notably poor. At least three Mu transposase-enabled integration site analysis replicates were required to capture all six integration sites in a cell line (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Since polyclonal CAR-T cell populations contain far more than six integration sites, integration site analysis is unlikely to capture all integration sites, especially those from rare clones. With single-cell EpiVIA, only ~200 integration sites were detected from 700 M read pairs in ~5,000 CAR-transduced T cells, which was notably far from saturation (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>). Hence, capture efficiencies should be better characterized, and protocol improvements are needed to assay rarer clones.</p></sec></sec><sec id=\"s3\"><title>CAR Detection at the Transcriptomic Level</title><p>After integration into the genome, the CAR vector is transcribed into mRNA. There are two main methods for detecting CAR mRNA: RNA-sequencing (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>) and RNAscope <italic>in situ</italic> hybridization (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>). These methods determine the abundance and subcellular location of the CAR mRNA, respectively. Detection of CAR mRNA can be more functionally relevant than detection of the CAR vector, since CAR mRNA is closer to CAR protein, which exerts biological functions.</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Transcriptomic CAR detection. RNA-sequencing (RNA-seq) and RNAscope <italic>in situ</italic> hybridization (RNAscope ISH) measure CAR mRNA abundance and subcellular localization, respectively. <bold>(A)</bold> With RNA-seq, CAR mRNA is first converted to cDNA, which is then fragmented and prepped for sequencing. Counting the number of reads that map to the CAR sequence measures CAR mRNA abundance. <bold>(B)</bold> With RNAscope ISH, the CAR mRNA is first hybridized with target-specific RNA probes. Subsequently, this complex is hybridized with the preamplifier, amplifier, and fluorescent probes to form a fluorescently labeled CAR mRNA complex for fluorescence microscopy.</p></caption><graphic xlink:href=\"fimmu-11-01770-g0003\"/></fig><sec><title>RNA-Sequencing</title><p>CAR mRNA abundance can be quantified by RNA-sequencing (RNA-seq, <xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>). CAR mRNA quantity depends upon genomic factors: VCN, viral promoter strength, local chromatin architecture, regulatory elements, and DNA methylation. Importantly, CAR mRNA quantity drives antigen-independent tonic signaling (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>) and amount of translated CARs on the cell surface influences antigen-sensitivity, NFAT signaling, and cytokine production (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). In general, RNA-seq quantifies mRNA by converting mRNA to cDNA via reverse transcription. The cDNA can subsequently be fragmented, sequenced, and aligned to gene sequences to measure mRNA abundance. RNA-seq can correlate CAR mRNA abundance with transcriptional profiles. For instance, Zhang et al. utilized RNA-seq on anti-CD19 CAR-T patient samples to measure correlation between CAR and <italic>CD19</italic> expression (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). These analyses also apply at the single-cell level. Sheih et al. utilized single-cell RNA-seq to quantify CAR mRNAs and interrogate transcriptional profiles in CD8<sup>+</sup> T cells from CAR-T infusion products. CAR mRNA quantification helped to distinguish between CAR-transduced vs. non-transduced cells in their single-cell dataset (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). Prospectively, RNA-seq may also help characterize how viral promoters influence CAR transcription and <italic>in vivo</italic> differentiation. For example, with anti-CD19 CAR-T cell therapy, Kymriah employs the elongation factor 1-α (EF-1α) promoter while Yescarta employs the murine stem cell virus (MSCV) promoter. Although the EF-1α promoter drives higher and more consistent murine <italic>in vivo</italic> transcription than the MSCV promoter (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>), no studies have determined whether this difference influences CAR-T cell functionality, differentiation, or clinical outcomes.</p><p>RNA-seq is now a routinely employed biological assay with many published protocols, commercial kits, and analysis pipelines. By integrating the genomic factors that influence CAR expression into a single readout, RNA-seq for the CAR mRNA arguably provides more clinically and biologically relevant data than qPCR for the CAR vector. Furthermore, in single-cell RNA-seq datasets, CAR mRNA quantification can help differentiate CAR-T cells from non-CAR-T cells during analysis (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). However, mRNA-seq notably cannot capture factors that influence CAR translation, such as ribosome, initiation factors, and amino acid availability. Therefore, flow cytometry or western blotting for the CAR protein may be superior methods for determining CAR functionality.</p></sec><sec><title>RNAscope <italic>in situ</italic> Hybridization</title><p>Both the quantity and subcellular localization of the CAR mRNA can be determined by RNAscope <italic>in situ</italic> hybridization (RNAscope ISH, <xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>). RNAscope ISH utilizes RNA-specific oligonucleotide probes that anneal with a targeted RNA molecule in fixed and permeabilized cells, to generate fluorescence signals for microscopy. Dual target probes (for specificity) and additional adaptor probes (for signal amplification) enable detection, localization, and visualization at the single-molecule level. Using orthogonal sets of probes, RNAscope ISH can be multiplexed—CAR mRNA can be simultaneously detected along with other target mRNA on the same slide (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). Furthermore, RNAscope ISH can correlate a CAR-T cell's CAR mRNA quantity with the CAR-T cell's relative location within a tissue sample. For instance, RNAscope ISH (to detect the CAR mRNA's 3′-untranslated region) was employed to visualize anti-EGFRvIII CAR-T cell infiltration into glioblastoma tumor sections before and after intravenous infusion. This study showed active trafficking of anti-EGFRvIII CAR-T cells into tumor regions, which correlated with EGFRvIII downmodulation on tumor cells (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Furthermore, RNAscope ISH was employed to show anti-ROR1 and anti-BCMA CAR-T cell biodistribution and tissue trafficking in xenograft tumor models (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>).</p><p>RNAscope ISH is a specialized tool with many strengths: (1) spatial resolution spanning the single-molecule and cellular levels; (2) ability to probe subcellular mRNA localization; (3) capability for multiplex detection; and (4) compatibility with microscopy. However, RNAscope ISH requires fixed and permeabilized cells, hence it cannot be used for live-cell RNA imaging. Studies have yet to take advantage of this method's unique strengths. For example, current studies use RNAscope ISH to distinguish CAR-T cells from non-CAR-T cells in tissue sections, with only limited use of its multiplexing capabilities. Furthermore, no studies have yet analyzed how CAR mRNA subcellular localization (e.g., near cell membrane) may influence CAR mRNA stability, degradation, or translation.</p></sec></sec><sec id=\"s4\"><title>CAR detection at the Proteomic Level</title><p>After translation from CAR mRNA, the CAR protein drives antigen-dependent signaling. Unlike detection at the genomic or transcriptomic levels, detection at the proteomic level can directly evaluate CAR functionality. The CAR protein can be detected by flow cytometry (with staining agents), luminescence (with Topanga reagent), immunoprecipitation (with staining agents), and microscopy (with fluorescent protein fusions). <xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref> depicts where each detection reagent acts.</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Proteomic and Organismal CAR Detection. <bold>(A)</bold> At the proteomic level, the CAR protein can be detected with staining agents (for flow cytometry and immunoprecipitation), Topanga reagent (for luminescence), or fused fluorescent proteins [for microscopy and flow cytometry; cyan fluorescent protein (CFP) is shown as an example]. The approximate location or binding site for each method is depicted on the cartoon. <bold>(B)</bold> At the organismal level, the biodistribution of CAR-T cells between organ compartments can be measured by bioluminescence imaging (BLI) or positron emission tomography (PET) scanning using a luciferase substrate or radiotracer, respectively. Furthermore, single CAR-T cells can be tracked in tissue with two-photon microscopy.</p></caption><graphic xlink:href=\"fimmu-11-01770-g0004\"/></fig><sec><title>Staining Agents for Flow Cytometry</title><p>The presence and quantity of the CAR protein on the cell surface can be assayed via fluorescent CAR-staining agents and flow cytometry. In addition to CAR protein quantitation, these staining agents also enable multicolor flow cytometry-based profiling and fluorescence-activated cell sorting. CAR-staining agents were instrumental in illuminating factors that impact CAR-T cell clinical efficacy, including T cell subset composition, CAR downmodulation after antigen engagement (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B32\" ref-type=\"bibr\">32</xref>), and CAR-T cell trogocytosis (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>). Although many CAR-staining agents exist, a comparison of sensitivity and specificity metrics between these staining agents has yet to be performed. Each staining agent's target site and general properties are summarized in <xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref> and <xref rid=\"T1\" ref-type=\"table\">Table 1</xref>, respectively.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>CAR staining reagents.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Property</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Anti-IgG antibodies</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Protein L</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Antigen-Fc</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Anti-idiotype antibody</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Anti-linker antibody</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">One-step staining</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Compatibility w/antibody panels</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inconsistent</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Yes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Compatibility w/FcX reagents</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inconsistent</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">No</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Some</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Yes</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reagent stability</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">High</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">High</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Often low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">High</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">High</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Specificity for CAR</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Low</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Low</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">High</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">High</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">High</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Access to academic labs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Easy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Easy</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Easy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Hard</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Hard</td></tr></tbody></table></table-wrap><p>Two CAR-staining agents target IgG-like fragments: polyclonal anti-IgG antibodies and Protein L. Polyclonal anti-IgG (often of goat origin) are commonly used as secondary antibodies to stain IgG-like fragments. As polyclonal reagents, they have significant batch-to-batch variation. Although they are provided by a variety of vendors, the polyclonal goat anti-mouse F(ab)<sub>2</sub> from Jackson ImmunoResearch Laboratories is the most widely used, and was historically used to characterize the anti-CD19 CAR in Yescarta (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). Protein L is a <italic>Peptostreptococcus magnus</italic> bacterial surface protein that binds to many immunoglobulin kappa (κ) light chains, including human V<sub>K</sub>I, V<sub>K</sub>III, V<sub>K</sub>IV, and murine V<sub>K</sub>I, without interfering with the immunoglobulin's antigen-binding site. In addition to whole antibodies, Protein L can also bind light chains on scFv (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). Zheng et al. optimized Protein L as a CAR-staining reagent for flow cytometric detection: biotinylated Protein L (1 μg per million lymphocytes in 200 μL) is applied followed by fluorophore-labeled streptavidin. Successfully staining was achieved with a variety of CARs, including CARs containing human scFv (anti-EGFRvIII, anti-VEGFR2), murine scFv (anti-CD19, anti-CSPG4), and humanized scFv (anti-HER2, anti-PSCA). However, their method requires multiple cell washes before Protein L staining, since carry-over immunoglobulin in serum or culture media must be strictly removed. Furthermore, their results suggest their method may have worse stain index compared to polyclonal anti-IgG antibodies (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). Protein L has been used to characterize masked CARs with tumor-specific activation (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>), CAR downmodulation (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>), tonic signaling (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>), and to activate CAR-T cells via Protein L that is bound on plates (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>).</p><p>Although both polyclonal anti-IgG antibodies and Protein L are relatively cheap CAR-staining reagents, they share significant shortcomings: (1) cross-reactivity with non-CAR IgG-like proteins on the cell surface, requiring stringent washing before and after staining; (2) incompatibility with antibodies and many FcX blocking reagents during multicolor flow cytometry, requiring multiple staining and washing steps; (3) cannot independently stain different CARs on a dual-CAR expressing T cell; and (4) cannot stain CARs with synthetic scFv.</p><p>Antigen-Fc is a CAR-staining agent that takes advantage of the CAR's binding affinity for its target antigen. Antigen-Fc are chimeric proteins with an N-terminal target antigen fused to a C-terminal Fc fragment (often from human IgG1). Due to the Fc fragment, antigen-Fc dimerizes under non-reducing conditions in solution and can be purified via Protein A beads. To stain CAR-T cells, antigen-Fc is applied, followed by a secondary staining step with fluorophore-labeled anti-Fc or anti-biotin/streptavidin (if the antigen-Fc was biotinylated). Alternatively, the antigen-Fc is directly conjugated with a fluorescent dye, which eliminates the secondary staining step. Biochemically, antigen-Fc binds with antibody-like specificity and affinity. Antigen-Fc has been used to evaluate novel CAR constructs (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>), to independently measure expression of each CAR in dual CAR-T cells (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>), and to activate CAR-T cells via antigen-Fc that is bound on plates (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>). Antigen-Fc, including CD19-Fc, HER2-Fc, and PSCA-Fc, are commercially available from many vendors.</p><p>CD19-Fc is of special interest, due to the success of anti-CD19 CAR-T cell therapy and availability of patient samples for research. De Oliveira et al. expressed CD19-Fc for CAR-T cell staining and found that exon 4 of the CD19 ectodomain (CD19ecto) is required for binding to the anti-CD19 (FMC63) CAR used in Yescarta and Kymriah. However, their staining results show inferior stain index than either polyclonal anti-IgG antibodies or Protein L, hinting at issues with protein quality (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>). Indeed, CD19ecto aggregates in higher-order disulfide-bonded oligomers and is notorious for being a difficult-to-express protein (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>). The crystal structure of CD19ecto bound to a B43-Fab shows that CD19ecto can form a unique elongated β-sandwich, which may be difficult to fold properly within overexpression systems (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>). In response to technical challenges with CD19ecto production, Lobner et al. expressed a novel chimera consisting of an N-terminal CD19ecto with a C-terminal human serum albumin domain 2 (AD2). Compared to CD19ecto, the CD19-AD2 chimera is easier to produce, monomeric, and effectively binds and stains the anti-CD19 (FMC63) CAR (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>).</p><p>In conclusion, although antigen-Fc are more CAR-specific than polyclonal anti-IgG antibodies and Protein L, antigen-Fc also have notable disadvantages: (1) more expensive; (2) possible decreased stability in solution compared to traditional antibodies; (3) may be incompatible with FcX blocking reagents; and (4) the Fc fragment may non-specifically bind Fc receptors. Future iterations of antigen-Fc may involve engineered Fc regions that enable compatibility with FcX blocking reagents and disable non-specific binding of Fc receptors. Furthermore, future iterations of antigen-Fc may also take advantage of higher valency binding. Antigen-Fc are analogous to MHC-multimers, which stain T cells via TCR-binding (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>, <xref rid=\"B46\" ref-type=\"bibr\">46</xref>). Research on MHC-multimers have shown that higher valency staining reagents improve sensitivity via higher avidity binding. For example, MHC-dodecamers (12-valency) and MHC-dextramers (>>4-valency) are more sensitive than MHC-tetramers (4-valency) (<xref rid=\"B47\" ref-type=\"bibr\">47</xref>, <xref rid=\"B48\" ref-type=\"bibr\">48</xref>). However, CAR-staining reagents with higher target valency than antigen-Fc (2-valency) have yet to be constructed. The potential sensitivity enhancement from higher avidity binding has yet to be determined. Higher sensitivity antigen-Fc variants may facilitate staining and analysis of CAR-T cells with low CAR expression due to genome position effects on the CAR vector or CAR downmodulation.</p><p>Another CAR-staining agent is anti-idiotype antibodies. Anti-idiotype antibodies specifically bind the variable regions of a particular scFv. Furthermore, anti-idiotype antibodies enable immunohistochemical staining and can potentially block CAR ligation in an <italic>in vivo</italic> setting. Jena et al. developed and characterized a novel monoclonal anti-idiotype antibody (clone 136.20.1) against the anti-CD19 (FMC63) scFv from immunized mice. Their results show 136.20.1 has a lower detection limit of 0.1%, is compatible with microscopy and immunohistochemistry, and inhibits the effector functions of anti-CD19 CAR-T cells (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>). This antibody has since been used to characterize CAR-T cells in preclinical studies (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B24\" ref-type=\"bibr\">24</xref>) and clinical trials (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>). Interestingly, 136.20.1 was also used to create a novel anti-idiotype CAR as a cellular antidote and kill switch during therapy (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>). Other anti-idiotype antibodies include clone 1A7, which can detect the anti-GD2 (14g2a) CAR (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>).</p><p>Notable advantages of anti-idiotype antibodies include high reagent stability, low background staining, compatibility with antibody panels in multicolor flow cytometry, and the ability to discriminate between different types of CARs. However, most anti-idiotype antibodies are difficult-to-obtain and commercially unavailable.</p><p>Finally, Kite Pharma developed rabbit monoclonal antibodies against two commonly used linkers in the CAR scFv: clone KIP-1 against the Whitlow linker and clone KIP-4 against the G<sub>4</sub>S linker. These linkers connect the heavy and light chains in the scFv. KIP-1 can detect (via flow cytometry) and activate CAR-T cells with the Whitlow linker (i.e., Kymriah and Yescarta) (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>). KIP-1 was subsequently used in Kite Pharma and Gilead-sponsored studies (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>). However, these linker antibodies are unlikely to be accessible to most academic labs without industry sponsorship.</p></sec><sec><title>Biochemical Assays</title><p>The CAR's presence and binding competence can be assayed by luminescence using the Topanga reagent. Gopalakrishnan et al. developed the Topanga reagent, a chimeric protein consisting of the N-terminal CAR-antigen fused to a C-terminal marine luciferase, NLuc. Incubation of the Topanga reagent with CAR-T cell mixtures facilitated luminescent detection. The Topanga reagent's exceptional sensitivity allows it to detect CAR-binding in a mixture of 0.001% CAR-T cells out of 1 million PBMCs (<xref rid=\"B55\" ref-type=\"bibr\">55</xref>). Although the Topanga reagent cannot determine percentage of CAR-expressing cells, it might be useful for quality control during manufacturing and testing the binding functionality of the CAR.</p><p>CAR signaling can be assayed by immunoprecipitation (IP) or co-immunoprecipitation (co-IP) using Protein L-conjugated beads for analysis of CAR post-translational modifications and interaction partners (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). Protein L-conjugated beads bind to the light chain of the CAR scFv, and can pull down CAR interaction partners for western blotting or mass spectrometry. Ramello et al. utilized this approach to pull down CAR immune complexes. Complexes were analyzed by tandem liquid chromatography-mass spectrometry, to identify 253 CAR interaction partners enriched within 15 canonical pathways. Notably, their analysis demonstrated that 2nd-generation CARs associate with a constitutively phosphorylated CD3ζ, which correlates with stronger phosphorylation of downstream signaling proteins (<xref rid=\"B56\" ref-type=\"bibr\">56</xref>). In addition to Protein L beads, CAR IP can be performed on epitope-tagged CAR receptors. Salter et al. used the 9-amino acid Strep-tag II to tag the CAR between the scFv and the hinge. Their co-IP analysis showed that endogenous Lck and CD28 differentially associate with CD28-based and 4-1BB-based CARs in the absence of signaling (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>). IP with epitope tags is expected to be more target-specific than IP with Protein L, since Protein L is known to interact with IgG-like molecules. However, these epitope tags must not interfere with CAR function. Furthermore, current FDA-approved anti-CD19 CAR designs do not have a convenient epitope tag for IP.</p></sec><sec><title>Microscopy</title><p>CAR trafficking and immunological synapse (IS) formation can be visualized by microscopy. Importantly, confocal and total internal reflection fluorescence (TIRF) microscopy can probe CAR signaling and inform CAR engineering.</p><p>Confocal microscopy visualizes the CAR with high spatial resolution. Effector and target cells can be adhered to glass slides, allowed to interact, and fixed prior to imaging. Using this approach, Davenport et al. found that CAR-T cells form non-classical IS with multifocal Lck microclusters that may facilitate serial killing (<xref rid=\"B58\" ref-type=\"bibr\">58</xref>). In addition, Long et al. utilized confocal microscopy with Cerulean-tagged CAR to show aggregation of the anti-GD2 CAR on the cell surface, which may contribute to antigen-independent tonic signaling (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>). However, this setup limits spatial resolution because the CAR IS lies on a vertical imaging plane formed through horizontal cell-cell interactions (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>). Xiong et al. circumvented this limitation using confocal microscopy with a vertical cell-pairing system, which flips the CAR IS onto a horizontal imaging plane. Their setup revealed that characteristics of the IS, including antigen clustering, lytic granule polarization, and distribution of key signaling molecules, predict CAR-T cell efficacy <italic>in vivo</italic> (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>).</p><p>In live cells, the CAR can be visualized at the molecular level via lipid bilayer experiments in conjunction with TIRF microscopy. TIRF microscopy excites fluorophores by inducing an evanescent field near the interface between two media with different refractive indices. In this setup, the CAR-T cell interacts with antigen on glass-supported lipid bilayers to form an IS on the horizontal plane. The evanescent field selectively excites fluorophores near this plane. CAR proteins at this interface can be directly or indirectly detected, via fluorescently labeled CAR or CAR antigen (<xref rid=\"B61\" ref-type=\"bibr\">61</xref>). Using TIRF, Xiaolei et al. characterized recruitment of CAR microclusters to the CAR IS, and found that the CAR IS disassembles quicker than the classical TCR IS (<xref rid=\"B62\" ref-type=\"bibr\">62</xref>).</p><p>With either TIRF or confocal microscopy, fluorescently labeling the CAR for direct detection is preferred over labeling the CAR antigen. Labeling the CAR allows CAR tracking outside of the IS and in resting CAR-T cells. One common method is to chimerically tag fluorescent proteins, such as green fluorescence protein derivatives, to the CAR C-terminus. In addition to enabling direct CAR visualization, this method also facilitates CAR quantification via flow cytometry. Walker et al. utilized cyan fluorescence protein-labeled CAR to measure anti-CD19 CAR downmodulation after antigen engagement. However, this tagging was not possible with all CAR constructs. They reported that an anti-ALK CAR tagged with cyan fluorescence protein failed to express (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Similarly, Morrissey et al. engineered enhanced green fluorescence protein-labeled CARs for phagocytosis that direct macrophages to engulf target cells. Trafficking of these CARs was studied via live-cell imaging (<xref rid=\"B63\" ref-type=\"bibr\">63</xref>). For CARs that are not amenable to fluorescent protein fusion, an alternative method is staining the extracellular regions of the CAR with fluorescent Fabs or scFvs immediately before microscopy. These Fabs should neither block the CAR antigen binding site nor influence CAR trafficking or mechanotransduction. Sasmal et al. utilized this method to label the TCR with an anti-TCRβ scFv for FRET studies (<xref rid=\"B64\" ref-type=\"bibr\">64</xref>). However, this method has yet to be applied onto CARs.</p><p>Finally, CARs have yet to be visualized with super-resolution microscopy or lattice light-sheet microscopy (LLSM). These emerging technologies can significantly improve spatial and temporal resolution. Rosenberg et al. utilized LLSM to visualize TCR dynamics, which were correlated with T cell signaling states (<xref rid=\"B65\" ref-type=\"bibr\">65</xref>, <xref rid=\"B66\" ref-type=\"bibr\">66</xref>). However, similar experiments to characterize CAR dynamics have not yet been performed. Nerreter et al. utilized stochastic optical reconstruction microscopy, a form of super-resolution microscopy, to image CD19 expression on multiple myeloma patient cancer cells, which was compared with flow cytometry data. Their analysis established a sensitivity threshold for CAR-T cell efficacy (<xref rid=\"B67\" ref-type=\"bibr\">67</xref>). However, similar studies to visualize the CAR itself have not been conducted.</p></sec></sec><sec id=\"s5\"><title>CAR Detection at the Organismal Level</title><p>After CAR-T cells are manufactured and intravenously infused, CAR-T cells proliferate and traffic between blood, lymph nodes, bone marrow, peripheral tissue, and tumor. <italic>In vivo</italic> detection and tracking of CAR-T cells can probe location-dependent phenotypes and elucidate models for therapy failure. There are three main methods for tracking CAR-T cells at the organismal level: bioluminescence imaging (BLI), positron emission tomography (PET) scanning, and two-photon microscopy (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>). All three methods can monitor CAR-T cells in organs. BLI and PET scanning utilize reporters and probes for visualization. A representative, but not exhaustive, list of applications of BLI and PET scanning is provided in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>. Two-photon microscopy utilizes fluorescence for visualization with single-cell resolution. Unlike CAR detection in the previous levels, CAR detection at the organismal level most directly studies CAR-T cells <italic>in vivo</italic>.</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Representative BLI and PET scanning applications.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Method type</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Reporter</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Probe</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Validation system</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Notes and reference</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BLI</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Firefly luciferase (FLuc)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D-luciferin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Anti-PSCA CAR-T cells in xenograft mouse model</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B68\" ref-type=\"bibr\">68</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BLI</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Firefly luciferase (FLuc)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">D-luciferin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">anti-HLA-A*02:01 CAR-Tregs in human allograft mouse model</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">(<xref rid=\"B69\" ref-type=\"bibr\">69</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Duplex BLI</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Renilla luciferase (RLuc) pavee Click beetle luciferase (CBRLuc)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Coelenterazine and D-luciferin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">anti-PSMA CAR-T cells in xenograft mouse model</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Signal diminished by poor substrate availability (<xref rid=\"B70\" ref-type=\"bibr\">70</xref>)</td></tr><tr style=\"border-bottom: thin solid #000000;\"><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Duplex BLI</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Stabilized color FLuc mutants</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Infraluciferin</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">anti-CD19 CAR-T cells in xenograft mouse model</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Used spectral unmixing (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PET scanning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Herpes simplex virus type 1 thymidine kinase (HSV1-TK)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><sup>18</sup>F-FHBG</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-13 zetakine CAR-T cells in clinical trial</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Clinical study (<xref rid=\"B72\" ref-type=\"bibr\">72</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PET Scanning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DOTA antibody reporter 1 (DAbR1)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><sup>86</sup>Y-AABD for imaging pavee <sup>177</sup>Lu-AABD for suicide</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">anti-CD19 CAR-T cells in xenograft mouse model</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Forms covalent bond between reporter and probe (<xref rid=\"B73\" ref-type=\"bibr\">73</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PET scanning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>E. coli</italic> dihydrofolate reductase enzyme (eDHFR)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><sup>18</sup>F-labeled trimethoprim (<sup>18</sup>F-TMP)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">anti-GD2 CAR-T cells in xenograft mouse model</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sensitivity of ~11,000 CD8<sup>+</sup> CAR-T cells per mm<sup>3</sup> (<xref rid=\"B74\" ref-type=\"bibr\">74</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PET Scanning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human somatostatin receptor 2 (SSTR2)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><sup>18</sup>F-NOTA-Octreotide (NOTAOCT)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ICAM-1-directed CAR-T cells in xenograft mouse model</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Background expression of SSTR2 in healthy human tissue (<xref rid=\"B75\" ref-type=\"bibr\">75</xref>, <xref rid=\"B76\" ref-type=\"bibr\">76</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PET scanning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Human sodium iodide symporter (hNIS)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><inline-formula><mml:math id=\"M1\"><mml:mmultiscripts><mml:mtext>T</mml:mtext><mml:mprescripts/><mml:none/><mml:mrow><mml:mtext>99m</mml:mtext></mml:mrow></mml:mmultiscripts><mml:msubsup><mml:mtext>cO</mml:mtext><mml:mn>4</mml:mn><mml:mo>−</mml:mo></mml:msubsup></mml:math></inline-formula></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">anti-PSMA CAR-T cells in xenograft mouse model</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Cheap and widely used radiotracer (<xref rid=\"B77\" ref-type=\"bibr\">77</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PET scanning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Prostate-specific membrane antigen (PSMA)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><sup>18</sup>F-DCFPyL</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">anti-CD19 CAR-T cells in xenograft mouse model</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reporter/probe used extensively in tracking prostate cancer (<xref rid=\"B78\" ref-type=\"bibr\">78</xref>)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PET scanning</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">None</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><sup>89</sup>Zr-<italic>p</italic>-isothiocyanatobenzyl-desferrioxamine (<sup>89</sup>Zr-DFO)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">anti-CD19 CAR-T cells in xenograft mouse model</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Physical labeling bypasses need for reporter; long half-life (<xref rid=\"B79\" ref-type=\"bibr\">79</xref>)</td></tr></tbody></table></table-wrap><sec><title>Bioluminescence Imaging</title><p><italic>In vivo</italic> BLI captures CAR-T cell biodistribution throughout an organism. Furthermore, BLI can probe for trafficking into solid tumors without needing to isolate the tumor for manual processing. To enable BLI, CAR-T cells must be co-transduced with luciferase. During imaging, luciferase substrate is injected, circulate and diffuse to CAR-T cells, and get processed by luciferase to emit light. The emitted light is captured using charge-coupled device (CCD) cameras, which convert light into electronic currents that localize the light source. Conventional luciferases come from terrestrial (i.e., North American firefly luciferase, FLuc) or marine (i.e., Renilla luciferase, RLuc) animals, which use D-luciferin or coelenterazine, respectively, along with O<sub>2</sub> and sometimes ATP. Newer luciferases are smaller and more sensitive (<xref rid=\"B80\" ref-type=\"bibr\">80</xref>).</p><p>BLI can track <italic>in vivo</italic> CAR-T cell expansion. Torres Chavez et al. utilized BLI to compare expansion kinetics of anti-CD19 CAR-T cells cultured with different sera during <italic>ex vivo</italic> transduction. BLI showed that human platelet lysate led to memory-like CAR-T cells, which exhibited superior <italic>in vivo</italic> expansion upon tumor re-challenge (<xref rid=\"B68\" ref-type=\"bibr\">68</xref>). Furthermore, BLI can track <italic>in vivo</italic> CAR-T cell trafficking. Dawson et al. utilized BLI to show trafficking of anti-HLA-A<sup>*</sup>02:01 CAR-Tregs, which migrated into transplanted human allograft skin tissue and associated draining lymph nodes. Functionally, these CAR-Tregs prevented allograft rejection in NSG mice (<xref rid=\"B69\" ref-type=\"bibr\">69</xref>).</p><p>Importantly, luciferase/substrate pairs can be multiplexed. For example, FLuc and RLuc can tag different cell populations in the same animal for imaging using different substrates. Serganova et al. used multiplex BLI to simultaneously track anti-PSMA CAR-T and PSMA<sup>+</sup> tumor cells with RLuc and click beetle luciferase, respectively, in a mouse model. Multiplex BLI revealed initial CAR-T cell sequestration in the lungs (<xref rid=\"B70\" ref-type=\"bibr\">70</xref>). However, this method requires sequential injection of luciferase substrates. The signal from the first injection must entirely disappear before the second injection, which requires careful optimization. Hence, Stowe et al. developed a different approach to BLI multiplexing: spectral unmixing. In their system, two cell populations are tagged with two distinct luciferases that share a common substrate: infraluciferin. These distinct luciferases generate light with dissimilar emission wavelengths. After infraluciferin injection, total BLI signal is captured, which is spectrally unmixed into two bioluminescence channels. Their method captured anti-CD19 CAR-T cells homing to and expanding within the lymphoma tumor in a mouse model (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>). BLI spectral unmixing eliminates the requirement for sequential substrate injection.</p><p>Strengths of BLI include accessibility of CCD cameras among core facilities, standardized and high-throughput protocols, affordability, and multiplexed live-cell imaging. Furthermore, engineered FLuc derivatives, such as AkaLumine-HCl can emit near-infrared light, for superior deep-tissue penetration (<xref rid=\"B81\" ref-type=\"bibr\">81</xref>). Hence, BLI is the method of choice for preclinical CAR-T cell experiments. However, it comes with notable weaknesses. BLI is not used in clinical trials because humans are too large for the emitted light to penetrate tissue efficiently. Furthermore, the luciferase reporter may be immunogenic. In mice, unlike intravital two-photon microscopy, BLI cannot track CAR-T cells at the single-cell level. Finally, the location and metabolism of the CAR-T cells may decrease ATP and O<sub>2</sub> availability, leading to diminished BLI signal. Substrate availability is even more limited in the tumor microenvironment (<xref rid=\"B70\" ref-type=\"bibr\">70</xref>). Engineered luciferases with superior enzymatic activity and tissue penetration only partially addresses these issues.</p></sec><sec><title>Positron Emission Tomography</title><p>Positron emission tomography (PET) scanning also captures CAR-T cell biodistribution throughout an organism. To enable PET, the CAR is co-transduced with a PET reporter, which can capture and accumulate a positron-emitting small molecule probe. For imaging, the probe is intravenously injected, which preferentially accumulates in CAR-T cells due to the co-expressed PET reporter. Emitted positrons colocalize with CAR-T cells, lose kinetic energy, combine with a nearby electron, get annihilated, and emit high-energy photons. The high energy photons are captured with a PET scanner (<xref rid=\"B82\" ref-type=\"bibr\">82</xref>).</p><p>Although many PET reporter/probe pairs have been developed to track CAR-T cells, only one pair has been tried on patients in a CAR-T cell clinical study (NCT00730613 and NCT01082926): herpes simplex virus type 1 thymidine kinase (HSV1-TK) paired with 9-[4-[<sup>18</sup>F]fluoro-3-(hydroxymethyl)butyl]guanine (<sup>18</sup>F-FHBG). HSV1-TK is a cytosolic viral kinase that selectively phosphorylates nucleoside analogs such as <sup>18</sup>F-FHBG. Phosphorylated <sup>18</sup>F-FHBG then accumulates intracellularly. The pharmacology and safety profile of <sup>18</sup>F-FHBG in humans are well-documented, and <sup>18</sup>F-FHBG is FDA-approved as an investigational new drug. In this clinical study, Keu et al. co-expressed HSV1-TK with an interleukin-13 (IL-13) zetakine CAR in CD8<sup>+</sup> T cells to treat recurrent high-grade glioma in seven patients. The glioma disrupts the blood-brain barrier, allowing <sup>18</sup>F-FHBG to diffuse into the tumor. PET scans show increased signal around the tumor after CAR-T cell infusion, which suggests active trafficking of CAR-T cells into the tumor. However, increased PET signal can also feasibly be due to increased non-specific vascular leakage or glioma progression, which this pilot study cannot address (<xref rid=\"B72\" ref-type=\"bibr\">72</xref>). Importantly, the HSV1-TK reporter may also function as a suicide switch by accumulating ganciclovir, a separate nucleoside analog which can induce apoptosis (<xref rid=\"B83\" ref-type=\"bibr\">83</xref>). This can be a critical safety mechanism for patients experiencing adverse CAR-T cell-related side effects, including cytokine release syndrome and pneumonia. The kinetics and utility of HSV1-TK as a suicide switch for CAR-T cells have yet to be clinically tested.</p><p>Other PET reporter/probe pairs have been developed in preclinical mouse models and are summarized in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>. Krebs et al. (<xref rid=\"B73\" ref-type=\"bibr\">73</xref>) used DOTA antibody reporter 1 (DAbR1), which binds irreversibly on the cell surface with <sup>86</sup>Y-labeled (<italic>S</italic>)-2-(4-acrylamidobenzyl)-DOTA (<sup>86</sup>Y-AABD). DAbR1 does not inhibit <italic>in vitro</italic> cytotoxicity, and PET scans show homing of CAR-T cells to the tumor. Furthermore, they predicted DAbR1 can be a suicide switch with <sup>177</sup>Lu-AABD, a heavier and more radioactive nuclide. Sellmyer et al. used <italic>Escherichia coli</italic> dihydrofolate reductase enzyme (eDHFR), which binds to <sup>18</sup>F-labeled trimethoprim (<sup>18</sup>F-TMP), to image anti-GD2 CAR-T cells in mouse xenograft models. PET scans show colocalization between anti-GD2 CAR-T cells and GD2<sup>+</sup> tumor, which was confirmed with bioluminescence. Finally, they calculated that their method can detect ~11,000 CD8<sup>+</sup> CAR-T cells per mm<sup>3</sup> (<xref rid=\"B74\" ref-type=\"bibr\">74</xref>). Park et al. used human somatostatin receptor 2 (SSTR2), which ensures intracellular accumulation of <sup>18</sup>F-NOTA-Octreotide (NOTAOCT), to track CAR-T cells of differing affinities to ICAM-1. PET scans captured CAR-T cell expansion and contraction kinetics (<xref rid=\"B75\" ref-type=\"bibr\">75</xref>). However, background expression of SSTR2 may preclude its use in clinical trials (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>). Emami-Shahri et al. used human sodium iodide symporter (hNIS), which is compatible with <inline-formula><mml:math id=\"M2\"><mml:mmultiscripts><mml:mtext>T</mml:mtext><mml:mprescripts/><mml:none/><mml:mrow><mml:mtext>99m</mml:mtext></mml:mrow></mml:mmultiscripts><mml:msubsup><mml:mtext>cO</mml:mtext><mml:mn>4</mml:mn><mml:mo>−</mml:mo></mml:msubsup></mml:math></inline-formula>, a cheap and widely used clinical radiotracer. Their results show trafficking of anti-PSMA CAR-T cells into the tumor, which was confirmed by IHC (<xref rid=\"B77\" ref-type=\"bibr\">77</xref>). Finally, Minn et al. (<xref rid=\"B78\" ref-type=\"bibr\">78</xref>) co-transduced CAR-T cells with PSMA, which interacts with <sup>18</sup>F-DCFPyL. Their results show divergence between CAR-T cell occupancy in blood, bone marrow, and tumor.</p><p>Furthermore, PET scanning can image physically labeled CAR-T cells, which bypasses the requirement for a PET reporter. CAR-T cells can be radiolabeled after manufacturing and prior to infusion. Lee et al. developed <sup>89</sup>Zr-<italic>p</italic>-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS, DFO), which covalently labels 70–79% of CAR-T cells with negligible impact on cell viability and proliferation. <sup>89</sup>Zr's long half-life (78.4 h) makes this nuclide suitable for long-term <italic>in vivo</italic> tracking. PET scanning captured these cells as they migrated between lung, liver, and spleen (<xref rid=\"B79\" ref-type=\"bibr\">79</xref>).</p><p>Strengths of PET scanning include accessibility of PET scanners in the clinic, penetration of positrons through tissue, and the dual use of PET reporter also as a suicide safety switch. Unlike BLI, PET scanning is widely used in the clinic. However, PET scanning shares some limitations with BLI, including lack of single-cell resolution and potential immunogenicity of the PET reporter. The latter limitation can be ameliorated with PET reporters that are based on endogenous human proteins (e.g., SSTR2 and hNIS). However, background expression of endogenous human proteins may also obscure results. Finally, unlike with BLI, PET scanning cannot multiplex different reporters, since all reporter/probe pairs emit positrons. Hence, PET scanning cannot simultaneously image both CAR-T and tumor cells.</p></sec><sec><title>Two-Photon Microscopy</title><p>Finally, two-photon microscopy can capture the distribution, motility, and functionality of CAR-T cells <italic>in vivo</italic> at the single-cell level. With two-photon microscopy, one fluorophore simultaneously absorbs multiple (usually two) units of near-infrared (NIR) photons and emits a single unit of fluorescence. Since NIR photons minimize scattering and multiphoton absorption occurs rarely in an area of high photon density, two-photon microscopy has deep tissue penetration, superior spatial resolution, and diminished photobleaching. These qualities are ideal for <italic>in vivo</italic> live-imaging mouse experiments to capture single CAR-T cells in action (<xref rid=\"B84\" ref-type=\"bibr\">84</xref>). Hence, out of the three CAR detection methods at the organismal level, two-photon microscopy is the most suitable for mechanistic studies at the cellular level.</p><p>Cazaux et al. utilized intravital two-photon microscopy to track GFP<sup>+</sup>CD8<sup>+</sup> anti-murine CD19 CAR-T cells in a syngeneic lymphoma mouse model. In addition to CAR-T cell motility, two-photon microscopy was also used to measure calcium flux and detect apoptosis in CAR-T cells and cancer cells, respectively, via Förster resonance energy transfer sensors. Two-photon microscopy demonstrated that: (1) B cells in circulation hindered CAR-T cells from trafficking to the bone marrow; (2) CAR-T cells killed both directly (through contact) and indirectly (through epitope spreading or cytokines); and (3) there is less immunosurveillance in lymph nodes than in bone marrow (<xref rid=\"B85\" ref-type=\"bibr\">85</xref>). Furthermore, Mulazzani et al. used intravital two-photon microscopy to compare GFP<sup>+</sup> anti-CD19 CAR-T cell infiltration into primary central nervous system lymphoma from intravenous and intracerebral CAR-T cell injection. Two-photon microscopy showed that intracerebral injection caused superior CAR-T cell infiltration and persistence, which was associated with long-term survival (<xref rid=\"B86\" ref-type=\"bibr\">86</xref>). In addition to genetically encoded fluorescent proteins, two-photon microscopy can also involve inorganic fluorophores. Ma et al. developed biodegradable polydopamine (PDA) nanodots with oxidation-induced fluorescence to track CAR-T cell targets <italic>in vivo</italic>. PDA can be endocytosed by target cells and oxidized intracellularly for imaging. They demonstrated proof-of-principle in dissected mouse tissue (<xref rid=\"B87\" ref-type=\"bibr\">87</xref>).</p><p>Two-photon microscopy is a powerful tool to capture <italic>in vivo</italic> CAR-T cell behavior and to generate novel hypotheses for CAR-T cell therapy failure and relapse. Its strengths (single-cell resolution, spatiotemporal resolution, tissue penetration) are ideal for mechanistic studies in mice. In addition, this method naturally links with other fluorescence-based tools, such as FRET sensors. However, unlike PET-based CAR tracking, two-photon microscopy cannot realistically be applied for clinical studies. Furthermore, two-photon microscopy requires equipment that might be inaccessible for many labs.</p></sec></sec><sec id=\"s6\"><title>Discussion and Outlook</title><p>In this review, we summarized CAR detection methods that operate at the genomic, transcriptomic, proteomic, and organismal levels. We have also identified key areas where CAR detection methods may be improved.</p><p>Based on the studies summarized in this review, we observed that development of new CAR detection methods has often proceeded through three phases. First, the new detection method is tested, validated, and optimized using CAR-T cells generated from a healthy donor's T cells after transduction with a known CAR construct. Under this controlled scenario, the method's accuracy (e.g., false negative and false positive incidences) and reproducibility (e.g., error across replicates) can be measured and optimized. Second, the optimized detection method is applied on clinical CAR-T cell samples from patients. Successful application on clinical samples demonstrates utility in a real-world scenario. Third, the results from the new detection method are compared with results obtained from existing detection methods. These three phases measure performance metrics and ensure utility for clinical studies.</p><p>Although no standard guidelines exist for developing new CAR detection methods, we believe the three aforementioned phases can serve as practical guidelines for development of future CAR detection methods. Furthermore, if the detection method is to be used for clinical or diagnostic purposes, we believe it should be accurate, reliable, reproducible, readily implemented, and easily interpreted. Results from one clinical laboratory should be replicable in another clinical laboratory. These suggestions are complementary with GCLP practices.</p><p>For CAR basic science studies, we believe that developing single-molecule microscopy-based CAR visualization will become increasingly important. The CAR has existed in its current form for years, but its molecular mechanisms are poorly understood or optimized. Although functionally similar to the TCR, the CAR traffics differently (<xref rid=\"B62\" ref-type=\"bibr\">62</xref>), and signaling is less efficient and sensitive (<xref rid=\"B88\" ref-type=\"bibr\">88</xref>). Furthermore, CAR-induced tonic signaling hastens CAR-T cell exhaustion (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>). We believe CAR signaling inefficiencies should be understood via microscopy-based CAR IS visualization. Mechanistic insights from CAR IS visualization with new technologies, such as super-resolution or lattice light-sheet microscopy, may inform engineering endeavors that improve CARs.</p><p>For CAR clinical studies, we believe that developing PET-based <italic>in vivo</italic> CAR-tracking methods will become increasingly important. Since multiple clinical trials aim to extend CAR-T cell therapy from hematological cancers to solid tumors, the ability to measure CAR-T cell trafficking into the tumor without the need for a biopsy is essential. This is particularly important for tumors at physically hard-to-access locations. The recent clinical trial that utilizes HSV1-TK as a PET reporter is a promising start, but confounding variables (vascular leakage and glioma progression) obscured conclusions drawn from their data (<xref rid=\"B72\" ref-type=\"bibr\">72</xref>). Newer CAR-T cell clinical studies that involve solid tumors should routinely employ PET scanning as both a research tool and on-treatment indicator of clinical efficacy. Meanwhile, other clinical studies should address the potential use of PET reporters as a CAR suicide safety switch.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>JH conceived the original concept. YH organized and wrote the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>We thank Dr. Nicholas Ankenbruck for helpful suggestions during manuscript preparations. All figures were created with <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.BioRender.com\">BioRender.com</ext-link>.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> We thank Phi Beta Psi, the Ullman Fund in Cancer Immunology, the Hoogland Lymphoma Research Pilot Projects, and Chicago Immunoengineering Innovation Center for financial support. YH was supported by the University of Chicago MSTP Training Grant (T32GM007281).</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>June</surname><given-names>CH</given-names></name><name><surname>O'Connor</surname><given-names>RS</given-names></name><name><surname>Kawalekar</surname><given-names>OU</given-names></name><name><surname>Ghassemi</surname><given-names>S</given-names></name><name><surname>Milone</surname><given-names>MC</given-names></name></person-group>. <article-title>CAR T cell immunotherapy for human cancer</article-title>. <source>Science.</source> (<year>2018</year>) <volume>359</volume>:<fpage>1361</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1126/science.aar6711</pub-id><pub-id pub-id-type=\"pmid\">29567707</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maude</surname><given-names>SL</given-names></name><name><surname>Laetsch</surname><given-names>TW</given-names></name><name><surname>Buechner</surname><given-names>J</given-names></name><name><surname>Rives</surname><given-names>S</given-names></name><name><surname>Boyer</surname><given-names>M</given-names></name><name><surname>Bittencourt</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia</article-title>. <source>N Engl J Med</source>. (<year>2018</year>) <volume>378</volume>:<fpage>439</fpage>–<lpage>48</lpage>. <pub-id pub-id-type=\"doi\">10.1056/NEJMoa1709866</pub-id><pub-id pub-id-type=\"pmid\">29385370</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Neelapu</surname><given-names>SS</given-names></name><name><surname>Locke</surname><given-names>FL</given-names></name><name><surname>Bartlett</surname><given-names>NL</given-names></name><name><surname>Lekakis</surname><given-names>LJ</given-names></name><name><surname>Miklos</surname><given-names>DB</given-names></name><name><surname>Jacobson</surname><given-names>CA</given-names></name><etal/></person-group>. <article-title>Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma</article-title>. <source>N Engl J Med</source>. (<year>2017</year>) <volume>377</volume>:<fpage>2531</fpage>–<lpage>44</lpage>. <pub-id pub-id-type=\"doi\">10.1056/NEJMoa1707447</pub-id><pub-id pub-id-type=\"pmid\">29226797</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>J</given-names></name><name><surname>Li</surname><given-names>W</given-names></name><name><surname>Huang</surname><given-names>K</given-names></name><name><surname>Zhang</surname><given-names>Y</given-names></name><name><surname>Kupfer</surname><given-names>G</given-names></name><name><surname>Zhao</surname><given-names>Q</given-names></name></person-group>. <article-title>Chimeric antigen receptor T cell (CAR-T) immunotherapy for solid tumors: lessons learned and strategies for moving forward</article-title>. <source>J Hematol Oncol</source>. (<year>2018</year>) <volume>11</volume>:<fpage>22</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s13045-018-0568-6</pub-id><pub-id pub-id-type=\"pmid\">29433552</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gumba</surname><given-names>H</given-names></name><name><surname>Musyoki</surname><given-names>J</given-names></name><name><surname>Mosobo</surname><given-names>M</given-names></name><name><surname>Lowe</surname><given-names>B</given-names></name></person-group>. <article-title>Implementation of good clinical laboratory practice in an immunology basic research laboratory</article-title>. <source>Am J Clin Pathol</source>. (<year>2019</year>) <volume>151</volume>:<fpage>270</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1093/ajcp/aqy138</pub-id><pub-id pub-id-type=\"pmid\">30339188</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>J</given-names></name><name><surname>Zhou</surname><given-names>G</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Zhao</surname><given-names>Q</given-names></name></person-group>. <article-title>Building potent chimeric antigen receptor T cells with CRISPR genome editing</article-title>. <source>Front Immunol</source>. (<year>2019</year>) <volume>10</volume>:<fpage>456</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2019.00456</pub-id><pub-id pub-id-type=\"pmid\">30941126</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hamada</surname><given-names>M</given-names></name><name><surname>Nishio</surname><given-names>N</given-names></name><name><surname>Okuno</surname><given-names>Y</given-names></name><name><surname>Suzuki</surname><given-names>S</given-names></name><name><surname>Kawashima</surname><given-names>N</given-names></name><name><surname>Muramatsu</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Integration mapping of piggybac-mediated CD19 chimeric antigen receptor T cells analyzed by novel tagmentation-assisted PCR</article-title>. <source>EBioMedicine</source>. (<year>2018</year>) <volume>34</volume>:<fpage>18</fpage>–<lpage>26</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ebiom.2018.07.008</pub-id><pub-id pub-id-type=\"pmid\">30082227</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fraietta</surname><given-names>JA</given-names></name><name><surname>Lacey</surname><given-names>SF</given-names></name><name><surname>Orlando</surname><given-names>EJ</given-names></name><name><surname>Pruteanu-Malinici</surname><given-names>I</given-names></name><name><surname>Gohil</surname><given-names>M</given-names></name><name><surname>Lundh</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia</article-title>. <source>Nat Med</source>. (<year>2018</year>) <volume>24</volume>:<fpage>563</fpage>–<lpage>71</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41591-018-0010-1</pub-id><pub-id pub-id-type=\"pmid\">29713085</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>H</given-names></name><name><surname>Du</surname><given-names>X</given-names></name><name><surname>Chen</surname><given-names>WH</given-names></name><name><surname>Lou</surname><given-names>J</given-names></name><name><surname>Xiao</surname><given-names>HL</given-names></name><name><surname>Pan</surname><given-names>YM</given-names></name><etal/></person-group>. <article-title>Establishment of a quantitative polymerase chain reaction assay for monitoring chimeric antigen receptor T cells in peripheral blood</article-title>. <source>Transplant Proc</source>. (<year>2018</year>) <volume>50</volume>:<fpage>104</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.transproceed.2017.11.028</pub-id><pub-id pub-id-type=\"pmid\">29407291</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kunz</surname><given-names>A</given-names></name><name><surname>Gern</surname><given-names>U</given-names></name><name><surname>Schmitt</surname><given-names>A</given-names></name><name><surname>Neuber</surname><given-names>B</given-names></name><name><surname>Wang</surname><given-names>L</given-names></name><name><surname>Hückelhoven-Krauss</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Optimized assessment of qPCR-based vector copy numbers as a safety parameter for GMP-grade CAR T cells and monitoring of frequency in patients</article-title>. <source>Mol Ther Methods Clin Dev</source>. (<year>2020</year>) <volume>17</volume>:<fpage>448</fpage>–<lpage>54</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.omtm.2020.02.003</pub-id><pub-id pub-id-type=\"pmid\">32201711</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ståhlberg</surname><given-names>A</given-names></name><name><surname>Kubista</surname><given-names>M</given-names></name></person-group>. <article-title>Technical aspects and recommendations for single-cell qPCR</article-title>. <source>Mol Aspects Med</source>. (<year>2018</year>) <volume>59</volume>:<fpage>28</fpage>–<lpage>35</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.mam.2017.07.004</pub-id><pub-id pub-id-type=\"pmid\">28756182</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vogelstein</surname><given-names>B</given-names></name><name><surname>Kinzler</surname><given-names>KW</given-names></name></person-group>. <article-title>Digital PCR</article-title>. <source>Proc Natl Acad Sci USA</source>. (<year>1999</year>) <volume>96</volume>:<fpage>9236</fpage>–<lpage>41</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.96.16.9236</pub-id><pub-id pub-id-type=\"pmid\">10430926</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fehse</surname><given-names>B</given-names></name><name><surname>Badbaran</surname><given-names>A</given-names></name><name><surname>Berger</surname><given-names>C</given-names></name><name><surname>Sonntag</surname><given-names>T</given-names></name><name><surname>Riecken</surname><given-names>K</given-names></name><name><surname>Geffken</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Digital PCR assays for precise quantification of CD19-CAR-T cells after treatment with axicabtagene ciloleucel</article-title>. <source>Mol Ther Methods Clin Dev</source>. (<year>2020</year>) <volume>16</volume>:<fpage>172</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.omtm.2019.12.018</pub-id><pub-id pub-id-type=\"pmid\">32055645</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Santeramo</surname><given-names>I</given-names></name><name><surname>Bagnati</surname><given-names>M</given-names></name><name><surname>Harvey</surname><given-names>EJ</given-names></name><name><surname>Hassan</surname><given-names>E</given-names></name><name><surname>Surmacz-Cordle</surname><given-names>B</given-names></name><name><surname>Marshall</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Vector copy distribution at a single cell level enhances analytical characterisation of gene-modified cell therapies</article-title>. <source>Mol Ther Methods Clin Dev</source>. (<year>2020</year>) <volume>17</volume>:<fpage>944</fpage>–<lpage>56</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.omtm.2020.04.016</pub-id><pub-id pub-id-type=\"pmid\">32420408</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nobles</surname><given-names>CL</given-names></name><name><surname>Sherrill-Mix</surname><given-names>S</given-names></name><name><surname>Everett</surname><given-names>JK</given-names></name><name><surname>Reddy</surname><given-names>S</given-names></name><name><surname>Fraietta</surname><given-names>JA</given-names></name><name><surname>Porter</surname><given-names>DL</given-names></name><etal/></person-group>. <article-title>CD19-targeting CAR T cell immunotherapy outcomes correlate with genomic modification by vector integration</article-title>. <source>J Clin Invest</source>. (<year>2020</year>) <volume>130</volume>:<fpage>673</fpage>–<lpage>85</lpage>. <pub-id pub-id-type=\"doi\">10.1172/JCI130144</pub-id><pub-id pub-id-type=\"pmid\">31845905</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fraietta</surname><given-names>JA</given-names></name><name><surname>Nobles</surname><given-names>CL</given-names></name><name><surname>Sammons</surname><given-names>MA</given-names></name><name><surname>Lundh</surname><given-names>S</given-names></name><name><surname>Carty</surname><given-names>SA</given-names></name><name><surname>Reich</surname><given-names>TJ</given-names></name><etal/></person-group>. <article-title>Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells</article-title>. <source>Nature</source>. (<year>2018</year>)<volume>558</volume>:<fpage>307</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41586-018-0178-z</pub-id><pub-id pub-id-type=\"pmid\">29849141</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hacien-Bey-Abina</surname><given-names>S</given-names></name><name><surname>Kalle</surname><given-names>CV</given-names></name><name><surname>Schmidt</surname><given-names>M</given-names></name><name><surname>McCormack</surname><given-names>MP</given-names></name><name><surname>Wulffraat</surname><given-names>N</given-names></name><name><surname>Leboulch</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>LMO2-Associated clonal T cell proliferation in two patients after gene therapy for SCID-X1</article-title>. <source>Science.</source> (<year>2003</year>) <volume>302</volume>:<fpage>415</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1126/science.1088547</pub-id><pub-id pub-id-type=\"pmid\">14564000</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>X</given-names></name><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Crise</surname><given-names>B</given-names></name><name><surname>Burgess</surname><given-names>SM</given-names></name></person-group>. <article-title>Transcription start regions in the human genome are favored targets for MLV integration</article-title>. <source>Science.</source> (<year>2003</year>) <volume>300</volume>:<fpage>1749</fpage>–<lpage>51</lpage>. <pub-id pub-id-type=\"doi\">10.1126/science.1083413</pub-id><pub-id pub-id-type=\"pmid\">12805549</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brady</surname><given-names>T</given-names></name><name><surname>Roth</surname><given-names>SL</given-names></name><name><surname>Malani</surname><given-names>N</given-names></name><name><surname>Wang</surname><given-names>GP</given-names></name><name><surname>Berry</surname><given-names>CC</given-names></name><name><surname>Leboulch</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>A method to sequence and quantify DNA integration for monitoring outcome in gene therapy</article-title>. <source>Nucl Acids Res</source>. (<year>2011</year>) <volume>39</volume>:<fpage>e72</fpage>. <pub-id pub-id-type=\"doi\">10.1093/nar/gkr140</pub-id><pub-id pub-id-type=\"pmid\">21415009</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Berry</surname><given-names>CC</given-names></name><name><surname>Nobles</surname><given-names>C</given-names></name><name><surname>Six</surname><given-names>E</given-names></name><name><surname>Wu</surname><given-names>Y</given-names></name><name><surname>Malani</surname><given-names>N</given-names></name><name><surname>Sherman</surname><given-names>E</given-names></name><etal/></person-group>. <article-title>INSPIIRED: quantification and visualization tools for analyzing integration site distributions</article-title>. <source>Mol Ther Methods Clin Dev</source>. (<year>2017</year>) <volume>4</volume>:<fpage>17</fpage>–<lpage>26</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.omtm.2016.11.003</pub-id><pub-id pub-id-type=\"pmid\">28344988</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sherman</surname><given-names>E</given-names></name><name><surname>Nobles</surname><given-names>C</given-names></name><name><surname>Berry</surname><given-names>CC</given-names></name><name><surname>Six</surname><given-names>E</given-names></name><name><surname>Wu</surname><given-names>Y</given-names></name><name><surname>Dryga</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>INSPIIRED: a pipeline for quantitative analysis of sites of new DNA integration in cellular genomes</article-title>. <source>Mol Ther Methods Clin Dev</source>. (<year>2017</year>) <volume>4</volume>:<fpage>39</fpage>–<lpage>49</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.omtm.2016.11.002</pub-id><pub-id pub-id-type=\"pmid\">28344990</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>W</given-names></name><name><surname>Fasolino</surname><given-names>M</given-names></name><name><surname>Cattau</surname><given-names>B</given-names></name><name><surname>Goldman</surname><given-names>N</given-names></name><name><surname>Kong</surname><given-names>W</given-names></name><name><surname>Frederick</surname><given-names>MA</given-names></name><etal/></person-group>. <article-title>Joint profiling of chromatin accessibility and CAR-T integration site analysis at population and single-cell levels</article-title>. <source>Proc Natl Acad Sci USA</source>. (<year>2020</year>) <volume>117</volume>:<fpage>5442</fpage>–<lpage>52</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1919259117</pub-id><pub-id pub-id-type=\"pmid\">32094195</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Berry</surname><given-names>CC</given-names></name><name><surname>Gillet</surname><given-names>NA</given-names></name><name><surname>Melamed</surname><given-names>A</given-names></name><name><surname>Gormley</surname><given-names>N</given-names></name><name><surname>Bangham</surname><given-names>CRM</given-names></name><name><surname>Bushman</surname><given-names>FD</given-names></name></person-group>. <article-title>Estimating abundances of retroviral insertion sites from DNA fragment length data</article-title>. <source>Bioinformatics</source>. (<year>2012</year>) <volume>28</volume>:<fpage>755</fpage>–<lpage>62</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bioinformatics/bts004</pub-id><pub-id pub-id-type=\"pmid\">22238265</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gomes-Silva</surname><given-names>D</given-names></name><name><surname>Mukherjee</surname><given-names>M</given-names></name><name><surname>Srinivasan</surname><given-names>M</given-names></name><name><surname>Krenciute</surname><given-names>G</given-names></name><name><surname>Dakhova</surname><given-names>O</given-names></name><name><surname>Zheng</surname><given-names>Y</given-names></name><etal/></person-group>. <article-title>Tonic 4-1BB costimulation in chimeric antigen receptors impedes T cell survival and is vector-dependent</article-title>. <source>Cell Rep</source>. (<year>2017</year>) <volume>21</volume>:<fpage>17</fpage>–<lpage>26</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.celrep.2017.09.015</pub-id><pub-id pub-id-type=\"pmid\">28978471</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Walker</surname><given-names>AJ</given-names></name><name><surname>Majzner</surname><given-names>RG</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Wanhainen</surname><given-names>K</given-names></name><name><surname>Long</surname><given-names>AH</given-names></name><name><surname>Nguyen</surname><given-names>SM</given-names></name><etal/></person-group>. <article-title>Tumor antigen and receptor densities regulate efficacy of a chimeric antigen receptor targeting anaplastic lymphoma kinase</article-title>. <source>Mol Ther</source>. (<year>2017</year>) <volume>25</volume>:<fpage>2189</fpage>–<lpage>201</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ymthe.2017.06.008</pub-id><pub-id pub-id-type=\"pmid\">28676342</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>Q</given-names></name><name><surname>Hu</surname><given-names>H</given-names></name><name><surname>Chen</surname><given-names>SY</given-names></name><name><surname>Liu</surname><given-names>CJ</given-names></name><name><surname>Hu</surname><given-names>FF</given-names></name><name><surname>Yu</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Transcriptome and regulatory network analyses of CD19-CAR-T immunotherapy for B-ALL</article-title>. <source>Genomics Proteomics Bioinform</source>. (<year>2019</year>) <volume>17</volume>:<fpage>190</fpage>–<lpage>200</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.gpb.2018.12.008</pub-id><pub-id pub-id-type=\"pmid\">31201998</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sheih</surname><given-names>A</given-names></name><name><surname>Voillet</surname><given-names>V</given-names></name><name><surname>Hanafi</surname><given-names>LA</given-names></name><name><surname>DeBerg</surname><given-names>HA</given-names></name><name><surname>Yajima</surname><given-names>M</given-names></name><name><surname>Hawkins</surname><given-names>R</given-names></name><etal/></person-group>. <article-title>Clonal kinetics and single-cell transcriptional profiling of CAR-T cells in patients undergoing CD19 CAR-T immunotherapy</article-title>. <source>Nat Commun</source>. (<year>2020</year>) <volume>11</volume>:<fpage>219</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41467-019-13880-1</pub-id><pub-id pub-id-type=\"pmid\">31924795</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>N</given-names></name><name><surname>Rajasekaran</surname><given-names>N</given-names></name><name><surname>Hou</surname><given-names>T</given-names></name><name><surname>Lisowski</surname><given-names>L</given-names></name><name><surname>Mellins</surname><given-names>ED</given-names></name></person-group>. <article-title>Comparison of transduction efficiency among various lentiviruses containing GFP reporter in bone marrow hematopoietic stem cell transplantation</article-title>. <source>Exp Hematol</source>. (<year>2013</year>) <volume>41</volume>:<fpage>934</fpage>–<lpage>43</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.exphem.2013.07.002</pub-id><pub-id pub-id-type=\"pmid\">23954710</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>F</given-names></name><name><surname>Flanagan</surname><given-names>J</given-names></name><name><surname>Su</surname><given-names>N</given-names></name><name><surname>Wang</surname><given-names>LC</given-names></name><name><surname>Bui</surname><given-names>S</given-names></name><name><surname>Nielson</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>RNAscope: a novel <italic>in situ</italic> RNA analysis platform for formalin-fixed, paraffin-embedded tissues</article-title>. <source>J Mol Diagnostics</source>. (<year>2012</year>) <volume>14</volume>:<fpage>22</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jmoldx.2011.08.002</pub-id><pub-id pub-id-type=\"pmid\">22166544</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>O'Rourke</surname><given-names>DM</given-names></name><name><surname>Nasrallah</surname><given-names>MP</given-names></name><name><surname>Desai</surname><given-names>A</given-names></name><name><surname>Melenhorst</surname><given-names>JJ</given-names></name><name><surname>Mansfield</surname><given-names>K</given-names></name><name><surname>Morrissette</surname><given-names>JJD</given-names></name><etal/></person-group>. <article-title>A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma</article-title>. <source>Sci Transl Med</source>. (<year>2017</year>) <volume>9</volume>:<fpage>eaaa0984</fpage>. <pub-id pub-id-type=\"doi\">10.1126/scitranslmed.aaa0984</pub-id><pub-id pub-id-type=\"pmid\">28724573</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Pimentel</surname><given-names>H</given-names></name><name><surname>Jarnagin</surname><given-names>H</given-names></name><name><surname>Zong</surname><given-names>H</given-names></name><name><surname>Todorov</surname><given-names>C</given-names></name><name><surname>Ganley</surname><given-names>K</given-names></name><name><surname>Eng</surname><given-names>F</given-names></name><etal/></person-group>\n<article-title>Abstract 2324: preclinical safety, biodistribution, and tumor infiltration analysis of CAR T cell targets using in situ hybridization technology</article-title>. In: <source>Immunology</source>. <publisher-loc>Atlanta, GA</publisher-loc>: <publisher-name>American Association for Cancer Research</publisher-name> (<year>2019</year>). p. <fpage>2324</fpage>\n<pub-id pub-id-type=\"doi\">10.1158/1538-7445.AM2019-2324</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yang</surname><given-names>Y</given-names></name><name><surname>Kohler</surname><given-names>ME</given-names></name><name><surname>Chien</surname><given-names>CD</given-names></name><name><surname>Sauter</surname><given-names>CT</given-names></name><name><surname>Jacoby</surname><given-names>E</given-names></name><name><surname>Yan</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>TCR engagement negatively affects CD8 but not CD4 CAR T cell expansion and leukemic clearance</article-title>. <source>Sci Transl Med</source>. (<year>2017</year>) <volume>9</volume>:<fpage>eaag1209</fpage>. <pub-id pub-id-type=\"doi\">10.1126/scitranslmed.aag1209</pub-id><pub-id pub-id-type=\"pmid\">29167392</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hamieh</surname><given-names>M</given-names></name><name><surname>Dobrin</surname><given-names>A</given-names></name><name><surname>Cabriolu</surname><given-names>A</given-names></name><name><surname>van der Stegen</surname><given-names>SJC</given-names></name><name><surname>Giavridis</surname><given-names>T</given-names></name><name><surname>Mansilla-Soto</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>CAR T cell trogocytosis and cooperative killing regulate tumour antigen escape</article-title>. <source>Nature</source>. (<year>2019</year>) <volume>568</volume>:<fpage>112</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41586-019-1054-1</pub-id><pub-id pub-id-type=\"pmid\">30918399</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kochenderfer</surname><given-names>JN</given-names></name><name><surname>Feldman</surname><given-names>SA</given-names></name><name><surname>Zhao</surname><given-names>Y</given-names></name><name><surname>Xu</surname><given-names>H</given-names></name><name><surname>Black</surname><given-names>MA</given-names></name><name><surname>Morgan</surname><given-names>RA</given-names></name><etal/></person-group>. <article-title>Construction and preclinical evaluation of an anti-CD19 chimeric antigen receptor</article-title>. <source>J Immunother</source>. (<year>2009</year>) <volume>32</volume>:<fpage>689</fpage>–<lpage>702</lpage>. <pub-id pub-id-type=\"doi\">10.1097/CJI.0b013e3181ac6138</pub-id><pub-id pub-id-type=\"pmid\">19561539</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nilson</surname><given-names>BHK</given-names></name><name><surname>Lögdberg</surname><given-names>L</given-names></name><name><surname>Kastern</surname><given-names>W</given-names></name><name><surname>Björck</surname><given-names>L</given-names></name><name><surname>Åkerström</surname><given-names>B</given-names></name></person-group>. <article-title>Purification of antibodies using protein L-binding framework structures in the light chain variable domain</article-title>. <source>J Immunol Methods</source>. (<year>1993</year>) <volume>164</volume>:<fpage>33</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1016/0022-1759(93)90273-A</pub-id><pub-id pub-id-type=\"pmid\">8360508</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zheng</surname><given-names>Z</given-names></name><name><surname>Chinnasamy</surname><given-names>N</given-names></name><name><surname>Morgan</surname><given-names>RA</given-names></name></person-group>. <article-title>Protein L: a novel reagent for the detection of chimeric antigen receptor (CAR) expression by flow cytometry</article-title>. <source>J Transl Med</source>. (<year>2012</year>) <volume>10</volume>:<fpage>29</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1479-5876-10-29</pub-id><pub-id pub-id-type=\"pmid\">22330761</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Han</surname><given-names>X</given-names></name><name><surname>Bryson</surname><given-names>PD</given-names></name><name><surname>Zhao</surname><given-names>Y</given-names></name><name><surname>Cinay</surname><given-names>GE</given-names></name><name><surname>Li</surname><given-names>S</given-names></name><name><surname>Guo</surname><given-names>Y</given-names></name><etal/></person-group>. <article-title>Masked chimeric antigen receptor for tumor-specific activation</article-title>. <source>Mol Ther</source>. (<year>2017</year>) <volume>25</volume>:<fpage>274</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ymthe.2016.10.011</pub-id><pub-id pub-id-type=\"pmid\">28129121</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Long</surname><given-names>AH</given-names></name><name><surname>Haso</surname><given-names>WM</given-names></name><name><surname>Shern</surname><given-names>JF</given-names></name><name><surname>Wanhainen</surname><given-names>KM</given-names></name><name><surname>Murgai</surname><given-names>M</given-names></name><name><surname>Ingaramo</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors</article-title>. <source>Nat Med</source>. (<year>2015</year>) <volume>21</volume>:<fpage>581</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nm.3838</pub-id><pub-id pub-id-type=\"pmid\">25939063</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sun</surname><given-names>M</given-names></name><name><surname>Shi</surname><given-names>H</given-names></name><name><surname>Liu</surname><given-names>C</given-names></name><name><surname>Liu</surname><given-names>J</given-names></name><name><surname>Liu</surname><given-names>X</given-names></name><name><surname>Sun</surname><given-names>Y</given-names></name></person-group>. <article-title>Construction and evaluation of a novel humanized HER2-specific chimeric receptor</article-title>. <source>Breast Cancer Res</source>. (<year>2014</year>) <volume>16</volume>:<fpage>R61</fpage>. <pub-id pub-id-type=\"doi\">10.1186/bcr3674</pub-id><pub-id pub-id-type=\"pmid\">24919843</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ruella</surname><given-names>M</given-names></name><name><surname>Barrett</surname><given-names>DM</given-names></name><name><surname>Kenderian</surname><given-names>SS</given-names></name><name><surname>Shestova</surname><given-names>O</given-names></name><name><surname>Hofmann</surname><given-names>TJ</given-names></name><name><surname>Perazzelli</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies</article-title>. <source>J Clin Invest</source>. (<year>2016</year>) <volume>126</volume>:<fpage>3814</fpage>–<lpage>26</lpage>. <pub-id pub-id-type=\"doi\">10.1172/JCI87366</pub-id><pub-id pub-id-type=\"pmid\">27571406</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Oliveira</surname><given-names>SN</given-names></name><name><surname>Wang</surname><given-names>J</given-names></name><name><surname>Ryan</surname><given-names>C</given-names></name><name><surname>Morrison</surname><given-names>SL</given-names></name><name><surname>Kohn</surname><given-names>DB</given-names></name><name><surname>Hollis</surname><given-names>RP</given-names></name></person-group>. <article-title>A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors</article-title>. <source>J Transl Med</source>. (<year>2013</year>) <volume>11</volume>:<fpage>23</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1479-5876-11-23</pub-id><pub-id pub-id-type=\"pmid\">23360526</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chang</surname><given-names>ZL</given-names></name><name><surname>Lorenzini</surname><given-names>MH</given-names></name><name><surname>Chen</surname><given-names>X</given-names></name><name><surname>Tran</surname><given-names>U</given-names></name><name><surname>Bangayan</surname><given-names>NJ</given-names></name><name><surname>Chen</surname><given-names>YY</given-names></name></person-group>. <article-title>Rewiring T-cell responses to soluble factors with chimeric antigen receptors</article-title>. <source>Nat Chem Biol</source>. (<year>2018</year>) <volume>14</volume>:<fpage>317</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nchembio.2565</pub-id><pub-id pub-id-type=\"pmid\">29377003</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Teplyakov</surname><given-names>A</given-names></name><name><surname>Obmolova</surname><given-names>G</given-names></name><name><surname>Luo</surname><given-names>J</given-names></name><name><surname>Gilliland</surname><given-names>GL</given-names></name></person-group>. <article-title>Crystal structure of B-cell co-receptor CD19 in complex with antibody B43 reveals an unexpected fold</article-title>. <source>Proteins Struct Funct Bioinforma</source>. (<year>2018</year>) <volume>86</volume>:<fpage>495</fpage>–<lpage>500</lpage>. <pub-id pub-id-type=\"doi\">10.1002/prot.25485</pub-id><pub-id pub-id-type=\"pmid\">29490423</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lobner</surname><given-names>E</given-names></name><name><surname>Wachernig</surname><given-names>A</given-names></name><name><surname>Gudipati</surname><given-names>V</given-names></name><name><surname>Mayrhofer</surname><given-names>P</given-names></name><name><surname>Salzer</surname><given-names>B</given-names></name><name><surname>Lehner</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Getting CD19 into shape: expression of natively folded “difficult-to-express” CD19 for staining and stimulation of CAR-T cells</article-title>. <source>Front Bioeng Biotechnol</source>. (<year>2020</year>) <volume>8</volume>:<fpage>49</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fbioe.2020.00049</pub-id><pub-id pub-id-type=\"pmid\">32117929</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Toebes</surname><given-names>M</given-names></name><name><surname>Rodenko</surname><given-names>B</given-names></name><name><surname>Ovaa</surname><given-names>H</given-names></name><name><surname>Schumacher</surname><given-names>TNM</given-names></name></person-group>. <article-title>Generation of peptide MHC class I monomers and multimers through ligand exchange</article-title>. <source>Curr Protoc Immunol</source>. (<year>2009</year>) Chapter 18:Unit 18.16. <pub-id pub-id-type=\"doi\">10.1002/0471142735.im1816s87</pub-id><pub-id pub-id-type=\"pmid\">19918947</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bentzen</surname><given-names>AK</given-names></name><name><surname>Hadrup</surname><given-names>SR</given-names></name></person-group>. <article-title>Evolution of MHC-based technologies used for detection of antigen-responsive T cells</article-title>. <source>Cancer Immunol Immunother</source>. (<year>2017</year>) <volume>66</volume>:<fpage>657</fpage>–<lpage>66</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00262-017-1971-5</pub-id><pub-id pub-id-type=\"pmid\">28314956</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>J</given-names></name><name><surname>Zeng</surname><given-names>X</given-names></name><name><surname>Sigal</surname><given-names>N</given-names></name><name><surname>Lund</surname><given-names>PJ</given-names></name><name><surname>Su</surname><given-names>LF</given-names></name><name><surname>Huang</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Detection, phenotyping, and quantification of antigen-specific T cells using a peptide-MHC dodecamer</article-title>. <source>Proc Natl Acad Sci USA</source>. (<year>2016</year>) <volume>113</volume>:<fpage>E1890</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1602488113</pub-id><pub-id pub-id-type=\"pmid\">26979955</pub-id></mixed-citation></ref><ref id=\"B48\"><label>48.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Batard</surname><given-names>P</given-names></name><name><surname>Peterson</surname><given-names>DA</given-names></name><name><surname>Devêvre</surname><given-names>E</given-names></name><name><surname>Guillaume</surname><given-names>P</given-names></name><name><surname>Cerottini</surname><given-names>JC</given-names></name><name><surname>Rimoldi</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Dextramers: new generation of fluorescent MHC class I/peptide multimers for visualization of antigen-specific CD8+ T cells</article-title>. <source>J Immunol Methods</source>. (<year>2006</year>) <volume>310</volume>:<fpage>136</fpage>–<lpage>48</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jim.2006.01.006</pub-id><pub-id pub-id-type=\"pmid\">16516226</pub-id></mixed-citation></ref><ref id=\"B49\"><label>49.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jena</surname><given-names>B</given-names></name><name><surname>Maiti</surname><given-names>S</given-names></name><name><surname>Huls</surname><given-names>H</given-names></name><name><surname>Singh</surname><given-names>H</given-names></name><name><surname>Lee</surname><given-names>DA</given-names></name><name><surname>Champlin</surname><given-names>RE</given-names></name><etal/></person-group>. <article-title>Chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials</article-title>. <source>PLoS ONE</source>. (<year>2013</year>) <volume>8</volume>:<fpage>e57838</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0057838</pub-id><pub-id pub-id-type=\"pmid\">23469246</pub-id></mixed-citation></ref><ref id=\"B50\"><label>50.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>DW</given-names></name><name><surname>Kochenderfer</surname><given-names>JN</given-names></name><name><surname>Stetler-Stevenson</surname><given-names>M</given-names></name><name><surname>Cui</surname><given-names>YK</given-names></name><name><surname>Delbrook</surname><given-names>C</given-names></name><name><surname>Feldman</surname><given-names>SA</given-names></name><etal/></person-group>. <article-title>T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial</article-title>. <source>Lancet</source>. (<year>2015</year>) <volume>385</volume>:<fpage>517</fpage>–<lpage>28</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0140-6736(14)61403-3</pub-id><pub-id pub-id-type=\"pmid\">25319501</pub-id></mixed-citation></ref><ref id=\"B51\"><label>51.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ruella</surname><given-names>M</given-names></name><name><surname>Barrett</surname><given-names>DM</given-names></name><name><surname>Shestova</surname><given-names>O</given-names></name><name><surname>Perazzelli</surname><given-names>J</given-names></name><name><surname>Posey</surname><given-names>AD</given-names></name><name><surname>Kozlowski</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>A cellular antidote to specifically deplete anti-CD19 chimeric antigen receptor (CAR19) positive cells</article-title>. <source>Blood</source>. (<year>2017</year>) <volume>130</volume>:<fpage>4463</fpage>. <pub-id pub-id-type=\"doi\">10.1182/blood.V130.Suppl_1.4463.4463</pub-id><pub-id pub-id-type=\"pmid\">31703119</pub-id></mixed-citation></ref><ref id=\"B52\"><label>52.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yvon</surname><given-names>E</given-names></name><name><surname>Del Vecchio</surname><given-names>M</given-names></name><name><surname>Savoldo</surname><given-names>B</given-names></name><name><surname>Hoyos</surname><given-names>V</given-names></name><name><surname>Dutour</surname><given-names>A</given-names></name><name><surname>Anichini</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Immunotherapy of metastatic melanoma using genetically engineered GD2-specific T cells</article-title>. <source>Clin Cancer Res</source>. (<year>2009</year>) <volume>15</volume>:<fpage>5852</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.1158/1078-0432.CCR-08-3163</pub-id><pub-id pub-id-type=\"pmid\">19737958</pub-id></mixed-citation></ref><ref id=\"B53\"><label>53.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Sievers</surname><given-names>SA</given-names></name><name><surname>Kelley</surname><given-names>KA</given-names></name><name><surname>Astrow</surname><given-names>SH</given-names></name><name><surname>Bot</surname><given-names>A</given-names></name><name><surname>Wiltzius</surname><given-names>JJ</given-names></name></person-group>\n<article-title>Abstract 1204: design and development of anti-linker antibodies for the detection and characterization of CAR T cells</article-title>. In: <source>Experimental and Molecular Therapeutics.</source>\n<publisher-loc>Atlanta, GA</publisher-loc>: <publisher-name>American Association for Cancer Research</publisher-name> (<year>2019</year>). p. <fpage>1204</fpage>\n<pub-id pub-id-type=\"doi\">10.1158/1538-7445.AM2019-1204</pub-id></mixed-citation></ref><ref id=\"B54\"><label>54.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>PH</given-names></name><name><surname>Lipschitz</surname><given-names>M</given-names></name><name><surname>Wright</surname><given-names>K</given-names></name><name><surname>Armand</surname><given-names>P</given-names></name><name><surname>Jacobson</surname><given-names>CA</given-names></name><name><surname>Roberts</surname><given-names>ZJ</given-names></name><etal/></person-group>\n<article-title>Analysis of CAR-T and immune cells within the tumor micro-environment of diffuse large B-cell lymphoma post CAR-T treatment by multiplex immunofluorescence</article-title>. <source>Blood</source>. (<year>2018</year>) <volume>132</volume>:<fpage>678</fpage>\n<pub-id pub-id-type=\"doi\">10.1182/blood-2018-99-113644</pub-id><pub-id pub-id-type=\"pmid\">30115634</pub-id></mixed-citation></ref><ref id=\"B55\"><label>55.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gopalakrishnan</surname><given-names>R</given-names></name><name><surname>Matta</surname><given-names>H</given-names></name><name><surname>Choi</surname><given-names>S</given-names></name><name><surname>Natarajan</surname><given-names>V</given-names></name><name><surname>Prins</surname><given-names>R</given-names></name><name><surname>Gong</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>A novel luciferase-based assay for the detection of chimeric antigen receptors</article-title>. <source>Sci Rep</source>. (<year>2019</year>) <volume>9</volume>:<fpage>1957</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-018-38258-z</pub-id><pub-id pub-id-type=\"pmid\">30760795</pub-id></mixed-citation></ref><ref id=\"B56\"><label>56.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ramello</surname><given-names>MC</given-names></name><name><surname>Benzaïd</surname><given-names>I</given-names></name><name><surname>Kuenzi</surname><given-names>BM</given-names></name><name><surname>Lienlaf-Moreno</surname><given-names>M</given-names></name><name><surname>Kandell</surname><given-names>WM</given-names></name><name><surname>Santiago</surname><given-names>DN</given-names></name><etal/></person-group>. <article-title>An immunoproteomic approach to characterize the CAR interactome and signalosome</article-title>. <source>Sci Signal</source>. (<year>2019</year>) <volume>12</volume>:<fpage>eaap9777</fpage>. <pub-id pub-id-type=\"doi\">10.1126/scisignal.aap9777</pub-id><pub-id pub-id-type=\"pmid\">30755478</pub-id></mixed-citation></ref><ref id=\"B57\"><label>57.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Salter</surname><given-names>AI</given-names></name><name><surname>Ivey</surname><given-names>RG</given-names></name><name><surname>Kennedy</surname><given-names>JJ</given-names></name><name><surname>Voillet</surname><given-names>V</given-names></name><name><surname>Rajan</surname><given-names>A</given-names></name><name><surname>Alderman</surname><given-names>EJ</given-names></name><etal/></person-group>. <article-title>Phosphoproteomic analysis of chimeric antigen receptor signaling reveals kinetic and quantitative differences that affect cell function</article-title>. <source>Sci Signal</source>. (<year>2018</year>) <volume>11</volume>:<fpage>eaat6753</fpage>. <pub-id pub-id-type=\"doi\">10.1126/scisignal.aat6753</pub-id><pub-id pub-id-type=\"pmid\">30131370</pub-id></mixed-citation></ref><ref id=\"B58\"><label>58.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Davenport</surname><given-names>AJ</given-names></name><name><surname>Cross</surname><given-names>RS</given-names></name><name><surname>Watson</surname><given-names>KA</given-names></name><name><surname>Liao</surname><given-names>Y</given-names></name><name><surname>Shi</surname><given-names>W</given-names></name><name><surname>Prince</surname><given-names>HM</given-names></name><etal/></person-group>. <article-title>Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity</article-title>. <source>Proc Natl Acad Sci USA</source>. (<year>2018</year>) <volume>115</volume>:<fpage>E2068</fpage>–<lpage>76</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1716266115</pub-id><pub-id pub-id-type=\"pmid\">29440406</pub-id></mixed-citation></ref><ref id=\"B59\"><label>59.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jang</surname><given-names>JH</given-names></name><name><surname>Huang</surname><given-names>Y</given-names></name><name><surname>Zheng</surname><given-names>P</given-names></name><name><surname>Jo</surname><given-names>MC</given-names></name><name><surname>Bertolet</surname><given-names>G</given-names></name><name><surname>Zhu</surname><given-names>MX</given-names></name><etal/></person-group>. <article-title>Imaging of cell–cell communication in a vertical orientation reveals high-resolution structure of immunological synapse and novel PD-1 dynamics</article-title>. <source>J Immunol</source>. (<year>2015</year>) <volume>195</volume>:<fpage>1320</fpage>–<lpage>30</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.1403143</pub-id><pub-id pub-id-type=\"pmid\">26123352</pub-id></mixed-citation></ref><ref id=\"B60\"><label>60.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xiong</surname><given-names>W</given-names></name><name><surname>Chen</surname><given-names>Y</given-names></name><name><surname>Kang</surname><given-names>X</given-names></name><name><surname>Chen</surname><given-names>Z</given-names></name><name><surname>Zheng</surname><given-names>P</given-names></name><name><surname>Hsu</surname><given-names>YH</given-names></name><etal/></person-group>. <article-title>Immunological synapse predicts effectiveness of chimeric antigen receptor cells</article-title>. <source>Mol Ther</source>. (<year>2018</year>) <volume>26</volume>:<fpage>963</fpage>–<lpage>75</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ymthe.2018.01.020</pub-id><pub-id pub-id-type=\"pmid\">29503199</pub-id></mixed-citation></ref><ref id=\"B61\"><label>61.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Libby</surname><given-names>KA</given-names></name><name><surname>Su</surname><given-names>X</given-names></name></person-group>\n<article-title>T-cell receptor signaling</article-title>. In: <person-group person-group-type=\"editor\"><name><surname>Liu</surname><given-names>C</given-names></name></person-group>, editor. <source>Methods in Molecular Biology.</source>\n<publisher-loc>New York, NY</publisher-loc>: <publisher-name>Humana</publisher-name> (<year>2020</year>). p. <fpage>153</fpage>–<lpage>60</lpage>.</mixed-citation></ref><ref id=\"B62\"><label>62.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Su</surname><given-names>X</given-names></name><name><surname>Vale</surname><given-names>R</given-names></name></person-group>\n<article-title>Mechanisms of chimeric antigen receptor (CAR) signaling during T cell activation</article-title>. <source>Biophys J</source>. (<year>2018</year>) <volume>114</volume>:<fpage>107a</fpage>−<lpage>8a</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bpj.2017.11.625</pub-id></mixed-citation></ref><ref id=\"B63\"><label>63.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morrissey</surname><given-names>MA</given-names></name><name><surname>Williamson</surname><given-names>AP</given-names></name><name><surname>Steinbach</surname><given-names>AM</given-names></name><name><surname>Roberts</surname><given-names>EW</given-names></name><name><surname>Kern</surname><given-names>N</given-names></name><name><surname>Headley</surname><given-names>MB</given-names></name><etal/></person-group>. <article-title>Chimeric antigen receptors that trigger phagocytosis</article-title>. <source>Elife</source>. (<year>2018</year>) <volume>7</volume>:<fpage>e36688</fpage>. <pub-id pub-id-type=\"doi\">10.7554/eLife.36688</pub-id><pub-id pub-id-type=\"pmid\">29862966</pub-id></mixed-citation></ref><ref id=\"B64\"><label>64.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sasmal</surname><given-names>DK</given-names></name><name><surname>Feng</surname><given-names>W</given-names></name><name><surname>Roy</surname><given-names>S</given-names></name><name><surname>Leung</surname><given-names>P</given-names></name><name><surname>He</surname><given-names>Y</given-names></name><name><surname>Cai</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>TCR–pMHC bond conformation controls TCR ligand discrimination</article-title>. <source>Cell Mol Immunol</source>. (<year>2020</year>) <volume>17</volume>:<fpage>203</fpage>–<lpage>17</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41423-019-0273-6</pub-id><pub-id pub-id-type=\"pmid\">31530899</pub-id></mixed-citation></ref><ref id=\"B65\"><label>65.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rosenberg</surname><given-names>J</given-names></name><name><surname>Huang</surname><given-names>J</given-names></name></person-group>. <article-title>Visualizing surface T-cell receptor dynamics four-dimensionally using lattice light-sheet microscopy</article-title>. <source>J Vis Exp</source>. (<year>2019</year>) <volume>2020</volume>:<fpage>e59914</fpage>. <pub-id pub-id-type=\"doi\">10.3791/59914</pub-id><pub-id pub-id-type=\"pmid\">32065118</pub-id></mixed-citation></ref><ref id=\"B66\"><label>66.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rosenberg</surname><given-names>J</given-names></name><name><surname>Cao</surname><given-names>G</given-names></name><name><surname>Borja-Prieto</surname><given-names>F</given-names></name><name><surname>Huang</surname><given-names>J</given-names></name></person-group>. <article-title>Lattice light-sheet microscopy multi-dimensional analyses (LaMDA) of T-cell receptor dynamics predict T-cell signaling states</article-title>. <source>Cell Syst</source>. (<year>2020</year>) <volume>10</volume>:<fpage>433</fpage>–<lpage>44.e5</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cels.2020.04.006</pub-id><pub-id pub-id-type=\"pmid\">32437685</pub-id></mixed-citation></ref><ref id=\"B67\"><label>67.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nerreter</surname><given-names>T</given-names></name><name><surname>Letschert</surname><given-names>S</given-names></name><name><surname>Götz</surname><given-names>R</given-names></name><name><surname>Doose</surname><given-names>S</given-names></name><name><surname>Danhof</surname><given-names>S</given-names></name><name><surname>Einsele</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Super-resolution microscopy reveals ultra-low CD19 expression on myeloma cells that triggers elimination by CD19 CAR-T</article-title>. <source>Nat Commun</source>. (<year>2019</year>) <volume>10</volume>:<fpage>3137</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41467-019-10948-w</pub-id><pub-id pub-id-type=\"pmid\">31316055</pub-id></mixed-citation></ref><ref id=\"B68\"><label>68.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Torres Chavez</surname><given-names>A</given-names></name><name><surname>McKenna</surname><given-names>MK</given-names></name><name><surname>Canestrari</surname><given-names>E</given-names></name><name><surname>Dann</surname><given-names>CT</given-names></name><name><surname>Ramos</surname><given-names>CA</given-names></name><name><surname>Lulla</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Expanding CAR T cells in human platelet lysate renders T cells with <italic>in vivo</italic> longevity</article-title>. <source>J Immunother Cancer</source>. (<year>2019</year>) <volume>7</volume>:<fpage>330</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s40425-019-0804-9</pub-id><pub-id pub-id-type=\"pmid\">31779709</pub-id></mixed-citation></ref><ref id=\"B69\"><label>69.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dawson</surname><given-names>NAJ</given-names></name><name><surname>Lamarche</surname><given-names>C</given-names></name><name><surname>Hoeppli</surname><given-names>RE</given-names></name><name><surname>Bergqvist</surname><given-names>P</given-names></name><name><surname>Fung</surname><given-names>VCW</given-names></name><name><surname>McIver</surname><given-names>E</given-names></name><etal/></person-group>. <article-title>Systematic testing and specificity mapping of alloantigen-specific chimeric antigen receptors in regulatory T cells</article-title>. <source>JCI Insight</source>. (<year>2019</year>) <volume>4</volume>:<fpage>e123672</fpage>. <pub-id pub-id-type=\"doi\">10.1172/jci.insight.123672</pub-id><pub-id pub-id-type=\"pmid\">30753169</pub-id></mixed-citation></ref><ref id=\"B70\"><label>70.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Serganova</surname><given-names>I</given-names></name><name><surname>Moroz</surname><given-names>E</given-names></name><name><surname>Cohen</surname><given-names>I</given-names></name><name><surname>Moroz</surname><given-names>M</given-names></name><name><surname>Mane</surname><given-names>M</given-names></name><name><surname>Zurita</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Enhancement of PSMA-directed CAR adoptive immunotherapy by PD-1/PD-L1 blockade</article-title>. <source>Mol Ther Oncolytics</source>. (<year>2017</year>) <volume>4</volume>:<fpage>41</fpage>–<lpage>54</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.omto.2016.11.005</pub-id><pub-id pub-id-type=\"pmid\">28345023</pub-id></mixed-citation></ref><ref id=\"B71\"><label>71.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stowe</surname><given-names>CL</given-names></name><name><surname>Burley</surname><given-names>TA</given-names></name><name><surname>Allan</surname><given-names>H</given-names></name><name><surname>Vinci</surname><given-names>M</given-names></name><name><surname>Kramer-Marek</surname><given-names>G</given-names></name><name><surname>Ciobota</surname><given-names>DM</given-names></name><etal/></person-group>. <article-title>Near-infrared dual bioluminescence imaging in mouse models of cancer using infraluciferin</article-title>. <source>Elife</source>. (<year>2019</year>) <volume>8</volume>:<fpage>e45801</fpage>. <pub-id pub-id-type=\"doi\">10.7554/eLife.45801</pub-id><pub-id pub-id-type=\"pmid\">31610848</pub-id></mixed-citation></ref><ref id=\"B72\"><label>72.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Keu</surname><given-names>KV</given-names></name><name><surname>Witney</surname><given-names>TH</given-names></name><name><surname>Yaghoubi</surname><given-names>S</given-names></name><name><surname>Rosenberg</surname><given-names>J</given-names></name><name><surname>Kurien</surname><given-names>A</given-names></name><name><surname>Magnusson</surname><given-names>R</given-names></name><etal/></person-group>. <article-title>Reporter gene imaging of targeted T cell immunotherapy in recurrent glioma</article-title>. <source>Sci Transl Med</source>. (<year>2017</year>) <volume>9</volume>:<fpage>eaag2196</fpage>. <pub-id pub-id-type=\"doi\">10.1126/scitranslmed.aag2196</pub-id><pub-id pub-id-type=\"pmid\">28100832</pub-id></mixed-citation></ref><ref id=\"B73\"><label>73.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krebs</surname><given-names>S</given-names></name><name><surname>Ahad</surname><given-names>A</given-names></name><name><surname>Carter</surname><given-names>LM</given-names></name><name><surname>Eyquem</surname><given-names>J</given-names></name><name><surname>Brand</surname><given-names>C</given-names></name><name><surname>Bell</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Antibody with infinite affinity for <italic>in vivo</italic> tracking of genetically engineered lymphocytes</article-title>. <source>J Nucl Med</source>. (<year>2018</year>) <volume>59</volume>:<fpage>1894</fpage>–<lpage>900</lpage>. <pub-id pub-id-type=\"doi\">10.2967/jnumed.118.208041</pub-id><pub-id pub-id-type=\"pmid\">29903928</pub-id></mixed-citation></ref><ref id=\"B74\"><label>74.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sellmyer</surname><given-names>MA</given-names></name><name><surname>Richman</surname><given-names>SA</given-names></name><name><surname>Lohith</surname><given-names>K</given-names></name><name><surname>Hou</surname><given-names>C</given-names></name><name><surname>Weng</surname><given-names>CC</given-names></name><name><surname>Mach</surname><given-names>RH</given-names></name><etal/></person-group>. <article-title>Imaging CAR T cell trafficking with eDFHR as a PET reporter gene</article-title>. <source>Mol Ther</source>. (<year>2019</year>) <volume>28</volume>:<fpage>42</fpage>–<lpage>51</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ymthe.2019.10.007</pub-id><pub-id pub-id-type=\"pmid\">31668558</pub-id></mixed-citation></ref><ref id=\"B75\"><label>75.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Park</surname><given-names>S</given-names></name><name><surname>Shevlin</surname><given-names>E</given-names></name><name><surname>Vedvyas</surname><given-names>Y</given-names></name><name><surname>Zaman</surname><given-names>M</given-names></name><name><surname>Park</surname><given-names>S</given-names></name><name><surname>Hsu</surname><given-names>YMS</given-names></name><etal/></person-group>. <article-title>Micromolar affinity CAR T cells to ICAM-1 achieves rapid tumor elimination while avoiding systemic toxicity</article-title>. <source>Sci Rep</source>. (<year>2017</year>) <volume>7</volume>:<fpage>14366</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-017-14749-3</pub-id><pub-id pub-id-type=\"pmid\">29085043</pub-id></mixed-citation></ref><ref id=\"B76\"><label>76.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bhanat</surname><given-names>E</given-names></name><name><surname>Koch</surname><given-names>CA</given-names></name><name><surname>Parmar</surname><given-names>R</given-names></name><name><surname>Garla</surname><given-names>V</given-names></name><name><surname>Vijayakumar</surname><given-names>V</given-names></name></person-group>. <article-title>Somatostatin receptor expression in non-classical locations – clinical relevance?</article-title>\n<source>Rev Endocr Metab Disord</source>. (<year>2018</year>) <volume>19</volume>:<fpage>123</fpage>–<lpage>32</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s11154-018-9470-3</pub-id><pub-id pub-id-type=\"pmid\">30324319</pub-id></mixed-citation></ref><ref id=\"B77\"><label>77.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Emami-Shahri</surname><given-names>N</given-names></name><name><surname>Foster</surname><given-names>J</given-names></name><name><surname>Kashani</surname><given-names>R</given-names></name><name><surname>Gazinska</surname><given-names>P</given-names></name><name><surname>Cook</surname><given-names>C</given-names></name><name><surname>Sosabowski</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Clinically compliant spatial and temporal imaging of chimeric antigen receptor T-cells</article-title>. <source>Nat Commun</source>. (<year>2018</year>) <volume>9</volume>:<fpage>1081</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41467-018-03524-1</pub-id><pub-id pub-id-type=\"pmid\">29540684</pub-id></mixed-citation></ref><ref id=\"B78\"><label>78.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Minn</surname><given-names>I</given-names></name><name><surname>Huss</surname><given-names>DJ</given-names></name><name><surname>Ahn</surname><given-names>HH</given-names></name><name><surname>Chinn</surname><given-names>TM</given-names></name><name><surname>Park</surname><given-names>A</given-names></name><name><surname>Jones</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Imaging CAR T cell therapy with PSMA-targeted positron emission tomography</article-title>. <source>Sci Adv</source>. (<year>2019</year>) <volume>5</volume>:<fpage>eaaw5096</fpage>. <pub-id pub-id-type=\"doi\">10.1126/sciadv.aaw5096</pub-id><pub-id pub-id-type=\"pmid\">31281894</pub-id></mixed-citation></ref><ref id=\"B79\"><label>79.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>SH</given-names></name><name><surname>Soh</surname><given-names>H</given-names></name><name><surname>Chung</surname><given-names>JH</given-names></name><name><surname>Cho</surname><given-names>EH</given-names></name><name><surname>Lee</surname><given-names>SJ</given-names></name><name><surname>Ju</surname><given-names>JM</given-names></name><etal/></person-group>. <article-title>Feasibility of real-time <italic>in vivo</italic> 89Zr-DFO-labeled CAR T-cell trafficking using PET imaging</article-title>. <source>PLoS ONE</source>. (<year>2020</year>) <volume>15</volume>:<fpage>e0223814</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0223814</pub-id><pub-id pub-id-type=\"pmid\">31910217</pub-id></mixed-citation></ref><ref id=\"B80\"><label>80.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mezzanotte</surname><given-names>L</given-names></name><name><surname>van</surname><given-names>‘t Root M</given-names></name><name><surname>Karatas</surname><given-names>H</given-names></name><name><surname>Goun</surname><given-names>EA</given-names></name><name><surname>Löwik</surname><given-names>CWGM</given-names></name></person-group>. <article-title><italic>In vivo</italic> molecular bioluminescence imaging: new tools and applications</article-title>. <source>Trends Biotechnol</source>. (<year>2017</year>) <volume>35</volume>:<fpage>640</fpage>–<lpage>52</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.tibtech.2017.03.012</pub-id><pub-id pub-id-type=\"pmid\">28501458</pub-id></mixed-citation></ref><ref id=\"B81\"><label>81.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuchimaru</surname><given-names>T</given-names></name><name><surname>Iwano</surname><given-names>S</given-names></name><name><surname>Kiyama</surname><given-names>M</given-names></name><name><surname>Mitsumata</surname><given-names>S</given-names></name><name><surname>Kadonosono</surname><given-names>T</given-names></name><name><surname>Niwa</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging</article-title>. <source>Nat Commun</source>. (<year>2016</year>) <volume>7</volume>:<fpage>11856</fpage>. <pub-id pub-id-type=\"doi\">10.1038/ncomms11856</pub-id><pub-id pub-id-type=\"pmid\">27297211</pub-id></mixed-citation></ref><ref id=\"B82\"><label>82.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alavi</surname><given-names>A</given-names></name><name><surname>Huang</surname><given-names>SS</given-names></name></person-group>\n<article-title>Positron emission tomography in medicine: an overview</article-title>. <source>Cancer Imaging</source>. (<year>2008</year>) <volume>31</volume>:<fpage>39</fpage>–<lpage>44</lpage>. <pub-id pub-id-type=\"doi\">10.1016/B978-012374212-4.50009-2</pub-id></mixed-citation></ref><ref id=\"B83\"><label>83.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eissenberg</surname><given-names>LG</given-names></name><name><surname>Rettig</surname><given-names>M</given-names></name><name><surname>Dehdashti</surname><given-names>F</given-names></name><name><surname>Piwnica-Worms</surname><given-names>D</given-names></name><name><surname>DiPersio</surname><given-names>JF</given-names></name></person-group>. <article-title>Suicide genes: monitoring cells in patients with a safety switch</article-title>. <source>Front Pharmacol</source>. (<year>2014</year>) <volume>5</volume>:<fpage>5</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.3389/fphar.2014.00241</pub-id><pub-id pub-id-type=\"pmid\">24550830</pub-id></mixed-citation></ref><ref id=\"B84\"><label>84.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ishii</surname><given-names>T</given-names></name><name><surname>Ishii</surname><given-names>M</given-names></name></person-group>. <article-title>Intravital two-photon imaging: a versatile tool for dissecting the immune system</article-title>. <source>Ann Rheum Dis</source>. (<year>2011</year>) <volume>70</volume>:<fpage>113</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1136/ard.2010.138156</pub-id><pub-id pub-id-type=\"pmid\">21339213</pub-id></mixed-citation></ref><ref id=\"B85\"><label>85.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cazaux</surname><given-names>M</given-names></name><name><surname>Grandjean</surname><given-names>CL</given-names></name><name><surname>Lemaître</surname><given-names>F</given-names></name><name><surname>Garcia</surname><given-names>Z</given-names></name><name><surname>Beck</surname><given-names>RJ</given-names></name><name><surname>Milo</surname><given-names>I</given-names></name><etal/></person-group>. <article-title>Single-cell imaging of CAR T cell activity <italic>in vivo</italic> reveals extensive functional and anatomical heterogeneity</article-title>. <source>J Exp Med</source>. (<year>2019</year>) <volume>216</volume>:<fpage>1038</fpage>–<lpage>49</lpage>. <pub-id pub-id-type=\"doi\">10.1084/jem.20182375</pub-id><pub-id pub-id-type=\"pmid\">30936262</pub-id></mixed-citation></ref><ref id=\"B86\"><label>86.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mulazzani</surname><given-names>M</given-names></name><name><surname>Fräßle</surname><given-names>SP</given-names></name><name><surname>von Mücke-Heim</surname><given-names>I</given-names></name><name><surname>Langer</surname><given-names>S</given-names></name><name><surname>Zhou</surname><given-names>X</given-names></name><name><surname>Ishikawa-Ankerhold</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Long-term <italic>in vivo</italic> microscopy of CAR T cell dynamics during eradication of CNS lymphoma in mice</article-title>. <source>Proc Natl Acad Sci USA</source>. (<year>2019</year>) <volume>116</volume>:<fpage>24275</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1903854116</pub-id><pub-id pub-id-type=\"pmid\">31712432</pub-id></mixed-citation></ref><ref id=\"B87\"><label>87.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ma</surname><given-names>B</given-names></name><name><surname>Liu</surname><given-names>F</given-names></name><name><surname>Zhang</surname><given-names>S</given-names></name><name><surname>Duan</surname><given-names>J</given-names></name><name><surname>Kong</surname><given-names>Y</given-names></name><name><surname>Li</surname><given-names>Z</given-names></name><etal/></person-group>. <article-title>Two-photon fluorescent polydopamine nanodots for CAR-T cell function verification and tumor cell/tissue detection</article-title>. <source>J Mater Chem B</source>. (<year>2018</year>) <volume>6</volume>:<fpage>6459</fpage>–<lpage>67</lpage>. <pub-id pub-id-type=\"doi\">10.1039/C8TB01930D</pub-id><pub-id pub-id-type=\"pmid\">32254653</pub-id></mixed-citation></ref><ref id=\"B88\"><label>88.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Harris</surname><given-names>DT</given-names></name><name><surname>Hager</surname><given-names>M V</given-names></name><name><surname>Smith</surname><given-names>SN</given-names></name><name><surname>Cai</surname><given-names>Q</given-names></name><name><surname>Stone</surname><given-names>JD</given-names></name><name><surname>Kruger</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Comparison of T cell activities mediated by human TCRs and CARs that use the same recognition domains</article-title>. <source>J Immunol</source>. (<year>2018</year>) <volume>200</volume>:<fpage>1088</fpage>–<lpage>100</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.1700236</pub-id><pub-id pub-id-type=\"pmid\">29288199</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Endocrinol.</journal-id><journal-title-group><journal-title>Frontiers in Endocrinology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2392</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849313</article-id><article-id pub-id-type=\"pmc\">PMC7431617</article-id><article-id pub-id-type=\"doi\">10.3389/fendo.2020.00549</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology</subject><subj-group><subject>Mini Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Pineal Neurosteroids: Biosynthesis and Physiological Functions</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Haraguchi</surname><given-names>Shogo</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/32894/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Tsutsui</surname><given-names>Kazuyoshi</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/10812/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Biochemistry, Showa University School of Medicine</institution>, <addr-line>Tokyo</addr-line>, <country>Japan</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Graduate School of Integrated Sciences for Life, Hiroshima University</institution>, <addr-line>Hiroshima</addr-line>, <country>Japan</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Vance L. Trudeau, University of Ottawa, Canada</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Vincent M. Cassone, University of Kentucky, United States; Maria Claudia Gonzalez Deniselle, CONICET Instituto de Biología y Medicina Experimental (IBYME), Argentina</p></fn><corresp id=\"c001\">*Correspondence: Shogo Haraguchi <email>shogo.haraguchi@gmail.com</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>549</elocation-id><history><date date-type=\"received\"><day>30</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>06</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Haraguchi and Tsutsui.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Haraguchi and Tsutsui</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Similar to the adrenal glands, gonads, and placenta, vertebrate brains also produce various steroids, which are known as “neurosteroids.” Neurosteroids are mainly synthesized in the hippocampus, hypothalamus, and cerebellum; however, it has recently been discovered that in birds, the pineal gland, a photosensitive region in the brain, produces more neurosteroids than other brain regions. A series of experiments using molecular and biochemical techniques have found that the pineal gland produces various neurosteroids, including sex steroids, <italic>de novo</italic> from cholesterol. For instance, allopregnanolone and 7α-hydroxypregnenolone are actively produced in the pineal gland, unlike in other brain regions. Pineal 7α-hydroxypregnenolone, an up-regulator of locomotion, enhances locomotor activity in response to light stimuli in birds. Additionally, pineal allopregnanolone acts on Purkinje cells in the cerebellum and prevents neuronal apoptosis within the developing cerebellum in juvenile birds. Furthermore, exposure to light during nighttime hours can cause loss of diurnal variations of pineal allopregnanolone synthesis during early posthatch life, eventually leading to cerebellar Purkinje cell death in juvenile birds. In light of these new findings, this review summarizes the biosynthesis and physiological functions of pineal neurosteroids. Given that the circadian rhythms of individuals in modern societies are constantly interrupted by artificial light exposure, these findings in birds, which are excellent model diurnal animals, may have direct implications for addressing problems regarding the mental health and brain development of humans.</p></abstract><kwd-group><kwd>allopregnanolone</kwd><kwd>7α-hydroxypregnenolone</kwd><kwd>neurosteroid</kwd><kwd>pineal gland</kwd><kwd>cerebellum</kwd><kwd>light</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Japan Society for the Promotion of Science<named-content content-type=\"fundref-id\">10.13039/501100001691</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"2\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"34\"/><page-count count=\"6\"/><word-count count=\"3350\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Similar to the gonads and placenta, vertebrate brains actively also produce various steroid hormones. These steroid hormones produced in the brain are named “neurosteroids.” The production of neurosteroids was demonstrated firstly in mammals, and then in other vertebrates (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>–<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Thus, neurosteroid production appears to be a universal feature of the brain in vertebrates.</p><p>It is known that neurosteroids are produced in glial cells and neurons of the central and peripheral nervous systems (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>, <xref rid=\"B5\" ref-type=\"bibr\">5</xref>). However, we have demonstrated that the pineal gland produces neurosteroids from cholesterol in birds during early posthatch period (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>–<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). Notably, allopregnanolone (also known as 3α,5α-tetrahydroprogesterone; 3α,5α-THP) and 7α-hydroxypregnenolone are the two major neurosteroids produced in the pineal gland (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Of these two, pineal allopregnanolone prevents the death of developing Purkinje cells (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B8\" ref-type=\"bibr\">8</xref>), and pineal 7α-hydroxypregnenolone functions as an up-regulator of locomotion, regulating locomotor activity in response to light stimuli in birds (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>).</p></sec><sec id=\"s2\"><title>Biosynthesis of Pineal Neurosteroids</title><p>The pineal glands of vertebrates respond to light stimuli and fulfill important functions in the organization of circadian rhythms. The secretion of melatonin, a major hormone produced by the pineal gland, shows a clear daily rhythm with its peak concentration occurring at night (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B9\" ref-type=\"bibr\">9</xref>). However, it was not known whether the pineal gland produces neurosteroids until recently. We have recently demonstrated that the pineal gland is a newly found neurosteroidogenic organ producing a variety of neurosteroids from cholesterol (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Biosynthetic pathways of pineal neurosteroids. Allopregnanolone and 7α-hydroxypregnenolone are the major neurosteroids produced in the pineal gland of birds. P450scc, cytochrome P450 side-chain cleavage enzyme; P4507α, cytochrome P450 7α-hydroxylase; 3β-HSD, 3β-hydroxysteroid dehydrogenase/Δ<sup>5</sup>-Δ<sup>4</sup>-isomerase; 3α-HSD, 3α-hydroxysteroid dehydrogenase/Δ<sup>5</sup>-Δ<sup>4</sup>-isomerase; 5α-reductase; 5β-reductase; P45017α,lyase, cytochrome P450 17α-hydroxylase/c17,20-lyase; 17β-HSD, 17β-hydroxysteroid dehydrogenase; and P450arom, cytochrome P450 aromatase.</p></caption><graphic xlink:href=\"fendo-11-00549-g0001\"/></fig><p>Pregnenolone is an anabolic intermediate of most endogenous steroid hormones and is produced from cholesterol through the mitochondrial cholesterol side chain cleavage enzyme cytochrome P450scc (P450scc; encoded by the <italic>Cyp11a</italic> gene). We have demonstrated by transcription-polymerase chain reaction (RT-PCR) that the pineal gland in juvenile birds expresses <italic>P450scc</italic> mRNA (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). The protein product of this mRNA is localized in the cells that form the follicular structures in the pineal glands of birds (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). We have demonstrated by high-performance liquid chromatography (HPLC) with radioactive flow detector analysis that <sup>3</sup>H-cholesterol is converted to radioactive pregnenolone when incubated with pineal gland extract from juvenile birds (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>). This observation has confirmed the presence of functional P450scc in the pineal gland (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>), which has also been detected by gas chromatography-mass spectrometry (GC/MS) (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Subsequent RT-PCR–based assessment has revealed that key steroidogenic enzymes, cytochrome P450 7α-hydroxylase (P4507α; encoded by the <italic>Cyp7b</italic> gene), 3α-hydroxysteroid dehydrogenase/Δ<sup>5</sup>-Δ<sup>4</sup>-isomerase (3α-HSD; encoded by the <italic>Hsd3a</italic> gene), 3β-hydroxysteroid dehydrogenase/Δ<sup>5</sup>-Δ<sup>4</sup>-isomerase (3β-HSD; encoded by the <italic>Hsd3b</italic> gene), 5α-reductase (encoded by the <italic>Srd5a</italic> gene), 5β-reductase (encoded by the <italic>Srd5b</italic> gene), cytochrome P450 17α-hydroxylase/c17,20-lyase (P45017α,lyase; encoded by the <italic>Cyp17</italic> gene), 17β-hydroxysteroid dehydrogenase (17β-HSD; encoded by the <italic>Hsd17b</italic> gene), and cytochrome P450 aromatase (P450arom; encoded by the <italic>Cyp19</italic> gene), are expressed in the pineal gland of birds (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p><p>We further demonstrated that steroid hormones are indeed present in the pineal gland. Incubation of <sup>3</sup>H-pregnenolone with pineal glands from posthatch birds generates 7α- and/or 7β-hydroxypregnenolone by the action of P4507α found in the pineal glands (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). In addition to these neurosteroid isomers, progesterone, allopregnanolone (3α, 5α-THP) and/or epipregnanolone (3β, 5β-THP), androstenedione, testosterone, 5α- and/or 5β-dihydrotestosterone, and estradiol-17β are also produced (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). These <italic>ex vivo</italic> observations have confirmed that the pineal glands in juvenile birds have the biosynthetic machinery for major steroid hormones, which have also been verified to be produced as neurosteroids <italic>in vivo</italic> (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Although HPLC analysis has failed to resolve the isomers of these hormones, such as 7α-/β-hydroxypregnenolone, allo/epipregnanolone, and 5α-/β-dihydrotestosterone, several sets of isomers have been successfully isolated by GC/MS analysis (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Especially, 7α-hydroxypregnenolone and allopregnanolone are actively released (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>).</p><p>Taken together, these findings indicate that the pineal gland in juvenile birds produces various neurosteroids from cholesterol. Accordingly, this is the first demonstration of neurosteroid synthesis in the pineal gland in a vertebrate.</p></sec><sec id=\"s3\"><title>Physiological Function of Pineal 7α-Hydroxypregnenolone in Light-Dependent Locomotion</title><p>The chick pineal gland is used as a model for studies on the light-dependent phase-shifting mechanism of the circadian clock (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). To search for genes involved in this mechanism, a differential GeneChip analysis has been performed. This transcriptomics analysis has identified the light-induced transcriptional activation of the full set of genes in the pineal gland involved in cholesterol biosynthesis (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). When the pineal gland was exposed to light, it produced cholesterol and 7α-hydroxypregnenolone <italic>ex vivo</italic>. Interestingly, this light-induced production of 7α-hydroxypregnenolone occurred only when the gland was exposed to light at early night but not at late night or during the daytime. During early night time, the circadian clock is sensitive to light, which causes phase-delay of the clock (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). Thus, the light-sensitive pineal production of 7α-hydroxypregnenolone appears to be regulated by the circadian clock.</p><p>In vertebrates, an intracerebroventricular injection of 7α-hydroxypregnenolone activates locomotor activities (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>–<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). Thus, the intracerebroventricular injection of 7α-hydroxypregnenolone was administered in a dose-dependent manner at early night in chicks (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). After the injection, chicks were placed individually for locomotor activity measurement in an open field apparatus for 20 min. Spontaneous locomotor activities of chicks were stimulated by the intracerebroventricular injection of 7α-hydroxypregnenolone in a dose-dependent manner (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). Furthermore, when chicks are exposed to light during early night time, their locomotor activities reach the daytime level (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). These results suggest that pineal 7α-hydroxypregnenolone reaches the target sites within the brain by volume transmission (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>) upon light exposure at early night.</p></sec><sec id=\"s4\"><title>Physiological Function of Pineal Allopregnanolone in Purkinje Cell Survival During Development</title><p>7α-Hydroxypregnenolone and allopregnanolone are actively released during early posthatch period compared with adulthood (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Therefore, 7α-hydroxypregnenolone and allopregnanolone may play key roles in birds during early posthatch period. In vertebrates, pinealectomy decreases cell number in the developing brain (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>). These findings suggest that these major neurosteroids secreted from the pineal gland are involved in the development of brain cells.</p><p>In chicks, pinealectomy decreases the concentration of allopregnanolone and the number of cerebellar Purkinje cells, whereas the supplementation of allopregnanolone to pinealectomized birds increases the concentration of allopregnanolone and recovers the number of Purkinje cells (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Thus, pineal allopregnanolone is considered to be an essential factor for the normal development of cerebellar Purkinje cells. It thus appears that pineal allopregnanolone functions as an essential factor for Purkinje cells during posthatch period.</p><p>In addition, pinealectomy in juvenile birds increases the expression of active caspase-3 in Purkinje cells, whereas allopregnanolone supplementation decreases the expression of active caspase-3 during posthatch period (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Thus, the neuroprotective action of pineal allopregnanolone on cerebellar Purkinje cells is exerted by suppressing the activation of caspase-3 (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>).</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>A schematic model of the effect of pineal allopregnanolone on Purkinje cell survival immediately after hatching under a 12/12 h light/dark cycle or with 1 h light exposure during the dark period (light-at-night condition). <bold>(Left)</bold> panel The normal cerebellar development under a 12/12 h light/dark cycle during the first week after hatching. Pineal allopregnanolone induces the expression of pituitary adenylate cyclase-activating polypeptide (PACAP), a neuroprotective factor, through the membrane progestin receptor α (mPRα) receptor binding mechanism in Purkinje cells. Subsequently, PACAP inhibits the activation of caspase-3 that facilitates the apoptosis of cerebellar Purkinje cells. <bold>(Right)</bold> panel The abnormal cerebellar development under the light-at-night condition during the first week after hatching. The light-at-night condition disrupts the diurnal rhythm in pineal allopregnanolone synthesis. Decreased pineal allopregnanolone synthesis leads to decreased expression of PACAP in Purkinje cells. Consequently, the active caspase-3 level increases, inducing the apoptosis of Purkinje cells in the cerebellum.</p></caption><graphic xlink:href=\"fendo-11-00549-g0002\"/></fig><p>Allopregnanolone acts mainly as a ligand of the γ-aminobutyric acid type A (GABA<sub>A</sub>) receptor and may also act as an agonist of the membrane progesterone receptors α (mPRα), as well as the mPRβ and mPRγ (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>–<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). Therefore, either mPR siRNA or isoallopregnanolone, an antagonist of allopregnanolone, was delivered into the cerebellum of posthatched chicks. It was found that the silencing of mPRα increases the number of Purkinje cells that express active caspase-3 in the cerebellum of chicks (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). Furthermore, to uncover the mechanism of neuroprotective action of allopregnanolone in cerebellar Purkinje cells, allopregnanolone action on the expression of neuroprotective/neurotoxic factors (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>–<xref rid=\"B26\" ref-type=\"bibr\">26</xref>) has been investigated. Pinealectomy decreases the mRNA levels of pituitary adenylate cyclase-activating polypeptide (PACAP), a neuroprotective factor, in the cerebellum of juvenile birds (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). It has been found that a daily injection of allopregnanolone in pinealectomized juvenile birds upregulates PACAP relative to the levels in control birds (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). These findings show that PACAP mediates the neuroprotective action of pineal allopregnanolone through mPRα receptor binding during cerebellar development (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>).</p></sec><sec id=\"s5\"><title>Light-at-Night Affects the Development of Cerebellum Through a Mechanism Mediated by Pineal Allopregnanolone Action</title><p>It is known that environmental stimuli affect the development of animals including humans. In vertebrate brain development, a natural light-dark cycle promotes better brain development than constant conditions, such as constant light or constant darkness (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>–<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). However, the molecular mechanisms that control how environmental light conditions affect brain development remain unclear. The pineal gland is a photosensitive organ. To investigate whether light conditions are involved in the synthesis of allopregnanolone in the pineal gland, the birds have been incubated under either a 12/12 h light/dark (LD) cycle or LD cycle with 1 h light exposure during the dark period (light-at-night). Consequently, it has been found that the allopregnanolone concentration and synthesis during the dark period are higher in the pineal glands of LD birds than in those of light-at-night birds (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>) (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). Furthermore, the number of cerebellar Purkinje cells is decreased by the light-at-night condition (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>) (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). It is therefore considered that pineal allopregnanolone is a critical metabolite that affects cerebellar development in vertebrates, depending on the environmental light conditions.</p></sec><sec sec-type=\"conclusions\" id=\"s6\"><title>Conclusions</title><p>This review summarized the recent data on pineal neurosteroids. Studies have indicated that the pineal gland produces neurosteroids from cholesterol in birds. Pineal 7α-hydroxypregnenolone regulates locomotion in response to light stimuli in birds. Pineal allopregnanolone prevents the death of developing Purkinje cells by suppressing neuronal apoptosis during development. In addition, circadian disruption by light exposure during nighttime leads to cell death of developing Purkinje cells through pineal allopregnanolone-dependent mechanisms in juvenile birds. These observations suggest that nighttime artificial light exposure in modern societies may also perturb the development of the human brain.</p><p>Almost all animals have circadian rhythms. However, modern life conditions chronically disrupt circadian rhythm through artificial light exposure. The disruption of circadian rhythm is associated with a decline in mental and physical health (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>–<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). The most potent circadian rhythm disruption is inappropriately timed bright light exposure (e.g., light-at-night). To investigate the effects of chronic circadian disruption in modern societies on mental and physical health, which is efficiently modeled by the light-at-night condition presented here, many studies have been conducted on mice. However, it is important for us to bear in mind that laboratory mice are mainly nocturnal animals, whereas humans are diurnal. Thus, birds are excellent animal models to uncover the effect of light-at-night on diurnal animals, including humans.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>SH and KT wrote the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by JSPS Grants-in-Aid for Scientific Research (KAKENHI) Grant Numbers JP15K18571 and JP19K09033.</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Baulieu</surname><given-names>EE</given-names></name></person-group>\n<article-title>Neurosteroids: of the nervous system, by the nervous system, for the nervous system</article-title>. <source>Rec Prog Hormone Res.</source> (<year>1997</year>) <volume>52</volume>:<fpage>1</fpage>–<lpage>32</lpage>.</mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tsutsui</surname><given-names>K</given-names></name><name><surname>Ukena</surname><given-names>K</given-names></name><name><surname>Takase</surname><given-names>M</given-names></name><name><surname>Kohchi</surname><given-names>C</given-names></name><name><surname>Lea</surname><given-names>RW</given-names></name></person-group>. <article-title>Neurosteroid biosynthesis in vertebrate brains</article-title>. <source>Comp Biochem Physiol C.</source> (<year>1999</year>) <volume>124</volume>:<fpage>121</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0742-8413(99)00065-1</pub-id><pub-id pub-id-type=\"pmid\">10622427</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Compagnone</surname><given-names>NA</given-names></name><name><surname>Mellon</surname><given-names>SH</given-names></name></person-group>. <article-title>Neurosteroids: biosynthesis and function of these novel neuromodulators</article-title>. <source>Front Neuroendocrinol.</source> (<year>2000</year>) <volume>21</volume>:<fpage>1</fpage>–<lpage>56</lpage>. <pub-id pub-id-type=\"doi\">10.1006/frne.1999.0188</pub-id><pub-id pub-id-type=\"pmid\">10662535</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tsutsui</surname><given-names>K</given-names></name><name><surname>Matsunaga</surname><given-names>M</given-names></name><name><surname>Miyabara</surname><given-names>H</given-names></name><name><surname>Ukena</surname><given-names>K</given-names></name></person-group>. <article-title>Neurosteroid biosynthesis in the quail brain</article-title>. <source>J Exp Zool.</source> (<year>2006</year>) <volume>305A</volume>:<fpage>733</fpage>–<lpage>42</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jez.a.302</pub-id><pub-id pub-id-type=\"pmid\">16902960</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Do-Rego</surname><given-names>JL</given-names></name><name><surname>Seong</surname><given-names>JY</given-names></name><name><surname>Burel</surname><given-names>D</given-names></name><name><surname>Leprince</surname><given-names>J</given-names></name><name><surname>Luu-The</surname><given-names>V</given-names></name><name><surname>Tsutsui</surname><given-names>K</given-names></name></person-group>. <article-title>Neurosteroid biosynthesis: enzymatic pathways and neuroendocrine regulation by neurotransmitters and neuropeptides</article-title>. <source>Front Neuroendocrinol.</source> (<year>2009</year>) <volume>30</volume>:<fpage>259</fpage>–<lpage>301</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.yfrne.2009.05.006</pub-id><pub-id pub-id-type=\"pmid\">19505496</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hatori</surname><given-names>M</given-names></name><name><surname>Hirota</surname><given-names>T</given-names></name><name><surname>Iitsuka</surname><given-names>M</given-names></name><name><surname>Kurabayashi</surname><given-names>N</given-names></name><name><surname>Haraguchi</surname><given-names>S</given-names></name><name><surname>Kokame</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Light-dependent and circadian clock-regulated activation of sterol regulatory element-binding protein, X-box-binding protein 1, and heat shock factor pathways</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2011</year>) <volume>108</volume>:<fpage>4864</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1015959108</pub-id><pub-id pub-id-type=\"pmid\">21383147</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haraguchi</surname><given-names>S</given-names></name><name><surname>Hara</surname><given-names>S</given-names></name><name><surname>Ubuka</surname><given-names>T</given-names></name><name><surname>Mita</surname><given-names>M</given-names></name><name><surname>Tsutsui</surname><given-names>K</given-names></name></person-group>. <article-title>Possible role of pineal allopregnanolone in Purkinje cell survival</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2012</year>) <volume>109</volume>:<fpage>21110</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1210804109</pub-id><pub-id pub-id-type=\"pmid\">23213208</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haraguchi</surname><given-names>S</given-names></name><name><surname>Kamata</surname><given-names>M</given-names></name><name><surname>Tokita</surname><given-names>T</given-names></name><name><surname>Tashiro</surname><given-names>KI</given-names></name><name><surname>Sato</surname><given-names>M</given-names></name><name><surname>Nozaki</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Light-at-night exposure affects brain development through pineal allopregnanolone-dependent mechanisms</article-title>. <source>eLife.</source> (<year>2019</year>) <volume>8</volume>:<fpage>e45306</fpage>. <pub-id pub-id-type=\"doi\">10.7554/eLife.45306.037</pub-id><pub-id pub-id-type=\"pmid\">31566568</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reiter</surname><given-names>RJ</given-names></name></person-group>. <article-title>Pineal melatonin: cell biology of its synthesis and of its physiological interactions</article-title>. <source>Endocr Rev.</source> (<year>1991</year>) <volume>12</volume>:<fpage>151</fpage>–<lpage>180</lpage>. <pub-id pub-id-type=\"doi\">10.1210/edrv-12-2-151</pub-id><pub-id pub-id-type=\"pmid\">1649044</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fukada</surname><given-names>Y</given-names></name><name><surname>Okano</surname><given-names>T</given-names></name></person-group>. <article-title>Circadian clock system in the pineal gland</article-title>. <source>Mol Neurobiol.</source> (<year>2002</year>) <volume>25</volume>:<fpage>19</fpage>–<lpage>30</lpage>. <pub-id pub-id-type=\"doi\">10.1385/MN:25:1:019</pub-id><pub-id pub-id-type=\"pmid\">11890455</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Matsunaga</surname><given-names>M</given-names></name><name><surname>Ukena</surname><given-names>K</given-names></name><name><surname>Baulieu</surname><given-names>EE</given-names></name><name><surname>Tsutsui</surname><given-names>K</given-names></name></person-group>. <article-title>7α-Hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts by means of the dopaminergic system</article-title>. <source>Proc Natl Acad Sci USA</source>. (<year>2004</year>) <volume>101</volume>:<fpage>17282</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.0407176101</pub-id><pub-id pub-id-type=\"pmid\">15569930</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tsutsui</surname><given-names>K</given-names></name><name><surname>Inoue</surname><given-names>K</given-names></name><name><surname>Miyabara</surname><given-names>H</given-names></name><name><surname>Suzuki</surname><given-names>S</given-names></name><name><surname>Ogura</surname><given-names>Y</given-names></name><name><surname>Haraguchi</surname><given-names>S</given-names></name></person-group>. <article-title>7α-Hydroxypregnenolone mediates melatonin action underlying diurnal locomotor rhythms</article-title>. <source>J Neurosci</source>. (<year>2008</year>) <volume>28</volume>:<fpage>2158</fpage>–<lpage>67</lpage>. <pub-id pub-id-type=\"doi\">10.1523/JNEUROSCI.3562-07.2008</pub-id><pub-id pub-id-type=\"pmid\">18305249</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haraguchi</surname><given-names>S</given-names></name><name><surname>Koyama</surname><given-names>T</given-names></name><name><surname>Hasunuma</surname><given-names>I</given-names></name><name><surname>Vaudry</surname><given-names>H</given-names></name><name><surname>Tsutsui</surname><given-names>K</given-names></name></person-group>. <article-title>Prolactin increases the synthesis of 7α-hydroxypregnenolone, a key factor for induction of locomotor activity, in breeding male newts</article-title>. <source>Endocrinology.</source> (<year>2010</year>) <volume>151</volume>:<fpage>2211</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.1210/en.2009-1229</pub-id><pub-id pub-id-type=\"pmid\">20219980</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haraguchi</surname><given-names>S</given-names></name><name><surname>Koyama</surname><given-names>T</given-names></name><name><surname>Hasunuma</surname><given-names>I</given-names></name><name><surname>Okuyama</surname><given-names>S</given-names></name><name><surname>Ubuka</surname><given-names>T</given-names></name><name><surname>Kikuyama</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Acute stress increases the synthesis of 7α-hydroxypregnenolone, a new key neurosteroid stimulating locomotor activity, through corticosterone action in newts</article-title>. <source>Endocrinology.</source> (<year>2012</year>) <volume>153</volume>:<fpage>794</fpage>–<lpage>805</lpage>. <pub-id pub-id-type=\"doi\">10.1210/en.2011-1422</pub-id><pub-id pub-id-type=\"pmid\">22128027</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haraguchi</surname><given-names>S</given-names></name><name><surname>Yamamoto</surname><given-names>Y</given-names></name><name><surname>Suzuki</surname><given-names>Y</given-names></name><name><surname>Hyung Chang</surname><given-names>J</given-names></name><name><surname>Koyama</surname><given-names>T</given-names></name><name><surname>Sato</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>7α-Hydroxypregnenolone, a key neuronal modulator of locomotion, stimulates upstream migration by means of the dopaminergic system in salmon</article-title>. <source>Sci Rep</source>. (<year>2015</year>) <volume>5</volume>:<fpage>12546</fpage>. <pub-id pub-id-type=\"doi\">10.1038/srep12546</pub-id><pub-id pub-id-type=\"pmid\">26220247</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Fillenz</surname><given-names>M</given-names></name></person-group>\n<article-title>Volume transmission in the brain: Novel mechanisms for neuronal transmission</article-title>. In: <person-group person-group-type=\"editor\"><name><surname>Fuxe</surname><given-names>K</given-names></name><name><surname>Agnati</surname><given-names>LF</given-names></name></person-group> editors. <source>Advances in Neurosciences</source>, <volume>Vol. 1</volume>\n<publisher-loc>New York, NY</publisher-loc>: <publisher-name>Raven Press</publisher-name> (<year>1991</year>). <fpage>p. 193</fpage>–<lpage>4</lpage>.</mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kilic</surname><given-names>E</given-names></name><name><surname>Hermann</surname><given-names>DM</given-names></name><name><surname>Isenmann</surname><given-names>S</given-names></name><name><surname>Bähr</surname><given-names>M</given-names></name></person-group>. <article-title>Effects of pinealectomy and melatonin on the retrograde degeneration of retinal ganglion cells in a novel model of intraorbital optic nerve transection in mice</article-title>. <source>J Pineal Res.</source> (<year>2002</year>) <volume>32</volume>:<fpage>106</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1034/j.1600-079x.2002.1823.x</pub-id><pub-id pub-id-type=\"pmid\">12071467</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tunç</surname><given-names>AT</given-names></name><name><surname>Turgut</surname><given-names>M</given-names></name><name><surname>Aslan</surname><given-names>H</given-names></name><name><surname>Sahin</surname><given-names>B</given-names></name><name><surname>Yurtseven</surname><given-names>ME</given-names></name><name><surname>Kaplan</surname><given-names>S</given-names></name></person-group>. <article-title>Neonatal pinealectomy induces Purkinje cell loss in the cerebellum of the chick: a stereological study</article-title>. <source>Brain Res.</source> (<year>2006</year>) <volume>1067</volume>:<fpage>95</fpage>–<lpage>102</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.brainres.2005.10.011</pub-id><pub-id pub-id-type=\"pmid\">16360649</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pang</surname><given-names>Y</given-names></name><name><surname>Dong</surname><given-names>J</given-names></name><name><surname>Thomas</surname><given-names>P</given-names></name></person-group>. <article-title>Characterization, neurosteroid binding and brain distribution of human membrane progesterone receptors δ and ε (mPRδ and mPRε) and mPRδ involvement in neurosteroid inhibition of apoptosis</article-title>. <source>Endocrinology.</source> (<year>2013</year>) <volume>154</volume>:<fpage>283</fpage>–<lpage>95</lpage>. <pub-id pub-id-type=\"doi\">10.1210/en.2012-1772</pub-id><pub-id pub-id-type=\"pmid\">23161870</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schumacher</surname><given-names>M</given-names></name><name><surname>Mattern</surname><given-names>C</given-names></name><name><surname>Ghoumari</surname><given-names>A</given-names></name><name><surname>Oudinet</surname><given-names>JP</given-names></name><name><surname>Liere</surname><given-names>P</given-names></name><name><surname>Labombarda</surname><given-names>F</given-names></name><etal/></person-group>. <article-title>Revisiting the roles of progesterone and allopregnanolone in the nervous system: resurgence of the progesterone receptors</article-title>. <source>Prog Neurobiol.</source> (<year>2014</year>) <volume>113</volume>:<fpage>6</fpage>–<lpage>39</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.pneurobio.2013.09.004</pub-id><pub-id pub-id-type=\"pmid\">24172649</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Belelli</surname><given-names>D</given-names></name><name><surname>Lambert</surname><given-names>JJ</given-names></name></person-group>. <article-title>Neurosteroids: endogenous regulators of the GABA<sub>A</sub> receptor</article-title>. <source>Nat Rev Neurosci.</source> (<year>2005</year>) <volume>6</volume>:<fpage>565</fpage>–<lpage>75</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrn1703</pub-id><pub-id pub-id-type=\"pmid\">15959466</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bernal</surname><given-names>J</given-names></name></person-group>. <article-title>Thyroid hormone receptors in brain development and function</article-title>. <source>Nat Clin Pract Endocrinol Metabol.</source> (<year>2007</year>) <volume>3</volume>:<fpage>249</fpage>–<lpage>59</lpage>. <pub-id pub-id-type=\"doi\">10.1038/ncpendmet0424</pub-id><pub-id pub-id-type=\"pmid\">17315033</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Falluel-Morel</surname><given-names>A</given-names></name><name><surname>Vaudry</surname><given-names>D</given-names></name><name><surname>Aubert</surname><given-names>N</given-names></name><name><surname>Galas</surname><given-names>L</given-names></name><name><surname>Benard</surname><given-names>M</given-names></name><name><surname>Basille</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Pituitary adenylate cyclase-activating polypeptide prevents the effects of ceramides on migration, neurite outgrowth, and cytoskeleton remodeling</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2005</year>) <volume>102</volume>:<fpage>2637</fpage>–<lpage>42</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.0409681102</pub-id><pub-id pub-id-type=\"pmid\">15695581</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Koibuchi</surname><given-names>N</given-names></name><name><surname>Chin</surname><given-names>WW</given-names></name></person-group>\n<article-title>Thyroid hormone action and brain development</article-title>. <source>Trends Endocrinol Metab.</source> (<year>2000</year>) <volume>11</volume>:<fpage>123</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S1043-2760(00)00238-1</pub-id><pub-id pub-id-type=\"pmid\">10754532</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vaudry</surname><given-names>D</given-names></name><name><surname>Falluel-Morel</surname><given-names>A</given-names></name><name><surname>Leuillet</surname><given-names>S</given-names></name><name><surname>Vaudry</surname><given-names>H</given-names></name><name><surname>Gonzalez</surname><given-names>BJ</given-names></name></person-group>. <article-title>Regulators of cerebellar granule cell development act through specific signaling pathways</article-title>. <source>Science.</source> (<year>2003</year>) <volume>300</volume>:<fpage>1532</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1126/science.1085260</pub-id><pub-id pub-id-type=\"pmid\">12791981</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sasahara</surname><given-names>K</given-names></name><name><surname>Shikimi</surname><given-names>H</given-names></name><name><surname>Haraguchi</surname><given-names>S</given-names></name><name><surname>Sakamoto</surname><given-names>H</given-names></name><name><surname>Honda</surname><given-names>S</given-names></name><name><surname>Harada</surname><given-names>N</given-names></name><etal/></person-group>. <article-title>Mode of action and functional significance of estrogen-inducing dendritic growth, Spinogenesis, and synaptogenesis in the developing Purkinje cell</article-title>. <source>J Neurosci.</source> (<year>2007</year>) <volume>27</volume>:<fpage>7408</fpage>–<lpage>17</lpage>. <pub-id pub-id-type=\"doi\">10.1523/JNEUROSCI.0710-07.2007</pub-id><pub-id pub-id-type=\"pmid\">17626201</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bakkum</surname><given-names>BW</given-names></name><name><surname>Benevento</surname><given-names>LA</given-names></name><name><surname>Cohen</surname><given-names>RS</given-names></name></person-group>. <article-title>Effects of light/dark- and dark-rearing on synaptic morphology in the superior colliculus and visual cortex of the postnatal and adult rat</article-title>. <source>J Neurosci Res.</source> (<year>1991</year>) <volume>28</volume>:<fpage>65</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.1002/jnr.490280107</pub-id><pub-id pub-id-type=\"pmid\">2041057</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brooks</surname><given-names>E</given-names></name><name><surname>Waters</surname><given-names>E</given-names></name><name><surname>Farrington</surname><given-names>L</given-names></name><name><surname>Canal</surname><given-names>MM</given-names></name></person-group>. <article-title>Differential hypothalamic tyrosine hydroxylase distribution and activation by light in adult mice reared under different light conditions during the suckling period</article-title>. <source>Brain Struct Funct.</source> (<year>2011</year>) <volume>216</volume>:<fpage>357</fpage>–<lpage>70</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00429-011-0318-9</pub-id><pub-id pub-id-type=\"pmid\">21509614</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dulcis</surname><given-names>D</given-names></name><name><surname>Spitzer</surname><given-names>NC</given-names></name></person-group>. <article-title>Illumination controls differentiation of dopamine neurons regulating behaviour</article-title>. <source>Nature.</source> (<year>2008</year>) <volume>456</volume>:<fpage>195</fpage>–<lpage>201</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature07569</pub-id><pub-id pub-id-type=\"pmid\">19005547</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Komuro</surname><given-names>Y</given-names></name><name><surname>Fahrion</surname><given-names>JK</given-names></name><name><surname>Hu</surname><given-names>T</given-names></name><name><surname>Ohno</surname><given-names>N</given-names></name><name><surname>Fenner</surname><given-names>KB</given-names></name><etal/></person-group>. <article-title>Light stimuli control neuronal migration by altering of insulin-like growth factor 1 (IGF-1) signaling</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2012</year>) <volume>109</volume>:<fpage>2630</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1111326109</pub-id><pub-id pub-id-type=\"pmid\">22308338</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ohta</surname><given-names>H</given-names></name><name><surname>Mitchell</surname><given-names>AC</given-names></name><name><surname>McMahon</surname><given-names>DG</given-names></name></person-group>. <article-title>Constant light disrupts the developing mouse biological clock</article-title>. <source>Pediatr Res.</source> (<year>2006</year>) <volume>60</volume>:<fpage>304</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1203/01.pdr.0000233114.18403.66</pub-id><pub-id pub-id-type=\"pmid\">16857759</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kantermann</surname><given-names>T</given-names></name><name><surname>Roenneberg</surname><given-names>T</given-names></name></person-group>\n<article-title>Is light-at-night a health risk factor or a health risk predictor?</article-title>\n<source>Chronobiol Int.</source> (<year>2009</year>) <volume>26</volume>:<fpage>1069</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1080/07420520903223984</pub-id><pub-id pub-id-type=\"pmid\">19731106</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>J</given-names></name><name><surname>Dauchy</surname><given-names>RT</given-names></name><name><surname>Tirrell</surname><given-names>PC</given-names></name><name><surname>Wu</surname><given-names>SS</given-names></name><name><surname>Lynch</surname><given-names>DT</given-names></name><name><surname>Jitawatanarat</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Light at night activates IGF-1R/PDK1 signaling and accelerates tumor growth in human breast cancer xenografts</article-title>. <source>Cancer Res.</source> (<year>2011</year>) <volume>71</volume>:<fpage>2622</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.1158/0008-5472.CAN-10-3837</pub-id><pub-id pub-id-type=\"pmid\">21310824</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Smarr</surname><given-names>BL</given-names></name><name><surname>Grant</surname><given-names>AD</given-names></name><name><surname>Perez</surname><given-names>L</given-names></name><name><surname>Zucker</surname><given-names>I</given-names></name><name><surname>Kriegsfeld</surname><given-names>LJ</given-names></name></person-group>. <article-title>Maternal and early-life circadian disruption have long-lasting negative consequences on offspring development and adult behavior in mice</article-title>. <source>Sci Rep.</source> (<year>2017</year>) <volume>7</volume>:<fpage>3326</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-017-03406-4</pub-id><pub-id pub-id-type=\"pmid\">28607386</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Immunol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Immunol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Immunol.</journal-id><journal-title-group><journal-title>Frontiers in Immunology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-3224</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849638</article-id><article-id pub-id-type=\"pmc\">PMC7431618</article-id><article-id pub-id-type=\"doi\">10.3389/fimmu.2020.01776</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Immunology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>HDAC6 Mediates Poly (I:C)-Induced TBK1 and Akt Phosphorylation in Macrophages</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Yan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/965631/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Ke</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/657250/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Fu</surname><given-names>Jian</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/783417/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Respiratory and Critical Care Medicine, The Second Hospital of Jilin University</institution>, <addr-line>Changchun</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Toxicology and Cancer Biology, College of Medicine, University of Kentucky</institution>, <addr-line>Lexington, KY</addr-line>, <country>United States</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Liwu Li, Virginia Tech, United States</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Jie Fan, University of Pittsburgh, United States; Narasaiah Kolliputi, University of South Florida, United States</p></fn><corresp id=\"c001\">*Correspondence: Ke Wang <email>wke@jlu.edu.cn</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1776</elocation-id><history><date date-type=\"received\"><day>24</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>03</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Wang, Wang and Fu.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Wang, Wang and Fu</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Macrophages are derived from monocytes in the bone marrow and play an important role in anti-viral innate immune responses. Macrophages produce cytokines such as interferons and IL-10 upon viral infection to modulate anti-viral immune responses. Type I interferons (IFNs) promote anti-viral defense. IL-10 is a suppressor cytokine that down-regulates anti-viral immune responses. HDAC6 is a tubulin deacetylase that can modulate microtubule dynamics and microtubule-mediated cell signaling pathways. In the present study, we investigated the potential role of HDAC6 in macrophage anti-viral responses by examining poly (I:C)-induced IFN-β and IL-10 production in mouse bone marrow-derived macrophages (BMDMs). We also investigated the role of HDAC6 in poly (I:C)-induced anti-viral signaling such as TBK1, GSK-3β, and Akt activation in mouse BMDMs. Our data showed that HDAC6 deletion enhanced poly (I:C)-induced INF-β expression in macrophages by up-regulating TBK1 activity and eliminating the inhibitory regulation of GSK-3β. Furthermore, HDAC6 deletion inhibited poly (I:C)-induced suppressor cytokine IL-10 production in the BMDMs, which was associated with the inhibition of Akt activation. Our results suggest that HDAC6 modulates IFN-β and IL-10 production in macrophages through its regulation of TBK1, GSK-3β, and Akt signaling. HDAC6 could act as a suppressor of anti-viral innate immune responses in macrophages.</p></abstract><kwd-group><kwd>innate immunity</kwd><kwd>infection</kwd><kwd>cytokine</kwd><kwd>acetylation</kwd><kwd>microtubule</kwd></kwd-group><counts><fig-count count=\"6\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"42\"/><page-count count=\"7\"/><word-count count=\"3827\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Viral infection is a major health burden worldwide (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>–<xref rid=\"B3\" ref-type=\"bibr\">3</xref>). The innate immune system, which is at the forefront against viral infection, is required to orchestrate ant-viral immune responses (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>–<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). Macrophages play a critical role in anti-viral innate immune responses (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>–<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Macrophages are derived from monocytes in the bone marrow (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). The monocytes differentiate into macrophages after entering the tissues through circulation (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Macrophages are activated during viral infection to launch anti-viral defense and to eliminate viral pathogens (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>–<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). Cytokines, chemokines, and anti-viral proteins produced by macrophages are needed for effective anti-viral defense (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>–<xref rid=\"B10\" ref-type=\"bibr\">10</xref>).</p><p>Type I interferons (IFNs) play an important role in anti-viral innate immune responses by up-regulating anti-viral defense (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>). Type I IFNs contain several subtypes such as IFN-α and IFN-β (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>). Their expression is dependent on the stimuli and cell types (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>). The expression of Type I IFNs is regulated at the transcriptional level by IFN regulatory factors (IRFs) (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>). IL-10 is a suppressor cytokine during viral infection (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>). IL-10 has been reported to down-regulate anti-viral immune responses and delay virus elimination (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Macrophages are mighty anti-viral effector cells of innate immunity (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>–<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). However, macrophages are also a major source of IL-10 production during viral infection (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Viral infection could induce IL-10 production in macrophages (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>). The increased suppressor cytokine IL-10 could then suppress anti-viral responses such as the type I IFN expression and allow the escape of virus from the immune defense (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Therefore, the timing and balance of IL-10 and Type I IFN expression could be important to control viral infection (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>).</p><p>TANK-binding kinase 1 (TBK1), an IKK-related serine/threonine kinase, is a critical player in anti-viral immunity (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>). TBK1 regulates anti-viral type I interferon production (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>). TBK1 activation by viral components such as double stranded RNA (dsRNA) binding to TLR3 leads to signaling transduction that induces type I IFN expression (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>). The activity of TBK1 is regulated by TBK1 phosphorylation (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>). Glycogen Synthase Kinase-3β (GSK-3β) is a key modulator of TBK1 activity (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>–<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). GSK-3β binds to TBK1 and induces TBK1 phosphorylation upon viral infection (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>–<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Several studies suggest that there are close interactions between GSK-3β and microtubules (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>–<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). HDAC6, a unique cytoplasmic class II deacetylase, is a well-established tubulin deacetylase that can modulate microtubule dynamics and microtubule-mediated cellular responses (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>–<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). However, the role of HDAC6 in anti-viral signaling and responses in macrophages remains largely unknown.</p><p>Polyinosinic-polycytidylic acid (Poly (I:C)), a synthetic analog of viral double stranded RNA (dsRNA) (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>, <xref rid=\"B29\" ref-type=\"bibr\">29</xref>), has been used as a dsRNA ligand of TLR3 to simulate viral infection (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>, <xref rid=\"B29\" ref-type=\"bibr\">29</xref>). TLR3 is expressed in many immune cells including macrophages (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>–<xref rid=\"B9\" ref-type=\"bibr\">9</xref>, <xref rid=\"B29\" ref-type=\"bibr\">29</xref>). Binding of poly (I:C) to TLR3 activates cell signaling pathways and induces type-1 interferon (IFN) responses (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>, <xref rid=\"B29\" ref-type=\"bibr\">29</xref>). In the present study, we investigated HDAC6 regulation of poly (I:C)-induced TBK1 activation, GSK-3β phosphorylation, and IFN-β expression in bone marrow-derived macrophages. Furthermore, AKT, which can modulate IL-10 production and GSK-3β activity (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>, <xref rid=\"B31\" ref-type=\"bibr\">31</xref>), has been reported to interact with microtubules (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B33\" ref-type=\"bibr\">33</xref>). In our studies, we also examined the role of HDAC6 in poly (I:C)-induced AKT phosphorylation and IL-10 production in macrophages.</p></sec><sec sec-type=\"methods\" id=\"s2\"><title>Methods</title><sec><title>Reagents</title><p>GSK-3β (Cat#12456), β-actin (Cat#5125), GAPDH (Cat#8884), AKT (Cat#4691), TBK1 (Cat#3504), phospho-TBK1 (Ser172) (Cat#5483), phospho-AKT (ser473) (Cat#4060), HDAC6 (Cat#7612), phospho-GSK3β (Cat#9323), and acetyl-α-tubulin (Lys40) (Cat#5335) antibodies were purchased from Cell Signaling Technology (Danvers, Massachusetts). Poly (I:C) was purchased from Sigma Aldrich (Cat#P9582; St. Louis, Missouri). M-CSF was obtained from R&D Systems (Cat#416-ML; Minneapolis, Minnesota).</p></sec><sec><title>Mouse Bone Marrow-Derived Macrophages</title><p>Wild type C57BL/6J mice and HDAC6 knockout C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, Maine). HDAC6 deletion in C57BL/6J mice is produced by CRISPR/Cas9-generated HDAC6 gene knock-out mutation. 9- to 15-week-old sex and age-matched HDAC6 knockout C57BL/6J mice and wild type C57BL/6J mice were used for the studies. All experiments and animal care procedures were approved by the Institutional Animal Care and Use Committee of the University of Kentucky. Mouse bone marrow-derived macrophages were prepared as described preciously (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). Briefly, bone marrow cells were flushed out from femurs and tibias of mice with cold DMEM medium containing 0.5 mM EDTA. The cell suspension was then passed through a cell strainer, centrifuged, and resuspended in DMEM medium containing 10% FBS and 20 ng/ml M-CSF. The cells were seeded into a cell culture plate and maintained in an incubator with 5% CO<sub>2</sub> at 37°C for 5 d with fresh medium added every other day. After day 5, fully differentiated cells were stimulated with poly (I:C).</p></sec><sec><title>Immunoblotting and ELISA Assays</title><p>Immunoblotting assays were performed as described previously (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>, <xref rid=\"B36\" ref-type=\"bibr\">36</xref>). Briefly, the protein samples were separated by SDS-PAGE electrophoresis and transferred to polyvinylidenedifluoride membranes. After probing with primary and secondary antibodies, the membranes were developed using a Clarity Western ECL Substrate (Cat#1705061; Bio-Rad, Hercules, California). IFN-β and IL-10 levels in the cell culture supernatant of BMDMs were examined by the mouse IFN-β ELISA kit (Cat# 42410; PBL Assay Science; Piscataway, New Jersey) and mouse IL-10 ELISA kit (Cat#431414; Biolegend; San Diego, California) according to the manufacturer's protocol.</p></sec><sec><title>Statistical Analysis</title><p>All experiments were repeated three times. Data are expressed as mean ± SEM. The Student's <italic>t</italic>-test was used for comparisons of two groups. ANOVA and <italic>post hoc</italic> multiple comparison tests were employed for multiple groups. Statistical significance was assigned to a <italic>P</italic> < 0.05.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><p>To investigate the role of HDAC6 in poly (I:C)-induced type I IFN responses, we first examined the effects of HDAC6 deletion on poly (I:C)-induced IFN-β production in macrophages. BMDMs from the HDAC6 knockout and wild type mice were challenged with poly (I:C). Our data showed that HDAC6 deletion markedly enhanced poly (I:C)-induced IFN-β production in the BMDMs (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>HDAC6 deletion enhances poly (I:C)-induced IFN-β production in BMDMs. BMDMs from the HDAC6 knockout and wild type mice (300,000 cells/well) were challenged without or with 100 μg/ml poly (I:C) for 12 h. Experiments were repeated three times. IFN-β production was assessed by ELISA. *<italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"fimmu-11-01776-g0001\"/></fig><p>We then conducted experiments to assess the role of HDAC6 in poly (I:C)-induced suppressor cytokine IL-10 production in macrophages. BMDMs from the HDAC6 knockout and wild type mice were challenged with poly (I:C). Our data showed that HDAC6 deletion inhibited poly (I:C)-induced IL-10 production in the BMDMs (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>).</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>HDAC6 deletion suppresses poly (I:C)-induced IL-10 production in BMDMs. BMDMs from the HDAC6 knockout and wild type mice (300,000 cells/well) were challenged without or with 100 μg/ml poly (I:C) for 12 h. Experiments were repeated three times. IL-10 production was assessed by ELISA. *<italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"fimmu-11-01776-g0002\"/></fig><p>Microtubule is involved in modulating a variety of cell signaling pathways (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B33\" ref-type=\"bibr\">33</xref>). Tubulin acetylation status controls microtubule stability and dynamics (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B33\" ref-type=\"bibr\">33</xref>, <xref rid=\"B37\" ref-type=\"bibr\">37</xref>). Tubulin is an endogenous substrate of HDAC6 (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B27\" ref-type=\"bibr\">27</xref>). We conducted immunoblotting assays to examine the effects of HDAC6 deletion on tubulin acetylation status in macrophages. Our results showed that HDAC6 deletion caused hyperacetylation of α-tubulin in the BMDMs (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>).</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>HDAC6 deletion induces robust α-tubulin acetylation in BMDMs. BMDMs from the HDAC6 knockout and wild type mice were challenged without or with 20 μg/ml poly (I:C) for 30 min. Experiments were repeated three times. Representative blots of HDAC6 expression and α-tubulin acetylation (ace-α-tubulin).</p></caption><graphic xlink:href=\"fimmu-11-01776-g0003\"/></fig><p>TBK1 activation up-regulates type I IFN expression and anti-viral immunity (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>). To assess the effects of HDAC6 deletion on anti-viral signaling in macrophages, we examined TBK1 modulation by HDAC6. HDAC6 deletion enhanced poly (I:C)-induced TBK1 activation in the BMDMs as showed by the increased TBK1 phosphorylation at Ser172 of its kinase activation loop (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>).</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>HDAC6 deletion promotes poly (I:C)-induced TBK1 activation in BMDMs. BMDMs from the HDAC6 knockout and wild type mice were challenged without or with 20 μg/ml poly (I:C) for 30 min. Experiments were repeated three times. Representative blots and densitometry analysis of TBK1 phosphorylation at Ser172. *<italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"fimmu-11-01776-g0004\"/></fig><p>GSK-3β interacts with microtubules and directly binds to TBK1 to modulate TBK1 activity (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>–<xref rid=\"B20\" ref-type=\"bibr\">20</xref>, <xref rid=\"B24\" ref-type=\"bibr\">24</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>). GSK-3β function is suppressed by an inhibitory phosphorylation at Ser9 (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>–<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). To investigate the potential involvement of GSK-3β in HDAC6- and tubulin acetylation-mediated TBK1 modulation, we examined the effects of HDAC6 deletion on GSK-3β phosphorylation at Ser9. Our data showed that HDAC6 deletion led to a marked reduction of GSK-3β phosphorylation at Ser9 in BMDMs (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>), indicating that HDAC6 deletion increases GSK-3β activity.</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>HDAC6 deletion eliminates the inhibitory regulation of GSK-3β in BMDMs. BMDMs from the HDAC6 knockout and wild type mice were challenged without or with 20 μg/ml poly (I:C) for 15 min. Experiments were repeated three times. Representative blots and densitometry analysis of GSK-3β phosphorylation at Ser9. *<italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"fimmu-11-01776-g0005\"/></fig><p>Akt is another cell signaling molecule that interacts with microtubules (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B33\" ref-type=\"bibr\">33</xref>). Akt has been reported to modulate IL-10 expression and GSK-3β activity (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>, <xref rid=\"B31\" ref-type=\"bibr\">31</xref>). To assess the potential modulation of Akt by HDAC6 and tubulin acetylation, we conducted experiments to examine the effects of HDAC6 deletion on Akt activation by examining Akt phosphorylation at Ser473 that is known to mediate Akt activation. In our studies, HDAC6 deletion inhibited poly (I:C)-induced Akt phosphorylayion at Ser473 (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>).</p><fig id=\"F6\" position=\"float\"><label>Figure 6</label><caption><p>HDAC6 deletion inhibits poly (I:C)-induced Akt activation in BMDMs. BMDMs from the HDAC6 knockout and wild type mice were challenged without or with 20 μg/ml poly (I:C) for 15 min. Experiments were repeated three times. Representative blots and densitometry analysis of Akt phosphorylation at Ser473. *<italic>P</italic> < 0.05.</p></caption><graphic xlink:href=\"fimmu-11-01776-g0006\"/></fig></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Macrophages play a central role in defending against viral infection (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>–<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). We investigated the role of HDAC6 in poly (I:C)-induced anti-viral Type I interferon (IFN) and the suppressor cytokine IL-10 production in bone marrow-derived macrophages. Type I interferon (IFN) responses are fundamental in host innate immunity against viruses (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>), whereas the production of IL-10 by macrophages during viral infection has been reported to downregulate type I interferon anti-viral immune responses and hamper virus elimination (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>). The interactions of the type I interferon- and IL-10-mediated pathways could play an important role in modulating innate immune responses during viral infection (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>). One previous study indicates that HDAC6 could modulate anti-viral responses in different cell types including monocytes and fibroblasts (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>). However, <italic>in</italic> our studies, poly (I:C)-induced IFN-β production was increased by HDAC6 deletion in BMDMs, whereas poly (I:C)-induced IL-10 production was inhibited by HDAC6 deletion in BMDMs. Our data suggest that HDAC6 could function as a suppressor of anti-viral responses in macrophages by suppressing anti-viral cytokine IFN-β expression while elevating the suppressor cytokine IL-10 expression.</p><p>HDAC6 has been reported to modulate many signaling pathways that could modulate anti-viral immune responses including NF-κB, ERK, and inflammasome activation (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B40\" ref-type=\"bibr\">40</xref>, <xref rid=\"B41\" ref-type=\"bibr\">41</xref>). We examined some key signaling components in anti-viral responses. TBK1 is a key kinase in anti-viral innate immunity that activates the transcription factor IRF3 to induce type I IFN production (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Autophosphorylation of TBK1 at Ser172 within its kinase activation loop is needed for its activation (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>). TBK1-mediated anti-viral immune responses can be regulated by controlling Ser172 autophosphorylation (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Poly (I:C) induces type 1 interferon (IFN) responses through its binding to TLR3 and activating TBK1 (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B28\" ref-type=\"bibr\">28</xref>). In our studies, HDAC6 deletion led to a marked increase of poly (I:C)-induced TBK1 autophosphorylation at Ser172, which correlates with the increased IFN-β production in the macrophages. Our data indicate that HDAC6 could inhibit TBK1 activity during dsRNA viral infection to suppress type 1 interferon (IFN) responses.</p><p>GSK-3β is a major modulator of TBK1 activity (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>). GSK-3β binds to TBK1 and enhances TBK1 activity by facilitating its auto-phosphorylation at Ser172 during viral infection, which then up-regulates type I IFN responses (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Phosphorylation of GSK-3β at Ser9 has been known to inhibit GSK-3β function (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>, <xref rid=\"B42\" ref-type=\"bibr\">42</xref>). HDAC6 deletion led to a reduction of the inhibitory GSK3β phosphorylation at Ser9 in macrophages during poly (I:C) challenge, which was associated with the increased TBK1 activation. Our results suggest that HDAC6 can regulate anti-viral responses through GSK-3β. GSK3β has been reported to interact with microtubules (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>–<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). Our results showed that HDAC6 deletion led to a robust α-tubulin acetylation in macrophages. HDAC6, by modulating tubulin acetylation status, could suppress GSK-3β function and promote GSK-3β Ser9 phosphorylation through its control of microtubule dynamics.</p><p>Akt has been reported to modulate IL-10 expression and GSK-3β activity (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>, <xref rid=\"B31\" ref-type=\"bibr\">31</xref>). Akt interacts with microtubules, and their interactions are regulated by tubulin acetylation (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B33\" ref-type=\"bibr\">33</xref>). HDAC6 modulates a wide range of cellular responses and signaling through deacetylation of tubulin and microtubules (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>–<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). HDAC6 deletion inhibited poly (I:C)-induced Akt activation as demonstrated by the reduced Akt phosphorylation within the carboxy terminus at Ser473. Our data suggest that HDAC6 could modulate Akt activation in macrophages during viral infection. The inhibition of Akt activation by HDAC6 deletion could contribute to the decrease of poly (I:C)-induced IL-10 production in the macrophages. Furthermore, Akt-mediated phosphorylation of GSK-3β at Ser9 has been reported to inhibit GSK-3β activity (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). The inhibition of poly (I:C)-induced Akt activation by HDAC6 deletion could also contribute to the elimination of inhibitory phosphorylation of GSK-3β at Ser9.</p><p>In summary, our data indicate that HDAC6 deletion enhances poly (I:C)-induced INF-β expression by up-regulating TBK1 activity in macrophages, which is accomplished by eliminating the inhibitory regulation of GSK-3β. Furthermore, HDAC6 deletion inhibits poly (I:C)-induced suppressor cytokine IL-10 production in macrophages, which is associated with the decreased Akt activation. Our results suggest that HDAC6 could act as a suppressor of anti-viral innate immune responses in macrophages.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>All datasets presented in this study are included in the article.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Kentucky.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>YW designed the study, performed the experiments, edited the manuscript, and analyzed the data. KW designed the study, analyzed the data, and edited the manuscript. JF designed the study, analyzed the data, and wrote the manuscript.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Te</surname><given-names>HS</given-names></name><name><surname>Jensen</surname><given-names>DM</given-names></name></person-group>. <article-title>Epidemiology of hepatitis B and C viruses: a global overview</article-title>. <source>Clin Liver Dis.</source> (<year>2010</year>) <volume>14</volume>:<fpage>1</fpage>–<lpage>21</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cld.2009.11.009</pub-id><pub-id pub-id-type=\"pmid\">20123436</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shi</surname><given-names>T</given-names></name><name><surname>Denouel</surname><given-names>A</given-names></name><name><surname>Tietjen</surname><given-names>AK</given-names></name><name><surname>Campbell</surname><given-names>I</given-names></name><name><surname>Moran</surname><given-names>E</given-names></name><name><surname>Li</surname><given-names>X</given-names></name><etal/></person-group>. <article-title>Global disease burden estimates of respiratory syncytial virus-associated acute respiratory infection in older adults in 2015: a systematic review and meta-analysis</article-title>. <source>J Infect Dis</source>. (<year>2019</year>). <pub-id pub-id-type=\"doi\">10.1093/infdis/jiz059</pub-id>. [Epub ahead of print].<pub-id pub-id-type=\"pmid\">30880339</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gower</surname><given-names>E</given-names></name><name><surname>Estes</surname><given-names>C</given-names></name><name><surname>Blach</surname><given-names>S</given-names></name><name><surname>Razavi-Shearer</surname><given-names>K</given-names></name><name><surname>Razavi</surname><given-names>H</given-names></name></person-group>. <article-title>Global epidemiology and genotype distribution of the hepatitis C virus infection</article-title>. <source>J Hepatol.</source> (<year>2014</year>) <volume>61</volume>:<fpage>S45</fpage>–<lpage>57</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jhep.2014.07.027</pub-id><pub-id pub-id-type=\"pmid\">25086286</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Silvin</surname><given-names>A</given-names></name><name><surname>Manel</surname><given-names>N</given-names></name></person-group>. <article-title>Innate immune sensing of HIV infection</article-title>. <source>Curr Opin Immunol.</source> (<year>2015</year>) <volume>32</volume>:<fpage>54</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.coi.2014.12.003</pub-id><pub-id pub-id-type=\"pmid\">25617674</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lunemann</surname><given-names>A</given-names></name><name><surname>Rowe</surname><given-names>M</given-names></name><name><surname>Nadal</surname><given-names>D</given-names></name></person-group>. <article-title>Innate immune recognition of EBV</article-title>. <source>Curr Top Microbiol Immunol.</source> (<year>2015</year>) <volume>391</volume>:<fpage>265</fpage>–<lpage>87</lpage>. <pub-id pub-id-type=\"doi\">10.1007/978-3-319-22834-1_9</pub-id><pub-id pub-id-type=\"pmid\">26428378</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Saha</surname><given-names>B</given-names></name><name><surname>Szabo</surname><given-names>G</given-names></name></person-group>. <article-title>Innate immune cell networking in hepatitis C virus infection</article-title>. <source>J Leukoc Biol.</source> (<year>2014</year>) <volume>96</volume>:<fpage>757</fpage>–<lpage>66</lpage>. <pub-id pub-id-type=\"doi\">10.1189/jlb.4MR0314-141R</pub-id><pub-id pub-id-type=\"pmid\">25001860</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cassol</surname><given-names>E</given-names></name><name><surname>Cassetta</surname><given-names>L</given-names></name><name><surname>Alfano</surname><given-names>M</given-names></name><name><surname>Poli</surname><given-names>G</given-names></name></person-group>. <article-title>Macrophage polarization and HIV-1 infection</article-title>. <source>J Leukoc Biol.</source> (<year>2010</year>) <volume>87</volume>:<fpage>599</fpage>–<lpage>608</lpage>. <pub-id pub-id-type=\"doi\">10.1189/jlb.1009673</pub-id><pub-id pub-id-type=\"pmid\">20042468</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alfano</surname><given-names>M</given-names></name><name><surname>Graziano</surname><given-names>F</given-names></name><name><surname>Genovese</surname><given-names>L</given-names></name><name><surname>Poli</surname><given-names>G</given-names></name></person-group>. <article-title>Macrophage polarization at the crossroad between HIV-1 infection and cancer development</article-title>. <source>Arterioscler Thromb Vasc Biol.</source> (<year>2013</year>) <volume>33</volume>:<fpage>1145</fpage>–<lpage>52</lpage>. <pub-id pub-id-type=\"doi\">10.1161/ATVBAHA.112.300171</pub-id><pub-id pub-id-type=\"pmid\">23677880</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ellermann-Eriksen</surname><given-names>S</given-names></name></person-group>. <article-title>Macrophages and cytokines in the early defence against herpes simplex virus</article-title>. <source>Virol J.</source> (<year>2005</year>) <volume>2</volume>:<fpage>59</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1743-422X-2-59</pub-id><pub-id pub-id-type=\"pmid\">16076403</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nyman</surname><given-names>TA</given-names></name><name><surname>Matikainen</surname><given-names>S</given-names></name></person-group>. <article-title>Proteomics to study macrophage response to viral infection</article-title>. <source>J Proteomics.</source> (<year>2018</year>) <volume>180</volume>:<fpage>99</fpage>–<lpage>107</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jprot.2017.06.018</pub-id><pub-id pub-id-type=\"pmid\">28647517</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kruize</surname><given-names>Z</given-names></name><name><surname>Kootstra</surname><given-names>NA</given-names></name></person-group>. <article-title>The role of macrophages in HIV-1 persistence and pathogenesis</article-title>. <source>Front Microbiol.</source> (<year>2019</year>) <volume>10</volume>:<fpage>2828</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fmicb.2019.02828</pub-id><pub-id pub-id-type=\"pmid\">31866988</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ginhoux</surname><given-names>F</given-names></name><name><surname>Jung</surname><given-names>S</given-names></name></person-group>. <article-title>Monocytes and macrophages: developmental pathways and tissue homeostasis</article-title>. <source>Nat Rev Immunol.</source> (<year>2014</year>) <volume>14</volume>:<fpage>392</fpage>–<lpage>404</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nri3671</pub-id><pub-id pub-id-type=\"pmid\">24854589</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Takaoka</surname><given-names>A</given-names></name><name><surname>Yanai</surname><given-names>H</given-names></name></person-group>. <article-title>Interferon signalling network in innate defence</article-title>. <source>Cell Microbiol.</source> (<year>2006</year>) <volume>8</volume>:<fpage>907</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1462-5822.2006.00716.x</pub-id><pub-id pub-id-type=\"pmid\">16681834</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McNab</surname><given-names>F</given-names></name><name><surname>Mayer-Barber</surname><given-names>K</given-names></name><name><surname>Sher</surname><given-names>A</given-names></name><name><surname>Wack</surname><given-names>A</given-names></name><name><surname>O'Garra</surname><given-names>A</given-names></name></person-group>. <article-title>Type I interferons in infectious disease</article-title>. <source>Nat Rev Immunol.</source> (<year>2015</year>) <volume>15</volume>:<fpage>87</fpage>–<lpage>103</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nri3787</pub-id><pub-id pub-id-type=\"pmid\">25614319</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Couper</surname><given-names>KN</given-names></name><name><surname>Blount</surname><given-names>DG</given-names></name><name><surname>Riley</surname><given-names>EM</given-names></name></person-group>. <article-title>IL-10: the master regulator of immunity to infection</article-title>. <source>J Immunol.</source> (<year>2008</year>) <volume>180</volume>:<fpage>5771</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.180.9.5771</pub-id><pub-id pub-id-type=\"pmid\">18424693</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zdrenghea</surname><given-names>MT</given-names></name><name><surname>Makrinioti</surname><given-names>H</given-names></name><name><surname>Muresan</surname><given-names>A</given-names></name><name><surname>Johnston</surname><given-names>SL</given-names></name><name><surname>Stanciu</surname><given-names>LA</given-names></name></person-group>. <article-title>The role of macrophage IL-10/innate IFN interplay during virus-induced asthma</article-title>. <source>Rev Med Virol.</source> (<year>2015</year>) <volume>25</volume>:<fpage>33</fpage>–<lpage>49</lpage>. <pub-id pub-id-type=\"doi\">10.1002/rmv.1817</pub-id><pub-id pub-id-type=\"pmid\">25430775</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhao</surname><given-names>W</given-names></name></person-group>. <article-title>Negative regulation of TBK1-mediated antiviral immunity</article-title>. <source>FEBS Lett.</source> (<year>2013</year>) <volume>587</volume>:<fpage>542</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.febslet.2013.01.052</pub-id><pub-id pub-id-type=\"pmid\">23395611</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Louis</surname><given-names>C</given-names></name><name><surname>Burns</surname><given-names>C</given-names></name><name><surname>Wicks</surname><given-names>I</given-names></name></person-group>. <article-title>TANK-binding kinase 1-dependent responses in health and autoimmunity</article-title>. <source>Front Immunol.</source> (<year>2018</year>) <volume>9</volume>:<fpage>434</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2018.00434</pub-id><pub-id pub-id-type=\"pmid\">29559975</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lei</surname><given-names>CQ</given-names></name><name><surname>Zhong</surname><given-names>B</given-names></name><name><surname>Zhang</surname><given-names>Y</given-names></name><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>S</given-names></name><name><surname>Shu</surname><given-names>HB</given-names></name></person-group>. <article-title>Glycogen synthase kinase 3beta regulates IRF3 transcription factor-mediated antiviral response via activation of the kinase TBK1</article-title>. <source>Immunity.</source> (<year>2010</year>) <volume>33</volume>:<fpage>878</fpage>–<lpage>89</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.immuni.2010.11.021</pub-id><pub-id pub-id-type=\"pmid\">21145761</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhou</surname><given-names>FQ</given-names></name><name><surname>Snider</surname><given-names>WD</given-names></name></person-group>. <article-title>Cell biology. GSK-3beta and microtubule assembly in axons</article-title>. <source>Science.</source> (<year>2005</year>) <volume>308</volume>:<fpage>211</fpage>–<lpage>14</lpage>. <pub-id pub-id-type=\"doi\">10.1126/science.1110301</pub-id><pub-id pub-id-type=\"pmid\">15825222</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ciani</surname><given-names>L</given-names></name><name><surname>Salinas</surname><given-names>PC</given-names></name></person-group>. <article-title>c-Jun N-terminal kinase (JNK) cooperates with Gsk3beta to regulate dishevelled-mediated microtubule stability</article-title>. <source>BMC Cell Biol.</source> (<year>2007</year>) <volume>8</volume>:<fpage>27</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1471-2121-8-27</pub-id><pub-id pub-id-type=\"pmid\">17608927</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Namekata</surname><given-names>K</given-names></name><name><surname>Harada</surname><given-names>C</given-names></name><name><surname>Guo</surname><given-names>X</given-names></name><name><surname>Kimura</surname><given-names>A</given-names></name><name><surname>Kittaka</surname><given-names>D</given-names></name><name><surname>Watanabe</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Dock3 stimulates axonal outgrowth via GSK-3beta-mediated microtubule assembly</article-title>. <source>J Neurosci.</source> (<year>2012</year>) <volume>32</volume>:<fpage>264</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1523/JNEUROSCI.4884-11.2012</pub-id><pub-id pub-id-type=\"pmid\">22219288</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eng</surname><given-names>CH</given-names></name><name><surname>Huckaba</surname><given-names>TM</given-names></name><name><surname>Gundersen</surname><given-names>GG</given-names></name></person-group>. <article-title>The formin mDia regulates GSK3β through novel PKCs to promote microtubule stabilization but not MTOC reorientation in migrating fibroblasts</article-title>. <source>Mol Biol Cell.</source> (<year>2006</year>) <volume>17</volume>:<fpage>5004</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.1091/mbc.e05-10-0914</pub-id><pub-id pub-id-type=\"pmid\">16987962</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kumar</surname><given-names>P</given-names></name><name><surname>Lyle</surname><given-names>KS</given-names></name><name><surname>Gierke</surname><given-names>S</given-names></name><name><surname>Matov</surname><given-names>A</given-names></name><name><surname>Danuser</surname><given-names>G</given-names></name><name><surname>Wittmann</surname><given-names>T</given-names></name></person-group>. <article-title>GSK3β phosphorylation modulates CLASP-microtubule association and lamella microtubule attachment</article-title>. <source>J Cell Biol.</source> (<year>2009</year>) <volume>184</volume>:<fpage>895</fpage>–<lpage>908</lpage>. <pub-id pub-id-type=\"doi\">10.1083/jcb.200901042</pub-id><pub-id pub-id-type=\"pmid\">19289791</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Matthias</surname><given-names>P</given-names></name><name><surname>Yoshida</surname><given-names>M</given-names></name><name><surname>Khochbin</surname><given-names>S</given-names></name></person-group>. <article-title>HDAC6 a new cellular stress surveillance factor</article-title>. <source>Cell Cycle.</source> (<year>2008</year>) <volume>7</volume>:<fpage>7</fpage>–<lpage>10</lpage>. <pub-id pub-id-type=\"doi\">10.4161/cc.7.1.5186</pub-id><pub-id pub-id-type=\"pmid\">18196966</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Seidel</surname><given-names>C</given-names></name><name><surname>Schnekenburger</surname><given-names>M</given-names></name><name><surname>Dicato</surname><given-names>M</given-names></name><name><surname>Diederich</surname><given-names>M</given-names></name></person-group>. <article-title>Histone deacetylase 6 in health and disease</article-title>. <source>Epigenomics.</source> (<year>2015</year>) <volume>7</volume>:<fpage>103</fpage>–<lpage>18</lpage>. <pub-id pub-id-type=\"doi\">10.2217/epi.14.69</pub-id><pub-id pub-id-type=\"pmid\">25687470</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Shin</surname><given-names>D</given-names></name><name><surname>Kwon</surname><given-names>SH</given-names></name></person-group>. <article-title>Histone deacetylase 6 plays a role as a distinct regulator of diverse cellular processes</article-title>. <source>FEBS J.</source> (<year>2013</year>) <volume>280</volume>:<fpage>775</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1111/febs.12079</pub-id><pub-id pub-id-type=\"pmid\">23181831</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Matsumoto</surname><given-names>M</given-names></name><name><surname>Oshiumi</surname><given-names>H</given-names></name><name><surname>Seya</surname><given-names>T</given-names></name></person-group>. <article-title>Antiviral responses induced by the TLR3 pathway</article-title>. <source>Rev Med Virol.</source> (<year>2011</year>) <volume>21</volume>:<fpage>67</fpage>–<lpage>77</lpage>. <pub-id pub-id-type=\"doi\">10.1002/rmv.680</pub-id><pub-id pub-id-type=\"pmid\">21312311</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Matsumoto</surname><given-names>M</given-names></name><name><surname>Seya</surname><given-names>T</given-names></name></person-group>. <article-title>TLR3: interferon induction by double-stranded RNA including poly(I:C)</article-title>. <source>Adv Drug Deliv Rev.</source> (<year>2008</year>) <volume>60</volume>:<fpage>805</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.addr.2007.11.005</pub-id><pub-id pub-id-type=\"pmid\">18262679</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ha</surname><given-names>SD</given-names></name><name><surname>Ng</surname><given-names>D</given-names></name><name><surname>Pelech</surname><given-names>SL</given-names></name><name><surname>Kim</surname><given-names>SO</given-names></name></person-group>. <article-title>Critical role of the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase-3 signaling pathway in recovery from anthrax lethal toxin-induced cell cycle arrest and MEK cleavage in macrophages</article-title>. <source>J Biol Chem.</source> (<year>2007</year>) <volume>282</volume>:<fpage>36230</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M707622200</pub-id><pub-id pub-id-type=\"pmid\">17951252</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martin</surname><given-names>M</given-names></name><name><surname>Schifferle</surname><given-names>RE</given-names></name><name><surname>Cuesta</surname><given-names>N</given-names></name><name><surname>Vogel</surname><given-names>SN</given-names></name><name><surname>Katz</surname><given-names>J</given-names></name><name><surname>Michalek</surname><given-names>SM</given-names></name></person-group>. <article-title>Role of the phosphatidylinositol 3 kinase-Akt pathway in the regulation of IL-10 and IL-12 by porphyromonas gingivalis lipopolysaccharide</article-title>. <source>J Immunol.</source> (<year>2003</year>) <volume>171</volume>:<fpage>717</fpage>–<lpage>25</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.171.2.717</pub-id><pub-id pub-id-type=\"pmid\">12847238</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shah</surname><given-names>N</given-names></name><name><surname>Kumar</surname><given-names>S</given-names></name><name><surname>Zaman</surname><given-names>N</given-names></name><name><surname>Pan</surname><given-names>CC</given-names></name><name><surname>Bloodworth</surname><given-names>JC</given-names></name><name><surname>Lei</surname><given-names>W</given-names></name><etal/></person-group>. <article-title>TAK1 activation of alpha-TAT1 and microtubule hyperacetylation control AKT signaling and cell growth</article-title>. <source>Nat Commun.</source> (<year>2018</year>) <volume>9</volume>:<fpage>1696</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41467-018-04121-y</pub-id><pub-id pub-id-type=\"pmid\">29703898</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Giustiniani</surname><given-names>J</given-names></name><name><surname>Daire</surname><given-names>V</given-names></name><name><surname>Cantaloube</surname><given-names>I</given-names></name><name><surname>Durand</surname><given-names>G</given-names></name><name><surname>Pous</surname><given-names>C</given-names></name><name><surname>Perdiz</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Tubulin acetylation favors Hsp90 recruitment to microtubules and stimulates the signaling function of the Hsp90 clients Akt/PKB and p53</article-title>. <source>Cell Signal.</source> (<year>2009</year>) <volume>21</volume>:<fpage>529</fpage>–<lpage>39</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cellsig.2008.12.004</pub-id><pub-id pub-id-type=\"pmid\">19136058</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>X</given-names></name><name><surname>Goncalves</surname><given-names>R</given-names></name><name><surname>Mosser</surname><given-names>DM</given-names></name></person-group>. <article-title>The isolation and characterization of murine macrophages</article-title>. <source>Curr Protoc Immunol.</source> (<year>2008</year>) <volume>Chapter 14</volume>:<fpage>Unit 14.11</fpage>. <pub-id pub-id-type=\"doi\">10.1002/0471142735.im1401s83</pub-id><pub-id pub-id-type=\"pmid\">19016445</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>L</given-names></name><name><surname>Zhou</surname><given-names>X</given-names></name><name><surname>Shetty</surname><given-names>S</given-names></name><name><surname>Hou</surname><given-names>G</given-names></name><name><surname>Wang</surname><given-names>Q</given-names></name><name><surname>Fu</surname><given-names>J</given-names></name></person-group>. <article-title>HDAC6 inhibition blocks inflammatory signaling and caspase-1 activation in LPS-induced acute lung injury</article-title>. <source>Toxicol Appl Pharmacol.</source> (<year>2019</year>) <volume>370</volume>:<fpage>178</fpage>–<lpage>83</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.taap.2019.03.017</pub-id><pub-id pub-id-type=\"pmid\">30910594</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gao</surname><given-names>R</given-names></name><name><surname>Ma</surname><given-names>Z</given-names></name><name><surname>Hu</surname><given-names>Y</given-names></name><name><surname>Chen</surname><given-names>J</given-names></name><name><surname>Shetty</surname><given-names>S</given-names></name><name><surname>Fu</surname><given-names>J</given-names></name></person-group>. <article-title>Sirt1 restrains lung inflammasome activation in a murine model of sepsis</article-title>. <source>Am J Physiol Lung Cell Mol Physiol.</source> (<year>2015</year>) <volume>308</volume>:<fpage>L847</fpage>–<lpage>53</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajplung.00274.2014</pub-id><pub-id pub-id-type=\"pmid\">25659903</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>L</given-names></name><name><surname>Yang</surname><given-names>XJ</given-names></name></person-group>. <article-title>Tubulin acetylation: responsible enzymes, biological functions and human diseases</article-title>. <source>Cell Mol Life Sci.</source> (<year>2015</year>) <volume>72</volume>:<fpage>4237</fpage>–<lpage>55</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00018-015-2000-5</pub-id><pub-id pub-id-type=\"pmid\">26227334</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ko</surname><given-names>R</given-names></name><name><surname>Lee</surname><given-names>SY</given-names></name></person-group>. <article-title>Glycogen synthase kinase 3beta in Toll-like receptor signaling</article-title>. <source>BMB Rep.</source> (<year>2016</year>) <volume>49</volume>:<fpage>305</fpage>–<lpage>10</lpage>. <pub-id pub-id-type=\"doi\">10.5483/BMBRep.2016.49.6.059</pub-id><pub-id pub-id-type=\"pmid\">26996345</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Choi</surname><given-names>SJ</given-names></name><name><surname>Lee</surname><given-names>HC</given-names></name><name><surname>Kim</surname><given-names>JH</given-names></name><name><surname>Park</surname><given-names>SY</given-names></name><name><surname>Kim</surname><given-names>TH</given-names></name><name><surname>Lee</surname><given-names>WK</given-names></name><etal/></person-group>. <article-title>HDAC6 regulates cellular viral RNA sensing by deacetylation of RIG-I</article-title>. <source>EMBO J.</source> (<year>2016</year>) <volume>35</volume>:<fpage>429</fpage>–<lpage>42</lpage>. <pub-id pub-id-type=\"doi\">10.15252/embj.201592586</pub-id><pub-id pub-id-type=\"pmid\">26746851</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moreno-Gonzalo</surname><given-names>O</given-names></name><name><surname>Ramirez-Huesca</surname><given-names>M</given-names></name><name><surname>Blas-Rus</surname><given-names>N</given-names></name><name><surname>Cibrian</surname><given-names>D</given-names></name><name><surname>Saiz</surname><given-names>ML</given-names></name><name><surname>Jorge</surname><given-names>I</given-names></name><etal/></person-group>. <article-title>HDAC6 controls innate immune and autophagy responses to TLR-mediated signalling by the intracellular bacteria Listeria monocytogenes</article-title>. <source>PLoS Pathog.</source> (<year>2017</year>) <volume>13</volume>:<fpage>e1006799</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.ppat.1006799</pub-id><pub-id pub-id-type=\"pmid\">29281743</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hwang</surname><given-names>I</given-names></name><name><surname>Lee</surname><given-names>E</given-names></name><name><surname>Jeon</surname><given-names>SA</given-names></name><name><surname>Yu</surname><given-names>JW</given-names></name></person-group>. <article-title>Histone deacetylase 6 negatively regulates NLRP3 inflammasome activation</article-title>. <source>Biochem Biophys Res Commun.</source> (<year>2015</year>) <volume>467</volume>:<fpage>973</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbrc.2015.10.033</pub-id><pub-id pub-id-type=\"pmid\">26471297</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cross</surname><given-names>DA</given-names></name><name><surname>Alessi</surname><given-names>DR</given-names></name><name><surname>Cohen</surname><given-names>P</given-names></name><name><surname>Andjelkovich</surname><given-names>M</given-names></name><name><surname>Hemmings</surname><given-names>BA</given-names></name></person-group>. <article-title>Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B</article-title>. <source>Nature</source>. (<year>1995</year>) <volume>378</volume>:<fpage>785</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1038/378785a0</pub-id><pub-id pub-id-type=\"pmid\">8524413</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Endocrinol.</journal-id><journal-title-group><journal-title>Frontiers in Endocrinology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2392</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849300</article-id><article-id pub-id-type=\"pmc\">PMC7431619</article-id><article-id pub-id-type=\"doi\">10.3389/fendo.2020.00516</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Fundamental Concepts and Novel Aspects of Polycystic Ovarian Syndrome: Expert Consensus Resolutions</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Aversa</surname><given-names>Antonio</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/42679/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>La Vignera</surname><given-names>Sandro</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/360159/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Rago</surname><given-names>Rocco</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Gambineri</surname><given-names>Alessandra</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Nappi</surname><given-names>Rossella E.</given-names></name><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1046310/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Calogero</surname><given-names>Aldo E.</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/360161/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ferlin</surname><given-names>Alberto</given-names></name><xref ref-type=\"aff\" rid=\"aff6\"><sup>6</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/42613/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Experimental and Clinical Medicine, University “Magna Graecia”</institution>, <addr-line>Catanzaro</addr-line>, <country>Italy</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Clinical and Experimental Medicine, University of Catania</institution>, <addr-line>Catania</addr-line>, <country>Italy</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Physiopathology of Reproduction and Andrology Unit, Sandro Pertini Hospital</institution>, <addr-line>Rome</addr-line>, <country>Italy</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Medical and Surgical Science, University Alma Mater Studiorum</institution>, <addr-line>Bologna</addr-line>, <country>Italy</country></aff><aff id=\"aff5\"><sup>5</sup><institution>Research Center for Reproductive Medicine, Gynecological Endocrinology and Menopause, IRCCS San Matteo Foundation, University of Pavia</institution>, <addr-line>Pavia</addr-line>, <country>Italy</country></aff><aff id=\"aff6\"><sup>6</sup><institution>Department of Clinical and Experimental Sciences, University of Brescia</institution>, <addr-line>Brescia</addr-line>, <country>Italy</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Daniele Santi, University of Modena and Reggio Emilia, Italy</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Ljiljana Marina, University of Belgrade, Serbia; Biagio Cangiano, University of Milan, Italy</p></fn><corresp id=\"c001\">*Correspondence: Alberto Ferlin <email>alberto.ferlin@unibs.it</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Reproduction, a section of the journal Frontiers in Endocrinology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>516</elocation-id><history><date date-type=\"received\"><day>15</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>26</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Aversa, La Vignera, Rago, Gambineri, Nappi, Calogero and Ferlin.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Aversa, La Vignera, Rago, Gambineri, Nappi, Calogero and Ferlin</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Polycystic ovary syndrome (PCOS) is a very common endocrine and metabolic disorder with the involvement of both genetic and environmental factors. Although much has been clarified on its pathogenesis, diagnosis, clinical manifestations, and therapy, there are still areas of uncertainty. To address fundamental concepts, novel aspects and hypotheses, and future perspectives, including the possible additional benefits of treatment with nutraceuticals, an expert consensus panel formed by endocrinologists and gynecologists was established. After an independent review of the literature, the panel convened electronically on February 3, 2020, and six resolutions were created, debated, and agreed upon discussion, and finally approved in their final form in a consensus livestream meeting held on April 15. The summary of the resolutions are: (1) PCOS is a well-established medical condition that negatively affects reproduction, general health, sexual health, and quality of life; (2) the symptoms and signs of PCOS appear early in life especially in female newborns from PCOS carriers; (3) women with PCOS have significantly increased risk of pregnancy-related complications including gestational diabetes mellitus; (4) a male PCOS equivalent exists, and it may impact on metabolic health and probably on reproduction; (5) the evidence supports that medical therapy for PCOS is effective, rational, and evidence-based; (6) the evidence supports a major research initiative to explore possible benefits of nutraceutical therapy for PCOS. The proposed resolutions may be regarded as points of agreement based on the current scientific evidence available.</p></abstract><kwd-group><kwd>PCOS</kwd><kwd>medical therapy</kwd><kwd>nutraceuticals</kwd><kwd>PCOS carriers</kwd><kwd>male PCOS</kwd><kwd>consensus</kwd></kwd-group><counts><fig-count count=\"0\"/><table-count count=\"2\"/><equation-count count=\"0\"/><ref-count count=\"187\"/><page-count count=\"16\"/><word-count count=\"14445\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Polycystic ovary syndrome (PCOS) is a very common endocrine disorder in women of reproductive age, with a reported prevalence ranging from 6 to 15% (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Its etiology involves both genetic and environmental factors. Typically, women with PCOS show clinical and biochemical hyperandrogenism, oligoanovulation, and micropolycystic morphology of the ovaries (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). However, PCOS diagnosis relies on specific criteria that differ according to the scientific association that released them (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Criteria for the diagnosis of polycystic ovary syndrome (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>NIH/NICHD</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>ESHRE/ASRM 2004</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Androgen Excess Society 2006</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Includes all of the following criteria:</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Includes two of the following criteria:</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Includes all of the following criteria:</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Clinical and/or biochemical signs of hyperandrogenism</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Clinical and/or biochemical signs of hyperandrogenism</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Clinical and/or biochemical signs of hyperandrogenism</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Menstrual dysfunction</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Oligo-ovulation or anovulation<break/> • Polycystic ovaries</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Ovarian dysfunction and/or polycystic ovaries</td></tr></tbody></table><table-wrap-foot><p><italic>ESHRE/ASRM, European Society for Human Reproduction and Embryology/American Society for Reproductive Medicine; NIH/NICHD, National Institutes of Health/National Institute of Child Health and Human Development</italic>.</p></table-wrap-foot></table-wrap><p>The diagnostic criteria do not consider the widely recognized dysmetabolic background of PCOS. Indeed, many patients have impaired insulin action [up to 75% of them are insulin-resistant (IR)], hyperinsulinemia, and overweight/obesity, and this appears to play a crucial role in the pathogenesis of PCOS. Two major PCOS phenotypes can be distinguished: the overweight/obese and the lean one. Their estimated prevalence is ≈80 and ≈20%, respectively (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Insulin resistance is a fundamental pathogenic component of PCOS, both in lean and overweight–obese patients. The expression of the syndrome differs between lean and overweight–obese PCOS. Worryingly, some patients with the lean phenotype may not show symptoms such as irregular menses or acne (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>, <xref rid=\"B6\" ref-type=\"bibr\">6</xref>), and this increases the chance of underdiagnosis or misdiagnosis. Women with PCOS are exposed to metabolic alterations, endothelial dysfunction, and cardiovascular risk factors, independently from obesity, even if obesity aggravates the phenotype (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Therefore, a timely diagnosis and proper management should be warranted.</p><p>Although much has been clarified in recent years on the pathogenesis, diagnosis, clinical manifestations, and therapy of PCOS, there are still many doubts and consequent uncertainties in the choice of the therapeutic approach in clinical practice.</p><p>To address some important concerns of PCOS and especially its possible treatment with nutraceuticals, a panel of Italian experts convened webinar on February 3, 2020, after an independent review of the literature. The panel included clinicians dealing with this condition such as endocrinologists and gynecologists. Six resolutions were proposed, debated, and agreed on after a thorough discussion. Then the resolutions were written, discussed in their final version, and approved in a final consensus webinar held on April 15.</p><p>The summary of the resolutions and the condensed expert opinions are reported in <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>.</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Summary of resolutions and expert opinion.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Resolution</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Condensed expert opinion</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PCOS is a well-established medical condition that negatively affects reproduction, general health, sexual health, and quality of life.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• PCOS is a multifaceted disease with an impact on various aspects of a woman's life, such as aesthetics, reproduction, metabolism, psychological well-being, and sexuality.<break/> • Phenotypization is fundamental for providing a tailored therapy.</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">2</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">The symptoms and signs of PCOS appear early in life especially in female newborns from PCOS carriers.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Daughters of PCOS women inherit certain characteristics that become more evident across puberty.<break/> • Early recognition of PCOS in adolescence is fundamental to set up individualized strategies to ameliorate symptoms and to counteract reproductive and metabolic risks associated with this condition.</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">3</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Women with PCOS have significantly increased risk of pregnancy-related complications including gestational diabetes.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Women with PCOS have an increased risk of GDM than controls, especially if obesity/metabolic syndrome are present, and should be carefully investigated and monitored during early pregnancy with OGTT.<break/> • Changes in intestinal microbiota during pregnancy may contribute to the onset of metabolic dysfunction in both the mother and the offspring.</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">A male PCOS equivalent seems to exist, and it may impact on metabolic health and probably on reproduction.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• Male PCOS equivalent may be diagnosed in presence of PCOS-like hormonal pattern, metabolic abnormalities, overweight/obesity, and/or clinical signs of hyperandrogenism, above all in patients aged <35 years with a family history positive for PCOS.<break/> • The metabolic and hormonal profile should be assessed in first-degree male relatives of PCOS women and in men with early-onset AGA. This may help to prevent the risk of T2DM and CVD later in life.<break/> • Further studies are needed to confirm the existence of a male PCOS equivalent and to evaluate its impact on the testicular function.</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">The evidence supports that medical therapy for women with PCOS is effective, rational, and evidence-based.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• No single unified treatment for PCOS is available, and treatment should be individualized.<break/> • Targets for pharmacological treatment include biochemical and clinical androgen excess, menstrual irregularities, anovulation, insulin resistance, and metabolic profile.<break/> • Lifestyle counseling should be provided in all cases.<break/> • COCPs are the first-line treatment for long-term management of menstrual irregularities and hyperandrogenism.<break/> • Metformin should be recommended in overweight/obese adult PCOS women and considered in adolescents with PCOS for the management of weight, insulin resistance, and metabolic abnormalities.</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">The evidence supports a major research initiative to explore possible benefits of nutraceutical therapy for PCOS.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">• MI and DCI show different insulin-mimetic properties. Inositol administration should be aimed to keep unaltered the MI/DCI ratio.<break/> • Treatment with MI/ALA combination may ameliorate hyperinsulinemia, decrease oxidative stress markers at oocyte level, and normalize endometrial inflammasome in PCOS women with idiopathic recurrent pregnancy loss.<break/> • The hormonal and clinical profile of overweight/obese women with PCOS may benefit from prolonged use of MI/ALA combination, such as a higher recovery of class II oocytes during ART.<break/> • NAFLD may be associated with PCOS. A timely diagnosis is warranted to avoid the NAFLD-related long-term complications. A nutraceutical approach could be useful in the treatment of NAFLD.<break/> • Hyperomocysteinemia may be associated with selected PCOS patients. Treatment with folic acid should be started to avoid the long-term consequences on the cardiovascular system.<break/> • Nutraceuticals, associated with diet and lifestyle modifications, can be important therapeutic option to manage pregnancy-related complications in PCOS pregnant patients.</td></tr></tbody></table><table-wrap-foot><p><italic>AGA, androgenic alopecia; ALA, α-lipoic acid; ART, assisted reproductive techniques; COCP, combined oral contraceptive pills; DCI, D-chiro-inositol; GDM, gestational diabetes mellitus; MI, myoinositol; NAFLD, non-alcoholic fatty liver disease; OGTT, oral glucose tolerance test; T2DM, type 2 diabetes mellitus</italic>.</p></table-wrap-foot></table-wrap><sec><title>Resolution 1: PCOS is a Well-Established Medical Condition That Negatively Affects Reproduction, General Health, Sexual Health, and Quality of Life</title><p>Polycystic ovary syndrome is the most frequent disorder in women of reproductive age, and it is also one of the most well-studied. Nevertheless, many unresolved questions regarding its pathogenesis, diagnosis, clinical manifestations, acute and chronic complications, and treatment are still debated (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). It is one of the most common causes of menstrual irregularities, such as amenorrhea, oligomenorrhea, and polymenorrhea, and is the primary cause of anovulatory infertility (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). Infertility is aggravated by a high percentage of miscarriages, as well as by low pregnancy and live birth rates after assisted reproductive technologies (ARTs) (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). In addition, women with PCOS tend to produce a greater number of oocytes during ART and to have higher rates of ovarian hyperstimulation syndrome and/or multiple gestations compared with non-PCOS infertile women (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Furthermore, increased pregnancy complications and obstetric and neonatal risks are also associated with PCOS. These include gestational diabetes mellitus (GDM), preeclampsia, pregnancy-induced hypertension, postpartum hemorrhage and infection, preterm delivery, meconium aspiration, stillbirth, operative deliveries, and shoulder dystocia (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>, <xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B11\" ref-type=\"bibr\">11</xref>). The presence of obesity exacerbates all these complications and produces a blunted responsiveness to ovulation induction in proportion to its severity and, particularly, when the abdominal phenotype of obesity (visceral fat accumulation) is present (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>–<xref rid=\"B15\" ref-type=\"bibr\">15</xref>).</p><p>The pathophysiological mechanisms by which PCOS negatively impacts on fertility are complex and not completely understood. Undoubtedly, hyperandrogenism, the consequent hyperestrogenemia, IR, and compensatory hyperinsulinemia play an important role acting on both the ovary and the endometrium (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>–<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). In addition, there is emerging evidence that proinflammatory cytokines and oxidative stress may directly impact on oocyte quality and may induce endothelial dysfunction, thus contributing to infertility (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B12\" ref-type=\"bibr\">12</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Some recent <italic>ex vivo</italic> and <italic>in vivo</italic> studies have also demonstrated that proinflammatory cytokines inhibit follicle-stimulating hormone (FSH) and luteinizing hormone (LH) receptor expression, thus impairing the regular follicular development and its luteinization. They also inhibit FSH-induced 17β-estradiol and progesterone secretion from granulosa cells obtained by preovulatory follicles and stimulate testosterone production from theca cells, all mechanisms that contribute to impair ovulation and, therefore, infertility (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>–<xref rid=\"B22\" ref-type=\"bibr\">22</xref>).</p><p>Polycystic ovary syndrome is not only a reproductive endocrinopathy, but it is also a metabolic endocrinopathy, for the high prevalence of overweight/abdominal obesity, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), type 2 diabetes mellitus (T2DM), and metabolic syndrome (MetS) (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B23\" ref-type=\"bibr\">23</xref>, <xref rid=\"B24\" ref-type=\"bibr\">24</xref>). Obesity, hyperandrogenism, and IR are known risk factors for increasing NAFLD and T2DM occurrence in PCOS (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>, <xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B26\" ref-type=\"bibr\">26</xref>). Furthermore, these metabolic alterations are well-known additional risk factors for cardiovascular diseases (CVDs), although strong evidence in PCOS is lacking. Additionally, chronic low-grade inflammation is a risk factor for IR and T2DM (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B27\" ref-type=\"bibr\">27</xref>–<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). In addition, many recent studies have clearly shown that PCOS is associated with anxiety (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>–<xref rid=\"B36\" ref-type=\"bibr\">36</xref>) and depression (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>–<xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B37\" ref-type=\"bibr\">37</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>), and with a poorer quality of life (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B34\" ref-type=\"bibr\">34</xref>, <xref rid=\"B39\" ref-type=\"bibr\">39</xref>–<xref rid=\"B42\" ref-type=\"bibr\">42</xref>), even in adolescence and young age (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>). Both physical and mental distress may contribute to decrease the quality of life in PCOS (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>, <xref rid=\"B44\" ref-type=\"bibr\">44</xref>, <xref rid=\"B45\" ref-type=\"bibr\">45</xref>) regardless of body mass index (BMI), hyperandrogenism, and socioeconomic status (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>). However, the presence of obesity, menstrual disorders, clinical hyperandrogenism (acne, alopecia, hirsutism), and infertility may impact on the quality of life (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>, <xref rid=\"B31\" ref-type=\"bibr\">31</xref>, <xref rid=\"B34\" ref-type=\"bibr\">34</xref>, <xref rid=\"B35\" ref-type=\"bibr\">35</xref>, <xref rid=\"B46\" ref-type=\"bibr\">46</xref>–<xref rid=\"B49\" ref-type=\"bibr\">49</xref>). Thus, infertility and hirsutism have been associated with both anxiety (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>, <xref rid=\"B27\" ref-type=\"bibr\">27</xref>, <xref rid=\"B50\" ref-type=\"bibr\">50</xref>) and depression (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>, <xref rid=\"B46\" ref-type=\"bibr\">46</xref>) and alopecia with anxiety (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>), whereas obesity and acne with depression (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>, <xref rid=\"B31\" ref-type=\"bibr\">31</xref>, <xref rid=\"B34\" ref-type=\"bibr\">34</xref>). Accordingly, both weight loss and oral contraceptive use significantly improve several physical and mental domains related to quality of life, depressive symptoms, and anxiety disorders (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>, <xref rid=\"B52\" ref-type=\"bibr\">52</xref>). A high percentage of eating disorders, such as anorexia nervosa, bulimia nervosa, and binge eating disorder, have also been observed in PCOS (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>, <xref rid=\"B53\" ref-type=\"bibr\">53</xref>), particularly in the presence of anxiety and depression (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>).</p><p>More contradictory results have been found regarding the association between PCOS and sexual dysfunctions, because some studies described a high prevalence of sexual dysfunctions (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>, <xref rid=\"B46\" ref-type=\"bibr\">46</xref>, <xref rid=\"B49\" ref-type=\"bibr\">49</xref>, <xref rid=\"B54\" ref-type=\"bibr\">54</xref>–<xref rid=\"B56\" ref-type=\"bibr\">56</xref>), whereas others did not (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>, <xref rid=\"B57\" ref-type=\"bibr\">57</xref>–<xref rid=\"B64\" ref-type=\"bibr\">64</xref>). The studies that have demonstrated a high prevalence (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>, <xref rid=\"B50\" ref-type=\"bibr\">50</xref>–<xref rid=\"B53\" ref-type=\"bibr\">53</xref>) described defect in arousal, poor lubrification (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>) and pain during intercourse (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>, <xref rid=\"B53\" ref-type=\"bibr\">53</xref>), and high degree of sexual dissatisfaction (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>, <xref rid=\"B50\" ref-type=\"bibr\">50</xref>–<xref rid=\"B52\" ref-type=\"bibr\">52</xref>). All the aspects of sexual dysfunction described in PCOS are exacerbated by the presence of obesity (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>, <xref rid=\"B50\" ref-type=\"bibr\">50</xref>, <xref rid=\"B51\" ref-type=\"bibr\">51</xref>, <xref rid=\"B61\" ref-type=\"bibr\">61</xref>, <xref rid=\"B65\" ref-type=\"bibr\">65</xref>), alopecia, and infertility, regardless of its duration (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>). Contradictory results have been described regarding the association between sexual dysfunction and androgen circulating levels, with some studies demonstrating a positive correlation (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>), others negative (<xref rid=\"B61\" ref-type=\"bibr\">61</xref>, <xref rid=\"B66\" ref-type=\"bibr\">66</xref>), whereas others show no significant association (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>). Hirsutism (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>, <xref rid=\"B64\" ref-type=\"bibr\">64</xref>), acne (<xref rid=\"B67\" ref-type=\"bibr\">67</xref>), and a sense of low attractiveness (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>, <xref rid=\"B51\" ref-type=\"bibr\">51</xref>) resulted mostly associated with low sexual satisfaction. As expected, PCOS women with the lowest sexual satisfaction exhibited an even stronger predilection toward anxiety and depression than the other women (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>, <xref rid=\"B50\" ref-type=\"bibr\">50</xref>, <xref rid=\"B51\" ref-type=\"bibr\">51</xref>).</p><p>Altogether, these data demonstrate that PCOS is appropriately defined syndrome for the complexity and heterogeneity of the clinical manifestations, associated comorbidities, and clinical consequences that accompany the woman during reproductive and postreproductive age, thus impacting on the quality of life.</p><sec><title>Expert Opinion</title><list list-type=\"bullet\"><list-item><p>Polycystic ovary syndrome is a multifaceted disease with an impact on various aspects of women's life, such as aesthetics, reproduction, metabolism, psychological well-being, and sexuality.</p></list-item><list-item><p>The lean PCOS phenotype has a high risk of underdiagnosis and misdiagnosis with respect to the overweight/obese phenotype.</p></list-item><list-item><p>The phenotypization of the woman with PCOS is fundamental for providing a tailored therapy, by taking advantage of the wide and diversified therapeutic availability.</p></list-item></list></sec></sec><sec><title>Resolution 2: The Symptoms and Signs of PCOS Appear Early in Life Especially in Female Newborns From PCOS Carriers</title><p>Nowadays, PCOS is a lifelong medical condition requiring a multispecialist vision for early diagnosis and an effective management and treatment plan over time (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). Preconception care, mainly in infertility clinics, is the golden moment to identify PCOS carriers because PCOS-like outcomes, especially in female offspring, result from both genetic and epigenetic mechanisms (<xref rid=\"B68\" ref-type=\"bibr\">68</xref>, <xref rid=\"B69\" ref-type=\"bibr\">69</xref>). Indeed, specific PCOS-susceptibility loci may explain family predisposition and the variable clinical presentation of PCOS, including neuroendocrine, reproductive, and metabolic abnormalities (<xref rid=\"B70\" ref-type=\"bibr\">70</xref>). On the other hand, maternal–fetal interactions account for early signs of hyperandrogenism in offspring of PCOS carriers, resulting not only from heritability, but also from the intrauterine androgen excess of maternal and/or fetal origin, with the contribution of a dysfunctional placenta (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>). Overexposure to androgens <italic>in utero</italic> influences the activity of multiple pathways regulating gonadotropin-releasing hormone (GnRH) pulsatility, follicular development, ovarian steroidogenesis, and insulin–glucose homeostasis (<xref rid=\"B72\" ref-type=\"bibr\">72</xref>).</p><p>Glucose intolerance and hyperinsulinemia in metabolically unhealthy PCOS mothers further contribute to the prenatal hyperandrogenic milieu, which plays a leading role in reprogramming female offspring for reproductive, behavioral, and metabolic PCOS-like traits with some gender differences (<xref rid=\"B73\" ref-type=\"bibr\">73</xref>). On the other hand, injuries to the fetoplacental unit deriving from gestational-related conditions, such as hypertension and/or T2DM, or unhealthy habits (smoking, lack of exercise, etc.) may have an influence on weight at birth reprogramming metabolic function even in offspring of mothers without PCOS (<xref rid=\"B74\" ref-type=\"bibr\">74</xref>).</p><p>During infancy and adolescence, this “metabolic memory” may likely induce a non-genetic inheritance of PCOS-like features, linked to overweight and/or abdominal obesity, which are still preventable with healthy habits (adequate diet, active lifestyle, etc.) (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>, <xref rid=\"B75\" ref-type=\"bibr\">75</xref>). Altogether, endocrine and metabolic events in intrauterine life become evident early postnatally (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>) and may give origin to a high rate of overt signs and symptoms of PCOS manifesting at puberty and progressing with age (<xref rid=\"B77\" ref-type=\"bibr\">77</xref>). Apart from subtle signs of a compromised cardiometabolic health, predominantly in female offspring (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>) the diagnosis of PCOS in mothers and/or elevated fetal testosterone has been also linked to pervasive developmental disabilities, autism spectrum disorder, and attention-deficit/hyperactivity disorder, an evidence recently confirmed in a US cohort of infants of PCOS mothers (<xref rid=\"B78\" ref-type=\"bibr\">78</xref>).</p><p>Polycystic ovary syndrome diagnosis during infancy and childhood is virtually impossible because symptoms are missing. However, the measurement of anti-Müllerian hormone (AMH), a surrogate marker of ovarian hyperandrogenism produced by small antral follicles (<xref rid=\"B79\" ref-type=\"bibr\">79</xref>), in daughters of women with PCOS may represent a reliable proxy. Indeed, AMH values are higher across puberty and correlate with LH and testosterone plasma levels, indicating multifollicular ovarian morphology resulting from enhanced recruitment, decreased depletion or even from follicular growing arrest, and reduced rate of atresia (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B80\" ref-type=\"bibr\">80</xref>). Newborns with low and high birth weights have higher AMH levels than normal birth weight infants tested at 2–3 months of age (<xref rid=\"B81\" ref-type=\"bibr\">81</xref>). However, body weight abnormalities at birth do not seem associated with having a PCOS mother (<xref rid=\"B82\" ref-type=\"bibr\">82</xref>). On the other hand, birth weight and thinness at birth independently predict symptoms of PCOS in adulthood (<xref rid=\"B83\" ref-type=\"bibr\">83</xref>). Many coexisting elements, such as maternal obesity, pregnancy complications, and other comorbidity make it difficult to identify the real contribution of PCOS mothers to health and development of offspring (<xref rid=\"B84\" ref-type=\"bibr\">84</xref>).</p><p>In any case, it is more likely that daughters of PCOS women have more abdominal fat distribution and signs of hyperandrogenism compared with control daughters across pubertal maturation (<xref rid=\"B85\" ref-type=\"bibr\">85</xref>). Increased glucose-stimulated insulin levels are also a consistent phenotype in the daughters of women with PCOS in mid to late puberty (<xref rid=\"B86\" ref-type=\"bibr\">86</xref>). Moreover, 5α-reductase, the key enzyme in androgen metabolism, catalyzing the irreversible conversion of testosterone to dihydrotestosterone (DHT) into the skin, is significantly increased during early childhood in daughters of women with PCOS (<xref rid=\"B87\" ref-type=\"bibr\">87</xref>). On the other hand, IR <italic>per se</italic> amplifies hyperandrogenic signs by enhancing the activity of 5α-reductase at the target levels (<xref rid=\"B88\" ref-type=\"bibr\">88</xref>).</p><p>Even though no causal relationship between excessive adiposity and early puberty has been clearly demonstrated, it is likely that these girls can accelerate their growth and maturation in a homeostatic attempt to reduce their abdominal obesity, showing a higher tendency to early pubarche and eventually early menarche (<xref rid=\"B89\" ref-type=\"bibr\">89</xref>). On the other hand, an excess of adipose tissue during adolescence undoubtedly increases the possibility of developing multifollicular ovarian morphology resembling PCOS-like aspect. This is also a sign of reproductive axis immaturity, and therefore, it is not considered a diagnostic criterion within 2 years after menarche. Irregular menses that persist 2 years after menarche may be a sign of PCOS, although they may continue up to the fifth year after menarche without developing PCOS. Circulating androgens reach adult levels generally by age 15 years, and hirsutism may increase progressively over the time (<xref rid=\"B90\" ref-type=\"bibr\">90</xref>). In adolescence, IR and hyperinsulinemia have a widespread impact on reproductive function by multiple mechanisms, including a synergic role with LH to stimulate the secretion of androgens from the ovarian theca. Moreover, insulin, by lowering liver sex hormone–binding globulin (SHBG) production, increases androgen bioavailability at target organs, further contributing to clinical signs of hyperandrogenism (<xref rid=\"B91\" ref-type=\"bibr\">91</xref>).</p><p>Finally, IR, independently from the extent of obesity and magnitude of androgen concentrations, may be present even in lean PCOS. However, transient hyperinsulinemia is typical at puberty and may amplify the individual predisposition to develop PCOS (<xref rid=\"B92\" ref-type=\"bibr\">92</xref>).</p><p>Collectively, these data indicate that clinical signs of hyperandrogenism observed in adolescent PCOS, in particular acne, are likely influenced not only by the abundance of DHT at the level of the pilosebaceous unit, but also by different abnormal insulin signaling in concert with nutritional aspects (<xref rid=\"B93\" ref-type=\"bibr\">93</xref>). Symptoms of androgen excess may be quite common in adolescents and may cause significant distress well before a final diagnosis of PCOS can be formulated. To ameliorate hyperandrogenic signs, it is essential to find therapeutic strategies to fill the gap between the physiological changes of puberty and the clear clinical picture that becomes evident some years following menarche (<xref rid=\"B94\" ref-type=\"bibr\">94</xref>). Therefore, knowing that an intergenerational risk for PCOS exists serves to prevent its onset from intrauterine to adult life.</p><sec><title>Expert Opinion</title><list list-type=\"bullet\"><list-item><p>Polycystic ovary syndrome is a clear example of transgenerational disease that results from genetic and epigenetic mechanisms from prenatal life to adulthood.</p></list-item><list-item><p>Daughters of women with PCOS inherit certain characteristics that become more evident across puberty. These include abdominal fat distribution and signs of hyperandrogenism.</p></list-item><list-item><p>Early recognition of PCOS in adolescence is fundamental to set up individualized strategies to ameliorate symptoms and to counteract reproductive and metabolic risks associated with this condition.</p></list-item></list></sec></sec><sec><title>Resolution 3: Women With PCOS Have Significantly Increased Risk of Pregnancy-Related Complications Including Gestational Diabetes</title><p>Polycystic ovary syndrome is a primary risk factor for adverse pregnancy outcomes. A meta-analysis conducted by Kjerulff et al. (<xref rid=\"B95\" ref-type=\"bibr\">95</xref>) indicated that pregnancy in PCOS patients is associated with increased risk of GDM, pregnancy-induced hypertension, preeclampsia, preterm delivery, and small-for-gestational age, and according to various data, the risk of miscarriage in PCOS women is reported to be three times higher than in healthy women (<xref rid=\"B96\" ref-type=\"bibr\">96</xref>). Indeed, PCOS could be included as a risk factor for GDM (<xref rid=\"B97\" ref-type=\"bibr\">97</xref>).</p><p>During pregnancy, a physiologic insulin insensitivity occurs because of the release of placental hormones. These hormones promote nutrient utilization by the fetus, but on the other hand, IR associated with pregnancy is the main pathogenic mechanism leading the development of GDM in predisposed women such as PCOS. All lipoprotein subclasses and lipids are markedly increased in pregnant women, and the most pronounced differences are observed for the intermediate-density, low-density (LDL), and high-density lipoprotein (HDL) triglyceride concentrations (<xref rid=\"B98\" ref-type=\"bibr\">98</xref>). All these metabolic alterations lead to an increase in the plasma concentration of circulating proinflammatory cytokines, such as tumor necrosis factor α and interleukin 6 (IL-6), with reduction of plasma anti-inflammatory molecule levels such as adiponectin and IL-10 (<xref rid=\"B99\" ref-type=\"bibr\">99</xref>). Furthermore, inflammatory mediator overexpression, together with an increase of reactive oxygen species (ROS), could lead to metabolic alterations and vascular disease and induce inhibition of the insulin signaling pathway, thus resulting in IR, reduced insulin gene expression, and, consequently, reduced β-cell insulin secretion and GDM (<xref rid=\"B100\" ref-type=\"bibr\">100</xref>). In many cases, these factors are already present in women with PCOS, and therefore pregnancy can be the final hit in the onset of DM.</p><p>According to the current Italian guidelines on GDM, PCOS women carrying high risk (i.e., obesity, previous macrosomia or GDM, fasting blood sugar 100–125 mg/dL at the beginning of pregnancy) should be investigated by oral glucose tolerance test (OGTT) between the 16th and 18th week of gestation and, if normal, by repeating it between the 24th and 28th week of pregnancy (<xref rid=\"B101\" ref-type=\"bibr\">101</xref>).</p><p>A crucial role of the intestinal microbiota in pregnancy-related GDM has been suggested. In the first trimester, the composition of the gut microbiota is similar to that of a non-pregnant woman. In the following months, the variability of microorganisms decreases, and there is an increase in the populations of <italic>Proteobacteria</italic> and <italic>Actinobacteria</italic>; <italic>Bifidobacteria</italic> belong to the latter and play a pivotal role in the defense against pathogenic bacteria, in strengthening the intestinal barrier, and in the nutrients' metabolism. The intestine of a pregnant woman can become more permeable favoring the so-called “bacterial translocation” so that the fetus can come into contact with microorganisms of maternal origin (microbes have been found in the blood of the umbilical cord, in the amniotic fluid, and even in the meconium) (<xref rid=\"B102\" ref-type=\"bibr\">102</xref>–<xref rid=\"B104\" ref-type=\"bibr\">104</xref>). Furthermore, according to the most widespread theories, the baby's intestine is colonized during childbirth by the bacteria of the maternal microbiota. If the pregnant woman is in dysbiosis, the fetus will not receive bifid but other bacterial strains, which could lead to a greater exposure of the newborn to diseases. This imbalance can also lead to a greater absorption of calories with a consequent weight increase of the pregnant woman and a greater risk of developing GDM. Even the newborn could more easily develop childhood diabetes, allergies, and childhood obesity (<xref rid=\"B102\" ref-type=\"bibr\">102</xref>–<xref rid=\"B104\" ref-type=\"bibr\">104</xref>).</p><sec><title>Expert Opinion</title><list list-type=\"bullet\"><list-item><p>Women with PCOS have an increased risk of GDM than controls, especially if obesity/MetS are present, and should be carefully investigated and monitored during early pregnancy with OGTT.</p></list-item><list-item><p>Changes in intestinal microbiota during pregnancy may contribute to the onset of metabolic dysfunction in both the mother and the offspring.</p></list-item></list></sec></sec><sec><title>Resolution 4: A Male PCOS Equivalent Seems to Exist, and it May Impact on Metabolic Health and Probably on Reproduction</title><p>The genetic background of PCOS (<xref rid=\"B105\" ref-type=\"bibr\">105</xref>) suggests the existence of a male PCOS equivalent (<xref rid=\"B106\" ref-type=\"bibr\">106</xref>). Indeed, even male relatives inherit the same genetic factors predisposing to female PCOS. However, little is known about the putative presence of a male-PCOS and its possible consequences in men.</p><p>Kinship of PCOS women has an increased risk to develop metabolic abnormalities and CVDs, independently of the gender. Indeed, their siblings have a higher prevalence of IR, hyperinsulinemia, dyslipidemia, and hypertension already at a young age (<40 years) (<xref rid=\"B107\" ref-type=\"bibr\">107</xref>–<xref rid=\"B109\" ref-type=\"bibr\">109</xref>). Young first-degree male relatives have endothelial dysfunction that is not present in female PCOS patients (<xref rid=\"B110\" ref-type=\"bibr\">110</xref>). Therefore, the male relatives have an increased metabolic and cardiovascular risk. Furthermore, obesity, MetS, T2DM, and CVDs are more frequently diagnosed in male and female first-degree relatives of PCOS women compared to those with a negative PCOS family history (<xref rid=\"B107\" ref-type=\"bibr\">107</xref>, <xref rid=\"B108\" ref-type=\"bibr\">108</xref>).</p><p>Only few studies have explored hormonal abnormalities in the first-degree male relatives of women with PCOS. They have been shown to have higher dehydroepiandrosterone (DHEAS) level (<xref rid=\"B111\" ref-type=\"bibr\">111</xref>, <xref rid=\"B112\" ref-type=\"bibr\">112</xref>), which suggests the presence of a similar steroidogenic abnormality reported in their sisters. In addition, they have higher levels of AMH, LH, and FSH compared with controls (<xref rid=\"B113\" ref-type=\"bibr\">113</xref>). Moreover, a higher LH and FSH response to GnRH stimulation test has been reported in these men compared to controls (<xref rid=\"B114\" ref-type=\"bibr\">114</xref>), suggesting the presence of an abnormal GnRH-induced gonadotropin release.</p><p>The presence of metabolic, cardiovascular, and hormonal alterations in the male relative of women with PCOS supports the existence of a male PCOS equivalent. Accordingly, several authors have shown the occurrence of early-onset (<35 years) androgenetic alopecia (AGA) [grade ≥III, Hamilton–Norwood scale (<xref rid=\"B115\" ref-type=\"bibr\">115</xref>, <xref rid=\"B116\" ref-type=\"bibr\">116</xref>)] in male relatives of PCOS women (<xref rid=\"B117\" ref-type=\"bibr\">117</xref>, <xref rid=\"B118\" ref-type=\"bibr\">118</xref>). Therefore, early-onset AGA has been proposed as a clinical sign of the male PCOS equivalent. On this account, the hormonal and metabolic pattern has been evaluated in men with early-onset AGA, independently from the kinship with PCOS women. A meta-analysis carried out in 1,009 unrelated men showed increased LH and DHEAS, decreased SHBG, a downward trend for FSH, and an upward trend for the LH/FSH ratio in patients with early-onset AGA compared with controls (<xref rid=\"B119\" ref-type=\"bibr\">119</xref>), therefore resembling the female PCOS hormonal pattern. The same meta-analysis found a significant increase of insulin levels and Homeostatic Model Assessment (HOMA) index, total and LDL cholesterol, and triglycerides in patients with respect to controls, already before the age of 35 years. Unfortunately, there are no data on the reproductive function in men with early-onset AGA. Although it is plausible that the reproductive potential of subjects with “male PCOS phenotype” might be compromised similarly to women with PCOS, one could also speculate that fertility could be even improved in these subjects, highlighting therefore an intriguing evolutionary paradox.</p><p>By contrast, a large amount of data has been produced on the metabolic, cardiovascular, and prostatic sequelae in aging men with early-onset AGA. A hospital-based analysis reported a higher risk for myocardial infarction in 665 patients with respect to 772 controls; the risk was higher for severe vertex [Relative risk(RR), 3.4] compared with frontal AGA (RR, 0.9) (<xref rid=\"B120\" ref-type=\"bibr\">120</xref>). A retrospective study carried out in 22,071 men aged 40–84 years found an increased prevalence of T2DM in patients with severe vertex AGA than those belonging to other hair-pattern categories (<xref rid=\"B121\" ref-type=\"bibr\">121</xref>). Moreover, the Framingham Study showed a positive correlation between AGA progression and coronary heart disease (CHD) (<xref rid=\"B122\" ref-type=\"bibr\">122</xref>). The NHANES I Epidemiologic Follow-up Study (3,932 men aged 25–73 years) reported that severe AGA was positively associated with mortality for CHD in men before 55 years of age (<xref rid=\"B123\" ref-type=\"bibr\">123</xref>). Furthermore, a meta-analysis on 29,254 participants confirmed the positive correlation between AGA and CHD, IR, hyperinsulinemia, and MetS in both genders (<xref rid=\"B124\" ref-type=\"bibr\">124</xref>). The level of the evidence is high enough that AGA has been already proposed as an independent predictor of mortality for T2DM and CVDs (<xref rid=\"B125\" ref-type=\"bibr\">125</xref>). Finally, prostate inflammation, hyperplasia, and even cancer have been reported in men with early-onset AGA (<xref rid=\"B120\" ref-type=\"bibr\">120</xref>). Current evidence supports a relationship between prostate inflammation and hyperplasia with IR and hyperinsulinemia, whose mechanisms are not clear yet. It might be speculated that prostate diseases represent long-term consequence of male PCOS equivalent, whose pathogenesis includes metabolic abnormalities.</p><p>In conclusion, PCOS syndrome is not simply a primarily ovarian disorder, because the core of its pathogenesis is a metabolic dysfunction, as also suggested by the long-term complications found in these patients. The genetic predisposition to develop a syndrome with a negative impact on metabolism can occur also in men. Current evidence indicates the existence of metabolic, cardiovascular, and hormonal abnormalities in the first-degree males of PCOS women. The higher prevalence of early-onset AGA found in these men supports that it may represent a phenotypic feature of the male PCOS equivalent. Accordingly, patients with early-onset AGA show a PCOS-like hormonal pattern, increased insulin, HOMA index, total and LDL cholesterol, and triglycerides compared to age-matched controls. Similar to female PCOS, long-term consequences of early-onset AGA (male PCOS equivalent) include T2DM, CVDs, and a higher mortality for CHD. Hence, the male PCOS syndrome might be defined as an endocrine syndrome with a metabolic background predisposing to the development of T2DM, CVDs, and prostatic diseases later in life.</p><sec><title>Expert Opinion</title><list list-type=\"bullet\"><list-item><p>Male PCOS equivalent may be diagnosed in presence of PCOS-like hormonal pattern (increased DHEAS, AMH, LH, LH/FSH ratio, high calculated free testosterone), metabolic abnormalities (IR, hyperinsulinemia, low SHBG levels, hyperglycemia), overweight/obesity, and/or clinical signs of hyperandrogenism (mainly early-onset AGA), above all in patients younger than 35 years with a family history positive for PCOS.</p></list-item><list-item><p>The metabolic and hormonal profile should be assessed in first-degree male relatives of PCOS women and in men with early-onset AGA. This may help to prevent the risk of T2DM and CVD later in life.</p></list-item><list-item><p>Further studies are needed to confirm the existence of a male PCOS equivalent and to evaluate its impact on the testicular function.</p></list-item></list></sec></sec><sec><title>Resolution 5: Evidence Supports That Medical Therapy for Women With PCOS is Effective, Rational, and Evidence-Based</title><p>Pharmacological treatments commonly used in women with PCOS are off-label because neither the Food and Drug Administration nor the European Medicines Agency has ever approved a specific drug for PCOS treatment. However, they are widely used and their efficacy is evidence-based and rational (<xref rid=\"B74\" ref-type=\"bibr\">74</xref>).</p><p>Therapeutic approaches aim to improve disease-related symptoms and should therefore target hyperandrogenism, the consequences of ovarian dysfunction (menstrual irregularities, infertility), and/or the associated metabolic disorders. Apart from ovulation induction and treatment of infertility and topical-aesthetic therapies for hirsutism and acne, pharmacological treatment is recommended when the first-line approach represented by lifestyle modifications (diet and/or physical activity) is not able alone to ameliorate the symptoms and signs of PCOS. The evidence for medical therapy of PCOS is summarized in a recent report from 37 societies and organizations that published the first international evidence-based guideline for the assessment and management of PCOS (<xref rid=\"B94\" ref-type=\"bibr\">94</xref>).</p><p>Combined oral contraceptive pills (COCPs) are first-line pharmacological management for menstrual irregularity and hyperandrogenism, whereas metformin (alone or in addition to COCPs) is recommended for the management of metabolic alterations associated with PCOS (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>, <xref rid=\"B126\" ref-type=\"bibr\">126</xref>). The rational and efficacy of COCPs treatment are linked to their ability to decrease the pulse frequency of GnRH (and therefore the secretion of FSH and LH), the ability of progestin combined to lack of estrogen positive feedback to suppress midcycle LH surge and LH levels (and thus ovarian androgen production), and to the ability of different estrogen-progestin combinations to increase SHBG, thereby reducing bioavailable free androgens. Also, some progestins have antiandrogenic properties, because of their antagonizing effects on the androgen receptor and to the inhibition of 5α-reductase activity.</p><p>Combined oral contraceptive pills exert a class effect on PCOS. However, in order to reduce the thromboembolic risk, the lowest effective estrogen doses (such as 20–30 μg of ethinylestradiol) or the use of natural estradiol should be preferred. Even though clinical trials (<xref rid=\"B127\" ref-type=\"bibr\">127</xref>) almost always use COCPs containing antiandrogen progestins (due to higher effect on hyperandrogenism symptoms), the guidelines do not recommend a specific progestin. A recent meta-analysis showed that COCPs containing cyproterone acetate are more effective in suppressing gonadotropins, leading to a decrease in androgen levels (<xref rid=\"B128\" ref-type=\"bibr\">128</xref>). Another meta-analysis showed that COCPs containing drospirenone have a more potent effect than COCPs containing chlormadinone acetate in the reduction of circulating androgen-related hirsutism (<xref rid=\"B129\" ref-type=\"bibr\">129</xref>). A risk–benefit assessment should take place when selecting a specific COCPs in the individual woman to minimize potential metabolic and thromboembolic consequences. Indeed, third-generation and antiandrogenic progestins are the drugs of choice for hirsutism, alopecia, and acne, but may carry higher risks as compared to the second generation. In combination with COCPs, antiandrogens—considering their potential hepatotoxicity and other side effects—should only be considered in PCOS when androgen-related alopecia and/or hirsutism have to be treated, after 6 months or more failure of COCPs and cosmetic therapy (<xref rid=\"B130\" ref-type=\"bibr\">130</xref>). Antiandrogens must be used with effective contraception, to avoid male fetal undervirilization.</p><p>Metformin, in addition to lifestyle changes, should be recommended in adult women with BMI ≥25 kg/m<sup>2</sup> and considered in adolescents with PCOS for the management of weight (<xref rid=\"B131\" ref-type=\"bibr\">131</xref>) and metabolic abnormalities. It is most beneficial in high metabolic risk groups including patients with T2DM risk factors or impaired glucose tolerance and non-obese IR PCOS women; it should be started at a low dose (with 500-mg increments 1–2 weekly), using extended-release preparations, to minimize gastrointestinal side effects. Conclusive data suggest that metformin alone has benefits for adult women for management of weight, hormonal (testosterone), and metabolic outcomes (fasting glucose and insulin, LDL cholesterol), especially for women with BMI ≥25 kg/m<sup>2</sup>, whereas COCPs are more effective than metformin for menstrual regulation, and metformin combined with COCPs may be useful for the management of metabolic features (<xref rid=\"B95\" ref-type=\"bibr\">95</xref>). Finally, there is reliable evidence regarding the use of metformin for anthropometric (reduction of body weight and decrease of waist) outcomes and COCPs for hyperandrogenism in women with PCOS (<xref rid=\"B132\" ref-type=\"bibr\">132</xref>). In fact, in overweight women with PCOS, metformin plus lifestyle changes might be the best intervention to improve IR and total triglycerides, whereas COCPs plus lifestyle changes appear to be the best intervention for the reduction of total cholesterol and LDL cholesterol, therefore suggesting a combined treatment with metformin and COCPs in these patients (<xref rid=\"B133\" ref-type=\"bibr\">133</xref>).</p><p>A recent meta-analysis (<xref rid=\"B134\" ref-type=\"bibr\">134</xref>) suggested that orlistat, an antiobesity drug that decreases fat absorption from the intestine, is more effective than metformin in weight loss, LDL and cholesterol level, and IR. Another meta-analysis aimed at evaluating the efficacy and safety of glucagon-like peptide 1 (GLP-1) receptor agonists showed that GLP-1 receptor agonists were more effective than metformin in improving insulin sensitivity and reducing BMI, suggesting that GLP-1 receptor agonists might be a good choice for obese patients with PCOS, especially those with IR (<xref rid=\"B135\" ref-type=\"bibr\">135</xref>). However, for both orlistat and GLP-1 receptor agonists, the available evidence is of low quality and therefore inconclusive.</p><sec><title>Expert Opinion</title><list list-type=\"bullet\"><list-item><p>No single unified treatment for PCOS is available. Treatment should be individualized and adapted to the patient. Treatment should aim to improve those symptoms and signs that represent the patient's real needs and can be changed over time.</p></list-item><list-item><p>Targets for pharmacological treatment include biochemical and clinical androgen excess, menstrual irregularities, anovulation, insulin resistance, and metabolic profile.</p></list-item><list-item><p>Lifestyle counseling should be provided in all cases given the deleterious effects of abdominal adiposity and obesity on the cardiometabolic risk profile.</p></list-item><list-item><p>Combined oral contraceptive pills should be used as a first-line treatment for long-term management of menstrual irregularities and hyperandrogenism.</p></list-item><list-item><p>No specific COCPs are recommended. However, those containing the lowest effective doses of estrogens (20–30 μg ethinylestradiol) or preparations with natural estrogens should be preferred.</p></list-item><list-item><p>Combined oral contraceptive pills containing an antiandrogenic progestin are the drugs of choice for hirsutism, alopecia, and acne.</p></list-item><list-item><p>Metformin should be recommended in overweight/obese adult PCOS women and considered in adolescents and lean IR PCOS women for the management of weight, IR, and metabolic abnormalities.</p></list-item></list></sec></sec><sec><title>Resolution 6: Evidence Supporting the Possible Benefits of Nutraceutical Therapy in PCOS</title><p>Several nutraceuticals have been investigated for their possible benefits in the management of PCOS-related IR, anovulation, liver inflammation and hyperhomocysteinemia. They include inositols, α-lipoic acid (ALA), silybin, resveratrol, vitamin D, vitamin E, and folic acid, whose evidences are summarized in the following resolution.</p><p>Myoinositol (MI) and <sc>d</sc>-chiro-inositol (DCI) are both implicated in the modulation of insulin signaling on steroid and ovarian folliculogenesis. In recent years, several studies have shown their effectiveness in patients with PCOS (<xref rid=\"B136\" ref-type=\"bibr\">136</xref>). The choice of the inositol to be prescribed to women with PCOS should be guided as much as possible by an evidence-based background. Both MI and DCI show insulin-mimetic properties and decrease postprandial blood glucose, but display a different peripheral action (<xref rid=\"B137\" ref-type=\"bibr\">137</xref>). Particularly, DCI acts on glycogen synthesis at the level of the skeletal muscle (<xref rid=\"B138\" ref-type=\"bibr\">138</xref>) by up-regulating GLUT4 expression (<xref rid=\"B130\" ref-type=\"bibr\">130</xref>). Myoinositol is involved in glucose uptake and FSH signaling in the ovary, whereas DCI influences the insulin-dependent synthesis of androgens (<xref rid=\"B139\" ref-type=\"bibr\">139</xref>). Epimerase converts MI into DCI in an insulin-sensitive manner and IR dramatically reduces the amount of epimerization (<xref rid=\"B140\" ref-type=\"bibr\">140</xref>) leading to a deficiency of DCI-dependent insulin-sensitive properties in PCOS patients. In human ovaries, ~99% of the intracellular pool of inositol is constituted of MI, and the remaining part of DCI (<xref rid=\"B132\" ref-type=\"bibr\">132</xref>, <xref rid=\"B141\" ref-type=\"bibr\">141</xref>). An imbalance in the ovarian concentration between MI and DCI can compromise the FSH pulsatility rate so that we highlight the importance of keeping unaltered the MI/DCI ratio, rather than restoring only one of the two inositols (<xref rid=\"B142\" ref-type=\"bibr\">142</xref>, <xref rid=\"B143\" ref-type=\"bibr\">143</xref>).</p><p>Recent therapeutic evidence addressed the importance of the association between ALA and MI or DCI. α-Lipoic acid is a powerful antioxidant and enzymatic cofactor of the mitochondrial respiratory chain, in turn, capable of increasing insulin sensitivity. It is believed to directly scavenge ROS and reactive nitrogen species (RNS), both <italic>in vitro</italic> and <italic>in vivo</italic>. α-Lipoic acid regenerates essential antioxidant molecules, that is, coenzyme Q10, vitamin C, vitamin E, and chelates several heavy metals involved in oxidative processes. Also, ALA can repair oxidative stress-damaged proteins, lipids, and DNA (<xref rid=\"B144\" ref-type=\"bibr\">144</xref>). Also, it is supposed to improve glycemic control due to the ALA-induced GLUT-4 expression with subsequent uptake of glucose within tissues (<xref rid=\"B145\" ref-type=\"bibr\">145</xref>). Also, systematic review and meta-analysis suggested that ALA might be able to decrease serum leptin (<xref rid=\"B146\" ref-type=\"bibr\">146</xref>) concentrations especially in younger adults depending on the longer time of assumption; also, a significant increase in serum levels of adiponectin in studies, which lasted for more than 8 weeks (<xref rid=\"B146\" ref-type=\"bibr\">146</xref>). α-Lipoic acid possibly decreases both adipose tissue leptin and circulating leptin mRNA levels through enhanced peroxisome proliferator-activated receptor-γ activity, which has an important role in decreasing leptin gene expression and in determining appropriate insulin signaling (<xref rid=\"B147\" ref-type=\"bibr\">147</xref>). This effect may be considered important given a recent meta-analysis suggesting that ALA decreased body weight among participants with obesity; therefore, it might be possible that ALA by AMPK activation and weight reduction decreases leptin and increases adiponectin levels (<xref rid=\"B146\" ref-type=\"bibr\">146</xref>, <xref rid=\"B148\" ref-type=\"bibr\">148</xref>), thus representing a very promising molecule in reducing some frequent features of PCOS, that is, increased body weight and inflammation (<xref rid=\"B145\" ref-type=\"bibr\">145</xref>).</p><p>Several studies support the use of ALA (<xref rid=\"B149\" ref-type=\"bibr\">149</xref>, <xref rid=\"B150\" ref-type=\"bibr\">150</xref>) for its direct and indirect antioxidant effects through the regeneration of other antioxidants and its anti-inflammatory activities exerted by inhibiting some cytokines. Some authors have shown that the endometrial inflammasome, in which prevails the overexpression/activation of NALP-3, is associated with idiopathic recurrent pregnancy loss (RPL) and that the combined treatment can re-modulate the endometrial pathway of NALP-3 with a decrease of the concentrations of apoptotic cytokines (<xref rid=\"B151\" ref-type=\"bibr\">151</xref>). In a group of PCOS patients, treatment with ALA and DCI led to a non-significant improvement of clinical and metabolic features, that is, insulin, BMI, HDL, and menstrual cyclicity, compared to an untreated control group (<xref rid=\"B152\" ref-type=\"bibr\">152</xref>). Masharani and colleagues highlighted the benefits of ALA on glucose uptake in lean PCOS patients, although this evidence is limited by the poor number of patients treated (<xref rid=\"B153\" ref-type=\"bibr\">153</xref>). In the pilot cohort study by De Cicco and colleagues, 40 patients with normoinsulinemic overweight PCOS were treated for 6 months with the MI plus ALA combination and the results showed a decrease in BMI, waist-hip ratio, hirsutism score, AMH, ovarian volume, and antral follicle count, and an increase in the number of menstrual cycles (from 2 to 5) (<xref rid=\"B154\" ref-type=\"bibr\">154</xref>). Genazzani and colleagues demonstrated that the combined administration of ALA and MI to obese PCOS women, grouped by the presence or absence of familiarity with type 1 or T2DM, led to an improvement of insulin sensitivity in patients with T2DM familiarity and an improvement in the insulin response to OGTT in both groups, unlike what was observed after treatment with MI alone (<xref rid=\"B155\" ref-type=\"bibr\">155</xref>).</p><p>In PCOS women with familiarity for T2DM and therefore with a reduced expression of lipoic acid synthase and epimerase, the combination of ALA plus MI markedly reactivated the stimulus on GLUT4 and therefore was able to improve insulin sensitivity (<xref rid=\"B156\" ref-type=\"bibr\">156</xref>). Also, the treatment with ALA (400 mg/d) improved the metabolic features especially in the presence of T2DM. Numerous evidence show that the treatment with MI and/or DCI improves reproductive outcomes in terms of spontaneous induction of ovulation and leads to a modification of the standard treatments used for ART, that is, a reduction in either the gonadotropin units required for the controlled ovarian hyperstimulation (COH) or in the overall number of days required to reach the maximal stimulation (<xref rid=\"B157\" ref-type=\"bibr\">157</xref>). At the same time, an improvement in oocyte quality and pregnancy rate has been observed both in humans (<xref rid=\"B158\" ref-type=\"bibr\">158</xref>–<xref rid=\"B161\" ref-type=\"bibr\">161</xref>) and in animal models (<xref rid=\"B162\" ref-type=\"bibr\">162</xref>). A randomized trial evaluated the effects of MI alone or in combination with ALA in a population of non-obese PCOS women undergoing ART. Significant reductions in BMI, insulin (baseline and after OGTT), ovarian volume, and gonadotropin units used for COH were observed. The oocyte quality was inversely correlated with the decrease of the BMI, resulting in a greater recovery of M-II oocytes, the formation of classes I and II embryos, and the increase in the pregnancy rate (<xref rid=\"B163\" ref-type=\"bibr\">163</xref>).</p><p>Evidence has shown the role of nutraceuticals in treating NAFLD. Silybin seems effective to treat PCOS-related NAFLD, which is defined as the clustering of triglycerides in macro- or micro-vesicles in more than 5% of hepatocytes. NAFLD is frequently present in PCOS women, with a diagnosis rate of up to 40% in the lean phenotype (<xref rid=\"B164\" ref-type=\"bibr\">164</xref>) and nearly 50% of obese PCOS women (<xref rid=\"B165\" ref-type=\"bibr\">165</xref>). It may develop into cirrhosis and hepatic failure, and therefore, its identification and treatment are of importance in patients with PCOS. A large body of <italic>in vitro</italic> evidence supports the direct benefit of incubation with silybin on hepatocytes in NAFLD models, mainly due to its capacity to reverse lipid accumulation in the liver (<xref rid=\"B166\" ref-type=\"bibr\">166</xref>–<xref rid=\"B168\" ref-type=\"bibr\">168</xref>). A controlled study in 90 NAFLD patients and 30 healthy controls reported the effectiveness of 6-month administration of silybin, vitamin D, and vitamin E on NAFLD fibrosis score, metabolic markers, oxidative stress, and endothelial dysfunction (<xref rid=\"B169\" ref-type=\"bibr\">169</xref>). Finally, the use of the polyphenol resveratrol is associated with the reduction of circulating tryglicerides (<xref rid=\"B170\" ref-type=\"bibr\">170</xref>). Altogether these preliminary reports suggest that the role of nutraceuticals in the treatment of PCOS may be extended to NAFLD, though focused randomized controlled trials (RCTs) need to be accomplished.</p><p>Hyperhomocysteinemia occurs in some patients with PCOS, in particular in those with a greater metabolic derangement (women with IR or hyperinsulinemia) (<xref rid=\"B171\" ref-type=\"bibr\">171</xref>). A genomic study evaluated the effect of the G2706A and G1051A polymorphisms of the <italic>ABCA1</italic> gene on homocysteine serum levels in a cohort of 98 PCOS patients and 93 healthy controls. Significantly higher homocysteine levels were found in PCOS patients with the <italic>ABCA</italic> G1051A mutant genotype than heterozygotes or wild type (<xref rid=\"B172\" ref-type=\"bibr\">172</xref>). Interestingly, among patients with PCOS, homocysteine levels may predict pregnancy outcome, being higher in PCOS patients with recurrent pregnancy loss compared with fertile PCOS controls (<xref rid=\"B173\" ref-type=\"bibr\">173</xref>). Hyperhomocysteinemia predicts the risk of developing atherosclerosis in patients with MetS (<xref rid=\"B174\" ref-type=\"bibr\">174</xref>) and it is a risk factor for ischemic heart disease and stroke (<xref rid=\"B175\" ref-type=\"bibr\">175</xref>). A meta-analysis has shown that folic acid lowers the risk of stroke by 10% and of overall CVD by 4% in patients with hyperomocysteinemia (<xref rid=\"B176\" ref-type=\"bibr\">176</xref>). This evidence justify the assessment of homocysteine and its possible treatment in PCOS patients.</p><p>Diet and healthy lifestyles are universally recognized treatments for obesity and IR before and during pregnancy in PCOS women (<xref rid=\"B177\" ref-type=\"bibr\">177</xref>). Remarkably, in most countries, none of the oral hypoglycemic agents are allowed during the gestational period and insulin remains the only therapeutic option to manage GDM. However, in the last years, in a variety of experimental models, inositol and antioxidant supplementation have shown insulin-sensitizing, anti-inflammatory, and antioxidant properties, providing an important therapeutic opportunity for women with PCOS and GDM (<xref rid=\"B178\" ref-type=\"bibr\">178</xref>, <xref rid=\"B179\" ref-type=\"bibr\">179</xref>). On the other hand, combined inositol treatment improves blood pressure, glucose, and leptin levels in pregnant women with metabolic-like syndrome phenotype (<xref rid=\"B180\" ref-type=\"bibr\">180</xref>). Promising data from RCTs of MI dietary supplementation have shown positive results in terms of ameliorating IR, the incidence of GDM, and its adverse outcomes (<xref rid=\"B181\" ref-type=\"bibr\">181</xref>). Interesting studies report that MI can prevent the development of GDM by improving glucose metabolism in PCOS patients (<xref rid=\"B126\" ref-type=\"bibr\">126</xref>, <xref rid=\"B182\" ref-type=\"bibr\">182</xref>). In this condition, a diet rich in fat-soluble vitamins, fiber, and antioxidants may play a positive role (<xref rid=\"B183\" ref-type=\"bibr\">183</xref>). For example, chronic consumption of quercetin, a flavonoid antioxidant, seems to alleviate fasting and postprandial hyperglycemia in animal models of DM, in part by inhibiting α-glucosidase activity (<xref rid=\"B184\" ref-type=\"bibr\">184</xref>). α-Lipoic acid as a supplemental agent has recently been proposed in T2DM, obesity, and pregnancies complicated by GDM (<xref rid=\"B156\" ref-type=\"bibr\">156</xref>). α-Lipoic acid can decrease either glycemic or inflammation levels especially in dysmetabolic patients with an increased T2DM risk occurrence (<xref rid=\"B144\" ref-type=\"bibr\">144</xref>). A recent meta-analysis indicated that it decreases body weight and leptin levels and increases adiponectin levels in obese participants (<xref rid=\"B146\" ref-type=\"bibr\">146</xref>). Other antioxidants, such as epigallocatechin 3-gallate, vitamin E, and resveratrol, have been suggested to have similar effects (<xref rid=\"B170\" ref-type=\"bibr\">170</xref>). Vitamin D replacement may have some beneficial effects on IR (<xref rid=\"B185\" ref-type=\"bibr\">185</xref>).</p><p>Finally, a meta-analysis of 11 RCTs including 719 pregnant women with GDM recently analyzed the effect of a 4- to 8-week-long probiotic administration on pregnancy outcome, glycemic control, blood lipid profile, inflammation, and oxidative stress. <italic>Lactobacillus</italic> and <italic>Bifidobacterium</italic> were prescribed in almost all RCTs. Probiotic administration resulted in a lower risk of offspring's hyperbilirubinemia. The authors also found the significant improvement of maternal glycemia, lipid profile, inflammation, and oxidative stress (<xref rid=\"B186\" ref-type=\"bibr\">186</xref>). In conclusion, inositols, ALA, vitamins, antioxidant supplementation, and multiple combinations of these compounds, associated with diet and lifestyle modifications, can be an important therapeutic option to manage PCOS and pregnancy-related complications. Evaluation of the safety of these compounds and their combinations concludes that they are safe, and there is no evidence of adverse events both in mothers and fetuses (<xref rid=\"B187\" ref-type=\"bibr\">187</xref>).</p><sec><title>Expert Opinion</title><list list-type=\"bullet\"><list-item><p>Myoinositol and DCI show different insulin-mimetic properties. Inositol administration should be aimed to keep unaltered the MI/DCI ratio.</p></list-item><list-item><p>Treatment with MI/ALA combination may ameliorate hyperinsulinemia, decrease oxidative stress markers at oocyte level, and normalize endometrial inflammasome in PCOS women with idiopathic recurrent pregnancy loss.</p></list-item><list-item><p>The hormonal and clinical profile of overweight/obese women with PCOS may benefit from prolonged use of MI/ALA combination, such as a higher recovery of class II oocytes during ART.</p></list-item><list-item><p>Non-alcoholic fatty liver disease may be associated with PCOS. A timely diagnosis is warranted to avoid the NAFLD-related long-term complications. A nutraceutical approach could be useful in the treatment of NAFLD.</p></list-item><list-item><p>Hyperhomocysteinemia may be associated with selected PCOS patients. Treatment with folic acid should be started to avoid the long-term consequences on the cardiovascular system.</p></list-item><list-item><p>Nutraceuticals, associated with diet and lifestyle modifications, can be important therapeutic option to manage pregnancy-related complications in PCOS pregnant patients.</p></list-item><list-item><p>Gut microbiota integrity is important to prevent pregnancy-related complications in PCOS women.</p></list-item></list></sec></sec></sec><sec sec-type=\"discussion\" id=\"s2\"><title>Discussion</title><p>Polycystic ovary syndrome is a clinically heterogeneous syndrome. Given the wide range of available therapeutic choices, it is important to recognize the exact phenotype (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>) to provide the best evidence-based approach. Fetal exposure to a hyperinsulinemic and hyperandrogenic uterine environment leads to epigenetic changes (<xref rid=\"B82\" ref-type=\"bibr\">82</xref>) that, in addition to the genetic background, confer the susceptibility of developing a metabolic derangement in the offspring (male and female) of PCOS women. This represents the rational basis to look for PCOS early in life, allowing a timely diagnosis already in adolescence. Women with PCOS are exposed to an increased metabolic (and, probably, cardiovascular) risk later in life (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Also, still in the youth, PCOS women have a higher risk of developing GDM compared to their counterparts, and therefore, proper counseling has to be made in those women who wish to become pregnant (<xref rid=\"B97\" ref-type=\"bibr\">97</xref>). Interestingly, the evidence pointed out to the possible existence of a male PCOS equivalent (<xref rid=\"B106\" ref-type=\"bibr\">106</xref>), which, similarly to the classic female phenotype (<xref rid=\"B119\" ref-type=\"bibr\">119</xref>), deserves to be early detected to avoid its long-term cardiometabolic (<xref rid=\"B125\" ref-type=\"bibr\">125</xref>) and, possibly, reproductive complications.</p><p>Several therapeutic choices are currently available for the management of PCOS women. The assessment of anthropometric data, biochemical and clinical androgen excess, menstrual irregularities, oligoanovulation, IR, and metabolic profile are required to adopt the best effective therapeutic strategy (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>). Therapeutic options range from lifestyle changes (<xref rid=\"B48\" ref-type=\"bibr\">48</xref>) and aesthetic interventions to nutraceuticals and, finally, medications. Lifestyle counseling is required for a proper PCOS management in all patients. Although no specific COCP is recommended, this document suggests a prescription of the lowest effective estrogen doses (<xref rid=\"B74\" ref-type=\"bibr\">74</xref>). Those containing neutral/antiandrogenic progestin or antiandrogens should be preferred in the case of hirsutism, alopecia, and acne (<xref rid=\"B130\" ref-type=\"bibr\">130</xref>). Metformin should be recommended in overweight/obese adult PCOS women and considered in adolescents with PCOS for the management of weight, IR, and metabolic abnormalities. Furthermore, metformin may be an option to improve fertility, especially in IR overweight PCOS women.</p><p>Nutraceutical therapy seems to represent a challenge for the treatment of IR-PCOS women, with particular attention to restoring the MI/DCI ratio, which is unbalanced because of the insulin-dependent epimerase dysregulation, especially in obese patients (<xref rid=\"B140\" ref-type=\"bibr\">140</xref>), and possible usefulness of ALA. Many other supplemental elements are also available for the treatment of additional PCOS features, such as NAFLD or hyperomocysteinemia. However, further unbiased RCTs should be warranted to provide evidence-based data on the correct use of nutraceutical and appropriate timing in different phenotypes of PCOS patients.</p><p>This expert panel strongly encourages well-performed RCTs focused on analyzing the uncovered issues related to the application of nutraceuticals in these patients.</p></sec><sec id=\"s3\"><title>Author Contributions</title><p>All authors contributed to conceptualization, methodology, bibliographic search and literature review, original draft preparation, review, editing, and have read and agreed to the published version of the manuscript.</p></sec><sec id=\"s4\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past co-authorship with the authors RR, AC, and AF.</p></sec></body><back><ack><p>Rossella Cannarella (Catania), Rosita A. Condorelli (Catania), Alessandro Dal Lago (Rome), Paolo Facondo (Brescia), Arianna Novellis (Bologna), Letizia Pezzaioli (Brescia), Giulia Stincardini (Pavia), Lara Tiranini (Pavia), Margherita Vergine (Catanzaro).</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> The authors declare that this study received funding from Uriach Srl. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>HF</given-names></name><name><surname>Chen</surname><given-names>HS</given-names></name><name><surname>Rao</surname><given-names>DP</given-names></name><name><surname>Gong</surname><given-names>J</given-names></name></person-group>. <article-title>Association between polycystic ovary syndrome and the risk of pregnancy complications: a PRISMA-compliant systematic review and meta-analysis</article-title>. <source>Medicine.</source> (<year>2016</year>) <volume>95</volume>:<fpage>e4863</fpage>. <pub-id pub-id-type=\"doi\">10.1097/MD.0000000000004863</pub-id><pub-id pub-id-type=\"pmid\">28002314</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Van Hooff</surname><given-names>MH</given-names></name><name><surname>Voorhorst</surname><given-names>FJ</given-names></name><name><surname>Kaptein</surname><given-names>MB</given-names></name><name><surname>Hirasing</surname><given-names>RA</given-names></name><name><surname>Koppenaal</surname><given-names>C</given-names></name><name><surname>Schoemaker</surname><given-names>J</given-names></name></person-group>. <article-title>Predictive value of menstrual cycle pattern, body mass index, hormone levels and polycystic ovaries at age 15 years for oligo-amenorrhoea at age 18 years</article-title>. <source>Hum Reprod.</source> (<year>2004</year>) <volume>19</volume>:<fpage>383</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.1093/humrep/deh079</pub-id><pub-id pub-id-type=\"pmid\">14747186</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Goodman</surname><given-names>NF</given-names></name><name><surname>Cobin</surname><given-names>RH</given-names></name><name><surname>Futterweit</surname><given-names>W</given-names></name><name><surname>Glueck</surname><given-names>JS</given-names></name><name><surname>Legro</surname><given-names>RS</given-names></name><name><surname>Carmina</surname><given-names>E</given-names></name></person-group>\n<article-title>American association of clinical endocrinologists, American college of endocrinology, and androgen excess and PCOS society disease state clinical review: guide to the best practices in the evaluation and treatment of polycystic ovary syndrome Part 2</article-title>. <source>Endocr Pract.</source> (<year>2015</year>) <volume>21</volume>:<fpage>1415</fpage>–<lpage>26</lpage>. <pub-id pub-id-type=\"doi\">10.4158/EP15748.DSCPT2</pub-id><pub-id pub-id-type=\"pmid\">26642102</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Toosy</surname><given-names>S</given-names></name><name><surname>Sodi</surname><given-names>R</given-names></name><name><surname>Pappachan</surname><given-names>JM</given-names></name></person-group>. <article-title>Lean polycystic ovary syndrome (PCOS): an evidence-based practical approach</article-title>. <source>J Diabetes Metab Disord.</source> (<year>2018</year>) <volume>17</volume>:<fpage>277</fpage>–<lpage>85</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s40200-018-0371-5</pub-id><pub-id pub-id-type=\"pmid\">30918863</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nestler</surname><given-names>JE</given-names></name><name><surname>Jakubowicz</surname><given-names>DJ</given-names></name></person-group>. <article-title>Lean women with polycystic ovary syndrome respond to insulin reduction with decreases in ovarian P450c17 alpha activity and serum androgens</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>1997</year>) <volume>82</volume>:<fpage>4075</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.82.12.4075</pub-id><pub-id pub-id-type=\"pmid\">9398716</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Doh</surname><given-names>E</given-names></name><name><surname>Mbanya</surname><given-names>A</given-names></name><name><surname>Kemfang-Ngowa</surname><given-names>JD</given-names></name><name><surname>Dohbit</surname><given-names>S</given-names></name><name><surname>Tchana-Sinou</surname><given-names>M</given-names></name><name><surname>Foumane</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>The relationship between adiposity and insulin sensitivity in african women living with the polycystic ovarian syndrome: a clamp study</article-title>. <source>Int J Endocrinol.</source> (<year>2016</year>) <volume>2016</volume>:<fpage>9201701</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2016/9201701</pub-id><pub-id pub-id-type=\"pmid\">27672393</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sprung</surname><given-names>VS</given-names></name><name><surname>Jones</surname><given-names>H</given-names></name><name><surname>Pugh</surname><given-names>CJ</given-names></name><name><surname>Aziz</surname><given-names>NF</given-names></name><name><surname>Daousi</surname><given-names>C</given-names></name><name><surname>Kemp</surname><given-names>GJ</given-names></name><etal/></person-group>\n<article-title>Endothelial dysfunction in hyperandrogenic polycystic ovary syndrome is not explained by either obesity or ectopic fat deposition</article-title>. <source>Clin Sci.</source> (<year>2014</year>) <volume>126</volume>:<fpage>67</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1042/CS20130186</pub-id><pub-id pub-id-type=\"pmid\">23826984</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Conway</surname><given-names>G</given-names></name><name><surname>Dewailly</surname><given-names>D</given-names></name><name><surname>Diamanti-Kandarakis</surname><given-names>E</given-names></name><name><surname>Escobar-Morreale</surname><given-names>HF</given-names></name><name><surname>Franks</surname><given-names>S</given-names></name><name><surname>Gambineri</surname><given-names>A</given-names></name><etal/></person-group>\n<article-title>ESE PCOS special interest group</article-title>. The polycystic ovary syndrome: a position statement from the European society of endocrinology. <source>Eur J Endocrinol.</source> (<year>2014</year>) <volume>171</volume>:<fpage>P1</fpage>–<lpage>29</lpage>. <pub-id pub-id-type=\"doi\">10.1530/EJE-14-0253</pub-id><pub-id pub-id-type=\"pmid\">24849517</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Usadi</surname><given-names>RS</given-names></name><name><surname>Legro</surname><given-names>RS</given-names></name></person-group>. <article-title>Reproductive impact of polycystic ovary syndrome</article-title>. <source>Curr Opin Endocrinol Diabetes Obes.</source> (<year>2012</year>) <volume>19</volume>:<fpage>5050</fpage>–<lpage>511</lpage>. <pub-id pub-id-type=\"doi\">10.1097/MED.0b013e328359ff92</pub-id><pub-id pub-id-type=\"pmid\">23108198</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fauser</surname><given-names>BC</given-names></name><name><surname>Tarlatzis</surname><given-names>BC</given-names></name><name><surname>Rebar</surname><given-names>RW</given-names></name><name><surname>Lergo</surname><given-names>RS</given-names></name><name><surname>Balen</surname><given-names>AH</given-names></name><name><surname>Lobo</surname><given-names>R</given-names></name><etal/></person-group>. <article-title>Consensus on women's health aspects of polycystic ovary syndrome (PCOS): the amsterdam ESHRE/ASRM-Sponsored 3rd PCOS consensus workshop group</article-title>. <source>Fertil Steril.</source> (<year>2012</year>) <volume>97</volume>:<fpage>28</fpage>–<lpage>38.e25</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fertnstert.2011.09.024</pub-id><pub-id pub-id-type=\"pmid\">22153789</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Palomba</surname><given-names>S</given-names></name><name><surname>Falbo</surname><given-names>A</given-names></name><name><surname>Daolio</surname><given-names>J</given-names></name><name><surname>Battagila</surname><given-names>FA</given-names></name><name><surname>La Sala</surname><given-names>GB</given-names></name></person-group>. <article-title>Pregnancy complications in infertile patients with polycystic ovary syndrome: updated evidence</article-title>. <source>Minerva Ginecol.</source> (<year>2018</year>) <volume>70</volume>:<fpage>754</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.23736/S0026-4784.18.04230-2</pub-id><pub-id pub-id-type=\"pmid\">29644330</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pasquali</surname><given-names>R</given-names></name><name><surname>Gambineri</surname><given-names>A</given-names></name><name><surname>Pagotto</surname><given-names>U</given-names></name></person-group>. <article-title>The impact of obesity on reproduction in women with polycystic ovary syndrome</article-title>. <source>Br J Obstet Gynecol.</source> (<year>2006</year>) <volume>113</volume>:<fpage>1148</fpage>–<lpage>59</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1471-0528.2006.00990.x</pub-id><pub-id pub-id-type=\"pmid\">16827825</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Balen</surname><given-names>AH</given-names></name><name><surname>Conway</surname><given-names>GS</given-names></name><name><surname>Kaltsas</surname><given-names>G</given-names></name><name><surname>Techatrasak</surname><given-names>K</given-names></name><name><surname>Manning</surname><given-names>PJ</given-names></name><name><surname>West</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>Polycystic ovary syndrome: the spectrum of the disorder in 1741 patients</article-title>. <source>Hum Reprod.</source> (<year>1995</year>) <volume>10</volume>:<fpage>2107</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1093/oxfordjournals.humrep.a136243</pub-id><pub-id pub-id-type=\"pmid\">8567849</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pasquali</surname><given-names>R</given-names></name><name><surname>Patton</surname><given-names>L</given-names></name><name><surname>Gambineri</surname><given-names>A</given-names></name></person-group>. <article-title>Obesity and infertility</article-title>. <source>Curr Opin Endocrinol Diabetes Obes.</source> (<year>2007</year>) <volume>14</volume>:<fpage>482</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1097/MED.0b013e3282f1d6cb</pub-id><pub-id pub-id-type=\"pmid\">17982356</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jungheim</surname><given-names>ES</given-names></name><name><surname>Lanzendorf</surname><given-names>SE</given-names></name><name><surname>Odem</surname><given-names>RR</given-names></name><name><surname>Moley</surname><given-names>KH</given-names></name><name><surname>Chang</surname><given-names>AS</given-names></name><name><surname>Ratts</surname><given-names>VS</given-names></name><etal/></person-group>. <article-title>Morbid obesity is associated with lower clinical pregnancy rates after <italic>in vitro</italic> fertilization in women with polycystic ovary syndrome</article-title>. <source>Fertil Steril.</source> (<year>2009</year>) <volume>92</volume>:<fpage>256</fpage>–<lpage>61</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fertnstert.2008.04.063</pub-id><pub-id pub-id-type=\"pmid\">18692801</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gambineri</surname><given-names>A</given-names></name><name><surname>Laudisio</surname><given-names>D</given-names></name><name><surname>Marocco</surname><given-names>C</given-names></name><name><surname>Radellini</surname><given-names>S</given-names></name><name><surname>Colao</surname><given-names>A</given-names></name><name><surname>Savastano</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Obesity programs of nutrition, education, research and assessment (OPERA) group</article-title>. Female infertility: which role for obesity? <source>Int J Obes Suppl.</source> (<year>2019</year>) <volume>9</volume>:<fpage>65</fpage>–<lpage>72</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41367-019-0009-1</pub-id><pub-id pub-id-type=\"pmid\">31391925</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ferreira</surname><given-names>SR</given-names></name><name><surname>Motta</surname><given-names>AB</given-names></name></person-group>. <article-title>Uterine function: from normal to polycystic ovarian syndrome alterations</article-title>. <source>Curr Med Chem.</source> (<year>2018</year>) <volume>25</volume>:<fpage>1792</fpage>–<lpage>804</lpage>. <pub-id pub-id-type=\"doi\">10.2174/0929867325666171205144119</pub-id><pub-id pub-id-type=\"pmid\">29210631</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rosenfield</surname><given-names>RL</given-names></name><name><surname>Ermann</surname><given-names>DA</given-names></name></person-group>. <article-title>The pathogenesis of polycystic ovary syndrome (PCOS): the hypothesis of PCOS as functional ovarian hyperandrogenism reivisted</article-title>. <source>Endocr Rev.</source> (<year>2016</year>) <volume>37</volume>:<fpage>467</fpage>–<lpage>520</lpage>. <pub-id pub-id-type=\"doi\">10.1210/er.2015-1104</pub-id><pub-id pub-id-type=\"pmid\">27459230</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Astapova</surname><given-names>O</given-names></name><name><surname>Minor</surname><given-names>BMN</given-names></name><name><surname>Hammes</surname><given-names>SR</given-names></name></person-group>. <article-title>Physiological and pathological androgen actions in the ovary</article-title>. <source>Endocrinology.</source> (<year>2019</year>) <volume>160</volume>:<fpage>1166</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1210/en.2019-00101</pub-id><pub-id pub-id-type=\"pmid\">30912811</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Popovic</surname><given-names>M</given-names></name><name><surname>Sartorius</surname><given-names>G</given-names></name><name><surname>Christ-Crain</surname><given-names>M</given-names></name></person-group>. <article-title>Chronic low-grade inflammation in polycystic ovary syndrome: is there a (patho)-physiological role for interleukin-1?</article-title>\n<source>Semin Immunopathol.</source> (<year>2019</year>) <volume>41</volume>:<fpage>447</fpage>–<lpage>59</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00281-019-00737-4</pub-id><pub-id pub-id-type=\"pmid\">31139895</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shorakes</surname><given-names>S</given-names></name><name><surname>Teede</surname><given-names>H</given-names></name><name><surname>DeCourten</surname><given-names>B</given-names></name><name><surname>Lambert</surname><given-names>G</given-names></name><name><surname>Boyle</surname><given-names>J</given-names></name><name><surname>Moran</surname><given-names>LJ</given-names></name></person-group>. <article-title>The emerging role of chronic low-grade inflammation in the pathophysiology of polycystic ovary syndrome</article-title>. <source>Semin Reprod Med.</source> (<year>2015</year>) <volume>33</volume>:<fpage>257</fpage>–<lpage>69</lpage>. <pub-id pub-id-type=\"doi\">10.1055/s-0035-1556568</pub-id><pub-id pub-id-type=\"pmid\">26132930</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Repaci</surname><given-names>A</given-names></name><name><surname>Gambineri</surname><given-names>A</given-names></name><name><surname>Pasquali</surname><given-names>R</given-names></name></person-group>. <article-title>The role of low-grade inflammation in the polycystic ovary syndrome</article-title>. <source>Mol Cell Endocrinol.</source> (<year>2011</year>) <volume>335</volume>:<fpage>30</fpage>–<lpage>41</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.mce.2010.08.002</pub-id><pub-id pub-id-type=\"pmid\">20708064</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gambineri</surname><given-names>A</given-names></name><name><surname>Repaci</surname><given-names>A</given-names></name><name><surname>Patton</surname><given-names>L</given-names></name><name><surname>Grassi</surname><given-names>I</given-names></name><name><surname>Pocognoli</surname><given-names>P</given-names></name><name><surname>Cognigni</surname><given-names>GE</given-names></name><etal/></person-group>. <article-title>Prominent role of low HDL-cholesterol in explaining the high prevalence of the metabolic syndrome in polycystic ovary syndrome</article-title>. <source>Nutr Metab Cardiovasc Dis.</source> (<year>2009</year>) <volume>19</volume>:<fpage>797</fpage>–<lpage>804</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.numecd.2009.01.007</pub-id><pub-id pub-id-type=\"pmid\">19359152</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gambineri</surname><given-names>A</given-names></name><name><surname>Patton</surname><given-names>L</given-names></name><name><surname>Altieri</surname><given-names>P</given-names></name><name><surname>Pagotto</surname><given-names>U</given-names></name><name><surname>Pizzi</surname><given-names>C</given-names></name><name><surname>Manzoli</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Polycystic ovary syndrome is a risk factor for type 2 diabetes: results from a long-term prospective study</article-title>. <source>Diabetes.</source> (<year>2012</year>) <volume>61</volume>:<fpage>2369</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.2337/db11-1360</pub-id><pub-id pub-id-type=\"pmid\">22698921</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pasquali</surname><given-names>R</given-names></name><name><surname>Gambineri</surname><given-names>A</given-names></name></person-group>. <article-title>Glucose intolerance states in women with the polycystic ovary syndrome</article-title>. <source>J Endocrinol Invest.</source> (<year>2013</year>) <volume>36</volume>:<fpage>648</fpage>–<lpage>53</lpage>. <pub-id pub-id-type=\"doi\">10.1007/BF03346757</pub-id><pub-id pub-id-type=\"pmid\">24105073</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rocha</surname><given-names>ALL</given-names></name><name><surname>Faria</surname><given-names>LC</given-names></name><name><surname>Guimarães</surname><given-names>TCM</given-names></name><name><surname>Moreira</surname><given-names>GV</given-names></name><name><surname>Cândido</surname><given-names>AL</given-names></name><name><surname>Couto</surname><given-names>CA</given-names></name><etal/></person-group>. <article-title>Non-alcoholic fatty liver disease in women with polycystic ovary syndrome: systematic review and meta-analysis</article-title>. <source>J Endocrinol Invest.</source> (<year>2017</year>) <volume>40</volume>:<fpage>1279</fpage>–<lpage>88</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s40618-017-0708-9</pub-id><pub-id pub-id-type=\"pmid\">28612285</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ojeda-Ojeda</surname><given-names>M</given-names></name><name><surname>Martinez-García</surname><given-names>MÁ</given-names></name><name><surname>Alpanés</surname><given-names>M</given-names></name><name><surname>Luque-Ramírez</surname><given-names>M</given-names></name><name><surname>Escobar-Morreale</surname><given-names>HF</given-names></name></person-group>. <article-title>Association of TLR2 S450S and ICAM1 K469E polymorphisms with polycystic ovary syndrome (PCOS) and obesity</article-title>. <source>J Reprod Immunol.</source> (<year>2016</year>) <volume>113</volume>:<fpage>9</fpage>–<lpage>15</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jri.2015.09.072</pub-id><pub-id pub-id-type=\"pmid\">26498675</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Donath</surname><given-names>MY</given-names></name></person-group>. <article-title>Targeting inflammation in the treatment of type 2 diabetes: time to start</article-title>. <source>Nat Rev Drug Discov.</source> (<year>2014</year>) <volume>13</volume>:<fpage>465</fpage>–<lpage>76</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrd4275</pub-id><pub-id pub-id-type=\"pmid\">24854413</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jager</surname><given-names>J</given-names></name><name><surname>Grémeaux</surname><given-names>T</given-names></name><name><surname>Cormont</surname><given-names>M</given-names></name><name><surname>Le Marchand-Brustel</surname><given-names>Y</given-names></name><name><surname>Tanti</surname><given-names>JF</given-names></name></person-group>. <article-title>Interleukin-1beta-induced insulin resistance in adipocytes through down-regulation of insulin receptor substrate-1 expression</article-title>. <source>Endocrinology.</source> (<year>2007</year>) <volume>148</volume>:<fpage>241</fpage>–<lpage>51</lpage>. <pub-id pub-id-type=\"doi\">10.1210/en.2006-0692</pub-id><pub-id pub-id-type=\"pmid\">17038556</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chaudhari</surname><given-names>AP</given-names></name><name><surname>Mazumdar</surname><given-names>K</given-names></name><name><surname>Mehta</surname><given-names>PD</given-names></name></person-group>. <article-title>Anxiety, depression, and quality of life in women with polycystic ovarian syndrome</article-title>. <source>Indian J Psychol Med.</source> (<year>2018</year>) <volume>40</volume>:<fpage>239</fpage>–<lpage>46</lpage>. <pub-id pub-id-type=\"doi\">10.4103/IJPSYM.IJPSYM_561_17</pub-id><pub-id pub-id-type=\"pmid\">29875531</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tan</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>Q-Y</given-names></name><name><surname>Feng</surname><given-names>G-M</given-names></name><name><surname>Li</surname><given-names>XY</given-names></name><name><surname>Huang</surname><given-names>W</given-names></name></person-group>. <article-title>Increased risk of psychiatric disorders in women with polycystic ovary syndrome in southwest China</article-title>. <source>Chin Med J.</source> (<year>2017</year>) <volume>130</volume>:<fpage>262</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.4103/0366-6999.198916</pub-id><pub-id pub-id-type=\"pmid\">28139507</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ozcan Dag</surname><given-names>Z</given-names></name><name><surname>Alpua</surname><given-names>M</given-names></name><name><surname>Isik</surname><given-names>Y</given-names></name><name><surname>Buturak</surname><given-names>SV</given-names></name><name><surname>Tulmac</surname><given-names>OB</given-names></name><name><surname>Turkel</surname><given-names>Y</given-names></name></person-group>. <article-title>The evaluation of temperament and quality of life in patients with polycystic ovary syndrome</article-title>. <source>Gynecol Endocrinol.</source> (<year>2017</year>) <volume>33</volume>:<fpage>250</fpage>–<lpage>3</lpage>. <pub-id pub-id-type=\"doi\">10.1080/09513590.2016.1254610</pub-id><pub-id pub-id-type=\"pmid\">27908217</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Annagür</surname><given-names>BB</given-names></name><name><surname>Kerimoglu</surname><given-names>ÖS</given-names></name><name><surname>Tazegül</surname><given-names>A</given-names></name><name><surname>Gündüz</surname><given-names>S</given-names></name><name><surname>Gençoglu</surname><given-names>BB</given-names></name></person-group>. <article-title>Psychiatric comorbidity in women with polycystic ovary syndrome</article-title>. <source>J Obstet Gynaecol Res</source>. (<year>2015</year>) <volume>41</volume>:<fpage>1229</fpage>–<lpage>33</lpage>. <pub-id pub-id-type=\"doi\">10.1111/jog.12696</pub-id><pub-id pub-id-type=\"pmid\">25833092</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Deeks</surname><given-names>AA</given-names></name><name><surname>Gibson-Helm</surname><given-names>ME</given-names></name><name><surname>Paul</surname><given-names>E</given-names></name><name><surname>Teede</surname><given-names>HJ</given-names></name></person-group>. <article-title>Is having polycystic ovary syndrome a predictor of poor psychological function including anxiety and depression?</article-title>\n<source>Hum Reprod Oxf Engl.</source> (<year>2011</year>) <volume>26</volume>:<fpage>1399</fpage>–<lpage>407</lpage>. <pub-id pub-id-type=\"doi\">10.1093/humrep/der071</pub-id><pub-id pub-id-type=\"pmid\">21436137</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cinar</surname><given-names>N</given-names></name><name><surname>Kizilarslanoglu</surname><given-names>MC</given-names></name><name><surname>Harmanci</surname><given-names>A</given-names></name><name><surname>Aksoy</surname><given-names>DY</given-names></name><name><surname>Bozdag</surname><given-names>G</given-names></name><name><surname>Demir</surname><given-names>B</given-names></name><etal/></person-group>. <article-title>Depression, anxiety and cardiometabolic risk in polycystic ovary syndrome</article-title>. <source>Hum Reprod.</source> (<year>2011</year>) <volume>26</volume>:<fpage>3339</fpage>–<lpage>45</lpage>. <pub-id pub-id-type=\"doi\">10.1093/humrep/der338</pub-id><pub-id pub-id-type=\"pmid\">21984577</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Scaruffi</surname><given-names>E</given-names></name><name><surname>Gambineri</surname><given-names>A</given-names></name><name><surname>Cattaneo</surname><given-names>S</given-names></name><name><surname>Turra</surname><given-names>J</given-names></name><name><surname>Vettor</surname><given-names>R</given-names></name><name><surname>Mioni</surname><given-names>R</given-names></name></person-group>. <article-title>Personality and psychiatric disorders in women affected by polycystic ovary syndrome</article-title>. <source>Front Endocrinol.</source> (<year>2014</year>) <volume>5</volume>:<fpage>185</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fendo.2014.00185</pub-id><pub-id pub-id-type=\"pmid\">25429283</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Çoban</surname><given-names>ÖG</given-names></name><name><surname>Tulaci</surname><given-names>ÖD</given-names></name><name><surname>Adanir</surname><given-names>AS</given-names></name><name><surname>Önder</surname><given-names>A</given-names></name></person-group>. <article-title>Psychiatric disorders, self-esteem, and quality of life in adolescents with polycystic ovary syndrome</article-title>. <source>J Pediatr Adolesc Gynecol.</source> (<year>2019</year>) <volume>32</volume>:<fpage>600</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jpag.2019.07.008</pub-id><pub-id pub-id-type=\"pmid\">31362114</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stapinska-Syniec</surname><given-names>A</given-names></name><name><surname>Grabowska</surname><given-names>K</given-names></name><name><surname>Szpotanska-Sikorska</surname><given-names>M</given-names></name><name><surname>Pietrzak</surname><given-names>B</given-names></name></person-group>. <article-title>Depression, sexual satisfaction, and other psychological issues in women with polycystic ovary syndrome</article-title>. <source>Gynecol Endocrinol.</source> (<year>2018</year>) <volume>34</volume>:<fpage>597</fpage>–<lpage>600</lpage>. <pub-id pub-id-type=\"doi\">10.1080/09513590.2018.1427713</pub-id><pub-id pub-id-type=\"pmid\">29336189</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shafti</surname><given-names>V</given-names></name><name><surname>Shahbazi</surname><given-names>S</given-names></name></person-group>. <article-title>Comparing sexual function and quality of life in polycystic ovary syndrome and healthy women</article-title>. <source>J Fam Reprod Health.</source> (<year>2016</year>) <volume>10</volume>:<fpage>92</fpage>–<lpage>98</lpage>.<pub-id pub-id-type=\"pmid\">27648099</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Karjula</surname><given-names>S</given-names></name><name><surname>Morin-Papunen</surname><given-names>L</given-names></name><name><surname>Franks</surname><given-names>S</given-names></name><name><surname>Auvinen</surname><given-names>J</given-names></name><name><surname>Järvelin</surname><given-names>MR</given-names></name><name><surname>Tapanainen</surname><given-names>JS</given-names></name><etal/></person-group>. <article-title>Population-based data at ages 31 and 46 show decreased HRQoL and life satisfaction in women with PCOS symptoms</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2020</year>) <volume>105</volume>:<fpage>1814</fpage>–<lpage>26</lpage>. <pub-id pub-id-type=\"doi\">10.1210/clinem/dgz256</pub-id><pub-id pub-id-type=\"pmid\">31970392</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rzonca</surname><given-names>E</given-names></name><name><surname>Bien</surname><given-names>A</given-names></name><name><surname>Wdowiak</surname><given-names>A</given-names></name><name><surname>Szymanski</surname><given-names>R</given-names></name><name><surname>Iwanowicz-Palus</surname><given-names>G</given-names></name></person-group>. <article-title>Determinants of quality of life and satisfaction with life in women with polycystic ovary syndrome</article-title>. <source>Int J Environ Res Public Health.</source> (<year>2018</year>) <volume>15</volume>:<fpage>376</fpage>. <pub-id pub-id-type=\"doi\">10.3390/ijerph15020376</pub-id><pub-id pub-id-type=\"pmid\">29470449</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Panico</surname><given-names>A</given-names></name><name><surname>Messina</surname><given-names>G</given-names></name><name><surname>Lupoli</surname><given-names>GA</given-names></name><name><surname>Lupoli</surname><given-names>R</given-names></name><name><surname>Cacciapuoti</surname><given-names>M</given-names></name><name><surname>Moscatelli</surname><given-names>F</given-names></name><etal/></person-group>. <article-title>Quality of life in overweight (obese) and normal-weight women with polycystic ovary syndrome</article-title>. <source>Patient Prefer Adherence.</source> (<year>2017</year>) <volume>11</volume>:<fpage>423</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.2147/PPA.S119180</pub-id><pub-id pub-id-type=\"pmid\">28280314</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Guidi</surname><given-names>J</given-names></name><name><surname>Gambineri</surname><given-names>A</given-names></name><name><surname>Zanotti</surname><given-names>L</given-names></name><name><surname>Fanelli</surname><given-names>F</given-names></name><name><surname>Fava</surname><given-names>GA</given-names></name><name><surname>Pasquali</surname><given-names>R</given-names></name></person-group>\n<article-title>Psychological aspects of hyperandrogenic states in late adolescent and young women</article-title>. <source>Clin Endocrinol.</source> (<year>2015</year>) <volume>83</volume>:<fpage>872</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1111/cen.12783</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shishehgar</surname><given-names>F</given-names></name><name><surname>Ramezani Tehrani</surname><given-names>F</given-names></name><name><surname>Mirmiran</surname><given-names>P</given-names></name><name><surname>Hajian</surname><given-names>S</given-names></name><name><surname>Baghestani</surname><given-names>AR</given-names></name></person-group>. <article-title>Comparison of the association of excess weight on health related quality of life of women with polycystic ovary syndrome: an age- and bmi-matched case control study</article-title>. <source>PLoS ONE.</source> (<year>2016</year>) <volume>11</volume>:<fpage>e0162911</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0162911</pub-id><pub-id pub-id-type=\"pmid\">27736861</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Coffey</surname><given-names>S</given-names></name><name><surname>Bano</surname><given-names>G</given-names></name><name><surname>Mason</surname><given-names>HD</given-names></name></person-group>. <article-title>Health-related quality of life in women with polycystic ovary syndrome: a comparison with the general population using the polycystic ovary syndrome questionnaire (PCOSQ) and the short form-36 (SF-36)</article-title>. <source>Gynecol Endocrinol.</source> (<year>2006</year>) <volume>22</volume>:<fpage>80</fpage>–<lpage>86</lpage>. <pub-id pub-id-type=\"doi\">10.1080/09513590600604541</pub-id><pub-id pub-id-type=\"pmid\">16603432</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kogure</surname><given-names>GS</given-names></name><name><surname>Ribeiro</surname><given-names>VB</given-names></name><name><surname>Lopes</surname><given-names>IP</given-names></name><name><surname>Furtado</surname><given-names>CLM</given-names></name><name><surname>Kodato</surname><given-names>S</given-names></name><name><surname>Silva de Sá</surname><given-names>MF</given-names></name><etal/></person-group>. <article-title>Body image and its relationships with sexual functioning, anxiety, and depression in women with polycystic ovary syndrome</article-title>. <source>J Affect Disord.</source> (<year>2019</year>) <volume>253</volume>:<fpage>385</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jad.2019.05.006</pub-id><pub-id pub-id-type=\"pmid\">31082731</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Angin</surname><given-names>P</given-names></name><name><surname>Yoldemir</surname><given-names>T</given-names></name><name><surname>Atasayan</surname><given-names>K</given-names></name></person-group>. <article-title>Quality of life among infertile PCOS patients</article-title>. <source>Arch Gynecol Obstet.</source> (<year>2019</year>) <volume>300</volume>:<fpage>461</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00404-019-05202-z</pub-id><pub-id pub-id-type=\"pmid\">31172306</pub-id></mixed-citation></ref><ref id=\"B48\"><label>48.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Behboodi Moghadam</surname><given-names>Z</given-names></name><name><surname>Fereidooni</surname><given-names>B</given-names></name><name><surname>Saffari</surname><given-names>M</given-names></name><name><surname>Montazeri</surname><given-names>A</given-names></name></person-group>. <article-title>Polycystic ovary syndrome and its impact on Iranian women's quality of life: a population-based study</article-title>. <source>BMC Womens Health.</source> (<year>2018</year>) <volume>18</volume>:<fpage>164</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s12905-018-0658-1</pub-id><pub-id pub-id-type=\"pmid\">30305063</pub-id></mixed-citation></ref><ref id=\"B49\"><label>49.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Benetti-Pinto</surname><given-names>CL</given-names></name><name><surname>Ferreira</surname><given-names>SR</given-names></name><name><surname>Antunes</surname><given-names>A</given-names></name><name><surname>Yela</surname><given-names>DA</given-names></name></person-group>. <article-title>The influence of body weight on sexual function and quality of life in women with polycystic ovary syndrome</article-title>. <source>Arch Gynecol Obstet.</source> (<year>2015</year>) <volume>291</volume>:<fpage>451</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00404-014-3423-1</pub-id><pub-id pub-id-type=\"pmid\">25138126</pub-id></mixed-citation></ref><ref id=\"B50\"><label>50.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Asik</surname><given-names>M</given-names></name><name><surname>Altinbas</surname><given-names>K</given-names></name><name><surname>Eroglu</surname><given-names>M</given-names></name><name><surname>Karaahmet</surname><given-names>E</given-names></name><name><surname>Erbag</surname><given-names>G</given-names></name><name><surname>Ertekin</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Evaluation of affective temperament and anxiety-depression levels of patients with polycystic ovary syndrome</article-title>. <source>J Affect Disord.</source> (<year>2015</year>) <volume>185</volume>:<fpage>214</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jad.2015.06.043</pub-id><pub-id pub-id-type=\"pmid\">26241866</pub-id></mixed-citation></ref><ref id=\"B51\"><label>51.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dokras</surname><given-names>A</given-names></name><name><surname>Sarwer</surname><given-names>DB</given-names></name><name><surname>Allison</surname><given-names>KC</given-names></name><name><surname>Milman</surname><given-names>L</given-names></name><name><surname>Kris-Etherton</surname><given-names>PM</given-names></name><name><surname>Kunselman</surname><given-names>AR</given-names></name><etal/></person-group>. <article-title>Weight loss and lowering androgens predict improvements in health-related quality of life in women with PCOS</article-title>. <source>J Clin Endocrinol Metab</source>. (<year>2016</year>) <volume>101</volume>:<fpage>2966</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2016-1896</pub-id><pub-id pub-id-type=\"pmid\">27253669</pub-id></mixed-citation></ref><ref id=\"B52\"><label>52.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Harris-Glocker</surname><given-names>M</given-names></name><name><surname>Davidson</surname><given-names>K</given-names></name><name><surname>Kochman</surname><given-names>L</given-names></name><name><surname>Guzick</surname><given-names>D</given-names></name><name><surname>Hoeger</surname><given-names>K</given-names></name></person-group>\n<article-title>Improvement in quality-of-life questionnaire measures in obese adolescent females with polycystic ovary syndrome treated with lifestyle changes and oral contraceptives, with or without metformin</article-title>. <source>Fertil Steril.</source> (<year>2010</year>) <volume>93</volume>:<fpage>1016</fpage>–<lpage>19</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fertnstert.2009.08.006</pub-id><pub-id pub-id-type=\"pmid\">19781696</pub-id></mixed-citation></ref><ref id=\"B53\"><label>53.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>I</given-names></name><name><surname>Cooney</surname><given-names>LG</given-names></name><name><surname>Saini</surname><given-names>S</given-names></name><name><surname>Smith</surname><given-names>ME</given-names></name><name><surname>Sammel</surname><given-names>MD</given-names></name><name><surname>Allison</surname><given-names>KC</given-names></name><etal/></person-group>. <article-title>Increased risk of disordered eating in polycystic ovary syndrome</article-title>. <source>Fertil Steril.</source> (<year>2017</year>) <volume>107</volume>:<fpage>796</fpage>–<lpage>802</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fertnstert.2016.12.014</pub-id><pub-id pub-id-type=\"pmid\">28104244</pub-id></mixed-citation></ref><ref id=\"B54\"><label>54.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hashemi</surname><given-names>S</given-names></name><name><surname>Ramezani Tehrani</surname><given-names>F</given-names></name><name><surname>Farahmand</surname><given-names>M</given-names></name><name><surname>Bahri Khomami</surname><given-names>M</given-names></name></person-group>. <article-title>Association of PCOS and its clinical signs with sexual function among Iranian women affected by PCOS</article-title>. <source>J Sex Med</source>. (<year>2014</year>) <volume>11</volume>:<fpage>2508</fpage>–<lpage>14</lpage>. <pub-id pub-id-type=\"doi\">10.1111/jsm.12627</pub-id><pub-id pub-id-type=\"pmid\">24995944</pub-id></mixed-citation></ref><ref id=\"B55\"><label>55.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Månsson</surname><given-names>M</given-names></name><name><surname>Norström</surname><given-names>K</given-names></name><name><surname>Holte</surname><given-names>J</given-names></name><name><surname>Landin-Wilhelmsen</surname><given-names>K</given-names></name><name><surname>Dahlgren</surname><given-names>E</given-names></name><name><surname>Landén</surname><given-names>M</given-names></name></person-group>. <article-title>Sexuality and psychological wellbeing in women with polycystic ovary syndrome compared with healthy controls</article-title>. <source>Eur J Obstet Gynecol Reprod Biol.</source> (<year>2011</year>) <volume>155</volume>:<fpage>161</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ejogrb.2010.12.012</pub-id><pub-id pub-id-type=\"pmid\">21232840</pub-id></mixed-citation></ref><ref id=\"B56\"><label>56.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Elsenbruch</surname><given-names>S</given-names></name><name><surname>Hahn</surname><given-names>S</given-names></name><name><surname>Kowalsky</surname><given-names>D</given-names></name><name><surname>Offner</surname><given-names>AH</given-names></name><name><surname>Schedlowski</surname><given-names>M</given-names></name><name><surname>Mann</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Quality of life, psychosocial well-being, and sexual satisfaction in women with polycystic ovary syndrome</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2003</year>) <volume>88</volume>:<fpage>5801</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2003-030562</pub-id><pub-id pub-id-type=\"pmid\">14671172</pub-id></mixed-citation></ref><ref id=\"B57\"><label>57.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Noroozzadeh</surname><given-names>M</given-names></name><name><surname>Tehrani</surname><given-names>FR</given-names></name><name><surname>Mobarakabadi</surname><given-names>SS</given-names></name><name><surname>Farahmand</surname><given-names>M</given-names></name><name><surname>Dovom</surname><given-names>MR</given-names></name></person-group>. <article-title>Sexual function and hormonal profiles in women with and without polycystic ovary syndrome: a population-based study</article-title>. <source>Int J Impot Res.</source> (<year>2017</year>) <volume>29</volume>:<fpage>1</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1038/ijir.2016.35</pub-id><pub-id pub-id-type=\"pmid\">27654035</pub-id></mixed-citation></ref><ref id=\"B58\"><label>58.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kowalczyk</surname><given-names>R</given-names></name><name><surname>Skrzypulec-Plinta</surname><given-names>V</given-names></name><name><surname>Nowosielski</surname><given-names>K</given-names></name></person-group>. <article-title>Sexuality in women with polycystic ovary syndrome</article-title>. <source>Ginekol Pol.</source> (<year>2015</year>) <volume>86</volume>:<fpage>100</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.17772/gp/1995</pub-id><pub-id pub-id-type=\"pmid\">25807833</pub-id></mixed-citation></ref><ref id=\"B59\"><label>59.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zueff</surname><given-names>LN</given-names></name><name><surname>Lara</surname><given-names>LA</given-names></name><name><surname>Vieira</surname><given-names>CS</given-names></name><name><surname>de Paula Martins</surname><given-names>W</given-names></name><name><surname>Ferriani</surname><given-names>RA</given-names></name></person-group>\n<article-title>Body composition characteristics predict sexual functioning in obese women with or without PCOS</article-title>. <source>J Sex Marital Ther</source>. (<year>2015</year>) <volume>41</volume>:<fpage>227</fpage>–<lpage>37</lpage>. <pub-id pub-id-type=\"doi\">10.1080/0092623X.2013.864369</pub-id><pub-id pub-id-type=\"pmid\">24274091</pub-id></mixed-citation></ref><ref id=\"B60\"><label>60.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ferraresi</surname><given-names>SR</given-names></name><name><surname>Lara</surname><given-names>LA</given-names></name><name><surname>Reis</surname><given-names>RM</given-names></name><name><surname>Rosa e Silva</surname><given-names>AC</given-names></name></person-group>. <article-title>Changes in sexual function among women with polycystic ovary syndrome: a pilot study</article-title>. <source>J Sex Med.</source> (<year>2013</year>) <volume>10</volume>:<fpage>467</fpage>–<lpage>73</lpage>. <pub-id pub-id-type=\"doi\">10.1111/jsm.12011</pub-id><pub-id pub-id-type=\"pmid\">23210985</pub-id></mixed-citation></ref><ref id=\"B61\"><label>61.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ercan</surname><given-names>CM</given-names></name><name><surname>Coksuer</surname><given-names>H</given-names></name><name><surname>Aydogan</surname><given-names>U</given-names></name><name><surname>Alanbay</surname><given-names>I</given-names></name><name><surname>Keskin</surname><given-names>U</given-names></name><name><surname>Karasahin</surname><given-names>KE</given-names></name><etal/></person-group>. <article-title>Sexual dysfunction assessment and hormonal correlations in patients with polycystic ovary syndrome</article-title>. <source>Int J Impot Res.</source> (<year>2013</year>) <volume>25</volume>:<fpage>127</fpage>–<lpage>32</lpage>. <pub-id pub-id-type=\"doi\">10.1038/ijir.2013.2</pub-id><pub-id pub-id-type=\"pmid\">23407282</pub-id></mixed-citation></ref><ref id=\"B62\"><label>62.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stovall</surname><given-names>DW</given-names></name><name><surname>Scriver</surname><given-names>JL</given-names></name><name><surname>Clayton</surname><given-names>AH</given-names></name><name><surname>Williams</surname><given-names>CD</given-names></name><name><surname>Pastore</surname><given-names>LM</given-names></name></person-group>. <article-title>Sexual function in women with polycystic ovary syndrome</article-title>. <source>J Sex Med.</source> (<year>2012</year>) <volume>9</volume>:<fpage>224</fpage>–<lpage>30</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1743-6109.2011.02539.x</pub-id><pub-id pub-id-type=\"pmid\">22082203</pub-id></mixed-citation></ref><ref id=\"B63\"><label>63.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Battaglia</surname><given-names>C</given-names></name><name><surname>Nappi</surname><given-names>RE</given-names></name><name><surname>Mancini</surname><given-names>F</given-names></name><name><surname>Cianciosi</surname><given-names>A</given-names></name><name><surname>Persico</surname><given-names>N</given-names></name><name><surname>Busacchi</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>PCOS, sexuality, and clitoral vascularisation: a pilot study</article-title>. <source>J Sex Med.</source> (<year>2008</year>) <volume>5</volume>:<fpage>2886</fpage>–<lpage>94</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1743-6109.2008.01010.x</pub-id><pub-id pub-id-type=\"pmid\">19090942</pub-id></mixed-citation></ref><ref id=\"B64\"><label>64.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Diamond</surname><given-names>MP</given-names></name><name><surname>Legro</surname><given-names>RS</given-names></name><name><surname>Coutifaris</surname><given-names>C</given-names></name><name><surname>Alvero</surname><given-names>R</given-names></name><name><surname>Robinson</surname><given-names>RD</given-names></name><name><surname>Casson</surname><given-names>PA</given-names></name><etal/></person-group>. <article-title>Sexual function in infertile women with polycystic ovary syndrome and unexplained infertility</article-title>. <source>Am J Obstet Gynecol.</source> (<year>2017</year>) <volume>217</volume>:<fpage>191.e1</fpage>–<lpage>191.e19</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ajog.2017.04.034</pub-id><pub-id pub-id-type=\"pmid\">28455078</pub-id></mixed-citation></ref><ref id=\"B65\"><label>65.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eftekhar</surname><given-names>T</given-names></name><name><surname>Sohrabvand</surname><given-names>F</given-names></name><name><surname>Zabandan</surname><given-names>N</given-names></name><name><surname>Shariat</surname><given-names>M</given-names></name><name><surname>Haghollahi</surname><given-names>F</given-names></name><name><surname>Ghahghaei-Nezamabadi</surname><given-names>A</given-names></name></person-group>. <article-title>Sexual dysfunction in patients with polycystic ovary syndrome and its affected domains</article-title>. <source>Iran J Reprod Med.</source> (<year>2014</year>) <volume>12</volume>:<fpage>539</fpage>–<lpage>46</lpage>.<pub-id pub-id-type=\"pmid\">25408703</pub-id></mixed-citation></ref><ref id=\"B66\"><label>66.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rellini</surname><given-names>AH</given-names></name><name><surname>Stratton</surname><given-names>N</given-names></name><name><surname>Tonani</surname><given-names>S</given-names></name><name><surname>Santamaria</surname><given-names>V</given-names></name><name><surname>Brambilla</surname><given-names>E</given-names></name><name><surname>Nappi</surname><given-names>RE</given-names></name></person-group>. <article-title>Differences in sexual desire between women with clinical versus biochemical signs of hyperandrogenism in polycystic ovarian syndrome</article-title>. <source>Horm Behav.</source> (<year>2013</year>) <volume>63</volume>:<fpage>65</fpage>–<lpage>71</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.yhbeh.2012.10.013</pub-id><pub-id pub-id-type=\"pmid\">23127604</pub-id></mixed-citation></ref><ref id=\"B67\"><label>67.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Frène</surname><given-names>V</given-names></name><name><surname>Verhofstadt</surname><given-names>L</given-names></name><name><surname>Loeys</surname><given-names>T</given-names></name><name><surname>Stuyver</surname><given-names>I</given-names></name><name><surname>Buysse</surname><given-names>A</given-names></name><name><surname>De Sutter</surname><given-names>P</given-names></name></person-group>. <article-title>Sexual and relational satisfaction in couples where the woman has polycystic ovary syndrome: a dyadic analysis</article-title>. <source>Hum Reprod Oxf Engl.</source> (<year>2015</year>) <volume>30</volume>:<fpage>625</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.1093/humrep/deu342</pub-id><pub-id pub-id-type=\"pmid\">25534460</pub-id></mixed-citation></ref><ref id=\"B68\"><label>68.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Christ</surname><given-names>JP</given-names></name><name><surname>Gunning</surname><given-names>MN</given-names></name><name><surname>Meun</surname><given-names>C</given-names></name><name><surname>Eijkemans</surname><given-names>MJC</given-names></name><name><surname>van Rijn</surname><given-names>BB</given-names></name><name><surname>Bonsel</surname><given-names>GJ</given-names></name><etal/></person-group>. <article-title>Pre-conception characteristics predict obstetrical and neonatal outcomes in women with polycystic ovary syndrome</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2019</year>) <volume>104</volume>:<fpage>809</fpage>–<lpage>18</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2018-01787</pub-id><pub-id pub-id-type=\"pmid\">30590587</pub-id></mixed-citation></ref><ref id=\"B69\"><label>69.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Abbott</surname><given-names>DH</given-names></name><name><surname>Barnett</surname><given-names>DK</given-names></name><name><surname>Bruns</surname><given-names>CM</given-names></name><name><surname>Dumesic</surname><given-names>DA</given-names></name></person-group>. <article-title>Androgen excess fetal programming of female reproduction: a developmental aetiology for polycystic ovary syndrome?</article-title>\n<source>Hum Reprod Update.</source> (<year>2005</year>) <volume>11</volume>:<fpage>357</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.1093/humupd/dmi013</pub-id><pub-id pub-id-type=\"pmid\">15941725</pub-id></mixed-citation></ref><ref id=\"B70\"><label>70.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Day</surname><given-names>F</given-names></name><name><surname>Karaderi</surname><given-names>T</given-names></name><name><surname>Jones</surname><given-names>MR</given-names></name><name><surname>Meun</surname><given-names>C</given-names></name><name><surname>He</surname><given-names>C</given-names></name><name><surname>Drong</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Large-scale genome-wide meta-analysis of polycystic ovary syndrome suggests shared genetic architecture for different diagnosis criteria</article-title>. <source>PLoS Genet</source>. (<year>2019</year>) <volume>15</volume>:<fpage>e1008517</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pgen.1007813</pub-id><pub-id pub-id-type=\"pmid\">31805045</pub-id></mixed-citation></ref><ref id=\"B71\"><label>71.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dumesic</surname><given-names>DA</given-names></name><name><surname>Hoyos</surname><given-names>LR</given-names></name><name><surname>Chazenbalk</surname><given-names>GD</given-names></name><name><surname>Naik</surname><given-names>R</given-names></name><name><surname>Padmanabhan</surname><given-names>V</given-names></name><name><surname>Abbott</surname><given-names>DH</given-names></name></person-group>. <article-title>Mechanisms of intergenerational transmission of polycystic ovary syndrome</article-title>. <source>Reproduction.</source> (<year>2020</year>) <volume>159</volume>:<fpage>R1</fpage>–<lpage>R13</lpage>. <pub-id pub-id-type=\"doi\">10.1530/REP-19-0197</pub-id><pub-id pub-id-type=\"pmid\">31376813</pub-id></mixed-citation></ref><ref id=\"B72\"><label>72.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Filippou</surname><given-names>P</given-names></name><name><surname>Homburg</surname><given-names>R</given-names></name></person-group>. <article-title>Is foetal hyperexposure to androgens a cause of PCOS?</article-title>\n<source>Hum Reprod Update</source>. (<year>2017</year>) <volume>23</volume>:<fpage>421</fpage>–<lpage>32</lpage>. <pub-id pub-id-type=\"doi\">10.1093/humupd/dmx013</pub-id><pub-id pub-id-type=\"pmid\">28531286</pub-id></mixed-citation></ref><ref id=\"B73\"><label>73.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Risal</surname><given-names>S</given-names></name><name><surname>Pei</surname><given-names>Y</given-names></name><name><surname>Lu</surname><given-names>H</given-names></name><name><surname>Manti</surname><given-names>M</given-names></name><name><surname>Fornes</surname><given-names>R</given-names></name><name><surname>Pui</surname><given-names>HP</given-names></name><etal/></person-group>. <article-title>Prenatal androgen exposure and transgenerational susceptibility to polycystic ovary syndrome</article-title>. <source>Nat Med.</source> (<year>2019</year>) <volume>25</volume>:<fpage>1894</fpage>–<lpage>904</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41591-019-0666-1</pub-id><pub-id pub-id-type=\"pmid\">31792459</pub-id></mixed-citation></ref><ref id=\"B74\"><label>74.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Escobar-Morreale</surname><given-names>HF</given-names></name></person-group>. <article-title>Polycystic ovary syndrome: definition, aetiology, diagnosis and treatment</article-title>. <source>Nat Rev Endocrinol.</source> (<year>2018</year>) <volume>14</volume>:<fpage>270</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrendo.2018.24</pub-id><pub-id pub-id-type=\"pmid\">29569621</pub-id></mixed-citation></ref><ref id=\"B75\"><label>75.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yessoufou</surname><given-names>A</given-names></name><name><surname>Moutairou</surname><given-names>K</given-names></name></person-group>. <article-title>Maternal diabetes in pregnancy: early and long-term outcomes on the offspring and the concept of “metabolic memory”</article-title>. <source>Exp Diabetes Res.</source> (<year>2011</year>) <volume>2011</volume>:<fpage>218598</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2011/218598</pub-id><pub-id pub-id-type=\"pmid\">22144985</pub-id></mixed-citation></ref><ref id=\"B76\"><label>76.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gunning</surname><given-names>MN</given-names></name><name><surname>Sir Petermann</surname><given-names>T</given-names></name><name><surname>Crisosto</surname><given-names>N</given-names></name><name><surname>van Rijn</surname><given-names>BB</given-names></name><name><surname>de Wilde</surname><given-names>MA</given-names></name><name><surname>Christ</surname><given-names>JP</given-names></name><etal/></person-group>. <article-title>Cardiometabolic health in offspring of women with PCOS compared to healthy controls: a systematic review and individual participant data meta-analysis</article-title>. <source>Hum Reprod Update.</source> (<year>2020</year>) <volume>26</volume>:<fpage>103</fpage>–<lpage>17</lpage>. <pub-id pub-id-type=\"doi\">10.1093/humupd/dmz036</pub-id><pub-id pub-id-type=\"pmid\">31867675</pub-id></mixed-citation></ref><ref id=\"B77\"><label>77.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Welt</surname><given-names>CK</given-names></name><name><surname>Carmina</surname><given-names>E</given-names></name></person-group>. <article-title>Clinical review: lifecycle of polycystic ovary syndrome (PCOS): from in utero to menopause</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2013</year>) <volume>98</volume>:<fpage>4629</fpage>–<lpage>38</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2013-2375</pub-id><pub-id pub-id-type=\"pmid\">24064685</pub-id></mixed-citation></ref><ref id=\"B78\"><label>78.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bell</surname><given-names>GA</given-names></name><name><surname>Sundaram</surname><given-names>R</given-names></name><name><surname>Mumford</surname><given-names>SL</given-names></name><name><surname>Park</surname><given-names>H</given-names></name><name><surname>Mills</surname><given-names>J</given-names></name><name><surname>Bell</surname><given-names>EM</given-names></name><etal/></person-group>. <article-title>Maternal polycystic ovarian syndrome and early offspring development</article-title>. <source>Hum Reprod.</source> (<year>2018</year>) <volume>33</volume>:<fpage>1307</fpage>–<lpage>15</lpage>. <pub-id pub-id-type=\"doi\">10.1093/humrep/dey087</pub-id><pub-id pub-id-type=\"pmid\">29668891</pub-id></mixed-citation></ref><ref id=\"B79\"><label>79.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dewailly</surname><given-names>D</given-names></name><name><surname>Pigny</surname><given-names>P</given-names></name><name><surname>Soudan</surname><given-names>B</given-names></name><name><surname>Catteau-Jonard</surname><given-names>S</given-names></name><name><surname>Decanter</surname><given-names>C</given-names></name><name><surname>Poncelet</surname><given-names>E</given-names></name><etal/></person-group>. <article-title>Reconciling the definitions of polycystic ovary syndrome: the ovarian follicle number and serum anti-müllerian hormone concentrations aggregate with the markers of hyperandrogenism</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2010</year>) <volume>95</volume>:<fpage>4399</fpage>–<lpage>405</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2010-0334</pub-id><pub-id pub-id-type=\"pmid\">20610596</pub-id></mixed-citation></ref><ref id=\"B80\"><label>80.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sir-Petermann</surname><given-names>T</given-names></name><name><surname>Codner</surname><given-names>E</given-names></name><name><surname>Maliqueo</surname><given-names>M</given-names></name><name><surname>Echiburú</surname><given-names>B</given-names></name><name><surname>Hitschfeld</surname><given-names>C</given-names></name><name><surname>Crisosto</surname><given-names>N</given-names></name><etal/></person-group>. <article-title>Increased anti-Müllerian hormone serum concentrations in prepubertal daughters of women with polycystic ovary syndrome</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2006</year>) <volume>91</volume>:<fpage>3105</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2005-2693</pub-id><pub-id pub-id-type=\"pmid\">16720659</pub-id></mixed-citation></ref><ref id=\"B81\"><label>81.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sir-Petermann</surname><given-names>T</given-names></name><name><surname>Márquez</surname><given-names>L</given-names></name><name><surname>Cárcamo</surname><given-names>M</given-names></name><name><surname>Hitschfeld</surname><given-names>C</given-names></name><name><surname>Codner</surname><given-names>E</given-names></name><name><surname>Maliqueo</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Effects of birth weight on anti-mullerian hormone serum concentrations in infant girls</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2010</year>) <volume>95</volume>:<fpage>903</fpage>–<lpage>10</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2009-1771</pub-id><pub-id pub-id-type=\"pmid\">19934359</pub-id></mixed-citation></ref><ref id=\"B82\"><label>82.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Legro</surname><given-names>RS</given-names></name><name><surname>Roller</surname><given-names>RL</given-names></name><name><surname>Dodson</surname><given-names>WC</given-names></name><name><surname>Stetter</surname><given-names>CM</given-names></name><name><surname>Kunselman</surname><given-names>AR</given-names></name><name><surname>Dunaif</surname><given-names>A</given-names></name></person-group>. <article-title>Associations of birthweight and gestational age with reproductive and metabolic phenotypes in women with polycystic ovarian syndrome and their first-degree relatives</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2010</year>) <volume>95</volume>:<fpage>789</fpage>–<lpage>99</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2009-1849</pub-id><pub-id pub-id-type=\"pmid\">19965924</pub-id></mixed-citation></ref><ref id=\"B83\"><label>83.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Davies</surname><given-names>MJ</given-names></name><name><surname>March</surname><given-names>WA</given-names></name><name><surname>Willson</surname><given-names>KJ</given-names></name><name><surname>Giles</surname><given-names>LC</given-names></name><name><surname>Moore</surname><given-names>VM</given-names></name></person-group>. <article-title>Birthweight and thinness at birth independently predict symptoms of polycystic ovary syndrome in adulthood</article-title>. <source>Hum Reprod.</source> (<year>2012</year>) <volume>27</volume>:<fpage>1475</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.1093/humrep/des027</pub-id><pub-id pub-id-type=\"pmid\">22373955</pub-id></mixed-citation></ref><ref id=\"B84\"><label>84.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vanky</surname><given-names>E</given-names></name><name><surname>Engen Hanem</surname><given-names>LG</given-names></name><name><surname>Abbott</surname><given-names>DH</given-names></name></person-group>. <article-title>Children born to women with polycystic ovary syndrome-short- and long-term impacts on health and development</article-title>. <source>Fertil Steril.</source> (<year>2019</year>) <volume>111</volume>:<fpage>1065</fpage>–<lpage>75</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fertnstert.2019.03.015</pub-id><pub-id pub-id-type=\"pmid\">31056313</pub-id></mixed-citation></ref><ref id=\"B85\"><label>85.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Legro</surname><given-names>RS</given-names></name><name><surname>Kunselman</surname><given-names>AR</given-names></name><name><surname>Stetter</surname><given-names>CM</given-names></name><name><surname>Gnatuk</surname><given-names>CL</given-names></name><name><surname>Estes</surname><given-names>SJ</given-names></name><name><surname>Brindle</surname><given-names>E</given-names></name><etal/></person-group>\n<article-title>Normal pubertal development in daughters of women with PCOS: a controlled study</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2017</year>) <volume>102</volume>:<fpage>122</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2016-2707</pub-id><pub-id pub-id-type=\"pmid\">27778640</pub-id></mixed-citation></ref><ref id=\"B86\"><label>86.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kent</surname><given-names>SC</given-names></name><name><surname>Gnatuk</surname><given-names>CL</given-names></name><name><surname>Kunselman</surname><given-names>AR</given-names></name><name><surname>Demers</surname><given-names>LM</given-names></name><name><surname>Lee</surname><given-names>PA</given-names></name><name><surname>Legro</surname><given-names>RS</given-names></name></person-group>. <article-title>Hyperandrogenism and hyperinsulinism in children of women with polycystic ovary syndrome: a controlled study</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2008</year>) <volume>93</volume>:<fpage>1662</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2007-1958</pub-id><pub-id pub-id-type=\"pmid\">18270257</pub-id></mixed-citation></ref><ref id=\"B87\"><label>87.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Zegher</surname><given-names>F</given-names></name><name><surname>López-Bermejo</surname><given-names>A</given-names></name><name><surname>Ibáñez</surname><given-names>L</given-names></name></person-group>. <article-title>Central obesity, faster maturation, and 'PCOS' in girls</article-title>. <source>Trends Endocrinol Metab.</source> (<year>2018</year>) <volume>29</volume>:<fpage>815</fpage>–<lpage>18</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.tem.2018.09.005</pub-id><pub-id pub-id-type=\"pmid\">30297320</pub-id></mixed-citation></ref><ref id=\"B88\"><label>88.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Torchen</surname><given-names>LC</given-names></name><name><surname>Idkowiak</surname><given-names>J</given-names></name><name><surname>Fogel</surname><given-names>NR</given-names></name><name><surname>O'Neil</surname><given-names>DM</given-names></name><name><surname>Shackleton</surname><given-names>CH</given-names></name><name><surname>Arlt</surname><given-names>W</given-names></name><etal/></person-group>. <article-title>Evidence for increased 5α-reductase activity during early childhood in daughters of women with polycystic ovary syndrome</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2016</year>) <volume>101</volume>:<fpage>2069</fpage>–<lpage>75</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2015-3926</pub-id><pub-id pub-id-type=\"pmid\">26990942</pub-id></mixed-citation></ref><ref id=\"B89\"><label>89.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>C</given-names></name><name><surname>Wei</surname><given-names>K</given-names></name><name><surname>Jiang</surname><given-names>Z</given-names></name></person-group>. <article-title>5α-reductase activity in women with polycystic ovary syndrome: a systematic review and meta-analysis</article-title>. <source>Reprod Biol Endocrinol</source>. (<year>2017</year>) <volume>15</volume>:<fpage>21</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s12958-017-0242-9</pub-id><pub-id pub-id-type=\"pmid\">28347315</pub-id></mixed-citation></ref><ref id=\"B90\"><label>90.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Azziz</surname><given-names>R</given-names></name></person-group>. <article-title>Polycystic ovary syndrome</article-title>. <source>Obstet Gynecol.</source> (<year>2018</year>) <volume>132</volume>:<fpage>321</fpage>–<lpage>36</lpage>. <pub-id pub-id-type=\"doi\">10.1097/AOG.0000000000002698</pub-id><pub-id pub-id-type=\"pmid\">29995717</pub-id></mixed-citation></ref><ref id=\"B91\"><label>91.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ibáñez</surname><given-names>L</given-names></name><name><surname>Oberfield</surname><given-names>SE</given-names></name><name><surname>Witchel</surname><given-names>S</given-names></name><name><surname>Auchus</surname><given-names>RJ</given-names></name><name><surname>Chang</surname><given-names>RJ</given-names></name><name><surname>Codner</surname><given-names>E</given-names></name><etal/></person-group>\n<article-title>An international consortium update: pathophysiology, diagnosis, and treatment of polycystic ovarian syndrome in adolescence</article-title>. <source>Horm Res Paediatr.</source> (<year>2017</year>) <volume>88</volume>:<fpage>371</fpage>–<lpage>95</lpage>. <pub-id pub-id-type=\"doi\">10.1159/000479371</pub-id><pub-id pub-id-type=\"pmid\">29156452</pub-id></mixed-citation></ref><ref id=\"B92\"><label>92.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Witchel</surname><given-names>SF</given-names></name><name><surname>Oberfield</surname><given-names>SE</given-names></name><name><surname>Peña</surname><given-names>AS</given-names></name></person-group>. <article-title>Polycystic ovary syndrome: pathophysiology, presentation, and treatment with emphasis on adolescent girls</article-title>. <source>J Endocr Soc.</source> (<year>2019</year>) <volume>3</volume>:<fpage>1545</fpage>–<lpage>73</lpage>. <pub-id pub-id-type=\"doi\">10.1210/js.2019-00078</pub-id><pub-id pub-id-type=\"pmid\">31384717</pub-id></mixed-citation></ref><ref id=\"B93\"><label>93.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Melnik</surname><given-names>BC</given-names></name></person-group>. <article-title>Acne vulgaris: the metabolic syndrome of the pilosebaceous follicle</article-title>. <source>Clin Dermatol.</source> (<year>2018</year>) <volume>36</volume>:<fpage>29</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.clindermatol.2017.09.006</pub-id><pub-id pub-id-type=\"pmid\">29241749</pub-id></mixed-citation></ref><ref id=\"B94\"><label>94.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Teede</surname><given-names>HJ</given-names></name><name><surname>Misso</surname><given-names>ML</given-names></name><name><surname>Costello</surname><given-names>MF</given-names></name><name><surname>Dokras</surname><given-names>A</given-names></name><name><surname>Laven</surname><given-names>J</given-names></name><name><surname>Moran</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome</article-title>. <source>Hum Reprod</source>. (<year>2019</year>) <volume>33</volume>:<fpage>1602</fpage>–<lpage>18</lpage>. <pub-id pub-id-type=\"doi\">10.1093/humrep/dey256</pub-id><pub-id pub-id-type=\"pmid\">30052961</pub-id></mixed-citation></ref><ref id=\"B95\"><label>95.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kjerulff</surname><given-names>LE</given-names></name><name><surname>Sanchez-Ramos</surname><given-names>L</given-names></name><name><surname>Duffy</surname><given-names>D</given-names></name></person-group>. <article-title>Pregnancy outcomes in women with polycystic ovary syndrome: a metaanalysis</article-title>. <source>Am J Obstet Gynecol.</source> (<year>2011</year>) <volume>204</volume>:<fpage>558.e1</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ajog.2011.03.021</pub-id><pub-id pub-id-type=\"pmid\">21752757</pub-id></mixed-citation></ref><ref id=\"B96\"><label>96.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Katulski</surname><given-names>K</given-names></name><name><surname>Czyzyk</surname><given-names>A</given-names></name><name><surname>Podfigurna-Stopa</surname><given-names>A</given-names></name><name><surname>Genazzani</surname><given-names>AR</given-names></name><name><surname>Meczekalski</surname><given-names>B</given-names></name></person-group>. <article-title>Pregnancy complications in polycystic ovary syndrome patients</article-title>. <source>Gynecol Endocrinol.</source> (<year>2015</year>) <volume>31</volume>:<fpage>87</fpage>–<lpage>91</lpage>. <pub-id pub-id-type=\"doi\">10.3109/09513590.2014.974535</pub-id><pub-id pub-id-type=\"pmid\">25356655</pub-id></mixed-citation></ref><ref id=\"B97\"><label>97.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mustaniemi</surname><given-names>S</given-names></name><name><surname>Vääräsmäki</surname><given-names>M</given-names></name><name><surname>Eriksson</surname><given-names>JG</given-names></name><name><surname>Gissler</surname><given-names>M</given-names></name><name><surname>Laivuori</surname><given-names>H</given-names></name><name><surname>Ijäs</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Polycystic ovary syndrome and risk factors for gestational diabetes</article-title>. <source>Endocr Connect.</source> (<year>2018</year>) <volume>7</volume>:<fpage>859</fpage>–<lpage>69</lpage>. <pub-id pub-id-type=\"doi\">10.1530/EC-18-0076</pub-id><pub-id pub-id-type=\"pmid\">29858213</pub-id></mixed-citation></ref><ref id=\"B98\"><label>98.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>Q</given-names></name><name><surname>Würtz</surname><given-names>P</given-names></name><name><surname>Auro</surname><given-names>K</given-names></name><name><surname>Mäkinen</surname><given-names>V-P</given-names></name><name><surname>Kangas</surname><given-names>AJ</given-names></name><name><surname>Soininen</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Metabolic profiling of pregnancy: cross-sectional and longitudinal evidence</article-title>. <source>BMC Med</source>. (<year>2016</year>) <volume>14</volume>:<fpage>205</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s12916-016-0733-0</pub-id><pub-id pub-id-type=\"pmid\">27955712</pub-id></mixed-citation></ref><ref id=\"B99\"><label>99.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Formoso</surname><given-names>G</given-names></name><name><surname>Baldassarre</surname><given-names>MPA</given-names></name><name><surname>Ginestra</surname><given-names>F</given-names></name><name><surname>Carlucci</surname><given-names>MA</given-names></name><name><surname>Bucci</surname><given-names>I</given-names></name><name><surname>Consoli</surname><given-names>A</given-names></name></person-group>. <article-title>Inositol and antioxidant supplementation: safety and efficacy in pregnancy</article-title>. <source>Diabetes Metab Res Rev.</source> (<year>2019</year>) <volume>35</volume>:<fpage>e3154</fpage>. <pub-id pub-id-type=\"doi\">10.1002/dmrr.3154</pub-id><pub-id pub-id-type=\"pmid\">30889626</pub-id></mixed-citation></ref><ref id=\"B100\"><label>100.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wilcox</surname><given-names>G</given-names></name></person-group>. <article-title>Impact of pregnancy on inborn errors of metabolism</article-title>. <source>Rev Endocr Metab Disord</source>. (<year>2018</year>) <volume>19</volume>:<fpage>13</fpage>–<lpage>33</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s11154-018-9455-2</pub-id><pub-id pub-id-type=\"pmid\">30198059</pub-id></mixed-citation></ref><ref id=\"B101\"><label>101.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vitacolonna</surname><given-names>E</given-names></name><name><surname>Succurro</surname><given-names>E</given-names></name><name><surname>Lapolla</surname><given-names>A</given-names></name><name><surname>Scavini</surname><given-names>M</given-names></name><name><surname>Bonomo</surname><given-names>M</given-names></name><name><surname>Di Cianni</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Guidelines for the screening and diagnosis of gestational diabetes in Italy from 2010 to 2019: critical issues and the potential for improvement</article-title>. <source>Acta Diabetol.</source> (<year>2019</year>) <volume>56</volume>:<fpage>1159</fpage>–<lpage>67</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00592-019-01397-4</pub-id><pub-id pub-id-type=\"pmid\">31396699</pub-id></mixed-citation></ref><ref id=\"B102\"><label>102.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xu</surname><given-names>Y</given-names></name><name><surname>Zhang</surname><given-names>M</given-names></name><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Sun</surname><given-names>Z</given-names></name><name><surname>Ran</surname><given-names>L</given-names></name><name><surname>Ban</surname><given-names>Y</given-names></name><etal/></person-group>. <article-title>Differential intestinal and oral microbiota features associated with gestational diabetes and maternal inflammation</article-title>. <source>Am J Physiol Endocrinol Metab.</source> (<year>2019</year>) <volume>319</volume>:<fpage>E247</fpage>–<lpage>E53</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpendo.00266.2019</pub-id><pub-id pub-id-type=\"pmid\">31891538</pub-id></mixed-citation></ref><ref id=\"B103\"><label>103.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cui</surname><given-names>M</given-names></name><name><surname>Qi</surname><given-names>C</given-names></name><name><surname>Yang</surname><given-names>L</given-names></name><name><surname>Zhang</surname><given-names>M</given-names></name><name><surname>Wang</surname><given-names>H</given-names></name><name><surname>She</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>A pregnancy complication-dependent change in SIgA-targeted microbiota during third trimester</article-title>. <source>Food Funct.</source> (<year>2020</year>) <volume>11</volume>:<fpage>1513</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1039/C9FO02919B</pub-id><pub-id pub-id-type=\"pmid\">31994568</pub-id></mixed-citation></ref><ref id=\"B104\"><label>104.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ponzo</surname><given-names>V</given-names></name><name><surname>Ferrocino</surname><given-names>I</given-names></name><name><surname>Zarovska</surname><given-names>A</given-names></name><name><surname>Amenta</surname><given-names>MB</given-names></name><name><surname>Leone</surname><given-names>F</given-names></name><name><surname>Monzeglio</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>The microbiota composition of the offspring of patients with gestational diabetes mellitus (GDM)</article-title>. <source>PLoS ONE.</source> (<year>2019</year>) <volume>14</volume>:<fpage>e0226545</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0226545</pub-id><pub-id pub-id-type=\"pmid\">31841548</pub-id></mixed-citation></ref><ref id=\"B105\"><label>105.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Calogero</surname><given-names>AE</given-names></name><name><surname>Calabrò</surname><given-names>V</given-names></name><name><surname>Catanuso</surname><given-names>M</given-names></name><name><surname>Condorelli</surname><given-names>RA</given-names></name><name><surname>La Vignera</surname><given-names>S</given-names></name></person-group>. <article-title>Understanding polycystic ovarian syndrome pathogenesis: an updated of its genetic aspects</article-title>. <source>J Endocrinol Invest</source>. (<year>2011</year>) <volume>34</volume>:<fpage>630</fpage>–<lpage>44</lpage>. <pub-id pub-id-type=\"doi\">10.3275/7746</pub-id><pub-id pub-id-type=\"pmid\">21606667</pub-id></mixed-citation></ref><ref id=\"B106\"><label>106.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cannarella</surname><given-names>R</given-names></name><name><surname>Condorelli</surname><given-names>RA</given-names></name><name><surname>Mongioì</surname><given-names>LM</given-names></name><name><surname>La Vignera</surname><given-names>S</given-names></name><name><surname>Calogero</surname><given-names>AE</given-names></name></person-group>. <article-title>Does a male polycystic ovarian syndrome equivalent exist?</article-title>\n<source>J Endocrinol Invest</source>. (<year>2018</year>) <volume>41</volume>:<fpage>49</fpage>–<lpage>57</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s40618-017-0728-5</pub-id><pub-id pub-id-type=\"pmid\">28711970</pub-id></mixed-citation></ref><ref id=\"B107\"><label>107.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Norman</surname><given-names>RJ</given-names></name><name><surname>Masters</surname><given-names>S</given-names></name><name><surname>Hague</surname><given-names>W</given-names></name></person-group>. <article-title>Hyperinsulinemia is common in family members of women with polycystic ovary syndrome</article-title>. <source>Fertil Steril</source>. (<year>1996</year>) <volume>66</volume>:<fpage>942</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0015-0282(16)58687-7</pub-id><pub-id pub-id-type=\"pmid\">8941059</pub-id></mixed-citation></ref><ref id=\"B108\"><label>108.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Benítez</surname><given-names>R</given-names></name><name><surname>Sir-Petermann</surname><given-names>T</given-names></name><name><surname>Palomino</surname><given-names>A</given-names></name><name><surname>Angel</surname><given-names>B</given-names></name><name><surname>Maliqueo</surname><given-names>M</given-names></name><name><surname>Pérez</surname><given-names>F</given-names></name><etal/></person-group>\n<article-title>Prevalence of metabolic disorders among family members of patients with polycystic ovary syndrome</article-title>. <source>Rev Med Chil</source>. (<year>2001</year>) <volume>129</volume>:<fpage>707</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.4067/S0034-98872001000700001</pub-id><pub-id pub-id-type=\"pmid\">11552437</pub-id></mixed-citation></ref><ref id=\"B109\"><label>109.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yilmaz</surname><given-names>M</given-names></name><name><surname>Bukan</surname><given-names>N</given-names></name><name><surname>Ersoy</surname><given-names>R</given-names></name><name><surname>Karakoç</surname><given-names>A</given-names></name><name><surname>Yetkin</surname><given-names>I</given-names></name><name><surname>Ayvaz</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Glucose intolerance, insulin resistance and cardiovascular risk factors in first degree relatives of women with polycystic ovary syndrome</article-title>. <source>Hum Reprod</source>. (<year>2005</year>) <volume>20</volume>:<fpage>2414</fpage>–<lpage>20</lpage>. <pub-id pub-id-type=\"doi\">10.1093/humrep/dei070</pub-id><pub-id pub-id-type=\"pmid\">15890734</pub-id></mixed-citation></ref><ref id=\"B110\"><label>110.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kaushal</surname><given-names>R</given-names></name><name><surname>Parchure</surname><given-names>N</given-names></name><name><surname>Bano</surname><given-names>G</given-names></name><name><surname>Kaski</surname><given-names>JC</given-names></name><name><surname>Nussey</surname><given-names>SS</given-names></name></person-group>. <article-title>Insulin resistance and endothelial dysfunction in the brothers of Indian subcontinent Asian women with polycystic ovaries</article-title>. <source>Clin Endocrinol</source>. (<year>2004</year>) <volume>60</volume>:<fpage>322</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-2265.2004.01981.x</pub-id><pub-id pub-id-type=\"pmid\">15008997</pub-id></mixed-citation></ref><ref id=\"B111\"><label>111.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Legro</surname><given-names>RS</given-names></name><name><surname>Kunselman</surname><given-names>AR</given-names></name><name><surname>Demers</surname><given-names>L</given-names></name><name><surname>Wang</surname><given-names>SC</given-names></name><name><surname>Bentley-Lewis</surname><given-names>R</given-names></name><name><surname>Dunaif</surname><given-names>A</given-names></name></person-group>. <article-title>Elevated dehydroepiandrosterone sulfate levels as the reproductive phenotype in the brothers of women with polycystic ovary syndrome</article-title>. <source>J Clin Endocrinol Metab</source>. (<year>2002</year>) <volume>87</volume>:<fpage>2134</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jcem.87.5.8387</pub-id><pub-id pub-id-type=\"pmid\">11994353</pub-id></mixed-citation></ref><ref id=\"B112\"><label>112.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lenarcik</surname><given-names>A</given-names></name><name><surname>Bidzinska-Speichert</surname><given-names>B</given-names></name><name><surname>Tworowska-Bardzinska</surname><given-names>U</given-names></name><name><surname>Krepuła</surname><given-names>K</given-names></name></person-group>. <article-title>Hormonal abnormalities in first-degree relatives of women with polycystic ovary syndrome (PCOS)</article-title>. <source>Endokrynologia Polska</source>. (<year>2011</year>) <volume>62</volume>:<fpage>129</fpage>–<lpage>33</lpage>.<pub-id pub-id-type=\"pmid\">21528474</pub-id></mixed-citation></ref><ref id=\"B113\"><label>113.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Torchen</surname><given-names>LC</given-names></name><name><surname>Kumar</surname><given-names>A</given-names></name><name><surname>Kalra</surname><given-names>B</given-names></name><name><surname>Savjani</surname><given-names>G</given-names></name><name><surname>Sisk</surname><given-names>R</given-names></name><name><surname>Legro</surname><given-names>RS</given-names></name><etal/></person-group>\n<article-title>Increased antimullarian hormone levels and other reproductive endocrine changes in adult male relatives of women with polycystic ovary syndrome</article-title>. <source>Fertil Steril</source>. (<year>2016</year>) <volume>106</volume>:<fpage>50</fpage>–<lpage>55</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fertnstert.2016.03.029</pub-id><pub-id pub-id-type=\"pmid\">27042970</pub-id></mixed-citation></ref><ref id=\"B114\"><label>114.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>DM</given-names></name><name><surname>Torchen</surname><given-names>LC</given-names></name><name><surname>Sung</surname><given-names>Y</given-names></name><name><surname>Paparodis</surname><given-names>R</given-names></name><name><surname>Legro</surname><given-names>RS</given-names></name><name><surname>Grebe</surname><given-names>SK</given-names></name><etal/></person-group>. <article-title>Evidence for gonadotrophin secretory and steroidogenic abnormalities in brothers of women with polycystic ovary syndrome</article-title>. <source>Hum Reprod</source>. (<year>2014</year>) <volume>29</volume>:<fpage>2764</fpage>–<lpage>72</lpage>. <pub-id pub-id-type=\"doi\">10.1093/humrep/deu282</pub-id><pub-id pub-id-type=\"pmid\">25336708</pub-id></mixed-citation></ref><ref id=\"B115\"><label>115.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hamilton</surname><given-names>JB</given-names></name></person-group>. <article-title>Patterned loss of hair in man: types and incidence</article-title>. <source>Ann NY Acad Sci</source>. (<year>1951</year>) <volume>53</volume>:<fpage>708</fpage>–<lpage>28</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1749-6632.1951.tb31971.x</pub-id><pub-id pub-id-type=\"pmid\">14819896</pub-id></mixed-citation></ref><ref id=\"B116\"><label>116.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Norwood</surname><given-names>OT</given-names></name></person-group>. <article-title>Male pattern baldness: classification and incidence</article-title>. <source>South Med J</source>. (<year>1975</year>) <volume>68</volume>:<fpage>1359</fpage>–<lpage>65</lpage>. <pub-id pub-id-type=\"doi\">10.1097/00007611-197511000-00009</pub-id><pub-id pub-id-type=\"pmid\">1188424</pub-id></mixed-citation></ref><ref id=\"B117\"><label>117.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lunde</surname><given-names>O</given-names></name><name><surname>Magnus</surname><given-names>P</given-names></name><name><surname>Sandvik</surname><given-names>L</given-names></name><name><surname>Høglo</surname><given-names>S</given-names></name></person-group>. <article-title>Familial clustering in the polycystic ovarian syndrome</article-title>. <source>Gynecol Obstet Invest</source>. (<year>1989</year>) <volume>28</volume>:<fpage>23</fpage>–<lpage>30</lpage>. <pub-id pub-id-type=\"doi\">10.1159/000293493</pub-id><pub-id pub-id-type=\"pmid\">2777131</pub-id></mixed-citation></ref><ref id=\"B118\"><label>118.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dusková</surname><given-names>M</given-names></name><name><surname>Cermáková</surname><given-names>I</given-names></name><name><surname>Hill</surname><given-names>M</given-names></name><name><surname>Vanková</surname><given-names>M</given-names></name><name><surname>Sámalíková</surname><given-names>P</given-names></name><name><surname>Stárka</surname><given-names>L</given-names></name></person-group>. <article-title>What may be the markers of the male equivalent of polycystic ovary syndrome?</article-title>\n<source>Physiol Res</source>. (<year>2004</year>) <volume>53</volume>:<fpage>287</fpage>–<lpage>95</lpage>.<pub-id pub-id-type=\"pmid\">15209536</pub-id></mixed-citation></ref><ref id=\"B119\"><label>119.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cannarella</surname><given-names>R</given-names></name><name><surname>La Vignera</surname><given-names>S</given-names></name><name><surname>Condorelli</surname><given-names>RA</given-names></name><name><surname>Calogero</surname><given-names>AE</given-names></name></person-group>. <article-title>Glycolipid and hormonal profiles in young men with early-onset androgenetic alopecia: a meta-analysis</article-title>. <source>Sci Rep</source>. (<year>2017</year>) <volume>7</volume>:<fpage>7801</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-017-08528-3</pub-id><pub-id pub-id-type=\"pmid\">28798373</pub-id></mixed-citation></ref><ref id=\"B120\"><label>120.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lesko</surname><given-names>SM</given-names></name><name><surname>Rosenberg</surname><given-names>L</given-names></name><name><surname>Shapiro</surname><given-names>S</given-names></name></person-group>. <article-title>A case-control study of baldness in relation to myocardial infarction in men</article-title>. <source>JAMA</source>. (<year>1993</year>) <volume>269</volume>:<fpage>998</fpage>–<lpage>1003</lpage>. <pub-id pub-id-type=\"doi\">10.1001/jama.1993.03500080046030</pub-id><pub-id pub-id-type=\"pmid\">8429606</pub-id></mixed-citation></ref><ref id=\"B121\"><label>121.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lotufo</surname><given-names>PA</given-names></name><name><surname>Chae</surname><given-names>CU</given-names></name><name><surname>Ajani</surname><given-names>UA</given-names></name><name><surname>Hennekens</surname><given-names>CH</given-names></name><name><surname>Manson</surname><given-names>JE</given-names></name></person-group>. <article-title>Male pattern baldness and coronary heart disease: the physicians' health study</article-title>. <source>Arch Intern Med</source>. (<year>2000</year>) <volume>160</volume>:<fpage>165</fpage>–<lpage>71</lpage>. <pub-id pub-id-type=\"doi\">10.1001/archinte.160.2.165</pub-id><pub-id pub-id-type=\"pmid\">10647754</pub-id></mixed-citation></ref><ref id=\"B122\"><label>122.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Herrera</surname><given-names>CR</given-names></name><name><surname>D' Agostino</surname><given-names>RB</given-names></name><name><surname>Gerstman</surname><given-names>BB</given-names></name><name><surname>Bosco</surname><given-names>LA</given-names></name><name><surname>Belanger</surname><given-names>AJ</given-names></name></person-group>. <article-title>Baldness and coronary heart disease rates in men from the framingham study</article-title>. <source>Am J Epidemiol</source>. (<year>1995</year>) <volume>142</volume>:<fpage>828</fpage>–<lpage>33</lpage>. <pub-id pub-id-type=\"doi\">10.1093/oxfordjournals.aje.a117722</pub-id><pub-id pub-id-type=\"pmid\">7572959</pub-id></mixed-citation></ref><ref id=\"B123\"><label>123.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ford</surname><given-names>ES</given-names></name><name><surname>Freedman</surname><given-names>DS</given-names></name><name><surname>Byers</surname><given-names>T</given-names></name></person-group>\n<article-title>Baldness and ischemic heart disease in a national sample of men</article-title>. <source>Am J Epidemiol</source>. (<year>1996</year>) <volume>143</volume>:<fpage>651</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1093/oxfordjournals.aje.a008797</pub-id><pub-id pub-id-type=\"pmid\">8651226</pub-id></mixed-citation></ref><ref id=\"B124\"><label>124.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Trieu</surname><given-names>N</given-names></name><name><surname>Eslick</surname><given-names>GD</given-names></name></person-group>. <article-title>Alopecia and its association with coronary heart disease and cardiovascular risk factors: a meta-analysis</article-title>. <source>Int J Cardiol</source>. (<year>2014</year>) <volume>176</volume>:<fpage>687</fpage>–<lpage>95</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijcard.2014.07.079</pub-id><pub-id pub-id-type=\"pmid\">25150481</pub-id></mixed-citation></ref><ref id=\"B125\"><label>125.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Su</surname><given-names>LH</given-names></name><name><surname>Chen</surname><given-names>LS</given-names></name><name><surname>Lin</surname><given-names>SC</given-names></name><name><surname>Chen</surname><given-names>HH</given-names></name></person-group>. <article-title>Association of androgenetic alopecia with mortality from diabetes mellitus and heart disease</article-title>. <source>JAMA Dermatol</source>. (<year>2013</year>) <volume>149</volume>:<fpage>601</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1001/jamadermatol.2013.130</pub-id><pub-id pub-id-type=\"pmid\">23677087</pub-id></mixed-citation></ref><ref id=\"B126\"><label>126.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>D' Anna</surname><given-names>R</given-names></name><name><surname>Scilipoti</surname><given-names>A</given-names></name><name><surname>Giordano</surname><given-names>D</given-names></name><name><surname>Caruso</surname><given-names>C</given-names></name><name><surname>Cannata</surname><given-names>ML</given-names></name><name><surname>Interdonato</surname><given-names>ML</given-names></name><etal/></person-group>. <article-title>Myo-Inositol supplementation and onset of gestational diabetes mellitus in pregnant women with a family history of type 2 diabetes: a prospective, randomized, placebo-controlled study</article-title>. <source>Diabetes Care.</source> (<year>2013</year>) <volume>36</volume>:<fpage>854e7</fpage>. <pub-id pub-id-type=\"doi\">10.2337/dc12-1371</pub-id><pub-id pub-id-type=\"pmid\">23340885</pub-id></mixed-citation></ref><ref id=\"B127\"><label>127.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tay</surname><given-names>CT</given-names></name><name><surname>Joham</surname><given-names>AE</given-names></name><name><surname>Hiam</surname><given-names>DS</given-names></name><name><surname>Gadalla</surname><given-names>MA</given-names></name><name><surname>Pundir</surname><given-names>J</given-names></name><name><surname>Thangaratinam</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Pharmacological and surgical treatment of nonreproductive outcomes in polycystic ovary syndrome: an overview of systematic reviews</article-title>. <source>Clin Endocrinol.</source> (<year>2018</year>) <volume>89</volume>:<fpage>535</fpage>–<lpage>53</lpage>. <pub-id pub-id-type=\"doi\">10.1111/cen.13753</pub-id><pub-id pub-id-type=\"pmid\">29846959</pub-id></mixed-citation></ref><ref id=\"B128\"><label>128.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Amiri</surname><given-names>M</given-names></name><name><surname>Ramezani Tehrani</surname><given-names>F</given-names></name><name><surname>Nahidi</surname><given-names>F</given-names></name><name><surname>Kabir</surname><given-names>A</given-names></name><name><surname>Azizi</surname><given-names>F</given-names></name></person-group>. <article-title>Comparing the effects of combined oral contraceptives containing progestins with low androgenic and antiandrogenic activities on the hypothalamic-pituitary-gonadal axis in patients with polycystic ovary syndrome: systematic review and meta-analysis</article-title>. <source>JMIR Res Protoc.</source> (<year>2018</year>) <volume>7</volume>:<fpage>e113</fpage>. <pub-id pub-id-type=\"doi\">10.2196/resprot.9024</pub-id><pub-id pub-id-type=\"pmid\">29695378</pub-id></mixed-citation></ref><ref id=\"B129\"><label>129.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Menshawy</surname><given-names>A</given-names></name><name><surname>Ismail</surname><given-names>A</given-names></name><name><surname>Abdel-Maboud</surname><given-names>M</given-names></name><name><surname>El-Din</surname><given-names>AA</given-names></name><name><surname>Elgebaly</surname><given-names>A</given-names></name><name><surname>Gadelkarim</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Effect of chlormadinone acetate versus drospirenone-containing oral contraceptives on the endocrinal features of women with polycystic ovary syndrome: systematic review and meta-analysis of randomized clinical trials</article-title>. <source>J Gynecol Obstet Hum Reprod.</source> (<year>2019</year>) <volume>48</volume>:<fpage>763</fpage>–<lpage>70</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jogoh.2019.03.025</pub-id><pub-id pub-id-type=\"pmid\">30940512</pub-id></mixed-citation></ref><ref id=\"B130\"><label>130.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gao</surname><given-names>YF</given-names></name><name><surname>Zhang</surname><given-names>MN</given-names></name><name><surname>Wang</surname><given-names>TX</given-names></name><name><surname>Wu</surname><given-names>TC</given-names></name><name><surname>Ai</surname><given-names>RD</given-names></name><name><surname>Zhang</surname><given-names>ZS</given-names></name></person-group>. <article-title>Hypoglycemic effect of D-chiro-inositol in type 2 diabetes mellitus rats through the PI3K/Akt signaling pathway</article-title>. <source>Mol Cell Endocrinol.</source> (<year>2016</year>) <volume>433</volume>:<fpage>26</fpage>–<lpage>34</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.mce.2016.05.013</pub-id><pub-id pub-id-type=\"pmid\">27212205</pub-id></mixed-citation></ref><ref id=\"B131\"><label>131.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Naderpoor</surname><given-names>N</given-names></name><name><surname>Shorakae</surname><given-names>S</given-names></name><name><surname>De Courten</surname><given-names>B</given-names></name><name><surname>Misso</surname><given-names>ML</given-names></name><name><surname>Moran</surname><given-names>LJ</given-names></name><name><surname>Teede</surname><given-names>HJ</given-names></name></person-group>. <article-title>Metformin and lifestyle modification in polycystic ovary syndrome: systematic review and meta-analysis</article-title>. <source>Hum Reprod Update.</source> (<year>2016</year>) <volume>22</volume>:<fpage>408</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1093/humupd/dmv063</pub-id><pub-id pub-id-type=\"pmid\">27118195</pub-id></mixed-citation></ref><ref id=\"B132\"><label>132.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bharti</surname><given-names>K</given-names></name><name><surname>Kalra</surname><given-names>S</given-names></name><name><surname>Sharma</surname><given-names>JB</given-names></name></person-group>. <article-title>The inositols and polycystic ovary syndrome</article-title>. <source>Indian J Endocrinol Metab</source>. (<year>2017</year>) <volume>20</volume>:<fpage>720</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.4103/2230-8210.189231</pub-id><pub-id pub-id-type=\"pmid\">27730087</pub-id></mixed-citation></ref><ref id=\"B133\"><label>133.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>A</given-names></name><name><surname>Mo</surname><given-names>T</given-names></name><name><surname>Li</surname><given-names>Q</given-names></name><name><surname>Shen</surname><given-names>C</given-names></name><name><surname>Liu</surname><given-names>M</given-names></name></person-group>. <article-title>The effectiveness of metformin, oral contraceptives, and lifestyle modification in improving the metabolism of overweight women with polycystic ovary syndrome: a network meta-analysis</article-title>. <source>Endocrine.</source> (<year>2019</year>) <volume>64</volume>:<fpage>220</fpage>–<lpage>32</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s12020-019-01860-w</pub-id><pub-id pub-id-type=\"pmid\">30911997</pub-id></mixed-citation></ref><ref id=\"B134\"><label>134.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Panda</surname><given-names>SR</given-names></name><name><surname>Jain</surname><given-names>M</given-names></name><name><surname>Jain</surname><given-names>S</given-names></name><name><surname>Saxena</surname><given-names>R</given-names></name><name><surname>Hota</surname><given-names>S</given-names></name></person-group>. <article-title>Effect of orlistat versus metformin in various aspects of polycystic ovarian syndrome: a systematic review of randomized control trials</article-title>. <source>J Obstet Gynaecol India.</source> (<year>2018</year>) <volume>68</volume>:<fpage>336</fpage>–<lpage>43</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s13224-018-1140-6</pub-id><pub-id pub-id-type=\"pmid\">30224835</pub-id></mixed-citation></ref><ref id=\"B135\"><label>135.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Han</surname><given-names>Y</given-names></name><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>He</surname><given-names>B</given-names></name></person-group>. <article-title>GLP-1 receptor agonists versus metformin in PCOS: a systematic review and meta-analysis</article-title>. <source>Reprod Biomed Online.</source> (<year>2019</year>) <volume>39</volume>:<fpage>332</fpage>–<lpage>42</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.rbmo.2019.04.017</pub-id><pub-id pub-id-type=\"pmid\">31229399</pub-id></mixed-citation></ref><ref id=\"B136\"><label>136.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Laganà</surname><given-names>AS</given-names></name><name><surname>Rossetti</surname><given-names>P</given-names></name><name><surname>Buscema</surname><given-names>M</given-names></name><name><surname>La Vignera</surname><given-names>S</given-names></name><name><surname>Condorelli</surname><given-names>RA</given-names></name><name><surname>Gullo</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Metabolism and ovarian function in PCOS women: a therapeutic approach with inositols</article-title>. <source>Int J Endocrinol.</source> (<year>2016</year>) <volume>2016</volume>:<fpage>6306410</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2016/6306410</pub-id><pub-id pub-id-type=\"pmid\">27579037</pub-id></mixed-citation></ref><ref id=\"B137\"><label>137.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Croze</surname><given-names>ML</given-names></name><name><surname>Soulage</surname><given-names>CO</given-names></name></person-group>. <article-title>Potential role and therapeutic interests of myo-inositol in metabolic diseases</article-title>. <source>Biochimie.</source> (<year>2013</year>) <volume>95</volume>:<fpage>1811</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.biochi.2013.05.011</pub-id><pub-id pub-id-type=\"pmid\">23764390</pub-id></mixed-citation></ref><ref id=\"B138\"><label>138.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Larner</surname><given-names>J</given-names></name></person-group>. <article-title>D-chiro-inositol-its functional role in insulin action and its deficit in insulin resistance</article-title>. <source>Int J Exp Diabetes Res.</source> (<year>2002</year>) <volume>3</volume>:<fpage>47</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.1080/15604280212528</pub-id><pub-id pub-id-type=\"pmid\">11900279</pub-id></mixed-citation></ref><ref id=\"B139\"><label>139.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nestler</surname><given-names>JE</given-names></name><name><surname>Unfer</surname><given-names>V</given-names></name></person-group>. <article-title>Reflections on inositol(s) for PCOS therapy: steps toward success</article-title>. <source>Gynecol Endocrinol.</source> (<year>2015</year>) <volume>31</volume>:<fpage>501</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.3109/09513590.2015.1054802</pub-id><pub-id pub-id-type=\"pmid\">26177098</pub-id></mixed-citation></ref><ref id=\"B140\"><label>140.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Laganà</surname><given-names>AS</given-names></name><name><surname>Garzon</surname><given-names>S</given-names></name><name><surname>Casarin</surname><given-names>J</given-names></name><name><surname>Franchi</surname><given-names>M</given-names></name><name><surname>Ghezzi</surname><given-names>F</given-names></name></person-group>. <article-title>Inositol in polycystic ovary syndrome: restoring fertility through a pathophysiology-based approach</article-title>. <source>Trends Endocrinol Metab.</source> (<year>2018</year>) <volume>29</volume>:<fpage>768</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.tem.2018.09.001</pub-id><pub-id pub-id-type=\"pmid\">30270194</pub-id></mixed-citation></ref><ref id=\"B141\"><label>141.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Carlomagno</surname><given-names>G</given-names></name><name><surname>Unfer</surname><given-names>V</given-names></name><name><surname>Benvenga</surname><given-names>S</given-names></name><name><surname>Nestler</surname><given-names>JE</given-names></name></person-group>. <article-title>Myo-inositol: with or without</article-title>. <source>Endocrine J.</source> (<year>2016</year>) <volume>62</volume>:<fpage>297</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1507/endocrj.EJ14-0566</pub-id><pub-id pub-id-type=\"pmid\">25739338</pub-id></mixed-citation></ref><ref id=\"B142\"><label>142.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Condorelli</surname><given-names>RA</given-names></name><name><surname>Calogero</surname><given-names>AE</given-names></name><name><surname>Di Mauro</surname><given-names>M</given-names></name><name><surname>Mongioi</surname><given-names>LM</given-names></name><name><surname>Cannarella</surname><given-names>R</given-names></name><name><surname>Rosta</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Androgen excess and metabolic disorders in women with PCOS: beyond the body mass index</article-title>. <source>J Endocrinol Invest.</source> (<year>2018</year>) <volume>41</volume>:<fpage>383</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s40618-017-0762-3</pub-id><pub-id pub-id-type=\"pmid\">28942551</pub-id></mixed-citation></ref><ref id=\"B143\"><label>143.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Condorelli</surname><given-names>RA</given-names></name><name><surname>Calogero</surname><given-names>AE</given-names></name><name><surname>Di Mauro</surname><given-names>M</given-names></name><name><surname>La Vignera</surname><given-names>S</given-names></name></person-group>. <article-title>PCOS and diabetes mellitus: from insulin resistance to altered beta pancreatic function, a link in evolution</article-title>. <source>Gynecol Endocrinol.</source> (<year>2017</year>) <volume>33</volume>:<fpage>665</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1080/09513590.2017.1342240</pub-id><pub-id pub-id-type=\"pmid\">28644709</pub-id></mixed-citation></ref><ref id=\"B144\"><label>144.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rahimlou</surname><given-names>M</given-names></name><name><surname>Asadi</surname><given-names>M</given-names></name><name><surname>Banaei Jahromi</surname><given-names>N</given-names></name><name><surname>Mansoori</surname><given-names>A</given-names></name></person-group>. <article-title>Alpha-lipoic acid (ALA) supplementation effect on glycemic and inflammatory biomarkers: a systematic review and meta-analysis</article-title>. <source>Clin Nutr ESPEN</source>. (<year>2019</year>) <volume>32</volume>:<fpage>16</fpage>–<lpage>28</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.clnesp.2019.03.015</pub-id><pub-id pub-id-type=\"pmid\">31221283</pub-id></mixed-citation></ref><ref id=\"B145\"><label>145.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Padmalayam</surname><given-names>I</given-names></name><name><surname>Hasham</surname><given-names>S</given-names></name><name><surname>Saxena</surname><given-names>U</given-names></name><name><surname>Pillarisetti</surname><given-names>S</given-names></name></person-group>. <article-title>Lipoic acid synthase (LASY): a novel role in inflammation, mitochondrial function, and insulin resistance</article-title>. <source>Diabetes.</source> (<year>2009</year>) <volume>58</volume>:<fpage>600</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.2337/db08-0473</pub-id><pub-id pub-id-type=\"pmid\">19074983</pub-id></mixed-citation></ref><ref id=\"B146\"><label>146.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Namazi</surname><given-names>N</given-names></name><name><surname>Larijani</surname><given-names>B</given-names></name><name><surname>Azadbakht</surname><given-names>L</given-names></name></person-group>. <article-title>Alpha-lipoic acid supplement in obesity treatment: a systematic review and meta-analysis of clinical trials</article-title>. <source>Clin Nutr</source>. (<year>2018</year>) <volume>37</volume>:<fpage>419</fpage>–<lpage>28</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.clnu.2017.06.002</pub-id><pub-id pub-id-type=\"pmid\">28629898</pub-id></mixed-citation></ref><ref id=\"B147\"><label>147.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pershadsingh</surname><given-names>HA</given-names></name></person-group>. <article-title>Alpha-lipoic acid: physiologic mechanisms and indications for the treatment of metabolic syndrome</article-title>. <source>Expert Opin Investig Drugs</source>. (<year>2007</year>) <volume>16</volume>:<fpage>291</fpage>–<lpage>302</lpage>. <pub-id pub-id-type=\"doi\">10.1517/13543784.16.3.291</pub-id><pub-id pub-id-type=\"pmid\">17302524</pub-id></mixed-citation></ref><ref id=\"B148\"><label>148.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haghighatdoost</surname><given-names>F</given-names></name><name><surname>Gholami</surname><given-names>A</given-names></name><name><surname>Hariri</surname><given-names>M</given-names></name></person-group>. <article-title>Alpha-lipoic acid effect on leptin and adiponectin concentrations: a systematic review and meta-analysis of randomized controlled trials</article-title>. <source>Eur J Clin Pharmacol.</source> (<year>2020</year>) <volume>76</volume>:<fpage>649</fpage>–<lpage>57</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00228-020-02844-w</pub-id><pub-id pub-id-type=\"pmid\">32040596</pub-id></mixed-citation></ref><ref id=\"B149\"><label>149.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Konrad</surname><given-names>D</given-names></name></person-group>\n<article-title>Utilization of the insuli-signaling network in the metabolic actions of α-lipoic acid- reduction or oxidation?</article-title>\n<source>Antioxid Redox Sign.</source> (<year>2005</year>) <volume>7</volume>:<fpage>1032</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1089/ars.2005.7.1032</pub-id></mixed-citation></ref><ref id=\"B150\"><label>150.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Golbidi</surname><given-names>S</given-names></name><name><surname>Badran</surname><given-names>M</given-names></name><name><surname>Laher</surname><given-names>I</given-names></name></person-group>. <article-title>Diabetes and alpha lipoic acid</article-title>. <source>Front Pharmacol.</source> (<year>2011</year>) <volume>2</volume>:<fpage>69</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fphar.2011.00069</pub-id><pub-id pub-id-type=\"pmid\">22125537</pub-id></mixed-citation></ref><ref id=\"B151\"><label>151.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Di Nicuolo</surname><given-names>F</given-names></name><name><surname>D' Ippolito</surname><given-names>S</given-names></name><name><surname>Castellani</surname><given-names>R</given-names></name><name><surname>Rossi</surname><given-names>ED</given-names></name><name><surname>Masciullo</surname><given-names>V</given-names></name><name><surname>Specchia</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Effect of alpha-lipoic acid and myoinositol on endometrial inflammasome from recurrent pregnancy loss women</article-title>. <source>Am J Reprod Immunol.</source> (<year>2019</year>) <volume>82</volume>:<fpage>e13153</fpage>. <pub-id pub-id-type=\"doi\">10.1111/aji.13153</pub-id><pub-id pub-id-type=\"pmid\">31148259</pub-id></mixed-citation></ref><ref id=\"B152\"><label>152.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cianci</surname><given-names>A</given-names></name><name><surname>Panella</surname><given-names>M</given-names></name><name><surname>Fichera</surname><given-names>M</given-names></name><name><surname>Falduzzi</surname><given-names>C</given-names></name><name><surname>Bartolo</surname><given-names>M</given-names></name><name><surname>Caruso</surname><given-names>S</given-names></name></person-group>. <article-title>D-chiro-inositol and alpha lipoic acid treatment of metabolic and menses disorders in women with PCOS</article-title>. <source>Gynecol Endocrinol</source>. (<year>2015</year>) <volume>31</volume>:<fpage>483</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.3109/09513590.2015.1014784</pub-id><pub-id pub-id-type=\"pmid\">25893270</pub-id></mixed-citation></ref><ref id=\"B153\"><label>153.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Masharani</surname><given-names>U</given-names></name><name><surname>Gjerde</surname><given-names>C</given-names></name><name><surname>Evans</surname><given-names>JL</given-names></name><name><surname>Youngren</surname><given-names>JF</given-names></name><name><surname>Goldfine</surname><given-names>ID</given-names></name></person-group>. <article-title>Effects of controlled-release alpha lipoic acid in lean, nondiabetic patients with polycystic ovary syndrome</article-title>. <source>J Diabetes Sci Technol.</source> (<year>2010</year>) <volume>4</volume>:<fpage>359</fpage>–<lpage>64</lpage>. <pub-id pub-id-type=\"doi\">10.1177/193229681000400218</pub-id><pub-id pub-id-type=\"pmid\">20307398</pub-id></mixed-citation></ref><ref id=\"B154\"><label>154.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Cicco</surname><given-names>S</given-names></name><name><surname>Immediata</surname><given-names>V</given-names></name><name><surname>Romualdi</surname><given-names>D</given-names></name><name><surname>Policola</surname><given-names>C</given-names></name><name><surname>Tropea</surname><given-names>A</given-names></name><name><surname>Di Florio</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>Myoinositol combined with alpha-lipoic acid may improve the clinical and endocrine features of polycystic ovary syndrome through an insulin-independent action</article-title>. <source>Gynecol Endocrinol.</source> (<year>2017</year>) <volume>33</volume>:<fpage>698</fpage>–<lpage>701</lpage>. <pub-id pub-id-type=\"doi\">10.1080/09513590.2017.1313972</pub-id><pub-id pub-id-type=\"pmid\">28434274</pub-id></mixed-citation></ref><ref id=\"B155\"><label>155.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gennazzani</surname><given-names>AD</given-names></name><name><surname>Despini</surname><given-names>G</given-names></name><name><surname>Santagni</surname><given-names>S</given-names></name><name><surname>Prati</surname><given-names>A</given-names></name><name><surname>Rattighieri</surname><given-names>E</given-names></name><name><surname>Chierchia</surname><given-names>E</given-names></name><etal/></person-group>\n<article-title>Effects of a combination of alpha lipoic acid and myo-inoitol on insulin dynamics in overweight/obese patients with PCOS</article-title>. <source>Endocrinol Metab Synd</source>. (<year>2014</year>) <volume>3</volume>:<fpage>3</fpage>\n<pub-id pub-id-type=\"doi\">10.4172/2161-1017.1000140</pub-id></mixed-citation></ref><ref id=\"B156\"><label>156.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Genazzani</surname><given-names>AD</given-names></name><name><surname>Shefer</surname><given-names>K</given-names></name><name><surname>Della Casa</surname><given-names>D</given-names></name><name><surname>Prati</surname><given-names>A</given-names></name><name><surname>Napolitano</surname><given-names>A</given-names></name><name><surname>Manzo</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Modulatory effects of alpha-lipoic acid (ALA) administration on insulin sensitivity in obese PCOS patients</article-title>. <source>J Endocrinol Invest.</source> (<year>2018</year>) <volume>41</volume>:<fpage>583</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s40618-017-0782-z</pub-id><pub-id pub-id-type=\"pmid\">29090431</pub-id></mixed-citation></ref><ref id=\"B157\"><label>157.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Colazingari</surname><given-names>S</given-names></name><name><surname>Treglia</surname><given-names>M</given-names></name><name><surname>Najjar</surname><given-names>R</given-names></name><name><surname>Bevilacqua</surname><given-names>A</given-names></name></person-group>. <article-title>The combined therapy myo-inositol plus D-chiro-inositol, rather than D-chiro-inositol, is able to improve IVF outcomes: results from a randomized controlled trial</article-title>. <source>Arch Gynecol Obstet.</source> (<year>2013</year>) <volume>288</volume>:<fpage>1405</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00404-013-2855-3</pub-id><pub-id pub-id-type=\"pmid\">23708322</pub-id></mixed-citation></ref><ref id=\"B158\"><label>158.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tang</surname><given-names>T</given-names></name><name><surname>Lord</surname><given-names>JM</given-names></name><name><surname>Norman</surname><given-names>RJ</given-names></name><name><surname>Balen</surname><given-names>AH</given-names></name></person-group>. <article-title>Insulin-sensitising drugs (metformin, rosiglitazone, pioglitazone, D-chiro-inositol) for women with polycystic ovary syndrome, oligo amenorrhoea and subfertility</article-title>. <source>Cochrane Database Syst Rev.</source> (<year>2012</year>) <volume>11</volume>:<fpage>CD003053</fpage>. <pub-id pub-id-type=\"doi\">10.1002/14651858.CD003053.pub5</pub-id><pub-id pub-id-type=\"pmid\">29183107</pub-id></mixed-citation></ref><ref id=\"B159\"><label>159.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Palomba</surname><given-names>S</given-names></name><name><surname>Falbo</surname><given-names>A</given-names></name><name><surname>Carrillo</surname><given-names>L</given-names></name><name><surname>Villani</surname><given-names>MT</given-names></name><name><surname>Orio</surname><given-names>F</given-names></name><name><surname>Russo</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>Metformin reduces risk of ovarian hyperstimulation syndrome in patients with polycystic ovary syndrome during gonadotropin-stimulated <italic>in vitro</italic> fertilization cycles: a randomized, controlled trial</article-title>. <source>Fertil Steril.</source> (<year>2011</year>) <volume>96</volume>:<fpage>1384</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fertnstert.2011.09.020</pub-id><pub-id pub-id-type=\"pmid\">21982727</pub-id></mixed-citation></ref><ref id=\"B160\"><label>160.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Papaleo</surname><given-names>E</given-names></name><name><surname>Unfer</surname><given-names>V</given-names></name><name><surname>Baillergeon</surname><given-names>JP</given-names></name><name><surname>Fusi</surname><given-names>F</given-names></name><name><surname>Occhi</surname><given-names>F</given-names></name><name><surname>de Santis</surname><given-names>L</given-names></name></person-group>. <article-title>Myo-inositol may improve oocyte quality in intracytoplasmatic sperm injection cycles</article-title>. A prospective, controlled, randomized trial. <source>Fertil Steril.</source> (<year>2009</year>) <volume>91</volume>:<fpage>1750</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fertnstert.2008.01.088</pub-id><pub-id pub-id-type=\"pmid\">18462730</pub-id></mixed-citation></ref><ref id=\"B161\"><label>161.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ciotta</surname><given-names>L</given-names></name><name><surname>Stracquadanio</surname><given-names>M</given-names></name><name><surname>Pagano</surname><given-names>I</given-names></name><name><surname>Carbonaro</surname><given-names>A</given-names></name><name><surname>Palumbo</surname><given-names>M</given-names></name><name><surname>Gulino</surname><given-names>F</given-names></name></person-group>. <article-title>Effects of myoinositol supplementation on oocyte's quality in PCOS patients: a double blind trial</article-title>. <source>Eur Rev Med Pharmacol Sci.</source> (<year>2011</year>) <volume>15</volume>:<fpage>509</fpage>–<lpage>14</lpage>.<pub-id pub-id-type=\"pmid\">21744744</pub-id></mixed-citation></ref><ref id=\"B162\"><label>162.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Truong</surname><given-names>T</given-names></name><name><surname>Gardner</surname><given-names>DK</given-names></name></person-group>. <article-title>Antioxidants improve IVF outcome and subsequent embryo development in the mouse</article-title>. <source>Hum Reprod.</source> (<year>2017</year>) <volume>32</volume>:<fpage>2404</fpage>–<lpage>13</lpage>. <pub-id pub-id-type=\"doi\">10.1093/humrep/dex330</pub-id><pub-id pub-id-type=\"pmid\">29136144</pub-id></mixed-citation></ref><ref id=\"B163\"><label>163.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rago</surname><given-names>R</given-names></name><name><surname>Marcucci</surname><given-names>I</given-names></name><name><surname>Leto</surname><given-names>G</given-names></name><name><surname>Caponecchia</surname><given-names>L</given-names></name><name><surname>Salacone</surname><given-names>P</given-names></name><name><surname>Bonanni</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Effect of myo-inositol and alpha-lipoic acid on oocyte quality in polycystic ovary syndrome non-obese women undergoing <italic>in vitro</italic> fertilization: a pilot study</article-title>. <source>J Biol Regul Homeost Agents.</source> (<year>2015</year>) <volume>29</volume>:<fpage>913</fpage>–<lpage>23</lpage>.<pub-id pub-id-type=\"pmid\">26753656</pub-id></mixed-citation></ref><ref id=\"B164\"><label>164.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ciotta</surname><given-names>L</given-names></name><name><surname>Pagano</surname><given-names>I</given-names></name><name><surname>Stracquadanio</surname><given-names>M</given-names></name><name><surname>Formuso</surname><given-names>C</given-names></name></person-group>. <article-title>Polycystic ovarian syndrome incidence in young women with non-alcoholic fatty liver disease</article-title>. <source>Minerva Ginecol.</source> (<year>2011</year>) <volume>63</volume>:<fpage>429</fpage>–<lpage>37</lpage>.<pub-id pub-id-type=\"pmid\">21926952</pub-id></mixed-citation></ref><ref id=\"B165\"><label>165.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cree-Green</surname><given-names>M</given-names></name><name><surname>Bergman</surname><given-names>BC</given-names></name><name><surname>Coe</surname><given-names>GV</given-names></name><name><surname>Newnes</surname><given-names>L</given-names></name><name><surname>Baumgartner</surname><given-names>AD</given-names></name><name><surname>Bacon</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Hepatic steatosis is common in adolescents with obesity and PCOS and relates to <italic>de novo</italic> lipogenesis but not insulin resistance</article-title>. <source>Obesity.</source> (<year>2016</year>) <volume>24</volume>:<fpage>2399</fpage>–<lpage>406</lpage>. <pub-id pub-id-type=\"doi\">10.1002/oby.21651</pub-id><pub-id pub-id-type=\"pmid\">27804265</pub-id></mixed-citation></ref><ref id=\"B166\"><label>166.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Grasselli</surname><given-names>E</given-names></name><name><surname>Baldini</surname><given-names>F</given-names></name><name><surname>Vecchione</surname><given-names>G</given-names></name><name><surname>Oliveira</surname><given-names>PJ</given-names></name><name><surname>Sardão</surname><given-names>VA</given-names></name><name><surname>Voci</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Excess fructose and fatty acids trigger a model of non alcoholic fatty liver disease progression <italic>in vitro</italic>: protective effect of the flavonoid silybin</article-title>. <source>Int J Mol Med.</source> (<year>2019</year>) <volume>44</volume>:<fpage>705</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.3892/ijmm.2019.4234</pub-id><pub-id pub-id-type=\"pmid\">31173180</pub-id></mixed-citation></ref><ref id=\"B167\"><label>167.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lama</surname><given-names>S</given-names></name><name><surname>Vanacore</surname><given-names>D</given-names></name><name><surname>Diano</surname><given-names>N</given-names></name><name><surname>Nicolucci</surname><given-names>C</given-names></name><name><surname>Errico</surname><given-names>S</given-names></name><name><surname>Dallio</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Ameliorative effect of Silybin on bisphenol A induced oxidative stress, cell proliferation and steroid hormones oxidation in HepG2 cell cultures</article-title>. <source>Sci Rep.</source> (<year>2019</year>) <volume>9</volume>:<fpage>3228</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-019-40105-8</pub-id><pub-id pub-id-type=\"pmid\">30824780</pub-id></mixed-citation></ref><ref id=\"B168\"><label>168.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vecchione</surname><given-names>G</given-names></name><name><surname>Grasselli</surname><given-names>E</given-names></name><name><surname>Cioffi</surname><given-names>F</given-names></name><name><surname>Baldini</surname><given-names>F</given-names></name><name><surname>Oliveira</surname><given-names>PJ</given-names></name><name><surname>Sardão</surname><given-names>VA</given-names></name><etal/></person-group>\n<article-title>The nutraceutic Silybin counteracts excess lipid accumulation and ongoing oxidative stress in an <italic>in vitro</italic> model of non-alcoholic fatty liver disease progression</article-title>. <source>Front Nutr.</source> (<year>2017</year>) <volume>4</volume>:<fpage>42</fpage>\n<pub-id pub-id-type=\"doi\">10.3389/fnut.2017.00042</pub-id><pub-id pub-id-type=\"pmid\">28971098</pub-id></mixed-citation></ref><ref id=\"B169\"><label>169.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Federico</surname><given-names>A</given-names></name><name><surname>Dallio</surname><given-names>M</given-names></name><name><surname>Masarone</surname><given-names>M</given-names></name><name><surname>Gravina</surname><given-names>AG</given-names></name><name><surname>Di Sarno</surname><given-names>R</given-names></name><name><surname>Tuccillo</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>Evaluation of the effect derived from silybin with vitamin d and vitamin e administration on clinical, metabolic, endothelial dysfunction, oxidative stress parameters, and serological worsening markers in nonalcoholic fatty liver disease patients</article-title>. <source>Oxid Med Cell Longev.</source> (<year>2019</year>) <volume>2019</volume>:<fpage>8742075</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2019/8742075</pub-id><pub-id pub-id-type=\"pmid\">31737175</pub-id></mixed-citation></ref><ref id=\"B170\"><label>170.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tran</surname><given-names>HT</given-names></name><name><surname>Liong</surname><given-names>S</given-names></name><name><surname>Lim</surname><given-names>R</given-names></name><name><surname>Barker</surname><given-names>G</given-names></name><name><surname>Lappas</surname><given-names>M</given-names></name></person-group>. <article-title>Resveratrol ameliorates the chemical and microbial induction of inflammation and IR in human placenta, adipose tissue and skeletal muscle</article-title>. <source>PLoS ONE.</source> (<year>2017</year>) <volume>12</volume>:<fpage>e0173373</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0173373</pub-id><pub-id pub-id-type=\"pmid\">28278187</pub-id></mixed-citation></ref><ref id=\"B171\"><label>171.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Diwaker</surname><given-names>A</given-names></name><name><surname>Kishore</surname><given-names>D</given-names></name></person-group>. <article-title>Evaluation of plasma homocysteine levels in patients of PCOS</article-title>. <source>J Assoc Physicians India</source>. (<year>2018</year>) <volume>66</volume>:<fpage>17</fpage>–<lpage>20</lpage>.<pub-id pub-id-type=\"pmid\">31317701</pub-id></mixed-citation></ref><ref id=\"B172\"><label>172.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Karadeniz</surname><given-names>M</given-names></name><name><surname>Erdogan</surname><given-names>M</given-names></name><name><surname>Ayhan</surname><given-names>Z</given-names></name><name><surname>Yalcin</surname><given-names>M</given-names></name><name><surname>Olukman</surname><given-names>M</given-names></name><name><surname>Cetinkalp</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Effect Of G2706A and G1051A polymorphisms of the ABCA1 gene on the lipid, oxidative stress and homocystein levels in Turkish patients with polycystic ovary syndrome</article-title>. <source>Lipids Health Dis.</source> (<year>2011</year>) <volume>10</volume>:<fpage>193</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1476-511X-10-193</pub-id><pub-id pub-id-type=\"pmid\">22035022</pub-id></mixed-citation></ref><ref id=\"B173\"><label>173.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Asanidze</surname><given-names>E</given-names></name><name><surname>Kristesashvili</surname><given-names>J</given-names></name><name><surname>Andguladze</surname><given-names>S</given-names></name></person-group>. <article-title>Correlation between levels of homocysteine, anti-müllerian hormone and insulin resistance in PCOS patients with recurrent miscarriage</article-title>. <source>Georgian Med News.</source> (<year>2019</year>) <volume>290</volume>:<fpage>25</fpage>–<lpage>9</lpage>.<pub-id pub-id-type=\"pmid\">31322509</pub-id></mixed-citation></ref><ref id=\"B174\"><label>174.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Piazzolla</surname><given-names>G</given-names></name><name><surname>Candigliota</surname><given-names>M</given-names></name><name><surname>Fanelli</surname><given-names>M</given-names></name><name><surname>Castrovilli</surname><given-names>A</given-names></name><name><surname>Berardi</surname><given-names>E</given-names></name><name><surname>Antonica</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Hyperhomocysteinemia is an independent risk factor of atherosclerosis in patients with metabolic syndrome</article-title>. <source>Diabetol Metab Syndr.</source> (<year>2019</year>) <volume>11</volume>:<fpage>87</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s13098-019-0484-0</pub-id><pub-id pub-id-type=\"pmid\">31673296</pub-id></mixed-citation></ref><ref id=\"B175\"><label>175.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Singh</surname><given-names>Y</given-names></name><name><surname>Samuel</surname><given-names>VP</given-names></name><name><surname>Dahiya</surname><given-names>S</given-names></name><name><surname>Gupta</surname><given-names>G</given-names></name><name><surname>Gillhotra</surname><given-names>R</given-names></name><name><surname>Mishra</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Combinational effect of angiotensin receptor blocker and folic acid therapy on uric acid and creatinine level in hyperhomocysteinemia-associated hypertension</article-title>. <source>Biotechnol Appl Biochem.</source> (<year>2019</year>) <volume>66</volume>:<fpage>715</fpage>–<lpage>19</lpage>. <pub-id pub-id-type=\"doi\">10.1002/bab.1799</pub-id><pub-id pub-id-type=\"pmid\">31314127</pub-id></mixed-citation></ref><ref id=\"B176\"><label>176.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Totzeck</surname><given-names>M</given-names></name><name><surname>Mincu</surname><given-names>RI</given-names></name><name><surname>Rassaf</surname><given-names>T</given-names></name></person-group>. <article-title>Cardiovascular adverse events in patients with cancer treated with bevacizumab: a meta-analysis of more than 20 000 patients</article-title>. <source>J Am Heart Assoc.</source> (<year>2017</year>) <volume>6</volume>:<fpage>e006278</fpage>. <pub-id pub-id-type=\"doi\">10.1161/JAHA.117.006278</pub-id><pub-id pub-id-type=\"pmid\">28862931</pub-id></mixed-citation></ref><ref id=\"B177\"><label>177.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shepherd</surname><given-names>E</given-names></name><name><surname>Gomersall</surname><given-names>JC</given-names></name><name><surname>Tieu</surname><given-names>J</given-names></name><name><surname>Han</surname><given-names>S</given-names></name><name><surname>Crowther</surname><given-names>CA</given-names></name><name><surname>Middleton</surname><given-names>P</given-names></name></person-group>\n<article-title>Combined diet and exercise interventions for preventing gestational diabetes mellitus</article-title>. <source>Cochrane Database Syst Rev.</source> (<year>2017</year>) <volume>2017</volume>:<fpage>CD010443</fpage>\n<pub-id pub-id-type=\"doi\">10.1002/14651858.CD010443.pub3</pub-id></mixed-citation></ref><ref id=\"B178\"><label>178.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morley</surname><given-names>LC</given-names></name><name><surname>Tang</surname><given-names>T</given-names></name><name><surname>Yasmin</surname><given-names>E</given-names></name><name><surname>Norman</surname><given-names>RJ</given-names></name><name><surname>Balen</surname><given-names>AH</given-names></name></person-group>. <article-title>Insulin-sensitising drugs (metformin, rosiglitazone, pioglitazone, D-chiro-inositol) for women with polycystic ovary syndrome, oligo amenorrhoea and subfertility</article-title>. <source>Cochrane Database Syst Rev.</source> (<year>2017</year>) <volume>11</volume>:<fpage>CD003053</fpage>. <pub-id pub-id-type=\"doi\">10.1002/14651858.CD003053.pub6</pub-id><pub-id pub-id-type=\"pmid\">29183107</pub-id></mixed-citation></ref><ref id=\"B179\"><label>179.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Showell</surname><given-names>MG</given-names></name><name><surname>Mackenzie-Proctor</surname><given-names>R</given-names></name><name><surname>Jordan</surname><given-names>V</given-names></name><name><surname>Hodgson</surname><given-names>R</given-names></name><name><surname>Farquhar</surname><given-names>C</given-names></name></person-group>. <article-title>Inositol for subfertile women with polycystic ovary syndrome</article-title>. <source>Cochrane Database Syst Rev.</source> (<year>2018</year>) <volume>2018</volume>:<fpage>CD012378</fpage>. <pub-id pub-id-type=\"doi\">10.1002/14651858.CD012378.pub2</pub-id><pub-id pub-id-type=\"pmid\">30570133</pub-id></mixed-citation></ref><ref id=\"B180\"><label>180.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ferrari</surname><given-names>F</given-names></name><name><surname>Facchinetti</surname><given-names>F</given-names></name><name><surname>Ontiveros</surname><given-names>AE</given-names></name><name><surname>Roberts</surname><given-names>RP</given-names></name><name><surname>Saade</surname><given-names>MM</given-names></name><name><surname>Blackwell</surname><given-names>SC</given-names></name><etal/></person-group>. <article-title>The effect of combined inositol supplementation on maternal metabolic profile in pregnancies complicated by metabolic syndrome and obesity</article-title>. <source>Am J Obstet Gynecol.</source> (<year>2016</year>) <volume>215</volume>:<fpage>503.e1</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ajog.2016.05.038</pub-id><pub-id pub-id-type=\"pmid\">27255472</pub-id></mixed-citation></ref><ref id=\"B181\"><label>181.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Celentano</surname><given-names>C</given-names></name><name><surname>Matarrelli</surname><given-names>B</given-names></name><name><surname>Mattei</surname><given-names>PA</given-names></name><name><surname>Pavone</surname><given-names>G</given-names></name><name><surname>Vitacolonna</surname><given-names>E</given-names></name><name><surname>Liberati</surname><given-names>M</given-names></name></person-group>. <article-title>Myo-inositol supplementation to prevent gestational diabetes mellitus</article-title>. <source>Curr Diab Rep.</source> (<year>2016</year>) <volume>16</volume>:<fpage>30</fpage>. <pub-id pub-id-type=\"doi\">10.1007/s11892-016-0726-6</pub-id><pub-id pub-id-type=\"pmid\">26898405</pub-id></mixed-citation></ref><ref id=\"B182\"><label>182.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>D'Anna</surname><given-names>R</given-names></name><name><surname>Di Benedetto</surname><given-names>V</given-names></name><name><surname>Rizzo</surname><given-names>P</given-names></name><name><surname>Raffone</surname><given-names>E</given-names></name><name><surname>Interdonato</surname><given-names>ML</given-names></name><name><surname>Corrado</surname><given-names>F</given-names></name><etal/></person-group>. <article-title>Myo-inositol may prevent gestational diabetes in PCOS women</article-title>. <source>Gynecol Endocrinol.</source> (<year>2012</year>) <volume>28</volume>:<fpage>440</fpage>–<lpage>2</lpage>. <pub-id pub-id-type=\"doi\">10.3109/09513590.2011.633665</pub-id><pub-id pub-id-type=\"pmid\">22122627</pub-id></mixed-citation></ref><ref id=\"B183\"><label>183.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ponzo</surname><given-names>V</given-names></name><name><surname>Fedele</surname><given-names>D</given-names></name><name><surname>Goitre</surname><given-names>I</given-names></name><name><surname>Leone</surname><given-names>F</given-names></name><name><surname>Lezo</surname><given-names>A</given-names></name><name><surname>Monzeglio</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>Diet-Gut microbiota interactions and gestational diabetes mellitus (GDM)</article-title>. <source>Nutrients.</source> (<year>2019</year>) <volume>11</volume>:<fpage>330</fpage>. <pub-id pub-id-type=\"doi\">10.3390/nu11020330</pub-id><pub-id pub-id-type=\"pmid\">30717458</pub-id></mixed-citation></ref><ref id=\"B184\"><label>184.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kim</surname><given-names>JH</given-names></name><name><surname>Kang</surname><given-names>MJ</given-names></name><name><surname>Choi</surname><given-names>HN</given-names></name><name><surname>Jeong</surname><given-names>SM</given-names></name><name><surname>Lee</surname><given-names>YM</given-names></name><name><surname>Kim</surname><given-names>JI</given-names></name><etal/></person-group>. <article-title>Quercetin attenuates fasting and postprandial hyperglycemia in animal models of diabetes mellitus</article-title>. <source>Nutr Res Pract.</source> (<year>2011</year>) <volume>5</volume>:<fpage>107</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.4162/nrp.2011.5.2.107</pub-id><pub-id pub-id-type=\"pmid\">21556223</pub-id></mixed-citation></ref><ref id=\"B185\"><label>185.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Günalan</surname><given-names>E</given-names></name><name><surname>Yaba</surname><given-names>A</given-names></name><name><surname>Yilmaz</surname><given-names>B</given-names></name></person-group>. <article-title>The effect of nutrient supplementation in the management of polycystic ovary syndrome-associated metabolic dysfunctions: a critical review</article-title>. <source>J Turk Ger Gynecol Assoc.</source> (<year>2018</year>) <volume>19</volume>:<fpage>220</fpage>–<lpage>32</lpage>. <pub-id pub-id-type=\"doi\">10.4274/jtgga.2018.0077</pub-id><pub-id pub-id-type=\"pmid\">30299265</pub-id></mixed-citation></ref><ref id=\"B186\"><label>186.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Ma</surname><given-names>S</given-names></name><name><surname>Wu</surname><given-names>S</given-names></name><name><surname>Guo</surname><given-names>C</given-names></name><name><surname>Long</surname><given-names>S</given-names></name><name><surname>Tan</surname><given-names>H</given-names></name></person-group>. <article-title>Effects of probiotic supplement in pregnant women with gestational diabetes mellitus: a systematic review and meta-analysis of randomized controlled trials</article-title>. <source>J Diabetes Res.</source> (<year>2019</year>) <volume>2019</volume>:<fpage>5364730</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2019/5364730</pub-id><pub-id pub-id-type=\"pmid\">31583250</pub-id></mixed-citation></ref><ref id=\"B187\"><label>187.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Malvasi</surname><given-names>A</given-names></name><name><surname>Kosmas</surname><given-names>I</given-names></name><name><surname>Mynbaev</surname><given-names>OA</given-names></name><name><surname>Sparic</surname><given-names>R</given-names></name><name><surname>Gustapane</surname><given-names>S</given-names></name><name><surname>Guido</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Can trans resveratrol plus d-chiro-inositol and myo-inositol improve maternal metabolic profile in overweight pregnant patients?</article-title>\n<source>Clin Ter.</source> (<year>2017</year>) <volume>168</volume>:<fpage>e240</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.7417/T.2017.2013</pub-id><pub-id pub-id-type=\"pmid\">28703838</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Immunol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Immunol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Immunol.</journal-id><journal-title-group><journal-title>Frontiers in Immunology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-3224</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849642</article-id><article-id pub-id-type=\"pmc\">PMC7431620</article-id><article-id pub-id-type=\"doi\">10.3389/fimmu.2020.01781</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Immunology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Administration of Amyloid Precursor Protein Gene Deleted Mouse ESC-Derived Thymic Epithelial Progenitors Attenuates Alzheimer's Pathology</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Zhao</surname><given-names>Jin</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/942776/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Su</surname><given-names>Min</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/959789/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Lin</surname><given-names>Yujun</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Liu</surname><given-names>Haiyan</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>He</surname><given-names>Zhixu</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Lai</surname><given-names>Laijun</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"aff\" rid=\"aff5\"><sup>5</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/599895/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Guizhou Provincial Key Laboratory for Regenerative Medicine, Tissue Engineering and Stem Cell Research Center, Department of Immunology, School of Basic Medical Sciences, Guizhou Medical University</institution>, <addr-line>Guiyang</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Key Laboratory of Adult Stem Cell Translational Research, Chinese Academy of Medical Sciences</institution>, <addr-line>Guiyang</addr-line>, <country>China</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Allied Health Sciences, University of Connecticut</institution>, <addr-line>Storrs, CT</addr-line>, <country>United States</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Pediatrics, Affiliated Hospital of Zunyi Medical University</institution>, <addr-line>Zunyi</addr-line>, <country>China</country></aff><aff id=\"aff5\"><sup>5</sup><institution>University of Connecticut Stem Cell Institute, University of Connecticut</institution>, <addr-line>Storrs, CT</addr-line>, <country>United States</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Nicolai Stanislas Van Oers, University of Texas Southwestern Medical Center, United States</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Liqi Li, National Institutes of Health (NIH), United States; Dong-Ming Su, University of North Texas Health Science Center, United States</p></fn><corresp id=\"c001\">*Correspondence: Zhixu He <email>hzx@gmc.edu.cn</email></corresp><corresp id=\"c002\">Laijun Lai <email>laijun.lai@uconn.edu</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1781</elocation-id><history><date date-type=\"received\"><day>17</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>03</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Zhao, Su, Lin, Liu, He and Lai.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Zhao, Su, Lin, Liu, He and Lai</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Alzheimer's disease (AD) is a devastating neurodegenerative disorder and the most common cause of dementia in older adults. Although amyloid-beta (Aβ) plaque deposition and chronic neuroinflammation in the central nervous system (CNS) contribute to AD pathology, neither Aβ plaque removal nor anti-inflammatory therapy has shown much clinical success, suggesting that the combinational therapies for the disease-causative factors may be needed for amelioration. Recent data also suggest that systemic immunity in AD should be boosted, rather than suppressed, to drive an immune-dependent cascade needed for Aβ clearance and brain repair. Thymic epithelial cells (TECs) not only play a critical role in supporting T cell development but also mediate the deletion of autoreactive T cells by expressing autoantigens. We have reported that embryonic stem cells (ESCs) can be selectively induced to differentiate into thymic epithelial progenitors (TEPs) <italic>in vitro</italic> that further develop into TECs <italic>in vivo</italic> to support T cell development. We show here that transplantation of mouse ESC (mESC)-TEPs into AD mice reduced cerebral Aβ plaque load and improved cognitive performance, in correlation with an increased number of T cells, enhanced choroid plexus (CP) gateway activity, and increased number of macrophages in the brain. Furthermore, transplantation of the amyloid precursor protein (APP) gene deleted mESC-TEPs (APP<sup>−/−</sup>) results in more effective reduction of AD pathology as compared to wild-type (APP<sup>+/+</sup>) mESC-TEPs. This is associated with the generation of Aβ-specific T cells, which leads to an increase of anti-Aβ antibody (Ab)-producing B cells in the spleen and enhanced levels of anti-Aβ antibodies in the serum, as well as an increase of Aβ phagocytosing macrophages in the CNS. Our results suggest that transplantation of APP<sup>−/−</sup> human ESC- or induced pluripotent stem cell (iPSC)-derived TEPs may provide a new tool to mitigate AD in patients.</p></abstract><kwd-group><kwd>Alzheimer's disease</kwd><kwd>amyloid-beta</kwd><kwd>thymic epithelial cells</kwd><kwd>embryonic stem cells</kwd><kwd>amyloid precursor protein</kwd><kwd>T cells</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Institutes of Health<named-content content-type=\"fundref-id\">10.13039/100000002</named-content></funding-source></award-group><award-group><funding-source id=\"cn002\">Connecticut Innovations<named-content content-type=\"fundref-id\">10.13039/100004829</named-content></funding-source></award-group><award-group><funding-source id=\"cn003\">National Natural Science Foundation of China<named-content content-type=\"fundref-id\">10.13039/501100001809</named-content></funding-source></award-group><award-group><funding-source id=\"cn004\">Guizhou Science and Technology Department<named-content content-type=\"fundref-id\">10.13039/501100004001</named-content></funding-source></award-group><award-group><funding-source id=\"cn005\">Guizhou Medical University<named-content content-type=\"fundref-id\">10.13039/501100010265</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"8\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"87\"/><page-count count=\"16\"/><word-count count=\"10147\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Alzheimer's disease (AD) is a devastating age-related neurodegenerative disorder, affecting over 34 million people worldwide (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). AD is characterized by progressive loss of memory and cognitive functions (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>–<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). The cognitive decline in AD is associated with hallmark protein aggregates, amyloid-beta (Aβ) plaques and neurofibrillary tangles, which are accompanied by neuroinflammation, and synaptic and neuronal loss (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>–<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Aβ plaques play a central role in the pathogenesis of AD and are generated from proteolytic cleavage of amyloid precursor protein (APP) (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Aβ can accelerate neuronal cell death and neuronal tangle formation, affect synaptic function adversely and eventually cause neuron loss (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>–<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B8\" ref-type=\"bibr\">8</xref>). The accumulated Aβ plaques and neuroinflammation have led to numerous attempts over the years to treat AD, either by removing the Aβ plaques, or by systemic anti-inflammatory drug administration to arrest brain inflammation. However, the drugs tested thus far for AD have largely failed in the clinic (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B9\" ref-type=\"bibr\">9</xref>–<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). These failures suggest that, although removal of Aβ plaques may be important, this approach alone is not enough to arrest or reverse cognitive loss. Furthermore, recent data also suggest that mitigating neuroinflammation in AD necessitates stimulation, rather than suppression of the immune system, to drive an immune-dependent cascade needed for the Aβ clearance and brain repair (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>). It has been shown that anti-inflammation is an active mechanism mediated by recruitment of circulating immune cells to sites of brain pathology (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>–<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). In addition, systemic immune deficiency is associated with cognitive dysfunction (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>) and accelerated AD pathology (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B20\" ref-type=\"bibr\">20</xref>).</p><p>T cells are the major component of the immune system. Multiple lines of evidence have suggested that T cells play an important role in the CNS maintenance and repair. For example, systemic T cell deficiency is associated with increased neuronal loss in animal models of CNS injury or AD (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B21\" ref-type=\"bibr\">21</xref>). Systemic T cells not only participate in CNS repair, but are also needed for life-long brain plasticity (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B22\" ref-type=\"bibr\">22</xref>, <xref rid=\"B23\" ref-type=\"bibr\">23</xref>). Both T cells and monocyte-derived macrophages recognizing brain antigens are required for coping with and helping heal brain damage during central nervous system (CNS) injuries (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>–<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). T cells present in the periphery play an important role in adaptive–innate immunity cross-talk and help in CNS repair (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B20\" ref-type=\"bibr\">20</xref>). Furthermore, it has been suggested that autoreactive T cells that recognize CNS-specific antigens augment the recruitment of monocyte-derived macrophages to the brain (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>, <xref rid=\"B26\" ref-type=\"bibr\">26</xref>).</p><p>The thymus is the primary organ for T cell generation. It, however, undergoes age-dependent thymic involution, resulting in decreased numbers of T cells in the elderly. This reduction has direct etiological linkages with many diseases (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>–<xref rid=\"B34\" ref-type=\"bibr\">34</xref>), including acceleration of the development and progression of AD (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). T cell development in the thymus depends on the thymic microenvironment, in which thymic epithelial cells (TECs) are the major component (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>–<xref rid=\"B41\" ref-type=\"bibr\">41</xref>). However, TECs undergo both qualitative and quantitative loss over time, which is believed to be the major factor responsible for age-dependent thymic involution (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>–<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). It is well-known that embryonic stem cells (ESCs) have the dual ability to propagate indefinitely <italic>in vitro</italic> in an undifferentiated state and to differentiate into many types of cells (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>). We have reported that ESCs can be selectively induced to generate TEPs <italic>in vitro</italic> (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>–<xref rid=\"B46\" ref-type=\"bibr\">46</xref>). When transplanted into young or old mice, the ESC-TEPs further develop into TECs, reconstitute the normal thymic architecture, and promote T cell generation, resulting in increased number of functional T cell in the periphery (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>–<xref rid=\"B46\" ref-type=\"bibr\">46</xref>).</p><p>We hypothesized that AD aged mice and patients have a very severe defect in the thymic microenvironment and that transplantation of ESC-TEPs into AD mice would rejuvenate the aged thymic microenvironment, leading to an increased number of functional T cell in the periphery, resulting in attenuated AD pathology. It is well-known that TECs, especially medullary TECs (mTECs), are involved in the deletion of autoreactive T cells. We have demonstrated that transplantation of ESC-TEPs expressing disease-causative self-antigen results in the deletion of the antigen-specific autoreactive T cells (<xref rid=\"B47\" ref-type=\"bibr\">47</xref>, <xref rid=\"B48\" ref-type=\"bibr\">48</xref>). Our hypothesis further proposes that transplantation of APP gene-deleted ESC-TEPs would lead to the generation of Aβ-specific autoreactive T cells that could help the production of other Aβ-specific immune cells to clear the Aβ plaques in the CNS.</p><p>We show here that transplantation of APP gene deleted (APP<sup>−/−</sup>) or their wild-type (APP<sup>+/+</sup>) mouse ESC (mESCs)-derived-TEPs results in enhanced thymopoiesis, increased T cell number, especially IFN-γ-producing cells, in the periphery, enhanced choroid plexus (CP) gateway activity, and enhanced recruitment of macrophages into the brain. Consequently, these mice have reduced Aβ deposits in the brain and improved cognitive performance. Furthermore, transplantation of APP<sup>−/−</sup> mESC-TEPs has a greater effect than that of APP<sup>+/+</sup> mESC-TEPs in clearance of Aβ deposits in the CNS and reversal of cognitive decline. This is related to the generation of Aβ-specific T cells, increased numbers of anti-Aβ antibody (Ab)-producing B cells in the spleen, increased levels of anti-Aβ Ab in the serum, and enhanced function of macrophages to phagocytose Aβ in the brain. Our results suggest that human ESC (hESC)- or induced pluripotent stem cell (iPSC)-derived TEPs, especially APP<sup>−/−</sup> hESC or iPSC-TEPs, may serve as a novel tool to modify AD pathology.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Mice</title><p>3xTg-AD, APP/PS1, C57BL/6 (B6) mice were purchased from Jackson Laboratory. The mice were used in accordance with a protocol approved by the Institutional Animal Care and Use Committee of the University of Connecticut.</p></sec><sec><title>Cell Culture</title><p>B6 mESC line (from Cyagen, Santa Clara, CA) were cultured in ESGRO Complete Plus Serum-free Clonal Grade Medium with GSK3β inhibitor supplement (Millipore, Temecula, CA). For TEP differentiation, mESCs were first induced to differentiate into definitive endoderm, and then TEPs in the presence of BMP-4, FGF 7, FGF10, and EGF, as well as rFOXN1 and rHOXA3 protein as we previously described (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>).</p></sec><sec><title>Genome Editing</title><p>The APP gene in mESCs was knocked out by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas9) genome editing. B6 mESCs were transfected with APP-specific double nickase plasmids or control double nickase plasmids (from Santa Cruz Biotechnology). The cells were screened to obtain APP<sup>−/−</sup> and APP<sup>+/+</sup> mESCs. The information of the plasmids and gRNA sequences are shown in <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Figure 1</xref>.</p></sec><sec><title>Intrathymic Injection</title><p>Mice were anesthetized and injected with 5 × 10<sup>4</sup> cells in 10–20 μl PBS into the thymus posterior to the upper sternum using a 26–28 gauge needle as described (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>).</p></sec><sec><title>Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Real-Time Qualitative RT-PCR (qRT- PCR)</title><p>Total RNA was extracted from tissues or cells using a Nucleo Spin RNA II kit (Macherey-Nagel, Düren, Germany). The RNA was converted into complementary DNA using High Capacity cDNA Reverse Transcription Kit (Invitrogen, USA). RT-PCR was performed with GoTaq® Green Master Mix (Promega, USA). qRT- PCR was performed with the Power SYBR green master mix (Applied Biosystems, UK) using the 7500 real-time PCR system (Applied Biosystems, UK). The primer sequences are shown in <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Table 1</xref>.</p></sec><sec><title>Western Blot Analysis</title><p>GFP<sup>+</sup> mESC-TECs were purified from the thymocytes using a magnetic-activated cell sorter immunomagnetic separation system (Mitenyi Biotec). The cells were collected and lysed. Equal amounts of denatured proteins were loaded onto a 4–12% Bis-Tris gel (Invitrogen, Carlsbad, CA), electrophoresed and transferred onto a PVDF membrane (Invitrogen). The membranes were blocked with 5% nonfat milk in TBST (mixture of Tris-Buffered Saline and Tween 20), and incubated with anti-mouse APP monoclonal antibody (Invitrogen) at 4 degree overnight. The membranes were then incubated with goat anti-mouse IgG HRP-conjugated secondary antibody and developed with a SuperSignal West Pico chemiluminescence substrate (Thermo Scientific, Rockford, IL).</p></sec><sec><title>Immunohistochemistry</title><p>The brain tissues were incubated in a fixative solution, embedded in OCT medium, snap frozen, and subsequently cut into 6 μm sections. The cultured cells were incubated with primary antibodies. The following primary antibodies were used: mouse anti-Aβ (clone 6E10,) and rabbit anti-GFAP (Biolegend, USA). After washing, the sections were incubated with fluorochrome-conjugated secondary antibody, counterstained with 4′, 6′-diamidino-2-phenylindole (DAPI) and observed under a Nikon A1R Spectral Confocal microscope (Nikon, Kanagawa, Japan). To quantify the staining intensity, total cells and background fluorescence intensity were measured using ImageJ software (NIH, USA), and the intensity of specific staining was calculated as described (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>).</p></sec><sec><title>Flow Cytometry Analysis</title><p>To analyze TECs, the thymi were incubated at 37°C in 0.01 (w/v) liberase (Roche, Nutley NJ) and 0.02% (w/v) DNAse I (Roche) with regular and gentle agitation as described (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>). A single-cell suspension of tissues was stained with fluorochrome-conjugated antibodies directly or indirectly as described (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>). For intracellular staining, the cells were first permeabilized with a BD Cytofix/Cytoperm solution for 20 min at 4°C. The following antibodies were used: CD4, CD8, EpCAM1, CD45, CD11b, F4/80, IFNγ, Ly6c, and SRA1 (BioLegend, San Diego, CA, or ThermoFisher Scientific), Keratin (K) 5 (Covance, Dallas, TX), and K8 (US Biological, Salem, MA). The samples were analyzed on an LSRFortessa X-20 Cell Analyzer (BD Biosciences). Data analysis was performed using FlowJo software (Ashland, OR).</p></sec><sec><title>T Cell Proliferation Assay</title><p>Splenocytes were stained with 5 μM CFSE (ThermoFisher Scientific) for 15 min. at 37°C. The cells were then cultured in a 96-well flat-bottom plate in the presence of plate-bound Aβ40 or Aβ42 (Anaspec, USA) and anti-CD3 antibody for 3 days. The cells were then stained with anti-PE labeled-CD4 and APC labeled-CD8 antibodies and analyzed for CFSE levels by T cells using flow cytometry.</p></sec><sec><title>ELISA Assay for Anti-Aβ40 or Anti-Aβ42 Antibody</title><p>Aβ40 or Aβ42 (Anaspec, USA) was coated on 96-well microplates overnight at 4°C, then blocked with blocking buffer (2% BSA+5% goat serum in PBS) for 2 h at room temperature. The serum samples diluted into 1:1000 were added to the plates and incubated 2 h at room temperature. After washing, HRP-conjugated goat anti-mouse IgG (Biolegend) was added to the plates and incubated for 1 h. The reaction was developed by TMB substrate (Thermo Scientific, USA) and stopped with 0.1 N HCl. The microplate was read at 450 nm under a microplate reader (Bio-Tek, ELX800, USA). The antibody concentrations were calculated using a standard curve generated with known concentrations of anti-Aβ antibody.</p></sec><sec><title>Soluble Aβ Protein (sAβ) Isolation and Quantification</title><p>Brain parenchyma was dissected, snap-frozen and kept at −75°C until homogenization. The samples were homogenized, and the supernatants were collected and detected for the concentrations of Aβ<sub>1−40</sub> and Aβ<sub>1−42</sub> by ELISA as described (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>).</p></sec><sec><title>B Cell ELISpot Assay</title><p>MultiScreen-IP plates (Millipore, Billerica, MA) were washed with 70% ethanol, rinsed three times with PBS, coated with Aβ40 (4 μg/ml) or Aβ42 (4 μg/ml) at 4°C overnight. The plates were blocked with blocking buffer (2% BSA in RPMI medium). 1 × 10<sup>4</sup> splenocytes were added into the plates and incubated for 48 h. The plates were washed 6 times with 0.25% Tween 20 (Sigma, USA) in PBS, incubated with HRP-conjugated goat anti-mouse IgG (H+L) (Biolegend) for 1 h, developed with a DAB Peroxidase Substrate Kit (Vector, USA), and counted for ELISpots (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>).</p></sec><sec><title>Amyloid Phagocytosis Assay</title><p>HiLyte Fluor 647 Beta-Amyloid (1–42) (Anaspec) was resuspended in Tris/EDTA (pH 8.2) at 20 mM and then incubated in the dark for 3 days at 37°C to promote aggregation. Macrophages in suspension were pretreated in low serum medium as described (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>). The HiLyte Fluor 647 Beta-Amyloid was added and incubated for 5 h. Cells were stained with macrophage markers; amyloid phagocytosis by the macrophages was determined by flow cytometry (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>).</p></sec><sec><title>Barnes Maze</title><p>Barnes Maze was conducted as previously described (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>, <xref rid=\"B55\" ref-type=\"bibr\">55</xref>). Briefly, each mouse was placed in the center of the maze and subjected to aversive stimuli. Mice were trained 4 training trials per day for 5 days, and a probe test was performed 24 h after the last training trial. The latency and number of errors were recorded for the training tail and probe test.</p></sec><sec><title>Novel Object Recognition (NOR) Test</title><p>A NOR test was conducted as previously described (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>–<xref rid=\"B56\" ref-type=\"bibr\">56</xref>). Briefly, mice were trained by allowing them to explore two identical objects placed at opposite ends of the arena for 10 min. 24 h later, mice were tested with one copy of the familiar object and one novel object of similar dimensions for 3 min. The time spent on exploring and sniffing of each object was recorded. The NOR index represents the percentage of time mice spent exploring the novel object.</p></sec><sec><title>Statistical Analysis</title><p><italic>P</italic>-values were based on the two-sided Student's <italic>T</italic>-test. A confidence level above 95% (<italic>p</italic> < 0.05) was determined to be significant.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><list list-type=\"simple\"><list-item><p>1. Deletion of the APP gene in mESCs to generate APP<sup>−/−</sup> mESCs and generation of TEPs from APP<sup>−/−</sup> and APP<sup>+/+</sup> mESCs <italic>in vitro</italic>.</p></list-item></list><p>Since Aβ is produced from proteolytic cleavage of APP, we deleted the APP gene in mESCs using CRISPR and Cas9 genome editing. B6 mESCs were transfected with APP-specific double nickase plasmids that contain the APP-specific-guide RNAs, and the Cas9 nuclease and GFP genes. The cells were screened in puromycin to obtain APP<sup>−/−</sup> mESCs. The gene deletion was confirmed by RT-PCR with one of primers spanning the gRNA region (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). The cells that were transfected with control double nickase plasmids containing non-targeting scrambled gRNA, Cas9, and GFP genes were used as a control (APP<sup>+/+</sup> mESCs).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Characterization of APP<sup>−/−</sup> and APP<sup>+/+</sup> mESCs. <bold>(A)</bold> The expression of the APP mRNA in APP<sup>−/−</sup>and APP<sup>+/+</sup> mESCs was measured by RT-PCR. <bold>(B)</bold> APP<sup>−/−</sup> and APP<sup>+/+</sup> mESCs were assessed for the expression of the pluripotent marker AP. <bold>(C)</bold> APP<sup>−/−</sup> and APP<sup>+/+</sup> mESCs were induced to differentiate into TEPs <italic>in vitro</italic>. The number of mESC-derived TEPs (EpCAM1<sup>+</sup>K5<sup>+</sup>K8<sup>+</sup>) were analyzed by flow cytometry. <bold>(D)</bold> APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-EpCAM1<sup>+</sup> TEPs were injected into the thymus of syngeneic C57BL/6 mice. Two months later, GFP<sup>+</sup> mESC-TECs were purified from the thymus, and analyzed for the expression of APP protein by Western blot. The data are presented from 3 independent experiments.</p></caption><graphic xlink:href=\"fimmu-11-01781-g0001\"/></fig><p>Both APP<sup>−/−</sup> and APP <sup>+/+</sup> mESCs were positive for alkaline phosphatase (AP) activity, indicating that the mESCs were in an undifferentiated state (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). We then induced the APP <sup>−/−</sup> and APP<sup>+/+</sup> mESCs to differentiate into TEPs <italic>in vitro</italic> following our protocol (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>). After the differentiation, both APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-derived cells contained comparable numbers of EpCAM1 positive cells that co-expressed K5 and K8, a phenotype of TEPs (<xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref>). We purified EpCAM1<sup>+</sup> TEPs from APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-derived cells and injected an equal number of the TEPs into the thymus of syngeneic mice. Two months later, the mESC-TEPs generated comparable numbers of GFP<sup>+</sup> mESC-derived TECs that accounted for 51–58% of total TECs.</p><p>Western blot analysis showed that purified GFP<sup>+</sup> APP<sup>+/+</sup> mESC-TECs expressed APP protein, whereas GFP<sup>+</sup> APP<sup>−/−</sup> mESC-TECs did not (<xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Figure 2</xref>). Together, these results indicate that the APP gene has been deleted in the APP<sup>−/−</sup> mESCs, and that the deletion does not affect the differentiation ability of mESCs into TEPs and TECs.</p><list list-type=\"simple\"><list-item><p>2. Both APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-TEP-transplanted AD mice have an improved cognitive performance, and APP<sup>−/−</sup> mESC-TEP-transplanted mice perform better than APP<sup>+/+</sup> mESC-TEP-transplanted mice.</p></list-item></list><p>To determine whether transplantation of APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-TEPs affects cognitive performance in AD mice, 3XTg-AD mice aged 12 months, an age of advanced cerebral pathology, were injected intrathymically (i.t.) with APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEPs. APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-derived EpCAM1<sup>−</sup> non-TEPs (control cells) were used as controls. Two months later, the mice were evaluated for spatial learning and memory. It has been reported that the Barnes maze, a hippocampal-dependent spatial task (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>, <xref rid=\"B58\" ref-type=\"bibr\">58</xref>), is the most sensitive test for detecting cognitive deficits in 3XTg-AD mice (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>). We found that both APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-TEP-treated mice had significantly greater Barnes maze learning curves than control cell-treated mice (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>). Furthermore, APP<sup>−/−</sup> mESC-TEP-treated mice performed better than APP<sup>+/+</sup> mESC-TEP-treated mice, almost reaching the performance level observed in wild-type (WT) non-AD mice (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>). Since there were no significant differences between APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-EpCAM1<sup>−</sup> control cell-transplanted mice in all of the results in this paper (data not shown), we pooled the data from the two groups and named this group as control cell (Ctrl)-transplanted mice.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Transplantation of APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-TEPs improves cognitive performance in AD mice and APP<sup>−/−</sup> mESC-TEPs are more effective than APP<sup>+/+</sup> mESC-TEPs. <bold>(A–D)</bold> 3XTg-AD mice (12-month-old) were injected i.t. with APP<sup>−/−</sup> EpCAM1<sup>+</sup> mESC-TEPs, APP<sup>+/+</sup> EpCAM1<sup>+</sup> mESC-TEPs, or control cells (5 × 10<sup>4</sup> cells/mouse). Age-matched untreated WT mice were also used as controls. Two months later, the mice were evaluated for cognitive performance by Barnes Maze and Object Recognition tests. <bold>(A)</bold> The escape latency during the training period, and <bold>(B,C)</bold> the escape latency and the number of errors committed during the probe trial are shown. <bold>(D)</bold> NOR index was determined as the time spent interacting with the novel object divided by the total time of exploration during the testing phase. The data are expressed as mean ± SD from one of three independent experiments with similar results (four to eight mice per group in each experiment). *<italic>p</italic> < 0.05 vs. control cell group, **<italic>p</italic> < 0.05 vs. APP<sup>+/+</sup> mESC-TEP group.</p></caption><graphic xlink:href=\"fimmu-11-01781-g0002\"/></fig><p>Both APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-TEP-treated mice also had decreased latency to find the target zone during the probe trial conducted 24 h after the final training session (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>), indicating an improved memory performance. In addition, the number of errors committed in the APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEP-treated mice was also significantly reduced, as compared to control cell-treated mice (<xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>).</p><p>The NOR test is to study learning and memory in rodents based on their spontaneous tendency to have more interactions with a novel than with a familiar object (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>). NOR is a more cortically-dependent novel object recognition preference task (<xref rid=\"B57\" ref-type=\"bibr\">57</xref>, <xref rid=\"B58\" ref-type=\"bibr\">58</xref>). In agreement with the results in the Barnes maze task, APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEP-treated mice performed significantly better than control cell-treated mice (<xref ref-type=\"fig\" rid=\"F2\">Figure 2D</xref>). In all of these studies (<xref ref-type=\"fig\" rid=\"F2\">Figures 2A–D</xref>), APP<sup>−/−</sup> mESC-TEP-treated mice performed significantly better than APP<sup>+/+</sup> mESC-TEP-treated mice. Together, our data suggest that transplantation of APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEPs results in improved spatial learning and memory in 3xTg-AD mice and that transplantation of APP<sup>−/−</sup> mESC-TEPs demonstrates greater effectiveness than APP<sup>+/+</sup> mESC-TEPs.</p><list list-type=\"simple\"><list-item><p>3. Both APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-TEP-transplanted AD mice have reduced AD pathology with greater reduction in APP<sup>−/−</sup> mESC-TEP-transplanted mice.</p></list-item></list><p>We then determined whether transplantation of APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEPs leads to improved AD pathology. After the Barnes maze and NOR tests (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>), the brains were harvested and immunohistochemical analysis performed. We found that both APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-TEP-transplanted 3XTg-AD mice had a reduced cerebral Aβ plaque load in the hippocampus, specifically in the dentate gyrus (DG) and in the cerebral cortex (layer V) (<xref ref-type=\"fig\" rid=\"F3\">Figures 3A–C</xref>), areas showing robust Aβ-plaque pathology in AD mice. Comparatively, APP<sup>−/−</sup> mESC-TEP-transplanted mice had the greater reduction in Aβ-plaque load (<xref ref-type=\"fig\" rid=\"F3\">Figures 3A–C</xref>). Astrogliosis, as assessed by glial fibrillary acid protein (GFAP) immunoreactivity, was also reduced in APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-TEP-treated mice, as compared to control cell-treated mice (<xref ref-type=\"fig\" rid=\"F3\">Figures 3A,D</xref>). Again, APP<sup>−/−</sup> mESC-TEP-transplanted mice had, by comparison, a greater reduction in GFAP immunoreactivity (<xref ref-type=\"fig\" rid=\"F3\">Figures 3A,D</xref>).</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Transplantation of APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEPs attenuates AD pathology and APP<sup>−/−</sup> mESC-TEPs are more effective than APP<sup>+/+</sup> mESC-TEPs. <bold>(A–C)</bold> 3XTg-AD mice (12-month-old) were injected i.t. with APP<sup>−/−</sup> mESC-TEPs, APP<sup>+/+</sup> mESC-TEPs, or control cells as in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>. Two and a half months later, the mice were evaluated for <bold>(A–D)</bold> brain pathology, and <bold>(E)</bold> soluble Aβ levels. <bold>(A–D)</bold> The brain sections were immunostained for Aβ (in red), GFAP (in green) and Hoechst nuclear staining. Mean Aβ area and plaque numbers in the hippocampal DG and the cortex fifth layer, and GFAP immunoreactivity in the hippocampus were measured. <bold>(A)</bold> Representative immunofluorescent images, and <bold>(B–D)</bold> quantitative analysis of Aβ and GFAP. <bold>(E)</bold> Levels of soluble Aβ1–40 and Aβ1–42 in the cerebral brain parenchyma of the mice were quantified by ELISA. <bold>(F,G)</bold> 14-month-old APP/PS1 mice were injected i.t. with APP<sup>−/−</sup> mESC-TEPs, APP<sup>+/+</sup> mESC-TEPs, or control cells. Two months later, the mice were evaluated for brain pathology. <bold>(F)</bold> Representative immunofluorescent images, and (G) quantitative analysis of Aβ. The data are expressed as mean ± SD from one of three independent experiments with similar results (four to eight mice per group per experiment). *<italic>p</italic> < 0.05 vs. control cell group, **<italic>p</italic> < 0.05 vs. APP<sup>+/+</sup> mESC-TEP group.</p></caption><graphic xlink:href=\"fimmu-11-01781-g0003\"/></fig><p>Since impaired synaptic plasticity and memory deficits in AD are associated with elevated cerebral soluble Aβ1-40/Aβ1-42 (sAβ) levels (<xref rid=\"B61\" ref-type=\"bibr\">61</xref>), we then measured sAβ levels in the AD mice by ELISA. Consistent with the immunohistochemical results, both APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-TEP-treated mice had reduced cerebral sAβ, as compared to control cell-treated mice (<xref ref-type=\"fig\" rid=\"F3\">Figure 3E</xref>). Transplantation of APP<sup>−/−</sup> mESC-TEPs demonstrated the greater reduction (<xref ref-type=\"fig\" rid=\"F3\">Figure 3E</xref>).</p><p>We likewise examined the effect of mESC-TEPs in another AD model, APP/PS1 mice, which develop Aβ-plaque pathology at a more advanced age than do 3XTg-AD mice. Transplantation of APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEPs reduced hippocampal Aβ plaque load, as compared to control cell-treated mice, and APP<sup>−/−</sup> mESC-TEP-treated mice had more Aβ plaque load reduction than APP<sup>+/+</sup> mESC-TEP-treated mice (<xref ref-type=\"fig\" rid=\"F3\">Figures 3F,G</xref>). Taken together, our results suggest that transplantation of APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEPs into AD mice results in clearance of Ab plaques and reversal of cognitive decline, and APP<sup>−/−</sup> mESC-TEP-treated mice perform better than APP<sup>+/+</sup> mESC-TEP-treated mice.</p><list list-type=\"simple\"><list-item><p>4. Both APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-TEP-transplanted AD mice have increased T cell numbers, and APP<sup>−/−</sup> mESC-TEP-transplanted mice have enhanced T cell proliferation in response to Aβ stimulation.</p></list-item></list><p>We have previously demonstrated that transplantation of mESC-TEPs results in enhanced thymopoiesis and increased T cell numbers in the spleen (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>–<xref rid=\"B45\" ref-type=\"bibr\">45</xref>). Consistent with the previous reports, APP<sup>+/+</sup> or APP<sup>−/−</sup> mESC-TEP-transplanted mice had increased numbers of thymocytes in the thymus and T cells in the spleen compared to control cell-treated mice. The number of T cells in the thymus and the spleen between APP<sup>+/+</sup> and APP<sup>−/−</sup> mESC-TEP-transplanted mice were not significantly different (<xref ref-type=\"fig\" rid=\"F4\">Figures 4A,B</xref>).</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Transplantation of APP<sup>+/+</sup> or APP<sup>−/−</sup> mESC-TEPs leads to increased T cell number, and enhanced T cell proliferative response to Aβ proteins. 3XTg-AD mice (12-month-old) were injected i.t. with APP<sup>−/−</sup> mESC-TEPs, APP<sup>+/+</sup> mESC-TEPs, or control cells as in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>. Two and a half months later, the thymus and spleen were harvested. The number of <bold>(A)</bold> total thymocytes and their subsets, and <bold>(B)</bold> CD4 and CD8 T cells in the spleen were analyzed by flow cytometry. <bold>(C,D)</bold> The splenocytes (normalized to 1 × 10<sup>5</sup> T cells/well) from the AD mice were labeled with CFSE and cultured with Aβ40 or Aβ42 in the presence of anti-CD3 antibody for 3 days. The cells were stained with mouse anti-CD4 and CD8 antibodies and analyzed for CFSE levels by CD4<sup>+</sup> and CD8<sup>+</sup> T cells, respectively. Black line: APP<sup>−/−</sup> mESC-TEP-transplanted mice; black dash line: APP<sup>+/+</sup> mESC-TEP-transplanted mice; gray line: control cell-transplanted mice; gray shadow: unstimulated T cells. <bold>(E,F)</bold> The splenocytes were analyzed for the percentage of IFNγ-producing CD4 T cells. <bold>(C,E)</bold> Representative flow cytometric profiles, and <bold>(D,F)</bold> statistical analyses are shown. The data are expressed as mean ± SD from one of three independent experiments with similar results (4–8 mice per group in each experiment). *<italic>p</italic> < 0.05 vs. control cell group, **<italic>p</italic> < 0.05 vs. APP<sup>+/+</sup> mESC-TEP group.</p></caption><graphic xlink:href=\"fimmu-11-01781-g0004\"/></fig><p>Thymocytes can be divided into four major subsets: CD4 and CD8 double negative (DN), double positive (DP), CD4 single positive (SP), and CD8 SP thymocytes. DN thymoctyes can be further divided into DN1 to DN4 subsets based on the expression of CD44 and CD25. APP<sup>+/+</sup> or APP<sup>−/−</sup> mESC-TEP-transplanted mice had decreased percentages of DN subsets (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Figure 3</xref>), suggesting improved thymocyte development. Because APP<sup>+/+</sup> or APP<sup>−/−</sup> mESC-TEP-transplanted mice had increased numbers of total thymocytes, and numbers of each thymocyte subsets in these mice were higher than those in control cell-treated mice (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Figure 3</xref>). We also analyzed the percentage and number of regulatory T cells (Tregs) in the thymus and the spleen. Although the percentages of Tregs were not significant different among the groups, the number of Tregs in APP<sup>+/+</sup> or APP<sup>−/−</sup> mESC-TEP-transplanted AD mice was higher than WT or control cell-transplanted AD mice (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Figures 3</xref>, <xref ref-type=\"supplementary-material\" rid=\"SM1\">4</xref>). Similarly, the percentages of CD11c<sup>+</sup> dendritic cells (DCs) and CD19<sup>+</sup> B cells in the spleen were not significant different among the groups (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Figure 4</xref>).</p><p>We then determined the proliferation of the splenic T cells in response to Aβ40 and Aβ42 protein stimulation in the presence of anti-CD3 antibody <italic>in vitro</italic>. The proliferation of CD4 and CD8 T cells from APP<sup>+/+</sup> mESC-TEP-transplanted mice was slightly higher than that from control cell-transplanted mice (<xref ref-type=\"fig\" rid=\"F4\">Figures 4C,D</xref>), which may be due to enhanced T cell function after transplantation of mESC-TEPs. Furthermore, the proliferation of both CD4 and CD8 T cells from APP<sup>−/−</sup> mESC-TEP-transplanted mice was significantly higher than that from APP<sup>+/+</sup> mESC-TEP-transplanted mice (<xref ref-type=\"fig\" rid=\"F4\">Figures 4C,D</xref>). The latter results suggest that Aβ-specific autoreactive T cells might not be deleted in the thymus of APP<sup>−/−</sup> mESC-TEP-transplanted mice, leading to presence of Aβ-specific autoreactive T cells in the periphery, resulting in greater proliferation response. Of note, although the proliferation of both CD4 and CD8 T cells in response to anti-CD3 antibody alone (without Aβ40 or Aβ42 peptide) from APP<sup>+/+</sup> or APP<sup>−/−</sup> mESC-TEP-transplanted mice was greater than that from control cell-transplanted mice, there was no significant difference between APP<sup>+/+</sup> and APP<sup>−/−</sup> mESC-TEP-transplanted groups (data not shown).</p><p>It has been reported that PD-1 blockage reduced AD pathology involves an IFNγ-dependent immunological response (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). We then analyzed IFNγ-producing T cells in the spleen and found that the percentage of IFNγ-producing CD4 T cells in APP<sup>+/+</sup> or APP<sup>−/−</sup> mESC-TEP-transplanted AD mice was significantly higher than that in control cell-treated mice (<xref ref-type=\"fig\" rid=\"F4\">Figures 4E,F</xref>). This is consistent with our previous reports that transplantation of mESC-TEPs leads to the generation of functional T cells including enhanced production of IFNγ (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>, <xref rid=\"B45\" ref-type=\"bibr\">45</xref>). Compared with APP<sup>+/+</sup> mESC-TEP-transplanted mice, the percentage of IFNγ-producing CD4 T cells in APP<sup>−/−</sup> mESC-TEP-transplanted mice showed a larger increase, which is probably due to enhanced anti-Aβ autoimmunity in APP<sup>−/−</sup> mESC-TEP-transplanted AD mice. Furthermore, more IFNγ was detected in the supernatant of cultured splenocyts from APP<sup>−/−</sup> mESC-TEP-transplanted AD mice in response to Aβ40 or Aβ42 protein stimulation (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Figure 5</xref>).</p><list list-type=\"simple\"><list-item><p>5. APP<sup>−/−</sup> mESC-TEP-transplanted mice have an increased number of anti-Aβ Ab-producing B cells in the spleen and increased level of anti-Aβ Ab in the serum.</p></list-item></list><p>It is well-known that T cells can help B cell functions. We then determined whether the enhanced T cell proliferation to Aβ stimulation in mESC-TEP-transplanted AD mice led to increased production of anti-Aβ Ab-producing B cells. We used Aβ40 and Aβ42 as antigens for an ELISpot assay to measure anti-Aβ Ab-producing B cells in the spleen. The number of anti-Aβ Ab-producing B cells in APP<sup>+/+</sup> mESC-TEP-transplanted mice was higher than that in control cell-transplanted mice, while the number of anti-Aβ Ab-producing B cells in APP<sup>−/−</sup> mESC-TEP- transplanted mice was higher than that in APP<sup>+/+</sup> mESC-TEP-transplanted mice (<xref ref-type=\"fig\" rid=\"F5\">Figures 5A,B</xref>).</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>Transplantation of APP<sup>−/−</sup> mESC-TEPs results in an increased number of anti-Aβ Ab-producing B cells in the spleen and increased levels of anti-Aβ Abs in the serum. 3XTg-AD mice were injected i.t. with APP<sup>−/−</sup> mESC-TEPs, APP<sup>+/+</sup> mESC-TEPs, or control cells as in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>. Two and half months later, the spleen and serum were harvested. <bold>(A,B)</bold> The splenocytes (normalized to 1 × 10<sup>4</sup> CD19<sup>+</sup> B cells/well) were placed on culture plates coated with Aβ40 (4 μg/ml) or Aβ42 (4 μg/ml). Anti-Aβ Ab-producing B cells were measured by an ELISpot assay. <bold>(C)</bold> The levels of anti-Aβ40 and anti-Aβ42 antibodies in the serum were measured by ELISA. The data are expressed as mean ± SD from one of three independent experiments with similar results (4–8 mice per group in each experiment). *<italic>p</italic> < 0.05 vs. control cell group, **<italic>p</italic> < 0.05 vs. APP<sup>+/+</sup> mESC-TEP group.</p></caption><graphic xlink:href=\"fimmu-11-01781-g0005\"/></fig><p>We also analyzed the levels of anti-Aβ Abs in the serum. Consistent with the ELISpot results, the levels of both anti-Aβ40 and anti-Aβ42 antibodies in the serum of APP<sup>+/+</sup> mESC-TEP-treated mice were higher than those in control cell-treated mice, and the levels of these antibodies in APP<sup>−/−</sup> mESC-TEP-treated mice were significantly higher than those in APP<sup>+/+</sup> mESC-TEP-treated mice (<xref ref-type=\"fig\" rid=\"F5\">Figure 5C</xref>). The results suggest that increased T cell number in APP<sup>+/+</sup> or APP<sup>−/−</sup> mESC-TEP-treated mice, especially Aβ-specific T cells in APP<sup>−/−</sup> mESC-TEP-treated mice, help to generate anti-Aβ Ab-producing B cells that secret anti-Aβ Abs into the serum.</p><list list-type=\"simple\"><list-item><p>6. Transplantation of both APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-TEPs enhances the brain's choroid plexus (CP) activity.</p></list-item></list><p>The CP, the epithelial layer that forms the blood–CSF barrier, is a selective gateway for leukocyte entry to the CNS (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). AD mice have a defect in the CP gateway, as indicated by significantly lower levels of leukocyte homing and trafficking molecule expression in the CP (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). In contrast, IFNγ signaling enhances the expression of leukocyte trafficking molecules (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). Since we have demonstrated that IFNγ-producing T cells were increased in the spleen of APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEP-treated AD mice (<xref ref-type=\"fig\" rid=\"F4\">Figure 4E</xref>), we analyzed IFNγ availability at the CP in these mice. qRT-PCR analysis revealed a higher IFNγ mRNA expression level in the CP of APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEP-transplanted AD mice, compared with control cell-treated mice (<xref ref-type=\"fig\" rid=\"F6\">Figure 6A</xref>). Flow cytometric examination confirmed a significantly higher percentage of IFNγ-producing CD4<sup>+</sup> immune cells in this compartment in APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEP-transplanted AD mice (<xref ref-type=\"fig\" rid=\"F6\">Figure 6B</xref>). Again, the IFNγ mRNA expression levels and the percentage of IFNγ-producing CD4<sup>+</sup> immune cells in the CP APP<sup>−/−</sup> mESC-TEP-transplanted AD mice were higher than those in APP<sup>+/+</sup> mESC-TEP-transplanted AD mice (<xref ref-type=\"fig\" rid=\"F6\">Figures 6A,B</xref>), consistent with the results for the percentage of IFNγ-producing CD4<sup>+</sup> splenic T cells among the mice (<xref ref-type=\"fig\" rid=\"F4\">Figure 4E</xref>).</p><fig id=\"F6\" position=\"float\"><label>Figure 6</label><caption><p>Transplantation of APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEPs increases IFNγ availability and leukocyte trafficking molecule expression in the CP. 3XTg-AD mice were injected i.t. with APP<sup>−/−</sup> mESC-TEPs, APP<sup>+/+</sup> mESC-TEPs, or control cells as in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>. Two and half months later, the CP was harvested. <bold>(A)</bold> mRNA levels of IFNγ were analyzed by qRT-PCR. <bold>(B)</bold> Flow cytometric analysis for the percentage of IFNγ-producing cells in CD4<sup>+</sup> cells, (left) representative flow cytometric profiles, and (right) statistical analyses. <bold>(C)</bold> mRNA levels of icam1, ccl2, vcam1, cxcl10 were analyzed by qRT-PCR. The expression levels of the genes in control cell-treated mice are defined as 1. The data are expressed as mean ± SD from one of three independent experiments with similar results (4–8 mice per group in each experiment). *<italic>p</italic> < 0.05 vs. control cell group, **<italic>p</italic> < 0.05 vs. APP<sup>+/+</sup> mESC-TEP group.</p></caption><graphic xlink:href=\"fimmu-11-01781-g0006\"/></fig><p>Since increased IFNγ availability can enhance CP activity in this compartment (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>), we analyzed the expression of leukocyte homing and trafficking molecules, including intercellular adhesion molecule 1 (icam1), chemokine C-C motif ligand 2 (ccl2), vascular cell adhesion molecule 1 (vcam1), and C-X-C motif chemokine 10 (cxcl10) in the CP. As shown in <xref ref-type=\"fig\" rid=\"F6\">Figure 6C</xref>, the mRNA expression levels of these leukocyte trafficking molecules in the CP of APP<sup>+/+</sup> mESC-TEP-treated AD mice were higher than those in control cell-treated mice. The mRNA expression levels of these molecules (except vcam1) in the CP of APP<sup>−/−</sup> mESC-TEP-treated AD mice were higher than those in APP<sup>+/+</sup> mESC-TEP-treated mice (<xref ref-type=\"fig\" rid=\"F6\">Figure 6C</xref>). These results suggest that administration of APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEPs results in an increased CP activity, which is likely due to the increased IFNγ availability in this compartment.</p><list list-type=\"simple\"><list-item><p>7. APP<sup>−/−</sup> mESC-TEP-transplanted mice have an increased number of Aβ phagocytosing macrophages in the brain and the spleen.</p></list-item></list><p>Increased CP activity can result in recruitment of monocyte-derived macrophages to the brain to attenuate AD pathology (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Since transplantation of APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEPs led to an increased CP activity, we analyzed whether there was an increased number of monocyte-derived macrophages in the brain. It has been shown that CD45<sup>hi</sup>/CD11b<sup>+</sup> cells represent a myeloid population enriched with CNS-infiltrating monocyte-derived macrophages in the brain (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>). We found both APP<sup>−/−</sup> and APP<sup>+/+</sup> mESC-TEP-transplanted AD mice had an elevated proportion of CD45<sup>hi</sup>/CD11b<sup>+</sup> cells in the brain, as compared to control cell-treated mice (<xref ref-type=\"fig\" rid=\"F7\">Figures 7A,B</xref>). The proportion of CD45<sup>hi</sup>/CD11b<sup>+</sup> cells in the brain of APP<sup>−/−</sup> mESC-TEP-transplanted AD mice was higher than that in APP<sup>+/+</sup> mESC-TEP-transplanted mice. Furthermore, the CD45<sup>hi</sup>/CD11b<sup>+</sup> cells in APP<sup>−/−</sup> mESC-TEP-transplanted mice had a higher percentage of lymphocyte antigen 6c (Ly6C) positive cells than those in APP<sup>+/+</sup> mESC-TEP-transplanted mice (<xref ref-type=\"fig\" rid=\"F7\">Figure 7C</xref>). The CD45<sup>hi</sup>/CD11b<sup>+</sup> cells in APP<sup>−/−</sup> mESC-TEP-transplanted mice also expressed higher levels of chemokine receptor ccr2 and scavenger receptor A (SRA1) (<xref ref-type=\"fig\" rid=\"F7\">Figures 7D,E</xref>). It has been reported that Ly6C and ccr2 are related to myeloid cell neuroprotection in AD (<xref rid=\"B62\" ref-type=\"bibr\">62</xref>), whereas SRA1 is an Aβ-binding scavenger receptor associated with Aβ-plaque clearance (<xref rid=\"B63\" ref-type=\"bibr\">63</xref>). We also analyzed the phagocytosis ability of CD45<sup>hi</sup>/CD11b<sup>+</sup> cells and found that the cells in APP<sup>−/−</sup> mESC-TEP-transplanted mice had a higher ability to phagocytose Aβ42 than those in APP<sup>+/+</sup> mESC-TEP-transplanted mice (<xref ref-type=\"fig\" rid=\"F7\">Figure 7F</xref>).</p><fig id=\"F7\" position=\"float\"><label>Figure 7</label><caption><p>Transplantation of APP<sup>−/−</sup> mESC-TEPs results in an increased number of Aβ phagocytosing macrophages in the brain and the spleen. 3XTg-AD mice were injected i.t. with APP<sup>−/−</sup> mESC-TEPs, APP<sup>+/+</sup> mESC-TEPs or control cells as in <xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>. Two and half months later, <bold>(A–F)</bold> the brain and <bold>(G,H)</bold> the spleen were harvested. <bold>(A–C)</bold> The brain was analyzed for the percentage of CD45<sup>hi</sup>/CD11b<sup>+</sup> cells and the expression of Ly6C by CD45<sup>hi</sup> or CD45<sup>lo</sup> cells. <bold>(A)</bold> Flow cytometry gating strategy is shown. <bold>(B,C)</bold> Statistical analysis of the percentages of <bold>(B)</bold> CD45<sup>hi</sup>/CD11b <sup>+</sup> cells in CD11b <sup>+</sup> cells and <bold>(C)</bold> Ly6C<sup>+</sup> in CD45<sup>hi</sup>/CD11b <sup>+</sup> cells. <bold>(D,E)</bold> The brain was analyzed for <bold>(D)</bold> the expression of ccr2 and SRA1 mRNA by qRT-PCR (the expression level of the genes in control cell-treated mice is defined as 1), and <bold>(E)</bold> the expression of SRA1 protein by CD45<sup>hi</sup>CD11b <sup>+</sup> cells using flow cytometry. <bold>(F)</bold> CD45<sup>hi</sup>/CD11b <sup>+</sup> cells were isolated from brains and analyzed for phagocytosis using HF647 Aβ42. <bold>(G,H)</bold> The splenocytes were analyzed for <bold>(G)</bold> the percentage of F4/80<sup>+</sup> macrophages and <bold>(H)</bold> the ability of F4/80<sup>+</sup> macrophages to phagocytose Aβ42. The data are expressed as mean ± SD from one of three independent experiments with similar results (4–8 mice per group in each experiment). *<italic>p</italic> < 0.05 vs. control cell group, **<italic>p</italic> < 0.05 vs. APP<sup>+/+</sup> mESC-TEP group.</p></caption><graphic xlink:href=\"fimmu-11-01781-g0007\"/></fig><p>We then analyzed macrophages in the spleen and found the percentages of F4/80<sup>+</sup> macrophages in APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEP-transplanted AD mice were higher than that those in control cell-treated mice (<xref ref-type=\"fig\" rid=\"F7\">Figure 7G</xref>). Although the percentage of F4/80<sup>+</sup> macrophages in APP<sup>−/−</sup> mESC-TEP-transplanted mice was slightly higher than that in APP<sup>+/+</sup> mESC-TEP-transplanted mice, the difference did not reach statistical significance (<xref ref-type=\"fig\" rid=\"F7\">Figure 7G</xref>). Furthermore, F4/80<sup>+</sup> macrophages in APP<sup>+/+</sup> mESC-TEP-transplanted mice were more able to phagocytose Aβ42 than those in control cell-transplanted mice, and the macrophages in APP<sup>−/−</sup> mESC-TEP-transplanted mice had a greater ability than those in APP<sup>+/+</sup> mESC-TEP-transplanted mice (<xref ref-type=\"fig\" rid=\"F7\">Figure 7H</xref>), in agreement with the data for the macrophage function in the brain (<xref ref-type=\"fig\" rid=\"F7\">Figure 7F</xref>).</p></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>AD is hallmarked by the accumulation of Aβ plaques in the brain, which can adversely affect synaptic function and eventually cause neuron loss (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>–<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B8\" ref-type=\"bibr\">8</xref>). The brain has been traditionally considered a site of immune privilege and exempt from systemic immune surveillance (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B24\" ref-type=\"bibr\">24</xref>). It is now accepted that neuro-immunological cross-talk, in which circulating immune cells enter the CNS, play an important role in brain tissue maintenance and repair, especially in pathological conditions (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B24\" ref-type=\"bibr\">24</xref>–<xref rid=\"B26\" ref-type=\"bibr\">26</xref>, <xref rid=\"B64\" ref-type=\"bibr\">64</xref>–<xref rid=\"B66\" ref-type=\"bibr\">66</xref>). Like the situation in cancer immunology, onset of AD may reflect systemic immune suppression and the loss of immune surveillance (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B67\" ref-type=\"bibr\">67</xref>), impairing the ability to mount an immune response to fight brain pathology (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>, <xref rid=\"B68\" ref-type=\"bibr\">68</xref>). For example, it has been shown that AD severity is greater in immunocompromised mice (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). In contrast, replacement of the missing adaptive immune populations, such as T cells and B cells, can dramatically reduce AD pathology (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). Boosting recruitment of monocyte-derived macrophages to sites of brain pathology also facilitates Aβ plaque clearance and relieves AD pathology (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B69\" ref-type=\"bibr\">69</xref>–<xref rid=\"B72\" ref-type=\"bibr\">72</xref>). Therefore, systemic immunity in AD should be driven, rather than suppressed, to initiate an immune-dependent cascade to dissipate the Aβ clearance and repair the brain (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>).</p><p>It is well-known that the thymus, the primary organ for T cell generation, undergoes a profound atrophy with age, a process termed thymic involution, resulting in decreased numbers of T cells in older adults. The reduced T cell number in older adults is likely to contribute AD development and progression. Indeed, in this study, we have shown that transplantation of either APP<sup>−/−</sup> or APP<sup>+/+</sup> mESC-TEPs enhances thymopoiesis that results in increased number of T cells, especially IFNγ-producing T cells in the spleen and the CP, leading to enhanced CP activity and increased number of macrophages in the brain. In addition, these mice also have an increased number of macrophages in the spleen. It has been shown that increased IFNγ availability in the CP can enhance the CP activity (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>). Both APP<sup>+/+</sup> and APP<sup>−/−</sup> mESC-TEP-transplanted mice have enhanced expression of leukocyte homing and trafficking molecules icam1, vcam1, cxcl10, and ccl2 in the CP, which may be due to the increased IFNγ availability in this compartment. It is likely that the enhanced CP activity leads to increased migration of macrophages into the brain, resulting in an increased number of macrophages in this organ. It is also possible that increased T cell numbers in the spleen aid the macrophages, increasing their number in the spleen, likewise contributing to the increase in the brain. Although the improved thymopoiesis and an increased number of immune cells in the periphery (especially macrophages in the brain) attenuate AD pathology, they are insufficient for reduction of cerebral Aβ plaque load and for improving cognitive performance as indicated by the data that transplantation of APP<sup>+/+</sup> mESC-TEPs is less efficient than that of APP<sup>−/−</sup> mESC-TEPs.</p><p>Compared to APP<sup>+/+</sup> mESC-TEP-transplanted mice, APP<sup>−/−</sup> mESC-TEP-transplanted mice have increased Aβ-induced T cell proliferation, increased anti-Aβ Ab-producing B cells in the spleen and anti-Aβ Abs in the serum, as well as increased Aβ phagocytosing macrophages in the brain. Since TECs expressing self-antigens play a critical role in deleting autoreactive T cells specific to the antigens, transplantation of APP<sup>−/−</sup> mESC-TEPs could result in the failure to delete Aβ-specific autoreactive T cells in the thymus, leading to the presence of the autoreactive T cells in the periphery. This is supported by the data that T cells from APP<sup>−/−</sup> mESC-TEP-transplanted mice have increased proliferation in response to Aβ stimulation. The Aβ-specific autoreactive T cells may then help to produce anti-Aβ Ab-producing B cells that secret anti-Aβ Abs into the serum and to produce Aβ phagocytosing macrophages that migrate into the brain. Together, these Aβ-specific immune cells and Abs reduce the AD pathology. Our results support the notion that breaking Aβ-specific immune tolerance is a novel target for AD immunotherapy (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>).</p><p>Studies have shown that adaptive–innate immunity cross talk is important in ameliorating AD progression, in which T cells are critical (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). CD4 T cells are essential in the activation of B cells to secrete antibodies to mediate humoral immune responses (<xref rid=\"B73\" ref-type=\"bibr\">73</xref>, <xref rid=\"B74\" ref-type=\"bibr\">74</xref>). Antibody response to an antigen requires help from the antigen-specific T cells. B cell antigen receptor usually delivers an antigen to intracellular sites where it is degraded and returned to the B cell surface as the peptide bound to MHC II molecule. The peptide:MHC II complex is recognized by the antigen-specific helper T cells, inducing the B cells to develop into antibody-secreting cells. It is possible that Aβ-specific autoreactive T cells generated in APP<sup>−/−</sup> mESC-TEP-transplanted AD mice recognize the Aβ peptide:MHC II on B cells, and stimulate the B cells to proliferate and differentiate into plasma cells secreting anti-Aβ antibodies. Consequently, the anti-Aβ antibodies neutralize the toxin of Aβ and/or facilitate uptake of Aβ by macrophages by coating to Aβ to enhance the recognition by Fc receptors on macrophages.</p><p>CD4 T cells are also important in activating macrophages (<xref rid=\"B75\" ref-type=\"bibr\">75</xref>). Once activated, the macrophages phagocytose the related antigens. It has been shown that recruitment of circulating monocyte-derived macrophages can modify AD pathology (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B76\" ref-type=\"bibr\">76</xref>–<xref rid=\"B78\" ref-type=\"bibr\">78</xref>) by removing misfolded protein including Aβ-plaques (<xref rid=\"B69\" ref-type=\"bibr\">69</xref>, <xref rid=\"B79\" ref-type=\"bibr\">79</xref>, <xref rid=\"B80\" ref-type=\"bibr\">80</xref>), balancing the local inflammatory milieu (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>, <xref rid=\"B80\" ref-type=\"bibr\">80</xref>), reducing gliosis (<xref rid=\"B81\" ref-type=\"bibr\">81</xref>), and protecting synaptic structures (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>, <xref rid=\"B82\" ref-type=\"bibr\">82</xref>, <xref rid=\"B83\" ref-type=\"bibr\">83</xref>). Since activated macrophages can cause local tissue damage (<xref rid=\"B84\" ref-type=\"bibr\">84</xref>–<xref rid=\"B87\" ref-type=\"bibr\">87</xref>), it is important that the macrophage activity is strictly regulated by antigen-specific T cells. It will be of interest to determine whether Aβ-specific autoreactive T cells in APP<sup>−/−</sup> ESC-TEP-transplanted AD mice only activate the macrophages that specifically phagocytose Aβ, avoiding unnecessary local tissue damage.</p><p>In summary, we have demonstrated that transplantation of APP<sup>+/+</sup> or APP<sup>−/−</sup> mESC-TEPs into AD mice attenuates AD pathology, which is associated with enhanced systemic IFNγ-producing T cells and CP gateway activity with increased expression levels of leukocyte homing and trafficking molecules, as well as increased number of macrophages in the CNS. Furthermore, transplantation of APP<sup>−/−</sup> mESC-TEPs has significantly greater effectiveness. This is related to the generation of T cells reactive with Aβ, which accompanied by increased number of anti-Aβ Ab-producing B cells in the spleen and enhanced level of anti-Aβ Ab in the serum, as well as an increased number of Aβ phagocytosing macrophages in the brain (<xref ref-type=\"fig\" rid=\"F8\">Figure 8</xref>). Our results suggest that transplantation of APP<sup>−/−</sup> human ESC-TEPs or iPSC-TEPs has the potential to be used in the prevention and treatment of AD patients.</p><fig id=\"F8\" position=\"float\"><label>Figure 8</label><caption><p>Diagrams showing the mechanisms by which transplantation of APP<sup>+/+</sup> or APP<sup>−/−</sup> mESC-TEPs into AD mice attenuates AD pathology. <bold>(A)</bold> Schematic of transplantation of mESC-derived cells and analyses of the AD mice. <bold>(B,C)</bold> Diagrams of the mechanisms of transplantation of <bold>(B)</bold> APP<sup>+/+</sup> or <bold>(C)</bold> APP<sup>−/−</sup> mESC-TEPs into AD mice reduces Aβ plaque load and increases cognitive performance.</p></caption><graphic xlink:href=\"fimmu-11-01781-g0008\"/></fig></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The datasets generated for this study are available on request to the corresponding author.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The animal study was reviewed and approved by Institutional Animal Care and Use Committee of the University of Connecticut.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>JZ designed experiments, performed experiments, analyzed data, and wrote the manuscript. MS performed experiments and analyzed data. YL and HL performed experiments. ZH designed experiments, analyzed data, and supervised the study. LL designed experiments, analyzed data, supervised the study and wrote the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by grants from NIH (1R01AI123131, to LL), Connecticut Regenerative Medicine Research Fund (16-RMB-UCONN-02, to LL), Guizhou Medical University Graduate Student Innovation Program (Guiyi YJSCXJH [2019] 001, to JZ), National Natural Science Foundation of China (81871313, to ZH), and Guizhou Province Science and Technology Project (Qian Ke He [2016]4002, [2019]5406, to ZH).</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s9\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fimmu.2020.01781/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fimmu.2020.01781/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Data_Sheet_1.PDF\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><collab>Alzheimer's disease facts and figures</collab></person-group>\n<article-title>Alzheimer's & dementia</article-title>. <source>J Alzheimer's Assoc.</source> (<year>2016</year>) <volume>12</volume>:<fpage>459</fpage>–<lpage>509</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jalz.2016.03.001</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hardy</surname><given-names>J</given-names></name><name><surname>Selkoe</surname><given-names>DJ</given-names></name></person-group>. <article-title>The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics</article-title>. <source>Science.</source> (<year>2002</year>) <volume>297</volume>:<fpage>353</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1126/science.1072994</pub-id><pub-id pub-id-type=\"pmid\">12130773</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weiner</surname><given-names>HL</given-names></name><name><surname>Frenkel</surname><given-names>D</given-names></name></person-group>. <article-title>Immunology and immunotherapy of Alzheimer's disease</article-title>. <source>Nat Rev Immunol.</source> (<year>2006</year>) <volume>6</volume>:<fpage>404</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nri1843</pub-id><pub-id pub-id-type=\"pmid\">16639431</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Baruch</surname><given-names>K</given-names></name><name><surname>Deczkowska</surname><given-names>A</given-names></name><name><surname>Rosenzweig</surname><given-names>N</given-names></name><name><surname>Tsitsou-Kampeli</surname><given-names>A</given-names></name><name><surname>Sharif</surname><given-names>AM</given-names></name><name><surname>Matcovitch-Natan</surname><given-names>O</given-names></name><etal/></person-group>. <article-title>PD-1 immune checkpoint blockade reduces pathology and improves memory in mouse models of Alzheimer's disease</article-title>. <source>Nat Med.</source> (<year>2016</year>) <volume>22</volume>:<fpage>135</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nm.4022</pub-id><pub-id pub-id-type=\"pmid\">26779813</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ballard</surname><given-names>C</given-names></name><name><surname>Gauthier</surname><given-names>S</given-names></name><name><surname>Corbett</surname><given-names>A</given-names></name><name><surname>Brayne</surname><given-names>C</given-names></name><name><surname>Aarsland</surname><given-names>D</given-names></name><name><surname>Jones</surname><given-names>E</given-names></name></person-group>. <article-title>Alzheimer's disease</article-title>. <source>Lancet.</source> (<year>2011</year>) <volume>377</volume>:<fpage>1019</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0140-6736(10)61349-9</pub-id><pub-id pub-id-type=\"pmid\">21371747</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Guenette</surname><given-names>S</given-names></name><name><surname>Strecker</surname><given-names>P</given-names></name><name><surname>Kins</surname><given-names>S</given-names></name></person-group>. <article-title>APP protein family signaling at the synapse: insights from intracellular APP-binding proteins</article-title>. <source>Front Mol Neurosci.</source> (<year>2017</year>) <volume>10</volume>:<fpage>87</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fnmol.2017.00087</pub-id><pub-id pub-id-type=\"pmid\">28424586</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hamley</surname><given-names>IW</given-names></name></person-group>. <article-title>The amyloid beta peptide: a chemist's perspective. Role in Alzheimer's and fibrillization</article-title>. <source>Chem Rev.</source> (<year>2012</year>) <volume>112</volume>:<fpage>5147</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.1021/cr3000994</pub-id><pub-id pub-id-type=\"pmid\">22813427</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eisele</surname><given-names>YS</given-names></name><name><surname>Duyckaerts</surname><given-names>C</given-names></name></person-group>. <article-title>Propagation of Ass pathology: hypotheses, discoveries, and yet unresolved questions from experimental and human brain studies</article-title>. <source>Acta Neuropathol.</source> (<year>2016</year>) <volume>131</volume>:<fpage>5</fpage>–<lpage>25</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00401-015-1516-y</pub-id><pub-id pub-id-type=\"pmid\">26715565</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wyss-Coray</surname><given-names>T</given-names></name><name><surname>Rogers</surname><given-names>J</given-names></name></person-group>. <article-title>Inflammation in Alzheimer disease-a brief review of the basic science and clinical literature</article-title>. <source>Cold Spring Harbor Perspect Med.</source> (<year>2012</year>) <volume>2</volume>:<fpage>a006346</fpage>. <pub-id pub-id-type=\"doi\">10.1101/cshperspect.a006346</pub-id><pub-id pub-id-type=\"pmid\">22315714</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arvanitakis</surname><given-names>Z</given-names></name><name><surname>Grodstein</surname><given-names>F</given-names></name><name><surname>Bienias</surname><given-names>JL</given-names></name><name><surname>Schneider</surname><given-names>JA</given-names></name><name><surname>Wilson</surname><given-names>RS</given-names></name><name><surname>Kelly</surname><given-names>JF</given-names></name><etal/></person-group>. <article-title>Relation of NSAIDs to incident AD, change in cognitive function, AD pathology</article-title>. <source>Neurology.</source> (<year>2008</year>) <volume>70</volume>:<fpage>2219</fpage>–<lpage>25</lpage>. <pub-id pub-id-type=\"doi\">10.1212/01.wnl.0000313813.48505.86</pub-id><pub-id pub-id-type=\"pmid\">18519870</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martin</surname><given-names>BK</given-names></name><name><surname>Szekely</surname><given-names>C</given-names></name><name><surname>Brandt</surname><given-names>J</given-names></name><name><surname>Piantadosi</surname><given-names>S</given-names></name><name><surname>Breitner</surname><given-names>JC</given-names></name><name><surname>Craft</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Cognitive function over time in the Alzheimer's Disease Anti-inflammatory Prevention Trial (ADAPT): results of a randomized, controlled trial of naproxen and celecoxib</article-title>. <source>Arch Neurol.</source> (<year>2008</year>) <volume>65</volume>:<fpage>896</fpage>–<lpage>905</lpage>. <pub-id pub-id-type=\"doi\">10.1001/archneur.2008.65.7.nct70006</pub-id><pub-id pub-id-type=\"pmid\">18474729</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Doody</surname><given-names>RS</given-names></name><name><surname>Raman</surname><given-names>R</given-names></name><name><surname>Farlow</surname><given-names>M</given-names></name><name><surname>Iwatsubo</surname><given-names>T</given-names></name><name><surname>Vellas</surname><given-names>B</given-names></name><name><surname>Joffe</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>A phase 3 trial of semagacestat for treatment of Alzheimer's disease</article-title>. <source>N Engl J Med.</source> (<year>2013</year>) <volume>369</volume>:<fpage>341</fpage>–<lpage>50</lpage>. <pub-id pub-id-type=\"doi\">10.1056/NEJMoa1210951</pub-id><pub-id pub-id-type=\"pmid\">23883379</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Baruch</surname><given-names>K</given-names></name><name><surname>Rosenzweig</surname><given-names>N</given-names></name><name><surname>Kertser</surname><given-names>A</given-names></name><name><surname>Deczkowska</surname><given-names>A</given-names></name><name><surname>Sharif</surname><given-names>AM</given-names></name><name><surname>Spinrad</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Breaking immune tolerance by targeting Foxp3(+) regulatory T cells mitigates Alzheimer's disease pathology</article-title>. <source>Nat Commun.</source> (<year>2015</year>) <volume>6</volume>:<fpage>7967</fpage>. <pub-id pub-id-type=\"doi\">10.1038/ncomms8967</pub-id><pub-id pub-id-type=\"pmid\">26284939</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schwartz</surname><given-names>M</given-names></name><name><surname>Baruch</surname><given-names>K</given-names></name></person-group>. <article-title>Breaking peripheral immune tolerance to CNS antigens in neurodegenerative diseases: boosting autoimmunity to fight-off chronic neuroinflammation</article-title>. <source>J Autoimmun.</source> (<year>2014</year>) <volume>54</volume>:<fpage>8</fpage>–<lpage>14</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jaut.2014.08.002</pub-id><pub-id pub-id-type=\"pmid\">25199710</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kunis</surname><given-names>G</given-names></name><name><surname>Baruch</surname><given-names>K</given-names></name><name><surname>Rosenzweig</surname><given-names>N</given-names></name><name><surname>Kertser</surname><given-names>A</given-names></name><name><surname>Miller</surname><given-names>O</given-names></name><name><surname>Berkutzki</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>IFN-gamma-dependent activation of the brain's choroid plexus for CNS immune surveillance and repair</article-title>. <source>Brain.</source> (<year>2013</year>) <volume>136</volume>:<fpage>3427</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1093/brain/awt259</pub-id><pub-id pub-id-type=\"pmid\">24088808</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schwartz</surname><given-names>M</given-names></name><name><surname>Arad</surname><given-names>M</given-names></name><name><surname>Ben-Yehuda</surname><given-names>H</given-names></name></person-group>. <article-title>Potential immunotherapy for Alzheimer disease and age-related dementia</article-title>. <source>Dialogues Clin Neurosci.</source> (<year>2019</year>) <volume>21</volume>:<fpage>21</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.31887/DCNS.2019.21.1/mschwartz</pub-id><pub-id pub-id-type=\"pmid\">31607777</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Theriault</surname><given-names>P</given-names></name><name><surname>ElAli</surname><given-names>A</given-names></name><name><surname>Rivest</surname><given-names>S</given-names></name></person-group>. <article-title>The dynamics of monocytes and microglia in Alzheimer's disease</article-title>. <source>Alzheimer's Res Ther.</source> (<year>2015</year>) <volume>7</volume>:<fpage>41</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s13195-015-0125-2</pub-id><pub-id pub-id-type=\"pmid\">25878730</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ziv</surname><given-names>Y</given-names></name><name><surname>Ron</surname><given-names>N</given-names></name><name><surname>Butovsky</surname><given-names>O</given-names></name><name><surname>Landa</surname><given-names>G</given-names></name><name><surname>Sudai</surname><given-names>E</given-names></name><name><surname>Greenberg</surname><given-names>N</given-names></name><etal/></person-group>. <article-title>Immune cells contribute to the maintenance of neurogenesis and spatial learning abilities in adulthood</article-title>. <source>Nat Neurosci.</source> (<year>2006</year>) <volume>9</volume>:<fpage>268</fpage>–<lpage>75</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nn1629</pub-id><pub-id pub-id-type=\"pmid\">16415867</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Marsh</surname><given-names>SE</given-names></name><name><surname>Abud</surname><given-names>EM</given-names></name><name><surname>Lakatos</surname><given-names>A</given-names></name><name><surname>Karimzadeh</surname><given-names>A</given-names></name><name><surname>Yeung</surname><given-names>ST</given-names></name><name><surname>Davtyan</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>The adaptive immune system restrains Alzheimer's disease pathogenesis by modulating microglial function</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2016</year>) <volume>113</volume>:<fpage>E1316</fpage>–<lpage>25</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1525466113</pub-id><pub-id pub-id-type=\"pmid\">26884167</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rosenzweig</surname><given-names>N</given-names></name><name><surname>Dvir-Szternfeld</surname><given-names>R</given-names></name><name><surname>Tsitsou-Kampeli</surname><given-names>A</given-names></name><name><surname>Keren-Shaul</surname><given-names>H</given-names></name><name><surname>Ben-Yehuda</surname><given-names>H</given-names></name><name><surname>Weill-Raynal</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>PD-1/PD-L1 checkpoint blockade harnesses monocyte-derived macrophages to combat cognitive impairment in a tauopathy mouse model</article-title>. <source>Nat Commun.</source> (<year>2019</year>) <volume>10</volume>:<fpage>465</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41467-019-08352-5</pub-id><pub-id pub-id-type=\"pmid\">30692527</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yoles</surname><given-names>E</given-names></name><name><surname>Hauben</surname><given-names>E</given-names></name><name><surname>Palgi</surname><given-names>O</given-names></name><name><surname>Agranov</surname><given-names>E</given-names></name><name><surname>Gothilf</surname><given-names>A</given-names></name><name><surname>Cohen</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Protective autoimmunity is a physiological response to CNS trauma</article-title>. <source>J Neurosci.</source> (<year>2001</year>) <volume>21</volume>:<fpage>3740</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1523/JNEUROSCI.21-11-03740.2001</pub-id><pub-id pub-id-type=\"pmid\">11356861</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Miller</surname><given-names>AH</given-names></name></person-group>. <article-title>Depression and immunity: a role for T cells?</article-title>\n<source>Brain Behav Immunol.</source> (<year>2010</year>) <volume>24</volume>:<fpage>1</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbi.2009.09.009</pub-id><pub-id pub-id-type=\"pmid\">19818725</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wolf</surname><given-names>SA</given-names></name><name><surname>Steiner</surname><given-names>B</given-names></name><name><surname>Akpinarli</surname><given-names>A</given-names></name><name><surname>Kammertoens</surname><given-names>T</given-names></name><name><surname>Nassenstein</surname><given-names>C</given-names></name><name><surname>Braun</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>CD4-positive T lymphocytes provide a neuroimmunological link in the control of adult hippocampal neurogenesis</article-title>. <source>J Immunol.</source> (<year>2009</year>) <volume>182</volume>:<fpage>3979</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.0801218</pub-id><pub-id pub-id-type=\"pmid\">19299695</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Louveau</surname><given-names>A</given-names></name><name><surname>Harris</surname><given-names>TH</given-names></name><name><surname>Kipnis</surname><given-names>J</given-names></name></person-group>. <article-title>Revisiting the mechanisms of CNS immune privilege</article-title>. <source>Trends Immunol.</source> (<year>2015</year>) <volume>36</volume>:<fpage>569</fpage>–<lpage>77</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.it.2015.08.006</pub-id><pub-id pub-id-type=\"pmid\">26431936</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moalem</surname><given-names>G</given-names></name><name><surname>Leibowitz-Amit</surname><given-names>R</given-names></name><name><surname>Yoles</surname><given-names>E</given-names></name><name><surname>Mor</surname><given-names>F</given-names></name><name><surname>Cohen</surname><given-names>IR</given-names></name><name><surname>Schwartz</surname><given-names>M</given-names></name></person-group>. <article-title>Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy</article-title>. <source>Nat Med.</source> (<year>1999</year>) <volume>5</volume>:<fpage>49</fpage>–<lpage>55</lpage>. <pub-id pub-id-type=\"doi\">10.1038/4734</pub-id><pub-id pub-id-type=\"pmid\">9883839</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shechter</surname><given-names>R</given-names></name><name><surname>London</surname><given-names>A</given-names></name><name><surname>Varol</surname><given-names>C</given-names></name><name><surname>Raposo</surname><given-names>C</given-names></name><name><surname>Cusimano</surname><given-names>M</given-names></name><name><surname>Yovel</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Infiltrating blood-derived macrophages are vital cells playing an anti-inflammatory role in recovery from spinal cord injury in mice</article-title>. <source>PLoS Med.</source> (<year>2009</year>) <volume>6</volume>:<fpage>e1000113</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pmed.1000113</pub-id><pub-id pub-id-type=\"pmid\">19636355</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rapalino</surname><given-names>O</given-names></name><name><surname>Lazarov-Spiegler</surname><given-names>O</given-names></name><name><surname>Agranov</surname><given-names>E</given-names></name><name><surname>Velan</surname><given-names>GJ</given-names></name><name><surname>Yoles</surname><given-names>E</given-names></name><name><surname>Fraidakis</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Implantation of stimulated homologous macrophages results in partial recovery of paraplegic rats</article-title>. <source>Nat Med.</source> (<year>1998</year>) <volume>4</volume>:<fpage>814</fpage>–<lpage>21</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nm0798-814</pub-id><pub-id pub-id-type=\"pmid\">9662373</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>London</surname><given-names>A</given-names></name><name><surname>Itskovich</surname><given-names>E</given-names></name><name><surname>Benhar</surname><given-names>I</given-names></name><name><surname>Kalchenko</surname><given-names>V</given-names></name><name><surname>Mack</surname><given-names>M</given-names></name><name><surname>Jung</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Neuroprotection and progenitor cell renewal in the injured adult murine retina requires healing monocyte-derived macrophages</article-title>. <source>J Exp Med.</source> (<year>2011</year>) <volume>208</volume>:<fpage>23</fpage>–<lpage>39</lpage>. <pub-id pub-id-type=\"doi\">10.1084/jem.20101202</pub-id><pub-id pub-id-type=\"pmid\">21220455</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Benowitz</surname><given-names>LI</given-names></name><name><surname>Popovich</surname><given-names>PG</given-names></name></person-group>. <article-title>Inflammation and axon regeneration</article-title>. <source>Curr Opin Neurol.</source> (<year>2011</year>) <volume>24</volume>:<fpage>577</fpage>–<lpage>83</lpage>. <pub-id pub-id-type=\"doi\">10.1097/WCO.0b013e32834c208d</pub-id><pub-id pub-id-type=\"pmid\">21968547</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dorshkind</surname><given-names>K</given-names></name><name><surname>Swain</surname><given-names>S</given-names></name></person-group>. <article-title>Age-associated declines in immune system development and function: causes, consequences, and reversal</article-title>. <source>Curr Opin Immunol.</source> (<year>2009</year>) <volume>21</volume>:<fpage>404</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.coi.2009.07.001</pub-id><pub-id pub-id-type=\"pmid\">19632102</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Swain</surname><given-names>SL</given-names></name><name><surname>Nikolich-Zugich</surname><given-names>J</given-names></name></person-group>. <article-title>Key research opportunities in immune system aging</article-title>. <source>J Gerontol A Biol Sci Med Sci.</source> (<year>2009</year>) <volume>64</volume>:<fpage>183</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1093/gerona/gln068</pub-id><pub-id pub-id-type=\"pmid\">19251915</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Taub</surname><given-names>DD</given-names></name><name><surname>Longo</surname><given-names>DL</given-names></name></person-group>. <article-title>Insights into thymic aging and regeneration</article-title>. <source>Immunol Rev.</source> (<year>2005</year>) <volume>205</volume>:<fpage>72</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.0105-2896.2005.00275.x</pub-id><pub-id pub-id-type=\"pmid\">15882346</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lynch</surname><given-names>HE</given-names></name><name><surname>Goldberg</surname><given-names>GL</given-names></name><name><surname>Chidgey</surname><given-names>A</given-names></name><name><surname>van den Brink</surname><given-names>MR</given-names></name><name><surname>Boyd</surname><given-names>R</given-names></name><name><surname>Sempowski</surname><given-names>GD</given-names></name></person-group>. <article-title>Thymic involution and immune reconstitution</article-title>. <source>Trends Immunol.</source> (<year>2009</year>) <volume>30</volume>:<fpage>366</fpage>–<lpage>73</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.it.2009.04.003</pub-id><pub-id pub-id-type=\"pmid\">19540807</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aspinall</surname><given-names>R</given-names></name><name><surname>Pitts</surname><given-names>D</given-names></name><name><surname>Lapenna</surname><given-names>A</given-names></name><name><surname>Mitchell</surname><given-names>W</given-names></name></person-group>. <article-title>Immunity in the elderly: the role of the thymus</article-title>. <source>J Comp Pathol.</source> (<year>2010</year>) <volume>142</volume>(<issue>Suppl. 1</issue>):<fpage>S111</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jcpa.2009.10.022</pub-id><pub-id pub-id-type=\"pmid\">19954794</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chidgey</surname><given-names>A</given-names></name><name><surname>Dudakov</surname><given-names>J</given-names></name><name><surname>Seach</surname><given-names>N</given-names></name><name><surname>Boyd</surname><given-names>R</given-names></name></person-group>. <article-title>Impact of niche aging on thymic regeneration and immune reconstitution</article-title>. <source>Semin Immunol.</source> (<year>2007</year>) <volume>19</volume>:<fpage>331</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.smim.2007.10.006</pub-id><pub-id pub-id-type=\"pmid\">18024073</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Anderson</surname><given-names>G</given-names></name><name><surname>Takahama</surname><given-names>Y</given-names></name></person-group>. <article-title>Thymic epithelial cells: working class heroes for T cell development and repertoire selection</article-title>. <source>Trends Immunol.</source> (<year>2012</year>) <volume>33</volume>:<fpage>256</fpage>–<lpage>63</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.it.2012.03.005</pub-id><pub-id pub-id-type=\"pmid\">22591984</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zediak</surname><given-names>VP</given-names></name><name><surname>Bhandoola</surname><given-names>A</given-names></name></person-group>. <article-title>Aging and T cell development: interplay between progenitors and their environment</article-title>. <source>Semin Immunol.</source> (<year>2005</year>) <volume>17</volume>:<fpage>337</fpage>–<lpage>46</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.smim.2005.05.004</pub-id><pub-id pub-id-type=\"pmid\">15961315</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ciofani</surname><given-names>M</given-names></name><name><surname>Zuniga-Pflucker</surname><given-names>JC</given-names></name></person-group>. <article-title>The thymus as an inductive site for T lymphopoiesis</article-title>. <source>Annu Rev Cell Dev Biol.</source> (<year>2007</year>) <volume>23</volume>:<fpage>463</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1146/annurev.cellbio.23.090506.123547</pub-id><pub-id pub-id-type=\"pmid\">17506693</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mondino</surname><given-names>A</given-names></name><name><surname>Khoruts</surname><given-names>A</given-names></name><name><surname>Jenkins</surname><given-names>MK</given-names></name></person-group>. <article-title>The anatomy of T-cell activation and tolerance</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>1996</year>) <volume>93</volume>:<fpage>2245</fpage>–<lpage>52</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.93.6.2245</pub-id><pub-id pub-id-type=\"pmid\">8637857</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Houssaint</surname><given-names>E</given-names></name><name><surname>Flajnik</surname><given-names>M</given-names></name></person-group>. <article-title>The role of thymic epithelium in the acquisition of tolerance</article-title>. <source>Immunol Today.</source> (<year>1990</year>) <volume>11</volume>:<fpage>357</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.1016/0167-5699(90)90141-U</pub-id><pub-id pub-id-type=\"pmid\">2222760</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sun</surname><given-names>L</given-names></name><name><surname>Guo</surname><given-names>J</given-names></name><name><surname>Brown</surname><given-names>R</given-names></name><name><surname>Amagai</surname><given-names>T</given-names></name><name><surname>Zhao</surname><given-names>Y</given-names></name><name><surname>Su</surname><given-names>DM</given-names></name></person-group>. <article-title>Declining expression of a single epithelial cell-autonomous gene accelerates age-related thymic involution</article-title>. <source>Aging Cell.</source> (<year>2010</year>) <volume>9</volume>:<fpage>347</fpage>–<lpage>57</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1474-9726.2010.00559.x</pub-id><pub-id pub-id-type=\"pmid\">20156205</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Murry</surname><given-names>CE</given-names></name><name><surname>Keller</surname><given-names>G</given-names></name></person-group>. <article-title>Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development</article-title>. <source>Cell.</source> (<year>2008</year>) <volume>132</volume>:<fpage>661</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cell.2008.02.008</pub-id><pub-id pub-id-type=\"pmid\">18295582</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hu</surname><given-names>R</given-names></name><name><surname>Liu</surname><given-names>Y</given-names></name><name><surname>Su</surname><given-names>M</given-names></name><name><surname>Song</surname><given-names>Y</given-names></name><name><surname>Rood</surname><given-names>D</given-names></name><name><surname>Lai</surname><given-names>L</given-names></name></person-group>. <article-title>Transplantation of donor-origin mouse embryonic stem cell-derived thymic epithelial progenitors prevents the development of chronic graft-versus-host disease in mice</article-title>. <source>Stem Cells Transl Med.</source> (<year>2017</year>) <volume>6</volume>:<fpage>121</fpage>–<lpage>30</lpage>. <pub-id pub-id-type=\"doi\">10.5966/sctm.2016-0012</pub-id><pub-id pub-id-type=\"pmid\">28170174</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lai</surname><given-names>L</given-names></name><name><surname>Cui</surname><given-names>C</given-names></name><name><surname>Jin</surname><given-names>J</given-names></name><name><surname>Hao</surname><given-names>Z</given-names></name><name><surname>Zheng</surname><given-names>Q</given-names></name><name><surname>Ying</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Mouse embryonic stem cell-derived thymic epithelial cell progenitors enhance T-cell reconstitution after allogeneic bone marrow transplantation</article-title>. <source>Blood.</source> (<year>2011</year>) <volume>118</volume>:<fpage>3410</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1182/blood-2011-03-340794</pub-id><pub-id pub-id-type=\"pmid\">21791423</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lai</surname><given-names>L</given-names></name><name><surname>Jin</surname><given-names>J</given-names></name></person-group>. <article-title>Generation of thymic epithelial cell progenitors by mouse embryonic stem cells</article-title>. <source>Stem Cells.</source> (<year>2009</year>) <volume>27</volume>:<fpage>3012</fpage>–<lpage>20</lpage>. <pub-id pub-id-type=\"doi\">10.1002/stem.238</pub-id><pub-id pub-id-type=\"pmid\">19824081</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Su</surname><given-names>M</given-names></name><name><surname>Hu</surname><given-names>R</given-names></name><name><surname>Jin</surname><given-names>J</given-names></name><name><surname>Yan</surname><given-names>Y</given-names></name><name><surname>Song</surname><given-names>Y</given-names></name><name><surname>Sullivan</surname><given-names>R</given-names></name><etal/></person-group>. <article-title>Efficient <italic>in vitro</italic> generation of functional thymic epithelial progenitors from human embryonic stem cells</article-title>. <source>Sci Rep.</source> (<year>2015</year>) <volume>5</volume>:<fpage>9882</fpage>. <pub-id pub-id-type=\"doi\">10.1038/srep09882</pub-id><pub-id pub-id-type=\"pmid\">26044259</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Su</surname><given-names>M</given-names></name><name><surname>Lin</surname><given-names>Y</given-names></name><name><surname>Cui</surname><given-names>C</given-names></name><name><surname>Tian</surname><given-names>X</given-names></name><name><surname>Lu</surname><given-names>X</given-names></name><name><surname>He</surname><given-names>Z</given-names></name><etal/></person-group>. <article-title>ESC-derived thymic epithelial cells expressing MOG prevents EAE by central and peripheral tolerance mechanisms</article-title>. <source>Cell Immunol.</source> (<year>2017</year>) <volume>322</volume>:<fpage>84</fpage>–<lpage>91</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cellimm.2017.10.007</pub-id><pub-id pub-id-type=\"pmid\">29074250</pub-id></mixed-citation></ref><ref id=\"B48\"><label>48.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Su</surname><given-names>M</given-names></name><name><surname>Song</surname><given-names>Y</given-names></name><name><surname>He</surname><given-names>Z</given-names></name><name><surname>Hu</surname><given-names>R</given-names></name><name><surname>Rood</surname><given-names>D</given-names></name><name><surname>Lai</surname><given-names>L</given-names></name></person-group>. <article-title>Administration of embryonic stem cell-derived thymic epithelial progenitors expressing MOG induces antigen-specific tolerance and ameliorates experimental autoimmune encephalomyelitis</article-title>. <source>J Autoimmun.</source> (<year>2015</year>) <volume>58</volume>:<fpage>36</fpage>–<lpage>47</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jaut.2015.01.002</pub-id><pub-id pub-id-type=\"pmid\">25618825</pub-id></mixed-citation></ref><ref id=\"B49\"><label>49.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Song</surname><given-names>Y</given-names></name><name><surname>Su</surname><given-names>M</given-names></name><name><surname>Zhu</surname><given-names>J</given-names></name><name><surname>Di</surname><given-names>W</given-names></name><name><surname>Liu</surname><given-names>Y</given-names></name><name><surname>Hu</surname><given-names>R</given-names></name><etal/></person-group>. <article-title>FOXN1 recombinant protein enhances T-cell regeneration after hematopoietic stem cell transplantation in mice</article-title>. <source>Eur J Immunol.</source> (<year>2016</year>) <volume>46</volume>:<fpage>1518</fpage>–<lpage>28</lpage>. <pub-id pub-id-type=\"doi\">10.1002/eji.201546196</pub-id><pub-id pub-id-type=\"pmid\">27125859</pub-id></mixed-citation></ref><ref id=\"B50\"><label>50.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Seach</surname><given-names>N</given-names></name><name><surname>Wong</surname><given-names>K</given-names></name><name><surname>Hammett</surname><given-names>M</given-names></name><name><surname>Boyd</surname><given-names>RL</given-names></name><name><surname>Chidgey</surname><given-names>AP</given-names></name></person-group>. <article-title>Purified enzymes improve isolation and characterization of the adult thymic epithelium</article-title>. <source>J Immunol Methods.</source> (<year>2012</year>) <volume>385</volume>:<fpage>23</fpage>–<lpage>34</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jim.2012.07.023</pub-id><pub-id pub-id-type=\"pmid\">22910002</pub-id></mixed-citation></ref><ref id=\"B51\"><label>51.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lai</surname><given-names>L</given-names></name><name><surname>Zhang</surname><given-names>M</given-names></name><name><surname>Goldschneider</surname><given-names>I</given-names></name></person-group>. <article-title>Recombinant IL-7/HGFbeta efficiently induces transplantable murine hematopoietic stem cells</article-title>. <source>J Clin Invest.</source> (<year>2012</year>) <volume>122</volume>:<fpage>3552</fpage>–<lpage>62</lpage>. <pub-id pub-id-type=\"doi\">10.1172/JCI46055</pub-id><pub-id pub-id-type=\"pmid\">22996694</pub-id></mixed-citation></ref><ref id=\"B52\"><label>52.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lai</surname><given-names>L</given-names></name><name><surname>Jin</surname><given-names>J</given-names></name><name><surname>Goldschneider</surname><given-names>I</given-names></name></person-group>. <article-title><italic>In vivo</italic> antitumor activity of a recombinant IL-7/HGFbeta hybrid cytokine in mice</article-title>. <source>Cancer Res.</source> (<year>2011</year>) <volume>71</volume>:<fpage>61</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1158/0008-5472.CAN-10-3198</pub-id><pub-id pub-id-type=\"pmid\">21084268</pub-id></mixed-citation></ref><ref id=\"B53\"><label>53.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Melchior</surname><given-names>B</given-names></name><name><surname>Garcia</surname><given-names>AE</given-names></name><name><surname>Hsiung</surname><given-names>BK</given-names></name><name><surname>Lo</surname><given-names>KM</given-names></name><name><surname>Doose</surname><given-names>JM</given-names></name><name><surname>Thrash</surname><given-names>JC</given-names></name><etal/></person-group>. <article-title>Dual induction of TREM2 and tolerance-related transcript, Tmem176b, in amyloid transgenic mice: implications for vaccine-based therapies for Alzheimer's disease</article-title>. <source>ASN Neuro.</source> (<year>2010</year>) <volume>2</volume>:<fpage>e00037</fpage>. <pub-id pub-id-type=\"doi\">10.1042/AN20100010</pub-id><pub-id pub-id-type=\"pmid\">20640189</pub-id></mixed-citation></ref><ref id=\"B54\"><label>54.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martinez-Coria</surname><given-names>H</given-names></name><name><surname>Yeung</surname><given-names>ST</given-names></name><name><surname>Ager</surname><given-names>RR</given-names></name><name><surname>Rodriguez-Ortiz</surname><given-names>CJ</given-names></name><name><surname>Baglietto-Vargas</surname><given-names>D</given-names></name><name><surname>LaFerla</surname><given-names>FM</given-names></name></person-group>. <article-title>Repeated cognitive stimulation alleviates memory impairments in an Alzheimer's disease mouse model</article-title>. <source>Brain Res Bull.</source> (<year>2015</year>) <volume>117</volume>:<fpage>10</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.brainresbull.2015.07.001</pub-id><pub-id pub-id-type=\"pmid\">26162480</pub-id></mixed-citation></ref><ref id=\"B55\"><label>55.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Clinton</surname><given-names>LK</given-names></name><name><surname>Billings</surname><given-names>LM</given-names></name><name><surname>Green</surname><given-names>KN</given-names></name><name><surname>Caccamo</surname><given-names>A</given-names></name><name><surname>Ngo</surname><given-names>J</given-names></name><name><surname>Oddo</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Age-dependent sexual dimorphism in cognition and stress response in the 3xTg-AD mice</article-title>. <source>Neurobiol Dis.</source> (<year>2007</year>) <volume>28</volume>:<fpage>76</fpage>–<lpage>82</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.nbd.2007.06.013</pub-id><pub-id pub-id-type=\"pmid\">17659878</pub-id></mixed-citation></ref><ref id=\"B56\"><label>56.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>St-Amour</surname><given-names>I</given-names></name><name><surname>Pare</surname><given-names>I</given-names></name><name><surname>Tremblay</surname><given-names>C</given-names></name><name><surname>Coulombe</surname><given-names>K</given-names></name><name><surname>Bazin</surname><given-names>R</given-names></name><name><surname>Calon</surname><given-names>F</given-names></name></person-group>. <article-title>IVIg protects the 3xTg-AD mouse model of Alzheimer's disease from memory deficit and Aβ pathology</article-title>. <source>J Neuroinflamm.</source> (<year>2014</year>) <volume>11</volume>:<fpage>54</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1742-2094-11-54</pub-id><pub-id pub-id-type=\"pmid\">24655894</pub-id></mixed-citation></ref><ref id=\"B57\"><label>57.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cohen</surname><given-names>SJ</given-names></name><name><surname>Stackman</surname><given-names>RW</given-names><suffix>Jr</suffix></name></person-group>. <article-title>Assessing rodent hippocampal involvement in the novel object recognition task</article-title>. a review. <source>Behav Brain Res.</source> (<year>2015</year>) <volume>285</volume>:<fpage>105</fpage>–<lpage>17</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbr.2014.08.002</pub-id><pub-id pub-id-type=\"pmid\">25169255</pub-id></mixed-citation></ref><ref id=\"B58\"><label>58.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Maras</surname><given-names>PM</given-names></name><name><surname>Molet</surname><given-names>J</given-names></name><name><surname>Chen</surname><given-names>Y</given-names></name><name><surname>Rice</surname><given-names>C</given-names></name><name><surname>Ji</surname><given-names>SG</given-names></name><name><surname>Solodkin</surname><given-names>A</given-names></name><etal/></person-group>\n<article-title>Preferential loss of dorsal-hippocampus synapses underlies memory impairments provoked by short, multimodal stress</article-title>. <source>Mol Psychiatry.</source> (<year>2014</year>) <volume>19</volume>:<fpage>811</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.1038/mp.2014.12</pub-id><pub-id pub-id-type=\"pmid\">24589888</pub-id></mixed-citation></ref><ref id=\"B59\"><label>59.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stover</surname><given-names>KR</given-names></name><name><surname>Campbell</surname><given-names>MA</given-names></name><name><surname>van Winssen</surname><given-names>CM</given-names></name><name><surname>Brown</surname><given-names>RE</given-names></name></person-group>. <article-title>Early detection of cognitive deficits in the 3xTg-AD mouse model of Alzheimer's disease</article-title>. <source>Behav Brain Res.</source> (<year>2015</year>) <volume>289</volume>:<fpage>29</fpage>–<lpage>38</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbr.2015.04.012</pub-id><pub-id pub-id-type=\"pmid\">25896362</pub-id></mixed-citation></ref><ref id=\"B60\"><label>60.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arsenault</surname><given-names>D</given-names></name><name><surname>Julien</surname><given-names>C</given-names></name><name><surname>Tremblay</surname><given-names>C</given-names></name><name><surname>Calon</surname><given-names>F</given-names></name></person-group>. <article-title>DHA improves cognition and prevents dysfunction of entorhinal cortex neurons in 3xTg-AD mice</article-title>. <source>PLoS ONE.</source> (<year>2011</year>) <volume>6</volume>:<fpage>e17397</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0017397</pub-id><pub-id pub-id-type=\"pmid\">21383850</pub-id></mixed-citation></ref><ref id=\"B61\"><label>61.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shankar</surname><given-names>GM</given-names></name><name><surname>Li</surname><given-names>S</given-names></name><name><surname>Mehta</surname><given-names>TH</given-names></name><name><surname>Garcia-Munoz</surname><given-names>A</given-names></name><name><surname>Shepardson</surname><given-names>NE</given-names></name><name><surname>Smith</surname><given-names>I</given-names></name><etal/></person-group>. <article-title>Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory</article-title>. <source>Nat Med.</source> (<year>2008</year>) <volume>14</volume>:<fpage>837</fpage>–<lpage>42</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nm1782</pub-id><pub-id pub-id-type=\"pmid\">18568035</pub-id></mixed-citation></ref><ref id=\"B62\"><label>62.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Naert</surname><given-names>G</given-names></name><name><surname>Rivest</surname><given-names>S</given-names></name></person-group>. <article-title>A deficiency in CCR2+ monocytes: the hidden side of Alzheimer's disease</article-title>. <source>J Mol Cell Biol.</source> (<year>2013</year>) <volume>5</volume>:<fpage>284</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1093/jmcb/mjt028</pub-id><pub-id pub-id-type=\"pmid\">23892208</pub-id></mixed-citation></ref><ref id=\"B63\"><label>63.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>El Khoury</surname><given-names>J</given-names></name><name><surname>Hickman</surname><given-names>SE</given-names></name><name><surname>Thomas</surname><given-names>CA</given-names></name><name><surname>Cao</surname><given-names>L</given-names></name><name><surname>Silverstein</surname><given-names>SC</given-names></name><name><surname>Loike</surname><given-names>JD</given-names></name></person-group>. <article-title>Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils</article-title>. <source>Nature.</source> (<year>1996</year>) <volume>382</volume>:<fpage>716</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1038/382716a0</pub-id><pub-id pub-id-type=\"pmid\">8751442</pub-id></mixed-citation></ref><ref id=\"B64\"><label>64.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schwartz</surname><given-names>M</given-names></name></person-group>. <article-title>Can immunotherapy treat neurodegeneration?</article-title>\n<source>Science.</source> (<year>2017</year>) <volume>357</volume>:<fpage>254</fpage>–<lpage>55</lpage>. <pub-id pub-id-type=\"doi\">10.1126/science.aai8231</pub-id><pub-id pub-id-type=\"pmid\">28729500</pub-id></mixed-citation></ref><ref id=\"B65\"><label>65.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Derecki</surname><given-names>NC</given-names></name><name><surname>Cardani</surname><given-names>AN</given-names></name><name><surname>Yang</surname><given-names>CH</given-names></name><name><surname>Quinnies</surname><given-names>KM</given-names></name><name><surname>Crihfield</surname><given-names>A</given-names></name><name><surname>Lynch</surname><given-names>KR</given-names></name><etal/></person-group>. <article-title>Regulation of learning and memory by meningeal immunity: a key role for IL-4</article-title>. <source>J Exp Med.</source> (<year>2010</year>) <volume>207</volume>:<fpage>1067</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.1084/jem.20091419</pub-id><pub-id pub-id-type=\"pmid\">20439540</pub-id></mixed-citation></ref><ref id=\"B66\"><label>66.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shechter</surname><given-names>R</given-names></name><name><surname>Miller</surname><given-names>O</given-names></name><name><surname>Yovel</surname><given-names>G</given-names></name><name><surname>Rosenzweig</surname><given-names>N</given-names></name><name><surname>London</surname><given-names>A</given-names></name><name><surname>Ruckh</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Recruitment of beneficial M2 macrophages to injured spinal cord is orchestrated by remote brain choroid plexus</article-title>. <source>Immunity.</source> (<year>2013</year>) <volume>38</volume>:<fpage>555</fpage>–<lpage>69</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.immuni.2013.02.012</pub-id><pub-id pub-id-type=\"pmid\">23477737</pub-id></mixed-citation></ref><ref id=\"B67\"><label>67.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schwartz</surname><given-names>M</given-names></name><name><surname>Ziv</surname><given-names>Y</given-names></name></person-group>. <article-title>Immunity to self and self-maintenance: what can tumor immunology teach us about ALS and Alzheimer's disease?</article-title>\n<source>Trends Pharmacol Sci.</source> (<year>2008</year>) <volume>29</volume>:<fpage>287</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.tips.2008.03.006</pub-id><pub-id pub-id-type=\"pmid\">18453002</pub-id></mixed-citation></ref><ref id=\"B68\"><label>68.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schwartz</surname><given-names>M</given-names></name><name><surname>Baruch</surname><given-names>K</given-names></name></person-group>. <article-title>The resolution of neuroinflammation in neurodegeneration: leukocyte recruitment via the choroid plexus</article-title>. <source>EMBO J.</source> (<year>2014</year>) <volume>33</volume>:<fpage>7</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.1002/embj.201386609</pub-id><pub-id pub-id-type=\"pmid\">24357543</pub-id></mixed-citation></ref><ref id=\"B69\"><label>69.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Simard</surname><given-names>AR</given-names></name><name><surname>Soulet</surname><given-names>D</given-names></name><name><surname>Gowing</surname><given-names>G</given-names></name><name><surname>Julien</surname><given-names>JP</given-names></name><name><surname>Rivest</surname><given-names>S</given-names></name></person-group>. <article-title>Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease</article-title>. <source>Neuron.</source> (<year>2006</year>) <volume>49</volume>:<fpage>489</fpage>–<lpage>502</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuron.2006.01.022</pub-id><pub-id pub-id-type=\"pmid\">16476660</pub-id></mixed-citation></ref><ref id=\"B70\"><label>70.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Koronyo-Hamaoui</surname><given-names>M</given-names></name><name><surname>Ko</surname><given-names>MK</given-names></name><name><surname>Koronyo</surname><given-names>Y</given-names></name><name><surname>Azoulay</surname><given-names>D</given-names></name><name><surname>Seksenyan</surname><given-names>A</given-names></name><name><surname>Kunis</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Attenuation of AD-like neuropathology by harnessing peripheral immune cells: local elevation of IL-10 and MMP-9</article-title>. <source>J Neurochem.</source> (<year>2009</year>) <volume>111</volume>:<fpage>1409</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1471-4159.2009.06402.x</pub-id><pub-id pub-id-type=\"pmid\">19780903</pub-id></mixed-citation></ref><ref id=\"B71\"><label>71.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Koronyo</surname><given-names>Y</given-names></name><name><surname>Salumbides</surname><given-names>BC</given-names></name><name><surname>Sheyn</surname><given-names>J</given-names></name><name><surname>Pelissier</surname><given-names>L</given-names></name><name><surname>Li</surname><given-names>S</given-names></name><name><surname>Ljubimov</surname><given-names>V</given-names></name><etal/></person-group>. <article-title>Therapeutic effects of glatiramer acetate and grafted CD115(+) monocytes in a mouse model of Alzheimer's disease</article-title>. <source>Brain.</source> (<year>2015</year>) <volume>138</volume>:<fpage>2399</fpage>–<lpage>422</lpage>. <pub-id pub-id-type=\"doi\">10.1093/brain/awv150</pub-id><pub-id pub-id-type=\"pmid\">26049087</pub-id></mixed-citation></ref><ref id=\"B72\"><label>72.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mildner</surname><given-names>A</given-names></name><name><surname>Schlevogt</surname><given-names>B</given-names></name><name><surname>Kierdorf</surname><given-names>K</given-names></name><name><surname>Bottcher</surname><given-names>C</given-names></name><name><surname>Erny</surname><given-names>D</given-names></name><name><surname>Kummer</surname><given-names>MP</given-names></name><etal/></person-group>. <article-title>Distinct and non-redundant roles of microglia and myeloid subsets in mouse models of Alzheimer's disease</article-title>. <source>J Neurosci.</source> (<year>2011</year>) <volume>31</volume>:<fpage>11159</fpage>–<lpage>71</lpage>. <pub-id pub-id-type=\"doi\">10.1523/JNEUROSCI.6209-10.2011</pub-id><pub-id pub-id-type=\"pmid\">21813677</pub-id></mixed-citation></ref><ref id=\"B73\"><label>73.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parker</surname><given-names>DC</given-names></name></person-group>. <article-title>The functions of antigen recognition in T cell-dependent B cell activation</article-title>. <source>Semin Immunol.</source> (<year>1993</year>) <volume>5</volume>:<fpage>413</fpage>–<lpage>20</lpage>. <pub-id pub-id-type=\"doi\">10.1006/smim.1993.1047</pub-id><pub-id pub-id-type=\"pmid\">8123796</pub-id></mixed-citation></ref><ref id=\"B74\"><label>74.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parker</surname><given-names>DC</given-names></name></person-group>\n<article-title>T cell-dependent B cell activation</article-title>. <source>Annu Rev Immunol.</source> (<year>1993</year>) <volume>11</volume>:<fpage>331</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.1146/annurev.iy.11.040193.001555</pub-id><pub-id pub-id-type=\"pmid\">8476565</pub-id></mixed-citation></ref><ref id=\"B75\"><label>75.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stout</surname><given-names>RD</given-names></name><name><surname>Bottomly</surname><given-names>K</given-names></name></person-group>. <article-title>Antigen-specific activation of effector macrophages by IFN-gamma producing (TH1) T cell clones. Failure of IL-4-producing (TH2) T cell clones to activate effector function in macrophages</article-title>. <source>J Immunol.</source> (<year>1989</year>) <volume>142</volume>:<fpage>760</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"pmid\">2464024</pub-id></mixed-citation></ref><ref id=\"B76\"><label>76.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Raposo</surname><given-names>C</given-names></name><name><surname>Graubardt</surname><given-names>N</given-names></name><name><surname>Cohen</surname><given-names>M</given-names></name><name><surname>Eitan</surname><given-names>C</given-names></name><name><surname>London</surname><given-names>A</given-names></name><name><surname>Berkutzki</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>CNS repair requires both effector and regulatory T cells with distinct temporal and spatial profiles</article-title>. <source>J Neurosci.</source> (<year>2014</year>) <volume>34</volume>:<fpage>10141</fpage>–<lpage>55</lpage>. <pub-id pub-id-type=\"doi\">10.1523/JNEUROSCI.0076-14.2014</pub-id><pub-id pub-id-type=\"pmid\">25080578</pub-id></mixed-citation></ref><ref id=\"B77\"><label>77.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Beers</surname><given-names>DR</given-names></name><name><surname>Henkel</surname><given-names>JS</given-names></name><name><surname>Zhao</surname><given-names>W</given-names></name><name><surname>Wang</surname><given-names>J</given-names></name><name><surname>Appel</surname><given-names>SH</given-names></name></person-group>. <article-title>CD4+ T cells support glial neuroprotection, slow disease progression, and modify glial morphology in an animal model of inherited ALS</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2008</year>) <volume>105</volume>:<fpage>15558</fpage>–<lpage>63</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.0807419105</pub-id><pub-id pub-id-type=\"pmid\">18809917</pub-id></mixed-citation></ref><ref id=\"B78\"><label>78.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kunis</surname><given-names>G</given-names></name><name><surname>Baruch</surname><given-names>K</given-names></name><name><surname>Miller</surname><given-names>O</given-names></name><name><surname>Schwartz</surname><given-names>M</given-names></name></person-group>. <article-title>Immunization with a myelin-derived antigen activates the brain's choroid plexus for recruitment of immunoregulatory cells to the CNS and attenuates disease progression in a mouse model of ALS</article-title>. <source>J Neurosci.</source> (<year>2015</year>) <volume>35</volume>:<fpage>6381</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1523/JNEUROSCI.3644-14.2015</pub-id><pub-id pub-id-type=\"pmid\">25904790</pub-id></mixed-citation></ref><ref id=\"B79\"><label>79.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wisniewski</surname><given-names>HM</given-names></name><name><surname>Barcikowska</surname><given-names>M</given-names></name><name><surname>Kida</surname><given-names>E</given-names></name></person-group>. <article-title>Phagocytosis of beta/A4 amyloid fibrils of the neuritic neocortical plaques</article-title>. <source>Acta Neuropathol.</source> (<year>1991</year>) <volume>81</volume>:<fpage>588</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1007/BF00310142</pub-id><pub-id pub-id-type=\"pmid\">1858487</pub-id></mixed-citation></ref><ref id=\"B80\"><label>80.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Akiyama</surname><given-names>H</given-names></name><name><surname>Kondo</surname><given-names>H</given-names></name><name><surname>Mori</surname><given-names>H</given-names></name><name><surname>Kametani</surname><given-names>F</given-names></name><name><surname>Nishimura</surname><given-names>T</given-names></name><name><surname>Ikeda</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>The amino-terminally truncated forms of amyloid beta-protein in brain macrophages in the ischemic lesions of Alzheimer's disease patients</article-title>. <source>Neurosci Lett.</source> (<year>1996</year>) <volume>219</volume>:<fpage>115</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0304-3940(96)13197-9</pub-id><pub-id pub-id-type=\"pmid\">8971793</pub-id></mixed-citation></ref><ref id=\"B81\"><label>81.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rolls</surname><given-names>A</given-names></name><name><surname>Shechter</surname><given-names>R</given-names></name><name><surname>Schwartz</surname><given-names>M</given-names></name></person-group>. <article-title>The bright side of the glial scar in CNS repair</article-title>. <source>Nat Rev Neurosci.</source> (<year>2009</year>) <volume>10</volume>:<fpage>235</fpage>–<lpage>41</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrn2591</pub-id><pub-id pub-id-type=\"pmid\">19229242</pub-id></mixed-citation></ref><ref id=\"B82\"><label>82.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Simard</surname><given-names>AR</given-names></name><name><surname>Rivest</surname><given-names>S</given-names></name></person-group>. <article-title>Neuroprotective properties of the innate immune system and bone marrow stem cells in Alzheimer's disease</article-title>. <source>Mol Psychiatry.</source> (<year>2006</year>) <volume>11</volume>:<fpage>327</fpage>–<lpage>35</lpage>. <pub-id pub-id-type=\"doi\">10.1038/sj.mp.4001809</pub-id><pub-id pub-id-type=\"pmid\">16491130</pub-id></mixed-citation></ref><ref id=\"B83\"><label>83.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Butovsky</surname><given-names>O</given-names></name><name><surname>Koronyo-Hamaoui</surname><given-names>M</given-names></name><name><surname>Kunis</surname><given-names>G</given-names></name><name><surname>Ophir</surname><given-names>E</given-names></name><name><surname>Landa</surname><given-names>G</given-names></name><name><surname>Cohen</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Glatiramer acetate fights against Alzheimer's disease by inducing dendritic-like microglia expressing insulin-like growth factor 1</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2006</year>) <volume>103</volume>:<fpage>11784</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.0604681103</pub-id><pub-id pub-id-type=\"pmid\">16864778</pub-id></mixed-citation></ref><ref id=\"B84\"><label>84.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Duffield</surname><given-names>JS</given-names></name></person-group>. <article-title>The inflammatory macrophage: a story of Jekyll and Hyde</article-title>. <source>Clin Sci.</source> (<year>2003</year>) <volume>104</volume>:<fpage>27</fpage>–<lpage>38</lpage>. <pub-id pub-id-type=\"doi\">10.1042/cs1040027</pub-id><pub-id pub-id-type=\"pmid\">12519085</pub-id></mixed-citation></ref><ref id=\"B85\"><label>85.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>James</surname><given-names>DG</given-names></name></person-group>. <article-title>A clinicopathological classification of granulomatous disorders</article-title>. <source>Postgrad Med J.</source> (<year>2000</year>) <volume>76</volume>:<fpage>457</fpage>–<lpage>65</lpage>. <pub-id pub-id-type=\"doi\">10.1136/pmj.76.898.457</pub-id><pub-id pub-id-type=\"pmid\">10908370</pub-id></mixed-citation></ref><ref id=\"B86\"><label>86.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Labow</surname><given-names>RS</given-names></name><name><surname>Meek</surname><given-names>E</given-names></name><name><surname>Santerre</surname><given-names>JP</given-names></name></person-group>. <article-title>Model systems to assess the destructive potential of human neutrophils and monocyte-derived macrophages during the acute and chronic phases of inflammation</article-title>. <source>J Biomed Mater Res.</source> (<year>2001</year>) <volume>54</volume>:<fpage>189</fpage>–<lpage>97</lpage>. <pub-id pub-id-type=\"doi\">10.1002/1097-4636(200102)54:2<189::AID-JBM5>3.0.CO;2-8</pub-id><pub-id pub-id-type=\"pmid\">11093178</pub-id></mixed-citation></ref><ref id=\"B87\"><label>87.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wigginton</surname><given-names>JE</given-names></name><name><surname>Kirschner</surname><given-names>D</given-names></name></person-group>. <article-title>A model to predict cell-mediated immune regulatory mechanisms during human infection with Mycobacterium tuberculosis</article-title>. <source>J Immunol.</source> (<year>2001</year>) <volume>166</volume>:<fpage>1951</fpage>–<lpage>67</lpage>. <pub-id pub-id-type=\"doi\">10.4049/jimmunol.166.3.1951</pub-id><pub-id pub-id-type=\"pmid\">11160244</pub-id></mixed-citation></ref></ref-list><glossary><def-list><title>Abbreviations</title><def-item><term>AD</term><def><p>Alzheimer's disease</p></def></def-item><def-item><term>Aβ</term><def><p>amyloid-beta</p></def></def-item><def-item><term>CNS</term><def><p>central nervous system</p></def></def-item><def-item><term>TECs</term><def><p>thymic epithelial cells</p></def></def-item><def-item><term>ESCs</term><def><p>embryonic stem cells</p></def></def-item><def-item><term>TEPs</term><def><p>thymic epithelial progenitors</p></def></def-item><def-item><term>CP</term><def><p>choroid plexus</p></def></def-item><def-item><term>APP</term><def><p>e amyloid precursor protein</p></def></def-item><def-item><term>Ab</term><def><p>antibody</p></def></def-item><def-item><term>iPSCs</term><def><p>induced pluripotent stem cell</p></def></def-item><def-item><term>CRISPR</term><def><p>clustered Regularly Interspaced Short Palindromic Repeats</p></def></def-item><def-item><term>Cas9</term><def><p>CRISPR-associated protein</p></def></def-item><def-item><term>qRT-PCR</term><def><p>real-time qualitative RT-PCR</p></def></def-item><def-item><term>K5</term><def><p>keratin 5</p></def></def-item><def-item><term>K8</term><def><p>keratin 8</p></def></def-item><def-item><term>NOR</term><def><p>Novel object recognition</p></def></def-item><def-item><term>sAβ</term><def><p>soluble Aβ protein</p></def></def-item><def-item><term>i.t.</term><def><p>intrathymically</p></def></def-item><def-item><term>WT</term><def><p>wild-type</p></def></def-item><def-item><term>Ctrl</term><def><p>control cell</p></def></def-item><def-item><term>DG</term><def><p>dentate gyrus</p></def></def-item><def-item><term>GFAP</term><def><p>glial fibrillary acid protein</p></def></def-item><def-item><term>icam1</term><def><p>intercellular adhesion molecule 1</p></def></def-item><def-item><term>ccl2</term><def><p>chemokine C-C motif ligand 2</p></def></def-item><def-item><term>vcam1</term><def><p>vascular cell adhesion molecule 1</p></def></def-item><def-item><term>cxcl10</term><def><p>C-X-C motif chemokine 10</p></def></def-item><def-item><term>SRA1</term><def><p>scavenger receptor A.</p></def></def-item></def-list></glossary></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"review-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Endocrinol.</journal-id><journal-title-group><journal-title>Frontiers in Endocrinology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2392</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849302</article-id><article-id pub-id-type=\"pmc\">PMC7431621</article-id><article-id pub-id-type=\"doi\">10.3389/fendo.2020.00519</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology</subject><subj-group><subject>Review</subject></subj-group></subj-group></article-categories><title-group><article-title>Metformin May Contribute to Inter-individual Variability for Glycemic Responses to Exercise</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Malin</surname><given-names>Steven K.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/567011/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Stewart</surname><given-names>Nathan R.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Kinesiology, University of Virginia</institution>, <addr-line>Charlottesville, VA</addr-line>, <country>United States</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Division of Endocrinology and Metabolism, University of Virginia</institution>, <addr-line>Charlottesville, VA</addr-line>, <country>United States</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Robert M. Berne Cardiovascular Research Center, University of Virginia</institution>, <addr-line>Charlottesville, VA</addr-line>, <country>United States</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: John P. Thyfault, University of Kansas Medical Center, United States</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Camila Manrique-Acevedo, University of Missouri, United States; Nathan C. Winn, Vanderbilt University, United States; Louise Lantier, Vanderbilt University, United States</p></fn><corresp id=\"c001\">*Correspondence: Steven K. Malin <email>skm6n@virginia.edu</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Clinical Diabetes, a section of the journal Frontiers in Endocrinology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>519</elocation-id><history><date date-type=\"received\"><day>30</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>26</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Malin and Stewart.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Malin and Stewart</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Metformin and exercise independently improve glycemic control. Metformin traditionally is considered to reduce hepatic glucose production, while exercise training is thought to stimulate skeletal muscle glucose disposal. Collectively, combining treatments would lead to the anticipation for additive glucose regulatory effects. Herein, we discuss recent literature suggesting that metformin may inhibit, enhance or have no effect on exercise mediated benefits toward glucose regulation, with particular emphasis on insulin sensitivity. Importantly, we address issues surrounding the impact of metformin on exercise induced glycemic benefit across multiple insulin sensitive tissues (e.g., skeletal muscle, liver, adipose, vasculature, and the brain) in effort to illuminate potential sources of inter-individual glycemic variation. Therefore, the review identifies gaps in knowledge that require attention in order to optimize medical approaches that improve care of people with elevated blood glucose levels and are at risk of cardiovascular disease.</p></abstract><kwd-group><kwd>pre-diabetes</kwd><kwd>type 2 diabetes</kwd><kwd>metabolic syndrome</kwd><kwd>insulin resistance</kwd><kwd>exercise</kwd><kwd>weight loss</kwd></kwd-group><counts><fig-count count=\"1\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"130\"/><page-count count=\"13\"/><word-count count=\"11686\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Nearly 34.2 million individuals in the U.S. have type 2 diabetes, and ~88 million men and women have prediabetes (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Perhaps more concerningly is the observation that new cases of type 2 diabetes have increased significantly among U.S. youth, particularly non-Hispanic black people (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). This is clinically concerning because people with hyperglycemia are at greatly elevated risk for not only retinopathy, nephropathy, renal disease, but also cardiovascular disease (CVD). Blood glucose regulation is considered to be a complex balance between endogenous glucose production and peripheral glucose uptake. Insulin resistance of organs regulating these processes is considered to a be a primary defect. In particular, insulin resistance contributes to compensatory hyperinsulinemia via taxation on the pancreatic beta-cells to secrete insulin. Over time, however, the beta-cells begin to “fail” and cannot compensate for the ambient levels of systemic insulin resistance resulting in severe hyperglycemia. Therefore, targeting insulin resistance is a reasonable approach to the prevention, treatment, and management of type 2 diabetes.</p><p>Although randomized clinical trials show the efficacy of exercise to treat type 2 diabetes (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>) as well as prevent the progression from prediabetes to type 2 diabetes (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>, <xref rid=\"B4\" ref-type=\"bibr\">4</xref>), there is large inter-individual heterogeneity in response to conventional exercise aerobic (up to 5 d/wk at 60–85% HR<sub>max</sub>) and strength (up to 2 d/wk at 60–80% 1-repetition max). Moreover, the optimal dose of exercise to improve glycemic control remains to be elucidated (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>–<xref rid=\"B7\" ref-type=\"bibr\">7</xref>), and exercise adherence remains low. Patients with prediabetes and/or type 2 diabetes often exhibit multiple pathophysiological abnormalities that contribute to the approximate 20% lower aerobic capacity compared to those without dysglycemia (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). These include: mitochondrial dysfunction, poor muscle perfusion, and low cardiac function in addition to declines in pancreatic insulin secretion and sensitivity. Together, these are mechanisms contributing to decreased oxidative capacity and may help explain barriers to starting exercise interventions (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). Subsequently, many individuals may require pharmacological therapy to manage blood glucose concentrations. The American Diabetes Association suggests that in addition to lifestyle modification, metformin be considered the “first-line” pharmacological treatment to manage blood glucose in those with type 2 diabetes as well as those with prediabetes and at least 1 CVD risk factor (e.g., hypertension, elevated triacylglycerol, low HDL, etc.) (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). Not surprisingly, metformin is the most widely used prescription drug to treat hyperglycemia in adults with type 2 diabetes (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). In addition, metformin has gained interest in cancer prevention/treatment (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>) as well as lifespan within aging (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>). This highlights that metformin is a multi-faceted drug with health effects. Despite the widespread popularity of metformin, the interaction with exercise has received little attention. If anything, the overarching thought is that recommending exercise plus metformin will enhance glycemic control, and be better than either intervention alone. Herein, we highlight recent data describing whether co-prescribing metformin with exercise blunts, enhances, or has negligible effects on glucose regulation for ultimate CVD risk reduction. In this review, we focus on the multiple tissues (i.e., skeletal muscle, liver, adipose, vasculature, and brain) that metformin may affect during exercise training to influence cardiometabolic health (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Lastly, we hypothesize that combining metformin with exercise may induce cellular processes that regulate metabolic adaptation in relation to glycemia.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Summary of exercise and/or metformin interactions. Exercise lowers blood glucose mainly through increases in AMPK production in most organs excluding the brain, where production is decreased; and the vasculature, where adaptations are largely driven by nitric oxide (NO) <bold>(A)</bold>. Metformin alone also improves glycemic control through similar mechanisms, primarily by decreasing hepatic glucose production. Metformin also decreases reactive oxygen species (ROS) production which is suspected to improve tissue glycemic control as well as memory and cognitive function in the brain <bold>(B)</bold>. The combination of metformin with exercise has blunted effects on skeletal muscle glucose uptake and visceral adiposity. The effects of metformin with exercise on the liver, vasculature, and brain are still largely unknown <bold>(C)</bold>. We hypothesize that the combination of metformin and exercise are not necessarily additive in terms of glycemic control. Metformin blunts the beneficial adaptations that are typically seen with aerobic and/or resistance training in skeletal muscle tissue. VO2max, maximal oxygen consumption; FFM, fat-free mass; AMPK, adenosine monophosphate kinase; IHTG, intrahepatic triglycerides; FFA, free fatty acids; NO, nitic oxide.</p></caption><graphic xlink:href=\"fendo-11-00519-g0001\"/></fig></sec><sec id=\"s2\"><title>Impact of Metformin on Exercise Mediated Glycemic Control</title><p>Regulation of blood glucose is a tightly controlled process through “cross-talk” of pancreatic insulin secretion and insulin action on several tissues, including but not limited to: skeletal muscle, liver, adipose tissue, vasculature, and the brain. Fasting plasma glucose is maintained by endogenous (principally hepatic) glucose production, as glucose disposal by skeletal muscle and adipose tissue is minimal (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>). Following mixed-meal absorption, insulin levels rise in response to carbohydrate (and to smaller extents protein) to reduce liver glucose production and lipolysis as well as stimulate blood flow to skeletal muscle for glucose uptake (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). Moreover, insulin acts on the brain to provide additional regulation of endogenous glucose production as well as inhibit additional food intake (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Thus, considering treatments that impact liver and/or skeletal muscle glucose metabolism should, in theory, lead to enhanced glycemic control.</p><p>Current exercise prescription advised by the American College of Sports Medicine and the American Diabetes Association for men and women with prediabetes or T2D is to perform either 150 min/week or more of moderate intensity or 75 min/week of vigorous intensity aerobic exercise. The Look AHEAD study is a landmark clinical trial that reported a ≥ 7% reduction in weight loss through a nutrition intervention in combination with ≥175 min exercise/wk, reduced CVD risk by ~0.7%/yr (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>), although more work is needed to understand the utility of lifestyle treatment for vasculature related events/mortality. Exercise can consist of either aerobic or resistance form, although the combination may result in the best HbA1c reductions (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). While there is still much debate as to whether exercise intensity is critical for glycemic control (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>), we and others have shown either no effect (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>) or that moderate intensity may have slightly better effects (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). Regardless, more recent work has suggested “exercise snacks” may be a novel approach to combat post-prandial hyperglycemia, and further work examining time of day to exercise is warranted (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>).</p><p>Metformin predominantly reduces circulating glucose by lowering hepatic glucose production (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B21\" ref-type=\"bibr\">21</xref>), although it has also been reported to increase peripheral insulin sensitivity in some but not all work (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>, <xref rid=\"B23\" ref-type=\"bibr\">23</xref>). In the landmark U.S. diabetes prevention program, metformin was shown to decrease the incidence of T2D by 31% (1,700 mg/d) in adults with impaired glucose compared with 58% following lifestyle modification (7% weight loss and 150 min/wk of physical activity) (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Although these findings suggest lifestyle was better than metformin alone, the Indian Diabetes Prevention Program (IDDP) observed that both regular physical activity (recommended >30 min/d) and metformin (500 mg/d) reduced the progression of impaired glucose tolerance to T2D in native Asian Indians (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>).</p><p>Exercise and metformin both increase 5-adenosine monophosphate kinase (AMPK). This is important because AMPK is one of the several mechanisms by which each therapy act to suppress hepatic glucose output and increase insulin-stimulated glucose disposal (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B23\" ref-type=\"bibr\">23</xref>). As a result, it would be fair to expect a greater benefit to glycemic control since two of the major organs regulating blood glucose would be impacted compared with either treatment alone. However, the literature on co-prescribing lifestyle modification with metformin on blood glucose is equivocal (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Indeed, some (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>) have shown that lifestyle modification plus metformin resulted in more weight loss than lifestyle modification alone, and the weight loss was associated with lower 2-h circulating glucose levels. This is somewhat consistent with recent work by Erickson et al. (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>) showing that post-meal exercise and metformin resulted in the lowest peak post-prandial glucose excursion compared with either treatment alone in people with hyperglycemia. Furthermore, Ortega et al. sought to test the effects of combining metformin with exercise on free-living glycemic control in individuals with prediabetes or T2D (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). The results of this later work demonstrated that high intensity interval exercise in combination with metformin therapy lowered interstitial fluid glucose to a greater extent than exercise alone. Interestingly, others have suggested that in people with T2D treated with metformin that timing exercise 30 to 60 min following drug ingestion may impact plasma glucose and insulin to a greater extent than exercising 90 min after ingestion (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). The Diabetes Aerobic and Resistance Exercise (DARE) trial, however, showed that people with T2D on metformin plus lifestyle modification had similar HbA1c improvements when compared with individuals on lifestyle modification only (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). This is consistent with the IDDP since it was shown that the combination therapy of metformin and lifestyle modification had equivalent effects to reduce the progression from prediabetes to T2D (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). Notwithstanding this, a retrospective analysis of the Look AHEAD study demonstrated that people with type 2 diabetes treated with metformin prior to and during intensive lifestyle therapy had smaller improvements in fasting plasma glucose and HbA1c compared with those undergoing lifestyle therapy only (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Further, the work by Boulé et al. (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>) tested the effect of metformin on glycemic control in response to a single bout of submaximal aerobic exercise at ~33, 67, and 79% of VO<sub>2</sub>peak and resistance exercise (i.e., leg extension and flexion) in individuals with T2D. Their results implied that metformin blunted reductions in post-prandial blood glucose concentrations during a standardized meal. Together, most (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>, <xref rid=\"B27\" ref-type=\"bibr\">27</xref>) but not all (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>, <xref rid=\"B31\" ref-type=\"bibr\">31</xref>) studies showing an additive effect of metformin plus exercise studied individuals who were already prescribed metformin. In contrast, we showed previously (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>) that 12 weeks of metformin plus exercise training prospectively in naïve users had no effect on fasting plasma glucose in adults with prediabetes. This is consistent with newer work (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>, <xref rid=\"B34\" ref-type=\"bibr\">34</xref>) whereby in normoglycemic insulin-resistant adults, fasting or postprandial glucose levels did not appear to be negatively affected by metformin. Somewhat surprisingly, however, is the observation that no randomized clinical trial has been designed to date to test the effectiveness of exercise plus metformin on glycemic control. Given that some work shows opposing (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>), additive (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>, <xref rid=\"B27\" ref-type=\"bibr\">27</xref>), or null findings (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>–<xref rid=\"B34\" ref-type=\"bibr\">34</xref>), it is reasonable to suggest that metformin contributes to inter-individual glycemic response differences (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Summary of clinical trials examining the impact of metformin in combination with exercise on glycemic control compared to exercise alone.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Study details</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Prescription/Population</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>FPG</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>2-h Postprandial</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>HbA1c</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ramachandran et al. (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Walk or cycle >30 min/d + 1,000 mg/d metformin for ~3 years in overweight/obese adults</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↔</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↔ during OGTT</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sharoff et al. (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">An acute bout of cycle ergometry for 30 min @ 65% VO<sub>2</sub>Peak + 2–3 weeks of 2,000 mg/d metformin treatment prior in overweight/obese adults</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↔</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Love-Osborne et al. (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Self-chosen life style change + 1,700 mg/d metformin for 6 months in overweight/obese adolescents</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↔</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↔ during OGTT</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Erickson et al. (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Postmeal exercise (5 x 10-min bouts of treadmill walking at 60% VO<sub>2</sub>Peak) + 1,000–2,000 mg/d metformin in overweight/obese adults</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↓ during MMTT</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ortega et al. (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">An acute bout of exercise + physician prescribed dose of metformin in insulin-resistant adults</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↔ during OGTT</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Boulé et al. (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">~33 submaximal exercise bouts lasting 3–15 min @ 67–79% VO<sub>2</sub>Peak + 28 d of 2,000 mg/d metformin treatment prior in adults with T2D.</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑ during MMTT</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Terada and Boulé (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Nutrition intervention/with ≥175 min exercise/wk + metformin therapy in overweight adults with T2D</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↑</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Malin et al. (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">60–75 min of moderate-high intensity concurrent training 3 d/wk + of 2,000 mg/d metformin administration for 12 weeks in adults with prediabetes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↔</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Konopka et al. (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">45 min of moderate-high intensity cycle ergometry 3 d/wk + 2,000 mg/d metformin for 12 weeks in older adults with prediabetes</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↔</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↔</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Walton et al. (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>)</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">PRT + 1,700 mg/d metformin for 14 weeks in healthy older adults</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">↔</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">N/A</td></tr></tbody></table><table-wrap-foot><p><italic>FPG, fasting plasma glucose; OGTT, oral glucose tolerance test; MMTT, mixed meal tolerance test; N/A, Not applicable because the measurement was either not reported or measured; ↑, higher; ↓, lower; ↔, no difference</italic>.</p></table-wrap-foot></table-wrap></sec><sec id=\"s3\"><title>Effect of Metformin on Exercise-Mediated Skeletal Muscle Insulin Sensitizing Effects</title><p>Exercise improves glycemic control through both skeletal muscle insulin-dependent and insulin-independent mechanisms (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). Subsequently, contraction mediated mechanisms favoring glucose uptake last for ~3–6 h following a single bout of exercise. In time, insulin-sensitizing effects take over to explain improved glucose control (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). Habitual exercise (i.e., lifestyle change) is recommended to reduce T2D risk in part by maintaining skeletal muscle glucose disposal.</p><p>Metformin is suggested to stimulate skeletal muscle glucose uptake and oxidation (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>). Moreover, metformin has been shown to lower intramuscular triglyceride content and bioactive acyl-chain bioactive lipids (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>, <xref rid=\"B39\" ref-type=\"bibr\">39</xref>) through in part elevations in fat oxidation. Together, these observations indicate that metformin has effects on skeletal muscle energy metabolism that favor glucose homeostasis.</p><p>Because metformin is advised as a first-line pharmacological agent, we conducted a double-blind, randomized control trial to test the effect of exercise training with and without metformin on insulin sensitivity in people with prediabetes (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). For 12 weeks, individuals were randomized to either: placebo, metformin, exercise training with placebo, or exercise training with metformin. All people were provided metformin at 2,000 mg/d or a placebo, while those randomized to exercise underwent a progressive aerobic and resistance training program at 70% of their individual heart rate peak and 1-repetition max, respectively. Insulin sensitivity was determined about 28 h post-exercise via the euglycemic-hyperinsulinemic clamp with glucose isotope tracers. Tracers were utilized to determine the effects of metformin on skeletal muscle insulin sensitivity as well as hepatic glucose production. The primary results showed that metformin blunted exercise mediated increases in insulin-stimulated skeletal muscle glucose uptake by ~30%, suggesting that metformin diminishes both single and repeated bouts of exercise benefit on glucose metabolism (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>, <xref rid=\"B31\" ref-type=\"bibr\">31</xref>, <xref rid=\"B32\" ref-type=\"bibr\">32</xref>). Although to date no follow up studies have been conducted using stable isotopes to understand skeletal muscle insulin-stimulated glucose disposal, recent work has tested the effect of metformin on aerobic or resistance exercise skeletal muscle cellular adaptation (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>, <xref rid=\"B34\" ref-type=\"bibr\">34</xref>). The results of these studies collectively show that metformin opposes skeletal muscle mitochondrial adaptations as well as inhibits fat-free mass accretion (see below <italic>Cell Mechanisms</italic> for further discussion), which were directly correlated with attenuated gains in aerobic fitness as well as strength. Together, these findings highlight that blunted fitness adaptation may relate to the reduced skeletal muscle insulin sensitivity response. In either case, this smaller gain in insulin sensitivity following the combination of exercise and metformin treatment does not apparently lead to stark blood glucose elevations (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>, <xref rid=\"B31\" ref-type=\"bibr\">31</xref>, <xref rid=\"B32\" ref-type=\"bibr\">32</xref>). Further work is warranted to better understand how the combination of drug-exercise therapies contributes to glycemic control across exercise doses, particularly in people with T2D. For instance, recent work demonstrated that metformin increased carbohydrate utilization during high intensity interval exercise in insulin resistant adults when compared to exercise alone (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). This may be of clinical relevance since carbohydrate use during exercise was related to insulin sensitivity as measured by the intravenous glucose tolerance test. The findings of Ortega et al. (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>) also suggest that exercise intensity may interact with metformin to positively influence insulin-stimulated glucose uptake when compared with moderate intensity exercise (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>, <xref rid=\"B40\" ref-type=\"bibr\">40</xref>). Whether exercise intensity interacts with metformin to affect skeletal muscle insulin sensitivity in clinical populations remains to be tested to help understand if muscle is the primary driver of glycemic variation responses.</p></sec><sec id=\"s4\"><title>Effect of Metformin on Exercise-Mediated Liver Insulin Sensitizing Effects</title><p>Hepatic glucose production results from gluconeogenesis and/or glycogenolysis, and people with impaired fasting glucose display elevated hepatic glucose production (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>, <xref rid=\"B41\" ref-type=\"bibr\">41</xref>), or inappropriately normal levels given the prevailing hyperinsulinemia (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>). Indeed, people at risk for or with T2D, in particular, have impaired responses to insulin (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>). This highlights that the liver becomes insulin resistant and plays roles in both fasting and fed states. While fasting glucose (and insulin) may serve as a proxy for hepatic glucose production, and study of hepatokines, liver fat, or liver enzymes (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>) may provide indirect estimates of hepatic function, use of stable isotopes along with hyperinsulinemic-clamps represent ideal methodologies to depict the role of the liver on glycemic control.</p><p>The exercise impact on hepatic glucose production is generally positive. One to seven days of aerobic exercise has been shown in people with T2D to increase hepatic insulin sensitivity (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>, <xref rid=\"B45\" ref-type=\"bibr\">45</xref>). Exercise training studies of ~12 weeks have also demonstrated favorable effects on hepatic insulin sensitivity (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>), with at least some of the effect being related to improved hepatokines (i.e., fetuin-A) (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>). However, it is worth noting that others have suggested that re-feeding calories expended from exercise negates these liver insulin-sensitizing benefits of exercise in adults with excess weight/insulin resistance (<xref rid=\"B47\" ref-type=\"bibr\">47</xref>). It cannot be ruled out though that discrepancies between short-term training studies may relate to exercise intensity, as higher intensity exercise activates AMPK in hepatocytes (<xref rid=\"B48\" ref-type=\"bibr\">48</xref>). As a result, it seems that energy deficit, at least partially, created by exercise is an important mechanism improving hepatic insulin sensitivity.</p><p>Metformin improves hepatic insulin sensitivity. The mechanism by which metformin lowers hepatic glucose production is mainly thought to be through activation of AMPK and reduction in gluconeogenic enzymes (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>), although some suggest antagonism of glucagon may be important (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>). In addition, metformin is considered to increase fat oxidation in hepatocytes, thereby reducing the potential delirious effects of lipids on insulin signaling (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>). Recent work has suggested that metformin may benefit conditions of hepatic steatosis. In particular, although metformin-induced similar reductions in the hepatic triglyceride content of Otsuka Long-Evans Tokushima Fatty (OLETF) rats under caloric restriction, compared to caloric restriction alone, the combined treatment lowered hepatic-derived inflammation more (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>). Additionally, metformin augmented the benefits of caloric restriction on lowering post-prandial circulating glucose in rodents, suggesting that metformin may impact the liver during energy deficit reduce diabetes and non-alcoholic fatty liver disease risk (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>). This observation of greater glycemic benefit was in parallel to greater beta-oxidation and mitochondrial mitophagy (i.e., BNIP3).</p><p>To date, we are aware of only one study in humans that has systematically tested the effect of combining metformin with exercise on hepatic glucose production (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). In this study, we showed that 12 weeks of metformin, exercise, or the combination of therapies maintained hepatic glucose production as measured by stable isotopes despite reductions in fasting plasma insulin. This highlights that all treatments improved hepatic insulin sensitivity in middle-aged adults with prediabetes. Thus, it would seem the liver is unlikely to explain glycemic variation post-exercise. Further work in humans is required to understand, nevertheless, how exercise and metformin interact to affect hepatic function given that fatty liver disease is prominent in people with obesity and T2D, and fatty liver disease plays a critical role in the development of CVD.</p></sec><sec id=\"s5\"><title>Influence of Metformin and Exercise Adipose Tissue Insulin Action</title><p>Adipose tissue is the primary supplier of plasma free fatty acids (FFA). FFAs provide energy to working tissues, including skeletal muscle and liver primarily during fasting states. In response to mixed meals (i.e., carbohydrate, protein, and fat), insulin suppresses lipolysis due to a feedback loop with the pancreas (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>), and lowers circulating FFA to enable insulin action on the peripheral for glycemic control. However, when adipose tissue becomes resistant to the action of insulin, FFA concentrations rise in circulation and play an important role in the development of insulin resistance (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>). In fact, the release of FFAs from adipose tissue contributes, not only to declines in skeletal muscle and hepatic insulin sensitivity but also to endothelial dysfunction and reduced β-cell function in obesity, prediabetes, and T2D (<xref rid=\"B55\" ref-type=\"bibr\">55</xref>–<xref rid=\"B57\" ref-type=\"bibr\">57</xref>). The reason FFAs contribute to this multi-tissue insulin resistance is beyond the scope of this review, but likely relates to elevated plasma FFA concentrations being linked with reduced mitochondrial function and metabolic flexibility (<xref rid=\"B58\" ref-type=\"bibr\">58</xref>), Therefore, it would be reasonable to expect aerobic exercise interventions designed to improve oxidative capacity to not only protect against FFA-induced insulin resistance but also improve adipose insulin action.</p><p>Exercise confers several benefits to adipose tissue that include reductions in not only total fat mass but also visceral adiposity (<xref rid=\"B59\" ref-type=\"bibr\">59</xref>). A consequence of this improved body fat mass has been proposed to decrease circulating FFAs as well as inflammatory mediators referred to as adipokines. Indeed, we have shown that changes in circulating FFAs following moderate intensity training are directly related to improved peripheral insulin sensitivity (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>) and short-term interval or continuous exercise increases adipose insulin sensitivity in adults with prediabetes (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). While reductions in body fat following exercise training may be a key explanation for reducing circulating FFAs (<xref rid=\"B60\" ref-type=\"bibr\">60</xref>) in relation to improved peripheral insulin sensitivity and CVD risk reduction, fat loss is not required for improved adipocyte function. In fact, we recently showed that energy deficit, but not fat mass reduction, is important for improving adipokine profiles during caloric restriction (<xref rid=\"B61\" ref-type=\"bibr\">61</xref>). Moreover, Heiston et al. demonstrated that just 2 weeks of aerobic interval or continuous exercise increased adiponectin and lowered leptin prior to clinically meaningful weight loss or reductions in fat mass in older adults with prediabetes (<xref rid=\"B62\" ref-type=\"bibr\">62</xref>). Regardless, prior work (<xref rid=\"B63\" ref-type=\"bibr\">63</xref>) showed that hepatic insulin sensitivity was increased more following exercise training with a hypocaloric diet than when compared with a eucaloric diet during lipid-infusion. This suggests that in addition to exercise, calorie restriction may protect the liver from obesity-driven insulin resistance more so than training alone, despite comparable peripheral insulin sensitivity (<xref rid=\"B64\" ref-type=\"bibr\">64</xref>). Taken together, exercise, with or without caloric restriction, is an effective treatment for improving adipose tissue function.</p><p>Metformin is known to induce weight loss in adults with obesity, prediabetes, and T2D (<xref rid=\"B65\" ref-type=\"bibr\">65</xref>). Metformin reduces circulating FFA in part through inhibiting lipolysis (<xref rid=\"B66\" ref-type=\"bibr\">66</xref>). In fact, in murine adipocytes, metformin activated AMPK and blunted ANP as well as catecholamine-stimulated lipolysis (<xref rid=\"B67\" ref-type=\"bibr\">67</xref>, <xref rid=\"B68\" ref-type=\"bibr\">68</xref>). Interestingly, elevated and/or blunted reductions in circulating FFAs have been reported after metformin plus exercise treatment during rest, exercise, or insulin-stimulated conditions compared to exercise alone (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>, <xref rid=\"B31\" ref-type=\"bibr\">31</xref>, <xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B69\" ref-type=\"bibr\">69</xref>). While recent work suggests that oral metformin administration does not impact subcutaneous adipose tissue lipolysis during submaximal exercise in young lean men (<xref rid=\"B70\" ref-type=\"bibr\">70</xref>), it remains possible that in clinical populations alterations in either adipose lipolysis or reduced clearance as well as esterification may contribute to higher plasma FFAs. In either case, the elevated FFAs have been correlated attenuated gains in insulin sensitivity following metformin plus exercise therapy (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>, <xref rid=\"B32\" ref-type=\"bibr\">32</xref>). This may be clinically important as intrahepatic fat accumulation was lowered more after a diet and exercise than when lifestyle therapy was combined with metformin in obese adolescents (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>). The blunted improvement in hepatic steatosis in these adolescents is consistent with the view that elevated FFAs from adipose tissue travel through the portal vein to the liver for increasing hepatic lipid storage. Collectively, this work highlights that adipose-derived metabolism may play a role in CVD risk following the co-prescription of metformin and exercise.</p></sec><sec id=\"s6\"><title>Influence of Metformin and Exercise Vasculature Function</title><p>Insulin promotes vasodilation in large conduit arteries and resistance arterioles as well as microvasculature perfusion (<xref rid=\"B72\" ref-type=\"bibr\">72</xref>). Conduit and resistance arteries are important for the delivery of nutrients and oxygen to metabolically active tissues, whereas the microvasculature provides a critical role in the exchange of these substances. In turn, adequate insulin-stimulated blood flow and endothelial function are essential for glucose regulation. However, during periods of physical inactivity and/or nutrient excess, hyperinsulinemia develops and has been related to elevated endothelin-1 (ET-1) mediated vasoconstriction. This impaired glucose delivery may not only increase risk for T2D but also contribute to endothelial dysfunction through lower nitric oxide bioavailability. Interestingly, people with insulin resistance have been noted to have normal fasting vascular function, but impaired conduit or microvascular insulin action (<xref rid=\"B73\" ref-type=\"bibr\">73</xref>). This demonstrates that mechanisms underlying disease states may be unique in the fasted vs. insulin-stimulated state.</p><p>Habitual physical activity elevated insulin-mediated skeletal muscle glucose disposal and limb blood flow (<xref rid=\"B65\" ref-type=\"bibr\">65</xref>, <xref rid=\"B74\" ref-type=\"bibr\">74</xref>). The dose at which exercise impacts vascular insulin sensitivity, however, is less clear. Although recent work suggests that interval exercise improves flow-mediated dilation (FMD), which measures large conduit arteries, more than continuous exercise in sedentary people (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B12\" ref-type=\"bibr\">12</xref>, <xref rid=\"B75\" ref-type=\"bibr\">75</xref>) not all studies agree (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>). Interestingly, we recently studied the effect of interval vs. continuous exercise on fasting and post-prandial arterial stiffness as well as endothelial function as measured by FMD in older adults with prediabetes (<xref rid=\"B77\" ref-type=\"bibr\">77</xref>, <xref rid=\"B78\" ref-type=\"bibr\">78</xref>). We found that 2 weeks of high intensity interval or moderate continuous exercise reduced post-prandial arterial stiffness but had no overall effect on fasting or post-prandial FMD. Nonetheless, when examination of responder compared with non-responder analysis was performed, it was shown that continuous exercise elicited a 57% response rate to raise FMD compared with only 42% with interval exercise (<xref rid=\"B78\" ref-type=\"bibr\">78</xref>). This latter finding is consistent with work showing that either a single bout or short-term exercise training at moderate continuous intensity can promote vasodilation after glucose-induced insulin stimulation in adults with and without T2D (<xref rid=\"B79\" ref-type=\"bibr\">79</xref>–<xref rid=\"B82\" ref-type=\"bibr\">82</xref>). Therefore, exercise appears to exert unique effects on the vasculature in fasted compared with fed (or insulin-stimulated) states based on the intensity at which exercise is performed in clinical populations. While these studies tested vascular function under a glucose load, no study to date has investigated the effect of lipid infusion on endothelial function before or during insulin-stimulation following training. However, aerobic fitness has been directly correlated with the preservation of insulin-stimulated microcirculatory function in healthy young adults (<xref rid=\"B83\" ref-type=\"bibr\">83</xref>). Moreover, in healthy inactive young adults, 12 weeks of interval exercise was shown to increase brachial artery conduit artery function more so than continuous training alone during a high fat meal (<xref rid=\"B84\" ref-type=\"bibr\">84</xref>). Together, fitness mediated mechanisms may be important for opposing FFA-induced vs. glucose-induced skeletal muscle vascular insulin resistance.</p><p>Metformin improves brachial artery FMD in people with type 1 diabetes (<xref rid=\"B85\" ref-type=\"bibr\">85</xref>) and polycystic ovarian syndrome (<xref rid=\"B86\" ref-type=\"bibr\">86</xref>). Moreover, metformin treatment for 4 weeks increases forearm blood flow and glucose uptake following a 75 g glucose load in people with T2D (<xref rid=\"B87\" ref-type=\"bibr\">87</xref>). Interestingly, this improvement in forearm blood flow corresponded with improved glucose tolerance and lower FFA levels, suggesting lower gluco-lipid toxicity may contribute to improved endothelial function. Given that insulin-mediated glucose uptake is more closely associated with microvascular blood flow than total flow (<xref rid=\"B88\" ref-type=\"bibr\">88</xref>), it is important to understand the role of metformin on microvasculature function. To date, no data exist in humans studying the impact of metformin on microcirculatory function. Recently, Bradley et al. though showed that 2 weeks of metformin treatment improved microvascular responses during a euglycemic-hyperinsulinemic clamp in the muscle of high-fat fed rat (<xref rid=\"B89\" ref-type=\"bibr\">89</xref>). In particular, metformin lowered body weight and FFAs as well as improved insulin-stimulated muscle Akt phosphorylation, which confirms improved insulin signaling. Although there was no change in muscle AMPK phosphorylation, these findings suggest that metformin impacts nutrient exchange with skeletal muscle for glucose uptake. This is consistent with the notion that metformin increases eNOS phosphorylation in cultured endothelial cells (<xref rid=\"B90\" ref-type=\"bibr\">90</xref>). While work in human microvasculature insulin sensitivity awaits further investigation, metformin appears to have a direct effect on vasculature insulin action in skeletal muscle.</p><p>Traditionally, chronic exercise reduces CVD risk by decreasing blood pressure, triglycerides (TG), and inflammation (<xref rid=\"B91\" ref-type=\"bibr\">91</xref>). Metformin is not only used to treat T2D but also it is suggested to lower CVD risk (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Indeed, the UK Prospective Diabetes Study (UKPDS) was a multi-center trial demonstrating that using pharmacological agents like metformin reduced HbA1c by ~11% over 10 years as well as lowered microvasculature endpoints (e.g., retinal photocoagulation) (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>, <xref rid=\"B92\" ref-type=\"bibr\">92</xref>, <xref rid=\"B93\" ref-type=\"bibr\">93</xref>). However, there are few data from randomized trials examining if metformin alters the vasculature adaptation to exercise. From our observations of blunted insulin sensitivity following the combined treatment (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>), we studied the impact metformin would have on exercise-mediated improvements in CVD risk factors (i.e., blood pressure, inflammation, and blood lipids) (<xref rid=\"B94\" ref-type=\"bibr\">94</xref>). Interestingly, metformin or exercise training monotherapies lowered systolic blood pressure and C-reactive protein (CRP) by ~7–8 and 20–25%, respectively, in people with prediabetes. When metformin and exercise were combined though, blunted reductions in systolic blood pressure and CRP were observed. These data were in line with others reporting that combining metformin with a low-fat diet and increase physical activity program had no further improvement in blood pressure (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>). Furthermore, our observations were confirmed in obese insulin resistant adolescents whereby the metformin plus lifestyle modification blunted reductions in CRP as well as fibrinogen (<xref rid=\"B71\" ref-type=\"bibr\">71</xref>). Taken together, the metformin plus exercise therapy has strong clinical potential to oppose the reversal of chronic disease, including hypertension and metabolic syndrome. Further work is required for elucidating the vascularture mechanism(s) (e.g., FMD or angiogenesis) by which metformin interacts with exercise to lower or prevent CVD risk in people at risk for T2D.</p></sec><sec id=\"s7\"><title>Metformin and Exercise on Brain Insulin Sensitivity</title><p>Insulin impacts the central nervous system by regulating hepatic glucose production, food intake and adipose metabolism, vasodilation/vasoconstriction of blood vessels as well as pancreatic insulin secretion and skeletal muscle insulin sensitivity (<xref rid=\"B95\" ref-type=\"bibr\">95</xref>, <xref rid=\"B96\" ref-type=\"bibr\">96</xref>). Although these effects of insulin are clearly important for systemic glucose control, more recent work highlights that insulin also impacts memory, mood, and cognition (<xref rid=\"B97\" ref-type=\"bibr\">97</xref>, <xref rid=\"B98\" ref-type=\"bibr\">98</xref>). Interestingly, Williams et al. (<xref rid=\"B99\" ref-type=\"bibr\">99</xref>) demonstrated direct effects of insulin on memory using intravenous insulin administration via a hyperinsulinemic-euglycemic clamp in 12 healthy older adults. In particular, this improvement in memory was related to increased blood oxygen level-dependent (BOLD) signaling as measured by functional MRI (fMRI) during the clamp (<xref rid=\"B99\" ref-type=\"bibr\">99</xref>). Furthermore, improved memory was best in those individuals with the highest systemic insulin sensitivity. This suggests that declines in insulin sensitivity may contribute to brain pathology in the hypothalamus (<xref rid=\"B95\" ref-type=\"bibr\">95</xref>). Not surprisingly, this may relate to cognitive decline (<xref rid=\"B100\" ref-type=\"bibr\">100</xref>), cerebral atrophy (<xref rid=\"B101\" ref-type=\"bibr\">101</xref>) as well as low brain blood flow and metabolism across aging (<xref rid=\"B102\" ref-type=\"bibr\">102</xref>). Additionally, this altered brain insulin action may be a key pathological factor in regulating glycemic control in individuals with obesity, T2D, aging, and Alzheimer's disease (<xref rid=\"B103\" ref-type=\"bibr\">103</xref>, <xref rid=\"B104\" ref-type=\"bibr\">104</xref>).</p><p>During exercise brain glucose uptake declines in an intensity-based manner (<xref rid=\"B105\" ref-type=\"bibr\">105</xref>). This is likely the result of increased substrate availability (e.g., lactate) that allows glucose to be used by other tissues, such as skeletal muscle and red blood cells, for energy production. Conversely, aerobic interval exercise (4 x 4 min > 90% VO<sub>2</sub>peak) for 3 d/wk combined with moderate intensity exercise (70% VO<sub>2</sub>peak) for 2d/wk training has been demonstrated to increase basal glucose uptake in brain regions critical to cognitive function in young and older adults (<xref rid=\"B106\" ref-type=\"bibr\">106</xref>). Interestingly, the latter findings were observed in the parietal-temporal and caudate regions, which are linked to Alzheimer's disease. In either case, there remains limited data in humans with obesity or T2D confirming the effects of exercise on brain insulin sensitivity in relation to glucose metabolism. It was shown that lifestyle modification inducing weight loss, including increased physical activity and low-fat diet, increased brain insulin sensitivity in people with obesity as assessed by intranasal insulin spray (<xref rid=\"B107\" ref-type=\"bibr\">107</xref>). Moreover, Honkala et al. (<xref rid=\"B108\" ref-type=\"bibr\">108</xref>) demonstrated in sedentary middle-aged adults with insulin resistance sprint interval training for 2 weeks lowered insulin-stimulated glucose uptake in the temporal cortex, cingulate gyrus, cerebellum as well as global regions when compared with moderate continuous training. This intensity-based effect was observed despite both exercise intensities raising whole-body insulin sensitivity. This later finding of discordance with brain and periphery insulin action following high intensity exercise on tissue-specific glucose uptake, is consistent with the observation that people with increased brain glucose uptake in response to insulin have decreased insulin-stimulated skeletal muscle glucose disposal (<xref rid=\"B108\" ref-type=\"bibr\">108</xref>). Because exercise is known to increase skeletal muscle insulin sensitivity, it is paramount to understand the role exercise dose on affecting insulin-mediated brain glucose metabolism. Recently, wheel running in obese rats with T2D indicated that exercise was capable of improving insulin-stimulated posterior cerebral artery vasodilation in association with nitric oxide and reduced ET-1 signaling (<xref rid=\"B109\" ref-type=\"bibr\">109</xref>). Moreover, Ruegsegger et al. reported that exercise improved brain insulin sensitivity of rodents fed a high-fat diet (<xref rid=\"B110\" ref-type=\"bibr\">110</xref>). The mechanism by which exercise increased brain insulin sensitivity appears related to increased ATP and reduced ROS generation by mitochondria. Additional work is warranted to understand this brain-skeletal muscle “cross-talk” in order to better understand glycemic control responses to exercise.</p><p>Metformin has been suggested as a potential treatment for cognitive impairment (<xref rid=\"B111\" ref-type=\"bibr\">111</xref>). Because metformin has been shown to promote peripheral insulin sensitivity, it would be reasonable to expect an impact on the brain. A recent pilot trial was conducted whereby metformin was administered in patients with Alzheimer's disease (<xref rid=\"B112\" ref-type=\"bibr\">112</xref>). It was reported that metformin was linked to improved learning, memory, and attention in individuals with mild cognitive impairment. The reason metformin may improve this cognitive function in humans remains to be elucidated, but work in high-fat-fed rodents suggests that increased brain insulin sensitivity, as well as cerebral and hippocampal mitochondrial function, may play a role (<xref rid=\"B113\" ref-type=\"bibr\">113</xref>). In addition, metformin is capable of crossing the blood-brain barrier and regulating tau phosphorylation in mouse models, thereby minimizing risk for Alzheimer's disease (<xref rid=\"B114\" ref-type=\"bibr\">114</xref>).</p><p>To date, no studies have examined how metformin in combination with exercise affects brain regulation of glycemic control. This may be important given the collective body of literature demonstrates that metformin attenuates skeletal muscle insulin sensitivity (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>, <xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B54\" ref-type=\"bibr\">54</xref>), and skeletal muscle is a key tissue proposed to secrete myokines that affect brain function and cognition (<xref rid=\"B115\" ref-type=\"bibr\">115</xref>). Further work in this area is warranted to provide an improved understanding of how exercise and/or metformin benefit not only glycemic control but also reduce T2D and dementia risk in aging adults.</p></sec><sec id=\"s8\"><title>Cellular Mechanism by Which Metformin Impacts Exercise Adaptation</title><p>Most agree that exercise or metformin therapy alone confer favorable effects on cellular pathways that regulate glycemic control across tissues for T2D and CVD risk reduction. The major concern at hand is the notion that 1 + 1 = 2 when considering exercise and metformin for cardiometabolic health. It now appears clear that the mechanism(s) by which exercise and metformin act to affect health interact on some yet to be determined pathway(s) that influences adaptation.</p><p>Aerobic fitness (i.e., VO<sub>2</sub>peak) is related to reduced risk for developing T2D independent of age and family diabetes history (<xref rid=\"B76\" ref-type=\"bibr\">76</xref>). Not surprisingly, elevations in VO<sub>2</sub>peak have been implicated in metabolic adaptations (e.g., mitochondrial biogenesis, oxidative enzymes) that are strongly associated with elevated insulin sensitivity (<xref rid=\"B91\" ref-type=\"bibr\">91</xref>). A reason metformin could constrain gains in aerobic fitness relates to the observation that metformin partially inhibits Complex 1 of the mitochondrial electron transport system (<xref rid=\"B116\" ref-type=\"bibr\">116</xref>). In turn, we examined the impact metformin has on VO<sub>2</sub>peak 10 weeks of exercise training in individuals with prediabetes (<xref rid=\"B69\" ref-type=\"bibr\">69</xref>). Exercise training alone significantly enhanced VO<sub>2</sub>peak by nearly 20%, while metformin plus exercise only increased by ~10%. This attenuated aerobic fitness adaptation has public health relevance since the combined treatment resulted in people exercising at a higher percentage of their post-training VO<sub>2</sub>peak of roughly 5% and consequently, people reported a higher perception of effort (via the Borg Scale) (<xref rid=\"B69\" ref-type=\"bibr\">69</xref>). This observation is consistent with new work highlighting that even acute administration of metformin raised perceptions of effort during exercise (<xref rid=\"B117\" ref-type=\"bibr\">117</xref>). Interestingly, new work highlights in older adults that 12 weeks of metformin treatment blunted the improvement in aerobic fitness by ~50% (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>), which is consistent with our work in middle-aged adults with prediabetes (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). The implication of these findings is important as an increased perception of effort could lead to possibly a decrease in either long-term exercise adherence and/or changes in non-exercise physical activity behavior, thereby independently or collectively negatively influencing cardiometabolic health. However, it is worth acknowledging that not all studies confirm that metformin decreases VO<sub>2</sub>peak. In fact, some have shown metformin to raise exercise tolerance in people with coronary artery disease (<xref rid=\"B118\" ref-type=\"bibr\">118</xref>).</p><p>A possible reason metformin interacts with exercise-mediated skeletal muscle adaptation relates to lowering mitochondrial ROS generation (<xref rid=\"B119\" ref-type=\"bibr\">119</xref>). We previously hypothesized that skeletal muscle contraction induced ROS generation is an important mediator of glucose and insulin metabolism adaptation, in part based on literature showing anti-oxidants blunt exercise health benefit (<xref rid=\"B120\" ref-type=\"bibr\">120</xref>). Newer literature supports this idea suggesting that blunting NADPH oxidase 2 (NOX2)-mediated ROS, which is responsible for GLUT-4 translocation, blunts glucose uptake during muscle contraction in both human and mouse models (<xref rid=\"B121\" ref-type=\"bibr\">121</xref>). But, because metformin counters ROS signaling (<xref rid=\"B119\" ref-type=\"bibr\">119</xref>) in muscle, it is possible that the post-exercise cellular signals important for mitochondrial capacity (e.g., PGC-1a), blood flow (e.g., nitric oxide mediated endothelial function), glucose uptake (GLUT-4 translocation), as well as brain glucose metabolism that contribute to multi-organ insulin sensitivity, are blunted. This hypothesis was somewhat supported by prior work, whereby Sharoff et al. showed that metformin blunted the rise in AMPK activity during the immediate post-exercise period in insulin resistant adults, and this skeletal muscle observation directly correlated with attenuated insulin sensitivity (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). However, new work suggests that acute metformin treatment for 4 days did not affect AMPK activity during exercise in skeletal muscle or adipose tissue of lean healthy men. However, a novel observation was that metformin concentrations were detected in skeletal muscle, and it was proposed that longer duration (e.g., 5 days vs. 12 weeks) may be needed to elicit change in AMPK and/or mitochondrial content (<xref rid=\"B117\" ref-type=\"bibr\">117</xref>). We recognize though that not all studies support the action of metformin to reduce complex I of the mitochondria and impact indirectly AMPK, and this is an area of much debate (<xref rid=\"B122\" ref-type=\"bibr\">122</xref>). Indeed, recent work highlights that metformin may impede both the malate-aspartate as well as the glycerol-phosphate shuttle, thereby together increasing the cytosolic NADH:NAD+ ratio and allosterically inhibiting energetic processes that would support tissue function (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>). Interestingly, it was proposed that metformin may impact immune function in older adults following resistance training, and alleviate inflammatory mediated processes that may hinder muscle accretion in response to resistance exercise (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). This is consistent with the notion that metformin promotes polarization from M1 pro-inflammatory macrophages to M2 anti-inflammatory macrophages (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>) as well as induces autophagy to attenuate Th2 immune cell activation and inflammation (<xref rid=\"B123\" ref-type=\"bibr\">123</xref>). However, the results of the recent MASTERS trial showed no effect of metformin on resistance training-induced inflammation in skeletal muscle, despite the observation that lean body mass gains were blunted in relation to strength following the combined therapy compared with resistance exercise training alone. This was shown to parallel AMPK activation as well as inhibition of p70S6K1 phosphorylation (an immediate target of mTOR) (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). An additional or alternative explanation for the blunted muscle accretion post-training in the latter study may result from newer work showing that metformin reduces skeletal muscle autophagy and/or cell proliferation in C2C12 myotubes (<xref rid=\"B124\" ref-type=\"bibr\">124</xref>, <xref rid=\"B125\" ref-type=\"bibr\">125</xref>), although data in humans following exercise training is unknown. Taken together, with possible influences of gastrointestinal adaptations with metformin of gut microbiota, bile acids, and/or GLP-1 (<xref rid=\"B65\" ref-type=\"bibr\">65</xref>), additional work is required to understand the exact cellular mechanisms by which metformin interacts with exercise across tissues for optimization of glycemic control. In fact, it is important to acknowledge that there are no suggestions for altered fasting glucose or liver insulin action in response to exercise plus metformin. Moreover, although elevated FFA levels have been detected following the combined therapy, no studies have been specifically designed to understand adipose insulin sensitivity following exercise plus metformin treatment. Nor has there been work examining the interaction of exercise and metformin on vasculature or brain insulin sensitivity to understand the importance of blood delivery and neural control of glucose metabolism. At this time, skeletal muscle appears to be a primary tissue regulating blood glucose, and additional cellular work is warranted to understand if these combined therapies lead to over-taxation of bioenergetic pathways that result in mal-adaptation. This may be particularly important since new work suggests that exercise may alter the pharmacokinetics and increase the bio-availability of metformin in circulation (<xref rid=\"B126\" ref-type=\"bibr\">126</xref>).</p></sec><sec id=\"s9\"><title>Clinical Considerations and Conclusions</title><p>Developing precise exercise programs for maximal glycemic control remains to be identified. The collective literature suggests that, if anything, metformin attenuates the effects of exercise at improving insulin sensitivity at the level of skeletal muscle. Moreover, alterations in blood glucose, hypertension as well as inflammation have been noted. While no study to date has shown blood glucose to worsen as reflected by higher blood glucose concentrations relative to the start of the combined treatment, the literature highlights that there are either null, additive, or blunted effects on glycemia. The reason for this variability is not entirely clear but may relate to studies whereby people are habitual vs. naive metformin users or the outcome of interest. In either case, it is clear the magnitude of benefit will vary based on what tissue or outcome is of interest. Systemic studies determining the benefit of different exercise doses as well as risk factors of people (age, hypertension, dementia, T2D, etc.) co-prescribed metformin would enable individualized treatments that favor glycemic control. For instance, to date a basic biologic question is whether men or women respond differently to exercise plus metformin therapy based on underlying differences in aerobic fitness as well as muscle mass/fiber composition. Further, these gains in aerobic fitness and muscle mass are not only relevant to aging men and women with or without chronic disease, but also children and adolescents. It is well accepted that peak fitness/bone/muscle occur near the 3rd decade of life. But the effect of prescribing metformin with exercise in children and adolescents have on the rate of gain in these fitness outcomes is largely unknown in boys and girls. With emerging literature suggesting that off label or prophylactic use of metformin may be effective for weight management and obesity prevention in adolescents (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>, <xref rid=\"B71\" ref-type=\"bibr\">71</xref>, <xref rid=\"B127\" ref-type=\"bibr\">127</xref>) more children may be provided metformin and recommended to exercise. This raises potential concern toward altered maturation growth rates and cardiometabolic risk during youth as well as then for later in life health risk compared with youth advised to exercise only with proper nutrition (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>, <xref rid=\"B71\" ref-type=\"bibr\">71</xref>). Thus, health care providers should be aware of these potential interactions to strike balance between current disease risk with long-term well-being. We also recognize that people are not often prescribed only one medication, and further work is warranted to tease out the effects of multiple pharmacological agents or even dietary supplements (e.g., metformin with GLP-1 agonists, SGLT-2 inhibitors, statins, antioxidants, etc.) in combination with exercise to gain a better understanding on glucose metabolism. However, it is important to acknowledge that recent work has suggested that other glycemic medications, including GLP-1 agonists and SGLT-2 inhibitors, have been shown to interact with exercise (<xref rid=\"B128\" ref-type=\"bibr\">128</xref>–<xref rid=\"B130\" ref-type=\"bibr\">130</xref>). This highlights the potential for medications to interfere or add with exercise-mediated glycemic benefit. Thus, there is potential for people to be at risk for developing T2D or cardiovascular abnormalities when co-prescribed treatments compared with those treated with exercise alone over time. Large-randomized clinical trials are critically needed to determine the effects combining exercise, with or without diet, and medications for improved evidenced-based practice.</p></sec><sec id=\"s10\"><title>Author Contributions</title><p>SM wrote the majority of the review with NS providing edits. SM and NS collaborated on writing on the metformin and exercise on brain insulin sensitivity section. NS drafted the figure with SM providing edits.</p></sec><sec id=\"s11\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>We would like to thank Emily M. Heiston, Udeyvir Cheema and Anna Ballanytne for helpful discussions related to topics herein.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> SM was supported by National Institutes of Health RO1-HL130296.</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><collab>National Diabetes Statistics Report 2020 | CDC</collab></person-group> Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.cdc.gov/diabetes/library/features/diabetes-stat-report.html\">https://www.cdc.gov/diabetes/library/features/diabetes-stat-report.html</ext-link> (accessed April 23, 2020)</mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Church</surname><given-names>TS</given-names></name><name><surname>Blair</surname><given-names>SN</given-names></name><name><surname>Cocreham</surname><given-names>S</given-names></name><name><surname>Johannsen</surname><given-names>N</given-names></name><name><surname>Johnson</surname><given-names>W</given-names></name><name><surname>Kramer</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Effects of aerobic and resistance training on hemoglobin A1c levels in patients with type 2 diabetes: a randomized controlled trial</article-title>. <source>J Am Med Assoc.</source> (<year>2010</year>) <volume>304</volume>:<fpage>2253</fpage>–<lpage>62</lpage>. <pub-id pub-id-type=\"doi\">10.1001/jama.2010.1710</pub-id><pub-id pub-id-type=\"pmid\">21098771</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tuomilehto</surname><given-names>J</given-names></name><name><surname>Lindström</surname><given-names>J</given-names></name><name><surname>Eriksson</surname><given-names>JG</given-names></name><name><surname>Valle</surname><given-names>TT</given-names></name><name><surname>Hamäläinen</surname><given-names>H</given-names></name><name><surname>Ianne-Parikka</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance</article-title>. <source>N Engl J Med.</source> (<year>2001</year>) <volume>344</volume>:<fpage>1343</fpage>–<lpage>50</lpage>. <pub-id pub-id-type=\"doi\">10.1056/NEJM200105033441801</pub-id><pub-id pub-id-type=\"pmid\">11333990</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Knowler</surname><given-names>WC</given-names></name><name><surname>Barrett-Connor</surname><given-names>E</given-names></name><name><surname>Fowler</surname><given-names>SE</given-names></name><name><surname>Hamman</surname><given-names>RF</given-names></name><name><surname>Lachin</surname><given-names>JM</given-names></name><name><surname>Walker</surname><given-names>EA</given-names></name><etal/></person-group>\n<article-title>Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin</article-title>. <source>N Engl J Med.</source> (<year>2002</year>) <volume>346</volume>:<fpage>393</fpage>–<lpage>403</lpage>. <pub-id pub-id-type=\"doi\">10.1056/NEJMoa012512</pub-id><pub-id pub-id-type=\"pmid\">11832527</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Houmard</surname><given-names>JA</given-names></name><name><surname>Tanner</surname><given-names>CJ</given-names></name><name><surname>Slentz</surname><given-names>CA</given-names></name><name><surname>Duscha</surname><given-names>BD</given-names></name><name><surname>McCartney</surname><given-names>JS</given-names></name><name><surname>Kraus</surname><given-names>WE</given-names></name></person-group>. <article-title>Effect of the volume and intensity of exercise training on insulin sensitivity</article-title>. <source>J Appl Physiol.</source> (<year>2004</year>) <volume>96</volume>:<fpage>101</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1152/japplphysiol.00707.2003</pub-id><pub-id pub-id-type=\"pmid\">12972442</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dubé</surname><given-names>JJ</given-names></name><name><surname>Allison</surname><given-names>KF</given-names></name><name><surname>Rousson</surname><given-names>V</given-names></name><name><surname>Goodpaster</surname><given-names>BH</given-names></name><name><surname>Amati</surname><given-names>F</given-names></name></person-group>. <article-title>Exercise dose and insulin sensitivity: relevance for diabetes prevention</article-title>. <source>Med Sci Sports Exerc.</source> (<year>2012</year>) <volume>44</volume>:<fpage>793</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1249/MSS.0b013e31823f679f</pub-id><pub-id pub-id-type=\"pmid\">22051572</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Malin</surname><given-names>SK</given-names></name><name><surname>Solomon</surname><given-names>TPJ</given-names></name><name><surname>Blaszczak</surname><given-names>A</given-names></name><name><surname>Finnegan</surname><given-names>S</given-names></name><name><surname>Filion</surname><given-names>J</given-names></name><name><surname>Kirwan</surname><given-names>JP</given-names></name></person-group>. <article-title>Pancreatic β-cell function increases in a linear dose-response manner following exercise training in adults with prediabetes</article-title>. <source>Am J Physiol Endocrinol Metab.</source> (<year>2013</year>) <volume>305</volume>:<fpage>E1248</fpage>–<lpage>54</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpendo.00260.2013</pub-id><pub-id pub-id-type=\"pmid\">24045867</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Regensteiner</surname><given-names>JG</given-names></name><name><surname>Sippel</surname><given-names>J</given-names></name><name><surname>McFarling</surname><given-names>ET</given-names></name><name><surname>Wolfel</surname><given-names>EE</given-names></name><name><surname>Hiatt</surname><given-names>WR</given-names></name></person-group>\n<article-title>Effects of non-insulin-dependent diabetes on oxygen consumption during treadmill exercise</article-title>. <source>Med Sci Sports Exerc.</source> (<year>1995</year>) <volume>27</volume>:<fpage>875</fpage>–<lpage>81</lpage>. <pub-id pub-id-type=\"doi\">10.1249/00005768-199506000-00012</pub-id><pub-id pub-id-type=\"pmid\">7658949</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wahl</surname><given-names>MP</given-names></name><name><surname>Scalzo</surname><given-names>RL</given-names></name><name><surname>Regensteiner</surname><given-names>JG</given-names></name><name><surname>Reusch</surname><given-names>JEB</given-names></name></person-group>. <article-title>Mechanisms of aerobic exercise impairment in diabetes: a narrative review</article-title>. <source>Front Endocrinol.</source> (<year>2018</year>) <volume>9</volume>:<fpage>181</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fendo.2018.00181</pub-id><pub-id pub-id-type=\"pmid\">29720965</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><collab>American Diabetes Association</collab></person-group>. <article-title>9. Pharmacologic approaches to glycemic treatment: Standards of medical care in diabetes 2019</article-title>. <source>Diabetes Care.</source> (<year>2019</year>) <volume>42</volume>:<fpage>S90</fpage>–<lpage>102</lpage>. <pub-id pub-id-type=\"doi\">10.2337/dc19-S009</pub-id><pub-id pub-id-type=\"pmid\">30559235</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Viollet</surname><given-names>B</given-names></name><name><surname>Guigas</surname><given-names>B</given-names></name><name><surname>Sanz Garcia</surname><given-names>N</given-names></name><name><surname>Leclerc</surname><given-names>J</given-names></name><name><surname>Foretz</surname><given-names>M</given-names></name><name><surname>Andreelli</surname><given-names>F</given-names></name></person-group>. <article-title>Cellular and molecular mechanisms of metformin: an overview</article-title>. <source>Clin Sci.</source> (<year>2012</year>) <volume>122</volume>:<fpage>253</fpage>–<lpage>70</lpage>. <pub-id pub-id-type=\"doi\">10.1042/CS20110386</pub-id><pub-id pub-id-type=\"pmid\">22117616</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pollak</surname><given-names>M</given-names></name></person-group>. <article-title>Metformin and pancreatic cancer: a clue requiring investigation</article-title>. <source>Clin Cancer Res.</source> (<year>2012</year>) <volume>18</volume>:<fpage>2723</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1158/1078-0432.CCR-12-0694</pub-id><pub-id pub-id-type=\"pmid\">22465829</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Novelle</surname><given-names>MG</given-names></name><name><surname>Ali</surname><given-names>A</given-names></name><name><surname>Diéguez</surname><given-names>C</given-names></name><name><surname>Bernier</surname><given-names>M</given-names></name><name><surname>de Cabo</surname><given-names>R</given-names></name></person-group>. <article-title>Metformin: a hopeful promise in aging research</article-title>. <source>Cold Spring Harb Perspect Med.</source> (<year>2016</year>) <volume>6</volume>:<fpage>a025932</fpage>. <pub-id pub-id-type=\"doi\">10.1101/cshperspect.a025932</pub-id><pub-id pub-id-type=\"pmid\">26931809</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jenkins</surname><given-names>NT</given-names></name><name><surname>Padilla</surname><given-names>J</given-names></name><name><surname>Arce-Esquivel</surname><given-names>AA</given-names></name><name><surname>Bayless</surname><given-names>DS</given-names></name><name><surname>Martin</surname><given-names>JS</given-names></name><name><surname>Leidy</surname><given-names>HJ</given-names></name><etal/></person-group>. <article-title>Effects of endurance exercise training, metformin, and their combination on Adipose tissue leptin and IL-10 secretion in OLETF rats</article-title>. <source>J Appl Physiol.</source> (<year>2012</year>) <volume>113</volume>:<fpage>1873</fpage>–<lpage>83</lpage>. <pub-id pub-id-type=\"doi\">10.1152/japplphysiol.00936.2012</pub-id><pub-id pub-id-type=\"pmid\">23019312</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Baron</surname><given-names>AD</given-names></name></person-group>. <article-title>Hemodynamic actions of insulin</article-title>. <source>Am J Physiol Endocrinol Metab.</source> (<year>1994</year>) <volume>267</volume>:<fpage>E187</fpage>–<lpage>202</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpendo.1994.267.2.e187</pub-id><pub-id pub-id-type=\"pmid\">8074198</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ahima</surname><given-names>RS</given-names></name><name><surname>Antwi</surname><given-names>DA</given-names></name></person-group>. <article-title>Brain regulation of appetite and satiety</article-title>. <source>Endocrinol Metab Clin North Am.</source> (<year>2008</year>) <volume>37</volume>:<fpage>811</fpage>–<lpage>23</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ecl.2008.08.005</pub-id><pub-id pub-id-type=\"pmid\">19026933</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ryan</surname><given-names>D</given-names></name><name><surname>Espeland</surname><given-names>M</given-names></name><name><surname>Foster</surname><given-names>G</given-names></name><name><surname>Haffner</surname><given-names>S</given-names></name><name><surname>Hubbard</surname><given-names>V</given-names></name><name><surname>Johnson</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Look AHEAD (Action for Health in Diabetes): design and methods for a clinical trial of weight loss for the prevention of cardiovascular disease in type 2 diabetes</article-title>. <source>Control Clin Trials.</source> (<year>2003</year>) <volume>24</volume>:<fpage>610</fpage>–<lpage>28</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0197-2456(03)00064-3</pub-id><pub-id pub-id-type=\"pmid\">14500058</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Solomon</surname><given-names>TPJ</given-names></name><name><surname>Eves</surname><given-names>FF</given-names></name><name><surname>Laye</surname><given-names>MJ</given-names></name></person-group>. <article-title>Targeting postprandial hyperglycemia with physical activity may reduce cardiovascular disease risk</article-title>. But what should we do, and when is the right time to move? <source>Front Cardiovasc Med.</source> (<year>2018</year>) <volume>5</volume>:<fpage>99</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fcvm.2018.00099</pub-id><pub-id pub-id-type=\"pmid\">30073171</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gilbertson</surname><given-names>NM</given-names></name><name><surname>Eichner</surname><given-names>NZM</given-names></name><name><surname>Francois</surname><given-names>M</given-names></name><name><surname>Gaitan</surname><given-names>JM</given-names></name><name><surname>Heiston</surname><given-names>EM</given-names></name><name><surname>Weltman</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Glucose tolerance is linked to postprandial fuel use independent of exercise dose</article-title>. <source>Med Sci Sports Exerc.</source> (<year>2018</year>) <volume>50</volume>:<fpage>2058</fpage>–<lpage>66</lpage>. <pub-id pub-id-type=\"doi\">10.1249/MSS.0000000000001667</pub-id><pub-id pub-id-type=\"pmid\">29762253</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Slentz</surname><given-names>CA</given-names></name><name><surname>Bateman</surname><given-names>LA</given-names></name><name><surname>Willis</surname><given-names>LH</given-names></name><name><surname>Granville</surname><given-names>EO</given-names></name><name><surname>Piner</surname><given-names>LW</given-names></name><name><surname>Samsa</surname><given-names>GP</given-names></name><etal/></person-group>. <article-title>Effects of exercise training alone vs a combined exercise and nutritional lifestyle intervention on glucose homeostasis in prediabetic individuals: a randomised controlled trial</article-title>. <source>Diabetologia.</source> (<year>2016</year>) <volume>59</volume>:<fpage>2088</fpage>–<lpage>98</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00125-016-4051-z</pub-id><pub-id pub-id-type=\"pmid\">27421729</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mikus</surname><given-names>CR</given-names></name><name><surname>Boyle</surname><given-names>LJ</given-names></name><name><surname>Borengasser</surname><given-names>SJ</given-names></name><name><surname>Oberlin</surname><given-names>DJ</given-names></name><name><surname>Naples</surname><given-names>SP</given-names></name><name><surname>Fletcher</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Simvastatin impairs exercise training adaptations</article-title>. <source>J Am Coll Cardiol.</source> (<year>2013</year>) <volume>62</volume>:<fpage>709</fpage>–<lpage>14</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jacc.2013.02.074</pub-id><pub-id pub-id-type=\"pmid\">23583255</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lehtovirta</surname><given-names>M</given-names></name><name><surname>Forsén</surname><given-names>B</given-names></name><name><surname>Gullström</surname><given-names>M</given-names></name><name><surname>Häggblom</surname><given-names>M</given-names></name><name><surname>Eriksson</surname><given-names>JG</given-names></name><name><surname>Taskinen</surname><given-names>MR</given-names></name><etal/></person-group>. <article-title>Metabolic effects of metformin in patients with impaired glucose tolerance</article-title>. <source>Diabet Med.</source> (<year>2001</year>) <volume>18</volume>:<fpage>578</fpage>–<lpage>83</lpage>. <pub-id pub-id-type=\"doi\">10.1046/j.1464-5491.2001.00539.x</pub-id><pub-id pub-id-type=\"pmid\">11553189</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sharoff</surname><given-names>CG</given-names></name><name><surname>Hagobian</surname><given-names>TA</given-names></name><name><surname>Malin</surname><given-names>SK</given-names></name><name><surname>Chipkin</surname><given-names>SR</given-names></name><name><surname>Yu</surname><given-names>H</given-names></name><name><surname>Hirshman</surname><given-names>MF</given-names></name><etal/></person-group>. <article-title>Combining short-term metformin treatment and one bout of exercise does not increase insulin action in insulin-resistant individuals</article-title>. <source>Am J Physiol Endocrinol Metab.</source> (<year>2010</year>) <volume>298</volume>:<fpage>E815</fpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpendo.00517.2009</pub-id><pub-id pub-id-type=\"pmid\">20071560</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ramachandran</surname><given-names>A</given-names></name><name><surname>Snehalatha</surname><given-names>C</given-names></name><name><surname>Mary</surname><given-names>S</given-names></name><name><surname>Mukesh</surname><given-names>B</given-names></name><name><surname>Bhaskar</surname><given-names>AD</given-names></name><name><surname>Vijay</surname><given-names>V</given-names></name></person-group>. <article-title>The Indian Diabetes Prevention Programme shows that lifestyle modification and metformin prevent type 2 diabetes in Asian Indian subjects with impaired glucose tolerance (IDPP-1)</article-title>. <source>Diabetologia.</source> (<year>2006</year>) <volume>49</volume>:<fpage>289</fpage>–<lpage>97</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00125-005-0097-z</pub-id><pub-id pub-id-type=\"pmid\">16391903</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Love-Osborne</surname><given-names>K</given-names></name><name><surname>Sheeder</surname><given-names>J</given-names></name><name><surname>Zeitler</surname><given-names>P</given-names></name></person-group>. <article-title>Addition of metformin to a lifestyle modification program in adolescents with insulin resistance</article-title>. <source>J Pediatr.</source> (<year>2008</year>) <volume>152</volume>:<fpage>817</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jpeds.2008.01.018</pub-id><pub-id pub-id-type=\"pmid\">18492523</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Erickson</surname><given-names>ML</given-names></name><name><surname>Little</surname><given-names>JP</given-names></name><name><surname>Gay</surname><given-names>JL</given-names></name><name><surname>McCully</surname><given-names>KK</given-names></name><name><surname>Jenkins</surname><given-names>NT</given-names></name></person-group>. <article-title>Postmeal exercise blunts postprandial glucose excursions in people on metformin monotherapy</article-title>. <source>J Appl Physiol.</source> (<year>2017</year>) <volume>123</volume>:<fpage>444</fpage>–<lpage>50</lpage>. <pub-id pub-id-type=\"doi\">10.1152/japplphysiol.00213.2017</pub-id><pub-id pub-id-type=\"pmid\">28522762</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ortega</surname><given-names>JF</given-names></name><name><surname>Hamouti</surname><given-names>N</given-names></name><name><surname>Fernández-Elías</surname><given-names>VE</given-names></name><name><surname>de Prada</surname><given-names>MVG</given-names></name><name><surname>Martínez-Vizcaíno</surname><given-names>V</given-names></name><name><surname>Mora-Rodríguez</surname><given-names>R</given-names></name></person-group>. <article-title>Metformin does not attenuate the acute insulin-sensitizing effect of a single bout of exercise in individuals with insulin resistance</article-title>. <source>Acta Diabetol.</source> (<year>2014</year>) <volume>51</volume>:<fpage>749</fpage>–<lpage>55</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00592-014-0580-4</pub-id><pub-id pub-id-type=\"pmid\">24682492</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>T</given-names></name><name><surname>Lu</surname><given-names>C</given-names></name><name><surname>Schumann</surname><given-names>M</given-names></name><name><surname>Le</surname><given-names>S</given-names></name><name><surname>Yang</surname><given-names>Y</given-names></name><name><surname>Zhuang</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Timing of exercise affects glycemic control in type 2 diabetes patients treated with metformin</article-title>. <source>J Diabetes Res.</source> (<year>2018</year>) <volume>2018</volume>:<fpage>2483273</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2018/2483273</pub-id><pub-id pub-id-type=\"pmid\">29785399</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boulé</surname><given-names>NG</given-names></name><name><surname>Kenny</surname><given-names>GP</given-names></name><name><surname>Larose</surname><given-names>J</given-names></name><name><surname>Khandwala</surname><given-names>F</given-names></name><name><surname>Kuzik</surname><given-names>N</given-names></name><name><surname>Sigal</surname><given-names>RJ</given-names></name></person-group>. <article-title>Does metformin modify the effect on glycaemic control of aerobic exercise, resistance exercise or both?</article-title>\n<source>Diabetologia.</source> (<year>2013</year>) <volume>56</volume>:<fpage>2378</fpage>–<lpage>82</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00125-013-3026-6</pub-id><pub-id pub-id-type=\"pmid\">23975325</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Terada</surname><given-names>T</given-names></name><name><surname>Boulé</surname><given-names>NG</given-names></name></person-group>. <article-title>Does metformin therapy influence the effects of intensive lifestyle intervention?</article-title> Exploring the interaction between first line therapies in the Look AHEAD trial. <source>Metabolism.</source> (<year>2019</year>) <volume>94</volume>:<fpage>39</fpage>–<lpage>46</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.metabol.2019.01.004</pub-id><pub-id pub-id-type=\"pmid\">30653978</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boulé</surname><given-names>NG</given-names></name><name><surname>Robert</surname><given-names>C</given-names></name><name><surname>Bell</surname><given-names>GJ</given-names></name><name><surname>Johnson</surname><given-names>ST</given-names></name><name><surname>Bell</surname><given-names>RC</given-names></name><name><surname>Lewanczuk</surname><given-names>RZ</given-names></name><etal/></person-group>. <article-title>Metformin and exercise in type 2 diabetes</article-title>. <source>Diabetes Care.</source> (<year>2011</year>) <volume>34</volume>:<fpage>1469</fpage>–<lpage>74</lpage>. <pub-id pub-id-type=\"doi\">10.2337/DC10-2207</pub-id><pub-id pub-id-type=\"pmid\">21602430</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Malin</surname><given-names>SK</given-names></name><name><surname>Gerber</surname><given-names>R</given-names></name><name><surname>Chipkin</surname><given-names>SR</given-names></name><name><surname>Braun</surname><given-names>B</given-names></name></person-group>. <article-title>Independent and combined effects of exercise training and metformin on insulin sensitivity in individuals with prediabetes</article-title>. <source>Diabetes Care.</source> (<year>2012</year>) <volume>35</volume>:<fpage>131</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.2337/dc11-0925</pub-id><pub-id pub-id-type=\"pmid\">22040838</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Konopka</surname><given-names>AR</given-names></name><name><surname>Laurin</surname><given-names>JL</given-names></name><name><surname>Schoenberg</surname><given-names>HM</given-names></name><name><surname>Reid</surname><given-names>JJ</given-names></name><name><surname>Castor</surname><given-names>WM</given-names></name><name><surname>Wolff</surname><given-names>CA</given-names></name><etal/></person-group>. <article-title>Metformin inhibits mitochondrial adaptations to aerobic exercise training in older adults</article-title>. <source>Aging Cell.</source> (<year>2019</year>) <volume>18</volume>:<fpage>e12880</fpage>. <pub-id pub-id-type=\"doi\">10.1111/acel.12880</pub-id><pub-id pub-id-type=\"pmid\">30548390</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Walton</surname><given-names>RG</given-names></name><name><surname>Dungan</surname><given-names>CM</given-names></name><name><surname>Long</surname><given-names>DE</given-names></name><name><surname>Tuggle</surname><given-names>SC</given-names></name><name><surname>Kosmac</surname><given-names>K</given-names></name><name><surname>Peck</surname><given-names>BD</given-names></name><etal/></person-group>. <article-title>Metformin blunts muscle hypertrophy in response to progressive resistance exercise training in older adults: a randomized, double-blind, placebo-controlled, multicenter trial: the MASTERS trial</article-title>. <source>Aging Cell.</source> (<year>2019</year>) <volume>18</volume>:<fpage>e13039</fpage>. <pub-id pub-id-type=\"doi\">10.1111/acel.13039</pub-id><pub-id pub-id-type=\"pmid\">31557380</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hawley</surname><given-names>JA</given-names></name><name><surname>Lessard</surname><given-names>SJ</given-names></name></person-group>. <article-title>Exercise training-induced improvements in insulin action</article-title>. <source>Acta Physiol.</source> (<year>2008</year>) <volume>192</volume>:<fpage>127</fpage>–<lpage>35</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1748-1716.2007.01783.x</pub-id><pub-id pub-id-type=\"pmid\">18171435</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Holloszy</surname><given-names>JO</given-names></name></person-group>\n<article-title>Invited review: exercise-induced increase in muscle insulin sensitivity</article-title>. <source>J Appl Physiol.</source> (<year>2005</year>) <volume>99</volume>:<fpage>338</fpage>–<lpage>43</lpage>. <pub-id pub-id-type=\"doi\">10.1152/japplphysiol.00123.2005</pub-id><pub-id pub-id-type=\"pmid\">16036907</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Stumvoll</surname><given-names>M</given-names></name><name><surname>Häring</surname><given-names>HU</given-names></name><name><surname>Matthaei</surname><given-names>S</given-names></name></person-group>. <article-title>Metformin</article-title>. <source>Endocr Res.</source> (<year>2006</year>) <volume>32</volume>:<fpage>39</fpage>–<lpage>57</lpage>. <pub-id pub-id-type=\"doi\">10.1080/07435800701743828</pub-id><pub-id pub-id-type=\"pmid\">18271504</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>C</given-names></name><name><surname>Liu</surname><given-names>F</given-names></name><name><surname>Yuan</surname><given-names>Y</given-names></name><name><surname>Wu</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>H</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Metformin suppresses lipid accumulation in skeletal muscle by promoting fatty acid oxidation</article-title>. <source>Clin Lab.</source> (<year>2014</year>) <volume>60</volume>:<fpage>887</fpage>–<lpage>96</lpage>. <pub-id pub-id-type=\"doi\">10.7754/Clin.Lab.2013.130531</pub-id><pub-id pub-id-type=\"pmid\">25016691</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zabielski</surname><given-names>P</given-names></name><name><surname>Chacinska</surname><given-names>M</given-names></name><name><surname>Charkiewicz</surname><given-names>K</given-names></name><name><surname>Baranowski</surname><given-names>M</given-names></name><name><surname>Gorski</surname><given-names>J</given-names></name><name><surname>Blachnio-Zabielska</surname><given-names>AU</given-names></name></person-group>. <article-title>Effect of metformin on bioactive lipid metabolism in insulin-resistant muscle</article-title>. <source>J Endocrinol.</source> (<year>2017</year>) <volume>233</volume>:<fpage>329</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1530/JOE-16-0381</pub-id><pub-id pub-id-type=\"pmid\">28522731</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Malin</surname><given-names>SK</given-names></name><name><surname>Stephens</surname><given-names>BR</given-names></name><name><surname>Sharoff</surname><given-names>CG</given-names></name><name><surname>Hagobian</surname><given-names>TA</given-names></name><name><surname>Chipkin</surname><given-names>SR</given-names></name><name><surname>Braun</surname><given-names>B</given-names></name></person-group>. <article-title>Metformin's effect on exercise and postexercise substrate oxidation</article-title>. <source>Int J Sport Nutr Exerc Metab.</source> (<year>2010</year>) <volume>20</volume>:<fpage>63</fpage>–<lpage>71</lpage>. <pub-id pub-id-type=\"doi\">10.1123/ijsnem.20.1.63</pub-id><pub-id pub-id-type=\"pmid\">20190353</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Linden</surname><given-names>MA</given-names></name><name><surname>Fletcher</surname><given-names>JA</given-names></name><name><surname>Matthew Morris</surname><given-names>E</given-names></name><name><surname>Meers</surname><given-names>GM</given-names></name><name><surname>Kearney</surname><given-names>ML</given-names></name><name><surname>Crissey</surname><given-names>JM</given-names></name><etal/></person-group>. <article-title>Combining metformin and aerobic exercise training in the treatment of type 2 diabetes and NAFLD in OLETF rats</article-title>. <source>Am J Physiol Endocrinol Metab.</source> (<year>2014</year>) <volume>306</volume>:<fpage>E300</fpage>–<lpage>10</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpendo.00427.2013</pub-id><pub-id pub-id-type=\"pmid\">24326426</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Basu</surname><given-names>R</given-names></name><name><surname>Chandramouli</surname><given-names>V</given-names></name><name><surname>Dicke</surname><given-names>B</given-names></name><name><surname>Landau</surname><given-names>B</given-names></name><name><surname>Rizza</surname><given-names>R</given-names></name></person-group>. <article-title>Obesity and type 2 diabetes impair insulin-induced suppression of glycogenolysis as well as gluconeogenesis</article-title>. <source>Diabetes.</source> (<year>2005</year>) <volume>54</volume>:<fpage>1942</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.2337/diabetes.54.7.1942</pub-id><pub-id pub-id-type=\"pmid\">15983193</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Winnick</surname><given-names>JJ</given-names></name><name><surname>Sherman</surname><given-names>WM</given-names></name><name><surname>Habash</surname><given-names>DL</given-names></name><name><surname>Stout</surname><given-names>MB</given-names></name><name><surname>Failla</surname><given-names>ML</given-names></name><name><surname>Belury</surname><given-names>MA</given-names></name><etal/></person-group>\n<article-title>Short-term aerobic exercise training in obese humans with type 2 diabetes mellitus improves whole-body insulin sensitivity through gains in peripheral, not hepatic insulin sensitivity</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2008</year>) <volume>93</volume>:<fpage>771</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2007-1524</pub-id><pub-id pub-id-type=\"pmid\">18073312</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Devlin</surname><given-names>JT</given-names></name><name><surname>Hirshman</surname><given-names>M</given-names></name><name><surname>Horton</surname><given-names>ED</given-names></name><name><surname>Horton</surname><given-names>ES</given-names></name></person-group>. <article-title>Enhanced peripheral and splanchnic insulin sensitivity in NIDDM men after single bout of exercise</article-title>. <source>Diabetes.</source> (<year>1987</year>) <volume>36</volume>:<fpage>434</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.2337/diab.36.4.434</pub-id><pub-id pub-id-type=\"pmid\">3102297</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kirwan</surname><given-names>JP</given-names></name><name><surname>Solomon</surname><given-names>TPJ</given-names></name><name><surname>Wojta</surname><given-names>DM</given-names></name><name><surname>Staten</surname><given-names>MA</given-names></name><name><surname>Holloszy</surname><given-names>JO</given-names></name></person-group>. <article-title>Effects of 7 days of exercise training on insulin sensitivity and responsiveness in type 2 diabetes mellitus</article-title>. <source>Am J Physiol Endocrinol Metab.</source> (<year>2009</year>) <volume>297</volume>:<fpage>E151</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpendo.00210.2009</pub-id><pub-id pub-id-type=\"pmid\">19383872</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Malin</surname><given-names>SK</given-names></name><name><surname>Del Rincon</surname><given-names>JP</given-names></name><name><surname>Huang</surname><given-names>H</given-names></name><name><surname>Kirwan</surname><given-names>JP</given-names></name></person-group>. <article-title>Exercise-induced lowering of fetuin-A may increase hepatic insulin sensitivity</article-title>. <source>Med Sci Sports Exerc.</source> (<year>2014</year>) <volume>46</volume>:<fpage>2085</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1249/MSS.0000000000000338</pub-id><pub-id pub-id-type=\"pmid\">24637346</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Black</surname><given-names>SE</given-names></name><name><surname>Mitchell</surname><given-names>E</given-names></name><name><surname>Freedson</surname><given-names>PS</given-names></name><name><surname>Chipkin</surname><given-names>SR</given-names></name><name><surname>Braun</surname><given-names>B</given-names></name></person-group>. <article-title>Improved insulin action following short-term exercise training: role of energy and carbohydrate balance</article-title>. <source>J Appl Physiol.</source> (<year>2005</year>) <volume>99</volume>:<fpage>2285</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1152/japplphysiol.00291.2005</pub-id><pub-id pub-id-type=\"pmid\">16081626</pub-id></mixed-citation></ref><ref id=\"B48\"><label>48.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Camacho</surname><given-names>RC</given-names></name><name><surname>Donahue</surname><given-names>EP</given-names></name><name><surname>James</surname><given-names>FD</given-names></name><name><surname>Berglund</surname><given-names>ED</given-names></name><name><surname>Wasserman</surname><given-names>DH</given-names></name></person-group>. <article-title>Energy state of the liver during short-term and exhaustive exercise in C57BL/6J mice</article-title>. <source>Am J Physiol Endocrinol Metab.</source> (<year>2006</year>) <volume>290</volume>:<fpage>E405</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpendo.00385.2005</pub-id><pub-id pub-id-type=\"pmid\">16219665</pub-id></mixed-citation></ref><ref id=\"B49\"><label>49.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Foretz</surname><given-names>M</given-names></name><name><surname>Guigas</surname><given-names>B</given-names></name><name><surname>Viollet</surname><given-names>B</given-names></name></person-group>. <article-title>Understanding the glucoregulatory mechanisms of metformin in type 2 diabetes mellitus</article-title>. <source>Nat Rev Endocrinol.</source> (<year>2019</year>) <volume>15</volume>:<fpage>569</fpage>–<lpage>89</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41574-019-0242-2</pub-id><pub-id pub-id-type=\"pmid\">31439934</pub-id></mixed-citation></ref><ref id=\"B50\"><label>50.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Miller</surname><given-names>RA</given-names></name><name><surname>Chu</surname><given-names>Q</given-names></name><name><surname>Xie</surname><given-names>J</given-names></name><name><surname>Foretz</surname><given-names>M</given-names></name><name><surname>Viollet</surname><given-names>B</given-names></name><name><surname>Birnbaum</surname><given-names>MJ</given-names></name></person-group>. <article-title>Biguanides suppress hepatic glucagon signalling by decreasing production of cyclic AMP</article-title>. <source>Nature</source>. (<year>2013</year>) <volume>494</volume>:<fpage>256</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature11808</pub-id><pub-id pub-id-type=\"pmid\">23292513</pub-id></mixed-citation></ref><ref id=\"B51\"><label>51.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mazza</surname><given-names>A</given-names></name><name><surname>Fruci</surname><given-names>B</given-names></name><name><surname>Garinis</surname><given-names>GA</given-names></name><name><surname>Giuliano</surname><given-names>S</given-names></name><name><surname>Malaguarnera</surname><given-names>R</given-names></name><name><surname>Belfiore</surname><given-names>A</given-names></name></person-group>. <article-title>The role of metformin in the management of NAFLD</article-title>. <source>Exp Diabetes Res</source>. (<year>2012</year>) <volume>2012</volume>:<fpage>716404</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2012/716404</pub-id><pub-id pub-id-type=\"pmid\">22194737</pub-id></mixed-citation></ref><ref id=\"B52\"><label>52.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Linden</surname><given-names>MA</given-names></name><name><surname>Lopez</surname><given-names>KT</given-names></name><name><surname>Fletcher</surname><given-names>JA</given-names></name><name><surname>Morris</surname><given-names>EM</given-names></name><name><surname>Meers</surname><given-names>GM</given-names></name><name><surname>Siddique</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Combining metformin therapy with caloric restriction for the management of type 2 diabetes and nonalcoholic fatty liver disease in obese rats</article-title>. <source>Appl Physiol Nutr Metab.</source> (<year>2015</year>) <volume>40</volume>:<fpage>1038</fpage>–<lpage>47</lpage>. <pub-id pub-id-type=\"doi\">10.1139/apnm-2015-0236</pub-id><pub-id pub-id-type=\"pmid\">26394261</pub-id></mixed-citation></ref><ref id=\"B53\"><label>53.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jensen</surname><given-names>MD</given-names></name><name><surname>Nielsen</surname><given-names>S</given-names></name></person-group>. <article-title>Insulin dose response analysis of free fatty acid kinetics</article-title>. <source>Metabolism.</source> (<year>2007</year>) <volume>56</volume>:<fpage>68</fpage>–<lpage>76</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.metabol.2006.08.022</pub-id><pub-id pub-id-type=\"pmid\">17161228</pub-id></mixed-citation></ref><ref id=\"B54\"><label>54.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Clarson</surname><given-names>CL</given-names></name><name><surname>Mahmud</surname><given-names>FH</given-names></name><name><surname>Baker</surname><given-names>JE</given-names></name><name><surname>Clark</surname><given-names>HE</given-names></name><name><surname>Mckay</surname><given-names>WM</given-names></name><name><surname>Schauteet</surname><given-names>VD</given-names></name><etal/></person-group>\n<article-title>Metformin in combination with structured lifestyle intervention improved body mass index in obese adolescents, but did not improve insulin resistance</article-title>. <source>Endocrine.</source> (<year>2009</year>) <volume>36</volume>:<fpage>141</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s12020-009-9196-9</pub-id><pub-id pub-id-type=\"pmid\">19387874</pub-id></mixed-citation></ref><ref id=\"B55\"><label>55.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Denis McGarry</surname><given-names>J</given-names></name></person-group>. <article-title>Dysregulation of fatty acid metabolism in the etiology of type 2 diabetes</article-title>. <source>Diabetes.</source> (<year>2002</year>) <volume>51</volume>:<fpage>7</fpage>–<lpage>18</lpage>. <pub-id pub-id-type=\"doi\">10.2337/diabetes.51.1.7</pub-id><pub-id pub-id-type=\"pmid\">11756317</pub-id></mixed-citation></ref><ref id=\"B56\"><label>56.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cusi</surname><given-names>K</given-names></name><name><surname>Kashyap</surname><given-names>S</given-names></name><name><surname>Gastaldelli</surname><given-names>A</given-names></name><name><surname>Bajaj</surname><given-names>M</given-names></name><name><surname>Cersosimo</surname><given-names>E</given-names></name></person-group>. <article-title>Effects on insulin secretion and insulin action of a 48-h reduction of plasma free fatty acids with acipimox in nondiabetic subjects genetically predisposed to type 2 diabetes</article-title>. <source>Am J Physiol Endocrinol Metab.</source> (<year>2007</year>) <volume>292</volume>:<fpage>E1775</fpage>–<lpage>81</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpendo.00624.2006</pub-id><pub-id pub-id-type=\"pmid\">17299078</pub-id></mixed-citation></ref><ref id=\"B57\"><label>57.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tönjes</surname><given-names>A</given-names></name><name><surname>Fasshauer</surname><given-names>M</given-names></name><name><surname>Kratzsch</surname><given-names>J</given-names></name><name><surname>Stumvoll</surname><given-names>M</given-names></name><name><surname>Bluher</surname><given-names>M</given-names></name></person-group>. <article-title>Adipokine pattern in subjects with impaired fasting glucose and impaired glucose tolerance in comparison to normal glucose tolerance and diabetes</article-title>. <source>PLoS ONE.</source> (<year>2010</year>) <volume>5</volume>:<fpage>e13911</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0013911</pub-id><pub-id pub-id-type=\"pmid\">21085476</pub-id></mixed-citation></ref><ref id=\"B58\"><label>58.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van de Weijer</surname><given-names>T</given-names></name><name><surname>Sparks</surname><given-names>LM</given-names></name><name><surname>Phielix</surname><given-names>E</given-names></name><name><surname>Meex</surname><given-names>RC</given-names></name><name><surname>van Herpen</surname><given-names>NA</given-names></name><name><surname>Hesselink</surname><given-names>MKC</given-names></name><etal/></person-group>. <article-title>Relationships between mitochondrial function and metabolic flexibility in type 2 diabetes mellitus</article-title>. <source>PLoS ONE.</source> (<year>2013</year>) <volume>8</volume>:<fpage>e51648</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0051648</pub-id><pub-id pub-id-type=\"pmid\">23418416</pub-id></mixed-citation></ref><ref id=\"B59\"><label>59.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brennan</surname><given-names>AM</given-names></name><name><surname>Day</surname><given-names>AG</given-names></name><name><surname>Cowan</surname><given-names>TE</given-names></name><name><surname>Clarke</surname><given-names>GJ</given-names></name><name><surname>Lamarche</surname><given-names>B</given-names></name><name><surname>Ross</surname><given-names>R</given-names></name></person-group>. <article-title>Individual response to standardized exercise: total and abdominal adipose tissue</article-title>. <source>Med. Sci. Sports Exerc.</source> (<year>2019</year>) <volume>52</volume>:<fpage>490</fpage>–<lpage>7</lpage>\n<pub-id pub-id-type=\"doi\">10.1249/MSS.0000000000001930</pub-id><pub-id pub-id-type=\"pmid\">31479006</pub-id></mixed-citation></ref><ref id=\"B60\"><label>60.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Solomon</surname><given-names>TPJ</given-names></name><name><surname>Sistrun</surname><given-names>SN</given-names></name><name><surname>Krishnan</surname><given-names>RK</given-names></name><name><surname>Del Aguila</surname><given-names>LF</given-names></name><name><surname>Marchetti</surname><given-names>CM</given-names></name><name><surname>O'Carroll</surname><given-names>SM</given-names></name><etal/></person-group>. <article-title>Exercise and diet enhance fat oxidation and reduce insulin resistance in older obese adults</article-title>. <source>J Appl Physiol.</source> (<year>2008</year>) <volume>104</volume>:<fpage>1313</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1152/japplphysiol.00890.2007</pub-id><pub-id pub-id-type=\"pmid\">18323464</pub-id></mixed-citation></ref><ref id=\"B61\"><label>61.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gilbertson</surname><given-names>NM</given-names></name><name><surname>Eichner</surname><given-names>NZM</given-names></name><name><surname>Heiston</surname><given-names>EM</given-names></name><name><surname>Gaitán</surname><given-names>JM</given-names></name><name><surname>Francois</surname><given-names>ME</given-names></name><name><surname>Mehaffey</surname><given-names>JH</given-names></name><etal/></person-group>. <article-title>A low-calorie diet with or without interval exercise training improves adiposopathy in obese women</article-title>. <source>Appl Physiol Nutr Metab.</source> (<year>2019</year>) <volume>44</volume>:<fpage>1057</fpage>–<lpage>64</lpage>. <pub-id pub-id-type=\"doi\">10.1139/apnm-2018-0717</pub-id><pub-id pub-id-type=\"pmid\">30785773</pub-id></mixed-citation></ref><ref id=\"B62\"><label>62.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Heiston</surname><given-names>EM</given-names></name><name><surname>Eichner</surname><given-names>NZ</given-names></name><name><surname>Gilbertson</surname><given-names>NM</given-names></name><name><surname>Malin</surname><given-names>SK</given-names></name></person-group>. <article-title>Exercise improves adiposopathy, insulin sensitivity and metabolic syndrome severity independent of intensity</article-title>. <source>Exp Physiol</source>. (<year>2020</year>) <volume>105</volume>:<fpage>632</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1113/EP088158</pub-id><pub-id pub-id-type=\"pmid\">32020676</pub-id></mixed-citation></ref><ref id=\"B63\"><label>63.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Haus</surname><given-names>JM</given-names></name><name><surname>Solomon</surname><given-names>TPJ</given-names></name><name><surname>Marchetti</surname><given-names>CM</given-names></name><name><surname>Edmison</surname><given-names>JM</given-names></name><name><surname>González</surname><given-names>F</given-names></name><name><surname>Kirwan</surname><given-names>JP</given-names></name></person-group>. <article-title>Free fatty acid-induced hepatic insulin resistance is attenuated following lifestyle intervention in obese individuals with impaired glucose tolerance</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2010</year>) <volume>95</volume>:<fpage>323</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2009-1101</pub-id><pub-id pub-id-type=\"pmid\">19906790</pub-id></mixed-citation></ref><ref id=\"B64\"><label>64.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Solomon</surname><given-names>TPJ</given-names></name><name><surname>Haus</surname><given-names>JM</given-names></name><name><surname>Marchetti</surname><given-names>CM</given-names></name><name><surname>Stanley</surname><given-names>WC</given-names></name><name><surname>Kirwan</surname><given-names>JP</given-names></name></person-group>. <article-title>Effects of exercise training and diet on lipid kinetics during free fatty acid-induced insulin resistance in older obese humans with impaired glucose tolerance</article-title>. <source>Am J Physiol Metab.</source> (<year>2009</year>) <volume>297</volume>:<fpage>E552</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpendo.00220.2009</pub-id><pub-id pub-id-type=\"pmid\">19531640</pub-id></mixed-citation></ref><ref id=\"B65\"><label>65.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Malin</surname><given-names>SK</given-names></name><name><surname>Kashyap</surname><given-names>SR</given-names></name></person-group>. <article-title>Effects of metformin on weight loss: potential mechanisms</article-title>. <source>Curr Opin Endocrinol Diabetes Obes.</source> (<year>2014</year>) <volume>21</volume>:<fpage>323</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1097/MED.0000000000000095</pub-id><pub-id pub-id-type=\"pmid\">25105996</pub-id></mixed-citation></ref><ref id=\"B66\"><label>66.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Flechtner-Mors</surname><given-names>M</given-names></name><name><surname>Ditschuneit</surname><given-names>HH</given-names></name><name><surname>Jenkinson</surname><given-names>CP</given-names></name><name><surname>Alt</surname><given-names>A</given-names></name><name><surname>Adler</surname><given-names>G</given-names></name></person-group>. <article-title>Metformin inhibits catecholamine-stimulated lipolysis in obese, hyperinsulinemic, hypertensive subjects in subcutaneous adipose tissue: an <italic>in situ</italic> microdialysis study</article-title>. <source>Diabet Med.</source> (<year>1999</year>) <volume>16</volume>:<fpage>1000</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1046/j.1464-5491.1999.00189.x</pub-id><pub-id pub-id-type=\"pmid\">10656228</pub-id></mixed-citation></ref><ref id=\"B67\"><label>67.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhang</surname><given-names>T</given-names></name><name><surname>He</surname><given-names>J</given-names></name><name><surname>Xu</surname><given-names>C</given-names></name><name><surname>Zu</surname><given-names>L</given-names></name><name><surname>Jiang</surname><given-names>H</given-names></name><name><surname>Pu</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Mechanisms of metformin inhibiting lipolytic response to isoproterenol in primary rat adipocytes</article-title>. <source>J Mol Endocrinol.</source> (<year>2009</year>) <volume>42</volume>:<fpage>57</fpage>–<lpage>66</lpage>. <pub-id pub-id-type=\"doi\">10.1677/JME-08-0130</pub-id><pub-id pub-id-type=\"pmid\">18955435</pub-id></mixed-citation></ref><ref id=\"B68\"><label>68.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bourron</surname><given-names>O</given-names></name><name><surname>Daval</surname><given-names>M</given-names></name><name><surname>Hainault</surname><given-names>I</given-names></name><name><surname>Hajduch</surname><given-names>E</given-names></name><name><surname>Servant</surname><given-names>JM</given-names></name><name><surname>Gautier</surname><given-names>JF</given-names></name><etal/></person-group>. <article-title>Biguanides and thiazolidinediones inhibit stimulated lipolysis in human adipocytes through activation of AMP-activated protein kinase</article-title>. <source>Diabetologia.</source> (<year>2010</year>) <volume>53</volume>:<fpage>768</fpage>–<lpage>78</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00125-009-1639-6</pub-id><pub-id pub-id-type=\"pmid\">20043143</pub-id></mixed-citation></ref><ref id=\"B69\"><label>69.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Malin</surname><given-names>SK</given-names></name><name><surname>Braun</surname><given-names>B</given-names></name></person-group>. <article-title>Effect of metformin on substrate utilization after exercise training in adults with impaired glucose tolerance</article-title>. <source>Appl Physiol Nutr Metab.</source> (<year>2013</year>) <volume>38</volume>:<fpage>427</fpage>–<lpage>30</lpage>. <pub-id pub-id-type=\"doi\">10.1139/apnm-2012-0433</pub-id><pub-id pub-id-type=\"pmid\">23713537</pub-id></mixed-citation></ref><ref id=\"B70\"><label>70.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krauzová</surname><given-names>E</given-names></name><name><surname>Tuma</surname><given-names>P</given-names></name><name><surname>de Glisezinski</surname><given-names>I</given-names></name><name><surname>Štich</surname><given-names>V</given-names></name><name><surname>Šiklová</surname><given-names>M</given-names></name></person-group>. <article-title>Metformin does not inhibit exercise-induced lipolysis in adipose tissue in young healthy lean men</article-title>. <source>Front Physiol.</source> (<year>2018</year>) <volume>9</volume>:<fpage>604</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fphys.2018.00604</pub-id><pub-id pub-id-type=\"pmid\">29875699</pub-id></mixed-citation></ref><ref id=\"B71\"><label>71.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mauras</surname><given-names>N</given-names></name><name><surname>DelGiorno</surname><given-names>C</given-names></name><name><surname>Hossain</surname><given-names>J</given-names></name><name><surname>Bird</surname><given-names>K</given-names></name><name><surname>Killen</surname><given-names>K</given-names></name><name><surname>Merinbaum</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Metformin use in children with obesity and normal glucose tolerance-effects on cardiovascular markers and intrahepatic fat</article-title>. <source>J Pediatr Endocrinol Metab.</source> (<year>2012</year>) <volume>25</volume>:<fpage>33</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1515/jpem-2011-0450</pub-id><pub-id pub-id-type=\"pmid\">22570948</pub-id></mixed-citation></ref><ref id=\"B72\"><label>72.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Barrett</surname><given-names>EJ</given-names></name><name><surname>Wang</surname><given-names>H</given-names></name><name><surname>Upchurch</surname><given-names>CT</given-names></name><name><surname>Liu</surname><given-names>Z</given-names></name></person-group>. <article-title>Insulin regulates its own delivery to skeletal muscle by feed-forward actions on the vasculature</article-title>. <source>Am J Physiol Endocrinol Metab.</source> (<year>2011</year>) <volume>301</volume>:<fpage>E252</fpage>. <pub-id pub-id-type=\"doi\">10.1152/ajpendo.00186.2011</pub-id><pub-id pub-id-type=\"pmid\">21610226</pub-id></mixed-citation></ref><ref id=\"B73\"><label>73.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jahn</surname><given-names>LA</given-names></name><name><surname>Hartline</surname><given-names>L</given-names></name><name><surname>Rao</surname><given-names>N</given-names></name><name><surname>Logan</surname><given-names>B</given-names></name><name><surname>Kim</surname><given-names>JJ</given-names></name><name><surname>Aylor</surname><given-names>K</given-names></name><etal/></person-group>\n<article-title>Insulin enhances endothelial function throughout the arterial tree in healthy but not metabolic syndrome subjects</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2016</year>) <volume>101</volume>:<fpage>1198</fpage>–<lpage>206</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2015-3293</pub-id><pub-id pub-id-type=\"pmid\">26756115</pub-id></mixed-citation></ref><ref id=\"B74\"><label>74.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>De Filippis</surname><given-names>E</given-names></name><name><surname>Cusi</surname><given-names>K</given-names></name><name><surname>Ocampo</surname><given-names>G</given-names></name><name><surname>Berria</surname><given-names>R</given-names></name><name><surname>Buck</surname><given-names>S</given-names></name><name><surname>Consoli</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Exercise-induced improvement in vasodilatory function accompanies increased insulin sensitivity in obesity and type 2 diabetes mellitus</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2006</year>) <volume>91</volume>:<fpage>4903</fpage>–<lpage>10</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2006-1142</pub-id><pub-id pub-id-type=\"pmid\">17018657</pub-id></mixed-citation></ref><ref id=\"B75\"><label>75.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Paulsen</surname><given-names>G</given-names></name><name><surname>Cumming</surname><given-names>KT</given-names></name><name><surname>Holden</surname><given-names>G</given-names></name><name><surname>Hallén</surname><given-names>J</given-names></name><name><surname>Rønnestad</surname><given-names>BR</given-names></name><name><surname>Sveen</surname><given-names>O</given-names></name><etal/></person-group>. <article-title>Vitamin C and E supplementation hampers cellular adaptation to endurance training in humans: a double-blind, randomised, controlled trial</article-title>. <source>J Physiol.</source> (<year>2014</year>) <volume>592</volume>:<fpage>1887</fpage>–<lpage>901</lpage>. <pub-id pub-id-type=\"doi\">10.1113/jphysiol.2013.267419</pub-id><pub-id pub-id-type=\"pmid\">24492839</pub-id></mixed-citation></ref><ref id=\"B76\"><label>76.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wei</surname><given-names>M</given-names></name><name><surname>Gibbons</surname><given-names>LW</given-names></name><name><surname>Mitchell</surname><given-names>TL</given-names></name><name><surname>Kampert</surname><given-names>JB</given-names></name><name><surname>Lee</surname><given-names>CD</given-names></name><name><surname>Blair</surname><given-names>SN</given-names></name></person-group>. <article-title>The association between cardiorespiratory fitness and impaired fasting glucose and type 2 diabetes mellitus in men</article-title>. <source>Ann Intern Med.</source> (<year>1999</year>) <volume>130</volume>:<fpage>89</fpage>–<lpage>96</lpage>. <pub-id pub-id-type=\"doi\">10.7326/0003-4819-130-2-199901190-00002</pub-id><pub-id pub-id-type=\"pmid\">10068380</pub-id></mixed-citation></ref><ref id=\"B77\"><label>77.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eichner</surname><given-names>NZM</given-names></name><name><surname>Gaitán</surname><given-names>JM</given-names></name><name><surname>Gilbertson</surname><given-names>NM</given-names></name><name><surname>Khurshid</surname><given-names>M</given-names></name><name><surname>Weltman</surname><given-names>A</given-names></name><name><surname>Malin</surname><given-names>SK</given-names></name></person-group>. <article-title>Postprandial augmentation index is reduced in adults with prediabetes following continuous and interval exercise training</article-title>. <source>Exp Physiol.</source> (<year>2019</year>) <volume>104</volume>:<fpage>264</fpage>–<lpage>71</lpage>. <pub-id pub-id-type=\"doi\">10.1113/EP087305</pub-id><pub-id pub-id-type=\"pmid\">30537411</pub-id></mixed-citation></ref><ref id=\"B78\"><label>78.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Malin</surname><given-names>SK</given-names></name><name><surname>Gilbertson</surname><given-names>NM</given-names></name><name><surname>Eichner</surname><given-names>NZM</given-names></name><name><surname>Heiston</surname><given-names>E</given-names></name><name><surname>Miller</surname><given-names>S</given-names></name><name><surname>Weltman</surname><given-names>A</given-names></name></person-group>. <article-title>Impact of short-term continuous and interval exercise training on endothelial function and glucose metabolism in prediabetes</article-title>. <source>J Diabetes Res</source>. (<year>2019</year>) <volume>2019</volume>:<fpage>4912174</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2019/4912174</pub-id><pub-id pub-id-type=\"pmid\">31976336</pub-id></mixed-citation></ref><ref id=\"B79\"><label>79.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Weiss</surname><given-names>EP</given-names></name><name><surname>Arif</surname><given-names>H</given-names></name><name><surname>Villareal</surname><given-names>DT</given-names></name><name><surname>Marzetti</surname><given-names>E</given-names></name><name><surname>Holloszy</surname><given-names>JO</given-names></name></person-group>. <article-title>Endothelial function after high-sugar food ingestion is improved by endurance exercise performed on the previous day</article-title>. <source>Am J Clin Nutr</source>. (<year>2008</year>) <volume>88</volume>:<fpage>51</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1093/ajcn/88.1.51</pub-id><pub-id pub-id-type=\"pmid\">18614723</pub-id></mixed-citation></ref><ref id=\"B80\"><label>80.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fujita</surname><given-names>S</given-names></name><name><surname>Rasmussen</surname><given-names>BB</given-names></name><name><surname>Cadenas</surname><given-names>JG</given-names></name><name><surname>Drummond</surname><given-names>MJ</given-names></name><name><surname>Glynn</surname><given-names>EL</given-names></name><name><surname>Sattler</surname><given-names>FR</given-names></name><etal/></person-group>. <article-title>Aerobic exercise overcomes the age-related insulin resistance of muscle protein metabolism by improving endothelial function and Akt/mammalian target of rapamycin signaling</article-title>. <source>Diabetes.</source> (<year>2007</year>) <volume>56</volume>:<fpage>1615</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.2337/db06-1566</pub-id><pub-id pub-id-type=\"pmid\">17351147</pub-id></mixed-citation></ref><ref id=\"B81\"><label>81.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhu</surname><given-names>W</given-names></name><name><surname>Zhong</surname><given-names>C</given-names></name><name><surname>Yu</surname><given-names>Y</given-names></name><name><surname>Li</surname><given-names>K</given-names></name></person-group>. <article-title>Acute effects of hyperglycaemia with and without exercise on endothelial function in healthy young men</article-title>. <source>Eur J Appl Physiol.</source> (<year>2007</year>) <volume>99</volume>:<fpage>585</fpage>–<lpage>91</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00421-006-0378-3</pub-id><pub-id pub-id-type=\"pmid\">17206439</pub-id></mixed-citation></ref><ref id=\"B82\"><label>82.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mikus</surname><given-names>CR</given-names></name><name><surname>Fairfax</surname><given-names>ST</given-names></name><name><surname>Libla</surname><given-names>JL</given-names></name><name><surname>Boyle</surname><given-names>LJ</given-names></name><name><surname>Vianna</surname><given-names>LC</given-names></name><name><surname>Oberlin</surname><given-names>DJ</given-names></name><etal/></person-group>. <article-title>Seven days of aerobic exercise training improves conduit artery blood flow following glucose ingestion in patients with type 2 diabetes</article-title>. <source>J Appl Physiol.</source> (<year>2011</year>) <volume>111</volume>:<fpage>657</fpage>–<lpage>64</lpage>. <pub-id pub-id-type=\"doi\">10.1152/japplphysiol.00489.2011</pub-id><pub-id pub-id-type=\"pmid\">21737826</pub-id></mixed-citation></ref><ref id=\"B83\"><label>83.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eggleston</surname><given-names>EM</given-names></name><name><surname>Jahn</surname><given-names>LA</given-names></name><name><surname>Barrett</surname><given-names>EJ</given-names></name></person-group>. <article-title>Hyperinsulinemia rapidly increases human muscle microvascular perfusion but fails to increase muscle insulin clearance: evidence that a saturable process mediates muscle insulin uptake</article-title>. <source>Diabetes.</source> (<year>2007</year>) <volume>56</volume>:<fpage>2958</fpage>–<lpage>63</lpage>. <pub-id pub-id-type=\"doi\">10.2337/db07-0670</pub-id><pub-id pub-id-type=\"pmid\">17720897</pub-id></mixed-citation></ref><ref id=\"B84\"><label>84.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ramírez-Vélez</surname><given-names>R</given-names></name><name><surname>Correa-Rodríguez</surname><given-names>M</given-names></name><name><surname>Tordecilla-Sanders</surname><given-names>A</given-names></name><name><surname>Aya-Aldana</surname><given-names>V</given-names></name><name><surname>Izquierdo</surname><given-names>M</given-names></name><name><surname>Correa-Bautista</surname><given-names>JE</given-names></name><etal/></person-group>. <article-title>Exercise and postprandial lipemia: effects on vascular health in inactive adults</article-title>. <source>Lipids Health Dis.</source> (<year>2018</year>) <volume>17</volume>:<fpage>69</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s12944-018-0719-3</pub-id><pub-id pub-id-type=\"pmid\">29615070</pub-id></mixed-citation></ref><ref id=\"B85\"><label>85.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pitocco</surname><given-names>D</given-names></name><name><surname>Zaccardi</surname><given-names>F</given-names></name><name><surname>Tarzia</surname><given-names>P</given-names></name><name><surname>Milo</surname><given-names>M</given-names></name><name><surname>Scavone</surname><given-names>G</given-names></name><name><surname>Rizzo</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Metformin improves endothelial function in type 1 diabetic subjects: a pilot, placebo-controlled randomized study</article-title>. <source>Diabetes, Obes Metab.</source> (<year>2013</year>) <volume>15</volume>:<fpage>427</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.1111/dom.12041</pub-id><pub-id pub-id-type=\"pmid\">23167274</pub-id></mixed-citation></ref><ref id=\"B86\"><label>86.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jensterle</surname><given-names>M</given-names></name><name><surname>Weber</surname><given-names>M</given-names></name><name><surname>Pfeifer</surname><given-names>M</given-names></name><name><surname>Prezelj</surname><given-names>J</given-names></name><name><surname>Pfutzner</surname><given-names>A</given-names></name><name><surname>Janez</surname><given-names>A</given-names></name></person-group>. <article-title>Assessment of insulin resistance in young women with polycystic ovary syndrome</article-title>. <source>Int J Gynecol Obstet.</source> (<year>2008</year>) <volume>102</volume>:<fpage>137</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ijgo.2008.03.017</pub-id><pub-id pub-id-type=\"pmid\">18504045</pub-id></mixed-citation></ref><ref id=\"B87\"><label>87.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Magalhães</surname><given-names>FO</given-names></name><name><surname>Gouveia</surname><given-names>LMB</given-names></name><name><surname>Torquato</surname><given-names>MTCG</given-names></name><name><surname>Paccola</surname><given-names>GMGF</given-names></name><name><surname>Piccinato</surname><given-names>CE</given-names></name><name><surname>Foss</surname><given-names>MC</given-names></name></person-group>. <article-title>Metformin increases blood flow and forearm glucose uptake in a group of non-obese type 2 diabetes patients</article-title>. <source>Horm Metab Res.</source> (<year>2006</year>) <volume>38</volume>:<fpage>513</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1055/s-2006-949522</pub-id><pub-id pub-id-type=\"pmid\">16941277</pub-id></mixed-citation></ref><ref id=\"B88\"><label>88.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vincent</surname><given-names>MA</given-names></name><name><surname>Clerk</surname><given-names>LH</given-names></name><name><surname>Lindner</surname><given-names>JR</given-names></name><name><surname>Klibanov</surname><given-names>AL</given-names></name><name><surname>Clark</surname><given-names>MG</given-names></name><name><surname>Rattigan</surname><given-names>S</given-names></name><etal/></person-group>. <article-title>Microvascular recruitment is an early insulin effect that regulates skeletal muscle glucose uptake <italic>in vivo</italic></article-title>. <source>Diabetes.</source> (<year>2004</year>) <volume>53</volume>:<fpage>1418</fpage>–<lpage>23</lpage>. <pub-id pub-id-type=\"doi\">10.2337/diabetes.53.6.1418</pub-id><pub-id pub-id-type=\"pmid\">15161743</pub-id></mixed-citation></ref><ref id=\"B89\"><label>89.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bradley</surname><given-names>EA</given-names></name><name><surname>Premilovac</surname><given-names>D</given-names></name><name><surname>Betik</surname><given-names>AC</given-names></name><name><surname>Hu</surname><given-names>D</given-names></name><name><surname>Attrill</surname><given-names>E</given-names></name><name><surname>Richards</surname><given-names>SM</given-names></name><etal/></person-group>. <article-title>Metformin improves vascular and metabolic insulin action in insulin-resistant muscle</article-title>. <source>J Endocrinol.</source> (<year>2019</year>) <volume>243</volume>:<fpage>85</fpage>–<lpage>96</lpage>. <pub-id pub-id-type=\"doi\">10.1530/JOE-19-0067</pub-id><pub-id pub-id-type=\"pmid\">31394501</pub-id></mixed-citation></ref><ref id=\"B90\"><label>90.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>An</surname><given-names>H</given-names></name><name><surname>Wei</surname><given-names>R</given-names></name><name><surname>Ke</surname><given-names>J</given-names></name><name><surname>Yang</surname><given-names>J</given-names></name><name><surname>Liu</surname><given-names>Y</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name><etal/></person-group>. <article-title>Metformin attenuates fluctuating glucose-induced endothelial dysfunction through enhancing GTPCH1-mediated eNOS recoupling and inhibiting NADPH oxidase</article-title>. <source>J Diabetes Complic.</source> (<year>2016</year>) <volume>30</volume>:<fpage>1017</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jdiacomp.2016.04.018</pub-id><pub-id pub-id-type=\"pmid\">27217019</pub-id></mixed-citation></ref><ref id=\"B91\"><label>91.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kraus</surname><given-names>WE</given-names></name><name><surname>Slentz</surname><given-names>CA</given-names></name></person-group>. <article-title>Exercise training, lipid regulation, and insulin action: a tangled web of cause and effect</article-title>. <source>Obesity.</source> (<year>2009</year>) <volume>17</volume>:<fpage>S21</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1038/oby.2009.384</pub-id><pub-id pub-id-type=\"pmid\">19927141</pub-id></mixed-citation></ref><ref id=\"B92\"><label>92.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>King</surname><given-names>P</given-names></name><name><surname>Peacock</surname><given-names>I</given-names></name><name><surname>Donnelly</surname><given-names>R</given-names></name></person-group>. <article-title>The UK Prospective Diabetes Study (UKPDS): clinical and therapeutic implications for type 2 diabetes</article-title>. <source>Br J Clin Pharmacol.</source> (<year>1999</year>) <volume>48</volume>:<fpage>643</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1046/j.1365-2125.1999.00092.x</pub-id><pub-id pub-id-type=\"pmid\">10594464</pub-id></mixed-citation></ref><ref id=\"B93\"><label>93.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wilding</surname><given-names>JPH</given-names></name></person-group>. <article-title>The importance of weight management in type 2 diabetes mellitus</article-title>. <source>Int J Clin Pract.</source> (<year>2014</year>) <volume>68</volume>:<fpage>682</fpage>–<lpage>91</lpage>. <pub-id pub-id-type=\"doi\">10.1111/ijcp.12384</pub-id><pub-id pub-id-type=\"pmid\">24548654</pub-id></mixed-citation></ref><ref id=\"B94\"><label>94.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Malin</surname><given-names>SK</given-names></name><name><surname>Nightingale</surname><given-names>J</given-names></name><name><surname>Choi</surname><given-names>S-E</given-names></name><name><surname>Chipkin</surname><given-names>SR</given-names></name><name><surname>Braun</surname><given-names>B</given-names></name></person-group>. <article-title>Metformin modifies the exercise training effects on risk factors for cardiovascular disease in impaired glucose tolerant adults</article-title>. <source>Obesity.</source> (<year>2013</year>) <volume>21</volume>:<fpage>93</fpage>–<lpage>100</lpage>. <pub-id pub-id-type=\"doi\">10.1002/oby.20235</pub-id><pub-id pub-id-type=\"pmid\">23505172</pub-id></mixed-citation></ref><ref id=\"B95\"><label>95.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cetinkalp</surname><given-names>S</given-names></name><name><surname>Simsir</surname><given-names>I</given-names></name><name><surname>Ertek</surname><given-names>S</given-names></name></person-group>. <article-title>Insulin resistance in brain and possible therapeutic approaches</article-title>. <source>Curr Vasc Pharmacol.</source> (<year>2014</year>) <volume>12</volume>:<fpage>553</fpage>–<lpage>64</lpage>. <pub-id pub-id-type=\"doi\">10.2174/1570161112999140206130426</pub-id><pub-id pub-id-type=\"pmid\">23627981</pub-id></mixed-citation></ref><ref id=\"B96\"><label>96.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kullmann</surname><given-names>S</given-names></name><name><surname>Heni</surname><given-names>M</given-names></name><name><surname>Hallschmid</surname><given-names>M</given-names></name><name><surname>Fritsche</surname><given-names>A</given-names></name><name><surname>Preissl</surname><given-names>H</given-names></name><name><surname>Häring</surname><given-names>HU</given-names></name></person-group>. <article-title>Brain insulin resistance at the crossroads of metabolic and cognitive disorders in humans</article-title>. <source>Physiol Rev.</source> (<year>2016</year>) <volume>96</volume>:<fpage>1169</fpage>–<lpage>209</lpage>. <pub-id pub-id-type=\"doi\">10.1152/physrev.00032.2015</pub-id><pub-id pub-id-type=\"pmid\">27489306</pub-id></mixed-citation></ref><ref id=\"B97\"><label>97.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Benedict</surname><given-names>C</given-names></name><name><surname>Hallschmid</surname><given-names>M</given-names></name><name><surname>Hatke</surname><given-names>A</given-names></name><name><surname>Schultes</surname><given-names>B</given-names></name><name><surname>Fehm</surname><given-names>HL</given-names></name><name><surname>Born</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Intranasal insulin improves memory in humans</article-title>. <source>Psychoneuroendocrinology.</source> (<year>2004</year>) <volume>29</volume>:<fpage>1326</fpage>–<lpage>34</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.psyneuen.2004.04.003</pub-id><pub-id pub-id-type=\"pmid\">15288712</pub-id></mixed-citation></ref><ref id=\"B98\"><label>98.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Malin</surname><given-names>SK</given-names></name><name><surname>Kirwan</surname><given-names>JP</given-names></name></person-group>. <article-title>Fasting hyperglycaemia blunts the reversal of impaired glucose tolerance after exercise training in obese older adults</article-title>. <source>Diabetes, Obes Metab.</source> (<year>2012</year>) <volume>14</volume>:<fpage>835</fpage>–<lpage>41</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1463-1326.2012.01608.x</pub-id><pub-id pub-id-type=\"pmid\">22510250</pub-id></mixed-citation></ref><ref id=\"B99\"><label>99.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Williams</surname><given-names>VJ</given-names></name><name><surname>Trombetta</surname><given-names>BA</given-names></name><name><surname>Jafri</surname><given-names>RZ</given-names></name><name><surname>Koenig</surname><given-names>AM</given-names></name><name><surname>Wennick</surname><given-names>CD</given-names></name><name><surname>Carlyle</surname><given-names>BC</given-names></name><etal/></person-group>. <article-title>Task-related fMRI BOLD response to hyperinsulinemia in healthy older adults</article-title>. <source>JCI Insight</source>. (<year>2019</year>) <volume>4</volume>:<fpage>e129700</fpage>. <pub-id pub-id-type=\"doi\">10.1172/jci.insight.129700</pub-id><pub-id pub-id-type=\"pmid\">31211691</pub-id></mixed-citation></ref><ref id=\"B100\"><label>100.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Burns</surname><given-names>JM</given-names></name><name><surname>Honea</surname><given-names>RA</given-names></name><name><surname>Vidoni</surname><given-names>ED</given-names></name><name><surname>Hutfles</surname><given-names>LJ</given-names></name><name><surname>Brooks</surname><given-names>WM</given-names></name><name><surname>Swerdlow</surname><given-names>RH</given-names></name></person-group>. <article-title>Insulin is differentially related to cognitive decline and atrophy in Alzheimer's disease and aging</article-title>. <source>Biochim Biophys Acta.</source> (<year>2012</year>) <volume>1822</volume>:<fpage>333</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbadis.2011.06.011</pub-id><pub-id pub-id-type=\"pmid\">21745566</pub-id></mixed-citation></ref><ref id=\"B101\"><label>101.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Willette</surname><given-names>AA</given-names></name><name><surname>Xu</surname><given-names>G</given-names></name><name><surname>Johnson</surname><given-names>SC</given-names></name><name><surname>Birdsill</surname><given-names>AC</given-names></name><name><surname>Jonaitis</surname><given-names>EM</given-names></name><name><surname>Sager</surname><given-names>MA</given-names></name><etal/></person-group>. <article-title>Insulin resistance, brain atrophy, and cognitive performance in late middle-aged adults</article-title>. <source>Diabetes Care.</source> (<year>2013</year>) <volume>36</volume>:<fpage>443</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.2337/dc12-0922</pub-id><pub-id pub-id-type=\"pmid\">23069842</pub-id></mixed-citation></ref><ref id=\"B102\"><label>102.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Willette</surname><given-names>AA</given-names></name><name><surname>Modanlo</surname><given-names>N</given-names></name><name><surname>Kapogiannis</surname><given-names>D</given-names></name></person-group>. <article-title>Insulin resistance predicts medial temporal hypermetabolism in mild cognitive impairment conversion to Alzheimer disease</article-title>. <source>Diabetes.</source> (<year>2015</year>) <volume>64</volume>:<fpage>1933</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.2337/db14-1507</pub-id><pub-id pub-id-type=\"pmid\">25576061</pub-id></mixed-citation></ref><ref id=\"B103\"><label>103.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chiti</surname><given-names>F</given-names></name><name><surname>Dobson</surname><given-names>CM</given-names></name></person-group>. <article-title>Protein misfolding, amyloid formation, and human disease: a summary of progress over the last decade</article-title>. <source>Annu Rev Biochem.</source> (<year>2017</year>) <volume>86</volume>:<fpage>27</fpage>–<lpage>68</lpage>. <pub-id pub-id-type=\"doi\">10.1146/annurev-biochem-061516-045115</pub-id><pub-id pub-id-type=\"pmid\">28498720</pub-id></mixed-citation></ref><ref id=\"B104\"><label>104.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arnold</surname><given-names>SE</given-names></name><name><surname>Arvanitakis</surname><given-names>Z</given-names></name><name><surname>Macauley-Rambach</surname><given-names>SL</given-names></name><name><surname>Koenig</surname><given-names>AM</given-names></name><name><surname>Wang</surname><given-names>HY</given-names></name><name><surname>Ahima</surname><given-names>RS</given-names></name><etal/></person-group>. <article-title>Brain insulin resistance in type 2 diabetes and Alzheimer disease: concepts and conundrums</article-title>. <source>Nat Rev Neurol.</source> (<year>2018</year>) <volume>14</volume>:<fpage>168</fpage>–<lpage>81</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrneurol.2017.185</pub-id><pub-id pub-id-type=\"pmid\">29377010</pub-id></mixed-citation></ref><ref id=\"B105\"><label>105.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kemppainen</surname><given-names>J</given-names></name><name><surname>Aalto</surname><given-names>S</given-names></name><name><surname>Fujimoto</surname><given-names>T</given-names></name><name><surname>Kalliokoski</surname><given-names>KK</given-names></name><name><surname>Långsjö</surname><given-names>J</given-names></name><name><surname>Oikonen</surname><given-names>V</given-names></name><etal/></person-group>. <article-title>High intensity exercise decreases global brain glucose uptake in humans</article-title>. <source>J Physiol.</source> (<year>2005</year>) <volume>568</volume>:<fpage>323</fpage>–<lpage>32</lpage>. <pub-id pub-id-type=\"doi\">10.1113/jphysiol.2005.091355</pub-id><pub-id pub-id-type=\"pmid\">16037089</pub-id></mixed-citation></ref><ref id=\"B106\"><label>106.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Robinson</surname><given-names>MM</given-names></name><name><surname>Lowe</surname><given-names>VJ</given-names></name><name><surname>Nair</surname><given-names>KS</given-names></name></person-group>. <article-title>Increased brain glucose uptake after 12 weeks of aerobic high-intensity interval training in young and older adults</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2018</year>) <volume>103</volume>:<fpage>221</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2017-01571</pub-id><pub-id pub-id-type=\"pmid\">29077855</pub-id></mixed-citation></ref><ref id=\"B107\"><label>107.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kullmann</surname><given-names>S</given-names></name><name><surname>Valenta</surname><given-names>V</given-names></name><name><surname>Wagner</surname><given-names>R</given-names></name><name><surname>Tschritter</surname><given-names>O</given-names></name><name><surname>Machann</surname><given-names>J</given-names></name><name><surname>Häring</surname><given-names>H-U</given-names></name><etal/></person-group>. <article-title>Brain insulin sensitivity is linked to adiposity and body fat distribution</article-title>. <source>Nat Commun</source>. (<year>2020</year>) <volume>11</volume>:<fpage>1841</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41467-020-15686-y</pub-id><pub-id pub-id-type=\"pmid\">32296068</pub-id></mixed-citation></ref><ref id=\"B108\"><label>108.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Honkala</surname><given-names>SM</given-names></name><name><surname>Johansson</surname><given-names>J</given-names></name><name><surname>Motiani</surname><given-names>KK</given-names></name><name><surname>Eskelinen</surname><given-names>JJ</given-names></name><name><surname>Virtanen</surname><given-names>KA</given-names></name><name><surname>Löyttyniemi</surname><given-names>E</given-names></name><etal/></person-group>. <article-title>Short-term interval training alters brain glucose metabolism in subjects with insulin resistance</article-title>. <source>J Cereb Blood Flow Metab.</source> (<year>2018</year>) <volume>38</volume>:<fpage>1828</fpage>–<lpage>38</lpage>. <pub-id pub-id-type=\"doi\">10.1177/0271678X17734998</pub-id><pub-id pub-id-type=\"pmid\">28959911</pub-id></mixed-citation></ref><ref id=\"B109\"><label>109.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Olver</surname><given-names>TD</given-names></name><name><surname>Mcdonald</surname><given-names>MW</given-names></name><name><surname>Klakotskaia</surname><given-names>D</given-names></name><name><surname>Richardson</surname><given-names>RA</given-names></name><name><surname>Jasperse</surname><given-names>JL</given-names></name><name><surname>Melling</surname><given-names>CWJ</given-names></name><etal/></person-group>. <article-title>A chronic physical activity treatment in obese rats normalizes the contributions of ET-1 and NO to insulin-mediated posterior cerebral artery vasodilation</article-title>. <source>J Appl Physiol.</source> (<year>2017</year>) <volume>122</volume>:<fpage>1040</fpage>–<lpage>50</lpage>. <pub-id pub-id-type=\"doi\">10.1152/japplphysiol.00811.2016</pub-id><pub-id pub-id-type=\"pmid\">28183819</pub-id></mixed-citation></ref><ref id=\"B110\"><label>110.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ruegsegger</surname><given-names>GN</given-names></name><name><surname>Creo</surname><given-names>AL</given-names></name><name><surname>Cortes</surname><given-names>TM</given-names></name><name><surname>Dasari</surname><given-names>S</given-names></name><name><surname>Nair</surname><given-names>KS</given-names></name></person-group>. <article-title>Altered mitochondrial function in insulin-deficient and insulin-resistant states</article-title>. <source>J Clin Invest.</source> (<year>2018</year>) <volume>128</volume>:<fpage>3671</fpage>–<lpage>81</lpage>. <pub-id pub-id-type=\"doi\">10.1172/JCI120843</pub-id><pub-id pub-id-type=\"pmid\">30168804</pub-id></mixed-citation></ref><ref id=\"B111\"><label>111.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yarchoan</surname><given-names>M</given-names></name><name><surname>Arnold</surname><given-names>SE</given-names></name></person-group>. <article-title>Repurposing diabetes drugs for brain insulin resistance in Alzheimer disease</article-title>. <source>Diabetes.</source> (<year>2014</year>) <volume>63</volume>:<fpage>2253</fpage>–<lpage>61</lpage>. <pub-id pub-id-type=\"doi\">10.2337/db14-0287</pub-id><pub-id pub-id-type=\"pmid\">24931035</pub-id></mixed-citation></ref><ref id=\"B112\"><label>112.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Koenig</surname><given-names>AM</given-names></name><name><surname>Mechanic-Hamilton</surname><given-names>D</given-names></name><name><surname>Xie</surname><given-names>SX</given-names></name><name><surname>Combs</surname><given-names>MF</given-names></name><name><surname>Cappola</surname><given-names>AR</given-names></name><name><surname>Xie</surname><given-names>L</given-names></name><etal/></person-group>. <article-title>Effects of the insulin sensitizer metformin in alzheimer disease: pilot data from a randomized placebo-controlled crossover study</article-title>. <source>Alzheimer Dis Assoc Disord.</source> (<year>2017</year>) <volume>31</volume>:<fpage>107</fpage>–<lpage>13</lpage>. <pub-id pub-id-type=\"doi\">10.1097/WAD.0000000000000202</pub-id><pub-id pub-id-type=\"pmid\">28538088</pub-id></mixed-citation></ref><ref id=\"B113\"><label>113.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ruegsegger</surname><given-names>GN</given-names></name><name><surname>Vanderboom</surname><given-names>PM</given-names></name><name><surname>Dasari</surname><given-names>S</given-names></name><name><surname>Klaus</surname><given-names>KA</given-names></name><name><surname>Kabiraj</surname><given-names>P</given-names></name><name><surname>McCarthy</surname><given-names>CB</given-names></name><etal/></person-group>. <article-title>Exercise and metformin counteract altered mitochondrial function in the insulin resistant brain</article-title>. <source>JCI Insight</source>. (<year>2019</year>) <volume>4</volume>:<fpage>e130681</fpage>. <pub-id pub-id-type=\"doi\">10.1172/jci.insight.130681</pub-id><pub-id pub-id-type=\"pmid\">31534057</pub-id></mixed-citation></ref><ref id=\"B114\"><label>114.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kickstein</surname><given-names>E</given-names></name><name><surname>Krauss</surname><given-names>S</given-names></name><name><surname>Thornhill</surname><given-names>P</given-names></name><name><surname>Rutschow</surname><given-names>D</given-names></name><name><surname>Zeller</surname><given-names>R</given-names></name><name><surname>Sharkey</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Biguanide metformin acts on tau phosphorylation via mTOR/protein phosphatase 2A (PP2A) signaling</article-title>. <source>Proc Natl Acad Sci USA.</source> (<year>2010</year>) <volume>107</volume>:<fpage>21830</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.0912793107</pub-id><pub-id pub-id-type=\"pmid\">21098287</pub-id></mixed-citation></ref><ref id=\"B115\"><label>115.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wrann</surname><given-names>CD</given-names></name><name><surname>White</surname><given-names>JP</given-names></name><name><surname>Salogiannnis</surname><given-names>J</given-names></name><name><surname>Laznik-Bogoslavski</surname><given-names>D</given-names></name><name><surname>Wu</surname><given-names>J</given-names></name><name><surname>Ma</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Exercise induces hippocampal BDNF through a PGC-1α/FNDC5 pathway</article-title>. <source>Cell Metab.</source> (<year>2013</year>) <volume>18</volume>:<fpage>649</fpage>–<lpage>59</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cmet.2013.09.008</pub-id><pub-id pub-id-type=\"pmid\">24120943</pub-id></mixed-citation></ref><ref id=\"B116\"><label>116.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brunmair</surname><given-names>B</given-names></name><name><surname>Staniek</surname><given-names>K</given-names></name><name><surname>Gras</surname><given-names>F</given-names></name><name><surname>Scharf</surname><given-names>N</given-names></name><name><surname>Althaym</surname><given-names>A</given-names></name><name><surname>Clara</surname><given-names>R</given-names></name><etal/></person-group>. <article-title>Thiazolidinediones, like metformin, inhibit respiratory complex I: a common mechanism contributing to their antidiabetic actions?</article-title>\n<source>Diabetes.</source> (<year>2004</year>) <volume>53</volume>:<fpage>1052</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.2337/diabetes.53.4.1052</pub-id><pub-id pub-id-type=\"pmid\">15047621</pub-id></mixed-citation></ref><ref id=\"B117\"><label>117.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kristensen</surname><given-names>JM</given-names></name><name><surname>Lillelund</surname><given-names>C</given-names></name><name><surname>Kjøbsted</surname><given-names>R</given-names></name><name><surname>Birk</surname><given-names>JB</given-names></name><name><surname>Andersen</surname><given-names>NR</given-names></name><name><surname>Nybo</surname><given-names>L</given-names></name><etal/></person-group>\n<article-title>Metformin does not compromise energy status in human skeletal muscle at rest or during acute exercise: a randomised, crossover trial</article-title>. <source>Physiol Rep.</source> (<year>2019</year>) <volume>7</volume>:<fpage>e14307</fpage>\n<pub-id pub-id-type=\"doi\">10.14814/phy2.14307</pub-id><pub-id pub-id-type=\"pmid\">31833226</pub-id></mixed-citation></ref><ref id=\"B118\"><label>118.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jadhav</surname><given-names>S</given-names></name><name><surname>Ferrell</surname><given-names>W</given-names></name><name><surname>Greer</surname><given-names>IA</given-names></name><name><surname>Petrie</surname><given-names>JR</given-names></name><name><surname>Cobbe</surname><given-names>SM</given-names></name><name><surname>Sattar</surname><given-names>N</given-names></name></person-group>. <article-title>Effects of metformin on microvascular function and exercise tolerance in women with angina and normal coronary arteries</article-title>. A Randomized, Double-Blind, Placebo-Controlled Study. <source>J Am Coll Cardiol.</source> (<year>2006</year>) <volume>48</volume>:<fpage>956</fpage>–<lpage>63</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jacc.2006.04.088</pub-id><pub-id pub-id-type=\"pmid\">16949486</pub-id></mixed-citation></ref><ref id=\"B119\"><label>119.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kane</surname><given-names>DA</given-names></name><name><surname>Anderson</surname><given-names>EJ</given-names></name><name><surname>Price</surname><given-names>JW</given-names></name><name><surname>Woodlief</surname><given-names>TL</given-names></name><name><surname>Lin</surname><given-names>C</given-names></name><name><surname>Te Bikman</surname><given-names>BT</given-names></name><etal/></person-group>. <article-title>Metformin selectively attenuates mitochondrial H2O2 emission without affecting respiratory capacity in skeletal muscle of obese rats</article-title>. <source>Free Radic Biol Med.</source> (<year>2010</year>) <volume>49</volume>:<fpage>1082</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.freeradbiomed.2010.06.022</pub-id><pub-id pub-id-type=\"pmid\">20600832</pub-id></mixed-citation></ref><ref id=\"B120\"><label>120.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gliemann</surname><given-names>L</given-names></name><name><surname>Schmidt</surname><given-names>JF</given-names></name><name><surname>Olesen</surname><given-names>J</given-names></name><name><surname>Biensø</surname><given-names>RS</given-names></name><name><surname>Peronard</surname><given-names>SL</given-names></name><name><surname>Grandjean</surname><given-names>SU</given-names></name><etal/></person-group>. <article-title>Resveratrol blunts the positive effects of exercise training on cardiovascular health in aged men</article-title>. <source>J Physiol.</source> (<year>2013</year>) <volume>591</volume>:<fpage>5047</fpage>–<lpage>59</lpage>. <pub-id pub-id-type=\"doi\">10.1113/jphysiol.2013.258061</pub-id><pub-id pub-id-type=\"pmid\">23878368</pub-id></mixed-citation></ref><ref id=\"B121\"><label>121.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Henríquez-Olguin</surname><given-names>C</given-names></name><name><surname>Knudsen</surname><given-names>JR</given-names></name><name><surname>Raun</surname><given-names>SH</given-names></name><name><surname>Li</surname><given-names>Z</given-names></name><name><surname>Dalbram</surname><given-names>E</given-names></name><name><surname>Treebak</surname><given-names>JT</given-names></name><etal/></person-group>. <article-title>Cytosolic ROS production by NADPH oxidase 2 regulates muscle glucose uptake during exercise</article-title>. <source>Nat Commun.</source> (<year>2019</year>) <volume>10</volume>:<fpage>4623</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41467-019-12523-9</pub-id><pub-id pub-id-type=\"pmid\">31604916</pub-id></mixed-citation></ref><ref id=\"B122\"><label>122.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Foretz</surname><given-names>M</given-names></name><name><surname>Hébrard</surname><given-names>S</given-names></name><name><surname>Leclerc</surname><given-names>J</given-names></name><name><surname>Zarrinpashneh</surname><given-names>E</given-names></name><name><surname>Soty</surname><given-names>M</given-names></name><name><surname>Mithieux</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state</article-title>. <source>J Clin Invest.</source> (<year>2010</year>) <volume>120</volume>:<fpage>2355</fpage>–<lpage>69</lpage>. <pub-id pub-id-type=\"doi\">10.1172/JCI40671</pub-id><pub-id pub-id-type=\"pmid\">20577053</pub-id></mixed-citation></ref><ref id=\"B123\"><label>123.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Park</surname><given-names>MJ</given-names></name><name><surname>Lee</surname><given-names>SY</given-names></name><name><surname>Moon</surname><given-names>SJ</given-names></name><name><surname>Son</surname><given-names>HJ</given-names></name><name><surname>Lee</surname><given-names>SH</given-names></name><name><surname>Kim</surname><given-names>EK</given-names></name><etal/></person-group>. <article-title>Metformin attenuates graft-versus-host disease via restricting mammalian target of rapamycin/signal transducer and activator of transcription 3 and promoting adenosine monophosphate–activated protein kinase-autophagy for the balance between T helper 17 and Tregs</article-title>. <source>Transl Res.</source> (<year>2016</year>) <volume>173</volume>:<fpage>115</fpage>–<lpage>30</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.trsl.2016.03.006</pub-id><pub-id pub-id-type=\"pmid\">27126953</pub-id></mixed-citation></ref><ref id=\"B124\"><label>124.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Crocker</surname><given-names>CL</given-names></name><name><surname>Baumgarner</surname><given-names>BL</given-names></name><name><surname>Kinsey</surname><given-names>ST</given-names></name></person-group>. <article-title>β-guanidinopropionic acid and metformin differentially impact autophagy, mitochondria and cellular morphology in developing C2C12 muscle cells</article-title>. <source>J. Muscle Res. Cell Motil.</source> (<year>2019</year>). <pub-id pub-id-type=\"doi\">10.1007/s10974-019-09568-0</pub-id>. [Epub ahead of print].<pub-id pub-id-type=\"pmid\">31836952</pub-id></mixed-citation></ref><ref id=\"B125\"><label>125.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wolff</surname><given-names>CA</given-names></name><name><surname>Reid</surname><given-names>JJ</given-names></name><name><surname>Musci</surname><given-names>RV</given-names></name><name><surname>Bruns</surname><given-names>DR</given-names></name><name><surname>Linden</surname><given-names>MA</given-names></name><name><surname>Konopka</surname><given-names>AR</given-names></name><etal/></person-group>\n<article-title>Differential effects of rapamycin and metformin in combination with rapamycin on mechanisms of proteostasis in cultured skeletal myotubes</article-title>. <source>J Gerontol Ser A Biol Sci Med Sci</source>. (<year>2020</year>) <volume>75</volume>:<fpage>32</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1093/gerona/glz058</pub-id><pub-id pub-id-type=\"pmid\">30820523</pub-id></mixed-citation></ref><ref id=\"B126\"><label>126.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nikolaidis</surname><given-names>S</given-names></name><name><surname>Virgiliou</surname><given-names>C</given-names></name><name><surname>Vekiou</surname><given-names>M</given-names></name><name><surname>Skari</surname><given-names>A</given-names></name><name><surname>Kechagidou</surname><given-names>A</given-names></name><name><surname>Gika</surname><given-names>H</given-names></name><etal/></person-group>. <article-title>Effect of exercise on key pharmacokinetic parameters related to metformin absorption in healthy humans: A pilot study</article-title>. <source>Scand J Med Sci Sport</source>. (<year>2020</year>) <volume>30</volume>:<fpage>858</fpage>–<lpage>64</lpage>. <pub-id pub-id-type=\"doi\">10.1111/sms.13628</pub-id><pub-id pub-id-type=\"pmid\">31975547</pub-id></mixed-citation></ref><ref id=\"B127\"><label>127.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Czepiel</surname><given-names>KS</given-names></name><name><surname>Perez</surname><given-names>NP</given-names></name><name><surname>Campoverde Reyes</surname><given-names>KJ</given-names></name><name><surname>Sabharwal</surname><given-names>S</given-names></name><name><surname>Stanford</surname><given-names>FC</given-names></name></person-group>. <article-title>Pharmacotherapy for the treatment of overweight and obesity in children, adolescents, and young adults in a large health system in the US</article-title>. <source>Front Endocrinol</source>. (<year>2020</year>) <volume>11</volume>:<fpage>290</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fendo.2020.00290</pub-id><pub-id pub-id-type=\"pmid\">32477270</pub-id></mixed-citation></ref><ref id=\"B128\"><label>128.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jørgensen</surname><given-names>PG</given-names></name><name><surname>Jensen</surname><given-names>MT</given-names></name><name><surname>Mensberg</surname><given-names>P</given-names></name><name><surname>Storgaard</surname><given-names>H</given-names></name><name><surname>Nyby</surname><given-names>S</given-names></name><name><surname>Jensen</surname><given-names>JS</given-names></name><etal/></person-group>. <article-title>Effect of exercise combined with glucagon-like peptide-1 receptor agonist treatment on cardiac function: a randomized double-blind placebo-controlled clinical trial</article-title>. <source>Diabetes Obes Metab.</source> (<year>2017</year>) <volume>19</volume>:<fpage>1040</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1111/dom.12900</pub-id><pub-id pub-id-type=\"pmid\">28188972</pub-id></mixed-citation></ref><ref id=\"B129\"><label>129.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mensberg</surname><given-names>P</given-names></name><name><surname>Nyby</surname><given-names>S</given-names></name><name><surname>Jørgensen</surname><given-names>PG</given-names></name><name><surname>Storgaard</surname><given-names>H</given-names></name><name><surname>Jensen</surname><given-names>MT</given-names></name><name><surname>Sivertsen</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Near-normalization of glycaemic control with glucagon-like peptide-1 receptor agonist treatment combined with exercise in patients with type 2 diabetes</article-title>. <source>Diabetes, Obes Metab.</source> (<year>2017</year>) <volume>19</volume>:<fpage>172</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.1111/dom.12797</pub-id><pub-id pub-id-type=\"pmid\">27717126</pub-id></mixed-citation></ref><ref id=\"B130\"><label>130.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Newman</surname><given-names>AA</given-names></name><name><surname>Grimm</surname><given-names>NC</given-names></name><name><surname>Wilburn</surname><given-names>JR</given-names></name><name><surname>Schoenberg</surname><given-names>HM</given-names></name><name><surname>Raj Trikha</surname><given-names>SJ</given-names></name><name><surname>Luckasen</surname><given-names>GJ</given-names></name><etal/></person-group>. <article-title>Influence of sodium glucose cotransporter 2 inhibition on physiological adaptation to endurance exercise training</article-title>. <source>J Clin Endocrinol Metab.</source> (<year>2019</year>) <volume>104</volume>:<fpage>1953</fpage>–<lpage>66</lpage>. <pub-id pub-id-type=\"doi\">10.1210/jc.2018-01741</pub-id><pub-id pub-id-type=\"pmid\">30597042</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Endocrinol (Lausanne)</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Endocrinol.</journal-id><journal-title-group><journal-title>Frontiers in Endocrinology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-2392</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849284</article-id><article-id pub-id-type=\"pmc\">PMC7431622</article-id><article-id pub-id-type=\"doi\">10.3389/fendo.2020.00494</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Endocrinology</subject><subj-group><subject>Clinical Trial</subject></subj-group></subj-group></article-categories><title-group><article-title>Randomized Clinical Trial: Bergamot Citrus and Wild Cardoon Reduce Liver Steatosis and Body Weight in Non-diabetic Individuals Aged Over 50 Years</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Ferro</surname><given-names>Yvelise</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/507289/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Montalcini</surname><given-names>Tiziana</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/470370/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Mazza</surname><given-names>Elisa</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Foti</surname><given-names>Daniela</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/141432/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Angotti</surname><given-names>Elvira</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Gliozzi</surname><given-names>Micaela</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Nucera</surname><given-names>Saverio</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Paone</surname><given-names>Sara</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/943686/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Bombardelli</surname><given-names>Ezio</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Aversa</surname><given-names>Ilaria</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Musolino</surname><given-names>Vincenzo</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1011177/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Mollace</surname><given-names>Vincenzo</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Pujia</surname><given-names>Arturo</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/979752/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Health Science, University Magna Grecia</institution>, <addr-line>Catanzaro</addr-line>, <country>Italy</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Clinical and Experimental Medicine, University Magna Grecia</institution>, <addr-line>Catanzaro</addr-line>, <country>Italy</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Medical and Surgical Science, University Magna Grecia</institution>, <addr-line>Catanzaro</addr-line>, <country>Italy</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Plantexresearch Srl</institution>, <addr-line>Milan</addr-line>, <country>Italy</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Sungsoon Fang, Yonsei University, South Korea</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Kyeongkyu Kim, Salk Institute for Biological Studies, United States; Hyon-Seung Yi, Chungnam National University, South Korea; Youngsup Song, University of Ulsan, South Korea</p></fn><corresp id=\"c001\">*Correspondence: Tiziana Montalcini <email>tmontalcini@unicz.it</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Obesity, a section of the journal Frontiers in Endocrinology</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†These authors have contributed equally to this work</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>494</elocation-id><history><date date-type=\"received\"><day>10</day><month>1</month><year>2020</year></date><date date-type=\"accepted\"><day>22</day><month>6</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Ferro, Montalcini, Mazza, Foti, Angotti, Gliozzi, Nucera, Paone, Bombardelli, Aversa, Musolino, Mollace and Pujia.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Ferro, Montalcini, Mazza, Foti, Angotti, Gliozzi, Nucera, Paone, Bombardelli, Aversa, Musolino, Mollace and Pujia</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p><bold>Background:</bold> Non-alcoholic fatty liver disease is the most common cause of liver-related morbidity and mortality in the world. However, no effective pharmacological treatment for this condition has been found.</p><p><bold>Purpose:</bold> This study evaluated the effect of a nutraceutical containing bioactive components from Bergamot citrus and wild cardoon as a treatment for individuals with fatty liver disease. The primary outcome measure was the change in liver fat content.</p><p><bold>Methods:</bold> A total of 102 patients with liver steatosis were enrolled in a double-blind placebo controlled clinical trial. The intervention group received a nutraceutical containing a Bergamot polyphenol fraction and <italic>Cynara Cardunculus</italic> extract, 300 mg/day for 12 weeks. The control group received a placebo daily. Liver fat content, by transient elastography, serum transaminases, lipids and glucose were measured at the baseline and the end of the study.</p><p><bold>Results:</bold> We found a greater liver fat content reduction in the participants taking the nutraceutical rather than placebo (−48.2 ± 39 vs. −26.9 ± 43 dB/m, <italic>p</italic> = 0.02); The percentage CAP score reduction was statistically significant in those with android obesity, overweight/obesity as well as in women. However, after adjustment for weight change, the percentage CAP score reduction was statistically significant only in those over 50 years (44 vs. 78% in placebo and nutraceutical, respectively, <italic>p</italic> = 0.007).</p><p><bold>Conclusions:</bold> This specific nutraceutical containing bioactive components from Bergamot and wild cardoon reduced the liver fat content during 12 weeks in individuals with liver steatosis over 50 years. If confirmed, this nutraceutical could become the cornerstone treatment of patients affected by liver steatosis.</p><p><bold>Clinical Trial Registration:</bold>\n<ext-link ext-link-type=\"uri\" xlink:href=\"http://www.isrctn.com\">www.isrctn.com</ext-link>, identifier ISRCTN12833814.</p></abstract><kwd-group><kwd>nutraceuticals</kwd><kwd>liver steatosis</kwd><kwd>lipid</kwd><kwd>flavonoids</kwd><kwd>liver elastography</kwd></kwd-group><counts><fig-count count=\"3\"/><table-count count=\"4\"/><equation-count count=\"0\"/><ref-count count=\"57\"/><page-count count=\"12\"/><word-count count=\"9604\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Non-alcoholic fatty liver disease (NAFLD) is becoming the leading cause of liver damage in Western countries (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). NAFLD is a serious concern because the incidence of hepatocellular carcinoma associated with NAFLD has increased 10-fold in the past decades (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). Despite the genetic heri-tability predisposition to progressive NAFLD (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>), the factors leading to the progression from simple liver steatosis to severe liver scarring and cirrhosis are not fully clear. Several studies have shown that inflammatory markers, such as Tumor Necrosis Factor alpha (TNF-α) and other cytokines, play an important role in the pathogenesis of the NAFLD (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Oxidative stress is one of the key mediators of hepatic damage (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Additional factors involved in the pathogenesis of NAFLD are obesity and obesity-related metabolic disorders (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Furthermore, it has been demonstrated that an impaired lipophagy can lead to excessive tissue lipid accumulation making this pathway a new potential therapeutic target in NAFLD (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>).</p><p>There are currently no approved drugs to treat patients with fatty liver disease. The cornerstone of NAFLD treatment is lifestyle intervention and weight loss (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). Although most of the data on this issue are inconclusive, many dietary natural compounds isolated from fruits and vegetables have been proposed as promising agents capable of reversing hepatic steatosis (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>). It has been demonstrated that Bergamot (<italic>Citrus bergamia Risso et Poiteau</italic>) flavonoids, in the form of Bergamot polyphenol fraction (BPF) supplementation, are able to stimulate lipophagy and prevent pathogenic fat accumulation in diet-induced NAFLD rats (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Hesperidin, one of these flavonoids, prevents several form of liver damage (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>, <xref rid=\"B13\" ref-type=\"bibr\">13</xref>) as well as hepatocarcinogenesis (<xref rid=\"B14\" ref-type=\"bibr\">14</xref>).</p><p>However, molecules that have choleretic properties could offer additional benefits in the treatment of liver steatosis. Wild cardoon (<italic>Cynara cardunculus L.</italic>, the wild ancestor of the globe artichoke) extracts are rich in sesquiterpenes such as cynaropicrin, which possess significant choleretic proprieties thereby improving liver function (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). Bergamot polyphenols do not seem to have a choleretic effect. Wild cardoon also possess anti-inflammatory proprieties (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>–<xref rid=\"B18\" ref-type=\"bibr\">18</xref>).</p><p>Since currently we have no single ingredients working efficiently in NAFLD, the synergic effect of several natural molecules, which possess different proprieties, would represents an original approach in reducing the liver fat content.</p><p>In this study our aim was to test the effect of a new nutraceutical containing natural bioactive components from Bergamot and wild Cardoon, as a treatment for patients with liver steatosis.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Subjects</title><p>We enrolled adult individuals invited by newspaper advertisements to be screened for the possible presence of liver steatosis by transient elastography (TE). A population of 102 subjects with NAFLD, aged between 30 and 75 and attending the Clinical Nutrition Unit of the “Mater Domini” Azienda Uni-versity Hospital in Catanzaro, Italy, were enrolled (study duration between February 11, 2019 and June 24, 2019), who were not taking nutraceuticals, supplements or functional food. Local ethical committee at the “Mater Domini” Azienda University Hospital approved the protocol (219/2018/CE, approved September 24, 2018) which was funded by Italian Ministry of University and Research (MIUR, Nutramed Project, PON 03PE000_78_1). The study is listed on the ISRCTN registry (study ID ISRCTN12833814).</p><p>According to the protocol of the study, we excluded subjects with past and current alcohol abuse [> 20 g of alcohol per day; 350 mL (12 oz) of beer, 120 mL (4 oz) of wine, and 45 mL (1.5 oz) of hard liquor each contain 10 g of alcohol], who had clinical and laboratory signs of chronic hepatitis B and/or C virus infection or allergies to cardoon, artichoke or maize or with triglycerides concentration over 250 mg/dl (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>) and subjects affected by diabetes. Furthermore, we excluded individuals with autoimmune or cholestatic liver disease, liver cirrhosis, pregnancy, nephrotic syndrome, chronic renal failure, gastroesophageal reflux, cancer, and those taking amiodarone, antiretroviral agents, corticosteroids, methotrexate, tamoxifen, valproate, as ascertained from their clinical records. The study's protocol allowed to enroll only long-term lipid-lowering drugs users (more than 6 weeks).</p></sec><sec><title>Study Design</title><p>Patients were randomly assigned in a 1:1 ratio to receive either a nutraceutical from Bergamot and wild cardoon (abbreviated, BC) or a placebo for up to 12 weeks (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>- Flow-chart of the study). Computer-generated random numbers were used for the simple randomization of subjects.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Flow-chart of the study.</p></caption><graphic xlink:href=\"fendo-11-00494-g0001\"/></fig><p>Eighty-six subjects completed the entire 12 weeks of the study. The study's treatments were as follows:</p><list list-type=\"order\"><list-item><p>BC (provided by Herbal & Antioxidant SRL, Bianco, RC, Italy): one capsule containing a combination product containing bergamot polyphenolic fraction (BPF®), and wild type <italic>Cynara Cardunculus</italic> extract (CyC) plus excipients including PUFA and a mixture of bergamot pulp and albedo derivative]. (registered Patents RM2008A000615, PCT/IB2009/055061 and 102017000040866); (batch number 18R049, expiration date 10/2020).</p></list-item><list-item><p>Placebo: one capsule containing maltodextrin plus excipients including PUFA and a mixture of bergamot pulp and albedo derivative; (batch number 18R050, expiration date 10/2020).</p></list-item></list><p>In this study, the primary outcome measure was the change in liver fat content, measured as “controlled attenuation parameter” (CAP) by TE (Fibroscan), and/or other markers of fatty liver, after 12 weeks of treatment. Secondary outcomes were the changes from baseline in plasma lipids and inflammatory markers after 12 weeks of treatment.</p><p>Participants received oral and written recommendations to adhere to a Mediterranean dietary pattern, without energy restriction (except in the obese) by a registered dietitians (RD) (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). Y.F. enrolled the participants. A RD assigned participants to interventions. Both the experimenters and participants were blind to who received the nutraceutical or the placebo.</p><p>In order to create a negative energy balance, a 400–500 calorie restriction from baseline energy intake was prescribed for overweight/obese individuals.</p><p>Written informed consent was obtained from all participants. The investigation conforms to the principles outlined in the Declaration of Helsinki.</p><p>The product BC has been carried out according to EU Directive 2004/9/EC and Directive 2004/9/EC for Good Laboratory Practice Guidelines (GLP) as well as OECD Guidelines for Repeated Dose 28 and 90-day Oral Toxicity Study in Rodents.</p></sec><sec><title>Preparation of BC Formulation</title><p>The specification sheet with the most relevant active ingredients of BPF and CyC are reported in the supplementary material (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figures 1</xref>, <xref ref-type=\"supplementary-material\" rid=\"SM1\">2</xref>). Briefly, bergamot juice (BJ) was obtained from peeled-off fruits by industrial pressing and squeezing. The juice was oil fraction depleted by stripping, clarified by ultra-filtration and loaded on suitable polystyrene resin columns absorbing polyphenol compounds of MW between 300 and 600 Da (Mitsubishi). Polyphenol fraction was eluted by a mild KOH solution. Moreover, the phytocomplex was then neutralized by filtration on cationic resin at acidic pH. Finally, it was vacuum dried and minced to the desired particle size to obtain a powder. In particular, powder was micronized and co-grinded with bergamot albedo fibers when given alone or in combination. BPF powder was analyzed by HPLC for flavonoid and other polyphenol content. In addition, toxicological analyses were performed including heavy metal, pesticide, phthalate and sinephrine content which revealed the absence of known toxic compounds. Standard microbiological tests detected no mycotoxins and bacteria. The same procedure was used for the production of CyC extract. Fibers obtained by bergamot albedo were used micronized and co-grinded with plant extracts as excipients for final formulations. All materials were provided by HEAD srl (Bianco, Italy.) Finally, to obtain a formulation containing both extracts, 150 mg of BPF powder were combined with 150 mg of CyC and were encapsulated in capsules containing 300 mg of excipients represented by albedo fibers micronized and co-grinded with plant extracts (Seris srl, Cuneo, Italy). The final formulation contained 5% of cynaropicrin. Capsules containing 600 mg maltodextrin were prepared for placebo studies. All capsules were put into bottles containing 56 capsules (for 4 week) and packaged in color-coded plastic bags containing 4 bottles (for 16 weeks) for distribution to participants. All procedures have been performed according to the European Community Guidelines concerning dietary supplements (for toxicological reports and pharmacokinetic studies see <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Material</xref>).</p></sec></sec><sec><title>Dietary Intake Assessment (see <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Tables 1, 2</xref>)</title><sec><title>Liver Transient Elastography (TE)</title><p>CAP is a novel non-invasive measure of NAFLD. TE can quantify liver steatosis by CAP assessment and measure liver stiffness (Fibroscan® Echosense SASU, Paris, France) (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). Both CAP and stiffness score were obtained simultaneously and in the same volume of liver parenchyma. All patients were evaluated using the 3.5 MHz standard M probe on the right lobe of the liver through intercostal spaces with the patient lying supine and placing the right arm behind the head to facilitate access to the right upper quadrant of the abdomen. The tip of the probe transducer was placed on the skin between the rib bones at the level of the right lobe of the liver. All scans were performed by the same investigator. Liver stiffness was expressed by the median value (in kPa) of 10 measurements performed between 25 and 65 mm depth. Only results with 10 valid shots and interquartile range (IQR)/median liver stiffness ratio<30% were included. The cut-off value for defining the presence of fibrosis was liver stiffness >7 kPa.</p><p>We assessed CAP score using only the M probe because the CAP algorithm is specific to this device. Ten successful measurements were performed on each patient, and only cases with 10 successful acquisitions were taken into account for this study. The success rate was calculated as the number of successful measurements divided by the total number of measurements. The ratio of the IQR of liver stiffness to the median (IQR/MLSM) was calculated as an indicator of variability. The final CAP score (ranged from 100 to 400 decibels per meter (dBm–), was the median of individual measurements. The ratio of IQR in CAP values to the median (IQR/M CAP) was used as an indicator of variability for the final CAP. The diagnosis of NAFLD was based on a CAP > 216 dB/m. In order to identify each steatosis grade, three different cut-offs were used: CAP between 216 and 252 dB/m for the diagnosis of S1 grade, CAP between 253 and 296 dB/m for the diagnosis of S2 grade, and CAP > 296 dB/m for the diagnosis of S3 grade (severe) (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>, <xref rid=\"B23\" ref-type=\"bibr\">23</xref>).</p></sec><sec><title>Anthropometric Measurements and Cardiovascular Risk Factors Assessment</title><p>Body weight, BMI and waist and hip circumferences (WC and HC) were measured as previously described (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). Obesity was defined by the presence of a body mass index (BMI) ≥ 30 kg/m<sup>2</sup>.</p><p>We assessed the presence of the classical cardiovascular (CV) risk factors from clinical records and patient interview (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>, <xref rid=\"B24\" ref-type=\"bibr\">24</xref>, <xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Blood pressure was determined at the time of the three visits (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). The following criteria were used to define and exclude diabetes: fasting blood glucose ≥ 126 mg/dL or antidiabetic treatment (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). Metabolic Syndrome (MS) definition was based on the National Cholesterol Education Program's (NCEP) Adult Treatment Panel III report (ATP III). Individuals with at least three or more abnormalities were identified as having MS (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>).</p><p>According to World Health Organization criteria (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>), android obesity was defined as a Waist-hip-ratio (WHR) above 0.90 for males and above 0.85 for females.</p></sec><sec><title>Biochemical Evaluation</title><p>Venous blood was collected after fasting overnight into vacutainer tubes (Becton & Dickinson, Plymouth, England) and centrifuged within 4 h. Serum glucose, total cholesterol, high density lipoprotein (HDL)-cholesterol, triglycerides, creatinine, ALT, AST, GGT, and insulin were measured by chemiluminescent immunoassay on COBAS 8000 (Roche, Switzerland), according to the manufacturer's instructions. LDL- C level was calculated by the Friedewald formula (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>).</p><p>Blood count analysis was performed using ADVIA 2120i (Siemens Healthcare Diagnostics, Marburg, Germany).</p><p>Homeostatic model assessment (HOMA) index was calculated for assessing β-cell function and insulin resistance (IR) from fasting glucose and insulin concentrations (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). The serum concentrations of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) were determined by sandwich enzyme-linked immunosorbent assay (ELISA) (R&D Systems Inc., Minneapolis, USA) according to the manufacturer's instructions.</p></sec><sec><title>Safety Parameters and Adverse Events</title><p>We measured several parameters of general health such as blood pressure as well as serum glucose, creatinine, total bilirubin, and lipids.</p><p>We used a patient-reported outcome questionnaire for the measurement of adverse events (AEs). The questionnaire investigated the presence of any new symptoms after entering the study that could be related to the intervention. We assessed the nature of the ADs such as those regarding severity.</p></sec><sec><title>Data Analysis</title><p>Data are reported as mean ± standard deviation (SD).</p><p>It was taken into account a mean CAP value of 268.6 ± 52 dB/m for individuals with liver steatosis (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Thus, to detect a CAP score reduction of at least 12%, with an effect size (ES = mean CAP difference/ baseline SD) of 0.62, with 80% power on a two-sided level of significance of 0.05, a minimum of 44 subjects for each group were required. Considering a 10% of drop-out, we enrolled 102 patients (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p><p>A Chi square test was performed to analyze the prevalence between groups and an independent unpaired samples <italic>t</italic>-test was used to compare the difference between means. Specifically, we calculated the changes in variables and compared the means of these changes between treatment groups. Changes in the clinical characteristics from baseline to follow-up (within group variation) were calculated using paired Student's <italic>t</italic>-test (two tailed). We used the Bonferroni adjustment method for multiple testing correction (General Linear Model -GLM). Indeed, a repeated measure ANOVA was performed to evaluate time-group difference in CAP score (ANOVA/GLM for grouped data).</p><p>We used both an indirect assessment method (i.e., pill count) and patient interviews to assess adherence. We performed intention-to-treat (ITT) as well as on-treatment (OT) analyses, defining OT as those participants taking more than 80% of the prescribed treatment.</p><p>We performed several <italic>post-hoc</italic> analyses (not pre-specified) in the following subgroups of participants: ITT; OT; overweight/obese; men; women; with metabolic syndrome; over 50 years; with age ≤ 50 years; with gynoid obesity; with android obesity.</p><p>Significant differences were assumed to be present at <italic>p</italic> < 0.05 (two-tailed). All comparisons were performed using SPSS 22.0 for Windows (IBM Corporation, New York, NY, United States).</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><p>Ninety-five subjects completed the first part of the study. Four subjects were lost within 6 weeks in the placebo group and three subjects in the BC group (one due to suspected cancer and one due to low back pain in BC; <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Eighty-six subjects completed the study (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). One participant was lost due to suspected cancer and one due to aphthous oral ulcer in the placebo group). The mean age of the population was 51 ± 9 years. A total of 52 (61%) were male. The mean basal LDL-C and CAP score was 119 ± 33 mg/dl and 289 ± 39 dB/m, respectively.</p><sec><title>Dietary Intake Assessment</title><p>Nutrient intake assessment and dietary intake changes during the study are showed in <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Table 2</xref>.</p></sec><sec><title>Baseline Demographic and Clinical Characteristics of Participants According to the Treatments</title><p><xref rid=\"T1\" ref-type=\"table\">Table 1</xref> shows the basal clinical characteristics of participants according to the allocation (<italic>n</italic> = 102). The groups were comparable for all of the characteristics. About half of the population had hyperlipidemia.</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Baseline demographic and clinical characteristics of participants according to the treatments.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Variables</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Placebo (<italic>n</italic> = 51)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>BC (<italic>n</italic> = 51)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>p-value</italic></bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Age (years)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">51 ± 11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53 ± 9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.43</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weight (Kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80 ± 11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80 ± 12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.94</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BMI (Kg/m<sup>2</sup>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29 ± 4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.86</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">WC (cm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">97 ± 8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99 ± 10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.29</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HC (cm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">107 ± 7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">105 ± 7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.30</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FM (Kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25 ± 8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25 ± 7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.80</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SBP (mmHg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">113 ± 17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">112 ± 14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.97</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DBP (mmHg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">71 ± 14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">73 ± 11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.70</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP score (dB/m)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">285 ± 40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">294 ± 39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.27</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IQR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11 ± 6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11 ± 5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.95</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Stiffness (kPa)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.3 ± 1.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.9 ± 1.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.15</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IQR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14 ± 5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14 ± 7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.53</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Glucose (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92 ± 8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">93 ± 8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.74</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insulin (mU/L)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.9 ± 4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10 ± 5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.84</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HOMA-IR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.3 ± 1.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.3 ± 1.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.74</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TC (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">196 ± 39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">193 ± 39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.66</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TG (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">115 ± 48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">107 ± 53</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.42</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HDL-C (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">51 ± 13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">53 ± 12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.48</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">LDL-C (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">122 ± 34</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">118 ± 34</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.60</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Non-HDL-C (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">145 ± 36</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">140 ± 39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.49</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AST (IU/L)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23 ± 15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21 ± 6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.23</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ALT (IU/L)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32 ± 27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23 ± 12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">γGT (UI/L)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27 ± 20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25 ± 17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.59</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Creatinine (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.82 ± 0.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.83 ± 0.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.80</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Uric Acid (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.1 ± 1.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.2 ± 1.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.70</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total bilirubin (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.61 ± 0.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.62 ± 0.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.90</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"4\" rowspan=\"1\"><bold>Prevalence</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gender (Female,%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">45</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">43</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Menopause (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">68</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Smokers (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.19</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Obesity (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.77</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">MS (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.37</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hypertension (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hyperlipidemia (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">57</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">49</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.55</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Antihypertensive drugs (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lipid-lowering agents (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.38</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Antiplatelet agents (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.71</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Liver steatosis S1 grade (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.20</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Liver steatosis S2 grade (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.20</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Liver steatosis S3 grade (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">49</td><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Liver Fibrosis (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.71</td></tr></tbody></table><table-wrap-foot><p><italic>BMI, body mass index; WC, waist circumference; HC, hip circumference; FM, fat mass; SBP, systolic blood pressure; DBP, diastolic blood pressure; CAP, controlled attenuation parameter; IQR, interquartile range; HOMA-IR, homeostatic model assessment of insulin resistance; TC, total cholesterol; TG, triglycerides; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; AST, aspartate aminotransferase; ALT, alanine aminotransferase; γGT, gamma glutamyltransferase</italic>.</p></table-wrap-foot></table-wrap><p>The nutrient profile of the overall diet of the population according to the treatments is shown in <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Table 1</xref>. At enrolment, nutrient intake of the two groups were comparable (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Table 2</xref>). Furthermore, <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Table 2</xref>, shows the dietary changes during the study. Only overweight/obese individuals reached a 400–500 caloric restriction from the baseline intake. At the end of the intervention, the two groups were comparable <italic>(t-test)</italic>.</p><p><xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Figure 3</xref> shows individual body weight reduction according to the treatment.</p></sec><sec><title>Clinical Characteristics Changes at Follow-Up and Outcome of the Study</title><p><xref rid=\"T2\" ref-type=\"table\">Table 2</xref> shows the baseline and follow-up clinical characteristics of participants who completed the study (12 weeks) according to the treatment group (<italic>n</italic> = 86). At baseline, the groups were comparable for all of the characteristics (see <italic>unpaired t-test</italic> between treatments). LDL-C, HDL- C, non-HDL-C, TC, HOMA- IR, AST, and γGT decreased only in the participants taking BC (LDL- C from 116 ± 32 to 107 ± 30 mg/dl, <italic>p</italic> = 0.002, <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>).</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Baseline and follow-up clinical characteristics of participants according to the treatments (Intention To Treat analysis).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" colspan=\"3\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Placebo (</bold><italic><bold>n</bold></italic>\n<bold>= 41)</bold></th><th valign=\"top\" align=\"center\" colspan=\"3\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>BC (</bold><italic><bold>n</bold></italic>\n<bold>= 45)</bold></th><th rowspan=\"1\" colspan=\"1\"/><th rowspan=\"1\" colspan=\"1\"/></tr><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Variables</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Basal</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Follow-up</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>p</italic>-value</bold><break/>\n<bold>(paired <italic>t</italic>-test)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Basal</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Follow-up</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>p</italic>-value</bold><break/>\n<bold>(paired <italic>t</italic>-test)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>p</italic>-value</bold><break/>\n<bold>(unpaired <italic>t</italic>-test between basal values)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>p</italic>-value (unpaired <italic>t</italic>-test between follow-up values)</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weight (Kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80 ± 11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">77 ± 11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80 ± 12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">76 ± 12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.87</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.64</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BMI (Kg/m<sup>2</sup>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28.7 ± 4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.7 ± 4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.2 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27.6 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.53</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.76</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">WC (cm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">97 ± 8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">93 ± 8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99 ± 10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">95 ± 9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.23</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.52</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HC (cm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">107 ± 7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">103 ± 6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">106 ± 7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">102 ± 7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.52</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.50</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FM (Kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25 ± 8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22 ± 8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25 ± 7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21 ± 7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.93</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.54</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">SBP (mmHg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">111 ± 17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110 ± 13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.73</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">111 ± 14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">109 ± 12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.31</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.97</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.70</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DBP (mmHg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">71 ± 15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">71 ± 9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.91</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">73 ± 11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">72 ± 10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.90</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP score (dB/m)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">283 ± 40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">256 ± 52</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">295 ± 38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">247 ± 42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.37</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Stiffness (kPa)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.1 ± 1.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.6 ± 1.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.046</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.0 ± 1.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.7 ± 1.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.68</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.74</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Glucose (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92 ± 8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92 ± 8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">93 ± 8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92 ± 13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.85</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.95</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insulin (mU/L)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.2 ± 4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.9 ± 5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.021</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.2 ± 5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.3 ± 5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.016</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.86</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.50</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HOMA-IR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.3 ± 1.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.1 ± 1.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.4 ± 1.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.0 ± 1.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.034</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.76</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.63</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TC (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">194 ± 40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">189 ± 42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">188 ± 34</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">177 ± 33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.13</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TG (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">113 ± 48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">110 ± 55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.72</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">102 ± 47</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">101 ± 49</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.84</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.28</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.38</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HDL-C (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50 ± 12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">49 ± 11</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">52 ± 12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">49 ± 13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.002</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.99</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">LDL-C (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">122 ± 35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">117 ± 36</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">116 ± 32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">107 ± 30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.002</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.15</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Non-HDL-C (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">144 ± 38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">139 ± 42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.34</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">136 ± 36</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">127 ± 34</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.003</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.13</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AST (IU/L)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21 ± 9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20 ± 5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21 ± 6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19 ± 4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.015</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.71</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.76</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ALT (IU/L)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">28 ± 21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22 ± 9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.007</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24 ± 13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19 ± 8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.29</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.14</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">γGT (UI/L)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27 ± 21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25 ± 22</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">26 ± 18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">22 ± 18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.011</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.76</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.56</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Creatinine (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.82 ± 0.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.85 ± 0.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.034</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.83 ± 0.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.83 ± 0.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.63</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.68</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.63</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Uric Acid (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.2 ± 1.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.3 ± 1.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.2 ± 1.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.1 ± 1.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.33</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total bilirubin (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.62 ± 0.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.69 ± 0.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.046</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.64 ± 0.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.65 ± 0.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.88</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.82</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.85</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">WBCs (x10<sup>3</sup>/uL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.3 ± 1.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.0 ± 1.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.4 ± 1.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6.0 ± 1.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.91</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.89</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lymphocyte (x10<sup>3</sup>/uL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.91 ± 0.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.96 ± 0.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.06 ± 0.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.98 ± 0.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.47</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.31</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.95</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neutrophil (x10<sup>3</sup>/uL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.68 ± 1.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.37 ± 0.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.01 ± 1.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.90 ± 0.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.60</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.87</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.027</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Monocyte (x10<sup>3</sup>/uL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.37 ± 0.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.36 ± 0.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.39 ± 0.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.38 ± 0.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.75</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.63</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"9\" rowspan=\"1\"><italic><bold>Cytokine evaluation</bold></italic></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-1β (pg/mL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.08 ± 0.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.53 ± 0.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.11 ± 0.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.75 ± 0.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.85</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.24</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-6 (pg/mL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.68 ± 0.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.57 ± 1.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.92 ± 0.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.03 ± 1.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.12</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TNF-α (pg/mL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.08 ± 0.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.53 ± 0.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.56 ± 3.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.04 ± 2.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.002</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.16</td></tr></tbody></table><table-wrap-foot><p><italic>BMI, body mass index; WC, waist circumference; HC, hip circumference; FM, fat mass; SBP, Systolic Blood Pressure; DBP, Diastolic Blood Pressure; CAP, controlled attenuation parameter; HOMA-IR, homeostatic model assessment of insulin resistance; TC, total cholesterol; TG, triglycerides; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; AST, aspartate aminotransferase; ALT, alanine aminotransferase; γGT, gamma glutamyltransferase; WBCs, white blood cells; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor α</italic>.</p></table-wrap-foot></table-wrap><p>The CAP score, body weight, insulin and ALT decreased in the participants taking BC as well as in participants in the placebo group (in BC group, CAP score dropped from 295 ± 38 to 247 ± 42 dB/m, <italic>p</italic> < 0.001; paired <italic>t</italic>-test; <xref rid=\"T2\" ref-type=\"table\">Table 2</xref>).</p><p><xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref> shows the individual CAP score reduction for the participants in each group.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Individual CAP score reduction according to the treatments.</p></caption><graphic xlink:href=\"fendo-11-00494-g0002\"/></fig><p>The changes in the clinical parameters after each treatment period are shown in <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>. The change in the serum concentration of the white blood cells, IL-1β, IL-6, and TNF-α did not differ between groups (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>).</p><table-wrap id=\"T3\" position=\"float\"><label>Table 3</label><caption><p>Changes in clinical parameters at follow-up according to the treatments (unpaired <italic>t</italic>-test).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Variables</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Placebo (<italic>n</italic> = 41)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>BC (<italic>n</italic> = 45)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>p-value</italic></bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Follow-up duration (days)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">88 ± 4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">88 ± 6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.94</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Adherence to treatment (≥ 80%, %)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weight (Kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.7 ± 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.2 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.004</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">BMI (Kg/m<sup>2</sup>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.9 ± 0.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.6 ± 0.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.003</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">WC (cm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−3.7 ± 4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.9 ± 4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.16</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HC (cm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−3.2 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−3.2 ± 4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.99</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">FM (kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.4 ± 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−3.3 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.07</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP score (dB/m)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−26.9 ± 43</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−48.2 ± 39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.020</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Stiffness (kPa)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.46 ± 1.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.26 ± 1.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.49</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Glucose (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.07 ± 7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.3 ± 10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.91</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Insulin (mU/L)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.2 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.9 ± 5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.41</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HOMA-IR</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.2 ± 0.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.4 ± 1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.43</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TC (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−5.2 ± 32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−11.9 ± 21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.26</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TG (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.2 ± 40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.0 ± 34</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.85</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HDL-C (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.5 ± 6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.8 ± 6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.06</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">LDL-C (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.3 ± 28</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−8.9 ± 18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.36</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Non-HDL-C (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.3 ± 27</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−9.1 ± 19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.42</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">AST (IU/L)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.9 ± 7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.8 ± 5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.48</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ALT (IU/L)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−3.5 ± 28</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.0 ± 17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.76</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">γGT (UI/L)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.6 ± 9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−3.8 ± 9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.55</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Uric Acid (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.13 ± 0.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.09 ± 0.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.18</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Total bilirubin (mg/dL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.07 ± 0.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.01 ± 0.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.22</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">WBCs (x 10<sup>3</sup>/uL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.37 ± 1.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.35 ± 1.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.94</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Lymphocyte (x 10<sup>3</sup>/uL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.04 ± 0.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.07 ± 0.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.36</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Neutrophil (x 10<sup>3</sup>/uL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.31 ± 1.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.11 ± 1.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.50</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Monocyte (x 10<sup>3</sup>/uL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.01 ± 0.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.01 ± 0.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.97</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-1β (pg/mL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.56 ± 0.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.36 ± 0.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.29</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">IL-6 (pg/mL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.42 ± 0.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.48 ± 0.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.56</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TNF-α (pg/mL)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.73 ± 2.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.52 ± 2.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.71</td></tr></tbody></table><table-wrap-foot><p><italic>BMI, body mass index; WC, waist circumference; HC, hip circumference; FM, fat mass; CAP, controlled attenuation parameter; HOMA-IR, homeostatic model assessment of insulin resistance; TC, total cholesterol; TG, triglycerides; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; AST, aspartate aminotransferase; ALT, alanine aminotransferase; γGT, gamma glutamyltransferase; WBCs, white blood cells; IL-1β, interleukin-1β; IL-6, interleukin-6; TNF-α, tumor necrosis factor α</italic>.</p></table-wrap-foot></table-wrap><p>BC significantly lowered body weight, BMI and CAP score compared to the placebo [absolute difference: body weight−2.7 ± 2 <italic>vs</italic>. −4.2 ± 3 kg (<italic>p</italic> = 0.004) and CAP score −26.9 ± 43 <italic>vs</italic>. −48.2 ± 39 dB/m (<italic>p</italic> = 0.002) in the placebo and BC group, respectively (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). The mean CAP score decreased significantly over time in the BC group vs. the placebo group (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>).</p></sec><sec><title>Subgroup Analysis</title><p><xref rid=\"T4\" ref-type=\"table\">Table 4</xref> shows the changes in the clinical parameters in subgroups according to the treatments. The percentage CAP score reduction was statistically significant in those with android obesity or in overweight/obese individuals (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). Furthermore, CAP score reduction was higher in the OT rather than ITT analysis (−17.1 <italic>vs</italic>. −15.9% in OT <italic>vs</italic>. ITT analysis) as well as a higher CAP score reduction was found in women rather than in men (<italic>p</italic> = 0.03, <xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). However, after adjustment for weight change, the percentage CAP score reduction was statistically significant only in those over 50 years.</p><table-wrap id=\"T4\" position=\"float\"><label>Table 4</label><caption><p>Changes in clinical parameters in the subgroups according to the treatments.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Variables</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Placebo (<italic>n</italic> = 41)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>BC (<italic>n</italic> = 45)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>p-value</italic></bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" colspan=\"4\" rowspan=\"1\"><bold>ITT</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weight (Kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.7 ± 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.2 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.004</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP score (dB/m)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−26.9 ± 43</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−48.2 ± 39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.020</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−9.2 ± 16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−15.9 ± 13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.036</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Improvement (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">51</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">76</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.025</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"4\" rowspan=\"1\"><bold>On-Treatment</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weight (Kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.7 ± 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.4 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.002</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP score (dB/m)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−25.7 ± 45</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−51.7 ± 38</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.007</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−8.7 ± 17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−17.1 ± 12</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.013</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Improvement (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">79</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.010</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"4\" rowspan=\"1\"><bold>Women</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weight (Kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.6 ± 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.3 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.063</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP score (dB/m)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−22.3 ± 47</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−54.1 ± 35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.034</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−8 ± 18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−17.8 ± 10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.067</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Improvement (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">56</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">83</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.13</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"4\" rowspan=\"1\"><bold>Men</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weight (Kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.8 ± 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.2 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.030</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP score (dB/m)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−29.9 ± 42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−44.3 ± 42</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.225</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−9.9 ± 15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−14.7 ± 14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.249</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Improvement (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">70</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.15</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"4\" rowspan=\"1\"><bold>Age ≤ 50 years</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weight (Kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.9 ± 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.1 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.17</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP score (dB/m)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−43.4 ± 37</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−39.5 ± 43</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.75</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−15.7 ± 13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−13.9 ± 15</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.67</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Improvement (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">67</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">73</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.74</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"4\" rowspan=\"1\"><bold>Age > 50 years</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weight (Kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.4 ± 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.4 ± 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.004</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP score (dB/m)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−9.7 ± 44</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−56.5 ± 34</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><0.001</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><italic>a</italic>CAP score (dB/m)<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−13.7 ± 9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−52.9 ± 8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.004</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.3 ± 16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−17.9 ± 10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.001</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Improvement (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.006</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"4\" rowspan=\"1\"><bold>Android Obesity</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weight (Kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.8 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.4 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.016</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP score (dB/m)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−23.7 ± 45</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−51.2 ± 39</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.014</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−8.1 ± 17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−16.8 ± 13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.027</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Improvement (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">44</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.007</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"4\" rowspan=\"1\"><bold>Gynoid Obesity</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weight (Kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.5 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−3.8 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.22</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP score (dB/m)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−33.3 ± 41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−37.6 ± 41</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.80</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−11.4 ± 16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−12.9 ± 14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.80</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Improvement (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">64</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">60</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"4\" rowspan=\"1\"><bold>Metabolic Syndrome</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weight (Kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.8 ± 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−3.4 ± 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.43</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP score (dB/m)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−9.1 ± 54</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−47 ± 54</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.13</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.3 ± 20</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−14.2 ± 16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.16</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Improvement (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">55</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">45</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.65</td></tr><tr><td valign=\"top\" align=\"left\" colspan=\"4\" rowspan=\"1\"><bold>Overweight & Obesity</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Weight (Kg)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−3.0 ± 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.5 ± 3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.005</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP score (dB/m)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−25.6 ± 46</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−50.7 ± 40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.018</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CAP (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−8.4 ± 17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−16.5 ± 18</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.031</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Improvement (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">44</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">78</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.007</td></tr></tbody></table><table-wrap-foot><p><italic>CAP, controlled attenuation parameter</italic>.</p><fn id=\"TN1\"><label>*</label><p><italic>CAP score adjusted for body weight change</italic>.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Disease Risk Analysis</title><p><xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref> shows non-improvement or liver steatosis progression risk according to the subgroups in individuals taking BC. The nutraceutical reduced the risk in the following subgroups: over 50 years, with android obesity, with overweight/obesity (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>).</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Non-improvement or progression liver steatosis risk.</p></caption><graphic xlink:href=\"fendo-11-00494-g0003\"/></fig></sec><sec><title>Adverse Events</title><p>The findings are reported in the <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Figure 4</xref>. The participants reported a total of 24 AEs, all of grade 1 (mild).</p></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>Our clinical trial examined the effects of a new nutraceutical containing a combination of polyphenols from Bergamot citrus and terpenes and other flavonoids from wild cardoon (abbreviated, BC), in reducing the liver fat content in non-diabetic individuals with NAFLD.</p><p>BC significantly reduced the CAP score by 7% (-9% in OT analysis; −26.9 ± 43 and −48 ± 39 dB/m in the placebo and BC, respectively), after 12 weeks (<xref rid=\"T3\" ref-type=\"table\">Tables 3</xref>, <xref rid=\"T4\" ref-type=\"table\">4</xref>). The main finding was that BC reduced the CAP score by 15%, in the participants over 50 years (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>).</p><p>The changes observed in the CAP score were in the range of those obtained in one double-blind, randomized, placebo-controlled study with hesperidin supplementation (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). In that study, hesperidin supplementation, compared with placebo, was associated with a significant reduction in CAP score (~-31 vs ~-51 in the placebo and Hesperidin group, respectively) (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). However, in that study participants were recruited only with NAFLD grades 2 and 3, while in our study we enrolled all individuals who had some degree of steatosis (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). Furthermore, to date, we have no single ingredients working efficiently in NAFLD. Several studies performed in animal models of liver steatosis confirm our results (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>, <xref rid=\"B33\" ref-type=\"bibr\">33</xref>). It has been demonstrated that a mixture of natural citrus polyphenols from Bergamot prevents pathogenic fat accumulation in the liver of rats (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>, <xref rid=\"B34\" ref-type=\"bibr\">34</xref>). However, no further studies exist on the effects of nutraceuticals on NAFLD patients.</p><p>The findings from studies in several cellular models have indicated the ability of dietary polyphenols to counteract ROS production and its associated oxidative damage by controlling mitochondrial membrane potential and oxidative phosphorylation (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>, <xref rid=\"B34\" ref-type=\"bibr\">34</xref>). Dietary polyphenols can also act indirectly by up-regulating endogenous antioxidant defenses (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>, <xref rid=\"B36\" ref-type=\"bibr\">36</xref>). Furthermore, these molecules act as potent inducer of lipophagy in animal models of steatosis (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>).</p><p>Citrus fruits are notably rich in flavonoid compounds. However, among different citrus fruits, Bergamot contains the highest concentrations of total flavone glycosides, including both flavone <italic>O</italic>- and <italic>C</italic>-glycosides (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>).</p><p>A study has demonstrated that in most citrus juices, vicenin-2 is, by far, the most abundant flavone derivative with the highest level in Bergamot cultivars, followed by other fruits (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>).</p><p>Although pears (<italic>Pyrus</italic> spp) are dietary sources of bioactive components such as polyphenols and triterpenic acids (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>), the chemical composition of these fruits significantly differ from Bergamot and Wild Cardoon. Indeed, chlorogenic acid, quinic acid and arbutin are the primary polyphenols and ursolic acid is the predominant triterpenoid in thinned pears, whereas chlorogenic acid and most of the flavan-3-ols, are the main antioxidants in young pears (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>).</p><p>Cynaropicrin from other source, such as <italic>Saussurea amara</italic>, a Mongolian medicinal plant, provokes a dose-dependent increase in bile flow in the isolated rat liver perfusion system, thus confirm our findings (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>).</p><p>Using a patented extraction technology through collaborative works of various research institutions, BC contains the highest concentration available of these potent compounds. BC is composed of several, well-known, biologically active phenols, such as naringine, neohesperidine, neoeriocitrine, brutieridine, melitidine, cynaropicrin, cholotogenic acid. The largest representative sesquiterpene lactones in wild cardoon is cynaropicrin which possesses choleretic, anti-inflammatory and anti-hyperlipidemic proprieties (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>). Cynaropicrin has potent suppressive effects on TNF-α (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>). In addition, wild cardoon recovers other biologically active compounds, such as caffeoylquinic acids, luteolin, and apigenin derivatives, all of which have potential important effects on human health (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>). In this study, a specification sheet of the product including the mean concentration of the most relevant active ingredients as well as the HPLC traces for both bergamot and CyC extract was provided (see <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Material</xref>). Since currently we have no single molecules working efficiently in NAFLD, the synergic effect of all these polyphenols would represent a novel approach in reducing the liver fat content.</p><p>Of interest, in the present study the nutraceutical BC, compared with placebo, was associated with a significant body weight reduction (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref> and <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Figure 3</xref>) (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). In individuals with NAFLD, interventions aimed at weight loss were associated with improvements in blood biomarkers of liver disease, as well as instrumental and histological markers, such as liver stiffness and steatosis (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). CAP score reduction seems to be explained by weight loss in our population, but not among subjects aged >50 years (<italic>post-hoc</italic> analyses, <xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). Therefore, BC improved CAP score in adult population.</p><p>Our subgroup analyses yield more than one significant interaction. These significant interactions might, however, be associated with each other, and thus explained by a common factor, Thus, CAP score reduction in participants with android obesity as well as overweight-obesity could also be due to weight loss. Although such <italic>post-hoc</italic> analyses might not carry the same weight of evidence as the primary prespecified analysis, they offer the opportunity to explore hypotheses that might not be immediately envisaged at the time of study design and reflect the dynamic nature of clinical research (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>). The potential value of <italic>post-hoc</italic> analyses in hypothesis generation cannot be entirely discounted, thus, the outcomes might inform the design of future studies.</p><p>Some clarification is needed regarding the lack of an expected significant difference in LDL-cholesterol reduction between groups (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>). First, the main purpose of this study was not blood lipid reduction. In addition, only half of the population had hyperlipidaemia and about 15% had taken lipid-lowering agents. The use of these agents did not influence the CAP score differences (data not shown). However, in participants taking BC, we found that LDL-cholesterol dropped from 116 ± 32 to 107 ± 30 mg/dl (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>).</p><p>Our population had only mild hypercholesterolemia (<xref rid=\"T1\" ref-type=\"table\">Tables 1</xref>, <xref rid=\"T2\" ref-type=\"table\">2</xref>), which limited the efficacy of BC. Moreover, it is well-known that hypercholesterolemia and inflammation are linked in a vicious cycle in which the excess of cholesterol induces an inflammatory response that, in turn, accelerates cholesterol deposition and amplifies inflammation (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>).</p><p>Cytokines play a key role in inflammatory diseases and a link with hypercholesterolemia and atherosclerosis has emerged mainly for the IL-6, IL-1, and TNFα pathways (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>).</p><p>All these mechanisms can explain, in our study, both the lack of the lipid-lowering and antiinflammatory effects. In fact, in this study the change in serum concentration of the white blood cells, IL-1β, IL-6, and TNF-α did not differ between groups after 12 weeks of treatment (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). Based on these results, how should we interpret the effects on CAP? The potential effects of BC on the liver would not be limited to antiinflammatory and antioxidant actions. It has been dimonstrated that BPF would prevent NAFLD via stimulation of lipophagy (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Mitochondria-associated membranes or mitochondria-endoplasmic reticulum contact sites have been implicated in the formation of autophagosomes (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>). Polyphenols exert a modulatory action on several mitochondrial processes, not necessarily inflammatory-related, such as biogenesis, membrane potential maintenance, electron transport chain, ATP synthesis and cell death triggering (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). Future studies aimed at discriminating the extent to which a cytoprotective effect of BC on the liver is a consequence of its direct modulatory action on the formerly-referred mitochondrial processes or whether it also critically arises from antiflammatory/antioxidant actions, are warranted.</p><p>We found a significant CAP score reduction especially in women (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). This finding is linked to a different redox state between genders (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>–<xref rid=\"B47\" ref-type=\"bibr\">47</xref>). These findings should be taken into account in future studies.</p><p>Despite from a statistical point of view not significant, placebo group had a higher AST concentration at baseline, than BC group (<italic>p</italic> = 0.06). However, these values refer to the whole enrolled population. We performed the subsequent ITT as well as OT analyses and this initial basal difference disappeared (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). Furthermore, it is unlikely, from a clinical point of view, that a similar slightly difference solely in AST translates in a significant CAP change.</p><p>BC is a safe product. The most frequent AE in BC was diarrhea but was reported only in three participants. We found 12 AEs for each groups, all of grade 1 (mild). Previously, Gliozzi et al. (<xref rid=\"B48\" ref-type=\"bibr\">48</xref>) prescribed 1,300 mg/day of BPF to 107 patients with NAFLD and metabolic syndrome for 120 days. Furthermore, in another study 32 subjects with mixed hyperlipidemia received 1,500 mg of bergamot combined with hypocaloric diet (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>). None of these studies, testing a high concentration of BPF, reported any type of adverse events. In a study carried out in individuals infected by hepatitis C virus, <italic>CyC</italic> extract normalized ALT and AST, as well as the level of bilirubin (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>). Furthermore, only a moderately interaction with human drug-metabolizing enzymes, such as CYP1A2, CYP2D6, CYP2E1, and CYP3A4, was observed (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>). In our tests of potential toxicity, no incidence of significant treatment-related clinical abnormalities was found with BC throughout the studies (see <xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplemental Material</xref>).</p><p>In this study, the pattern of liver enzymes change may provide insight into the differential effect of diet and BC on the liver. A minimal weight loss is always associated with ALT reduction. For every 5% weight loss, a four greater likelihood of ALT normalization was observed (<xref rid=\"B51\" ref-type=\"bibr\">51</xref>). This concept can explain the ALT reduction in both treatments. ALT reduction could suggest an enhanced hepatic lipid mobilization. In the liver, ALT is localized solely in the cellular cytoplasm, whereas AST is both cytosolic and mitochondrial (80% of total activity). In our study, AST reduced solely in BC group. Due to the peculiar intralobular distribution of AST (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>), our finding may suggest a deep, intralobular effect of BC.</p><p>However, in the present study BC, compared with placebo, was associated with a significant body weight reduction but CAP score reduction was explained by weight loss only in subjects aged <50 years (<xref rid=\"T4\" ref-type=\"table\">Table 4</xref>). Consequently, BC improved CAP score independent of weight loss solely in adult individuals. Cell culture, animal, and some human studies suggest that consumption of polyphenols (from foods or supplements) changes energy metabolism and may facilitate weight loss or prevent weight gain (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>). These potential effects may occur through a variety of mechanisms: stimulation of catabolic pathways in adipose tissue and liver, reduction of obesity-related inflammation, increase in the uptake of glucose by skeletal muscles and others (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>).</p><p>This study has some limitations. Of course, findings after a 12-week intervention cannot accurately reflect the long-term effects of BC in NAFLD patients. We found a low AEs rate (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>). Although the results of this study confirmed our assumption, further studies with higher dosages and longer intervention periods are needed to confirm our findings. Unfortunately we did not assess the oxidative stress markers. Another limitation is that CAP score might not be proper to evaluated steatosis stages in detail. Liver biopsy is still considered the gold standard for the diagnosis of fatty liver disease. However, because liver biopsy is an invasive procedure, in the context of the follow-up of individuals with NAFLD, it is not the most appropriate method (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>).</p><p>Thus, the first-line examination is abdominal ultrasound. However, ultrasound has low sensitivity for the detection of steatosis when it affects <20% of the liver or in individuals with severe obesity. Magnetic resonance spectroscopy is costly, therefore being used only for research purposes. TE by CAP measurement has been shown to detect steatosis with good sensitivity and specificity (<xref rid=\"B55\" ref-type=\"bibr\">55</xref>). Other prospective studies are mandatory before definitively recommending this technique for the prediction of steatosis grades. However, the hypothetical bias in our study would pertain to both treatments.</p><p>Another limitation of this study is related to the interpretation of the results from <italic>post-hoc</italic> analyses which should be conducted with caution as they serve only in hypothesis generation.</p><p>However, this study has important strengths. Most of the currently available data on nutraceuticals pertains to <italic>in vitro</italic> and animal studies, whose observations and conclusions do not always extrapolate directly to humans. Our study is a randomized controlled trial, thus, as “gold standard” in evidence-based medicine, may really detect clinically relevant conclusions on the effects of this new nutraceutical. Moreover, this type of study decreases patient and observer bias. Furthermore, knowledge translation into clinical practice may rapidly occurs.</p><p>An important strength of this study is related to the stability of the active ingredients in BC. In this study we provided the batch number, which suggest that, the manufacturing activities are in accordance with the international procedures (<xref rid=\"B56\" ref-type=\"bibr\">56</xref>, <xref rid=\"B57\" ref-type=\"bibr\">57</xref>). The batch used in this experiment has an expiration date (10–2020) and within this period the stability of the active ingredients is guaranteed. Furthermore, BC phytocomplex is analyzed for its integrity and stability every 2 months and, the flavonoid profile is intact overtime.</p></sec><sec sec-type=\"conclusions\" id=\"s5\"><title>Conclusion</title><p>The results of this study demonstrate that BC significantly reduces the CAP score by 15% but, from a subgroup analysis, we can confirm this finding only in the participants over 50 years. In this population with mild hypercholesterolemia, we did not observe any lipid-lowering and anti-inflammatory effect. Treatment with BC was well-tolerated and was not associated with an increased risk for adverse events. BC would constitute a promising complement to non-pharmacological measures that are commonly used to counteract the onset and progression of NAFLD, at least in individuals over 50 years. Future studies confirming our results and addressing whether long-term BC treatment can reduce the severity of NAFLD would be important for the future.</p></sec><sec sec-type=\"data-availability\" id=\"s6\"><title>Data Availability Statement</title><p>The datasets generated for this study are available on request to the corresponding author.</p></sec><sec id=\"s7\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by Mater Domini Azienda University Hospital. The patients/participants provided their written informed consent to participate in this study.</p></sec><sec id=\"s8\"><title>Author Contributions</title><p>YF and EM: responsibility for the integrity of the work and methodology. TM and AP: conceptualization and original draft preparation. SP and EM: data curation. TM: writing. AP, VMu, EB, SN, and VMo: review & editing. DF, IA, and EA: laboratory Investigation. MG and VMo: funding acquisition. All authors approved the final version and contributed to the manuscript preparation and interpretation of data.</p></sec><sec id=\"s9\"><title>Conflict of Interest</title><p>EB was employed by the company Plantexresearch srl, Milano. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>We thanks Herbal & Antioxidant SRL, Bianco, RC, Italy, for given us the nutraceutical. YF has served as consultant for the Nutramed Project and has received a grant.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This study was funded by Italian Ministry of University and Research, grant number: Nutramed Project, PON 03PE000_78_1.</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s10\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fendo.2020.00494/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fendo.2020.00494/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Data_Sheet_1.zip\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ekstedt</surname><given-names>M</given-names></name><name><surname>Franzén</surname><given-names>LE</given-names></name><name><surname>Mathiesen</surname><given-names>UL</given-names></name><name><surname>Thorelius</surname><given-names>L</given-names></name><name><surname>Holmqvist</surname><given-names>M</given-names></name><name><surname>Bodemar</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Long-term follow-up of patients with NAFLD and elevated liver enzymes</article-title>. <source>Hepatology.</source> (<year>2006</year>) <volume>44</volume>:<fpage>865</fpage>–<lpage>73</lpage>. <pub-id pub-id-type=\"doi\">10.1002/hep.21327</pub-id><pub-id pub-id-type=\"pmid\">17006923</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dyson</surname><given-names>J</given-names></name><name><surname>Jaques</surname><given-names>B</given-names></name><name><surname>Chattopadyhay</surname><given-names>D</given-names></name><name><surname>Lochan</surname><given-names>R</given-names></name><name><surname>Graham</surname><given-names>J</given-names></name><name><surname>Das</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Hepatocellular cancer: the impact of obesity, type 2 diabetes and a multidisciplinary team</article-title>. <source>J Hepatol.</source> (<year>2014</year>) <volume>60</volume>:<fpage>110</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jhep.2013.08.011</pub-id><pub-id pub-id-type=\"pmid\">23978719</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Younossi</surname><given-names>ZM</given-names></name><name><surname>Koenig</surname><given-names>AB</given-names></name><name><surname>Abdelatif</surname><given-names>D</given-names></name><name><surname>Fazel</surname><given-names>Y</given-names></name><name><surname>Henry</surname><given-names>L</given-names></name><name><surname>Wymer</surname><given-names>M</given-names></name></person-group>\n<article-title>Global epidemiology of non-alcoholic fatty liver disease-Meta-ana¬lytic assessment of prevalence, incidence and outcomes</article-title>. <source>Hepatology.</source> (<year>2016</year>) <volume>64</volume>:<fpage>73</fpage>–<lpage>84</lpage>. <pub-id pub-id-type=\"doi\">10.1002/hep.28431</pub-id><pub-id pub-id-type=\"pmid\">26707365</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manco</surname><given-names>M</given-names></name><name><surname>Marcellini</surname><given-names>M</given-names></name><name><surname>Giannone</surname><given-names>G</given-names></name><name><surname>Nobili</surname><given-names>V</given-names></name></person-group>. <article-title>Correlation of serum TNF-alpha levels and histologic liver injury scores in pediatric nonalcoholic fatty liver disease</article-title>. <source>Am J Clin Pathol.</source> (<year>2007</year>) <volume>127</volume>:<fpage>954</fpage>–<lpage>60</lpage>. <pub-id pub-id-type=\"doi\">10.1309/6VJ4DWGYDU0XYJ8Q</pub-id><pub-id pub-id-type=\"pmid\">17509993</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sumida</surname><given-names>Y</given-names></name><name><surname>Niki</surname><given-names>E</given-names></name><name><surname>Naito</surname><given-names>Y</given-names></name><name><surname>Yoshikawa</surname><given-names>T</given-names></name></person-group>. <article-title>Involvement of free radicals and oxidative stress in NAFLD/NASH</article-title>. <source>Free Radic Res.</source> (<year>2013</year>) <volume>47</volume>:<fpage>869</fpage>–<lpage>80</lpage>. <pub-id pub-id-type=\"doi\">10.3109/10715762.2013.837577</pub-id><pub-id pub-id-type=\"pmid\">24004441</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Czaja</surname><given-names>MJ</given-names></name></person-group>. <article-title>Liver injury in the setting of steatosis: crosstalk between adipokine and cytokine</article-title>. <source>Hepatology.</source> (<year>2004</year>) <volume>40</volume>:<fpage>19</fpage>–<lpage>22</lpage>. <pub-id pub-id-type=\"doi\">10.1002/hep.20328</pub-id><pub-id pub-id-type=\"pmid\">15239081</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mehta</surname><given-names>K</given-names></name><name><surname>Van Thiel</surname><given-names>DH</given-names></name><name><surname>Shah</surname><given-names>N</given-names></name><name><surname>Mobarhan</surname><given-names>S</given-names></name></person-group>. <article-title>Nonalcoholic fatty liver disease: pathogenesis and the role of antioxidants</article-title>. <source>Nutr Rev.</source> (<year>2002</year>) <volume>60</volume>:<fpage>289</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1301/002966402320387224</pub-id><pub-id pub-id-type=\"pmid\">12296456</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>K</given-names></name><name><surname>Czaja</surname><given-names>MJ</given-names></name></person-group>. <article-title>Regulation of lipid stores and metabolism by lipophagy</article-title>. <source>Cell Death Differ.</source> (<year>2013</year>) <volume>20</volume>:<fpage>3</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1038/cdd.2012.63</pub-id><pub-id pub-id-type=\"pmid\">22595754</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Koutoukidis</surname><given-names>DA</given-names></name><name><surname>Astbury</surname><given-names>NM</given-names></name><name><surname>Tudor</surname><given-names>KE</given-names></name><name><surname>Morris</surname><given-names>E</given-names></name><name><surname>Henry</surname><given-names>JA</given-names></name><name><surname>Noreik</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Association of weight loss interventions with changes in biomarkers of nonalcoholic fatty liver disease: a systematic review and meta-analysis</article-title>. <source>JAMA Intern Med.</source> (<year>2019</year>) <volume>179</volume>:<fpage>1303</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1001/jamainternmed.2019.2248</pub-id><pub-id pub-id-type=\"pmid\">31260026</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ferramosca</surname><given-names>A</given-names></name><name><surname>Di Giacomo</surname><given-names>M</given-names></name><name><surname>Zara</surname><given-names>V</given-names></name></person-group>. <article-title>Antioxidant dietary approach in treatment of fatty liver: new insights and updates</article-title>. <source>World J Gastroenterol.</source> (<year>2017</year>) <volume>23</volume>:<fpage>4146</fpage>–<lpage>57</lpage>. <pub-id pub-id-type=\"doi\">10.3748/wjg.v23.i23.4146</pub-id><pub-id pub-id-type=\"pmid\">28694655</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parafati</surname><given-names>M</given-names></name><name><surname>Lascala</surname><given-names>A</given-names></name><name><surname>Morittu</surname><given-names>VM</given-names></name><name><surname>Trimboli</surname><given-names>F</given-names></name><name><surname>Rizzuto</surname><given-names>A</given-names></name><name><surname>Brunelli</surname><given-names>E</given-names></name><etal/></person-group>. <article-title>Bergamot polyphenol fraction prevents nonalcoholic fatty liver disease via stimulation of lipophagy in cafeteria diet-induced rat model of metabolic syndrome</article-title>. <source>J Nutr Biochem.</source> (<year>2015</year>) <volume>26</volume>:<fpage>938</fpage>–<lpage>48</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jnutbio.2015.03.008</pub-id><pub-id pub-id-type=\"pmid\">26025327</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Siddiqi</surname><given-names>A</given-names></name><name><surname>Hasan</surname><given-names>SK</given-names></name><name><surname>Nafees</surname><given-names>S</given-names></name><name><surname>Rashid</surname><given-names>S</given-names></name><name><surname>Saidullah</surname><given-names>B</given-names></name><name><surname>Sultana</surname><given-names>S</given-names></name></person-group>. <article-title>Chemopreventive efficacy of hesperidin against chemically induced nephrotoxicity and renal carcinogenesis via amelioration of oxidative stress and modulation of multiple molecular pathways</article-title>. <source>Exp Mol Pathol.</source> (<year>2015</year>) <volume>99</volume>:<fpage>641</fpage>–<lpage>53</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.yexmp.2015.11.012</pub-id><pub-id pub-id-type=\"pmid\">26551080</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>El-Sisi</surname><given-names>AEE</given-names></name><name><surname>Sokar</surname><given-names>SS</given-names></name><name><surname>Shebl</surname><given-names>AM</given-names></name><name><surname>Mohamed</surname><given-names>DZ</given-names></name></person-group>. <article-title>Antifibrotic effect of diethylcarbamazine combined with hesperidin against ethanol induced liver fibrosis in rats</article-title>. <source>Biomed Pharmacother.</source> (<year>2017</year>) <volume>89</volume>:<fpage>1196</fpage>–<lpage>206</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.biopha.2017.03.013</pub-id><pub-id pub-id-type=\"pmid\">28320086</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mahmoud</surname><given-names>AM</given-names></name><name><surname>Mohammed</surname><given-names>HM</given-names></name><name><surname>Khadrawy</surname><given-names>SM</given-names></name><name><surname>Galaly</surname><given-names>SR</given-names></name></person-group>. <article-title>Hesperidin protects against chemically induced hepatocarcinogenesis via modulation of Nrf2/ARE/HO-1, PPARγ and TGF-β1/Smad3 signaling, and amelioration of oxidative stress and inflammation</article-title>. <source>Chem Biol Interact.</source> (<year>2017</year>) <volume>277</volume>:<fpage>146</fpage>–<lpage>58</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cbi.2017.09.015</pub-id><pub-id pub-id-type=\"pmid\">28935427</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Glasl</surname><given-names>S</given-names></name><name><surname>Tsendayush</surname><given-names>D</given-names></name><name><surname>Batchimeg</surname><given-names>U</given-names></name><name><surname>Holec</surname><given-names>N</given-names></name><name><surname>Wurm</surname><given-names>E</given-names></name><name><surname>Kletter</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>Choleretic effects of the mongolian medicinal plant saussurea amara in the isolated perfused rat liver</article-title>. <source>Planta Med.</source> (<year>2007</year>) <volume>73</volume>:<fpage>59</fpage>–<lpage>66</lpage>. <pub-id pub-id-type=\"doi\">10.1055/s-2006-957063</pub-id><pub-id pub-id-type=\"pmid\">17177133</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Elsebai</surname><given-names>MF</given-names></name><name><surname>Mocan</surname><given-names>A</given-names></name><name><surname>Atanasov</surname><given-names>AG</given-names></name></person-group>. <article-title>Cynaropicrin: A Comprehensive Research Review and Therapeutic Potential As an Anti-Hepatitis C Virus Agent</article-title>. <source>Front Pharmacol.</source> (<year>2016</year>) <volume>7</volume>:<fpage>472</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fphar.2016.00472</pub-id><pub-id pub-id-type=\"pmid\">28008316</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cho</surname><given-names>JY</given-names></name><name><surname>Park</surname><given-names>J</given-names></name><name><surname>Yoo</surname><given-names>ES</given-names></name><name><surname>Baik</surname><given-names>KU</given-names></name><name><surname>Jung</surname><given-names>JH</given-names></name><name><surname>Lee</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Inhibitory effect of sesquiterpene lactones from saussurea lappa on tumor necrosis factor-alpha production in murine macrophage-like cells</article-title>. <source>Planta Med.</source> (<year>1998</year>) <volume>64</volume>:<fpage>594</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1055/s-2006-957528</pub-id><pub-id pub-id-type=\"pmid\">9810262</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kukic</surname><given-names>J</given-names></name><name><surname>Popovic</surname><given-names>V</given-names></name><name><surname>Petrovic</surname><given-names>S</given-names></name><name><surname>Mucaji</surname><given-names>P</given-names></name><name><surname>Ciric</surname><given-names>A</given-names></name><name><surname>Stojkovic</surname><given-names>D</given-names></name><etal/></person-group>\n<article-title>Antioxidant and antimicrobial activity of cynara cardunculus extracts</article-title>. <source>Food Chem.</source> (<year>2008</year>) <volume>107</volume>:<fpage>861</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.foodchem.2007.09.005</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parker</surname><given-names>HM</given-names></name><name><surname>Cohn</surname><given-names>JS</given-names></name><name><surname>O'Connor</surname><given-names>HT</given-names></name><name><surname>Garg</surname><given-names>ML</given-names></name><name><surname>Caterson</surname><given-names>ID</given-names></name><name><surname>George</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Effect of fish oil supplementation on hepatic and visceral fat in overweight men: a randomized controlled trial</article-title>. <source>Nutrients.</source> (<year>2019</year>) <volume>11</volume>:<fpage>E475</fpage>. <pub-id pub-id-type=\"doi\">10.3390/nu11020475</pub-id><pub-id pub-id-type=\"pmid\">30813440</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mazza</surname><given-names>E</given-names></name><name><surname>Fava</surname><given-names>A</given-names></name><name><surname>Ferro</surname><given-names>Y</given-names></name><name><surname>Rotundo</surname><given-names>S</given-names></name><name><surname>Romeo</surname><given-names>S</given-names></name><name><surname>Bosco</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Effect of the replacement of dietary vegetable oils with a low dose of extravirgin olive oil in the mediterranean diet on cognitive functions in the elderly</article-title>. <source>J Transl Med.</source> (<year>2018</year>) <volume>16</volume>:<fpage>10</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s12967-018-1386-x</pub-id><pub-id pub-id-type=\"pmid\">29351799</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ferro</surname><given-names>Y</given-names></name><name><surname>Carè</surname><given-names>I</given-names></name><name><surname>Mazza</surname><given-names>E</given-names></name><name><surname>Provenzano</surname><given-names>F</given-names></name><name><surname>Colica</surname><given-names>C</given-names></name><name><surname>Torti</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>Protein and vitamin B6 intake are associated with liver steatosis assessed by transient elastography, especially in obese individuals</article-title>. <source>Clin Mol Hepatol.</source> (<year>2017</year>) <volume>23</volume>:<fpage>249</fpage>–<lpage>59</lpage>. <pub-id pub-id-type=\"doi\">10.3350/cmh.2017.0019</pub-id><pub-id pub-id-type=\"pmid\">28750503</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>de</surname><given-names>Lédinghen V</given-names></name><name><surname>Vergniol</surname><given-names>J</given-names></name><name><surname>Foucher</surname><given-names>J</given-names></name><name><surname>Merrouche</surname><given-names>W</given-names></name><name><surname>le Bail</surname><given-names>B</given-names></name></person-group>. <article-title>Non-invasive diagnosis of liver steatosis using controlled attenuation parameter (CAP) and transient elastography</article-title>. <source>Liver Int.</source> (<year>2012</year>) <volume>32</volume>:<fpage>911</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1478-3231.2012.02820.x</pub-id><pub-id pub-id-type=\"pmid\">22672642</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Myers</surname><given-names>RP</given-names></name><name><surname>Pollett</surname><given-names>A</given-names></name><name><surname>Kirsch</surname><given-names>R</given-names></name><name><surname>Pomier-Layrargues</surname><given-names>G</given-names></name><name><surname>Beaton</surname><given-names>M</given-names></name><name><surname>Levstik</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Controlled Attenuation Parameter (CAP): a noninvasive method for the detection of hepatic steatosis based on transient elastography</article-title>. <source>Liver Int.</source> (<year>2012</year>) <volume>32</volume>:<fpage>902</fpage>–<lpage>10</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1478-3231.2012.02781.x</pub-id><pub-id pub-id-type=\"pmid\">22435761</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Psaty</surname><given-names>BM</given-names></name><name><surname>Furberg</surname><given-names>CD</given-names></name><name><surname>Kuller</surname><given-names>LH</given-names></name><name><surname>Bild</surname><given-names>DE</given-names></name><name><surname>Rautaharju</surname><given-names>PM</given-names></name><name><surname>Polak</surname><given-names>JF</given-names></name><etal/></person-group>. <article-title>Traditional risk factors and subclinical disease measures as predictors of first myocardial infarction in older adults: the cardiovascular health study</article-title>. <source>Arch Intern Med.</source> (<year>1999</year>) <volume>59</volume>:<fpage>1339</fpage>–<lpage>47</lpage>. <pub-id pub-id-type=\"doi\">10.1001/archinte.159.12.1339</pub-id><pub-id pub-id-type=\"pmid\">10386510</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><collab>Centers for Disease Control and Prevention (CDC)</collab></person-group>. <article-title>Use of cessation methods among smokers aged 16–24 years—United States, 2003</article-title>. <source>MMWR Morb Mortal Wkly Rep</source>. (<year>2006</year>) <volume>55</volume>:<fpage>1351</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"pmid\">17183227</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><collab>Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults</collab></person-group>\n<article-title>Executive summary of the third report of the National Cholesterol Education Program (NCEP) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel III)</article-title>. <source>JAMA.</source> (<year>2001</year>) <volume>285</volume>:<fpage>2486</fpage>–<lpage>97</lpage>. <pub-id pub-id-type=\"doi\">10.1001/jama.285.19.2486</pub-id><pub-id pub-id-type=\"pmid\">11368702</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nishida</surname><given-names>C</given-names></name><name><surname>Ko</surname><given-names>GT</given-names></name><name><surname>Kumanyika</surname><given-names>S</given-names></name></person-group>. <article-title>Body fat distribution and noncommunicable diseases in populations: overview of the 2008 WHO expert consultation on waist circumference and waist-hip ratio</article-title>. <source>Eur J Clin Nutr.</source> (<year>2010</year>) <volume>64</volume>:<fpage>2</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1038/ejcn.2009.139</pub-id><pub-id pub-id-type=\"pmid\">19935820</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Montalcini</surname><given-names>T</given-names></name><name><surname>Gorgone</surname><given-names>G</given-names></name><name><surname>Federico</surname><given-names>D</given-names></name><name><surname>Ceravolo</surname><given-names>R</given-names></name><name><surname>Emanuele</surname><given-names>V</given-names></name><name><surname>Sesti</surname><given-names>G</given-names></name><etal/></person-group>. <article-title>Association of LDL cholesterol with carotid atherosclerosis in menopausal women affected by the metabolic syndrome</article-title>. <source>Nutr Metab Cardiovasc Dis.</source> (<year>2005</year>) <volume>15</volume>:<fpage>368</fpage>–<lpage>72</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.numecd.2004.10.003</pub-id><pub-id pub-id-type=\"pmid\">16216723</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Matthews</surname><given-names>DR</given-names></name><name><surname>Hosker</surname><given-names>JP</given-names></name><name><surname>Rudenski</surname><given-names>AS</given-names></name><name><surname>Naylor</surname><given-names>BA</given-names></name><name><surname>Treacher</surname><given-names>DF</given-names></name><name><surname>Turner</surname><given-names>RC</given-names></name></person-group>. <article-title>Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man</article-title>. <source>Diabetologia.</source> (<year>1985</year>) <volume>28</volume>:<fpage>412</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1007/BF00280883</pub-id><pub-id pub-id-type=\"pmid\">3899825</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ferraioli</surname><given-names>G</given-names></name><name><surname>Tinelli</surname><given-names>C</given-names></name><name><surname>Lissandrin</surname><given-names>R</given-names></name><name><surname>Zicchetti</surname><given-names>M</given-names></name><name><surname>Faliva</surname><given-names>M</given-names></name><name><surname>Perna</surname><given-names>S</given-names></name><etal/></person-group>\n<article-title>Correlation of the controlled attenuation parameter with indices of liver steatosis in overweight or obese individuals: a pilot study</article-title>. <source>Eur J Gastroenterol Hepatol.</source> (<year>2015</year>) <volume>27</volume>:<fpage>305</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.1097/MEG.0000000000000287</pub-id><pub-id pub-id-type=\"pmid\">25629575</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cheraghpour</surname><given-names>M</given-names></name><name><surname>Imani</surname><given-names>H</given-names></name><name><surname>Ommi</surname><given-names>S</given-names></name><name><surname>Alavian</surname><given-names>SM</given-names></name><name><surname>Karimi-Shahrbabak</surname><given-names>E</given-names></name><name><surname>Hedayati</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Hesperidin improves hepatic steatosis, hepatic enzymes, and metabolic and inflammatory parameters in patients with nonalcoholic fatty liver disease: a randomized, placebo-controlled, double-blind clinical trial</article-title>. <source>Phytother Res.</source> (<year>2019</year>) <volume>33</volume>:<fpage>2118</fpage>–<lpage>25</lpage>. <pub-id pub-id-type=\"doi\">10.1002/ptr.6406</pub-id><pub-id pub-id-type=\"pmid\">31264313</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhu</surname><given-names>W</given-names></name><name><surname>Chen</surname><given-names>S</given-names></name><name><surname>Chen</surname><given-names>R</given-names></name><name><surname>Peng</surname><given-names>Z</given-names></name><name><surname>Wan</surname><given-names>J</given-names></name><name><surname>Wu</surname><given-names>B</given-names></name></person-group>. <article-title>Taurine and tea polyphenols combination ameliorate nonalcoholic steatohepatitis in rats</article-title>. <source>BMC Complement Altern Med.</source> (<year>2017</year>) <volume>17</volume>:<fpage>455</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s12906-017-1961-3</pub-id><pub-id pub-id-type=\"pmid\">28886741</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Parafati</surname><given-names>M</given-names></name><name><surname>Lascala</surname><given-names>A</given-names></name><name><surname>La Russa</surname><given-names>D</given-names></name><name><surname>Mignogna</surname><given-names>C</given-names></name><name><surname>Trimboli</surname><given-names>F</given-names></name><name><surname>Morittu</surname><given-names>VM</given-names></name><etal/></person-group>. <article-title>Bergamot polyphenols boost therapeutic effects of the diet on non-alcoholic steatohepatitis (NASH) induced by “junk food”: evidence for anti-inflammatory activity</article-title>. <source>Nutrients.</source> (<year>2018</year>) <volume>1</volume>:<fpage>10</fpage>. <pub-id pub-id-type=\"doi\">10.3390/nu10111604</pub-id><pub-id pub-id-type=\"pmid\">30388763</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Serrano</surname><given-names>JC</given-names></name><name><surname>Cassanye</surname><given-names>A</given-names></name><name><surname>Martín-Gari</surname><given-names>M</given-names></name><name><surname>Granado-Serrano</surname><given-names>AB</given-names></name><name><surname>Portero-Otín</surname><given-names>M</given-names></name></person-group>. <article-title>Effect of dietary bioactive compounds on mitochondrial and metabolic flexibility</article-title>. <source>Diseases.</source> (<year>2016</year>) <volume>4</volume>:<fpage>E14</fpage>. <pub-id pub-id-type=\"doi\">10.3390/diseases4010014</pub-id><pub-id pub-id-type=\"pmid\">28933394</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Olayinka</surname><given-names>ET</given-names></name><name><surname>Ore</surname><given-names>A</given-names></name><name><surname>Adeyemo</surname><given-names>OA</given-names></name><name><surname>Ola</surname><given-names>OS</given-names></name><name><surname>Olotu</surname><given-names>OO</given-names></name><name><surname>Echebiri</surname><given-names>RC</given-names></name></person-group>. <article-title>Quercetin, a flavonoid antioxidant, ameliorated procarbazine-induced oxidative damage to murine tissues</article-title>. <source>Antioxidants.</source> (<year>2015</year>) <volume>4</volume>:<fpage>304</fpage>–<lpage>21</lpage>. <pub-id pub-id-type=\"doi\">10.3390/antiox4020304</pub-id><pub-id pub-id-type=\"pmid\">26783707</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sandoval-Acuña</surname><given-names>C</given-names></name><name><surname>Ferreira</surname><given-names>J</given-names></name><name><surname>Speisky</surname><given-names>H</given-names></name></person-group>. <article-title>Polyphenols and mitochondria: anupdate on their increasingly emerging ROS-scavenging independent actions</article-title>. <source>Arch Biochem Biophys.</source> (<year>2014</year>) <volume>559</volume>:<fpage>75</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.abb.2014.05.017</pub-id><pub-id pub-id-type=\"pmid\">24875147</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hostetler</surname><given-names>GL</given-names></name><name><surname>Ralston</surname><given-names>RA</given-names></name><name><surname>Schwartz</surname><given-names>SJ</given-names></name></person-group>. <article-title>Flavones: food sources, bioavailability, metabolism, and bioactivity</article-title>. <source>Adv Nutr</source>. (<year>2017</year>) <volume>8</volume>:<fpage>423</fpage>–<lpage>35</lpage>. <pub-id pub-id-type=\"doi\">10.3945/an.116.012948</pub-id><pub-id pub-id-type=\"pmid\">28507008</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Barreca</surname><given-names>D</given-names></name><name><surname>Mandalari</surname><given-names>G</given-names></name><name><surname>Calderaro</surname><given-names>A</given-names></name><name><surname>Smeriglio</surname><given-names>A</given-names></name><name><surname>Trombetta</surname><given-names>D</given-names></name><name><surname>Felice</surname><given-names>MR</given-names></name><etal/></person-group>. <article-title>Citrus Flavones: an update on sources, biological functions, and health promoting properties</article-title>. <source>Plants</source>. (<year>2020</year>) <volume>9</volume>:<fpage>288</fpage>. <pub-id pub-id-type=\"doi\">10.3390/plants9030288</pub-id><pub-id pub-id-type=\"pmid\">32110931</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sun</surname><given-names>L</given-names></name><name><surname>Tao</surname><given-names>S</given-names></name><name><surname>Zhang</surname><given-names>S</given-names></name></person-group>. <article-title>Characterization and quantification of polyphenols and triterpenoids in thinned young fruits of ten pear varieties by UPLC-Q TRAP-MS/MS</article-title>. <source>Molecules</source>. (<year>2019</year>) <volume>24</volume>:<fpage>159</fpage>. <pub-id pub-id-type=\"doi\">10.3390/molecules24010159</pub-id><pub-id pub-id-type=\"pmid\">30609827</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Curran-Everett</surname><given-names>D</given-names></name><name><surname>Milgrom</surname><given-names>H</given-names></name></person-group>. <article-title><italic>Post-hoc</italic> data analysis: benefits and limitations</article-title>. <source>Curr Opin Allergy Clin Immunol.</source> (<year>2013</year>) <volume>13</volume>:<fpage>223</fpage>–<lpage>4</lpage>. <pub-id pub-id-type=\"doi\">10.1097/ACI.0b013e3283609831</pub-id><pub-id pub-id-type=\"pmid\">23571411</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mollace</surname><given-names>V</given-names></name><name><surname>Sacco</surname><given-names>I</given-names></name><name><surname>Janda</surname><given-names>E</given-names></name><name><surname>Malara</surname><given-names>C</given-names></name><name><surname>Ventrice</surname><given-names>D</given-names></name><name><surname>Colica</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>Hypolipemic and hypoglycaemic activity of bergamot polyphenols: from animal models to human studies</article-title>. <source>Fitoterapia.</source> (<year>2011</year>) <volume>82</volume>:<fpage>309</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.fitote.2010.10.014</pub-id><pub-id pub-id-type=\"pmid\">21056640</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Catapano</surname><given-names>AL</given-names></name><name><surname>Pirillo</surname><given-names>A</given-names></name><name><surname>Norata</surname><given-names>GD</given-names></name></person-group>. <article-title>Vascular inflammation and low-density lipoproteins: is cholesterol the link? A lesson from the clinical trials</article-title>. <source>Br J Pharmacol.</source> (<year>2017</year>) <volume>174</volume>:<fpage>3973</fpage>–<lpage>85</lpage>. <pub-id pub-id-type=\"doi\">10.1111/bph.13805</pub-id><pub-id pub-id-type=\"pmid\">28369752</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hamasaki</surname><given-names>M</given-names></name><name><surname>Furuta</surname><given-names>N</given-names></name><name><surname>Matsuda</surname><given-names>A</given-names></name><name><surname>Nezu</surname><given-names>A</given-names></name><name><surname>Yamamoto</surname><given-names>A</given-names></name><name><surname>Fujita</surname><given-names>N</given-names></name><etal/></person-group>. <article-title>Autophagosomes form at ER-mitochondria contact sites</article-title>. <source>Nature</source>. (<year>2013</year>) <volume>495</volume>:<fpage>389</fpage>–<lpage>93</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature11910</pub-id><pub-id pub-id-type=\"pmid\">23455425</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Borrás</surname><given-names>C</given-names></name><name><surname>Sastre</surname><given-names>J</given-names></name><name><surname>García-Sala</surname><given-names>D</given-names></name><name><surname>Lloret</surname><given-names>A</given-names></name><name><surname>Pallardó</surname><given-names>FV</given-names></name><name><surname>Viña</surname><given-names>J</given-names></name></person-group>. <article-title>Mitochondria from females exhibit higher antioxidant gene expression and lower oxidative damage than males</article-title>. <source>Free Radic Biol Med.</source> (<year>2003</year>) <volume>34</volume>:<fpage>546</fpage>–<lpage>52</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0891-5849(02)01356-4</pub-id><pub-id pub-id-type=\"pmid\">12614843</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ide</surname><given-names>T</given-names></name><name><surname>Tsutsui</surname><given-names>H</given-names></name><name><surname>Ohashi</surname><given-names>N</given-names></name><name><surname>Hayashidani</surname><given-names>S</given-names></name><name><surname>Suematsu</surname><given-names>N</given-names></name><name><surname>Tsuchihashi</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Greater oxidative stress in healthy young men compared with premenopausal women</article-title>. <source>Arterioscler Thromb Vasc Biol.</source> (<year>2002</year>) <volume>22</volume>:<fpage>438</fpage>–<lpage>42</lpage>. <pub-id pub-id-type=\"doi\">10.1161/hq0302.104515</pub-id><pub-id pub-id-type=\"pmid\">11884287</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Block</surname><given-names>G</given-names></name><name><surname>Dietrich</surname><given-names>M</given-names></name><name><surname>Norkus</surname><given-names>EP</given-names></name><name><surname>Morrow</surname><given-names>JD</given-names></name><name><surname>Hudes</surname><given-names>M</given-names></name><name><surname>Caan</surname><given-names>B</given-names></name><etal/></person-group>. <article-title>Factors associated with oxidative stress in human populations</article-title>. <source>Am J Clin Nutr.</source> (<year>2002</year>) <volume>156</volume>:<fpage>274</fpage>–<lpage>85</lpage>. <pub-id pub-id-type=\"doi\">10.1093/aje/kwf029</pub-id><pub-id pub-id-type=\"pmid\">12142263</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Keaney</surname><given-names>JF</given-names><suffix>Jr</suffix></name><name><surname>Larson</surname><given-names>MG</given-names></name><name><surname>Vasan</surname><given-names>RS</given-names></name><name><surname>Wilson</surname><given-names>PW</given-names></name><name><surname>Lipinska</surname><given-names>I</given-names></name><name><surname>Corey</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Obesity and systemic oxidative stress: clinical correlates of oxidative stress in the framingham study</article-title>. <source>Arterioscler Thromb Vasc Biol.</source> (<year>2003</year>) <volume>23</volume>:<fpage>434</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1161/01.ATV.0000058402.34138.11</pub-id><pub-id pub-id-type=\"pmid\">12615693</pub-id></mixed-citation></ref><ref id=\"B48\"><label>48.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gliozzi</surname><given-names>M</given-names></name><name><surname>Carresi</surname><given-names>C</given-names></name><name><surname>Musolino</surname><given-names>V</given-names></name><name><surname>Palma</surname><given-names>E</given-names></name><name><surname>Muscoli</surname><given-names>C</given-names></name><name><surname>Vitale</surname><given-names>C</given-names></name><etal/></person-group>\n<article-title>The effect of bergamot-derived polyphenolic fraction on LDL small dense particles and non alcoholic fatty liver disease in patients with metabolic syndrome</article-title>. <source>Adv Biol Chem.</source> (<year>2014</year>) <volume>4</volume>:<fpage>129</fpage>\n<pub-id pub-id-type=\"doi\">10.4236/abc.2014.42017</pub-id></mixed-citation></ref><ref id=\"B49\"><label>49.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Elsebai</surname><given-names>MF</given-names></name><name><surname>Abass</surname><given-names>K</given-names></name><name><surname>Hakkola</surname><given-names>J</given-names></name><name><surname>Atawia</surname><given-names>AR</given-names></name><name><surname>Farag</surname><given-names>MA</given-names></name></person-group>. <article-title>The wild Egyptian artichoke as a promising functional food for the treatment of hepatitis C virus as revealed via UPLC-MS and clinical trials</article-title>. <source>Food Funct.</source> (<year>2016</year>) <volume>7</volume>:<fpage>3006</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.1039/C6FO00656F</pub-id><pub-id pub-id-type=\"pmid\">27296047</pub-id></mixed-citation></ref><ref id=\"B50\"><label>50.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Di Lorenzo</surname><given-names>C</given-names></name><name><surname>Ceschi</surname><given-names>A</given-names></name><name><surname>Kupferschmidt</surname><given-names>H</given-names></name><name><surname>Lüde</surname><given-names>S</given-names></name><name><surname>De Souza Nascimento</surname><given-names>E</given-names></name><name><surname>Dos Santos</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Adverse effects of plant food supplements and botanical preparations: a systematic review with critical evaluation of causality</article-title>. <source>Br J Clin Pharmacol.</source> (<year>2015</year>) <volume>79</volume>:<fpage>578</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.1111/bcp.12519</pub-id><pub-id pub-id-type=\"pmid\">25251944</pub-id></mixed-citation></ref><ref id=\"B51\"><label>51.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Suzuki</surname><given-names>A</given-names></name><name><surname>Lindor</surname><given-names>K</given-names></name><name><surname>St Saver</surname><given-names>J</given-names></name><name><surname>Lymp</surname><given-names>J</given-names></name><name><surname>Mendes</surname><given-names>F</given-names></name><name><surname>Muto</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Effect of changes on body weight and lifestyle in nonalcoholic fatty liver disease</article-title>. <source>J Hepatol.</source> (<year>2005</year>) <volume>43</volume>:<fpage>1060</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jhep.2005.06.008</pub-id><pub-id pub-id-type=\"pmid\">16140415</pub-id></mixed-citation></ref><ref id=\"B52\"><label>52.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Seeto</surname><given-names>RK</given-names></name><name><surname>Fenn</surname><given-names>B</given-names></name><name><surname>Rockey</surname><given-names>DC</given-names></name></person-group>. <article-title>Ischemic hepatitis: clinical presentation and pathogenesis</article-title>. <source>Am J Med.</source> (<year>2000</year>) <volume>109</volume>:<fpage>109</fpage>–<lpage>13</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0002-9343(00)00461-7</pub-id><pub-id pub-id-type=\"pmid\">10967151</pub-id></mixed-citation></ref><ref id=\"B53\"><label>53.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Meydani</surname><given-names>M</given-names></name><name><surname>Hasan</surname><given-names>ST</given-names></name></person-group>. <article-title>Dietary polyphenols and obesity</article-title>. <source>Nutrients.</source> (<year>2010</year>) <volume>2</volume>:<fpage>737</fpage>–<lpage>51</lpage>. <pub-id pub-id-type=\"doi\">10.3390/nu2070737</pub-id><pub-id pub-id-type=\"pmid\">22254051</pub-id></mixed-citation></ref><ref id=\"B54\"><label>54.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cosgrove</surname><given-names>D</given-names></name><name><surname>Piscaglia</surname><given-names>F</given-names></name><name><surname>Bamber</surname><given-names>J</given-names></name><name><surname>Bojunga</surname><given-names>J</given-names></name><name><surname>Correas</surname><given-names>JM</given-names></name><name><surname>Gilja</surname><given-names>OH</given-names></name><etal/></person-group>. <article-title>EFSUMB guidelines and recommendations on the clinical use of ultrasound elastography. Part 2: clinical applications</article-title>. <source>Ultraschall Med.</source> (<year>2013</year>) <volume>34</volume>:<fpage>238</fpage>–<lpage>53</lpage>. <pub-id pub-id-type=\"doi\">10.1055/s-0033-1335375</pub-id><pub-id pub-id-type=\"pmid\">23605169</pub-id></mixed-citation></ref><ref id=\"B55\"><label>55.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shi</surname><given-names>KQ</given-names></name><name><surname>Tang</surname><given-names>JZ</given-names></name><name><surname>Zhu</surname><given-names>XL</given-names></name><name><surname>Ying</surname><given-names>L</given-names></name><name><surname>Li</surname><given-names>DW</given-names></name><name><surname>Gao</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Controlled attenuation parameter for the detection of steatosis severity in chronic liver disease: a meta-analysis of diagnostic accuracy</article-title>. <source>J Gastroenterol Hepatol.</source> (<year>2014</year>) <volume>29</volume>:<fpage>1149</fpage>–<lpage>58</lpage>. <pub-id pub-id-type=\"doi\">10.1111/jgh.12519</pub-id><pub-id pub-id-type=\"pmid\">24476011</pub-id></mixed-citation></ref><ref id=\"B56\"><label>56.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><collab>WHO Technical Reports Series</collab></person-group> (<year>1996</year>), n. 863. Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://extranet.who.int/prequal/content/who-technical-report-series\">https://extranet.who.int/prequal/content/who-technical-report-series</ext-link></mixed-citation></ref><ref id=\"B57\"><label>57.</label><mixed-citation publication-type=\"webpage\"><person-group person-group-type=\"author\"><collab>WHO Technical Reports Series</collab></person-group> (<year>2016</year>), n.999. Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://extranet.who.int/prequal/content/who-technical-report-series\">https://extranet.who.int/prequal/content/who-technical-report-series</ext-link></mixed-citation></ref></ref-list><glossary><def-list><title>Abbreviations</title><def-item><term>NAFLD</term><def><p>Non-alcoholic fatty liver disease</p></def></def-item><def-item><term>BPF</term><def><p>polyphenolic fraction</p></def></def-item><def-item><term>BF</term><def><p>bergamot and wild cardoon</p></def></def-item><def-item><term>TE</term><def><p>transient elastography</p></def></def-item><def-item><term>BMI</term><def><p>body mass index</p></def></def-item><def-item><term>WC</term><def><p>waist circumference</p></def></def-item><def-item><term>HC</term><def><p>hip circumference</p></def></def-item><def-item><term>FM</term><def><p>fat mass</p></def></def-item><def-item><term>SBP</term><def><p>systolic blood pressure</p></def></def-item><def-item><term>DBP</term><def><p>diastolic blood pressure</p></def></def-item><def-item><term>CAP</term><def><p>controlled attenuation parameter</p></def></def-item><def-item><term>IQR</term><def><p>interquartile range</p></def></def-item><def-item><term>HOMA-IR</term><def><p>homeostatic model assessment of insulin resistance</p></def></def-item><def-item><term>TC</term><def><p>total cholesterol</p></def></def-item><def-item><term>TG</term><def><p>triglycerides</p></def></def-item><def-item><term>HDL-C</term><def><p>high density lipoprotein cholesterol</p></def></def-item><def-item><term>LDL-C</term><def><p>low density lipoprotein cholesterol</p></def></def-item><def-item><term>AST</term><def><p>aspartate aminotransferase</p></def></def-item><def-item><term>ALT</term><def><p>alanine aminotransferase</p></def></def-item><def-item><term>γGT</term><def><p>gamma glutamyltransferase.</p></def></def-item></def-list></glossary></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Pediatr</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Pediatr</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Pediatr.</journal-id><journal-title-group><journal-title>Frontiers in Pediatrics</journal-title></journal-title-group><issn pub-type=\"epub\">2296-2360</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850561</article-id><article-id pub-id-type=\"pmc\">PMC7431623</article-id><article-id pub-id-type=\"doi\">10.3389/fped.2020.00463</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Pediatrics</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Short-Term Consequences of Pediatric Anti-cancer Treatment Regarding Blood Pressure, Motor Performance, Physical Activity and Reintegration Into Sports Structures</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Keiser</surname><given-names>Tina</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Gaser</surname><given-names>Dominik</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/971855/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Peters</surname><given-names>Christiane</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/965992/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Oberhoffer-Fritz</surname><given-names>Renate</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/641081/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Kesting</surname><given-names>Sabine</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/636506/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>von Luettichau</surname><given-names>Irene</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/900711/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Sports Medicine and Exercise, Justus-Liebig University Gießen</institution>, <addr-line>Gießen</addr-line>, <country>Germany</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Sport and Health Sciences, Institute of Preventive Pediatrics, Technical University of Munich</institution>, <addr-line>Munich</addr-line>, <country>Germany</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Pediatrics and Children's Cancer Research Center, Kinderklinik München Schwabing, TUM School of Medicine, Technical University of Munich</institution>, <addr-line>Munich</addr-line>, <country>Germany</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Kirsten K. Ness, St. Jude Children's Research Hospital, United States</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Seth E. Karol, St. Jude Children's Research Hospital, United States; Jacques Grill, Institut Gustave Roussy, France</p></fn><corresp id=\"c001\">*Correspondence: Sabine Kesting <email>sabine.kesting@tum.de</email></corresp><corresp id=\"c002\">Irene von Luettichau <email>Irene.Teichert-vonLuettichau@mri.tum.de</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Pediatric Oncology, a section of the journal Frontiers in Pediatrics</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†These authors share senior authorship</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>8</volume><elocation-id>463</elocation-id><history><date date-type=\"received\"><day>29</day><month>1</month><year>2020</year></date><date date-type=\"accepted\"><day>02</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Keiser, Gaser, Peters, Oberhoffer-Fritz, Kesting and von Luettichau.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Keiser, Gaser, Peters, Oberhoffer-Fritz, Kesting and von Luettichau</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p><bold>Background:</bold> Cardiovascular diseases in childhood cancer survivors are known late sequelae following treatment. Arterial stiffness, pulse wave velocity (PWV) and central systolic blood pressure (cSBP) are potential predictors to assess the status of cardiovascular health. Frequent inpatient stays and reduced physical activity (PA) during treatment can lead to noticeable impairments regarding motor skills and physical performance. The present study examined parameters of cardiovascular health, motor performance and the status of integration into sports structures shortly after cessation of treatment.</p><p><bold>Methods:</bold> A cross-sectional, monocentric study was conducted from April to June 2019. Participants (6–18 yrs, mixed cancer entities) during maintenance therapy and follow-up care were recruited. Peripheral and central systolic/diastolic blood pressure (pSBP, pDBP, cSBP) and PWV were assessed using the Mobil-O-Graph®. The MOON test (MOtor performance in pediatric ONcology) was used to scale motor performance. PA levels and status of integration into sports structures were assessed with a questionnaire referring to the KiGGS study. All measured data were compared to published reference values.</p><p><bold>Results:</bold> Forty participants (11.3 ± 3.8 years, 50% female) were recruited 1.6 ± 1.8 years post-treatment. PSBP (z-score: 0.87 ± 0.67, <italic>p</italic> = 0.003), pDBP (0.83 ± 1.94, <italic>p</italic> = 0.033) and cSBP (≥8 years: 0.60 ± 1.29, <italic>p</italic> = 0.011) were significantly increased compared to reference values. PWV was also elevated, but not significantly. Motor performance was reduced in almost all motor abilities. Thirty-six percent of the examined group did not participate in physical education at school to the full extent. Only 17% reported 1 hour of daily moderate-to-vigorous PA as recommended for children and adolescents by the World Health Organization. Half of the participants were active sports club members before treatment, but one third did not resume their former membership.</p><p><bold>Conclusion:</bold> Increased cardiovascular parameters and impaired motor performance shortly after cessation of treatment, physical inactivity, and low rates of integration into regular sports programs highlight the support needed. Young cancer patients should receive early support in coping with physical limitations preferably soon after diagnosis. Motor deficits could be reduced by applying targeted interventions. Furthermore, a regular sports therapy program during in- and outpatient care could increase engagement in PA to possibly counteract risk factors and improve cardiovascular health.</p></abstract><kwd-group><kwd>childhood cancer</kwd><kwd>cardiovascular health</kwd><kwd>motor performance</kwd><kwd>physical activity</kwd><kwd>sports</kwd><kwd>reintegration</kwd><kwd>blood pressure</kwd><kwd>arterial stiffness</kwd></kwd-group><counts><fig-count count=\"4\"/><table-count count=\"5\"/><equation-count count=\"0\"/><ref-count count=\"54\"/><page-count count=\"13\"/><word-count count=\"10281\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Extensive research and optimized treatment regimens resulted in an increase of the 5-year survival rate to 85% in the USA (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>) and of the 15-year survival rate to 82% for patients under the age of 15 in Germany (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>). However, childhood cancer is a rare disease. It contributes only around 1% to all malignant diseases in developed countries (<xref rid=\"B3\" ref-type=\"bibr\">3</xref>). Worldwide, 215,000 children under the age of 15 and 85,000 adolescents aged between 15 and 19 are diagnosed with cancer every year (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). As a consequence of the success in the treatment of childhood cancer, the importance of survival quality and prevention of late sequelae have received more attention during the last years.</p><p>Known negative long-term consequences of intensive treatment for childhood and adolescent cancer patients often include adverse effects on the cardiovascular system (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Cardiovascular diseases are the most frequently reported causes of death in childhood cancer survivors following secondary tumors (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). Arterial stiffness, pulse wave velocity (PWV) and central systolic blood pressure (cSBP) are potential predictors for cardiovascular diseases frequently investigated in medical research to evaluate the status of a patient's cardiovascular health (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B8\" ref-type=\"bibr\">8</xref>).</p><p>PWV describes the velocity of the pressure wave in the aorta, which spreads from the left ventricle through the arterial vascular system during systole (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>). Non-invasive investigation of the PWV, via ultrasound or oscillometric methods, provides information on the elasticity of the vascular system and enables early recognition of damages in the vessels. Thus, in order to detect potential structural modifications in the vascular system and indicators for arterial stiffness at an early stage, this subclinical parameter should be surveyed continually. Previous data indicate a positive correlation of PWV with arterial vascular stiffness (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>, <xref rid=\"B10\" ref-type=\"bibr\">10</xref>). Moreover, elevated PWV reflecting subclinical vascular damage was shown in pediatric patients after hematopoietic stem cell transplantation (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). On the contrary, another study investigated elevated blood pressure levels, but no statistically significant variation for PWV in pediatric cancer survivors compared to healthy children and adolescents (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>).</p><p>In addition to the above-mentioned late sequelae, several problems already arise during treatment and often persist throughout survivorship. For instance, frequent long-term inpatient stays and reduced physical activity during treatment can lead to noticeably reduced physical performance of childhood cancer survivors and reintegration into sports structures might be affected throughout rehabilitation process (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>).</p><p>In healthy populations, reduced physical activity leads to negative consequences for cardiovascular health (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). Additionally, the necessary use of anthracyclines in almost 60% of applied therapy regimens in childhood cancer increases the risk of cardiovascular morbidity and mortality eight-fold compared to age-matched patients not receiving anthracyclines, indicating the importance of reducing such long-term consequences (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>).</p><p>Due to a poor state of health and impaired immune function, sports options such as physical education at school, engagement in sports clubs or recreational sports are no longer feasible during therapy. Consequently, reintegration after cessation of treatment is associated with even more barriers due to disease- and treatment-related impairments (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>). Circumstances of anti-cancer treatment can lead to inactivity, resulting in deficits of fine and gross motor skills, reduced muscle strength, and poor physical fitness following treatment (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B20\" ref-type=\"bibr\">20</xref>). Especially, motor performance in pediatric bone tumor patients often remains reduced until at least 2 years after cessation of treatment (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). Impairments of physical performance have been shown to persist throughout survivorship (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>), which may complicate the survivors' reintegration into both social and sports structures as well as the development of a long-term active lifestyle (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>, <xref rid=\"B24\" ref-type=\"bibr\">24</xref>).</p><p>According to a questionnaire-based study, childhood cancer survivors' reintegration rate into physical education at school is very low, especially after treatment for bone tumors (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). The lack of comprehensive offers of physical activity promotion and motor development might exacerbate motor impairments and problems of reintegration into sports structures (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>).</p><p>Von Korn et al. (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>) examined motor performance using the Fitnessgram® as well as peripheral blood pressure, central blood pressure and PWV using the Mobil-O-Graph® in children after treatment for childhood cancer (<italic>n</italic> = 92, aged 12.5 ± 4.2 years, 3.6 ± 2.8 years post-diagnosis). Their results show reduced motor performance of childhood cancer survivors compared to reference values of healthy children. However, no correlation could be drawn regarding cardiovascular parameters and motor performance (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>).</p><p>The present cross-sectional study aimed at investigating various parameters of cardiovascular health, motor performance, and status of physical activity in children and adolescents shortly after cessation of anti-cancer treatment or during ongoing oral maintenance therapy. The collection of such data is of considerable importance for the early detection of health implications related to both disease and treatment. Moreover, the findings will help to support the development of preventive strategies regarding the health of children and adolescents treated for cancer. Appropriate strategies during (primary and secondary prevention) and following cancer treatment (tertiary prevention) need improvements.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Design</title><p>The cross-sectional, monocentric study was performed over a period of 3 months (April–June 2019) at our institution. The assessment of cardiovascular parameters using the Mobil-O-Graph® was followed by the MOON test (MOtor performance in pediatric ONcology) to evaluate motor performance. Finally, the participants completed a standardized questionnaire referring to the KiGGS study (German Health Interview and Examination Survey for Children and Adolescents) to collect data regarding their current level of physical activity and status of integration into sports structures. The Ethics Committee of the School of Medicine of the Technical University of Munich approved the study (project number 148/19 S-SR). Participation was voluntary and informed written consent was signed by each participant, as well as by his or her legal guardian. All data was collected encoded (pseudonym) and in accordance with privacy policy standards.</p></sec><sec><title>Participants</title><p>Prior to addressing the participants, all eligible children and adolescents were screened using the electronic patient record (SAP® ERP). Patients were recruited during routine follow-up visits. The following inclusion criteria were applied: (1) children and adolescents during maintenance therapy and follow-up care of a pediatric oncological disease and (2) currently aged between 6 and 18 years. No restriction was applied regarding the period post-treatment. Exclusion criteria were: (1) medical contraindications such as fever, acute infection, orthopedic restrictions and mental retardation, (2) insufficient knowledge of the German language, and (3) absence of written informed consent. The attending physician confirmed participation for all recruited children and adolescents. Following these inclusion criteria, 81 children and adolescents were initially found eligible (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Flow chart of recruitment. Out of 81 participants eligible, 31 could not be addressed due to several medical examinations in different departments of the hospital and resulting in missing time slots. Longer periods post-treatment are associated with fewer appointments for follow-ups and more medical examinations take place in 1 day. This does not necessarily mean that the children who could not be included in the study due to missing time slots are medically more complex. Ten participants refused participation. Thus, the sample included 40 participants.</p></caption><graphic xlink:href=\"fped-08-00463-g0001\"/></fig></sec><sec><title>Outcome Measures</title><sec><title>Anamnestic and Anthropometric Data</title><p>Anamnestic and clinical data (i.e., type of cancer, treatment regime, end of therapy) was obtained from the electronic patient record (SAP® ERP). The nursing staff assessed anthropometric data (height and weight) during routine medical examination (seca 701 electronic column scale, seca 216 mechanical measuring rod). Body mass index (BMI) was calculated as a ratio of body weight (kg) per square body height (m<sup>2</sup>). By using the reference values of a healthy German cohort (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>), BMI was converted into percentiles and classified in underweight <10th percentile, normal weight 10th−90th percentile and overweight >90th percentile (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>).</p></sec><sec><title>Cardiovascular Parameters</title><p>Prior to the measurement, the participants had to rest for at least 10 min in a supine position. PWV, central blood pressure and peripheral blood pressure were assessed using the Mobil-O-Graph®, (I.E.M. GmbH, Stolberg, Germany and HMS Client-Server Version 5.1) an oscillometric, non-invasive method. Measurements were performed on the left upper arm. The cuff was inflated twice with a rest of 30 s in between. Cuff size was chosen according to the circumference of the participant's left upper arm. An ARCSolver Algorithm calculated the cSBP indirectly as well as the PWV from recorded brachial pulse. Raw data was transformed into z-scores and compared by using z-scores of a healthy reference cohort (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). PSBP and pDBP were compared to references from the national cohort of 4.529 children and adolescents (KiGGS study) (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). For the assessment of PWV and cSBP values, references from Elmenhorst et al. (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>) of 1.445 healthy children and young adults were used. To evaluate the results of the parameters cSBP and PWV, the examined participants were separated into two groups (<8 years and ≥8 years). According to the age distribution of the reference values, participants <8 years were compared to height-matched reference values and participants ≥8 years were compared to age-matched references.</p><p>The measurement using the Mobil-O-Graph® was previously used several times in pediatric patients (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>–<xref rid=\"B31\" ref-type=\"bibr\">31</xref>) and was also successfully applied in childhood cancer survivors (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>).</p></sec><sec><title>Motor Performance</title><p>To quantify motor performance, the MOON test (MOtor performance in pediatric ONcology) was applied (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). The test assesses motor abilities, coordination, speed, flexibility and strength and consists of eight test items: <italic>eye-hand coordination</italic> (inserting pins), <italic>static balance</italic> (static stand), <italic>upper extremity coordination</italic> (throwing at a target), <italic>speed</italic> (reaction test), <italic>muscular endurance</italic> (sit-to-stand), <italic>flexibility</italic> (stand and reach), <italic>hand grip strength</italic> (hand-held dynamometry), and <italic>muscular explosive strength</italic> (medicine ball shot). The test lasts 20 min on average. Data of each item was compared to published age- and sex-matched reference values of a healthy population within an age range of 6 to 17 years (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). Data of participants older than 17 years was compared to reference values of healthy 17-year-olds, since reference values of healthy 18-year-olds are not available. Calculation of a total score is not possible within this tool. Instead, each item was analyzed individually and the percentage deviation to reference values was computed.</p></sec><sec><title>Physical Activity and Reintegration Into Sports Structures After Acute Treatment</title><p>Physical activity levels and status of integration into sports structures were assessed with a standardized, self-reporting questionnaire referring to the KiGGS study (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). The questionnaire was supplemented by several disease- and treatment-related aspects in accordance with the study of Kesting et al. (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>) to investigate potential barriers regarding reintegration and participation in sports activities (e.g., barriers with respect to exemption from physical education at school or non-participation in sports clubs, sports therapy offers during treatment). The KiGGS study offers the reference values of healthy children and adolescents (<italic>n</italic> = 4.529) for comparison of our data.</p></sec></sec><sec><title>Data Analysis</title><p>Cardiovascular parameters were analyzed and compared to the healthy reference population with the one sample <italic>t</italic>-test. Motor performance was analyzed using the Wilcoxon signed-rank test in comparison to age- and sex-matched reference values. Pearson correlation was applied to calculate possible associations between motor performance and cardiovascular parameters, BMI and the period post-treatment. The Mann-Whitney-<italic>U</italic>-test was performed to evaluate anthracycline-mediated effects on cardiovascular health as well as differences within subgroups regarding different entities and levels of physical activity, motor performance, physical education at school, and achievement of physical activity recommendations.</p><p>Explorative two-sided statistical tests were conducted and <italic>p</italic> ≤ 0.05 was considered statistically significant. No adjustment for multiple comparison was conducted. Correlations coefficient (ρ) were classified according to Cohen (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>).</p><p>Descriptive statistics were calculated with Microsoft Excel (version 15.39) for demographic characteristics and medical data. GraphPad Prism (version 8) was used to perform all further statistical analyses. Data analysis was performed in consultation with the Institute of Medical Informatics, Statistics and Epidemiology of the Technical University of Munich.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>Participants</title><p>Out of 81 eligible children and adolescents who met the inclusion criteria, a total of 40 participants (50% female) with various cancer entities were recruited and examined (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>Table 1</label><caption><p>Anthropometric and medical characteristics of the participants (<italic>n</italic> = 40).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Characteristics</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>N</italic> (%)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Mean ± SD</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Median</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Range</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Age at diagnosis (years)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40 (100)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.26 ± 4.32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0–16</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Age at assessment (years)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40 (100)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11.28 ± 3.80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6–18</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   <8 years</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10 (25)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   ≥8 years</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30 (75)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Period post-diagnosis (years)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40 (100)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.81 ± 3.17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.2–14</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Period post-treatment (years)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40 (100)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.56 ± 1.79</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.04</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.2–10.33</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   <1 year</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19 (48)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   1–5 years</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">20 (50)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   >5 years</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (3)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Leukemia/Lymphoma</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18 (45)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Bone tumor</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (5)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Brain tumor</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7 (18)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Other solid tumors<xref ref-type=\"table-fn\" rid=\"TN1\"><sup>*</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13 (33)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Body mass index<xref ref-type=\"table-fn\" rid=\"TN2\"><sup>•</sup></xref> (kg/m<sup>2</sup>)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40 (100)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17.63 ± 3.26</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.2–27.50</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Underweight</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9 (23)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Normal weight</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25 (63)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Overweight</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6 (15)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Chemotherapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27 (68)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Anthracycline application</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">25 (63)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Cumulative dose (mg/m<sup>2</sup>)</td><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">207 ± 81</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">227</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">92–354</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Radiotherapy</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13 (33)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">   Chest-directed radiation</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (10)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Anthracycline + chest radiation</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4 (10)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Surgical tumor resection</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19 (48)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Relapse</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12 (30)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><p><italic>Results are given in Mean ± SD (M, median; Range)</italic>.</p><fn id=\"TN1\"><label>*</label><p><italic>Other solid tumors: alveolar rhabdomyosarcoma (n = 1), carcinoid tumor of the appendix (n = 2), nephroblastoma (n = 3), focal nodular hyperplasia liver (n = 1), mature cystic teratoma ovary (n = 2), thoracic ganglioneuroma (n = 2), papillary thyroid carcinoma (n = 1), neuroblastoma (n = 2)</italic>.</p></fn><fn id=\"TN2\"><label>•</label><p><italic>BMI was converted into percentiles and classified in underweight <10th percentile, normal weight 10th−90th percentile and overweight >90th percentile (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>)</italic>.</p></fn><p><italic>Thirteen participants received a combination of anthracyclines (mainly the combination of Doxorubicin and Daunorubicin)</italic>.</p></table-wrap-foot></table-wrap></sec><sec><title>Performed Assessments</title><p>For various reasons, not all tests could be realized with all participants. In 6/40 participants (15%), cardiovascular parameters could not be evaluated due to missing time slots during routine appointment and a lack of willingness to prolong the outpatient visit. Six of the 40 participants could not perform the MOON test due to medical limitations (current orthopedic restrictions, <italic>n</italic> = 2), examination-related limitations, i.e., a drain tube in the crook of the arm for follow-up MRT (<italic>n</italic> = 2) and abandonment due to lack of time (<italic>n</italic> = 2). The central venous device has already been explanted in all participants prior to our study.</p><p>Of the remaining 34 participants, not everyone performed every test item: Two participants could not perform the test item <italic>speed</italic> due to a drain tube in the crook of the arm. Four participants could not accomplish the test item <italic>muscular explosive strength</italic> due to orthopedic restrictions as well as examination-related limitations. Three participants did not perform the item <italic>muscular endurance of the legs</italic> (<italic>n</italic> = 1 had crutches, <italic>n</italic> = 1 severe muscular deficit in the legs, <italic>n</italic> = 1 lack of time). Two participants could not perform the test item <italic>hand grip strength</italic> with both hands due to lack of time (<italic>n</italic> = 1) and infusion needle in the crook of the arm (<italic>n</italic> = 2). The test item <italic>upper extremity coordination</italic> was measured in <italic>n</italic> = 13 participants because reference values are provided for children between 6 and 10 years only.</p></sec><sec><title>Cardiovascular Health</title><p>In 34/40 participants, all parameters were assessed. Based on the underlying reference values for cSBP and PWV of Elmenhorst et al. (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>), the group was separated into participants aged <8 years (height-matched reference values) and ≥8 years (age-matched reference values). PSBP (z-score: 0.87 ± 1.67, <italic>p</italic> = 0.003), pDBP (z-score: 0.83 ± 1.94, <italic>p</italic> = 0.033) as well as cSBP values (≥8 years: z-score: 0.60 ± 1.29, <italic>p</italic> = 0.011) were significantly increased compared to reference values of healthy children and adolescents (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). PWV was elevated, but not significantly (<8 years: z-score: 1.15 ± 2.89, <italic>p</italic> = 0.374; ≥8 years: z-score: 0.55 ± 1.90, <italic>p</italic> = 0.127).</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Cardiovascular parameters shown in z-scores and compared to published reference values (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>). pSBP, peripheral systolic blood pressure; pDBP, peripheral diastolic blood pressure; PWV, pulse wave velocity; cSBP, central systolic blood pressure. *Significant values (<italic>p</italic> ≤ 0.05).</p></caption><graphic xlink:href=\"fped-08-00463-g0002\"/></fig><p>Comparison of cardiovascular parameters of 23 participants who received anthracyclines during intense therapy with a cumulative dose of 207 ± 81 mg/m<sup>2</sup> and subjects who did not receive cardiotoxic agents did not show statistically significant differences.</p></sec><sec><title>Motor Performance</title><p>The participants' (<italic>n</italic> = 34/40, 85%) motor performance was reduced in almost all motor abilities compared to the reference values of healthy children and adolescents (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>). Significant impairments became obvious in the following dimensions: <italic>muscular explosive strength</italic> (<italic>p</italic> < 0.001), <italic>upper extremity coordination</italic> (<italic>p</italic> = 0.032), <italic>muscular endurance of the legs</italic> (<italic>p</italic> = 0.020) and <italic>hand grip strength</italic> on the right hand (<italic>p</italic> = 0.002). The performance of <italic>eye-hand coordination, speed, flexibility</italic>, and <italic>hand grip strength</italic> on the left hand were also reduced, but not significantly. In the test item <italic>static stand</italic>, the study participants performed slightly better compared to the reference population. For <italic>upper extremity coordination</italic> (throwing at a target) only reference values from children aged 6–10 years are available. Therefore, comparison of the collected data was only possible with the same age group (<italic>n</italic> = 13/40, 33%). Data for older participants was not collected.</p><table-wrap id=\"T2\" position=\"float\"><label>Table 2</label><caption><p>Results of the MOON-test compared to reference values (<italic>n</italic> = 34).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Motor ability</bold></th><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Test item</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>N</italic></bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Mean ± SD of difference to reference values (%)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Media<italic>n</italic> (%)<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>#</sup></xref></bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>p</italic>-value</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Eye-hand coordination</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Inserting pins (time)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">34</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−2.12 ± 13.74</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.08</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.599</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Static balance<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>#</sup></xref><xref ref-type=\"table-fn\" rid=\"TN4\"><sup>ϕ</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Static stand (contacts)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">34</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.79 ± 7.49<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.99<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.184</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Speed</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Reaction test (time)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.73 ± 15.48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.71</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.111</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Upper extremity coordination<xref ref-type=\"table-fn\" rid=\"TN5\"><sup>θ</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Throwing at a target (points)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−26.23 ± 39.16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−37.50</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.032</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Flexibility<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>#</sup></xref></td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Stand and reach (cm)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">34</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.71 ± 8.60<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.89<xref ref-type=\"table-fn\" rid=\"TN3\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.278</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Muscular explosive strength</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Medicine ball shot (meter)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−20.60 ± 13.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−22.16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><0.001</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Muscular endurance legs</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sit-to-stand (sec)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">31</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−7.33 ± 24.80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−8.58</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.020</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hand grip strength</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hand-held dynamometry (kg)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Right</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−15.41 ± 23.59</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−24.65</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.002</bold></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Left</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−8.85 ± 0.17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−13.87</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.074</td></tr></tbody></table><table-wrap-foot><p><italic>All results were compared to the reference values of each single test item (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). Speed could only be measured in 32 participants, muscular explosive strength in 30 participants and hand grip strength in 32 (right) and 33 (left) participants, due to lack of time, orthopedic restrictions or drain tube in the crook of the arm</italic>.</p><fn id=\"TN3\"><label>#</label><p><italic>For the test items static balance and flexibility, the absolute differences were used, as the measured values would have fluctuated around zero and would have given oversized percentages</italic>.</p></fn><fn id=\"TN4\"><label>ϕ</label><p><italic>Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail; in this context, a negative difference to reference values represents fewer contacts and therefore better results</italic>.</p></fn><fn id=\"TN5\"><label>θ</label><p><italic>Although all participants completed this test item, reference values are provided for children between 6 and 10 years only</italic>.</p></fn><p><italic>M, median; abs, indicates absolute</italic>.</p><p><italic>Bold numbers indicate significant values (p ≤ 0.05)</italic>.</p></table-wrap-foot></table-wrap><p>Comparing motor performance of participants diagnosed with leukemia/lymphoma (<italic>n</italic> = 18/40, 45%) and participants diagnosed with brain tumors (<italic>n</italic> = 7/40, 18%) revealed some differences (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). Children and adolescents treated for brain tumors performed significantly worse in <italic>eye-hand coordination</italic> than participants treated for leukemia/lymphoma (<italic>p</italic> = 0.005). Moreover, the performances in all other tested motoric dimensions of participants diagnosed with brain tumor were deteriorated compared to participants treated for leukemia/lymphomas, but not significantly.</p><table-wrap id=\"T3\" position=\"float\"><label>Table 3</label><caption><p>Results of the MOON-test comparing participants treated for leukemia/lymphoma and participants treated for brain tumors compared to reference values.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" colspan=\"3\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Leukemia/Lymphoma (<italic>n</italic> = 18/40)</bold></th><th valign=\"top\" align=\"center\" colspan=\"3\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\"><bold>Brain tumor (<italic>n</italic> = 7/40)</bold></th><th rowspan=\"1\" colspan=\"1\"/></tr><tr><th valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"><bold>Motor ability</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>N</italic></bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Mean ± SD (%)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Median (%)<xref ref-type=\"table-fn\" rid=\"TN7\"><sup>#</sup></xref></bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>N</italic></bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Mean ± SD (%)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Median (%)<xref ref-type=\"table-fn\" rid=\"TN7\"><sup>#</sup></xref></bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold><italic>p</italic>-value</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Eye-hand coordination</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.31 ± 10.40</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.083</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−19.97 ± 17.32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−19.19</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>0.005</bold></td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Static balance<xref ref-type=\"table-fn\" rid=\"TN7\"><sup>#</sup></xref><xref ref-type=\"table-fn\" rid=\"TN6\"><sup>ϕ</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.55 ± 8.35<xref ref-type=\"table-fn\" rid=\"TN7\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.70</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.79 ± 7.567<xref ref-type=\"table-fn\" rid=\"TN7\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.5<xref ref-type=\"table-fn\" rid=\"TN7\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.195</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Speed</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−5.74 ± 12.32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−3.48</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−11.60 ± 26.16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">>0.999</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Flexibility<xref ref-type=\"table-fn\" rid=\"TN7\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.61 ± 9.59<xref ref-type=\"table-fn\" rid=\"TN7\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−0.89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.75 ± 6.00<xref ref-type=\"table-fn\" rid=\"TN7\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.96<xref ref-type=\"table-fn\" rid=\"TN7\"><sup>#</sup></xref></td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.550</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Muscular explosive strength</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−19.95 ± 16.91</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−23.79</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−20.53 ± 8.04</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−19.95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.519</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Muscular endurance legs</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−1.891 ± 30.83</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−4.450</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−13.35 ± 9.80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−8.89</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.445</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hand grip strength right</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−7.62 ± 24.21</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−9.70</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−20.08 ± 14.82</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−28.95</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.398</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hand grip strength left</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−3.28 ± 34.33</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−3.35</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−19.98 ± 35.36</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">−16.49</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.595</td></tr></tbody></table><table-wrap-foot><p><italic>All results were compared to the reference values of each single test item (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>)</italic>.</p><fn id=\"TN6\"><label>ϕ</label><p><italic>Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail; in this context, a negative difference to reference values represents fewer contacts and therefore better results</italic>.</p></fn><fn id=\"TN7\"><label>#</label><p><italic>For the test items static balance and flexibility, the absolute differences were used as the measured values would have fluctuated around zero and would have given oversized percentages</italic>.</p></fn><p><italic>Due to insufficient number of participants, the comparison regarding the test item upper extremity coordination was disregarded (reference values only provided for children aged 6 and 10 years)</italic>.</p><p><italic>M, median; abs, indicates absolute</italic>.</p><p><italic>Bold numbers indicate significant values (p ≤ 0.05)</italic>.</p></table-wrap-foot></table-wrap><p>To determine influencing factors on motor performance, the correlation between motor performance results and BMI as well as the period post-treatment was performed. With increasing BMI, values of <italic>static balance</italic> deteriorated significantly (ρ = 0.418, <italic>p</italic> = 0.014) which corresponds to a moderate to high correlation (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>). A negative difference to reference values means fewer contacts to the ground and therefore better performance in static balance. Furthermore, some non-significant correlations were found. Deteriorated <italic>eye-hand coordination</italic> (ρ = −0.257, <italic>p</italic> = 0.143) and <italic>flexibility</italic> (ρ = −0.117, <italic>p</italic> = 0.512) were also associated with a higher BMI. However, superior values in <italic>upper extremity coordination</italic> (ρ = 0.317, <italic>p</italic> = 0.315), <italic>muscular explosive strength</italic> (ρ = 0.139, <italic>p</italic> = 0.474), <italic>hand grip strength</italic> (right: ρ = 0.170, <italic>p</italic> = 0.359; left: ρ = 0.306, <italic>p</italic> = 0.089) and <italic>muscle endurance of the legs</italic> (ρ = 0.170, <italic>p</italic> = 0.362) were associated with increased BMI. The test item <italic>speed</italic> showed no association with BMI (ρ = 0.029, <italic>p</italic> = 0877).</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Pearson correlation between static balance and BMI (kg/m<sup>2</sup>). • leukemia/lymphoma, ◦ bone tumor, △ brain tumor, ◆ other solid tumors. Horizontal line indicates the reference values (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). The absolute difference from reference values is given (number of contacts to the ground). A negative difference means fewer ground contacts and therefore better performance. Thirty four participants performed the test.</p></caption><graphic xlink:href=\"fped-08-00463-g0003\"/></fig><p>A longer period post-treatment was significantly associated with decreased <italic>eye-hand coordination</italic> (ρ = −0.353, <italic>p</italic> = 0.041), corresponding to a moderate correlation (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>) (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). Especially participants treated for a brain tumor with a longer period post-treatment showed deteriorated values in <italic>eye-hand coordination</italic>. Speed performance was deteriorated in participants with longer post-treatment period (ρ = −0.329, <italic>p</italic> = 0.066).</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Pearson correlation between eye-hand coordination and period post-treatment (months). • leukemia/lymphoma, ◦ bone tumor, △ brain tumor, ◆ other solid tumors. Horizontal line indicates the reference values (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Thirty four participants performed the test item.</p></caption><graphic xlink:href=\"fped-08-00463-g0004\"/></fig></sec><sec><title>Physical Activity and Reintegration Into Sports Structures</title><p>According to the self-reported questionnaire, 36% (<italic>n</italic> = 13/36) did not participate in physical education at school to full extend: 28% (<italic>n</italic> = 10/36) were not admitted to school sports activities and 8% (<italic>n</italic> = 3/36) were partly excluded (<xref rid=\"T4\" ref-type=\"table\">Table 4.1</xref>). Neither of the two participants treated for bone tumor was taking part in physical education at school (<italic>n</italic> = 2/36, 6%), whereas children with other tumors participated to a notably higher rate. Treatment-related muscular deficits (<italic>n</italic> = 2/36, 6%) and osteonecrosis (<italic>n</italic> = 3, 8%) were the most common reasons for participants not taking part in physical education at school.</p><table-wrap id=\"T4\" position=\"float\"><label>Table 4.1</label><caption><p>Participation in physical education at school subdivided into entities (<italic>n</italic> = 36).</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Participation</bold><break/><bold><italic>N</italic> (%)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Partial exemption</bold><break/><bold><italic>N</italic> (%)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>Full exemption</bold><break/><bold><italic>N</italic> (%)</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Entire group (<italic>n</italic> = 36)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">23 (64%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3 (8%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10 (28%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Leukemia/Lymphoma (<italic>n</italic> = 16)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9 (56%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (6%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6 (38%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Bone tumor (<italic>n</italic> = 2)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (100%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Brain tumor (<italic>n</italic> = 6)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3 (50%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2 (33%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (17%)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Other solid tumors (<italic>n</italic> = 12)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11 (92%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0 (0%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1 (8%)</td></tr></tbody></table><table-wrap-foot><p><italic>Abs. N, number. Four out of 40 children were still attending kindergarten and could not answer the question regarding participation in physical education at school</italic>.</p></table-wrap-foot></table-wrap><p>Only 17% (<italic>n</italic> = 7/40) reported moderate-to-vigorous physical activity for 60 min daily as generally recommended by the WHO for healthy children and adolescents (<xref rid=\"T5\" ref-type=\"table\">Table 4.2</xref>). This percentage is comparable to the achievements in the healthy reference population (15%, <italic>n</italic> = 4.529).</p><table-wrap id=\"T5\" position=\"float\"><label>Table 4.2</label><caption><p>Physical activity and engagement in sports club and recreational sports.</p></caption><table frame=\"hsides\" rules=\"groups\"><thead><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\"><bold>Entire group</bold></th><th valign=\"top\" align=\"center\" style=\"border-bottom: thin solid #000000;\" rowspan=\"1\" colspan=\"1\"><bold>Reference population<xref ref-type=\"table-fn\" rid=\"TN8\"><sup>*</sup></xref></bold></th></tr><tr><th rowspan=\"1\" colspan=\"1\"/><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>(<italic>n</italic> = 40)</bold></th><th valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><bold>(<italic>n</italic> = 4.529)</bold></th></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Physical activity guidelines</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">WHO (60 min/day)</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Daily physical activity</td><td rowspan=\"1\" colspan=\"1\"/><td rowspan=\"1\" colspan=\"1\"/></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Daily walking distance <1 km</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Daily walking distance 1–5 km</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">80%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">76%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Daily walking distance >5 km</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sports club activity (currently)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">58%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Former membership</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">27%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">19%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Recreational sports activity</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">89%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">61%</td></tr></tbody></table><table-wrap-foot><p><italic>Abs. N, number</italic>.</p><fn id=\"TN8\"><label>*</label><p><italic>Reference values derived from the national cohort of healthy children and adolescents in the KiGGS study (German Health Interviews and Examination Survey for Children and Adolescents) (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>)</italic>.</p></fn></table-wrap-foot></table-wrap><p>Every second participant questioned was an active member in a sports club, whereas 27% (<italic>n</italic> = 11/40) did not return to a sports club following cancer treatment. Almost one-third, 23% (<italic>n</italic> = 9/40), has never been a sports club member. Reasons for not engaging in sports club activities of 20 participants were: no interest/fun (40%, <italic>n</italic> = 8/20), physical weakness (<italic>n</italic> = 4/20, 20%), no time (<italic>n</italic> = 3/20, 15 %), anxiety (15%, <italic>n</italic> = 3/20), and physician-based prohibition due to clear medical reasons (10%, <italic>n</italic> = 2/20). Nearly all participants, <italic>n</italic> = 34/36 (98%), were active in recreational sports.</p><p>Further analyses pointed toward differences in physical activity and sports club participation, especially between participants with brain tumors and leukemia/lymphomas. The number of participants in recreational sports was reported high in both groups: leukemia/lymphoma (88%) and brain tumor (100%). In contrast, a difference was found in sports club activity. Sixty-six percent of leukemia/lymphoma patients were members of a sports club, whereas only 28% of participants with a brain tumor were active in a sports club. On the other hand, almost half (43%) of the children treated for brain tumor and 23% of the children treated for leukemia/lymphoma were former members.</p><p>Concerning possible correlations between motor abilities and physical education at school, participation in sports clubs or recreational sports (defined as active/inactive), as well as meeting the physical activity recommendations no significant associations could be determined. Likewise, the comparison of motor abilities of participants receiving sports therapy during treatment did not show any correlation. Almost half of the participants (45%, <italic>n</italic> = 18/40) took part in a sports therapy programme during treatment, which was mainly offered as care and varied greatly in terms of training interventions without any standardization.</p></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>The results of our study clearly present evidence for deteriorated cardiovascular function in children and adolescents shortly after cessation of cancer treatment. Increased pSBP and increased pDBP are risk factors for cardiovascular diseases, regarding guidelines for arterial hypertension (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). Potential cardiovascular consequences such as stroke, sudden death, heart failure and peripheral artery disease due to elevated blood pressure values are described in the aforementioned guidelines as well as in the literature (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>, <xref rid=\"B35\" ref-type=\"bibr\">35</xref>). Accordingly, childhood cancer survivors with elevated blood pressure are at risk to experience such cardiovascular late effects. Regarding 10-years survivors of childhood cancer, a higher prevalence of hypertension is assumed (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>) and cardiovascular disease-related deaths are eight times more likely in childhood cancer survivors compared to the general population (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>). Our findings support previous study results, which depict complications such as increased blood pressure, prehypertension and hypertension in children and adolescents treated for cancer (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>, <xref rid=\"B38\" ref-type=\"bibr\">38</xref>).</p><p>This study aimed at investigating specific parameters that could serve as early predictors for potential damage to the cardiovascular system. Recent evidence of cSBP, as a suitable parameter to determine the elasticity of blood vessels, suggests that cSBP is more closely related to cardiovascular events in the future than brachial blood pressure (<xref rid=\"B39\" ref-type=\"bibr\">39</xref>, <xref rid=\"B40\" ref-type=\"bibr\">40</xref>). Increased cSBP in participants in our study may result from early changes in arterial wall stiffness. As a further parameter to detect early impairments in elasticity of the vascular system, PWV was investigated. In contrast to prior studies (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>, <xref rid=\"B8\" ref-type=\"bibr\">8</xref>), no decisive change was observed in PWV. While anthracyclines have been found to result in cytotoxic and cardiotoxic effects, these consequences have been described as a clear influence on arterial stiffness and impaired endothelial function (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>, <xref rid=\"B42\" ref-type=\"bibr\">42</xref>). In our study, we did not find any associations between increased blood pressure or PWV and anthracycline-containing chemotherapy in more than half of the participants with a cumulative dose of 207 ± 81 mg/m<sup>2</sup> (range 92–354 mg/m<sup>2</sup>). Potentially, these consequences found in previous studies were not yet shown in our study cohort due to a shorter period post-treatment compared to other studies (<xref rid=\"B41\" ref-type=\"bibr\">41</xref>, <xref rid=\"B42\" ref-type=\"bibr\">42</xref>). Although cSBP was increased as a sign for changes in arterial wall stiffness, consequences regarding clear outcomes might develop later and in older subjects (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>) and probably more sensitive methods need to be applied.</p><p>Although the measuring methods and instruments as well as their manufacturers differ from previous studies to this one, a comparison of the results is drawn in the following. Measurements with SphygmoCor (AtCor Medical, West Ryde, Australia) showed significantly higher values of PWV in participants >18 years of age (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). However, no indication for higher PWV in participants receiving anthracyclines was found (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). Another recent study (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>) examined PWV using Skidmore Medical Limited (Bristol, United Kingdom; Version 4). Their results show that PWV was elevated in 6% of children and adolescent treated for cancer after hematopoietic cell transplantation. The authors concluded that a larger waist circumference and the time of transplantation prior to the age of twelve years was associated with increased PWV (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). An ultrasound-based study showed increased arterial stiffness of the carotid artery compared to a control group in survivors of leukemia >5 years after cancer diagnosis (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>).</p><p>Nevertheless, one recent study using the Mobil-O-Graph® as well, described results similar to our findings (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>): The study examined n = 92 children and adolescents treated for cancer, aged 12.5 ± 4.2 years and 3.6 ± 2.8 post-diagnosis. Results show an increase in pSBP, but no significant changes in cSBP or PWV were found, independent of receiving anthracyclines. Accordingly, while our findings lend additional support to an increase in pSBP, and no significant changes in PWV, they do illustrate a significant elevation of cSBP, independent of receiving anthracyclines.</p><p>Our experience in performing the measurement with the Mobil-O-Graph® is consistently positive. Considering the compliance of the measurement with acceptance from all participants in our study was captured. The duration of the measurement was clearly accepted and did not raise any problems. Furthermore, the instrument was used with adolescents as well as very young children without encountering any difficulties.</p><p>Discrepancies of the cardiovascular outcomes in different studies and the use of diverse measurement methods support the need for further research to gain knowledge and additional insights to finally standardize the methods.</p><p>Considering the period of time post-diagnosis and post-treatment in various studies, differences in the results are discernible. In our study, participants were screened after a shorter period post-diagnosis (2.81 ± 3.17 years) and post-treatment (1.56 ± 1.79 years) compared to previous ones and showed elevated values of PWV. On the contrary, another study investigated elevated blood pressure levels, but no statistically significant variation for PWV in pediatric cancer patients compared to healthy children and adolescents was found (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Therefore, longitudinal studies are necessary to better quantify potential temporal damages following cancer-related treatment and, in particular, changes in PWV to examine any association with the period of time post-treatment.</p><p>Our study demonstrated impairments in children and adolescents after anti-cancer treatment in nearly all dimensions of motor performance compared to healthy children and adolescents. The only exception was the test item <italic>static stand</italic>. In this test, participants performed better compared to the healthy reference values. It should be noted that this result would be even more distinct without the two outliers shown in <xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>. One participant after treatment for leukemia and one participant after treatment for a brain tumor showed values remarkably high and therefore many contacts to the ground compared to all others and the reference values. However, the small sample of children treated for a brain tumor participating in our study showed clearly reduced static balance overall (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref>) and these impairments are well-known in this group of patients (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>). Furthermore, the test item examining eye-hand coordination (inserting pins) revealed low performance in participants treated for brain tumors compared to all others and to the reference values. According to <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>, this seems to persist for months after cessation of treatment. However, these results should not be over interpreted due to the small number of participants and the three outliers. These three participants treated for a brain tumor with the longest periods post-treatment performed distinctly worse, but for useful results and clear interpretations a larger number of participants with long periods post-treatment needs to be examined. Measured impairments could be related to a very low level of physical activity, decreased strength and overall coordination. Thus, a lack of motoric development in these participants already begins during acute treatment (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>). In addition, the intense treatment with severe surgery that is required often in this group of patients should be considered. With regard to the medical treatment brain tumor patients usually receive, radiation therapy should be addressed. A total of <italic>n</italic> = 5/7 participants were exposed to radiation therapy. This might be an additional reason for impairments compared to other patients and healthy children. Furthermore, negative outcomes regarding motor performance due to additional radiation therapy in young brain tumor patients has been shown by Ottensmeier et al. (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>). Cranial radiation therapy can be part of the treatment regime for leukemia patients as well and, therefore, could show an impact on motor skills in this group. In our study, only three out of 18 participants treated for leukemia received cranial radiation therapy. Due to this small number we refrained from any interpretations.</p><p>Our study showed impairments especially in the motoric dimensions of strength and coordination in the whole sample. Similar results were found by another study investigating pediatric cancer patients at the end of acute treatment, also using the MOON test (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). The motor abilities strength and coordination are essential to perform activities of daily living. Therefore, it seems to be of particular importance to train strength and coordination under supervised conditions already during and after treatment to counteract the loss of strength and coordination in survivors of childhood cancer. Our study did not show a clear difference in motor performance comparing participants who took part in a sports program during treatment and those, who did not (45 vs. 55%). The sports therapy program was implemented in June 2016 and offered twice a week in the beginning. Content and duration of interventions were not standardized and most of the participants took part in the care program and not in a study. Therefore, no conclusion can be drawn regarding effectiveness of our sports therapy program in the beginning, because that was not the aim of this screening. Until now, clear training recommendations and concepts for specific strength and coordination training for children and adolescents with cancer are still missing. Those instructions need to be developed and disseminated considering individuality and treatment. This would enable a more focused approach to the problem of deteriorated strength and coordination during and after therapy. <italic>Upper extremity coordination</italic> (throwing at a target) was tested in 13 participants aged from 6 to 10 years and found to be significantly deteriorated in our study. However, considering the small sample size studies with larger numbers of participants are needed on this motor dimension. According to subjective appraisal, participants did not have enough power to hit the target from the marked line. This assumption is reflected in the deteriorated values of <italic>muscular explosive strength</italic> and <italic>hand grip strength</italic>.</p><p>Regarding coherences between static balance and BMI as well as the period post-treatment, both variables appeared to influence single dimensions of motor performance. Supporting the results of a recent study with patients treated for bone cancer (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>), our study showed that higher BMI was associated with poorer performance in <italic>static balance</italic>, and a longer period post-treatment was associated with deteriorated eye-hand coordination. Functional limitations with increasing BMI were shown in healthy children and adolescents as well (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>). Eye-hand coordination is necessary for manual writing and handicraft work and most relevant for reintegration into school life as well as daily activities. Motoric skills like eye-hand coordination as well as flexibility should also be trained slightly during cancer treatment to counteract possible restrictions. With simple, targeted exercise interventions like throwing balls or plain strength exercises, motor skills of children and adolescents treated for cancer could continuously be trained during treatment. Exercises during intensive treatment have already been shown feasible (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>–<xref rid=\"B48\" ref-type=\"bibr\">48</xref>) and well-received (<xref rid=\"B49\" ref-type=\"bibr\">49</xref>) in pediatric cancer patients. These findings correspond to our own observation since the beginning of our sports program.</p><p>Contrary to expectations, the findings of our study regarding WHO recommendations for physical activity in participants were comparable with reference values of healthy children and adolescents. But it is worth mentioning that only a low percentage of healthy peers achieve those recommendations according to the results of the KiGGS study due to a shift from usual daily physical activity in younger children to predominantly recreational sports activities in adolescents that are not performed every day (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Every second participant was active in a sports club. Surprisingly, more participants than healthy children and adolescents were former sports club members. This result points to the fact that many children and adolescents treated for cancer do not return to a sports club early after treatment. In contrast, a higher number (nearly 90%) of participants compared to references (barley two-thirds), were active in recreational sports. The high participation rate in recreational, non-structured sports as well as a low number of currently active members of sports clubs could be referred on one hand to a short period-post treatment and a resulting lack of social integration. On the other hand, an individual training in this period might be even more useful and the level of intensity in a training in larger groups cannot be achieved yet. Further investigations regarding the main reasons are necessary.</p><p>Most children and adolescents in this study participated in physical education at school after intensive treatment and only few were exempted partly or to full extend. All participants treated for a bone tumor were exempted to the full extent and these findings agree with the results of Kesting et al. (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). Due to the small number of patients treated for bone tumors (<italic>n</italic> = 2) in our study, no further conclusions can be drawn. Nevertheless, future studies, should examine this entity more closely with regard to participation in physical activities/education in school, in order to define and decrease possible barriers. Participants treated for a brain tumor had a low participation in physical education at school. This result is similar to a prior study as well, where the main reason for full exemption in all entities was medical advice against sports participation by the physician (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). In contrast, this reason was mentioned only once in our study. Another study investigating patients with acute lymphoblastic leukemia almost 5 years following treatment showed that nearly all children and adolescents are participating in physical education at school. Barriers of non-participation included exhaustion or fear of injury (<xref rid=\"B50\" ref-type=\"bibr\">50</xref>). The lower participation rate in physical education at school in our study compared to the high participation rate in the previously described study is probably shown due to a shorter period post-treatment.</p><p>All in all, it should be noted, that shortly after treatment, the return to any kind of sports may not be considered as priority one for all patients and their families. Especially, during return to school, physical education might not be as important for the child's school career, but could help to reintegrate and socialize with its friends and peers.</p><p>With respect to existing barriers, the most frequently mentioned reason for full exemption from physical education at school in our study was osteonecrosis. This demonstrates that non-participation in physical education at school is not only dependent on judgement of medical or school staff, but on intra-individual reasons as well. To reduce these barriers in the future, individual and specific advice from educated trainers and therapists is needed on a regular basis and especially at the end of acute treatment and as a support during the return to normality.</p><p>Lastly, participants' motor performance was deteriorated maybe due to non-participation in physical education at school and no sufficient other type of physical activity to improve motor skills. However, these findings were not statistically significant.</p><p>To counteract impaired motor performance and deteriorated cardiovascular values, exercise and sports therapy during and after treatment should be an integral part of every department for pediatric oncology as suggested in recent reviews (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>, <xref rid=\"B51\" ref-type=\"bibr\">51</xref>). In turn, support and advice can enable children and adolescents to increase their physical activity, which might help to facilitate reintegration into sports structures and social structures after cessation of treatment.</p><p>Training during and after treatment should preferably be supervised and controlled. Findings from a recent study (<xref rid=\"B52\" ref-type=\"bibr\">52</xref>) investigating exercise in patients undergoing anti-cancer treatment, further support the promotion of exercise during and after treatment to gain physiological and functional benefits, as well as to improve quality of life. Another study published practical applications for the use of exercise for pediatric cancer patients and outlined guidelines for incorporating physical activity and exercise into daily practice in hospitals (<xref rid=\"B53\" ref-type=\"bibr\">53</xref>) that are in accordance with our own experiences in our program.</p><p>To achieve improvements in motor abilities and to counteract their decline, it is particularly important for children to remain active beyond medical treatment. This will facilitate reintegration into physical education at school, sports structures, as well as social structures.</p><p>In addition, it seems to be useful to offer professional support and advice for trainers in sports clubs on a larger scale. This enables trainers to work and qualify on disease-specific peculiarities and resilience. Therefore, better integration of formerly cancer-treated children and adolescents into sports clubs might be possible. Moreover, there should also be more training opportunities for sports teachers on how to differentiate the strain level of chronically ill children during physical education at school.</p><p>Nevertheless, trends were detected: participants who were not involved in physical education at school, who were engaging in neither sports club activities nor recreational sports, who did not meet the WHO physical activity recommendations and who did not receive sports therapy during treatment performed worse in almost all motoric abilities.</p></sec><sec id=\"s5\"><title>Limitations</title><p>This study shows some limitations that are quite common and known in research within this specific sample. The group of examined participants is very heterogeneous regarding age, variety of entities, applied treatment regimens, and the range of physical impairments. Subgroup analyses point out specifics regarding different entities and show problems in groups that are usually understudied (e.g., brain tumor patients). The disadvantages of self-reporting tools like the questionnaire assessing physical activity are known (<xref rid=\"B54\" ref-type=\"bibr\">54</xref>). Recall-bias and social desirability might have distorted data, but assessment of several aspects (e.g., status of integration in sport structures and barriers) is only possible using self-reporting. Application of objective tools for cardiovascular parameters and motor performance provided reliable data. However, all measurements were performed only once. Repeated measurements could increase reliability. Recruitment was a challenge in this study. Of 81 children and adolescents eligible, 31 could not be addressed due to several medical examinations in different departments within follow-up care on 1 day and therefore only very short time slots. Most of them have appointments every 3 months or less frequent. Longer periods post-treatment are associated with fewer appointments for follow-ups. Even though this does not necessarily mean that these children are medically more complex, data of these children might have influenced the results and this aspect needs to be considered as a limitation of our study. In addition, more participants treated for a bone tumor should be studied regarding their physical impairments. However, we were able to show subjective and objective data of participants treated for different types of cancer and with various problems and needs.</p></sec><sec sec-type=\"conclusions\" id=\"s6\"><title>Conclusions</title><p>This study focused on cardiovascular and motor impairments of children and adolescents during maintenance and follow-up care. The analysis of their status of integration into sports structures as well as participation in physical education at school enables identification and definition of potential barriers and provides insights on how to best development of effective and helpful strategies. Affected children and adolescents should receive early support in handling their physical limitations, already during and following treatment. Early motor deficits should be revealed and reduced by applying targeted sports interventions. Implementation of sports therapy during and shortly after treatment could reduce arising therapy-related late effects, such as cardiovascular diseases. Presumably, these interventions targeted at reducing cardiovascular damage and implemented by sports therapists should be (1) initiated prior to the application of cardiotoxic agents, (2) a holistic approach including a high amount of endurance training, and (3) performed under cardiological monitoring for safety reasons and dose finding of sports interventions. Generally, children and adolescents with cancer as well as their parents should be supported and advised in every phase of treatment. Moreover, they should be encouraged to be physically active and to develop a long-term active lifestyle.</p><p>Therefore, sports programs should be included in pediatric oncology team efforts as a meaningful, cost-effective preventive approach in terms of late effects associated with physical inactivity.</p></sec><sec sec-type=\"data-availability\" id=\"s7\"><title>Data Availability Statement</title><p>The datasets generated for this study are available on request to the corresponding author.</p></sec><sec id=\"s8\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by The Ethics Committee of the School of Medicine of the Technical University of Munich. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.</p></sec><sec id=\"s9\"><title>Author Contributions</title><p>SK and DG were responsible for conception and design of the study and the coordination of data collection. IL supervised the medical support and gave important input for drafting and revising the manuscript. TK was responsible for examination and collecting data, analyzing, and processing data. CP gave important input for the concept. RO-F gave important input for drafting and revising the manuscript. TK and SK wrote the manuscript with input from all authors, who read, and approved the final version of the manuscript.</p></sec><sec id=\"s10\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><ack><p>The authors gratefully acknowledge all children and adolescents and their parents for participation. Furthermore, the authors are thankful for the supporting staff of the Department of Pediatric Hematology and Oncology at the Kinderklinik München Schwabing. We thank Maria Keiser for proofreading and language editing.</p></ack><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Noone</surname><given-names>A</given-names></name><name><surname>Howlader</surname><given-names>N</given-names></name><name><surname>Krapcho</surname><given-names>M</given-names></name><name><surname>Miller</surname><given-names>D</given-names></name><name><surname>Brest</surname><given-names>A</given-names></name><name><surname>Yu</surname><given-names>M</given-names></name><etal/></person-group>\n<source>Screenshot.pdf. SEER Cancer Statistics Review, 1975-2015.</source>\n<publisher-loc>Bethesda, MD</publisher-loc>: <publisher-name>National Cancer Institute</publisher-name> (<year>2018</year>).</mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kaatsch</surname><given-names>P</given-names></name><name><surname>Spix</surname><given-names>C</given-names></name><name><surname>Grabow</surname><given-names>D</given-names></name><name><surname>Becker</surname><given-names>C</given-names></name></person-group>\n<source>German Childhood Cancer Registry - Annual Report</source>. (<year>2018</year>) (1980-2017). Institute of Medical Biostatistics, Epidemiology and Informatics (IMBEI) at the University Medical Center of the Johannes Gutenberg University Mainz (<year>2019</year>). Apr. [German Childhood Cancer Registry - Annual Report (<year>2018</year>). (1980-2017)].</mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bhakta</surname><given-names>N</given-names></name><name><surname>Force</surname><given-names>LM</given-names></name><name><surname>Allemani</surname><given-names>C</given-names></name><name><surname>Atun</surname><given-names>R</given-names></name><name><surname>Bray</surname><given-names>F</given-names></name><name><surname>Coleman</surname><given-names>MP</given-names></name><etal/></person-group>. <article-title>Childhood cancer burden: a review of global estimates</article-title>. <source>Lancet Oncol.</source> (<year>2019</year>) <volume>20</volume>:<fpage>e42</fpage>–<lpage>53</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S1470-2045(18)30761-7</pub-id><pub-id pub-id-type=\"pmid\">30614477</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Steliarova-Foucher</surname><given-names>E</given-names></name><name><surname>Colombet</surname><given-names>M</given-names></name><name><surname>Ries</surname><given-names>LAG</given-names></name><name><surname>Moreno</surname><given-names>F</given-names></name><name><surname>Dolya</surname><given-names>A</given-names></name><name><surname>Bray</surname><given-names>F</given-names></name><etal/></person-group>. <article-title>International incidence of childhood cancer, 2001–10: a population-based registry study</article-title>. <source>Lancet Oncol.</source> (<year>2017</year>) <volume>18</volume>:<fpage>719</fpage>–<lpage>31</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S1470-2045(17)30186-9</pub-id><pub-id pub-id-type=\"pmid\">28410997</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hudson</surname><given-names>MM</given-names></name><name><surname>Ness</surname><given-names>KK</given-names></name><name><surname>Gurney</surname><given-names>JG</given-names></name><name><surname>Mulrooney</surname><given-names>DA</given-names></name><name><surname>Chemaitilly</surname><given-names>W</given-names></name><name><surname>Krull</surname><given-names>KR</given-names></name><etal/></person-group>. <article-title>Clinical ascertainment of health outcomes among adults treated for childhood cancer</article-title>. <source>JAMA.</source> (<year>2013</year>) <volume>309</volume>:<fpage>2371</fpage>–<lpage>81</lpage>. <pub-id pub-id-type=\"doi\">10.1001/jama.2013.6296</pub-id><pub-id pub-id-type=\"pmid\">23757085</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mertens</surname><given-names>AC</given-names></name><name><surname>Liu</surname><given-names>Q</given-names></name><name><surname>Neglia</surname><given-names>JP</given-names></name><name><surname>Wasilewski</surname><given-names>K</given-names></name><name><surname>Leisenring</surname><given-names>W</given-names></name><name><surname>Armstrong</surname><given-names>GT</given-names></name><etal/></person-group>. <article-title>Cause-specific late mortality among 5-year survivors of childhood cancer: the childhood cancer survivor study</article-title>. <source>JNCI J Natl Cancer Inst.</source> (<year>2008</year>) <volume>100</volume>:<fpage>1368</fpage>–<lpage>79</lpage>. <pub-id pub-id-type=\"doi\">10.1093/jnci/djn310</pub-id><pub-id pub-id-type=\"pmid\">18812549</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boutouyrie</surname><given-names>P</given-names></name><name><surname>Tropeano</surname><given-names>AI</given-names></name><name><surname>Asmar</surname><given-names>R</given-names></name><name><surname>Gautier</surname><given-names>I</given-names></name><name><surname>Benetos</surname><given-names>A</given-names></name><name><surname>Lacolley</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Aortic stiffness is an independent predictor of primary coronary events in hypertensive patients: a longitudinal study</article-title>. <source>Hypertension.</source> (<year>2002</year>) <volume>39</volume>:<fpage>10</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1161/hy0102.099031</pub-id><pub-id pub-id-type=\"pmid\">11799071</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krystal</surname><given-names>JI</given-names></name><name><surname>Reppucci</surname><given-names>M</given-names></name><name><surname>Mayr</surname><given-names>T</given-names></name><name><surname>Fish</surname><given-names>JD</given-names></name><name><surname>Sethna</surname><given-names>C</given-names></name></person-group>. <article-title>Arterial stiffness in childhood cancer survivors: arterial stiffness in childhood cancer survivors</article-title>. <source>Pediatr Blood Cancer.</source> (<year>2015</year>) <volume>62</volume>:<fpage>1832</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1002/pbc.25547</pub-id><pub-id pub-id-type=\"pmid\">25895119</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Baulmann</surname><given-names>J</given-names></name><name><surname>Nürnberger</surname><given-names>J</given-names></name><name><surname>Slany</surname><given-names>J</given-names></name><name><surname>Schmieder</surname><given-names>R</given-names></name><name><surname>Schmidt-Trucksäss</surname><given-names>A</given-names></name><name><surname>Baumgart</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Arterielle Gefäßsteifigkeit und Pulswellenanalyse</article-title>. <source>DMW Dtsch Med Wochenschr.</source> (<year>2010</year>) <volume>135</volume>:<fpage>S4</fpage>–<lpage>14</lpage>. <pub-id pub-id-type=\"doi\">10.1055/s-0030-1249183</pub-id><pub-id pub-id-type=\"pmid\">20180172</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>DeLoach</surname><given-names>SS</given-names></name><name><surname>Townsend</surname><given-names>RR</given-names></name></person-group>. <article-title>Vascular stiffness: its measurement and significance for epidemiologic and outcome studies</article-title>. <source>Clin J Am Soc Nephrol.</source> (<year>2008</year>) <volume>3</volume>:<fpage>184</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.2215/CJN.03340807</pub-id><pub-id pub-id-type=\"pmid\">18178784</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Borchert-Mörlins</surname><given-names>B</given-names></name><name><surname>Memaran</surname><given-names>N</given-names></name><name><surname>Sauer</surname><given-names>M</given-names></name><name><surname>Maecker-Kolhoff</surname><given-names>B</given-names></name><name><surname>Sykora</surname><given-names>K-W</given-names></name><name><surname>Blöte</surname><given-names>R</given-names></name><etal/></person-group>. <article-title>Cardiovascular risk factors and subclinical organ damage after hematopoietic stem cell transplantation in pediatric age</article-title>. <source>Bone Marrow Transplant.</source> (<year>2018</year>) <volume>53</volume>:<fpage>983</fpage>–<lpage>92</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41409-018-0104-x</pub-id><pub-id pub-id-type=\"pmid\">29426831</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>von Korn</surname><given-names>P</given-names></name><name><surname>Müller</surname><given-names>J</given-names></name><name><surname>Quell</surname><given-names>C</given-names></name><name><surname>Tenius</surname><given-names>L</given-names></name><name><surname>Oberhoffer</surname><given-names>R</given-names></name><name><surname>Feuchtinger</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>Health-related physical fitness and arterial stiffness in childhood cancer survivors</article-title>. <source>Front Cardiovasc Med.</source> (<year>2019</year>) <volume>6</volume>:<fpage>63</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fcvm.2019.00063</pub-id><pub-id pub-id-type=\"pmid\">31157240</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fuemmeler</surname><given-names>BF</given-names></name><name><surname>Pendzich</surname><given-names>MK</given-names></name><name><surname>Clark</surname><given-names>K</given-names></name><name><surname>Lovelady</surname><given-names>C</given-names></name><name><surname>Rosoff</surname><given-names>P</given-names></name><name><surname>Blatt</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Diet, physical activity, and body composition changes during the first year of treatment for childhood acute leukemia and lymphoma</article-title>. <source>J Pediatr Hematol Oncol.</source> (<year>2013</year>) <volume>35</volume>:<fpage>437</fpage>–<lpage>43</lpage>. <pub-id pub-id-type=\"doi\">10.1097/MPH.0b013e318279cd3e</pub-id><pub-id pub-id-type=\"pmid\">23211695</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ness</surname><given-names>KK</given-names></name><name><surname>Leisenring</surname><given-names>WM</given-names></name><name><surname>Huang</surname><given-names>S</given-names></name><name><surname>Hudson</surname><given-names>MM</given-names></name><name><surname>Gurney</surname><given-names>JG</given-names></name><name><surname>Whelan</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Predictors of inactive lifestyle among adult survivors of childhood cancer: a report from the childhood cancer survivor study</article-title>. <source>Cancer.</source> (<year>2009</year>) <volume>115</volume>:<fpage>1984</fpage>–<lpage>94</lpage>. <pub-id pub-id-type=\"doi\">10.1002/cncr.24209</pub-id><pub-id pub-id-type=\"pmid\">19224548</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Booth</surname><given-names>FW</given-names></name><name><surname>Roberts</surname><given-names>CK</given-names></name><name><surname>Thyfault</surname><given-names>JP</given-names></name><name><surname>Ruegsegger</surname><given-names>GN</given-names></name><name><surname>Toedebusch</surname><given-names>RG</given-names></name></person-group>. <article-title>Role of inactivity in chronic diseases: evolutionary insight and pathophysiological mechanisms</article-title>. <source>Physiol Rev.</source> (<year>2017</year>) <volume>97</volume>:<fpage>1351</fpage>–<lpage>402</lpage>. <pub-id pub-id-type=\"doi\">10.1152/physrev.00019.2016</pub-id><pub-id pub-id-type=\"pmid\">28814614</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van Dalen</surname><given-names>EC</given-names></name><name><surname>Raphaël</surname><given-names>MF</given-names></name><name><surname>Caron</surname><given-names>HN</given-names></name><name><surname>Kremer</surname><given-names>LC</given-names></name></person-group>\n<article-title>Treatment including anthracyclines versus treatment not including anthracyclines for childhood cancer</article-title>. <source>Cochrane Database Syst Rev.</source> (<year>2009</year>) CD006647. <pub-id pub-id-type=\"doi\">10.1002/14651858.CD006647.pub2.</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boman</surname><given-names>KK</given-names></name><name><surname>Hörnquist</surname><given-names>L</given-names></name><name><surname>de Graaff</surname><given-names>L</given-names></name><name><surname>Rickardsson</surname><given-names>J</given-names></name><name><surname>Lannering</surname><given-names>B</given-names></name><name><surname>Gustafsson</surname><given-names>G</given-names></name></person-group>. <article-title>Disability, body image and sports/physical activity in adult survivors of childhood CNS tumors: population-based outcomes from a cohort study</article-title>. <source>J Neurooncol.</source> (<year>2013</year>) <volume>112</volume>:<fpage>99</fpage>–<lpage>106</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s11060-012-1039-5</pub-id><pub-id pub-id-type=\"pmid\">23354653</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ness</surname><given-names>KK</given-names></name><name><surname>Mertens</surname><given-names>AC</given-names></name><name><surname>Hudson</surname><given-names>MM</given-names></name><name><surname>Wall</surname><given-names>MM</given-names></name><name><surname>Leisenring</surname><given-names>WM</given-names></name><name><surname>Oeffinger</surname><given-names>KC</given-names></name><etal/></person-group>. <article-title>Limitations on physical performance and daily activities among long-term survivors of childhood cancer</article-title>. <source>Ann Intern Med.</source> (<year>2005</year>) <volume>143</volume>:<fpage>639</fpage>–<lpage>47</lpage>. <pub-id pub-id-type=\"doi\">10.7326/0003-4819-143-9-200511010-00007</pub-id><pub-id pub-id-type=\"pmid\">16263886</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Winter</surname><given-names>C</given-names></name><name><surname>Rosenbaum</surname><given-names>D</given-names></name></person-group>. <article-title>Sportinterventionsprogramme in der pädiatrischen Onkologie</article-title>. <source>Dtsch Z Für Sportmed.</source> (<year>2014</year>). <pub-id pub-id-type=\"doi\">10.5960/dzsm.2012.085</pub-id><pub-id pub-id-type=\"pmid\">9648386</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Götte</surname><given-names>M</given-names></name><name><surname>Kesting</surname><given-names>SV</given-names></name><name><surname>Winter</surname><given-names>CC</given-names></name><name><surname>Rosenbaum</surname><given-names>D</given-names></name><name><surname>Boos</surname><given-names>J</given-names></name></person-group>. <article-title>Motor performance in children and adolescents with cancer at the end of acute treatment phase</article-title>. <source>Eur J Pediatr.</source> (<year>2015</year>) <volume>174</volume>:<fpage>791</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00431-014-2460-x</pub-id><pub-id pub-id-type=\"pmid\">25428233</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kesting</surname><given-names>SV</given-names></name><name><surname>Götte</surname><given-names>M</given-names></name><name><surname>Seidel</surname><given-names>CC</given-names></name><name><surname>Rosenbaum</surname><given-names>D</given-names></name><name><surname>Boos</surname><given-names>J</given-names></name></person-group>. <article-title>Motor performance after treatment for pediatric bone tumors</article-title>. <source>J Pediatr Hematol Oncol.</source> (<year>2015</year>) <volume>37</volume>:<fpage>509</fpage>–<lpage>14</lpage>. <pub-id pub-id-type=\"doi\">10.1097/MPH.0000000000000396</pub-id><pub-id pub-id-type=\"pmid\">26207777</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wilson</surname><given-names>C</given-names></name><name><surname>Gawade</surname><given-names>P</given-names></name><name><surname>Ness</surname><given-names>K</given-names></name></person-group>. <article-title>Impairments that influence physical function among survivors of childhood cancer</article-title>. <source>Children.</source> (<year>2015</year>) <volume>2</volume>:<fpage>1</fpage>–<lpage>36</lpage>. <pub-id pub-id-type=\"doi\">10.3390/children2010001</pub-id><pub-id pub-id-type=\"pmid\">25692094</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kesting</surname><given-names>SV</given-names></name><name><surname>Götte</surname><given-names>M</given-names></name><name><surname>Seidel</surname><given-names>CC</given-names></name><name><surname>Rosenbaum</surname><given-names>D</given-names></name><name><surname>Boos</surname><given-names>J</given-names></name></person-group>. <article-title>One in four questioned children faces problems regarding reintegration into physical education at school after treatment for pediatric cancer: problems of reintegration into physical education</article-title>. <source>Pediatr Blood Cancer.</source> (<year>2016</year>) <volume>63</volume>:<fpage>737</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1002/pbc.25852</pub-id><pub-id pub-id-type=\"pmid\">26681662</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eime</surname><given-names>RM</given-names></name><name><surname>Young</surname><given-names>JA</given-names></name><name><surname>Harvey</surname><given-names>JT</given-names></name><name><surname>Charity</surname><given-names>MJ</given-names></name><name><surname>Payne</surname><given-names>WR</given-names></name></person-group>. <article-title>A systematic review of the psychological and social benefits of participation in sport for children and adolescents: informing development of a conceptual model of health through sport</article-title>. <source>Int J Behav Nutr Phys Act.</source> (<year>2013</year>) <volume>10</volume>:<fpage>98</fpage>. <pub-id pub-id-type=\"doi\">10.1186/1479-5868-10-98</pub-id><pub-id pub-id-type=\"pmid\">23945179</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Bös</surname><given-names>K</given-names></name><name><surname>Worth</surname><given-names>A</given-names></name><name><surname>Opper</surname><given-names>E</given-names></name><name><surname>Oberger</surname><given-names>J</given-names></name><name><surname>Woll</surname><given-names>A</given-names></name></person-group>. <article-title>Motorik-Modul: eine Studie zur motorischen Leistungsfähigkeit und körperlich-sportlichen Aktivität von Kindern und Jugendlichen in Deutschland; Abschlussbericht zum Forschungsprojekt. 1. Aufl., Stand: Januar</article-title> (<year>2009</year>). <publisher-loc>Baden-Baden</publisher-loc>: <publisher-name>Nomos-Verl</publisher-name>; (<year>2009</year>). <fpage>426</fpage> p. (Forschungsreihe / Bundesministerium für Familie, Senioren, Frauen und Jugend).</mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Neuhauser</surname><given-names>H</given-names></name><name><surname>Schienkiewitz</surname><given-names>A</given-names></name><name><surname>Schaffrath Rosario</surname><given-names>A</given-names></name><name><surname>Dortschy</surname><given-names>R</given-names></name><name><surname>Kurth</surname><given-names>BM</given-names></name></person-group>\n<source>Referenzperzentile für anthropometrische Maßzahlen und Blutdruck aus der Studie zur Gesundheit von Kindern und Jugendlichen in Deutschland (KiGGS)</source>. <publisher-loc>Berlin</publisher-loc>: <publisher-name>erweiterte Auflage, Robert Koch-Institut</publisher-name> (<year>2013</year>).</mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Elmenhorst</surname><given-names>J</given-names></name><name><surname>Hulpke-Wette</surname><given-names>M</given-names></name><name><surname>Barta</surname><given-names>C</given-names></name><name><surname>Dalla Pozza</surname><given-names>R</given-names></name><name><surname>Springer</surname><given-names>S</given-names></name><name><surname>Oberhoffer</surname><given-names>R</given-names></name></person-group>. <article-title>Percentiles for central blood pressure and pulse wave velocity in children and adolescents recorded with an oscillometric device</article-title>. <source>Atherosclerosis.</source> (<year>2015</year>) <volume>238</volume>:<fpage>9</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.atherosclerosis.2014.11.005</pub-id><pub-id pub-id-type=\"pmid\">25461733</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Alber</surname><given-names>V</given-names></name></person-group>\n<source>Gefäßsteifigkeitsindices bei Kindern mit Adipositas, Diabetes und Fettstoffwechselstörung- Vergleich mit einem Normkollektiv</source>. <publisher-loc>Munich</publisher-loc>: <publisher-name>Doctoral Dissertation, TUM School of Medicine, Technical University of Munich</publisher-name> (<year>2017</year>). p. <fpage>104</fpage>.</mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Duarte</surname><given-names>SV</given-names></name><name><surname>de Souza Rajão</surname><given-names>J</given-names></name><name><surname>Pinho</surname><given-names>JF</given-names></name><name><surname>dos Santos</surname><given-names>LM</given-names></name><name><surname>Alves-Neves</surname><given-names>CM</given-names></name><name><surname>Magalhães</surname><given-names>GS</given-names></name><etal/></person-group>. <article-title>Changes in aortic pulse wave components, pulse pressure amplification, and hemodynamic parameters of children and adolescents with type 1 diabetes</article-title>. <source>Pediatr Diabetes.</source> (<year>2019</year>) <volume>20</volume>:<fpage>202</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1111/pedi.12782</pub-id><pub-id pub-id-type=\"pmid\">30259609</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hock</surname><given-names>J</given-names></name><name><surname>Häcker</surname><given-names>AL</given-names></name><name><surname>Reiner</surname><given-names>B</given-names></name><name><surname>Oberhoffer</surname><given-names>R</given-names></name><name><surname>Hager</surname><given-names>A</given-names></name><name><surname>Ewert</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Functional outcome in contemporary children and young adults with tetralogy of Fallot after repair</article-title>. <source>Arch Dis Child.</source> (<year>2019</year>) <volume>104</volume>:<fpage>129</fpage>–<lpage>33</lpage>. <pub-id pub-id-type=\"doi\">10.1136/archdischild-2017-314733</pub-id><pub-id pub-id-type=\"pmid\">29970581</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tokgöz</surname><given-names>ST</given-names></name><name><surname>Yilmaz</surname><given-names>D</given-names></name><name><surname>Tokgöz</surname><given-names>Y</given-names></name><name><surname>Çelik</surname><given-names>B</given-names></name><name><surname>Bulut</surname><given-names>Y</given-names></name></person-group>. <article-title>The evaluation of arterial stiffness of essential hypertension and white coat hypertension in children: a case-control study</article-title>. <source>Cardiol Young.</source> (<year>2018</year>) <volume>28</volume>:<fpage>403</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1017/S1047951117002025</pub-id><pub-id pub-id-type=\"pmid\">29223189</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Götte</surname><given-names>M</given-names></name><name><surname>Kesting</surname><given-names>S</given-names></name><name><surname>Albrecht</surname><given-names>C</given-names></name><name><surname>Worth</surname><given-names>A</given-names></name><name><surname>Bös</surname><given-names>K</given-names></name><name><surname>Boos</surname><given-names>J</given-names></name></person-group>. <article-title>MOON-test - determination of motor performance in the pediatric oncology</article-title>. <source>Klin Pädiatr.</source> (<year>2013</year>) <volume>225</volume>:<fpage>133</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1055/s-0033-1343411</pub-id><pub-id pub-id-type=\"pmid\">23599231</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Cohen</surname><given-names>J</given-names></name></person-group>. <source>Statistical Power Analysis for the Behavioral Sciences. 2nd ed.</source>\n<publisher-loc>Hillsdale, NJ</publisher-loc>: <publisher-name>L. Erlbaum Associates</publisher-name> (<year>1988</year>). p. <fpage>567</fpage>.</mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mancia</surname><given-names>G</given-names></name><name><surname>Fagard</surname><given-names>R</given-names></name><name><surname>Narkiewicz</surname><given-names>K</given-names></name><name><surname>Redón</surname><given-names>J</given-names></name><name><surname>Zanchetti</surname><given-names>A</given-names></name><name><surname>Böhm</surname><given-names>M</given-names></name><etal/></person-group>\n<article-title>2013 ESH/ESC Guidelines for the management of arterial hypertension: the task force for the management of arterial hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC)</article-title>. <source>J Hypertens.</source> (<year>2013</year>) <volume>31</volume>:<fpage>1281</fpage>–<lpage>357</lpage>. <pub-id pub-id-type=\"doi\">10.1097/01.hjh.0000431740.32696.cc</pub-id><pub-id pub-id-type=\"pmid\">23817082</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lewington</surname><given-names>S</given-names></name><name><surname>Clarke</surname><given-names>R</given-names></name><name><surname>Qizilbash</surname><given-names>N</given-names></name><name><surname>Peto</surname><given-names>R</given-names></name><name><surname>Collins</surname><given-names>R</given-names></name></person-group>. <article-title>Age-specific relevance of usual blood pressure to vascular mortality: a meta-analysis of individual data for one million adults in 61 prospective studies</article-title>. <source>Lancet.</source> (<year>2002</year>) <volume>360</volume>:<fpage>1903</fpage>–<lpage>13</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0140-6736(02)11911-8</pub-id><pub-id pub-id-type=\"pmid\">12493255</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gibson</surname><given-names>TM</given-names></name><name><surname>Li</surname><given-names>Z</given-names></name><name><surname>Green</surname><given-names>DM</given-names></name><name><surname>Armstrong</surname><given-names>GT</given-names></name><name><surname>Mulrooney</surname><given-names>DA</given-names></name><name><surname>Srivastava</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Blood pressure status in adult survivors of childhood cancer: a report from the St. Jude lifetime cohort study</article-title>. <source>Cancer Epidemiol Biomarkers Prev.</source> (<year>2017</year>) <volume>26</volume>:<fpage>1705</fpage>–<lpage>13</lpage>. <pub-id pub-id-type=\"doi\">10.1158/1055-9965.EPI-17-0510</pub-id><pub-id pub-id-type=\"pmid\">29167278</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mertens</surname><given-names>AC</given-names></name><name><surname>Yasui</surname><given-names>Y</given-names></name><name><surname>Neglia</surname><given-names>JP</given-names></name><name><surname>Potter</surname><given-names>JD</given-names></name><name><surname>Nesbit</surname><given-names>M</given-names></name><name><surname>Ruccione</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Late mortality experience in five-year survivors of childhood and adolescent cancer: the childhood cancer survivor study</article-title>. <source>J Clin Oncol.</source> (<year>2013</year>) <volume>19</volume>:<fpage>3163</fpage>–<lpage>72</lpage>. <pub-id pub-id-type=\"doi\">10.1200/JCO.2001.19.13.3163</pub-id><pub-id pub-id-type=\"pmid\">11432882</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Veringa</surname><given-names>SJE</given-names></name><name><surname>van</surname><given-names>Dulmen-den Broeder E</given-names></name><name><surname>Kaspers</surname><given-names>GJL</given-names></name><name><surname>Veening</surname><given-names>MA</given-names></name></person-group>. <article-title>Blood pressure and body composition in long-term survivors of childhood acute lymphoblastic leukemia</article-title>. <source>Pediatr Blood Cancer.</source> (<year>2012</year>) <volume>58</volume>:<fpage>278</fpage>–<lpage>82</lpage>. <pub-id pub-id-type=\"doi\">10.1002/pbc.23251</pub-id><pub-id pub-id-type=\"pmid\">21793179</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Roman</surname><given-names>MJ</given-names></name><name><surname>Devereux</surname><given-names>RB</given-names></name><name><surname>Kizer</surname><given-names>JR</given-names></name><name><surname>Lee</surname><given-names>ET</given-names></name><name><surname>Galloway</surname><given-names>JM</given-names></name><name><surname>Ali</surname><given-names>T</given-names></name><etal/></person-group>. <article-title>Central pressure more strongly relates to vascular disease and outcome than does brachial pressure: the strong heart study</article-title>. <source>Hypertension.</source> (<year>2007</year>) <volume>50</volume>:<fpage>197</fpage>–<lpage>203</lpage>. <pub-id pub-id-type=\"doi\">10.1161/HYPERTENSIONAHA.107.089078</pub-id><pub-id pub-id-type=\"pmid\">17485598</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pini</surname><given-names>R</given-names></name><name><surname>Cavallini</surname><given-names>MC</given-names></name><name><surname>Palmieri</surname><given-names>V</given-names></name><name><surname>Marchionni</surname><given-names>N</given-names></name><name><surname>Di Bari</surname><given-names>M</given-names></name><name><surname>Devereux</surname><given-names>RB</given-names></name><etal/></person-group>\n<article-title>Central but not brachial blood pressure predicts cardiovascular events in an unselected geriatric population</article-title>. <source>J Am Coll Cardiol.</source> (<year>2008</year>) <volume>51</volume>:<fpage>2432</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jacc.2008.03.031</pub-id><pub-id pub-id-type=\"pmid\">18565402</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jenei</surname><given-names>Z</given-names></name><name><surname>Bárdi</surname><given-names>E</given-names></name><name><surname>Magyar</surname><given-names>MT</given-names></name><name><surname>Horváth</surname><given-names>Á</given-names></name><name><surname>Paragh</surname><given-names>G</given-names></name><name><surname>Kiss</surname><given-names>C</given-names></name></person-group>. <article-title>Anthracycline causes impaired vascular endothelial function and aortic stiffness in long term survivors of childhood cancer</article-title>. <source>Pathol Oncol Res.</source> (<year>2013</year>) <volume>19</volume>:<fpage>375</fpage>–<lpage>83</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s12253-012-9589-6</pub-id><pub-id pub-id-type=\"pmid\">23242567</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chaosuwannakit</surname><given-names>N</given-names></name><name><surname>D'Agostino</surname><given-names>R</given-names></name><name><surname>Hamilton</surname><given-names>CA</given-names></name><name><surname>Lane</surname><given-names>KS</given-names></name><name><surname>Ntim</surname><given-names>WO</given-names></name><name><surname>Lawrence</surname><given-names>J</given-names></name><etal/></person-group>. <article-title>Aortic stiffness increases upon receipt of anthracycline chemotherapy</article-title>. <source>J Clin Oncol.</source> (<year>2010</year>) <volume>28</volume>:<fpage>166</fpage>–<lpage>72</lpage>. <pub-id pub-id-type=\"doi\">10.1200/JCO.2009.23.8527</pub-id><pub-id pub-id-type=\"pmid\">19901105</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dengel</surname><given-names>DR</given-names></name><name><surname>Kelly</surname><given-names>AS</given-names></name><name><surname>Zhang</surname><given-names>L</given-names></name><name><surname>Hodges</surname><given-names>JS</given-names></name><name><surname>Baker</surname><given-names>KS</given-names></name><name><surname>Steinberger</surname><given-names>J</given-names></name></person-group>. <article-title>Signs of early sub-clinical atherosclerosis in childhood cancer survivors: endothelial function in cancer survivors</article-title>. <source>Pediatr Blood Cancer.</source> (<year>2014</year>) <volume>61</volume>:<fpage>532</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1002/pbc.24829</pub-id><pub-id pub-id-type=\"pmid\">24155068</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ottensmeier</surname><given-names>H</given-names></name><name><surname>Schlegel</surname><given-names>PG</given-names></name><name><surname>Eyrich</surname><given-names>M</given-names></name><name><surname>Wolff</surname><given-names>JE</given-names></name><name><surname>Juhnke</surname><given-names>B-O</given-names></name><name><surname>von Hoff</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Treatment of children under 4 years of age with medulloblastoma and ependymoma in the HIT2000/HIT-REZ 2005 trials: neuropsychological outcome 5 years after treatment</article-title>. <source>PLoS ONE.</source> (<year>2020</year>) <volume>15</volume>:<fpage>e0227693</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0227693</pub-id><pub-id pub-id-type=\"pmid\">31971950</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lopes</surname><given-names>VP</given-names></name><name><surname>Stodden</surname><given-names>DF</given-names></name><name><surname>Bianchi</surname><given-names>MM</given-names></name><name><surname>Maia</surname><given-names>JAR</given-names></name><name><surname>Rodrigues</surname><given-names>LP</given-names></name></person-group>. <article-title>Correlation between BMI and motor coordination in children</article-title>. <source>J Sci Med Sport.</source> (<year>2012</year>) <volume>15</volume>:<fpage>38</fpage>–<lpage>43</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jsams.2011.07.005</pub-id><pub-id pub-id-type=\"pmid\">21831708</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rustler</surname><given-names>V</given-names></name><name><surname>Hagerty</surname><given-names>M</given-names></name><name><surname>Daeggelmann</surname><given-names>J</given-names></name><name><surname>Marjerrison</surname><given-names>S</given-names></name><name><surname>Bloch</surname><given-names>W</given-names></name><name><surname>Baumann</surname><given-names>FT</given-names></name></person-group>. <article-title>Exercise interventions for patients with pediatric cancer during inpatient acute care: a systematic review of literature</article-title>. <source>Pediatr Blood Cancer.</source> (<year>2017</year>) <volume>64</volume>:<fpage>e26567</fpage>. <pub-id pub-id-type=\"doi\">10.1002/pbc.26567</pub-id><pub-id pub-id-type=\"pmid\">28423225</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Braam</surname><given-names>KI</given-names></name><name><surname>van der Torre</surname><given-names>P</given-names></name><name><surname>Takken</surname><given-names>T</given-names></name><name><surname>Veening</surname><given-names>MA</given-names></name><name><surname>van</surname><given-names>Dulmen-den Broeder E</given-names></name><name><surname>Kaspers</surname><given-names>GJ</given-names></name></person-group>\n<article-title>Physical exercise training interventions for children and young adults during and after treatment for childhood cancer</article-title>. <source>Cochrane Database Syst Rev.</source> (<year>2016</year>) <volume>2016</volume>:<fpage>CD008796</fpage>\n<pub-id pub-id-type=\"doi\">10.1002/14651858.CD008796.pub3</pub-id></mixed-citation></ref><ref id=\"B48\"><label>48.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Speyer</surname><given-names>E</given-names></name><name><surname>Herbinet</surname><given-names>A</given-names></name><name><surname>Vuillemin</surname><given-names>A</given-names></name><name><surname>Briançon</surname><given-names>S</given-names></name><name><surname>Chastagner</surname><given-names>P</given-names></name></person-group>. <article-title>Effect of adapted physical activity sessions in the hospital on health-related quality of life for children with cancer: a cross-over randomized trial</article-title>. <source>Pediatr Blood Cancer.</source> (<year>2010</year>) <volume>55</volume>:<fpage>1160</fpage>–<lpage>6</lpage>. <pub-id pub-id-type=\"doi\">10.1002/pbc.22698</pub-id><pub-id pub-id-type=\"pmid\">20672368</pub-id></mixed-citation></ref><ref id=\"B49\"><label>49.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Götte</surname><given-names>M</given-names></name><name><surname>Kesting</surname><given-names>S</given-names></name><name><surname>Winter</surname><given-names>C</given-names></name><name><surname>Rosenbaum</surname><given-names>D</given-names></name><name><surname>Boos</surname><given-names>J</given-names></name></person-group>. <article-title>Experience of barriers and motivations for physical activities and exercise during treatment of pediatric patients with cancer: exercise barriers during cancer treatment</article-title>. <source>Pediatr Blood Cancer.</source> (<year>2014</year>) <volume>61</volume>:<fpage>1632</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1002/pbc.25071</pub-id><pub-id pub-id-type=\"pmid\">24753116</pub-id></mixed-citation></ref><ref id=\"B50\"><label>50.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Alias</surname><given-names>H</given-names></name><name><surname>Mohd Nazi</surname><given-names>NA</given-names></name><name><surname>Lau Sie Chong</surname><given-names>D</given-names></name></person-group>. <article-title>Participation in physical activity and physical education in school among children with acute lymphoblastic leukemia after intensive chemotherapy</article-title>. <source>Front Pediatr.</source> (<year>2019</year>) <volume>7</volume>:<fpage>73</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fped.2019.00073</pub-id><pub-id pub-id-type=\"pmid\">30937299</pub-id></mixed-citation></ref><ref id=\"B51\"><label>51.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Morales</surname><given-names>JS</given-names></name><name><surname>Valenzuela</surname><given-names>PL</given-names></name><name><surname>Rincón-Castanedo</surname><given-names>C</given-names></name><name><surname>Takken</surname><given-names>T</given-names></name><name><surname>Fiuza-Luces</surname><given-names>C</given-names></name><name><surname>Santos-Lozano</surname><given-names>A</given-names></name></person-group>. <article-title>Exercise training in childhood cancer: a systematic review and meta-analysis of randomized controlled trials</article-title>. <source>Cancer Treat Rev.</source> (<year>2018</year>) <volume>70</volume>:<fpage>154</fpage>–<lpage>67</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ctrv.2018.08.012</pub-id><pub-id pub-id-type=\"pmid\">30218787</pub-id></mixed-citation></ref><ref id=\"B52\"><label>52.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Santa Mina</surname><given-names>D</given-names></name><name><surname>Sabiston</surname><given-names>CM</given-names></name><name><surname>Au</surname><given-names>D</given-names></name><name><surname>Fong</surname><given-names>AJ</given-names></name><name><surname>Capozzi</surname><given-names>LC</given-names></name><name><surname>Langelier</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Connecting people with cancer to physical activity and exercise programs: a pathway to create accessibility and engagement</article-title>. <source>Curr Oncol.</source> (<year>2018</year>) <volume>25</volume>:<fpage>149</fpage>–<lpage>62</lpage>. <pub-id pub-id-type=\"doi\">10.3747/co.25.3977</pub-id><pub-id pub-id-type=\"pmid\">29719431</pub-id></mixed-citation></ref><ref id=\"B53\"><label>53.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Spreafico</surname><given-names>F</given-names></name><name><surname>Murelli</surname><given-names>M</given-names></name><name><surname>Timmons</surname><given-names>BW</given-names></name><name><surname>Massimino</surname><given-names>M</given-names></name><name><surname>Barr</surname><given-names>R</given-names></name></person-group>. <article-title>Sport activities and exercise as part of routine cancer care in children and adolescents</article-title>. <source>Pediatr Blood Cancer.</source>\n<volume>66</volume>:<fpage>e27826</fpage>. <pub-id pub-id-type=\"doi\">10.1002/pbc.27826</pub-id><pub-id pub-id-type=\"pmid\">31115152</pub-id></mixed-citation></ref><ref id=\"B54\"><label>54.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Trost</surname><given-names>SG</given-names></name></person-group>\n<article-title>State of the art reviews: measurement of physical activity in children and adolescents</article-title>. <source>Am J Lifestyle Med.</source> (<year>2007</year>) <volume>1</volume>:<fpage>299</fpage>–<lpage>314</lpage>. <pub-id pub-id-type=\"doi\">10.1177/1559827607301686</pub-id></mixed-citation></ref></ref-list><glossary><def-list><title>Abbreviations</title><def-item><term>PWV</term><def><p>Pulse Wave Velocity</p></def></def-item><def-item><term>WHO</term><def><p>World Health Organization</p></def></def-item><def-item><term>cSBP</term><def><p>central systolic blood pressure</p></def></def-item><def-item><term>pSBP</term><def><p>peripheral systolic blood pressure</p></def></def-item><def-item><term>pDBP</term><def><p>peripheral diastolic blood pressure</p></def></def-item><def-item><term>BMI</term><def><p>Body mass index</p></def></def-item><def-item><term>MOON</term><def><p>Motor performance in pediatric oncology</p></def></def-item><def-item><term>KiGGS</term><def><p>German Health Interviews and Examination Survey for Children and Adolescents.</p></def></def-item></def-list></glossary></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Neurosci</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Neurosci</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Neurosci.</journal-id><journal-title-group><journal-title>Frontiers in Neuroscience</journal-title></journal-title-group><issn pub-type=\"ppub\">1662-4548</issn><issn pub-type=\"epub\">1662-453X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32848578</article-id><article-id pub-id-type=\"pmc\">PMC7431624</article-id><article-id pub-id-type=\"doi\">10.3389/fnins.2020.00822</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Neuroscience</subject><subj-group><subject>Brief Research Report</subject></subj-group></subj-group></article-categories><title-group><article-title>A Panel of Synapse-Related Genes as a Biomarker for Gliomas</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Ji</surname><given-names>Xiangwen</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/923271/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zhang</surname><given-names>Hongwei</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/1004874/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Cui</surname><given-names>Qinghua</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/946363/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Biomedical Informatics, Center for Non-coding RNA Medicine, MOE Key Lab of Cardiovascular Sciences, School of Basic Medical Sciences, Peking University</institution>, <addr-line>Beijing</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Department of Physiology and Pathophysiology, Center for Non-coding RNA Medicine, MOE Key Lab of Cardiovascular Sciences, School of Basic Medical Sciences, Peking University</institution>, <addr-line>Beijing</addr-line>, <country>China</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Neurosurgery, Sanbo Brain Hospital, Capital Medical University</institution>, <addr-line>Beijing</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Dongqing Wei, Shanghai Jiao Tong University, China</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Oksana Sorokina, University of Edinburgh, United Kingdom; Anatoly Sorokin, University of Liverpool, United Kingdom</p></fn><corresp id=\"c001\">*Correspondence: Qinghua Cui, <email>cuiqinghua@hsc.pku.edu.cn</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Systems Biology, a section of the journal Frontiers in Neuroscience</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>14</volume><elocation-id>822</elocation-id><history><date date-type=\"received\"><day>04</day><month>3</month><year>2020</year></date><date date-type=\"accepted\"><day>14</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Ji, Zhang and Cui.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Ji, Zhang and Cui</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Gliomas are the most common primary brain cancers. In recent years, <italic>IDH</italic> mutation and 1p/19q codeletion have been suggested as biomarkers for the diagnosis, treatment, and prognosis of gliomas. However, these biomarkers are only effective for a part of glioma patients, and thus more biomarkers are still emergently needed. Recently, an electrochemical communication between normal neurons and glioma cells by neuro-glioma synapse has been reported. Moreover, it was discovered that breast-to-brain metastasis tumor cells have pseudo synapses with neurons, and these synapses were indicated to promote tumor progression and metastasis. Based on the above observations, we first curated a panel of 17 synapse-related genes and then proposed a metric, synapse score to quantify the “stemness” for each sample of 12 glioma gene expression datasets from TCGA, CGGA, and GEO. Strikingly, synapse score showed excellent predictive ability for the prognosis, diagnosis, and grading of gliomas. Moreover, being compared with the two established biomarkers, <italic>IDH</italic> mutation and 1p/19q codeletion, synapse score demonstrated independent and better predictive performance. In conclusion, this study proposed a quantitative method, synapse score, as an efficient biomarker for monitoring gliomas.</p></abstract><kwd-group><kwd>glioma</kwd><kwd>synapse</kwd><kwd>biomarker</kwd><kwd>survival</kwd><kwd>WHO grade</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Natural Science Foundation of China<named-content content-type=\"fundref-id\">10.13039/501100001809</named-content></funding-source><award-id rid=\"cn001\">81670462</award-id><award-id rid=\"cn001\">81970440</award-id><award-id rid=\"cn001\">81921001</award-id></award-group></funding-group><counts><fig-count count=\"3\"/><table-count count=\"1\"/><equation-count count=\"0\"/><ref-count count=\"38\"/><page-count count=\"8\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Brain and other nervous system cancers are estimated to take up 1.4% of new cancers but 2.9% of cancer deaths in 2019 (<xref rid=\"B8\" ref-type=\"bibr\">Brain and Other Nervous System Cancer, 2019</xref>). Gliomas are the most frequent of these cancers, including astrocytoma (including glioblastoma), oligodendroglioma, ependymoma, oligoastrocytoma (mixed glioma), malignant glioma, not otherwise specified (NOS) glioma, and a few rare histologies (<xref rid=\"B25\" ref-type=\"bibr\">Ostrom et al., 2016</xref>). The World Health Organization (WHO) classified gliomas into grades I to IV and introduced biomarkers of <italic>IDH</italic> mutation and 1p/19q codeletion in the 2016 edition (<xref rid=\"B20\" ref-type=\"bibr\">Louis et al., 2007</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Wesseling and Capper, 2018</xref>). Glioblastoma (WHO grade IV) accounts for about half of gliomas, with a median survival of less than 2 years (<xref rid=\"B15\" ref-type=\"bibr\">Gramatzki et al., 2016</xref>; <xref rid=\"B25\" ref-type=\"bibr\">Ostrom et al., 2016</xref>). Gliomas with lower grade have a diverse prognosis, either progressing to be as poor as glioblastoma or living more than 10 years after effective treatment (<xref rid=\"B28\" ref-type=\"bibr\">Ruda and Soffietti, 2017</xref>).</p><p>Over the years, with the fast improvement of omics and big data technology, RNA sequencing has been developing toward lower cost and higher throughput, producing a large amount of biological and medical data, which provides great convenience for life science research (<xref rid=\"B6\" ref-type=\"bibr\">Bolouri et al., 2016</xref>). Impelled by advantage of big data analysis, numerous biomarkers have been found in the diagnosis and prognosis of gliomas (<xref rid=\"B19\" ref-type=\"bibr\">Kros et al., 2015</xref>). Gene set enrichment analysis (GSEA) provides a facility to extract effective information from a large number of RNA expression data (<xref rid=\"B29\" ref-type=\"bibr\">Subramanian et al., 2005</xref>). Moreover, single sample GSEA (ssGSEA) can calculate without group information and give every sample an enrichment score (<xref rid=\"B3\" ref-type=\"bibr\">Barbie et al., 2009</xref>). The Biomarkers such as <italic>IDH</italic> mutation and 1p/19q codeletion provided help for monitoring the development and prognosis of gliomas but are only effective for a part of patients (<xref rid=\"B1\" ref-type=\"bibr\">Aibaidula et al., 2017</xref>). Therefore, given the enormous severity of gliomas, more biomarkers are emergently needed.</p><p>It is recently reported that neuron and glioma have electrochemical communication through AMPA receptor-dependent synapses between presynaptic neurons and postsynaptic glioma cells (<xref rid=\"B33\" ref-type=\"bibr\">Venkataramani et al., 2019</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Venkatesh et al., 2019</xref>). These observations suggest that the neural synaptic electrochemical connections promote glioma progression. Simultaneously, an appearance of glutamatergic “pseudo-tripartite” synapses between breast-to-brain metastasis tumor cells and neurons was observed (<xref rid=\"B38\" ref-type=\"bibr\">Zeng et al., 2019</xref>). Based on these anatomical and cytological findings, we hypothesized that the synapse-related genes can be used as a biomarker for glioma prognosis. To confirm this hypothesis, here we first curated a list of genes involved in synapse-related functions and then performed ssGSEA analysis for glioma gene expression datasets from the Cancer Genome Atlas (TCGA), the Chinese Glioma Genome Atlas (CGGA), and the Gene Expression Omnibus (GEO). Strikingly, these synapse-related genes were found to be an independent and effective biomarker for gliomas.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Gene Expression Datasets and Analysis</title><p>RNAseq data, normalized in fragments per kilo-base per million mapped fragments, as well as sample and clinical information were obtained from TCGA data portal<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup>. WHO grade, <italic>IDH</italic> mutation status, and 1p/19q codeletion status were obtained from the study by <xref rid=\"B10\" ref-type=\"bibr\">Ceccarelli et al. (2016)</xref>. CGGA<sup><xref ref-type=\"fn\" rid=\"footnote2\">2</xref></sup> provides tumor gene expression data for thousands of glioma patients (including one microarray and two RNAseq batches), as well as corresponding clinical data. The calculation and presentation of the results will be conducted separately due to different platforms and batches. In addition, glioma microarray gene expression profiling data (GSE4290, GSE16011, GSE50161, GSE52009, GSE54004, GSE61374, and GSE107850) were available at GEO datasets<sup><xref ref-type=\"fn\" rid=\"footnote3\">3</xref></sup>. Gene expression data were structured with gene symbols as row names and sample IDs as column names; duplicate gene symbols were averaged using their median value.</p></sec><sec id=\"S2.SS2\"><title>Synapse-Related Genes Screening</title><p>Gene ontology (GO) terms, which were related to synapse, neuron, neurotransmitter transport, glutamate receptor, or cell junction, were selected from NCBI<sup><xref ref-type=\"fn\" rid=\"footnote4\">4</xref></sup>. Using ssGSEA, we calculated enrichment scores (ESs) for each GO term and each sample in two CGGA RNAseq batches. The ssGSEA algorithm was performed by python (v3.6.8) package gseapy (v0.9.13), which is a python wrapper for GSEA and ssGSEA. The minimum number of genes in the gene set was set as 10, and the maximum was 1,000. As a result, 163 of 581 terms were retained. Cox regression models were used to calculate the hazard ratios (HRs) and <italic>p</italic>-values for ESs of each GO term. We used CoxPHFitter from python package lifelines (v0.23.7) to fit Cox models. Default parameters were used except the data frame and the column names of survival times and events. <italic>P</italic>-values were adjusted using Benjamini-Hochberg method. The false discovery rates (FDRs) of the two batches are multiplied to calculate the combined FDR (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary File S1</xref>). The terms with different directions in two batches (HR < 1 in one batch and HR > 1 in the other) were excluded. Terms with top 10 smallest combined FDR values, except “peripheral nervous system neuron development” (GO:0048935) as gliomas are located in the central nervous system, are used for subsequent analysis (ionotropic glutamate receptor signaling pathway, AMPA glutamate receptor complex, regulation of short-term neuronal synaptic plasticity, dopaminergic synapse, synapse maturation, excitatory postsynaptic potential, parallel fiber to Purkinje cell synapse, synapse organization, and regulation of AMPA receptor activity). Next, we evaluated the HRs and <italic>p</italic>-values of 171 genes (eight genes are not in the datasets) from these nine GO terms and calculated the combined FDRs (<xref ref-type=\"supplementary-material\" rid=\"DS2\">Supplementary File S2</xref>). One hundred forty-four genes were filtered out with the same directions in two data batches. Then we obtained ESs of genes with top <italic>n</italic> (<italic>n</italic> = 1, 2, …, 144) smallest combined FDRs for each sample in two data batches. After we evaluated the combined FDRs of every gene set, the gene set with the top 17 genes were selected (<xref ref-type=\"supplementary-material\" rid=\"DS3\">Supplementary File S3</xref>). Finally, using this synapse-related 17-gene set, we performed ssGSEA (default parameters) and calculated ESs for samples of TCGA, CGGA, and GEO datasets. We defined the ES as synapse score.</p></sec><sec id=\"S2.SS3\"><title>Statistical Analysis</title><p>Kaplan–Meier (K–M) curves and Cox proportional hazards regression were performed by R packages survival (v2.44-1.1) and survminer (v0.4.6) and python package lifelines (v0.23.7). Log rank test was used to calculate the difference between two K–M curves. Significance of difference between two groups of continuous variables was analyzed by two-sided Wilcoxon rank sum test. Receiver operating characteristic (ROC) curve and area under ROC curve (AUROC) were processed by R package pROC (<xref rid=\"B27\" ref-type=\"bibr\">Robin et al., 2011</xref>) (v1.15.3). All statistical significances above were calculated by R (v3.5.2). Spearman’s correlation analysis was applied to evaluate the correlation using python package scipy (v1.2.1). <italic>P</italic>-values < 0.05 were considered significant.</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>The Screening of Synapse-Related Genes</title><p>In order to investigate whether synapse-related genes can be biomarkers for glioma patients, we first curated a list of GO terms associated with synapse, neuron, neurotransmitter transport, glutamate receptor, or cell junction. After excluding the terms with less than 10 or more than 1,000 genes, 163 terms were retained. Then we evaluated the survival prediction performances of these gene sets in two CGGA RNAseq batches using ssGSEA and Cox regression (<xref ref-type=\"supplementary-material\" rid=\"DS1\">Supplementary File S1</xref>). Most (118/163) of the terms were found to have HR < 1 in both data batches. The 10 best performed terms were further selected, and “peripheral nervous system neuron development” (GO:0048935) is excluded as gliomas are located in the central nervous system (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>). As a result, 171 genes were collected.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>The screening of synapse-related genes. <bold>(A)</bold> The survival prognostic performances of 163 synapse-related gene ontology terms. The chosen terms were labeled. <bold>(B)</bold> The prognostic performances of 171 collected genes. The finally selected genes were labeled. <bold>(C)</bold> The prognostic performances of gene sets with different sizes. Min<sub><italic>one gene</italic></sub>: the best performance of single collected gene. The prognostic performances were evaluated by hazard ratios (HRs) of Cox regression. FDRs were calculated by Benjamini-Hochberg method. <bold>(D)</bold> The distribution of combined FDRs of 10,000 random 17-gene sets. Arrow: the combined FDR of our synapse-related 17-gene set.</p></caption><graphic xlink:href=\"fnins-14-00822-g001\"/></fig><p>Afterward, we assessed the prognostic performances of these genes using Cox regression (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref> and <xref ref-type=\"supplementary-material\" rid=\"DS2\">Supplementary File S2</xref>). 144 genes were filtered out with the same directions (both HRs < 1 or both HRs > 1) in two data batches. To further trim the gene set, we calculated ESs of gene sets which include the top <italic>n</italic> (<italic>n</italic> = 1, 2, …, 144) best performed genes and evaluated their survival prognostic abilities (<xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref> and <xref ref-type=\"supplementary-material\" rid=\"DS3\">Supplementary File S3</xref>). In most (142/144) cases, the gene sets performed better than any of the 144 genes on its own. Finally, the gene set with 17 genes was selected (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref> and <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table S1</xref>), including profilin 1 (<italic>PFN1</italic>), SH3 and multiple ankyrin repeat domains 2 (<italic>SHANK2</italic>), calcium voltage-gated channel auxiliary subunit gamma 2 (<italic>CACNG2</italic>), tenascin R (<italic>TNR</italic>), shisa family member 7 (<italic>SHISA7</italic>), cholinergic receptor nicotinic beta 2 subunit (<italic>CHRNB2</italic>), glutamate ionotropic receptor NMDA type subunit 3A (<italic>GRIN3A</italic>), mitogen-activated protein kinase eight interacting protein 2 (<italic>MAPK8IP2</italic>), glutamate ionotropic receptor delta type subunit 1 (<italic>GRID1</italic>), unc-13 homolog A (<italic>UNC13A</italic>), LDL receptor-related protein 4 (<italic>LRP4</italic>), syntabulin (<italic>SYBU</italic>), solute carrier family 16 member 3 (<italic>SLC16A3</italic>), dystrophin-related protein 2 (<italic>DRP2</italic>), glutamate ionotropic receptor kainate type subunit 4 (<italic>GRIK4</italic>), glutamate ionotropic receptor NMDA type subunit 2C (<italic>GRIN2C</italic>), and immunoglobin superfamily member 21 (<italic>IGSF21</italic>).</p><p>As the next few gene sets, neurotransmitter uptake (GO:0001504), glutamate receptor signaling pathway (GO:0007215), NMDA selective glutamate receptor complex (GO:0017146), and glutamate receptor activity (GO:0008066) have combined FDRs of similar magnitudes (<5 × 10<sup>–35</sup>); the choice of top 10 terms could be too arbitrary. It may be useful to include them in subsequent analyses. We took these terms into consideration one by one and performed the same steps of screening and trimming described above. The inclusion of the term neurotransmitter uptake did not change the final result, and the same 17 genes were screened out. As for the other three terms, they all resulted in a 20-gene set, adding potassium voltage-gated channel subfamily B member 1 (<italic>KCNB1</italic>), nicastrin (<italic>NCSTN</italic>), and phospholipase C beta 1 (<italic>PLCB1</italic>) to the previous 17-gene set. But its combined <italic>p</italic>-value (2.38 × 10<sup>–64</sup>) was a little worse than the previous 17-gene set (3.70 × 10<sup>–66</sup>). Although there are still many significant terms, like focal adhesion (GO:0005925) at #15, we could not consider more due to the time complexity of subsequent screening and trimming. Finally, we decided to use the 17-gene set for future validations.</p><p>To further verify the efficiency of the 17-gene set, a permutation experiment was performed. After randomly selecting 10,000 sets with 17 genes from all the 23,271 genes that exist in both batches of datasets, we tested their prognostic abilities by ssGSEA and Cox regression. As a result, the combined FDR of the selected 17-gene set ranked first in all random gene sets ascendingly (<xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref> and <xref ref-type=\"supplementary-material\" rid=\"DS4\">Supplementary File S4</xref>).</p></sec><sec id=\"S3.SS2\"><title>The Panel of Synapse-Related Genes Serves as a Novel Biomarker for Gliomas</title><p>Using the 17 collected synapse-related genes, we performed ssGSEA to TCGA, CGGA, and GEO datasets, and the ESs, defined as synapse score, were used for survival analysis. The results show that glioma patients with higher synapse scores have longer overall survival time (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). Cox regression analysis also shows the same results (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). Moreover, patients with higher WHO grade have significantly lower synapse scores (<xref ref-type=\"fig\" rid=\"F3\">Figure 3</xref> and <xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1</xref>), which agrees with the survival analysis. In addition, it is worthy to mention that there were normal brain samples in datasets GSE4290 (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>), GSE16011 (<xref ref-type=\"fig\" rid=\"F3\">Figure 3D</xref>), and GSE50161 (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1c</xref>). The synapse scores of normal samples were significantly higher than glioma samples, suggesting that the synapse score shows an ability to distinguish between glioma and normal brain tissue by giving a cutoff value, which reveals a potential diagnostic application of synapse score. ROC analyses were further used to evaluate the diagnostic ability; the areas under the curve (AUCs) of GSE4290, GSE16011, and GSE50161 datasets are 0.89, 0.94, and 0.99, respectively (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S2</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Kaplan–Meier curve of overall survival. <bold>(A)</bold> TCGA lower grade glioma (LGG) and glioblastoma multiforme (GBM). <bold>(B)</bold> CGGA Microarray. <bold>(C)</bold> GSE107850 from GEO datasets. Group was separated by the median value of synapse scores. Differences between two curves were estimated by log-rank test.</p></caption><graphic xlink:href=\"fnins-14-00822-g002\"/></fig><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>The predictive ability of synapse score adjusted using WHO grade, <italic>IDH</italic> mutation, and 1p/19q codeletion.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Datasets</td><td valign=\"top\" align=\"center\" colspan=\"4\" rowspan=\"1\">Hazard ratio (95% CI) (no. of samples)<hr/></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Unadjusted</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Grade-adjusted</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\"><italic>IDH</italic> Status-adjusted</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1p/19q Codeletion-adjusted</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CGGA Microarray</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0379 (0.0151–0.0947)*** (<italic>n</italic> = 298)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.270 (0.0852–0.856)* (<italic>n</italic> = 295)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0645 (0.0249–0.167)*** (<italic>n</italic> = 296)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.0491 (9.04 × 10<sup>–3</sup> to 0.267)*** (<italic>n</italic> = 91)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CGGA RNAseq batch 1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.77 × 10<sup>–5</sup> (1.15 × 10<sup>–5</sup> to 2.89 × 10<sup>–4</sup>)*** (<italic>n</italic> = 311)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.04 × 10<sup>–3</sup> (1.57 × 10<sup>–4</sup> to 6.85 × 10<sup>–3</sup>)*** (<italic>n</italic> = 307)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.97 × 10<sup>–5</sup> (3.49 × 10<sup>–6</sup> to 2.52 × 10<sup>–4</sup>)*** (<italic>n</italic> = 310)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.93 × 10<sup>–4</sup> (4.61 × 10<sup>–5</sup> to 1.86 × 10<sup>–3</sup>)*** (<italic>n</italic> = 303)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">CGGA RNAseq batch 2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.31 × 10<sup>–4</sup> (9.18 × 10<sup>–5</sup> to 1.20 × 10<sup>–3</sup>)*** (<italic>n</italic> = 619)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.17 × 10<sup>–3</sup> (1.99 × 10<sup>–3</sup> to 0.0423)*** (<italic>n</italic> = 619)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5.62 × 10<sup>–3</sup> (1.16 × 10<sup>–3</sup> to 0.0272)*** (<italic>n</italic> = 574)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7.85 × 10<sup>–4</sup> (1.72 × 10<sup>–4</sup> to 3.58 × 10<sup>–3</sup>)*** (<italic>n</italic> = 555)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">TCGA GBM + LGG</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.84 × 10<sup>–7</sup> (4.00 × 10<sup>–8</sup> to 2.02 × 10<sup>–6</sup>)*** (<italic>n</italic> = 695)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8.18 × 10<sup>–3</sup> (4.13 × 10<sup>–4</sup> to 0.162)** (<italic>n</italic> = 634)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4.83 × 10<sup>–3</sup> (2.96 × 10<sup>–4</sup> to 0.0788)*** (<italic>n</italic> = 685)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9.48 × 10<sup>–7</sup> (1.03 × 10<sup>–7</sup> to 8.77 × 10<sup>–6</sup>)*** (<italic>n</italic> = 688)</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">GSE107850</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.53 × 10<sup>–4</sup> (2.20 × 10<sup>–6</sup> to 0.0107)*** (<italic>n</italic> = 195)</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3.09 × 10<sup>–4</sup> (3.03 × 10<sup>–6</sup> to 0.0314)*** (<italic>n</italic> = 180)</td><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/></tr></tbody></table><table-wrap-foot><attrib><italic>Hazard ratio (HR) and 95% confidence interval (95% CI) of synapse score using univariate and multivariate Cox proportional hazards regression models for gliomas were shown. HR with 95% CI that does not include one is considered significant. *p < 0.05, **p < 0.01, ***p < 0.001.</italic></attrib></table-wrap-foot></table-wrap><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Synapse scores were significantly lower in higher grade gliomas. <bold>(A)</bold> TCGA lower grade glioma (LGG) and glioblastoma multiforme (GBM). <bold>(B)</bold> CGGA RNAseq batch 1. <bold>(C)</bold> GSE4290. <bold>(D)</bold> GSE16011. Significances of difference between two groups were analyzed by two-side Wilcoxon rank sum test. *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, ***<italic>p</italic> < 0.001.</p></caption><graphic xlink:href=\"fnins-14-00822-g003\"/></fig></sec><sec id=\"S3.SS3\"><title>Comparison of Synapse Score With Established Biomarkers</title><p><italic>IDH</italic> mutation and 1p/19q codeletion are two established biomarkers for gliomas. Both biomarkers provided great help for monitoring glioma development, but both are effective on only some patients. Therefore, it is interesting to explore whether synapse score is an independent biomarker and whether synapse score is better than the established biomarkers or not. For doing so, we first analyzed the relationship of synapse scores with <italic>IDH</italic> mutation and 1p/19q codeletion status. We found that <italic>IDH</italic>-mut gliomas were associated with significantly higher synapse scores than <italic>IDH</italic>-wt ones (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figures S3a–d</xref>). And 1p/19q codeletion gliomas represent higher synapse scores than non-codeletion ones (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figures S3e–h</xref>). Moreover, after removing the effects of the two established biomarkers using multivariate Cox regression model, we revealed that synapse score is an independent biomarker for predicting prolonged overall survival in gliomas (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). In addition, the grading ability of synapse score is also independent of <italic>IDH</italic> mutation and 1p/19q codeletion (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S4</xref>). Finally, we compared the survival predictive performance of synapse score, <italic>IDH</italic> mutation, and 1p/19q codeletion status (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table S2</xref>). In most instances, synapse score outperforms <italic>IDH</italic> mutation and 1p/19q codeletion.</p></sec></sec><sec id=\"S4\"><title>Discussion</title><p>Given the recently revealed roles of neuro-glioma synapse in glioma development, here we curated a panel of 17 synapse-related genes and proposed the synapse score as a biomarker for the prognosis, grading, and diagnosis of gliomas. The synapse score was validated by more than 3,000 samples of 12 datasets from TCGA, CGGA, and GEO.</p><p>AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptor, one type of glutamate receptors, was focused on in recent studies of neuron-glioma synapses (<xref rid=\"B33\" ref-type=\"bibr\">Venkataramani et al., 2019</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Venkatesh et al., 2019</xref>). In our study, several AMPA glutamate receptor-related terms, such as ionotropic glutamate receptor signaling pathway (GO:0035235), AMPA glutamate receptor complex (GO:0032281), and regulation of AMPA receptor activity (GO:2000311), were filtered out to have strong survival predictive capacities, suggesting a significant role of AMPA receptor in gliomas.</p><p>In addition to AMPA receptor, other ionotropic glutamate receptor genes are also used in the 17-gene set, including N-Methyl-<sc>D</sc>-aspartate (NMDA) receptor [<italic>GRIN3A</italic> (<xref rid=\"B21\" ref-type=\"bibr\">Marco et al., 2013</xref>), <italic>GRIN2C</italic> (<xref rid=\"B12\" ref-type=\"bibr\">Collingridge et al., 2009</xref>)], kainate receptor [<italic>GRIK4</italic> (<xref rid=\"B2\" ref-type=\"bibr\">Arora et al., 2018</xref>)], and non-classical glutamate receptor such as glutamate delta-1 receptor [<italic>GRID1</italic> (<xref rid=\"B16\" ref-type=\"bibr\">Gupta et al., 2015</xref>)], suggesting that other ionotropic glutamate receptors also perform important functions in gliomas. Meanwhile, a gene from other synaptic receptors such as nicotinic acetylcholine receptor [<italic>CHRNB2</italic> (<xref rid=\"B13\" ref-type=\"bibr\">Diaz-Otero et al., 2008</xref>)] was also collected. More genes do not belong to receptors, and they perform neuronal-specific synthesis and glycosylation [<italic>TNR</italic> (<xref rid=\"B37\" ref-type=\"bibr\">Woodworth et al., 2004</xref>)], signal transduction [<italic>MAPK8IP2</italic> (<xref rid=\"B18\" ref-type=\"bibr\">Kennedy et al., 2007</xref>)], neurotransmission [<italic>UNC13A</italic> (<xref rid=\"B26\" ref-type=\"bibr\">Reddy-Alla et al., 2017</xref>), <italic>LRP4</italic> (<xref rid=\"B30\" ref-type=\"bibr\">Sun et al., 2016</xref>)], synapse formation [<italic>LRP4</italic> (<xref rid=\"B17\" ref-type=\"bibr\">Karakatsani et al., 2017</xref>)], inhibitory synapse differentiation [<italic>IGSF21</italic> (<xref rid=\"B31\" ref-type=\"bibr\">Tanabe et al., 2017</xref>)], and other functions in synapses (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table S1</xref>).</p><p>Many of the selected genes have been found to be associated with neurological diseases, including autism [<italic>SHANK2</italic> (<xref rid=\"B22\" ref-type=\"bibr\">Monteiro and Feng, 2017</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Won et al., 2012</xref>)], chronic pain [<italic>CACNG2</italic> (<xref rid=\"B7\" ref-type=\"bibr\">Bortsov et al., 2019</xref>; <xref rid=\"B24\" ref-type=\"bibr\">Nissenbaum et al., 2010</xref>)], epilepsy [<italic>CHRNB2</italic> (<xref rid=\"B13\" ref-type=\"bibr\">Diaz-Otero et al., 2008</xref>)], Huntington’s disease [<italic>GRIN3A</italic> (<xref rid=\"B21\" ref-type=\"bibr\">Marco et al., 2013</xref>)], and neurodegenerative diseases [<italic>SYBU</italic> (<xref rid=\"B4\" ref-type=\"bibr\">Bereczki et al., 2018</xref>)] (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table S1</xref>). However, only a few genes have been studied in gliomas. For example, <italic>PFN1</italic> has been found to be involved in tumor angiogenesis in glioblastoma (<xref rid=\"B14\" ref-type=\"bibr\">Fan et al., 2014</xref>) and was also found to be associated with poor prognosis in our study (HR > 1) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). According to a proteomics study of gliomas (<xref rid=\"B5\" ref-type=\"bibr\">Bi et al., 2017</xref>), <italic>TNR</italic> is down-regulated in glioblastomas. A similar result was found in our study, that low expression of this gene was correlated with poor prognosis (HR < 1). These studies validate our findings and suggest the research and application values of other synapse-related genes in gliomas.</p><p>When screening GO terms, there are 17 terms with the opposite directions (HR > 1 in one batch and HR < 1 in the other). Interestingly, all of these terms are negative in the batch 1 dataset and positive in the batch two dataset. There are 4 terms that are not significant in both datasets (FDR ≥ 0.05), which may be random effects. In addition, 10 terms are only significant in batch one, while one term is only significant in batch two, which may be caused by batch effect and differences of samples. Moreover, there are two terms that are significant but have opposite directions in two batches (neuroblast proliferation and neuron maturation). Given their low FDR ranking (FDR1: 140th, FDR2: 126th for neuroblast proliferation, FDR1: 144th, FDR2: 121st for neuron maturation out of 163 terms, ascendingly), these could be false positives. The practical effects of these terms need to be widely validated in future studies.</p><p>There are 121 significant (FDR1 < 0.05 and FDR2 < 0.05) synapse-related terms with the same direction of HRs in two batches of datasets, suggesting important roles of synapse-related genes in gliomas. But we could not consider all of the terms and genes due to time complexity. Finding the best gene set is a non-deterministic polynomial-time (NP) hard problem. In this paper, we used heuristic algorithms to find the optimal gene set by adding genes one by one in ascending order of combined FDR. It is known that heuristic algorithms do not always get the best results. There could be a gene set and a machine learning method with better prognostic ability using the synapse-related genes. Although our 17-gene set may not be the best result, it is still validated by a permutation experiment and 10 additional datasets and showed better prognostic capability than traditional biomarkers, <italic>IDH</italic> mutation, and 1p/19q codeletion, revealing the extensive research and application value of synapse-related genes in gliomas.</p><p>In spite of its ability as glioma biomarker for the identified synapse-related gene panel, it should be especially noted that the result seems the opposite of existing knowledge. That is, it was reported that neuro-glioma synapse could promote tumor progression and metastasis (<xref rid=\"B33\" ref-type=\"bibr\">Venkataramani et al., 2019</xref>; <xref rid=\"B34\" ref-type=\"bibr\">Venkatesh et al., 2019</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Zeng et al., 2019</xref>), which thus can infer that synapse-related genes should result in a poorer prognosis. However, we revealed it is associated with a better but not poorer prognosis. One possible reason is that the more severe the disease is, the less the normal neurons exist. Molecular processes may play different roles in various cells, organs, and diseases. For example, as an important discovery in glioma research, <italic>IDH</italic> mutation is identified as one of the early events of gliomas, and the epigenetic changes caused by <italic>IDH</italic> mutation are considered as a main tumor driver (<xref rid=\"B32\" ref-type=\"bibr\">Turkalp et al., 2014</xref>). Nevertheless, clinical studies have found that <italic>IDH</italic> mutation can lead to a longer survival time (<xref rid=\"B9\" ref-type=\"bibr\">Cancer Genome Atlas Research Network et al., 2015</xref>). Similarly, immunotherapy, which has been widely used, was criticized for producing serious side effects (<xref rid=\"B23\" ref-type=\"bibr\">Moslehi et al., 2018</xref>). These instances suggest that the synapse-related gene panel could also have multiple aspects.</p><p>Analogously, <italic>IDH</italic>-mut and 1p/19q codeletion are typically biomarkers that promote glioma progression but benefit prognosis. Existing studies have focused on mechanisms that promote glioma, but the reasons for better prognosis are generally reported by clinical studies, such as better chemoradiotherapy sensitivity (<xref rid=\"B11\" ref-type=\"bibr\">Chen et al., 2017</xref>). We conjectured that synapses, <italic>IDH</italic> mutation, and 1p/19q codeletion shared a part of the mechanism that resulted in the observed phenomenon. The causations in synapses, mutations, and gliomas remain to be explored.</p><p>In summary, although the mechanism is unclear, we revealed that the proposed synapse score is an independent and potentially better biomarker for glioma overall survival and shows a predictive capacity in different grade gliomas and normal brain tissues, which could be useful in the prognosis, grading, and diagnosis of gliomas.</p></sec><sec sec-type=\"data-availability\" id=\"S5\"><title>Data Availability Statement</title><p>Publicly available datasets were analyzed in this study. This data can be found here: <ext-link ext-link-type=\"uri\" xlink:href=\"https://portal.gdc.cancer.gov/\">https://portal.gdc.cancer.gov/</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"http://firebrowse.org/\">http://firebrowse.org/</ext-link>, <ext-link ext-link-type=\"uri\" xlink:href=\"http://www.cgga.org.cn/\">http://www.cgga.org.cn/</ext-link>, and <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/gds/\">https://www.ncbi.nlm.nih.gov/gds/</ext-link>.</p></sec><sec id=\"S6\"><title>Author Contributions</title><p>QC conceived the project. XJ performed the analysis and conducted the experiments. XJ, HZ, and QC wrote the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by grants from the Natural Science Foundation of China (81670462, 81970440, and 81921001 to QC).</p></fn></fn-group><fn-group><fn id=\"footnote1\"><label>1</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"https://portal.gdc.cancer.gov/\">https://portal.gdc.cancer.gov/</ext-link></p></fn><fn id=\"footnote2\"><label>2</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"http://www.cgga.org.cn/\">http://www.cgga.org.cn/</ext-link></p></fn><fn id=\"footnote3\"><label>3</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ncbi.nlm.nih.gov/gds/\">https://www.ncbi.nlm.nih.gov/gds/</ext-link></p></fn><fn id=\"footnote4\"><label>4</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"https://ftp.ncbi.nih.gov/gene/DATA/\">https://ftp.ncbi.nih.gov/gene/DATA/</ext-link></p></fn></fn-group><sec id=\"S8\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fnins.2020.00822/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fnins.2020.00822/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"FS1\"><media xlink:href=\"Image_1.PDF\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS1\"><media xlink:href=\"Table_1.PDF\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"DS1\"><media xlink:href=\"Data_Sheet_1.XLSX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"DS2\"><media xlink:href=\"Data_Sheet_2.XLSX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"DS3\"><media xlink:href=\"Data_Sheet_3.XLSX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"DS4\"><media xlink:href=\"Data_Sheet_4.XLSX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Aibaidula</surname><given-names>A.</given-names></name><name><surname>Chan</surname><given-names>A. K.</given-names></name><name><surname>Shi</surname><given-names>Z.</given-names></name><name><surname>Li</surname><given-names>Y.</given-names></name><name><surname>Zhang</surname><given-names>R.</given-names></name><name><surname>Yang</surname><given-names>R.</given-names></name></person-group> (<year>2017</year>). <article-title>Adult IDH wild-type lower-grade gliomas should be further stratified.</article-title>\n<source><italic>Neuro Oncol.</italic></source>\n<volume>19</volume>\n<fpage>1327</fpage>–<lpage>1337</lpage>. <pub-id pub-id-type=\"doi\">10.1093/neuonc/nox078</pub-id>\n<pub-id pub-id-type=\"pmid\">28575485</pub-id></mixed-citation></ref><ref id=\"B2\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arora</surname><given-names>V.</given-names></name><name><surname>Pecoraro</surname><given-names>V.</given-names></name><name><surname>Aller</surname><given-names>M. I.</given-names></name><name><surname>Roman</surname><given-names>C.</given-names></name><name><surname>Paternain</surname><given-names>A. V.</given-names></name><name><surname>Lerma</surname><given-names>J.</given-names></name></person-group> (<year>2018</year>). <article-title>Increased Grik4 gene dosage causes imbalanced circuit output and human disease-related behaviors.</article-title>\n<source><italic>Cell Rep.</italic></source>\n<volume>23</volume>\n<fpage>3827</fpage>–<lpage>3838</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.celrep.2018.05.086</pub-id>\n<pub-id pub-id-type=\"pmid\">29949767</pub-id></mixed-citation></ref><ref id=\"B3\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Barbie</surname><given-names>D. A.</given-names></name><name><surname>Tamayo</surname><given-names>P.</given-names></name><name><surname>Boehm</surname><given-names>J. S.</given-names></name><name><surname>Kim</surname><given-names>S. Y.</given-names></name><name><surname>Moody</surname><given-names>S. E.</given-names></name><name><surname>Dunn</surname><given-names>I. F.</given-names></name></person-group> (<year>2009</year>). <article-title>Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1.</article-title>\n<source><italic>Nature</italic></source>\n<volume>462</volume>\n<fpage>108</fpage>–<lpage>112</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature08460</pub-id>\n<pub-id pub-id-type=\"pmid\">19847166</pub-id></mixed-citation></ref><ref id=\"B4\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bereczki</surname><given-names>E.</given-names></name><name><surname>Branca</surname><given-names>R. M.</given-names></name><name><surname>Francis</surname><given-names>P. T.</given-names></name><name><surname>Pereira</surname><given-names>J. B.</given-names></name><name><surname>Baek</surname><given-names>J. H.</given-names></name><name><surname>Hortobagyi</surname><given-names>T.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Synaptic markers of cognitive decline in neurodegenerative diseases: a proteomic approach.</article-title>\n<source><italic>Brain</italic></source>\n<volume>141</volume>\n<fpage>582</fpage>–<lpage>595</lpage>. <pub-id pub-id-type=\"doi\">10.1093/brain/awx352</pub-id>\n<pub-id pub-id-type=\"pmid\">29324989</pub-id></mixed-citation></ref><ref id=\"B5\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bi</surname><given-names>B. B.</given-names></name><name><surname>Li</surname><given-names>F.</given-names></name><name><surname>Guo</surname><given-names>J. S.</given-names></name><name><surname>Li</surname><given-names>C. L.</given-names></name><name><surname>Jing</surname><given-names>R. R.</given-names></name><name><surname>Lv</surname><given-names>X.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Label-free quantitative proteomics unravels the importance of rna processing in glioma malignancy.</article-title>\n<source><italic>Neuroscience</italic></source>\n<volume>351</volume>\n<fpage>84</fpage>–<lpage>95</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuroscience.2017.03.023</pub-id>\n<pub-id pub-id-type=\"pmid\">28341197</pub-id></mixed-citation></ref><ref id=\"B6\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bolouri</surname><given-names>H.</given-names></name><name><surname>Zhao</surname><given-names>L. P.</given-names></name><name><surname>Holland</surname><given-names>E. C.</given-names></name></person-group> (<year>2016</year>). <article-title>Big data visualization identifies the multidimensional molecular landscape of human gliomas.</article-title>\n<source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source>\n<volume>113</volume>\n<fpage>5394</fpage>–<lpage>5399</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.1601591113</pub-id>\n<pub-id pub-id-type=\"pmid\">27118839</pub-id></mixed-citation></ref><ref id=\"B7\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bortsov</surname><given-names>A. V.</given-names></name><name><surname>Devor</surname><given-names>M.</given-names></name><name><surname>Kaunisto</surname><given-names>M. A.</given-names></name><name><surname>Kalso</surname><given-names>E.</given-names></name><name><surname>Brufsky</surname><given-names>A.</given-names></name><name><surname>Kehlet</surname><given-names>H.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>CACNG2 polymorphisms associate with chronic pain after mastectomy.</article-title>\n<source><italic>Pain</italic></source>\n<volume>160</volume>\n<fpage>561</fpage>–<lpage>568</lpage>. <pub-id pub-id-type=\"doi\">10.1097/j.pain.0000000000001432</pub-id>\n<pub-id pub-id-type=\"pmid\">30371558</pub-id></mixed-citation></ref><ref id=\"B8\"><mixed-citation publication-type=\"book\"><collab>Brain and Other Nervous System Cancer</collab> (<year>2019</year>). <source><italic>Cancer Stat Facts.</italic></source>\n<publisher-loc>Atlanta</publisher-loc>: <publisher-name>CDC</publisher-name>.</mixed-citation></ref><ref id=\"B9\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cancer Genome Atlas Research Network, Brat</surname><given-names>D. J.</given-names></name><name><surname>Verhaak</surname><given-names>R. G.</given-names></name><name><surname>Aldape</surname><given-names>K. D.</given-names></name><name><surname>Yung</surname><given-names>W. K.</given-names></name><name><surname>Salama</surname><given-names>S. R.</given-names></name><etal/></person-group> (<year>2015</year>). <article-title>Comprehensive, integrative genomic analysis of diffuse lower-grade gliomas.</article-title>\n<source><italic>N. Engl. J. Med.</italic></source>\n<volume>372</volume>\n<fpage>2481</fpage>–<lpage>2498</lpage>. <pub-id pub-id-type=\"doi\">10.1056/nejmoa1402121</pub-id>\n<pub-id pub-id-type=\"pmid\">26061751</pub-id></mixed-citation></ref><ref id=\"B10\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ceccarelli</surname><given-names>M.</given-names></name><name><surname>Barthel</surname><given-names>F. P.</given-names></name><name><surname>Malta</surname><given-names>T. M.</given-names></name><name><surname>Sabedot</surname><given-names>T. S.</given-names></name><name><surname>Salama</surname><given-names>S. R.</given-names></name><name><surname>Murray</surname><given-names>B. A.</given-names></name></person-group> (<year>2016</year>). <article-title>Molecular profiling reveals biologically discrete subsets and pathways of progression in diffuse glioma.</article-title>\n<source><italic>Cell</italic></source>\n<volume>164</volume>\n<fpage>550</fpage>–<lpage>563</lpage>.<pub-id pub-id-type=\"pmid\">26824661</pub-id></mixed-citation></ref><ref id=\"B11\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>R.</given-names></name><name><surname>Smith-Cohn</surname><given-names>M.</given-names></name><name><surname>Cohen</surname><given-names>A. L.</given-names></name><name><surname>Colman</surname><given-names>H.</given-names></name></person-group> (<year>2017</year>). <article-title>Glioma subclassifications and their clinical significance.</article-title>\n<source><italic>Neurotherapeutics</italic></source>\n<volume>14</volume>\n<fpage>284</fpage>–<lpage>297</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s13311-017-0519-x</pub-id>\n<pub-id pub-id-type=\"pmid\">28281173</pub-id></mixed-citation></ref><ref id=\"B12\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Collingridge</surname><given-names>G. L.</given-names></name><name><surname>Olsen</surname><given-names>R. W.</given-names></name><name><surname>Peters</surname><given-names>J.</given-names></name><name><surname>Spedding</surname><given-names>M.</given-names></name></person-group> (<year>2009</year>). <article-title>A nomenclature for ligand-gated ion channels.</article-title>\n<source><italic>Neuropharmacology</italic></source>\n<volume>56</volume>\n<fpage>2</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuropharm.2008.06.063</pub-id>\n<pub-id pub-id-type=\"pmid\">18655795</pub-id></mixed-citation></ref><ref id=\"B13\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Diaz-Otero</surname><given-names>F.</given-names></name><name><surname>Quesada</surname><given-names>M.</given-names></name><name><surname>Morales-Corraliza</surname><given-names>J.</given-names></name><name><surname>Martinez-Parra</surname><given-names>C.</given-names></name><name><surname>Gomez-Garre</surname><given-names>P.</given-names></name><name><surname>Serratosa</surname><given-names>J. M.</given-names></name></person-group> (<year>2008</year>). <article-title>Autosomal dominant nocturnal frontal lobe epilepsy with a mutation in the CHRNB2 gene.</article-title>\n<source><italic>Epilepsia</italic></source>\n<volume>49</volume>\n<fpage>516</fpage>–<lpage>520</lpage>.<pub-id pub-id-type=\"pmid\">17900292</pub-id></mixed-citation></ref><ref id=\"B14\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fan</surname><given-names>Y.</given-names></name><name><surname>Potdar</surname><given-names>A. A.</given-names></name><name><surname>Gong</surname><given-names>Y. Q.</given-names></name><name><surname>Eswarappa</surname><given-names>S. M.</given-names></name><name><surname>Donnola</surname><given-names>S.</given-names></name><name><surname>Lathia</surname><given-names>J. D.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Profilin-1 phosphorylation directs angiocrine expression and glioblastoma progression through HIF-1 alpha accumulation.</article-title>\n<source><italic>Nat. Cell Biol.</italic></source>\n<volume>16</volume>\n<fpage>445</fpage>–<lpage>U144</lpage>.<pub-id pub-id-type=\"pmid\">24747440</pub-id></mixed-citation></ref><ref id=\"B15\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gramatzki</surname><given-names>D.</given-names></name><name><surname>Dehler</surname><given-names>S.</given-names></name><name><surname>Rushing</surname><given-names>E. J.</given-names></name><name><surname>Zaugg</surname><given-names>K.</given-names></name><name><surname>Hofer</surname><given-names>S.</given-names></name><name><surname>Yonekawa</surname><given-names>Y.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>Glioblastoma in the Canton of Zurich, Switzerland revisited: 2005 to 2009.</article-title>\n<source><italic>Cancer</italic></source>\n<volume>122</volume>\n<fpage>2206</fpage>–<lpage>2215</lpage>. <pub-id pub-id-type=\"doi\">10.1002/cncr.30023</pub-id>\n<pub-id pub-id-type=\"pmid\">27088883</pub-id></mixed-citation></ref><ref id=\"B16\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gupta</surname><given-names>S. C.</given-names></name><name><surname>Yadav</surname><given-names>R.</given-names></name><name><surname>Pavuluri</surname><given-names>R.</given-names></name><name><surname>Morley</surname><given-names>B. J.</given-names></name><name><surname>Stairs</surname><given-names>D. J.</given-names></name><name><surname>Dravid</surname><given-names>S. M.</given-names></name></person-group> (<year>2015</year>). <article-title>Essential role of GluD1 in dendritic spine development and GluN2B to GluN2A NMDAR subunit switch in the cortex and hippocampus reveals ability of GluN2B inhibition in correcting hyperconnectivity.</article-title>\n<source><italic>Neuropharmacology</italic></source>\n<volume>93</volume>\n<fpage>274</fpage>–<lpage>284</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.neuropharm.2015.02.013</pub-id>\n<pub-id pub-id-type=\"pmid\">25721396</pub-id></mixed-citation></ref><ref id=\"B17\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Karakatsani</surname><given-names>A.</given-names></name><name><surname>Marichal</surname><given-names>N.</given-names></name><name><surname>Urban</surname><given-names>S.</given-names></name><name><surname>Kalamakis</surname><given-names>G.</given-names></name><name><surname>Ghanem</surname><given-names>A.</given-names></name><name><surname>Schick</surname><given-names>A.</given-names></name></person-group> (<year>2017</year>). <article-title>Neuronal LRP4 regulates synapse formation in the developing CNS.</article-title>\n<source><italic>Development</italic></source>\n<volume>144</volume>\n<fpage>4604</fpage>–<lpage>4615</lpage>. <pub-id pub-id-type=\"doi\">10.1242/dev.150110</pub-id>\n<pub-id pub-id-type=\"pmid\">29061639</pub-id></mixed-citation></ref><ref id=\"B18\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kennedy</surname><given-names>N. J.</given-names></name><name><surname>Martin</surname><given-names>G.</given-names></name><name><surname>Ehrhardt</surname><given-names>A. G.</given-names></name><name><surname>Cavanagh-Kyros</surname><given-names>J.</given-names></name><name><surname>Kuan</surname><given-names>C. Y.</given-names></name><name><surname>Rakic</surname><given-names>P.</given-names></name><etal/></person-group> (<year>2007</year>). <article-title>Requirement of JIP scaffold proteins for NMDA-mediated signal transduction.</article-title>\n<source><italic>Gene Dev.</italic></source>\n<volume>21</volume>\n<fpage>2336</fpage>–<lpage>2346</lpage>. <pub-id pub-id-type=\"doi\">10.1101/gad.1563107</pub-id>\n<pub-id pub-id-type=\"pmid\">17875667</pub-id></mixed-citation></ref><ref id=\"B19\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kros</surname><given-names>J. M.</given-names></name><name><surname>Mustafa</surname><given-names>D. M.</given-names></name><name><surname>Dekker</surname><given-names>L. J.</given-names></name><name><surname>Sillevis Smitt</surname><given-names>P. A.</given-names></name><name><surname>Luider</surname><given-names>T. M.</given-names></name><name><surname>Zheng</surname><given-names>P. P.</given-names></name></person-group> (<year>2015</year>). <article-title>Circulating glioma biomarkers.</article-title>\n<source><italic>Neuro Oncol.</italic></source>\n<volume>17</volume>\n<fpage>343</fpage>–<lpage>360</lpage>.<pub-id pub-id-type=\"pmid\">25253418</pub-id></mixed-citation></ref><ref id=\"B20\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Louis</surname><given-names>D. N.</given-names></name><name><surname>Ohgaki</surname><given-names>H.</given-names></name><name><surname>Wiestler</surname><given-names>O. D.</given-names></name><name><surname>Cavenee</surname><given-names>W. K.</given-names></name><name><surname>Burger</surname><given-names>P. C.</given-names></name><name><surname>Jouvet</surname><given-names>A.</given-names></name><etal/></person-group> (<year>2007</year>). <article-title>The 2007 WHO classification of tumours of the central nervous system.</article-title>\n<source><italic>Acta Neuropathol.</italic></source>\n<volume>114</volume>\n<fpage>97</fpage>–<lpage>109</lpage>.<pub-id pub-id-type=\"pmid\">17618441</pub-id></mixed-citation></ref><ref id=\"B21\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Marco</surname><given-names>S.</given-names></name><name><surname>Giralt</surname><given-names>A.</given-names></name><name><surname>Petrovic</surname><given-names>M. M.</given-names></name><name><surname>Pouladi</surname><given-names>M. A.</given-names></name><name><surname>Martinez-Turrillas</surname><given-names>R.</given-names></name><name><surname>Martinez-Hernandez</surname><given-names>J.</given-names></name></person-group> (<year>2013</year>). <article-title>Suppressing aberrant GluN3A expression rescues synaptic and behavioral impairments in Huntington’s disease models.</article-title>\n<source><italic>Nat. Med.</italic></source>\n<volume>19</volume>\n<fpage>1030</fpage>–<lpage>1038</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nm.3246</pub-id>\n<pub-id pub-id-type=\"pmid\">23852340</pub-id></mixed-citation></ref><ref id=\"B22\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Monteiro</surname><given-names>P.</given-names></name><name><surname>Feng</surname><given-names>G. P.</given-names></name></person-group> (<year>2017</year>). <article-title>SHANK proteins: roles at the synapse and in autism spectrum disorder.</article-title>\n<source><italic>Nat. Rev. Neurosci.</italic></source>\n<volume>18</volume>\n<fpage>147</fpage>–<lpage>157</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrn.2016.183</pub-id>\n<pub-id pub-id-type=\"pmid\">28179641</pub-id></mixed-citation></ref><ref id=\"B23\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moslehi</surname><given-names>J. J.</given-names></name><name><surname>Salem</surname><given-names>J. E.</given-names></name><name><surname>Sosman</surname><given-names>J. A.</given-names></name><name><surname>Lebrun-Vignes</surname><given-names>B.</given-names></name><name><surname>Johnson</surname><given-names>D. B.</given-names></name></person-group> (<year>2018</year>). <article-title>Increased reporting of fatal immune checkpoint inhibitor-associated myocarditis.</article-title>\n<source><italic>Lancet</italic></source>\n<volume>391</volume>:<issue>933</issue>\n<pub-id pub-id-type=\"doi\">10.1016/s0140-6736(18)30533-6</pub-id></mixed-citation></ref><ref id=\"B24\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Nissenbaum</surname><given-names>J.</given-names></name><name><surname>Devor</surname><given-names>M.</given-names></name><name><surname>Seltzer</surname><given-names>Z.</given-names></name><name><surname>Gebauer</surname><given-names>M.</given-names></name><name><surname>Michaelis</surname><given-names>M.</given-names></name><name><surname>Tal</surname><given-names>M.</given-names></name></person-group> (<year>2010</year>). <article-title>Susceptibility to chronic pain following nerve injury is genetically affected by CACNG2.</article-title>\n<source><italic>Genome Res.</italic></source>\n<volume>20</volume>\n<fpage>1180</fpage>–<lpage>1190</lpage>. <pub-id pub-id-type=\"doi\">10.1101/gr.104976.110</pub-id>\n<pub-id pub-id-type=\"pmid\">20688780</pub-id></mixed-citation></ref><ref id=\"B25\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ostrom</surname><given-names>Q. T.</given-names></name><name><surname>Gittleman</surname><given-names>H.</given-names></name><name><surname>Xu</surname><given-names>J.</given-names></name><name><surname>Kromer</surname><given-names>C.</given-names></name><name><surname>Wolinsky</surname><given-names>Y.</given-names></name><name><surname>Kruchko</surname><given-names>C.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the united states in 2009-2013.</article-title>\n<source><italic>Neuro Oncol.</italic></source>\n<volume>18</volume>\n<fpage>v1</fpage>–<lpage>v75</lpage>. <pub-id pub-id-type=\"doi\">10.1093/neuonc/now207</pub-id>\n<pub-id pub-id-type=\"pmid\">28475809</pub-id></mixed-citation></ref><ref id=\"B26\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reddy-Alla</surname><given-names>S.</given-names></name><name><surname>Bohme</surname><given-names>M. A.</given-names></name><name><surname>Reynolds</surname><given-names>E.</given-names></name><name><surname>Beis</surname><given-names>C.</given-names></name><name><surname>Grasskamp</surname><given-names>A. T.</given-names></name><name><surname>Mampell</surname><given-names>M. M.</given-names></name></person-group> (<year>2017</year>). <article-title>Stable positioning of Unc13 restricts synaptic vesicle fusion to defined release sites to promote synchronous neurotransmission.</article-title>\n<source><italic>Neuron</italic></source>\n<volume>95</volume>\n<fpage>1350</fpage>–<lpage>1364</lpage>.<pub-id pub-id-type=\"pmid\">28867551</pub-id></mixed-citation></ref><ref id=\"B27\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Robin</surname><given-names>X.</given-names></name><name><surname>Turck</surname><given-names>N.</given-names></name><name><surname>Hainard</surname><given-names>A.</given-names></name><name><surname>Tiberti</surname><given-names>N.</given-names></name><name><surname>Lisacek</surname><given-names>F.</given-names></name><name><surname>Sanchez</surname><given-names>J. C.</given-names></name><etal/></person-group> (<year>2011</year>). <article-title>pROC: an open-source package for R and S+ to analyze and compare ROC curves.</article-title>\n<source><italic>BMC Bioinformatics</italic></source>\n<volume>12</volume>:<issue>77</issue>. <pub-id pub-id-type=\"doi\">10.1186/1471-2105-12-77</pub-id>\n<pub-id pub-id-type=\"pmid\">21414208</pub-id></mixed-citation></ref><ref id=\"B28\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ruda</surname><given-names>R.</given-names></name><name><surname>Soffietti</surname><given-names>R.</given-names></name></person-group> (<year>2017</year>). <article-title>Controversies in management of low-grade gliomas in light of new data from clinical trials.</article-title>\n<source><italic>Neuro Oncol.</italic></source>\n<volume>19</volume>\n<fpage>143</fpage>–<lpage>144</lpage>. <pub-id pub-id-type=\"doi\">10.1093/neuonc/now275</pub-id>\n<pub-id pub-id-type=\"pmid\">28391299</pub-id></mixed-citation></ref><ref id=\"B29\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Subramanian</surname><given-names>A.</given-names></name><name><surname>Tamayo</surname><given-names>P.</given-names></name><name><surname>Mootha</surname><given-names>V. K.</given-names></name><name><surname>Mukherjee</surname><given-names>S.</given-names></name><name><surname>Ebert</surname><given-names>B. L.</given-names></name><name><surname>Gillette</surname><given-names>M. A.</given-names></name><etal/></person-group> (<year>2005</year>). <article-title>Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.</article-title>\n<source><italic>Proc. Natl. Acad. Sci. U.S.A.</italic></source>\n<volume>102</volume>\n<fpage>15545</fpage>–<lpage>15550</lpage>. <pub-id pub-id-type=\"doi\">10.1073/pnas.0506580102</pub-id>\n<pub-id pub-id-type=\"pmid\">16199517</pub-id></mixed-citation></ref><ref id=\"B30\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sun</surname><given-names>X. D.</given-names></name><name><surname>Li</surname><given-names>L.</given-names></name><name><surname>Liu</surname><given-names>F.</given-names></name><name><surname>Huang</surname><given-names>Z. H.</given-names></name><name><surname>Bean</surname><given-names>J. C.</given-names></name><name><surname>Jiao</surname><given-names>H. F.</given-names></name></person-group> (<year>2016</year>). <article-title>Lrp4 in astrocytes modulates glutamatergic transmission.</article-title>\n<source><italic>Nat. Neurosci.</italic></source>\n<volume>19</volume>\n<fpage>1010</fpage>–<lpage>1018</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nn.4326</pub-id>\n<pub-id pub-id-type=\"pmid\">27294513</pub-id></mixed-citation></ref><ref id=\"B31\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tanabe</surname><given-names>Y.</given-names></name><name><surname>Naito</surname><given-names>Y.</given-names></name><name><surname>Vasuta</surname><given-names>C.</given-names></name><name><surname>Lee</surname><given-names>A. K.</given-names></name><name><surname>Soumounou</surname><given-names>Y.</given-names></name><name><surname>Linhoff</surname><given-names>M. W.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>IgSF21 promotes differentiation of inhibitory synapses via binding to neurexin2 alpha.</article-title>\n<source><italic>Nat. Commun.</italic></source>\n<volume>8</volume>:<issue>408</issue>.</mixed-citation></ref><ref id=\"B32\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Turkalp</surname><given-names>Z.</given-names></name><name><surname>Karamchandani</surname><given-names>J.</given-names></name><name><surname>Das</surname><given-names>S.</given-names></name></person-group> (<year>2014</year>). <article-title>IDH mutation in glioma: new insights and promises for the future.</article-title>\n<source><italic>JAMA Neurol.</italic></source>\n<volume>71</volume>\n<fpage>1319</fpage>–<lpage>1325</lpage>.<pub-id pub-id-type=\"pmid\">25155243</pub-id></mixed-citation></ref><ref id=\"B33\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Venkataramani</surname><given-names>V.</given-names></name><name><surname>Tanev</surname><given-names>D.</given-names><suffix>I</suffix></name><name><surname>Strahle</surname><given-names>C.</given-names></name><name><surname>Studier-Fischer</surname><given-names>A.</given-names></name><name><surname>Fankhauser</surname><given-names>L. T.</given-names></name></person-group> (<year>2019</year>). <article-title>Glutamatergic synaptic input to glioma cells drives brain tumour progression.</article-title>\n<source><italic>Nature</italic></source>\n<volume>573</volume>\n<fpage>532</fpage>–<lpage>538</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41586-019-1564-x</pub-id>\n<pub-id pub-id-type=\"pmid\">31534219</pub-id></mixed-citation></ref><ref id=\"B34\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Venkatesh</surname><given-names>H. S.</given-names></name><name><surname>Morishita</surname><given-names>W.</given-names></name><name><surname>Geraghty</surname><given-names>A. C.</given-names></name><name><surname>Silverbush</surname><given-names>D.</given-names></name><name><surname>Gillespie</surname><given-names>S. M.</given-names></name><name><surname>Arzt</surname><given-names>M.</given-names></name></person-group> (<year>2019</year>). <article-title>Electrical and synaptic integration of glioma into neural circuits.</article-title>\n<source><italic>Nature</italic></source>\n<volume>573</volume>\n<fpage>539</fpage>–<lpage>545</lpage>.<pub-id pub-id-type=\"pmid\">31534222</pub-id></mixed-citation></ref><ref id=\"B35\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wesseling</surname><given-names>P.</given-names></name><name><surname>Capper</surname><given-names>D.</given-names></name></person-group> (<year>2018</year>). <article-title>WHO 2016 Classification of gliomas.</article-title>\n<source><italic>Neuropathol. Appl. Neurobiol.</italic></source>\n<volume>44</volume>\n<fpage>139</fpage>–<lpage>150</lpage>. <pub-id pub-id-type=\"doi\">10.1111/nan.12432</pub-id>\n<pub-id pub-id-type=\"pmid\">28815663</pub-id></mixed-citation></ref><ref id=\"B36\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Won</surname><given-names>H.</given-names></name><name><surname>Lee</surname><given-names>H. R.</given-names></name><name><surname>Gee</surname><given-names>H. Y.</given-names></name><name><surname>Mah</surname><given-names>W.</given-names></name><name><surname>Kim</surname><given-names>J. I.</given-names></name><name><surname>Lee</surname><given-names>J.</given-names></name></person-group> (<year>2012</year>). <article-title>Autistic-like social behaviour in Shank2-mutant mice improved by restoring NMDA receptor function.</article-title>\n<source><italic>Nature</italic></source>\n<volume>486</volume>\n<fpage>261</fpage>–<lpage>265</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nature11208</pub-id>\n<pub-id pub-id-type=\"pmid\">22699620</pub-id></mixed-citation></ref><ref id=\"B37\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Woodworth</surname><given-names>A.</given-names></name><name><surname>Pesheva</surname><given-names>P.</given-names></name><name><surname>Fiete</surname><given-names>D.</given-names></name><name><surname>Baenziger</surname><given-names>J. U.</given-names></name></person-group> (<year>2004</year>). <article-title>Neuronal-specific synthesis and glycosylation of tenascin-R.</article-title>\n<source><italic>J. Biol. Chem.</italic></source>\n<volume>279</volume>\n<fpage>10413</fpage>–<lpage>10421</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.m312466200</pub-id>\n<pub-id pub-id-type=\"pmid\">14681222</pub-id></mixed-citation></ref><ref id=\"B38\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zeng</surname><given-names>Q.</given-names></name><name><surname>Michael</surname><given-names>I. P.</given-names></name><name><surname>Zhang</surname><given-names>P.</given-names></name><name><surname>Saghafinia</surname><given-names>S.</given-names></name><name><surname>Knott</surname><given-names>G.</given-names></name><name><surname>Jiao</surname><given-names>W.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Synaptic proximity enables NMDAR signalling to promote brain metastasis.</article-title>\n<source><italic>Nature</italic></source>\n<volume>573</volume>\n<fpage>526</fpage>–<lpage>531</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41586-019-1576-6</pub-id>\n<pub-id pub-id-type=\"pmid\">31534217</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Oncol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Oncol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Oncol.</journal-id><journal-title-group><journal-title>Frontiers in Oncology</journal-title></journal-title-group><issn pub-type=\"epub\">2234-943X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32850455</article-id><article-id pub-id-type=\"pmc\">PMC7431625</article-id><article-id pub-id-type=\"doi\">10.3389/fonc.2020.01407</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Oncology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>BRE Promotes Esophageal Squamous Cell Carcinoma Growth by Activating AKT Signaling</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Jin</surname><given-names>Fujun</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/793097/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zhu</surname><given-names>Yexuan</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Chen</surname><given-names>Jingyi</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Rongze</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Wang</surname><given-names>Yiliang</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/647674/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wu</surname><given-names>Yanting</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Zhou</surname><given-names>Pengjun</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Song</surname><given-names>Xiaowei</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Ren</surname><given-names>Zhe</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Dong</surname><given-names>Jun</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref><xref ref-type=\"corresp\" rid=\"c002\"><sup>*</sup></xref></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Guangzhou Jinan Biomedicine Research and Development Center, College of Life Science and Technology, Institute of Biomedicine, Jinan University</institution>, <addr-line>Guangzhou</addr-line>, <country>China</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University</institution>, <addr-line>Guangzhou</addr-line>, <country>China</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department of Pathophysiology, School of Medicine, GHM Institute of CNS Regeneration, Jinan University</institution>, <addr-line>Guangzhou</addr-line>, <country>China</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Song-Qiang Xie, Linzhou Cancer Hospital, China</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Hamid Morjani, Université de Reims Champagne-Ardenne, France; Yanquan Zhang, University of Kentucky, United States</p></fn><corresp id=\"c001\">*Correspondence: Zhe Ren <email>rz62@163.com</email></corresp><corresp id=\"c002\">Jun Dong <email>dongjunbox@163.com</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†These authors have contributed equally to this work</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>10</volume><elocation-id>1407</elocation-id><history><date date-type=\"received\"><day>30</day><month>1</month><year>2020</year></date><date date-type=\"accepted\"><day>03</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Jin, Zhu, Chen, Wang, Wang, Wu, Zhou, Song, Ren and Dong.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Jin, Zhu, Chen, Wang, Wang, Wu, Zhou, Song, Ren and Dong</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Brain and reproductive organ-expressed protein (BRE) is aberrantly expressed in multiple cancers; however, its expression pattern in human esophageal squamous cell carcinoma (ESCC) and its role in ESCC progression remain unclear. In this study, we aimed to investigate the expression pattern of BRE in human ESCC and its role in ESCC progression. BRE was overexpressed in ESCC tissues compared with that in the adjacent non-tumor tissues. Forced expression of BRE was sufficient to enhance ESCC cell growth by promoting cell cycle progression and anti-apoptosis. Silencing of BRE suppressed these malignant phenotypes of ESCC cells. Mechanistic evaluation revealed that BRE overexpression activated the phosphorylation of AKT, and inhibition of the AKT pathway by MK2206 decreased the BRE-induced cell growth and apoptotic resistance in ESCC cells, highlighting the critical role of AKT signaling in mediating the effects of BRE. Moreover, the effects of BRE on ESCC cell growth and AKT activation were verified in a xenograft model <italic>in vivo</italic>. The present results show that BRE is overexpressed in ESCC tissues and contributes to the growth of ESCC cells by activating AKT signaling both <italic>in vitro</italic> and <italic>in vivo</italic> and provide insight into the role of BRE in AKT signaling and ESCC pathogenesis.</p></abstract><kwd-group><kwd>esophageal squamous cell carcinoma</kwd><kwd>brain and reproductive organ-expressed protein</kwd><kwd>AKT</kwd><kwd>apoptosis</kwd><kwd>cell cycle</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">National Natural Science Foundation of China<named-content content-type=\"fundref-id\">10.13039/501100001809</named-content></funding-source><award-id rid=\"cn001\">81471235</award-id><award-id rid=\"cn001\">81802193</award-id></award-group><award-group><funding-source id=\"cn002\">Natural Science Foundation of Guangdong Province<named-content content-type=\"fundref-id\">10.13039/501100003453</named-content></funding-source><award-id rid=\"cn002\">2018A030313604</award-id><award-id rid=\"cn002\">2019A1515012024</award-id></award-group></funding-group><counts><fig-count count=\"6\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"32\"/><page-count count=\"11\"/><word-count count=\"5490\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignant tumors and a serious threat to human health; it is the eighth most common malignant cancer and sixth leading cause of cancer-related mortality worldwide (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>–<xref rid=\"B3\" ref-type=\"bibr\">3</xref>). Although diagnosis and treatment strategies have improved, the 5-year survival rate of patients with ESCC remains <20% (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). Late diagnosis, early metastasis, and a lack of targeted chemotherapeutic drugs contribute to the low survival rates of patients with ESCC (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>). Therefore, it is important to identify regulatory factors contributing to ESCC progression to develop new drug intervention strategies for this lethal disease.</p><p>BRE is a stress-responsive gene that is expressed as multiple mRNA isoforms in human cells (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>, <xref rid=\"B7\" ref-type=\"bibr\">7</xref>). Differential expression of the BRE gene has been reported in response to various stress and biological signals, such as ionizing radiation and chorionic gonadotropin treatments (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>). BRE is involved in numerous biological phenomena including apoptosis (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>), DNA damage repair (<xref rid=\"B10\" ref-type=\"bibr\">10</xref>), cell differentiation (<xref rid=\"B11\" ref-type=\"bibr\">11</xref>), and tissue repair (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). BRE exerts anti-apoptotic effects by interacting with TNF-R1 and Fas to inhibit apoptotic activation (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>). Furthermore, BRE is indispensable for the integrity of the DNA damage repair-related BRCA1-A complex, wherein BRE interacts with MERIT40 to help form BRCA1 foci at DNA damage sites (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>, <xref rid=\"B16\" ref-type=\"bibr\">16</xref>). In humans, BRE overexpression is associated with the aggressiveness of liver and lung carcinomas (<xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B18\" ref-type=\"bibr\">18</xref>). However, BRE overexpression also helps predict favorable relapse-free survival in patients with breast cancer and adults with acute myeloid leukemia (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B20\" ref-type=\"bibr\">20</xref>). Thus, the role of BRE in tumorigenesis potentially depends on the tumor subtype. Chen et al. reported that BRE was overexpressed in a malignantly transformed esophageal carcinoma cell line in comparison with that in an immortalized human esophageal epithelial cell line (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). However, BRE expression in patients with ESCC, and its regulatory functions and role in ESCC progression remain unclear.</p><p>In this study, we aimed to investigate the expression pattern of BRE is human ESCC and its role in ESCC progression. Functional analyses of BRE knockdown or overexpression in four ESCC cells and mechanistic assays were carried out, followed by <italic>in vivo</italic> analysis using a mouse xenograft model to analyze the expression pattern of BRE in ESCC and determine its role in ESCC progression.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Cell Culture and Lentiviral Infection</title><p>ESCC cell lines KYSE140, KYSE450, KYSE510, Eca109, and TE-1 were obtained from the Chinese Academy of Sciences Cell Bank and maintained in RPIM-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco) at 37°C and 5% CO<sub>2</sub>. For lentiviral infection, ESCC cells were infected with lentivirus at a multiplicity of infection of 20 in the presence of 5 μg/mL polybrene (Sigma, USA). Specific lentiviral short hairpin (shRNA) targeting the human BRE gene and scrambled control shRNA were purchased from Cyagen Biosciences (Guangzhou, China). The sequences were shBRE sense: TGT ACT TGT CAC CTC GAA T; shBRE antisense: ATT CGA GGT GAC AAG TAC A; Scramble sense: TTC TCC GAA CGT GTC ACG T; Scramble antisense: ACG TGA CAC GTT CGG AGA A. The stable BRE knockdown or overexpression ESCC cell lines were selected with 5 μg/ml of puromycin for 2 weeks and the stable cell lines were used in the subsequent cellular experiments.</p></sec><sec><title>CCK-8 and Edu Assay</title><p>To analyze cell viability, ESCC cells with BRE knockdown or overexpression were plated into 96-well plates at a density of 1 × 10<sup>4</sup> cells/well. After 24, 48, 72, or 96 h, the medium was replaced with medium containing 10% CCK-8 reagent and incubated at 37°C for 1 h, after which the absorbance was measured using a microplate reader at 450 nm. The OD<sub>450</sub> value for each time point was used to generate a growth curve. The proliferation of ESCC cells was measured using a commercial Cell-Light Edu <italic>in vitro</italic> Kit (Ribobio, China) in accordance with the manufacturer's instructions.</p></sec><sec><title>Apoptosis and Cell Cycle Assay</title><p>Apoptotic cells were analyzed using the Annexin V/PI Apoptosis Detection Kit (Keygentec, China). For cell cycle analysis, the cells were synchronized in G0/G1-phase by serum-starvation for 24 h and then released and harvested after 12 h. The cells were then collected and stained using the Cell Cycle Analysis Kit (Beyotime, China) in accordance with the manufacturer's instructions. A total of 2 × 10<sup>5</sup> cells was counted via flow cytometry (Calibur, BD Biosciences, USA) and the data were analyzed using FlowJo software.</p></sec><sec><title>Clone Formation Assay</title><p>To evaluate the clone formation potential of ESCC cells, 200 cells were plated into each well of a 6-well plate, and the plate was incubated at 37°C and 5% CO<sub>2</sub> in an incubator for 2 weeks. The medium was replaced every 3 d. After 2 weeks, the clones were stained with 0.5% crystal violet for 30 min and enumerated.</p></sec><sec><title>Western Blotting</title><p>Cultured ESCC cells and patient samples were homogenized in RIPA lysis buffer (Beyotime) in the presence of 1 mM PMSF (Beyotime) and lysed on ice for 30 min, followed by centrifugation at 14,000 × g for 10 min at 4°C to harvest the supernatant. Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime) and normalized. Proteins were separated via SDS-PAGE and analyzed using the standard western blotting protocol. GAPDH was used as the internal control. The following primary antibodies were used: anti-BRE (#ab177960, 1:1000, Abcam, UK), anti-AKT (#2920, 1:1000, Cell Signaling Technology, USA), anti-p-AKT (#4060, 1:1000, Cell Signaling Technology), anti-mTOR (#2983, 1:1000, Cell Signaling Technology), anti-p-mTOR (#5536, 1:1000, Cell Signaling Technology), anti-PTEN (#9188, 1:1000, Cell Signaling Technology, USA), anti-p110 (#4249, 1:1000, Cell Signaling Technology), anti-p85 (#4292, 1:1000, Cell Signaling Technology), and anti-GAPDH (#5174, 1:1000, Cell Signaling Technology) antibodies.</p></sec><sec><title>Quantitative Real-Time PCR Analysis</title><p>The total RNA was extracted from cultured ESCC cells and patient samples with TRIzol reagent (Invitrogen, USA) in accordance with the manufacturer's protocols. An equal amount total RNA was reverse-transcribed into cDNA using the PrimeScript RT Master Mix Kit (TAKARA, Japan). The q-PCR assays were performed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA) using the SYBR Premix Ex Taq II Kit in accordance with the manufacturer's instructions (TAKARA). Relative gene expression levels were normalized to those of the housekeeping gene GAPDH. Gene-specific primer pairs used herein were the following: BRE sense, GAA GCT GCC CGT AGA TTT CA; BRE antisense, GTG GCT TCA GTG TCC TCA AA; PTEN sense, TAG ACC AGT GGC ACT GTT GT; PTEN antisense, TGG CAG ACC ACA AAC TGA GGA T; GADPH sense, AGC CTC AAG ATC ATC AGC AAT G; GADPH antisense, CAC GAT ACC AAA GTT GTC ATG GAT.</p></sec><sec><title>Immunohistochemical Analysis</title><p>Patient ESCC tumor tissue and matched peri-tumor normal esophageal tissues were obtained from Sun Yet-Sen University Cancer Center. Written informed consent was obtained from all ESCC patients before the study. The use of the clinical specimens for research purposes was approved by the Ethics Committee of Jinan University. Tissue samples were fixed with 4% paraformaldehyde and embedded in paraffin and sectioned for immunohistochemical staining using the standard protocols. Primary antibodies against BRE were obtained from Abcam (ab177960, 1:50), and the staining results were scored by three pathologists without any information regarding tissue features. A semi-quantitative immunoreactive score (IRS) was used to evaluate the expression of the BRE proteins. Briefly, IRS was calculated by multiplying the staining intensity (graded as: 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining) with the percentage of positively stained cells (0 = no stained cell, 1 = <10% of stained cells, 2 = 10–50% of stained cells, 3 = 51–80% of stained cells, and 4 = >80% of stained cells). An IRS <4 was defined as low expression, IRS between 4 and 8 was defined as medium expression, and IRS≤ 8 was defined as high expression (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>, <xref rid=\"B23\" ref-type=\"bibr\">23</xref>).</p></sec><sec><title>Xenograft Models</title><p>To analyze the <italic>in vivo</italic> growth of ESCC cells, 2 × 10<sup>5</sup> Eca109 or YSE140 cells were resuspended in 200 μl of 50% Matrigel PBS (Corning, USA) and injected subcutaneously into 5-week-old female BALB/c nude mice, six mice for each group. The tumor volume was measured every 3 days as described previously (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>). After 3 weeks, the mice were euthanized, and the tumors were harvested. Mouse handling and experimental protocols were approved by the Experiment Animal Care Committee and the Ethics Committee of Jinan University (Guangzhou, China).</p></sec><sec><title>Statistical Analysis</title><p>Data are expressed as the mean ± SEM values, and between-group comparisons were carried out using the paired samples Student's <italic>t</italic>-test for patients IHC samples scores or independent samples Student's <italic>t</italic>-test. The threshold for statistical significance was set to <sup>*</sup><italic>p</italic> < 0.05; <sup>**</sup><italic>p</italic> < 0.01.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title>BRE Is Frequently Overexpressed in ESCC Tissue</title><p>To examine BRE expression in ESCC, immunohistochemical analysis was performed in 50 pairs of ESCC and matched tumor-adjacent normal tissue. The IHC analysis of the cell proliferation marker Ki67 showed that the proliferation of the tumor tissues was significantly higher than that of tumor-adjacent normal tissues (<xref ref-type=\"fig\" rid=\"F1\">Figure 1A</xref>), thus identified the profile of the samples we collected. Based on the IHC density, the tissues samples were divided into three groups in accordance with BRE expression levels (high, medium, and low) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1B</xref>). The immunohistochemical analysis revealed that BRE was significantly upregulated in ESCC tissues than in the tumor-adjacent normal esophageal tissues (<xref ref-type=\"fig\" rid=\"F1\">Figure 1C</xref>). Consistently, 58% of ESCC samples (29/50) displayed BRE upregulation, whereas only 22% (11/50) of peri-tumor normal esophageal samples displayed BRE upregulation (<xref ref-type=\"fig\" rid=\"F1\">Figure 1D</xref>). Furthermore, we detected BRE expression in eight ESCC samples via western blotting and q-PCR analysis, as shown in <xref ref-type=\"fig\" rid=\"F1\">Figures 1E,F</xref>, and BRE protein was clearly overexpressed in ESCC samples compared with that in the tumor-adjacent normal esophageal tissues. Finally, BRE expression in five ESCC cell lines and the normal human esophageal epithelial cell (HEEC) line was analyzed by western blotting; concurrent with clinical samples, BRE was also significantly upregulated in ESCC cell lines (<xref ref-type=\"fig\" rid=\"F1\">Figure 1G</xref>).</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>BRE was overexpressed in ESCC tissues. <bold>(A)</bold> Representative images of IHC analysis of Ki67 staining in ESCC and tumor-adjacent normal tissue. <bold>(B)</bold> Representative photographs of IHC analysis with high, medium, and low intensities of BRE staining in ESCC and tumor-adjacent normal tissue. <bold>(C)</bold> Immunohistochemistry score of BRE in 50 pairs of ESCC and peri-tumor tissues. <bold>(D)</bold> Percentage of samples with high, medium, and low BRE protein levels in 50 pairs of ESCC and peri-tumor tissues. <bold>(E)</bold> BRE protein levels in eight ESCC (T) and peri-tumor tissues (P); <bold>(F)</bold>\n<italic>BRE</italic> gene expression levels in eight paired ESCC and tumor-adjacent tissues. <bold>(G)</bold> BRE protein levels in five ESCC cell lines and normal esophageal epithelial cell lines HEEC. Data are expressed as mean ± SEM values; paired Student's <italic>t</italic>-test was used to evaluate significant differences, **<italic>p</italic> < 0.01.</p></caption><graphic xlink:href=\"fonc-10-01407-g0001\"/></fig></sec><sec><title>BRE Promotes ESCC Cell Proliferation</title><p>To investigate the functions of BRE in ESCC, we knocked down and overexpressed the BRE genes in ESCC cell lines, using lentiviruses. The CCK-8 assay revealed that the viability of two ESCC cell lines, KYSE450 and KYESE140, was significantly decreased when BRE was knocked down (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>), whereas BRE overexpression significantly increased the viability of the Eca109 and TE-1 ESCC cell lines (<xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). Furthermore, the cell proliferation rate was analyzed using the EdU assay. As shown in <xref ref-type=\"fig\" rid=\"F2\">Figure 2C</xref>, the knockdown of BRE expression considerably decreased the percentage of EdU-positive proliferating cells in KYSE450 and KYESE140 cell lines. However, BRE overexpression significantly promoted the proliferation of Eca109 and TE-1 ESCC cells (<xref ref-type=\"fig\" rid=\"F2\">Figure 2D</xref>). Concurrently, the colony formation assay results showed that BRE knockdown inhibited self-renewal in ESCC cells, whereas BRE overexpression exhibited the opposite effects (<xref ref-type=\"fig\" rid=\"F2\">Figure 2E</xref>). Meanwhile, the cell cycle analysis revealed that the knockdown of BRE in KYSE450 and KYESE140 cells significantly decreased the percentage of cells in the S phase and increased the percentage of cells in the G0/G1 phase (<xref ref-type=\"fig\" rid=\"F3\">Figures 3A,B</xref>). In contrast, BRE overexpression significantly increased the percentage of Eca109 and TE-1 cells in the S phase (<xref ref-type=\"fig\" rid=\"F3\">Figures 3C,D</xref>). These results show that BRE promotes cell cycle progression to induce the growth of ESCC cells.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>BRE promotes ESCC cell proliferation. <bold>(A,B)</bold> The viability of ESCC cell lines after BRE knockdown or overexpression was determined using the CCK-8 assay. Data are expressed as mean ± SEM; two-way ANOVA was used to evaluate significant differences, **<italic>p</italic> < 0.01; <bold>(C,D)</bold> Representative photographs of EdU-incorporated cells after BRE knockdown or overexpression. <bold>(E)</bold> Representative photographs of clone formation assays in different ESCC cell lines after BRE knockdown or overexpression.</p></caption><graphic xlink:href=\"fonc-10-01407-g0002\"/></fig><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>BRE promotes cell cycle progression of ESCC cells. Cell cycle distribution of ESCC cell lines after BRE knockdown <bold>(A)</bold> or overexpression <bold>(C)</bold> were analyzed via flow cytometry. <bold>(B,D)</bold> Quantitative data of the flow cytometry, <italic>n</italic> = 3; data are expressed as mean ± SEM. The two-way ANOVA was used to evaluate significant differences, *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01.</p></caption><graphic xlink:href=\"fonc-10-01407-g0003\"/></fig></sec><sec><title>BRE Inhibits Apoptosis in ESCC Cells</title><p>We performed the flow cytometry analysis to determine the effect of BRE on apoptosis in different ESCC cells. As shown in <xref ref-type=\"fig\" rid=\"F4\">Figures 4A,B</xref>, BRE knockdown in KYESE140 and KYSE450 cells significantly increased the percentage of apoptotic cells from 6.9 to 8.5% in KYSE140 cells and from 7.2 to 16.5% in KYSE450 cells. Upon lentiviral overexpression of BRE, apoptotic cell death was considerably inhibited. BRE overexpression downregulated the percentage of apoptotic cells from 9.8 to 7.6% in TE-1 cells and from 5.6 to 3.9% in Eca109 cells (<xref ref-type=\"fig\" rid=\"F4\">Figures 4C,D</xref>). Moreover, in the presence of apoptosis-inducing anti-tumor drug cisplatin, BRE knockdown more strongly induced apoptotic cell death in both cell lines. The percentage of apoptotic cells increased from 16.1 to 27.2% in KYSE140 cells and from 22.4 to 39.3% in KYSE450 cells (<xref ref-type=\"fig\" rid=\"F4\">Figures 4A,B</xref>). As expected, BRE overexpression inhibited the apoptosis of ESCC cells, and the percentage of apoptotic cells decreased from 55 to 40% in TE-1 cells and from 50.1 to 27.1% in Eca109 cells (<xref ref-type=\"fig\" rid=\"F4\">Figures 4C,D</xref>).</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>BRE influences apoptosis of ESCC cells. The apoptotic cells of different ESCC cell lines after <bold>(A)</bold> BRE knockdown or <bold>(C)</bold> overexpression were analyzed by flow cytometry. <bold>(B,D)</bold> Quantitative data of flow cytometry, <italic>n</italic> = 3; data are expressed as mean ± SEM. The two-way ANOVA was used to evaluate significant differences, *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01.</p></caption><graphic xlink:href=\"fonc-10-01407-g0004\"/></fig></sec><sec><title>BRE Regulates AKT Signaling to Modulate ESCC Growth and Apoptosis</title><p>AKT signaling is widely involved in regulating cancer cell growth and inhibition of apoptosis (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Meanwhile, through analysis of the published microarray data we found that BRE knockdown downregulated the AKT signaling pathways (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). Therefore, we investigated the effect of BRE on the AKT signaling pathways through western blotting in four ESCC cell lines. As shown in <xref ref-type=\"fig\" rid=\"F5\">Figure 5A</xref>, BRE knockdown inhibited the phosphorylation of AKT and its down-stream target mTOR without influencing the total protein level of both AKT and mTOR. However, when BRE was overexpressed in Eca109 and TE-1 cells, AKT and mTOR phosphorylation were considerably upregulated (<xref ref-type=\"fig\" rid=\"F5\">Figure 5B</xref>). Furthermore, to determine whether AKT is responsible for BRE-induced cell growth and apoptotic resistance in ESCC cells, we used the widely used AKT inhibitor MK2206 to inhibit the activity of AKT in BRE-overexpressing cells. The results showed that AKT inhibition significantly diminished the cell growth (<xref ref-type=\"fig\" rid=\"F5\">Figure 5C</xref>) and apoptotic resistance (<xref ref-type=\"fig\" rid=\"F5\">Figures 5D,E</xref>) in both BRE-overexpressing Eca109 and TE-1 cells. Meanwhile, BRE overexpression also significantly reversed the cytotoxicity of the AKT inhibitors (<xref ref-type=\"fig\" rid=\"F5\">Figures 5C–E</xref>). These results indicate that BRE could modulate the AKT pathway to regulate ESCC growth and survival. Next, to reveal the detailed mechanisms of action of BRE in AKT activation, we detected the expression levels of AKT upstream regulators. The WB experiment results showed that knockdown of BRE significantly increased the expression of PTEN, a negative regulator of AKT activation (<xref ref-type=\"fig\" rid=\"F5\">Figure 5F</xref>). Contrarily, overexpression of BRE significantly decreased the expression of PTEN (<xref ref-type=\"fig\" rid=\"F5\">Figure 5F</xref>). However, both knockdown and overexpression of BRE had no significant influence on the expression of PI3K, a positive regulator of AKT activation (<xref ref-type=\"fig\" rid=\"F5\">Figure 5F</xref>). Furthermore, we found that BRE could regulate the expression of PTEN (<xref ref-type=\"fig\" rid=\"F5\">Figure 5G</xref>). These results indicate that BRE could negatively regulate the expression of PTEN to modulate AKT activation.</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>BRE positively modulates the AKT pathway. Expression of molecules of the AKT signaling pathway upon BRE knockdown <bold>(A)</bold> or overexpression <bold>(B)</bold> was analyzed by western blotting. <bold>(C)</bold> The viability of BRE overexpressed Eca109 and TE-1 cells with or without 10 μM MK2206, an AKT inhibitor, treatment were analyzed using the CCK-8 assay, <italic>n</italic> = 3. Data are expressed as mean ± SEM. The two-way ANOVA was used to evaluate significant differences, n.s, not significant, **<italic>p</italic> < 0.01. <bold>(D)</bold> Eca109 and TE-1 cells with or without BRE overexpression were treated with 10 μM cisplatin for 24 h; one BRE overexpression group was simultaneously treated with 10 μM MK2206, and the percentage of apoptotic cells was analyzed by flow cytometry. <bold>(E)</bold> Quantitative data of flow cytometry, <italic>n</italic> = 3. <bold>(F)</bold> Expression of molecules of the AKT signaling upstream regulators upon BRE knockdown or overexpression was analyzed by western blotting. <bold>(G)</bold> qPCR analysis of PTEN gene expression in BRE knockdown or overexpression ESCC cells, <italic>n</italic> = 3. Data are expressed as mean ± SEM; Student's <italic>t</italic>-test was used to evaluate significant differences, *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01.</p></caption><graphic xlink:href=\"fonc-10-01407-g0005\"/></fig></sec><sec><title>BRE Promotes ESCC Growth <italic>in vivo</italic></title><p>We evaluated the function of BRE <italic>in vivo</italic>. KYSE140 cells with a stable BRE knockdown and Eca109 cells with stable BRE overexpression were subcutaneously injected into the flanks of nude mice to induce the development of xenograft tumors. Our data show that the <italic>in vivo</italic> growth of KYSE140 cells was greatly reduced upon BRE knockdown (<xref ref-type=\"fig\" rid=\"F6\">Figure 6A</xref>). While, the growth of BRE-overexpressing Eca109 cells was accelerated in comparison with the control group (<xref ref-type=\"fig\" rid=\"F6\">Figure 6B</xref>). Next, we evaluated the pathologic changes induced by BRE knockdown or overexpression through Ki67 IHC staining of the xenograft tumor sections (<xref ref-type=\"fig\" rid=\"F6\">Figures 6C–F</xref>). The results showed that BRE considerably increased the percentage of Ki67+ cells and promoted ESCC growth <italic>in vivo</italic>. Finally, we detected the expression of p-AKT, p-mTOR, PTEN and cleaved-Caspase3 in xenograft tumors via IHC analysis. Our results showed that BRE knockdown considerably downregulated the expression of p-AKT and p-mTOR, but increased the expression of PTEN and apoptosis markers cleaved-Caspase3 in xenograft tumors formed by BRE knockdown KYSE140 cells (<xref ref-type=\"fig\" rid=\"F6\">Figures 6C,D</xref>). In contrast, the expression of p-AKT and p-mTOR was significantly enhanced, whereas the expression of PTEN and cleaved-Caspase3 was notably decreased in BRE-overexpressed tissues (<xref ref-type=\"fig\" rid=\"F6\">Figures 6E,F</xref>). Taken together, these results showed that BRE activated AKT signaling and inhibited the apoptosis of the ESCC xenograft tumor tissues and indicated that BRE plays an important role in promoting the growth of ESCC <italic>in vivo</italic>.</p><fig id=\"F6\" position=\"float\"><label>Figure 6</label><caption><p>BRE promotes ESCC growth <italic>in vivo</italic>. Xenograft tumor growth curve of xenograft tumors generated via BRE knockdown in KYESE140 cells <bold>(A)</bold> or in BRE-overexpressing Eca109 cells <bold>(B)</bold>, <italic>n</italic> = 6, and data are expressed as mean ± SEM; two-way ANOVA was used to evaluate significant differences, *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01. Representative photographs of immunohistochemical analysis of Ki67, p-AKT, p-mTOR, PTEN, Cleaved-Caspase3 in xenograft tumor sections formed by BRE knockdown in KYESE140 cells <bold>(C)</bold> or BRE-overexpressing Eca109 cells <bold>(E)</bold>. <bold>(D,F)</bold> The immunoreactive areas in the IHC images were quantified using ImagePro Plus 6.0 software (Media Cybernetics, Silver Spring, MD). The integrated optical density (IOD) values were represented as the mean ± SEM. Student's <italic>t</italic>-test was used to evaluate significant differences, **<italic>p</italic> < 0.01.</p></caption><graphic xlink:href=\"fonc-10-01407-g0006\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>BRE is a highly conserved protein expressed nearly ubiquitously in all tissues in humans and mice (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>) and is frequently upregulated in many tumors, thus regulating the development of different cancers (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>, <xref rid=\"B28\" ref-type=\"bibr\">28</xref>). Chen et al. reported that BRE was overexpressed in a malignantly transformed esophageal carcinoma cell line compared with that in an immortalized human esophageal epithelial cell line (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>). However, the functions of BRE in human ESCC and its potential regulatory mechanism remain largely unclear.</p><p>In this study, we analyzed BRE protein expression in 50 paired ESCC and peri-tumor normal esophageal tissues <italic>via</italic> immunohistochemical analysis and reported that BRE is frequently upregulated in ESCC tissues compared with that in tumor-adjacent normal esophageal tissues. These results were verified by western blotting in eight paired ESCC and peri-tumor normal esophageal tissues. Furthermore, the q-PCR analysis revealed that BRE expression was consistently upregulated in ESCC tissues. Thus, BRE may play a similar role in ESCC and HCC (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>) in regulating the growth and apoptosis of ESCC cells. Therefore, we performed knockdown and overexpression experiments to determine the role of BRE in ESCC progression <italic>in vitro</italic>. Our results showed that BRE knockdown significantly inhibited the growth of ESCC cells by inhibiting cell proliferation and cell cycle progression and inducing apoptosis. However, when BRE was ectopically overexpressed, ESCC cell growth was significantly increased. Furthermore, overexpression inhibited antitumor drug-induced apoptosis in ESCC cells, indicating that BRE promotes chemotherapeutic resistance in ESCC cells. Finally, we examined the functions of BRE in ESCC growth <italic>in vivo</italic> and found that BRE overexpression accelerates ESCC cell growth. These results show that BRE positively regulates ESCC cell growth both <italic>in vitro</italic> and <italic>in vivo</italic>. Furthermore, these results are consistent with those of studies on HCC and lung cancers (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>, <xref rid=\"B17\" ref-type=\"bibr\">17</xref>, <xref rid=\"B28\" ref-type=\"bibr\">28</xref>).</p><p>Our results show that BRE positively modulates AKT phosphorylation in ESCC cells. BRE knockdown significantly downregulated p-AKT and p-mTOR, an important downstream target of p-AKT signaling. In contrast, both p-AKT and p-mTOR were significantly upregulated in BRE-overexpressing ESCC cells. Moreover, by treating BRE-overexpressed ESCC cells with an AKT inhibitor, we further confirmed that AKT signaling was required for cell growth and apoptotic resistance in BRE-overexpressing cells, which is consistent with studies reporting that AKT potently promotes ESCC cell progression and patient survival (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>, <xref rid=\"B30\" ref-type=\"bibr\">30</xref>). Tang et al. reported that the <italic>Akt-3</italic> gene was upregulated upon BRE knockdown in C2C12 cells (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). Our results show that BRE does not influence total AKT expression; however, it significantly promotes AKT phosphorylation in ESCC cells. These results show that AKT regulation by BRE depends on the cell type. Furthermore, the results indicated that BRE could inhibit the expression of the AKT upstream negative regulator PTEN, without any influence on the expression of the AKT upstream positive regulator PI3K. In our previous study, we had reported that BRE could negatively regulate the stability of p53 (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). Considering that PTEN was a target gene of the p53, we hypothesize that BRE may modulate the gene expression of PTEN through p53 in ESCC cells.</p><p>In conclusion, our study shows that BRE is frequently overexpressed in ESCC tissues, and overexpression of BRE in ESCC cells activated the AKT signaling pathway, thereby increasing ESCC cell growth and decreasing apoptosis. These data are potentially applicable for the development of ESCC interventions and treatments. However, the limitations of this study are the relatively small number of patients examined and lack of clinical data regarding the disease progression. Thus, the correlation between BRE expression and ESCC disease progression requires further analysis. Meanwhile, the detailed regulatory roles of BRE on AKT activation and PTEN expression also need further exploration.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The datasets generated for this study are available on request to the corresponding author.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by The Ethics Committee of Jinan University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by The Experiment Animal Care Committee and the Ethics Committee of Jinan University.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>FJ, ZR, and JD: conceptualization and supervision. YZ, FJ, RW, and JC: investigation. FJ, YWa, and YWu: formal analysis. PZ and XS: resources. FJ: original draft preparation. ZR and JD: manuscript review and editing. All authors read and approved the final manuscript.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This study was funded by the National Natural Science Foundation of China (81802193 and 81471235), the Natural Science Foundation of Guangdong Province, grant number (2018A030313604 and 2019A1515012024), and the Open Project of Key Laboratory of Ministry of Education (2016RF01).</p></fn></fn-group><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rustgi</surname><given-names>AK</given-names></name><name><surname>El-Serag</surname><given-names>HB</given-names></name></person-group>. <article-title>Esophageal carcinoma</article-title>. <source>N Engl J Med.</source> (<year>2014</year>) <volume>371</volume>:<fpage>2499</fpage>–<lpage>509</lpage>. <pub-id pub-id-type=\"doi\">10.1056/NEJMra1314530</pub-id><pub-id pub-id-type=\"pmid\">25539106</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Torre</surname><given-names>LA</given-names></name><name><surname>Siegel</surname><given-names>RL</given-names></name><name><surname>Ward</surname><given-names>EM</given-names></name><name><surname>Jemal</surname><given-names>A</given-names></name></person-group>. <article-title>Global cancer incidence and mortality rates and trends–an update</article-title>. <source>Cancer Epidemiol Biomark Preven.</source> (<year>2015</year>) <volume>25</volume>:<fpage>16</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.1158/1055-9965.epi-15-0578</pub-id><pub-id pub-id-type=\"pmid\">26667886</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ohashi</surname><given-names>S</given-names></name><name><surname>Miyamoto</surname><given-names>S</given-names></name><name><surname>Kikuchi</surname><given-names>O</given-names></name><name><surname>Goto</surname><given-names>T</given-names></name><name><surname>Amanuma</surname><given-names>Y</given-names></name><name><surname>Muto</surname><given-names>M</given-names></name></person-group>. <article-title>Recent advances from basic and clinical studies of esophageal squamous cell carcinoma</article-title>. <source>Gastroenterology.</source> (<year>2015</year>) <volume>149</volume>:<fpage>1700</fpage>–<lpage>15</lpage>. <pub-id pub-id-type=\"doi\">10.1053/j.gastro.2015.08.054</pub-id><pub-id pub-id-type=\"pmid\">26376349</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>O' Sullivan</surname><given-names>KE</given-names></name><name><surname>Michielsen</surname><given-names>AJ</given-names></name><name><surname>O' Regan</surname><given-names>E</given-names></name><name><surname>Cathcart</surname><given-names>MC</given-names></name><name><surname>Moore</surname><given-names>G</given-names></name><name><surname>Breen</surname><given-names>E</given-names></name><etal/></person-group>. <article-title>pSTAT3 levels have divergent expression patterns and associations with survival in squamous cell carcinoma and adenocarcinoma of the oesophagus</article-title>. <source>Int J Mol Sci.</source> (<year>2018</year>) <volume>19</volume>:<fpage>15</fpage>. <pub-id pub-id-type=\"doi\">10.3390/ijms19061720</pub-id><pub-id pub-id-type=\"pmid\">29890775</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kuo</surname><given-names>KT</given-names></name><name><surname>Chen</surname><given-names>CL</given-names></name><name><surname>Chou</surname><given-names>TY</given-names></name><name><surname>Yeh</surname><given-names>CT</given-names></name><name><surname>Lee</surname><given-names>WH</given-names></name><name><surname>Wang</surname><given-names>LS</given-names></name></person-group>. <article-title>Nm23H1 mediates tumor invasion in esophageal squamous cell carcinoma by regulation of CLDN1 through the AKT signaling</article-title>. <source>Oncogenesis.</source> (<year>2016</year>) <volume>5</volume>:<fpage>e239</fpage>. <pub-id pub-id-type=\"doi\">10.1038/oncsis.2016.46</pub-id><pub-id pub-id-type=\"pmid\">27376780</pub-id></mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kar Keung Ching</surname><given-names>A</given-names></name><name><surname>Shan Li</surname><given-names>P</given-names></name><name><surname>Li</surname><given-names>Q</given-names></name><name><surname>Chung Lap Chan</surname><given-names>B</given-names></name><name><surname>Yeuk Hon Chan</surname><given-names>J</given-names></name><name><surname>Leong Lim</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Expression of human BRE in multiple isoforms</article-title>. <source>Biochem Biophys Res Commun.</source> (<year>2001</year>) <volume>288</volume>:<fpage>535</fpage>–<lpage>45</lpage>. <pub-id pub-id-type=\"doi\">10.1006/bbrc.2001.5801</pub-id><pub-id pub-id-type=\"pmid\">11676476</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tang</surname><given-names>MK</given-names></name><name><surname>Liu</surname><given-names>G</given-names></name><name><surname>Hou</surname><given-names>Z</given-names></name><name><surname>Chui</surname><given-names>YL</given-names></name><name><surname>Chan</surname><given-names>JY</given-names></name><name><surname>Lee</surname><given-names>KK</given-names></name></person-group>. <article-title>Livers overexpressing BRE transgene are under heightened state of stress-response, as revealed by comparative proteomics</article-title>. <source>Proteom Clin Appl.</source> (<year>2009</year>) <volume>3</volume>:<fpage>1362</fpage>–<lpage>70</lpage>. <pub-id pub-id-type=\"doi\">10.1002/prca.200900097</pub-id><pub-id pub-id-type=\"pmid\">21136956</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chan</surname><given-names>JY</given-names></name><name><surname>Li</surname><given-names>L</given-names></name><name><surname>Miao</surname><given-names>J</given-names></name><name><surname>Cai</surname><given-names>DQ</given-names></name><name><surname>Lee</surname><given-names>KK</given-names></name><name><surname>Chui</surname><given-names>YL</given-names></name></person-group>. <article-title>Differential expression of a novel gene BRE (TNFRSF1A modulator/BRCC45) in response to stress and biological signals</article-title>. <source>Mol Biol Rep.</source> (<year>2010</year>) <volume>37</volume>:<fpage>363</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s11033-009-9796-8</pub-id><pub-id pub-id-type=\"pmid\">19757177</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chui</surname><given-names>YL</given-names></name><name><surname>Ma</surname><given-names>CH</given-names></name><name><surname>Li</surname><given-names>W</given-names></name><name><surname>Xu</surname><given-names>Z</given-names></name><name><surname>Yao</surname><given-names>Y</given-names></name><name><surname>Lin</surname><given-names>FK</given-names></name><etal/></person-group>. <article-title>Anti-apoptotic protein BRE/BRCC45 attenuates apoptosis through maintaining the expression of caspase inhibitor XIAP in mouse Lewis lung carcinoma D122 cells</article-title>. <source>Apoptosis.</source> (<year>2014</year>) <volume>19</volume>:<fpage>829</fpage>–<lpage>40</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s10495-013-0963-y</pub-id><pub-id pub-id-type=\"pmid\">24395041</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Biswas</surname><given-names>K</given-names></name><name><surname>Philip</surname><given-names>S</given-names></name><name><surname>Yadav</surname><given-names>A</given-names></name><name><surname>Martin</surname><given-names>BK</given-names></name><name><surname>Burkett</surname><given-names>S</given-names></name><name><surname>Singh</surname><given-names>V</given-names></name><etal/></person-group>. <article-title>BRE/BRCC45 regulates CDC25A stability by recruiting USP7 in response to DNA damage</article-title>. <source>Nat Commun.</source> (<year>2018</year>) <volume>9</volume>:<fpage>537</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41467-018-03020-6</pub-id><pub-id pub-id-type=\"pmid\">29416040</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jin</surname><given-names>F</given-names></name><name><surname>Wang</surname><given-names>Y</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name><name><surname>Wu</surname><given-names>Y</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name><name><surname>Liu</surname><given-names>Q</given-names></name><etal/></person-group>. <article-title>Bre enhances osteoblastic differentiation by promoting the Mdm2-mediated degradation of p53</article-title>. <source>Stem Cells.</source> (<year>2017</year>) <volume>35</volume>:<fpage>1760</fpage>–<lpage>72</lpage>. <pub-id pub-id-type=\"doi\">10.1002/stem.2620</pub-id><pub-id pub-id-type=\"pmid\">28436570</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Xiao</surname><given-names>L</given-names></name><name><surname>Lee</surname><given-names>KK</given-names></name></person-group>. <article-title>BRE facilitates skeletal muscle regeneration by promoting satellite cell motility and differentiation</article-title>. <source>Biol Open.</source> (<year>2016</year>) <volume>5</volume>:<fpage>100</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1242/bio.012450</pub-id><pub-id pub-id-type=\"pmid\">26740569</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gu</surname><given-names>C</given-names></name><name><surname>Castellino</surname><given-names>A</given-names></name><name><surname>Chan</surname><given-names>JY</given-names></name><name><surname>Chao</surname><given-names>MV</given-names></name></person-group>. <article-title>BRE: a modulator of TNF-alpha action</article-title>. <source>FASEB J.</source> (<year>1998</year>) <volume>12</volume>:<fpage>1101</fpage>–<lpage>8</lpage>.<pub-id pub-id-type=\"pmid\">9737713</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>Q</given-names></name><name><surname>Ching</surname><given-names>AK</given-names></name><name><surname>Chan</surname><given-names>BC</given-names></name><name><surname>Chow</surname><given-names>SK</given-names></name><name><surname>Lim</surname><given-names>PL</given-names></name><name><surname>Ho</surname><given-names>TC</given-names></name><etal/></person-group>. <article-title>A death receptor-associated anti-apoptotic protein, BRE, inhibits mitochondrial apoptotic pathway</article-title>. <source>J Biol Chem.</source> (<year>2004</year>) <volume>279</volume>:<fpage>52106</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M408678200</pub-id><pub-id pub-id-type=\"pmid\">15465831</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hu</surname><given-names>X</given-names></name><name><surname>Kim</surname><given-names>JA</given-names></name><name><surname>Castillo</surname><given-names>A</given-names></name><name><surname>Huang</surname><given-names>M</given-names></name><name><surname>Liu</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>B</given-names></name></person-group>. <article-title>NBA1/MERIT40 and BRE interaction is required for the integrity of two distinct</article-title>. <source>J Biol Chem.</source> (<year>2011</year>) <volume>286</volume>:<fpage>11734</fpage>–<lpage>45</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M110.200857</pub-id><pub-id pub-id-type=\"pmid\">21282113</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Feng</surname><given-names>L</given-names></name><name><surname>Huang</surname><given-names>J</given-names></name><name><surname>Chen</surname><given-names>J</given-names></name></person-group>. <article-title>MERIT40 facilitates BRCA1 localization and DNA damage repair</article-title>. <source>Genes Dev.</source> (<year>2009</year>) <volume>23</volume>:<fpage>719</fpage>–<lpage>28</lpage>. <pub-id pub-id-type=\"doi\">10.1101/gad.1770609</pub-id><pub-id pub-id-type=\"pmid\">19261748</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chan</surname><given-names>BC</given-names></name><name><surname>Li</surname><given-names>Q</given-names></name><name><surname>Chow</surname><given-names>SK</given-names></name><name><surname>Ching</surname><given-names>AK</given-names></name><name><surname>Liew</surname><given-names>CT</given-names></name><name><surname>Lim</surname><given-names>PL</given-names></name><etal/></person-group>. <article-title>BRE enhances in vivo growth of tumor cells</article-title>. <source>Biochem Biophys Res Commun.</source> (<year>2005</year>) <volume>326</volume>:<fpage>268</fpage>–<lpage>73</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbrc.2004.11.013</pub-id><pub-id pub-id-type=\"pmid\">15582573</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chui</surname><given-names>YL</given-names></name><name><surname>Ching</surname><given-names>AK</given-names></name><name><surname>Chen</surname><given-names>S</given-names></name><name><surname>Yip</surname><given-names>FP</given-names></name><name><surname>Rowlands</surname><given-names>DK</given-names></name><name><surname>James</surname><given-names>AE</given-names></name><etal/></person-group>. <article-title>BRE over-expression promotes growth of hepatocellular carcinoma</article-title>. <source>Biochem Biophys Res Commun.</source> (<year>2010</year>) <volume>391</volume>:<fpage>1522</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bbrc.2009.12.111</pub-id><pub-id pub-id-type=\"pmid\">20035718</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Noordermeer</surname><given-names>SM</given-names></name><name><surname>Sanders</surname><given-names>MA</given-names></name><name><surname>Gilissen</surname><given-names>C</given-names></name><name><surname>Tonnissen</surname><given-names>E</given-names></name><name><surname>van der Heijden</surname><given-names>A</given-names></name><name><surname>Dohner</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>High BRE expression predicts favorable outcome in adult acute myeloid leukemia, in particular among MLL-AF9-positive patients</article-title>. <source>Blood.</source> (<year>2011</year>) <volume>118</volume>:<fpage>5613</fpage>–<lpage>21</lpage>. <pub-id pub-id-type=\"doi\">10.1182/blood-2011-06-359182</pub-id><pub-id pub-id-type=\"pmid\">21937695</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Noordermeer</surname><given-names>SM</given-names></name><name><surname>Wennemers</surname><given-names>M</given-names></name><name><surname>Bergevoet</surname><given-names>SM</given-names></name><name><surname>van der Heijden</surname><given-names>A</given-names></name><name><surname>Tonnissen</surname><given-names>E</given-names></name><name><surname>Sweep</surname><given-names>FC</given-names></name><etal/></person-group>. <article-title>Expression of the BRCA1 complex member BRE predicts disease free survival in breast cancer</article-title>. <source>Breast Cancer Res Treat.</source> (<year>2012</year>) <volume>135</volume>:<fpage>125</fpage>–<lpage>33</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s10549-012-2122-5</pub-id><pub-id pub-id-type=\"pmid\">22706632</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>HB</given-names></name><name><surname>Pan</surname><given-names>K</given-names></name><name><surname>Tang</surname><given-names>MK</given-names></name><name><surname>Chui</surname><given-names>YL</given-names></name><name><surname>Chen</surname><given-names>L</given-names></name><name><surname>Su</surname><given-names>ZJ</given-names></name><etal/></person-group>. <article-title>Comparative proteomic analysis reveals differentially expressed proteins regulated by a potential tumor promoter, BRE, in human esophageal carcinoma cells</article-title>. <source>Biochem Cell Biol.</source> (<year>2008</year>) <volume>86</volume>:<fpage>302</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1139/o08-069</pub-id><pub-id pub-id-type=\"pmid\">18756325</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>SH</given-names></name><name><surname>Lu</surname><given-names>HI</given-names></name><name><surname>Huang</surname><given-names>WT</given-names></name><name><surname>Tien</surname><given-names>WY</given-names></name><name><surname>Lan</surname><given-names>YC</given-names></name><name><surname>Lin</surname><given-names>WC</given-names></name><etal/></person-group>. <article-title>The prognostic significance of histone demethylase UTX in esophageal squamous cell carcinoma</article-title>. <source>Int J Mol Sci.</source> (<year>2018</year>) <volume>19</volume>:<fpage>297</fpage>. <pub-id pub-id-type=\"doi\">10.3390/ijms19010297</pub-id><pub-id pub-id-type=\"pmid\">29351209</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>DC</given-names></name><name><surname>Zhang</surname><given-names>MF</given-names></name><name><surname>Su</surname><given-names>SG</given-names></name><name><surname>Fang</surname><given-names>HY</given-names></name><name><surname>Wang</surname><given-names>XH</given-names></name><name><surname>He</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>HEY2, a target of miR-137, indicates poor outcomes and promotes cell proliferation and migration in hepatocellular carcinoma</article-title>. <source>Oncotarget.</source> (<year>2016</year>) <volume>7</volume>:<fpage>38052</fpage>–<lpage>63</lpage>. <pub-id pub-id-type=\"doi\">10.18632/oncotarget.9343</pub-id><pub-id pub-id-type=\"pmid\">27191260</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Song</surname><given-names>S</given-names></name><name><surname>Honjo</surname><given-names>S</given-names></name><name><surname>Jin</surname><given-names>J</given-names></name><name><surname>Chang</surname><given-names>SS</given-names></name><name><surname>Scott</surname><given-names>AW</given-names></name><name><surname>Chen</surname><given-names>Q</given-names></name><etal/></person-group>. <article-title>The hippo coactivator YAP1 mediates EGFR overexpression and confers chemoresistance in esophageal cancer</article-title>. <source>Clin Cancer Res.</source> (<year>2015</year>) <volume>21</volume>:<fpage>2580</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1158/1078-0432.CCR-14-2191</pub-id><pub-id pub-id-type=\"pmid\">25739674</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>LoRusso</surname><given-names>PM</given-names></name></person-group>. <article-title>Inhibition of the PI3K/AKT/mTOR pathway in solid tumors</article-title>. <source>J Clin Oncol.</source> (<year>2016</year>) <volume>34</volume>:<fpage>3803</fpage>–<lpage>15</lpage>. <pub-id pub-id-type=\"doi\">10.1200/Jco.2014.59.0018</pub-id><pub-id pub-id-type=\"pmid\">27621407</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>E</given-names></name><name><surname>Tang</surname><given-names>MK</given-names></name><name><surname>Yao</surname><given-names>Y</given-names></name><name><surname>Yau</surname><given-names>WWY</given-names></name><name><surname>Lo</surname><given-names>LM</given-names></name><name><surname>Yang</surname><given-names>XS</given-names></name><etal/></person-group>. <article-title>Silencing BRE expression in human umbilical cord perivascular (HUCPV) progenitor cells accelerates osteogenic and chondrogenic differentiation</article-title>. <source>PLoS ONE.</source> (<year>2013</year>) <volume>8</volume>:<fpage>e67896</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0067896</pub-id><pub-id pub-id-type=\"pmid\">23935848</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ching</surname><given-names>AK</given-names></name><name><surname>Li</surname><given-names>Q</given-names></name><name><surname>Lim</surname><given-names>PL</given-names></name><name><surname>Chan</surname><given-names>JY</given-names></name><name><surname>Chui</surname><given-names>YL</given-names></name></person-group>. <article-title>Expression of a conserved mouse stress-modulating gene, Bre: comparison with the human ortholog</article-title>. <source>DNA Cell Biol.</source> (<year>2003</year>) <volume>22</volume>:<fpage>497</fpage>–<lpage>504</lpage>. <pub-id pub-id-type=\"doi\">10.1089/10445490360708900</pub-id><pub-id pub-id-type=\"pmid\">14565866</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chan</surname><given-names>BC</given-names></name><name><surname>Ching</surname><given-names>AK</given-names></name><name><surname>To</surname><given-names>KF</given-names></name><name><surname>Leung</surname><given-names>JC</given-names></name><name><surname>Chen</surname><given-names>S</given-names></name><name><surname>Li</surname><given-names>Q</given-names></name><etal/></person-group>. <article-title>BRE is an antiapoptotic protein <italic>in vivo</italic> and overexpressed in human hepatocellular carcinoma</article-title>. <source>Oncogene.</source> (<year>2008</year>) <volume>27</volume>:<fpage>1208</fpage>–<lpage>17</lpage>. <pub-id pub-id-type=\"doi\">10.1038/sj.onc.1210733</pub-id><pub-id pub-id-type=\"pmid\">17704801</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manning</surname><given-names>BD</given-names></name><name><surname>Toker</surname><given-names>A</given-names></name></person-group>. <article-title>AKT/PKB signaling: navigating the network</article-title>. <source>Cell.</source> (<year>2017</year>) <volume>169</volume>:<fpage>381</fpage>–<lpage>405</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cell.2017.04.001</pub-id><pub-id pub-id-type=\"pmid\">28431241</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>J</given-names></name><name><surname>Lan</surname><given-names>T</given-names></name><name><surname>Zhang</surname><given-names>W</given-names></name><name><surname>Dong</surname><given-names>L</given-names></name><name><surname>Kang</surname><given-names>N</given-names></name><name><surname>Fu</surname><given-names>M</given-names></name><etal/></person-group>. <article-title>Dasatinib enhances cisplatin sensitivity in human esophageal squamous cell carcinoma (ESCC) cells via suppression of PI3K/AKT and Stat3 pathways</article-title>. <source>Arch Biochem Biophys.</source> (<year>2015</year>) <volume>575</volume>:<fpage>38</fpage>–<lpage>45</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.abb.2014.11.008</pub-id><pub-id pub-id-type=\"pmid\">25843419</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tang</surname><given-names>MK</given-names></name><name><surname>Wang</surname><given-names>CM</given-names></name><name><surname>Shan</surname><given-names>SW</given-names></name><name><surname>Chui</surname><given-names>YL</given-names></name><name><surname>Ching</surname><given-names>AK</given-names></name><name><surname>Chow</surname><given-names>PH</given-names></name><etal/></person-group>. <article-title>Comparative proteomic analysis reveals a function of the novel death receptor-associated protein BRE in the regulation of prohibitin and p53 expression and proliferation</article-title>. <source>Proteomics.</source> (<year>2006</year>) <volume>6</volume>:<fpage>2376</fpage>–<lpage>85</lpage>. <pub-id pub-id-type=\"doi\">10.1002/pmic.200500603</pub-id><pub-id pub-id-type=\"pmid\">16518872</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jin</surname><given-names>F</given-names></name><name><surname>Wang</surname><given-names>Y</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name><name><surname>Wu</surname><given-names>Y</given-names></name><name><surname>Wang</surname><given-names>X</given-names></name><name><surname>Liu</surname><given-names>Q</given-names></name><etal/></person-group>. <article-title>Bre enhances osteoblastic differentiation by promoting the Mdm2-mediated degradation of p53</article-title>. <source>Stem Cells.</source> (<year>2017</year>) <volume>35</volume>:<fpage>1760</fpage>–<lpage>72</lpage>. <pub-id pub-id-type=\"doi\">10.1002/stem.2620</pub-id>\n<pub-id pub-id-type=\"pmid\">28436570</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Immunol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Immunol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Immunol.</journal-id><journal-title-group><journal-title>Frontiers in Immunology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-3224</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849645</article-id><article-id pub-id-type=\"pmc\">PMC7431626</article-id><article-id pub-id-type=\"doi\">10.3389/fimmu.2020.01811</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Immunology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Household Contacts of Leprosy Patients in Endemic Areas Display a Specific Innate Immunity Profile</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>van Hooij</surname><given-names>Anouk</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/508177/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Tió-Coma</surname><given-names>Maria</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/884774/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Verhard</surname><given-names>Els M.</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Khatun</surname><given-names>Marufa</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Alam</surname><given-names>Khorshed</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Tjon Kon Fat</surname><given-names>Elisa</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>de Jong</surname><given-names>Danielle</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Sufian Chowdhury</surname><given-names>Abu</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/980418/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Corstjens</surname><given-names>Paul</given-names></name><xref ref-type=\"aff\" rid=\"aff3\"><sup>3</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Richardus</surname><given-names>Jan Hendrik</given-names></name><xref ref-type=\"aff\" rid=\"aff4\"><sup>4</sup></xref></contrib><contrib contrib-type=\"author\"><name><surname>Geluk</surname><given-names>Annemieke</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/137714/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>Department of Infectious Diseases, Leiden University Medical Center</institution>, <addr-line>Leiden</addr-line>, <country>Netherlands</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Rural Health Program, The Leprosy Mission International Bangladesh</institution>, <addr-line>Dhaka</addr-line>, <country>Bangladesh</country></aff><aff id=\"aff3\"><sup>3</sup><institution>Department Cell and Chemical Biology, Leiden University Medical Center</institution>, <addr-line>Leiden</addr-line>, <country>Netherlands</country></aff><aff id=\"aff4\"><sup>4</sup><institution>Department of Public Health, Erasmus MC, University Medical Center Rotterdam</institution>, <addr-line>Rotterdam</addr-line>, <country>Netherlands</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Malcolm Scott Duthie, HDT Biotech Corporation, United States</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Roberta Olmo Pinheiro, Oswaldo Cruz Foundation, Brazil; Yumi Maeda, National Institute of Infectious Diseases, Japan</p></fn><corresp id=\"c001\">*Correspondence: Annemieke Geluk <email>a.geluk@lumc.nl</email></corresp><fn fn-type=\"other\" id=\"fn001\"><p>This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology</p></fn><fn fn-type=\"other\" id=\"fn002\"><p>†These authors have contributed equally to this work</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1811</elocation-id><history><date date-type=\"received\"><day>15</day><month>5</month><year>2020</year></date><date date-type=\"accepted\"><day>07</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 van Hooij, Tió-Coma, Verhard, Khatun, Alam, Tjon Kon Fat, de Jong, Sufian Chowdhury, Corstjens, Richardus and Geluk.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>van Hooij, Tió-Coma, Verhard, Khatun, Alam, Tjon Kon Fat, de Jong, Sufian Chowdhury, Corstjens, Richardus and Geluk</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Leprosy is a chronic infectious disease, caused by <italic>Mycobacterium leprae</italic>, that can lead to severe life-long disabilities. The transmission of <italic>M. leprae</italic> is continuously ongoing as witnessed by the stable new case detection rate. The majority of exposed individuals does, however, not develop leprosy and is protected from infection by innate immune mechanisms. In this study the relation between innate immune markers and <italic>M. leprae</italic> infection as well as the occurrence of leprosy was studied in household contacts (HCs) of leprosy patients with high bacillary loads. Serum proteins associated with innate immunity (ApoA1, CCL4, CRP, IL-1Ra, IL-6, IP-10, and S100A12) were determined by lateral flow assays (LFAs) in conjunction with the presence of <italic>M. leprae</italic> DNA in nasal swabs (NS) and/or slit-skin smears (SSS). The HCs displayed ApoA1 and S100A12 levels similar to paucibacillary patients and could be differentiated from endemic controls based on the levels of these markers. In the 31 households included the number (percentage) of HCs that were concomitantly diagnosed with leprosy, or tested positive for <italic>M. leprae</italic> DNA in NS and SSS, was not equally divided. Specifically, households where <italic>M. leprae</italic> infection and leprosy disease was not observed amongst members of the household were characterized by higher S100A12 and lower CCL4 levels in whole blood assays of HCs in response to <italic>M. leprae</italic>. Lateral flow assays provide a convenient diagnostic tool to quantitatively measure markers of the innate immune response and thereby detect individuals which are likely infected with <italic>M. leprae</italic> and at risk of developing disease or transmitting bacteria. Low complexity diagnostic tests measuring innate immunity markers can therefore be applied to help identify who should be targeted for prophylactic treatment.</p></abstract><kwd-group><kwd>innate immunity</kwd><kwd>lateral flow test</kwd><kwd>diagnostics</kwd><kwd><italic>M. leprae</italic></kwd><kwd>UCP-LFA</kwd><kwd>leprosy</kwd></kwd-group><counts><fig-count count=\"5\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"47\"/><page-count count=\"12\"/><word-count count=\"7109\"/></counts></article-meta></front><body><sec sec-type=\"intro\" id=\"s1\"><title>Introduction</title><p>Leprosy is a debilitating disease that is one of the leading causes of long-term nerve damage worldwide (<xref rid=\"B1\" ref-type=\"bibr\">1</xref>). Multidrug therapy (MDT) effectively kills <italic>Mycobacterium leprae</italic>, the causative agent of leprosy, providing an effective cure when treatment is initiated timely (<xref rid=\"B2\" ref-type=\"bibr\">2</xref>, <xref rid=\"B3\" ref-type=\"bibr\">3</xref>). To achieve elimination of leprosy, however, it is vital to not only treat adequately and timely but also to prevent transmission (<xref rid=\"B4\" ref-type=\"bibr\">4</xref>). The stable new case detection rates in many leprosy endemic countries (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>) indicate that MDT insufficiently reduces transmission of <italic>M. leprae</italic>. Recognition of the often subtle cardinal clinical signs is of major importance for leprosy diagnosis (<xref rid=\"B6\" ref-type=\"bibr\">6</xref>). The declaration of the WHO in 2000 that leprosy had been eliminated as a public health problem (<xref rid=\"B7\" ref-type=\"bibr\">7</xref>), however, caused a reduction of leprosy control activities. The reduced intensity in case detection activities and training in the diagnosis and treatment of leprosy results in many cases that remain undetected for several years (<xref rid=\"B8\" ref-type=\"bibr\">8</xref>), allowing the transmission of <italic>M. leprae</italic> to continue.</p><p>Contacts close to leprosy patients have a higher risk of acquiring the infection, especially when the patients carry high bacillary loads (<xref rid=\"B9\" ref-type=\"bibr\">9</xref>–<xref rid=\"B11\" ref-type=\"bibr\">11</xref>). Fortunately, the majority of exposed individuals is naturally immune to <italic>M. leprae</italic> infection (<xref rid=\"B12\" ref-type=\"bibr\">12</xref>). Host immunity also determines the clinical phenotype of leprosy, ranging from paucibacillary (PB) patients with a strong proinflammatory response (Th1/Th17) leading to bacterial control to multibacillary (MB) patients with an anti-inflammatory immune response (Th2) producing large quantities of antibodies but unable to control the bacteria (<xref rid=\"B13\" ref-type=\"bibr\">13</xref>, <xref rid=\"B14\" ref-type=\"bibr\">14</xref>). In the innate immune response macrophages are critical mediators that define the course of <italic>M. leprae</italic> infection and clinical outcome. In PB patients IL-15 induces antimicrobial activity and the vitamin D-dependent antimicrobial program in macrophages restricting bacterial dissemination (proinflammatory M1 macrophages) (<xref rid=\"B15\" ref-type=\"bibr\">15</xref>). In contrast, in MB patients a scavenger receptor program is induced by IL-10, leading to foam cell formation by increased phagocytosis of mycobacteria and oxidized lipids, and persistence of <italic>M. leprae</italic> (anti-inflammatory M2 macrophages) (<xref rid=\"B16\" ref-type=\"bibr\">16</xref>, <xref rid=\"B17\" ref-type=\"bibr\">17</xref>).</p><p>Markers of the innate immune response can thus be helpful to identify <italic>M. leprae</italic> infected individuals who are prone to develop leprosy disease and thereby, since they are unable to kill and remove <italic>M. leprae</italic>, contribute to the ongoing transmission. No practical tools are yet available to identify individuals that should be prioritized for prophylactic treatment. Recently, biomarkers for leprosy and <italic>M. leprae</italic> infection were identified (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>), including serum proteins that play a role in innate immunity. For example, S100A12 is required to decrease <italic>M. leprae</italic> viability in infected macrophages (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). CCL4 and IP-10 attract innate immune cells such as natural killer (NK) cells and monocytes, whereas IL-1Ra-stimulated monocytes turn into M2 macrophages that produce high levels of the anti-inflammatory cytokine IL-10 (<xref rid=\"B21\" ref-type=\"bibr\">21</xref>).</p><p>Two other identified biomarkers (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>) that play a role in the innate immune system were contrasting acute phase proteins: anti-inflammatory ApoA1 and pro-inflammatory CRP. ApoA1 inhibits the recruitment of monocytes and macrophage chemotaxis (<xref rid=\"B22\" ref-type=\"bibr\">22</xref>), whereas CRP can recognize pathogens and activate the classical complement pathway (<xref rid=\"B23\" ref-type=\"bibr\">23</xref>). Together with αPGL-I IgM, the well-established biomarker for MB leprosy (<xref rid=\"B24\" ref-type=\"bibr\">24</xref>), the identified biomarkers were implemented in quantitative up-converting phosphor lateral flow assays (UCP-LFAs) (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>). These user-friendly tests are applicable in resource-limited settings, essential for diagnostic tools in large-scale contact screening of leprosy contacts, and provide quantitative results. The latter allows monitoring of drug treatment as well as discriminating high from low responders.</p><p>Previously, we analyzed nasal swabs (NS) and slit-skin smears (SSS) of household contacts (HCs) of MB leprosy patients with high bacillary loads for the presence of <italic>M. leprae</italic> DNA (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Here we analyzed the same individuals to examine the correlation of the presence of <italic>M. leprae</italic> DNA with the levels of innate immune markers. <italic>M. leprae</italic> DNA in NS indicates colonization of the HC with the bacterium, but not invasion of the tissue. Detection of <italic>M. leprae</italic> DNA in SSS does indicate that a HC is infected. In this study, levels of ApoA1, CCL4, CRP, IL-1Ra, IL-6, IP-10, αPGL-I IgM, and S100A12 were determined by UCP-LFAs in supernatants of 24 h <italic>M. leprae</italic> antigen-stimulated whole blood assays (WBA) addressing newly diagnosed MB patients with a high bacteriological index (BI) and their HCs in Bangladesh.</p></sec><sec sec-type=\"materials and methods\" id=\"s2\"><title>Materials and Methods</title><sec><title>Study Participants</title><p>The cohort used in this study originates from four districts in Bangladesh (Nilphamari, Rangpur, Panchagar, and Thakurgaon) and has been extensively described previously (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). The prevalence of leprosy in these districts was 0.9 per 10,000 and the new case detection rate 1.18 per 10,000 (Rural health program, the leprosy mission Bangladesh, yearly district activity report 2018).</p><p>Between July 2017 and May 2018, newly diagnosed leprosy patients (index case; <italic>n</italic> = 31) with BI ≥2 and between 3 and 15 HCs per index case (<italic>n</italic> = 279) were recruited (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Leprosy was diagnosed based on clinical and bacteriological observations and classified as MB or PB as described by the WHO (<xref rid=\"B5\" ref-type=\"bibr\">5</xref>) and the BI was determined. HCs were examined as well for signs and symptoms of leprosy upon recruitment and followed up yearly for surveillance of new case occurrence for ≥24 months after sample collection.</p><p>Control individuals without known contact to leprosy or TB patients and without clinical disease symptoms from the same leprosy endemic area (EC) were included and assessed for the absence of clinical signs and symptoms of leprosy and TB. Staff of leprosy or TB clinics were excluded as EC.</p></sec><sec><title>Household Contacts</title><p>The coding system used to describe physical and genetic distance of contacts from the patient has been extensively described previously (<xref rid=\"B26\" ref-type=\"bibr\">26</xref>). In short, four categories of physical distance are relevant for this study:</p><list list-type=\"simple\"><list-item><p>- KR: contacts living under the same roof and the same kitchen</p></list-item><list-item><p>- K: contacts living under a separate roof but using the same kitchen</p></list-item><list-item><p>- R: contacts living under the same roof, not using the same kitchen</p></list-item><list-item><p>- N1: next-door neighbors</p></list-item></list><p>In this study the KR and R group were considered as one group.</p><p>For genetic distance seven categories were defined: spouse (M), child (C), parent (P), sibling (B), other relative (O), relative in-law (CL, PL, BL, or OL), and not family related (N). CL, PL, and OL were considered as one group in this study, referred to by OL.</p></sec><sec><title>Ethics</title><p>This study was performed according to the Helsinki Declaration (version Fortaleza, Brazil, October 2013). The studies involving human participants were reviewed and approved by the Bangladesh Medical Research Council/National Research Ethics Committee (BMRC/NREC/2010-2013/1534). Participants were informed about the study-objectives, the samples and their right to refuse to take part or withdraw from the study without consequences for their treatment. Written informed consent was obtained before enrolment. All patients received treatment according to national guidelines.</p></sec><sec><title>Sample Collection</title><p>SSS from the earlobe and NS were collected for detection of <italic>M. leprae</italic> DNA as described previously (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). For the WBA, 4 ml venous blood was drawn and 1 ml was applied directly to a microtube precoated with 10 μg <italic>M. leprae</italic> whole cell sonicate (WCS) or without stimulus (Med). After 24 h incubation at 37°C the microtube was frozen at −20°C, shipped to the LUMC and stored at −80°C until further analysis.</p></sec><sec><title>DNA Isolation and RLEP PCR/qPCR</title><p>DNA isolated from the NS and SSS was used to perform RLEP PCR and qPCR as described previously (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Presence of <italic>M. leprae</italic> DNA was considered if a sample was positive for RLEP qPCR with a Ct lower than 37.5 or was positive for RLEP PCR at least in two out of three independently performed PCRs to avoid false positives.</p></sec><sec><title>UCP-LFAs</title><p>Levels of αPGL-I IgM, CRP, IP-10, S100A12, ApoA1, IL-6, IL-1Ra, and CCL4 in WBA supernatant were analyzed using UCP-LFAs. αPGL-I IgM, CRP, IP-10, S100A12, and ApoA1 UCP-LFAs have been described previously (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>). IL-6, IL-1Ra, and CCL4 UCP-LFAs were produced similarly, with a Test line of 200 ng MQ2-39C3 (IL-6; BioLegend, San Diego, USA), AF280 (IL-1Ra), and clone 24006 (CCL4) (R&D systems, Minneapolis, USA) and a Flow Control line with 100 ng Goat-anti-Rat (IL-6; R5130, Sigma-Aldrich), Goat-anti-Mouse (IL-1Ra; M8642; Sigma-Aldrich), and Rabbit-anti-Goat (CCL4; G4018, Sigma-Aldrich). Complementary antibodies were conjugated to the UCP particles, MQ2-13A5 (BioLegend, San Diego, USA), clone 10309 (IL-1Ra), and AF-271-NA (CCL4) (R&D systems, Minneapolis, USA). Yttrium fluoride upconverting nano materials (200 nm, NaYF4:Yb3+,Er 3+) functionalized with polyacrylic acid were obtained from Intelligent Material Solutions Inc. (Princeton, New Jersey, USA).</p><p>To perform the UCP-LFAs WBA supernatant was diluted 5-fold (IP-10, IL-1Ra and CCL4), 50-fold (IL-6, αPGL-I IgM and S100A12), 500-fold (CRP) and 5,000-fold (ApoA1) in high salt buffer (100 mM Tris pH 8, 270 mM NaCl, 1% (w/v) BSA, 1% (v/v) Triton X-100). As WCS stimulation does not affect the levels of ApoA1, CRP, and αPGL-I IgM these three markers were only determined in medium. Strips were analyzed using a UCP dedicated benchtop reader (UPCON; Labrox, Finland). Results are displayed as the ratio value between Test and Flow-Control signal based on relative fluorescence units (RFUs; excitation at 980 nm and emission at 550 nm) measured at the respective lines.</p></sec><sec><title>Statistical Analysis</title><p>GraphPad Prism version 8.1.1 for Windows (GraphPad Software, San Diego CA, USA) was used to perform Mann-Whitney <italic>U</italic>-tests, Kruskal-Wallis with Dunn's correction for multiple testing, Wilcoxon matched-pairs signed rank test, plot receiver operating characteristic (ROC) curves, and calculate the area under curve (AUC). The Pearson correlation coefficient and the corresponding <italic>p</italic>-values and heatmap were also determined using GraphPad Prism.</p></sec></sec><sec sec-type=\"results\" id=\"s3\"><title>Results</title><sec><title><italic>M. leprae</italic> DNA in Nasal Swabs/Slit-Skin Smears and the Occurrence of Leprosy in HCs</title><p>The presence of <italic>M. leprae</italic> DNA in NS and SSS of HCs was assessed in 31 households of MB index cases with BI ≥2 (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>) (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Out of 279 HCs, 29 were diagnosed with leprosy upon first physical investigation at intake, and four were diagnosed with PB leprosy during follow-up. Of the patients diagnosed at intake the majority (93%) had a low bacillary load: 22 were PB and seven were MB, of whom five with BI 0 (MB/BT) and two with BI ≥4 (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure 1</xref>). The HCs diagnosed with leprosy at intake (DevLep) were not evenly distributed over the different households: in 14 households none of the HCs had developed leprosy, whereas in the other 17 households, 9–42% suffered from leprosy (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Applying previous results on the presence of <italic>M. leprae</italic> DNA (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>), indicated that in 10 households <italic>M. leprae</italic> DNA was not detected in any of the HCs in NS and in 13 households all HCs were negative in the SSS. Of the households where <italic>M. leprae</italic> DNA was detected, percentages of colonization varied from 7 to 100% (NS) and for infection from 10 to 66% (SSS; <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). The proportion of <italic>M. leprae</italic> DNA presence in NS or SSS and identified leprosy in HCs upon first physical screening thus varies between households even if the index cases have similarly high bacillary loads.</p><fig id=\"F1\" position=\"float\"><label>Figure 1</label><caption><p>Percentage of <italic>M. leprae</italic> DNA positive nasal swabs/slit-skin smears and occurrence of leprosy in contacts per household. <bold>(A)</bold> Table indicates the number of household contacts per index case, the percentage of contacts that were diagnosed with leprosy during contact screening (%DevLep) and the percentage of contacts with <italic>M. leprae</italic> DNA detected in nasal swabs (%NS+) and slit-skin smears (%SSS+). The characteristics of the index case of each household (HH) are also indicated in this table. RLEP+ indicates whether <italic>M. leprae</italic> DNA was detected in the NS or SSS of the index case, the corresponding Ct values are indicative of the amount of <italic>M. leprae</italic> bacilli in NS and SSS. A low Ct value corresponds to high amounts of bacteria. BI, bacteriological index; NA, Not applicable. <bold>(B)</bold> On the <italic>x</italic>-axis the percentage range of household contacts (HCs) diagnosed with leprosy during contact screening (DevLep; dark red bars), that were <italic>M. leprae</italic> DNA positive in nasal swabs (NS+; yellow bars) or slit-skin smears (SSS+; orange bars) is indicated. The <italic>y</italic>-axis depicts the number of households for the percentage range indicated on the <italic>x</italic>-axis. The number of households within each percentage range was determined using the data table from <bold>(A)</bold>.</p></caption><graphic xlink:href=\"fimmu-11-01811-g0001\"/></fig></sec><sec><title>ApoA1 and S100A12 Levels Differentiate HCs From EC</title><p>Levels of αPGL-I IgM, CRP, IP-10, S100A12, ApoA1, IL-6, IL-1Ra, and CCL4 were determined by UCP-LFA in WBA supernatant. Levels of these eight markers in patients (<italic>n</italic> = 62; 38 MB and 24 PB), HCs (<italic>n</italic> = 244) and EC (<italic>n</italic> = 20) without known contact to leprosy patients were compared. Stimulation with <italic>M. leprae</italic> WCS had a significant impact on the CCL4, IL-1Ra, and IL-6 levels (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure 2</xref>). Significant differences between the groups were observed for αPGL-I IgM, S100A12<sub>Med</sub>, S100A12<sub>WCS</sub>, ApoA1, and CRP (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>). Compared to EC, the AUC values for αPGL-I IgM and CRP were significant only for MB patients, whereas ApoA1 and S100A12 levels significantly differed in both MB and PB patients. In HCs, however, the levels of S100A12 were comparable to those in (MB and PB) patients with similar AUCs (ranging from 0.85 to 0.91; <xref ref-type=\"fig\" rid=\"F2\">Figure 2B</xref>). Interestingly, the difference in ApoA1 levels between EC was more profound for HC (AUC:0.81; <italic>p</italic> < 0.0001) than for PB (AUC:0.76; <italic>p</italic> = 0.0039) or MB patients (AUC: 0.7; <italic>p</italic> = 0.0126). As described for other cohorts previously (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>), MB patients can be discriminated from HCs based on αPGL-I IgM (<italic>p</italic> < 0.0001) and CRP (<italic>p</italic> = 0.0024), but these markers cannot differentiate PB patients from HCs with similar rates of <italic>M. leprae</italic> DNA presence in NS and SSS (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). These data thus indicate that PB patients and HCs respond similarly to <italic>M. leprae</italic>.</p><fig id=\"F2\" position=\"float\"><label>Figure 2</label><caption><p>Differentiation of leprosy patients and household contacts (HC) from endemic controls (EC) by immune markers. Whole blood without stimulus (Med) or stimulated with <italic>M. leprae</italic> whole cell sonicate (WCS) was frozen after 24 h. Levels of 8 proteins (αPGL-I IgM, S100A12, ApoA1, CCL4, IP-10, IL-6, IL-1Ra, and CRP) were assessed by up-converting phosphor lateral flow assays (UCP-LFAs) in these whole blood assay supernatants for 31 households of index cases with multibacillary (MB) leprosy (bacteriological index ≥2). <bold>(A)</bold> UCP-LFA ratio values were calculated by dividing the peak area of the test line (T) by the peak area of the flow control line (FC; <italic>y</italic>-axis). As ratio values are marker dependent the <italic>y-</italic>axis scale differs per marker. The levels of MB (orange circles) and paucibacillary (PB; blue circles) patients, household contacts (HC; green circles) and endemic controls (EC; gray circles) were compared using the Kruskal-Wallis test with Dunn's correction for multiple testing. The data of CCL4, IP-10, IL-6, and IL-1Ra were not shown as no significant differences were observed in the levels of these proteins between groups. <italic>P</italic>-values: *<italic>p</italic> ≤ 0.05, **<italic>p</italic> ≤ 0.01, ****<italic>p</italic> ≤ 0.0001. <bold>(B)</bold> Receiver operating characteristic (ROC) curves were computed comparing the levels of αPGL-I IgM, CRP, S100A12, ApoA1 in multibacillary (MB) /paucibacillary (PB) patients and HC to EC. These levels were determined by up-converting phosphor lateral flow assays in supernatant of 24 h <italic>M. leprae</italic> antigen-stimulated whole blood assays (WBA; medium = Med, <italic>M. leprae</italic> whole cell sonicate = WCS). A summary of the areas under the curve (AUC) for MB (orange), PB (blue) and HC (green) is depicted in the spider plot showing the markers in which significant differences were observed (lower right panel).</p></caption><graphic xlink:href=\"fimmu-11-01811-g0002\"/></fig></sec><sec><title>S100A12 and CCL4 Response Is Associated With the Occurrence of Leprosy in Households</title><p>The relationship between disease and infection/colonization status in households was examined into more detail by determining the correlation between the immune markers and the percentage of HCs with detectable <italic>M. leprae</italic> DNA in NS (%NS) and SSS (%SSS) or diagnosed with leprosy (%DevLep) (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>). A highly significant (<italic>p</italic> < 0.0001) positive correlation was identified for the %DevLep with CCL4<sub>WCS</sub> and a negative correlation for %SSS with S100A12<sub>Med</sub> and S100A12<sub>WCS</sub> (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Table 1</xref>). For a subset of individuals qPCR Ct values were available indicative of the quantity of <italic>M. leprae</italic> DNA in NS (<italic>n</italic> = 105) or SSS (<italic>n</italic> = 71). These Ct values showed an inverse correlation with αPGL-I IgM antibodies in this cohort, indicating a strong positive correlation between the amount of <italic>M. leprae</italic> and the PGL-I antibody titer (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). For IL-1Ra<sub>Med</sub>/IL-1Ra<sub>WCS</sub> and inversely for CRP, a significant correlation was observed with the Ct values for both NS and SSS as well (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Table 1</xref>).</p><fig id=\"F3\" position=\"float\"><label>Figure 3</label><caption><p>Correlation of leprosy disease and M. leprae infection/colonization status in households with innate immune markers. <bold>(A)</bold> Whole blood without stimulus (Med) or stimulated with <italic>M. leprae</italic> whole cell sonicate (WCS) was frozen after 24 h. Levels of 8 proteins (αPGL-I IgM, S100A12, ApoA1, CCL4, IP-10, IL-6, IL-1Ra, and CRP) were assessed by up-converting phosphor lateral flow assays (UCP-LFAs) in supernatants of WBA for 31 households of index cases with multibacillary (MB) leprosy (bacterial index ≥2). The proportion of household contacts (HCs) diagnosed with leprosy upon first clinical examination (%DevLep) or with <italic>M. leprae</italic> DNA presence in nasal swabs (%NS) or slit-skin smears (%SSS) was calculated per household. These percentages and the RLEP Ct values determined by qPCR in NS and SSS were correlated with the levels of the assessed immune markers. The heatmap indicates the correlation coefficient (R), ranging from −1 (green) to 1 (orange) as determined using GraphPad Prism. Significant correlations (<italic>p</italic> < 0.05) are indicated with an asterisk (*), highly significant (<italic>p</italic> < 0.0001) are indicated with a black asterisk (*). <bold>(B)</bold> Significantly different (<italic>p</italic> < 0.05) levels of immune markers observed in HCs of <italic>M. leprae</italic> DNA positive (NS<sub>Pos</sub>) and negative (NS<sub>Neg</sub>) households. Ratio values (<italic>y</italic>-axis) represent the level of the assessed marker and were determined by dividing the signal of the test line (T) by the signal of the flow control (FC) line of the up-converting phosphor lateral flow assays. <bold>(C)</bold> Significantly different (<italic>p</italic> < 0.05) levels of immune markers observed in HCs of <italic>M. leprae</italic> DNA positive (SSS<sub>Pos</sub>) and negative (SSS<sub>Neg</sub>) households. <bold>(D)</bold> Significantly different (<italic>p</italic> < 0.05) levels of immune markers between HCs living in households where leprosy was diagnosed among contacts (DevLep) and in households where leprosy was not observed (NoLep).</p></caption><graphic xlink:href=\"fimmu-11-01811-g0003\"/></fig><p>A cross-sectional analysis was performed to compare households in which HCs developed leprosy to households where this was not observed. The same analysis was performed for households where <italic>M. leprae</italic> DNA was present in NS or SSS of HCs. In households where <italic>M. leprae</italic> DNA was detected in NS significantly lower levels of S100A12<sub>Med</sub> (<italic>p</italic> < 0.0001) and S100A12<sub>WCS</sub> (<italic>p</italic> = 0.0005) and higher levels of IL-1Ra<sub>WCS</sub> were observed (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>). S100A12 levels were also significantly lower in households where <italic>M. leprae</italic> DNA was detected in SSS (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>; <italic>p</italic> < 0.0001). CCL4 levels were higher in these households, especially in response to <italic>M. leprae</italic> WCS (<italic>p</italic> < 0.0001). Higher levels of CCL4<sub>WCS</sub> were also observed in the households where HCs of the primary index case were diagnosed with leprosy upon first physical investigation at intake (<italic>p</italic> = 0.0002) as well as increased levels of CRP (<italic>p</italic> = 0.025; <xref ref-type=\"fig\" rid=\"F3\">Figure 3D</xref>).</p><p>The levels of CCL4 and S100A12 showed a significant result in both the correlation and cross-sectional analysis, indicating an association of these markers with leprosy and/or <italic>M. leprae</italic> infection among HCs.</p></sec><sec><title><italic>M. leprae</italic> Colonization in HCs Correlates With Physical Distance to the Index Case</title><p>To examine the influence of the characteristics of the index case (all MB patients with high bacillary loads) on the development of leprosy and <italic>M. leprae</italic> colonization (NS) or infection (SSS) in HCs, a correlation and cross-sectional analysis was performed (<xref ref-type=\"supplementary-material\" rid=\"SM1\">Supplementary Figure 3</xref>). Cross-sectionally, higher S100A12<sub>Med</sub> levels were observed in index cases without detectable <italic>M. leprae</italic> DNA in NS of their HCs (<italic>p</italic> = 0.035). No other significant differences were observed in index cases for the other markers nor in the amount of bacteria in SSS or NS. Thus, characteristics of the index case in this cohort have little influence on the observed differences between the households (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>).</p><p>The influence of genetic relationship and physical distance of HCs to the index case was also examined. HCs were stratified by genetic distance against the percentage of leprosy and <italic>M. leprae</italic> DNA presence in NS and SSS in these groups (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>). Development of leprosy was most frequently observed in spouses (37%), followed by siblings (23%) and siblings in law (17%) (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>). Spouses also showed the highest frequency of <italic>M. leprae</italic> presence in NS and/or SSS (58%), followed by children (42%), and parents (41%) (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>). Spouses, children, and parents live in the closest proximity of patients (<xref ref-type=\"fig\" rid=\"F4\">Figure 4C</xref>; living under the same roof or sharing a kitchen) and thus have the highest level of exposure. Physical distance indeed correlated significantly (<italic>p</italic> = 0.003; <italic>R</italic><sup>2</sup> = 0.8) with the %NS<sub>Pos</sub> (colonization), though this was not observed for the development of leprosy in HCs (<italic>p</italic> = 0.07; <italic>R</italic><sup>2</sup> = 0.44).</p><fig id=\"F4\" position=\"float\"><label>Figure 4</label><caption><p>Stratification of household contacts by genetic distance to the index case. Eight different groups were classified for genetic distance: spouse (M), child (C), parent (P), sibling (B), other relative (O), brother/sister in law (BL), other relatives in law (OL), and not family related (N). <bold>(A)</bold> Percentage of individuals diagnosed with leprosy upon first clinical examination (DevLep; orange) stratified by genetic distance and ranked by percentage. <bold>(B)</bold> Percentage of <italic>M. leprae</italic> DNA presence in nasal swabs (NS; yellow), slit-skin smears (SSS; red) or both (NS + SSS; dark red) stratified by genetic distance. <bold>(C)</bold> Distribution of physical distance (Roof/kitchen = dark blue, kitchen = blue, Neighbor = gray) to the index case stratified by genetic distance.</p></caption><graphic xlink:href=\"fimmu-11-01811-g0004\"/></fig><p>The levels of the innate immune markers were also stratified by genetic distance. Based on the median levels of the assessed markers, the HC groups that were diagnosed with leprosy clustered apart from the HC groups that did not show symptoms of disease (<xref ref-type=\"fig\" rid=\"F5\">Figure 5</xref>). Across the groups with different genetic distance to the index case, similar innate immune mechanisms seem to play a role in the development of leprosy in HCs. Additionally, the index case group clustered apart from all HC groups rendering the assessed markers useful for leprosy diagnostics.</p><fig id=\"F5\" position=\"float\"><label>Figure 5</label><caption><p>Contacts diagnosed with leprosy upon first clinical screening cluster together based on their immune response, irrespective of genetic distance. Whole blood without stimulus (=Med) or stimulated with <italic>M. leprae</italic> whole cell sonicate (=WCS) was frozen after 24 h. Levels of 8 proteins (αPGL-I IgM, S100A12, ApoA1, CCL4, IP-10, IL-6, IL-1Ra, and CRP) were assessed by up-converting phosphor lateral flow assays in supernatants of whole blood assays (WBA) for 31 households of index cases with multibacillary (MB) leprosy (bacteriological index ≥2). The heatmap shows clustering based on average linkage performed by heatmapper (<xref rid=\"B27\" ref-type=\"bibr\">27</xref>) of the median level of eight serum protein markers in contacts diagnosed with leprosy upon first clinical screening of the HCs (DevLep) and without leprosy (NoLep) stratified by genetic distance; spouse (M), child (C); parent (P); sibling (B); other relative (O); brother/sister in law (BL); other relatives in law (OL) and not family related (N). The z-score indicates the deviation from the average level of the marker across groups, higher Z-scores are indicated in yellow and lower Z-scores in blue. Red = index case, yellow = contacts diagnosed with leprosy; green = household contacts without leprosy.</p></caption><graphic xlink:href=\"fimmu-11-01811-g0005\"/></fig></sec></sec><sec sec-type=\"discussion\" id=\"s4\"><title>Discussion</title><p>To examine the link between innate immunity and <italic>M. leprae</italic> colonization/infection in HCs, immune markers were assessed in 24 h <italic>M. leprae</italic> antigen-stimulated WBAs by UCP-LFAs. Even though all HCs were exposed to comparable levels of <italic>M. leprae</italic>, as all 31 index cases were MB patients with BI ≥2, there was a difference in the percentage of <italic>M. leprae</italic> DNA presence in NS/SSS and the occurrence of leprosy cases between households. Characteristics of the index case, such as the amount of <italic>M. leprae</italic> bacilli in NS or the αPGL-I antibody titer, had little influence on the development of leprosy nor on <italic>M. leprae</italic> colonization/infection in other household members. Physical distance of HCs to the index case was, however, significantly correlated with <italic>M. leprae</italic> colonization, though not with <italic>M. leprae</italic> infection or development of leprosy demonstrating the role of innate immune responses to remove bacteria.</p><p>In this study, S100A12 was associated with a protective response to <italic>M. leprae</italic> colonization/infection in HCs. As previously demonstrated (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>), S100A12 also remained a useful marker to discriminate leprosy patients from EC. S100A12 has a dual role inducing both proinflammatory and antimicrobial effects by interacting with different receptors, such as RAGE and TLR4 (<xref rid=\"B28\" ref-type=\"bibr\">28</xref>). RAGE expression is associated with disease severity and levels of proinflammatory cytokines in active tuberculosis (TB) (<xref rid=\"B29\" ref-type=\"bibr\">29</xref>). Contrary, RAGE is protective against the development of pulmonary TB in mouse models (<xref rid=\"B30\" ref-type=\"bibr\">30</xref>) in line with reduction of antimicrobial activity in human macrophages upon TLR2/1 ligand activation by S100A12 knockdown (<xref rid=\"B20\" ref-type=\"bibr\">20</xref>). S100A12 thus seems to protect exposed individuals from <italic>M. leprae</italic> colonization and infection, but once infected, S100A12 can contribute to maintain a detrimental, pro-inflammatory state in leprosy patients.</p><p>ApoA1 levels in HCs were similar to those in PB patients, suggesting that ApoA1 plays a role in limiting bacterial growth. This is in line with the finding that PB patients showed a similar low rate of <italic>M. leprae</italic> DNA presence in NS and SSS as HCs (<xref rid=\"B25\" ref-type=\"bibr\">25</xref>). Increased levels of ApoA1 have been observed in cells exposed to activated complement, where ApoA1 inhibits the formation of the membrane attack complex thereby contributing to complement clearance (<xref rid=\"B31\" ref-type=\"bibr\">31</xref>). Decreased levels are associated with destructive chronic inflammation, as ApoA1 exerts anti-inflammatory effects (<xref rid=\"B32\" ref-type=\"bibr\">32</xref>). The effects of ApoA1 do, however, not only rely on the protein level but also on the functionality, oxidative modification can for instance transform ApoA1 to an inflammatory agent (<xref rid=\"B33\" ref-type=\"bibr\">33</xref>). The role and functionality of ApoA1 in leprosy thus remains to be further elucidated. The influence of ApoA1 on lipid metabolism is of interest as dysfunctional high-density lipoprotein (involved in cholesterol transport to the liver of which the main protein is ApoA1) related to altered ApoA1 levels has been observed in MB patients (<xref rid=\"B34\" ref-type=\"bibr\">34</xref>). Moreover, it was suggested that <italic>M. leprae</italic> can directly affect ApoA1 biosynthesis.</p><p>Other markers in this study were associated with <italic>M. leprae</italic> colonization (IL-1Ra), whereas CCL4 was associated with infection and disease. These responses were most profound upon stimulation with <italic>M. leprae</italic> WCS, reflecting the innate immune response of these individuals to mycobacterial antigens. Interestingly, in whole blood of BCG-vaccinated infants the production of IL-1Ra and CCL4 was decreased upon stimulation of several TLRs (<xref rid=\"B35\" ref-type=\"bibr\">35</xref>). This observed response can be a result of BCG-induced trained innate immunity, which is immunological memory of the innate immune response that leads to an enhanced response to a subsequent trigger (<xref rid=\"B36\" ref-type=\"bibr\">36</xref>). Moreover, in Systemic Lupus Erythematosus (SLE) a pathogenic three-marker signature, including high levels of IL-1Ra and CCL4, was identified in monocytes (<xref rid=\"B37\" ref-type=\"bibr\">37</xref>). The signature was associated with the immune dysregulation in this autoimmune disease, in which flares occur similar to leprosy reactions (<xref rid=\"B38\" ref-type=\"bibr\">38</xref>). High levels of IL-1Ra and CCL4 thus seem indicative of pathogenic innate immune responses, corroborating earlier results on the identification of IL-1Ra and CCL4 as biomarkers associated with a pathogenic immune response to <italic>M. leprae</italic> (<xref rid=\"B18\" ref-type=\"bibr\">18</xref>, <xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B39\" ref-type=\"bibr\">39</xref>).</p><p>One of the challenges of application of host immune markers for diagnostics is the influence of co-morbidities or co-infections on biomarker levels. Helminth infections dampen the Th1 response and increase the risk for MB leprosy (<xref rid=\"B40\" ref-type=\"bibr\">40</xref>, <xref rid=\"B41\" ref-type=\"bibr\">41</xref>). A biomarker study to examine the influence of helminth co-infection in leprosy patients is currently ongoing. Moreover, the influence on biomarker levels of co-morbidities, such as diabetes mellitus which is known to increase the risk of active TB (<xref rid=\"B42\" ref-type=\"bibr\">42</xref>), on the disease outcome should be further studied. Another issue impeding straightforward implementation of biomarkers is that inflammatory markers are not disease-specific. For example, S100A12 has been described as biomarker for rheumatoid arthritis (<xref rid=\"B43\" ref-type=\"bibr\">43</xref>), TB (<xref rid=\"B44\" ref-type=\"bibr\">44</xref>) as well as inflammatory bowel disease (<xref rid=\"B45\" ref-type=\"bibr\">45</xref>). As the UCP-LFA allows quantitative measurement of biomarkers it would be interesting to compare disease-specific S100A12 levels for these conditions. Taking into account the multiple factors that influence host immune responses, a biomarker signature that combines several innate immune markers is required to identify individuals at risk of developing leprosy. This signature should also be evaluated in other inflammatory conditions.</p><p>In conclusion: Frequent exposure of HCs to <italic>M. leprae</italic> results in a continuously active innate immune response. This allows differentiation of HCs from EC by user-friendly diagnostic tests measuring specific serum protein levels. If the innate immune response is sufficient, pathogens, and pathogen-infected cells are being successfully removed. However, prolonged (intense) activation can lead to an immune response directed against the host (<xref rid=\"B46\" ref-type=\"bibr\">46</xref>). The resemblance of the innate immune response of PB patients and HCs observed in this and previous studies (<xref rid=\"B19\" ref-type=\"bibr\">19</xref>, <xref rid=\"B39\" ref-type=\"bibr\">39</xref>) indicates that PB leprosy can be a result of an imbalance in innate immunity. HCs that do not develop disease seem to effectively clear the bacteria without overactivation of the innate immune response. Elucidation of this delicate balance in innate immune responses by quantitation of appropriate biomarker signatures (<xref rid=\"B47\" ref-type=\"bibr\">47</xref>) can contribute to the identification of individuals at risk of developing leprosy upon <italic>M. leprae</italic> exposure. To gain more insight in this balance longitudinal analysis is required, which is currently ongoing. Diagnostic user-friendly rapid tests, as applied in this study, that allow quantitative measurement of combinations of innate immune markers represent useful tools to identify individuals that could benefit from prophylactic treatment.</p></sec><sec sec-type=\"data-availability\" id=\"s5\"><title>Data Availability Statement</title><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p></sec><sec id=\"s6\"><title>Ethics Statement</title><p>The studies involving human participants were reviewed and approved by the local ethical board in Bangladesh (BMRC/NREC/2010-2013/1534). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.</p></sec><sec id=\"s7\"><title>Author Contributions</title><p>AG, AH, and JR: designed research. AS, KA, and MK: enrolled patients, performed, and registered clinical diagnosis. AH, MT-C, EV, DJ, ET, and MK: performed experiments. PC and KA: resources. AH, MT-C, and AG: analyzed the data. AH and AG: wrote the paper. All authors: critically reviewed and agreed with the manuscript.</p></sec><sec id=\"s8\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer RP declared a past co-authorship with the authors MT-C and AG to the handling Editor.</p></sec></body><back><ack><p>We thank all patients and control individuals for their voluntary participation.</p></ack><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This study was supported by an R2STOP Research grant from Effect hope/The Leprosy Mission Canada, the Order of Malta-Grants-for-Leprosy-Research (MALTALEP), the Q.M. Gastmann-Wichers Foundation (to AG), and the Leprosy Research Initiative (LRI) together with the Turing Foundation (ILEP#: 703.15.07). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</p></fn></fn-group><sec sec-type=\"supplementary-material\" id=\"s9\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fimmu.2020.01811/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fimmu.2020.01811/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Data_Sheet_1.pdf\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><label>1.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Breen</surname><given-names>DP</given-names></name><name><surname>Deeb</surname><given-names>J</given-names></name><name><surname>Vaidya</surname><given-names>S</given-names></name><name><surname>Lockwood</surname><given-names>DN</given-names></name><name><surname>Radunovic</surname><given-names>A</given-names></name></person-group>. <article-title>Leprosy: a ‘common’ and curable cause of peripheral neuropathy with skin lesions</article-title>. <source>J R Coll Physicians Edinb.</source> (<year>2015</year>) <volume>45</volume>:<fpage>38</fpage>–<lpage>42</lpage>. <pub-id pub-id-type=\"doi\">10.4997/JRCPE.2015.109</pub-id><pub-id pub-id-type=\"pmid\">25874829</pub-id></mixed-citation></ref><ref id=\"B2\"><label>2.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><collab>World Health Organization</collab></person-group>\n<article-title>Global leprosy situation, 2010</article-title>. <source>Wkly Epidemiol Rec.</source> (<year>2010</year>) <volume>85</volume>:<fpage>337</fpage>–<lpage>48</lpage>.<pub-id pub-id-type=\"pmid\">20830851</pub-id></mixed-citation></ref><ref id=\"B3\"><label>3.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lazo-Porras</surname><given-names>M</given-names></name><name><surname>Prutsky</surname><given-names>GJ</given-names></name><name><surname>Barrionuevo</surname><given-names>P</given-names></name><name><surname>Tapia</surname><given-names>JC</given-names></name><name><surname>Ugarte-Gil</surname><given-names>C</given-names></name><name><surname>Ponce</surname><given-names>OJ</given-names></name><etal/></person-group>. <article-title>World Health Organization (WHO) antibiotic regimen against other regimens for the treatment of leprosy: a systematic review and meta-analysis</article-title>. <source>BMC Infect Dis.</source> (<year>2020</year>) <volume>20</volume>:<fpage>62</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s12879-019-4665-0</pub-id><pub-id pub-id-type=\"pmid\">31959113</pub-id></mixed-citation></ref><ref id=\"B4\"><label>4.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Smith</surname><given-names>CS</given-names></name><name><surname>Aerts</surname><given-names>A</given-names></name><name><surname>Kita</surname><given-names>E</given-names></name><name><surname>Virmond</surname><given-names>M</given-names></name></person-group>. <article-title>Time to define leprosy elimination as zero leprosy transmission?</article-title>\n<source>Lancet Infect Dis.</source> (<year>2016</year>) <volume>16</volume>:<fpage>398</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S1473-3099(16)00087-6</pub-id><pub-id pub-id-type=\"pmid\">27036335</pub-id></mixed-citation></ref><ref id=\"B5\"><label>5.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><collab>World Health Organisation</collab></person-group>\n<article-title>Global leprosy update, 2018: moving towards a leprosy-free world</article-title>. <source>Wkly Epidemiol Rec.</source> (<year>2019</year>) <volume>94</volume>(<issue>35/36</issue>):<fpage>389</fpage>–<lpage>412</lpage>.</mixed-citation></ref><ref id=\"B6\"><label>6.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eichelmann</surname><given-names>K</given-names></name><name><surname>Gonzalez Gonzalez</surname><given-names>SE</given-names></name><name><surname>Salas-Alanis</surname><given-names>JC</given-names></name><name><surname>Ocampo-Candiani</surname><given-names>J</given-names></name></person-group>. <article-title>Leprosy. an update: definition, pathogenesis, classification, diagnosis, and treatment</article-title>. <source>Actas Dermosifiliogr</source>. (<year>2013</year>) <volume>104</volume>:<fpage>554</fpage>–<lpage>63</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.adengl.2012.03.028</pub-id><pub-id pub-id-type=\"pmid\">23870850</pub-id></mixed-citation></ref><ref id=\"B7\"><label>7.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Roset Bahmanyar</surname><given-names>E</given-names></name><name><surname>Smith</surname><given-names>WC</given-names></name><name><surname>Brennan</surname><given-names>P</given-names></name><name><surname>Cummings</surname><given-names>R</given-names></name><name><surname>Duthie</surname><given-names>M</given-names></name><name><surname>Richardus</surname><given-names>JH</given-names></name><etal/></person-group>. <article-title>Leprosy diagnostic test development as a prerequisite towards elimination: requirements from the user's perspective</article-title>. <source>PLoS Negl Trop Dis.</source> (<year>2016</year>) <volume>10</volume>:<fpage>e0004331</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pntd.0004331</pub-id><pub-id pub-id-type=\"pmid\">26866699</pub-id></mixed-citation></ref><ref id=\"B8\"><label>8.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Smith</surname><given-names>WC</given-names></name><name><surname>van Brakel</surname><given-names>W</given-names></name><name><surname>Gillis</surname><given-names>T</given-names></name><name><surname>Saunderson</surname><given-names>P</given-names></name><name><surname>Richardus</surname><given-names>JH</given-names></name></person-group>. <article-title>The missing millions: a threat to the elimination of leprosy</article-title>. <source>PLoS Negl Trop Dis.</source> (<year>2015</year>) <volume>9</volume>:<fpage>e0003658</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pntd.0003658</pub-id><pub-id pub-id-type=\"pmid\">25905706</pub-id></mixed-citation></ref><ref id=\"B9\"><label>9.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bakker</surname><given-names>MI</given-names></name><name><surname>Hatta</surname><given-names>M</given-names></name><name><surname>Kwenang</surname><given-names>A</given-names></name><name><surname>Van Mosseveld</surname><given-names>P</given-names></name><name><surname>Faber</surname><given-names>WR</given-names></name><name><surname>Klatser</surname><given-names>PR</given-names></name><etal/></person-group>. <article-title>Risk factors for developing leprosy–a population-based cohort study in Indonesia</article-title>. <source>Lepr Rev.</source> (<year>2006</year>) <volume>77</volume>:<fpage>48</fpage>–<lpage>61</lpage>. <pub-id pub-id-type=\"pmid\">16715690</pub-id></mixed-citation></ref><ref id=\"B10\"><label>10.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Goulart</surname><given-names>IM</given-names></name><name><surname>Bernardes Souza</surname><given-names>DO</given-names></name><name><surname>Marques</surname><given-names>CR</given-names></name><name><surname>Pimenta</surname><given-names>VL</given-names></name><name><surname>Goncalves</surname><given-names>MA</given-names></name><name><surname>Goulart</surname><given-names>LR</given-names></name></person-group>. <article-title>Risk and protective factors for leprosy development determined by epidemiological surveillance of household contacts</article-title>. <source>Clin Vaccine Immunol.</source> (<year>2008</year>) <volume>15</volume>:<fpage>101</fpage>–<lpage>5</lpage>. <pub-id pub-id-type=\"doi\">10.1128/CVI.00372-07</pub-id><pub-id pub-id-type=\"pmid\">17989339</pub-id></mixed-citation></ref><ref id=\"B11\"><label>11.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sales</surname><given-names>AM</given-names></name><name><surname>Ponce de Leon</surname><given-names>A</given-names></name><name><surname>Duppre</surname><given-names>NC</given-names></name><name><surname>Hacker</surname><given-names>MA</given-names></name><name><surname>Nery</surname><given-names>JA</given-names></name><name><surname>Sarno</surname><given-names>EN</given-names></name><etal/></person-group>. <article-title>Leprosy among patient contacts: a multilevel study of risk factors</article-title>. <source>PLoS Negl Trop Dis.</source> (<year>2011</year>) <volume>5</volume>:<fpage>e1013</fpage>. <pub-id pub-id-type=\"doi\">10.1371/annotation/fc90a4dd-99a7-4c48-9dee-0494a0180038</pub-id><pub-id pub-id-type=\"pmid\">21423643</pub-id></mixed-citation></ref><ref id=\"B12\"><label>12.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Joyce</surname><given-names>MP</given-names></name></person-group>. <article-title>Historic aspects of human susceptibility to leprosy and the risk of conjugal transmission</article-title>. <source>Mem Inst Oswaldo Cruz.</source> (<year>2012</year>) <volume>107</volume>(<issue>Suppl. 1</issue>):<fpage>17</fpage>–<lpage>21</lpage>. <pub-id pub-id-type=\"doi\">10.1590/S0074-02762012000900004</pub-id><pub-id pub-id-type=\"pmid\">23283448</pub-id></mixed-citation></ref><ref id=\"B13\"><label>13.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sansonetti</surname><given-names>P</given-names></name><name><surname>Lagrange</surname><given-names>PH</given-names></name></person-group>. <article-title>The immunology of leprosy: speculations on the leprosy spectrum</article-title>. <source>Rev Infect Dis.</source> (<year>1981</year>) <volume>3</volume>:<fpage>422</fpage>–<lpage>69</lpage>. <pub-id pub-id-type=\"doi\">10.1093/clinids/3.3.422</pub-id><pub-id pub-id-type=\"pmid\">7025147</pub-id></mixed-citation></ref><ref id=\"B14\"><label>14.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Saini</surname><given-names>C</given-names></name><name><surname>Ramesh</surname><given-names>V</given-names></name><name><surname>Nath</surname><given-names>I</given-names></name></person-group>. <article-title>CD4+ Th17 cells discriminate clinical types and constitute a third subset of non Th1, Non Th2 T cells in human leprosy</article-title>. <source>PLoS Negl Trop Dis.</source> (<year>2013</year>) <volume>7</volume>:<fpage>e2338</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pntd.0002338</pub-id><pub-id pub-id-type=\"pmid\">23936569</pub-id></mixed-citation></ref><ref id=\"B15\"><label>15.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Modlin</surname><given-names>RL</given-names></name></person-group>. <article-title>The innate immune response in leprosy</article-title>. <source>Curr Opin Immunol.</source> (<year>2010</year>) <volume>22</volume>:<fpage>48</fpage>–<lpage>54</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.coi.2009.12.001</pub-id><pub-id pub-id-type=\"pmid\">20060279</pub-id></mixed-citation></ref><ref id=\"B16\"><label>16.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Montoya</surname><given-names>D</given-names></name><name><surname>Modlin</surname><given-names>RL</given-names></name></person-group>. <article-title>Learning from leprosy: insight into the human innate immune response</article-title>. <source>Adv Immunol.</source> (<year>2010</year>) <volume>105</volume>:<fpage>1</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1016/S0065-2776(10)05001-7</pub-id><pub-id pub-id-type=\"pmid\">20510728</pub-id></mixed-citation></ref><ref id=\"B17\"><label>17.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Mattos</surname><given-names>KA</given-names></name><name><surname>Oliveira</surname><given-names>VC</given-names></name><name><surname>Berrêdo-Pinho</surname><given-names>M</given-names></name><name><surname>Amaral</surname><given-names>JJ</given-names></name><name><surname>Antunes</surname><given-names>LC</given-names></name><name><surname>Melo</surname><given-names>RC</given-names></name><etal/></person-group>. <article-title>Mycobacterium leprae intracellular survival relies on cholesterol accumulation in infected macrophages: a potential target for new drugs for leprosy treatment</article-title>. <source>Cell Microbiol.</source> (<year>2014</year>) <volume>16</volume>:<fpage>797</fpage>–<lpage>815</lpage>. <pub-id pub-id-type=\"doi\">10.1111/cmi.12279</pub-id><pub-id pub-id-type=\"pmid\">24552180</pub-id></mixed-citation></ref><ref id=\"B18\"><label>18.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van Hooij</surname><given-names>A</given-names></name><name><surname>Tjon Kon Fat</surname><given-names>EM</given-names></name><name><surname>Batista da Silva</surname><given-names>M</given-names></name><name><surname>Carvalho Bouth</surname><given-names>R</given-names></name><name><surname>Cunha Messias</surname><given-names>AC</given-names></name><name><surname>Gobbo</surname><given-names>AR</given-names></name><etal/></person-group>. <article-title>Evaluation of immunodiagnostic tests for leprosy in Brazil, China and Ethiopia</article-title>. <source>Sci Rep.</source> (<year>2018</year>) <volume>8</volume>:<fpage>17920</fpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-018-36323-1</pub-id><pub-id pub-id-type=\"pmid\">30560920</pub-id></mixed-citation></ref><ref id=\"B19\"><label>19.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van Hooij</surname><given-names>A</given-names></name><name><surname>van den Eeden</surname><given-names>S</given-names></name><name><surname>Richardus</surname><given-names>R</given-names></name><name><surname>Tjon Kon Fat</surname><given-names>E</given-names></name><name><surname>Wilson</surname><given-names>L</given-names></name><name><surname>Franken</surname><given-names>K</given-names></name><etal/></person-group>. <article-title>Application of new host biomarker profiles in quantitative point-of-care tests facilitates leprosy diagnosis in the field</article-title>. <source>EBioMedicine.</source> (<year>2019</year>) <volume>47</volume>:<fpage>301</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ebiom.2019.08.009</pub-id><pub-id pub-id-type=\"pmid\">31422044</pub-id></mixed-citation></ref><ref id=\"B20\"><label>20.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Realegeno</surname><given-names>S</given-names></name><name><surname>Kelly-Scumpia</surname><given-names>KM</given-names></name><name><surname>Dang</surname><given-names>AT</given-names></name><name><surname>Lu</surname><given-names>J</given-names></name><name><surname>Teles</surname><given-names>R</given-names></name><name><surname>Liu</surname><given-names>PT</given-names></name><etal/></person-group>. <article-title>S100A12 is part of the antimicrobial network against mycobacterium leprae in human macrophages</article-title>. <source>PLoS Pathog.</source> (<year>2016</year>) <volume>12</volume>:<fpage>e1005705</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.ppat.1005705</pub-id><pub-id pub-id-type=\"pmid\">27355424</pub-id></mixed-citation></ref><ref id=\"B21\"><label>21.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Arango Duque</surname><given-names>G</given-names></name><name><surname>Descoteaux</surname><given-names>A</given-names></name></person-group>. <article-title>Macrophage cytokines: involvement in immunity and infectious diseases</article-title>. <source>Front Immunol.</source> (<year>2014</year>) <volume>5</volume>:<fpage>491</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2014.00491</pub-id><pub-id pub-id-type=\"pmid\">25339958</pub-id></mixed-citation></ref><ref id=\"B22\"><label>22.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Iqbal</surname><given-names>AJ</given-names></name><name><surname>Barrett</surname><given-names>TJ</given-names></name><name><surname>Taylor</surname><given-names>L</given-names></name><name><surname>McNeill</surname><given-names>E</given-names></name><name><surname>Manmadhan</surname><given-names>A</given-names></name><name><surname>Recio</surname><given-names>C</given-names></name><etal/></person-group>. <article-title>Acute exposure to apolipoprotein A1 inhibits macrophage chemotaxis <italic>in vitro</italic> and monocyte recruitment <italic>in vivo</italic></article-title>. <source>Elife.</source> (<year>2016</year>) <volume>5</volume>:<fpage>e15190</fpage>. <pub-id pub-id-type=\"doi\">10.7554/eLife.15190.020</pub-id><pub-id pub-id-type=\"pmid\">27572261</pub-id></mixed-citation></ref><ref id=\"B23\"><label>23.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Peisajovich</surname><given-names>A</given-names></name><name><surname>Marnell</surname><given-names>L</given-names></name><name><surname>Mold</surname><given-names>C</given-names></name><name><surname>Du Clos</surname><given-names>TW</given-names></name></person-group>. <article-title>C-reactive protein at the interface between innate immunity and inflammation</article-title>. <source>Expert Rev Clin Immunol.</source> (<year>2008</year>) <volume>4</volume>:<fpage>379</fpage>–<lpage>90</lpage>. <pub-id pub-id-type=\"doi\">10.1586/1744666X.4.3.379</pub-id><pub-id pub-id-type=\"pmid\">20476927</pub-id></mixed-citation></ref><ref id=\"B24\"><label>24.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Espinosa</surname><given-names>OA</given-names></name><name><surname>Benevides Ferreira</surname><given-names>SM</given-names></name><name><surname>Longhi Palacio</surname><given-names>FG</given-names></name><name><surname>Cortela</surname><given-names>D</given-names></name><name><surname>Ignotti</surname><given-names>E</given-names></name></person-group>. <article-title>Accuracy of Enzyme-Linked Immunosorbent Assays (ELISAs) in detecting antibodies against mycobacterium leprae in leprosy patients: a systematic review and meta-analysis</article-title>. <source>Can J Infect Dis Med Microbiol.</source> (<year>2018</year>) <volume>2018</volume>:<fpage>9828023</fpage>. <pub-id pub-id-type=\"doi\">10.1155/2018/9828023</pub-id><pub-id pub-id-type=\"pmid\">30622658</pub-id></mixed-citation></ref><ref id=\"B25\"><label>25.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tio-Coma</surname><given-names>M</given-names></name><name><surname>Avanzi</surname><given-names>C</given-names></name><name><surname>Verhard</surname><given-names>EM</given-names></name><name><surname>Pierneef</surname><given-names>L</given-names></name><name><surname>van Hooij</surname><given-names>A</given-names></name><name><surname>Benjak</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Detection of new <italic>Mycobacterium leprae</italic> subtype in Bangladesh by genomic characterization to explore transmission patterns</article-title>. <source>medRxiv [Preprint].</source> (<year>2020</year>). <pub-id pub-id-type=\"doi\">10.1101/2020.03.05.20031450</pub-id><pub-id pub-id-type=\"pmid\">32612587</pub-id></mixed-citation></ref><ref id=\"B26\"><label>26.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moet</surname><given-names>FJ</given-names></name><name><surname>Pahan</surname><given-names>D</given-names></name><name><surname>Schuring</surname><given-names>RP</given-names></name><name><surname>Oskam</surname><given-names>L</given-names></name><name><surname>Richardus</surname><given-names>JH</given-names></name></person-group>. <article-title>Physical distance, genetic relationship, age, and leprosy classification are independent risk factors for leprosy in contacts of patients with leprosy</article-title>. <source>J Infect Dis.</source> (<year>2006</year>) <volume>193</volume>:<fpage>346</fpage>–<lpage>53</lpage>. <pub-id pub-id-type=\"doi\">10.1086/499278</pub-id><pub-id pub-id-type=\"pmid\">16388481</pub-id></mixed-citation></ref><ref id=\"B27\"><label>27.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Babicki</surname><given-names>S</given-names></name><name><surname>Arndt</surname><given-names>D</given-names></name><name><surname>Marcu</surname><given-names>A</given-names></name><name><surname>Liang</surname><given-names>Y</given-names></name><name><surname>Grant</surname><given-names>JR</given-names></name><name><surname>Maciejewski</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Heatmapper: web-enabled heat mapping for all</article-title>. <source>Nucleic Acids Res.</source> (<year>2016</year>) <volume>44</volume>:<fpage>W147</fpage>–<lpage>53</lpage>. <pub-id pub-id-type=\"doi\">10.1093/nar/gkw419</pub-id><pub-id pub-id-type=\"pmid\">27190236</pub-id></mixed-citation></ref><ref id=\"B28\"><label>28.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bagheri</surname><given-names>V</given-names></name></person-group>\n<article-title>S100A12: friend or foe in pulmonary tuberculosis?</article-title>\n<source>Cytokine.</source> (<year>2017</year>) <volume>92</volume>:<fpage>80</fpage>–<lpage>2</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cyto.2017.01.009</pub-id><pub-id pub-id-type=\"pmid\">28110121</pub-id></mixed-citation></ref><ref id=\"B29\"><label>29.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lui</surname><given-names>G</given-names></name><name><surname>Wong</surname><given-names>CK</given-names></name><name><surname>Ip</surname><given-names>M</given-names></name><name><surname>Chu</surname><given-names>YJ</given-names></name><name><surname>Yung</surname><given-names>IM</given-names></name><name><surname>Cheung</surname><given-names>CS</given-names></name><etal/></person-group>. <article-title>HMGB1/RAGE signaling and pro-inflammatory cytokine responses in non-HIV adults with active pulmonary tuberculosis</article-title>. <source>PLoS ONE</source>. (<year>2016</year>) <volume>11</volume>:<fpage>e0159132</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0159132</pub-id><pub-id pub-id-type=\"pmid\">27434276</pub-id></mixed-citation></ref><ref id=\"B30\"><label>30.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van Zoelen</surname><given-names>MA</given-names></name><name><surname>Wieland</surname><given-names>CW</given-names></name><name><surname>van der Windt</surname><given-names>GJ</given-names></name><name><surname>Florquin</surname><given-names>S</given-names></name><name><surname>Nawroth</surname><given-names>PP</given-names></name><name><surname>Bierhaus</surname><given-names>A</given-names></name><etal/></person-group>. <article-title>Receptor for advanced glycation end products is protective during murine tuberculosis</article-title>. <source>Mol Immunol.</source> (<year>2012</year>) <volume>52</volume>:<fpage>183</fpage>–<lpage>9</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.molimm.2012.05.014</pub-id><pub-id pub-id-type=\"pmid\">22698798</pub-id></mixed-citation></ref><ref id=\"B31\"><label>31.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hamilton</surname><given-names>KK</given-names></name><name><surname>Zhao</surname><given-names>J</given-names></name><name><surname>Sims</surname><given-names>PJ</given-names></name></person-group>. <article-title>Interaction between apolipoproteins A-I and A-II and the membrane attack complex of complement. affinity of the apoproteins for polymeric C9</article-title>. <source>J Biol Chem.</source> (<year>1993</year>) <volume>268</volume>:<fpage>3632</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"pmid\">8429039</pub-id></mixed-citation></ref><ref id=\"B32\"><label>32.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Georgila</surname><given-names>K</given-names></name><name><surname>Vyrla</surname><given-names>D</given-names></name><name><surname>Drakos</surname><given-names>E</given-names></name></person-group>. <article-title>Apolipoprotein A-I (ApoA-I), immunity, inflammation and cancer</article-title>. <source>Cancers.</source> (<year>2019</year>) <volume>11</volume>:<fpage>1097</fpage>. <pub-id pub-id-type=\"doi\">10.3390/cancers11081097</pub-id><pub-id pub-id-type=\"pmid\">31374929</pub-id></mixed-citation></ref><ref id=\"B33\"><label>33.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang</surname><given-names>Y</given-names></name><name><surname>DiDonato</surname><given-names>JA</given-names></name><name><surname>Levison</surname><given-names>BS</given-names></name><name><surname>Schmitt</surname><given-names>D</given-names></name><name><surname>Li</surname><given-names>L</given-names></name><name><surname>Wu</surname><given-names>Y</given-names></name><etal/></person-group>. <article-title>An abundant dysfunctional apolipoprotein A1 in human atheroma</article-title>. <source>Nat Med.</source> (<year>2014</year>) <volume>20</volume>:<fpage>193</fpage>–<lpage>203</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nm.3459</pub-id><pub-id pub-id-type=\"pmid\">24464187</pub-id></mixed-citation></ref><ref id=\"B34\"><label>34.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lemes</surname><given-names>RMR</given-names></name><name><surname>Silva</surname><given-names>C</given-names></name><name><surname>Marques</surname><given-names>MÂM</given-names></name><name><surname>Atella</surname><given-names>GC</given-names></name><name><surname>Nery</surname><given-names>J</given-names></name><name><surname>Nogueira</surname><given-names>MRS</given-names></name><etal/></person-group>. <article-title>Altered composition and functional profile of high-density lipoprotein in leprosy patients</article-title>. <source>PLoS Negl Trop Dis.</source> (<year>2020</year>) <volume>14</volume>:<fpage>e0008138</fpage>. <pub-id pub-id-type=\"doi\">10.1371/journal.pntd.0008138</pub-id><pub-id pub-id-type=\"pmid\">32226013</pub-id></mixed-citation></ref><ref id=\"B35\"><label>35.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Freyne</surname><given-names>B</given-names></name><name><surname>Donath</surname><given-names>S</given-names></name><name><surname>Germano</surname><given-names>S</given-names></name><name><surname>Gardiner</surname><given-names>K</given-names></name><name><surname>Casalaz</surname><given-names>D</given-names></name><name><surname>Robins-Browne</surname><given-names>RM</given-names></name><etal/></person-group>. <article-title>Neonatal BCG vaccination influences cytokine responses to toll-like receptor ligands and heterologous antigens</article-title>. <source>J Infect Dis.</source> (<year>2018</year>) <volume>217</volume>:<fpage>1798</fpage>–<lpage>808</lpage>. <pub-id pub-id-type=\"doi\">10.1093/infdis/jiy069</pub-id><pub-id pub-id-type=\"pmid\">29415180</pub-id></mixed-citation></ref><ref id=\"B36\"><label>36.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Smith</surname><given-names>SG</given-names></name><name><surname>Kleinnijenhuis</surname><given-names>J</given-names></name><name><surname>Netea</surname><given-names>MG</given-names></name><name><surname>Dockrell</surname><given-names>HM</given-names></name></person-group>. <article-title>Whole blood profiling of bacillus calmette-guerin-induced trained innate immunity in infants identifies epidermal growth factor, IL-6, platelet-derived growth factor-AB/BB, and natural killer cell activation</article-title>. <source>Front Immunol.</source> (<year>2017</year>) <volume>8</volume>:<fpage>644</fpage>. <pub-id pub-id-type=\"doi\">10.3389/fimmu.2017.00644</pub-id><pub-id pub-id-type=\"pmid\">28634479</pub-id></mixed-citation></ref><ref id=\"B37\"><label>37.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>O'Gorman</surname><given-names>WE</given-names></name><name><surname>Kong</surname><given-names>DS</given-names></name><name><surname>Balboni</surname><given-names>IM</given-names></name><name><surname>Rudra</surname><given-names>P</given-names></name><name><surname>Bolen</surname><given-names>CR</given-names></name><name><surname>Ghosh</surname><given-names>D</given-names></name><etal/></person-group>. <article-title>Mass cytometry identifies a distinct monocyte cytokine signature shared by clinically heterogeneous pediatric SLE patients</article-title>. <source>J Autoimmun.</source> (<year>2017</year>). <pub-id pub-id-type=\"doi\">10.1016/j.jaut.2017.03.010.</pub-id> [Epub ahead of print]. <pub-id pub-id-type=\"pmid\">28389038</pub-id></mixed-citation></ref><ref id=\"B38\"><label>38.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ribeiro</surname><given-names>FM</given-names></name><name><surname>Gomez</surname><given-names>VE</given-names></name><name><surname>Albuquerque</surname><given-names>EM</given-names></name><name><surname>Klumb</surname><given-names>EM</given-names></name><name><surname>Shoenfeld</surname><given-names>Y</given-names></name></person-group>. <article-title>Lupus and leprosy: beyond the coincidence</article-title>. <source>Immunol Res.</source> (<year>2015</year>) <volume>61</volume>:<fpage>160</fpage>–<lpage>3</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s12026-014-8596-y</pub-id><pub-id pub-id-type=\"pmid\">25412733</pub-id></mixed-citation></ref><ref id=\"B39\"><label>39.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>van Hooij</surname><given-names>A</given-names></name><name><surname>Tjon Kon Fat</surname><given-names>EM</given-names></name><name><surname>Richardus</surname><given-names>R</given-names></name><name><surname>van den Eeden</surname><given-names>SJ</given-names></name><name><surname>Wilson</surname><given-names>L</given-names></name><name><surname>de Dood</surname><given-names>CJ</given-names></name><etal/></person-group>. <article-title>Quantitative lateral flow strip assays as user-friendly tools to detect biomarker profiles for leprosy</article-title>. <source>Sci Rep.</source> (<year>2016</year>) <volume>6</volume>:<fpage>34260</fpage>. <pub-id pub-id-type=\"doi\">10.1038/srep34260</pub-id><pub-id pub-id-type=\"pmid\">27682181</pub-id></mixed-citation></ref><ref id=\"B40\"><label>40.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Diniz</surname><given-names>LM</given-names></name><name><surname>Magalhães</surname><given-names>EF</given-names></name><name><surname>Pereira</surname><given-names>FE</given-names></name><name><surname>Dietze</surname><given-names>R</given-names></name><name><surname>Ribeiro-Rodrigues</surname><given-names>R</given-names></name></person-group>. <article-title>Presence of intestinal helminths decreases T helper type 1 responses in tuberculoid leprosy patients and may increase the risk for multi-bacillary leprosy</article-title>. <source>Clin Exp Immunol.</source> (<year>2010</year>) <volume>161</volume>:<fpage>142</fpage>–<lpage>50</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-2249.2010.04164.x</pub-id><pub-id pub-id-type=\"pmid\">20491787</pub-id></mixed-citation></ref><ref id=\"B41\"><label>41.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hagge</surname><given-names>DA</given-names></name><name><surname>Parajuli</surname><given-names>P</given-names></name><name><surname>Kunwar</surname><given-names>CB</given-names></name><name><surname>Rana</surname><given-names>D</given-names></name><name><surname>Thapa</surname><given-names>R</given-names></name><name><surname>Neupane</surname><given-names>KD</given-names></name><etal/></person-group>. <article-title>Opening a can of worms: leprosy reactions and complicit soil-transmitted helminths</article-title>. <source>EBioMedicine</source>. (<year>2017</year>) <volume>23</volume>:<fpage>119</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.ebiom.2017.08.026</pub-id><pub-id pub-id-type=\"pmid\">28882756</pub-id></mixed-citation></ref><ref id=\"B42\"><label>42.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ugarte-Gil</surname><given-names>C</given-names></name><name><surname>Alisjahbana</surname><given-names>B</given-names></name><name><surname>Ronacher</surname><given-names>K</given-names></name><name><surname>Riza</surname><given-names>AL</given-names></name><name><surname>Koesoemadinata</surname><given-names>RC</given-names></name><name><surname>Malherbe</surname><given-names>ST</given-names></name><etal/></person-group>. <article-title>Diabetes mellitus among pulmonary tuberculosis patients from 4 tuberculosis-endemic countries: the TANDEM study</article-title>. <source>Clin Infect Dis.</source> (<year>2020</year>) <volume>70</volume>:<fpage>780</fpage>–<lpage>8</lpage>. <pub-id pub-id-type=\"doi\">10.1093/cid/ciz284</pub-id><pub-id pub-id-type=\"pmid\">30958536</pub-id></mixed-citation></ref><ref id=\"B43\"><label>43.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liao</surname><given-names>H</given-names></name><name><surname>Wu</surname><given-names>J</given-names></name><name><surname>Kuhn</surname><given-names>E</given-names></name><name><surname>Chin</surname><given-names>W</given-names></name><name><surname>Chang</surname><given-names>B</given-names></name><name><surname>Jones</surname><given-names>MD</given-names></name><etal/></person-group>. <article-title>Use of mass spectrometry to identify protein biomarkers of disease severity in the synovial fluid and serum of patients with rheumatoid arthritis</article-title>. <source>Arthritis Rheum.</source> (<year>2004</year>) <volume>50</volume>:<fpage>3792</fpage>–<lpage>803</lpage>. <pub-id pub-id-type=\"doi\">10.1002/art.20720</pub-id><pub-id pub-id-type=\"pmid\">15593230</pub-id></mixed-citation></ref><ref id=\"B44\"><label>44.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Berrocal-Almanza</surname><given-names>LC</given-names></name><name><surname>Goyal</surname><given-names>S</given-names></name><name><surname>Hussain</surname><given-names>A</given-names></name><name><surname>Klassert</surname><given-names>TE</given-names></name><name><surname>Driesch</surname><given-names>D</given-names></name><name><surname>Grozdanovic</surname><given-names>Z</given-names></name><etal/></person-group>. <article-title>S100A12 is up-regulated in pulmonary tuberculosis and predicts the extent of alveolar infiltration on chest radiography: an observational study</article-title>. <source>Sci Rep.</source> (<year>2016</year>) <volume>6</volume>:<fpage>31798</fpage>. <pub-id pub-id-type=\"doi\">10.1038/srep31798</pub-id><pub-id pub-id-type=\"pmid\">27539060</pub-id></mixed-citation></ref><ref id=\"B45\"><label>45.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Manolakis</surname><given-names>AC</given-names></name><name><surname>Kapsoritakis</surname><given-names>AN</given-names></name><name><surname>Tiaka</surname><given-names>EK</given-names></name><name><surname>Potamianos</surname><given-names>SP</given-names></name></person-group>. <article-title>Calprotectin, calgranulin C, and other members of the s100 protein family in inflammatory bowel disease</article-title>. <source>Dig Dis Sci.</source> (<year>2011</year>) <volume>56</volume>:<fpage>1601</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s10620-010-1494-9</pub-id><pub-id pub-id-type=\"pmid\">21203903</pub-id></mixed-citation></ref><ref id=\"B46\"><label>46.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Blach-Olszewska</surname><given-names>Z</given-names></name><name><surname>Leszek</surname><given-names>J</given-names></name></person-group>. <article-title>Mechanisms of over-activated innate immune system regulation in autoimmune and neurodegenerative disorders</article-title>. <source>Neuropsychiatr Dis Treat.</source> (<year>2007</year>) <volume>3</volume>:<fpage>365</fpage>–<lpage>72</lpage>. <pub-id pub-id-type=\"pmid\">19300567</pub-id></mixed-citation></ref><ref id=\"B47\"><label>47.</label><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Khadge</surname><given-names>S</given-names></name><name><surname>Banu</surname><given-names>S</given-names></name><name><surname>Bobosha</surname><given-names>K</given-names></name><name><surname>van der Ploeg-van Schip</surname><given-names>JJ</given-names></name><name><surname>Goulart</surname><given-names>IM</given-names></name><name><surname>Thapa</surname><given-names>P</given-names></name><etal/></person-group>. <article-title>Longitudinal immune profiles in type 1 leprosy reactions in Bangladesh, Brazil, Ethiopia and Nepal</article-title>. <source>BMC Infect Dis.</source> (<year>2015</year>) <volume>15</volume>:<fpage>477</fpage>. <pub-id pub-id-type=\"doi\">10.1186/s12879-015-1128-0</pub-id><pub-id pub-id-type=\"pmid\">26510990</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Microbiol</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Microbiol</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Microbiol.</journal-id><journal-title-group><journal-title>Frontiers in Microbiology</journal-title></journal-title-group><issn pub-type=\"epub\">1664-302X</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849484</article-id><article-id pub-id-type=\"pmc\">PMC7431627</article-id><article-id pub-id-type=\"doi\">10.3389/fmicb.2020.01943</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Microbiology</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Commonalities and Differences in the Transcriptional Response of the Model Fungus <italic>Saccharomyces cerevisiae</italic> to Different Commercial Graphene Oxide Materials</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Laguna-Teno</surname><given-names>Felix</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/990811/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Suarez-Diez</surname><given-names>Maria</given-names></name><xref ref-type=\"aff\" rid=\"aff2\"><sup>2</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/13183/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Tamayo-Ramos</surname><given-names>Juan Antonio</given-names></name><xref ref-type=\"aff\" rid=\"aff1\"><sup>1</sup></xref><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/455013/overview\"/></contrib></contrib-group><aff id=\"aff1\"><sup>1</sup><institution>International Research Centre in Critical Raw Materials-ICCRAM, University of Burgos</institution>, <addr-line>Burgos</addr-line>, <country>Spain</country></aff><aff id=\"aff2\"><sup>2</sup><institution>Laboratory of Systems and Synthetic Biology, Wageningen University & Research</institution>, <addr-line>Wageningen</addr-line>, <country>Netherlands</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Markus Proft, Institute of Biomedicine of Valencia (CSIC), Spain</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Monika Mortimer, China Jiliang University, China; Intawat Nookaew, University of Arkansas for Medical Sciences, United States</p></fn><corresp id=\"c001\">*Correspondence: Juan Antonio Tamayo-Ramos, <email>ja.tamayoramos@gmail.com</email></corresp><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Fungi and Their Interactions, a section of the journal Frontiers in Microbiology</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>1943</elocation-id><history><date date-type=\"received\"><day>24</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>23</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Laguna-Teno, Suarez-Diez and Tamayo-Ramos.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Laguna-Teno, Suarez-Diez and Tamayo-Ramos</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Graphene oxide has become a very appealing nanomaterial during the last years for many different applications, but its possible impact in different biological systems remains unclear. Here, an assessment to understand the toxicity of different commercial graphene oxide nanomaterials on the unicellular fungal model organism <italic>Saccharomyces cerevisiae</italic> was performed. For this task, an RNA purification protocol was optimized to avoid the high nucleic acid absorption capacity of graphene oxide. The developed protocol is based on a sorbitol gradient separation process for the isolation of adequate ribonucleic acid levels (in concentration and purity) from yeast cultures exposed to the carbon derived nanomaterial. To pinpoint potential toxicity mechanisms and pathways, the transcriptome of <italic>S. cerevisiae</italic> exposed to 160 mg L<sup>–1</sup> of monolayer graphene oxide (GO) and graphene oxide nanocolloids (GOC) was studied and compared. Both graphene oxide products induced expression changes in a common group of genes (104), many of them related to iron homeostasis, starvation and stress response, amino acid metabolism and formate catabolism. Also, a high number of genes were only differentially expressed in either GO (236) or GOC (1077) exposures, indicating that different commercial products can induce specific changes in the physiological state of the fungus.</p></abstract><kwd-group><kwd><italic>Saccharomyces cerevisiae</italic></kwd><kwd>biological response</kwd><kwd>commercial graphene oxide</kwd><kwd>chelating agent</kwd><kwd>RNA isolation</kwd><kwd>transcriptomics</kwd><kwd>differential expression</kwd></kwd-group><counts><fig-count count=\"4\"/><table-count count=\"0\"/><equation-count count=\"0\"/><ref-count count=\"73\"/><page-count count=\"12\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Graphene oxide is a nanomaterial of great industrial interest, and many public and private initiatives have been launched during the last decade for the development of new technologies around this 2D carbon derived product (<xref rid=\"B58\" ref-type=\"bibr\">Shapira et al., 2016</xref>). New applications based on graphene oxide are expected to increase the chance of its environmental release, which could lead to unsafe human and ecosystem exposure levels. Therefore, any possible risks associated to graphene oxide applications and release need to be well-understood (<xref rid=\"B18\" ref-type=\"bibr\">Fadeel et al., 2018</xref>), particularly considering its morphological and physical properties, which suggest a potential risk to the health of humans and the environment. In fact, attention is being drawn to the safety assessment of carbon derived nanomaterials in different biological systems, including graphene oxide, by the scientific community.</p><p>In most cases, graphene oxide risk assessment studies have been focused on mammalian cell lines and laboratory animals, where different mechanisms associated to its potential toxicity have been determined, namely, physical destruction, induction of oxidative stress, DNA damage, inflammatory response, apoptosis, autophagy, and necrosis (<xref rid=\"B55\" ref-type=\"bibr\">Sanchez et al., 2012</xref>; <xref rid=\"B40\" ref-type=\"bibr\">Ou et al., 2016</xref>; <xref rid=\"B17\" ref-type=\"bibr\">Ema et al., 2017</xref>). Although the biological impact of the nanomaterial has been also studied on microbial systems, only a limited number of studies have explored the toxicity mechanisms based on gene expression analysis (<xref rid=\"B8\" ref-type=\"bibr\">Chen et al., 2017</xref>; <xref rid=\"B70\" ref-type=\"bibr\">Yu et al., 2017</xref>; <xref rid=\"B72\" ref-type=\"bibr\">Zhu et al., 2017</xref>). Graphene has strong cytotoxicity toward bacteria (<xref rid=\"B32\" ref-type=\"bibr\">Liu et al., 2011</xref>), while little has been reported on its antifungal activity (<xref rid=\"B3\" ref-type=\"bibr\">Asadi Shahi et al., 2019</xref>). Most research works studying fungal interactions with graphene derivatives have focused on the nanomaterial functionalization with antifungal compounds, the development of cost-effective methods for surface modified graphene, or the integration of the cellular physiology with electrical read outs (<xref rid=\"B26\" ref-type=\"bibr\">Kempaiah et al., 2011</xref>; <xref rid=\"B27\" ref-type=\"bibr\">Khanra et al., 2012</xref>; <xref rid=\"B29\" ref-type=\"bibr\">Li et al., 2013</xref>; <xref rid=\"B62\" ref-type=\"bibr\">Valentini et al., 2016</xref>; <xref rid=\"B19\" ref-type=\"bibr\">Farzanegan et al., 2018</xref>). Still, the specific fungal responses to the presence of graphene oxide in the environment are poorly understood, as well as the possible physiological changes or the induction of specific toxicity pathways. Few research works have been done using the fungal genetic model <italic>Saccharomyces cerevisiae</italic>, to understand the potential toxicity of graphene oxide and other carbon derived nanomaterials (<xref rid=\"B5\" ref-type=\"bibr\">Bayat et al., 2014</xref>; <xref rid=\"B70\" ref-type=\"bibr\">Yu et al., 2017</xref>; <xref rid=\"B72\" ref-type=\"bibr\">Zhu et al., 2017</xref>, <xref rid=\"B71\" ref-type=\"bibr\">2018</xref>), highlighting the need for more thorough studies assessing the global cellular response.</p><p>The yeast <italic>S. cerevisiae</italic> is one of the most widely used eukaryotic systems to understand basic molecular processes, therefore it is an ideal model to identify potential toxicity pathways induced by graphene oxide in fungi. Previous reports studying the toxicological effects of graphene oxide in this unicellular organism, show that an acute exposure leads to significant effects on cell viability and proliferation, due to mitochondria-mediated apoptosis, which could be associated with oxidative stress (<xref rid=\"B72\" ref-type=\"bibr\">Zhu et al., 2017</xref>). Also, at sublethal concentrations, cell growth and metabolism were reduced, possibly due to the iron chelating properties of graphene oxide (<xref rid=\"B70\" ref-type=\"bibr\">Yu et al., 2017</xref>). In this regard, a relevant binding capacity for metal ions and positively charged organic molecules has been assigned to this nanomaterial, through electrostatic interaction and coordination (<xref rid=\"B1\" ref-type=\"bibr\">Ali et al., 2019</xref>). This feature allows its use in the efficient removal of potentially toxic elements from contaminated aqueous media, but it could also impact nutrient bioavailability for the organisms present in a certain environment. Additionally, previous studies have reported that distinct graphene oxide products can have different reactivity against biological systems and biomolecules, possibly due to differences in their elemental composition or in morphological features (<xref rid=\"B2\" ref-type=\"bibr\">Antón-Millán et al., 2018</xref>; <xref rid=\"B30\" ref-type=\"bibr\">Li et al., 2018</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Domi et al., 2019</xref>). Therefore, to assess whether different commercial graphene oxide products could induce different toxicity responses in <italic>S. cerevisiae</italic>, two graphene derivatives: monolayer graphene oxide (GO) and graphene oxide nanocolloids (GOC) were selected and the global transcriptional response of the yeast was compared. For this task, an optimized protocol for RNA isolation from fungal cells exposed to graphene oxide, was developed too.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><sec id=\"S2.SS1\"><title>Materials and Reagents</title><p>Most of the chemicals and reagents were purchased from Sigma-Aldrich and Thermo Fisher Scientific. The graphene derivatives were obtained from different suppliers as well: Graphene oxide nanocolloids (GOC; ref: 795534; lot: MKCD9594) were purchased from Sigma-Aldrich, and monolayer graphene oxide (GO; C309/GORB014/D1) was purchased from Graphenea. Working stock suspensions of both nanomaterial types were obtained using ultrapure water, at a final concentration of 1000 mg L<sup>–1</sup>, and were sonicated using a Branson Sonifier Cell Disruptor Model SLPe, for 5 min, using an amplitude of 40%.</p><sec id=\"S2.SS1.SSS1\"><title><italic>S. cerevisiae</italic> Cells Exposure to Graphene Oxide Nanomaterials and RNA Isolation</title><p><italic>S. cerevisiae</italic> cells were pre-grown on YPD medium in an orbital shaker (185 rpm, 30°C) until an O.D.<sub>600 nm</sub> = 1 was reached. Cells were harvested, washed with PBS and resuspended in 50 mL (O.D.<sub>600 nm</sub> = 1) of fresh YPD medium containing 160 mg L<sup>–1</sup> of either GO or GOC, or without the presence of nanoparticles (negative control). Exposure cultures were performed in sterile 250 mL Erlenmeyer flasks, for 24 h (185 rpm, 30°C), growing two biological replicates per condition. Afterward, <italic>S. cerevisiae</italic> cells were harvested, resuspended with cold PBS and separated from the nanomaterials following the gradient centrifugation protocol described in the first paragraph of the Results and Discussion section, employing a Thermo ST 16R Sorvall centrifuge. All separation steps were performed at 4°C. Once yeast cells were separated from the graphene oxide nanoparticles, RNA isolation was performed using Thermo Fisher Scientific reagents, following the TRIzol<sup><italic>TM</italic></sup> Plus RNA Purification Kit user guide (Pub. No. MAN0000561), with minor modifications. Briefly, yeast aliquots were pelleted by centrifugation (13,000 g, 4°C) and were subsequently resuspended in 1 mL of TRIzol<sup><italic>TM</italic></sup> reagent and transferred to commercial 2 mL tubes prefilled with glass beads (Lysing Matrix C; MP). Yeast samples were disrupted using a FastPrep-24 Instrument (MP). After disruption, 200 μL of chloroform were added and the mix was homogenated for 10 s. The mix was poured into Phasemaker tubes (2 mL) and centrifuged at 13,000 g in a table-top centrifuge. The RNA present in the water phase was purified using the PureLink<sup><italic>TM</italic></sup> RNA Mini Kit (Thermo), following the manufacturer’s instructions.</p></sec></sec><sec id=\"S2.SS2\"><title>RNA Quality Control and Sequencing</title><p>RNA integrity was assessed with an Agilent 2100 system, and only high quality samples (RIN value ≥ 8) were selected for whole transcriptome shotgun sequencing. Total RNA was sent for whole transcriptome sequencing to Novogene Bioinformatics Technology Co. Ltd. (HongKong, China). After mRNA purification (starting with 1 μg of total RNA per sample), sequencing libraries were generated using NEB Next<sup>®</sup> Ultra<sup><italic>TM</italic></sup> RNA Library Prep Kit for Illumina<sup>®</sup> (NEB, United States) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. First, mRNA purification was done using poly-T oligo-attached magnetic beads, and fragmentation was carried out using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer (5X). Subsequently, first strand cDNA was synthesized with random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-) and second strand cDNA synthesis was done with DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of preferentially 150–200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, United States). Then 3 μl USER Enzyme (NEB, United States) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Subsequently, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. After sample preparation, the clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer’s instructions, followed by the library preparations sequencing on an Ilumina Hiseq4000 and 125 bp/150 bp paired-end reads were generated. Afterward, the original raw data from Illumina was transformed to Sequenced Reads by base calling and data quality control was done with the Casava v1.8 software.</p></sec><sec id=\"S2.SS3\"><title>RNA-Seq Data Processing and Analysis</title><p>Reads were pre-processed using FastqPuri for quality control and adapter, contamination and quality filtering (<xref rid=\"B45\" ref-type=\"bibr\">Pérez-Rubio et al., 2019</xref>). Reads with adapter contamination were removed, as well as the ones with 50% of the bases with quality below 20. Also, reads with a percentage of unidentified bases greater than 10% were also removed. Latest assembly of the reference genome for this strain was retrieved from Ensembl, revision 97, genome accession number (<ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"GCA_000146045.2\">GCA_000146045.2</ext-link>). Reads were mapped to the genome using Star v2.7.2b (<xref rid=\"B13\" ref-type=\"bibr\">Dobin et al., 2013</xref>). The genome was indexed specifying the read length to improve accuracy. The mapping was done using two pass method. Number of reads for each genome feature were retrieved using featureCounts (<xref rid=\"B31\" ref-type=\"bibr\">Liao et al., 2014</xref>). Total number of reads are summarized in the <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 1</xref>. Data have been submitted to the European Nucleotide Archive and can be found under accession number <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"PRJEB34525\">PRJEB34525</ext-link>. Read counts per gene were normalized and differential expression was computed using DESeq2 v 1.24, with default parameters except for the alpha threshold that was set to 0.05 (<xref rid=\"B34\" ref-type=\"bibr\">Love et al., 2014</xref>). Variance stabilizing transformation considering the experimental design was performed using the “rlog” command prior to principal component analysis. Enrichment analysis for selected groups of genes were performed using the hypergeometric function to model the background probability and the Benjamini–Hochberg procedure was used to control the false discovery rate (FDR) and correct for multiple testing. Gene ontology enrichment was performed using clusterProfiler v3.12.0, topGO and DOSE (<xref rid=\"B68\" ref-type=\"bibr\">Yu et al., 2012</xref>). Annotation files, both GAF and OBO were downloaded from Gene Ontology, release “2019-04-17” and pathway information was retrieved from KEGG (<xref rid=\"B25\" ref-type=\"bibr\">Kanehisa, 2000</xref>). The version 3.6.0 of R was used to perform the statistical analysis and visualizations were done with ggplot2 v3.2.1 (<xref rid=\"B66\" ref-type=\"bibr\">Wickham, 2009</xref>). Additional information about each gene was obtained from The Saccharomyces Genome Database (SGD) (<xref rid=\"B11\" ref-type=\"bibr\">Cherry et al., 2012</xref>).</p></sec></sec><sec id=\"S3\"><title>Results and Discussion</title><sec id=\"S3.SS1\"><title>Optimization of RNA Isolation From <italic>S. cerevisiae</italic> Cells Exposed to Different Commercial Graphene Oxide Products</title><p>The ability of graphene oxide to adsorb single-stranded nucleic acids (<xref rid=\"B44\" ref-type=\"bibr\">Park et al., 2013</xref>) is a burden for the isolation of RNA from cells that have been exposed to the nanomaterial. In the presence of this nanomaterial, the obtention of high-quality total RNA from <italic>S. cerevisiae</italic> cells, in enough amounts to be used for RNAseq analysis, can only be achieved if a nanoparticles-cells separation step is introduced prior to the start of the RNA isolation protocol. In fact, we failed in isolating total RNA from <italic>S. cerevisiae</italic> cells (strain BY4741) after an exposure experiment to GO and GOC, when they were not previously separated from the nanomaterials. Zhu and collaborators used a density gradient centrifugation protocol (<xref rid=\"B73\" ref-type=\"bibr\">Zhu et al., 2016</xref>), to separate graphene oxide nanoparticles from yeast cells for RNA purification (<xref rid=\"B72\" ref-type=\"bibr\">Zhu et al., 2017</xref>). However, considering the high mRNA turnover of some genes, we considered that the reported separation protocol used a too long centrifugation step (30 min) that might affect RNA integrity. Therefore, we decided to optimize the graphene oxide-cells separation protocol by speeding up the process, modifying the gradient centrifugation protocol, using a Thermo ST 16R Sorvall centrifuge, managing to efficiently separate the yeast cells from GO and GOC, by employing the following steps: once the cells exposure to the selected nanomaterials was finished, cells were harvested by centrifugation (5000 rpm, 4°C; acceleration: 9, deceleration: 9) and resuspended in cold PBS (2.5 mL). The resuspended cells were carefully overlayed in a concentrated sorbitol solution (4.2 M; 3 mL) prepared in PBS too and contained in disposable 15 mL tubes previously stored on ice. Subsequently, a gradient centrifugation was performed (5000 rpm, 4°C; acceleration: 9, deceleration: 5). As displayed in <xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>, the separation between yeast cells and the graphene oxide nanoparticles was possible with the described optimized protocol, and the isolation of high quality total RNA for transcriptomics analysis was successful.</p><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p><italic>S. cerevisiae</italic> cells separation from graphene oxide through density gradient centrifugation.</p></caption><graphic xlink:href=\"fmicb-11-01943-g001\"/></fig></sec><sec id=\"S3.SS2\"><title>Transcriptional Response of <italic>S. cerevisiae</italic> Cells to Different Graphene Oxide Products</title><p>To assess the impact of commercial graphene oxide products, GO and GOC, on <italic>S. cerevisiae</italic> cells, a comparative transcriptomics analysis was done. In a previous study, we characterized both nanomaterials and observed differences in their composition and their oxidative stress inducing capacity in yeast, although it was challenging to associate their toxicological potential to their physico-chemical characteristics due to the many different variables that could be involved, such as lateral dimension, surface structure, functional groups, purity and protein corona (<xref rid=\"B14\" ref-type=\"bibr\">Domi et al., 2019</xref>). GO and GOC showed a wide lateral size distribution (from the nanometric to the micrometric scale), with a flake thickness of 1–2 nm. Their chemical composition analysis, performed through ATR-FTIR, ICP-MS, and XPS, revealed both nanomaterials were similar in oxygen functional groups content, while significant differences in the concentration of metals, metalloids and non-metal elements were observed between both nanomaterials. The content of metallic and metalloid elements in both nanomaterials was low, but higher in GOC, while S species were more abundant in GO. The presence of organosulfate groups in graphene oxide is responsible for part of the reactivity of this nanomaterial type, such as in the immobilization of adsorbed species (<xref rid=\"B15\" ref-type=\"bibr\">Eigler et al., 2013</xref>). However, we could not get insights on the type of S species (e.g., organic or inorganic) present in both graphene oxide products. Overall, any of the differences observed between both nanomaterial types, as well as other non-identified factors, could be responsible for the distinct toxicological response displayed by the cellular systems used as toxicity models at viability, vitality and oxidative stress levels. In case of <italic>S. cerevisiae</italic>, GO showed a higher capacity than GOC to induce oxidative stress, while differences observed in viability after the exposure to both nanomaterials were not significant (<xref rid=\"B14\" ref-type=\"bibr\">Domi et al., 2019</xref>). The study of the global transcriptional response of <italic>S. cerevisiae</italic> cells to the presence of each nanoproduct could provide additional insights into the common and/or product-specific molecular mechanisms behind their toxicity inducing factors. With this purpose, we decided to expose yeast cells to GO and GOC for 24 h and to study their global transcriptional signature. Concentrations of GO and GOC (160 mg L<sup>–1</sup>) were selected based on ranges used in similar studies assessing the toxicological impact of graphene derivatives in different organisms including fungi (<xref rid=\"B14\" ref-type=\"bibr\">Domi et al., 2019</xref>; <xref rid=\"B61\" ref-type=\"bibr\">Suarez-Diez et al., 2020</xref>), and specifically the works of <xref rid=\"B70\" ref-type=\"bibr\">Yu et al. (2017)</xref> and <xref rid=\"B72\" ref-type=\"bibr\">Zhu et al. (2017)</xref>, that analyze the impact of graphene oxides, similar to the ones here studied. Yeast cells total RNA was isolated as described in the previous section and it was analyzed using the Illumina sequencing system (further details can be found in the “Materials and Methods” section). The obtained reads were mapped to the <italic>S. cerevisiae</italic> BY4741 genome. <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 1</xref> provides summarizing information on this process. The reads that could be uniquely mapped to the <italic>S. cerevisiae</italic> genome ranged between 89.3 and 92.4%, and 84.1–86.1% of the reads mapped to exonic regions in the genome. These numbers indicate the high quality of the RNA generated using our optimized protocol.</p><p>Principal Component Analysis (PCA) was performed to analyze the variability among the generated samples (<xref ref-type=\"fig\" rid=\"F2\">Figure 2</xref>). In this analysis, only the top 500 genes with most variability were considered to reduced noise associated to biological variability. Similar results are obtained when all genes are considered, as shown in the PCA plot displayed in <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Figure 1</xref>. Obvious clustering can be seen between samples corresponding to biological replicates. The dimensionality reduction can be considered adequate, as most of the variability (77%) is along the X axis, the first principal component (PC1). Samples corresponding to control (non-exposure) and exposure to 160 ml L<sup>–1</sup> GO are closer together in terms of transcriptomic response than they are to samples exposed to the same concentration of GOC. This similarity becomes even more apparent if we consider that the difference between the control and the GO samples is mainly along the second PC, whereas differences between GOC exposed and control samples appear along both PC1 and PC2. This can be interpreted as GOC having more variability than GO against the control, as the PC1 axis carries much more variability than the PC2 one. GOC exposure shows a much higher transcriptomic response than GO exposure, even though in both cases the same compound is used.</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Principal Component Analysis plot of the transcriptomic response of <italic>S. cerevisiae</italic> to two different graphene oxide products at 160 mg L<sup>–1</sup> (Control: non-exposed cells, GO: exposed to monolayer graphene, and GOC: exposed to graphene oxide nanocolloids). Only the top 500 genes with most variability were considered.</p></caption><graphic xlink:href=\"fmicb-11-01943-g002\"/></fig><p>Afterward, both exposure conditions were studied individually to visualize the transcriptional impact of each compound on <italic>S. cerevisiae</italic>. Differentially expressed genes were defined (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 2</xref>) by a False Discovery Rate (FDR) lower than 0.05. To further reduce the number of false positives associated to the relatively low number of replicates, we have imposed a threshold on fold change, so that only differentially expressed genes higher or lower than 1.5 and 1/1.5, respectively (corresponding to ± 0.585 in log<sub>2</sub>), were considered biologically meaningful (<xref rid=\"B56\" ref-type=\"bibr\">Schurch et al., 2016</xref>). The obtained data was displayed in volcano plots (<xref ref-type=\"fig\" rid=\"F3\">Figures 3A,B</xref>), where clear differences between both exposure conditions in number of genes differentially expressed could be observed. Yeast cells exposure to GOC induced more than three times more differentially expressed genes than GO exposure (1181 and 340 genes, respectively). Surprisingly, only a small part of the differentially expressed genes in both conditions was common (104, of which 60 were upregulated and 44 downregulated. This indicates a very specific transcriptional response of <italic>S. cerevisiae</italic> to each type of graphene oxide.</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>Volcano plots displaying the fold change (log2) of differentially expressed genes of GO vs. the control <bold>(A)</bold> and GOC vs. the control <bold>(B)</bold>, and Venn diagrams showing common and specific upregulated <bold>(C)</bold> and downregulated <bold>(D)</bold> genes between the different exposure conditions. The genes were considered significantly differentially expressed if they had a fold change higher than 1.5 (upregulated) or lower than 1/1.5 (downregulated), and an FDR lower than 0.05.</p></caption><graphic xlink:href=\"fmicb-11-01943-g003\"/></fig><p>Following the previous exploratory analysis, Gene Ontology enrichment and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of the differentially expressed genes were done. Both tests were performed separately for up and downregulated genes to study which biological functions were specifically altered upon yeast cells exposure to each nanomaterial, as well as to identify the common cellular response. Gene Ontology enrichment analyses were performed for each of the three ontologies: biological process (BP), molecular function (MF) and cellular component (CC). An overview of the results for the BP ontology is shown in <xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>, whereas full results are available in <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 3</xref>. This supplementary table also provides a full list of all the genes associated to the corresponding gene ontology terms and their functional annotation. The results of KEGG pathway enrichment analysis can be found in the <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 4</xref>. Enrichments were considered significant whenever FDR <0.05.</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Overview of gene ontology enrichment analysis of <italic>S. cerevisiae</italic>\n<bold>(A)</bold> upregulated and <bold>(B)</bold> downregulated genes. Blue and orange bars indicate terms enriched among the genes exclusively differentially expressed upon GOC or GO exposures respectively, while gray is used for terms found among the genes commonly differentially expressed in both exposure conditions. Bar size indicates the % of genes in the whole genome annotated to the corresponding term that have been found in each of the exposures. Gene ontology entries have been grouped in terms broadly related to <bold>(A)</bold> Group I: metal bioavailability, Group II: cell wall structure, and Group III metabolism and in <bold>(B)</bold> Group I: amino acid metabolism, Group II: protein translation, Group III: mitochondria, and Group IV: vacuolar acidification. All selected terms have an FDR lower than 0.05.</p></caption><graphic xlink:href=\"fmicb-11-01943-g004\"/></fig><p>Amongst the common upregulated genes (60, as shown in <xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>), there is a significant enrichment in genes associated to the Gene Ontoloy term “cellular iron ion homeostasis” (inside Group I, <xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>). The seven genes associated to this term commonly upregulated are involved in functions related to iron uptake at the cell surface, iron efflux from vacuole to cytosol and in metabolic adaptation to low iron conditions. YDR270W (<italic>CCC2</italic>) encodes a P-type copper-transporting ATPase necessary for the proper uptake of iron (<xref rid=\"B20\" ref-type=\"bibr\">Fu et al., 1995</xref>). The expression of YOL158C (<italic>ENB1</italic>), YHL047C (<italic>ARN2</italic>), and YOR384W (<italic>FRE5</italic>) is related to non-reductive and reductive iron transport systems (<xref rid=\"B36\" ref-type=\"bibr\">Martins et al., 1998</xref>; <xref rid=\"B21\" ref-type=\"bibr\">Heymann et al., 1999</xref>; <xref rid=\"B47\" ref-type=\"bibr\">Philpott et al., 2002</xref>). YLR136C (<italic>TIS11</italic>) and YLR205C (<italic>HMX1</italic>) are involved in mRNA and heme degradation, respectively mediating homeostatic changes, such as making heme iron available for metabolic needs, and reducing iron flux into respiratory complexes (<xref rid=\"B50\" ref-type=\"bibr\">Protchenko and Philpott, 2003</xref>; <xref rid=\"B51\" ref-type=\"bibr\">Puig et al., 2005</xref>). The expression of the mentioned genes is higher in iron starvation conditions, and it is controlled by the Aft1p and Aft2p regulators (<xref rid=\"B54\" ref-type=\"bibr\">Rutherford et al., 2003</xref>). The YLR136C (<italic>CTH2</italic>) gene, whose expression is also controlled by the Aft1/2 regulon and contributes to remodeling yeast metabolism by suppressing pathways employing many iron-containing enzymes, was also upregulated in the presence of GO and GOC (<xref rid=\"B37\" ref-type=\"bibr\">Matsuo et al., 2017</xref>). A role for <italic>CTH2</italic> in increasing resistance to ROS when this gene is overexpressed has been proposed (<xref rid=\"B37\" ref-type=\"bibr\">Matsuo et al., 2017</xref>). In case of YMR134W (<italic>ERG29</italic>), its function is related to ergosterol biosynthesis and has been tied to iron metabolism too (<xref rid=\"B39\" ref-type=\"bibr\">Moretti-Almeida et al., 2013</xref>). The common upregulation of YER037W (<italic>PHM8</italic>), which is involved in lysophosphatidic acid hydrolysis in response to phosphate starvation, suggests low availability of this nutrient too (<xref rid=\"B63\" ref-type=\"bibr\">Vardi et al., 2014</xref>). In addition, a common upregulation of transcriptional response to the presence of both nanomaterials was observed for genes annotated to the term “cellular aldehyde metabolic process” (<xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>, Group III): YGR256W (<italic>GND2</italic>), YMR095C (<italic>SNO1</italic>), YMR096W (<italic>SNZ1</italic>), and YNL117W (<italic>MLS1</italic>). <italic>GND2</italic> encodes a phosphogluconate dehydrogenase, which is induced in stress conditions and it could have a protective role against oxidative stress (<xref rid=\"B24\" ref-type=\"bibr\">Izawa et al., 1998</xref>). <italic>SNO1</italic> and <italic>SNZ1</italic> are members of a stationary phase-induced gene family, involved in pyridoxine (vitamin B6) biosynthesis, whose accumulation occurs as well in response to the limitation of specific nutrients and stress response to nucleotide imbalance (<xref rid=\"B41\" ref-type=\"bibr\">Padilla et al., 1998</xref>; <xref rid=\"B52\" ref-type=\"bibr\">Rodríguez-Navarro et al., 2002</xref>). Overexpression of these genes is also identified in the enrichment of the “Vitamin B6 metabolism” KEGG metabolic pathway (see <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 4</xref>). Furthermore, the YDR019C (<italic>GCV1</italic>) and YMR189W (<italic>GCV2</italic>) genes, which code two of the three proteins involved in the glycine decarboxylase multienzyme complex, are induced by high levels of glycine or repressed in the presence of rich nutritional environments or high quality nitrogen sources (<xref rid=\"B59\" ref-type=\"bibr\">Sinclair et al., 1996</xref>; <xref rid=\"B48\" ref-type=\"bibr\">Piper et al., 2002</xref>). Alterations in nitrogen metabolism are also evident in the metabolic pathway enrichment analysis that shows dysregulation of metabolic pathways related to amino acid synthesis and degradation (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 4</xref>).</p><p>Regarding the 39 exclusively upregulated <italic>S. cerevisiae</italic> genes in the presence of monolayer graphene oxide (GO) (<xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>), additional genes related to metallic elements transport (“ion transport”; “transition metal ion transport,” shown in <xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>, Group I, and in <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 3</xref>) were overexpressed: YMR058W (<italic>FET3</italic>), part of the high affinity iron uptake system in the cell wall (<xref rid=\"B4\" ref-type=\"bibr\">Askwith et al., 1994</xref>) and YKL220C (<italic>FRE2</italic>), a ferric and cupric reductase, which reduces siderophore-bound iron and oxidized copper prior to uptake by transporters, are involved in iron uptake (<xref rid=\"B16\" ref-type=\"bibr\">Elena and Despina, 1994</xref>); YHL040C (<italic>ARN1</italic>) and YEL065W (<italic>ARN3</italic>) are members of the ARN family transporters, which specifically recognize siderophore-iron chelates, and are induced in conditions of low iron (<xref rid=\"B22\" ref-type=\"bibr\">Heymann et al., 2006</xref>); YOR382W (<italic>FIT2</italic>) and YOR383C (<italic>FIT3</italic>) are cell wall glycosylphosphatidylinositol-anchored mannoproteins involved in the retention of siderophore-iron in the cell wall (<xref rid=\"B49\" ref-type=\"bibr\">Protchenko et al., 2001</xref>); YOR316C (<italic>COT1</italic>) is a vacuolar transporter that mediates zinc transport, but its expression levels are controlled by the iron regulon in yeast (<xref rid=\"B46\" ref-type=\"bibr\">Philpott and Protchenko, 2008</xref>); and YER053C (<italic>PIC2</italic>) belongs to the mitochondrial carrier family (MCF), involved in phosphate and copper transport (<xref rid=\"B64\" ref-type=\"bibr\">Vest et al., 2013</xref>).</p><p>Besides iron and other genes involved in metal homeostasis, the upregulation of YMR195W (<italic>ICYI</italic>), which is induced by amino acid starvation (<xref rid=\"B28\" ref-type=\"bibr\">Kleinschmidt et al., 2005</xref>), was observed too. It is also interesting to highlight the upregulation of two stress response genes: YMR175W (<italic>SIP18</italic>), regulated by osmotic stress, and YCR021C (<italic>HSP30</italic>), induced by heat shock and entry to the stationary phase (<xref rid=\"B43\" ref-type=\"bibr\">Panaretou and Piper, 1992</xref>; <xref rid=\"B38\" ref-type=\"bibr\">Miralles and Serrano, 1995</xref>; see <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 2</xref>). Few genes related to one carbon metabolism [YER081W (<italic>SER3</italic>) and YCL064C (<italic>CHA1</italic>)] and glycogen metabolism [YMR105C (<italic>PGM2</italic>), YIL050W (<italic>PCL7</italic>), and YJL137C (<italic>GLG2</italic>)] were found upregulated too in the GO condition.</p><p>Amongst the high number of exclusive upregulated genes (510, as shown in <xref ref-type=\"fig\" rid=\"F3\">Figure 3C</xref>) in yeast cells exposed to graphene nanocolloids (GOC), 13 of those were specifically related to the ergosterol biosynthetic process (see <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 3</xref>). Genes from the mentioned pathway were found to be overexpressed during iron starvation conditions in a previous study (<xref rid=\"B51\" ref-type=\"bibr\">Puig et al., 2005</xref>). Other authors studying the metabolic response to iron deficiency in <italic>S. cerevisiae</italic> only observed small changes in the transcript levels of <italic>EFG</italic> genes, but specific alterations in the ergosterol and sphingolipid biosynthetic pathways steps involving heme and diiron enzymes were found (<xref rid=\"B57\" ref-type=\"bibr\">Shakoury-Elizeh et al., 2010</xref>). The presence of GOC activated additional specific and general responses related to the low availability of other nutrients, such as zinc, phosphate, nitrogen and pyrimidine (YML123C, YBL042C, YJL056C, YPR035W, YOR030W, YLR014C, YNR002C, YKR042W, YIL101C, YGL180W).</p><p>The upregulation of many genes involved in the maintenance of cell wall integrity (see <xref ref-type=\"fig\" rid=\"F4\">Figure 4A</xref>, Group II, and <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 3</xref>), some of them induced in response to stress, such as YDR077W (<italic>SED1</italic>), YGR189C (<italic>CRH1</italic>), YLR194C (<italic>NCW2</italic>), YPR026W (<italic>ATH1</italic>), and YJL159W (<italic>HSP150</italic>), was also observed. Aggregation, morphological alterations, gemmation disturbance and in some cases cellular damage has been reported upon exposure of <italic>S. cerevisiae</italic> cells to graphene oxide (<xref rid=\"B72\" ref-type=\"bibr\">Zhu et al., 2017</xref>). Similarly, alterations at cell wall integrity at molecular level were also recently observed in the filamentous fungus <italic>Fusarium graminearum</italic> in the presence of different graphene oxide concentrations (<xref rid=\"B65\" ref-type=\"bibr\">Wang et al., 2019</xref>). Previous reports have highlighted the ability of the nanomaterial to intertwine with unicellular microbial systems (bacteria and fungal spores), probably causing structural damages of cell wall and plasma membranes (<xref rid=\"B9\" ref-type=\"bibr\">Chen et al., 2014</xref>).</p><p>Several genes described to have a role upon oxidative stress showed to be upregulated in the presence of GOC: YDL010W (<italic>GRX6</italic>), YGR154C (<italic>GTO1</italic>), YPL061W (<italic>ALD6</italic>), YKL086W (<italic>SRX1</italic>), YGR023W (<italic>MTL1</italic>), and YLR380W (<italic>CSR1</italic>) (see <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 2</xref> for exact values of fold change). Many studies have identified the production of reactive oxygen species as a common mechanism of carbon derived nanomaterials (graphene derivatives, carbon nanotubes, etc.) to induce cell toxicity in microbial and unicellular systems (<xref rid=\"B10\" ref-type=\"bibr\">Chen et al., 2019</xref>; <xref rid=\"B35\" ref-type=\"bibr\">Madannejad et al., 2019</xref>). Therefore, similar responses at transcriptional level have been described in research works studying the interaction between microorganisms and carbon derived nanomaterials (<xref rid=\"B73\" ref-type=\"bibr\">Zhu et al., 2016</xref>; <xref rid=\"B8\" ref-type=\"bibr\">Chen et al., 2017</xref>; <xref rid=\"B70\" ref-type=\"bibr\">Yu et al., 2017</xref>; <xref rid=\"B61\" ref-type=\"bibr\">Suarez-Diez et al., 2020</xref>). An additional detailed inspection of the results also showed the overexpression of a significant number of genes involved in alpha-amino acid biosynthetic process (23), antibiotic metabolic process (19), alcohol biosynthetic process (18), one-carbon metabolic process (8), and purine nucleobase biosynthetic process (8), which suggest that GOC induced severe changes in the physiological state of the yeast.</p><p>In relation to the significantly downregulated genes found in <italic>S. cerevisiae</italic> cells exposed to GO and GOC, 44 of them where common to both conditions (<xref ref-type=\"fig\" rid=\"F3\">Figure 3D</xref>), most of them with functions related to biosynthetic and metabolic processes related to amino acids biosynthesis, some of them shown in <xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>, Group I, such as the “isoleucine biosynthetic process,” “arginine biosynthetic process,” “aromatic amino acid family biosynthetic process,” and the “ornithine metabolic process,” which could be associated to deficiencies in specific iron-dependent enzymes involved in amino acid biosynthesis or the aforementioned low availability of nitrogen (<xref rid=\"B57\" ref-type=\"bibr\">Shakoury-Elizeh et al., 2010</xref>). For instance, the synthesis of branched-chain amino acids is subjected to iron availability due to the Fe/S proteins specifically involved in the pathway (<xref rid=\"B23\" ref-type=\"bibr\">Ihrig et al., 2010</xref>). One of them, the dihydroxyacid dehydratase YJR016C (<italic>ILV3</italic>), which catalyzes the third step in the common pathway leading to biosynthesis of leucine, isoleucine and valine, was downregulated upon exposure to both nanomaterials.</p><p>The transcriptional changes in genes associated to low nutrient availability in the presence of GO and GOC could be related to the capacity of these nanomaterials to adsorb biomolecules and ions, lowering their availability for biological systems. On one hand, the high protein adsorption capacity of GO and GOC has been recently described, which could have an impact on nitrogen availability in yeast cells (<xref rid=\"B2\" ref-type=\"bibr\">Antón-Millán et al., 2018</xref>; <xref rid=\"B14\" ref-type=\"bibr\">Domi et al., 2019</xref>). Also, iron sequestration by graphene oxide in yeast growth medium was previously described in a similar study were <italic>S. cerevisiae</italic> cells were exposed to a non-commercial sample of the nanomaterial (<xref rid=\"B70\" ref-type=\"bibr\">Yu et al., 2017</xref>). The ability of graphene oxide to adsorb iron was shown to be significantly higher than that of reduced graphene oxide, probably due to the difference in oxygen containing groups on the surface of both nanomaterial types, which makes the former nanomaterial type more reactive. The same observations were done by <xref rid=\"B2\" ref-type=\"bibr\">Antón-Millán et al. (2018)</xref> and <xref rid=\"B14\" ref-type=\"bibr\">Domi et al. (2019)</xref>, when comparing the protein adsorption capacity of graphene oxide with that of lower oxygen containing carbon derived nanomaterials, such as polycarboxylate functionalized graphene nanoplatelets and reduced graphene oxide, respectively. Nevertheless, <xref rid=\"B61\" ref-type=\"bibr\">Suarez-Diez et al. (2020)</xref> also observed transcriptional evidence of metal ions deficiency (including iron) when yeast cells were exposed for 2 h to high concentrations of polycarboxylate functionalized graphene nanoplatelets (800 mg L<sup>–1</sup>), but not when their concentration was five times lower. The present study confirms previous observations done by <xref rid=\"B70\" ref-type=\"bibr\">Yu et al. (2017)</xref> and <xref rid=\"B61\" ref-type=\"bibr\">Suarez-Diez et al. (2020)</xref> at transcriptomics level, suggesting that nutrient sequestration by graphene derived nanoparticles could provoke potential adverse effects on the physiological state of microbial systems.</p><p>As previously described for the upregulated genes in both exposure conditions, most of downregulated genes were specific for GO or GOC (<xref ref-type=\"fig\" rid=\"F3\">Figure 3D</xref>). In case of the GO condition, 197 genes were exclusively downregulated, most of them with functions related to the rRNA processing and ribosomal assembly (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>, Group II, and <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 3</xref>). Ribosomal protein (RP) genes, coding for structural components of cytoplasmic ribosomes, and ribosome biogenesis (Ribi) genes, are among the largest yeast regulons and are subjected to strict transcriptional regulation through various nutrient and stress signaling pathways (<xref rid=\"B7\" ref-type=\"bibr\">Bosio et al., 2017</xref>). In fact, different toxicology studies in <italic>S. cerevisiae</italic> have reported similar results in response to different stress inducing conditions (<xref rid=\"B69\" ref-type=\"bibr\">Yu et al., 2010</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Bereketoglu et al., 2017</xref>; <xref rid=\"B60\" ref-type=\"bibr\">Soontorngun, 2017</xref>). In particular, iron starvation has been recently shown to be responsible for the decrease in the transcription rates of RP and RiBi genes, through the inhibition of one of the major nutrient-sensing kinase pathways, such as the target of rapamycin complex 1 (TORC1) (<xref rid=\"B53\" ref-type=\"bibr\">Romero et al., 2019</xref>). However, since TORC1 activity is also regulated in response to different nutrient limiting conditions (carbon, nitrogen, phosphate) and other harmful stressors (high salt, redox stress, a shift to a higher temperature, or caffeine) (<xref rid=\"B33\" ref-type=\"bibr\">Loewith and Hall, 2011</xref>), the potential inhibition of this complex when yeast is exposed to graphene oxide nanomaterials could be due to more environmental factors in addition to iron limiting conditions. In this regard, it is interesting to remark that yeast cells exposed for 2 h to 160 mg L<sup>–1</sup> of polycarboxylate functionalized graphene nanoplatelets did not show transcriptional evidence of iron starvation or other nutritional stresses, but similarly to what was observed in the present study, a high number of RP and Ribi genes were found to be downregulated too (<xref rid=\"B61\" ref-type=\"bibr\">Suarez-Diez et al., 2020</xref>).</p><p>In relation to GOC, a higher number of genes (567) were exclusively downregulated in response to this nanomaterial (<xref ref-type=\"fig\" rid=\"F3\">Figure 3D</xref>). Many of them were linked to mitochondria and mitochondrial activity, as indicated in <xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>, Group III, and in <xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 3</xref>. For instance, mitochondrial translation genes (36), mitochondrial transport genes (17) and respiratory complex assembly genes (12) were significantly downregulated. Many toxicity studies have reported the ability of nanomaterials from different origin to damage mitochondrial structure and function (<xref rid=\"B67\" ref-type=\"bibr\">Wu et al., 2020</xref>). In particular, SWCNTs, MWCNTs and graphene oxide have been reported to reduce mitochondrial membrane potential in yeast (<xref rid=\"B73\" ref-type=\"bibr\">Zhu et al., 2016</xref>, <xref rid=\"B72\" ref-type=\"bibr\">2017</xref>, <xref rid=\"B71\" ref-type=\"bibr\">2018</xref>), as in many other eukaryotic cellular models (<xref rid=\"B67\" ref-type=\"bibr\">Wu et al., 2020</xref>). The reduction of mitochondrial activity has been related to decreased ROS production associated to mitochondrial respiratory reactions, through a mechanism mediated by <italic>CTH2</italic>, which is activated in response to iron-scarcity conditions (<xref rid=\"B37\" ref-type=\"bibr\">Matsuo et al., 2017</xref>). Additionally, the function of a high number of genes was associated to chromosome segregation (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 3</xref>). This mechanism is blocked in <italic>S. cerevisiae</italic> when DNA replication is challenged. For instance, several genes controlled by the CLB2 cluster (e.g., YGR108W, YDR146C, YGL116W, YIL158W, YNL058C, etc.) were downregulated, a process that has been associated as well to DNA replication stress induced by genotoxic conditions (<xref rid=\"B42\" ref-type=\"bibr\">Palou et al., 2015</xref>). Interestingly, genes related to the process of vacuolar acidification (11) showed a lower expression level too (<xref ref-type=\"fig\" rid=\"F4\">Figure 4B</xref>, Group IV). Several genes of the proton pump vacuolar ATPase (V-ATPase) complex, which controls intracellular and extracellular pH, were found to be downregulated (YGR020C, YBR127C, YEL051W, YEL027W, YHR026W, YHR039C-A, YCL005W-A) as well as some connected to its function (YKL119C, YHR060W). V-ATPases acidify endosomes and lysosomes by pumping protons from the cytoplasm to their lumen, promoting iron mobilization and utilization (<xref rid=\"B12\" ref-type=\"bibr\">Diab and Kane, 2013</xref>). If regulated incorrectly, iron may react with H<sub>2</sub>O<sub>2</sub>, generating hydroxyl radicals and provoking cellular damage. A lower activity of this complex generates an iron deprivation signal, inducing the iron regulon. Also, an acidic cytosolic environment could promote iron bioavailability (<xref rid=\"B12\" ref-type=\"bibr\">Diab and Kane, 2013</xref>).</p><p>The results obtained in the present study show common and distinct cellular responses to two very similar commercial graphene oxide products, indicating that small disparities in manufacturing processes can result in a specific and divergent responses to these nanomaterials from biological systems. Small undetected distinct morphological features or observed differences in elemental composition might influence the nanomaterials reactivity, allowing them to elicit common and specific transcriptional responses in yeast. Both nanomaterials induced common and specific responses associated to iron scarcity and other stress factors. Significant common and specific changes in genes linked to homeostasis and ribosomal indicate major changes in the physiological state of yeast cells in the presence of these nanomaterials. The reported results contribute to understand the physiological response of fungal cells to the presence of graphene oxide, highlighting the relevance of determining the biological response of potentially exposed organisms to specific commercial nanomaterials.</p></sec></sec><sec sec-type=\"data-availability\" id=\"S4\"><title>Data Availability Statement</title><p>The transcriptomics datasets generated and analyzed for this study can be found in the European Nucleotide Archive under accession number <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"PRJEB34525\">PRJEB34525</ext-link>.</p></sec><sec id=\"S5\"><title>Author Contributions</title><p>JT-R conceived and designed the work. JT-R, MS-D, and FL-T performed the experiments, analyzed and interpreted the data, and critically revised the manuscript for intellectual content. JT-R and FL-T drafted the manuscript. All authors contributed to the article and approved the submitted version.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> This work was supported by the European Union’s H2020 Research and Innovation Programme under the Marie Skłodowska-Curie Grant Agreement Nos. 691095 and 734873 and Junta de Castilla y Leon-FEDER under Grant Nos. BU079U16 and UBU-16-B.</p></fn></fn-group><ack><p>We would like to thank Sonia Martel and Rocío Barros for their invaluable contribution.</p></ack><sec id=\"S8\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fmicb.2020.01943/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fmicb.2020.01943/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"TS1\"><media xlink:href=\"Data_Sheet_1.zip\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ali</surname><given-names>I.</given-names></name><name><surname>Basheer</surname><given-names>A. A.</given-names></name><name><surname>Mbianda</surname><given-names>X. Y.</given-names></name><name><surname>Burakov</surname><given-names>A.</given-names></name><name><surname>Galunin</surname><given-names>E.</given-names></name><name><surname>Burakova</surname><given-names>I.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Graphene based adsorbents for remediation of noxious pollutants from wastewater.</article-title>\n<source><italic>Environ. Int.</italic></source>\n<volume>127</volume>\n<fpage>160</fpage>–<lpage>180</lpage>. <pub-id pub-id-type=\"doi\">10.1016/J.ENVINT.2019.03.029</pub-id>\n<pub-id pub-id-type=\"pmid\">30921668</pub-id></mixed-citation></ref><ref id=\"B2\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Antón-Millán</surname><given-names>N.</given-names></name><name><surname>García-Tojal</surname><given-names>J.</given-names></name><name><surname>Marty-Roda</surname><given-names>M.</given-names></name><name><surname>Garroni</surname><given-names>S.</given-names></name><name><surname>Cuesta-López</surname><given-names>S.</given-names></name><name><surname>Tamayo-Ramos</surname><given-names>J. A.</given-names></name></person-group> (<year>2018</year>). <article-title>Influence of Three Commercial Graphene Derivatives on the Catalytic Properties of a <italic>Lactobacillus plantarum</italic> α- l -Rhamnosidase When Used as Immobilization Matrices.</article-title>\n<source><italic>ACS Appl. Mater. Interfaces</italic></source>\n<volume>10</volume>\n<fpage>18170</fpage>–<lpage>18182</lpage>. <pub-id pub-id-type=\"doi\">10.1021/acsami.7b18844</pub-id>\n<pub-id pub-id-type=\"pmid\">29732878</pub-id></mixed-citation></ref><ref id=\"B3\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Asadi Shahi</surname><given-names>S.</given-names></name><name><surname>Roudbar Mohammadi</surname><given-names>S.</given-names></name><name><surname>Roudbary</surname><given-names>M.</given-names></name><name><surname>Delavari</surname><given-names>H.</given-names></name></person-group> (<year>2019</year>). <article-title>A new formulation of graphene oxide/fluconazole compound as a promising agent against <italic>Candida albicans</italic>.</article-title>\n<source><italic>Prog. Biomater.</italic></source>\n<volume>8</volume>\n<fpage>43</fpage>–<lpage>50</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s40204-019-0109-106</pub-id><pub-id pub-id-type=\"pmid\">30859396</pub-id></mixed-citation></ref><ref id=\"B4\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Askwith</surname><given-names>C.</given-names></name><name><surname>Eide</surname><given-names>D.</given-names></name><name><surname>Van Ho</surname><given-names>A.</given-names></name><name><surname>Bernard</surname><given-names>P. S.</given-names></name><name><surname>Li</surname><given-names>L.</given-names></name><name><surname>Davis-Kaplan</surname><given-names>S.</given-names></name><etal/></person-group> (<year>1994</year>). <article-title>The FET3 gene of <italic>S. cerevisiae</italic> encodes a multicopper oxidase required for ferrous iron uptake.</article-title>\n<source><italic>Cell</italic></source>\n<volume>76</volume>\n<fpage>403</fpage>–<lpage>410</lpage>. <pub-id pub-id-type=\"doi\">10.1016/0092-8674(94)90346-90348</pub-id><pub-id pub-id-type=\"pmid\">8293473</pub-id></mixed-citation></ref><ref id=\"B5\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bayat</surname><given-names>N.</given-names></name><name><surname>Rajapakse</surname><given-names>K.</given-names></name><name><surname>Marinsek-Logar</surname><given-names>R.</given-names></name><name><surname>Drobne</surname><given-names>D.</given-names></name><name><surname>Cristobal</surname><given-names>S.</given-names></name></person-group> (<year>2014</year>). <article-title>The effects of engineered nanoparticles on the cellular structure and growth of <italic>Saccharomyces cerevisiae</italic>.</article-title>\n<source><italic>Nanotoxicology</italic></source>\n<volume>8</volume>\n<fpage>363</fpage>–<lpage>373</lpage>. <pub-id pub-id-type=\"doi\">10.3109/17435390.2013.788748</pub-id>\n<pub-id pub-id-type=\"pmid\">23521755</pub-id></mixed-citation></ref><ref id=\"B6\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bereketoglu</surname><given-names>C.</given-names></name><name><surname>Arga</surname><given-names>K. Y.</given-names></name><name><surname>Eraslan</surname><given-names>S.</given-names></name><name><surname>Mertoglu</surname><given-names>B.</given-names></name></person-group> (<year>2017</year>). <article-title>Genome reprogramming in <italic>Saccharomyces cerevisiae</italic> upon nonylphenol exposure.</article-title>\n<source><italic>Physiol. Genomics</italic></source>\n<volume>49</volume>\n<fpage>549</fpage>–<lpage>566</lpage>. <pub-id pub-id-type=\"doi\">10.1152/physiolgenomics.00034.2017</pub-id>\n<pub-id pub-id-type=\"pmid\">28887370</pub-id></mixed-citation></ref><ref id=\"B7\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Bosio</surname><given-names>M. C.</given-names></name><name><surname>Fermi</surname><given-names>B.</given-names></name><name><surname>Dieci</surname><given-names>G.</given-names></name></person-group> (<year>2017</year>). <article-title>Transcriptional control of yeast ribosome biogenesis: a multifaceted role for general regulatory factors.</article-title>\n<source><italic>Transcription</italic></source>\n<volume>8</volume>\n<fpage>254</fpage>–<lpage>260</lpage>. <pub-id pub-id-type=\"doi\">10.1080/21541264.2017.1317378</pub-id>\n<pub-id pub-id-type=\"pmid\">28448767</pub-id></mixed-citation></ref><ref id=\"B8\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>J.</given-names></name><name><surname>Li</surname><given-names>S.</given-names></name><name><surname>Luo</surname><given-names>J.</given-names></name><name><surname>Zhang</surname><given-names>Y.</given-names></name><name><surname>Ding</surname><given-names>W.</given-names></name></person-group> (<year>2017</year>). <article-title>Graphene oxide induces toxicity and alters energy metabolism and gene expression in <italic>Ralstonia solanacearum</italic>.</article-title>\n<source><italic>J. Nanosci. Nanotechnol.</italic></source>\n<volume>17</volume>\n<fpage>186</fpage>–<lpage>195</lpage>. <pub-id pub-id-type=\"doi\">10.1166/jnn.2017.12575</pub-id>\n<pub-id pub-id-type=\"pmid\">29617100</pub-id></mixed-citation></ref><ref id=\"B9\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>J.</given-names></name><name><surname>Peng</surname><given-names>H.</given-names></name><name><surname>Wang</surname><given-names>X.</given-names></name><name><surname>Shao</surname><given-names>F.</given-names></name><name><surname>Yuan</surname><given-names>Z.</given-names></name><name><surname>Han</surname><given-names>H.</given-names></name></person-group> (<year>2014</year>). <article-title>Graphene oxide exhibits broad-spectrum antimicrobial activity against bacterial phytopathogens and fungal conidia by intertwining and membrane perturbation.</article-title>\n<source><italic>Nanoscale</italic></source>\n<volume>6</volume>\n<fpage>1879</fpage>–<lpage>1889</lpage>. <pub-id pub-id-type=\"doi\">10.1039/c3nr04941h</pub-id>\n<pub-id pub-id-type=\"pmid\">24362636</pub-id></mixed-citation></ref><ref id=\"B10\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>M.</given-names></name><name><surname>Sun</surname><given-names>Y.</given-names></name><name><surname>Liang</surname><given-names>J.</given-names></name><name><surname>Zeng</surname><given-names>G.</given-names></name><name><surname>Li</surname><given-names>Z.</given-names></name><name><surname>Tang</surname><given-names>L.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Understanding the influence of carbon nanomaterials on microbial communities.</article-title>\n<source><italic>Environ. Int.</italic></source>\n<volume>126</volume>\n<fpage>690</fpage>–<lpage>698</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.envint.2019.02.005</pub-id>\n<pub-id pub-id-type=\"pmid\">30875562</pub-id></mixed-citation></ref><ref id=\"B11\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Cherry</surname><given-names>J. M.</given-names></name><name><surname>Hong</surname><given-names>E. L.</given-names></name><name><surname>Amundsen</surname><given-names>C.</given-names></name><name><surname>Balakrishnan</surname><given-names>R.</given-names></name><name><surname>Binkley</surname><given-names>G.</given-names></name><name><surname>Chan</surname><given-names>E. T.</given-names></name><etal/></person-group> (<year>2012</year>). <article-title>Saccharomyces Genome Database: the genomics resource of budding yeast.</article-title>\n<source><italic>Nucleic Acids Res.</italic></source>\n<volume>40</volume>\n<fpage>D700</fpage>–<lpage>D705</lpage>. <pub-id pub-id-type=\"doi\">10.1093/nar/gkr1029</pub-id>\n<pub-id pub-id-type=\"pmid\">22110037</pub-id></mixed-citation></ref><ref id=\"B12\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Diab</surname><given-names>H. I.</given-names></name><name><surname>Kane</surname><given-names>P. M.</given-names></name></person-group> (<year>2013</year>). <article-title>Loss of vacuolar H+-ATPase (V-ATPase) activity in yeast generates an iron deprivation signal that is moderated by induction of the peroxiredoxin TSA2.</article-title>\n<source><italic>J. Biol. Chem.</italic></source>\n<volume>288</volume>\n<fpage>11366</fpage>–<lpage>11377</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M112.419259</pub-id>\n<pub-id pub-id-type=\"pmid\">23457300</pub-id></mixed-citation></ref><ref id=\"B13\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Dobin</surname><given-names>A.</given-names></name><name><surname>Davis</surname><given-names>C. A.</given-names></name><name><surname>Schlesinger</surname><given-names>F.</given-names></name><name><surname>Drenkow</surname><given-names>J.</given-names></name><name><surname>Zaleski</surname><given-names>C.</given-names></name><name><surname>Jha</surname><given-names>S.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>STAR: ultrafast universal RNA-seq aligner.</article-title>\n<source><italic>Bioinformatics</italic></source>\n<volume>29</volume>\n<fpage>15</fpage>–<lpage>21</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bioinformatics/bts635</pub-id>\n<pub-id pub-id-type=\"pmid\">23104886</pub-id></mixed-citation></ref><ref id=\"B14\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Domi</surname><given-names>B.</given-names></name><name><surname>Rumbo</surname><given-names>C.</given-names></name><name><surname>García-Tojal</surname><given-names>J.</given-names></name><name><surname>Elena Sima</surname><given-names>L.</given-names></name><name><surname>Negroiu</surname><given-names>G.</given-names></name><name><surname>Tamayo-Ramos</surname><given-names>J. A.</given-names></name></person-group> (<year>2019</year>). <article-title>Interaction analysis of commercial graphene oxide nanoparticles with unicellular systems and biomolecules.</article-title>\n<source><italic>Int. J. Mol. Sci.</italic></source>\n<volume>21</volume>:<issue>205</issue>. <pub-id pub-id-type=\"doi\">10.3390/ijms21010205</pub-id>\n<pub-id pub-id-type=\"pmid\">31892228</pub-id></mixed-citation></ref><ref id=\"B15\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Eigler</surname><given-names>S.</given-names></name><name><surname>Dotzer</surname><given-names>C.</given-names></name><name><surname>Hof</surname><given-names>F.</given-names></name><name><surname>Bauer</surname><given-names>W.</given-names></name><name><surname>Hirsch</surname><given-names>A.</given-names></name></person-group> (<year>2013</year>). <article-title>Sulfur Species in Graphene Oxide.</article-title>\n<source><italic>Chem. A Eur. J.</italic></source>\n<volume>19</volume>\n<fpage>9490</fpage>–<lpage>9496</lpage>. <pub-id pub-id-type=\"doi\">10.1002/chem.201300387</pub-id>\n<pub-id pub-id-type=\"pmid\">23780799</pub-id></mixed-citation></ref><ref id=\"B16\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Elena</surname><given-names>G.</given-names></name><name><surname>Despina</surname><given-names>A.</given-names></name></person-group> (<year>1994</year>). <article-title>Two distinctly regulated genes are required for ferric reduction, the first step of iron uptake in <italic>Saccharomyces cerevisiae</italic>.</article-title>\n<source><italic>Mol. Cell. Biol.</italic></source>\n<volume>14</volume>\n<fpage>3065</fpage>–<lpage>3073</lpage>. <pub-id pub-id-type=\"doi\">10.1128/MCB.14.5.3065.Updated</pub-id><pub-id pub-id-type=\"pmid\">8164662</pub-id></mixed-citation></ref><ref id=\"B17\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ema</surname><given-names>M.</given-names></name><name><surname>Gamo</surname><given-names>M.</given-names></name><name><surname>Honda</surname><given-names>K.</given-names></name></person-group> (<year>2017</year>). <article-title>A review of toxicity studies on graphene-based nanomaterials in laboratory animals.</article-title>\n<source><italic>Regul. Toxicol. Pharmacol.</italic></source>\n<volume>85</volume>\n<fpage>7</fpage>–<lpage>24</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.yrtph.2017.01.011</pub-id>\n<pub-id pub-id-type=\"pmid\">28161457</pub-id></mixed-citation></ref><ref id=\"B18\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fadeel</surname><given-names>B.</given-names></name><name><surname>Bussy</surname><given-names>C.</given-names></name><name><surname>Merino</surname><given-names>S.</given-names></name><name><surname>Vázquez</surname><given-names>E.</given-names></name><name><surname>Flahaut</surname><given-names>E.</given-names></name><name><surname>Mouchet</surname><given-names>F.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Safety assessment of graphene-based materials: focus on human health and the environment.</article-title>\n<source><italic>ACS Nano</italic></source>\n<volume>12</volume>\n<fpage>10582</fpage>–<lpage>10620</lpage>. <pub-id pub-id-type=\"doi\">10.1021/acsnano.8b04758</pub-id>\n<pub-id pub-id-type=\"pmid\">30387986</pub-id></mixed-citation></ref><ref id=\"B19\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Farzanegan</surname><given-names>A.</given-names></name><name><surname>Roudbary</surname><given-names>M.</given-names></name><name><surname>Falahati</surname><given-names>M.</given-names></name><name><surname>Khoobi</surname><given-names>M.</given-names></name><name><surname>Gholibegloo</surname><given-names>E.</given-names></name><name><surname>Farahyar</surname><given-names>S.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Synthesis, characterization and antifungal activity of a novel formulated nanocomposite containing Indolicidin and Graphene oxide against disseminated candidiasis.</article-title>\n<source><italic>J. Mycol. Med.</italic></source>\n<volume>28</volume>\n<fpage>628</fpage>–<lpage>636</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.mycmed.2018.07.009</pub-id>\n<pub-id pub-id-type=\"pmid\">30126717</pub-id></mixed-citation></ref><ref id=\"B20\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fu</surname><given-names>D.</given-names></name><name><surname>Beeler</surname><given-names>T. J.</given-names></name><name><surname>Dunn</surname><given-names>T. M.</given-names></name></person-group> (<year>1995</year>). <article-title>Sequence, mapping and disruption of CCC2, a gene that cross-complements the Ca2+-sensitive phenotype of csg1 mutants and encodes a P-type ATPase belonging to the Cu2+-ATPase subfamily.</article-title>\n<source><italic>Yeast</italic></source>\n<volume>11</volume>\n<fpage>283</fpage>–<lpage>292</lpage>. <pub-id pub-id-type=\"doi\">10.1002/yea.320110310</pub-id>\n<pub-id pub-id-type=\"pmid\">7785328</pub-id></mixed-citation></ref><ref id=\"B21\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Heymann</surname><given-names>P.</given-names></name><name><surname>Ernst</surname><given-names>J. F.</given-names></name><name><surname>Winkelmann</surname><given-names>G.</given-names></name></person-group> (<year>1999</year>). <article-title>Identification of a fungal triacetylfusarinine C siderophore transport gene (TAF1) in <italic>Saccharomyces cerevisiae</italic> as a member of the major facilitator superfamily.</article-title>\n<source><italic>Biometals</italic></source>\n<volume>12</volume>\n<fpage>301</fpage>–<lpage>306</lpage>. <pub-id pub-id-type=\"doi\">10.1023/a:1009252118050</pub-id><pub-id pub-id-type=\"pmid\">10816729</pub-id></mixed-citation></ref><ref id=\"B22\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Heymann</surname><given-names>P.</given-names></name><name><surname>Ernst</surname><given-names>J. F.</given-names></name><name><surname>Winkelmann</surname><given-names>G.</given-names></name></person-group> (<year>2006</year>). <article-title>Identification and substrate specificity of a ferrichrome-type siderophore transporter (Arn1p) in <italic>Saccharomyces cerevisiae</italic>.</article-title>\n<source><italic>FEMS Microbiol. Lett.</italic></source>\n<volume>186</volume>\n<fpage>221</fpage>–<lpage>227</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1574-6968.2000.tb09108.x</pub-id>\n<pub-id pub-id-type=\"pmid\">10802175</pub-id></mixed-citation></ref><ref id=\"B23\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ihrig</surname><given-names>J.</given-names></name><name><surname>Hausmann</surname><given-names>A.</given-names></name><name><surname>Hain</surname><given-names>A.</given-names></name><name><surname>Richter</surname><given-names>N.</given-names></name><name><surname>Hamza</surname><given-names>I.</given-names></name><name><surname>Lill</surname><given-names>R.</given-names></name><etal/></person-group> (<year>2010</year>). <article-title>Iron regulation through the back door: iron-dependent metabolite levels contribute to transcriptional adaptation to iron deprivation in <italic>Saccharomyces cerevisiae</italic>.</article-title>\n<source><italic>Eukaryot. Cell</italic></source>\n<volume>9</volume>\n<fpage>460</fpage>–<lpage>471</lpage>. <pub-id pub-id-type=\"doi\">10.1128/EC.00213-219</pub-id><pub-id pub-id-type=\"pmid\">20008079</pub-id></mixed-citation></ref><ref id=\"B24\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Izawa</surname><given-names>S.</given-names></name><name><surname>Maeda</surname><given-names>K.</given-names></name><name><surname>Miki</surname><given-names>T.</given-names></name><name><surname>Mano</surname><given-names>J.</given-names></name><name><surname>Inoue</surname><given-names>Y.</given-names></name><name><surname>Kimura</surname><given-names>A.</given-names></name></person-group> (<year>1998</year>). <article-title>Importance of glucose-6-phosphate dehydrogenase in the adaptive response to hydrogen peroxide in <italic>Saccharomyces cerevisiae</italic>.</article-title>\n<source><italic>Biochem. J.</italic></source>\n<volume>330</volume>\n<fpage>811</fpage>–<lpage>817</lpage>. <pub-id pub-id-type=\"doi\">10.1042/bj3300811</pub-id>\n<pub-id pub-id-type=\"pmid\">9480895</pub-id></mixed-citation></ref><ref id=\"B25\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kanehisa</surname><given-names>M.</given-names></name></person-group> (<year>2000</year>). <article-title>KEGG: Kyoto encyclopedia of genes and genomes.</article-title>\n<source><italic>Nucleic Acids Res.</italic></source>\n<volume>28</volume>\n<fpage>27</fpage>–<lpage>30</lpage>. <pub-id pub-id-type=\"doi\">10.1093/nar/28.1.27</pub-id>\n<pub-id pub-id-type=\"pmid\">10592173</pub-id></mixed-citation></ref><ref id=\"B26\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kempaiah</surname><given-names>R.</given-names></name><name><surname>Chung</surname><given-names>A.</given-names></name><name><surname>Maheshwari</surname><given-names>V.</given-names></name></person-group> (<year>2011</year>). <article-title>Graphene as cellular interface: electromechanical coupling with cells.</article-title>\n<source><italic>ACS Nano</italic></source>\n<volume>5</volume>\n<fpage>6025</fpage>–<lpage>6031</lpage>. <pub-id pub-id-type=\"doi\">10.1021/nn201791k</pub-id>\n<pub-id pub-id-type=\"pmid\">21675743</pub-id></mixed-citation></ref><ref id=\"B27\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Khanra</surname><given-names>P.</given-names></name><name><surname>Kuila</surname><given-names>T.</given-names></name><name><surname>Kim</surname><given-names>N. H.</given-names></name><name><surname>Bae</surname><given-names>S. H.</given-names></name><name><surname>Yu</surname><given-names>D.</given-names></name><name><surname>Lee</surname><given-names>J. H.</given-names></name></person-group> (<year>2012</year>). <article-title>Simultaneous bio-functionalization and reduction of graphene oxide by baker’s yeast.</article-title>\n<source><italic>Chem. Eng. J.</italic></source>\n<volume>183</volume>\n<fpage>526</fpage>–<lpage>533</lpage>. <pub-id pub-id-type=\"doi\">10.1016/J.CEJ.2011.12.075</pub-id></mixed-citation></ref><ref id=\"B28\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kleinschmidt</surname><given-names>M.</given-names></name><name><surname>Grundmann</surname><given-names>O.</given-names></name><name><surname>Blüthgen</surname><given-names>N.</given-names></name><name><surname>Mösch</surname><given-names>H. U.</given-names></name><name><surname>Braus</surname><given-names>G. H.</given-names></name></person-group> (<year>2005</year>). <article-title>Transcriptional profiling of <italic>Saccharomyces cerevisiae</italic> cells under adhesion-inducing conditions.</article-title>\n<source><italic>Mol. Genet. Genom.</italic></source>\n<volume>273</volume>\n<fpage>382</fpage>–<lpage>393</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00438-005-1139-1134</pub-id></mixed-citation></ref><ref id=\"B29\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>C.</given-names></name><name><surname>Wang</surname><given-names>X.</given-names></name><name><surname>Chen</surname><given-names>F.</given-names></name><name><surname>Zhang</surname><given-names>C.</given-names></name><name><surname>Zhi</surname><given-names>X.</given-names></name><name><surname>Wang</surname><given-names>K.</given-names></name><etal/></person-group> (<year>2013</year>). <article-title>The antifungal activity of graphene oxide–silver nanocomposites.</article-title>\n<source><italic>Biomaterials</italic></source>\n<volume>34</volume>\n<fpage>3882</fpage>–<lpage>3890</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.biomaterials.2013.02.001</pub-id>\n<pub-id pub-id-type=\"pmid\">23465487</pub-id></mixed-citation></ref><ref id=\"B30\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>R.</given-names></name><name><surname>Guiney</surname><given-names>L. M.</given-names></name><name><surname>Chang</surname><given-names>C. H.</given-names></name><name><surname>Mansukhani</surname><given-names>N. D.</given-names></name><name><surname>Ji</surname><given-names>Z.</given-names></name><name><surname>Wang</surname><given-names>X.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Surface Oxidation of Graphene Oxide Determines Membrane Damage, Lipid Peroxidation, and Cytotoxicity in Macrophages in a Pulmonary Toxicity Model.</article-title>\n<source><italic>ACS Nano</italic></source>\n<volume>12</volume>\n<fpage>1390</fpage>–<lpage>1402</lpage>. <pub-id pub-id-type=\"doi\">10.1021/acsnano.7b07737</pub-id>\n<pub-id pub-id-type=\"pmid\">29328670</pub-id></mixed-citation></ref><ref id=\"B31\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liao</surname><given-names>Y.</given-names></name><name><surname>Smyth</surname><given-names>G. K.</given-names></name><name><surname>Shi</surname><given-names>W.</given-names></name></person-group> (<year>2014</year>). <article-title>featureCounts: an efficient general purpose program for assigning sequence reads to genomic features.</article-title>\n<source><italic>Bioinformatics</italic></source>\n<volume>30</volume>\n<fpage>923</fpage>–<lpage>930</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bioinformatics/btt656</pub-id>\n<pub-id pub-id-type=\"pmid\">24227677</pub-id></mixed-citation></ref><ref id=\"B32\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Liu</surname><given-names>S.</given-names></name><name><surname>Zeng</surname><given-names>T. H.</given-names></name><name><surname>Hofmann</surname><given-names>M.</given-names></name><name><surname>Burcombe</surname><given-names>E.</given-names></name><name><surname>Wei</surname><given-names>J.</given-names></name><name><surname>Jiang</surname><given-names>R.</given-names></name><etal/></person-group> (<year>2011</year>). <article-title>Antibacterial activity of graphite, graphite oxide, graphene oxide, and reduced graphene oxide: membrane and oxidative stress.</article-title>\n<source><italic>ACS Nano</italic></source>\n<volume>5</volume>\n<fpage>6971</fpage>–<lpage>6980</lpage>. <pub-id pub-id-type=\"doi\">10.1021/nn202451x</pub-id>\n<pub-id pub-id-type=\"pmid\">21851105</pub-id></mixed-citation></ref><ref id=\"B33\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Loewith</surname><given-names>R.</given-names></name><name><surname>Hall</surname><given-names>M. N.</given-names></name></person-group> (<year>2011</year>). <article-title>Target of rapamycin (TOR) in nutrient signaling and growth control.</article-title>\n<source><italic>Genetics</italic></source>\n<volume>189</volume>\n<fpage>1177</fpage>–<lpage>1201</lpage>. <pub-id pub-id-type=\"doi\">10.1534/genetics.111.133363</pub-id>\n<pub-id pub-id-type=\"pmid\">22174183</pub-id></mixed-citation></ref><ref id=\"B34\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Love</surname><given-names>M. I.</given-names></name><name><surname>Huber</surname><given-names>W.</given-names></name><name><surname>Anders</surname><given-names>S.</given-names></name></person-group> (<year>2014</year>). <article-title>Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.</article-title>\n<source><italic>Genome Biol.</italic></source>\n<volume>15</volume>:<issue>550</issue>\n<pub-id pub-id-type=\"doi\">10.1186/s13059-014-0550-558</pub-id></mixed-citation></ref><ref id=\"B35\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Madannejad</surname><given-names>R.</given-names></name><name><surname>Shoaie</surname><given-names>N.</given-names></name><name><surname>Jahanpeyma</surname><given-names>F.</given-names></name><name><surname>Darvishi</surname><given-names>M. H.</given-names></name><name><surname>Azimzadeh</surname><given-names>M.</given-names></name><name><surname>Javadi</surname><given-names>H.</given-names></name></person-group> (<year>2019</year>). <article-title>Toxicity of carbon-based nanomaterials: reviewing recent reports in medical and biological systems.</article-title>\n<source><italic>Chem. Biol. Interact.</italic></source>\n<volume>307</volume>\n<fpage>206</fpage>–<lpage>222</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cbi.2019.04.036</pub-id>\n<pub-id pub-id-type=\"pmid\">31054282</pub-id></mixed-citation></ref><ref id=\"B36\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martins</surname><given-names>L. J.</given-names></name><name><surname>Jensen</surname><given-names>L. T.</given-names></name><name><surname>Simons</surname><given-names>J. R.</given-names></name><name><surname>Keller</surname><given-names>G. L.</given-names></name><name><surname>Winge</surname><given-names>D. R.</given-names></name></person-group> (<year>1998</year>). <article-title>Metalloregulation of FRE1 and FRE2 homologs in <italic>Saccharomyces cerevisiae</italic>.</article-title>\n<source><italic>J. Biol. Chem.</italic></source>\n<volume>273</volume>\n<fpage>23716</fpage>–<lpage>23721</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.273.37.23716</pub-id>\n<pub-id pub-id-type=\"pmid\">9726978</pub-id></mixed-citation></ref><ref id=\"B37\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Matsuo</surname><given-names>R.</given-names></name><name><surname>Mizobuchi</surname><given-names>S.</given-names></name><name><surname>Nakashima</surname><given-names>M.</given-names></name><name><surname>Miki</surname><given-names>K.</given-names></name><name><surname>Ayusawa</surname><given-names>D.</given-names></name><name><surname>Fujii</surname><given-names>M.</given-names></name></person-group> (<year>2017</year>). <article-title>Central roles of iron in the regulation of oxidative stress in the yeast <italic>Saccharomyces cerevisiae</italic>.</article-title>\n<source><italic>Curr. Genet.</italic></source>\n<volume>63</volume>\n<fpage>895</fpage>–<lpage>907</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00294-017-0689-684</pub-id><pub-id pub-id-type=\"pmid\">28289833</pub-id></mixed-citation></ref><ref id=\"B38\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Miralles</surname><given-names>V. J.</given-names></name><name><surname>Serrano</surname><given-names>R.</given-names></name></person-group> (<year>1995</year>). <article-title>A genomic locus in <italic>Saccharomyces cerevisiae</italic> with four genes up-regulated by osmotic stress.</article-title>\n<source><italic>Mol. Microbiol.</italic></source>\n<volume>17</volume>\n<fpage>653</fpage>–<lpage>662</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1365-2958.1995.mmi_17040653.x</pub-id><pub-id pub-id-type=\"pmid\">8801420</pub-id></mixed-citation></ref><ref id=\"B39\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Moretti-Almeida</surname><given-names>G.</given-names></name><name><surname>Netto</surname><given-names>L. E. S.</given-names></name><name><surname>Monteiro</surname><given-names>G.</given-names></name></person-group> (<year>2013</year>). <article-title>The essential gene YMR134W from <italic>Saccharomyces cerevisiae</italic> is important for appropriate mitochondrial iron utilization and the ergosterol biosynthetic pathway.</article-title>\n<source><italic>FEBS Lett.</italic></source>\n<volume>587</volume>\n<fpage>3008</fpage>–<lpage>3013</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.febslet.2013.07.024</pub-id>\n<pub-id pub-id-type=\"pmid\">23892078</pub-id></mixed-citation></ref><ref id=\"B40\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ou</surname><given-names>L.</given-names></name><name><surname>Song</surname><given-names>B.</given-names></name><name><surname>Liang</surname><given-names>H.</given-names></name><name><surname>Liu</surname><given-names>J.</given-names></name><name><surname>Feng</surname><given-names>X.</given-names></name><name><surname>Deng</surname><given-names>B.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>Toxicity of graphene-family nanoparticles: a general review of the origins and mechanisms.</article-title>\n<source><italic>Part. Fibre Toxicol.</italic></source>\n<volume>13</volume>:<issue>57</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12989-016-0168-y</pub-id>\n<pub-id pub-id-type=\"pmid\">27799056</pub-id></mixed-citation></ref><ref id=\"B41\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Padilla</surname><given-names>P. A.</given-names></name><name><surname>Fuge</surname><given-names>E. K.</given-names></name><name><surname>Crawford</surname><given-names>M. E.</given-names></name><name><surname>Errett</surname><given-names>A.</given-names></name><name><surname>Werner-Washburne</surname><given-names>M.</given-names></name></person-group> (<year>1998</year>). <article-title>The highly conserved, coregulated SNO and SNZ gene families in <italic>Saccharomyces cerevisiae</italic> respond to nutrient limitation.</article-title>\n<source><italic>J. Bacteriol.</italic></source>\n<volume>180</volume>\n<fpage>5718</fpage>–<lpage>5726</lpage>. <pub-id pub-id-type=\"doi\">10.1128/jb.180.21.5718-5726.1998</pub-id>\n<pub-id pub-id-type=\"pmid\">9791124</pub-id></mixed-citation></ref><ref id=\"B42\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Palou</surname><given-names>G.</given-names></name><name><surname>Palou</surname><given-names>R.</given-names></name><name><surname>Zeng</surname><given-names>F.</given-names></name><name><surname>Vashisht</surname><given-names>A. A.</given-names></name><name><surname>Wohlschlegel</surname><given-names>J. A.</given-names></name><name><surname>Quintana</surname><given-names>D. G.</given-names></name></person-group> (<year>2015</year>). <article-title>Three Different Pathways Prevent Chromosome Segregation in the Presence of DNA Damage or Replication Stress in Budding Yeast.</article-title>\n<source><italic>PLoS Genet.</italic></source>\n<volume>11</volume>:<issue>e1005468</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pgen.1005468</pub-id>\n<pub-id pub-id-type=\"pmid\">26332045</pub-id></mixed-citation></ref><ref id=\"B43\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Panaretou</surname><given-names>B.</given-names></name><name><surname>Piper</surname><given-names>P. W.</given-names></name></person-group> (<year>1992</year>). <article-title>The plasma membrane of yeast acquires a novel heat-shock protein (hsp30) and displays a decline in proton-pumping ATPase levels in response to both heat shock and the entry to stationary phase.</article-title>\n<source><italic>Eur. J. Biochem.</italic></source>\n<volume>206</volume>\n<fpage>635</fpage>–<lpage>640</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1432-1033.1992.tb16968.x</pub-id>\n<pub-id pub-id-type=\"pmid\">1535043</pub-id></mixed-citation></ref><ref id=\"B44\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Park</surname><given-names>J. S.</given-names></name><name><surname>Na</surname><given-names>H.-K.</given-names></name><name><surname>Min</surname><given-names>D.-H.</given-names></name><name><surname>Kim</surname><given-names>D.-E.</given-names></name></person-group> (<year>2013</year>). <article-title>Desorption of single-stranded nucleic acids from graphene oxide by disruption of hydrogen bonding.</article-title>\n<source><italic>Analyst</italic></source>\n<volume>138</volume>:<issue>1745</issue>. <pub-id pub-id-type=\"doi\">10.1039/c3an36493c</pub-id>\n<pub-id pub-id-type=\"pmid\">23361154</pub-id></mixed-citation></ref><ref id=\"B45\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pérez-Rubio</surname><given-names>P.</given-names></name><name><surname>Lottaz</surname><given-names>C.</given-names></name><name><surname>Engelmann</surname><given-names>J. C.</given-names></name></person-group> (<year>2019</year>). <article-title>FastqPuri: high-performance preprocessing of RNA-seq data.</article-title>\n<source><italic>BMC Bioinformatics</italic></source>\n<volume>20</volume>:<issue>226</issue>\n<pub-id pub-id-type=\"doi\">10.1186/s12859-019-2799-2790</pub-id></mixed-citation></ref><ref id=\"B46\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Philpott</surname><given-names>C. C.</given-names></name><name><surname>Protchenko</surname><given-names>O.</given-names></name></person-group> (<year>2008</year>). <article-title>Response to iron deprivation in <italic>Saccharomyces cerevisiae</italic>.</article-title>\n<source><italic>Eukaryot. Cell</italic></source>\n<volume>7</volume>\n<fpage>20</fpage>–<lpage>27</lpage>. <pub-id pub-id-type=\"doi\">10.1128/EC.00354-357</pub-id><pub-id pub-id-type=\"pmid\">17993568</pub-id></mixed-citation></ref><ref id=\"B47\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Philpott</surname><given-names>C. C.</given-names></name><name><surname>Protchenko</surname><given-names>O.</given-names></name><name><surname>Kim</surname><given-names>Y. W.</given-names></name><name><surname>Boretsky</surname><given-names>Y.</given-names></name><name><surname>Shakoury-Elizeh</surname><given-names>M.</given-names></name></person-group> (<year>2002</year>). <article-title>The response to iron deprivation in <italic>Saccharomyces cerevisiae</italic>: expression of siderophore-based systems of iron uptake.</article-title>\n<source><italic>Biochem. Soc. Trans.</italic></source>\n<volume>30</volume>\n<fpage>698</fpage>–<lpage>702</lpage>. <pub-id pub-id-type=\"doi\">10.1042/BST0300698</pub-id>\n<pub-id pub-id-type=\"pmid\">12196168</pub-id></mixed-citation></ref><ref id=\"B48\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Piper</surname><given-names>M. D. W.</given-names></name><name><surname>Hong</surname><given-names>S.-P.</given-names></name><name><surname>EiÃźing</surname><given-names>T.</given-names></name><name><surname>Sealey</surname><given-names>P.</given-names></name><name><surname>Dawes</surname><given-names>I. W.</given-names></name></person-group> (<year>2002</year>). <article-title>Regulation of the yeast glycine cleavage genes is responsive to the availability of multiple nutrients.</article-title>\n<source><italic>FEMS Yeast Res.</italic></source>\n<volume>2</volume>\n<fpage>59</fpage>–<lpage>71</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1567-1364.2002.tb00069.x</pub-id>\n<pub-id pub-id-type=\"pmid\">12702322</pub-id></mixed-citation></ref><ref id=\"B49\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Protchenko</surname><given-names>O.</given-names></name><name><surname>Ferea</surname><given-names>T.</given-names></name><name><surname>Rashford</surname><given-names>J.</given-names></name><name><surname>Tiedeman</surname><given-names>J.</given-names></name><name><surname>Brown</surname><given-names>P. O.</given-names></name><name><surname>Botstein</surname><given-names>D.</given-names></name><etal/></person-group> (<year>2001</year>). <article-title>Three Cell Wall Mannoproteins Facilitate the Uptake of Iron in <italic>Saccharomyces cerevisiae</italic>.</article-title>\n<source><italic>J. Biol. Chem.</italic></source>\n<volume>276</volume>\n<fpage>49244</fpage>–<lpage>49250</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M109220200</pub-id>\n<pub-id pub-id-type=\"pmid\">11673473</pub-id></mixed-citation></ref><ref id=\"B50\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Protchenko</surname><given-names>O.</given-names></name><name><surname>Philpott</surname><given-names>C. C.</given-names></name></person-group> (<year>2003</year>). <article-title>Regulation of intracellular heme levels by HMX1, a homologue of heme oxygenase, in <italic>Saccharomyces cerevisiae</italic>.</article-title>\n<source><italic>J. Biol. Chem.</italic></source>\n<volume>278</volume>\n<fpage>36582</fpage>–<lpage>36587</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M306584200</pub-id>\n<pub-id pub-id-type=\"pmid\">12840010</pub-id></mixed-citation></ref><ref id=\"B51\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Puig</surname><given-names>S.</given-names></name><name><surname>Askeland</surname><given-names>E.</given-names></name><name><surname>Thiele</surname><given-names>D. J.</given-names></name></person-group> (<year>2005</year>). <article-title>Coordinated remodeling of cellular metabolism during iron deficiency through targeted mRNA degradation.</article-title>\n<source><italic>Cell</italic></source>\n<volume>120</volume>\n<fpage>99</fpage>–<lpage>110</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.cell.2004.11.032</pub-id>\n<pub-id pub-id-type=\"pmid\">15652485</pub-id></mixed-citation></ref><ref id=\"B52\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rodríguez-Navarro</surname><given-names>S.</given-names></name><name><surname>Llorente</surname><given-names>B.</given-names></name><name><surname>Rodríguez-Manzaneque</surname><given-names>M. T.</given-names></name><name><surname>Ramne</surname><given-names>A.</given-names></name><name><surname>Uber</surname><given-names>G.</given-names></name><name><surname>Marchesan</surname><given-names>D.</given-names></name><etal/></person-group> (<year>2002</year>). <article-title>Functional analysis of yeast gene families involved in metabolism of vitamins B1 and B6.</article-title>\n<source><italic>Yeast</italic></source>\n<volume>19</volume>\n<fpage>1261</fpage>–<lpage>1276</lpage>. <pub-id pub-id-type=\"doi\">10.1002/yea.916</pub-id>\n<pub-id pub-id-type=\"pmid\">12271461</pub-id></mixed-citation></ref><ref id=\"B53\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Romero</surname><given-names>A. M.</given-names></name><name><surname>Ramos-Alonso</surname><given-names>L.</given-names></name><name><surname>Montellá-Manuel</surname><given-names>S.</given-names></name><name><surname>García-Martínez</surname><given-names>J.</given-names></name><name><surname>de la Torre-Ruiz</surname><given-names>M. Á</given-names></name><name><surname>Pérez-Ortín</surname><given-names>J. E.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>A genome-wide transcriptional study reveals that iron deficiency inhibits the yeast TORC1 pathway.</article-title>\n<source><italic>Biochim. Biophys. Acta</italic></source>\n<volume>1862</volume>:<issue>194414</issue>. <pub-id pub-id-type=\"doi\">10.1016/j.bbagrm.2019.194414</pub-id>\n<pub-id pub-id-type=\"pmid\">31394264</pub-id></mixed-citation></ref><ref id=\"B54\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rutherford</surname><given-names>J. C.</given-names></name><name><surname>Jaron</surname><given-names>S.</given-names></name><name><surname>Winge</surname><given-names>D. R.</given-names></name></person-group> (<year>2003</year>). <article-title>Aft1p and Aft2p mediate iron-responsive gene expression in yeast through related promoter elements.</article-title>\n<source><italic>J. Biol. Chem.</italic></source>\n<volume>278</volume>\n<fpage>27636</fpage>–<lpage>27643</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M300076200</pub-id>\n<pub-id pub-id-type=\"pmid\">12756250</pub-id></mixed-citation></ref><ref id=\"B55\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sanchez</surname><given-names>V. C.</given-names></name><name><surname>Jachak</surname><given-names>A.</given-names></name><name><surname>Hurt</surname><given-names>R. H.</given-names></name><name><surname>Kane</surname><given-names>A. B.</given-names></name></person-group> (<year>2012</year>). <article-title>Biological interactions of graphene-family nanomaterials: an interdisciplinary review.</article-title>\n<source><italic>Chem. Res. Toxicol.</italic></source>\n<volume>25</volume>\n<fpage>15</fpage>–<lpage>34</lpage>. <pub-id pub-id-type=\"doi\">10.1021/tx200339h</pub-id>\n<pub-id pub-id-type=\"pmid\">21954945</pub-id></mixed-citation></ref><ref id=\"B56\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Schurch</surname><given-names>N. J.</given-names></name><name><surname>Schofield</surname><given-names>P.</given-names></name><name><surname>Gierliński</surname><given-names>M.</given-names></name><name><surname>Cole</surname><given-names>C.</given-names></name><name><surname>Sherstnev</surname><given-names>A.</given-names></name><name><surname>Singh</surname><given-names>V.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?</article-title>\n<source><italic>RNA</italic></source>\n<volume>22</volume>\n<fpage>839</fpage>–<lpage>851</lpage>. <pub-id pub-id-type=\"doi\">10.1261/rna.053959.115</pub-id>\n<pub-id pub-id-type=\"pmid\">27022035</pub-id></mixed-citation></ref><ref id=\"B57\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shakoury-Elizeh</surname><given-names>M.</given-names></name><name><surname>Protchenko</surname><given-names>O.</given-names></name><name><surname>Berger</surname><given-names>A.</given-names></name><name><surname>Cox</surname><given-names>J.</given-names></name><name><surname>Gable</surname><given-names>K.</given-names></name><name><surname>Dunn</surname><given-names>T. M.</given-names></name><etal/></person-group> (<year>2010</year>). <article-title>Metabolic response to iron deficiency in <italic>Saccharomyces cerevisiae</italic>.</article-title>\n<source><italic>J. Biol. Chem.</italic></source>\n<volume>285</volume>\n<fpage>14823</fpage>–<lpage>14833</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M109.091710</pub-id>\n<pub-id pub-id-type=\"pmid\">20231268</pub-id></mixed-citation></ref><ref id=\"B58\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Shapira</surname><given-names>P.</given-names></name><name><surname>Gök</surname><given-names>A.</given-names></name><name><surname>Salehi</surname><given-names>F.</given-names></name></person-group> (<year>2016</year>). <article-title>Graphene enterprise: mapping innovation and business development in a strategic emerging technology.</article-title>\n<source><italic>J. Nanopart. Res.</italic></source>\n<volume>18</volume>:<issue>269</issue>\n<pub-id pub-id-type=\"doi\">10.1007/s11051-016-3572-3571</pub-id></mixed-citation></ref><ref id=\"B59\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Sinclair</surname><given-names>D. A.</given-names></name><name><surname>Hong</surname><given-names>S. P.</given-names></name><name><surname>Dawes</surname><given-names>I. W.</given-names></name></person-group> (<year>1996</year>). <article-title>Specific induction by glycine of the gene for the P-subunit of glycine decarboxylase from <italic>Saccharomyces cerevisiae</italic>.</article-title>\n<source><italic>Mol. Microbiol.</italic></source>\n<volume>19</volume>\n<fpage>611</fpage>–<lpage>623</lpage>. <pub-id pub-id-type=\"doi\">10.1046/j.1365-2958.1996.419947.x</pub-id>\n<pub-id pub-id-type=\"pmid\">8830251</pub-id></mixed-citation></ref><ref id=\"B60\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Soontorngun</surname><given-names>N.</given-names></name></person-group> (<year>2017</year>). <article-title>Reprogramming of nonfermentative metabolism by stress-responsive transcription factors in the yeast <italic>Saccharomyces cerevisiae</italic>.</article-title>\n<source><italic>Curr. Genet.</italic></source>\n<volume>63</volume>\n<fpage>1</fpage>–<lpage>7</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s00294-016-0609-z</pub-id>\n<pub-id pub-id-type=\"pmid\">27180089</pub-id></mixed-citation></ref><ref id=\"B61\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Suarez-Diez</surname><given-names>M.</given-names></name><name><surname>Porras</surname><given-names>S.</given-names></name><name><surname>Laguna-Teno</surname><given-names>F.</given-names></name><name><surname>Schaap</surname><given-names>P. J.</given-names></name><name><surname>Tamayo-Ramos</surname><given-names>J. A.</given-names></name></person-group> (<year>2020</year>). <article-title>Toxicological response of the model fungus <italic>Saccharomyces cerevisiae</italic> to different concentrations of commercial graphene nanoplatelets.</article-title>\n<source><italic>Sci. Rep.</italic></source>\n<volume>10</volume>\n<fpage>1</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.1038/s41598-020-60101-60107</pub-id><pub-id pub-id-type=\"pmid\">31913322</pub-id></mixed-citation></ref><ref id=\"B62\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Valentini</surname><given-names>L.</given-names></name><name><surname>Bittolo Bon</surname><given-names>S.</given-names></name><name><surname>Signetti</surname><given-names>S.</given-names></name><name><surname>Pugno</surname><given-names>N. M.</given-names></name></person-group> (<year>2016</year>). <article-title>Graphene-Based Bionic Composites with Multifunctional and Repairing Properties.</article-title>\n<source><italic>ACS Appl. Mater. Interfaces</italic></source>\n<volume>8</volume>\n<fpage>7607</fpage>–<lpage>7612</lpage>. <pub-id pub-id-type=\"doi\">10.1021/acsami.6b02530</pub-id>\n<pub-id pub-id-type=\"pmid\">26971362</pub-id></mixed-citation></ref><ref id=\"B63\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vardi</surname><given-names>N.</given-names></name><name><surname>Levy</surname><given-names>S.</given-names></name><name><surname>Gurvich</surname><given-names>Y.</given-names></name><name><surname>Polacheck</surname><given-names>T.</given-names></name><name><surname>Carmi</surname><given-names>M.</given-names></name><name><surname>Jaitin</surname><given-names>D.</given-names></name><etal/></person-group> (<year>2014</year>). <article-title>Sequential feedback induction stabilizes the phosphate starvation response in budding yeast.</article-title>\n<source><italic>Cell Rep.</italic></source>\n<volume>9</volume>\n<fpage>1122</fpage>–<lpage>1134</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.celrep.2014.10.002</pub-id>\n<pub-id pub-id-type=\"pmid\">25437565</pub-id></mixed-citation></ref><ref id=\"B64\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Vest</surname><given-names>K. E.</given-names></name><name><surname>Leary</surname><given-names>S. C.</given-names></name><name><surname>Winge</surname><given-names>D. R.</given-names></name><name><surname>Cobine</surname><given-names>P. A.</given-names></name></person-group> (<year>2013</year>). <article-title>Copper import into the mitochondrial matrix in <italic>Saccharomyces cerevisiae</italic> is mediated by Pic2, a mitochondrial carrier family protein.</article-title>\n<source><italic>J. Biol. Chem.</italic></source>\n<volume>288</volume>\n<fpage>23884</fpage>–<lpage>23892</lpage>. <pub-id pub-id-type=\"doi\">10.1074/jbc.M113.470674</pub-id>\n<pub-id pub-id-type=\"pmid\">23846699</pub-id></mixed-citation></ref><ref id=\"B65\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wang</surname><given-names>X.</given-names></name><name><surname>Liu</surname><given-names>C.</given-names></name><name><surname>Li</surname><given-names>H.</given-names></name><name><surname>Zhang</surname><given-names>H.</given-names></name><name><surname>Ma</surname><given-names>R.</given-names></name><name><surname>Zhang</surname><given-names>Q.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Metabonomics-assisted label-free quantitative proteomic and transcriptomic analysis reveals novel insights into the antifungal effect of graphene oxide for controlling: <italic>Fusarium graminearum</italic>.</article-title>\n<source><italic>Environ. Sci. Nano</italic></source>\n<volume>6</volume>\n<fpage>3401</fpage>–<lpage>3421</lpage>. <pub-id pub-id-type=\"doi\">10.1039/c9en00981g</pub-id></mixed-citation></ref><ref id=\"B66\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Wickham</surname><given-names>H.</given-names></name></person-group> (<year>2009</year>). <source><italic>Ggplot2: Elegant Graphics for Data Analysis.</italic></source>\n<publisher-loc>Berlin</publisher-loc>: <publisher-name>Springer</publisher-name>.</mixed-citation></ref><ref id=\"B67\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>D.</given-names></name><name><surname>Ma</surname><given-names>Y.</given-names></name><name><surname>Cao</surname><given-names>Y.</given-names></name><name><surname>Zhang</surname><given-names>T.</given-names></name></person-group> (<year>2020</year>). <article-title>Mitochondrial toxicity of nanomaterials.</article-title>\n<source><italic>Sci. Total Environ.</italic></source>\n<volume>702</volume>:<issue>134994</issue>. <pub-id pub-id-type=\"doi\">10.1016/j.scitotenv.2019.134994</pub-id>\n<pub-id pub-id-type=\"pmid\">31715400</pub-id></mixed-citation></ref><ref id=\"B68\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>G.</given-names></name><name><surname>Wang</surname><given-names>L. G.</given-names></name><name><surname>Han</surname><given-names>Y.</given-names></name><name><surname>He</surname><given-names>Q. Y.</given-names></name></person-group> (<year>2012</year>). <article-title>ClusterProfiler: an R package for comparing biological themes among gene clusters.</article-title>\n<source><italic>Omi. A J. Integr. Biol.</italic></source>\n<volume>16</volume>\n<fpage>284</fpage>–<lpage>287</lpage>. <pub-id pub-id-type=\"doi\">10.1089/omi.2011.0118</pub-id>\n<pub-id pub-id-type=\"pmid\">22455463</pub-id></mixed-citation></ref><ref id=\"B69\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>L.</given-names></name><name><surname>Guo</surname><given-names>N.</given-names></name><name><surname>Yang</surname><given-names>Y.</given-names></name><name><surname>Wu</surname><given-names>X.</given-names></name><name><surname>Meng</surname><given-names>R.</given-names></name><name><surname>Fan</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2010</year>). <article-title>Microarray analysis of p-anisaldehyde-induced transcriptome of <italic>Saccharomyces cerevisiae</italic>.</article-title>\n<source><italic>J. Ind. Microbiol. Biotechnol.</italic></source>\n<volume>37</volume>\n<fpage>313</fpage>–<lpage>322</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s10295-009-0676-y</pub-id>\n<pub-id pub-id-type=\"pmid\">20024600</pub-id></mixed-citation></ref><ref id=\"B70\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Yu</surname><given-names>Q.</given-names></name><name><surname>Zhang</surname><given-names>B.</given-names></name><name><surname>Li</surname><given-names>J.</given-names></name><name><surname>Du</surname><given-names>T.</given-names></name><name><surname>Yi</surname><given-names>X.</given-names></name><name><surname>Li</surname><given-names>M.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>Graphene oxide significantly inhibits cell growth at sublethal concentrations by causing extracellular iron deficiency.</article-title>\n<source><italic>Nanotoxicology</italic></source>\n<volume>11</volume>\n<fpage>1102</fpage>–<lpage>1114</lpage>. <pub-id pub-id-type=\"doi\">10.1080/17435390.2017.1398357</pub-id>\n<pub-id pub-id-type=\"pmid\">29119849</pub-id></mixed-citation></ref><ref id=\"B71\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhu</surname><given-names>S.</given-names></name><name><surname>Luo</surname><given-names>F.</given-names></name><name><surname>Li</surname><given-names>J.</given-names></name><name><surname>Zhu</surname><given-names>B.</given-names></name><name><surname>Wang</surname><given-names>G.-X.</given-names></name></person-group> (<year>2018</year>). <article-title>Biocompatibility assessment of single-walled carbon nanotubes using <italic>Saccharomyces cerevisiae</italic> as a model organism.</article-title>\n<source><italic>J. Nanobiotechnol.</italic></source>\n<volume>16</volume>:<issue>44</issue>\n<pub-id pub-id-type=\"doi\">10.1186/s12951-018-0370-371</pub-id></mixed-citation></ref><ref id=\"B72\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhu</surname><given-names>S.</given-names></name><name><surname>Luo</surname><given-names>F.</given-names></name><name><surname>Zhu</surname><given-names>B.</given-names></name><name><surname>Wang</surname><given-names>G.-X.</given-names></name></person-group> (<year>2017</year>). <article-title>Toxicological effects of graphene oxide on <italic>Saccharomyces cerevisiae</italic>.</article-title>\n<source><italic>Toxicol. Res.</italic></source>\n<volume>6</volume>\n<fpage>535</fpage>–<lpage>543</lpage>. <pub-id pub-id-type=\"doi\">10.1039/c7tx00103g</pub-id>\n<pub-id pub-id-type=\"pmid\">30090522</pub-id></mixed-citation></ref><ref id=\"B73\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhu</surname><given-names>S.</given-names></name><name><surname>Zhu</surname><given-names>B.</given-names></name><name><surname>Huang</surname><given-names>A.</given-names></name><name><surname>Hu</surname><given-names>Y.</given-names></name><name><surname>Wang</surname><given-names>G.</given-names></name><name><surname>Ling</surname><given-names>F.</given-names></name></person-group> (<year>2016</year>). <article-title>Toxicological effects of multi-walled carbon nanotubes on <italic>Saccharomyces cerevisiae</italic>: the uptake kinetics and mechanisms and the toxic responses.</article-title>\n<source><italic>J. Hazard. Mater.</italic></source>\n<volume>318</volume>\n<fpage>650</fpage>–<lpage>662</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jhazmat.2016.07.049</pub-id>\n<pub-id pub-id-type=\"pmid\">27475463</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
[
"<!DOCTYPE article\nPUBLIC \"-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD with MathML3 v1.2 20190208//EN\" \"JATS-archivearticle1-mathml3.dtd\">\n<article xmlns:xlink=\"http://www.w3.org/1999/xlink\" xmlns:mml=\"http://www.w3.org/1998/Math/MathML\" article-type=\"research-article\"><?properties open_access?><front><journal-meta><journal-id journal-id-type=\"nlm-ta\">Front Genet</journal-id><journal-id journal-id-type=\"iso-abbrev\">Front Genet</journal-id><journal-id journal-id-type=\"publisher-id\">Front. Genet.</journal-id><journal-title-group><journal-title>Frontiers in Genetics</journal-title></journal-title-group><issn pub-type=\"epub\">1664-8021</issn><publisher><publisher-name>Frontiers Media S.A.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type=\"pmid\">32849856</article-id><article-id pub-id-type=\"pmc\">PMC7431628</article-id><article-id pub-id-type=\"doi\">10.3389/fgene.2020.00922</article-id><article-categories><subj-group subj-group-type=\"heading\"><subject>Genetics</subject><subj-group><subject>Original Research</subject></subj-group></subj-group></article-categories><title-group><article-title>Sperm DNA Hypomethylation Proximal to Reproduction Pathway Genes in Maturing Elite Norwegian Red Bulls</article-title></title-group><contrib-group><contrib contrib-type=\"author\"><name><surname>Khezri</surname><given-names>Abdolrahman</given-names></name><xref ref-type=\"corresp\" rid=\"c001\"><sup>*</sup></xref><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/940532/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Narud</surname><given-names>Birgitte</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/959064/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Stenseth</surname><given-names>Else-Berit</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/959825/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Zeremichael</surname><given-names>Teklu Tewoldebrhan</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/959129/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Myromslien</surname><given-names>Frøydis Deinboll</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/961967/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Wilson</surname><given-names>Robert C.</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/959069/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Ahmad</surname><given-names>Rafi</given-names></name><xref ref-type=\"author-notes\" rid=\"fn002\"><sup>†</sup></xref><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/570281/overview\"/></contrib><contrib contrib-type=\"author\"><name><surname>Kommisrud</surname><given-names>Elisabeth</given-names></name><uri xlink:type=\"simple\" xlink:href=\"http://loop.frontiersin.org/people/959096/overview\"/></contrib></contrib-group><aff><institution>Department of Biotechnology, Inland Norway University of Applied Sciences</institution>, <addr-line>Hamar</addr-line>, <country>Norway</country></aff><author-notes><fn fn-type=\"edited-by\"><p>Edited by: Eveline M. Ibeagha-Awemu, Agriculture and Agri-Food Canada (AAFC), Canada</p></fn><fn fn-type=\"edited-by\"><p>Reviewed by: Shahin Eghbalsaied, Islamic Azad University, Isfahan, Iran; Jie Mei, Huazhong Agricultural University, China</p></fn><corresp id=\"c001\">*Correspondence: Abdolrahman Khezri, <email>abdolrahman.khezri@inn.no</email>; <email>khezri.vet@gmail.com</email></corresp><fn fn-type=\"other\" id=\"fn002\"><p><sup>†</sup>ORCID: Abdolrahman Khezri, <ext-link ext-link-type=\"uri\" xlink:href=\"https://orcid.org/0000-0003-1061-8229\">orcid.org/0000-0003-1061-8229</ext-link>; Rafi Ahmad, <ext-link ext-link-type=\"uri\" xlink:href=\"https://orcid.org/0000-0002-0383-7848\">orcid.org/0000-0002-0383-7848</ext-link></p></fn><fn fn-type=\"other\" id=\"fn004\"><p>This article was submitted to Livestock Genomics, a section of the journal Frontiers in Genetics</p></fn></author-notes><pub-date pub-type=\"epub\"><day>11</day><month>8</month><year>2020</year></pub-date><pub-date pub-type=\"collection\"><year>2020</year></pub-date><volume>11</volume><elocation-id>922</elocation-id><history><date date-type=\"received\"><day>27</day><month>4</month><year>2020</year></date><date date-type=\"accepted\"><day>24</day><month>7</month><year>2020</year></date></history><permissions><copyright-statement>Copyright © 2020 Khezri, Narud, Stenseth, Zeremichael, Myromslien, Wilson, Ahmad and Kommisrud.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Khezri, Narud, Stenseth, Zeremichael, Myromslien, Wilson, Ahmad and Kommisrud</copyright-holder><license xlink:href=\"http://creativecommons.org/licenses/by/4.0/\"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</license-p></license></permissions><abstract><p>Genomic selection in modern farming demands sufficient semen production in young bulls. Factors affecting semen quality and production capacity in young bulls are not well understood; DNA methylation, a complicated phenomenon in sperm cells, is one such factors. In this study, fresh and frozen-thawed semen samples from the same Norwegian Red (NR) bulls at both 14 and 17 months of age were examined for sperm chromatin integrity parameters, ATP content, viability, and motility. Furthermore, reduced representation bisulfite libraries constructed according to two protocols, the Ovation<sup>®</sup> RRBS Methyl-Seq System (Ovation method) and a previously optimized gel-free method and were sequenced to study the sperm DNA methylome in frozen-thawed semen samples. Sperm quality analyses indicated that sperm concentration, total motility and progressivity in fresh semen from 17 months old NR bulls were significantly higher compared to individuals at 14 months of age. The percentage of DNA fragmented sperm cells significantly decreased in both fresh and frozen-thawed semen samples in bulls with increasing age. Libraries from the Ovation method exhibited a greater percentage of read loss and shorter read size following trimming. Downstream analyses for reads obtained from the gel-free method revealed similar global sperm DNA methylation but differentially methylated regions (DMRs) between 14- and 17 months old NR bulls. The majority of identified DMRs were hypomethylated in 14 months old bulls. Most of the identified DMRs (69%) exhibited a less than 10% methylation difference while only 1.5% of DMRs exceeded a 25% methylation difference. Pathway analysis showed that genes annotated with DMRs having low methylation differences (less than 10%) and DMRs having between 10 and 25% methylation differences, could be associated with important hormonal signaling and sperm function relevant pathways, respectively. The current research shows that RRBS in parallel with routine sperm quality analyses could be informative in reproductive capacity of young NR bulls. Although global sperm DNA methylation levels in 14 and 17 months old NR bulls were similar, regions with low and varying levels of DNA methylation differences can be identified and linked with important sperm function and hormonal pathways.</p></abstract><kwd-group><kwd>Norwegian Red</kwd><kwd>bulls</kwd><kwd>puberty</kwd><kwd>sperm</kwd><kwd>DNA methylation</kwd><kwd>RRBS</kwd></kwd-group><funding-group><award-group><funding-source id=\"cn001\">Norges Forskningsråd<named-content content-type=\"fundref-id\">10.13039/501100005416</named-content></funding-source></award-group></funding-group><counts><fig-count count=\"6\"/><table-count count=\"3\"/><equation-count count=\"0\"/><ref-count count=\"54\"/><page-count count=\"14\"/><word-count count=\"0\"/></counts></article-meta></front><body><sec id=\"S1\"><title>Introduction</title><p>Epigenetics is a phenomenon where gene expression is regulated without any changes in DNA sequence, rather being modulated via changes in DNA methylation, histone post-translational modification, and interaction of transcriptional factors with small RNAs (<xref rid=\"B10\" ref-type=\"bibr\">Donkin and Barres, 2018</xref>). Epigenetic changes in sperm cells are even more complex compared to somatic cells for two main reasons. First, during primary phase of spermatogenesis, where germ cells develop to spermatids, DNA methylation is initially erased, becoming re-established later. Moreover, during spermiogenesis, where spermatids further differentiate to spermatozoa, the majority of histones are gradually replaced by protamines (<xref rid=\"B34\" ref-type=\"bibr\">O’Doherty and McGettigan, 2015</xref>; <xref rid=\"B30\" ref-type=\"bibr\">McSwiggin and O’Doherty, 2018</xref>). In recent years, different methods have been developed to study DNA methylation. Reduced representation bisulfite sequencing (RRBS) is an efficient and high-throughput method, allowing the study of DNA methylation profiles at single-base resolution, while experiment costs are kept low (<xref rid=\"B31\" ref-type=\"bibr\">Meissner et al., 2005</xref>). Previous studies have used RRBS to investigate DNA methylation profile in different bovine somatic tissues as well as bull sperm cells (<xref rid=\"B54\" ref-type=\"bibr\">Zhou et al., 2016</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Perrier et al., 2018</xref>).</p><p>Sexual maturation in bulls is a hormone-regulated process lasting up to 50 weeks of age (<xref rid=\"B40\" ref-type=\"bibr\">Rawlings et al., 2008</xref>). Previous research has demonstrated that semen quality is closely correlated with different environmental factors and animal age. For instance, it has been shown that sperm morphology, concentration and motility positively correlated with age in young tropical composite bulls (<xref rid=\"B14\" ref-type=\"bibr\">Fortes et al., 2012</xref>) and Austrian Simmental bulls (<xref rid=\"B15\" ref-type=\"bibr\">Fuerst-Waltl et al., 2006</xref>). Although several studies reported low methylation levels in different genomic features of bull sperm cells (<xref rid=\"B37\" ref-type=\"bibr\">Perrier et al., 2018</xref>; <xref rid=\"B53\" ref-type=\"bibr\">Zhou et al., 2018</xref>; <xref rid=\"B23\" ref-type=\"bibr\">Kiefer and Perrier, 2019</xref>), the methylation level is dynamic and recent evidence suggests that the bull sperm methylome correlates with age (<xref rid=\"B25\" ref-type=\"bibr\">Lambert et al., 2018</xref>).</p><p>Norwegian Red (NR) is a highly fertile breed with e.g., low incidence of calving difficulties and mastitis (<xref rid=\"B41\" ref-type=\"bibr\">Refsdal, 2007</xref>; <xref rid=\"B13\" ref-type=\"bibr\">Ferris et al., 2014</xref>). Historically the NR breeding program was based on progeny testing of sires. However, starting from 2016, genomic selection was implemented in the NR breeding program and top NR elite bulls are today selected based on genomic breeding values. Despite the fact that NR is widely employed for artificial insemination (AI) in Norway and a very good record of genetic information has been build up during the last 40 years, little is known about the NR sperm methylome. In addition, because of short generation intervals due to genomic selection, there is more demand hence more physiological pressure for semen production from young NR bulls. The objective of this research is to determine if sperm DNA methylome could provide additional information to age related sperm quality differences. For this purpose, sperm samples from the same NR bulls at 14 and 17 months of age were used for analyzing chromatin integrity, viability, ATP content, and motility parameters. Furthermore in order to assess the sperm DNA methylome, two different RRBS protocols including Ovation RRBS Methyl-Seq System (as a, simple, fast, and scalable solution) and a gel-free based RRBS protocol which previously was optimized to study boar sperm DNA methylome (<xref rid=\"B22\" ref-type=\"bibr\">Khezri et al., 2019</xref>), were implemented.</p></sec><sec sec-type=\"materials|methods\" id=\"S2\"><title>Materials and Methods</title><p>In the present study, the sperm quality traits including chromatin integrity parameters, ATP content, viability and motility parameters were analyzed in semen from NR elite bulls, at both 14 and 17 months of age. Furthermore, following comparison of two different protocols for constructing RRBS libraries, sperm DNA methylation in both age groups was analyzed.</p><sec id=\"S2.SS1\"><title>Semen Collection and Sample Preparation</title><p>Bulls in this study, were raised, cared for and fed standardized diet at Geno SA (Geno Breeding and AI Association, Hamar, Norway), AI station. Ejaculates were collected from nine young genomic selected NR bulls with unknown fertility at 14 and 17 months of age and processed by the breeding company Geno SA. Prior to dilution, sperm cell concentration in each ejaculate was calculated using an Accucell<sup>®</sup> spectrophotometer (IMV Technologies, L’Aigle, France). The semen was diluted in a two-step procedure using Biladyl extender (13500/0004-0006; Minitübe, GmbH, Tiefenbach, Germany). After first dilution, samples were taken for fresh semen analyses and simultaneously used for subjective quality control analysis. Ejaculates with motility above 70% and morphological abnormalities below 20% were further diluted with the glycerol-containing fraction (1:2) to a final concentration of 12 × 10<sup>6</sup> sperm cells per insemination dose, filled into 0.25 ml standard French mini straws (IMV, L’Aigle, France), and cryopreserved as previously described (<xref rid=\"B43\" ref-type=\"bibr\">Standerholen et al., 2014</xref>). Cryopreserved doses were later prepared for sperm quality analyses and DNA extraction by thawing the semen doses for 1 min in a water bath at 37°C. To minimize the influence of possible variation between straws, semen from two straws/ejaculate were pooled and mixed before analyses.</p></sec><sec id=\"S2.SS2\"><title>Sperm Quality Analyses</title><sec id=\"S2.SS2.SSS1\"><title>Sperm Chromatin Integrity Assessment</title><p>Sperm chromatin integrity assessment was performed using sperm chromatin structure assay (SCSA) (<xref rid=\"B12\" ref-type=\"bibr\">Evenson and Jost, 2000</xref>; <xref rid=\"B3\" ref-type=\"bibr\">Boe-Hansen et al., 2005</xref>). Using this assay, two different chromatin integrity parameters, including DNA fragmentation index (DFI) and high DNA stainability index (HDS), were measured.</p><p>In brief, both fresh and frozen-thawed semen samples were diluted to 2 × 10<sup>6</sup> cells/ml using TNE buffer (10 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, pH 7.4). Diluted samples were denatured for 30 s by adding an acid detergent solution (0.38 M NaCl, 80 mM HCl, 0.1% Triton-X 100, pH 1.2). Denatured samples were stained with acridine orange (AO) staining buffer (37 mM citric acid, 0.126 M Na<sub>2</sub>PO<sub>4</sub>, 1.1 mM EDTA, 0.15 M NaCl and 0.6 μg/ml of AO, pH 6.0) and were incubated for 3 min at room temperature. Two technical replicates were considered for each sample and 5000 sperm cells per replicate were analyzed using a pre-AO saturated flow cytometer equipped with a blue laser (488 nm) (Cell Lab QuantaTM SC MPL flow cytometer, Beckman Coulter, Fullerton, CA, United States). Laser stability was controlled (at the beginning of the experiment and after every fifth sample) using a bull reference sample with pre-identified DFI and by re-setting the mean green and red fluorescence signals to 425 ± 5 and 125 ± 5, respectively. Following AO staining, double- and single-stranded DNA emitted green (collected using a 525 nm band pass filter) and red fluorescence (collected using a 670 nm long pass filter), respectively. The percentage of red and green fluorescence was determined using the FCS Express 6 flow cytometry data analyzer software (<italic>Denovo</italic> Software, Los Angeles, CA, United States). Based on the ratio of red/(red + green), the DFI percentage was calculated. Furthermore, HDS sperm cells, which are considered as sperm cells with an incomplete chromatin condensation, were identified according to a high incorporation of AO into double-stranded DNA.</p></sec><sec id=\"S2.SS2.SSS2\"><title>Assessment of Sperm Viability</title><p>Sperm viability and data analysis were performed using the flow cytometry system described above and Kaluza<sup>®</sup> software, Version 2.1 (Beckman Coulter Ltd.). Frozen-thawed semen samples were diluted in SP-Talp media (105 mM NaCl, 3.1 mM KCl, 0.4 mM MgCl<sub>2</sub>, 2.0 mM CaCl<sub>2</sub>⋅2H<sub>2</sub>O, 0.3 mM NaH<sub>2</sub>PO<sub>4</sub>⋅H<sub>2</sub>O, 1 mM sodium pyruvate, 21.6 mM sodium lactate, 20 mM Hepes, 20 mM Hepes salt, 5 mM glucose, 50 μg/ml gentamycin) to a concentration of 1 × 10<sup>6</sup> sperm cells/ml. Two technical replicates were considered per sample. Sperm suspensions were stained with 0.48 μM propidium iodide (PI, Sigma-Aldrich) and incubated for 10 min prior to flow cytometric analysis. PI fluorescence was detected using a 670 nm long pass filter (FL3), and gating was performed to reveal sperm cells population (based on electronic volume) and percentages of living spermatozoa as previous described (<xref rid=\"B43\" ref-type=\"bibr\">Standerholen et al., 2014</xref>).</p></sec><sec id=\"S2.SS2.SSS3\"><title>Sperm ATP Content</title><p>The ATP content was measured in frozen-thawed semen samples using the CellTiter-Glo<sup>®</sup> Luminescent Cell Viability Assay (Promega; Madison, Wisconsin). A total volume of 60 μl of semen (3 × 10<sup>5</sup> sperm cells) was added to a white 96-well microtiter plate (NUNC<sup>®</sup>, Denmark) and mixed with 60 μl CellTiter-Glo<sup>®</sup> reagent. To induce cell lysis, the mixture was gently shaken for 2 min in a rotary shaker (IKA<sup>®</sup> MS 3 digital, United States), followed by 15 min incubation at room temperature to stabilize the luminescence. The bioluminescence signal was measured in relative luminescence units (RLU) using a FLUOstar OPTIMA multiwell plate reader (BMG LABTECH GmbH, Offenburg, Germany), equipped with MARS data analyzer software (Version 1.10, BMG LABTECH, Germany). Obtained RLU signals were converted to a corresponding ATP value in nM according to a prepared standard curve. ATP values obtained from the average of three technical replicates per sample. Then the value further corrected for the percentage of motile sperm cells before statistical analyses.</p></sec><sec id=\"S2.SS2.SSS4\"><title>Sperm Motility Analysis</title><p>Sperm motility analysis were performed using the SCA evolution CASA system (Microptic SL, Spain), equipped with a phase contrast Eclipse Ci-S/Ci-L microscope (Nikon, Japan), a BASLER Ace acA780-75 gc digital camera (Basler Vision Technologies, Ahrensburg, Germany) and Sperm class analyzer software (v 6.1.0.0). Fresh and thawed semen samples were incubated for 15 min at 37°C, and diluted (1:2) with pre-warmed PBS buffer (37°C) before analysis. A volume of 3 μl of diluted samples was loaded into the chamber of a pre-warmed (37°C) 20 μm depth Leja-4 slide (Leja products, Nieuw-Vennep, the Netherlands). Analyses were performed using two technical replicates per sample, under a 10x objective and on the pre-heated thermal stage (37°C) of the phase contrast microscope. Eight or more microscope fields with at least 800 cells per sample were analyzed. Bull sperm cells were detected based on head area (20–80 μm<sup>2</sup>) and camera setting of 45 frames per sec. The motility parameters measured were total motility, progressive motility, and hyperactive motility. In addition, other information regarding to sperm motion kinetics including curvilinear velocity (VCL, μm/s), straight-line velocity (VSL, μm/s), average path velocity (VAP, μm/s), straightness of the average path [STR (%) = VSL/VAP], linearity of the curvilinear path [LIN (%) = VSL/VCL], Wobble [WOB (%) = VAP/VCL], lateral displacement of sperm head (ALH, μm) and beat cross frequency (BCF, Hz) were obtained. Sperm cells were defined as static and progressive motile if VAP < 10 μm/s and STR > 70μm/s, respectively. Sperm cells with VCL > 80μm/s, ALH > 6.5μm and LIN < 65% were defined as sperm cells with hyperactive motility.</p></sec></sec><sec id=\"S2.SS3\"><title>RRBS Library Preparation</title><p>Prior to RRBS library construction, DNA from frozen-thawed sperm samples of seven bulls at, both 14 and 17 months of age, was extracted using a Maxwell 16 Benchtop instrument (Promega Corporation, United States) at Biobank AS, Hamar. Isolated DNA was quantified using Qubit dsDNA BR assay kit (Thermo Fisher Scientific, United States) and further diluted to 20 ng/μl in low TE media [10 mM Tris, pH 8.0 (Calbiochem, United States), 0.1 mM EDTA, pH 8.0 (Calbiochem, United States)]. Libraries for sperm DNA methylation analysis were constructed using the RRBS approach and according to two different RRBS protocols.</p><sec id=\"S2.SS3.SSS1\"><title>RRBS Library Preparation Using Ovation<sup>®</sup> RRBS Methyl-Seq System (Ovation Method)</title><p>In this method Ovation<sup>®</sup> RRBS Methyl-Seq System (NuGEN Technologies, San Carlos, CA, United States) was employed and RRBS libraries were constructed using 100 ng genomic DNA, according to the manufacturer’s protocol with slight modifications.</p><p>Briefly, genomic DNA was digested overnight at 37°C using <italic>Msp</italic>I and Taq α1 enzymes (New England Biolabs, United States). After digestion, AMPure XP beads (Beckman Coulter, United States) were added (2x) and samples were kept at room temperature for 30 min. Then by putting the samples on a side magnet, supernatant was removed and beads were washed twice with 100% EtOH. Dried beads were re-suspended in 10 μl elution buffer (Qiagen, Germany) and fragmented DNA was ligated with adapters by incubation at 25°C for 30 min followed by 70°C for 10 min. Adapter ligated fragments were final repaired at 60 and 70°C each for 10 min. The fragments were further size selected by adding 1.5x of 20% PEG 8000/2.5 M NaCl (Amresco Inc., United States) followed by incubation for 30 min at room temperature. The supernatant was removed as previously described and after washing the beads twice with 70% EtOH and drying, the beads were re-suspended in 25 μl elution buffer (Qiagen, Germany). Eluted products were subjected to bisulfite conversion using EpiTect kit (QIAGEN, Germany) following the manufacturer‘s protocol designated for DNA extracted from FFPE tissues. Bisulfite converted DNA, was amplified using 10 cycles of PCR. Amplified libraries were purified by adding 1x SPRI AMPure XP beads followed by incubation for 30 min at room temperature. Supernatant was removed, beads were washed with 70% EtOH and re-suspended in elution buffer. Eluted beads were further placed on a side magnet and purified libraries were transferred to a clean tube.</p></sec><sec id=\"S2.SS3.SSS2\"><title>RRBS Library Preparation Using a Gel-Free Multiplexed Method (Gel-Free Method)</title><p>In this method RRBS libraries were constructed using a gel-free multiplexed technique (<xref rid=\"B4\" ref-type=\"bibr\">Boyle et al., 2012</xref>), which we previously optimized it to study boar sperm DNA methylome (<xref rid=\"B22\" ref-type=\"bibr\">Khezri et al., 2019</xref>). The protocol was consisted of the following steps.</p><p>First, genomic DNA (100 ng) was digested as described in section “RRBS Library Preparation Using Ovation<sup>®</sup> RRBS Methyl-Seq System (Ovation Method).” Gap filling and A-tailing steps were carried out by adding 1 μl of Klenow fragment (New England Biolabs, United States) along with 1 μl of dNTP mixture containing 10 mM dATP, 1 mM dCTP, and 1 mM dGTP (New England Biolabs, United States) to fragmented DNA. The processed DNA was incubated for 20 min at 30°C followed by 20 min at 37°C. After incubation, fragmented DNA was size selected (300–500 bp) by adding a 2x AMPure XP beads (Beckman Coulter, United States). After incubation in room temperature for 30 min the supernatant was removed as previously described in section “RRBS Library Preparation Using Ovation<sup>®</sup> RRBS Methyl-Seq System (Ovation Method)” and beads were washed and re-suspended in 20 μl elution buffer (Qiagen, Germany). Adapter ligation was performed by adding 2 μl of NEXTflex<sup>TM</sup> Bisulfite-Seq barcodes (Bio Scientific Corporation, United States) and ligase mixture to eluted DNA followed by overnight incubation at 16°C. Adapter-ligated DNA again was size selected by adding 1.5x of 20% PEG 8000/2.5 M NaCl (Amresco Inc., United States) followed by incubation at room temperature for 30 min. The product was placed on a side magnet; supernatant was removed, beads were washed two times in 70% EtOH and were re-suspended in 25 μl elution buffer (Qiagen, Germany). Prior to fragment amplification, different PCR amplification cycles (10, 13, 16, and 19 cycles) were tested. PCR products were run on a gradient 4–20% Criterion precast polyacrylamide TBE gel (Thermo Fisher Scientific, United States). Gradient gel further stained with SybrGold (Thermo Fisher Scientific, United States) and the efficiency of protocol were evaluated based on the appearing DNA bands. Later, size selected DNA fragments were bisulfite converted and product was cleaned up according to recommended protocol in the QIAGEN EpiTect kit (<xref rid=\"B16\" ref-type=\"bibr\">Gu et al., 2011</xref>). At the last step, converted DNA, PCR amplified using 13 amplification steps (PCR Primer 1: 5′- AATGATACGGCGACCGAGATCTACAC-3′, PCR Primer 2: 5′-CAAGCAGAAGACGGCATACGAGAT-3′) and PCR product (libraries) were further cleaned up using 1x SPRI beads as described earlier for the Ovation method.</p></sec></sec><sec id=\"S2.SS4\"><title>Illumina Sequencing</title><p>Eluted RRBS libraries from both protocols were quantified using the PicoGreen dsDNA absorbance method and were sent to Norwegian Sequencing Center. Sequencing was performed using Illumina HiSeq 2500 in the paired-end (2 × 150 bp) mode.</p></sec><sec id=\"S2.SS5\"><title>Bioinformatics Analyses</title><sec id=\"S2.SS5.SSS1\"><title>Illumina Reads Quality Assessment and Trimming</title><p>The quality of paired-end Illumina reads first was assessed using fastQC software (v 0.11.8 for Linux). For reads obtained via the gel-free protocol, Illumina adapters and low-quality sequences (below 20 bp and Phred score of 30) were trimmed using Trim-galore software (v 0.4.4 for Linux) (<xref rid=\"B29\" ref-type=\"bibr\">Martin, 2011</xref>). For reads obtained via the Ovation protocol, manufacturer’s recommendations were followed for quality control and adapter trimming<sup><xref ref-type=\"fn\" rid=\"footnote1\">1</xref></sup>. Then, additional nucleotides from the 5′ ends of adapter-removed reads, were further trimmed using a NuGEN-developed ‘trimRRBSdiversityAdaptCustomers.py’ script in Python (2.7.5 for Linux).</p></sec><sec id=\"S2.SS5.SSS2\"><title>Mapping the Clean Reads With Reference Genome</title><p>Bull reference genome (<italic>bosTau 9</italic>) was downloaded from the UCSC database (<xref rid=\"B48\" ref-type=\"bibr\">UCSC, 2018</xref>) and was indexed using bismark_genome_preparation option in Bismark (v 0.19.0 for Linux) (<xref rid=\"B24\" ref-type=\"bibr\">Krueger and Andrews, 2011</xref>). After initial assessment of libraries (<xref rid=\"T2\" ref-type=\"table\">Table 2</xref>), only reads from the gel-free protocol were mapped to the reference genome. The mapping was carried out using Bismark and bowtie2 aligner (v 2.3.2 for Linux) (<xref rid=\"B24\" ref-type=\"bibr\">Krueger and Andrews, 2011</xref>) with following parameters [-n 0 −l 20 and –score-min (L, 0, −0.6)]. All covered Cs were used for calculation of global CpG methylation level in Bismark using following formula:% of global methylation = 100 <sup>∗</sup> number of methylated Cs/number of methylated Cs + number of unmethylated Cs.</p></sec><sec id=\"S2.SS5.SSS3\"><title>Differential Methylation Analysis</title><p>In this study differentially methylated regions were identified between control (17 months old bulls) and test (14 months old bulls) groups. In brief, SAM-sorted alignment files from Bismark were analyzed using the methylKit package (v 1.6.1) (<xref rid=\"B1\" ref-type=\"bibr\">Akalin et al., 2012</xref>) in Rstudio (v 1.1.453 for Linux). First, reads containing CpGs with more than 99.9th percentile coverage were filtered out. Every single C was considered to calculate differentially methylated regions (DMRs). For this purpose, the genome was divided in 1000 bp tiles with sliding step 1000 bp, containing at least three mutually covered Cs in the CpG context. Average DNA methylation of each tile was calculated and in order to determine DMRs with <italic>q-value</italic> < 0.05 (filtered DMRs onward), logistic regression with a sliding linear model to correct for multiple comparisons was applied. In this study, hypermethylation and hypomethylation are defined as any positive and negative value for DMRs in the test group compared to the control group, respectively. Later, DMRs were categorized as those with < 10% differential methylation (DMRs<sub><</sub><sub>10</sub><sub>%</sub>), those showing 10–25% (DMRs<sub>10</sub><sub>–</sub><sub>25</sub><sub>%</sub>) and those exhibiting > 25% methylation differences (DMRs<sub>></sub><sub>25</sub><sub>%</sub>) and were used for downstream analyses.</p></sec><sec id=\"S2.SS5.SSS4\"><title>Annotation of Differentially Methylated Regions</title><p>BED files containing gene and CpG annotation for the <italic>bosTau9</italic> assembly were downloaded from the UCSC table browser (<xref rid=\"B48\" ref-type=\"bibr\">UCSC, 2018</xref>). All DMRs with any level of hypo/hyper methylation were annotated with nearest (no specific cut off) transcriptional start site (TSS), genes elements (exons, introns, promoter, intergenic regions) and CpG features (CpG islands, CpG shore, other) using Genomation package (v 1.14.0) in Rstudio. Promoters and CpG shore were defined as ± 1000 bp and ± 2000 bp of the TSS and CpG islands, respectively.</p></sec><sec id=\"S2.SS5.SSS5\"><title>Pathway Analysis</title><p>Corresponding GenBank accession IDs to annotated TSSs, were submitted to DAVID Bioinformatics resources for functional annotation (<xref rid=\"B17\" ref-type=\"bibr\">Huang da et al., 2009</xref>) for Gene Ontology (GO) analysis. Gene enrichment for each identified pathway was calculated using Fisher’s exact test and <italic>p-value</italic> was Benjamini adjusted for multiple testing and set to 0.05.</p></sec></sec><sec id=\"S2.SS6\"><title>Statistical Analyses</title><p>Statistical analyses were performed in Rstudio (v 1.1.383 for windows). In order to compare sperm quality parameters in fresh and frozen-thawed samples from 14 and 17 months old bulls, linear mixed models within the lme4 package were established using quality parameters of sperm cells and bulls age as response and independent variables, respectively. In addition, animals, semen concentration at the time of semen collection and pedigree information were included as random effects. The level of significance (<italic>p-value</italic>) was set to 0.05 except for DFI and HDS where, in order to minimize type I error, <italic>p-values</italic> were Bonferroni adjusted to 0.025. Results were plotted using GraphPad Prism (v 6.01 for Windows, GraphPad Software, San Diego, CA, United States). Venn diagrams were constructed using Venny online platform (<xref rid=\"B35\" ref-type=\"bibr\">Oliveros, 2015</xref>). Pathway analysis results were plotted using ggplot2 package (v 3.1.0) in Rstudio (<xref rid=\"B50\" ref-type=\"bibr\">Wickham, 2016</xref>).</p></sec></sec><sec id=\"S3\"><title>Results</title><sec id=\"S3.SS1\"><title>Sperm Quality Analyses in Young Norwegian Red Bulls</title><p>Sperm quality analyses results showed that some of the parameters were significantly different between the 14 and 17- months old bulls (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). For instance, sperm concentration in 17 months old bulls was significantly higher compared to those 14 months old. Furthermore, both fresh and frozen-thawed samples from 14 months old bulls showed higher DFI compared to the 17 months old group. In addition, fresh sperm cells from 17 months old bulls showed significantly higher HDS (less condensed DNA) compared to those 14 months old. However, no significant changes in HDS between 14 and 17 months. of age were observed in frozen-thawed semen samples. The results further indicated that total sperm motility and progressivity in fresh semen from 17 months old bulls were significantly higher compared to 14 months old bulls. However, in frozen-thawed semen none of the sperm motility parameters was significantly different in bulls 14 months, compared to 17 months of age. Other sperm motion kinetic parameters showed no significance differences between 14 and 17 months old bulls (<xref ref-type=\"supplementary-material\" rid=\"TS1\">Supplementary Table 1</xref>).</p><table-wrap id=\"T1\" position=\"float\"><label>TABLE 1</label><caption><p>An overview of results (mean ± SEM) for different sperm quality parameters analyses for both fresh and frozen-thawed semen samples in 14 months (<italic>n</italic> = 9) and 17 months (<italic>n</italic> = 8) old Norwegian red bulls.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Fresh semen<hr/></td><td valign=\"top\" align=\"center\" colspan=\"2\" rowspan=\"1\">Frozen-thawed semen<hr/></td></tr><tr><td rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14 months</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17 months</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14 months</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17 months</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sperm concentration (10<sup>6</sup> cells/ml)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">974 ± 83.80</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1330 ± 133.40*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">DFI</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.78 ± 0.30</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.83 ± 0.20*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">2.02 ± 0.13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.64 ± 0.20*</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">HDS</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.67 ± 0.07</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.80 ± 0.10*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.64 ± 0.05</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">0.88 ± 0.15</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">ATP (nM/10<sup>6</sup> cells)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.22 ± 0.13</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">1.16 ± 0.06</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Viability (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">NA</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">62.15 ± 5.32</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">65.64 ± 2.85</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Motility (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">87.09 ± 1.51</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">98.10 ± 0.30*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">58.45 ± 5.10</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">63.47 ± 2.95</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Progressivity (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">72.90 ± 2.00</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">87.23 ± 1.00*</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">52.16 ± 5.52</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">59.26 ± 3.16</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Hyperactivity (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24.34 ± 1.83</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">24.21 ± 3.51</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10.44 ± 2.24</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.50 ± 1.47</td></tr></tbody></table><table-wrap-foot><attrib><italic><italic>Asterisks indicate a significant difference between age in fresh or frozen-thawed samples based on linear mixed model. *p < 0.05 for all parameters except for DFI and HDS, where *p < 0.025 (Bonferroni corrected). NA, data not available; DFI, DNA fragmentation index; HDS, high DNA stainability; nM, nanomolar.</italic></italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS2\"><title>Bioinformatics Analyses of RRBS Data</title><sec id=\"S3.SS2.SSS1\"><title>Comparison of RRBS Data Obtained Based on Ovation and Gel-Free Protocols</title><p><xref rid=\"T2\" ref-type=\"table\">Table 2</xref> compares the summary statistics for RRBS data obtained from two protocols. Surprisingly, no Illumina adapter contamination was detected for reverse reads in libraries constructed using Ovation method while both forward and reverse reads from RRBS libraries constructed based on gel-free method, showed Illumina adapter contamination. After quality control and trimming, 51% of reads were discarded in Ovation libraries, whereas quality control and trimming resulted in only 8% read loss in gel-free method. After trimming, reads with length < 50 bp and 100–150 bp were corresponding to 4 and 67% of all reads in libraries made according to gel-free protocol, respectively. Whereas reads with similar size in Ovation libraries were about 34 and 26% of total reads, respectively. This is particularly important, as longer reads tend to align better with reference genome in Bismark (<xref rid=\"B47\" ref-type=\"bibr\">Tran et al., 2014</xref>). Therefore, based on observed differences, we decided to conduct downstream analyses using RRBS libraries constructed based on the gel-free optimized method.</p><table-wrap id=\"T2\" position=\"float\"><label>TABLE 2</label><caption><p>An overview of summary statistic for RRBS libraries constructed based on Ovation RRBS Methyl-Seq and our previously optimized method (gel-free method).</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">RRBS protocol</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Adapter contamination</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Read loss after trimming</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Reads length (bp) distribution after trimming</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Ovation method</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Detected only in forward reads</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">51%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 50   34%</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50–99  40%</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100–150   26%</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Gel-free method</td><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Detected in both reads</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8%</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">< 50   4%</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">50–99  29%</td></tr><tr><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"justify\" rowspan=\"1\" colspan=\"1\"/><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">100–150   67%</td></tr></tbody></table><table-wrap-foot><attrib><italic><italic>Paired-end reads from both methods were quality checked using fastQC software and trimming for Ovation libraries was performed according to manufacturer’s instructions. Read loss percentage was calculated as number of reads after trimming/number of reads before trimming. Bp, base pair.</italic></italic></attrib></table-wrap-foot></table-wrap></sec><sec id=\"S3.SS2.SSS2\"><title>Basic Statistics of RRBS Libraries Constructed Based on Gel-Free Method</title><p>Using an in-house bioinformatics pipeline and after trimming the Illumina reads, 91% of reads were retrieved in libraries constructed based on the gel-free protocol. As shown in <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>, this was equal to an average of 7.7 million reads per sample, 33.1% unique mapping efficiency, 15.9x read coverage and 99.1% conversion rate. Overall, minimum variation was observed between samples from different individuals and age regarding to retrieved clean reads, mapping efficiency, global CpG methylation, and bisulfite conversion rate (<xref rid=\"T3\" ref-type=\"table\">Table 3</xref>). Furthermore, CpG statistic revealed that generated libraries in average covered 4.4 million CpG, with methylation average of 40%. Mixed models indicated that none of mapping efficiency, global CpG methylation level and conversion rate parameters were significantly different (<italic>p</italic> < 0.05) in 14 months compared to 17 months old bulls. Cluster analysis based on methylation value of CpG<sub><italic>W</italic></sub><sub>1000</sub> (i.e., CpGs that have fallen into a 1000 bp tiles across the genome) in each sample, showed that samples are distributed in two main clusters. However, within the main clusters, samples from the same individuals but different age always sub-clustered together (<xref ref-type=\"fig\" rid=\"F1\">Figure 1</xref>). Furthermore, Pearson’s correlation coefficient based on methylation value of CpG<sub><italic>W</italic></sub><sub>1000</sub> indicated a very high positive correlation between samples in term of global methylation profile (Pearson’s correlation coefficient ≥ 0.95) (<xref ref-type=\"supplementary-material\" rid=\"TS2\">Supplementary Table 2</xref>).</p><table-wrap id=\"T3\" position=\"float\"><label>TABLE 3</label><caption><p>An overview of basic statistic for RRBS libraries constructed based on the gel-free protocol.</p></caption><table frame=\"hsides\" rules=\"groups\" cellspacing=\"5\" cellpadding=\"5\"><thead><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">Sample ID</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Total reads</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Clean reads after trimming</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Read coverage (X)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Unique mapping efficiency (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Global CpG methylation (%)</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Number of covered CpGs</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">Bisulfite conversion rate (%)</td></tr></thead><tbody><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14 A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,092,814</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,424,375</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,287,779</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">17 A</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,769,052</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,037,908</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">29.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">38.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,161,727</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14 B</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,187,841</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,514,351</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.5</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">38.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3,867,873</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">17 B</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,916,740</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,242,150</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">30.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">37.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3,941,237</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14 C</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9,235,837</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,452,463</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">17.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,792,957</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">17 C</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,208,151</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,515,657</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">39.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,267,681</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14 D</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,608,751</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,932,141</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.3</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">39.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,529,706</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.3</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">17 D</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6,947,434</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6,395,163</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">12.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">32.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">39.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3,918,954</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14 E</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,745,029</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,098,249</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">14.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,230,165</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">17 E</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,179,041</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,538,949</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">15.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">34.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,384,431</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.2</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14 F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">9,561,827</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,631,458</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">18.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">33.9</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">41.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,855,729</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">99.0</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">17 F</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">11,204,211</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">10,288,338</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">21.6</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">34.8</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">41.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">5,383,672</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">98.9</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">14 G</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6,739,955</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">6,197,336</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">13.1</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">35.2</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">40.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">3,947,850</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">98.8</td></tr><tr><td valign=\"top\" align=\"left\" rowspan=\"1\" colspan=\"1\">17 G</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">8,406,205</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">7,643,950</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">16.4</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">37.0</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">42.7</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">4,819,724</td><td valign=\"top\" align=\"center\" rowspan=\"1\" colspan=\"1\">98.7</td></tr></tbody></table><table-wrap-foot><attrib><italic><italic>Libraries were constructed from sperm DNA collected from the same young Norwegian red bulls (n = 7) at two different ages. Letters A to G prefixed by 14 or 17, indicating different bulls of age 14 and 17 months, respectively. Clean reads were obtained after adapter and low-quality trimming of Illumina sequencing reads (total reads). Read coverage was calculated by number of bp in the clean reads/number of bp at in silico MspI-digested bosTau9 genome. Mapping efficiency shows the percentage of uniquely mapped clean reads with the reference genome. CpG methylation shows the percentage of global methylation in clean reads. Downstream analyses were performed based on all covered CpGs. Bisulfite conversion rate shows the proportion of Cs, which converted to uracil during bisulfite conversion process.</italic></italic></attrib></table-wrap-foot></table-wrap><fig id=\"F1\" position=\"float\"><label>FIGURE 1</label><caption><p>Hierarchical clustering of samples based on their CpG<sub><italic>W1000</italic></sub> methylation levels in sperm DNA from Norwegian red bulls. Letters A to G prefixed by 14 or 17, indicating different bulls of age 14 and 17 months, respectively.</p></caption><graphic xlink:href=\"fgene-11-00922-g001\"/></fig></sec><sec id=\"S3.SS2.SSS3\"><title>Differential Methylation Analysis</title><p>Differential methylation analysis were performed using a tile-based approach. This resulted in identification of 131,073 DMRs between test (14 months old) and control (17 months old) bulls. However, after setting the level of significance to <italic>q-value</italic> < 0.05, a total number of 6426 DMRs (filtered DMRs) were detected with varying levels of methylation ranging from 0 to 38%. Majority of filtered DMRs (60%) were found to be hypomethylated in the 14 months old group relative to the control group (<xref ref-type=\"fig\" rid=\"F2\">Figure 2A</xref>). Distribution of DMRs exhibiting varying degrees of methylation differences in hypomethylation and hypermethylation groups were similar; ∼70% of DMRs showed less than a 10% difference in methylation, and ca. 1.5% of DMRs had over a 25% difference in methylation levels (<xref ref-type=\"fig\" rid=\"F2\">Figures 2B,C</xref>).</p><fig id=\"F2\" position=\"float\"><label>FIGURE 2</label><caption><p>Numbers and levels of significant DMRs identified in the sperm DNA methylome between identical 14 months old bulls (test) and 17 months old bulls (control). <bold>(A)</bold> Total number of significant DMRs indicating that 60% are hypomethylated in the test group compared to the control group. <bold>(B,C)</bold> Different levels of hypo- and hypermethylated regions in the test group compared to the control group. Explanation of percentage ranges: DMRs<sub><</sub><sub>10%</sub> indicating regions with less than 10% difference in DNA methylation, DMRs<sub>10</sub><sub>–</sub><sub>25%</sub> indicating regions having between 10 and 25% difference in DNA methylation, DMRs<sub>></sub><sub>25%</sub> indicating regions having over 25% difference in DNA methylation.</p></caption><graphic xlink:href=\"fgene-11-00922-g002\"/></fig></sec><sec id=\"S3.SS2.SSS4\"><title>Annotation of Differentially Methylated Regions With Gene and CpG Features</title><p>In this study, all filtered DMRs with any level of methylation differences (DMRs<sub><</sub><sub>10</sub><sub>%</sub>, DMRs<sub>10</sub><sub>–</sub><sub>25</sub><sub>%</sub> and DMRs<sub>></sub><sub>25</sub><sub>%</sub>), were considered for downstream analyses. The filtered DMRs were annotated with gene and CpG features. The analyses showed that, on average, 65% of the filtered DMRs were present in the intergenic regions. Annotation of DMRs in both hypomethylation and hypermethylation groups within promoters and introns showed similar trend. For instance, DMRs<sub><</sub><sub>10</sub><sub>%</sub> and DMRs<sub>></sub><sub>25</sub><sub>%</sub> were the major groups that annotated within promoter and intron regions, respectively (<xref ref-type=\"fig\" rid=\"F3\">Figure 3A</xref>). For CpG features, on average, over 85% of filtered DMRs in both hypomethylation and hypermethylation groups were annotated within regions outside of CpG islands (CGI)/CpG shores. A majority of annotated DMRs within these external regions exhibited methylation differences exceeding 25%. Only around 15% of filtered DMRs were annotated within CGI/CpG shores and most showed less than a 10% methylation difference (<xref ref-type=\"fig\" rid=\"F3\">Figure 3B</xref>).</p><fig id=\"F3\" position=\"float\"><label>FIGURE 3</label><caption><p>An overview of the distribution of filtered DMRs in the <italic>bosTau9</italic> genome. <bold>(A)</bold> Annotation of the hypo- and hypermethylated regions with gene features. <bold>(B)</bold> Annotation of the hypo- and hypermethylated regions with CpG features. Hypo, hypomethylated regions; Hyper, hypermethylated regions; CGI, CpG island. Explanation of percentage ranges: DMRs<sub><</sub><sub>10%</sub> indicating regions with less than 10% difference in DNA methylation, DMRs<sub>10</sub><sub>–</sub><sub>25%</sub> indicating regions having between 10 and 25% difference in DNA methylation, DMRs<sub>></sub><sub>25%</sub> indicating regions having over 25% difference in DNA methylation.</p></caption><graphic xlink:href=\"fgene-11-00922-g003\"/></fig><p>Next, the nearest transcription start sites (TSSs) to filtered DMRs were extracted (<xref ref-type=\"fig\" rid=\"F4\">Figures 4A,B</xref>). This resulted in a greater number of TSSs in the hypomethylation group (2982 TSSs) compared to the hypermethylation group (2129 TSSs). However, in both hypomethylation and hypermethylation groups, a majority of TSSs were associated with DMRs<sub><</sub><sub>10</sub><sub>%</sub>, followed by DMRs<sub>10</sub><sub>–</sub><sub>25</sub><sub>%</sub> and DMRs<sub>></sub><sub>25</sub><sub>%</sub>. Furthermore, 474 TSSs associated to DMRs<sub><</sub><sub>10</sub><sub>%</sub> were annotated with both hypo- and hypermethylated regions. This number for the DMRs<sub>10</sub><sub>–</sub><sub>25</sub><sub>%</sub> was 156 TSSs and no commonly annotated TSS was found between hypo- and hypermethylated regions in the DMRs<sub>></sub><sub>25</sub><sub>%</sub> group (<xref ref-type=\"supplementary-material\" rid=\"FS1\">Supplementary Figure S1</xref> and <xref rid=\"T3\" ref-type=\"table\">Table 3</xref>).</p><fig id=\"F4\" position=\"float\"><label>FIGURE 4</label><caption><p>Annotation of the filtered DMRs with varying degrees of methylation difference to the nearest TSSs. Numbers inside the circles, showing the number of closest unique TSSs (duplicated TSSs removed) to DMRs in <bold>(A)</bold> hypomethylation and <bold>(B)</bold> hypermethylation groups. Explanation of percentage ranges: DMRs<sub><</sub><sub>10%</sub> indicating regions with less than 10% difference in DNA methylation, DMRs<sub>10</sub><sub>–</sub><sub>25%</sub> indicating regions having between 10 and 25% difference in DNA methylation, DMRs<sub>></sub><sub>25%</sub> indicating regions having over 25% difference in DNA methylation.</p></caption><graphic xlink:href=\"fgene-11-00922-g004\"/></fig></sec><sec id=\"S3.SS2.SSS5\"><title>Pathway Analysis</title><p>Genes whose TSSs were annotated with DMRs were first identified and subsequently subjected to pathway analysis. We were particularly interested in genes associated with biological processes and molecular functions related to sexual maturity such as androgen signaling, steroid hormone biosynthesis, steroid hormone receptor signaling, spermatogenesis and developmental growth. <xref ref-type=\"fig\" rid=\"F5\">Figures 5A,B</xref> show that the numbers of such genes were higher in the hypomethylation group compared to the hypermethylation group. Steroid hormone biosynthesis, identified exclusively in hypermethylation group. Moreover, genes whose TSSs were annotated with the DMRs<sub><</sub><sub>10</sub><sub>%</sub> and DMRs<sub>></sub><sub>25</sub><sub>%</sub> groups represented those functions to the greatest and least extent, respectively. In both hypo- and hypermethylation groups, functions including spermatogenesis, followed by steroid hormone receptor and energy homeostasis, were represented by the highest numbers of genes (<xref ref-type=\"supplementary-material\" rid=\"TS4\">Supplementary Table 4</xref>).</p><fig id=\"F5\" position=\"float\"><label>FIGURE 5</label><caption><p>Numbers of genes representing different molecular functions and biological processes related to sexual maturation in <bold>(A)</bold> hypomethylated (Hypo) and <bold>(B)</bold> hypermethylated (Hyper) groups and displayed according to their association with DMR-groups exhibiting varying degrees of methylation differences. Explanation of percentage ranges: DMRs<sub><</sub><sub>10%</sub> indicating regions with less than 10% difference in DNA methylation, DMRs<sub>10</sub><sub>–</sub><sub>25%</sub> indicating regions having between 10 and 25% difference in DNA methylation, DMRs<sub>></sub><sub>25%</sub> indicating regions having over 25% difference in DNA methylation.</p></caption><graphic xlink:href=\"fgene-11-00922-g005\"/></fig><p>As shown in <xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>, similar to biological processes and molecular functions, the majority of identified pathways (49 pathways) were in association with genes annotated with DMRs<sub><</sub><sub>10</sub><sub>%</sub>. Only eight pathways were linked to genes annotated with DMRs<sub>10</sub><sub>–</sub><sub>25</sub><sub>%</sub>. None of the identified pathways exhibited significant association with DMRs<sub>></sub><sub>25</sub><sub>%</sub>. Some of the hormonal pathways (gonadotropin-releasing hormone, estrogen and oxytocin signaling) and sperm function related pathways (disulfide bond and glycoprotein) were exclusively identified in the hypermethylation group of test samples (14 months old bulls). In other words, genes associated with those pathways were annotated with hypomethylated regions in more mature, 17 months old, bulls. Although the number of annotated TSSs to DMRs (with any level) was higher in hypomethylation groups compared to the hypermethylation groups (<xref ref-type=\"fig\" rid=\"F4\">Figure 4</xref>), the number of pathways represented by genes harboring those TSSs showed an opposite trend (<xref ref-type=\"fig\" rid=\"F6\">Figure 6</xref>). However, the majority of identified pathways in the DMRs<sub><</sub><sub>10</sub><sub>%</sub> hypomethylation group exhibited stronger <italic>p-values</italic> (<xref ref-type=\"fig\" rid=\"F6\">Figure 6A</xref>).</p><fig id=\"F6\" position=\"float\"><label>FIGURE 6</label><caption><p>Pathway analysis for annotated genes associated with <bold>(A)</bold> filtered DMRs with less than a 10% methylation difference and <bold>(B)</bold> filtered DMRs with a 10–25% methylation difference. No significant pathways were identified for annotated genes associated with DMRs over 25% methylation difference. GO terms are plotted in function of their Benjamini corrected <italic>p</italic>-value (<italic>x</italic>-axis) and fold enrichment (<italic>y</italic>-axis). Gene count size key shows the number of genes involved in that particular pathway. Hypo, hypomethylated regions (referring to TSSs annotated with hypomethylated regions in test group); Hyper, hypermethylated regions (referring to TSSs annotated to hypermethylated regions in test group).</p></caption><graphic xlink:href=\"fgene-11-00922-g006\"/></fig></sec></sec></sec><sec id=\"S4\"><title>Discussion</title><p>In this study, sperm quality was assessed in 14 and 17 months old NR bulls. Furthermore, DNA methylation patterns were elucidated in sperm cells from the same individuals using RRBS data generated from comparative library construction where two protocols were tested.</p><p>Our results showed that the number of sperm cells in ejaculates from NR bulls significantly increased with aging. These results support previous findings where total sperm count was higher in post-pubertal Holstein bulls compared to 4 months younger bulls (<xref rid=\"B9\" ref-type=\"bibr\">Devkota et al., 2008</xref>; <xref rid=\"B52\" ref-type=\"bibr\">Wu et al., 2020</xref>). Sperm DFI results (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>) indicate that DNA integrity also improves with aging. This can be explained by ongoing sperm nucleus development from 14 to 17 months of age as it has been reported that sperm DNA is more compact in older bulls rendering it less prone to fragmentation (<xref rid=\"B2\" ref-type=\"bibr\">Andrabi, 2007</xref>). In addition, sperm DFI was reduced in frozen-thawed semen samples compared to fresh samples in both age groups. It seems possible that sperm cells with higher DFI in fresh semen may not tolerate the cryopreservation process and became degraded during cryopreservation and thawing, hence not falling into the gate defined as sperm cells based on electronic volume in the flowcytometry analysis. However, the observed differences are small and less likely to be of biological importance. The HDS results presented here indicate that the degree of sperm chromatin compactness was reduced in fresh NR ejaculates at 17 compared to 14 months of age. This must, however, be further investigated using a larger number of samples. Furthermore, both total sperm motility and sperm progressivity were significantly increased in fresh semen from bulls of age 17 compared to 14 months. This trend was also observed for frozen-thawed samples, although the changes were not significant. These results are in agreement with observations from previous research where sperm cell motility in fresh samples were positively correlated with increasing age in Holstein bulls (<xref rid=\"B9\" ref-type=\"bibr\">Devkota et al., 2008</xref>; <xref rid=\"B32\" ref-type=\"bibr\">Murphy et al., 2018</xref>) and Nelore bulls (<xref rid=\"B42\" ref-type=\"bibr\">Reis et al., 2016</xref>). Overall, the results from sperm quality analyses show that sperm cells from NR bulls at 17 months of age displayed higher sperm quality compared to 14 months of age. Although sexual maturation lasting up to 50 weeks of age (<xref rid=\"B40\" ref-type=\"bibr\">Rawlings et al., 2008</xref>), previous evidences have documented that bull sperm quality increased even after puberty (<xref rid=\"B5\" ref-type=\"bibr\">Brito et al., 2004</xref>). Therefore, these results are likely to be related to the well-known sexual maturation process.</p><p>Read quality control is an important initial step in next generation sequencing data processing. The Trim-galore software has been recommended for trimming the low-quality reads in RRBS libraries (<xref rid=\"B51\" ref-type=\"bibr\">Wreczycka et al., 2017</xref>). Although RRBS libraries from the Ovation method were constructed, evaluated and trimmed according to criteria recommended by the manufacturer, surprisingly we did not detect any Illumina adapter sequences in the reverse reads of libraries. This observation might partially be explained by the sequencing technique and source of DNA. Although according to manufacture recommendation, Ovation method is compatible with paired-end sequencing, previous results employing this method, sequenced the libraries in a single-end mode, and not paired-end, mode (<xref rid=\"B38\" ref-type=\"bibr\">Pilsner et al., 2018</xref>; <xref rid=\"B6\" ref-type=\"bibr\">Chen et al., 2019</xref>; <xref rid=\"B36\" ref-type=\"bibr\">Paul et al., 2019</xref>; <xref rid=\"B39\" ref-type=\"bibr\">Rashid et al., 2020</xref>). The same studies applied this method to study DNA methylome in rat, mice and humans brain/sperm cells while, according to manufacturer’s recommendation, the Ovation method is designed to generate RRBS libraries from human genomic DNA (<xref rid=\"B33\" ref-type=\"bibr\">NuGEN, 2020</xref>). Length distribution after trimming also revealed that libraries constructed using the Ovation method had several peaks reflecting different fragment sizes specifically of short length, whereas reads from the gel-free method revealed only one major peak of fragments greater than 130 base pairs long. This is an important factor as it has been shown that longer reads align better and more specifically to a reference genome (<xref rid=\"B47\" ref-type=\"bibr\">Tran et al., 2014</xref>). To our knowledge, this was the first time the Ovation<sup>®</sup> RRBS Methyl-Seq system has been applied to study DNA methylation in bull sperm. However, further work is required to successfully adopt Ovation system for studying the bull sperm DNA methylome.</p><p>Basic statistics of sequencing results from the RRBS libraries constructed using the gel-free protocol indicated very consistent and reproducible bisulfite conversion between samples. The average conversion rate of 99.1% is higher and equal to previously published whole genome bisulfite sequencing (WGBS) (<xref rid=\"B11\" ref-type=\"bibr\">Duan et al., 2019</xref>) and RRBS results (<xref rid=\"B20\" ref-type=\"bibr\">Jiang et al., 2018</xref>) for bull sperm cells, respectively. Although in this study, the <italic>bosTau9</italic> genome was used as the reference genome and some relaxed alignment parameters were applied, the average mapping efficiency of 33.1% was much higher than previously reported results for RRBS libraries in bull sperm cells (<xref rid=\"B20\" ref-type=\"bibr\">Jiang et al., 2018</xref>).</p><p>Results further indicated a 40% global DNA methylation level in NR bull sperm cells. Previous studies showed that, in general, global DNA methylation level is low in bull sperm cells. For instance, using a luminometric methylation assay an average of 45% (<xref rid=\"B37\" ref-type=\"bibr\">Perrier et al., 2018</xref>) and using RRBS, an average of 35% (<xref rid=\"B20\" ref-type=\"bibr\">Jiang et al., 2018</xref>) global CpG methylation in bull sperm cells has been reported. Similarly, low global CpG methylation was also reported for ten different cattle tissues using RRBS (<xref rid=\"B54\" ref-type=\"bibr\">Zhou et al., 2016</xref>). Previously, we reported an average of 33% of global methylation in boar sperm cells using a gel-free RRBS technique (<xref rid=\"B22\" ref-type=\"bibr\">Khezri et al., 2019</xref>). However, it has been shown that global sperm DNA methylation in bulls can reach 75% as documented using WGBS (<xref rid=\"B53\" ref-type=\"bibr\">Zhou et al., 2018</xref>). One should keep in mind that the applied RRBS method in this study focuses on a small subset (CpG island) of the compact sperm genome where methylation levels are generally low (<xref rid=\"B44\" ref-type=\"bibr\">Suzuki and Bird, 2008</xref>). In addition, differences in global bull sperm DNA methylation described in the literature might be explained by different laboratory techniques, instrumental platforms, bioinformatics workflows, reference genome versions utilized for read alignment and interspecies differences in sperm DNA methylation patterns.</p><p>Here, no significant associations between sperm global CpG methylation and age were found. These findings are further supported by Pearson correlation and cluster analyses, where a high positive correlation between samples from both age groups was observed. In addition, samples from both age groups of the same individuals always clustered together, which suggests that, in this study, individual effects on global sperm DNA methylation are probably more pronounced than age effects. Considering uniform condition and environment for raising and feeding the bulls, it is least likely that individual differences in DNA methylation here is driven by environmental factors. However, in addition to environmental factors, it has been shown that individual differences in sperm DNA methylation may be explained by epigenetic polymorphism phenomenon and interindividual genetic diversity (<xref rid=\"B23\" ref-type=\"bibr\">Kiefer and Perrier, 2019</xref>). In agreement with global sperm DNA methylome results presented here, previous research reported that DNA methylation levels in bull sperm is dynamic during puberty, becoming stable after the age of 1 year (<xref rid=\"B25\" ref-type=\"bibr\">Lambert et al., 2018</xref>). In parallel with global methylation analysis, differential methylation analysis, showed an increasing trend of DNA methylation in the control group (sperm DNA from 17 months old bulls) compared to test group (sperm DNA from 14 months old bulls). Although, 70% of identified differentially methylated regions, displayed less than 10% methylation difference, we believe that this further highlights the possibility of an existing relationship between differentially methylated regions and sexual maturation in NR bulls. This hypothesis is supported by previous studies in Holstein bulls where more methylated regions were found in sperm cells from 16 months bulls compared to 10 months bulls (<xref rid=\"B25\" ref-type=\"bibr\">Lambert et al., 2018</xref>). Similar findings were reported in one Japanese black bull (at 14, 19, 28, 54, and 162 months of age), where authors identified eight CpGs that exhibited an age-dependent increase in their methylation levels (<xref rid=\"B45\" ref-type=\"bibr\">Takeda et al., 2019</xref>).</p><p>The distribution of DMRs demonstrated here showed that the majority lay within intergenic regions and regions outside CpG Islands/CpG shores. Similar trends have been reported in boar (<xref rid=\"B19\" ref-type=\"bibr\">Hwang et al., 2017</xref>; <xref rid=\"B22\" ref-type=\"bibr\">Khezri et al., 2019</xref>) and bull (<xref rid=\"B20\" ref-type=\"bibr\">Jiang et al., 2018</xref>; <xref rid=\"B37\" ref-type=\"bibr\">Perrier et al., 2018</xref>) sperm DNA. Previous research has shown that CpG Islands and CpG shores, in parallel with promoters, play an important role in regulation of transcription (<xref rid=\"B8\" ref-type=\"bibr\">Deaton and Bird, 2011</xref>; <xref rid=\"B28\" ref-type=\"bibr\">Long et al., 2017</xref>). Although only a small percentage of DMRs were annotated with CpG Island/CpG shores and promoters here, the majority of annotated DMRs exhibited less than a 10% methylation difference. This further suggests similar DNA methylation profiles in these regions in sperm samples from NR bulls at age 14 and 17 months.</p><p>GO analysis results for the DMR<sub>></sub><sub>25</sub><sub>%</sub> group further showed that molecular functions/biological processes such as energy homeostasis, developmental growth and androgen signaling could be driven by Cytochrome B5 Reductase 4 (<italic>CYB5R4</italic>), Phospholipase C Beta 1 (<italic>PLCB1</italic>) and NK3 homeobox 1 (<italic>NKX3-1</italic>) genes, respectively. However, these genes are not specific for reproduction or sexual maturation. For instance, previous research demonstrated that the <italic>CYB5R4</italic> gene could be consider as one of the candidate gene for quantitative trait locus studies for the oleic acid percentage in Japanese Black cattle (<xref rid=\"B21\" ref-type=\"bibr\">Kawaguchi et al., 2019</xref>). In other research, the <italic>PLCB1</italic> gene was identified in oxidative stress response and heat tolerance in Dehong humped cattle (<xref rid=\"B27\" ref-type=\"bibr\">Li et al., 2020</xref>). Furthermore, the transcription factor <italic>NKX3-1</italic> was proposed as a possible regulator of gene expression in the endometrium of cattle who received n-3 polyunsaturated fatty acid as a feed supplement (<xref rid=\"B49\" ref-type=\"bibr\">Waters et al., 2014</xref>). In addition, steroid hormone biosynthesis was the only biological process that was exclusively identified in the DMR<sub><</sub><sub>10</sub><sub>%</sub> hypomethylation group. Several genes also identified, such as cytochrome P450 superfamily members (<italic>CYP1B1, CYP11A1, and CYP2E1</italic>), steroid 5 alpha-reductase 2 (<italic>SRD5A2</italic>) and steroid sulfatase (<italic>STS</italic>) are annotated to be involve in steroid hormone biosynthesis. Given their annotated molecular functions/associated biological processes, genes identified in this study may contribute to age-dependent reproductive capacity in NR bulls.</p><p>Our analyses did not show any significant pathways connected to genes annotated with DMR<sub>></sub><sub>25</sub><sub>%</sub>. These findings are in line with previous research from Holstein bulls, where no significant DMR-associated pathways were found in sperm samples collected at 12 and 16 months of age (<xref rid=\"B25\" ref-type=\"bibr\">Lambert et al., 2018</xref>). For the DMR<sub>10</sub><sub>–</sub><sub>25</sub><sub>%</sub> group, a total number of eight significant pathways including sperm-relevant pathways such as “disulfide bond” and “glycoprotein” in 14 months old NR bulls were identified. “Disulfide bond” was exclusively identified in the hypermethylation group. It has been shown that disulfide bonds are essential for protamine function and DNA packaging in bull sperm chromatin (<xref rid=\"B18\" ref-type=\"bibr\">Hutchison et al., 2017</xref>). Although the number of bulls was limited here, fresh semen samples from 14 months old NR bulls exhibited higher degree of chromatin compaction compared to 17 months old bulls (<xref rid=\"T1\" ref-type=\"table\">Table 1</xref>). These results suggest a possible link between sperm DNA hypermethylation and DNA packaging via protamine function. Similar possible contribution of DNA methylation to nucleosome rigidity via histone function, has previously been suggested in human somatic cells (<xref rid=\"B7\" ref-type=\"bibr\">Choy et al., 2010</xref>; <xref rid=\"B26\" ref-type=\"bibr\">Lee and Lee, 2012</xref>). Furthermore, the pathway “glycoprotein” was identified in both hypo and hyper DMR<sub>10</sub><sub>–</sub><sub>25</sub><sub>%</sub> with a stronger <italic>p-values</italic> in the hypermethylation group. “Glycoproteins” have been identified in the sperm plasma membrane and play an important role in mammalian fertilization (<xref rid=\"B46\" ref-type=\"bibr\">Tecle and Gagneux, 2015</xref>). Further research is required to shed light on compositions of sperm glycoproteins during bull sexual maturation. The highest numbers of identified pathways with significant <italic>p-values</italic> were found to be related to genes annotated with DMR<sub><</sub><sub>10</sub><sub>%.</sub> In the study conducted by <xref rid=\"B25\" ref-type=\"bibr\">Lambert et al. (2018)</xref>, identified DMRs in sperm cells from bulls at 10 and 16 months of age were associated with pathways related to sperm function, including androgen hormone signaling. Here, we identified other hormonal pathways such as GnRH, estrogen and oxytocin signaling pathways, which were exclusively related to DMR<sub><</sub><sub>10</sub><sub>%</sub>. This further emphasizes the importance of hormonal signaling in development and sexual maturation. However, pathway analysis results need to be interpreted with caution for two main reasons. First, it has been recommended to avoid using differential DNA methylation level cut off percentages less than 5% in DMR-analysis due to the minimal effects on gene expression they exercise (<xref rid=\"B51\" ref-type=\"bibr\">Wreczycka et al., 2017</xref>). Second, a moderate number of genes annotated with DMRs overlapped between hypo- and hypermethylation groups. How transcriptional regulation can be exerted via TSSs proximal to both hypo- and hypermethylated regions is not clear, especially in sperm cells that are relatively transcriptionally silent. Therefore, further research using transcriptome analysis of <italic>in vitro</italic> produced embryos, fertilized with sperm cells from wider age groups of young NR bulls is recommended.</p></sec><sec id=\"S5\"><title>Conclusion</title><p>The purpose of the present research was to study the sperm DNA methylome, in parallel with sperm quality assessment, in similar NR bulls both at 14 and 17 months of age. Although the number of tested bulls were limited, the present study found that with increasing age of young bulls, sperm quality increased. Furthermore, a gel-free, multiplexed method to construct RRBS libraries from frozen-thawed bull sperm cells was found to be reproducible. The current results showed that sperm DNA methylation in 14- and 17-months-old NR bulls was similar globally, while marginally different regionally. Taken all together, identified DMRs even with low levels of methylation differences, in parallel with sperm quality results, offers some useful insight into the reproductive capacity of genomic selected young NR bulls.</p></sec><sec sec-type=\"data-availability\" id=\"S6\"><title>Data Availability Statement</title><p>The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.ebi.ac.uk/ena\">https://www.ebi.ac.uk/ena</ext-link>, <ext-link ext-link-type=\"DDBJ/EMBL/GenBank\" xlink:href=\"PRJEB37763\">PRJEB37763</ext-link>.</p></sec><sec id=\"S7\"><title>Ethics Statement</title><p>Ethical review and approval was not required for the animal study because sperm cells that we used in this research routinely collected from bulls owned by breeding company Geno in Norway. However, the bulls were housed and cared for according to international guidelines and regulations for keeping bulls in Norway, at Geno artificial insemination (AI) station, in Hamar, Norway.</p></sec><sec id=\"S8\"><title>Author Contributions</title><p>AK performed the sperm motility assay, bioinformatics and biostatistics analyses with inputs from RA as well as RW and wrote the manuscript. BN, E-BS, and TZ performed and drafted the viability assay, sperm chromatin integrity analysis and ATP content assay, respectively. E-BS prepared RRBS libraries with inputs from RW. FM and EK did conceptualization and original project draft. All authors were involved in the planning of the experiments and provided useful inputs, interpreted the data, read, edited, and approved the manuscript.</p></sec><sec id=\"conf1\"><title>Conflict of Interest</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></body><back><fn-group><fn fn-type=\"financial-disclosure\"><p><bold>Funding.</bold> The Research Council of Norway (Project No. 268048) and Regional Research Fund Inland, Norway (Project No. 257606), funded this research work. The funding bodies played no role in the design of the study and collection, analyses, and interpretation of data and in writing the manuscript.</p></fn></fn-group><ack><p>We kindly thank personnel at Geno AI station for collecting, evaluating, and processing the semen samples. We would also like to thank Janeth Mbuma for the sperm quality analyses she performed on a sub-set of samples. The Norwegian Sequencing Centre (www.sequencing.uio.no), a national technology platform hosted by Oslo University Hospital and the University of Oslo, supported by the Research Council of Norway and the Southeastern Regional Health Authority, provided the sequencing service.</p></ack><fn-group><fn id=\"footnote1\"><label>1</label><p><ext-link ext-link-type=\"uri\" xlink:href=\"https://github.com/nugentechnologies/NuMetRRBS\">https://github.com/nugentechnologies/NuMetRRBS</ext-link></p></fn></fn-group><sec id=\"S11\" sec-type=\"supplementary material\"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://www.frontiersin.org/articles/10.3389/fgene.2020.00922/full#supplementary-material\">https://www.frontiersin.org/articles/10.3389/fgene.2020.00922/full#supplementary-material</ext-link></p><supplementary-material content-type=\"local-data\" id=\"SM1\"><media xlink:href=\"Data_Sheet_1.DOCX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"FS1\"><media xlink:href=\"Presentation_1.PPTX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS1\"><media xlink:href=\"Table_1.XLSX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS2\"><media xlink:href=\"Table_2.XLSX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS3\"><media xlink:href=\"Table_3.XLSX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type=\"local-data\" id=\"TS4\"><media xlink:href=\"Table_4.XLSX\"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><title>References</title><ref id=\"B1\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Akalin</surname><given-names>A.</given-names></name><name><surname>Kormaksson</surname><given-names>M.</given-names></name><name><surname>Li</surname><given-names>S.</given-names></name><name><surname>Garrett-Bakelman</surname><given-names>F. E.</given-names></name><name><surname>Figueroa</surname><given-names>M. E.</given-names></name><name><surname>Melnick</surname><given-names>A.</given-names></name><etal/></person-group> (<year>2012</year>). <article-title>methylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles.</article-title>\n<source><italic>Genome Biol.</italic></source>\n<volume>13</volume>:<issue>R87</issue>. <pub-id pub-id-type=\"doi\">10.1186/gb-2012-13-10-r87</pub-id>\n<pub-id pub-id-type=\"pmid\">23034086</pub-id></mixed-citation></ref><ref id=\"B2\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Andrabi</surname><given-names>S. M. H.</given-names></name></person-group> (<year>2007</year>). <article-title>Mammalian sperm chromatin structure and assessment of DNA fragmentation.</article-title>\n<source><italic>J. Assist. Reprod. Genet.</italic></source>\n<volume>24</volume>\n<fpage>561</fpage>–<lpage>569</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s10815-007-9177-y</pub-id>\n<pub-id pub-id-type=\"pmid\">18008155</pub-id></mixed-citation></ref><ref id=\"B3\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boe-Hansen</surname><given-names>G. B.</given-names></name><name><surname>Ersboll</surname><given-names>A. K.</given-names></name><name><surname>Greve</surname><given-names>T.</given-names></name><name><surname>Christensen</surname><given-names>P.</given-names></name></person-group> (<year>2005</year>). <article-title>Increasing storage time of extended boar semen reduces sperm DNA integrity.</article-title>\n<source><italic>Theriogenology</italic></source>\n<volume>63</volume>\n<fpage>2006</fpage>–<lpage>2019</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.theriogenology.2004.09.006</pub-id>\n<pub-id pub-id-type=\"pmid\">15823356</pub-id></mixed-citation></ref><ref id=\"B4\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Boyle</surname><given-names>P.</given-names></name><name><surname>Clement</surname><given-names>K.</given-names></name><name><surname>Gu</surname><given-names>H.</given-names></name><name><surname>Smith</surname><given-names>Z. D.</given-names></name><name><surname>Ziller</surname><given-names>M.</given-names></name><name><surname>Fostel</surname><given-names>J. L.</given-names></name><etal/></person-group> (<year>2012</year>). <article-title>Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling.</article-title>\n<source><italic>Genome Biol.</italic></source>\n<volume>13</volume>:<issue>R92</issue>. <pub-id pub-id-type=\"doi\">10.1186/gb-2012-13-10-r92</pub-id>\n<pub-id pub-id-type=\"pmid\">23034176</pub-id></mixed-citation></ref><ref id=\"B5\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Brito</surname><given-names>L. F.</given-names></name><name><surname>Silva</surname><given-names>A. E.</given-names></name><name><surname>Unanian</surname><given-names>M. M.</given-names></name><name><surname>Dode</surname><given-names>M. A.</given-names></name><name><surname>Barbosa</surname><given-names>R. T.</given-names></name><name><surname>Kastelic</surname><given-names>J. P.</given-names></name></person-group> (<year>2004</year>). <article-title>Sexual development in early- and late-maturing Bos indicus and Bos indicus x <italic>Bos taurus</italic> crossbred bulls in Brazil.</article-title>\n<source><italic>Theriogenology</italic></source>\n<volume>62</volume>\n<fpage>1198</fpage>–<lpage>1217</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.theriogenology.2004.01.006</pub-id>\n<pub-id pub-id-type=\"pmid\">15325547</pub-id></mixed-citation></ref><ref id=\"B6\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Chen</surname><given-names>X.</given-names></name><name><surname>Zhang</surname><given-names>L.</given-names></name><name><surname>Wang</surname><given-names>C.</given-names></name></person-group> (<year>2019</year>). <article-title>Prenatal hypoxia-induced epigenomic and transcriptomic reprogramming in rat fetal and adult offspring hearts.</article-title>\n<source><italic>Sci. Data</italic></source>\n<volume>6</volume>:<issue>238</issue>. <pub-id pub-id-type=\"doi\">10.1038/s41597-019-0253-9</pub-id>\n<pub-id pub-id-type=\"pmid\">31664036</pub-id></mixed-citation></ref><ref id=\"B7\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Choy</surname><given-names>J. S.</given-names></name><name><surname>Wei</surname><given-names>S.</given-names></name><name><surname>Lee</surname><given-names>J. Y.</given-names></name><name><surname>Tan</surname><given-names>S.</given-names></name><name><surname>Chu</surname><given-names>S.</given-names></name><name><surname>Lee</surname><given-names>T.-H.</given-names></name></person-group> (<year>2010</year>). <article-title>DNA methylation increases nucleosome compaction and rigidity.</article-title>\n<source><italic>J. Am. Chem. Soc.</italic></source>\n<volume>132</volume>\n<fpage>1782</fpage>–<lpage>1783</lpage>. <pub-id pub-id-type=\"doi\">10.1021/ja910264z</pub-id>\n<pub-id pub-id-type=\"pmid\">20095602</pub-id></mixed-citation></ref><ref id=\"B8\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Deaton</surname><given-names>A. M.</given-names></name><name><surname>Bird</surname><given-names>A.</given-names></name></person-group> (<year>2011</year>). <article-title>CpG islands and the regulation of transcription.</article-title>\n<source><italic>Genes Dev.</italic></source>\n<volume>25</volume>\n<fpage>1010</fpage>–<lpage>1022</lpage>. <pub-id pub-id-type=\"doi\">10.1101/gad.2037511</pub-id>\n<pub-id pub-id-type=\"pmid\">21576262</pub-id></mixed-citation></ref><ref id=\"B9\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Devkota</surname><given-names>B.</given-names></name><name><surname>Koseki</surname><given-names>T.</given-names></name><name><surname>Matsui</surname><given-names>M.</given-names></name><name><surname>Sasaki</surname><given-names>M.</given-names></name><name><surname>Kaneko</surname><given-names>E.</given-names></name><name><surname>Miyamoto</surname><given-names>A.</given-names></name><etal/></person-group> (<year>2008</year>). <article-title>Relationships Among Age, Body Weight, scrotal circumference, semen quality and peripheral testosterone and estradiol concentrations in pubertal and postpubertal holstein bulls.</article-title>\n<source><italic>J. Vet. Med. Sci.</italic></source>\n<volume>70</volume>\n<fpage>119</fpage>–<lpage>121</lpage>. <pub-id pub-id-type=\"doi\">10.1292/jvms.70.119</pub-id>\n<pub-id pub-id-type=\"pmid\">18250585</pub-id></mixed-citation></ref><ref id=\"B10\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Donkin</surname><given-names>I.</given-names></name><name><surname>Barres</surname><given-names>R.</given-names></name></person-group> (<year>2018</year>). <article-title>Sperm epigenetics and influence of environmental factors.</article-title>\n<source><italic>Mo.l Metab.</italic></source>\n<volume>14</volume>\n<fpage>1</fpage>–<lpage>11</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.molmet.2018.02.006</pub-id>\n<pub-id pub-id-type=\"pmid\">29525406</pub-id></mixed-citation></ref><ref id=\"B11\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Duan</surname><given-names>J. E.</given-names></name><name><surname>Jiang</surname><given-names>Z. C.</given-names></name><name><surname>Alqahtani</surname><given-names>F.</given-names></name><name><surname>Mandoiu</surname><given-names>I.</given-names></name><name><surname>Dong</surname><given-names>H.</given-names></name><name><surname>Zheng</surname><given-names>X.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Methylome dynamics of bovine gametes and in vivo early embryos.</article-title>\n<source><italic>Front. Genet.</italic></source>\n<volume>10</volume>:<issue>512</issue>. <pub-id pub-id-type=\"doi\">10.3389/fgene.2019.00512</pub-id>\n<pub-id pub-id-type=\"pmid\">31191619</pub-id></mixed-citation></ref><ref id=\"B12\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Evenson</surname><given-names>D.</given-names></name><name><surname>Jost</surname><given-names>L.</given-names></name></person-group> (<year>2000</year>). <article-title>Sperm chromatin structure assay is useful for fertility assessment.</article-title>\n<source><italic>Methods Cell Sci.</italic></source>\n<volume>22</volume>\n<fpage>169</fpage>–<lpage>189</lpage>. <pub-id pub-id-type=\"doi\">10.1023/a:1009844109023</pub-id><pub-id pub-id-type=\"pmid\">11264952</pub-id></mixed-citation></ref><ref id=\"B13\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Ferris</surname><given-names>C. P.</given-names></name><name><surname>Patterson</surname><given-names>D. C.</given-names></name><name><surname>Gordon</surname><given-names>F. J.</given-names></name><name><surname>Watson</surname><given-names>S.</given-names></name><name><surname>Kilpatrick</surname><given-names>D. J.</given-names></name></person-group> (<year>2014</year>). <article-title>Calving traits, milk production, body condition, fertility, and survival of holstein-friesian and norwegian red dairy cattle on commercial dairy farms over 5 lactations.</article-title>\n<source><italic>J. Dairy Sci.</italic></source>\n<volume>97</volume>\n<fpage>5206</fpage>–<lpage>5218</lpage>. <pub-id pub-id-type=\"doi\">10.3168/jds.2013-7457</pub-id>\n<pub-id pub-id-type=\"pmid\">24952782</pub-id></mixed-citation></ref><ref id=\"B14\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fortes</surname><given-names>M. R. S.</given-names></name><name><surname>Holroyd</surname><given-names>R. G.</given-names></name><name><surname>Reverter</surname><given-names>A.</given-names></name><name><surname>Venus</surname><given-names>B. K.</given-names></name><name><surname>Satake</surname><given-names>N.</given-names></name><name><surname>Boe-Hansen</surname><given-names>G. B.</given-names></name></person-group> (<year>2012</year>). <article-title>The integrity of sperm chromatin in young tropical composite bulls.</article-title>\n<source><italic>Theriogenology</italic></source>\n<volume>78</volume>\n<fpage>326</fpage>–<lpage>333, 333.e1–.e4</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.theriogenology.2012.02.007</pub-id>\n<pub-id pub-id-type=\"pmid\">22494672</pub-id></mixed-citation></ref><ref id=\"B15\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Fuerst-Waltl</surname><given-names>B.</given-names></name><name><surname>Schwarzenbacher</surname><given-names>H.</given-names></name><name><surname>Perner</surname><given-names>C.</given-names></name><name><surname>Sölkner</surname><given-names>J.</given-names></name></person-group> (<year>2006</year>). <article-title>Effects of age and environmental factors on semen production and semen quality of Austrian Simmental bulls.</article-title>\n<source><italic>Anim. Reprod. Sci.</italic></source>\n<volume>95</volume>\n<fpage>27</fpage>–<lpage>37</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.anireprosci.2005.09.002</pub-id>\n<pub-id pub-id-type=\"pmid\">16207516</pub-id></mixed-citation></ref><ref id=\"B16\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Gu</surname><given-names>H.</given-names></name><name><surname>Smith</surname><given-names>Z. D.</given-names></name><name><surname>Bock</surname><given-names>C.</given-names></name><name><surname>Boyle</surname><given-names>P.</given-names></name><name><surname>Gnirke</surname><given-names>A.</given-names></name><name><surname>Meissner</surname><given-names>A.</given-names></name></person-group> (<year>2011</year>). <article-title>Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling.</article-title>\n<source><italic>Nat. Protoc.</italic></source>\n<volume>6</volume>\n<fpage>468</fpage>–<lpage>481</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nprot.2010.190</pub-id>\n<pub-id pub-id-type=\"pmid\">21412275</pub-id></mixed-citation></ref><ref id=\"B17\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Huang da</surname><given-names>W.</given-names></name><name><surname>Sherman</surname><given-names>B. T.</given-names></name><name><surname>Lempicki</surname><given-names>R. A.</given-names></name></person-group> (<year>2009</year>). <article-title>Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.</article-title>\n<source><italic>Nat. Protoc.</italic></source>\n<volume>4</volume>\n<fpage>44</fpage>–<lpage>57</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nprot.2008.211</pub-id>\n<pub-id pub-id-type=\"pmid\">19131956</pub-id></mixed-citation></ref><ref id=\"B18\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hutchison</surname><given-names>J. M.</given-names></name><name><surname>Rau</surname><given-names>D. C.</given-names></name><name><surname>DeRouchey</surname><given-names>J. E.</given-names></name></person-group> (<year>2017</year>). <article-title>Role of disulfide bonds on DNA packaging forces in bull sperm chromatin.</article-title>\n<source><italic>Biophys. J.</italic></source>\n<volume>113</volume>\n<fpage>1925</fpage>–<lpage>1933</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.bpj.2017.08.050</pub-id>\n<pub-id pub-id-type=\"pmid\">29117517</pub-id></mixed-citation></ref><ref id=\"B19\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Hwang</surname><given-names>J. H.</given-names></name><name><surname>An</surname><given-names>S. M.</given-names></name><name><surname>Kwon</surname><given-names>S.</given-names></name><name><surname>Park</surname><given-names>D. H.</given-names></name><name><surname>Kim</surname><given-names>T. W.</given-names></name><name><surname>Kang</surname><given-names>D. G.</given-names></name><etal/></person-group> (<year>2017</year>). <article-title>DNA methylation patterns and gene expression associated with litter size in Berkshire pig placenta.</article-title>\n<source><italic>PLoS One</italic></source>\n<volume>12</volume>:<issue>e0184539</issue>. <pub-id pub-id-type=\"doi\">10.1371/journal.pone.0184539</pub-id>\n<pub-id pub-id-type=\"pmid\">28880934</pub-id></mixed-citation></ref><ref id=\"B20\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Jiang</surname><given-names>Z.</given-names></name><name><surname>Lin</surname><given-names>J.</given-names></name><name><surname>Dong</surname><given-names>H.</given-names></name><name><surname>Zheng</surname><given-names>X.</given-names></name><name><surname>Marjani</surname><given-names>S. L.</given-names></name><name><surname>Duan</surname><given-names>J.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>DNA methylomes of bovine gametes and in vivo produced preimplantation embryos.</article-title>\n<source><italic>Biol. Reprod.</italic></source>\n<volume>99</volume>\n<fpage>949</fpage>–<lpage>959</lpage>. <pub-id pub-id-type=\"doi\">10.1093/biolre/ioy138</pub-id>\n<pub-id pub-id-type=\"pmid\">29912291</pub-id></mixed-citation></ref><ref id=\"B21\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kawaguchi</surname><given-names>F.</given-names></name><name><surname>Kigoshi</surname><given-names>H.</given-names></name><name><surname>Fukushima</surname><given-names>M.</given-names></name><name><surname>Iwamoto</surname><given-names>E.</given-names></name><name><surname>Kobayashi</surname><given-names>E.</given-names></name><name><surname>Oyama</surname><given-names>K.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Whole-genome resequencing to identify candidate genes for the QTL for oleic acid percentage in Japanese Black cattle.</article-title>\n<source><italic>Anim. Sci. J.</italic></source>\n<volume>90</volume>\n<fpage>467</fpage>–<lpage>472</lpage>. <pub-id pub-id-type=\"doi\">10.1111/asj.13179</pub-id>\n<pub-id pub-id-type=\"pmid\">30780197</pub-id></mixed-citation></ref><ref id=\"B22\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Khezri</surname><given-names>A.</given-names></name><name><surname>Narud</surname><given-names>B.</given-names></name><name><surname>Stenseth</surname><given-names>E.-B.</given-names></name><name><surname>Johannisson</surname><given-names>A.</given-names></name><name><surname>Myromslien</surname><given-names>F. D.</given-names></name><name><surname>Gaustad</surname><given-names>A. H.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>DNA methylation patterns vary in boar sperm cells with different levels of DNA fragmentation.</article-title>\n<source><italic>BMC Genomics</italic></source>\n<volume>20</volume>:<issue>897</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12864-019-6307-8</pub-id>\n<pub-id pub-id-type=\"pmid\">31775629</pub-id></mixed-citation></ref><ref id=\"B23\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Kiefer</surname><given-names>H.</given-names></name><name><surname>Perrier</surname><given-names>J.-P.</given-names></name></person-group> (<year>2019</year>). <article-title>DNA methylation in bull spermatozoa: evolutionary impacts, interindividual variability, and contribution to the embryo.</article-title>\n<source><italic>Can. J. Anim. Sci.</italic></source>\n<volume>100</volume>\n<fpage>1</fpage>–<lpage>16</lpage>. <pub-id pub-id-type=\"doi\">10.1139/cjas-2019-0071</pub-id></mixed-citation></ref><ref id=\"B24\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Krueger</surname><given-names>F.</given-names></name><name><surname>Andrews</surname><given-names>S. R.</given-names></name></person-group> (<year>2011</year>). <article-title>Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications.</article-title>\n<source><italic>Bioinformatics</italic></source>\n<volume>27</volume>\n<fpage>1571</fpage>–<lpage>1572</lpage>. <pub-id pub-id-type=\"doi\">10.1093/bioinformatics/btr167</pub-id>\n<pub-id pub-id-type=\"pmid\">21493656</pub-id></mixed-citation></ref><ref id=\"B25\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lambert</surname><given-names>S.</given-names></name><name><surname>Blondin</surname><given-names>P.</given-names></name><name><surname>Vigneault</surname><given-names>C.</given-names></name><name><surname>Labrecque</surname><given-names>R.</given-names></name><name><surname>Dufort</surname><given-names>I.</given-names></name><name><surname>Sirard</surname><given-names>M. A.</given-names></name></person-group> (<year>2018</year>). <article-title>Spermatozoa DNA methylation patterns differ due to peripubertal age in bulls.</article-title>\n<source><italic>Theriogenology</italic></source>\n<volume>106</volume>(<issue>Suppl. C</issue>), <fpage>21</fpage>–<lpage>29</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.theriogenology.2017.10.006</pub-id>\n<pub-id pub-id-type=\"pmid\">29031946</pub-id></mixed-citation></ref><ref id=\"B26\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Lee</surname><given-names>J. Y.</given-names></name><name><surname>Lee</surname><given-names>T.-H.</given-names></name></person-group> (<year>2012</year>). <article-title>Effects of DNA methylation on the structure of nucleosomes.</article-title>\n<source><italic>J. Am. Chem. Soc.</italic></source>\n<volume>134</volume>\n<fpage>173</fpage>–<lpage>175</lpage>. <pub-id pub-id-type=\"doi\">10.1021/ja210273w</pub-id>\n<pub-id pub-id-type=\"pmid\">22148575</pub-id></mixed-citation></ref><ref id=\"B27\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Li</surname><given-names>R.</given-names></name><name><surname>Li</surname><given-names>C.</given-names></name><name><surname>Chen</surname><given-names>H.</given-names></name><name><surname>Li</surname><given-names>R.</given-names></name><name><surname>Chong</surname><given-names>Q.</given-names></name><name><surname>Xiao</surname><given-names>H.</given-names></name><etal/></person-group> (<year>2020</year>). <article-title>Genome-wide scan of selection signatures in Dehong humped cattle for heat tolerance and disease resistance.</article-title>\n<source><italic>Anim. Genet.</italic></source>\n<volume>51</volume>\n<fpage>292</fpage>–<lpage>299</lpage>. <pub-id pub-id-type=\"doi\">10.1111/age.12896</pub-id>\n<pub-id pub-id-type=\"pmid\">31887783</pub-id></mixed-citation></ref><ref id=\"B28\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Long</surname><given-names>M. D.</given-names></name><name><surname>Smiraglia</surname><given-names>D. J.</given-names></name><name><surname>Campbell</surname><given-names>M. J.</given-names></name></person-group> (<year>2017</year>). <article-title>The genomic impact of DNA CpG methylation on gene expression; relationships in prostate cancer.</article-title>\n<source><italic>Biomolecules</italic></source>\n<volume>7</volume>:<issue>15</issue>. <pub-id pub-id-type=\"doi\">10.3390/biom7010015</pub-id>\n<pub-id pub-id-type=\"pmid\">28216563</pub-id></mixed-citation></ref><ref id=\"B29\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Martin</surname><given-names>M.</given-names></name></person-group> (<year>2011</year>). <article-title>Cutadapt removes adapter sequences from high-throughput sequencing reads.</article-title>\n<source><italic>EMBnet. J.</italic></source>\n<volume>17</volume>\n<fpage>10</fpage>–<lpage>12</lpage>. <pub-id pub-id-type=\"doi\">10.14806/ej.17.1.200</pub-id></mixed-citation></ref><ref id=\"B30\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>McSwiggin</surname><given-names>H. M.</given-names></name><name><surname>O’Doherty</surname><given-names>A. M.</given-names></name></person-group> (<year>2018</year>). <article-title>Epigenetic reprogramming during spermatogenesis and male factor infertility.</article-title>\n<source><italic>Reproduction</italic></source>\n<volume>156</volume>\n<fpage>R9</fpage>–<lpage>R21</lpage>. <pub-id pub-id-type=\"doi\">10.1530/REP-18-0009</pub-id>\n<pub-id pub-id-type=\"pmid\">29717022</pub-id></mixed-citation></ref><ref id=\"B31\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Meissner</surname><given-names>A.</given-names></name><name><surname>Gnirke</surname><given-names>A.</given-names></name><name><surname>Bell</surname><given-names>G. W.</given-names></name><name><surname>Ramsahoye</surname><given-names>B.</given-names></name><name><surname>Lander</surname><given-names>E. S.</given-names></name><name><surname>Jaenisch</surname><given-names>R.</given-names></name></person-group> (<year>2005</year>). <article-title>Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis.</article-title>\n<source><italic>Nucleic Acids Res.</italic></source>\n<volume>33</volume>\n<fpage>5868</fpage>–<lpage>5877</lpage>. <pub-id pub-id-type=\"doi\">10.1093/nar/gki901</pub-id>\n<pub-id pub-id-type=\"pmid\">16224102</pub-id></mixed-citation></ref><ref id=\"B32\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Murphy</surname><given-names>E. M.</given-names></name><name><surname>Kelly</surname><given-names>A. K.</given-names></name><name><surname>O’Meara</surname><given-names>C.</given-names></name><name><surname>Eivers</surname><given-names>B.</given-names></name><name><surname>Lonergan</surname><given-names>P.</given-names></name><name><surname>Fair</surname><given-names>S.</given-names></name></person-group> (<year>2018</year>). <article-title>Influence of bull age, ejaculate number, and season of collection on semen production and sperm motility parameters in Holstein Friesian bulls in a commercial artificial insemination centre.</article-title>\n<source><italic>J. Anim. Sci.</italic></source>\n<volume>96</volume>\n<fpage>2408</fpage>–<lpage>2418</lpage>. <pub-id pub-id-type=\"doi\">10.1093/jas/sky130</pub-id>\n<pub-id pub-id-type=\"pmid\">29767722</pub-id></mixed-citation></ref><ref id=\"B33\"><mixed-citation publication-type=\"book\"><collab>NuGEN</collab> (<year>2020</year>). <source><italic>User Guide for Ovation§RRBS Methyl-Seq System.</italic></source>\n<publisher-loc>Redwood City, CA</publisher-loc>: <publisher-name>Tecan Genomics, Inc</publisher-name>, <fpage>1</fpage>–<lpage>16</lpage>.</mixed-citation></ref><ref id=\"B34\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>O’Doherty</surname><given-names>A. M.</given-names></name><name><surname>McGettigan</surname><given-names>P. A.</given-names></name></person-group> (<year>2015</year>). <article-title>Epigenetic processes in the male germline.</article-title>\n<source><italic>Reprod. Fertil. Dev.</italic></source>\n<volume>27</volume>\n<fpage>725</fpage>–<lpage>738</lpage>. <pub-id pub-id-type=\"doi\">10.1071/RD14167</pub-id>\n<pub-id pub-id-type=\"pmid\">25200708</pub-id></mixed-citation></ref><ref id=\"B35\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Oliveros</surname><given-names>J. C.</given-names></name></person-group> (<year>2015</year>). <source><italic>Venny</italic>. <italic>An Interactive Tool for Comparing Lists With Venn’s Diagrams.</italic></source> Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://bioinfogp.cnb.csic.es/tools/venny/index.html\">https://bioinfogp.cnb.csic.es/tools/venny/index.html</ext-link>\n<comment>(accessed February 13, 2020)</comment>.</mixed-citation></ref><ref id=\"B36\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Paul</surname><given-names>J. L.</given-names></name><name><surname>Dashtipour</surname><given-names>K.</given-names></name><name><surname>Chen</surname><given-names>Z.</given-names></name><name><surname>Wang</surname><given-names>C.</given-names></name></person-group> (<year>2019</year>). <article-title>DNA methylome study of human cerebellar tissues identified genes and pathways possibly involved in essential tremor.</article-title>\n<source><italic>Precis. Clin. Med.</italic></source>\n<volume>2</volume>\n<fpage>221</fpage>–<lpage>234</lpage>. <pub-id pub-id-type=\"doi\">10.1093/pcmedi/pbz028</pub-id>\n<pub-id pub-id-type=\"pmid\">31886034</pub-id></mixed-citation></ref><ref id=\"B37\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Perrier</surname><given-names>J. P.</given-names></name><name><surname>Sellem</surname><given-names>E.</given-names></name><name><surname>Prezelin</surname><given-names>A.</given-names></name><name><surname>Gasselin</surname><given-names>M.</given-names></name><name><surname>Jouneau</surname><given-names>L.</given-names></name><name><surname>Piumi</surname><given-names>F.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>A multi-scale analysis of bull sperm methylome revealed both species peculiarities and conserved tissue-specific features.</article-title>\n<source><italic>BMC Genomics</italic></source>\n<volume>19</volume>:<issue>404</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12864-018-4764-0</pub-id>\n<pub-id pub-id-type=\"pmid\">29843609</pub-id></mixed-citation></ref><ref id=\"B38\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Pilsner</surname><given-names>J. R.</given-names></name><name><surname>Shershebnev</surname><given-names>A.</given-names></name><name><surname>Medvedeva</surname><given-names>Y. A.</given-names></name><name><surname>Suvorov</surname><given-names>A.</given-names></name><name><surname>Wu</surname><given-names>H.</given-names></name><name><surname>Goltsov</surname><given-names>A.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Peripubertal serum dioxin concentrations and subsequent sperm methylome profiles of young Russian adults.</article-title>\n<source><italic>Reprod. Toxicol.</italic></source>\n<volume>78</volume>\n<fpage>40</fpage>–<lpage>49</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.reprotox.2018.03.007</pub-id>\n<pub-id pub-id-type=\"pmid\">29550351</pub-id></mixed-citation></ref><ref id=\"B39\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rashid</surname><given-names>F.</given-names></name><name><surname>Ramakrishnan</surname><given-names>A.</given-names></name><name><surname>Fields</surname><given-names>C.</given-names></name><name><surname>Irudayaraj</surname><given-names>J.</given-names></name></person-group> (<year>2020</year>). <article-title>Acute PFOA exposure promotes epigenomic alterations in mouse kidney tissues.</article-title>\n<source><italic>Toxicol. Rep.</italic></source>\n<volume>7</volume>\n<fpage>125</fpage>–<lpage>132</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.toxrep.2019.12.010</pub-id>\n<pub-id pub-id-type=\"pmid\">31938689</pub-id></mixed-citation></ref><ref id=\"B40\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Rawlings</surname><given-names>N.</given-names></name><name><surname>Evans</surname><given-names>A.</given-names></name><name><surname>Chandolia</surname><given-names>R.</given-names></name><name><surname>Bagu</surname><given-names>E.</given-names></name></person-group> (<year>2008</year>). <article-title>Sexual Maturation in the Bull.</article-title>\n<source><italic>Reprod. Domest. Anim.</italic></source>\n<volume>43</volume>(<issue>Suppl. 2</issue>), <fpage>295</fpage>–<lpage>301</lpage>. <pub-id pub-id-type=\"doi\">10.1111/j.1439-0531.2008.01177.x</pub-id>\n<pub-id pub-id-type=\"pmid\">18638138</pub-id></mixed-citation></ref><ref id=\"B41\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Refsdal</surname><given-names>A. O.</given-names></name></person-group> (<year>2007</year>). <article-title>Reproductive performance of Norwegian cattle from 1985 to 2005: trends and seasonality.</article-title>\n<source><italic>Acta Vet. Scand.</italic></source>\n<volume>49</volume>:<issue>5</issue>. <pub-id pub-id-type=\"doi\">10.1186/1751-0147-49-5</pub-id>\n<pub-id pub-id-type=\"pmid\">17295930</pub-id></mixed-citation></ref><ref id=\"B42\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Reis</surname><given-names>L. S. L. S.</given-names></name><name><surname>Ramos</surname><given-names>A. A.</given-names></name><name><surname>Camargos</surname><given-names>A. S.</given-names></name><name><surname>Oba</surname><given-names>E.</given-names></name></person-group> (<year>2016</year>). <article-title>Integrity of the plasma membrane, the acrosomal membrane, and the mitochondrial membrane potential of sperm in Nelore bulls from puberty to sexual maturity.</article-title>\n<source><italic>Arq. Bras. Med. Vet. Zootec.</italic></source>\n<volume>68</volume>\n<fpage>620</fpage>–<lpage>628</lpage>.</mixed-citation></ref><ref id=\"B43\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Standerholen</surname><given-names>F. B.</given-names></name><name><surname>Myromslien</surname><given-names>F. D.</given-names></name><name><surname>Kommisrud</surname><given-names>E.</given-names></name><name><surname>Ropstad</surname><given-names>E.</given-names></name><name><surname>Waterhouse</surname><given-names>K. E.</given-names></name></person-group> (<year>2014</year>). <article-title>Comparison of electronic volume and forward scatter principles of cell selection using flow cytometry for the evaluation of acrosome and plasma membrane integrity of bull spermatozoa.</article-title>\n<source><italic>Cytometry A</italic></source>\n<volume>85</volume>\n<fpage>719</fpage>–<lpage>728</lpage>. <pub-id pub-id-type=\"doi\">10.1002/cyto.a.22474</pub-id>\n<pub-id pub-id-type=\"pmid\">24782360</pub-id></mixed-citation></ref><ref id=\"B44\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Suzuki</surname><given-names>M. M.</given-names></name><name><surname>Bird</surname><given-names>A.</given-names></name></person-group> (<year>2008</year>). <article-title>DNA methylation landscapes: provocative insights from epigenomics.</article-title>\n<source><italic>Nat. Rev. Genet.</italic></source>\n<volume>9</volume>\n<fpage>465</fpage>–<lpage>476</lpage>. <pub-id pub-id-type=\"doi\">10.1038/nrg2341</pub-id>\n<pub-id pub-id-type=\"pmid\">18463664</pub-id></mixed-citation></ref><ref id=\"B45\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Takeda</surname><given-names>K.</given-names></name><name><surname>Kobayashi</surname><given-names>E.</given-names></name><name><surname>Nishino</surname><given-names>K.</given-names></name><name><surname>Imai</surname><given-names>A.</given-names></name><name><surname>Adachi</surname><given-names>H.</given-names></name><name><surname>Hoshino</surname><given-names>Y.</given-names></name><etal/></person-group> (<year>2019</year>). <article-title>Age-related changes in DNA methylation levels at CpG sites in bull spermatozoa and in vitro fertilization-derived blastocyst-stage embryos revealed by combined bisulfite restriction analysis.</article-title>\n<source><italic>J. Reprod. Dev.</italic></source>\n<volume>65</volume>\n<fpage>305</fpage>–<lpage>312</lpage>. <pub-id pub-id-type=\"doi\">10.1262/jrd.2018-146</pub-id>\n<pub-id pub-id-type=\"pmid\">31061296</pub-id></mixed-citation></ref><ref id=\"B46\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tecle</surname><given-names>E.</given-names></name><name><surname>Gagneux</surname><given-names>P.</given-names></name></person-group> (<year>2015</year>). <article-title>Sugar-coated sperm: unraveling the functions of the mammalian sperm glycocalyx.</article-title>\n<source><italic>Mol. Reprod. Dev.</italic></source>\n<volume>82</volume>\n<fpage>635</fpage>–<lpage>650</lpage>. <pub-id pub-id-type=\"doi\">10.1002/mrd.22500</pub-id>\n<pub-id pub-id-type=\"pmid\">26061344</pub-id></mixed-citation></ref><ref id=\"B47\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Tran</surname><given-names>H.</given-names></name><name><surname>Porter</surname><given-names>J.</given-names></name><name><surname>Sun</surname><given-names>M.-A.</given-names></name><name><surname>Xie</surname><given-names>H.</given-names></name><name><surname>Zhang</surname><given-names>L.</given-names></name></person-group> (<year>2014</year>). <article-title>Objective and comprehensive evaluation of bisulfite short read mapping tools.</article-title>\n<source><italic>Adv. Bioinformat.</italic></source>\n<volume>2014</volume>\n<fpage>472045</fpage>–<lpage>472045</lpage>. <pub-id pub-id-type=\"doi\">10.1155/2014/472045</pub-id>\n<pub-id pub-id-type=\"pmid\">24839440</pub-id></mixed-citation></ref><ref id=\"B48\"><mixed-citation publication-type=\"journal\"><collab>UCSC</collab> (<year>2018</year>). <source><italic>Table Browser for bosTau 9 Assembly.</italic></source> Available online at: <ext-link ext-link-type=\"uri\" xlink:href=\"https://genome.ucsc.edu/cgi-bin/hgTables\">https://genome.ucsc.edu/cgi-bin/hgTables</ext-link>\n<comment>(accessed September 18, 2019)</comment>.</mixed-citation></ref><ref id=\"B49\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Waters</surname><given-names>S. M.</given-names></name><name><surname>Coyne</surname><given-names>G. S.</given-names></name><name><surname>Kenny</surname><given-names>D. A.</given-names></name><name><surname>Morris</surname><given-names>D. G.</given-names></name></person-group> (<year>2014</year>). <article-title>Effect of dietary n-3 polyunsaturated fatty acids on transcription factor regulation in the bovine endometrium.</article-title>\n<source><italic>Mol. Biol. Rep.</italic></source>\n<volume>41</volume>\n<fpage>2745</fpage>–<lpage>2755</lpage>. <pub-id pub-id-type=\"doi\">10.1007/s11033-014-3129-2</pub-id>\n<pub-id pub-id-type=\"pmid\">24449365</pub-id></mixed-citation></ref><ref id=\"B50\"><mixed-citation publication-type=\"book\"><person-group person-group-type=\"author\"><name><surname>Wickham</surname><given-names>H.</given-names></name></person-group> (<year>2016</year>). <source><italic>ggplot2: Elegant Graphics for Data Analysis.</italic></source>\n<publisher-loc>New York, NY</publisher-loc>: <publisher-name>Springer-Verlag</publisher-name>.</mixed-citation></ref><ref id=\"B51\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wreczycka</surname><given-names>K.</given-names></name><name><surname>Gosdschan</surname><given-names>A.</given-names></name><name><surname>Yusuf</surname><given-names>D.</given-names></name><name><surname>Gruning</surname><given-names>B.</given-names></name><name><surname>Assenov</surname><given-names>Y.</given-names></name><name><surname>Akalin</surname><given-names>A.</given-names></name></person-group> (<year>2017</year>). <article-title>Strategies for analyzing bisulfite sequencing data.</article-title>\n<source><italic>J. Biotechnol.</italic></source>\n<volume>261</volume>\n<fpage>105</fpage>–<lpage>115</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.jbiotec.2017.08.007</pub-id>\n<pub-id pub-id-type=\"pmid\">28822795</pub-id></mixed-citation></ref><ref id=\"B52\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Wu</surname><given-names>C.</given-names></name><name><surname>Blondin</surname><given-names>P.</given-names></name><name><surname>Vigneault</surname><given-names>C.</given-names></name><name><surname>Labrecque</surname><given-names>R.</given-names></name><name><surname>Sirard</surname><given-names>M.-A.</given-names></name></person-group> (<year>2020</year>). <article-title>The age of the bull influences the transcriptome and epigenome of blastocysts produced by IVF.</article-title>\n<source><italic>Theriogenology</italic></source>\n<volume>144</volume>\n<fpage>122</fpage>–<lpage>131</lpage>. <pub-id pub-id-type=\"doi\">10.1016/j.theriogenology.2019.12.020</pub-id>\n<pub-id pub-id-type=\"pmid\">31951983</pub-id></mixed-citation></ref><ref id=\"B53\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhou</surname><given-names>Y.</given-names></name><name><surname>Connor</surname><given-names>E. E.</given-names></name><name><surname>Bickhart</surname><given-names>D. M.</given-names></name><name><surname>Li</surname><given-names>C.</given-names></name><name><surname>Baldwin</surname><given-names>R. L.</given-names></name><name><surname>Schroeder</surname><given-names>S. G.</given-names></name><etal/></person-group> (<year>2018</year>). <article-title>Comparative whole genome DNA methylation profiling of cattle sperm and somatic tissues reveals striking hypomethylated patterns in sperm.</article-title>\n<source><italic>GigaScience</italic></source>\n<volume>7</volume>:<issue>giy039</issue>. <pub-id pub-id-type=\"doi\">10.1093/gigascience/giy039</pub-id>\n<pub-id pub-id-type=\"pmid\">29635292</pub-id></mixed-citation></ref><ref id=\"B54\"><mixed-citation publication-type=\"journal\"><person-group person-group-type=\"author\"><name><surname>Zhou</surname><given-names>Y.</given-names></name><name><surname>Xu</surname><given-names>L.</given-names></name><name><surname>Bickhart</surname><given-names>D. M.</given-names></name><name><surname>Abdel Hay</surname><given-names>E. H.</given-names></name><name><surname>Schroeder</surname><given-names>S. G.</given-names></name><name><surname>Connor</surname><given-names>E. E.</given-names></name><etal/></person-group> (<year>2016</year>). <article-title>Reduced representation bisulphite sequencing of ten bovine somatic tissues reveals DNA methylation patterns and their impacts on gene expression.</article-title>\n<source><italic>BMC Genomics</italic></source>\n<volume>17</volume>:<issue>779</issue>. <pub-id pub-id-type=\"doi\">10.1186/s12864-016-3116-1</pub-id>\n<pub-id pub-id-type=\"pmid\">27716143</pub-id></mixed-citation></ref></ref-list></back></article>\n"
] |
